Reproductive Techniques in Terrestrial Animals

### **Chapter 2**

## Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models

*Feda S. Aljaser*

### **Abstract**

The development in cryobiology in animal breeding had revolutionized the field of reproductive medicine. The main objective to preserve animal germplasm stems from variety of reasons such as conservation of endangered animal species, animal diversity, and an increased demand of animal models and/or genetically modified animals for research involving animal and human diseases. Cryopreservation has emerged as promising technique for fertility preservation and assisted reproduction techniques (ART) for production of animal breeds and genetically engineered animal species for research. Slow rate freezing and rapid freezing/vitrification are the two main methods of cryopreservation. Slow freezing is characterized by the phase transition (liquid turning into solid) when reducing the temperature below freezing point. Vitrification, on the other hand, is a phenomenon in which liquid solidifies without the formation of ice crystals, thus the process is referred to as a glass transition or ice-free cryopreservation. The vitrification protocol applies high concentrations of cryoprotective agents (CPA) used to avoid cryoinjury. This chapter provides a brief overview of fundamentals of cryopreservation and established methods adopted in cryopreservation. Strategies involved in cryopreserving germ cells (sperm and egg freezing) are included in this chapter. Last section describes the frontiers and advancement of cryopreservation in some of the important animal models like rodents (mouse and rats) and in few large animals (sheep, cow etc).

**Keywords:** cryopreservation, fertility, reproduction, cryobiology

### **1. Introduction**

Ever since the human evolution and civilization, human has been exploiting animals either for food, transport and or as companions. The use of animals in biomedical and behavioral research has greatly increased scientific knowledge and has benefitted human health enormously. Tremendous advancement took place in the field of medical sciences with the usability of animals for experimental research. Currently, around 75–100 million vertebrates are used annually in research and testing [1]. The most frequently used animals are mice and rats that constitute approximately 95% of experimental animals; mouse being the most commonly used animal in biomedical research [1]. Animal models are used in research for wider

understanding of vital physiological processes in human and animals. Animal models are also useful in investigating various diseases including metabolic disorders such as diabetes, cardiovascular disease (CVD), disorders in reproductive endocrinology, infertility, cancer and infectious diseases [2–7]. Human lives endangered due to organ failure were restored after successful organ transplantation accomplished in animal models. Optimization of cryopreservation protocols will significantly facilitate organ transplantation and/or replacement. In addition, animals like dogs, pigs, cats, sheep, non-human primates (NHP) and fish are widely used for genetic and physiological studies in human health and disease [8]. The chapter is focused on the fundamentals of cryobiology and strategies in male (sperm) and female fertility (oocyte and ovarian tissue) cryopreservation. The last section is focused on the frontiers in cryopreservation of most widely used animal models like rodents and higher animals used in biomedical research and toxicological studies.

### **1.1 Necessity to cryopreserve animal's genetic resources?**

Large numbers of animal breeds worldwide are either extinct or endangered with few at the verge of extinction. Hence, it is crucial to develop and apply rescue strategies to ensure survival of these species for the future. One way is to preserve the genetic resources or the germplasm of these species for their maintenance and future development. Genetic diversity is another threat resulting from animal husbandry errors that can result in genetic drift of existing colonies and genetic contamination of lines. Furthermore, weather related natural disaster is a major threat to animal husbandry and vivarium, especially in case of experimental animals that require special care in breeding and rearing colonies. Cattle and breeding industries are modified for the large-scale production of the animal species. There is an urgent need to improve the efficiency and sustainability of producing animals for food in the face of the everincreasing world population. Improved understanding of mechanisms and challenges of reproductive technologies are vital for improving the viability of the livestock industry [9]. Hence, one solution to preserve animal species is by freezing. Cryopreservation has emerged as the most efficient and compatible method for freezing human and animal genetic resources [10]. Cryobiology is an integrated study of various biological and physical sciences. Semen, embryos, oocytes, somatic cells, nuclear DNA, and other types of biological material such as blood, serum, and tissue, can be stored using cryopreservation, in order to preserve genetic materials or for other applications [8, 11]. The primary benefit of cryopreservation is the ability to save germplasms for extended periods of time, and thus maintaining the genetic diversity of a species or a breed [12]. Also, germplasm of genetically engineered animals (GEA) and cell lines of various species can be preserved by cryopreservation method. Gene banks/cryobanks are established and contain repository of cryopreserved genetic resources to regenerate a particular population in the future [13–15]. Sperm cryopreservation has been successfully applied in various fields to benefit the mankind and animals. Of prime significance, assisted reproduction technology (ART), the forefront in infertility treatment today might be inconceivable without the efficient cryopreservation techniques.

### **1.2 History behind the discovery**

*Spallanzani's observation* in the beginning of eighteenth century snow cooling the sperm was a breakthrough in the field of biology. Sperm was found to maintain

### *Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models DOI: http://dx.doi.org/10.5772/intechopen.101750*

motility and viability even when exposed to cold temperature conditions [16]. Later, in the nineteenth century a major breakthrough occurred with the successful cattle insemination performed using cryopreserved samples. A successful protocol for sperm freezing and storage at low temperatures (−79°C) was developed by Polge *et al.,* [17]. Spermatozoon was the first mammalian cell to be successfully frozen [18]. This significant success in sperm freezing was associated with the discovery of glycerol as a cryoprotectant by Polge *et al* in 1949 [17]. Since 1970 and till date, cryopreservation would not be successful preservation procedure in reproductive medicine without the discovery of plastic straws and cryotubes that tolerate extreme low temperature used in packaging sperm. Mouse embryo freezing was reported using dimethylsulfoxide (DMSO) as a cryoprotectant in 1972 after successfully cryopreserving spermatozoa [19].

### **2. Art of freezing life**

### **2.1 Cryopreservation methods**

Method of cryopreservation and recovery involves following steps. Initially, cells are treated with cryoprotective agents (CPAs) to prevent cryoinjury/damage. Later, cells are cooled in a controlled manner at subzero temperatures, at which the metabolic processes of the cell stops. The recovery of the cells follows a reversed procedure; cells are rapidly thawed and then the CPAs are gradually removed. The viability of the cryopreserved sample is enhanced with the use of appropriate CPA type and concentration and the appropriate rate of freezing [10].

The two basic methods of cryopreservation include the conventional slow freezing (SF) and the rapid freezing method, known as vitrification (Vit). These protocols require different concentrations of CPAs and apply different cooling rates. **Figure 1** illustrates the two methods of cryopreservation.

### *2.1.1 Slow freezing*

Slow freezing involves progressive cooling of sample over a period of 2–4 h either manually or automatically using a semi programmable freezer. This method was developed by Behrman and Sawada [20]. In SF, a phase transition occurs from liquid to solid on temperatures below freezing point. Slow-cooling protocols involve the use of <1.0 M of cryoprotective agents (CPAs), such as glycerol or dimethyl sulphoxide (DMSO), which have minimal toxicity at lower temperatures with the use of a high-cost controlled rate freezer or a benchtop portable freezing container [21].

