Preface

We are pleased to present our new book *Reproductive Biology and Technology in Animals*. This book has been designed to provide innovative information on new reproductive technologies in different animal species, including sheep, gilts, pigs, and other invertebrates. It is intended for academics, scientists, and veterinarians to update and deepen their knowledge of the physiology of reproduction. Therefore, the motivation to publish a comprehensive volume on advances in biotechnology arose from the growing awareness of recent advances in sperm management, oocyte characteristics, various genomic aspects related to somatic cell nuclear transfer, and the microarchitecture of the female reproductive system in these animal species. Other aspects have also been considered that refer to the reproductive cycles present in gastropods and fish. This volume is the culmination of the efforts of several researchers, scientists, and scholars from around the world who are well known and respected in the various frontiers of research into the physiology of reproduction. We sincerely believe that the book will prove to be a useful contribution not only to science but also to the general public. We hope that this book will be a useful source of information from both a scientific and practical point of view. We are grateful to all the contributing authors and to all the members of the international review panel who helped us enormously with their contributions, time, critical thinking, and suggestions in producing this edited and peer-reviewed volume. The editors are also grateful to IntechOpen and its team members for the opportunity to publish this book. Finally, we thank our family members for their love, support, encouragement, and patience throughout the period of this work.

**II**

**Chapter 7 113**

*by Verónica Mitsui Saito-Quezada, Esther Uría-Galicia, José Luis Gómez-Márquez,* 

Reproductive Cycle of *Hexaplex princeps* (Broderip, 1833)

*and Isaías Hazarmabeth Salgado-Ugarte*

*Ana Bertha Villaseñor-Martínez, Ma. De Lourdes Jiménez-Badillo* 

#### **Juan Carlos Gardon Poggi**

Department of Animal Medicine and Surgery, University Catholic of Valencia "San Vicente Mártir", Valencia, Spain

#### **Katy Satué Ambrojo**

Faculty of Veterinary, Department of Animal Medicine and Surgery, University of CEU-Cardenal Herrera, Valencia, Spain

Section 1

Mammals

**1**

Section 1 Mammals

**Chapter 1**

**Abstract**

**1. Introduction**

until birth [1].

**3**

**2. Microstructure of cortex of the ovary**

Micro-architecture of the Female

The female reproductive system consists of the ovary, oviduct, uterus, and vagina. This chapter will discuss how these organs look like under the microscope and what types of ultrastructural tissues are present in it, how the shape and physiology of the tissues/cells change with the physiological activities including reproductive cycles, what type of alterations occurs in the ovary during ovulation and how its follicle and epithelium differ, and how the ovulation takes place. The chapter will also elaborate how the lining epithelium and the tract mucosa facilitate the fertilized ovum and conceptus. Also, the chapter is highlighting the architectural changes within the mucosa of the uterus during and after pregnancy and type

**Keywords:** ovary, reproduction, microstructures, cortex, mucosa, zygote, sperm

the follicles especially the graafian follicle including mature ova, and the

It is important to know the microstructure of the female reproductive system. In this chapter those microarchitectures are highlighted which played an important role in theriogenology right from release to fertilization and care of embryo and infants. The micro-architecture of the ovarian cortex, the microscopic structures of

histomorphology of the fimbriae and oviduct and its function in ova transportation, zygote transformation, and embryo implantation were highlighted. The microstructures of various microscopic layers of the uterus and its role in reproduction were addressed. The structure and function of the cervix and vulva and its role in reproduction are mentioned. Also, the most suitable type of ova and sperm for fertilization was also mentioned. The whole reproductive system is concerned with the production and transport of the ovum, facilitation in the transport of spermatozoa, the fusion of both gametes, and the accommodation of the embryo and fetus

The cortex is a wide peripheral part of the ovary that contains follicles in various stages [2]. The surface of the ovary is covered by a thin layer of cuboidal cells, also called as germinal epithelium. Next to the germinal epithelium in the inferior side is

a thick layer of connective tissue (CT) called the tunica albuginea. And the

of ovary and spermatozoa that is most suitable for fertilization.

Reproductive System

*Arbab Sikandar and Muhammad Ali*

#### **Chapter 1**

## Micro-architecture of the Female Reproductive System

*Arbab Sikandar and Muhammad Ali*

#### **Abstract**

The female reproductive system consists of the ovary, oviduct, uterus, and vagina. This chapter will discuss how these organs look like under the microscope and what types of ultrastructural tissues are present in it, how the shape and physiology of the tissues/cells change with the physiological activities including reproductive cycles, what type of alterations occurs in the ovary during ovulation and how its follicle and epithelium differ, and how the ovulation takes place. The chapter will also elaborate how the lining epithelium and the tract mucosa facilitate the fertilized ovum and conceptus. Also, the chapter is highlighting the architectural changes within the mucosa of the uterus during and after pregnancy and type of ovary and spermatozoa that is most suitable for fertilization.

**Keywords:** ovary, reproduction, microstructures, cortex, mucosa, zygote, sperm

#### **1. Introduction**

It is important to know the microstructure of the female reproductive system. In this chapter those microarchitectures are highlighted which played an important role in theriogenology right from release to fertilization and care of embryo and infants. The micro-architecture of the ovarian cortex, the microscopic structures of the follicles especially the graafian follicle including mature ova, and the histomorphology of the fimbriae and oviduct and its function in ova transportation, zygote transformation, and embryo implantation were highlighted. The microstructures of various microscopic layers of the uterus and its role in reproduction were addressed. The structure and function of the cervix and vulva and its role in reproduction are mentioned. Also, the most suitable type of ova and sperm for fertilization was also mentioned. The whole reproductive system is concerned with the production and transport of the ovum, facilitation in the transport of spermatozoa, the fusion of both gametes, and the accommodation of the embryo and fetus until birth [1].

#### **2. Microstructure of cortex of the ovary**

The cortex is a wide peripheral part of the ovary that contains follicles in various stages [2]. The surface of the ovary is covered by a thin layer of cuboidal cells, also called as germinal epithelium. Next to the germinal epithelium in the inferior side is a thick layer of connective tissue (CT) called the tunica albuginea. And the

remaining portion of the ovarian cortex is covered by the CT which contains the primordial follicles, surrounded by a flat follicular cell (squamous shaped). Another follicle a bit larger is called a primary follicle, internally lined by the simple and stratified cuboidal epithelium termed as granulosa cells. In the connective tissue of the cortex of the ovary, many blood vessels surround the developing follicles. The granulosa cells are more prominent in the larger follicle called as secondary follicle or also called as antral follicle [3]. The antrum is the cavity formation within the follicle, which splits the granulosa cells into two layers. The fluid within the antrum is called as liquor folliculi. A single large ovum is present in the follicle, and a single layer of granulosa cells which surrounds the oocyte is known as corona radiata [4]. There are acellular glycoprotein layers known as zona pellucida between the corona radiata and oocyte. The zona pellucida which is 3–5 μm in thickness is produced by the ova and the granulosa cells. Within the antrum the ova are anchored by a compact mass of cells called as cumulus oophorus. After releasing the ova, the granulosa cells are disorganized within the antrum with pyknotic nuclei (fragmentation of nuclei) in the remaining follicles. This condition of the inactive follicles is called as atretic follicle [5].

luteinizing hormone (LH). Theca interna is highly vascularized, and its function is to deliver hormone, nutrients, vitamins, and cofactors, which are necessary for

In the graafian follicle, the granulosa cells and oocyte have a specific shape and

It is rich in hyaluronan and proteoglycans. It helps in follicle ruptures. Ovulation

occurs under the influence of follicle-stimulating hormone (FSH) and LH [7]. During ovulation the ova are in the metaphase stage of the second meiotic division.

**4. Microstructure and physiology of the oviduct and how it facilitates**

The oviduct has several layers including the mucosa, muscular layer, and a connective serosa. The size of these layers depends on different regions of the oviduct. The myosalpinx is comprised of smooth muscle; it is thin in the region of the ampulla and multilayered thick in the region of the isthmus. The endosalpinx is a term used for a mucosa, which is lined with simple columnar ciliated epithelium [8]. In the ampulla, the lining ciliated cells are numerous compared with that of secretory cells. The lining mucosa forms folds upward that fill the tubular lumen [9]. Due to the abundance of upward-directed folds, the lining mucosa of the ampulla increased the surface area, assuming excessive ability for the metabolic conversation between the epithelial, luminal, and the underlining vascular compartments. The mucosa of the isthmus has a smaller number of folds, while the

Sperm and oocyte will not reach the oviduct at the same time. In some animals the oocyte reaches first, and, in some, sperm approaches initially to the site of fertilization. The movement of sperm toward the isthmus is promoted by the ciliary movement of the lining epithelium. The spermatozoa are stored and remain active in the oviduct for up to 72 hours, but in some species the time is extended for a month like in bats. The fallopian tube contains fluid secreted by the glandular epithelium, which has a positive impact on the viability of sperm and provides nutrition to the oocyte [8]. This fluid helps in the maturation of the oocyte in the oviduct. Sperm adhere to the epithelial wall of the fallopian tube which preserves

position [6]. There are four different types of granulosa cell layers:

growth of the oocyte and granulosa cells [4].

*Micro-architecture of the Female Reproductive System DOI: http://dx.doi.org/10.5772/intechopen.88023*

• The outermost is membrana granulosa.

• The intermediate is cumulus oophorus.

• Just opposed to oocyte is corona radiata.

**the sperm, ovum, and early-stage embryo**

abundant number of secretory cells is present there.

**3.3 Granulosa cells and oocyte**

• The inner most is periantral.

**4.1 Microstructure of the oviduct**

**4.2 Function of the oviduct**

**5**

**3.4 Follicular fluid**

#### **3. Microstructure modulation in graafian follicle**

A graafian follicle consists of a large cavity called antrum having fluid termed as follicular fluid. Graafian follicle is also known as antral follicle [3]. Before ovulation, secondary follicle undergoes first mitotic division, and as a result the graafian follicle is formed which has two N haploid chromosomes. The characteristic structural feature of graafian follicle is follicular antrum in which granulosa cell and oocyte are present. Antral follicle is a three-dimensional structure having a cavity (antrum) which is surrounded by different types of cells [6]. The antral follicle has six different histologic components which are as follows:


#### **3.1 Theca externa**

Theca externa consists of smooth muscle cells that are innervated by the autonomic nerve. The functional importance of theca externa is still unclear, but there is evidence that changes in the contractile activity of theca externa are involved in atresia and ovulation [5].

#### **3.2 Theca interna**

It is consisting of different cells which are present in the matrix of loose connective tissues and blood vessels. The function of theca interna cells is regulated by luteinizing hormone (LH). Theca interna is highly vascularized, and its function is to deliver hormone, nutrients, vitamins, and cofactors, which are necessary for growth of the oocyte and granulosa cells [4].

#### **3.3 Granulosa cells and oocyte**

remaining portion of the ovarian cortex is covered by the CT which contains the primordial follicles, surrounded by a flat follicular cell (squamous shaped). Another follicle a bit larger is called a primary follicle, internally lined by the simple and stratified cuboidal epithelium termed as granulosa cells. In the connective tissue of the cortex of the ovary, many blood vessels surround the developing follicles. The granulosa cells are more prominent in the larger follicle called as secondary follicle or also called as antral follicle [3]. The antrum is the cavity formation within the follicle, which splits the granulosa cells into two layers. The fluid within the antrum is called as liquor folliculi. A single large ovum is present in the follicle, and a single layer of granulosa cells which surrounds the oocyte is known as corona radiata [4]. There are acellular glycoprotein layers known as zona pellucida between the corona radiata and oocyte. The zona pellucida which is 3–5 μm in thickness is produced by the ova and the granulosa cells. Within the antrum the ova are anchored by a compact mass of cells called as cumulus oophorus. After releasing the ova, the granulosa cells are disorganized within the antrum with pyknotic nuclei (fragmentation of nuclei) in the remaining follicles. This condition of the inactive follicles is

A graafian follicle consists of a large cavity called antrum having fluid termed as follicular fluid. Graafian follicle is also known as antral follicle [3]. Before ovulation, secondary follicle undergoes first mitotic division, and as a result the graafian follicle is formed which has two N haploid chromosomes. The characteristic structural feature of graafian follicle is follicular antrum in which granulosa cell and oocyte are present. Antral follicle is a three-dimensional structure having a cavity (antrum) which is surrounded by different types of cells [6]. The antral follicle has

Theca externa consists of smooth muscle cells that are innervated by the autonomic nerve. The functional importance of theca externa is still unclear, but there is evidence that changes in the contractile activity of theca externa are involved in

It is consisting of different cells which are present in the matrix of loose connective tissues and blood vessels. The function of theca interna cells is regulated by

called as atretic follicle [5].

*Reproductive Biology and Technology in Animals*

• The theca externa

• The theca interna

• The basal lamina

• The granulosa cell

• The follicular fluid

atresia and ovulation [5].

• The oocyte

**3.1 Theca externa**

**3.2 Theca interna**

**4**

**3. Microstructure modulation in graafian follicle**

six different histologic components which are as follows:

In the graafian follicle, the granulosa cells and oocyte have a specific shape and position [6]. There are four different types of granulosa cell layers:


#### **3.4 Follicular fluid**

It is rich in hyaluronan and proteoglycans. It helps in follicle ruptures. Ovulation occurs under the influence of follicle-stimulating hormone (FSH) and LH [7]. During ovulation the ova are in the metaphase stage of the second meiotic division.

#### **4. Microstructure and physiology of the oviduct and how it facilitates the sperm, ovum, and early-stage embryo**

#### **4.1 Microstructure of the oviduct**

The oviduct has several layers including the mucosa, muscular layer, and a connective serosa. The size of these layers depends on different regions of the oviduct. The myosalpinx is comprised of smooth muscle; it is thin in the region of the ampulla and multilayered thick in the region of the isthmus. The endosalpinx is a term used for a mucosa, which is lined with simple columnar ciliated epithelium [8]. In the ampulla, the lining ciliated cells are numerous compared with that of secretory cells. The lining mucosa forms folds upward that fill the tubular lumen [9]. Due to the abundance of upward-directed folds, the lining mucosa of the ampulla increased the surface area, assuming excessive ability for the metabolic conversation between the epithelial, luminal, and the underlining vascular compartments. The mucosa of the isthmus has a smaller number of folds, while the abundant number of secretory cells is present there.

#### **4.2 Function of the oviduct**

Sperm and oocyte will not reach the oviduct at the same time. In some animals the oocyte reaches first, and, in some, sperm approaches initially to the site of fertilization. The movement of sperm toward the isthmus is promoted by the ciliary movement of the lining epithelium. The spermatozoa are stored and remain active in the oviduct for up to 72 hours, but in some species the time is extended for a month like in bats. The fallopian tube contains fluid secreted by the glandular epithelium, which has a positive impact on the viability of sperm and provides nutrition to the oocyte [8]. This fluid helps in the maturation of the oocyte in the oviduct. Sperm adhere to the epithelial wall of the fallopian tube which preserves

its viability and stops the premature capacitation. This enhances the chances of fertilization [1].

**5.2 Process of fertilization within the fallopian tube**

[11], which results in the formation of zygote.

*Micro-architecture of the Female Reproductive System DOI: http://dx.doi.org/10.5772/intechopen.88023*

**reproduction**

**6.1 Mucosa**

**6.2 Submucosa**

**7**

At the site of ampulla-isthmus junction, the capacitated and hyperactive spermatozoa crossed the layer of corona radiata and pierced the glycoprotein layer of zona pellucida. Afterward the fusion of the spermatozoa with the ova takes place

If we see the cross section of the uterus, it consists of four layers including the innermost mucosa, the submucosa, the muscularis, and the outermost serosa [9].

It is the innermost layer facing the lumen. It is lined with secretary layers of columnar epithelium. The embryo after a series of changes including zygote, twocell (blastomeres) stage, morula (16-cell stage), blastula (inner cell mass leads to embryo, and the outer cell mass leads to the formation of the placenta, with the formation of fluid filled cavity), and gastrula (where formation of three characteristic embryonic germ layers occurs) is implanted in the endometrial mucosa with a layer of trophoblast [1]. The trophoblastic cell layers are proliferated in the lamina propria (uterine stroma) forming lacunae. The lacunae form an open connection with the maternal sinusoid in the uterine stroma, which results in the establishment of the uteroplacental circulation. The endometrial glands develop from the mucosa and invaginate inward into the submucosa adopting a coiled shape. The endometrial

glands secrete PGF2 alpha at a critical time during estrous cycle that causes

helps in pushing the baby during parturition [8].

• Transportation of the secretary products

• Transportation of the gametes (sperm and ova)

luteolysis of corpus luteum if the animal is not pregnant [12]. The endometrium of ruminants has caruncles that are highly vascularized and provide a maternal portion of the placenta if attachment of the embryo occurs. While these caruncles are absent in sow and mare, instead endometrial folds are present. These endometrial folds provide uterine surface for the attachment of the placenta. So, the uterus is the place where attachment and nourishment of the fertilized ova take place, and it

After the mucosa, a layer of submucosa is present. It houses a rich blood supply, nerve endings, and the lymphatics in addition to the rich supply of mononuclear cells. It has a role in providing nutrition and maintaining coordination and has supporting effect to the mucosa [9]. The muscularis is present beneath the submucosa and consists of double arranged layers of smooth muscles like longitudinal muscles (outer layer) and inner circular muscles (inner layer). A very prominent third layer of smooth muscle in oblique arrangement can also be seen in the gravid uterus. These layers played a very important function by providing the uterus with the ability to contract, and that contraction is very important on the following basis:

**6. Histological layers, physiology of the uterus, and its role in**

Fertilization of the ovum results in the formation of zygotes. After all the embryonic changes in the zygote, the later embryo is transferred along the fallopian tube. Some hormone also helps in the transportation of the embryo. The production of the estradiol (E2) by the embryo also helps in its transportation. In the mare, the secretion of prostaglandin (PGE2) during the first week acts on the oviduct locally and causes the transfer of the embryo to the posterior region of the uterus. The 4- to 8-cell stage embryos in hamster secrete platelet-activating factor (PAF). The later factor also acts on the oviduct locally, resulting in the transfer of the embryo toward the uterine horns. It is important to note that in the hamster and the mare, the main factor, decisive whether and when the fertilized ovum will drive toward the uterine horns or not, is based on the condition of the fertilized eggs.

#### **4.3 Transportation of oocyte**

During ovulation the fimbriae attached to the surface of graafian follicle and sweep the ova further anterior toward the ampulla [8]. The coordinated contraction of the myosalpinx along the length of the fallopian tube and the ciliary beats of the epithelial cells results in the transportation of the oocyte to the site of fertilization [9].

#### **5. Microstructures and physiology of fimbriae**

Fimbriae are finger-like growth processes that are present at the terminal of each fallopian tube. They increase the surface area of the infundibulum and cause it to slip over the surface of the ovary when ovulation occurs. This activity of the fimbriae increases the possibility that the oozed-out oocyte will be captured after ovulation from the graafian follicle and transported through a wider opening called ostium into the ampulla of the oviduct [9]. Above the mucus membrane of the fallopian tube, there are three layers of tissues, viz., the innermost layer consists of spirally arranged fibers, the middle layer consists of fibers with circular arrangement, and the outermost sheath has longitudinally arranged fibers that end in fimbriae near ovaries, forming a funnel-shaped depository structure called the infundibulum. One fimbria out of all fimbriae is long enough to reach the external surface of the ovary, which is called as fimbria ovarica [10]. The fallopian tube at this point is lined by small epithelial cells with small, slender hair-like cilia pulsating unidirectionally inside the fallopian tube to guide and direct the ovum from the ovary to the uterus [9].

#### **5.1 Role of fimbriae in directing the ova to area of fertilization**

As there is no direct connection between ovaries and the oviduct, the egg is transported to the uterus in peritoneal fluid produced by fimbriae on the terminal of tube's opening. The specialized granulosa cells of oocyte (cumulus oophorous) at ovulation are sticky and help in adhering to the surface of fimbriae. After this the fimbriae through muscular control create negative pressure that picks up oocyte and moves it to the fallopian tube [9]. There are also some hormonal changes that control the picking up of oocyte [7].

#### **5.2 Process of fertilization within the fallopian tube**

At the site of ampulla-isthmus junction, the capacitated and hyperactive spermatozoa crossed the layer of corona radiata and pierced the glycoprotein layer of zona pellucida. Afterward the fusion of the spermatozoa with the ova takes place [11], which results in the formation of zygote.

#### **6. Histological layers, physiology of the uterus, and its role in reproduction**

If we see the cross section of the uterus, it consists of four layers including the innermost mucosa, the submucosa, the muscularis, and the outermost serosa [9].

#### **6.1 Mucosa**

its viability and stops the premature capacitation. This enhances the chances

tube. Some hormone also helps in the transportation of the embryo. The

Fertilization of the ovum results in the formation of zygotes. After all the embryonic changes in the zygote, the later embryo is transferred along the fallopian

production of the estradiol (E2) by the embryo also helps in its transportation. In the mare, the secretion of prostaglandin (PGE2) during the first week acts on the oviduct locally and causes the transfer of the embryo to the posterior region of the uterus. The 4- to 8-cell stage embryos in hamster secrete platelet-activating factor (PAF). The later factor also acts on the oviduct locally, resulting in the transfer of the embryo toward the uterine horns. It is important to note that in the hamster and the mare, the main factor, decisive whether and when the fertilized ovum will drive toward the uterine horns or not, is based on the condition of the

During ovulation the fimbriae attached to the surface of graafian follicle and

contraction of the myosalpinx along the length of the fallopian tube and the ciliary beats of the epithelial cells results in the transportation of the oocyte to the site of

Fimbriae are finger-like growth processes that are present at the terminal of each fallopian tube. They increase the surface area of the infundibulum and cause it to slip over the surface of the ovary when ovulation occurs. This activity of the fimbriae increases the possibility that the oozed-out oocyte will be captured after ovulation from the graafian follicle and transported through a wider opening called ostium into the ampulla of the oviduct [9]. Above the mucus membrane of the fallopian tube, there are three layers of tissues, viz., the innermost layer consists of spirally arranged fibers, the middle layer consists of fibers with circular arrangement, and the outermost sheath has longitudinally arranged fibers that end in fimbriae near ovaries, forming a funnel-shaped depository structure called the infundibulum. One fimbria out of all fimbriae is long enough to reach the external surface of the ovary, which is called as fimbria ovarica [10]. The fallopian tube at this point is lined by small epithelial cells with small, slender hair-like cilia pulsating unidirectionally inside the fallopian tube to guide and direct the ovum from the

sweep the ova further anterior toward the ampulla [8]. The coordinated

**5. Microstructures and physiology of fimbriae**

**5.1 Role of fimbriae in directing the ova to area of fertilization**

As there is no direct connection between ovaries and the oviduct, the egg is transported to the uterus in peritoneal fluid produced by fimbriae on the terminal of tube's opening. The specialized granulosa cells of oocyte (cumulus oophorous) at ovulation are sticky and help in adhering to the surface of fimbriae. After this the fimbriae through muscular control create negative pressure that picks up oocyte and moves it to the fallopian tube [9]. There are also some hormonal changes that

of fertilization [1].

*Reproductive Biology and Technology in Animals*

fertilized eggs.

fertilization [9].

ovary to the uterus [9].

**6**

control the picking up of oocyte [7].

**4.3 Transportation of oocyte**

It is the innermost layer facing the lumen. It is lined with secretary layers of columnar epithelium. The embryo after a series of changes including zygote, twocell (blastomeres) stage, morula (16-cell stage), blastula (inner cell mass leads to embryo, and the outer cell mass leads to the formation of the placenta, with the formation of fluid filled cavity), and gastrula (where formation of three characteristic embryonic germ layers occurs) is implanted in the endometrial mucosa with a layer of trophoblast [1]. The trophoblastic cell layers are proliferated in the lamina propria (uterine stroma) forming lacunae. The lacunae form an open connection with the maternal sinusoid in the uterine stroma, which results in the establishment of the uteroplacental circulation. The endometrial glands develop from the mucosa and invaginate inward into the submucosa adopting a coiled shape. The endometrial glands secrete PGF2 alpha at a critical time during estrous cycle that causes luteolysis of corpus luteum if the animal is not pregnant [12]. The endometrium of ruminants has caruncles that are highly vascularized and provide a maternal portion of the placenta if attachment of the embryo occurs. While these caruncles are absent in sow and mare, instead endometrial folds are present. These endometrial folds provide uterine surface for the attachment of the placenta. So, the uterus is the place where attachment and nourishment of the fertilized ova take place, and it helps in pushing the baby during parturition [8].

#### **6.2 Submucosa**

After the mucosa, a layer of submucosa is present. It houses a rich blood supply, nerve endings, and the lymphatics in addition to the rich supply of mononuclear cells. It has a role in providing nutrition and maintaining coordination and has supporting effect to the mucosa [9]. The muscularis is present beneath the submucosa and consists of double arranged layers of smooth muscles like longitudinal muscles (outer layer) and inner circular muscles (inner layer). A very prominent third layer of smooth muscle in oblique arrangement can also be seen in the gravid uterus. These layers played a very important function by providing the uterus with the ability to contract, and that contraction is very important on the following basis:


It also has a key role in the expulsion of the fetus and fetal membranes at the time of parturition.

#### **6.3 Serosa**

It is the outer covering of the uterus that consists of a single layer of squamous cells.

**8. Gross and microscopic anatomy of the vulva and its role in**

present in the vulvar region of the female reproductive system:

• Apocrine glands (scent glands)

*Micro-architecture of the Female Reproductive System DOI: http://dx.doi.org/10.5772/intechopen.88023*

• Eccrine glands (sweat glands)

**8.1 Microscopic anatomy of vulva**

**8.2 Role of the vulva in reproduction**

• Enabling sperm to enter in the body of female

• Providing the sexual pleasure

The vulva is the most outer part of the female reproductive track immediately external to the vaginae. It is composed of various structures, including the mons pubis, clitoris, labia minora, labia majora, vulvar vestibule, vestibulovaginal bulbs, urethral meatus, hymen, and Bartholin and Skene glands and ducts. Bartholin glands are like the bulbourethral glands in male. The female clitoris is like the penis of the males [13]. The hymen is the stratified squamous nonkeratinized epithelium. The lateral vulvar surfaces are made up of labia majora and are comprised of fibrous and adipose folds. The labia minora also consists of connective tissue in two folds containing few (if any) adipose tissues. The labia minora is bifurcated anteriorly. The vestibule is the zone between Hart's line and the hymen, which consists of stratified squamous nonkeratinized epithelium [9]. The following are the glands

Vaginal lining epithelium is stratified squamous and nonkeratinized, whereas the epithelium of the labia majora is stratified squamous and keratinized. Microscopic layers of the vulva are the mucosa, submucosa, muscularis, and serosa [14, 15]. Glands are present in the submucosal layer and the innermost layer is the

Being a gateway to the uterus, its primary role is to offer protection by closing the labia [16]. The external uterine orifice is protected by the vulva supported by thick large lips of labia majora along with small lips of labia minora. The urethra also opens in the vulva that is known as the urethral meatus, thus performing a function of urine passage. It also gives the pathway for sperm for entry in the body. Sometimes due to the folds and the moisture, the fungal infection may occur. The area between the opening of the vagina and the anus, below the labia majora, is called the perineum [12]. By observing the vulva, we can detect if the female is in estrous or not. If the vulva is thick and edematous, it is in the heat, and if not the female is not in the heat; this is due to the estrogen level in the female body. The penis like clitoris detects the nerve stimuli and performs the following

• Protecting the internal genital organs from infection causing organisms

**reproduction**

• Skene glands

mucosa.

functions:

**9**

• Sebaceous glands

#### **7. Gross and microscopic structures and functions of the cervix**

The whole reproductive tract is tubular except the ovary. The cervix is a thickwalled tubular structure where mucosa-submucosa-derived folds or rings are present, but their number varies from species to species. Only onefold is seen in bitch and queen, but multiple folds are seen in cow, ewe, sow, and mare. In cow and ewe, rings/folds are having interlocking finger-like projections. In pigs, rings interdigitate so interdigitation is seen in them. The cervix is soft in mare due to the presence of loose folds of the mucosa. In a bitch the cervix is smooth due to lack of folds. The primary function of the cervix is lubrication of the canal which is required during copulation [8]. Lubrication is by two types of secretions, e.g., the sulfomucin which is viscous (mobility is directed toward exterior (vagina) and inhibits sperm transport in the uterus), and the sialomucins which facilitate the sperm to move into the uterus. The cervix isolates the conceptus inside the uterus from the outer environment by the formation of cervical seal under the action of progesterone which thickens the mucus produced in the cervix and glues the folding together to prevent entry of microbes during pregnancy.

Like other tubular organs, it consists of four tunics/laminae, i.e., mucosa, submucosa, muscularis, and serosa/adventitia [9]. The mucosal tunics of the endocervix are lined by simple columnar epithelium along with the presence of mucus-producing cells called goblet cells. In some animal species, a few columnar cells are found ciliated and in simple tubular glands may be seen in the ruminant's mucosa. In the case of pigs, up to 90% of the mucosal lining epithelium is stratified squamous. Most of the ectocervical portion of the cervical mucosa is lined by the stratified squamous epithelium. Mucosa and the submucosa form folds into the cervical lumen. These folds vary in height, width, and thickness from species to species. The lamina propria of the mucosa and submucosa combines to form propria-submucosa. This propriasubmucosa is heavily infiltrated by dense irregular connective tissue and supply of rich blood vessels extended deep in the submucosa. The muscularis layer is composed of two smooth muscle layers. The inner layer is circular layer and the outer layer is longitudinal in arrangements. The myenteric plexus is present in the muscular layers. Some elastic fibers are also observed in this layer. Serosa/adventitia is a loose connective tissue layer present in the outer surface. This layer is surrounded by the mesothelium which is a layer of simple squamous epithelium. Gartner's ducts may be seen in serosal layer unilateral or bilateral.

#### **7.1 Functions of the cervix**

During estrus stage of estrous cycle, the mucosa is responsible for mucus secretion. Dense irregular connective tissue becomes edematous and forms areolar structure, i.e., loose connective tissue. Circular muscular layer along with elastic fiber is responsible for the involution of the cervix after parturition [8].

#### **8. Gross and microscopic anatomy of the vulva and its role in reproduction**

The vulva is the most outer part of the female reproductive track immediately external to the vaginae. It is composed of various structures, including the mons pubis, clitoris, labia minora, labia majora, vulvar vestibule, vestibulovaginal bulbs, urethral meatus, hymen, and Bartholin and Skene glands and ducts. Bartholin glands are like the bulbourethral glands in male. The female clitoris is like the penis of the males [13]. The hymen is the stratified squamous nonkeratinized epithelium. The lateral vulvar surfaces are made up of labia majora and are comprised of fibrous and adipose folds. The labia minora also consists of connective tissue in two folds containing few (if any) adipose tissues. The labia minora is bifurcated anteriorly. The vestibule is the zone between Hart's line and the hymen, which consists of stratified squamous nonkeratinized epithelium [9]. The following are the glands present in the vulvar region of the female reproductive system:


• Movement of early embryo to the appropriate location

*Reproductive Biology and Technology in Animals*

• Maintaining and nourishment of the embryo and fetus

time of parturition.

entry of microbes during pregnancy.

in serosal layer unilateral or bilateral.

**7.1 Functions of the cervix**

**8**

**6.3 Serosa**

It also has a key role in the expulsion of the fetus and fetal membranes at the

It is the outer covering of the uterus that consists of a single layer of squamous cells.

The whole reproductive tract is tubular except the ovary. The cervix is a thickwalled tubular structure where mucosa-submucosa-derived folds or rings are present, but their number varies from species to species. Only onefold is seen in bitch and queen, but multiple folds are seen in cow, ewe, sow, and mare. In cow and ewe, rings/folds are having interlocking finger-like projections. In pigs, rings interdigitate so interdigitation is seen in them. The cervix is soft in mare due to the presence of loose folds of the mucosa. In a bitch the cervix is smooth due to lack of folds. The primary function of the cervix is lubrication of the canal which is required during copulation [8]. Lubrication is by two types of secretions, e.g., the sulfomucin which is viscous (mobility is directed toward exterior (vagina) and inhibits sperm transport in the uterus), and the sialomucins which facilitate the sperm to move into the uterus. The cervix isolates the conceptus inside the uterus from the outer environment by the formation of cervical seal under the action of progesterone which thickens the mucus produced in the cervix and glues the folding together to prevent

Like other tubular organs, it consists of four tunics/laminae, i.e., mucosa, submucosa, muscularis, and serosa/adventitia [9]. The mucosal tunics of the endocervix are lined by simple columnar epithelium along with the presence of mucus-producing cells called goblet cells. In some animal species, a few columnar cells are found ciliated and in simple tubular glands may be seen in the ruminant's mucosa. In the case of pigs, up to 90% of the mucosal lining epithelium is stratified squamous. Most of the ectocervical portion of the cervical mucosa is lined by the stratified squamous epithelium. Mucosa and the submucosa form folds into the cervical lumen. These folds vary in height, width, and thickness from species to species. The lamina propria of the mucosa and submucosa combines to form propria-submucosa. This propriasubmucosa is heavily infiltrated by dense irregular connective tissue and supply of rich blood vessels extended deep in the submucosa. The muscularis layer is composed of two smooth muscle layers. The inner layer is circular layer and the outer layer is longitudinal in arrangements. The myenteric plexus is present in the muscular layers. Some elastic fibers are also observed in this layer. Serosa/adventitia is a loose connective tissue layer present in the outer surface. This layer is surrounded by the mesothelium which is a layer of simple squamous epithelium. Gartner's ducts may be seen

During estrus stage of estrous cycle, the mucosa is responsible for mucus secre-

tion. Dense irregular connective tissue becomes edematous and forms areolar structure, i.e., loose connective tissue. Circular muscular layer along with elastic

fiber is responsible for the involution of the cervix after parturition [8].

**7. Gross and microscopic structures and functions of the cervix**


#### **8.1 Microscopic anatomy of vulva**

Vaginal lining epithelium is stratified squamous and nonkeratinized, whereas the epithelium of the labia majora is stratified squamous and keratinized. Microscopic layers of the vulva are the mucosa, submucosa, muscularis, and serosa [14, 15]. Glands are present in the submucosal layer and the innermost layer is the mucosa.

#### **8.2 Role of the vulva in reproduction**

Being a gateway to the uterus, its primary role is to offer protection by closing the labia [16]. The external uterine orifice is protected by the vulva supported by thick large lips of labia majora along with small lips of labia minora. The urethra also opens in the vulva that is known as the urethral meatus, thus performing a function of urine passage. It also gives the pathway for sperm for entry in the body. Sometimes due to the folds and the moisture, the fungal infection may occur. The area between the opening of the vagina and the anus, below the labia majora, is called the perineum [12]. By observing the vulva, we can detect if the female is in estrous or not. If the vulva is thick and edematous, it is in the heat, and if not the female is not in the heat; this is due to the estrogen level in the female body. The penis like clitoris detects the nerve stimuli and performs the following functions:


#### **9. Type of sperm which is most favorable for fertilization**

zona pellucida is the innermost thick transparent glycoprotein membranes surrounding the plasma membranes. With a maturation of oocyte, multiple granulosal cells enlarge called cumulus oophorus [3]. This oocyte and granulosal cells form complex known as cumulus oocyte complex (COC). The cumulus of mature COC adheres to the surface of fimbriae as sticky substances. After the sex hormones signal the fimbriae, it contracts and releases oocyte. After release, oocyte has only 24–28 hours to fuse with sperm; otherwise, it is lost. Oocyte releases some chemicals that attract the sperm, and the sperm tries to penetrate two protective layers of oocyte. First it burrows to the cell of corona radiata and then move toward zona pellucida layer. Zona pellucida is composed of four glycoproteins ZP1, ZP2, ZP3, and ZP4. The primary ligand of sperm oocyte binding is ZP3 and ZP4. Both induce acrosomal reaction of sperm to complete fusion reaction and form a zygote [18].

These factors make the ovum gamete most favorable for fertilization.

mucosa with the fertilized ovum and conceptus.

*Micro-architecture of the Female Reproductive System DOI: http://dx.doi.org/10.5772/intechopen.88023*

hereby acknowledged in helping me in the write-up.

\* and Muhammad Ali2

\*Address all correspondence to: drarbab786@gmail.com;

The microarchitectural examination of the reproductive organs at microscopic level is very much important to recognize its structure and function during reproductive physiology. Furthermore, this chapter highlighted the dealing of the lining

The author would like to acknowledge the efforts of Prof. Dr. Ashiq Hussain Cheema for facilitating and guiding. Furthermore, Jr. lab attendant Mr. Saqib Ali, is

1 Sub-campus, Jhang, University of Veterinary and Animal Sciences, Lahore,

2 University College of Veterinary and Animal Sciences, The Islamia University of

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

**11. Conclusion**

**Acknowledgements**

**Author details**

Arbab Sikandar<sup>1</sup>

Bahawalpur, Pakistan

arbab.sikandar@uvas.edu.pk

provided the original work is properly cited.

Pakistan

**11**

As we know around 250 million sperm cells enter the female external genitalia, but just a few thousands can enter the fallopian tube, and only a single sperm will fertilize the ova [14]. Several problems and barriers came in the pathway of the spermatozoa to touch the final goal. These range from the low pH in and around the vagina, the mucus of the cervix, the narrowness of the uterotubal junction (the entrance of the cervix), the WBCs of the immune system which treat the spermatozoa as a foreign entity to destroy, cell-to-cell interactions, gene expression, phenotypic sperm traits, sperm motility defects, DNA status, lack of capacitation or morphological normality, and failure of abnormal spermatozoa to reach to the site of fertilization. The wall of seminiferous tubules which are the coiled structure is responsible to produce the sperm. For a spermatozoon, around 28–42 days is lapsed to cross the male reproductive system. In the female reproductive tract, sperm undergo changes that help in fertilization called activation and capacitation, but all sperm are not capacitated at the same time; therefore, all sperm are not able to fertilize the cells [16]. However, several mechanisms that aid this process are good motility, adequate morphology, and normal DNA status of the cell. A sperm which has normal head, nucleus, and tail and has a moderate motility is considerable to fertilize the egg [17]. The sperm are guided upon their journey by the chemical and the temperature signals. A sperm reservoir in the fallopian tubes, where sperms bind to the epithelial lining (columnar in shape) of the tubes, reduces the chance of fertilization by multiple sperm. During ovulation, the sperm are hyperactivated to help them to the penetration of the mucus in the fallopian tubes and the outer coating of the egg. Sperm outer membrane fuses with egg outer membrane to facilitate fertilization. Acrosomal reaction occurs in the sperm head once the spermatozoa reached the ova. Acrosin enzyme helps in acrosomal reaction. Hyaluronic acid enzyme has an important role in the permeability and motility of sperms and their interactions with gametes. Formation of a functional reservoir through epithelial binding of sperm to the oviductal isthmus reduces the likelihood of the polyspermic fertilization. Motility or hyperactivation helps sperm in penetrating the mucus in the fallopian tubes and the cumulus oophorus (corona radiata), and the acrosome exocytosis may assist penetration of oocyte zona pellucida that proceeds the fusion within oocyte plasma membrane. Zona pellucida then undergoes biochemical changes so that further sperm cannot penetrate the cell called zona block [17]. In addition to alteration of zona pellucida, the cortical reaction reduces the ability of oocyte plasma membrane to fuse with additional spermatozoa, thus causing vitelline block. Both zona block and vitelline block prevent polyspermy.

#### **10. The type of ova that is most favorable for fertilization**

Fertilization is a process in which male and female gametes fuse to form a zygote. Ovum is the female gamete, and its selection is the criteria of fertilization. There are many factors that are responsible for its selection including follicular dominance, cumulus oophorus formation, cumulus oocyte complex formation, fimbrial supportive structure, and zona pellucidal layer that enable sperm to fuse with ovum.

Follicular dominance is based on E2 production. The follicle, which has greater ability of E2 production, has greater capability of its dominance. Dominancy is responsible for onward ovulation. Ovulated egg is surrounded by two protective layers [9]. Corona radiata is the outermost layer containing granulosal cells, and

*Micro-architecture of the Female Reproductive System DOI: http://dx.doi.org/10.5772/intechopen.88023*

zona pellucida is the innermost thick transparent glycoprotein membranes surrounding the plasma membranes. With a maturation of oocyte, multiple granulosal cells enlarge called cumulus oophorus [3]. This oocyte and granulosal cells form complex known as cumulus oocyte complex (COC). The cumulus of mature COC adheres to the surface of fimbriae as sticky substances. After the sex hormones signal the fimbriae, it contracts and releases oocyte. After release, oocyte has only 24–28 hours to fuse with sperm; otherwise, it is lost. Oocyte releases some chemicals that attract the sperm, and the sperm tries to penetrate two protective layers of oocyte. First it burrows to the cell of corona radiata and then move toward zona pellucida layer. Zona pellucida is composed of four glycoproteins ZP1, ZP2, ZP3, and ZP4. The primary ligand of sperm oocyte binding is ZP3 and ZP4. Both induce acrosomal reaction of sperm to complete fusion reaction and form a zygote [18]. These factors make the ovum gamete most favorable for fertilization.

#### **11. Conclusion**

**9. Type of sperm which is most favorable for fertilization**

*Reproductive Biology and Technology in Animals*

**10. The type of ova that is most favorable for fertilization**

with ovum.