Main advantage of SF is the reduced risk of contamination during the procedure, without the need of highly skilled professionals. However, SF has many disadvantages. It is time consuming and expensive. There is a high risk of cryoinjury due to the formation of extracellular ice. Although, slow cooling considered a successful method, the success rate is considerably low and might not be suitable for all kinds of cells and tissues [22]. SF is a commonly used method for preservation of animal germplasm for majority of farm animals like sheep, cow, zebrafish etc. [23, 24]. Though with certain drawbacks, nonetheless SF was found more efficient method to cryopreserve ovine embryos in comparison to vitrification [25].

### **Figure 1.**

*Comparison of slow freezing and vitrification method.*

### *2.1.2 Vitrification or ice-free cryopreservation*

To circumvent the process of ice crystallization, cost and duration for cryopreservation in SF, an alternative technique called rapid freezing or vitrification (Vit) was developed. Vit is an ultrarapid cooling method with high cooling rate, which enables putting cells at cryogenic temperatures, forming what is known as ice-front status, while avoiding ice crystallization, hence also termed ice-free cryopreservation. Vitrification is now the recommended protocol for embryos and cells freezing. The process includes cooling the cells or tissue to cryogenic temperatures using liquid nitrogen, after their exposure to high concentrations of CPAs, with subsequent rapid cooling to avoid ice nucleation [11]. The cell suspensions are transformed directly from the aqueous phase to a glass state upon exposure to LN2. High CPA concentration and higher cooling and warming rates eliminates ice formation [26]. Several factors like viscosity or thickness of the sample, cooling and warming rates, sample volume, CPA type and concentration have considerable effect on the process. Thus, a balance of all the important factors is required for successful vitrification. Vitrification is of two types—equilibrium and nonequilibrium. Formulation of multimolar CPA mixtures and their injection into the cell suspensions is termed as Equilibrium vitrification. Non-equilibrium vitrification includes the use of carriers like plastic straws or cryoloops for obtaining minimum drop volume and carrier-free systems that employs higher freezing rate and lower CPA mix concentration. In comparison to slow freezing, vitrification is more advantageous as is associated with decreased risk of cryoinjury, thereby ensuring sufficiently higher cell survival rate [11]. To note, there is no

*Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models DOI: http://dx.doi.org/10.5772/intechopen.101750*

universal protocol followed for the animal species. However, germ cells from species like mouse ovary [27], fish embryos [28], ovarian sheep [29] and many other species has been cryopreserved by vitrification [24].

### **2.2 Other emerging cryopreservation techniques**

### *2.2.1 Controlled vitrification by liquidus tracking (LT)*

Liquidus tracking (LT) is a slow and controlled vitrification protocol with gradual increase in the cryoprotectant concentration simultaneously with continuous decrease of the temperature at subzero ranges in a specified rate. With LT, recovery and restoration of chondrocytes was achieved successfully from cryopreserved articular cartilage [30] and promising results has been reported in case of ovarian tissue cryopreservation [31]. The principle of LT is the dynamic control of CPA concentrations, throughout the cooling process, in order to maintain the cell just above its freezing point at all times, without the formation of ice [11]. An example, Ovarian Tissue Cryopreservation (OTC) by LT has been reported successful in restoration of ovarian function in sheep model [29].

### *2.2.2 Laser pulse vitrification*

Fowler and Toner proposed the applicability of laser light in cryopreservation process. A successful recovery of rapidly frozen red blood cells by vitrification was achieved without the use of CPAs. Principally, laser targets only the intracellular ice causing it to melt and resolidify into glassy state. After thawing and use of laser light, around 80% of cells treated remained viable [32]. In an attempt, Jin *et al.,* [33] reported full survivals of mouse oocytes after vitrification in 3-fold diluted media and ultra-rapid warming by an IR laser pulse [33].

### *2.2.3 Isochoric and hyperbaric cryopreservation*

New approach in the cryopreservation method is freezing under pressure. Previous method discussed above employed constant-standard pressure (isobaric) conditions near 1 atm of pressure. Isochoric (constant-volume) cooling provides means to significantly lower nonfrozen storage temperatures without any or with only minimum requirements for CPAs, achieving greater metabolic reduction without injury associated with freezing, CPA toxicity, or increased amounts of osmotic solutes. The isochoric cryopreservation is a two-phase equilibrium process, in which ice and liquid exist simultaneously at equilibrium under constant temperature and volume, while hyperbaric cryopreservation the solution is maintained in a single phase as liquid, the survival rate is however low in this method [34]. RBCs were successfully cryopreserved by this method [35]. Despite multiple attempts, scientists have not been able to cryopreserve and restore normal functions of complex bio-samples, such as mammalian tissues and organs.

### **2.3 Fundamentals of cryopreservation and cryoinjury**

Prior to the understanding of the role of cryoprotective agents (CPAs), the impact of subzero temperatures on healthy tissues and basic principles in cryobiology must be knowledged. As known, water is one of the most essential elements present in

every cell, tissue, and organ of the living organisms on earth. It constitutes around 80% of tissue mass [36]. On lowering temperature, water undergoes phase transition (liquid to solid) and results in ice crystallization. The formation of intracellular ice cause damage to cellular structure and its function consequently leads to cryoinjury. Freezing can cause two types of harmful effects on cells. The formation of ice crystals damages the cell membrane and thus regain of structurally intact cells on thawing would be difficult. Further, ice formation increases the solute concentration leading to osmotic imbalance and cellular damage. To minimize or to mitigate these effects, two protective actions viz. selection of effective cryoprotectant, and appropriate cooling and thawing rates must be undertaken.

### *2.3.1 Cryoprotective agents (CPAs)*

The discovery of CPA and its role in reducing cryoinjury was a significant step in cryopreservation success. Biological acceptability, cell penetration, low toxicity, are some of the properties, a CPA should possess. As mentioned in previous section, best survival rate of cells and tissues depends on the optimization of factors like cooling rate, warming rate, sample volume. CPA concentration is a major factor influencing the success of the cryopreservation [37]. Based on their penetrating capabilities through cell membrane, CPAs are classified into two categories- membrane permeable/permeating and membrane impermeable/non permeating.