**10**

Fertilization is a process in which male and female gametes fuse to form a zygote. Ovum is the female gamete, and its selection is the criteria of fertilization. There are many factors that are responsible for its selection including follicular dominance, cumulus oophorus formation, cumulus oocyte complex formation, fimbrial supportive structure, and zona pellucidal layer that enable sperm to fuse

Follicular dominance is based on E2 production. The follicle, which has greater ability of E2 production, has greater capability of its dominance. Dominancy is responsible for onward ovulation. Ovulated egg is surrounded by two protective layers [9]. Corona radiata is the outermost layer containing granulosal cells, and

As we know around 250 million sperm cells enter the female external genitalia, but just a few thousands can enter the fallopian tube, and only a single sperm will fertilize the ova [14]. Several problems and barriers came in the pathway of the spermatozoa to touch the final goal. These range from the low pH in and around the vagina, the mucus of the cervix, the narrowness of the uterotubal junction (the entrance of the cervix), the WBCs of the immune system which treat the spermatozoa as a foreign entity to destroy, cell-to-cell interactions, gene expression, phenotypic sperm traits, sperm motility defects, DNA status, lack of capacitation or morphological normality, and failure of abnormal spermatozoa to reach to the site of fertilization. The wall of seminiferous tubules which are the coiled structure is responsible to produce the sperm. For a spermatozoon, around 28–42 days is lapsed to cross the male reproductive system. In the female reproductive tract, sperm undergo changes that help in fertilization called activation and capacitation, but all sperm are not capacitated at the same time; therefore, all sperm are not able to fertilize the cells [16]. However, several mechanisms that aid this process are good motility, adequate morphology, and normal DNA status of the cell. A sperm which has normal head, nucleus, and tail and has a moderate motility is considerable to fertilize the egg [17]. The sperm are guided upon their journey by the chemical and the temperature signals. A sperm reservoir in the fallopian tubes, where sperms bind to the epithelial lining (columnar in shape) of the tubes, reduces the chance of fertilization by multiple sperm. During ovulation, the sperm are hyperactivated to help them to the penetration of the mucus in the fallopian tubes and the outer coating of the egg. Sperm outer membrane fuses with egg outer membrane to facilitate fertilization. Acrosomal reaction occurs in the sperm head once the spermatozoa reached the ova. Acrosin enzyme helps in acrosomal reaction. Hyaluronic acid enzyme has an important role in the permeability and motility of sperms and their interactions with gametes. Formation of a functional reservoir through epithelial binding of sperm to the oviductal isthmus reduces the likelihood of the polyspermic fertilization. Motility or hyperactivation helps sperm in penetrating the mucus in the fallopian tubes and the cumulus oophorus (corona radiata), and the acrosome exocytosis may assist penetration of oocyte zona pellucida that proceeds the fusion within oocyte plasma membrane. Zona pellucida then undergoes biochemical changes so that further sperm cannot penetrate the cell called zona block [17]. In addition to alteration of zona pellucida, the cortical reaction reduces the ability of oocyte plasma membrane to fuse with additional spermatozoa, thus causing vitelline block. Both zona block and vitelline block prevent polyspermy.

The microarchitectural examination of the reproductive organs at microscopic level is very much important to recognize its structure and function during reproductive physiology. Furthermore, this chapter highlighted the dealing of the lining mucosa with the fertilized ovum and conceptus.

#### **Acknowledgements**

The author would like to acknowledge the efforts of Prof. Dr. Ashiq Hussain Cheema for facilitating and guiding. Furthermore, Jr. lab attendant Mr. Saqib Ali, is hereby acknowledged in helping me in the write-up.

#### **Author details**

Arbab Sikandar<sup>1</sup> \* and Muhammad Ali2

1 Sub-campus, Jhang, University of Veterinary and Animal Sciences, Lahore, Pakistan

2 University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan

\*Address all correspondence to: drarbab786@gmail.com; arbab.sikandar@uvas.edu.pk

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Arrotéia KF, Garcia PV, Barbieri MF, Justino ML, Pereira LAV. The epididymis: Embryology, structure, function and its role in fertilization and infertility. In: Pereira LAV, ed. Embryology-Updates and Highlights on Classic Topics. Rijeka, Croatia: IntechOpen; 2012. DOI: 10.5772/2142

[2] Osvaldo-Decima L. Smooth muscle in the ovary of the rat and monkey. Journal of Ultrastructure Research. 1970;**30** (1–2):218-237

[3] Fortune JE. Ovarian follicular growth and development in mammals. Biology of Reproduction. 1994;**50**(2):225-232

[4] Hyttel PFTCH, Fair T, Callesen H, Greve T. Oocyte growth, capacitation and final maturation in cattle. Theriogenology. 1997;**47**(1):23-32

[5] Saidapur SK. Follicular atresia in the ovaries of nonmammalian vertebrates. In: International Review of Cytology. Vol. 54. New York: Academic Press; 1978. pp. 225-244

[6] Rodgers RJ, Irving-Rodgers HF. Morphological classification of bovine ovarian follicles. Reproduction. 2010; **139**(2):309

[7] Wiley C, Jahnke M, Redifer C, Gunn PJ, Dohlman T. Effects of endogenous progesterone during ovarian follicle superstimulation on embryo quality and quantity in beef cows. Theriogenology. 2019;**129**:54-60

[8] Padubidri VG, Daftary SN, editors. Shaw's Textbook of Gynecology-EBOOK. Elsevier, India: Elsevier Health Sciences; 2018

[9] Boyd KL, Muehlenbachs A, Rendi MH, Garcia RL, Gibson-Corley KN. Female reproductive system. In: Comparative Anatomy and Histology. London, United Kingdom: Academic Press; 2018. pp. 303-334

[10] Guan J, Watrelot A. Fallopian subtle pathology. Best Practice and Research Clinical Obstetrics and Gynaecology. 2019. https://doi.org/10.1016/j.bpobgyn. 2018.12.012

[11] Carlson BM. Human Embryology and Developmental Biology E-Book. New York: Elsevier Health Sciences; 2018

[12] Bazer FW, Burghardt RC, Johnson GA, Spencer TE, Wu G. Mechanisms for the establishment and maintenance of pregnancy: Synergies from scientific collaborations. Biology of Reproduction. 2018;**99**(1):225-241

[13] Baskin L, Shen J, Sinclair A, Cao M, Liu X, Liu G, et al. Development of the human penis and clitoris. Differentiation. 2018;**103**:74-85

[14] Vuarin P, Hingrat Y, Lesobre L, Jalme MS, Lacroix F, Sorci G. Sperm competition accentuates selection on ejaculate attributes. Biology Letters. 2019;**15**(3):20180889

[15] Kumar V. Histological, histochemical and scanning electron microscopic studies on the oviduct and uterus of jaffarabadi buffalo (*Bubalus bubalis*) during follicular and luteal phases 2514 (doctoral dissertation). Junagadh: JAU; 2018

[16] Lucas D, Fox J. The psychology of human sexuality. The psychology of human sexuality. In: Noba Textbook Series: Psychology. Champaign, IL: DEF Publishers; 2018

[17] Lehmann R. Matchmaking molecule for egg and sperm. Science. 2018; **361**(6406):974-975

[18] Hirohashi N, Yanagimachi R. Sperm acrosome reaction: Its site and role in fertilization. Biology of Reproduction. 2018

**13**

**Chapter 2**

**Abstract**

High Surveillance

*François J. Richard*

developmental competence.

**1. Introduction**

Oocyte Meiotic Resumption under

Germinal vesicle breakdown (GVBD) is the hallmark of oocyte meiotic resump-

tion. It occurs under minimal stimulation during in vitro maturation (IVM). Several factors have been described to be involved in the inhibition of oocyte meiotic resumption such as purine derivatives. This study was assessing whether adenosinergic and guanosinergic systems are functional and participating in the inhibition of oocyte maturation. The objectives of the present study were to evaluate the effect of two purines, adenosine (ADO) and guanosine (GUO), on in vitro oocyte meiotic resumption, cumulus cell expansion, and gap junction communication. Both ADO and GUO significantly inhibited GVBD oocytes. The inhibitory effect lasted 24 hours and was reversible for meiotic resumption and cumulus cell expansion. Both ADO and GUO increased gap junction communication in cumulus cells. Equine chorionic gonadotropin (eCG) and the adenylyl cyclase stimulator, forskolin (FK), were both supportive of ADO and GUO inhibitory effect. The results are suggesting both adenosinergic and guanosinergic systems efficient in inhibiting oocyte meiotic resumption. The use of these two systems as part of a pre-IVM culture period would be a novel strategy to explore in order to improve oocyte

**Keywords:** oocyte, purine, adenosine, guanosine, meiotic resumption

Oocyte meiosis begins during fetal development in large animals such as in cow, sow, and ewe. Once the sub-phases of the first prophase are completed, the oocyte meiosis stops at the dictyate stage. At this point, crossing over is a past event and chromatin is accessible for transcription. This G2/M phase transition of the cell cycle characterizes mammalian oocytes. The female gamete bears 4n chromosomes as long as the ovulatory LH peak generates its effect on the preovulatory follicle to induce oocyte meiotic resumption. Dysregulation of the oocyte cell cycle induced by c-MOS proto-oncogene after gene null mutation caused parthenogenic development of the oocyte and explained female mouse infertility [1]. This phenotype illustrates how important it is to appropriately control oocyte meiotic resumption. Oocyte meiotic resumption is a highly important physiological event for species survival since it refers to a successful reproduction by appropriately preparing the female gamete. This unique cell division has to occur at the right time and imply high surveillance. From an evolutionary point of view, the number of female gametes produced went from a large number, such as in frog, to a small number in mammals. Although several thousands of oocytes are found in the ovaries, only a

#### **Chapter 2**

**References**

(1–2):218-237

[1] Arrotéia KF, Garcia PV, Barbieri MF,

*Reproductive Biology and Technology in Animals*

[10] Guan J, Watrelot A. Fallopian subtle pathology. Best Practice and Research Clinical Obstetrics and Gynaecology. 2019. https://doi.org/10.1016/j.bpobgyn.

[11] Carlson BM. Human Embryology and Developmental Biology E-Book. New York: Elsevier Health Sciences; 2018

[12] Bazer FW, Burghardt RC, Johnson GA, Spencer TE, Wu G. Mechanisms for the establishment and maintenance of pregnancy: Synergies from scientific collaborations. Biology of Reproduction.

[13] Baskin L, Shen J, Sinclair A, Cao M, Liu X, Liu G, et al. Development of the

[14] Vuarin P, Hingrat Y, Lesobre L, Jalme MS, Lacroix F, Sorci G. Sperm competition accentuates selection on ejaculate attributes. Biology Letters.

[15] Kumar V. Histological, histochemical and scanning electron microscopic studies on the oviduct and uterus of jaffarabadi buffalo (*Bubalus bubalis*) during follicular

and luteal phases 2514 (doctoral dissertation). Junagadh: JAU; 2018

[16] Lucas D, Fox J. The psychology of human sexuality. The psychology of human sexuality. In: Noba Textbook Series: Psychology. Champaign, IL: DEF

[17] Lehmann R. Matchmaking molecule for egg and sperm. Science. 2018;

[18] Hirohashi N, Yanagimachi R. Sperm acrosome reaction: Its site and role in fertilization. Biology of Reproduction.

2018.12.012

2018;**99**(1):225-241

human penis and clitoris. Differentiation. 2018;**103**:74-85

2019;**15**(3):20180889

Publishers; 2018

**361**(6406):974-975

2018

[2] Osvaldo-Decima L. Smooth muscle in the ovary of the rat and monkey. Journal of Ultrastructure Research. 1970;**30**

[3] Fortune JE. Ovarian follicular growth and development in mammals. Biology of Reproduction. 1994;**50**(2):225-232

[4] Hyttel PFTCH, Fair T, Callesen H, Greve T. Oocyte growth, capacitation

[5] Saidapur SK. Follicular atresia in the ovaries of nonmammalian vertebrates. In: International Review of Cytology. Vol. 54. New York: Academic Press;

[6] Rodgers RJ, Irving-Rodgers HF. Morphological classification of bovine ovarian follicles. Reproduction. 2010;

[7] Wiley C, Jahnke M, Redifer C, Gunn PJ, Dohlman T. Effects of endogenous progesterone during ovarian follicle superstimulation on embryo quality and quantity in beef cows. Theriogenology.

[8] Padubidri VG, Daftary SN, editors. Shaw's Textbook of Gynecology-EBOOK. Elsevier, India: Elsevier Health

[9] Boyd KL, Muehlenbachs A, Rendi MH, Garcia RL, Gibson-Corley KN. Female reproductive system. In:

Histology. London, United Kingdom: Academic Press; 2018. pp. 303-334

Comparative Anatomy and

and final maturation in cattle. Theriogenology. 1997;**47**(1):23-32

1978. pp. 225-244

**139**(2):309

2019;**129**:54-60

Sciences; 2018

**12**

Justino ML, Pereira LAV. The epididymis: Embryology, structure, function and its role in fertilization and

infertility. In: Pereira LAV, ed. Embryology-Updates and Highlights on Classic Topics. Rijeka, Croatia: IntechOpen; 2012. DOI: 10.5772/2142

## Oocyte Meiotic Resumption under High Surveillance

*François J. Richard*

#### **Abstract**

Germinal vesicle breakdown (GVBD) is the hallmark of oocyte meiotic resumption. It occurs under minimal stimulation during in vitro maturation (IVM). Several factors have been described to be involved in the inhibition of oocyte meiotic resumption such as purine derivatives. This study was assessing whether adenosinergic and guanosinergic systems are functional and participating in the inhibition of oocyte maturation. The objectives of the present study were to evaluate the effect of two purines, adenosine (ADO) and guanosine (GUO), on in vitro oocyte meiotic resumption, cumulus cell expansion, and gap junction communication. Both ADO and GUO significantly inhibited GVBD oocytes. The inhibitory effect lasted 24 hours and was reversible for meiotic resumption and cumulus cell expansion. Both ADO and GUO increased gap junction communication in cumulus cells. Equine chorionic gonadotropin (eCG) and the adenylyl cyclase stimulator, forskolin (FK), were both supportive of ADO and GUO inhibitory effect. The results are suggesting both adenosinergic and guanosinergic systems efficient in inhibiting oocyte meiotic resumption. The use of these two systems as part of a pre-IVM culture period would be a novel strategy to explore in order to improve oocyte developmental competence.

**Keywords:** oocyte, purine, adenosine, guanosine, meiotic resumption

#### **1. Introduction**

Oocyte meiosis begins during fetal development in large animals such as in cow, sow, and ewe. Once the sub-phases of the first prophase are completed, the oocyte meiosis stops at the dictyate stage. At this point, crossing over is a past event and chromatin is accessible for transcription. This G2/M phase transition of the cell cycle characterizes mammalian oocytes. The female gamete bears 4n chromosomes as long as the ovulatory LH peak generates its effect on the preovulatory follicle to induce oocyte meiotic resumption. Dysregulation of the oocyte cell cycle induced by c-MOS proto-oncogene after gene null mutation caused parthenogenic development of the oocyte and explained female mouse infertility [1]. This phenotype illustrates how important it is to appropriately control oocyte meiotic resumption.

Oocyte meiotic resumption is a highly important physiological event for species survival since it refers to a successful reproduction by appropriately preparing the female gamete. This unique cell division has to occur at the right time and imply high surveillance. From an evolutionary point of view, the number of female gametes produced went from a large number, such as in frog, to a small number in mammals. Although several thousands of oocytes are found in the ovaries, only a

small percentage is ovulated, and even less are fertilized. Considering this selective restriction, a framework of the meiotic resumption process has developed. Throughout the evolutionary process, mechanisms have been added to ensure a very precise control of this crucial event related to species survival, which is the final phase of gamete preparation for fertilization. Interestingly, the role of EGF-like peptides fully fits with this notion. It is well-known that the LH peak induces oocyte meiotic resumption in the preovulatory follicles. However, the EGF-like peptides are also active participants in the ovulatory process, meiotic resumption, and cumulus cell expansion, clearly supporting an add-up to the LH surge. Going back to the control of oocyte meiotic resumption, although the contribution of cAMP and cGMP is well described, it is obvious that other mechanisms may still be involved and be discovered.

It has long been known that adenosine (ADO) is a molecule playing an important role in various physiological systems such as in the central nervous system and cardiac function [2]. On the other hand, there are very few studies on the role of ADO in mammalian ovarian follicle. ADO is known to act on specific receptors, to cross plasma membrane using transporters, and to be generated from functional catabolism by extracellular enzymes, a system called adenosinergic [3]. On the other hand, guanosine (GUO) has not been as popular in research. However, in recent years a new interest on GUO has revealed its importance in the effect of ADO on the functioning of the central nervous system [4]. Although no specific receptor for GUO has been yet identified, its physiological impact leaves no doubt. GUO has neuroprotective effects, it diminishes the apoptotic effects observed in Parkinson's disease, and it also has a protective role during a challenge with glutamate, during mitochondrial stress, and during ischemia [4–6]. Because GUO can also be generated by a functional catabolism using extracellular enzymes, these results support the existence of a so-called guanosinergic system.

ADO has also been identified in the follicular fluid with several other purine derivatives. Among these derivatives, hypoxanthine is the compound that has attracted the most studies in last three decades. In mice, ADO improves the inhibitory effect of hypoxanthine on the resumption of meiosis but has no effect when used alone, even at a dose of 5 mM [7]. In the rat, the effect of ADO is also minimal [8]. In cattle, ADO used at 200 μM slowed meiotic resumption [9]. There is one study reporting that it has not been able to measure GUO in follicular fluid [10]. In contrast, GUO showed a very potent effect on the inhibition of meiotic resumption in mice [10] and rat [8].

This study is proposing to assess whether an adenosinergic and guanosinergic system are functional and participating in the inhibition of oocyte meiotic resumption. Specifically, the research presented here aims to study the involvement of ADO and GUO in the physiology of the ovarian follicle by targeting their effect on in vitro meiotic resumption using swine as the animal model.

#### **2. Material and methods**

#### **2.1 Chemicals**

Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The adenylyl cyclase activator, forskolin (FK), was prepared as a millimolar stock solution and stored at −20°C as already described [11]. ADO and GUO were prepared from the stock powder directly in the culture medium on the day of the experiment. 8-Bromoadenosine (8-BrADO) and 8-Bromoguanosine (8-BrGUO) were dissolved in DMSO, and a DMSO control was run simultaneously.

**15**

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

solution having antibiotics and antimycotics at 37°C.

**2.4 Recovery of cumulus-oocyte complexes (COC)**

tion of the oocytes.

**2.2 Ovary collections**

**2.3 Maturation medium**

−20°C until used [11].

with 100% humidity.

**2.5 Selecting COC and denuding oocytes**

geneous cytoplasm were selected.

**2.6 Assessment of oocyte nuclear maturation stage**

The chemicals were added to the maturation medium a few hours prior to the addi-

As previously described, prepubertal gilt ovaries were collected from a local slaughterhouse [12]. In brief, they were placed in saline (0.9% NaCl containing antibiotics and antimycotics, 100,000 IU/L penicillin G, 100 mg/L streptomycin, 250 μg/L amphotericin B) and kept at 37°C. They were rinsed once in a fresh saline

Oocytes were matured in BSA-free North Carolina State University 23 (NCSU) medium [13] supplemented with 25 μM β-mercaptoethanol (Bio-Rad, Hercules, CA, USA), 0.1 mg/mL cysteine, 10% (v/v) porcine follicular fluid (PFF), and gonadotropins (2.5 IU/well for hCG [APL, Ayerst Laboratories Inc., Philadelphia, PA, USA] and 2.5 IU/well for eCG [Folligon, Intervet, Whitby, ON, Canada]) [12]. PFF was collected from follicles of 2–6 mm in diameter. After centrifugation (1500×*g*, 30 minutes), the supernatant was filtered (0.8 and 0.45 μm) and stored at

Cumulus-oocyte complexes were collected from follicles of 2–6 mm in diameter. They were aspirated with a 10-mL syringe and an 18G needle [11]. The follicular contents were pooled in 50-mL conical tubes (Falcon, Franklin Lakes, NJ, USA). The pellet was washed twice with HEPES-buffered Tyrode's medium containing 0.01% (w/v) polyvinyl alcohol (PVA-TLH) [14]. The COC were recovered under a stereomicroscope and transferred to a petri dish containing PVA-TLH. The COC were washed three times with PVA-TLH and then subjected to their respective treatments. Groups of 20–30 COC were placed in the wells of four-well multi-dishes (Nunc, Roskilde, Denmark) containing 500 μL of maturation medium. The COC were cultured at 38.5°C, 5% CO2 in 95% air atmosphere

The criteria of selection were COC with a minimum of three layers of clear and compact cumulus cells which surrounded the oocyte [12]. Those with dark, pyknotic, or expanded cumulus cells, and those containing oocytes with a very clear cytoplasm or of small diameter were rejected. The oocytes were denuded of their cumulus cells by drawing several times the COC into a pipette using PVA-TLH. Once denuded, the oocytes were rinsed in PVA-TLH, and those with a homo-

The oocyte nuclear maturation stage was evaluated following a 48-hour fixation period in a solution of ethanol and acetic acid (3:1). Using a phase contrast microscope at 100 and 400× magnification immediately after staining with 1% aceto-orcein [15] allows us to assess oocyte nuclear maturation stage. Those having a nuclear membrane were considered at the germinal vesicle (GV) stage, whereas

The chemicals were added to the maturation medium a few hours prior to the addition of the oocytes.

### **2.2 Ovary collections**

*Reproductive Biology and Technology in Animals*

the existence of a so-called guanosinergic system.

in vitro meiotic resumption using swine as the animal model.

and be discovered.

in mice [10] and rat [8].

**2. Material and methods**

**2.1 Chemicals**

small percentage is ovulated, and even less are fertilized. Considering this selective restriction, a framework of the meiotic resumption process has developed. Throughout the evolutionary process, mechanisms have been added to ensure a very precise control of this crucial event related to species survival, which is the final phase of gamete preparation for fertilization. Interestingly, the role of EGF-like peptides fully fits with this notion. It is well-known that the LH peak induces oocyte meiotic resumption in the preovulatory follicles. However, the EGF-like peptides are also active participants in the ovulatory process, meiotic resumption, and cumulus cell expansion, clearly supporting an add-up to the LH surge. Going back to the control of oocyte meiotic resumption, although the contribution of cAMP and cGMP is well described, it is obvious that other mechanisms may still be involved

It has long been known that adenosine (ADO) is a molecule playing an important role in various physiological systems such as in the central nervous system and cardiac function [2]. On the other hand, there are very few studies on the role of ADO in mammalian ovarian follicle. ADO is known to act on specific receptors, to cross plasma membrane using transporters, and to be generated from functional catabolism by extracellular enzymes, a system called adenosinergic [3]. On the other hand, guanosine (GUO) has not been as popular in research. However, in recent years a new interest on GUO has revealed its importance in the effect of ADO on the functioning of the central nervous system [4]. Although no specific receptor for GUO has been yet identified, its physiological impact leaves no doubt. GUO has neuroprotective effects, it diminishes the apoptotic effects observed in Parkinson's disease, and it also has a protective role during a challenge with glutamate, during mitochondrial stress, and during ischemia [4–6]. Because GUO can also be generated by a functional catabolism using extracellular enzymes, these results support

ADO has also been identified in the follicular fluid with several other purine derivatives. Among these derivatives, hypoxanthine is the compound that has attracted the most studies in last three decades. In mice, ADO improves the inhibitory effect of hypoxanthine on the resumption of meiosis but has no effect when used alone, even at a dose of 5 mM [7]. In the rat, the effect of ADO is also minimal [8]. In cattle, ADO used at 200 μM slowed meiotic resumption [9]. There is one study reporting that it has not been able to measure GUO in follicular fluid [10]. In contrast, GUO showed a very potent effect on the inhibition of meiotic resumption

This study is proposing to assess whether an adenosinergic and guanosinergic system are functional and participating in the inhibition of oocyte meiotic resumption. Specifically, the research presented here aims to study the involvement of ADO and GUO in the physiology of the ovarian follicle by targeting their effect on

Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The adenylyl cyclase activator, forskolin (FK), was prepared as a millimolar stock solution and stored at −20°C as already described [11]. ADO and GUO were prepared from the stock powder directly in the culture medium on the day of the experiment. 8-Bromoadenosine (8-BrADO) and 8-Bromoguanosine (8-BrGUO) were dissolved in DMSO, and a DMSO control was run simultaneously.

**14**

As previously described, prepubertal gilt ovaries were collected from a local slaughterhouse [12]. In brief, they were placed in saline (0.9% NaCl containing antibiotics and antimycotics, 100,000 IU/L penicillin G, 100 mg/L streptomycin, 250 μg/L amphotericin B) and kept at 37°C. They were rinsed once in a fresh saline solution having antibiotics and antimycotics at 37°C.

#### **2.3 Maturation medium**

Oocytes were matured in BSA-free North Carolina State University 23 (NCSU) medium [13] supplemented with 25 μM β-mercaptoethanol (Bio-Rad, Hercules, CA, USA), 0.1 mg/mL cysteine, 10% (v/v) porcine follicular fluid (PFF), and gonadotropins (2.5 IU/well for hCG [APL, Ayerst Laboratories Inc., Philadelphia, PA, USA] and 2.5 IU/well for eCG [Folligon, Intervet, Whitby, ON, Canada]) [12]. PFF was collected from follicles of 2–6 mm in diameter. After centrifugation (1500×*g*, 30 minutes), the supernatant was filtered (0.8 and 0.45 μm) and stored at −20°C until used [11].

#### **2.4 Recovery of cumulus-oocyte complexes (COC)**

Cumulus-oocyte complexes were collected from follicles of 2–6 mm in diameter. They were aspirated with a 10-mL syringe and an 18G needle [11]. The follicular contents were pooled in 50-mL conical tubes (Falcon, Franklin Lakes, NJ, USA). The pellet was washed twice with HEPES-buffered Tyrode's medium containing 0.01% (w/v) polyvinyl alcohol (PVA-TLH) [14]. The COC were recovered under a stereomicroscope and transferred to a petri dish containing PVA-TLH. The COC were washed three times with PVA-TLH and then subjected to their respective treatments. Groups of 20–30 COC were placed in the wells of four-well multi-dishes (Nunc, Roskilde, Denmark) containing 500 μL of maturation medium. The COC were cultured at 38.5°C, 5% CO2 in 95% air atmosphere with 100% humidity.

#### **2.5 Selecting COC and denuding oocytes**

The criteria of selection were COC with a minimum of three layers of clear and compact cumulus cells which surrounded the oocyte [12]. Those with dark, pyknotic, or expanded cumulus cells, and those containing oocytes with a very clear cytoplasm or of small diameter were rejected. The oocytes were denuded of their cumulus cells by drawing several times the COC into a pipette using PVA-TLH. Once denuded, the oocytes were rinsed in PVA-TLH, and those with a homogeneous cytoplasm were selected.

#### **2.6 Assessment of oocyte nuclear maturation stage**

The oocyte nuclear maturation stage was evaluated following a 48-hour fixation period in a solution of ethanol and acetic acid (3:1). Using a phase contrast microscope at 100 and 400× magnification immediately after staining with 1% aceto-orcein [15] allows us to assess oocyte nuclear maturation stage. Those having a nuclear membrane were considered at the germinal vesicle (GV) stage, whereas

those without a nuclear membrane were considered to have resumed meiosis. The oocytes were considered mature when they were in anaphase I, telophase I, and metaphase II.

#### **2.7 Cumulus-cumulus gap-FRAP assay to measure gap junction communications (GJC)**

After 4 hours of in vitro culture, COC were loaded with calcein-AM (39,69-di(*O*-acetyl)-29,79-bis(*N*,*N*-bis(carboxymethyl) amino methyl)-fluorescein) and tetra(acetoxymethyl ester) (Molecular Probes C-3100) in IVM medium containing 0.1 mg/mL PVA [16]. After 20 minutes at 38.5°C, the live COC were mounted on glass slides in the PVA-containing IVM medium. Fluorescence recovery after photobleaching (FRAP) assays were conducted using Nikon Eclipse TE2000-E inverted confocal microscope. The bleaching was performed for 5 minutes using laser pulses on a limited region of cumulus cells observed at a magnification of 90×. The COC were photographed at 60× before bleaching and every 3 minutes thereafter for 12 minutes. Fluorescence intensity was quantified using ImageJ software (National Institutes of Health, USA). A relative fluorescence value was achieved by dividing the raw fluorescence measurement in the bleached area by the mean fluorescence in two adjacent regions. This value was further divided by the fluorescence value of a region at the opposite end of the COC to correct for unintended bleaching caused by the laser excitation.

#### **2.8 Statistical analyses**

All values are presented with their corresponding SEM, and the number of replicates is indicated for each experiment (at least three). The data were analyzed by one-way ANOVA using GraphPad Prism v7.02 for Windows (GraphPad Software, San Diego, CA, USA). When ANOVA indicated a significant effect of treatment (*P* < 0.05), individual treatment differences were compared using Bonferroni's multiple comparison post-hoc test. Significant effects were identified by \* or different letters.

#### **3. Results**

In our hands, prior to in vitro maturation, more than 95% of swine oocytes have not started their nuclear maturation, that is, their meiotic resumption (<5% of GVDB oocytes), and cumulus cells have not expanded yet [12].

#### **3.1 Adenosine inhibits oocyte meiotic resumption**

The first experiment is aiming to assess ADO on inhibition of oocyte GVBD. When COC are cultured for 24 hours in control treatment, more than 94.3 ± 3.0% of the oocytes were in GVBD, that is, oocyte meiotic resumption took place (**Figure 1A**). The inhibitory effect of ADO was dose-dependent with only 13.9 ± 2.1% in GVBD after an exposure to ADO at 2.0 mM for 24 hours (**Figure 1A**). The EC50 of ADO on the percentage of GVBD oocyte is calculated as already described [11] and determined to be 1.3 mM. In addition, at 2.0 mM ADO inhibited cumulus cell expansion (**Figure 1B5**) compared with the control (**Figure 1B1**). When cultured for 24 hours as oocytes denuded of their cumulus cells (DO), ADO (2.0 mM) did not significantly inhibit oocyte GVBD compared with the control conditions (**Figure 1C**). These results support an inhibitory effect of ADO on both oocyte meiotic resumption and cumulus expansion when cultured as COC but not as DO.

**17**

**Figure 1.**

adenosine, 8-BrADO.

**3.3 Reversibility of GVBD inhibition**

*0.5 mM, (4) ADO 1.0 mM, and (5) ADO 2.0 mM.*

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

**3.2 Cell-permeable analog of adenosine (8-BrADO) inhibits GVBD**

The following experiment is performed to assess a membrane-permeable analog of ADO, 8-BrADO (mimicking the intracellular effect). When COC are cultured for 24 hours, 12.2 ± 2.7% of the oocytes were in GVBD when treated with 2.0 mM ADO and 15.9 ± 0.8% when treated with 2.0 mM 8-BrADO (**Figure 2A**). These two treatments are significantly different to the control treatment (88.7 ± 2.9%; **Figure 2A**). The inhibition of cumulus cell expansion is also observed in both treatments (**Figure 2B2** and **B3**). These results support that the inhibitory effect of ADO may be mimicked by the cell-permeable analog of

*Dose-response of ADO on both (A) the percentage of GVBD oocyte and (B) cumulus cell expansion. (C) Effect of ADO (2.0 mM) on the percentage of denuded oocytes in GVBD. Data are presented as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by \*. In (B) treatments were (1) Ct, (2) ADO 0.25 mM, (3) ADO* 

To assess the reversibility, the test compounds (ADO and 8-BrADO) are washed

out, and the COC are cultured under control conditions for a second 24-hour

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

#### **Figure 1.**

*Reproductive Biology and Technology in Animals*

**communications (GJC)**

caused by the laser excitation.

**2.8 Statistical analyses**

**3. Results**

metaphase II.

those without a nuclear membrane were considered to have resumed meiosis. The oocytes were considered mature when they were in anaphase I, telophase I, and

**2.7 Cumulus-cumulus gap-FRAP assay to measure gap junction** 

After 4 hours of in vitro culture, COC were loaded with calcein-AM (39,69-di(*O*-acetyl)-29,79-bis(*N*,*N*-bis(carboxymethyl) amino methyl)-fluorescein) and tetra(acetoxymethyl ester) (Molecular Probes C-3100) in IVM medium containing 0.1 mg/mL PVA [16]. After 20 minutes at 38.5°C, the live COC were mounted on glass slides in the PVA-containing IVM medium. Fluorescence recovery after photobleaching (FRAP) assays were conducted using Nikon Eclipse TE2000-E inverted confocal microscope. The bleaching was performed for 5 minutes using laser pulses on a limited region of cumulus cells observed at a magnification of 90×. The COC were photographed at 60× before bleaching and every 3 minutes thereafter for 12 minutes. Fluorescence intensity was quantified using ImageJ software (National Institutes of Health, USA). A relative fluorescence value was achieved by dividing the raw fluorescence measurement in the bleached area by the mean fluorescence in two adjacent regions. This value was further divided by the fluorescence value of a region at the opposite end of the COC to correct for unintended bleaching

All values are presented with their corresponding SEM, and the number of replicates is indicated for each experiment (at least three). The data were analyzed by one-way ANOVA using GraphPad Prism v7.02 for Windows (GraphPad Software, San Diego, CA, USA). When ANOVA indicated a significant effect of treatment (*P* < 0.05), individual treatment differences were compared using Bonferroni's multiple compari-

In our hands, prior to in vitro maturation, more than 95% of swine oocytes have

not started their nuclear maturation, that is, their meiotic resumption (<5% of

The first experiment is aiming to assess ADO on inhibition of oocyte GVBD. When COC are cultured for 24 hours in control treatment, more than 94.3 ± 3.0% of the oocytes were in GVBD, that is, oocyte meiotic resumption took place (**Figure 1A**). The inhibitory effect of ADO was dose-dependent with only 13.9 ± 2.1% in GVBD after an exposure to ADO at 2.0 mM for 24 hours (**Figure 1A**).

The EC50 of ADO on the percentage of GVBD oocyte is calculated as already described [11] and determined to be 1.3 mM. In addition, at 2.0 mM ADO inhibited cumulus cell expansion (**Figure 1B5**) compared with the control (**Figure 1B1**). When cultured for 24 hours as oocytes denuded of their cumulus cells (DO), ADO (2.0 mM) did not significantly inhibit oocyte GVBD compared with the control conditions (**Figure 1C**). These results support an inhibitory effect of ADO on both oocyte meiotic resumption and cumulus expansion when cultured as COC but not as DO.

GVDB oocytes), and cumulus cells have not expanded yet [12].

**3.1 Adenosine inhibits oocyte meiotic resumption**

son post-hoc test. Significant effects were identified by \* or different letters.

**16**

*Dose-response of ADO on both (A) the percentage of GVBD oocyte and (B) cumulus cell expansion. (C) Effect of ADO (2.0 mM) on the percentage of denuded oocytes in GVBD. Data are presented as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by \*. In (B) treatments were (1) Ct, (2) ADO 0.25 mM, (3) ADO 0.5 mM, (4) ADO 1.0 mM, and (5) ADO 2.0 mM.*

#### **3.2 Cell-permeable analog of adenosine (8-BrADO) inhibits GVBD**

The following experiment is performed to assess a membrane-permeable analog of ADO, 8-BrADO (mimicking the intracellular effect). When COC are cultured for 24 hours, 12.2 ± 2.7% of the oocytes were in GVBD when treated with 2.0 mM ADO and 15.9 ± 0.8% when treated with 2.0 mM 8-BrADO (**Figure 2A**). These two treatments are significantly different to the control treatment (88.7 ± 2.9%; **Figure 2A**). The inhibition of cumulus cell expansion is also observed in both treatments (**Figure 2B2** and **B3**). These results support that the inhibitory effect of ADO may be mimicked by the cell-permeable analog of adenosine, 8-BrADO.

#### **3.3 Reversibility of GVBD inhibition**

To assess the reversibility, the test compounds (ADO and 8-BrADO) are washed out, and the COC are cultured under control conditions for a second 24-hour

#### **Figure 2.**

*Comparison of the effect of ADO (2.0 mM) with the cell-permeable analog 8-BrADO (2.0 mM) on both the percentage of GVBD oocytes and cumulus cell expansion after (A–B) 24 hours and (C–D) reversibility of 24 hours (Rev). DMSO treatment at 0.1% is the control for 8-BrADO. (C) Reversibility: COC were first cultured for 24 hours according to treatment and then cultured for a second 24 hours in control culture medium (Ct) to assess the reversibility of the inhibition of oocyte meiotic resumption. Data are expressed as the mean ±SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of treatment at P < 0.05 is shown by different letters and \*. In (B), treatments were (1) Ct, (2) ADO, and (3) 8-BrADO. In (D) treatments were (1) Ct, (2) ADO, (3) DMSO, and (4) 8-BrADO.*

period. The ADO-treated oocytes resumed meiosis showing no statistical difference with the control treatment, whereas a significant lower percentage of GVBD oocytes is observed following treatment with 8-BrADO (**Figure 2C**). Cumulus cell expansion is compromised by 8-BrADO (**Figure 2D4**), supporting an impairment of oocyte maturation (nuclear maturation and cumulus cell expansion) when using the halogenate compound. However, cumulus cells are expanding following reversibility treatment of the ADO-treated COC (**Figure 2D2**), supporting the reversibility of the ADO inhibitory effect. The appropriate controls are presented in **Figure 2D1** and **D3**. These data are supportive of an adenosinergic system involved in the control of oocyte meiotic resumption.

**19**

**Figure 3.**

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

**assay**

**3.4 Gap junction communications measured by cumulus-cumulus gap-FRAP** 

tion [17, 18], the aim of the following experiment was to measure the impact of

Since gap junction communications are highly regulated during in vitro matura-

*Effect of ADO (A) on gap junction communication, (B) according to hormonal supplementation, and (C) according to FK supplementation. (A) The effect of ADO (2.0 mM) on gap junction communications in between cumulus cells measured by fluorescent recovery after photobleaching after 4 hours of in vitro culture. The COC were prepared using calcein-AM as fluorescent probe. The data were plotted as relative intensity and presented as the mean ±SEM of a minimum of three replicates. ADO-treated cumulus cells recover significantly more fluorescence than the control cumulus cells (P < 0.05). (B) The effect of ADO according to hormonal supplementation on the percentage of GVBD oocyte after 24 hours of in vitro maturation. ADO (2.0 mM) was added either with eCG or hCG or both eCG and hCG. Data are expressed as the mean ± SEM of a minimum of three replicates. Statistically significant effect of the treatment at P < 0.05 is shown by \*. (C) The effect of ADO (2.0 mM) in the presence of either eCG and hCG or the adenylyl cyclase activator forskolin (FK, 1.1 μM), for 24 hours on the percentage of GVBD oocyte. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses.* 

*Statistically significant effect of the treatment at P < 0.05 is shown by \*.*

*Reproductive Biology and Technology in Animals*

**18**

**Figure 2.**

in the control of oocyte meiotic resumption.

period. The ADO-treated oocytes resumed meiosis showing no statistical difference with the control treatment, whereas a significant lower percentage of GVBD oocytes is observed following treatment with 8-BrADO (**Figure 2C**). Cumulus cell expansion is compromised by 8-BrADO (**Figure 2D4**), supporting an impairment of oocyte maturation (nuclear maturation and cumulus cell expansion) when using the halogenate compound. However, cumulus cells are expanding following reversibility treatment of the ADO-treated COC (**Figure 2D2**), supporting the reversibility of the ADO inhibitory effect. The appropriate controls are presented in **Figure 2D1** and **D3**. These data are supportive of an adenosinergic system involved

*ADO, and (3) 8-BrADO. In (D) treatments were (1) Ct, (2) ADO, (3) DMSO, and (4) 8-BrADO.*

*Comparison of the effect of ADO (2.0 mM) with the cell-permeable analog 8-BrADO (2.0 mM) on both the percentage of GVBD oocytes and cumulus cell expansion after (A–B) 24 hours and (C–D) reversibility of 24 hours (Rev). DMSO treatment at 0.1% is the control for 8-BrADO. (C) Reversibility: COC were first cultured for 24 hours according to treatment and then cultured for a second 24 hours in control culture medium (Ct) to assess the reversibility of the inhibition of oocyte meiotic resumption. Data are expressed as the mean ±SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of treatment at P < 0.05 is shown by different letters and \*. In (B), treatments were (1) Ct, (2)* 

#### **3.4 Gap junction communications measured by cumulus-cumulus gap-FRAP assay**

Since gap junction communications are highly regulated during in vitro maturation [17, 18], the aim of the following experiment was to measure the impact of

#### **Figure 3.**

*Effect of ADO (A) on gap junction communication, (B) according to hormonal supplementation, and (C) according to FK supplementation. (A) The effect of ADO (2.0 mM) on gap junction communications in between cumulus cells measured by fluorescent recovery after photobleaching after 4 hours of in vitro culture. The COC were prepared using calcein-AM as fluorescent probe. The data were plotted as relative intensity and presented as the mean ±SEM of a minimum of three replicates. ADO-treated cumulus cells recover significantly more fluorescence than the control cumulus cells (P < 0.05). (B) The effect of ADO according to hormonal supplementation on the percentage of GVBD oocyte after 24 hours of in vitro maturation. ADO (2.0 mM) was added either with eCG or hCG or both eCG and hCG. Data are expressed as the mean ± SEM of a minimum of three replicates. Statistically significant effect of the treatment at P < 0.05 is shown by \*. (C) The effect of ADO (2.0 mM) in the presence of either eCG and hCG or the adenylyl cyclase activator forskolin (FK, 1.1 μM), for 24 hours on the percentage of GVBD oocyte. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by \*.*

ADO treatment on gap junction communications measured by cumulus-cumulus gap-FRAP assay using calcein-AM as already described [12, 16]. Gap junction communication in cumulus cells was assessed after a 4-hour incubation in the presence of ADO (2.0 mM). The results show that gap junction communications between cumulus cells is increased following ADO treatment compared with the control treatment (**Figure 3A**). These results support that ADO maintains functional gap junctional communications between cumulus cells.

#### **3.5 The contribution of eCG in adenosine treatment**

The goal of the following experiment was to assess the contribution of gonadotropins in ADO-treated COC. The effect of ADO in inhibiting GVBD oocytes is observed in the presence of either eCG or hCG, or both in combination (**Figure 3B**). Comparison of ADO treatment supplemented with these hormones revealed no significant difference between eCG and the combination of eCG and hCG. However, a significant decrease in the percentage of GVBD was measured

#### **Figure 4.**

*The effect of GUO (A) on the percentage of GVBD oocytes, (B) on cumulus cell expansion, (C) on reversibility, (D) on gap junction communication, and (E) according to FK supplementation. (A–B) Comparison of the effect of GUO (2.0 mM) and 8-BrGUO (2.0 mM) after 24 hours. (C) Reversibility: COC were first cultured for 24 hours in the presence of 2.0 mM of GUO and then cultured for a second 24 hours in control culture medium (Ct) to assess the reversibility of the inhibition of oocyte meiotic resumption. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by different letters. (D) The effect of GUO (2.0 mM) on gap junction communications in between cumulus cells measured by fluorescent recovery after photobleaching after 4 hours of in vitro culture. The data were plotted as relative intensity and presented as the mean ± SEM of a minimum of three replicates. GUO-treated cumulus cells recover significantly more fluorescence than the control cumulus cells (P < 0.05). (E) The effect of GUO (2.0 mM) in the presence of either eCG and hCG or the adenylyl cyclase activator forskolin (FK, 1.1 μM), for 24 hours on the percentage of GVBD oocyte. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by \*. In (B), treatments were (1) Ct, (2) GUO, and (3) 8-BrGUO.*

**21**

**Figure 5.**

*(4) ADO 2.0 mM and GUO 2.0 mM.*

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

**3.6 Adenylyl cyclase activator (forskolin)**

adenosinergic system.

between hCG alone and the combination of hCG and eCG (**Figure 3B**), showing the significant contribution of eCG to ADO-inhibiting GVBD, yet supporting an

Since eCG contributes to ADO inhibitory effect, the following experiment was to evaluate the involvement of cAMP while using the adenylyl cyclase activator, forskolin. On itself, forskolin is significantly decreasing the percentage of GVBD oocytes (**Figure 3C**). ADO was significantly inhibiting GVBD both in presence of eCG-hCG and forskolin (**Figure 3C**). However, forskolin was not significantly

*The effect of supplementing both ADO and GUO (2.0 mM each) on (A) the percentage of oocyte meiotic resumption and (B) cumulus cell expansion. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by different letters. (B) The treatments were (1) Ct, (2) ADO 2.0 mM, (3) GUO 2.0 mM,* 

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

between hCG alone and the combination of hCG and eCG (**Figure 3B**), showing the significant contribution of eCG to ADO-inhibiting GVBD, yet supporting an adenosinergic system.

#### **3.6 Adenylyl cyclase activator (forskolin)**

Since eCG contributes to ADO inhibitory effect, the following experiment was to evaluate the involvement of cAMP while using the adenylyl cyclase activator, forskolin. On itself, forskolin is significantly decreasing the percentage of GVBD oocytes (**Figure 3C**). ADO was significantly inhibiting GVBD both in presence of eCG-hCG and forskolin (**Figure 3C**). However, forskolin was not significantly

*Reproductive Biology and Technology in Animals*

junctional communications between cumulus cells.

**3.5 The contribution of eCG in adenosine treatment**

ADO treatment on gap junction communications measured by cumulus-cumulus gap-FRAP assay using calcein-AM as already described [12, 16]. Gap junction communication in cumulus cells was assessed after a 4-hour incubation in the presence of ADO (2.0 mM). The results show that gap junction communications between cumulus cells is increased following ADO treatment compared with the control treatment (**Figure 3A**). These results support that ADO maintains functional gap

The goal of the following experiment was to assess the contribution of gonadotropins in ADO-treated COC. The effect of ADO in inhibiting GVBD oocytes is observed in the presence of either eCG or hCG, or both in combination (**Figure 3B**). Comparison of ADO treatment supplemented with these hormones revealed no significant difference between eCG and the combination of eCG and hCG. However, a significant decrease in the percentage of GVBD was measured

*The effect of GUO (A) on the percentage of GVBD oocytes, (B) on cumulus cell expansion, (C) on reversibility, (D) on gap junction communication, and (E) according to FK supplementation. (A–B) Comparison of the effect of GUO (2.0 mM) and 8-BrGUO (2.0 mM) after 24 hours. (C) Reversibility: COC were first cultured for 24 hours in the presence of 2.0 mM of GUO and then cultured for a second 24 hours in control culture medium (Ct) to assess the reversibility of the inhibition of oocyte meiotic resumption. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by different letters. (D) The effect of GUO (2.0 mM) on gap junction communications in between cumulus cells measured by fluorescent recovery after photobleaching after 4 hours of in vitro culture. The data were plotted as relative intensity and presented as the mean ± SEM of a minimum of three replicates. GUO-treated cumulus cells recover significantly more fluorescence than the control cumulus cells (P < 0.05). (E) The effect of GUO (2.0 mM) in the presence of either eCG and hCG or the adenylyl cyclase activator forskolin (FK, 1.1 μM), for 24 hours on the percentage of GVBD oocyte. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by \*. In* 

*(B), treatments were (1) Ct, (2) GUO, and (3) 8-BrGUO.*

**20**

**Figure 4.**

#### **Figure 5.**

*The effect of supplementing both ADO and GUO (2.0 mM each) on (A) the percentage of oocyte meiotic resumption and (B) cumulus cell expansion. Data are expressed as the mean ± SEM of a minimum of three replicates. The number of oocytes used is illustrated in parentheses. Statistically significant effect of the treatment at P < 0.05 is shown by different letters. (B) The treatments were (1) Ct, (2) ADO 2.0 mM, (3) GUO 2.0 mM, (4) ADO 2.0 mM and GUO 2.0 mM.*

changing the percentage of GVBD compared with eCG-hCG (**Figure 3C**). The results are proposing the contribution of cAMP to ADO-inhibiting GVBD.

#### **3.7 Guanosine inhibits oocyte meiotic resumption**

High-performance liquid chromatography was used to detect the presence of GUO in porcine follicular fluid from follicles of 2–6 mm in diameter (data not shown). Since GUO was detected in PFF, we undertook to assess whether GUO could play a role in inhibiting GVBD. **Figure 4** shows the significant effect of GUO in inhibiting both GVBD and cumulus expansion after 24 hours, highlighting the contribution of GUO in oocyte maturation. However, the membrane-permeable analog 8-BrGUO did not significantly inhibit GVBD (**Figure 4A**). Cumulus cell expansion was observed (**Figure 4B3**), supporting the inefficacy of 8-BrGUO in inhibiting oocyte GVBD and cumulus cell expansion. The reversibility treatment showed that GUO-treated COC resumed meiosis without any statistical significance compared with the control treatment (**Figure 4C**), supporting the reversibility of GUO inhibitory effect on GVBD. As ADO, GUO was also significantly increasing gap junction communication as measured by gap-FRAP assay (**Figure 4D**). Finally, forskolin was not significantly improving the inhibitory effect of GUO compared to eCG-hCG supplementation as measured on the percentage of GVBD oocytes (**Figure 4E**). GUO was also reversibly inhibiting oocyte GVBD, thus supporting a guanosinergic system.

#### **3.8 The effect of using both ADO and GUO**

The final experiment evaluated the effect of supplementing both ADO and GUO at 2.0 mM each on GVBD oocytes using COC (**Figure 5A**). The results showed that ADO, GUO, and both ADO and GUO were all significantly decreasing GVDB percentage compared with the control treatment. Although a significant effect of adding ADO with GUO was observed when compared with GUO (**Figure 5A**), the impact on the percentage went from 28.6 ± 1.4% for GUO to 11.8 ± 2.5% for ADO and GUO. Cumulus cell expansion was inhibited by ADO (**Figure 5B2**), GUO (**Figure 5B3**), and the combination of ADO and GUO (**Figure 5B4**) when compared with the control (**Figure 5B1**).

#### **4. Discussion**

The present study showed that both ADO and GUO are efficient in reversibly inhibiting swine oocyte GVBD (**Figures 1** and **4**). Cumulus cell expansion is significantly inhibited after 24 hours in presence to either ADO or GUO (**Figures 1** and **4**), supporting that these two purine nucleosides inhibit oocyte maturation with low GVBD percentages and no cumulus cell expansion. Both ADO and GUO also increased GJC in cumulus cells (**Figures 3** and **4**). eCG and FK are both supporting the two purines' inhibitory effect (**Figures 3** and **4**). The data suggest that the inhibitory effect of these two purines on GVBD strengthens the involvement of both adenosinergic and guanosinergic systems in meiotic resumption.

Several years ago it was clearly shown that hypoxanthine, a purine derivative, was an important component of a low molecular weight fraction from porcine follicular fluid and efficient at inhibiting oocyte GVBD [19]. It was also reported that this fraction did not exclusively contain hypoxanthine [10]. Although hypoxanthine was efficient in the mouse and rat, the question was still not fully assessed regarding the efficacy of ADO and GUO on swine COC with respect to the inhibition of oocyte meiotic resumption.

**23**

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

From a broader perspective, it is known that ADO is involved in several biological functions such as nucleotide biosynthesis and cellular energy metabolism [19]. The cellular uptake of ADO played by two classes of nucleotide transporters (SLC28 and SLC29) regulates these biological functions. Inside the cell, ADO is rapidly metabolized and either converted to inosine or adenosine monophosphate through adenosine deaminase or adenosine kinase, respectively [20]. Alternatively, extracellular ADO may serve as a signaling molecule, which activates adenosine receptors (ADORA) on the cell membrane surface. Four different seven-transmembrane domain receptors have been described [21]. It has been reported that a low dose of ADO (0.2 mM) produced a transient delay in bovine oocyte GVBD [9]. After 21 hours in culture, neither ADO nor hypoxanthine resulted in an efficient inhibition of oocyte GVBD [9]. In the mouse, 4.0 mM of hypoxanthine was clearly efficient at inhibiting oocyte GVBD, while the use of ADO by itself did not produce a significant inhibition [10]. However, the efficacy was enhanced when ADO was used in combination with hypoxanthine [7]. This was also observed when ADO was used together with FSH [22], forskolin [23], or cAMP analogs such as 8-bromocAMP [8]. Although these results have been provided to support that adenosine uptake and metabolism contribute to the inhibition of GVBD [24], ample evidence shows that functional adenosine receptors are present on ovarian cells [25]. The measured concentration of ADO in murine follicular fluid was between 0.38 and 0.68 mM [7]. In the present study, the inhibitory effect on GVBD was dosedependent with an IC50 of 1.3 mM (**Figure 1**). Using 2.0 mM, ADO was efficient at reversibly inhibiting swine oocyte GVBD and cumulus cell expansion (**Figure 1**). Although hypoxanthine and ADO have been measured in PFF [10], GUO has never been reported. In the present study, GUO has been found in PFF from 2 to 6 mm diameter follicles (data not shown). While limited information is available in the literature regarding GUO, it has been described as having important functions as an intercellular messenger especially in the central nervous system [4]. The mechanism underlying the neuroprotective properties of GUO is still not fully understood. One working hypothesis is that GUO may exert its biological effect by synchronizing distinct signaling pathways that may be related to the activation of purinergic receptor and specific G-protein binding sites [26–28]. In this study, 2.0 mM of GUO is reversibly inhibiting GVBD and cumulus cell expansion (**Figure 5**). In the mouse, GUO (1.0 mM) was reported to inhibit GVBD, while the same concentration of either hypoxanthine or ADO was inefficient [10]. In rats, the reported order of potency of these nucleosides was GUO > hypoxanthine > ADO [8]. In the mouse, there is an assumption based on a synergistic effect of ADO and GUO on the inhibition oocyte GVBD [7]. In the present study, the combination at 2.0 mM significantly increased the effect of GUO (**Figure 5**). Thus, the inhibitory effect of the combination of both ADO and GUO are somewhat additive according to the concentration used. Although GUO is proposed to activate a specific G-protein-coupled receptor with the involvement of P1 receptor [29], it has been recently reported that GUO functions as an extracellular signaling molecule without the need for GUO receptors [30]. In vascular smooth muscle cells, extracellular GUO regulated extracellular ADO [30]. This proposed GUO-ADO mechanism further regulated cell proliferation in vitro [31] and decreased inflammation in vivo

[32], supporting the additive inhibitory effect observed on oocyte GVBD.

We recently demonstrated the regulation of gap junction communications between cumulus cells during in vitro maturation [16–18]. An increase in gap junction communication was evident after 4 hours of in vitro culture [17, 18]. This observation provides an indication of how cumulus cells respond to the treatment and not only looking at cumulus cell expansion. In the present study, the effect of both treatments, ADO and GUO, was to increase gap junction communications

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

*Reproductive Biology and Technology in Animals*

guanosinergic system.

**4. Discussion**

**3.8 The effect of using both ADO and GUO**

pared with the control (**Figure 5B1**).

**3.7 Guanosine inhibits oocyte meiotic resumption**

changing the percentage of GVBD compared with eCG-hCG (**Figure 3C**). The results are proposing the contribution of cAMP to ADO-inhibiting GVBD.

High-performance liquid chromatography was used to detect the presence of GUO in porcine follicular fluid from follicles of 2–6 mm in diameter (data not shown). Since GUO was detected in PFF, we undertook to assess whether GUO could play a role in inhibiting GVBD. **Figure 4** shows the significant effect of GUO in inhibiting both GVBD and cumulus expansion after 24 hours, highlighting the contribution of GUO in oocyte maturation. However, the membrane-permeable analog 8-BrGUO did not significantly inhibit GVBD (**Figure 4A**). Cumulus cell expansion was observed (**Figure 4B3**), supporting the inefficacy of 8-BrGUO in inhibiting oocyte GVBD and cumulus cell expansion. The reversibility treatment showed that GUO-treated COC resumed meiosis without any statistical significance compared with the control treatment (**Figure 4C**), supporting the reversibility of GUO inhibitory effect on GVBD. As ADO, GUO was also significantly increasing gap junction communication as measured by gap-FRAP assay (**Figure 4D**). Finally, forskolin was not significantly improving the inhibitory effect of GUO compared to eCG-hCG supplementation as measured on the percentage of GVBD oocytes (**Figure 4E**). GUO was also reversibly inhibiting oocyte GVBD, thus supporting a

The final experiment evaluated the effect of supplementing both ADO and GUO at 2.0 mM each on GVBD oocytes using COC (**Figure 5A**). The results showed that ADO, GUO, and both ADO and GUO were all significantly decreasing GVDB percentage compared with the control treatment. Although a significant effect of adding ADO with GUO was observed when compared with GUO (**Figure 5A**), the impact on the percentage went from 28.6 ± 1.4% for GUO to 11.8 ± 2.5% for ADO and GUO. Cumulus cell expansion was inhibited by ADO (**Figure 5B2**), GUO (**Figure 5B3**), and the combination of ADO and GUO (**Figure 5B4**) when com-

The present study showed that both ADO and GUO are efficient in reversibly inhibiting swine oocyte GVBD (**Figures 1** and **4**). Cumulus cell expansion is significantly inhibited after 24 hours in presence to either ADO or GUO (**Figures 1** and **4**), supporting that these two purine nucleosides inhibit oocyte maturation with low GVBD percentages and no cumulus cell expansion. Both ADO and GUO also increased GJC in cumulus cells (**Figures 3** and **4**). eCG and FK are both supporting the two purines' inhibitory effect (**Figures 3** and **4**). The data suggest that the inhibitory effect of these two purines on GVBD strengthens the involvement of

Several years ago it was clearly shown that hypoxanthine, a purine derivative, was an important component of a low molecular weight fraction from porcine follicular fluid and efficient at inhibiting oocyte GVBD [19]. It was also reported that this fraction did not exclusively contain hypoxanthine [10]. Although hypoxanthine was efficient in the mouse and rat, the question was still not fully assessed regarding the efficacy of ADO and GUO on swine COC with respect to the inhibition of oocyte meiotic resumption.

both adenosinergic and guanosinergic systems in meiotic resumption.

**22**

From a broader perspective, it is known that ADO is involved in several biological functions such as nucleotide biosynthesis and cellular energy metabolism [19]. The cellular uptake of ADO played by two classes of nucleotide transporters (SLC28 and SLC29) regulates these biological functions. Inside the cell, ADO is rapidly metabolized and either converted to inosine or adenosine monophosphate through adenosine deaminase or adenosine kinase, respectively [20]. Alternatively, extracellular ADO may serve as a signaling molecule, which activates adenosine receptors (ADORA) on the cell membrane surface. Four different seven-transmembrane domain receptors have been described [21]. It has been reported that a low dose of ADO (0.2 mM) produced a transient delay in bovine oocyte GVBD [9]. After 21 hours in culture, neither ADO nor hypoxanthine resulted in an efficient inhibition of oocyte GVBD [9]. In the mouse, 4.0 mM of hypoxanthine was clearly efficient at inhibiting oocyte GVBD, while the use of ADO by itself did not produce a significant inhibition [10]. However, the efficacy was enhanced when ADO was used in combination with hypoxanthine [7]. This was also observed when ADO was used together with FSH [22], forskolin [23], or cAMP analogs such as 8-bromocAMP [8]. Although these results have been provided to support that adenosine uptake and metabolism contribute to the inhibition of GVBD [24], ample evidence shows that functional adenosine receptors are present on ovarian cells [25]. The measured concentration of ADO in murine follicular fluid was between 0.38 and 0.68 mM [7]. In the present study, the inhibitory effect on GVBD was dosedependent with an IC50 of 1.3 mM (**Figure 1**). Using 2.0 mM, ADO was efficient at reversibly inhibiting swine oocyte GVBD and cumulus cell expansion (**Figure 1**).

Although hypoxanthine and ADO have been measured in PFF [10], GUO has never been reported. In the present study, GUO has been found in PFF from 2 to 6 mm diameter follicles (data not shown). While limited information is available in the literature regarding GUO, it has been described as having important functions as an intercellular messenger especially in the central nervous system [4]. The mechanism underlying the neuroprotective properties of GUO is still not fully understood. One working hypothesis is that GUO may exert its biological effect by synchronizing distinct signaling pathways that may be related to the activation of purinergic receptor and specific G-protein binding sites [26–28]. In this study, 2.0 mM of GUO is reversibly inhibiting GVBD and cumulus cell expansion (**Figure 5**). In the mouse, GUO (1.0 mM) was reported to inhibit GVBD, while the same concentration of either hypoxanthine or ADO was inefficient [10]. In rats, the reported order of potency of these nucleosides was GUO > hypoxanthine > ADO [8]. In the mouse, there is an assumption based on a synergistic effect of ADO and GUO on the inhibition oocyte GVBD [7]. In the present study, the combination at 2.0 mM significantly increased the effect of GUO (**Figure 5**). Thus, the inhibitory effect of the combination of both ADO and GUO are somewhat additive according to the concentration used. Although GUO is proposed to activate a specific G-protein-coupled receptor with the involvement of P1 receptor [29], it has been recently reported that GUO functions as an extracellular signaling molecule without the need for GUO receptors [30]. In vascular smooth muscle cells, extracellular GUO regulated extracellular ADO [30]. This proposed GUO-ADO mechanism further regulated cell proliferation in vitro [31] and decreased inflammation in vivo [32], supporting the additive inhibitory effect observed on oocyte GVBD.

We recently demonstrated the regulation of gap junction communications between cumulus cells during in vitro maturation [16–18]. An increase in gap junction communication was evident after 4 hours of in vitro culture [17, 18]. This observation provides an indication of how cumulus cells respond to the treatment and not only looking at cumulus cell expansion. In the present study, the effect of both treatments, ADO and GUO, was to increase gap junction communications

compared with the control (**Figures 3** and **4**). Since gap junction communications are known to play a primordial role in oocyte maturation [33, 34], this result supports a clear impact of the treatments on cumulus cell functions that may be beneficial for the oocyte.

This study also provides evidence of hormonal supplementation impacting the inhibition of GVBD oocytes by ADO (**Figure 3**). This ADO-mediated inhibition of GVBD oocytes is improved according to the supplementation. Although it is not the purpose of this study to understand how this effect is transduced into the cells, eCG clearly improved the effect of ADO in the presence of hCG. This FSH-type stimulation seemed to be sufficient since the effect of eCG alone was not significantly different from that of eCG and hCG (**Figure 3**). As it is well-known that cumulus cells have an efficient response to FSH [35, 36], the inhibitory effect of ADO is significantly increased by FSH. This effect has been reported in the rat where the percentage of GVBD oocyte treated with ADO was decreased in the presence of FSH [22]. In addition, the FSH-induced granulosa cell differentiation was reduced by ADO [37], supporting the involvement of ADO in FSH response.

Although different forms of adenylyl cyclase have been characterized in oocytes and in cumulus cells [38], forskolin, a known adenylyl cyclase activator, makes a significant contribution to the inhibition of spontaneous maturation in several species as observed for rat [39], bovine [40], and porcine [11] oocytes. In the present study, forskolin inhibited oocyte GVBD while treated with ADO (**Figure 3C**). Similar results were obtained in the presence of GUO (**Figure4E**). These results support that constant stimulation of adenylyl cyclase, which increases the intracellular concentration of cAMP [40], promoted the inhibitory effects of ADO and GUO. In this regard, the results are proposing the contribution of cAMP to both ADO- and GUO-inhibiting GVBD.

The effect of the two purines goes well with the current working model of inhibition of oocyte GVBD involving C-type natriuretic peptide (CNP) as an oocytemeiosis-inhibiting peptide [41, 42]. CNP is involved in inhibiting oocyte GVBD [41, 43, 44]. CNP produced by granulosa cells is a ligand for NPR2, a member of guanylyl cyclase receptor family. NPR2 stimulation by CNP increased intracellular concentration of cGMP, inhibited oocyte phosphodiesterase type 3A, and thus maintained high intra-oocyte concentration of cAMP. The contribution of both purine nucleosides supports adenosinergic and guanosinergic system in the inhibition of oocyte meiotic resumption.

#### **5. Conclusion**

In conclusion, this study puts forward the contribution of ADO and GUO as inhibitory for oocyte GVBD in vitro suggesting that both adenosinergic and guanosinergic systems are efficient in inhibiting oocyte meiotic resumption. The use of these two systems as part of a pre-IVM culture period would be a novel strategy to explore in order to improve oocyte developmental competence.

Finally, it should be emphasized that the signaling involved in oocyte meiotic resumption may be modulated through the contribution of different pathways. The adenosinergic and guanosinergic systems of which we have presented the contribution illustrate this situation for meiotic resumption. It is also to be expected that other studies will pave the way for additional contributions. For example, a preliminary study from our lab revealed that, as demonstrated in bovine [45], porcine theca cells secreted efficient factors involved in oocyte meiotic resumption. Without knowing these secreted elements, this result highlights that oocyte meiotic resumption is under the control of the ovarian follicular cells. Meiotic resumption is thus under high surveillance!

**25**

**Author details**

François J. Richard

Centre de recherche en reproduction, développement et santé intergénérationnelle, Département des Sciences Animales, Faculté des sciences de l'agriculture et de

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

l'alimentation, Université Laval, Québec, Québec, Canada

provided the original work is properly cited.

\*Address all correspondence to: francois.richard@fsaa.ulaval.ca

*Oocyte Meiotic Resumption under High Surveillance DOI: http://dx.doi.org/10.5772/intechopen.88291*

for his contribution to the experimental work.

The author has no conflict of interest to declare.

This work was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-04774). Appreciation is extended to Mario Mayes

**Acknowledgements**

**Conflict of interest**

### **Acknowledgements**

*Reproductive Biology and Technology in Animals*

beneficial for the oocyte.

compared with the control (**Figures 3** and **4**). Since gap junction communications are known to play a primordial role in oocyte maturation [33, 34], this result supports a clear impact of the treatments on cumulus cell functions that may be

by ADO [37], supporting the involvement of ADO in FSH response.

the contribution of cAMP to both ADO- and GUO-inhibiting GVBD.

explore in order to improve oocyte developmental competence.

tion of oocyte meiotic resumption.

thus under high surveillance!

**5. Conclusion**

This study also provides evidence of hormonal supplementation impacting the inhibition of GVBD oocytes by ADO (**Figure 3**). This ADO-mediated inhibition of GVBD oocytes is improved according to the supplementation. Although it is not the purpose of this study to understand how this effect is transduced into the cells, eCG clearly improved the effect of ADO in the presence of hCG. This FSH-type stimulation seemed to be sufficient since the effect of eCG alone was not significantly different from that of eCG and hCG (**Figure 3**). As it is well-known that cumulus cells have an efficient response to FSH [35, 36], the inhibitory effect of ADO is significantly increased by FSH. This effect has been reported in the rat where the percentage of GVBD oocyte treated with ADO was decreased in the presence of FSH [22]. In addition, the FSH-induced granulosa cell differentiation was reduced

Although different forms of adenylyl cyclase have been characterized in oocytes and in cumulus cells [38], forskolin, a known adenylyl cyclase activator, makes a significant contribution to the inhibition of spontaneous maturation in several species as observed for rat [39], bovine [40], and porcine [11] oocytes. In the present study, forskolin inhibited oocyte GVBD while treated with ADO (**Figure 3C**). Similar results were obtained in the presence of GUO (**Figure4E**). These results support that constant stimulation of adenylyl cyclase, which increases the intracellular concentration of cAMP [40], promoted the inhibitory effects of ADO and GUO. In this regard, the results are proposing

The effect of the two purines goes well with the current working model of inhibition of oocyte GVBD involving C-type natriuretic peptide (CNP) as an oocytemeiosis-inhibiting peptide [41, 42]. CNP is involved in inhibiting oocyte GVBD [41, 43, 44]. CNP produced by granulosa cells is a ligand for NPR2, a member of guanylyl cyclase receptor family. NPR2 stimulation by CNP increased intracellular concentration of cGMP, inhibited oocyte phosphodiesterase type 3A, and thus maintained high intra-oocyte concentration of cAMP. The contribution of both purine nucleosides supports adenosinergic and guanosinergic system in the inhibi-

In conclusion, this study puts forward the contribution of ADO and GUO as inhibitory for oocyte GVBD in vitro suggesting that both adenosinergic and guanosinergic systems are efficient in inhibiting oocyte meiotic resumption. The use of these two systems as part of a pre-IVM culture period would be a novel strategy to

Finally, it should be emphasized that the signaling involved in oocyte meiotic resumption may be modulated through the contribution of different pathways. The adenosinergic and guanosinergic systems of which we have presented the contribution illustrate this situation for meiotic resumption. It is also to be expected that other studies will pave the way for additional contributions. For example, a preliminary study from our lab revealed that, as demonstrated in bovine [45], porcine theca cells secreted efficient factors involved in oocyte meiotic resumption. Without knowing these secreted elements, this result highlights that oocyte meiotic resumption is under the control of the ovarian follicular cells. Meiotic resumption is

**24**

This work was supported by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-04774). Appreciation is extended to Mario Mayes for his contribution to the experimental work.

### **Conflict of interest**

The author has no conflict of interest to declare.

### **Author details**

François J. Richard Centre de recherche en reproduction, développement et santé intergénérationnelle, Département des Sciences Animales, Faculté des sciences de l'agriculture et de l'alimentation, Université Laval, Québec, Québec, Canada

\*Address all correspondence to: francois.richard@fsaa.ulaval.ca

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Neurochemistry. 2012;**121**:329-342. DOI: 10.1111/j.1471-4159.2012.07692.x

[28] Ciruela F. Guanosine behind the scene. Journal of Neurochemistry. 2013;**126**:425-427. DOI: 10.1111/

[29] Bettio LEB, Gil-Mohapel J, Rodrigues ALS. Guanosine and its role in neuropathologies. Purinergic Signal. 2016;**12**:411-426. DOI: 10.1007/

s11302-016-9509-4

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[26] Ciccarelli R, Ballerini P, Sabatino G, Rathbone MP, D'Onofrio M, Caciagli F, et al. Involvement of astrocytes in purine-mediated reparative processes in the brain. International Journal of Developmental Neuroscience. 2001;**19**:395-414. DOI: 10.1016/ S0736-5748(00)00084-8

[27] Thauerer B, Zur Nedden S, Baier-Bitterlich G. Purine nucleosides: Endogenous neuroprotectants in hypoxic brain. Journal of Neurochemistry. 2012;**121**:329-342. DOI: 10.1111/j.1471-4159.2012.07692.x

[28] Ciruela F. Guanosine behind the scene. Journal of Neurochemistry. 2013;**126**:425-427. DOI: 10.1111/ jnc.12328

[29] Bettio LEB, Gil-Mohapel J, Rodrigues ALS. Guanosine and its role in neuropathologies. Purinergic Signal. 2016;**12**:411-426. DOI: 10.1007/ s11302-016-9509-4

**26**

*Reproductive Biology and Technology in Animals*

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**29**

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10.1095/biolreprod54.1.22

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Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes. Biology of Reproduction. 2014;**91**:61. DOI: 10.1095/biolreprod.114.118869

*Reproductive Biology and Technology in Animals*

[30] Jackson EK, Cheng D, Jackson TC, Verrier JD, Gillespie DG. Extracellular guanosine regulates extracellular adenosine levels. American Journal of Physiology. Cell Physiology. 2013;**304**:C406-C421. DOI: 10.1152/

of adenosine on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells. Biology of Reproduction. 1984;**30**:1082-1090. DOI:

[38] Horner K, Livera G, Hinckley M, Trinh K, Storm D, Conti M. Rodent oocytes express an active adenylyl cyclase required for meiotic arrest. Developmental Biology. 2003;**258**:385-396. DOI: 10.1016/

[39] Dekel N, Aberdam E, Sherizly I. Spontaneous maturation in vitro of cumulus-enclosed rat oocytes is inhibited by forskolin. Biology of Reproduction. 1984;**31**:244-250. DOI:

[40] Bilodeau S, Fortier MA, Sirard MA. Effect of adenylate cyclase stimulation on meiotic resumption and cyclic AMP content of zona-free and cumulus-enclosed bovine oocytes in vitro. Reproduction. 1993;**97**:5-11.

[41] Zhang M, Su YQ, Sugiura K, Xia G, Eppig JJ. Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes. Science. 2010;**330**:366-369. DOI: 10.1126/

[42] Gilchrist RB, Luciano AM, Richani D, Zeng HT, Wang X, De Vos M, et al. Oocyte maturation and quality: Role of cyclic nucleotides. Reproduction. 2016;**152**:R143-R157. DOI: 10.1530/

[43] Santiquet N, Papillon-Dion É, Djender N, Guillemette C, Richard FJ. New elements in the c-type natriuretic peptide signaling pathway inhibiting swine in vitro oocyte meiotic resumption. Biology of Reproduction. 2014;**91**:16. DOI: 10.1095/biolreprod.113.114132

[44] Franciosi F, Coticchio G, Lodde V, Tessaro I, Modina SC, Fadini R, et al.

10.1095/biolreprod30.5.1082

s0012-1606(03)00134-9

10.1095/biolreprod31.2.244

DOI: 10.1530/jrf.0.0970005

science.1193573

REP-15-0606

ajpcell.00212.2012

phy2.24

[31] Jackson EK, Gillespie DG.

Regulation of cell proliferation by the guanosine-adenosine mechanism: Role of adenosine receptors. Physiological Reports. 2013;**1**:e00024. DOI: 10.1002/

[32] Jackson EK, Mi Z. The guanosineadenosine interaction exists in vivo. The Journal of Pharmacology and Experimental Therapeutics. 2014;**350**: 719-726. DOI: 10.1124/jpet.114.216978

[33] Gershon E, Plaks V, Aharon I, Galiani D, Reizel Y, Sela-Abramovich S, et al. Oocyte-directed depletion of connexin43 using the Cre-LoxP system leads to subfertility in female mice. Developmental Biology. 2008;**313**:1-12. DOI: 10.1016/j.ydbio.2007.08.041

[34] Conti M, Hsieh M, Musa Zamah A, Oh JS. Novel signaling mechanisms in the ovary during oocyte maturation and ovulation. Molecular and Cellular Endocrinology. 2012;**356**:65-73. DOI:

[35] Khan DR, Guillemette C, Sirard MA, Richard FJ. Characterization of FSH signalling networks in bovine cumulus cells: A perspective on oocyte competence acquisition. Molecular Human Reproduction. 2015;**21**:688-701.

[36] Ali A, Sirard MA. Protein kinases influence bovine oocyte competence during short-term treatment with recombinant human follicle stimulating hormone. Reproduction. 2005;**130**:303-

10.1016/j.mce.2011.11.002

DOI: 10.1093/molehr/gav032

310. DOI: 10.1530/rep.1.00387

[37] Knecht M, Darbon JM, Ranta T, Baukal A, Catt KJ. Inhibitory actions

**28**

[45] Richard FJ, Sirard MA. Effects of follicular cells on oocyte maturation. II: Theca cell inhibition of bovine oocyte maturation in vitro. Biology of Reproduction. 1996;**54**:22-28. DOI: 10.1095/biolreprod54.1.22

**31**

**1. Introduction**

**Chapter 3**

Lambs

**Abstract**

Effect of Pre and Post Weaning

Diet Quality on Puberty Age and

Tail Measures in Kurdish Female

To determine the value of pre and post weaning nutrition on puberty age, some hormonal concentrations and tail measures in ewe lambs, a total of 40 clinically health Kurdish female lambs (30±8.6 d and weighing 10.2±3.4 kg) were randomly allocated to one of two experimental diets in pre-weaning period: high quality diet (**HQD**, 2.50 Mcal ME/kg dry matter (**DM**) and 148 g CP/kg DM) or low quality diet (LQD, 2.02 Mcal ME/kg DM and 87 g CP/kg DM). At weaning, one half of lambs from each group was randomly separated and assigned to HQD or LQD. So there were four treatment groups in post-weaning period: H-H (HQD pre- and post-weaning); H-L (HQD pre-weaning and LQD post-weaning); L-H (LQD preweaning and HQD post-weaning) and L-L (LQD pre and post-weaning, control group). Within the post-weaning, serum progesterone concentrations was greater for ewe lambs fed at H-H group than for other groups (*P*<0.05). Serum insulin concentration was affected by the diet quality at both periods (*P*<0.05). Leptin concentration was affected by treatment and ewe lambs of L-H group had higher leptin concentrations (*P*<0.05). Diet plan in the pre-pubertal period was affected

**Keywords:** hormone, Kurdish lambs, milk, nutrition plan, reproductive performance

In tropical, semiarid, and arid areas, animal production is dependent on supple-

mental feeding, especially in the reproductive seasons due to the higher energy demand [1]. Reproduction efficiency can play a critical role in determining profit potential for livestock production systems. Most sheep breeds become sexually active in response to decreasing day length in the late summer to early autumn, which is an additional constraint to the timing of puberty in ewe lambs [2]. If a ewe lamb fails to achieve puberty in its first autumn, it will be delayed until the following breeding [3]. Breeding ewes to lamb at 1 year of age is a potential means of improving farm profitability and ewe lifetime performance by reducing the time

*Sedigheh Menatian, Hamidreza Mirzaei Alamouti,* 

*Farshid Fatahnia and Reza Masoumi*

tail measures in 120 and 210 days of ages (*P*<0.05).

#### **Chapter 3**

## Effect of Pre and Post Weaning Diet Quality on Puberty Age and Tail Measures in Kurdish Female Lambs

*Sedigheh Menatian, Hamidreza Mirzaei Alamouti, Farshid Fatahnia and Reza Masoumi*

#### **Abstract**

To determine the value of pre and post weaning nutrition on puberty age, some hormonal concentrations and tail measures in ewe lambs, a total of 40 clinically health Kurdish female lambs (30±8.6 d and weighing 10.2±3.4 kg) were randomly allocated to one of two experimental diets in pre-weaning period: high quality diet (**HQD**, 2.50 Mcal ME/kg dry matter (**DM**) and 148 g CP/kg DM) or low quality diet (LQD, 2.02 Mcal ME/kg DM and 87 g CP/kg DM). At weaning, one half of lambs from each group was randomly separated and assigned to HQD or LQD. So there were four treatment groups in post-weaning period: H-H (HQD pre- and post-weaning); H-L (HQD pre-weaning and LQD post-weaning); L-H (LQD preweaning and HQD post-weaning) and L-L (LQD pre and post-weaning, control group). Within the post-weaning, serum progesterone concentrations was greater for ewe lambs fed at H-H group than for other groups (*P*<0.05). Serum insulin concentration was affected by the diet quality at both periods (*P*<0.05). Leptin concentration was affected by treatment and ewe lambs of L-H group had higher leptin concentrations (*P*<0.05). Diet plan in the pre-pubertal period was affected tail measures in 120 and 210 days of ages (*P*<0.05).

**Keywords:** hormone, Kurdish lambs, milk, nutrition plan, reproductive performance

#### **1. Introduction**

In tropical, semiarid, and arid areas, animal production is dependent on supplemental feeding, especially in the reproductive seasons due to the higher energy demand [1]. Reproduction efficiency can play a critical role in determining profit potential for livestock production systems. Most sheep breeds become sexually active in response to decreasing day length in the late summer to early autumn, which is an additional constraint to the timing of puberty in ewe lambs [2]. If a ewe lamb fails to achieve puberty in its first autumn, it will be delayed until the following breeding [3]. Breeding ewes to lamb at 1 year of age is a potential means of improving farm profitability and ewe lifetime performance by reducing the time interval from birth to first lambing, subsequently reducing feed, labor, housing, and other costs associated with raising replacement animals [4, 5].

Sexual development is an important factor and can be manipulated by altering growth rates [6–8]. During the productive life of ewes, puberty period is critical for both animal health and performance. The onset of puberty in sheep is influenced by genetic and environmental factors such as nutrition, day length, temperature, and their interaction [9]. Ewe lambs growing at faster rates will exhibit their first estrus and are more likely to conceive at a lower age and have heavier body weight (BW) than ewe lambs growing at slower rates [10]. Because of the importance of BW, environmental factors that can affect the rate of growth before and after weaning are important determinants of age at puberty. Mulvaney et al. [11] reported that ewe lambs gaining 208 g/ day compared to 153 g/day were more likely to return to breeding, although overall pregnancy rates did not differ. Generally, faster growth is associated with enhanced reproductive performance in ewe lambs, i.e., earlier attainment of puberty, more intense estrous activity, and higher conception and lambing rates when mated [12].

The most economically important traits in sheep production are growth, reproductive performance, and milk production, and there is no study on the abovementioned characteristics in Kurdish ewe lambs; therefore, the objective of this study was to compare the effects of diet quality fed during the pre-weaning and post-weaning periods and potential interactions between pre- and post-weaning diets on skeletal growth, reproduction performance, hormone concentrations, and milk production during the first lactation in Kurdish ewe lambs.

#### **2. Materials and methods**

#### **2.1 Hormonal drugs**

Controlled internal drug release (CIDR) with 300 mg of progesterone, a progestagen analogue (InterAg, Hamilton, New Zealand), PMSG (Folligon; Intervet International BV, Boxmeer, the Netherlands), oxytocin (Oxytocin V, 10 IU/ml, Phoenix Pharm, Auckland, New Zealand), and commercially available kits of leptin (LDN, Germany, LOT: 150873), insulin (DiaMetra, Italy, LOT N: 3949C), and progesterone (DiaMetra. Italy. LOT N: 4026) were used.

#### **2.2 Locations, animals, and treatment schedule**

This study was performed at the Nomadic Management Department, Ilam Province, Iran (33°51´ N, 46°27′ E), from January 2013 to December 2015. All procedures involving animal care and management were approved by the University of Zanjan Animal Care Committee (proposal no. 1169739). A total of 40 clinically health Kurdish female lambs (30 ± 8.6 days and weighing 10.2 ± 3.4 kg) were used. At 30 days of age, lambs were randomly housed together with twice daily access to their mother's milk and were allocated to one of the two experimental treatments to achieve either high or low rates of BW gain during two consecutive periods, from 30 to 120 (pre-weaning period) and from 121 to 210 days of age (post-weaning period). They were kept in individual pens (1 × 2 m) for three consecutive days every 2 weeks for recording dry matter intake (DMI). In pre-weaning period, the lambs were fed high-quality diet (HQD, n = 20) or low-quality diet (LQD, n = 20), and at the weaning time, HQD and LQD fed lambs were re-randomized so that one half of lambs from each group is randomly allocated to HQD or LQD. So there were four treatment groups (n = 10) in post-weaning period: HQD pre- and post-weaning (H-H); HQD pre-weaning and LQD post-weaning (H-L); LQD pre-weaning and

**33**

**Table 1**.

*\**

**Table 1.**

*Effect of Pre and Post Weaning Diet Quality on Puberty Age and Tail Measures in Kurdish…*

**Composition (%) Pre- and post-weaning diets**

Alfalfa hay 445.1 — Wheat straw — 513.7 Ground barley 445.1 428.1 Soybean meal 59.3 — Calcium carbonate 5.9 6.8 Salt 5.0 5.0 Mineral and vitamin premix\* 39.6 46.4 DM 916.0 919.0 CP 148.0 87.0 EE 58.0 22.0 NDF 285.0 450.0 NFC 466.0 371.0 ME (Mcal/kg) 2.50 2.02

**HQD LQD**

HQD post-weaning (L-H); and LQD pre- and post-weaning (L-L, control group). The HQD and LQD were formulated according to recommended nutrient requirements for small ruminants [13] that received a diet that covered nutrient requirements including energy and protein needs for a 20 kg growing lamb with an average daily gain (ADG) of 200 and 100 g/day, respectively. Diets were formulated to have different metabolize energy (ME) and crude protein (CP) contents. The HQD and LQD were contained 2.50 and 2.02 Mcal ME /kg DM and 14.9 and 8.9% CP (DM basis), respectively. Rations were totally hand-mixed for each pen and offered in equal proportions twice daily at 09:00 and 16:00 in pre- and post-weaning period. The ingredients and chemical composition of the experimental diets are shown in

*Each kg (DM basis) of mineral and vitamin premix contained 180 g of Ca, 70 g of P, 35 g of K, 50 g of Na, 58 g of Cl, 30 g of Mg, 32 g of S, 5 g of Mn, 4 g of Fe, 3 g of Zn, 300 mg of Cu, 100 mg of I, 100 mg of Co, 20 mg of Se, 400,000 IU vitamin A, 100,000 IU vitamin D3, and 245 IU vitamin E. DM, dry matter; CP, crude protein; EE,* 

*ether extract; NFC, nonfiber carbohydrates = 100 – (CP + NDF + EE + ash); ME, metabolite energy.*

When ewe lambs reached 210 days old, estrus was induced and synchronized by CIDR. Animals were treated with CIDR for 14 days and were injected with 500 IU PMSG at the time of CIDR withdrawal. Twenty-four hours after CIDR withdrawal, all of ewe lambs were monitored for estrus detection by five intact fertile rams and were ultimately naturally bred. The rams remained with the ewe lambs until the termination of estrous signs. After serving, all ewe lambs were kept together in the same nutritional and managerial conditions and reared in the pasture until 2 weeks before expected parturition. Pregnancy diagnosis was determined by using of transabdominal ultrasound (Pie Medical, Falco 100, Netherlands) at 60 days after serving.