### *2.3.1.1 Permeating CPAs*

Permeating CPAs are smaller sized (typically less than 100 daltons), and amphiphilic in nature [38]. Owing to these properties these molecules can penetrate through the cell membrane easily, tend to equilibrate within the cytoplasm and replace the intracellular water in order to avoid excessive dehydration. Henceforth, they protect the cell from intracellular ice formation (IIF) and salt accumulation. Examples of CPAs in this category are: glycerol (the first agent discovered), dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propanediol (propylene glycol) [39]. The protective role of CPAs is due to hydrogen bonding with water molecules, and lowering the freezing point of water. As a result, less water molecules are available to interact with themselves to form critical nucleation sites required for crystal formation [40]. To minimize toxicity, vitrification mixtures are often added in a stepwise fashion at temperatures near 0°C. Addition of permeating agents prevent the formation of ice and permit cell storage at supercool temperatures. Besides vitrifying, few CPAs, example DMSO have properties to enhance the cell permeability in a dose dependent manner. DMSO of about 5% is reported to increase permeability by reducing the thickness of the cell membrane. DMSO at 10% concentration is more effective and commonly used as it induces water pore formation in biological membranes. Intracellular water can thus easily be replaced by CPAs to promote vitrification. At higher, toxic concentrations (40%) however, lipid bilayers begin to disintegrate [41]. Thus selection of appropriate CPA concentration is vital for maintaining structural integrity and viability after freezing.

### *2.3.1.2 Non-permeating agents*

Unlike permeating, non-permeating agents are covalently linked dimers, trimers or polymers with a larger size. They cannot pass through the cell membrane and exert

### *Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models DOI: http://dx.doi.org/10.5772/intechopen.101750*

their protective effect extracellularly. Most commonly used non-penetrating CPAs are polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), raffinose, sucrose, and trehalose [42, 43]. The mode of action of non-permeating agents is similar to permeating agents by controlling osmolarity but works extracellularly and at a lower degree.

List of different permeating and non-permeating cryoprotective agents used in gamete and embryo cryopreservation in different species is listed in **Table 1**.


### **Table 1.**

*List of different permeating and non-permeating cryoprotectants used in assisted reproductive technologies.*

Besides these commonly used CPAs, protein like sericin from silkworm and small antifreeze proteins derived from marine teleosts or fishes have also garnered attention as CPAs in cryobiology [11].

### *2.3.2 Cooling and thawing rates*

Choice of appropriate cooling and thawing rates is another vital step for a successful cryopreservation. Mazur [44] has previously demonstrated the significant correlation between cooling rates and survivability of various cells [44]. The cooling rate was directly proportional to intracellular ice formation and inversely proportional to survivability among various cells. **Figure 2** illustrates the survival of several cell types after cryopreservation in relation to the cooling rate. All cells exhibited a characteristic inverted U-shaped curve indicating that the survival rate increases with an increase in cooling rate upto a point after which the survival gradually decreases. Larger cells dehydrate slowly compared to smaller cells. Hence, in the light of this, rates of cooling and thawing should be adjusted. With an exception to larger cells, a cooling rate of approximately 1°C/min is often recommended. Controlled rate freezers that modulate chamber temperatures are used for this purpose. Following cryopreservation, cells are stored for future thawing and appropriate use.

Unlike cooling rate, thawing rate has been given inadequate attention. Nevertheless, it is advisable to warm or thaw cells rapidly to prevent recrystallization of ice [47]. This can be explained thermodynamically as vitrified state is quasi-stable and can change into a lower energy crystal structure on thawing. Currently, to achieve maximum viability it is suggested to transport cryovials on vapor LN2 and warmed rapidly in a 37°C water bath for 90–120 s [39]. Decrease in viability after post thaw recovery is inevitable, no matter how well the cells were stored and thawed. Care should be taken to remove dead cells timely using density media like Ficol or other methods from the recovered cells to increase the viability of recovered cell prior to use. Density gradients can be utilized to increase viable cell density although this method often involves exposing cells to additional, potentially-harmful centrifugation. Current strategies for identifying cells that remain viable after preservation utilize organic fluorophores, and dyes.

### **Figure 2.**

*An inverted U shape curve demonstrating relationship between cooling rate and cell survival (adapted from article by Cipri et al., [45]; figure created originally by Critser and Mobraaten [46] in ILAR Journal).*

*Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models DOI: http://dx.doi.org/10.5772/intechopen.101750*

### *2.3.3 Recent advances in post-cryo cell recovery*

The complete cryopreservation procedure is associated with great efforts to maintain cell viability and function. However, some of the cryopreserved cells often demonstrate decreased viability following thawing. Therefore, it is essential to remove dead cells and increase the viability of live cells. Currently, organic fluorophores are employed for viable cells selection. Viable cell selection using this method is commonly practiced in ART in animal reproduction example semen/spermatozoa of bull, buffalo, rabbit, alpaca, stallion/horse and many other species [48–50]. The earlier practice to achieve this, involved the use of density gradient. Although a little expensive, advanced technologies like magnetic affinity cell separation (MACS), and fluorescence activated cell sorting (FACS) appeared beneficial [51, 52]. Cryosurvival and recovery of sperm in species like stallion, bovine etc. using FACS has been investigated [53, 54]. New strategies like the introduction of nano-science had revolutionized the field of cryobiology and forwarded it to more higher stages as nanoparticle mediated cell sorting is non-destructive and more beneficial than FACS [55].

### **2.4 Sperm cryopreservation**

After the successful live birth with 21 years cryopreserved sperm sample, sperm viability in cryopreserved sample was evident [56]. Currently, sperm cryopreservation as a method of fertility preservation had gained tremendous significance and applicability in human and animals [57, 58]. Sperm survival rate was found to increase with glycerol as CPAs and increasing concentration of glycerol added at constant cooling rate for long term storages [59]. Sperm freezing can be done by one of the main two established methods; slow freezing or ultra-rapid freezing [60, 61]. Owing to the deleterious effects of SF on sperm physiochemical activity and motility, the rapid freezing approach was suggested to be the potential solution to preserve cells without allowing the nucleation of ice crystals. Currently, ultra-rapid freezing adopted ensures both intra and extracellular vitrification thus improving sperm survival [62].

### **2.5 Female fertility cryopreservation**

Like sperm, female germ cells can be frozen by cryopreservation. Fertility preservation in female cancer survivors is a major concern in oncology and assisted conception. Fertility in females can be preserved by freezing egg either embryo, oocytes and oocytes within ovarian tissue. The results with oocyte cryopreservation were unsatisfactory. Ovarian tissue cryopreservation on the other side have attracted the interest of the medical and scientific communities. Cryopreservation protocols for oocyte and ovarian tissue are discussed briefly below.