BW was measured every 2 weeks from 30 to 210 days of age. Feed offered and feed refusals of individual pens were weighed and recorded daily, and DM content

**2.3 Estrous synchronization and pregnancy diagnosis**

*Pre- and post-weaning treatments: HQD, high-quality diet; LQD, low-quality diet.*

*Ingredients and chemical composition of experimental diets.*

**2.4 Data collection and calculation**

*DOI: http://dx.doi.org/10.5772/intechopen.88647*


*Effect of Pre and Post Weaning Diet Quality on Puberty Age and Tail Measures in Kurdish… DOI: http://dx.doi.org/10.5772/intechopen.88647*

*Pre- and post-weaning treatments: HQD, high-quality diet; LQD, low-quality diet.*

*\* Each kg (DM basis) of mineral and vitamin premix contained 180 g of Ca, 70 g of P, 35 g of K, 50 g of Na, 58 g of Cl, 30 g of Mg, 32 g of S, 5 g of Mn, 4 g of Fe, 3 g of Zn, 300 mg of Cu, 100 mg of I, 100 mg of Co, 20 mg of Se, 400,000 IU vitamin A, 100,000 IU vitamin D3, and 245 IU vitamin E. DM, dry matter; CP, crude protein; EE, ether extract; NFC, nonfiber carbohydrates = 100 – (CP + NDF + EE + ash); ME, metabolite energy.*

#### **Table 1.**

*Reproductive Biology and Technology in Animals*

interval from birth to first lambing, subsequently reducing feed, labor, housing,

Sexual development is an important factor and can be manipulated by altering growth rates [6–8]. During the productive life of ewes, puberty period is critical for both animal health and performance. The onset of puberty in sheep is influenced by genetic and environmental factors such as nutrition, day length, temperature, and their interaction [9]. Ewe lambs growing at faster rates will exhibit their first estrus and are more likely to conceive at a lower age and have heavier body weight (BW) than ewe lambs growing at slower rates [10]. Because of the importance of BW, environmental factors that can affect the rate of growth before and after weaning are important determinants of age at puberty. Mulvaney et al. [11] reported that ewe lambs gaining 208 g/ day compared to 153 g/day were more likely to return to breeding, although overall pregnancy rates did not differ. Generally, faster growth is associated with enhanced reproductive performance in ewe lambs, i.e., earlier attainment of puberty, more intense estrous activity, and higher conception and lambing rates when mated [12]. The most economically important traits in sheep production are growth, reproductive performance, and milk production, and there is no study on the abovementioned characteristics in Kurdish ewe lambs; therefore, the objective of this study was to compare the effects of diet quality fed during the pre-weaning and post-weaning periods and potential interactions between pre- and post-weaning diets on skeletal growth, reproduction performance, hormone concentrations, and

and other costs associated with raising replacement animals [4, 5].

milk production during the first lactation in Kurdish ewe lambs.

progesterone (DiaMetra. Italy. LOT N: 4026) were used.

**2.2 Locations, animals, and treatment schedule**

Controlled internal drug release (CIDR) with 300 mg of progesterone, a progestagen analogue (InterAg, Hamilton, New Zealand), PMSG (Folligon; Intervet International BV, Boxmeer, the Netherlands), oxytocin (Oxytocin V, 10 IU/ml, Phoenix Pharm, Auckland, New Zealand), and commercially available kits of leptin (LDN, Germany, LOT: 150873), insulin (DiaMetra, Italy, LOT N: 3949C), and

This study was performed at the Nomadic Management Department, Ilam Province, Iran (33°51´ N, 46°27′ E), from January 2013 to December 2015. All procedures involving animal care and management were approved by the University of Zanjan Animal Care Committee (proposal no. 1169739). A total of 40 clinically health Kurdish female lambs (30 ± 8.6 days and weighing 10.2 ± 3.4 kg) were used. At 30 days of age, lambs were randomly housed together with twice daily access to their mother's milk and were allocated to one of the two experimental treatments to achieve either high or low rates of BW gain during two consecutive periods, from 30 to 120 (pre-weaning period) and from 121 to 210 days of age (post-weaning period). They were kept in individual pens (1 × 2 m) for three consecutive days every 2 weeks for recording dry matter intake (DMI). In pre-weaning period, the lambs were fed high-quality diet (HQD, n = 20) or low-quality diet (LQD, n = 20), and at the weaning time, HQD and LQD fed lambs were re-randomized so that one half of lambs from each group is randomly allocated to HQD or LQD. So there were four treatment groups (n = 10) in post-weaning period: HQD pre- and post-weaning (H-H); HQD pre-weaning and LQD post-weaning (H-L); LQD pre-weaning and

**2. Materials and methods**

**2.1 Hormonal drugs**

**32**

*Ingredients and chemical composition of experimental diets.*

HQD post-weaning (L-H); and LQD pre- and post-weaning (L-L, control group). The HQD and LQD were formulated according to recommended nutrient requirements for small ruminants [13] that received a diet that covered nutrient requirements including energy and protein needs for a 20 kg growing lamb with an average daily gain (ADG) of 200 and 100 g/day, respectively. Diets were formulated to have different metabolize energy (ME) and crude protein (CP) contents. The HQD and LQD were contained 2.50 and 2.02 Mcal ME /kg DM and 14.9 and 8.9% CP (DM basis), respectively. Rations were totally hand-mixed for each pen and offered in equal proportions twice daily at 09:00 and 16:00 in pre- and post-weaning period. The ingredients and chemical composition of the experimental diets are shown in **Table 1**.

#### **2.3 Estrous synchronization and pregnancy diagnosis**

When ewe lambs reached 210 days old, estrus was induced and synchronized by CIDR. Animals were treated with CIDR for 14 days and were injected with 500 IU PMSG at the time of CIDR withdrawal. Twenty-four hours after CIDR withdrawal, all of ewe lambs were monitored for estrus detection by five intact fertile rams and were ultimately naturally bred. The rams remained with the ewe lambs until the termination of estrous signs. After serving, all ewe lambs were kept together in the same nutritional and managerial conditions and reared in the pasture until 2 weeks before expected parturition. Pregnancy diagnosis was determined by using of transabdominal ultrasound (Pie Medical, Falco 100, Netherlands) at 60 days after serving.

#### **2.4 Data collection and calculation**

BW was measured every 2 weeks from 30 to 210 days of age. Feed offered and feed refusals of individual pens were weighed and recorded daily, and DM content of total mixed ration (TMR) and orts was determined to estimate DMI. ME and CP intake were calculated as DMI from each diet multiplied by their ME and CP contents, respectively [13]. DM, CP, and ether extract (EE) of experimental diets were measured according to the methods of AOAC [14]. The neutral detergent fiber (NDF) was measured according to the method described by Van Soest et al. [15] without α-amylase and sodium sulfite and was expressed exclusive of residual ash. Nonfibrous carbohydrates (NFC) were calculated according to NRC [16] dairy cattle model as 100 − (CP + NDF + EE + ash). Milk intake by ewe lambs was measured by the weigh-suckle-weigh method (WSW) in three consecutive days every 2 weeks from the start of study to weaning (30–120 days). At the start of WSW method at each suckling occasion (twice daily), ewe lambs were weighed, allowed to suckle the udder of their dams, and weighed again immediately after suckling. The difference between pre- and post-suckling weights was defined as milk intake (2 months [17]). On each milking occasion, ewes were milked by hand after intravenous injection of 1 IU synthetic oxytocin. Milk samples of dams in subsequent lactation were collected in three consecutive days every 2 weeks and analyzed for fat, protein, and lactose by using of MilkoScan 133B (Foss Electric, Hillerød, Denmark). Milk protein, fat, and lactose yields were calculated by multiplying milk yield from the respective day by protein, fat, and lactose contents of the milk for each ewe. Milk gross energy (GE) was calculated as GE = ((0.0547 × CP%) + (0.0929 × Fat%) + (0.0395 × Lactose%)) according to NRC [16]. The mean metabolize ability of the ewe milk GE is 0.94 [18]; therefore, milk ME content was calculated as GE × 0.94. Energy-corrected milk (ECM) and fat-corrected milk (6.5% FCM) were calculated as ECM = (0.327 × kg milk) + (12.95 × kg fat) + (7.2 × kg protein) and FCM = milk yield × (0.37 + (0.097 × Fat%)).

#### **2.5 Blood sampling and analysis**

Before the first meal of the day, blood samples (5 ml) were collected by jugular venipuncture from each lamb every 2 weeks from 90 days of age until puberty (age at puberty was assessed by serum concentrations of progesterone, where puberty was determined as the age when two consecutive blood samples contained at least 1 ng of progesterone/mL). Hence, samples were centrifuged for 15 min (3000 rpm), and sera were separated into 1.5 ml micro tubes and then placed in freezer (−20°C). Serum samples were tested for leptin, insulin, and progesterone by ELISA method. Standard commercial kits were used for analysis, and the procedures were adopted as recommended by the manufacturer of these kits.

#### **2.6 Statistical analyses**

Data were analyzed as a completely randomized design in factorial arrangement (2 × 2) by using the mixed model procedure of SAS software [19] with fixed effects of treatment and random effects of lamb nested in treatments:

$$\text{Yik} = \mu + \text{Di} + \text{Lk(Di)} + \text{eik} \tag{1}$$

**35**

studies.

*Effect of Pre and Post Weaning Diet Quality on Puberty Age and Tail Measures in Kurdish…*

Measurements obtained before administration of dietary treatments were used as covariates. The covariates were removed from the model one at a time, starting with the least significant. Least square means, standard error of means, and P-values are reported. Statistical differences were considered significant when

Kurdish ewe is the most popular indigenous dairy breed of sheep in the west of Iran. Its main characteristics are high prolificacy and high milk yield. Considering the high genetic potential of Kurdish sheep, it is important to ensure that appropriate management practices are implemented in these intensive production systems.

Discussing about the topic of sheep and lamb management over the last 40–50 years traditionally involved sheep management, growth development, and early weaning. In the last 10–20 years, the concept of "intensified feeding or accelerated growth" has become a focus of discussion, and during that time the concept

The results of intake and BW that were obtained from ewe lambs pre- and post-weaning are summarized in **Table 2**. Results showed that initial BW was similar between all experimental groups (P > 0.05), but ewe lambs fed with HQD would gain faster than ewe lambs fed with LQD in BW. And also accelerated BW during the prepubertal period was achieved in the current study affecting the age of puberty (**Table 3**); this is in agreement with the results of Rosales et al. [20] in ewe lambs. BW includes muscle and fat and thus questions interpretation. An important consideration is that BW per se is simply mass and so encompasses physiological or mechanistic process that would affect the reproductive system [21]. In 2009 and 2010, Kenyon et al. indicated that there is a clear positive relationship between BW and reproductive performance in ewe lambs that aligns with the results of present

Results revealed that the HQD treatment increased DMI, compared with the LQD treatment during pre-weaning period (P < 0.01). DMI in L-H group was lower than in H-H group, which is similar to the observations by Aguerre et al. [22]. DMI in H-L group was greater than in L-L group, lambs fed with the HQD in pre-weaning had greater DMI when they were fed with the LQD in post-weaning, and it seems that the increase in DMI let to larger body size. Ewe lambs from the H-L treatments also experienced reduced growth rates during post-weaning period, possible reasons for this result may be the larger body size, higher basal metabolism, higher energy, and protein requirements; with regard to rumen capacity and appetite of lambs, the LQD could not cover their needs at period post-weaning. Animals of L-H treatment could respond to diet quality changes but with lower rates than before weaning. Feeding LQD reduces the weight gain of growing animals and can result in greater growth rate once dietary conditions improve, and current results

Ewe lamb fresh milk intake and milk ME and CP intake were not affected by diet quality among treatments (P > 0.05, **Table 2**). During the post-weaning period, lambs of H-H treatment had higher (P < 0.01; **Table 2**) DMI, ME, and CP intake

The rates of ADG are shown in **Table 2**, indicating differences in the timing of responses to diet quality. Hence, responses to HQD were much greater at a younger

has been applied to research programs and on farm in various ways.

are in agreement with those reported by Drouillard et al. (1991).

compared with other lamb treatments (P > 0.05).

*DOI: http://dx.doi.org/10.5772/intechopen.88647*

**3. Results and discussion**

**3.1 Intake, growth, and puberty**

P < 0.05 and trends are discussed when P < 0.01.

where Yij = dependent variable; μ = mean; Di = fixed effect of dietary treatment i; Lk(Di) = effect of lamb k nested in the dietary treatment; εik = error.

For repeated measure date model:

$$\text{Yijk} = \mu + \text{Di} + \text{ti}\\ \text{mej} + \text{Di} \times \text{ti}\\ \text{mej} + \perp \text{k(Đi}\\ \text{t}\text{)} + \varepsilon \text{ijk} \tag{2}$$

where timej = effect of time j as a fixed effect.

*Effect of Pre and Post Weaning Diet Quality on Puberty Age and Tail Measures in Kurdish… DOI: http://dx.doi.org/10.5772/intechopen.88647*

Measurements obtained before administration of dietary treatments were used as covariates. The covariates were removed from the model one at a time, starting with the least significant. Least square means, standard error of means, and P-values are reported. Statistical differences were considered significant when P < 0.05 and trends are discussed when P < 0.01.

#### **3. Results and discussion**

*Reproductive Biology and Technology in Animals*

**2.5 Blood sampling and analysis**

**2.6 Statistical analyses**

as recommended by the manufacturer of these kits.

For repeated measure date model:

where timej = effect of time j as a fixed effect.

of treatment and random effects of lamb nested in treatments:

Lk(Di) = effect of lamb k nested in the dietary treatment; εik = error.

of total mixed ration (TMR) and orts was determined to estimate DMI. ME and CP intake were calculated as DMI from each diet multiplied by their ME and CP contents, respectively [13]. DM, CP, and ether extract (EE) of experimental diets were measured according to the methods of AOAC [14]. The neutral detergent fiber (NDF) was measured according to the method described by Van Soest et al. [15] without α-amylase and sodium sulfite and was expressed exclusive of residual ash. Nonfibrous carbohydrates (NFC) were calculated according to NRC [16] dairy cattle model as 100 − (CP + NDF + EE + ash). Milk intake by ewe lambs was measured by the weigh-suckle-weigh method (WSW) in three consecutive days every 2 weeks from the start of study to weaning (30–120 days). At the start of WSW method at each suckling occasion (twice daily), ewe lambs were weighed, allowed to suckle the udder of their dams, and weighed again immediately after suckling. The difference between pre- and post-suckling weights was defined as milk intake (2 months [17]). On each milking occasion, ewes were milked by hand after intravenous injection of 1 IU synthetic oxytocin. Milk samples of dams in subsequent lactation were collected in three consecutive days every 2 weeks and analyzed for fat, protein, and lactose by using of MilkoScan 133B (Foss Electric, Hillerød, Denmark). Milk protein, fat, and lactose yields were calculated by multiplying milk yield from the respective day by protein, fat, and lactose contents of the milk for each ewe. Milk gross energy (GE) was calculated as GE = ((0.0547 × CP%) + (0.0929 × Fat%) + (0.0395 × Lactose%)) according to NRC [16]. The mean metabolize ability of the ewe milk GE is 0.94 [18]; therefore, milk ME content was calculated as GE × 0.94. Energy-corrected milk (ECM) and fat-corrected milk (6.5% FCM) were calculated as ECM = (0.327 × kg milk) + (12.95 × kg fat) + (7.2 × kg protein) and FCM = milk yield × (0.37 + (0.097 × Fat%)).

Before the first meal of the day, blood samples (5 ml) were collected by jugular venipuncture from each lamb every 2 weeks from 90 days of age until puberty (age at puberty was assessed by serum concentrations of progesterone, where puberty was determined as the age when two consecutive blood samples contained at least 1 ng of progesterone/mL). Hence, samples were centrifuged for 15 min (3000 rpm), and sera were separated into 1.5 ml micro tubes and then placed in freezer (−20°C). Serum samples were tested for leptin, insulin, and progesterone by ELISA method. Standard commercial kits were used for analysis, and the procedures were adopted

Data were analyzed as a completely randomized design in factorial arrangement (2 × 2) by using the mixed model procedure of SAS software [19] with fixed effects

= μ + + ( ) + ε (1)

= μ + + + × + ( ) + ε (2)

where Yij = dependent variable; μ = mean; Di = fixed effect of dietary treatment i;

**34**

Kurdish ewe is the most popular indigenous dairy breed of sheep in the west of Iran. Its main characteristics are high prolificacy and high milk yield. Considering the high genetic potential of Kurdish sheep, it is important to ensure that appropriate management practices are implemented in these intensive production systems.

#### **3.1 Intake, growth, and puberty**

Discussing about the topic of sheep and lamb management over the last 40–50 years traditionally involved sheep management, growth development, and early weaning. In the last 10–20 years, the concept of "intensified feeding or accelerated growth" has become a focus of discussion, and during that time the concept has been applied to research programs and on farm in various ways.

The results of intake and BW that were obtained from ewe lambs pre- and post-weaning are summarized in **Table 2**. Results showed that initial BW was similar between all experimental groups (P > 0.05), but ewe lambs fed with HQD would gain faster than ewe lambs fed with LQD in BW. And also accelerated BW during the prepubertal period was achieved in the current study affecting the age of puberty (**Table 3**); this is in agreement with the results of Rosales et al. [20] in ewe lambs. BW includes muscle and fat and thus questions interpretation. An important consideration is that BW per se is simply mass and so encompasses physiological or mechanistic process that would affect the reproductive system [21]. In 2009 and 2010, Kenyon et al. indicated that there is a clear positive relationship between BW and reproductive performance in ewe lambs that aligns with the results of present studies.

Results revealed that the HQD treatment increased DMI, compared with the LQD treatment during pre-weaning period (P < 0.01). DMI in L-H group was lower than in H-H group, which is similar to the observations by Aguerre et al. [22]. DMI in H-L group was greater than in L-L group, lambs fed with the HQD in pre-weaning had greater DMI when they were fed with the LQD in post-weaning, and it seems that the increase in DMI let to larger body size. Ewe lambs from the H-L treatments also experienced reduced growth rates during post-weaning period, possible reasons for this result may be the larger body size, higher basal metabolism, higher energy, and protein requirements; with regard to rumen capacity and appetite of lambs, the LQD could not cover their needs at period post-weaning. Animals of L-H treatment could respond to diet quality changes but with lower rates than before weaning. Feeding LQD reduces the weight gain of growing animals and can result in greater growth rate once dietary conditions improve, and current results are in agreement with those reported by Drouillard et al. (1991).

Ewe lamb fresh milk intake and milk ME and CP intake were not affected by diet quality among treatments (P > 0.05, **Table 2**). During the post-weaning period, lambs of H-H treatment had higher (P < 0.01; **Table 2**) DMI, ME, and CP intake compared with other lamb treatments (P > 0.05).

The rates of ADG are shown in **Table 2**, indicating differences in the timing of responses to diet quality. Hence, responses to HQD were much greater at a younger


*Pre-weaning treatments: HQD, high-quality diet; LQD, low-quality diet. Post-weaning treatments: H-H, HQD pre- and post-weaning; H-L, HQD pre-weaning and LQD post-weaning; L-H, LQD pre-weaning and HQD postweaning; L-L, LQD pre- and post-weaning (control).*

*DM, dry matter; ME, metabolite energy; CP, crude protein; BW, body weight; ADG, average daily gain; and FCR, feed conversion ratio.*

#### **Table 2.**

*Effects of pre- and post-weaning diet quality on intake and body weight of ewe lambs (30–210 days of age).*

age, while responses to HQD were greater for ewe lambs that fed with LQD at preweaning and then received HQD at post-weaning, which is in agreement with other studies on sheep and cattle [23–25]. These authors indicate that increased severity of feed restriction is likely to increase the rate of growth after realimentation. Animals fed with HQD had higher BW and ADG compared with animals fed with LQD at pre-weaning period (P < 0.01). Lambs on the L-H group during the post-weaning had higher ADG than lambs on H-L and L-L groups (P < 0.01). However, lambs had higher ADG than H-H group, but this difference was not significant (153 vs. 138 g/day).

Growth rate and feed conversion ratio (FCR) are considered key production parameters by sheep farmers. Optimizing these parameters can better farm income by improving the production of the farm and/or by improving production efficiency (lambs produced per unit of feed consumed). Lambs consuming the HQD in pre-weaning had similar FCR with lambs fed with the LQD. Ewe lambs consuming the HQD in pre-weaning and LQD in post-weaning (H-L group) had greater FCR than other groups (**Table 2**). Ewe lambs in L-H group had lowest FCR and greater feed efficiency than H-H group.

Nutrition is a factor that influences the start of lamb's puberty and has an important effect on sexual maturity [9]. Most Kurdish ewe lambs achieved puberty by 210–240 days of age when their average live weight was approximately 35 kg or approximately 65% of their estimated mature live weight [26]. The average puberty age of H-H and L-H groups was lower than H-L and L-L groups, respectively (P < 0.05, **Table 2**). Based on the results of current study, ewe lambs with higher growth rate were more likely to achieve puberty. And also these lambs were heavier at weaning time and grew faster during the post-weaning period. The ewe lambs of H-L group were heavier than ewe lambs of L-L group at weaning time and

**37**

**Table 3.**

*(30–210 days of age).*

*Effect of Pre and Post Weaning Diet Quality on Puberty Age and Tail Measures in Kurdish…*

post-weaning period. These results are consistent with previous reports that faster growth results in more ewes achieving puberty at a younger age in female sheep [20]. Most of the ewe lambs of L-L group did not show puberty at 210 days of age, and therefore they were removed from the first reproduction table. The average puberty age of H-H sequence was lower than other treatments, with no significant

The mean of serum insulin and leptin concentrations of ewe lambs at pre- and post-weaning is shown in **Table 3**. Serum insulin concentrations was higher for lambs fed with HQD compared with lambs fed with LQD (4.45 vs. 2.09 μIU/ml, respectively, **Table 3**). Animals in H-H treatments had higher serum insulin concentration at 210 days of age compared with other treatments, while leptin concentration of ewe lambs of L-H group was higher than in other treatments at 210 days of age. These results indicated that maybe increasing weight gain at post-weaning contributed with higher fat fraction and also ultrasonography evidence from fat and muscle diameter of between 12- and 13-rib area support these results (the results

Little is known about the importance of leptin in the early postnatal period, despite its potential role in important processes such as mammary gland and appetite regulation and a variety of other effects in the body [21]. Leptin is synthesized and secreted primarily particularly in adipose tissue, in addition to multiple other sites of production including the mammary gland and leptin regulated by multiple hormones including somatotropin, insulin, and IGF-I [27]. Leptin concentration was positively associated with higher values for growth and fat accumulation and therefore with an improvement in reproductive performance [20]. The current results support this concept because leptin concentration was positively correlated with body fat; however, endocrine links to the reproductive control centers have not

The hormones insulin and leptin have primary roles in the control of reproduction in sheep. In times of decreased feeding, these hormones interact at the hypothalamus to reduce reproduction and enhance feeding [1]. Thus, nutrient reprioritization occurs in part at the expense of reproductive function as a survival mechanism. For example, reduced insulin occurs to spare glucose for central nervous system function, and reduced leptin occurs to allow stimulation of neuropeptide Y to in turn increase appetite as well as changes in anabolic hormones and,

**Item Pre-weaning treatments Post-weaning treatments**

n 20 20 10 10 10 10 Insulin (μIU/ml) 4.45 2.09 4.48 2.32 3.59 1.35 Leptin (ng/ml) 2.84 2.20 3.03 2.06 3.66 1.98 Progesterone (ng/ml) 1.38 0.76 2.98 1.85 2.42 0.95 *Pre-weaning treatments: HQD, high-quality diet; LQD, low-quality diet. Post-weaning treatments: H-H, HQD pre- and post-weaning; H-L, HQD pre-weaning and LQD post-weaning; L-H, LQD pre-weaning and HQD post-*

*Effects of pre- and post-weaning diet quality on serum insulin and leptin concentrations of ewe lambs* 

**HQD LQD HQD LQD**

**H-H H-L L-H L-L**

*DOI: http://dx.doi.org/10.5772/intechopen.88647*

interaction of per- and post-weaning (P < 0.05).

**3.2 Hormones**

were not reported).

been clearly identified.

*weaning; L-L, LQD pre- and post-weaning (control).*

*Effect of Pre and Post Weaning Diet Quality on Puberty Age and Tail Measures in Kurdish… DOI: http://dx.doi.org/10.5772/intechopen.88647*

post-weaning period. These results are consistent with previous reports that faster growth results in more ewes achieving puberty at a younger age in female sheep [20]. Most of the ewe lambs of L-L group did not show puberty at 210 days of age, and therefore they were removed from the first reproduction table. The average puberty age of H-H sequence was lower than other treatments, with no significant interaction of per- and post-weaning (P < 0.05).

#### **3.2 Hormones**

*Reproductive Biology and Technology in Animals*

**treatments**

**Intake Intake**

**BW BW**

**Item Post-weaning treatments**

H-H H-L L-H L-L

138 31 153 57

**HQD LQD HQD LQD**

3.49 2.44 CP (g/day) 228 104 194 76

n 20 20 n 10 10 10 10

DM (kg/d) 0.97 0.64 DM (kg/day) 1.54 1.21 1.31 0.87 Fresh milk (kg/d) 1.11 1.18 ME (Mcal/day) 3.85 2.42 3.27 1.76

CP (diet + milk, g/d) 187.4 103 Puberty age (day) 123 254 168 267

120 days (kg) 31.2 22.5 210 days (kg) 43.8 33.9 36.3 26.6

FCR (%) 4.13 4.69 FCR (%) 11.1 38.7 8.57 15.26 *Pre-weaning treatments: HQD, high-quality diet; LQD, low-quality diet. Post-weaning treatments: H-H, HQD pre- and post-weaning; H-L, HQD pre-weaning and LQD post-weaning; L-H, LQD pre-weaning and HQD post-*

*DM, dry matter; ME, metabolite energy; CP, crude protein; BW, body weight; ADG, average daily gain; and FCR,* 

(g/days)

235 136 ADG 121–210 days

**Item Pre-weaning** 

30 days (kg) 10.1 10.2

*weaning; L-L, LQD pre- and post-weaning (control).*

ME (diet + milk, Mcal/d)

 ADG 30–120 days (g/day)

*feed conversion ratio.*

**Table 2.**

age, while responses to HQD were greater for ewe lambs that fed with LQD at preweaning and then received HQD at post-weaning, which is in agreement with other studies on sheep and cattle [23–25]. These authors indicate that increased severity of feed restriction is likely to increase the rate of growth after realimentation. Animals fed with HQD had higher BW and ADG compared with animals fed with LQD at pre-weaning period (P < 0.01). Lambs on the L-H group during the post-weaning had higher ADG than lambs on H-L and L-L groups (P < 0.01). However, lambs had higher ADG than H-H group, but this difference was not significant (153 vs. 138 g/day). Growth rate and feed conversion ratio (FCR) are considered key production parameters by sheep farmers. Optimizing these parameters can better farm income by improving the production of the farm and/or by improving production efficiency (lambs produced per unit of feed consumed). Lambs consuming the HQD in pre-weaning had similar FCR with lambs fed with the LQD. Ewe lambs consuming the HQD in pre-weaning and LQD in post-weaning (H-L group) had greater FCR than other groups (**Table 2**). Ewe lambs in L-H group had lowest FCR and greater

*Effects of pre- and post-weaning diet quality on intake and body weight of ewe lambs (30–210 days of age).*

Nutrition is a factor that influences the start of lamb's puberty and has an important effect on sexual maturity [9]. Most Kurdish ewe lambs achieved puberty by 210–240 days of age when their average live weight was approximately 35 kg or approximately 65% of their estimated mature live weight [26]. The average puberty age of H-H and L-H groups was lower than H-L and L-L groups, respectively (P < 0.05, **Table 2**). Based on the results of current study, ewe lambs with higher growth rate were more likely to achieve puberty. And also these lambs were heavier at weaning time and grew faster during the post-weaning period. The ewe lambs of H-L group were heavier than ewe lambs of L-L group at weaning time and

**36**

feed efficiency than H-H group.

The mean of serum insulin and leptin concentrations of ewe lambs at pre- and post-weaning is shown in **Table 3**. Serum insulin concentrations was higher for lambs fed with HQD compared with lambs fed with LQD (4.45 vs. 2.09 μIU/ml, respectively, **Table 3**). Animals in H-H treatments had higher serum insulin concentration at 210 days of age compared with other treatments, while leptin concentration of ewe lambs of L-H group was higher than in other treatments at 210 days of age. These results indicated that maybe increasing weight gain at post-weaning contributed with higher fat fraction and also ultrasonography evidence from fat and muscle diameter of between 12- and 13-rib area support these results (the results were not reported).

Little is known about the importance of leptin in the early postnatal period, despite its potential role in important processes such as mammary gland and appetite regulation and a variety of other effects in the body [21]. Leptin is synthesized and secreted primarily particularly in adipose tissue, in addition to multiple other sites of production including the mammary gland and leptin regulated by multiple hormones including somatotropin, insulin, and IGF-I [27]. Leptin concentration was positively associated with higher values for growth and fat accumulation and therefore with an improvement in reproductive performance [20]. The current results support this concept because leptin concentration was positively correlated with body fat; however, endocrine links to the reproductive control centers have not been clearly identified.

The hormones insulin and leptin have primary roles in the control of reproduction in sheep. In times of decreased feeding, these hormones interact at the hypothalamus to reduce reproduction and enhance feeding [1]. Thus, nutrient reprioritization occurs in part at the expense of reproductive function as a survival mechanism. For example, reduced insulin occurs to spare glucose for central nervous system function, and reduced leptin occurs to allow stimulation of neuropeptide Y to in turn increase appetite as well as changes in anabolic hormones and,


*Pre-weaning treatments: HQD, high-quality diet; LQD, low-quality diet. Post-weaning treatments: H-H, HQD pre- and post-weaning; H-L, HQD pre-weaning and LQD post-weaning; L-H, LQD pre-weaning and HQD postweaning; L-L, LQD pre- and post-weaning (control).*

#### **Table 3.**

*Effects of pre- and post-weaning diet quality on serum insulin and leptin concentrations of ewe lambs (30–210 days of age).*


*a,b,c Means with distinct subscripts in the same row differ (P < 0.05). Pre-weaning treatments: HQD, high-quality diet; LQD, low-quality diet. Post-weaning treatments: H-H, HQD pre- and post-weaning; H-L, HQD pre-weaning and LQD post-weaning; L-H, LQD pre-weaning and HQD post-weaning; L-L, LQD pre- and post-weaning (control).*

#### **Table 4.**

*Effects of pre- and post-weaning diet quality on tail measures of ewe lambs (30–210 days of age).*

ultimately, reduce Kp and GnRH until feed is more readily available [21]. Rosales et al. [20] reported that leptin levels positively correlated with earlier puberty onset in ewe lambs. And also these authors reported that leptin concentration was not related to age at first estrus, but puberty and BW at first estrus were positively correlated with leptin concentration [20, 28, 29].

Progesterone can be used to monitor the pregnancy status and timing of puberty [30]. Within the post-weaning, serum progesterone concentrations were greater for ewe lambs fed at H-H group than for those fed the L-H, H-L, and L-L treatments, respectively (P < 0.05, **Table 3**). In conclusion, it was concluded that prepubertal plan plays an important role in secretion of progesterone, which leads to early sexual puberty. The present findings demonstrate that in the female lamb, LQD may impair the systems governing the luteinizing hormone that controls follicle growth and cycle hormone (progesterone secretion) and delays puberty.

#### **3.3 Tail measures**

At the 120 days of age, HQD lambs had higher tail circumference (P < 0.01; **Table 4**), than LQD lambs. At 210 days of age, after a period of feed restriction of H-L treatment, the average tail length, tail width, and tail circumference of lambs in the H-L group were lower than H-H lambs (P < 0.05; **Table 4**) but showed no difference compared with that of the L-H group (P > 0.05; **Table 4**). Animal growth has been defined as the net accretion of protein and fat in respective tissues, controlled by nutrition, environment, and genetic capacity to grow [31]. Hamouda and Atti [32] revealed that in young lambs, carcass adiposity and particularly fat tail reduce Barbarine meat value as the lamb grows. And also, fat stored in the body is an important energy source when food is rare. In the present work, results showed that better diet with greater energy and protein complementation was able to improve intake and growth performance.

#### **4. Conclusions**

The present study confirmed that a HQD improved the BW and ADG at weaning and breeding time. Based on the results of this research, weaning weight, previous nutrition plan, and current nutrition level are factors that determine puberty age, hormone concentration, and tail measures. To conclude, it was concluded that

**39**

**Author details**

Sedigheh Menatian1

and Reza Masoumi2

Zanjan, Iran

*Effects of Pre- and Post-Weaning Diet Quality on Puberty Age, Some Hormone Concentrations…*

prepubertal plan plays an important role in secretion of progesterone, insulin, and leptin, which can lead to early sexual puberty. And also these strategic plans should

The authors would like to acknowledge the Nomadic Management Department of Ilam Province (Iran) for providing research opportunities and for their help and

improve economic traits at the start of lamb's puberty in sheep husbandry.

\*, Hamidreza Mirzaei Alamouti2

1 Department of Animal Science, Faculty of Agriculture, Ilam University, Ilam, Iran

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Department of Animal Science, Faculty of Agriculture, Zanjan University,

\*Address all correspondence to: menatian@alumni.znu.ac.ir

provided the original work is properly cited.

, Farshid Fatahnia1

*DOI: http://dx.doi.org/10.5772/intechopen.88647*

**Acknowledgements**

collaborations in this work.

*Effects of Pre- and Post-Weaning Diet Quality on Puberty Age, Some Hormone Concentrations… DOI: http://dx.doi.org/10.5772/intechopen.88647*

prepubertal plan plays an important role in secretion of progesterone, insulin, and leptin, which can lead to early sexual puberty. And also these strategic plans should improve economic traits at the start of lamb's puberty in sheep husbandry.

### **Acknowledgements**

*Reproductive Biology and Technology in Animals*

Tail measures (cm)

*(control).*

**Table 4.**

correlated with leptin concentration [20, 28, 29].

improve intake and growth performance.

**3.3 Tail measures**

**4. Conclusions**

and cycle hormone (progesterone secretion) and delays puberty.

ultimately, reduce Kp and GnRH until feed is more readily available [21]. Rosales et al. [20] reported that leptin levels positively correlated with earlier puberty onset in ewe lambs. And also these authors reported that leptin concentration was not related to age at first estrus, but puberty and BW at first estrus were positively

*Effects of pre- and post-weaning diet quality on tail measures of ewe lambs (30–210 days of age).*

**Item Pre-weaning treatments Post-weaning treatments**

n 20 20 10 10 10 10

Tail length 30.95 27.15 38.00a 33.10b 36.00ab 29.26c Tail width 27.75 24.55 35.45a 31.34b 32.37ab 27.18c Tail circumference 56.75a 47.45b 67.24a 55.82b 59.16b 49.52c *a,b,c Means with distinct subscripts in the same row differ (P < 0.05). Pre-weaning treatments: HQD, high-quality diet; LQD, low-quality diet. Post-weaning treatments: H-H, HQD pre- and post-weaning; H-L, HQD pre-weaning and LQD post-weaning; L-H, LQD pre-weaning and HQD post-weaning; L-L, LQD pre- and post-weaning* 

**HQD LQD HQD LQD**

H-H H-L L-H L-L

Progesterone can be used to monitor the pregnancy status and timing of puberty [30]. Within the post-weaning, serum progesterone concentrations were greater for ewe lambs fed at H-H group than for those fed the L-H, H-L, and L-L treatments, respectively (P < 0.05, **Table 3**). In conclusion, it was concluded that prepubertal plan plays an important role in secretion of progesterone, which leads to early sexual puberty. The present findings demonstrate that in the female lamb, LQD may impair the systems governing the luteinizing hormone that controls follicle growth

At the 120 days of age, HQD lambs had higher tail circumference (P < 0.01; **Table 4**), than LQD lambs. At 210 days of age, after a period of feed restriction of H-L treatment, the average tail length, tail width, and tail circumference of lambs in the H-L group were lower than H-H lambs (P < 0.05; **Table 4**) but showed no difference compared with that of the L-H group (P > 0.05; **Table 4**). Animal growth has been defined as the net accretion of protein and fat in respective tissues, controlled by nutrition, environment, and genetic capacity to grow [31]. Hamouda and Atti [32] revealed that in young lambs, carcass adiposity and particularly fat tail reduce Barbarine meat value as the lamb grows. And also, fat stored in the body is an important energy source when food is rare. In the present work, results showed that better diet with greater energy and protein complementation was able to

The present study confirmed that a HQD improved the BW and ADG at weaning and breeding time. Based on the results of this research, weaning weight, previous nutrition plan, and current nutrition level are factors that determine puberty age, hormone concentration, and tail measures. To conclude, it was concluded that

**38**

The authors would like to acknowledge the Nomadic Management Department of Ilam Province (Iran) for providing research opportunities and for their help and collaborations in this work.

#### **Author details**

Sedigheh Menatian1 \*, Hamidreza Mirzaei Alamouti2 , Farshid Fatahnia1 and Reza Masoumi2

1 Department of Animal Science, Faculty of Agriculture, Ilam University, Ilam, Iran

2 Department of Animal Science, Faculty of Agriculture, Zanjan University, Zanjan, Iran

\*Address all correspondence to: menatian@alumni.znu.ac.ir

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

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[2] Rosa HJD, Bryant MJ. Seasonality of reproduction in sheep. Small Ruminant Research. 2003;**48**:155-171

[3] Kenyon PR, Thompson AN, Morris ST. Breeding ewe lambs successfully to improve lifetime performance. Small Ruminant Research. 2014;**118**:2-15

[4] Young JM, Thompson AN, Kennedy AJ. Bio economic modeling to identify the relative importance of a range of critical control points for prime lamb production systems in south-west Victoria. Animal Production Science. 2010;**50**:748-756

[5] Kenyon PR, van der Linden DS, West DM, Morris ST. The effect of breeding hoggets on lifetime performance. New Zealand Journal of Agricultural Research. 2011;**54**:321-330

[6] Ettema JF, Santos JEP. Impact of age at calving on lactation, reproduction, health, and income in first-parity Holsteins on commercial farms. Journal of Dairy Science. 2004;**87**:2730-2742

[7] Umberger SH. The effect of accelerated growth and fattening from early weaning to parturition on ewe reproduction and lactation [PhD thesis]. Raleigh: North Carolina State University; 1980

[8] Wallace J, Bourke D, Da Silva P, Aitken R. Nutrient partitioning during adolescent pregnancy. Reproduction. 2001;**122**:347-357

[9] Hernandez F, Elvira L, Gonzalez-Martin JV, Gonzalez-Bulnes A, Astiz S. Influence

of age at first lambing on reproductive and productive performance of Lacaune dairy sheep under an intensive management system. The Journal of Dairy Research. 2011;**78**:160-167

[10] Downing J, Lees JL. Unpublished data cited by J.L. Lees in a paper read at the British Council Course No. 729. Edinburgh, March 1978; 1977

[11] Mulvaney FJ, Morris ST, Kenyon PR, West DM, Morel PCH. Effect of live weight at the start of the breeding period and live weight gain during the breeding period and pregnancy on reproductive performance of hoggets and the live weight of their lambs. New Zealand Journal of Agricultural Research. 2010;**53**:355-364

[12] Dyrmundsson OR. Natural factors affecting puberty and reproductive performance in ewe lambs: A review. Livestock Production Science. 1981;**8**:55-65

[13] NRC. Nutrient Requirements of Small Ruminants: Sheep, Goats. Washington, DC: National Academies Press; 2007

[14] AOAC. Official Methods of Analysis. 16th ed. Washington, DC: Association of Official Analytical Chemists; 1995

[15] Van Soest PJ, Robertson JB, Lew BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science. 1991;**74**:3583-3597

[16] NRC. Nutrient Requirements of Dairy Cattle. 7th ed. Washington, DC: National Academies Press; 2001

**41**

*Effect of Pre and Post Weaning Diet Quality on Puberty Age and Tail Measures in Kurdish…*

growth hormone and related genes in lambs. Small Ruminant Research.