### *2.5.1 Oocyte cryopreservation*

For oocyte cryopreservation, currently acknowledged methods are SF using equilibrium freezing and Vit/non-equilibrium protocols. In cryopreserving oocytes by SF method, the protocol involves gradual cooling of the specimen to lower temperature (−150°C)with controlled slow cooling rates in presence of low concentration of DMSO (1.5 M) plus non-permeating sugars like sucrose or trehalose at 0.3 M concentration. Specimens are stored at −196°C in LN2. The survival rate achieved by SF remains relatively unsatisfactory. Evidence from earlier investigations indicates survival rates plateau around 70–80% with this method [63]. Hence, the recommended method for cryopreservation of oocytes is vitrification. Initially, oocytes are equilibrated with a solution containing PEG and DMSO at 7.5% v/v for 5–15 min. Prior to storing the cells in LN2, the cells are exposed to vitrification media (PEG and DMSO-15% v/v, -and sucrose (0.5 M) for a minute and stored. For thawing, CPAs are gradually removed to avoid ice crystal formation. Later, the cells are revived following incubation in culture medium [64]. Intriguing results were obtained in systematic analysis conducted by Arav and Natan. Vitrified oocytes were reported to have higher oocyte survival and fertility rates compared to slow-cooled oocytes. Furthermore, no differences were observed in pregnancy rate, formation of top quality embryo and fertilization between vitrified and fresh oocytes thus strongly signifying vitrification as the superior procedure for the oocytes cryopreservation [65]. During the past few years, a significant progress has been made in the cryopreservation of oocytes of different species using new vitrification methods. High rates of survival and development after solid-surface vitrification have been reported for *in vitro* matured oocytes from cows [66] and goats [67].

### *2.5.2 Ovarian tissue cryopreservation (OTC)*

Parrot's study, which resulted in first mice offspring was developed following ovarian tissue cryopreservation (at −79°C) and isografting was a breakthrough in the early 1960s [68, 69]. Later, with further discoveries of CPAs like DMSO, propanediol, and ethylene glycol, cryopreservation methods gradually improved. Like oocyte, ovarian tissues are also preserved by SF and Vit method, specially ovarian cortical tissue from mammals [70]. After the advent of Vit for oocyte cryopreservation and in comparison, to SF, ovarian tissue vitrification is now considered promising for ovarian cortical tissue cryopreservation [71, 72]. Ovarian tissue cryopreservation is successfully accomplished in many animal species like mice [68], ewe [73], cow including human [74] and other species.

**Table 2** shows the comparison between SF and vitrification protocol for ovarian tissue cryopreservation. Vitrification was suggested to have several advantages over


### **Table 2.**

*Comparison between SF and vitrification protocol.*

the conventional SF in characteristics reviewed. However, SF remains the standard clinical method until further reports show improved success rate for Vit to be applied in human clinical use over SF. Currently, the main purpose for OTC is fertility preservation known so far, especially in young women diagnosed with cancer or some genetic disease that destroys ovarian reserve. Access to immature oocytes from antral follicles and restoration of organ function have evoked new perspectives in utility of OTC for social reasons besides medical use.

### **2.6 Applications of fertility preservation**

Germ cell cryopreservation certainly had emerged as effective method for long term fertility preservation in the field of reproductive medicine in both human and animals. Germ cells cryopreservation would be applicable in restoring fertility in animals and humans and preservation of endangered animal species. Over a decade there has been increase in production of GEA models from cryopreserved animal genetic resources for disease investigations. The practice of genetic engineering has increased the number of mouse and rat lines to tenfold the actual number. Currently, sperm cryopreservation is a fundamental technique in assisted reproduction technology (ART) like artificial insemination (AI), *in vitro* fertilization (IVF), or intra-cytoplasmic sperm injection (ICSI). AI has relatively been the most important practice contributing to the advancement of animal production. Many advantages of AI are enhanced when semen is cryopreserved and stored for extended periods. Several species such as mice, rabbits, pigs, goats, cows, and sheep has been successful reproduced adopting egg and ovarian tissue cryopreservation [66–68, 73, 74]. After successful animal experimentation using cryopreserved germ cells, utility of fertility preservation is gradually extended to human. At present, cryopreservation of human oocytes and sperm has been carried out for medical or social reasons. Infertility in couples is solved to a considerable extend. Cancer patients who are at risk of fertility loss either due to radio or chemotherapy and women who wish to conceive in later ages can benefit from the cryopreservation technology and fertility restoration.

### **2.7 Frontiers in cryopreservation in animal models**

The birth of mouse from 50 years old cryopreserved embryo had revolutionized the field of animal reproduction. Later, to achieve greater reproductive outcomes, cryopreservation protocols have been continuously refined over the years. In the given section, few of the important animal models has been discussed.

### *2.7.1 Mouse*

Mouse is one of the most commonly used animal model for research. In early 1990s, the first attempt in freezing mouse was a grand success. Later, cryopreservation of mouse spermatozoa resulted in production of a large number of mouse inbred and hybrid strains [75, 76]. There is a growing demand for the production of genetically modified mouse strains since mouse has become the most profound model system to investigate the genetics and pathogenetics of human diseases. Moreover, knock-out projects started in Europe and USA over a decade. As life maintenance of the growing number of mice is difficult and uneconomical in animal laboratories, germplasm cryopreservation provides a valuable means of maintaining transgenic mouse strains used in biomedical research [77]. Moreover, animal gametes- sperm,

eggs, and embryos preservation are now successfully preserved and maintained in cryobanks owing to the cryopreservation technique. Historically, embryo cryopreservation served as the gold standard for maintaining transgenic mice strains with single, multiple mutations, or complex genetic background [78, 79]. However, it is often more expensive due to costly and time-consuming superovulation procedures and subsequent cryopreservation. However, mouse sperm cryopreservation for long-term storage is simple and inexpensive, and it only requires few donor animals for protecting those commonly used inbred strains (e.g., C57BL/6, FVB, and 129/Sv) with single mutations [80]. Thus, sperm cryopreservation provides an efficient management of these genetic resources by reducing maintenance space and cost and by safeguarding them against, for example, disease, breeding failure, and genetic drift.

Although current sperm cryopreservation protocols showed relatively high success, there has been variation in the sperm motility and IVF outcomes post thawing. Many researchers investigated and worked to develop the gold standard protocol for mouse sperm freezing. Nakagata's protocol became the most widely used by many research laboratories and clinical and scientific facilities around the world [80]. The initial freezing solution simply contains 18% dehydrated skim milk and 3% raffinose in water, and cooling is achieved in LN2 vapor phase for 5 min followed by plunging the samples into LN2 at −196°C. Since the introduction of this initial protocol, there have been few changes in an effort to improve post-thaw fertilization potential of mouse sperm.

For sperm cryopreservation in mouse, epididymal sperm collected from the cauda epididymis of matured male mice are suspended in sugar-based cryoprotectant, which is loaded in freezing straws and preserved at −196°C [81]. The cryopreserved sperm can be used for efficient fertilization using improved IVF systems featuring methyl-β-cyclodextrin (MBCD) and reduced glutathione (GSH) [82, 83].