[26] Ehtesham SH, Vakili AR. The effect of spent mushroom substrate on blood metabolites and weight gain in Kurdish male lambs. Entomology and Applied Science Letters. 2015;**2**(1):29-33

[27] Smith JL, Sheffield LG. Production and regulation of leptin in bovine mammary epithelial cells. Domestic Animal Endocrinology. 2002;**2**:145-155

[28] Kenyon PR, Smith SL, Morel PCH, Morris ST, West DM. The effect of the maturity and prior breeding activity of rams and body condition score of ewe hoggets on the reproductive performance of ewe hoggets. New Zealand Veterinary Journal.

[29] Kenyon PR, Morris ST, West DM. Proportion of rams and the condition of ewe lambs at joining influences their breeding performance. Animal Production Science. 2010;**50**:454-459

[30] Olfati A, Moghaddam GH, Moradi Kor N, Bakhtiari M. The relationship between progesterone and biochemical constituents of amniotic fluid with placenta traits in Iranian crossbred ewes (Arkhar-merino × Ghezel). Asian Pacific Journal of Tropical Medicine.

2014;**7**(Suppl 1):162-166

2016;**42**:1-9

2011;**95**:120-127

[31] MacGheea ME, Bradleya JS, McCoski SR, Reeg AM, Ealy AD, Johnson SE. Plane of nutrition affects growth rate, organ size and skeletal muscle satellite cell activity in newborn calves. Journal of Animal Physiology and Animal Nutrition.

[32] Hamouda MB, Atti N. Comparison

growth curves of lamb fat tail measurements and their relationship with body weight in Babarine sheep. Small Ruminant Research.

2014;**119**:39-44

2009;**57**:290-294

*DOI: http://dx.doi.org/10.5772/intechopen.88647*

Peterson SW, et al. Comparison of four techniques to estimate milk production in singleton-rearing non-dairy ewes. Small Ruminant Research. 2010;**90**:18-26

[18] Treacher TT, Caja G. Nutrition during lactation. In: Freer MH, Dove H, editors. Sheep Nutrition. Australia:

Release 8.0. Cary, NC, USA: SAS

[20] Rosales Nieto CA, Ferguson MB, Macleay CA, Briegel JR, Wood DA, Martin GB, et al. Ewe lambs with higher breeding values for growth achieve higher reproductive performance when mated at age 8 months. Theriogenology.

Whitlock BK, Sartin JL. Hypothalamic integration of nutrient status and reproduction in the sheep. Reprod. Dom. Anim. 2013;**48**(Suppl. 1):44-52

[22] Aguerre M, Cajarville C, Kozloski JV, Repetto GL. Intake and digestive responses by ruminants fed fresh temperate pasture supplemented with increased levels of sorghum grain: A comparison between cattle and sheep. Animal Feed Science and

[19] SAS Institute. SAS®/STAT Software,

CSIRO Publishing; 2002

Institute, Inc.; 2003

2013;**80**:427-435

[21] Daniel JA, Foradori CD,

Technology. 2013;**186**:12-19

[23] Abouheif M, Al-Owaimer A,

[24] Drouillard JS, Ferrell CL, Klopfenstein TJ, Britton RA. Compensatory growth following metabolizable protein or energy restrictions in beef steers. Journal of Animal Science. 1991;**69**:811-818

[25] Yang J, Hou X, Gao A, Wang H. Effect of dietary energy and protein restriction followed by realimentation on pituitary mRNA expression of

Kraidees M, Metwally H, Shafey T. Effect of restricted feeding and realimentation on feed performance and carcass characteristic of growing lambs. Rev. Bras. Zootecn. 2013;**42**:95-101

[17] Van der Linden DS, Lopez-Villalobos N, Kenyon PR, Thorstensen E, Jenkinson CMC,

*Effect of Pre and Post Weaning Diet Quality on Puberty Age and Tail Measures in Kurdish… DOI: http://dx.doi.org/10.5772/intechopen.88647*

Peterson SW, et al. Comparison of four techniques to estimate milk production in singleton-rearing non-dairy ewes. Small Ruminant Research. 2010;**90**:18-26

[18] Treacher TT, Caja G. Nutrition during lactation. In: Freer MH, Dove H, editors. Sheep Nutrition. Australia: CSIRO Publishing; 2002

[19] SAS Institute. SAS®/STAT Software, Release 8.0. Cary, NC, USA: SAS Institute, Inc.; 2003

[20] Rosales Nieto CA, Ferguson MB, Macleay CA, Briegel JR, Wood DA, Martin GB, et al. Ewe lambs with higher breeding values for growth achieve higher reproductive performance when mated at age 8 months. Theriogenology. 2013;**80**:427-435

[21] Daniel JA, Foradori CD, Whitlock BK, Sartin JL. Hypothalamic integration of nutrient status and reproduction in the sheep. Reprod. Dom. Anim. 2013;**48**(Suppl. 1):44-52

[22] Aguerre M, Cajarville C, Kozloski JV, Repetto GL. Intake and digestive responses by ruminants fed fresh temperate pasture supplemented with increased levels of sorghum grain: A comparison between cattle and sheep. Animal Feed Science and Technology. 2013;**186**:12-19

[23] Abouheif M, Al-Owaimer A, Kraidees M, Metwally H, Shafey T. Effect of restricted feeding and realimentation on feed performance and carcass characteristic of growing lambs. Rev. Bras. Zootecn. 2013;**42**:95-101

[24] Drouillard JS, Ferrell CL, Klopfenstein TJ, Britton RA. Compensatory growth following metabolizable protein or energy restrictions in beef steers. Journal of Animal Science. 1991;**69**:811-818

[25] Yang J, Hou X, Gao A, Wang H. Effect of dietary energy and protein restriction followed by realimentation on pituitary mRNA expression of

growth hormone and related genes in lambs. Small Ruminant Research. 2014;**119**:39-44

[26] Ehtesham SH, Vakili AR. The effect of spent mushroom substrate on blood metabolites and weight gain in Kurdish male lambs. Entomology and Applied Science Letters. 2015;**2**(1):29-33

[27] Smith JL, Sheffield LG. Production and regulation of leptin in bovine mammary epithelial cells. Domestic Animal Endocrinology. 2002;**2**:145-155

[28] Kenyon PR, Smith SL, Morel PCH, Morris ST, West DM. The effect of the maturity and prior breeding activity of rams and body condition score of ewe hoggets on the reproductive performance of ewe hoggets. New Zealand Veterinary Journal. 2009;**57**:290-294

[29] Kenyon PR, Morris ST, West DM. Proportion of rams and the condition of ewe lambs at joining influences their breeding performance. Animal Production Science. 2010;**50**:454-459

[30] Olfati A, Moghaddam GH, Moradi Kor N, Bakhtiari M. The relationship between progesterone and biochemical constituents of amniotic fluid with placenta traits in Iranian crossbred ewes (Arkhar-merino × Ghezel). Asian Pacific Journal of Tropical Medicine. 2014;**7**(Suppl 1):162-166

[31] MacGheea ME, Bradleya JS, McCoski SR, Reeg AM, Ealy AD, Johnson SE. Plane of nutrition affects growth rate, organ size and skeletal muscle satellite cell activity in newborn calves. Journal of Animal Physiology and Animal Nutrition. 2016;**42**:1-9

[32] Hamouda MB, Atti N. Comparison growth curves of lamb fat tail measurements and their relationship with body weight in Babarine sheep. Small Ruminant Research. 2011;**95**:120-127

**40**

*Reproductive Biology and Technology in Animals*

of age at first lambing on reproductive and productive performance of

Lacaune dairy sheep under an intensive management system. The Journal of Dairy Research. 2011;**78**:160-167

[10] Downing J, Lees JL. Unpublished data cited by J.L. Lees in a paper read at the British Council Course No. 729.

[12] Dyrmundsson OR. Natural factors affecting puberty and reproductive performance in ewe lambs: A review. Livestock Production Science.

[13] NRC. Nutrient Requirements of Small Ruminants: Sheep, Goats. Washington, DC: National Academies

[14] AOAC. Official Methods of Analysis. 16th ed. Washington, DC: Association of Official Analytical

[15] Van Soest PJ, Robertson JB, Lew BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science.

[16] NRC. Nutrient Requirements of Dairy Cattle. 7th ed. Washington, DC: National Academies Press; 2001

Lopez-Villalobos N, Kenyon PR, Thorstensen E, Jenkinson CMC,

Edinburgh, March 1978; 1977

[11] Mulvaney FJ, Morris ST, Kenyon PR, West DM, Morel PCH. Effect of live weight at the start of the breeding period and live weight gain during the breeding period and pregnancy on reproductive performance of hoggets and the live weight of their lambs. New Zealand Journal of Agricultural Research.

2010;**53**:355-364

1981;**8**:55-65

Press; 2007

Chemists; 1995

1991;**74**:3583-3597

[17] Van der Linden DS,

[1] Besson M, Aubin J, Komen H, Poelman M, Quillet E, Vandeputte M, et al. Environmental impacts of genetic improvement of growth rate and feed conversion ratio in fish farming under rearing density and nitrogen output limitations. Journal of Cleaner

Production. 2016;**116**:100-109

Research. 2003;**48**:155-171

2014;**118**:2-15

**References**

2010;**50**:748-756

[3] Kenyon PR, Thompson AN, Morris ST. Breeding ewe lambs successfully to improve lifetime

[4] Young JM, Thompson AN, Kennedy AJ. Bio economic modeling to identify the relative importance of a range of critical control points for prime lamb production systems in south-west Victoria. Animal Production Science.

[5] Kenyon PR, van der Linden DS, West DM, Morris ST. The effect of breeding hoggets on lifetime performance. New Zealand Journal of Agricultural Research. 2011;**54**:321-330

[6] Ettema JF, Santos JEP. Impact of age at calving on lactation, reproduction, health, and income in first-parity Holsteins on commercial farms. Journal of Dairy Science. 2004;**87**:2730-2742

[7] Umberger SH. The effect of accelerated growth and fattening from early weaning to parturition on ewe reproduction and lactation [PhD thesis]. Raleigh: North Carolina State University; 1980

[8] Wallace J, Bourke D, Da Silva P, Aitken R. Nutrient partitioning during adolescent pregnancy. Reproduction.

[9] Hernandez F, Elvira L, Gonzalez-Martin JV, Gonzalez-Bulnes A, Astiz S. Influence

2001;**122**:347-357

[2] Rosa HJD, Bryant MJ. Seasonality of reproduction in sheep. Small Ruminant

performance. Small Ruminant Research.

**43**

cortisol parameters.

**Chapter 4**

**Abstract**

Bioclimatic Influence on the

Cows in the Amazonian Biome

*Jefferson Viana Alves Diniz, Laine Oliveira da Silva,* 

*Marcos Nereu Luckner, Rafael Augusto Satrapa,* 

**Keywords:** embryo transfer, thermal stress, thermoregulation, THI

*José Antonio Dell'Aqua Junior and Eunice Oba*

*Marina Marie Bento Nogueira, Rosano Ramos de Freitas,* 

The objective of the research was to evaluate the effect of the climate, through the parameters of rectal temperature (RT), temperature and humidity index (THI), and plasma cortisol concentration, on the physiological responses of embryo-recipient cows in the Amazonian biome. For the conduction of the experiment in which 11 rural properties participated, 235 crossbred cows were used as embryo recipients. The embryos were obtained by means of the in vitro production technique (PIV). The recipients were divided into batches and submitted based on the simplification of the P36 protocol for fixed-time embryo transfer (FTET). To the day of embryo transfer, blood samples were collected by venipuncture of the coccygeal vein in tubes with anticoagulant. Plasma cortisol dosages were done by solid-phase radioimmunoassay (RIA) technique using commercial kits. The rectal temperature of each of the recipients submitted to the protocol was verified using a digital clinical thermometer, and the ambient temperature and the relative humidity of the air were evaluated in this moment, both with the aid of own digital equipment. Statistical analysis of the parameters evaluated was Pearson's correlation and Student's t-test at a significance level of 5%. In the analysis of variance, it was observed that there was a significant difference in plasma cortisol and THI among the groups, where lower mean values were found in the GP group. The Amazonian climate is an inducer of thermal stress, which can cause abnormalities in the estral cycle and changes in the synthesis of sex hormones and embryonic development and, consequently, negatively affect the pregnancy rate in embryo-recipient cows, even in races more adapted to the tropics, as demonstrated by the RT, THI, and plasma

Pregnancy Rate in Embryo-Recipient

**Chapter 4**

## Bioclimatic Influence on the Pregnancy Rate in Embryo-Recipient Cows in the Amazonian Biome

*Jefferson Viana Alves Diniz, Laine Oliveira da Silva, Marina Marie Bento Nogueira, Rosano Ramos de Freitas, Marcos Nereu Luckner, Rafael Augusto Satrapa, José Antonio Dell'Aqua Junior and Eunice Oba*

#### **Abstract**

The objective of the research was to evaluate the effect of the climate, through the parameters of rectal temperature (RT), temperature and humidity index (THI), and plasma cortisol concentration, on the physiological responses of embryo-recipient cows in the Amazonian biome. For the conduction of the experiment in which 11 rural properties participated, 235 crossbred cows were used as embryo recipients. The embryos were obtained by means of the in vitro production technique (PIV). The recipients were divided into batches and submitted based on the simplification of the P36 protocol for fixed-time embryo transfer (FTET). To the day of embryo transfer, blood samples were collected by venipuncture of the coccygeal vein in tubes with anticoagulant. Plasma cortisol dosages were done by solid-phase radioimmunoassay (RIA) technique using commercial kits. The rectal temperature of each of the recipients submitted to the protocol was verified using a digital clinical thermometer, and the ambient temperature and the relative humidity of the air were evaluated in this moment, both with the aid of own digital equipment. Statistical analysis of the parameters evaluated was Pearson's correlation and Student's t-test at a significance level of 5%. In the analysis of variance, it was observed that there was a significant difference in plasma cortisol and THI among the groups, where lower mean values were found in the GP group. The Amazonian climate is an inducer of thermal stress, which can cause abnormalities in the estral cycle and changes in the synthesis of sex hormones and embryonic development and, consequently, negatively affect the pregnancy rate in embryo-recipient cows, even in races more adapted to the tropics, as demonstrated by the RT, THI, and plasma cortisol parameters.

**Keywords:** embryo transfer, thermal stress, thermoregulation, THI

#### **1. Introduction**

Brazil is a predominantly tropical country, with high average temperatures during the year, generally causing thermal stress to the production animals and causing physiological imbalances that, in turn, cause an increase in the net energy requirements for maintenance, which, consequently, causes a decrease in the available energy for the productive processes [1]. The heat stress condition harms homeostasis and promotes endocrine changes that have negative effects on reproductive events in the bovine female [2].

The stressor agent, through the preoptic area of the central nervous system, acts on the neurosecretory cells of the paraventricular nucleus of the hypothalamus, and, from this stimulation, these cells produce the corticotrophin-releasing hormone (CRH), which promotes the secretion of the adrenocorticotrophic hormone (ACTH) by the adenohypophysis [3]. ACTH acts on the adrenal glands stimulating the secretion of corticosteroids, such as the hormone cortisol [4]. Thus, cows undergoing thermal stress stimulate with greater intensity the hypothalamicpituitary-adrenal axis, increasing the concentrations of ACTH and cortisol [5]. This hormone interferes with mechanisms related to fertility, such as resumption of estrous cycle, ovulation of a competent oocyte, and establishment of gestation [6].

However, for a long time, in trying to overcome barriers, we have sought the support of reproductive biotechnologies with a view to increasing production, through the use of techniques such as artificial insemination (AI), artificial insemination at fixed time (AIFT), and fertilization in vitro [7, 8]. Brazil has a great representation in the world scenario of in vitro embryo production, and this is mainly due to the work done with zebu breeds with which best results are obtained, probably due to their better adaptation to the tropics [9]. In this sense, the objective of the research was to evaluate the effect of the climate, through the parameters of rectal temperature (RT), temperature and humidity index (THI), and plasma cortisol concentration, on the physiological responses of embryo-recipient cows in the Amazonian biome.

#### **2. Material and methods**

The study was elaborated according to the ethics committee of the Faculdade de Medicina Veterinária e Zootecnia—Universidade Estadual Paulista Júlio de Mesquita Filho (case number 227/2011). For the conduction of the experiment in which 11 rural properties participated, distributed in 6 municipalities of the state of Acre (9°6′36″ S, 70°31′12″ W), 235 crossbred cows were used as embryo recipients (*Bos taurus taurus* × *Bos taurus indicus*), native of Acre, uni- or multiparous, aged between 3 and 6 years, non-lactating, raised in an extensive regime, with water and mineral salt ad libitum, and with body condition score between 3 and 4, on a scale of 1–5 [10].

The embryos were obtained by means of the in vitro production technique (PIV), using semen and oocytes from selected Gir dairy cattle. The recipients were divided into batches containing 10–12 animals and submitted to the same estrus induction/synchronization protocol. Based on the simplification of the P36 protocol for fixed-time embryo transfer (FTET) described by [11], the protocols were developed as follows: on a random day of the estrous cycle (D0), each of the recipients received 1 g of P4 by an intravaginal device and 2.5 mg of estradiol benzoate (EB) intramuscularly (IM). On the eighth day (D8), the P4 device was withdrawn, and 150 μg of D-cloprostenol (PGF2α), 400 IU of eCG, and 1 mg of BE were given

**45**

**Table 1.**

*pregnant.*

*Bioclimatic Influence on the Pregnancy Rate in Embryo-Recipient Cows in the Amazonian Biome*

Concomitantly to the day of embryo transfer, blood samples were collected by venipuncture of the coccygeal vein in tubes with anticoagulant. The obtained plasma was fractionated in 1.5 mL microtubes duly identified and stored at 20°C negative until sent to the laboratory for analysis. Plasma cortisol dosages were done

Prior to performing at the D16 ultrasonography evaluation, the rectal temperature of each of the recipients submitted to the protocol was verified using a digital clinical thermometer, and the ambient temperature and the relative humidity of the air were evaluated in this moment, both with the aid of own digital equipment. The temperature and humidity index was calculated according to [12], using the

where Tbs refers to the dry-bulb temperature (°C) and RH to relative humidity (%). Statistical analysis of the parameters evaluated was Pearson's correlation and

The rates of yield of the embryo recipients and of pregnancy after application of the protocol were 67.23 (158/235) and 34.18% (54/158), respectively. Regarding the ambient temperature (RT), the relative air humidity (RH), and the RT recorded during the 15 months of the survey, mean values are 30.07, 69.23, and 39.28°C,

It was found that there was a significant difference of the THI and RT between the two established groups: pregnant cows (GP) and nonpregnant cows (GNP), where the lowest mean values were found in the GP group. It was also found that both groups had a significant and positive correlation between the THI and RT

The climatic variables in this study refer to the typical days of the Amazon region, collected on nonconsecutive dates. It was verified that the GNP group for the THI parameter (81.83 ± 0.03) had values above the care range; the GP group presented values below the safety range. It was also found that the THI values (70.50 ± 0.10) of the GP group were below the established values of 72–78.

**Parameters GNP GP** THI 81.83 ± 0.03a 70.50 ± 0.10b RT(°C) 39.52 ± 0.24a 38.66 ± 0.28b Pearson correlation between the THI and RT (*r* = 0.73; *p* = 0.0208) (*r* = 0.85; *p* = 0.0126) *GNP, not pregnant group; GP, pregnant group; THI, temperature and humidity index; RT, rectal temperature. Distinct lowercase letters on the same line indicate significant differences by the Student's t-test at the 5% level of significance.*

*Correlation between THI and RT (average ± standard error) in the groups which are not pregnant and* 

\_ Tbs – 14.3

100 + 46.3

intramuscularly. On the 16th day (D16), each recipient received a transferred embryo (blastocyst, grade 1 or 2) after being previously diagnosed by ultrasound of a corpus luteum (LC) in one of the ovaries. On the 41st day (D41), the diagnosis of

by solid-phase radioimmunoassay (RIA) technique using commercial kits.

THI = 0.8 Tbs + RH

*DOI: http://dx.doi.org/10.5772/intechopen.87998*

Student's t-test at a significance level of 5%.

gestation (DG) was made.

formula

**3. Results**

respectively.

parameters (**Table 1**).

*Bioclimatic Influence on the Pregnancy Rate in Embryo-Recipient Cows in the Amazonian Biome DOI: http://dx.doi.org/10.5772/intechopen.87998*

intramuscularly. On the 16th day (D16), each recipient received a transferred embryo (blastocyst, grade 1 or 2) after being previously diagnosed by ultrasound of a corpus luteum (LC) in one of the ovaries. On the 41st day (D41), the diagnosis of gestation (DG) was made.

Concomitantly to the day of embryo transfer, blood samples were collected by venipuncture of the coccygeal vein in tubes with anticoagulant. The obtained plasma was fractionated in 1.5 mL microtubes duly identified and stored at 20°C negative until sent to the laboratory for analysis. Plasma cortisol dosages were done by solid-phase radioimmunoassay (RIA) technique using commercial kits.

Prior to performing at the D16 ultrasonography evaluation, the rectal temperature of each of the recipients submitted to the protocol was verified using a digital clinical thermometer, and the ambient temperature and the relative humidity of the air were evaluated in this moment, both with the aid of own digital equipment. The temperature and humidity index was calculated according to [12], using the formula \_

$$\text{THI} = \text{0.8 Tbs} + \text{RH} \frac{\text{Tbs} - 14.3}{100} + 46.3 \text{ s}$$

where Tbs refers to the dry-bulb temperature (°C) and RH to relative humidity (%). Statistical analysis of the parameters evaluated was Pearson's correlation and Student's t-test at a significance level of 5%.

#### **3. Results**

*Reproductive Biology and Technology in Animals*

events in the bovine female [2].

the Amazonian biome.

**2. Material and methods**

Brazil is a predominantly tropical country, with high average temperatures during the year, generally causing thermal stress to the production animals and causing physiological imbalances that, in turn, cause an increase in the net energy requirements for maintenance, which, consequently, causes a decrease in the available energy for the productive processes [1]. The heat stress condition harms homeostasis and promotes endocrine changes that have negative effects on reproductive

The stressor agent, through the preoptic area of the central nervous system, acts on the neurosecretory cells of the paraventricular nucleus of the hypothalamus, and, from this stimulation, these cells produce the corticotrophin-releasing hormone (CRH), which promotes the secretion of the adrenocorticotrophic hormone (ACTH) by the adenohypophysis [3]. ACTH acts on the adrenal glands stimulating the secretion of corticosteroids, such as the hormone cortisol [4]. Thus, cows undergoing thermal stress stimulate with greater intensity the hypothalamicpituitary-adrenal axis, increasing the concentrations of ACTH and cortisol [5]. This hormone interferes with mechanisms related to fertility, such as resumption of estrous cycle, ovulation of a competent oocyte, and establishment of gestation [6]. However, for a long time, in trying to overcome barriers, we have sought the support of reproductive biotechnologies with a view to increasing production, through the use of techniques such as artificial insemination (AI), artificial

insemination at fixed time (AIFT), and fertilization in vitro [7, 8]. Brazil has a great representation in the world scenario of in vitro embryo production, and this is mainly due to the work done with zebu breeds with which best results are obtained, probably due to their better adaptation to the tropics [9]. In this sense, the objective of the research was to evaluate the effect of the climate, through the parameters of rectal temperature (RT), temperature and humidity index (THI), and plasma cortisol concentration, on the physiological responses of embryo-recipient cows in

The study was elaborated according to the ethics committee of the Faculdade de Medicina Veterinária e Zootecnia—Universidade Estadual Paulista Júlio de Mesquita Filho (case number 227/2011). For the conduction of the experiment in which 11 rural properties participated, distributed in 6 municipalities of the state of Acre (9°6′36″ S, 70°31′12″ W), 235 crossbred cows were used as embryo recipients (*Bos taurus taurus* × *Bos taurus indicus*), native of Acre, uni- or multiparous, aged between 3 and 6 years, non-lactating, raised in an extensive regime, with water and mineral salt ad libitum, and with body condition score between 3 and 4, on a scale

The embryos were obtained by means of the in vitro production technique (PIV), using semen and oocytes from selected Gir dairy cattle. The recipients were divided into batches containing 10–12 animals and submitted to the same estrus induction/synchronization protocol. Based on the simplification of the P36 protocol for fixed-time embryo transfer (FTET) described by [11], the protocols were developed as follows: on a random day of the estrous cycle (D0), each of the recipients received 1 g of P4 by an intravaginal device and 2.5 mg of estradiol benzoate (EB) intramuscularly (IM). On the eighth day (D8), the P4 device was withdrawn, and 150 μg of D-cloprostenol (PGF2α), 400 IU of eCG, and 1 mg of BE were given

**1. Introduction**

**44**

of 1–5 [10].

The rates of yield of the embryo recipients and of pregnancy after application of the protocol were 67.23 (158/235) and 34.18% (54/158), respectively. Regarding the ambient temperature (RT), the relative air humidity (RH), and the RT recorded during the 15 months of the survey, mean values are 30.07, 69.23, and 39.28°C, respectively.

It was found that there was a significant difference of the THI and RT between the two established groups: pregnant cows (GP) and nonpregnant cows (GNP), where the lowest mean values were found in the GP group. It was also found that both groups had a significant and positive correlation between the THI and RT parameters (**Table 1**).

The climatic variables in this study refer to the typical days of the Amazon region, collected on nonconsecutive dates. It was verified that the GNP group for the THI parameter (81.83 ± 0.03) had values above the care range; the GP group presented values below the safety range. It was also found that the THI values (70.50 ± 0.10) of the GP group were below the established values of 72–78.


*GNP, not pregnant group; GP, pregnant group; THI, temperature and humidity index; RT, rectal temperature. Distinct lowercase letters on the same line indicate significant differences by the Student's t-test at the 5% level of significance.*

#### **Table 1.**

*Correlation between THI and RT (average ± standard error) in the groups which are not pregnant and pregnant.*


**Table 2.**

*Correlation between plasma cortisol concentrations and THI (average ± standard error) in the groups which are not pregnant and pregnant.*

In the analysis of variance, it was observed that there was a significant difference in plasma cortisol and THI among the groups, where lower mean values were found in the GP group. It was also found that both groups had a significant and positive correlation between cortisol and THI parameters (**Table 2**).

#### **4. Discussion**

On the yield of protocols, similar results have been reported by [13, 14], with rates of 72.9 and 65%, respectively. The results for pregnancy rate were also shown to be close to those described by [15], which reached 36.60% (108/295), using eCG in D8.

The mean value of the recorded RT was attested as higher than the maximum of 39.1°C, as mentioned by [16], also remaining outside the limits of normality described by [17]. On the other hand, [18] state that cattle of all races have an average rectal temperature of 38.3°C with some variations. However, the RT remained within the physiological values that according to [19] for adult cattle is between 37.5 and 39.3°C. Therefore, the RT of the NP group exceeded the thermoneutral zone in which the maintenance of homeothermia occurs with a maximum mobilization of the mechanisms of heat dissipation, responsible for thermoregulation, and this deviation of energy may be responsible for a decrease in reproductive performance.

The comfort zone for cattle is relatively small, whereas for the European breeds, it is between −1 and 16°C and for the zebu breeds between 10 and 27°C [20], with limit critical from 35°C [21, 22]. In other words, the high-temperature indices of the Amazon region present a great challenge to the animals, requiring them to develop adaptive mechanisms for heat dissipation. However, the temperature range that confers thermal comfort in which there is minimum energy expenditure to maintain the homeothermia also depends on the relative humidity of the air [1].

The animals of this experiment were found in the Amazon biome characterized by high temperature and humidity [21, 22]. Therefore, when the air humidity is low, evaporation is facilitated; otherwise the evaporation process becomes slow or even null, making maintenance of homeothermia difficult [23]. Thus, these conditions of heat and relative air humidity, almost always, are above the zone of thermal comfort for the animals, demanding, by them, energy expenditure in terms of thermoregulatory physiological mechanisms in the attempt of heat dissipation [24]. However, there are occasions in which the loss of body heat becomes ineffective, and then thermal stress of the animal occurs, which is often a limiting factor for development, production, and reproduction [25].

The reproductive efficiency of dairy cattle exposed to adverse climatic conditions is compromised when temperature and humidity are high and solar radiation is intense for most of the year [26], a recurrent situation in the Amazonian biome.

**47**

**5. Conclusion**

*Bioclimatic Influence on the Pregnancy Rate in Embryo-Recipient Cows in the Amazonian Biome*

In the case of the use of PIV, embryo development becomes impaired when cows suffer from heat stress on AI day or up to 7 days after the procedure, which leads to less embryonic viability [2], which may render the results of the technique unfeasible. In a study with Dutch cows by Martello et al. [19], THI values up to 74 correspond to the safety range and from 74 to 78 care range. Igono et al. [27], however, consider THI above 76 in any environment stressful for cows with high milk yield. The climatic variables presented in this study indicate mild thermal stress; thus, the highest values of THI found in the GNP group indicate an environmental situation favoring stress for the animals, where the thermal condition was above that considered comfort. These processes may cause reproductive performance below ideal, such as a decrease in conception rate during the hot season by 20–30% [28] and significant economic losses [29]. Climatic conditions may be an important contributor to the low fertility of dairy cows during the summer months, especially

Plasma concentrations of cortisol, above 10 ng/mL for zebu breeds, were similar to those reported by [31] for both groups. Therefore, because it is considered the stress hormone, its evaluation, although expensive, has become of great value for the establishment of the animal welfare condition [32]. Higher production of cortisol causes negative feedback in the hypothalamus, decreasing the synthesis of gonadotrophin-releasing hormone (GnRH), and consequently reduces the release of gonadotrophins FSH and LH, facts that, in the end, are responsible for the low production of the gonadal hormones [33]. The reduction of the latter, in turn, causes less manifesta-

tion of estrus, low conception rate, embryonic mortality, and abortions [34].

reduce the effects of the thermal environment on production animals [38].

The Amazonian climate is an inducer of thermal stress, which can cause abnormalities in the estral cycle and changes in the synthesis of sex hormones and embryonic development and, consequently, negatively affect the pregnancy rate in embryo-recipient cows, even in races more adapted to the tropics, as demonstrated by the RT, THI, and plasma cortisol parameters. Therefore, it is recommended to adopt measures that may reduce the effect of environmental conditions on the

maintenance and recognition of pregnancy [36].

reproductive performance of production animals.

The lower plasma cortisol concentration in the GP group possibly favored the embryonic quality and maintenance of gestation in relation to the GNP group. Garcia-Ispierto et al. [35] found that the probability of pregnancy loss increases by 1.05 times each unit increase in THI between days 21 and 30 gestations. Due to the results obtained, it is possible to observe that the animals of the GNP group were in a situation of thermal stress, due to the increase of the reported plasma cortisol and of the THI that can act negatively on the luteal function, consequently, and thus the

Embryonic development is very susceptible to thermal stress, especially on the third day of development, reducing the proportion of embryos that continue to evolve [37]. In addition, the reduction of embryonic growth is associated with lower levels of interferon-tau that acts on the inhibition of pulsatile secretion of prostaglandin F2α, that is, low levels of interferon-tau may not block the regression of the corpus luteum, making it difficult to maintain the gestation [35]; thus, animals that are under thermal stress are likely to present alterations in the embryonic production of interferon-tau, which causes losses in the maintenance of gestation. Therefore, the techniques used in breeding should be associated with measures that

*DOI: http://dx.doi.org/10.5772/intechopen.87998*

in high-yielding cows [30].

#### *Bioclimatic Influence on the Pregnancy Rate in Embryo-Recipient Cows in the Amazonian Biome DOI: http://dx.doi.org/10.5772/intechopen.87998*

In the case of the use of PIV, embryo development becomes impaired when cows suffer from heat stress on AI day or up to 7 days after the procedure, which leads to less embryonic viability [2], which may render the results of the technique unfeasible.

In a study with Dutch cows by Martello et al. [19], THI values up to 74 correspond to the safety range and from 74 to 78 care range. Igono et al. [27], however, consider THI above 76 in any environment stressful for cows with high milk yield. The climatic variables presented in this study indicate mild thermal stress; thus, the highest values of THI found in the GNP group indicate an environmental situation favoring stress for the animals, where the thermal condition was above that considered comfort. These processes may cause reproductive performance below ideal, such as a decrease in conception rate during the hot season by 20–30% [28] and significant economic losses [29]. Climatic conditions may be an important contributor to the low fertility of dairy cows during the summer months, especially in high-yielding cows [30].

Plasma concentrations of cortisol, above 10 ng/mL for zebu breeds, were similar to those reported by [31] for both groups. Therefore, because it is considered the stress hormone, its evaluation, although expensive, has become of great value for the establishment of the animal welfare condition [32]. Higher production of cortisol causes negative feedback in the hypothalamus, decreasing the synthesis of gonadotrophin-releasing hormone (GnRH), and consequently reduces the release of gonadotrophins FSH and LH, facts that, in the end, are responsible for the low production of the gonadal hormones [33]. The reduction of the latter, in turn, causes less manifestation of estrus, low conception rate, embryonic mortality, and abortions [34].

The lower plasma cortisol concentration in the GP group possibly favored the embryonic quality and maintenance of gestation in relation to the GNP group. Garcia-Ispierto et al. [35] found that the probability of pregnancy loss increases by 1.05 times each unit increase in THI between days 21 and 30 gestations. Due to the results obtained, it is possible to observe that the animals of the GNP group were in a situation of thermal stress, due to the increase of the reported plasma cortisol and of the THI that can act negatively on the luteal function, consequently, and thus the maintenance and recognition of pregnancy [36].

Embryonic development is very susceptible to thermal stress, especially on the third day of development, reducing the proportion of embryos that continue to evolve [37]. In addition, the reduction of embryonic growth is associated with lower levels of interferon-tau that acts on the inhibition of pulsatile secretion of prostaglandin F2α, that is, low levels of interferon-tau may not block the regression of the corpus luteum, making it difficult to maintain the gestation [35]; thus, animals that are under thermal stress are likely to present alterations in the embryonic production of interferon-tau, which causes losses in the maintenance of gestation. Therefore, the techniques used in breeding should be associated with measures that reduce the effects of the thermal environment on production animals [38].

#### **5. Conclusion**

The Amazonian climate is an inducer of thermal stress, which can cause abnormalities in the estral cycle and changes in the synthesis of sex hormones and embryonic development and, consequently, negatively affect the pregnancy rate in embryo-recipient cows, even in races more adapted to the tropics, as demonstrated by the RT, THI, and plasma cortisol parameters. Therefore, it is recommended to adopt measures that may reduce the effect of environmental conditions on the reproductive performance of production animals.

*Reproductive Biology and Technology in Animals*

tion between cortisol and THI parameters (**Table 2**).

development, production, and reproduction [25].

**4. Discussion**

*are not pregnant and pregnant.*

in D8.

**Table 2.**

In the analysis of variance, it was observed that there was a significant difference in plasma cortisol and THI among the groups, where lower mean values were found in the GP group. It was also found that both groups had a significant and positive correla-

*Correlation between plasma cortisol concentrations and THI (average ± standard error) in the groups which* 

**Parameters GNP GP** Cortisol (ng/mL) 17.78 ± 5.54a 13.78 ± 4.74b THI 81.83 ± 0.03a 70.50 ± 0.10b Pearson correlation between the cortisol and THI (*r* = 0.45; *p* = 0.0368) (*r* = 0.76; *p* = 0.0186) *GNP, not pregnant group; GP, pregnant group; THI, temperature and humidity index. Distinct lowercase letters on* 

*the same line indicate significant differences by the Student's t-test at the 5% level of significance.*

On the yield of protocols, similar results have been reported by [13, 14], with rates of 72.9 and 65%, respectively. The results for pregnancy rate were also shown to be close to those described by [15], which reached 36.60% (108/295), using eCG

The mean value of the recorded RT was attested as higher than the maximum of 39.1°C, as mentioned by [16], also remaining outside the limits of normality described by [17]. On the other hand, [18] state that cattle of all races have an average rectal temperature of 38.3°C with some variations. However, the RT remained within the physiological values that according to [19] for adult cattle is between 37.5 and 39.3°C. Therefore, the RT of the NP group exceeded the thermoneutral zone in which the maintenance of homeothermia occurs with a maximum mobilization of the mechanisms of heat dissipation, responsible for thermoregulation, and this deviation of energy may be responsible for a decrease in reproductive performance. The comfort zone for cattle is relatively small, whereas for the European breeds, it is between −1 and 16°C and for the zebu breeds between 10 and 27°C [20], with limit critical from 35°C [21, 22]. In other words, the high-temperature indices of the Amazon region present a great challenge to the animals, requiring them to develop adaptive mechanisms for heat dissipation. However, the temperature range that confers thermal comfort in which there is minimum energy expenditure to maintain the homeothermia also depends on the relative humidity of the air [1].

The animals of this experiment were found in the Amazon biome characterized by high temperature and humidity [21, 22]. Therefore, when the air humidity is low, evaporation is facilitated; otherwise the evaporation process becomes slow or even null, making maintenance of homeothermia difficult [23]. Thus, these conditions of heat and relative air humidity, almost always, are above the zone of thermal comfort for the animals, demanding, by them, energy expenditure in terms of thermoregulatory physiological mechanisms in the attempt of heat dissipation [24]. However, there are occasions in which the loss of body heat becomes ineffective, and then thermal stress of the animal occurs, which is often a limiting factor for

The reproductive efficiency of dairy cattle exposed to adverse climatic conditions is compromised when temperature and humidity are high and solar radiation is intense for most of the year [26], a recurrent situation in the Amazonian biome.

**46**

#### **Author details**

Jefferson Viana Alves Diniz1 \*, Laine Oliveira da Silva2 , Marina Marie Bento Nogueira2 , Rosano Ramos de Freitas2 , Marcos Nereu Luckner2 , Rafael Augusto Satrapa<sup>2</sup> , José Antonio Dell'Aqua Junior3 and Eunice Oba<sup>3</sup>

1 Instituto Federal do Acre—IFAC, Sena Madureira, AC, Brasil

2 Universidade Federal do Acre—UFAC, Rio Branco, AC, Brasil

3 Universidade Estadual Paulista Júlio de Mesquita Filho, Botucatu, SP, Brasil

\*Address all correspondence to: jefferson.diniz@ifac.edu.br

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**49**

2012;**22**:55-65

*Bioclimatic Influence on the Pregnancy Rate in Embryo-Recipient Cows in the Amazonian Biome*

in vitro fertilization technique in the last decade and its effect on Brazilian embryo industry and animal production. Acta Scientiae Veterinariae. 2010;**38**:661-674

[10] Ferreira AM, Torres CAA. Perda de peso corporal e cessação da atividade ovariana luteínica cíclica em vacas mestiças leiteiras. Pesquisa Agropecuária Brasileira.

[11] Barros CM, Nogueira MFG. Embryo

[12] Pires MFA, Ferreira AM, Saturnino HM, Teodoro RL. Taxa de gestação em fêmeas da raça Holandesa confinadas em free stall, no verão e inverno. Arquivo Brasileiro de Medicina Veterinária e

[13] Barreiros TRR, Blaschi W, Borsato EA, Ludwig HÊ, Silva DRM, Seneda MM. Comparação das taxas de prenhez entre receptoras com corpos lúteos cavitários ou compactos após protocolo de sincronização com cloprostenol ou transferência de embriões em tempo fixo. Semina: Ciências Agrárias.

transfer in *Bos indicus* cattle. Theriogenology. 2001;**56**:1483-1496

Zootecnia. 2002;**54**:57-63

2006;**27**:657-664

[14] Rodrigues CA, Ranieri AL, Teixeira AA, Vieira LM, Ferreira RM, Ayres H, et al. Eficiência reprodutiva de receptoras holandesas de alta produção sincronizadas para TETF com protocolos com ou sem estradiol e/ou eCG. In: XXIV Reunião anual da sociedade brasileira de tecnologia de embriões; Porto de Galinhas; 2010

[15] Baruselli PS, Ferreira RM, Sá FIlho MF, Nasser LFT, Rodrigues CA, Bó GA. Bovine embryo transfer recipient synchronization and management in tropical environments. Reproduction,

Fertility, and Development.

2010;**22**:67-74

1993;**28**:411-418

*DOI: http://dx.doi.org/10.5772/intechopen.87998*

**References**

Nobel; 2000

[1] Silva RG. Introdução a

Bioclimatologia Animal. São Paulo:

[3] Minton JE. Function of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system in models of acute stress in domestic farm animals. Journal of Animal Science.

ER, Wessinger EW, Karsch FJ. Endocrine basis for disruptive effect of cortisol on preovulatory events. Endocrinology. 2005;**146**:2107-2115

1994;**72**:1891-1898

[2] Macedo GG, Costa e Silva EV, Pinho RO, Assumpção TI, Jacomini JO, Santos RM, et al. O estresse por calor diminui a fertilidade de fêmeas bovinas por afetar o desenvolvimento oocitário e o embrionário. Revista Brasileira de Reprodução Animal. 2014;**38**:80-85

[4] Breen KM, Billings HJ, Wagenmaker

[5] Cooke RF, Arthington JD, Araujo DB, Lamb GC. Effects of acclimation to human interaction on performance, temperament, physiological responses, and pregnancy rates of Brahmancrossbred cows. Journal of Animal Science. 2009;**87**:4125-4132

[6] Dobson H, Tebble JE, Smith RF. Is stress really all that important? Theriogenology. 2001;**55**:65-73

[7] Baruselli PS, Reis EL, Marques MO, Nasser LF, Bó GA. The use of hormonal treatments to improve reproductive performance of anestrous beef cattle in tropical climates. Animal Reproduction

Science. 2004;**82-83**:479-486

[9] Viana JHM, Siqueira LG, Palhao MP, Camargo LS. Use of

[8] Vieira RJ. Biotécnicas aplicadas à reprodução bovina: Generalidades. Revista Acadêmica Ciência Animal.

*Bioclimatic Influence on the Pregnancy Rate in Embryo-Recipient Cows in the Amazonian Biome DOI: http://dx.doi.org/10.5772/intechopen.87998*

#### **References**

*Reproductive Biology and Technology in Animals*

**48**

**Author details**

Jefferson Viana Alves Diniz1

Rafael Augusto Satrapa<sup>2</sup>

Marina Marie Bento Nogueira2

\*, Laine Oliveira da Silva2

, José Antonio Dell'Aqua Junior3

3 Universidade Estadual Paulista Júlio de Mesquita Filho, Botucatu, SP, Brasil

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Instituto Federal do Acre—IFAC, Sena Madureira, AC, Brasil

2 Universidade Federal do Acre—UFAC, Rio Branco, AC, Brasil

\*Address all correspondence to: jefferson.diniz@ifac.edu.br

provided the original work is properly cited.

, Rosano Ramos de Freitas2

,

, Marcos Nereu Luckner2

and Eunice Oba<sup>3</sup>

,

[1] Silva RG. Introdução a Bioclimatologia Animal. São Paulo: Nobel; 2000

[2] Macedo GG, Costa e Silva EV, Pinho RO, Assumpção TI, Jacomini JO, Santos RM, et al. O estresse por calor diminui a fertilidade de fêmeas bovinas por afetar o desenvolvimento oocitário e o embrionário. Revista Brasileira de Reprodução Animal. 2014;**38**:80-85

[3] Minton JE. Function of the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system in models of acute stress in domestic farm animals. Journal of Animal Science. 1994;**72**:1891-1898

[4] Breen KM, Billings HJ, Wagenmaker ER, Wessinger EW, Karsch FJ. Endocrine basis for disruptive effect of cortisol on preovulatory events. Endocrinology. 2005;**146**:2107-2115

[5] Cooke RF, Arthington JD, Araujo DB, Lamb GC. Effects of acclimation to human interaction on performance, temperament, physiological responses, and pregnancy rates of Brahmancrossbred cows. Journal of Animal Science. 2009;**87**:4125-4132

[6] Dobson H, Tebble JE, Smith RF. Is stress really all that important? Theriogenology. 2001;**55**:65-73

[7] Baruselli PS, Reis EL, Marques MO, Nasser LF, Bó GA. The use of hormonal treatments to improve reproductive performance of anestrous beef cattle in tropical climates. Animal Reproduction Science. 2004;**82-83**:479-486

[8] Vieira RJ. Biotécnicas aplicadas à reprodução bovina: Generalidades. Revista Acadêmica Ciência Animal. 2012;**22**:55-65

[9] Viana JHM, Siqueira LG, Palhao MP, Camargo LS. Use of in vitro fertilization technique in the last decade and its effect on Brazilian embryo industry and animal production. Acta Scientiae Veterinariae. 2010;**38**:661-674

[10] Ferreira AM, Torres CAA. Perda de peso corporal e cessação da atividade ovariana luteínica cíclica em vacas mestiças leiteiras. Pesquisa Agropecuária Brasileira. 1993;**28**:411-418

[11] Barros CM, Nogueira MFG. Embryo transfer in *Bos indicus* cattle. Theriogenology. 2001;**56**:1483-1496

[12] Pires MFA, Ferreira AM, Saturnino HM, Teodoro RL. Taxa de gestação em fêmeas da raça Holandesa confinadas em free stall, no verão e inverno. Arquivo Brasileiro de Medicina Veterinária e Zootecnia. 2002;**54**:57-63

[13] Barreiros TRR, Blaschi W, Borsato EA, Ludwig HÊ, Silva DRM, Seneda MM. Comparação das taxas de prenhez entre receptoras com corpos lúteos cavitários ou compactos após protocolo de sincronização com cloprostenol ou transferência de embriões em tempo fixo. Semina: Ciências Agrárias. 2006;**27**:657-664

[14] Rodrigues CA, Ranieri AL, Teixeira AA, Vieira LM, Ferreira RM, Ayres H, et al. Eficiência reprodutiva de receptoras holandesas de alta produção sincronizadas para TETF com protocolos com ou sem estradiol e/ou eCG. In: XXIV Reunião anual da sociedade brasileira de tecnologia de embriões; Porto de Galinhas; 2010

[15] Baruselli PS, Ferreira RM, Sá FIlho MF, Nasser LFT, Rodrigues CA, Bó GA. Bovine embryo transfer recipient synchronization and management in tropical environments. Reproduction, Fertility, and Development. 2010;**22**:67-74

[16] Dukes HH, Swenson MJ. Fisiologia dos Animais Domésticos. 12th ed. Rio de Janeiro: Guanabara Koogan; 2006

[17] Robinson EN. Termorregulação. In: Cunningham JG, editor. Tratado de fisiologia veterinária. 2th ed. Rio de Janeiro: Guanabara Koogan; 1999

[18] McDowell RE, Lee DHK, Fohrman MH. The measurement of water evaporation from limited areas of a normal body surface. Journal of Animal Science. 1954;**13**:405-416

[19] Martello LS, Peixoto AP, Regis JEF, Nascimento JWB, Araujo TGP, Lisboa ACC. Respostas fisiológicas e produtivas de vacas holandesas em lactação submetidas a diferentes ambientes. Revista Brasileira de Zootecnia. 2004;**33**:181-191

[20] Santos SA, McManus C, Souza GS, Soriano BMA, Silva RAMS, Comastri Filho JA, et al. Variações da temperatura corporal e da pele de vacas e bezerros das raças Pantaneira e Nelore no Pantanal. Archivos de Zootecnia. 2005;**54**:237-244

[21] Azevedo M, Pires MFA, Saturnino HM, Lana AMQ, Sampaio IBM, Monteiro JBN, et al. Estimativa de níveis críticos superiores do índice de temperatura e umidade para vacas leiteiras 1/2, 3/4 e 7/8 Holandês-zebu em lactação. Revista Brasileira de Zootecnia. 2005;**34**:2000-2008

[22] Furtado DA, Peixoto AP, Regis JEF, Nascimento JWB, Araujo TGP, Lisboa ACC. Termorregulação e desempenho de tourinhos Sindi e Guzerá, no agreste paraibano. Revista Brasileira de Engenharia Agrícola e Ambiental. 2012;**16**:1022-1028

[23] Wilson DCS, Corbett AD, Bovell DL. A preliminary study of the short circuit current (Isc) responses of sweat gland cells from normal and anhidrotic horses to purinergic and adrenergic

agonists. Journal Compilation. 2007;**18**:152-160

[24] Baêta FC, Souza C. Ambiência em edificações rurais: conforto animal. Viçosa: UFV; 1997

[25] Silva RG, Gaudiosi MC. Termólise evaporativa em ovinos sob altas temperaturas. In: anais do Congresso Brasileiro de Biometeorologia; Jaboticabal. 1995. pp. 193-203

[26] Kamal R, Dutt T, Patel M, Dey A, Bharti PK, Chandran PC. Heat stress and effect of shade materials on hormonal and behavior response of dairy cattle: A review. Tropical Animal Health and Production. 2018;**50**:701-706

[27] Igono MO, Bjtvedt G, Sanford-Crane HT. Environmental profile and critical temperature effects on milk production of Holsteins cows in desert climate. International Journal of Biometeorology. 1992;**36**:77-87

[28] De Rensis F, Marconi P, Capelli T, Gatti F, Facciolongo F, Franzini S, et al. Fertility in postpartum dairy cows in winter or summer following estrus synchronization and fixed time AI after the induction of an LH surge with GnRH or hCG. Theriogenology. 2002;**58**:1675-1687

[29] Collier RJ, Dahl GE, Vanbaale MJ. Major advances associated with environmental effects on dairy cattle. Journal of Dairy Science. 2006;**89**:1244-1253

[30] Kadzere CT, Murphy MR, Silanikove N, Maltz E. Heat stress in lactating dairy cows: A review. Livestock Production Science. 2002;**77**:59-91

[31] Yoshida C, Nakao T. Response of plasma cortisol and progesterone after ACTH challenge in ovariectomized lactating dairy cows. Reproduction and Development. 2005;**51**:99-107

**51**

*Bioclimatic Influence on the Pregnancy Rate in Embryo-Recipient Cows in the Amazonian Biome*

*DOI: http://dx.doi.org/10.5772/intechopen.87998*

[32] Benatti LAT. Avaliação do cortisol, perfil hematológico e proteico na resposta dos bovinos ao estresse. Veterinary Journal. 2010;**159**:201-206

[33] Menezes SRS. Efeitos do clima na performance reprodutiva de bovinos leiteiros nos Açores. Reproduction and

[34] Ferro FRA, Cavalcanti Neto CC, Toledo Filho MR, Ferri STS, Montaldo YC. Efeito do estresse calórico no desempenho reprodutivo de vacas leiteiras. Revista Verde. 2010;**5**:01-25

[35] Garcia-Ispierto F, Lopez-Gatius G, Bech-Sabat P. Climate factors affecting conception rate of high producing dairy cows in northeastern Spain. Theriogenology. 2007;**67**:1379-1385

[36] Reis LSLS, Pardo PE, Oba E, Kronka SN, Frazatti-Gallina NM. Matricaria chamomilla CH12 decreases handling stressing Nelore calves. Journal of Veterinary Science. 2006;**7**:189-192

[37] Sartori R, Gumen A, Guenther JN, Souza AH, Caraviello DZ, Willtbank MC. Comparison of artificial

insemination versus embryo transfer in lactating dairy cows. Theriogenology.

[38] Habeeb AAM, Gad AE, El-Tarabany AA, Atta MAA. Negative effects of heat stress on growth and milk production of farm animals. Journal of Animal Husbandry and Dairy Sciences.

2006;**65**:1311-1321

2018;**2**:1-12

Development. 2010;**51**:99-107

*Bioclimatic Influence on the Pregnancy Rate in Embryo-Recipient Cows in the Amazonian Biome DOI: http://dx.doi.org/10.5772/intechopen.87998*

[32] Benatti LAT. Avaliação do cortisol, perfil hematológico e proteico na resposta dos bovinos ao estresse. Veterinary Journal. 2010;**159**:201-206

*Reproductive Biology and Technology in Animals*

[16] Dukes HH, Swenson MJ. Fisiologia dos Animais Domésticos. 12th ed. Rio de Janeiro: Guanabara Koogan; 2006

agonists. Journal Compilation.

[24] Baêta FC, Souza C. Ambiência em edificações rurais: conforto animal.

[25] Silva RG, Gaudiosi MC. Termólise evaporativa em ovinos sob altas temperaturas. In: anais do Congresso Brasileiro de Biometeorologia; Jaboticabal. 1995. pp. 193-203

[26] Kamal R, Dutt T, Patel M, Dey A, Bharti PK, Chandran PC. Heat stress and effect of shade materials on hormonal and behavior response of dairy cattle: A review. Tropical Animal Health and Production.

[27] Igono MO, Bjtvedt G, Sanford-Crane HT. Environmental profile and critical temperature effects on milk production of Holsteins cows in desert climate. International Journal of

Biometeorology. 1992;**36**:77-87

[28] De Rensis F, Marconi P, Capelli T, Gatti F, Facciolongo F, Franzini S, et al. Fertility in postpartum dairy cows in winter or summer following estrus synchronization and fixed time AI after the induction of an LH surge with GnRH or hCG. Theriogenology.

[29] Collier RJ, Dahl GE, Vanbaale MJ. Major advances associated with environmental effects on dairy cattle. Journal of Dairy Science.

2007;**18**:152-160

Viçosa: UFV; 1997

2018;**50**:701-706

2002;**58**:1675-1687

2006;**89**:1244-1253

[30] Kadzere CT, Murphy MR, Silanikove N, Maltz E. Heat stress in lactating dairy cows: A review. Livestock Production Science. 2002;**77**:59-91

Development. 2005;**51**:99-107

[31] Yoshida C, Nakao T. Response of plasma cortisol and progesterone after ACTH challenge in ovariectomized lactating dairy cows. Reproduction and

[17] Robinson EN. Termorregulação. In: Cunningham JG, editor. Tratado de fisiologia veterinária. 2th ed. Rio de Janeiro: Guanabara Koogan; 1999

[18] McDowell RE, Lee DHK, Fohrman MH. The measurement of water evaporation from limited areas of a normal body surface. Journal of Animal

[19] Martello LS, Peixoto AP, Regis JEF, Nascimento JWB, Araujo TGP, Lisboa ACC. Respostas fisiológicas e produtivas

de vacas holandesas em lactação submetidas a diferentes ambientes. Revista Brasileira de Zootecnia.

[20] Santos SA, McManus C, Souza GS, Soriano BMA, Silva RAMS, Comastri Filho JA, et al. Variações da temperatura corporal e da pele de vacas e bezerros das raças Pantaneira e Nelore no Pantanal. Archivos de Zootecnia.

[21] Azevedo M, Pires MFA, Saturnino HM, Lana AMQ, Sampaio IBM, Monteiro JBN, et al. Estimativa de níveis críticos superiores do índice de temperatura e umidade para vacas leiteiras 1/2, 3/4 e 7/8 Holandês-zebu em lactação. Revista Brasileira de Zootecnia.

[22] Furtado DA, Peixoto AP, Regis JEF, Nascimento JWB, Araujo TGP, Lisboa ACC. Termorregulação e desempenho de tourinhos Sindi e Guzerá, no agreste paraibano. Revista Brasileira de Engenharia Agrícola e Ambiental.

[23] Wilson DCS, Corbett AD, Bovell DL. A preliminary study of the short circuit current (Isc) responses of sweat gland cells from normal and anhidrotic horses to purinergic and adrenergic

Science. 1954;**13**:405-416

2004;**33**:181-191

2005;**54**:237-244

2005;**34**:2000-2008

2012;**16**:1022-1028

**50**

[33] Menezes SRS. Efeitos do clima na performance reprodutiva de bovinos leiteiros nos Açores. Reproduction and Development. 2010;**51**:99-107

[34] Ferro FRA, Cavalcanti Neto CC, Toledo Filho MR, Ferri STS, Montaldo YC. Efeito do estresse calórico no desempenho reprodutivo de vacas leiteiras. Revista Verde. 2010;**5**:01-25

[35] Garcia-Ispierto F, Lopez-Gatius G, Bech-Sabat P. Climate factors affecting conception rate of high producing dairy cows in northeastern Spain. Theriogenology. 2007;**67**:1379-1385

[36] Reis LSLS, Pardo PE, Oba E, Kronka SN, Frazatti-Gallina NM. Matricaria chamomilla CH12 decreases handling stressing Nelore calves. Journal of Veterinary Science. 2006;**7**:189-192

[37] Sartori R, Gumen A, Guenther JN, Souza AH, Caraviello DZ, Willtbank MC. Comparison of artificial insemination versus embryo transfer in lactating dairy cows. Theriogenology. 2006;**65**:1311-1321

[38] Habeeb AAM, Gad AE, El-Tarabany AA, Atta MAA. Negative effects of heat stress on growth and milk production of farm animals. Journal of Animal Husbandry and Dairy Sciences. 2018;**2**:1-12

**53**

**Chapter 5**

in Pigs

**Abstract**

Recent Advance in Genome

*Emi Inada, Issei Saitoh and Akihide Tanimoto*

derived embryos that have been reconstituted with normal nuclei.

gene-engineered, ribonucleoprotein

**1. Introduction**

**Keywords:** genome editing, CRISPR/Cas9, ZFNs, TALENs, pigs, gene modification, microinjection, electroporation, somatic cell nuclear transfer, knock out, knock in,

The domestic pig has been widely used as a large animal model in biomedical research, as it is similar to humans with respect to the size of body and internal organs, longevity, anatomy, physiology, and metabolic profile [1]. Modification of the porcine genome is also important for studying the mechanisms underlying genetic disorders, developing therapeutic drugs, and improving pig meat production yields [2, 3]. Over the past three decades, attempts have been made to modify the porcine genome using genetic engineering technology, starting after Gordon et al. [4] first reported DNA microinjection (MI)-based production of transgenic (Tg) mice. Hammer et al. [5] first reported the successful production of Tg piglets using the technique reported by Gordon et al. [4], but attaining this result was more difficult than for rodents, where pronuclei are clearly visible using an optical microscope.

Editing-Based Gene Modification

*Masahiro Sato, Kazuchika Miyoshi, Hiroaki Kawaguchi,* 

Recently, a series of genome editing technologies including ZFNs, TALENs, and CRISPR/Cas9 systems have enabled gene modification in the endogenous target genes of various organisms including pigs, which are important for agricultural and biomedical research. Owing to its simple application for gene knockout and ease of use, the CRISPR/Cas9 is now in common use worldwide. The most important aspect of this process is the selection of the method used to deliver genome editing components to embryos. In earlier stages, zygote microinjection of these components [single guide RNA (sgRNA) + DNA/mRNA for Cas9] into the cytoplasm and/ or nuclei of a zygote has been frequently employed. However, this method is always associated with the generation of mosaic embryos in which genome-edited and unedited cells are mixed together. To avoid this mosaic issue, *in vitro* electroporation of zygotes in the presence of sgRNA mixed with Cas9 protein, referred to as a ribonucleoprotein (RNP), is now in frequent use. This review provides a historical background of the production of genome-edited pigs and also presents current research concerning how genome editing is induced in somatic cell nuclear transfer-

#### **Chapter 5**

## Recent Advance in Genome Editing-Based Gene Modification in Pigs

*Masahiro Sato, Kazuchika Miyoshi, Hiroaki Kawaguchi, Emi Inada, Issei Saitoh and Akihide Tanimoto*

#### **Abstract**

Recently, a series of genome editing technologies including ZFNs, TALENs, and CRISPR/Cas9 systems have enabled gene modification in the endogenous target genes of various organisms including pigs, which are important for agricultural and biomedical research. Owing to its simple application for gene knockout and ease of use, the CRISPR/Cas9 is now in common use worldwide. The most important aspect of this process is the selection of the method used to deliver genome editing components to embryos. In earlier stages, zygote microinjection of these components [single guide RNA (sgRNA) + DNA/mRNA for Cas9] into the cytoplasm and/ or nuclei of a zygote has been frequently employed. However, this method is always associated with the generation of mosaic embryos in which genome-edited and unedited cells are mixed together. To avoid this mosaic issue, *in vitro* electroporation of zygotes in the presence of sgRNA mixed with Cas9 protein, referred to as a ribonucleoprotein (RNP), is now in frequent use. This review provides a historical background of the production of genome-edited pigs and also presents current research concerning how genome editing is induced in somatic cell nuclear transferderived embryos that have been reconstituted with normal nuclei.

**Keywords:** genome editing, CRISPR/Cas9, ZFNs, TALENs, pigs, gene modification, microinjection, electroporation, somatic cell nuclear transfer, knock out, knock in, gene-engineered, ribonucleoprotein

#### **1. Introduction**

The domestic pig has been widely used as a large animal model in biomedical research, as it is similar to humans with respect to the size of body and internal organs, longevity, anatomy, physiology, and metabolic profile [1]. Modification of the porcine genome is also important for studying the mechanisms underlying genetic disorders, developing therapeutic drugs, and improving pig meat production yields [2, 3]. Over the past three decades, attempts have been made to modify the porcine genome using genetic engineering technology, starting after Gordon et al. [4] first reported DNA microinjection (MI)-based production of transgenic (Tg) mice. Hammer et al. [5] first reported the successful production of Tg piglets using the technique reported by Gordon et al. [4], but attaining this result was more difficult than for rodents, where pronuclei are clearly visible using an optical microscope. In the case of porcine zygotes, pronuclei are difficult to see due to the presence of high lipid content in the cytoplasm. Researchers must briefly centrifuge zygotes to visualize the pronuclei prior to MI [5], which is labor-intensive and requires skill. Moreover, MI-mediated transgene integration into host chromosomes occurs randomly, which often causes gene silencing [6]. However, for precise and efficient genetic modification in the porcine genome, homologous recombination (HR)-based gene targeting technology may be recommended, which was first developed by Smithies' group in mice [7]. In this case, the use of germline-competent embryonic stem (ES) cells is a prerequisite. These ES cells are first transfected with a targeting vector and then recombinant ES clones showing successful targeting are obtained. This vector usually contains a gene of interest (GOI) to be integrated into the target locus, together with a selection marker gene such a neomycin resistance gene (*neo*), and DNA sequences of appropriate length, termed homology arms (HA), that correspond to the endogenous target gene, are placed at both ends of the DNA containing the GOI and a marker gene. Chimeric mice can be obtained through blastocyst injection with the targeted ES clones, and the resulting chimeric mice would contribute to produce heterozygous mice carrying mutated traits (GOI/selection marker gene) in the target locus [8]. Unfortunately, there are no germline-competent porcine ES cells, despite extensive efforts [9–13]. Thus, to date, production of gene-targeted pigs derived from recombinant porcine ES cells has not yet been successful.

In 1996, scientists at *the Roslin Institute* (Wilmut and colleagues) first succeeded in producing cloned sheep using somatic cell nuclear transfer (SCNT) technology. They used fetal fibroblasts [14] or adult mammary gland-derived fibroblasts [15] as SCNT donors. Notably, prior to these reports, an attempt to produce cloned embryos by the similar technique shown by the *Roslin's* group has been made by Prather et al. [16], who performed transfer of nuclei from two, four and eight-cell embryos to the enucleated oocytes and successfully produced one piglets that had been derived from the four-cell embryo's nucleus. This approach is called "blastomere transplantation," which is basically different from the somatic cell-based SCNT. Generally, fibroblasts used as SCNT donors can proliferate actively *in vitro*, and therefore are considered to be ideal cells for transgenesis or gene targeting. If these engineered fibroblasts are used for SCNT, the resulting cloned embryos and piglets should have the engineered traits in their genome. This possibility was first proven by *the scientists at the Roslin Institute* [17, 18] who successfully produced genetically engineered (GE) cloned sheep through SCNT using GE fetal fibroblasts. Since then, SCNT using GE cells as SCNT donors has been a common approach for production of knockout (KO) or Tg piglets [19, 20]. However, as mentioned below, the efficiency of producing cloned GE piglets is extremely low and the preparation of GE donor cells is laborious and time-consuming [21]. During the past two decades, production of only a few GE (KO) piglets has been reported by traditional approaches [22–27]. Moreover, almost all resulting KO piglets were heterozygous with respect to the KO allele, thus requiring additional tasks such as breeding (likely one or two generations), sequence targeting, or *in vitro* cell cloning to obtain homozygous KO animals [28], which is also laborious, expensive, and time-consuming.

However, this situation drastically changed when new gene-targeting technologies emerged for precisely manipulating mammalian genomes, called "second-generation genome editing." These technologies require the design of site-specific engineered nucleases which can be zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) nucleases, all of which induce a doublestranded break (DSB) at a specific site in the genome. This DSB facilitates genetic modification such as nonhomologous end-joining (NHEJ) and homology directed repair (HDR) [29], as described below. Using these genome-editing systems, many

**55**

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

the cytoplasm of zygotes, as described below in more detail.

**2. Background of second-generation genome editing**

GE piglets have been produced using SCNT of genome-edited cells, or direct microinjection of genome-editing components (including engineered endonucleases) into

As mentioned above, site-specific engineered nucleases are used in these genome-editing techniques. ZFNs, TALENs, and CRISPR/Cas9 can all bind to DNA and induce DSB, which triggers endogenous DNA repair. If the template DNA is absent, the DSB is repaired via the NHEJ pathway where insertion or deletion of nucleotides (hereinafter called "indels") can happen in the cleaved area. These indels often cause frameshift of the amino acid sequence, leading to the generation of abnormal proteins or formation of a premature stop codon leading to cessation of protein synthesis. If template DNA homologous to the target site is present, it is inserted into the cleaved area via a site-specific HR event which is called HDR. Generally, NHEJ occurs in cells independent of its cell cycle, but HDR occurs

The ZFN technique uses the ZF protein (which binds to the target DNA) and the endonuclease *Fok* I (which cleaves DNA) [31]. ZF protein has several protein motifs capable of recognizing specific sequences of three nucleotides and binding to them. Notably, Urnov et al. [32] first demonstrated that ZFN is effective to induce DNA editing at the endogenous target gene in mammalian cells. Its targeting efficiency was over 18% in the absence of drug selection, which is ~1000-fold higher than that

The TALEN technique uses proteins, termed transcription activator-like effectors (TALEs), which contain 33–35 amino acid repeats that flank a central DNA binding region (amino acids 12 and 13), and *Fok* I nuclease, as in ZFN, thus the term TALE nucleases (TALENs) [33–35]. Notably, the design and engineering of TALENs

CRISPR/Cas9 employs a short (20 bp) RNA sequence called single-guide RNA (sgRNA) which can bind to the specific chromosomal DNA site together with the Cas9 endonuclease [37–40]. Once bound, two independent nuclease domains in Cas9 each cleave one of the DNA strand's three bases upstream of the protospacer adjacent motif (PAM), introducing DSB at the target site of the host chromosome, which is then repaired by NHEJ. This system is different from the other genome editing tools such as ZFNs and TALENs, and thus synthesis of sgRNA is a *prerequisite* for this system. This development dramatically reduced both the complexity

**Table 1** lists instances of production of GE piglets with genome editing technology from 2011 to 2018. This section provides a brief explanation on the background

In 2011, three types of GE piglets were produced using ZFNs from different laboratories. All of these piglets were produced by SCNT using GE cells as a SCNT donor. The first report showing successful production of GE piglets involved the disruption of enhanced green fluorescent protein (*EGFP*) gene in a hemizygous manner. Whyte et al. [41] demonstrated that a ZFN pair efficiently inactivated the expression of *EGFP* that was integrated into the chromosomes of porcine fibroblasts via the NHEJ pathway with an efficiency of ~5%. From this experiment, it

is simpler than that of ZFN, and thus can be done faster [35, 36].

and time required for the design and implementation of gene editing.

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

primarily in dividing cells [30].

achieved by traditional gene targeting.

**3. History of GE in pigs**

of GE pig production.

*Reproductive Biology and Technology in Animals*

In the case of porcine zygotes, pronuclei are difficult to see due to the presence of high lipid content in the cytoplasm. Researchers must briefly centrifuge zygotes to visualize the pronuclei prior to MI [5], which is labor-intensive and requires skill. Moreover, MI-mediated transgene integration into host chromosomes occurs randomly, which often causes gene silencing [6]. However, for precise and efficient genetic modification in the porcine genome, homologous recombination (HR)-based gene targeting technology may be recommended, which was first developed by Smithies' group in mice [7]. In this case, the use of germline-competent embryonic stem (ES) cells is a prerequisite. These ES cells are first transfected with a targeting vector and then recombinant ES clones showing successful targeting are obtained. This vector usually contains a gene of interest (GOI) to be integrated into the target locus, together with a selection marker gene such a neomycin resistance gene (*neo*), and DNA sequences of appropriate length, termed homology arms (HA), that correspond to the endogenous target gene, are placed at both ends of the DNA containing the GOI and a marker gene. Chimeric mice can be obtained through blastocyst injection with the targeted ES clones, and the resulting chimeric mice would contribute to produce heterozygous mice carrying mutated traits (GOI/selection marker gene) in the target locus [8]. Unfortunately, there are no germline-competent porcine ES cells, despite extensive efforts [9–13]. Thus, to date, production of gene-targeted pigs

derived from recombinant porcine ES cells has not yet been successful.

In 1996, scientists at *the Roslin Institute* (Wilmut and colleagues) first succeeded in producing cloned sheep using somatic cell nuclear transfer (SCNT) technology. They used fetal fibroblasts [14] or adult mammary gland-derived fibroblasts [15] as SCNT donors. Notably, prior to these reports, an attempt to produce cloned embryos by the similar technique shown by the *Roslin's* group has been made by Prather et al. [16], who performed transfer of nuclei from two, four and eight-cell embryos to the enucleated oocytes and successfully produced one piglets that had been derived from the four-cell embryo's nucleus. This approach is called "blastomere transplantation," which is basically different from the somatic cell-based SCNT. Generally, fibroblasts used as SCNT donors can proliferate actively *in vitro*, and therefore are considered to be ideal cells for transgenesis or gene targeting. If these engineered fibroblasts are used for SCNT, the resulting cloned embryos and piglets should have the engineered traits in their genome. This possibility was first proven by *the scientists at the Roslin Institute* [17, 18] who successfully produced genetically engineered (GE) cloned sheep through SCNT using GE fetal fibroblasts. Since then, SCNT using GE cells as SCNT donors has been a common approach for production of knockout (KO) or Tg piglets [19, 20]. However, as mentioned below, the efficiency of producing cloned GE piglets is extremely low and the preparation of GE donor cells is laborious and time-consuming [21]. During the past two decades, production of only a few GE (KO) piglets has been reported by traditional approaches [22–27]. Moreover, almost all resulting KO piglets were heterozygous with respect to the KO allele, thus requiring additional tasks such as breeding (likely one or two generations), sequence targeting, or *in vitro* cell cloning to obtain homozygous KO animals [28], which is also laborious, expensive, and time-consuming. However, this situation drastically changed when new gene-targeting technologies emerged for precisely manipulating mammalian genomes, called "second-generation genome editing." These technologies require the design of site-specific engineered nucleases which can be zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) nucleases, all of which induce a doublestranded break (DSB) at a specific site in the genome. This DSB facilitates genetic modification such as nonhomologous end-joining (NHEJ) and homology directed repair (HDR) [29], as described below. Using these genome-editing systems, many

**54**

GE piglets have been produced using SCNT of genome-edited cells, or direct microinjection of genome-editing components (including engineered endonucleases) into the cytoplasm of zygotes, as described below in more detail.

#### **2. Background of second-generation genome editing**

As mentioned above, site-specific engineered nucleases are used in these genome-editing techniques. ZFNs, TALENs, and CRISPR/Cas9 can all bind to DNA and induce DSB, which triggers endogenous DNA repair. If the template DNA is absent, the DSB is repaired via the NHEJ pathway where insertion or deletion of nucleotides (hereinafter called "indels") can happen in the cleaved area. These indels often cause frameshift of the amino acid sequence, leading to the generation of abnormal proteins or formation of a premature stop codon leading to cessation of protein synthesis. If template DNA homologous to the target site is present, it is inserted into the cleaved area via a site-specific HR event which is called HDR. Generally, NHEJ occurs in cells independent of its cell cycle, but HDR occurs primarily in dividing cells [30].

The ZFN technique uses the ZF protein (which binds to the target DNA) and the endonuclease *Fok* I (which cleaves DNA) [31]. ZF protein has several protein motifs capable of recognizing specific sequences of three nucleotides and binding to them. Notably, Urnov et al. [32] first demonstrated that ZFN is effective to induce DNA editing at the endogenous target gene in mammalian cells. Its targeting efficiency was over 18% in the absence of drug selection, which is ~1000-fold higher than that achieved by traditional gene targeting.

The TALEN technique uses proteins, termed transcription activator-like effectors (TALEs), which contain 33–35 amino acid repeats that flank a central DNA binding region (amino acids 12 and 13), and *Fok* I nuclease, as in ZFN, thus the term TALE nucleases (TALENs) [33–35]. Notably, the design and engineering of TALENs is simpler than that of ZFN, and thus can be done faster [35, 36].

CRISPR/Cas9 employs a short (20 bp) RNA sequence called single-guide RNA (sgRNA) which can bind to the specific chromosomal DNA site together with the Cas9 endonuclease [37–40]. Once bound, two independent nuclease domains in Cas9 each cleave one of the DNA strand's three bases upstream of the protospacer adjacent motif (PAM), introducing DSB at the target site of the host chromosome, which is then repaired by NHEJ. This system is different from the other genome editing tools such as ZFNs and TALENs, and thus synthesis of sgRNA is a *prerequisite* for this system. This development dramatically reduced both the complexity and time required for the design and implementation of gene editing.

#### **3. History of GE in pigs**

**Table 1** lists instances of production of GE piglets with genome editing technology from 2011 to 2018. This section provides a brief explanation on the background of GE pig production.

In 2011, three types of GE piglets were produced using ZFNs from different laboratories. All of these piglets were produced by SCNT using GE cells as a SCNT donor. The first report showing successful production of GE piglets involved the disruption of enhanced green fluorescent protein (*EGFP*) gene in a hemizygous manner. Whyte et al. [41] demonstrated that a ZFN pair efficiently inactivated the expression of *EGFP* that was integrated into the chromosomes of porcine fibroblasts via the NHEJ pathway with an efficiency of ~5%. From this experiment, it


**57**

**Method** SCNT/MI

MI SCNT SCNT SCNT SCNT SCNT SCNT SCNT SCNT

CRISPR/Cas9 (KI)

ZFN/TALEN (indels)

ZFN mRNA

Using pig fetal fibroblasts transfected with TALEN or

Using pig fetal fibroblasts transfected with targeting

donor vector and two expression vectors for sgRNA

and Cas9

TALENs (indels)

plasmid

ZFNs (indels)

CRISPR/Cas9 (indels)

Using pig liver-derived cells transfected with two or

three plasmids expressing Cas9 and sgRNA targeting

to GGTA1, CMAH, or putative iGb3S genes

Using pig fetal fibroblasts transfected with ZFN

Using pig liver-derived cells transfected with TALEN

plasmid

ZFN (indels)

Using pig fetal fibroblasts transfected with ZFN

plasmid

TALENs (indels)

Using pig fetal fibroblasts transfected with TALEN plasmid

CRISPR/Cas9 (indels)

Using porcine fetal fibroblasts transfected with Cas9/

sgRNA expression plasmid

A total of three piglets were obtained; fibroblasts from all three animals were negative for class I SLA cell surface expression

Of 27 live cloned piglets obtained, nine were targeted with biallelic mutations in *RAG1*, three were targeted with biallelic mutations in *RAG2*, and 10 were targeted with a monoallelic mutation in *RAG2*

Three *GGTA1* null piglets showing loss of α-Gal epitope

*GGTA1*

[55]

expression were born

Of 10 fetuses obtained, five had mutations in both the

*GGTA1*, *CMAH*,

[56]

*putative iGb3S*

*GGTA1* and *CMAH* genes

The *MSTN*-mutant pigs grew normally, had increased

*MSTN*

[57]

muscle mass with decreased fat accumulation

Livers from *ASGR1*−/− pigs exhibit decreased human

One of the cloned pigs generated GalT/CMAH-double

Highly efficient KI (up to 54%) was achieved after drug

*H11*

[60]

selection; one cloned piglet obtained showed correct

targeting

*CMAH*

[59]

homozygous KO pigs

*ASGR1*

[58]

platelet uptake

CRISPR/Cas9 (indels)

Cytoplasmic MI of Cas9 mRNA/sgRNA toward *in vivo* fertilized zygotes

CRISPR/Cas9 (KI/

indels)

Using porcine fetal fibroblasts transfected with sgRNA-Cas9 plasmids + donor DNA/cytoplasmic MI of Cas9 mRNA/sgRNA toward IVF-derived zygotes

Of the *CD163* recipients, five delivered healthy piglets by cesarean section; 12 of the 13 piglets contained either a biallelic or homozygous deletion of *CD1D*

*eGFP*, *CD163*, *CD1D*

[51]

*vWF*

[52]

Ten of 16 resulting piglets had indels with an efficiency of 63% and were comprised by cells with monoallelic mutant; they can be a model for von Willebrand disease

*class I MHC* *RAG1*, *RAG2*

[54]

[53]

**Genome editing tool (mode for gene modification)**

**Method for gene modification**

**Outcome**

**Target gene**

**References**

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

#### *Reproductive Biology and Technology in Animals*


#### *Recent Advance in Genome Editing-Based Gene Modification in Pigs DOI: http://dx.doi.org/10.5772/intechopen.88022*

*Reproductive Biology and Technology in Animals*

**56**

**Method**

**Genome editing** 

**Method for gene modification**

**Outcome**

**Target gene**

**References**

**tool (mode for gene** 

**modification)**

SCNT SCNT SCNT

MI MI SCNT SCNT SCNT SCNT SCNT

ZFN (KI)

ZFN (indels)

Using porcine adult liver-derived cells transfected

with ZFN plasmid through the two-steps

Using porcine fibroblast cells transfected with ZFN

plasmid and donor DNA

ZFNs (indels)

Using porcine fetal fibroblasts transfected with ZFN

mRNA

TALENs (indels/KI)

mRNA + ssODN

Using porcine fibroblasts transfected with TALEN

TALENs (indels)

Using porcine fetal fibroblasts transfected with

TALEN plasmid

ZFN, TALEN (indels)

*vivo* fertilized zygotes

Cytoplasmic MI of ZFN or TALEN mRNA toward *in* 

TALEN (indels)

Cytoplasmic MI of TALEN mRNA toward IVF-

derived zygotes

ZFN (indels)

Using porcine fetal fibroblasts transfected with ZFN

plasmid

ZFN (indels)

ZFN (indels)

Using adult porcine ear fibroblasts hemizygous for the

Seven of nine embryos (Day 12) exhibited loss of

Of 10 live piglets delivered, two carried the predicted

*PPARγ*

[42]

ZFN-induced mutation; lower expression of both

*PPAR-γ1* and *PPAR-γ2* was observed in those clones

Of six fetuses, all completely lacked α-Gal epitopes

CI of TALEN mRNAs inducing gene KO in up to 75% of embryos; Of the 18 live-born clones, eight contained

monoallelic mutations and 10 contained biallelic

modifications of the *LDLR* gene

Of 39 piglets produced, eight carried TALEN-derived

*NF*-*kappaB* 

[45]

*subunit*

editing events (21%); of nine piglets produced, one

carried an editing event at the ZFN target site (11%)

Three piglets with biallelic mutations of the *GGTA1*

*GGTA1*

[46]

gene exhibited loss of α-Gal epitopes on the surface of

Of eight piglets born from *DAZL*-modified cells, three

*DAZL*, *APC*

[47]

are still born; of the six piglets from *APC*-modified

cells, only one alive

The resulting *IL2RG* KO pigs completely lacked a

*IL2RG*

[48]

thymus and were deficient in T and NK cells, similar to

human X-linked SCID patients

Four viable and healthy cloned pigs obtained exhibited

*GGTA1*, *CMAH*

[49]

disruption of the *GGTA1* and the *CMAH* loci

Successfully produced healthy monoallelic/biallelic

*CMAH*

[50]

CMAH KO pigs

cells

*GGTA1*

*LDLR*

[44]

[43]

*eGFP* transgene

[41]

fluorescence

eGFP transgene

Using porcine fibroblasts transfected with ZFN

plasmid


**59**

**Method**

SCNT SCNT SCNT SCNT SCNT SCNT SCNT SCNT

MI MI

CRISPR/Cas9 (indels)

Cytoplasmic MI of sgRNA-Cas9 encoding vector

toward *in vivo* fertilized zygotes

CRISPR/Cas9 (KI)

Cytoplasmic MI of Cas9 mRNA + sgRNA + ssODN

toward *in vivo* fertilized zygotes

TALENs (indels)

CRISPR/Cas9 (KI)

TALENs/Cas9 (KI)

Using fetal fibroblast cells transfected with Cas9/

sgRNA or TALEN vector + ssODN

Using fetal fibroblast cells transfected with sgRNA-

Cas9 encoding vector + ssODN

Using ear fibroblasts transfected with TALEN vectors

TALENs (indels)

plasmids

CRISPR/Cas9 (indels)

Cas9 encoding vector

Using dermal fibroblasts transfected with TALEN

Using fetal fibroblast cells transfected with sgRNA-

Four live *RUNX3* KO piglets with monoallelic mutation showed the lack of RUNX3 protein in their internal organ system

*MSTN* KO piglets exhibited a double-muscled

*MSTN*

[75]

phenotype, possessing a higher body weight and

longissimus muscle mass measuring 170% that of wildtype piglets, with double the number of muscle fibers Of seven cloned piglets, some expressed human insulin

One cloned stillborn piglet harbored the orthologous p.C313Y mutation at the *MSTN* locus

*APP*, *LRRK2*, MSTN

*GGTA1*

[78]

[77]

Thirty *GGTA1* biallelic KO piglets were successfully

delivered and grew normally.