Several scientist across the globe had investigated the role of varied antioxidants like monothioglycerol [84], methyl-β-cyclodextrin (MBCD) [82] and the latest refinement was the introduction of reduced glutathione (GSH) to protect spermatozoa against oxidative stress during IVF treatment [83] and increase the fertilization rates. C57BL/6 is a major inbred strain used for the production of transgenic mice, and also as a back-cross for targeted mutant mice [85]. Therefore, it is necessary to establish cryopreservation method for C57BL/6 mouse spermatozoa that could maintain a high fertilizing ability after thawing. Recently, a preincubation medium containing methyl-beta-cyclodextrin used demonstrated increased fertility [82]. Further, it is augmented that sperm freezed in a cryomedia containing 18% raffinose and 3% skim milk, increased fertilization rate [86]. Sperm banking can be used for mice, but in some instances it is also important to bank the female genome [87].

Further, ovarian gamete cryopreservation, as harvested oocytes or contained within the primordial follicles in the cortical patch tissue, can be used for long-term storage of the female germline [88]. Cryopreservation protocol for ovarian tissue based on the slow-cooling procedures was initially developed and used for mouse eggs and embryos in 1970s [10]. This process requires a biological controlled rate freezer or equivalent equipment. It is now documented that in the mouse both fresh and frozen thawed grafts have the potential to restore long-term fertility (i.e., for 1 year) to the graft recipient [89]. A more recent advancement in duration of graft function is in case of ovarian tissue transplanted with transplanted graft functional for 5 years, or more persisting for more than 9 years [90]. Slow freezing is overshadowed with the development of more advanced closed vitrification system, proven more beneficial in ovarian cryopreservation.

*Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models DOI: http://dx.doi.org/10.5772/intechopen.101750*

### *2.7.2 Rat*

Animal model that is also commonly involved in the scientific work is rat, mainly in basic biology, physiology, brain science, and medicine. Over 40 year ago, the success of IVF was reported in rats. Characteristics, such as a short gestation and a relatively short life span, docile behavior, and ready availability of animals with well-defined health and genetic backgrounds make rat an ideal experimental model. The rat is a standard species for toxicological, teratological, and carcinogenesis testing by the pharmaceutical industry and governmental regulatory agencies [91, 92]. Rats are still continued to be used for nutritional, neurological and endocrinology studies. Cryopreservation has not been performed in rats as often as it has in mice, but the technique is becoming more widespread, for the same reasons that it is used widely in mice. Cryopreservation can be an efficient method of maintaining the potential of raising live mice of the thousands of genetically modified genotypes currently available [93, 94]. Sperm cryopreservation has been similarly successful in rats. Offspring are obtained from thawed sperm using intrauterine insemination or via IVF. Seita *et al.,* [95] established for the first time a successful IVF system using cryopreserved rat sperm [95]. A generalized protocol for IVF in rats (and similar breeds) from cryopreserved sperm and oocytes is illustrated in **Figure 3**.

### *2.7.3 Rabbit*

AI in rabbit using cooled semen stored for short time is a commonest ART practice in country like Europe, where rabbit rearing is common [96, 97]. High fertility rates and proliferation are obtained in this process. However, with limiting factors like low productivity and issues with cryopreserved rabbit sperm, AI is used for experimental or genetic resource bank purposes. Although cryopreserved sperm is not used for commercial purposes at the present, there is a need for reliable methods of rabbit sperm resource banking, especially as this species is a valuable animal

### **Figure 3.**

*A schematic representation of Assisted Reproduction techniques and In vitro fertilization; IVF in rats from cryopreserved sperm and oocytes.* 

in therapeutics(for production of vaccines, antibodies, hormones) [98, 99]. The extent of cryoinjury varies with the species due to differences in the gamete plasma membrane composition among them and cell size as well [100]. Hence, there is no universal protocol followed for all the animal species. It is obligatory to standardize cryopreservation protocol, extender and CPAs levels and composition, for each single species or even breeds.

Researchers observed significantly varying outcomes with the type of CPAs used in cryopreserving rabbit sperm. The use of CPAs like DMSO or ethylene glycol is recommended to enhance the storage of rabbit sperm [101]. Few other studies observed ethylene glycol to be less toxic than glycerol in cryofreezing rabbit sperm. Despite lower toxicity, ethylene glycol failed to provide protection to sperm cells when compared to other DMSO. Importantly, CPAs concentration need to be optimized for sufficient protection. To note, addition of CPAs at 5°C instead of 37°C was reported effective in cryopreserving rabbit sperm as permeability increases with increased temperatures [102]. Data on the CPA type and concentration for optimal cryopreservation is contradictory. However, it is well documented that a balance between all the involved parameters is a key for successful freezing. Sperm quality varies with the type of extenders used. The extenders used for rabbit sperm cryopreservation include a mixture of permeable and non-permeable CPAs. The use of two permeable CPAs is a common practice [103]. Sperm frozen in acetamide extender was found to be of superior quality than sperm DMSO and glycerol or other mixtured extenders [104]. The mixture of CPAs with egg yolk has differential effect on the sperm quality. Sperm frozen in extender containing egg yolk and DMSO demonstrated better sperm quality than for sperm frozen in the extender that contained high DMSO and lacking egg yolk [102]. Hence, it is obvious that a balance of all the essential factors is a key for successful freezing.

Recently, Domingo *et al*., [105] studied comparison of different semen extenders and cryoprotectant agents to enhance cryopreservation of rabbit spermatozoa and found that the addition of dimethylformamide (DMF) to INRA 96® exerts a protective effect on the membrane of spermatozoa improving seminal quality [105]. Although many efforts have been made to optimize cryopreservation extenders and protocols for rabbit sperm, many questions remain unanswered. In addition to cryopreserving sperm, rodents serve as a model for ovarian tissues cryopreservation and transplantation procedures as for current and future application and clinical use in the human. The production of GE rodent models for disease research increased over a decade using gene editing technologies (like CRISPR/Cas9).

### *2.7.4 Cryopreservation from rodents to larger animals*

From rodents to larger animals, cryopreservation is proved to be beneficial in fertility preservation, transplantation and breeding livestock. Advances in cryopreservation pioneered with transplantation of cryopreserved mouse primordial follicles in 1993 by J. Carroll and R. Gosden [106] followed by recognition of sheep as a larger model to study ovarian function. Gosden, in collaboration with D. Baird [107], developed techniques with vascular anastomosis that formed the basis of transplantation of larger organs such as kidney in human. The development of ART techniques has gained significance in the production of commercially important farm animal breeds and a few exotic or endangered species. Livestock industry especially cattle had benefitted to a major extent from the application of cryopreserved semen

### *Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models DOI: http://dx.doi.org/10.5772/intechopen.101750*

or embryos over the past decades. This was also the case in experimenting gamete cryopreservation. Larger animal like sheep is greatly emphasized to study human diseases particularly respiratory diseases and lung cancer, since the anatomy and physiology of the sheep respiratory system is more similar to that of humans than rodents. Sheep has been proposed as a good model for vaccines, asthma pathogenesis and inhalation treatments [108]. The gradual decline of genetic diversity in domestic breeds imposes a major threat to livestock, hence, international community strives harder to conserve the livestock genetics. Semen from most of the mammalian species has been successfully frozen [109].