Of five piglets delivered alive, three exhibited pigmentary

*Sox10*

[79]

disorders with light-colored iris in eye, which was

observed in patients harboring *Sox10* mutations

Of six healthy fetuses recovered, four exhibited loss of

*GGTA1*

[80]

α-Gal epitope expression, indicating a biallelic KO of

*GGTA1*

*INS*

[76]

TALENs (indels)

Using porcine fetal fibroblasts co-transfected with TALEN and hDAF expression plasmids

TALENs (indels)

CRISPR/Cas9 (KI)

Using pig fetal fibroblasts transfected with Cas9-

sgRNA expression vector + donor DNA containing Cre/loxP system

Using pig fetal fibroblasts transfected with TALEN plasmids

Two male live piglets with mono-allelic *MSTN* KO obtained exhibited enhanced myofiber quantity, but the myofiber size remained unaltered

In total, 12 live and two stillborn piglets were collected; all fetuses and piglets exhibited homozygous *GGTA1*-null mutation

*GGTA1*

[73]

Six live-born piglets and three stillborn piglets were obtained; the piglets showed eight base mono-allelic mutations of *GGTA1* and *hDAF* expression

*RUNX3*

[74]

*MSTN*

[71]

**Genome editing tool (mode for gene modification)**

**Method for gene modification**

**Outcome**

**Target gene**

**References**

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

*GGTA1*

[72]

*Reproductive Biology and Technology in Animals*


#### *Recent Advance in Genome Editing-Based Gene Modification in Pigs DOI: http://dx.doi.org/10.5772/intechopen.88022*

*Reproductive Biology and Technology in Animals*

**58**

**Method**

**Genome editing** 

**Method for gene modification**

**Outcome**

**Target gene**

**References**

**tool (mode for gene** 

**modification)**

CRISPR/Cas9

Using pig fetal fibroblasts transfected with sgRNA,

Four cloned double KO piglets showing loss of

*TYR*, *PARK2*,

[61]

*PINK1*

*MITF*

[62]

expression for both *PARK2* and *PINK1* were produced

Two live-born piglets obtained showed the white coat-

color phenotype over its entire body

All 16 piglets born were healthy and carried the

*Alb*

[63]

expected KI allele; the KI allele was successfully

transmitted through germline

Bi-allelic modifications of pig *Npc1l1* were achieved at

*Npc1l1* *MSTN*

[65]

[64]

the efficiency as high as 100%

Of eight marker-gene-free cloned pigs with biallelic mutations obtained, some showed phenotypes similar

One triple knockout pig was obtained; Cells from this cloned pig exhibited reduced human IgM and IgG

*GGTA1*, *CMAH*,

[66]

*β4GalNT2*

*IgM JH* *MSTN*

[68]

[67]

binding

Three cloned piglets with biallelic mutation produced showed no antibody-producing B cells

The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted

gene disruption and efficient production of *Myostatin*

Of two piglets obtained, one piglets exhibited *DMD*

*DMD*

[69]

phenotype, as exemplified by degenerative and

disordered skeletal and cardiac muscle

The heterozygous *FBN1* mutant pigs obtained exhibited abnormal phenotype, which resembles MFS found in

*FBN1*

[70]

humans

mutant pigs

to DM

Cas9 expression plasmids

Cytoplasmic MI of Cas9 mRNA + sgRNA + ssODN

toward *in vivo* fertilized zygotes

Cytoplasmic MI of Cas9 mRNA + sgRNA + circular

vector toward *in vivo* fertilized zygotes

SCNT

MI MI MI SCNT SCNT SCNT

EP MI SCNT

ZFN (indels)

Using pig fetal fibroblasts transfected with ZFN-

encoding mRNA

CRISPR/Cas9 (indels)

*vivo* fertilized zygotes

Cytoplasmic MI of Cas9 mRNA and sgRNA toward *in* 

CRISPR/Cas9 (indels)

derived zygotes

CRISPR/Cas9 (indels)

Cas9 encoding vector

Using Cas9 protein and sgRNA (RNP) toward IVF-

Using pig fetal fibroblasts transfected with sgRNA-

CRISPR/Cas9 (indels)

Using liver-derived cells transfected with sgRNA-

Cas9 encoding vectors

CRISPR/Cas9 (indels)

Cas9 encoding vector

Using pig fetal fibroblasts transfected with sgRNA-

CRISPR/Cas9 (indels)

*vivo* fertilized zygotes

Cytoplasmic MI of Cas9 mRNA and sgRNA toward *in* 

CRISPR/Cas9 (KI)

CRISPR/Cas9 (KI)


**61**

**Method**

SCNT SCNT

MI MI MI MI SCNT

CRISPR/Cas9 (indels)

Using fetal fibroblasts transfected with sgRNA and

Cas9 expression vectors

(Handmade

cloning)

EP SCNT SCNT

CRISPR/Cas9 (KI)

CRISPR/Cas9 (KI)

Using fetal fibroblasts transfected with sgRNA- Cas9

plasmid + donor DNA

Using fetal fibroblasts transfected with sgRNA- Cas9

plasmid + donor DNA

CRISPR/Cas9

Using Cas9 protein and sgRNA (RNP) toward IVF-

derived zygotes

CRISPR/Cas9 (indels)

Cytoplasmic MI of Cas9 mRNA and dual sgRNAs

toward *in vivo* fertilized zygotes

CRISPR/Cas9 (indels)

plasmid

CRISPR/Cas9 (indels)

Cytoplasmic MI of Cas9 mRNA + sgRNAs toward *in vivo*-derived zygotes

Direct pronuclear microinjection of Cas9-gRNA

CRISPR/Cas9 (KI)

Cytoplasmic MI of RNP toward *in vivo* fertilized zygotes

CRISPR/Cas9 (KI)

Using fetal fibroblasts transfected with Cas9-gRNA plasmid and targeting vector

CRISPR/Cas9 (indels)

Using fetal fibroblasts transfected with sgRNA-Cas9 encoding vectors

Of 26 female piglets delivered, 23 piglets carried mutations in the *MSTN* locus; the bi-allelic KO pigs were viable and exhibited partial double-muscled phenotype

Twelve male piglets were born and expressed *UCP1* in a tissue-specific manner

A total of 18 fetuses/born piglets were obtained; successful insertion of pseudo *attP* sites within the *COL1A* locus was observed

Indels in 92–100% of the embryos analyzed; all resulting 12 piglets had biallelic edits of *TMRPSS2*

Of seven born piglets, one exhibited biallelic KO

phenotype and one did monoallelic KO one

Of nine fetuses examined, three exhibited bi-allelic

*PDX1*

[95]

mutations at the *PDX1* locus; in those fetuses pancreatic

primordium was highly disorganized

Eleven live bi-allelic *GGTA1*/*CMAH* double KO piglets

*GGTA1*, *CMAH*

[96]

were obtained with the identical phenotype

Of 11 piglets born, nine survived; six of nine carried mutations in *TP53*; three of the genome-edited pigs

*TP53*

[97]

(50%) exhibited various tumor phenotypes

Three piglets born grew and developed normally; all

*Rosa26*

[98]

these piglets had *fat-1* KI at the Rosa26 locus

Of seven naturally delivered piglets, six showed

*HTT*

[99]

successful KI; the KI allele was successfully transmitted

through germline

*GGTA1*

[94]

*TMPRSS2*

[93]

**Genome editing tool (mode for gene modification)**

**Method for gene modification**

**Outcome**

**Target gene**

*MSTN*

[90]

**References**

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

[91]

*UCP1* *COL1A*

[92]

#### *Reproductive Biology and Technology in Animals*


#### *Recent Advance in Genome Editing-Based Gene Modification in Pigs DOI: http://dx.doi.org/10.5772/intechopen.88022*

*Reproductive Biology and Technology in Animals*

**60**

**Method**

**Genome editing** 

**Method for gene modification**

**Outcome**

**Target gene**

**References**

**tool (mode for gene** 

**modification)**

CRISPR/Cas9 (indels)

Cytoplasmic MI of Cas9 mRNA + three types of

Of two live-born piglets delivered, one piglet

*Parkin*, *DJ-1*,

[81]

*PINK1*

showed biallelic modification of all three genes,

and another showed biallelic modification of the

*DJ-1* and *PINK1* genes and monoallelic mutations of

*parkin* gene

sgRNAs toward *in vivo* fertilized zygotes

MI SCNT-MI

SCNT

MI MI SCNT SCNT SCNT SCNT

CRISPR/Cas9 (indels)

encoding vectors

Using fetal fibroblasts transfected with sgRNA-Cas9

TALEN (indels)

plasmid

Using fetal fibroblasts transfected with TALEN

TALENs

CRISPR/Cas9 (indels)

Using fetal fibroblasts transfected with sgRNA and

Cas9 expression vectors

Using fetal fibroblasts transfected with TALEN

plasmid

CRISPR/Cas9 (indels)

*vivo* fertilized zygotes

Cytoplasmic MI of Cas9 mRNA + sgRNA toward *in* 

CRISPR/Cas9 (indels)

IVF-derived zygotes

Cytoplasmic MI of Cas9 mRNA + sgRNA toward

CRISPR/Cas9 (indels)

Using kidney fibroblasts transfected with ZFN vectors

mating

CRISPR/Cas9 (indels)

Cytoplasmic MI of RNP toward SCNT embryos

Six fetuses recovered revealed that all fetuses carried

*GRB10*

[82]

biallelic edits for the *GRB10* gene (6/6, 100%)

Two healthy normal females with *GGTA1*, *CMAH*

*GGTA1*, *CMAH*

[83]

double KO phenotypes are currently being raised for

Seventeen live piglets and two stillborn were produced;

*RAG2*, *IL2RG*

[84]

all had mutations in both genes (no pigs with wild-type

Eighteen piglets recovered showed either mono- or

*NANOS2*

[85]

bi-allelic modifications and no wild-type animals;

*NANOS2* KO pigs phenocopied KO mice with male

specific germline ablation

Six biallelic KO pigs with mutations in *ApoE* and *LDLR*

*ApoE*, *LDLR*

[86]

genes were obtained successfully in a single step.

All six live piglets obtained carried biallelic mutations in

A total of 18 live piglets were obtained; they showed

*MSTN*

[88]

hypermuscular characteristics

A total of 37 PERV-inactivated piglets were generated;

*PERV*

[89]

15 piglets remain alive

*P53*

[87]

the *P53* locus

sequence)


*Summary of production of genome-edited pigs.*

**63**

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

of GE animals was also shown to be successful in mice [101–104].

ing mono-allelic mutants.

**4. Delivery method**

from ovaries obtained from a slaughterhouse.

transfection.

Hai et al. [52] first demonstrated that GE pigs can be produced using the CRISPR/Cas9 system. They performed cytoplasmic MI with Cas9 mRNA and sgRNA targeted to *von Willebrand factor* gene (*vWF*) to produce a pig *model* for type 1 von Willebrand disease. In this study, 10 of 16 resulting piglets had indels with an efficiency of 63%, and most pigs contained more than two different alleles, suggest-

Successful knock-in (KI) of a GOI into the target locus was first reported in pigs by Ruan et al. [60] and Peng et al. [63]. Zhou et al. [61] demonstrated the production of SCNT-treated piglets with mutations in multiple genes after a single

Fischer et al. [83] first succeeded in producing GE pigs by cytoplasmic MI of a Cas9 protein/gRNA complex called a ribonucleoprotein (RNP). Furthermore, GE pigs could be efficiently produced by *in vitro* EP in the presence of RNP [68]. Sheets et al. [82] produced GE fetuses by cytoplasmic MI of RNP into oocytes reconstituted with intact cells using the SCNT technology. By this treatment, they reported highly efficient (100%) generation of bi-allelic modification in the resultant cloned fetuses. The significance of this approach is be that researchers can obtain GE pigs with a defined genetic background, even though the starting oocytes are derived

For the production of GE pigs, the choice of delivery method for genome editing

components in porcine zygotes is important. As shown in **Table 1**, the methods for the production of GE pigs achieved by delivering genome editing reagents at earlier stages of development can be largely divided into four groups: the first is MI of genome editing reagents (in a form of DNA, mRNA or protein) into zygotes (**Figure 1A**); the second is SCNT using GE cells as the SCNT donor (**Figure 1B**); the third is *in vitro* EP of zygotes in the presence of genome editing reagents (**Figure 1C**); the fourth is MI of genome editing reagents into SCNT-treated

was found that the endogenous NHEJ pathway is effective for inducing mutation in a porcine target gene. Furthermore, SCNT using GE cells with the mutated allele as a SCNT donor demonstrated that seven of the nine resulting cloned fetuses (at Day 12) stopped expressing EGFP. Yang et al. [42] co-transfected porcine fibroblasts with ZFN and pcDNA3.1 plasmids (providing *neo*) by electroporation (EP), performed SCNT using these GE cells, and finally obtained peroxisome proliferator-activated receptor γ (PPARγ) mono-allelic KO pigs, which are expected to generate a porcine cardiomyopathy model. Hauschild et al. [43] attempted to destroy *GGTA1*, an endogenous gene encoding an enzyme required for production of a xenogeneic antigen called α-Gal epitope, using ZFNs in porcine fetal cells. They found that α-Gal epitope-negative cells with the bi-allelic KO phenotype can be efficiently isolated by FACS, and the resultant GE cells have the potential to make cloned piglets. Importantly, this experiment suggests that it is possible to produce individuals with a bi-allelic KO phenotype with this technology. In other words, bi-allelic KO piglets can be directly created without breeding or subcloning, which contrasts with past instances where only heterozygous KO piglets have been produced through traditional gene targeting. Hauschild et al. [43] also showed that neither off-target cleavage nor integration of the ZFN-coding plasmid occurred. The successful production of genome-edited piglets with bi-allelic KO genotype obtained after cytoplasmic MI of *in vivo*-derived porcine zygotes using either ZFN or TALEN mRNA was first reported by Lillico et al. [45]. This MI-based production

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

#### *Recent Advance in Genome Editing-Based Gene Modification in Pigs DOI: http://dx.doi.org/10.5772/intechopen.88022*

*Reproductive Biology and Technology in Animals*

**62**

**Method**

**Genome editing** 

**Method for gene modification**

**Outcome**

**Target gene**

**References**

**tool (mode for gene** 

**modification)**

CRISPR/Cas9 (indels)

plasmid

Using fetal fibroblasts transfected with sgRNA- Cas9

Of a total of 17 piglets obtained, 12 appeared healthy; all

*FBXO40*

[100]

had mutations at the target locus

*Abbreviations: APC, adenomatous polyposis coli; Alb, albumin; GGTA1, α-1,3-galactosyltransferase gene; APP, amyloid precursor protein; ApoE, apolipoprotein E; ASGR1, asialoglycoprotein receptor;* 

*β4GalNT2, β1,4-N-acetylgalactosaminyl transferase; class I MHC, class I major histocompatibility complex; CRISPR/Cas9, clustered regulated interspaced short palindromic repeat and CRISPR-associated* 

*Cas; COL1A, collagen type I alpha 1 chain; CMAH, cytidine monophosphate-N-acetylneuraminic acid hydroxylase; DAZL, deleted in azoospermia-like; DM, double muscling; DMD, Duchenne muscular* 

*dystrophy; PARKIN, E3 ubiquitin ligase PARK2; EP, electroporation; eGFP, enhanced green fluorescent protein; FBXO40, F-box protein 40; FBN1, fibrillin-1; GM, gene-modified; GRB10, growth-hormone* 

*receptor binding protein-10; H11, Hipp11; hDAF, human decay-accelerating factor; HTT, huntingtin; indels, insertion or deletion of nucleotides; INS, insulin; IL2RG, interleukin-2 receptor gamma; IVF,* 

*in vitro fertilized; iGb3S, isogloboside 3 synthase; IgM JH, JH region of the pig IgM heavy chain; KI, knock-in; KO, knockout; LRRK2, leucine-rich repeat kinase 2; LDLR, low density lipoprotein receptor;* 

*MFS, Marfan syndrome; MI, microinjection; SCNT-MI, microinjection following somatic cell nuclear transfer; MITF, Microphthalmia-associated transcription factor; MSTN, myostatin; NANOS2, nanos* 

*C2HC-type zinc finger 2; fat-1, n-3 fatty acid desaturase; Npc1l1, Niemann-Pick C1-Like 1; PDX-1, pancreas duodenum homeobox 1; DJ-1, PARK7; PARK2, parkin; PPARγ, peroxisome proliferatoractivated receptor-gamma; PERV, porcine endogenous retrovirus; PINK1, PTEN-induced putative kinase 1; RNP, ribonucleoprotein; RUNX3, Runt-related transcription factor 3; sgRNA, single guide RNA;* 

*ssODN, single-stranded DNA oligonucleotides; SCNT, somatic cell nuclear transfer; Sox10, SRY (sex determining region Y)-box 10; TALENs, transcription activator-like effector nucleases; TMPRSS2,* 

*transmembrane protease, serine S1, member 2; TYR, tyrosinase; UCP1, uncoupling protein 1; vWF, von Willebrand factor; ZFNs, zinc finger nucleases.*

**Table 1.**

*Summary of production of genome-edited pigs.*

SCNT was found that the endogenous NHEJ pathway is effective for inducing mutation in a porcine target gene. Furthermore, SCNT using GE cells with the mutated allele as a SCNT donor demonstrated that seven of the nine resulting cloned fetuses (at Day 12) stopped expressing EGFP. Yang et al. [42] co-transfected porcine fibroblasts with ZFN and pcDNA3.1 plasmids (providing *neo*) by electroporation (EP), performed SCNT using these GE cells, and finally obtained peroxisome proliferator-activated receptor γ (PPARγ) mono-allelic KO pigs, which are expected to generate a porcine cardiomyopathy model. Hauschild et al. [43] attempted to destroy *GGTA1*, an endogenous gene encoding an enzyme required for production of a xenogeneic antigen called α-Gal epitope, using ZFNs in porcine fetal cells. They found that α-Gal epitope-negative cells with the bi-allelic KO phenotype can be efficiently isolated by FACS, and the resultant GE cells have the potential to make cloned piglets. Importantly, this experiment suggests that it is possible to produce individuals with a bi-allelic KO phenotype with this technology. In other words, bi-allelic KO piglets can be directly created without breeding or subcloning, which contrasts with past instances where only heterozygous KO piglets have been produced through traditional gene targeting. Hauschild et al. [43] also showed that neither off-target cleavage nor integration of the ZFN-coding plasmid occurred.

The successful production of genome-edited piglets with bi-allelic KO genotype obtained after cytoplasmic MI of *in vivo*-derived porcine zygotes using either ZFN or TALEN mRNA was first reported by Lillico et al. [45]. This MI-based production of GE animals was also shown to be successful in mice [101–104].

Hai et al. [52] first demonstrated that GE pigs can be produced using the CRISPR/Cas9 system. They performed cytoplasmic MI with Cas9 mRNA and sgRNA targeted to *von Willebrand factor* gene (*vWF*) to produce a pig *model* for type 1 von Willebrand disease. In this study, 10 of 16 resulting piglets had indels with an efficiency of 63%, and most pigs contained more than two different alleles, suggesting mono-allelic mutants.

Successful knock-in (KI) of a GOI into the target locus was first reported in pigs by Ruan et al. [60] and Peng et al. [63]. Zhou et al. [61] demonstrated the production of SCNT-treated piglets with mutations in multiple genes after a single transfection.

Fischer et al. [83] first succeeded in producing GE pigs by cytoplasmic MI of a Cas9 protein/gRNA complex called a ribonucleoprotein (RNP). Furthermore, GE pigs could be efficiently produced by *in vitro* EP in the presence of RNP [68]. Sheets et al. [82] produced GE fetuses by cytoplasmic MI of RNP into oocytes reconstituted with intact cells using the SCNT technology. By this treatment, they reported highly efficient (100%) generation of bi-allelic modification in the resultant cloned fetuses. The significance of this approach is be that researchers can obtain GE pigs with a defined genetic background, even though the starting oocytes are derived from ovaries obtained from a slaughterhouse.

#### **4. Delivery method**

For the production of GE pigs, the choice of delivery method for genome editing components in porcine zygotes is important. As shown in **Table 1**, the methods for the production of GE pigs achieved by delivering genome editing reagents at earlier stages of development can be largely divided into four groups: the first is MI of genome editing reagents (in a form of DNA, mRNA or protein) into zygotes (**Figure 1A**); the second is SCNT using GE cells as the SCNT donor (**Figure 1B**); the third is *in vitro* EP of zygotes in the presence of genome editing reagents (**Figure 1C**); the fourth is MI of genome editing reagents into SCNT-treated

#### *Reproductive Biology and Technology in Animals*

#### **Figure 1.**

*Several methods to create genome-edited pigs. (A) Microinjection (MI)-based method using zygotes. (B) Somatic cell nuclear transfer (SCNT)-based method using gene-engineered (GE) cells as a SCNT donor. (C) Electroporation (EP)-based method using zygotes. (D) MI-based method using the SCNT-treated embryos.*

embryos reconstituted using a normal cell (**Figure 1D**). Furthermore, we will provide a new approach based on *in vitro* EP of the SCNT-treated embryos reconstituted using a normal cell as the fifth group of methods for possible production of GE pigs (**Figure 2A**). In the following sections, each of these methods are described.

#### **4.1 MI**

MI is an important tool in the creation of GE piglets. To date, about 30% (17/60) of studies (**Table 1**) have employed this approach. For example, in the case of MI with CRISPR/Cas9-related mRNA, a single cytoplasmic MI of 2–10 pL containing 125 ng/ μL Cas9 mRNA and 12.5 ng/μL sgRNA was adopted [62]. Yu et al. [69] employed Cas9 mRNA (20 ng/μL) and sgRNA (10 ng/μL) mixtures for cytoplasmic MI.

Is MI of these components deleterious to the development of porcine zygotes? According to Hai et al. [52], the *in vitro* developmental efficiencies of embryos injected with Cas9 mRNA/sgRNA (~79%) and embryos injected with water (~77%) were both very high and comparable with each other, suggesting that the MI and the Cas9 mRNA/sgRNA had little effect on early embryonic development. On the contrary, Whitworth et al. [51] reported that a higher concentration of sgRNA induces toxicity in porcine embryos. According to Whitworth et al. [51], 10 ng/μL of sgRNA and Cas9 mRNA are recommended.

Selecting appropriate zygotes also appears to be an important factor in the production of GE pigs. For acquisition of viable zygotes, there are at least two methods. One is isolation of zygotes from oviducts of a female that has been inseminated, hereinafter called "*in vivo*-derived zygotes," and the other is acquisition of zygotes by *in vitro* fertilization (IVF) between *in vitro* matured oocytes (derived from the ovaries obtained from the slaughterhouse) and sperm. Generally, it is believed that the *in vivo*-derived zygotes exhibit superior development performance comparing to the IVF-derived zygotes [51, 62]. Indeed, the number of laboratories using

**65**

**Figure 2.**

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

IVF-derived zygotes for production of GE pigs is low (~30% (5/17); see **Table 1**). However, acquisition of viable *in vivo*-derived zygotes is laborious and often associated with the sacrifice of pregnant females, which appears to be one of the major

*A new method for production of genome-edited pigs. (A) EP-based method using the SCNT-treated embryos,* 

The frequent generation of individuals with mosaic genotypes is also a serious problem associated with MI-based GE pig production. Sato et al. [105] demonstrated that cytoplasmic MI of parthenogenetically activated porcine embryos (hereinafter called "parthenotes") with Cas9 mRNA + sgRNA caused frequent mosaicism in the offspring (blastocysts) with cells with mixed genotype, so-called normal wild-type cells and mutated cells, when they were subjected to cytoplasmic MI immediately after oocyte activation. Notably, Carlson et al. [44] suggested that 100% of bovine embryos exhibited fluorescence expression after cytoplasmic MI of *EGFP* mRNA, but only ~40% of porcine embryos did. It is probable that an endogenous system for translation to protein from mRNA may not be sufficiently established in those porcine embryos, especially at the stage immediately after fertilization or zygotic activation. Indeed, Sato et al. [106] demonstrated that this mosaicism can be partially improved when cytoplasmic MI is performed with oocytes 12 h after activation. In contrast, other researchers reported that only 10–20% of MI-treated embryos exhibited mosaicism [51, 84]. Notably, Whitworth et al. [51] performed cytoplasmic MI with Cas9 mRNA + sgRNA toward fertilized oocytes at 14 h postfertilization. In this context, the use of sgRNA and an RNP instead of Cas9 mRNA

goals for improvement in genetic engineering technology using pigs.

*which is termed "GENTEP." (B) Experimental outline for checking the validity of GENTEP.*

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

*Recent Advance in Genome Editing-Based Gene Modification in Pigs DOI: http://dx.doi.org/10.5772/intechopen.88022*

#### **Figure 2.**

*Reproductive Biology and Technology in Animals*

embryos reconstituted using a normal cell (**Figure 1D**). Furthermore, we will provide a new approach based on *in vitro* EP of the SCNT-treated embryos reconstituted using a normal cell as the fifth group of methods for possible production of GE pigs (**Figure 2A**). In the following sections, each of these methods are

*Several methods to create genome-edited pigs. (A) Microinjection (MI)-based method using zygotes. (B) Somatic cell nuclear transfer (SCNT)-based method using gene-engineered (GE) cells as a SCNT donor. (C) Electroporation (EP)-based method using zygotes. (D) MI-based method using the SCNT-treated embryos.*

MI is an important tool in the creation of GE piglets. To date, about 30% (17/60) of studies (**Table 1**) have employed this approach. For example, in the case of MI with CRISPR/Cas9-related mRNA, a single cytoplasmic MI of 2–10 pL containing 125 ng/ μL Cas9 mRNA and 12.5 ng/μL sgRNA was adopted [62]. Yu et al. [69] employed Cas9

Is MI of these components deleterious to the development of porcine zygotes? According to Hai et al. [52], the *in vitro* developmental efficiencies of embryos injected with Cas9 mRNA/sgRNA (~79%) and embryos injected with water (~77%) were both very high and comparable with each other, suggesting that the MI and the Cas9 mRNA/sgRNA had little effect on early embryonic development. On the contrary, Whitworth et al. [51] reported that a higher concentration of sgRNA induces toxicity in porcine embryos. According to Whitworth et al. [51], 10 ng/μL of sgRNA

Selecting appropriate zygotes also appears to be an important factor in the production of GE pigs. For acquisition of viable zygotes, there are at least two methods. One is isolation of zygotes from oviducts of a female that has been inseminated, hereinafter called "*in vivo*-derived zygotes," and the other is acquisition of zygotes by *in vitro* fertilization (IVF) between *in vitro* matured oocytes (derived from the ovaries obtained from the slaughterhouse) and sperm. Generally, it is believed that the *in vivo*-derived zygotes exhibit superior development performance comparing to the IVF-derived zygotes [51, 62]. Indeed, the number of laboratories using

mRNA (20 ng/μL) and sgRNA (10 ng/μL) mixtures for cytoplasmic MI.

and Cas9 mRNA are recommended.

**64**

described.

**4.1 MI**

**Figure 1.**

*A new method for production of genome-edited pigs. (A) EP-based method using the SCNT-treated embryos, which is termed "GENTEP." (B) Experimental outline for checking the validity of GENTEP.*

IVF-derived zygotes for production of GE pigs is low (~30% (5/17); see **Table 1**). However, acquisition of viable *in vivo*-derived zygotes is laborious and often associated with the sacrifice of pregnant females, which appears to be one of the major goals for improvement in genetic engineering technology using pigs.

The frequent generation of individuals with mosaic genotypes is also a serious problem associated with MI-based GE pig production. Sato et al. [105] demonstrated that cytoplasmic MI of parthenogenetically activated porcine embryos (hereinafter called "parthenotes") with Cas9 mRNA + sgRNA caused frequent mosaicism in the offspring (blastocysts) with cells with mixed genotype, so-called normal wild-type cells and mutated cells, when they were subjected to cytoplasmic MI immediately after oocyte activation. Notably, Carlson et al. [44] suggested that 100% of bovine embryos exhibited fluorescence expression after cytoplasmic MI of *EGFP* mRNA, but only ~40% of porcine embryos did. It is probable that an endogenous system for translation to protein from mRNA may not be sufficiently established in those porcine embryos, especially at the stage immediately after fertilization or zygotic activation. Indeed, Sato et al. [106] demonstrated that this mosaicism can be partially improved when cytoplasmic MI is performed with oocytes 12 h after activation. In contrast, other researchers reported that only 10–20% of MI-treated embryos exhibited mosaicism [51, 84]. Notably, Whitworth et al. [51] performed cytoplasmic MI with Cas9 mRNA + sgRNA toward fertilized oocytes at 14 h postfertilization. In this context, the use of sgRNA and an RNP instead of Cas9 mRNA

may be the key to solving this issue of mosaicism, as the Cas9 protein is more rapidly translated, folded, and complexed with sgRNAs prior to editing, unlike the Cas9 mRNA [107–109]. For example, in mice, delivering RNPs into zygotes causes rapid genome editing in the target locus, which also maximizes efficiency while minimizing mosaicism [110–112]. Indeed, Sheets et al. [82] demonstrated that after MI with RNP, 100% of piglets produced had the bi-allelic KO genotype.

Interestingly, Petersen et al. [80] demonstrated that cytoplasmic MI of DNA vectors coding for CRISPR/Cas9 targeting the porcine *GGTA1* gene enabled biallelic knockout of *GGTA1* in 7/12 fetuses and piglets (58.3%). As mentioned previously, it is difficult to visualize porcine pronuclei at zygote stage under normal conditions due to high lipid content in the cytoplasm. Researchers therefore must centrifuge them briefly prior to MI. The fact that cytoplasmic MI of DNA vectors can induce genome editing at a target locus may be beneficial for researchers, because preparation of plasmid DNA is easier than that of mRNA, and it is generally more resistant against degradation than mRNA. According to Petersen et al. [80], it currently remains unknown how the circular DNA plasmid translocates from the cytoplasm to the nucleus. They speculate that the SV40 nuclear translocation signal of the CRISPR/Cas9 plasmid could play an important role by facilitating nuclear translocation via association with ubiquitous transcription factors.

#### **4.2 SCNT**

SCNT using GE cells as an SCNT donor is another way to produce GE pigs. The merit of this approach is the use of *in vitro* cultivated cells such as fetal fibroblasts to which various genetic engineering techniques (i.e., introduction of multiple KO, KI, and transgenes) can be applied easily. After gene transfer, these cells are subjected to cell selection through drug selection or fluorescence activated cell sorting (FACS) to enrich GE cells as a pure population. Thus, it is highly probable that the resulting SCNT-derived GE founder pigs have a predictable genotype and low rates of mosaicism. Unfortunately, as mentioned previously, the efficiency of SCNT to produce cloned piglets is still very low. Much effort has been focused on improving the low efficiency associated with the SCNT, which includes improvement of the oocyte/ zygote culture system and application of chemical reagents to alter the epigenetic status of transferred nuclei. For improving the culture method, researchers have used vitamin C [113], α-tocopherol [114], melatonin [115] or alanyl-glutamine dipeptide (instead of glutamine) [116]. For altering the epigenetic status, researchers have used histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) [117, 118], valproic acid (VPA) [119–121], scriptaid [122–124], LBH589 (panobinostat) [125], oxamflatin [126], PXD101 (belinostat) [127], quisinostat [128], MGCD0103 [129], or histone methyltransferase inhibitors such as MM-102 [130]. Lin et al. [131] employed tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum (ER) stress, and demonstrated that TUDCA can enhance the developmental potential of porcine SCNT embryos by attenuating ER stress and reducing apoptosis. Wang et al. [132] demonstrated that administration of siRNA or microRNA-148a, both of which can suppress the function of DNA methyltransferase 1 (*DNMT1*) at a transcriptional level, is effective for enhancing the developmental potential of SCNT embryos. Furthermore, Matoba et al. [133] succeeded in drastically increasing SCNT efficiency by cytoplasmic MI of mRNA coding for histone demethylase (*Kdm4d*) in mice.

#### **4.3 EP**

EP is known to be a useful and powerful gene delivery tool enabling transfer of exogenous substances (i.e., DNA) into a cell and was first applied to rat zygotes for

**67**

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

genome editing by Kaneko et al. [134]. Since then, many researchers have successfully induced gene edits by using this technology in mice [135, 136], bovines [137] and pigs [68]. The merit of this technology is that it is simple, rapid and convenient for genome editing in zygotes, compared to the previous MI-based technique. Notably, about 30–50 zygotes can be edited with one pulse of EP. Furthermore, EP only requires a square pulse generator called an electroporator, and not a more

As mentioned previously, Tanihara et al. [68] first applied EP to porcine IVFderived zygotes and produced genome-edited pigs. They used CRISPR/Cas9-based RNP for knock-in of a target gene, and achieved reduced mosaicism and higher efficiency of genome-edited pig production with EP (30 V, square pulse 1.0 ms in duration repeated five times) using an electrode (#LF501PT1-20; BEX Co. Ltd., Tokyo, Japan) connected to a CUY21EDIT II electroporator (BEX Co. Ltd.). Notably, they reported no appreciable reduction in the developmental ability of the EP-treated embryos.

Although direct modification of zygotic genomes provides some advantages, SCNT also provides a significant advantage by permitting the isolation of cells containing precise modifications before the expense of animal production is incurred. As mentioned previously, Sheets et al. [82] successfully produced genome-edited cloned pigs by combining SCNT with CRISPR/Cas9 MI, which is beneficial for researchers as they do not need to manage a founder herd, and can eliminate the need for laborious *in vitro* culture and screening. In this study, all (6/6) of the resultant clone fetuses exhibited 100% bi-allelic modification. Unfortunately, they failed to describe successful production of *live birth piglets*, but it seems that this approach

Similar to the approach shown by Sheets et al. [82], we tried to obtain cloned GE piglets through *in vitro* EP in the SCNT-treated embryos, which is called Genome Editing via Nuclear Transfer and subsequent Electroporation or GENTEP (**Figure 2A**).

SCNT-derived embryos were obtained by inserting fetal fibroblasts derived from microminiature pigs (MMP) [138] into the perivitelline space between enucleated porcine oocytes (derived from ovaries obtained from a slaughterhouse) and zona pellucida, according to the method described by Miyoshi et al. [119] (**Figure 2B**). The resulting SCNT-derived embryos were then subjected to electric activation following electric fusion between an egg and a cell (**Figure 2B**). Six or 12 h after activation, the SCNT-treated embryos were subjected to *in vitro* EP in the presence of RNP targeted to the pig low density lipoprotein receptor (*LDLR*) gene (**Figure 2B**). Parthenotes (~6 h after electric activation) were also used for *in vitro* EP using tetramethylrhodamine-labeled dextran 3 kDa (used as an indicator for successful gene delivery) (as shown in **Figure 3A**) or as controls for immunocyto-

First, we examined whether the *in vitro* EP we used here is effective for successful gene delivery to porcine embryos and does not cause any deleterious effects on their embryonic development, using porcine parthenotes (6 h after activation). The EP procedure was based on the method described by Hashimoto and Takemoto [135]. An electroporation chamber (#LF610P4-4\_470; BEX Co. Ltd.), in which two platinum block electrodes were situated with a 1-mm gap between them (**Figure 1C**), was placed under a stereoscopic microscope and connected to

Some results obtained from GENTEP-related experiments are presented below.

chemistry using anti-LDLR antibody (as shown in **Figure 3B**).

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

expensive micromanipulator system.

is a powerful tool for GE pig production.

**4.4 MI after SCNT**

**4.5 EP after SCNT**

*Recent Advance in Genome Editing-Based Gene Modification in Pigs DOI: http://dx.doi.org/10.5772/intechopen.88022*

genome editing by Kaneko et al. [134]. Since then, many researchers have successfully induced gene edits by using this technology in mice [135, 136], bovines [137] and pigs [68]. The merit of this technology is that it is simple, rapid and convenient for genome editing in zygotes, compared to the previous MI-based technique. Notably, about 30–50 zygotes can be edited with one pulse of EP. Furthermore, EP only requires a square pulse generator called an electroporator, and not a more expensive micromanipulator system.

As mentioned previously, Tanihara et al. [68] first applied EP to porcine IVFderived zygotes and produced genome-edited pigs. They used CRISPR/Cas9-based RNP for knock-in of a target gene, and achieved reduced mosaicism and higher efficiency of genome-edited pig production with EP (30 V, square pulse 1.0 ms in duration repeated five times) using an electrode (#LF501PT1-20; BEX Co. Ltd., Tokyo, Japan) connected to a CUY21EDIT II electroporator (BEX Co. Ltd.). Notably, they reported no appreciable reduction in the developmental ability of the EP-treated embryos.

#### **4.4 MI after SCNT**

*Reproductive Biology and Technology in Animals*

may be the key to solving this issue of mosaicism, as the Cas9 protein is more rapidly translated, folded, and complexed with sgRNAs prior to editing, unlike the Cas9 mRNA [107–109]. For example, in mice, delivering RNPs into zygotes causes rapid genome editing in the target locus, which also maximizes efficiency while minimizing mosaicism [110–112]. Indeed, Sheets et al. [82] demonstrated that after MI with

Interestingly, Petersen et al. [80] demonstrated that cytoplasmic MI of DNA vectors coding for CRISPR/Cas9 targeting the porcine *GGTA1* gene enabled biallelic knockout of *GGTA1* in 7/12 fetuses and piglets (58.3%). As mentioned previously, it is difficult to visualize porcine pronuclei at zygote stage under normal conditions due to high lipid content in the cytoplasm. Researchers therefore must centrifuge them briefly prior to MI. The fact that cytoplasmic MI of DNA vectors can induce genome editing at a target locus may be beneficial for researchers, because preparation of plasmid DNA is easier than that of mRNA, and it is generally more resistant against degradation than mRNA. According to Petersen et al. [80], it currently remains unknown how the circular DNA plasmid translocates from the cytoplasm to the nucleus. They speculate that the SV40 nuclear translocation signal of the CRISPR/Cas9 plasmid could play an important role by facilitating nuclear translo-

SCNT using GE cells as an SCNT donor is another way to produce GE pigs. The merit of this approach is the use of *in vitro* cultivated cells such as fetal fibroblasts to which various genetic engineering techniques (i.e., introduction of multiple KO, KI, and transgenes) can be applied easily. After gene transfer, these cells are subjected to cell selection through drug selection or fluorescence activated cell sorting (FACS) to enrich GE cells as a pure population. Thus, it is highly probable that the resulting SCNT-derived GE founder pigs have a predictable genotype and low rates of mosaicism. Unfortunately, as mentioned previously, the efficiency of SCNT to produce cloned piglets is still very low. Much effort has been focused on improving the low efficiency associated with the SCNT, which includes improvement of the oocyte/ zygote culture system and application of chemical reagents to alter the epigenetic status of transferred nuclei. For improving the culture method, researchers have used vitamin C [113], α-tocopherol [114], melatonin [115] or alanyl-glutamine dipeptide (instead of glutamine) [116]. For altering the epigenetic status, researchers have used histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) [117, 118], valproic acid (VPA) [119–121], scriptaid [122–124], LBH589 (panobinostat) [125], oxamflatin [126], PXD101 (belinostat) [127], quisinostat [128], MGCD0103 [129], or histone methyltransferase inhibitors such as MM-102 [130]. Lin et al. [131] employed tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum (ER) stress, and demonstrated that TUDCA can enhance the developmental potential of porcine SCNT embryos by attenuating ER stress and reducing apoptosis. Wang et al. [132] demonstrated that administration of siRNA or microRNA-148a, both of which can suppress the function of DNA methyltransferase 1 (*DNMT1*) at a transcriptional level, is effective for enhancing the developmental potential of SCNT embryos. Furthermore, Matoba et al. [133] succeeded in drastically increasing SCNT efficiency by cytoplasmic MI of mRNA coding for histone demethylase (*Kdm4d*) in mice.

EP is known to be a useful and powerful gene delivery tool enabling transfer of exogenous substances (i.e., DNA) into a cell and was first applied to rat zygotes for

RNP, 100% of piglets produced had the bi-allelic KO genotype.

cation via association with ubiquitous transcription factors.

**66**

**4.3 EP**

**4.2 SCNT**

Although direct modification of zygotic genomes provides some advantages, SCNT also provides a significant advantage by permitting the isolation of cells containing precise modifications before the expense of animal production is incurred. As mentioned previously, Sheets et al. [82] successfully produced genome-edited cloned pigs by combining SCNT with CRISPR/Cas9 MI, which is beneficial for researchers as they do not need to manage a founder herd, and can eliminate the need for laborious *in vitro* culture and screening. In this study, all (6/6) of the resultant clone fetuses exhibited 100% bi-allelic modification. Unfortunately, they failed to describe successful production of *live birth piglets*, but it seems that this approach is a powerful tool for GE pig production.

#### **4.5 EP after SCNT**

Similar to the approach shown by Sheets et al. [82], we tried to obtain cloned GE piglets through *in vitro* EP in the SCNT-treated embryos, which is called Genome Editing via Nuclear Transfer and subsequent Electroporation or GENTEP (**Figure 2A**). Some results obtained from GENTEP-related experiments are presented below.

SCNT-derived embryos were obtained by inserting fetal fibroblasts derived from microminiature pigs (MMP) [138] into the perivitelline space between enucleated porcine oocytes (derived from ovaries obtained from a slaughterhouse) and zona pellucida, according to the method described by Miyoshi et al. [119] (**Figure 2B**). The resulting SCNT-derived embryos were then subjected to electric activation following electric fusion between an egg and a cell (**Figure 2B**). Six or 12 h after activation, the SCNT-treated embryos were subjected to *in vitro* EP in the presence of RNP targeted to the pig low density lipoprotein receptor (*LDLR*) gene (**Figure 2B**). Parthenotes (~6 h after electric activation) were also used for *in vitro* EP using tetramethylrhodamine-labeled dextran 3 kDa (used as an indicator for successful gene delivery) (as shown in **Figure 3A**) or as controls for immunocytochemistry using anti-LDLR antibody (as shown in **Figure 3B**).

First, we examined whether the *in vitro* EP we used here is effective for successful gene delivery to porcine embryos and does not cause any deleterious effects on their embryonic development, using porcine parthenotes (6 h after activation). The EP procedure was based on the method described by Hashimoto and Takemoto [135]. An electroporation chamber (#LF610P4-4\_470; BEX Co. Ltd.), in which two platinum block electrodes were situated with a 1-mm gap between them (**Figure 1C**), was placed under a stereoscopic microscope and connected to

#### **Figure 3.**

*Validity check of GENTEP. (A) EP of parthenotes (6 h after activation) in the presence of tetramethylrhodamine-labelled dextran 3 kDa. Porcine parthenotes were subjected to* in vitro *EP, and then cultured for 7 days up to blastocysts. Note that almost all of the EP-treated blastocysts are fluorescent (arrows in a and b), while intact parthenotes do not fluoresce (c and d). Bar = 100 μm. (B) Staining with anti-LDLR antibody. The intact parthenote (hatched blastocysts) exhibits the reactivity to the antibody (arrowheads in a–c), but not to the second antibody alone (arrowheads in d–f). No reactivity to the antibody was also seen in the GENTEP-derived blastocyst (arrowheads in g–i). Nuclear staining with DAPI was performed after staining with the second antibody. Note that porcine zona pellucida was slightly stained with the second antibody, since it was found to be reactive with the second antibody alone (see d–f). Bar = 100 μm.*

an electric pulse generator (CUY21EDITII Genome Editor™, BEX Co. Ltd.). About 20 parthenotes were placed into a 5-μL drop containing 2 μg/μL tetramethylrhodamine-dextran 3 kDa (#D3307; Thermo Fisher Scientific Inc., Waltham, MA, USA) in Opti-MEM (Invitrogen, Carlsbad, CA, USA) between the electrodes (**Figure 1C**). EP was performed under these conditions: 30 V, square pulses, 1.0 ms in duration at 99 ms intervals, repeated seven times. The EP-treated parthenotes were then cultured for 7 days up to blastocysts to evaluate the *in vitro* developmental rate and uptake of fluorescent dye into the embryos. Approximately 40% of the EP-treated parthenotes developed to the blastocyst stage and ~80% of them exhibited bright tetramethylrhodamine-derived fluorescence [arrows in **Figure 3A(a,b)**]. This result suggests that our EP condition is useful for effective delivery of a foreign substance into porcine embryos and not harmful for their development.