With regards to temperature tolerance, sperm from different species exhibits varied responses. Bovine sperm shows higher tolerance to low temperatures, while porcine and ovine sperm are more sensitive and at risk of cold shock when exposed to temperature between 5° and 22°C leading to rapid loss of vitality. Animal sperm is highly vulnerable to oxidative damage owing to the loss of antioxidase enzyme and increased fatty acid oxidation on freezing [110]. The stability and viability of sperm is enhanced by adding semen extenders during freezing. The first semen extender for bovine sperm preservation used was egg yolk-sodium citrate diluent (EYC) and was gradually replaced with tris-buffered egg yolk (TRISEY)or tris-fructose yolk-glycerol [111]. Most of the industries use tris and citrate as active components in bovine sperm extenders. Addition of compounds like vitamin E to semen extenders is found to increase the structural integrity of acrosome, and thereby preventing sperm from oxidative damage via its antioxidant properties [24].

### *2.7.5 Vitrification in larger animals*

Vitrification of embryos was invented in 1985 [112]. Later vitrification emerged as one of the powerful methods for cryopreserving embryo from farm animals including cattle, goat, sheep and pig [28, 29, 113, 114]. The birth of the first calf was achieved from frozen/thawed oocyte was reported in 1992 [115]. Vitrification showed high success in bovine oocyte cryopreservation in 1998 [116]. Applicability of macromolecules with lesser toxicity as CPAs, the use of cryotop and solid surface virtification emerged gradually over time to overcome cryoinjury. Treatment with docetaxel improved cryopreservation of bovine oocyte as its protective against cytoskeleton injury thus can potentially enhance survival rate of post thawed oocytes [117]. High rates of survival and development after solid-surface vitrification have been reported for *in vitro* matured oocytes from cows [66] and goats [67]. SSV uses a metal surface, precooled to −180°C by partial immersion into liquid nitrogen, to cool microdrops of vitrification solution containing the embryos or oocytes.

OTC has been reported successful in restoration of ovarian function in sheep model. Complete restoration of acute ovarian function and high rates of natural fertility with multiple live births, were obtained following whole ovary cryopreservation and autotransplantation of adult sheep ovaries [29]. Although ovarian tissue cryopreservation has developed from experiments in sheep in early 1990s, it is now becoming recognized as relatively successful procedure for OTC in human, particularly to preserve the fertility of cancer patients to avoid gonadotoxic damage resulting from the therapy [118]. While there has been considerable success with cryopreservation of oocytes, embryos and semen in farm animals, this technology still requires refinement and further optimizing studies.

### **3. Conclusion**

Based on the utility and need of the animals for varied purposes, fertility preservation is a prerequisite to be practiced in animal husbandry and animal house unit for production of animal models. Understanding of the fundamentals of cryopreservation allows the development of more efficient procedures for cryopreservation of germ cells and further expand their clinical applications and utility in livestock, which can also be transferred to human application. Although, the field of cryobiology has advanced over the years, further research remains required to optimize cryoprotectant concentration, cooling and thawing rates to aim high success in animal reproduction.

### **Author details**

Feda S. Aljaser1,2

1 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia

2 Chair of Medical and Molecular Genetics Research, Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia

\*Address all correspondence to: faljaser@ksu.edu.sa

© 2022 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.

*Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models DOI: http://dx.doi.org/10.5772/intechopen.101750*

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## **Chapter 3** Embryo Transfer

*Ștefan Gregore Ciornei*

### **Abstract**

Assisted reproductive technologies (ART) have made tremendous advances, in last years. Artificial insemination is a method for achieving slow genetic progress in populations of animals. Many large and small ruminants are bred by AI, and more than a half million embryos are transferred every year around the world. Most of the ruminants sires used for artificial insemination were derived from embryo transfer. Improvements of reproductive biotechnologies of controlling the estrous cycle and ovulation have resulted in more effective programs for AI, superovulation of donor, and the management of ET. In the ruminants, ET procedure is a timely alternative that can allow good conception rates to be obtained constant in a year. There have been great advances of this biotechnique with on aimed to intensify the genetic progress between generations of farm. The gains is possible with the development of advanced reproductive biotechnique. The best current strategy in applying biotechnology to farmers is to use AI with sexed semen, so farmers will enjoy and benefit. The use of ET together with cryopreserved sexed embryos has a very specific potential for donor replacement and genetic improvement of the herd. In this chapter, procedures of the MOET protocol were described step by step.

**Keywords:** embryotransfer (ET), ruminant, cow, estrus, IVEP, MOET, ART

### **1. Introduction**

The ruminants sector plays an important role in global socioeconomic directions. Therefore, it is necessary to research, to discover and to innovate and transfer knowledge to the farmer, for practices and alternatives that improve ruminants reproduction and production.

Increasing the efficiency of breeding and production of a farm herd is one of the great challenges for large ruminant producers. Recently and now, genetic selection programs have sought the characteristics needed to increase milk production, with gains by increasing the quantity and quality of milk. However, reproductive efficiency was neglected. In recent years, various publications have presented strategies to further increase milk and meat production and also to increase reproductive performance, which is a key factor for the efficient growth of ruminants [1].

In farmers, importance of a sustainable, economically viable production system can be obtained by maximizing reproductive efficiency of the ruminants herd.

This reproduction management can determine the profitability from the number of offspring produced, the genetic progress and the shorter interval between lactations periods.

### *Animal Reproduction*

The essential importance of precision reproductive care is therefore highlighted. This reproductive biotechnology (ET, AI) applied is capable of produce maximum production efficiency in animal farms with vulnerable populations, or in limited areas, in addition to improving animal welfare.

The first biotechnology of reproduction represented by artificial insemination (AI) is known as the simplest and lowest cost of reproductive biotechnology. This technique enhances the male's genetics, bringing slow genetic progress. However, dairy breeds in conditions of seasonality and climate change exhibit failures in estrus cycling and demonstration, which compromises AI results. In other words, with the discovery and description of FTAI protocols that synchronize follicular growth and induce ovulation, it is possible to achieve a high rate of inseminated animals without the need to observe the clinical and behavioral signs of estrus. [2], thus providing an increase in the conception rate and avoiding the occurrence of human errors in the detection of estrus, and calculating the optimal time for insemination. However, gametes an embryo can undergo degeneration in the extreme temperatures of summer [3]. The transfer of embryos produced in vivo (ET) became a strategy to avoid the deleterious effects of this period and provide a higher productive index than with AI [4].

With the beginning of the evolution of modern biotechnologies, the next step as major commercial progress in reproductive biotechnology was the transfer of embryos that appeared in the late 1970s. The ability to preserve, freeze and transport bovine embryos around the world has made ET an extremely useful technology for disease control, genetic rescue of valuable individuals and the development of new lines or breeds of animals.