Second, we performed CRISPR/Cas9-based genome editing (targeted to the endogenous *LDLR* gene) with porcine SCNT-treated embryos. We designed sgRNA capable of recognizing a 20 bp sequence spanning the translation initiation codon (ATG) upstream of the protospacer adjacent motif (PAM) sequence (CGG) on the

**69**

**Figure 4.**

*red. Sequence recognized by sgRNA is shown in blue.*

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

first exon of porcine *LDLR* (left panel of **Figure 4A**). The sgRNA was synthesized by Integrated DNA Technologies, Inc. (IDT; Coralville, Iowa, USA) as Alt-R™ CRISPR crRNA product. The crRNA and tracrRNA (purchased from IDT) were combined for annealing and then mixed with recombinant Cas9 protein (TaKaRa Shuzo Co. Ltd., Shiga, Japan) to form RNP, according to the method of Ohtsuka et al. [139]. The final concentrations of the components in RNP were 30 μM/mL (for crRNA/tracrRNA) and 1 mg/mL (for Cas9 protein). The SCNT-treated embryos 6 or 12 h after activation were transferred to a 5-μL drop (containing RNP in Opti-MEM) and immediately subjected to *in vitro* EP under these conditions: 30 V, square pulses, 1.0 ms in duration at 99 ms intervals (or 0.5 ms in duration at 99.5 ms intervals), both repeated seven times. After EP, the embryos were promptly cultivated in normal medium for 7 days up to blastocysts and then subjected to analysis of molecular biology (possible mutations in the first exon of *LDLR*) and immunocytochemistry (possible loss of LDLR protein synthesis) parameters, as described

*Molecular biological analysis of the GENTEP-treated embryos (termed GENTEP-1 to -11) at a single embryo level. (A) Structure of porcine* LDLR *gene and a target sequence recognized by sgRNA (left panel), and the results of nested PCR (right panel). The target sequence (shown in blue) spanning ATG (shown in red) is located on the first exon of* LDLR*. PAM, protospacer adjacent motif. Primers used for first PCR and nested PCR are shown above the* LDRL*. In the right panel, a part of the nested PCR products (lanes 1–9) loaded onto 2% agarose gel is shown. Arrow indicates the PCR products of 355 bp in size. M, 100-bp ladder markers. (B) Ideogram pattern in the GENTEP-1 and -2 samples obtained after direct sequencing of the nested PCR products using LDLR-2S primer. (C) Various indels found in each GENTEP-treated embryo. ATG is shown in* 

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

#### *Recent Advance in Genome Editing-Based Gene Modification in Pigs DOI: http://dx.doi.org/10.5772/intechopen.88022*

#### **Figure 4.**

*Reproductive Biology and Technology in Animals*

an electric pulse generator (CUY21EDITII Genome Editor™, BEX Co. Ltd.). About 20 parthenotes were placed into a 5-μL drop containing 2 μg/μL tetramethylrhodamine-dextran 3 kDa (#D3307; Thermo Fisher Scientific Inc., Waltham, MA, USA) in Opti-MEM (Invitrogen, Carlsbad, CA, USA) between the electrodes (**Figure 1C**). EP was performed under these conditions: 30 V, square pulses, 1.0 ms in duration at 99 ms intervals, repeated seven times. The EP-treated parthenotes were then cultured for 7 days up to blastocysts to evaluate the *in vitro* developmental rate and uptake of fluorescent dye into the embryos. Approximately 40% of the EP-treated parthenotes developed to the blastocyst stage and ~80% of them exhibited bright tetramethylrhodamine-derived fluorescence [arrows in **Figure 3A(a,b)**]. This result suggests that our EP condition is useful for effective delivery of a foreign substance

*tetramethylrhodamine-labelled dextran 3 kDa. Porcine parthenotes were subjected to* in vitro *EP, and then cultured for 7 days up to blastocysts. Note that almost all of the EP-treated blastocysts are fluorescent (arrows in a and b), while intact parthenotes do not fluoresce (c and d). Bar = 100 μm. (B) Staining with anti-LDLR antibody. The intact parthenote (hatched blastocysts) exhibits the reactivity to the antibody (arrowheads in a–c), but not to the second antibody alone (arrowheads in d–f). No reactivity to the antibody was also seen in the GENTEP-derived blastocyst (arrowheads in g–i). Nuclear staining with DAPI was performed after staining with the second antibody. Note that porcine zona pellucida was slightly stained with the second antibody, since it was found to be reactive with the second antibody alone (see d–f). Bar = 100 μm.*

*Validity check of GENTEP. (A) EP of parthenotes (6 h after activation) in the presence of* 

Second, we performed CRISPR/Cas9-based genome editing (targeted to the endogenous *LDLR* gene) with porcine SCNT-treated embryos. We designed sgRNA capable of recognizing a 20 bp sequence spanning the translation initiation codon (ATG) upstream of the protospacer adjacent motif (PAM) sequence (CGG) on the

into porcine embryos and not harmful for their development.

**68**

**Figure 3.**

*Molecular biological analysis of the GENTEP-treated embryos (termed GENTEP-1 to -11) at a single embryo level. (A) Structure of porcine* LDLR *gene and a target sequence recognized by sgRNA (left panel), and the results of nested PCR (right panel). The target sequence (shown in blue) spanning ATG (shown in red) is located on the first exon of* LDLR*. PAM, protospacer adjacent motif. Primers used for first PCR and nested PCR are shown above the* LDRL*. In the right panel, a part of the nested PCR products (lanes 1–9) loaded onto 2% agarose gel is shown. Arrow indicates the PCR products of 355 bp in size. M, 100-bp ladder markers. (B) Ideogram pattern in the GENTEP-1 and -2 samples obtained after direct sequencing of the nested PCR products using LDLR-2S primer. (C) Various indels found in each GENTEP-treated embryo. ATG is shown in red. Sequence recognized by sgRNA is shown in blue.*

first exon of porcine *LDLR* (left panel of **Figure 4A**). The sgRNA was synthesized by Integrated DNA Technologies, Inc. (IDT; Coralville, Iowa, USA) as Alt-R™ CRISPR crRNA product. The crRNA and tracrRNA (purchased from IDT) were combined for annealing and then mixed with recombinant Cas9 protein (TaKaRa Shuzo Co. Ltd., Shiga, Japan) to form RNP, according to the method of Ohtsuka et al. [139]. The final concentrations of the components in RNP were 30 μM/mL (for crRNA/tracrRNA) and 1 mg/mL (for Cas9 protein). The SCNT-treated embryos 6 or 12 h after activation were transferred to a 5-μL drop (containing RNP in Opti-MEM) and immediately subjected to *in vitro* EP under these conditions: 30 V, square pulses, 1.0 ms in duration at 99 ms intervals (or 0.5 ms in duration at 99.5 ms intervals), both repeated seven times. After EP, the embryos were promptly cultivated in normal medium for 7 days up to blastocysts and then subjected to analysis of molecular biology (possible mutations in the first exon of *LDLR*) and immunocytochemistry (possible loss of LDLR protein synthesis) parameters, as described


*1 EP in the presence of RNP [10 μM of crRNA/tracrRNA mixture (targeted to LDLR gene) + 0.3 μg/μL of Cas9 protein] is performed on SCNT-treated embryos 6 or 12 h after activation. The EP-treated embryos were then cultured for 7 days to the blastocyst stage for the presence of mutations in the target gene at molecular biological and immunocytochemical levels.*

*2 EP was performed under the electric condition of 30 V in voltage, 0.5 ms in length of square pulse with 99.5-ms intervals (0.5) or 1.0 ms in length of square pulse with 99-ms intervals (1.0), and seven times of pulse stimulation using an electroporation chamber (#LF610P4-4\_470; BEX Co. Ltd.) connected to an electric pulse generator (CUY21EDITII. Genome Editor™, BEX Co. Ltd.).*

#### **Table 2.**

*Summary of the properties of blastocysts derived from EP1 toward the SCNT-treated porcine embryos.*

in **Figure 2B**. In each group, 8–17% of the EP-treated embryos developed to blastocysts (**Table 2**). These rates appear to be comparable to the yield (24.2%) in experiments performed using intact MMP fetal fibroblasts as SCNT donors [119]. All of the blastocysts obtained were then fixed with 4% paraformaldehyde, and a section of these embryos was subjected to immunocytochemical staining using anti-LDLR antibody (**Figure 2B**). The EP-treated cloned blastocyst (termed GENTEP-1) was unreactive to anti-LDLR (arrows in **Figure 3B(g–i)**). In contrast, a parthenote (blastocyst) exhibited positive reactivity to anti-LDLR (arrow in **Figure 3B(a–c)**). Staining with the second antibody alone failed to react with the antibody (arrow in **Figure 3B(d–f )**). Furthermore, each of these fixed blastocysts was subjected to genomic DNA isolation to examine possible mutations at the individual embryo level (**Figure 2B**). Next, GenomiPhi-based whole genome amplification (WGA) was performed using the isolated genomic DNA as a template, as described previously [140]. PCR was then performed using the WGA products as a PCR template. The primer sets used are LDLR-S (5′-AAACCTCACATTGAAATGCTG-3′)/ LDLR-RV (5′-CCTAAACTCTCGCGCCCCCCT-3′) for the first round of PCR and LDLR-2S (5′-CTGCAAATGACTGGGGCCCCG-3′)/LDLR-2RV (5′-CTCCAACCACGTAAGAATGAC-3′) for nested PCR (left panel of **Figure 4A**). Nested PCR using the LDLR-2S/LDLR-2RV primer set yields 355-bp products (left panel of **Figure 4A**). The typical example when the nested PCR products (lanes 1–9) are loaded onto a 2% agarose gel is shown in the right panel of **Figure 4A**. Almost all of the samples tested exhibited 355-bp products, except for lane 3 showing bands of reduced size, suggesting occurrence of a large deletion (probably over 100 bp) around the *LDLR* sequence recognized by sgRNA. In **Figure 4B**, an example of the results obtained from direct sequencing of nested PCR products using LDLR-2S primer is shown. The sample GENTEP-1, which has been shown to exhibit loss of the reactivity to anti-LDLR (see **Figure 3B**-**g**-**i**), had a large deletion including a sequence spanning ATG and PAM. Notably, there was no appreciable overlapping in ideograms of the sample, suggesting a homozygous bi-allelic KO phenotype (**Figure 4C**). Subcloning of the PCR products derived from the sample GENTEP-1 into TA cloning vector and subsequent sequencing demonstrated that all six clones obtained exhibited the same sequence as the parental product (data not shown). When the remaining PCR products were sequenced it was found that almost all (82%, 9/11) of the samples exhibited the homozygous bi-allelic KO genotype (**Figure 4C**).

**71**

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

techniques or factors are described in greater detail.

**5. Other techniques and factors affecting efficacy of the genome** 

As shown above, genome editing tools such as ZFNs, TALENs and CRISPR/Cas9 are considered useful in enabling site-specific gene modification in livestock such as pigs. However, there are still several techniques and factors that influence performance which must be addressed. These include the single embryo assay, off-target cutting, multiplexed genome engineering, KI, and Cas9 pigs. In this section, these

To increasing the efficiency of genome editing systems, it is important to select suitable sets of ZFNs (or TALENs) or sgRNA (in the case of CRISPR/Cas9). Researchers therefore must check the efficiency of these reagents by introducing them into cultured cells, but at this point it remains unknown whether they will function *in vivo*. Unlike small animals such as mice and rats, large animals have long gestation periods and it is costly to prepare large animal recipients. Therefore, this testing *in vivo* appears to be difficult in larger animals such as pigs. To overcome this issue, a single embryo (blastocyst) assay to evaluate the operability of the genome editing reagents prepared was provided by Wang et al. [62] who later re-validated those sets using porcine parthenotes. To our knowledge, this assay was first developed using mice by Sakurai et al. [141] who

It may be required to confirm at a molecular level whether the genome-edited embryos have mutations. In this case, WGA has often been employed for amplifying the whole genome of an embryo (blastocyst) using genomic DNA isolated from a single embryo as the DNA template [140, 141], since the blastocyst DNA is often too small to generate a sufficient amount of PCR product. The effectiveness of WGAbased amplification of blastocyst DNA has already been confirmed by ours [142] and others [44]. The resulting products obtained after PCR using WGA-derived DNA as the template are then subjected to direct sequencing for identification of

Since sgRNA used in the CRISPR/Cas9 system can recognize only a short sequence (20 bp) at the target gene where Cas9 cleaves, other genes with a similar sequence to the sgRNA may be susceptible to Cas9-mediated DNA cleavage, which leads to the occasional generation of off-target cutting [29, 143]. This unintended

Several strategies to minimize off-target cutting have been employed including the use of the double nickase mutant form of Cas9, which induces a single-strand break instead of DSB [144]; the use of RNP, whose half-life is shorter than the duration of transcription of plasmid or viral nucleic acids [110, 145]; or the fusion of catalytically inactive Cas9 with *Fok* I nuclease domain (fCas9) to improve the DNA cleavage specificity [146]. Recently, it was reported that Cpf1, a putative Class 2 CRISPR effector, mediates target DNA editing differently from Cas9 [147]. It generates a 5-nucleotide staggered cut with a 5′ overhang, which is particularly advantageous in facilitating an NHEJ-based KI into a genome. Several unique enzymes that can decrease the probability of off-target cleavage have also been produced. For example, two engineered enzymes produced from SpCas9 from *Streptococcus pyogenes* with the goal of enhancing specificity, called eSpCas9 [148] and SpCas9-HF [149], are reported to reduce the probability of mismatched DNA binding. A hybrid

reported that it is useful for confirming the fidelity of sgRNAs used.

possible mutations in the target gene, as shown in **Figure 4B**.

cutting is considered a serious problem to be resolved.

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

**editing system**

**5.1 Single embryo assay**

**5.2 Off-target cleavage**

#### **5. Other techniques and factors affecting efficacy of the genome editing system**

As shown above, genome editing tools such as ZFNs, TALENs and CRISPR/Cas9 are considered useful in enabling site-specific gene modification in livestock such as pigs. However, there are still several techniques and factors that influence performance which must be addressed. These include the single embryo assay, off-target cutting, multiplexed genome engineering, KI, and Cas9 pigs. In this section, these techniques or factors are described in greater detail.

#### **5.1 Single embryo assay**

*Reproductive Biology and Technology in Animals*

**EP condition2**

**Stage at EP after activation of SCNTtreated embryos**

*immunocytochemical levels.*

*(CUY21EDITII. Genome Editor™, BEX Co. Ltd.).*

*Summary of the properties of blastocysts derived from EP1*

*1*

*2*

**Table 2.**

in **Figure 2B**. In each group, 8–17% of the EP-treated embryos developed to blastocysts (**Table 2**). These rates appear to be comparable to the yield (24.2%) in experiments performed using intact MMP fetal fibroblasts as SCNT donors [119]. All of the blastocysts obtained were then fixed with 4% paraformaldehyde, and a section of these embryos was subjected to immunocytochemical staining using anti-LDLR antibody (**Figure 2B**). The EP-treated cloned blastocyst (termed GENTEP-1) was unreactive to anti-LDLR (arrows in **Figure 3B(g–i)**). In contrast, a parthenote (blastocyst) exhibited positive reactivity to anti-LDLR (arrow in **Figure 3B(a–c)**). Staining with the second antibody alone failed to react with the antibody (arrow in **Figure 3B(d–f )**). Furthermore, each of these fixed blastocysts was subjected to genomic DNA isolation to examine possible mutations at the individual embryo level (**Figure 2B**). Next, GenomiPhi-based whole genome amplification (WGA) was performed using the isolated genomic DNA as a template, as described previously [140]. PCR was then performed using the WGA products as a PCR template. The primer sets used are LDLR-S (5′-AAACCTCACATTGAAATGCTG-3′)/ LDLR-RV (5′-CCTAAACTCTCGCGCCCCCCT-3′) for the first round of PCR and LDLR-2S (5′-CTGCAAATGACTGGGGCCCCG-3′)/LDLR-2RV

**Total number of SCNT-treated embryos examined**

6 h 0.5 12 8 (66.7) 1 (8.3)

12 h 0.5 33 21 (63.6) 5 (15.2)

*EP in the presence of RNP [10 μM of crRNA/tracrRNA mixture (targeted to LDLR gene) + 0.3 μg/μL of Cas9 protein] is performed on SCNT-treated embryos 6 or 12 h after activation. The EP-treated embryos were then cultured for 7 days to the blastocyst stage for the presence of mutations in the target gene at molecular biological and* 

*EP was performed under the electric condition of 30 V in voltage, 0.5 ms in length of square pulse with 99.5-ms intervals (0.5) or 1.0 ms in length of square pulse with 99-ms intervals (1.0), and seven times of pulse stimulation using an electroporation chamber (#LF610P4-4\_470; BEX Co. Ltd.) connected to an electric pulse generator* 

**No. of embryos cleaved to the twocell stage (%)**

 *toward the SCNT-treated porcine embryos.*

1.0 12 11 (91.7) 2 (16.7)

1.0 35 23 (65.7) 3 (8.6)

**No. of embryos developed to blastocysts (%)**

(5′-CTCCAACCACGTAAGAATGAC-3′) for nested PCR (left panel of **Figure 4A**). Nested PCR using the LDLR-2S/LDLR-2RV primer set yields 355-bp products (left panel of **Figure 4A**). The typical example when the nested PCR products (lanes 1–9) are loaded onto a 2% agarose gel is shown in the right panel of **Figure 4A**. Almost all of the samples tested exhibited 355-bp products, except for lane 3 showing bands of reduced size, suggesting occurrence of a large deletion (probably over 100 bp) around the *LDLR* sequence recognized by sgRNA. In **Figure 4B**, an example of the results obtained from direct sequencing of nested PCR products using LDLR-2S primer is shown. The sample GENTEP-1, which has been shown to exhibit loss of the reactivity to anti-LDLR (see **Figure 3B**-**g**-**i**), had a large deletion including a sequence spanning ATG and PAM. Notably, there was no appreciable overlapping in ideograms of the sample, suggesting a homozygous bi-allelic KO phenotype (**Figure 4C**). Subcloning of the PCR products derived from the sample GENTEP-1 into TA cloning vector and subsequent sequencing demonstrated that all six clones obtained exhibited the same sequence as the parental product (data not shown). When the remaining PCR products were sequenced it was found that almost all (82%, 9/11) of the samples exhibited the homozygous bi-allelic KO

**70**

genotype (**Figure 4C**).

To increasing the efficiency of genome editing systems, it is important to select suitable sets of ZFNs (or TALENs) or sgRNA (in the case of CRISPR/Cas9). Researchers therefore must check the efficiency of these reagents by introducing them into cultured cells, but at this point it remains unknown whether they will function *in vivo*. Unlike small animals such as mice and rats, large animals have long gestation periods and it is costly to prepare large animal recipients. Therefore, this testing *in vivo* appears to be difficult in larger animals such as pigs. To overcome this issue, a single embryo (blastocyst) assay to evaluate the operability of the genome editing reagents prepared was provided by Wang et al. [62] who later re-validated those sets using porcine parthenotes. To our knowledge, this assay was first developed using mice by Sakurai et al. [141] who reported that it is useful for confirming the fidelity of sgRNAs used.

It may be required to confirm at a molecular level whether the genome-edited embryos have mutations. In this case, WGA has often been employed for amplifying the whole genome of an embryo (blastocyst) using genomic DNA isolated from a single embryo as the DNA template [140, 141], since the blastocyst DNA is often too small to generate a sufficient amount of PCR product. The effectiveness of WGAbased amplification of blastocyst DNA has already been confirmed by ours [142] and others [44]. The resulting products obtained after PCR using WGA-derived DNA as the template are then subjected to direct sequencing for identification of possible mutations in the target gene, as shown in **Figure 4B**.

#### **5.2 Off-target cleavage**

Since sgRNA used in the CRISPR/Cas9 system can recognize only a short sequence (20 bp) at the target gene where Cas9 cleaves, other genes with a similar sequence to the sgRNA may be susceptible to Cas9-mediated DNA cleavage, which leads to the occasional generation of off-target cutting [29, 143]. This unintended cutting is considered a serious problem to be resolved.

Several strategies to minimize off-target cutting have been employed including the use of the double nickase mutant form of Cas9, which induces a single-strand break instead of DSB [144]; the use of RNP, whose half-life is shorter than the duration of transcription of plasmid or viral nucleic acids [110, 145]; or the fusion of catalytically inactive Cas9 with *Fok* I nuclease domain (fCas9) to improve the DNA cleavage specificity [146]. Recently, it was reported that Cpf1, a putative Class 2 CRISPR effector, mediates target DNA editing differently from Cas9 [147]. It generates a 5-nucleotide staggered cut with a 5′ overhang, which is particularly advantageous in facilitating an NHEJ-based KI into a genome. Several unique enzymes that can decrease the probability of off-target cleavage have also been produced. For example, two engineered enzymes produced from SpCas9 from *Streptococcus pyogenes* with the goal of enhancing specificity, called eSpCas9 [148] and SpCas9-HF [149], are reported to reduce the probability of mismatched DNA binding. A hybrid enzyme combining the Cas9-nickase and PmCDA1, an activation-induced cytidine deaminase (AID) ortholog, could perform targeted nucleotide substitution [150]. Furthermore, a CRISPR system using a new Cas-related enzyme called Cas13a that targets RNA has also been recently developed [151].

Notably, in the case of GE pigs and embryos, there have been no reports of offtarget mutagenesis as shown by the following papers: [43, 50, 61–64, 69, 74, 77, 78, 80, 86, 100, 105]. This suggests a very low probability of off target-cleavage in GE pigs.

#### **5.3 Multiplexed genome engineering**

The CRISPR/Cas9 system can confer multigene KO in one shot of gene delivery [152, 153]. This property is especially beneficial for the purpose of creating disease model animals, as certain types of diseases are known to be caused by multigene defects. Interestingly, Sakurai et al. [154] demonstrated that at least nine endogenous genes can be knocked out simultaneously through a single shot of cytoplasmic MI of 12 sgRNAs together with Cas9 mRNA into murine zygotes. In pigs, Zhou et al. [61] demonstrated successful generation of PARK2 (*parkin*) and PTEN-induced putative kinase 1 (*PINK1*) double-KO pigs through SCNT with GE fetal fibroblasts after co-transfection of Cas9, *PARK2*-sgRNA, and *PINK1*-sgRNA-expressing vectors by electroporation. The percentage of *PARK2*<sup>−</sup>/<sup>−</sup> /*PINK1*<sup>−</sup>/<sup>−</sup> double-KO cells was up to 38.1%. SCNT using these double-KO cells resulted in the birth of 20 cloned piglets. Of these, four piglets developed normally, and both parkin and PINK1 in those individuals were depleted at the protein level. Estrada et al. [66] also succeeded in obtaining one triple-KO cloned piglet with mutations in *GGTA1* (coding for α-1,3-galactosyltransferase), *CMAH* (coding for cytidine monophosphate-*N*-acetylneuraminic acid hydroxylase) and *β4GalNT2* (coding for β1,4-*N*-acetylgalactosaminyl transferase) after SCNT. Wang et al. [81] generated PARK7 (*DJ-1*)/*parkin*/*PINK1* triple-gene modified pigs using the CRISPR/Cas9 system in one step through direct zygote injection of Cas9 mRNA and three types of sgRNAs. According to Wang et al. [81], of two live-born piglets delivered, one piglet showed biallelic modification of all three genes, and another showed biallelic modification of the *DJ-1* and *PINK1* genes and monoallelic mutation of the *parkin* gene.

#### **5.4 KI**

As shown in **Table 1**, in 2015 successful KI in pigs was reported by several groups. For example, Wang et al. [62] performed MI with *in vivo* fertilized zygotes (derived from colored pigs) using Cas9 mRNA + sgRNA + single-stranded DNA oligonucleotides (ssODN), targeting microphthalmia-associated transcription factor (MITF), a master regulator gene of melanocyte development, and obtained two live-born piglets showing the white coat color phenotype over its entire body. Peng et al. [63] tried to create KI piglets with a MI approach using a circular vector as donor DNA. They designed an sgRNA targeting the starting codon region (including the adjacent 5′ and ATG) and generated a targeted fragment (donor for HR) with the insert flanked by 1-kb HA on both sides. They performed cytoplasmic MI of Cas9 mRNA + *in vitro* synthesized sgRNA + circular vector containing the targeting fragment, and finally obtained 16 live piglets, all of which were found to carry the expected KI allele. Notably, they confirmed expression of human albumin (Alb) protein generated from the KI allele in the plasma of these cloned pigs. This means that expression of a transgene (human Alb as GOI) is possible under the control of an endogenous promoter system (in this case, Alb promoter).

Ruan et al. [60] demonstrated production of GE pigs with successful KI of GOI into the target *Hipp11* (*H11*) locus, which is considered as "safe harbor" genomic locus that allows gene expression without disrupting internal gene function, like the *Rosa26*

**73**

**6. Conclusion**

**5.5 Cas9 pigs**

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

cloned piglet which was later confirmed to show correct targeting.

similar effects (promotion of HDR efficiency) in porcine cells.

locus. They utilized a positive and negative selection method to insert *GFP* into the *pH 11* locus in pig fetal fibroblast cells by electroporation. The targeting donor vector (4.2 kb in size) contains a reporter cassette with *neo* and GFP genes which are flanked by a 0.8-kb HA to the *H11* locus on each side with the diphtheria toxin A (DTA) gene at the 3′ end. Cells were transfected with the linearized donor vector and two expression vectors for sgRNA (targeted to the *H11* locus) and Cas9. After drug selection, they obtained GE cells with successful KI at the *H11* locus with efficiencies up to 54%. Next, they performed SCNT using these correctly targeted clones, and obtained one

Generally, it is believed that HDR-mediated KI is more difficult than NHEJ-based indels. For example, in proliferating human cells, NHEJ has been reported to repair 75% of DSBs, while HDR repaired the remaining 25% [155]. To enhance the HDR efficiency, several approaches are now being attempted. For examples, co-injection of murine zygotes with a mixture containing Cas9 mRNA, sgRNA, template ssODNs and Scr7 (an inhibitor for DNA ligase IV) significantly improved the efficiency of HDR-mediated insertional mutagenesis [156]. Chu et al. [157] also demonstrated usefulness of Scr7 for abolishing NHEJ activity and increasing HDR in both human and mouse cell lines. However, the function of Scr7 in promoting HDR remains controversial. Some researchers demonstrated that Scr7 failed to increase HDR rates in rabbit embryos [158] and porcine fetal fibroblasts [159]. On the contrary, Li et al. [160] demonstrated that Scr7 promoted HDR efficiency in porcine fetal fibroblasts. The same group also showed that other reagents L755507 (β-3 adrenergic receptor agonist) and resveratrol (small-molecule compound found in grapes) also showed

As mentioned previously, the current generation of gene-edited pigs has mostly been produced through either MI or SCNT approaches, which are both expensive and time-consuming. In mice, several Tg lines carrying a Cas9-expressing cassette have been created [154, 161, 162]. These Tg mice are thought to be useful animals for direct *in vivo* genome editing experiments, because successful delivery of the expression vectors of sgRNAs alone or RNA itself into selected tissues caused generation of genome-edited tissues. For example, Platt et al. [161] demonstrated that *in vivo* viral administration of Kirsten rat sarcoma viral oncogene homolog (*Kras*), transformation related protein 53 (*Trp53*), and serine/threonine-protein kinase 11 (*Stk11*)-gRNAs to the Cas9-expressing

line caused lung carcinomas within a short period. This suggests that if a Cas9-

This Cas9 pig line will be used for various studies as indicated above.

expressing pig is produced, it will provide an easy and efficient way to produce genetic modifications, which should substantially facilitate studying gene functions, modeling human diseases, and promoting agricultural productivity. Based on this concept, Wang et al. [163] first produced Cre-dependent Cas9-expressing pigs to enable efficient *in vivo* genome editing. They first transfected the linear-targeting donor containing Credependent Cas9-expression cassette and TALEN plasmids directed to *Rosa26* locus into porcine fetal fibroblasts and finally selected clones carrying KI cassette. These clones were then used for SCNT to produce cloned GE piglets. They showed that cells isolated from several organs of GE pigs exhibited Cre-induced activation of Cas9 expression.

Because pigs are similar to humans in physiological, anatomical, and genetic aspects, they are now seen as a leading animal model for biomedical research. Recent

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

#### *Recent Advance in Genome Editing-Based Gene Modification in Pigs DOI: http://dx.doi.org/10.5772/intechopen.88022*

locus. They utilized a positive and negative selection method to insert *GFP* into the *pH 11* locus in pig fetal fibroblast cells by electroporation. The targeting donor vector (4.2 kb in size) contains a reporter cassette with *neo* and GFP genes which are flanked by a 0.8-kb HA to the *H11* locus on each side with the diphtheria toxin A (DTA) gene at the 3′ end. Cells were transfected with the linearized donor vector and two expression vectors for sgRNA (targeted to the *H11* locus) and Cas9. After drug selection, they obtained GE cells with successful KI at the *H11* locus with efficiencies up to 54%. Next, they performed SCNT using these correctly targeted clones, and obtained one cloned piglet which was later confirmed to show correct targeting.

Generally, it is believed that HDR-mediated KI is more difficult than NHEJ-based indels. For example, in proliferating human cells, NHEJ has been reported to repair 75% of DSBs, while HDR repaired the remaining 25% [155]. To enhance the HDR efficiency, several approaches are now being attempted. For examples, co-injection of murine zygotes with a mixture containing Cas9 mRNA, sgRNA, template ssODNs and Scr7 (an inhibitor for DNA ligase IV) significantly improved the efficiency of HDR-mediated insertional mutagenesis [156]. Chu et al. [157] also demonstrated usefulness of Scr7 for abolishing NHEJ activity and increasing HDR in both human and mouse cell lines. However, the function of Scr7 in promoting HDR remains controversial. Some researchers demonstrated that Scr7 failed to increase HDR rates in rabbit embryos [158] and porcine fetal fibroblasts [159]. On the contrary, Li et al. [160] demonstrated that Scr7 promoted HDR efficiency in porcine fetal fibroblasts. The same group also showed that other reagents L755507 (β-3 adrenergic receptor agonist) and resveratrol (small-molecule compound found in grapes) also showed similar effects (promotion of HDR efficiency) in porcine cells.

#### **5.5 Cas9 pigs**

*Reproductive Biology and Technology in Animals*

**5.3 Multiplexed genome engineering**

percentage of *PARK2*<sup>−</sup>/<sup>−</sup>

targets RNA has also been recently developed [151].

/*PINK1*<sup>−</sup>/<sup>−</sup>

the *DJ-1* and *PINK1* genes and monoallelic mutation of the *parkin* gene.

an endogenous promoter system (in this case, Alb promoter).

enzyme combining the Cas9-nickase and PmCDA1, an activation-induced cytidine deaminase (AID) ortholog, could perform targeted nucleotide substitution [150]. Furthermore, a CRISPR system using a new Cas-related enzyme called Cas13a that

Notably, in the case of GE pigs and embryos, there have been no reports of offtarget mutagenesis as shown by the following papers: [43, 50, 61–64, 69, 74, 77, 78, 80, 86, 100, 105]. This suggests a very low probability of off target-cleavage in GE pigs.

The CRISPR/Cas9 system can confer multigene KO in one shot of gene delivery [152, 153]. This property is especially beneficial for the purpose of creating disease model animals, as certain types of diseases are known to be caused by multigene defects. Interestingly, Sakurai et al. [154] demonstrated that at least nine endogenous genes can be knocked out simultaneously through a single shot of cytoplasmic MI of 12 sgRNAs together with Cas9 mRNA into murine zygotes. In pigs, Zhou et al. [61] demonstrated successful generation of PARK2 (*parkin*) and PTEN-induced putative kinase 1 (*PINK1*) double-KO pigs through SCNT with GE fetal fibroblasts after co-transfection of Cas9, *PARK2*-sgRNA, and *PINK1*-sgRNA-expressing vectors by electroporation. The

double-KO cells resulted in the birth of 20 cloned piglets. Of these, four piglets developed normally, and both parkin and PINK1 in those individuals were depleted at the protein level. Estrada et al. [66] also succeeded in obtaining one triple-KO cloned piglet with mutations in *GGTA1* (coding for α-1,3-galactosyltransferase), *CMAH* (coding for cytidine monophosphate-*N*-acetylneuraminic acid hydroxylase) and *β4GalNT2* (coding for β1,4-*N*-acetylgalactosaminyl transferase) after SCNT. Wang et al. [81] generated PARK7 (*DJ-1*)/*parkin*/*PINK1* triple-gene modified pigs using the CRISPR/Cas9 system in one step through direct zygote injection of Cas9 mRNA and three types of sgRNAs. According to Wang et al. [81], of two live-born piglets delivered, one piglet showed biallelic modification of all three genes, and another showed biallelic modification of

As shown in **Table 1**, in 2015 successful KI in pigs was reported by several groups. For example, Wang et al. [62] performed MI with *in vivo* fertilized zygotes (derived from colored pigs) using Cas9 mRNA + sgRNA + single-stranded DNA oligonucleotides (ssODN), targeting microphthalmia-associated transcription factor (MITF), a master regulator gene of melanocyte development, and obtained two live-born piglets showing the white coat color phenotype over its entire body. Peng et al. [63] tried to create KI piglets with a MI approach using a circular vector as donor DNA. They designed an sgRNA targeting the starting codon region (including the adjacent 5′ and ATG) and generated a targeted fragment (donor for HR) with the insert flanked by 1-kb HA on both sides. They performed cytoplasmic MI of Cas9 mRNA + *in vitro* synthesized sgRNA + circular vector containing the targeting fragment, and finally obtained 16 live piglets, all of which were found to carry the expected KI allele. Notably, they confirmed expression of human albumin (Alb) protein generated from the KI allele in the plasma of these cloned pigs. This means that expression of a transgene (human Alb as GOI) is possible under the control of

Ruan et al. [60] demonstrated production of GE pigs with successful KI of GOI into the target *Hipp11* (*H11*) locus, which is considered as "safe harbor" genomic locus that allows gene expression without disrupting internal gene function, like the *Rosa26*

double-KO cells was up to 38.1%. SCNT using these

**72**

**5.4 KI**

As mentioned previously, the current generation of gene-edited pigs has mostly been produced through either MI or SCNT approaches, which are both expensive and time-consuming. In mice, several Tg lines carrying a Cas9-expressing cassette have been created [154, 161, 162]. These Tg mice are thought to be useful animals for direct *in vivo* genome editing experiments, because successful delivery of the expression vectors of sgRNAs alone or RNA itself into selected tissues caused generation of genome-edited tissues. For example, Platt et al. [161] demonstrated that *in vivo* viral administration of Kirsten rat sarcoma viral oncogene homolog (*Kras*), transformation related protein 53 (*Trp53*), and serine/threonine-protein kinase 11 (*Stk11*)-gRNAs to the Cas9-expressing line caused lung carcinomas within a short period. This suggests that if a Cas9 expressing pig is produced, it will provide an easy and efficient way to produce genetic modifications, which should substantially facilitate studying gene functions, modeling human diseases, and promoting agricultural productivity. Based on this concept, Wang et al. [163] first produced Cre-dependent Cas9-expressing pigs to enable efficient *in vivo* genome editing. They first transfected the linear-targeting donor containing Credependent Cas9-expression cassette and TALEN plasmids directed to *Rosa26* locus into porcine fetal fibroblasts and finally selected clones carrying KI cassette. These clones were then used for SCNT to produce cloned GE piglets. They showed that cells isolated from several organs of GE pigs exhibited Cre-induced activation of Cas9 expression. This Cas9 pig line will be used for various studies as indicated above.

#### **6. Conclusion**

Because pigs are similar to humans in physiological, anatomical, and genetic aspects, they are now seen as a leading animal model for biomedical research. Recent advances in genome editing technology have led to accelerated production of GE pigs within a relatively short time period, which is beneficial due to cost savings in propagation of GE animals and maintaining animals for breeding. Production of GE pigs can be largely categorized into two approaches, so-called MI/EP-mediated production of GE zygotes and SCNT using GE cells as the SCNT donor. There are advantages and drawbacks for both these approaches. For example, the former is simpler, more convenient, and cost-effective than the latter. However, the available genetic background is limited. In this context, the latter is beneficial for the flexibility of choosing any type of genetic background, because the genetic background of SCNT-derived cloned pigs is determined by that of donor cells used for SCNT. Unfortunately, the efficiency of SCNT is extremely low at present. MI/EP with SCNT-treated embryos may compensate for these disadvantages associated with MI/EP or SCNT-mediated production of GE piglets, if the efficiency of SCNT is greatly improved in future.

#### **Acknowledgements**

We thank Shogo Matsunaga for their support in the GENTEP-related experiment, shown in **Figures 3** and **4**. This study was partly supported by a grant (no. 19K06372 for Masahiro Sato; nos. 25450475 and 16K08085 for Kazuchika Miyoshi; no. 18K09839 for Emi Inada; no. 17H04412 for Issei Saitoh; no. 16H05176 for Akihide Tanimoto) from the Ministry of Education, Science, Sports, and Culture, Japan.

#### **Conflicts of interest**

The founding sponsors had no role in the design of the study, collection, analyses, or interpretation of data, writing of the manuscript, and decision to publish the results.

#### **Author contributions**

Masahiro Sato designed the study and drafted the manuscript; Kazuchika Miyoshi and Hiroaki Kawaguchi involved in the GENTEP-related experiment; Emi Inada and Issei Saitoh critically revised the manuscript; Akihide Tanimoto supervised the manuscript.

**75**

**Author details**

Masahiro Sato1

and Akihide Tanimoto6

University, Kagoshima, Japan

Niigata University, Niigata, Japan

provided the original work is properly cited.

\*, Kazuchika Miyoshi<sup>2</sup>

Sciences, Kagoshima University, Kagoshima, Japan

Dental Sciences, Kagoshima University, Kagoshima, Japan

Kagoshima University, Kagoshima, Japan

, Hiroaki Kawaguchi3

1 Section of Gene Expression Regulation, Frontier Science Research Center,

2 Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima

Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan

\*Address all correspondence to: masasato@m.kufm.kagoshima-u.ac.jp

3 Department of Hygiene and Health Promotion Medicine, Graduate School of

4 Department of Pediatric Dentistry, Graduate School of Medical and Dental

5 Division of Pediatric Dentistry, Graduate School of Medical and Dental Science,

6 Department of Pathology, Division of Oncology, Graduate School of Medical and

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Emi Inada4

, Issei Saitoh5

*Recent Advance in Genome Editing-Based Gene Modification in Pigs*

*DOI: http://dx.doi.org/10.5772/intechopen.88022*

*Recent Advance in Genome Editing-Based Gene Modification in Pigs DOI: http://dx.doi.org/10.5772/intechopen.88022*

#### **Author details**

*Reproductive Biology and Technology in Animals*

greatly improved in future.

**Acknowledgements**

**Conflicts of interest**

**Author contributions**

supervised the manuscript.

Culture, Japan.

results.

advances in genome editing technology have led to accelerated production of GE pigs within a relatively short time period, which is beneficial due to cost savings in propagation of GE animals and maintaining animals for breeding. Production of GE pigs can be largely categorized into two approaches, so-called MI/EP-mediated production of GE zygotes and SCNT using GE cells as the SCNT donor. There are advantages and drawbacks for both these approaches. For example, the former is simpler, more convenient, and cost-effective than the latter. However, the available genetic background is limited. In this context, the latter is beneficial for the flexibility of choosing any type of genetic background, because the genetic background of SCNT-derived cloned pigs is determined by that of donor cells used for SCNT. Unfortunately, the efficiency of SCNT is extremely low at present. MI/EP with SCNT-treated embryos may compensate for these disadvantages associated with MI/EP or SCNT-mediated production of GE piglets, if the efficiency of SCNT is

We thank Shogo Matsunaga for their support in the GENTEP-related experiment, shown in **Figures 3** and **4**. This study was partly supported by a grant (no. 19K06372 for Masahiro Sato; nos. 25450475 and 16K08085 for Kazuchika Miyoshi; no. 18K09839 for Emi Inada; no. 17H04412 for Issei Saitoh; no. 16H05176 for Akihide Tanimoto) from the Ministry of Education, Science, Sports, and

The founding sponsors had no role in the design of the study, collection, analyses, or interpretation of data, writing of the manuscript, and decision to publish the

Masahiro Sato designed the study and drafted the manuscript; Kazuchika Miyoshi and Hiroaki Kawaguchi involved in the GENTEP-related experiment; Emi Inada and Issei Saitoh critically revised the manuscript; Akihide Tanimoto

**74**

Masahiro Sato1 \*, Kazuchika Miyoshi<sup>2</sup> , Hiroaki Kawaguchi3 , Emi Inada4 , Issei Saitoh5 and Akihide Tanimoto6

1 Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan

2 Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima, Japan

3 Department of Hygiene and Health Promotion Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan

4 Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan

5 Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan

6 Department of Pathology, Division of Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan

\*Address all correspondence to: masasato@m.kufm.kagoshima-u.ac.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Section 2

Other Species