ET is a multifactorial protocol that depends on several carefully and correctly performed sequential steps. Poor performance in any of the steps directly affects the success rate of the final result, the conception rate and the number of weaned products.

The use of embryo transfer as a breeding technique is growing throughout Europe, even in countries with less embryo transfer tradition. Historically the entire embryo transfer process was carried out at a specialist centres but now experienced reproduction vets are starting to carry out the artificial insemination (AI), flushing and searching as ambulatory procedures for transfer into a suitable recipient. The most time consuming and difficult part of the in vivo embryo transfer process is synchronizing recipients and transferring the embryo into the most suitable recipient. Receptors must be selected with with the best chance of maintaining the pregnancy [5, 6].

Embryo transfer provided a means by which the number of conception products could be multiplied rapidly, with the same origin. However, embryo transfer veterinarians have developed technology for commercial use and taken techniques from the laboratory to the farm. There have been countless practical difficulties for practitioners in uniting and setting up the International Embryo Transfer Society (IETS) in order to facilitate the discussions and steps deemed necessary for progress. Currently, the vast majority of countries in the European Union have one or more embryo transfer associations, where these actions are reported and come to support and develop ET biotechnology (eg AETE, SIET, AET-d, AETF, ARET and others).

Embryo transfer (ET) is now commonly used to produce AI sires from the top producing cows and proven bulls for the dairy industry [7]. As a perspective, the new genomic techniques presented are increasingly used for the selection of embryo donors, and genomic analysis has become essential for the selection of bull dams that will be used in embryo transfer [8]. Although the economy sometimes seems to

### *Embryo Transfer DOI: http://dx.doi.org/10.5772/intechopen.99683*

exclude the use of embryo transfer techniques for anything other than gamete production, the commercial cattle industry benefits from the use of commercial males produced through well-designed MOET programs [9].

With the explosive development of this biotechnology, the techniques for obtaining embryos have been improved, the materials and consumables have become more efficient, the equipment more efficient, which makes the production cost of the embryo decrease and be higher quality. This desideratum is fully accepted by farmers and who apply this ET biotechnology more and more frequently [10].

Although there has been no appreciable increase the embryo production per poliovulated donor in last years, the importance of follicle wave dynamics [11] and methods for the synchronization of follicular wave emergence [12, 13], they simplified the protocols by which female poiliovulation could be achieved, leading to increased embryo production per application session. Currently, donor cows are hyper-stimulated more frequently than in the past (at an interval of 30–60 days) and thus more embryos can be produced per year, without changes in the current superstimulation protocol [14]. Other authors [15] have been interested in various factors that affect the viable production of embryos in animals and especially in dairy cows.

Potential embryo donors can be inseminated naturally or artificially (AI) and the embryos are normally collected non-surgically from 6 to 8 days after fertilization. After collection, the embryos must be identified and then evaluated morphologically. The evaluation procedure is done in an appropriate environment before the transfer. At this stage, they can be subjected to manipulations, such as splitting and sexing, and can be cooled or frozen for shorter periods or longer storage [16]. Discussion of donor superovulation, recipient synchronization, and embryo transfer should begin with a review of recent information on the physiology of female reproduction and the estrous cycle.

The reproductive genetic potential of every normal newborn calf is enormous. It is said that there are about 150,000 "eggs" or potential oocytes in a female and many billions of sperm produced by each male. We can say that through natural reproduction, only a small part of the reproductive potential of a valuable individual could be realized. The bull will be able to produce an average of 15 to 50 calves per year, and the cow will have an average calf per year. With the use of artificial insemination biotechnology, it is possible to exploit the large number of sperm produced by a genetically superior bull; however, the reproductive potential of the female with superior genetics was largely unused. It will produce on average 5–8-10 calves in its entire biological life through normal management programs. As artificial insemination has done for bulls, embryo transfer is a technique that can greatly increase the number of offspring that a genetically important cow can prove and produce. The main reason for the development of embryo transfer in cattle was to further the increase in genetic progress of the female.

### **2. Advantages of embryo transfer (ET)**


### **3. Sexual cycle physiology**

The intrinsic control of the bovine estrous cycle is coordinated by the interdependent secretion of hormones from structures such as: hypothalamus, anterior pituitary, ovaries and uterus [26]. These include gonadotropin-releasing hormone

### *Embryo Transfer DOI: http://dx.doi.org/10.5772/intechopen.99683*

(GnRH) from the hypothalamus, folliculostimulating hormone (FSH) and luteinizing hormone (LH) from the anterior pituitary gland, estrogen, progesterone and inhibin from the ovary and prostaglandin F2a (PGF) from the uterus.

During gestation, the multiplication phase of the oogonia results in the constitution at the birth of a stock of primordial follicles, the number of which in the cow is between 200 and 250,000. These primordial follicles have a diameter of between 30 and 40 microns. At the secondary and especially tertiary stage, a cavity appears. It then becomes possible to identify by ultrasound these follicles with a diameter of between 2 and 4 mm.

In cows, as in many other mammals, follicular growth develops in the form of waves. In a 21-day cycle, there are two waves. The presence of a third wave is not uncommon. It has the effect of lengthening the average length of a cycle by a few days (24 vs. 21 days). When the cycle has two waves (**Figure 1**), the emergence of one wave occurs on days 0–1 and 10–11 of the cycle. Day 0 corresponds to that of estrus. By emergence we mean the moment or by ultrasound, it is possible to distinguish in the mass of follicles recruited the one which will become dominant. During the 2.5 days after the emergence of a wave, the selected future dominant and dominated follicles continue to grow. The dominant follicle reaches at this time the average diameter of 8 or even 9 mm. This moment is called "follicular deviation" and characterizes the moment when the dominant follicle will be able to clearly distinguish itself from other growing follicles. Its diameter is therefore 2 mm greater than that of the other selected follicles. The follicle continues to grow until it reaches a diameter of 10 mm. Clinically, this dominance can be identified by ultrasound, or by Doppler ultrasound to identify changes in the vascularization of the follicle or by assaying hormones such as estradiol or follicular fluid inhibin. This dominance is therefore both morphological, ie exerted by the largest follicle and physiological, because it brings about an arrest in the development of the dominated follicles which go through a static phase before settling down. This physiological dominance also implies the appearance at the granular level of LH hormone receptors which will take over from FSH to ensure further growth of the dominant follicle. The period of physiological dominance is

### **Figure 1.**

*Physiology of the sexual cycle in cows, with two follicular waves.*

shorter than that of morphological dominance. Clinically, the identification of more than 10 follicles with a diameter of between 3 and 8 mm makes it possible to exclude the presence of a physiologically dominant follicle. Growth of the dominant follicle will continue until it reaches a maximum diameter of 16 mm.

This is followed by a so-called static plateau phase lasting more or less 6 days at the end of which the dominant follicle will begin to regress. Due to the gradual decrease in estradiol synthesis by the dominant follicle, there is a new release of FSH and the appearance on day 10 of new follicular emergence. This cannot be observed as long as the dominant follicle from the previous wave is in the growth or plateau phase. This new wave develops like the previous one through the dominance of a new follicle which will suppress the growth of the subordinate follicles which will become saturated. The follicle continues to grow. In proestrus he is no longer under the progesterone influence, so given the massive release of LH he can stop growing and then ovulate.

During the cycle, the follicular population is therefore distributed into several classes except that of the follicles in the reserve. A first class consists of recruited follicles. Their diameter is 2 to 4 mm. A second class is made up of growing follicles. These follicles can potentially become the ovulatory follicle. Their diameter is between 6 and 10 mm. The third class refers to the dominant follicle. Its diameter is between 10 and 16 mm. Finally, can we also identify the preovulatory follicle with a diameter greater than 15 mm. It will persist on the ovary for 5 to 6 days before regressing or ovulating (**Figures 1** and **2**) [5, 6, 10, 27].

In a cycle with three waves (**Figure 2**), the emergence of cows occurs on days 1, 10 and 17 respectively, with day 0 being estrus and therefore day 1 ovulation. The general wave pattern is comparable to that described for a cycle with two waves. Waves 1 and 2 are anovulatory. Only the third is normally ovulatory. It will be seen that the luteal phase like the cycle is of longer duration than for a cycle with two waves. Likewise, the interval between the onset of the ovulatory wave and ovulation is shorter (7 vs. 11 days).

The main mechanism of synchronization of the estrous cycle is ovulation, when the first follicular wave occurs [11]. A new hormone-secreting endocrine gland is

### **Figure 2.**

*Physiology of the sexual cycle in cows, with three follicular waves.*

### *Embryo Transfer DOI: http://dx.doi.org/10.5772/intechopen.99683*

formed instead of the ovulatory follicle and is called the corpus luteum (CL) it is formed in the following days (3–5) and in the absence of pregnancy, it wraps around day 16 or 17 of the cycle [26]. The most common hypothesis for CL regression is that the non-pregnant uterus secretes a luteolytic factor into the uterine venous blood. This substance is transferred through a local blood (veno-arterial) pathway to the ovarian artery through which it reaches the ovary and causes luteolysis [27].

After CL regression, a rapid decrease in serum progesterone concentrations to values lower than 1 ng/ml results, at the same time, the frequency of LH pulse increases and follicular development is further stimulated. The growth and maturation of the follicle that becomes preovulatory results in increased estradiol secretion, which causes local estrogenic changes in the oviduct and uterus, behavioral estrus and a preovulatory release of LH (around the time of estrus manifestation). The preovulatory LH peak results in the resumption of the oocyte meiosis process, and ovulation 24 to 32 hours later and the luteinization of the ovulated follicle to form a secretory corpus luteum hemoragicum. The growth and development of the hemorrhagic corpus in a fully functional CL results in progestative changes in the oviduct and uterus that are favorable for embryonic development and pregnancy. If pregnancy does not occur, the cycle resumes again with the disappearance of CL [6, 11, 23, 27].

### **3.1 Estrus synchronization, superovulation**

Estrus synchronization and superovulation are critical components of an embryo transfer program. These techniques involve the manipulation of the basic endocrine patterns, presented and described in this document [28]. The key to successful estrus synchronization is synchronous growth and ovulation of a viable dominant follicle and closely synchronized, rapid declines in circulating progesterone to values <1 ng/ ml [29]. If properly implemented, within the physiological constraints of their mechanism of action, current techniques for synchronization of estrus and ovulation are highly successful [30]. However, the variation in the dynamics of ovarian follicular waves makes it difficult to control the exact time of estrus and ovulation.

The goal of superstimulation treatments in cows is to obtain the maximum number of fertilized and transferable embryos with the highest possible probability of inducing and sustaining a pregnancy.

The variability of the ovarian response was related to differences in superstimulatory treatments, such as gonadotropin preparation, gonadotropin type, duration of treatment, timing of previous estrus treatment, total gonadotropin dose, and use of additional hormones in superstimulation. Protocol [4]. Additional, equally important, sources of variability are factors inherent in the animal and its environment. These factors may include nutritional status, reproductive history, age, season, breed, ovarian status at the time of treatment and perhaps most importantly, inherent numbers of antral follicles [5]. While considerable recent progress has been made in the study of bovine reproductive physiology, factors inherent to the donor animal that affect superovulatory response are only partially understood [13, 25, 30].

### **4. Embryo transfer procedures**

In farm animals, fertilized ova is removed from the uterus of their dam (the donor) and transferred to the uterus of other females (recipients) for development to term. Almost all commercial embryo transfers now use nonsurgical methods to recover the

**Figure 3.** *Stages of in vivo embryo transfer in large ruminants.*

embryos rather than surgical methods (only for small ruminants). The procedure requires multiple steps (**Figure 3**), a large amount of time, and a variable cost.

The stages of a direct/in vivo ET protocol are highlighted in the following mandatory steps [31]:


### **4.1 Donor selection**

The selection of the embryo donor candidate is based on two major criteria: (1) the genetic merit, generally evaluated by the owner and based on performance, and (2) the reproduction criteria interpreted and evaluated by the veterinarian. The donor must be in good physical condition, an average but growing BCS. It should be free of underlying conditions, be at least 50 to 60 days after calving and have a regular cycle. In general, cows with a history of reproductive problems, even minor ones, do not make good embryo donor animals.

### *Embryo Transfer DOI: http://dx.doi.org/10.5772/intechopen.99683*

Donors are further evaluated by careful examination of the cervix, uterus, and ovaries per rectum to determine if they lack adhesions to neighboring organ structures, and the presence of other palpable lesions. It is recommended to test the permeability of the cervical canal with a cervical dilator, especially if the donor is before the first calving - heifer. This prevents the occasional of being unable to negotiate the cervix after a series of costly hormonal injections.

Single or multiple embryos can be collected from ovulating or naturally superovulated cows. For optimal efficiency, 2 to 4 donors should be treated and synchronized with their recipients for each attempt/session; this allows the sharing of the recommended potential of 8–10 recipients per donor.

### **4.2 Superovulation**

Superovulation is and remains one of the least anticipated steps in the process of embryo production. The objective of superstimulation treatments in the cow is to obtain the maximum number of fertilized and transferable embryos with a high probability of producing pregnancies [32].

In the bovine tremendous variation in response occurs with age, breed, lactational status, nutritional status, season, and stage of the cycle at which treatment is initiated. Follicle stimulating hormone (FSH), which has a short half-life (Pluset, Folltropin-V, and others), necessitates twice-daily injections over a period of 4 to 5 days. Synthetic hormones with a long half-life (like PMSG), are administered in a single dose, but have other drawbacks. Treatment is start in the mid-luteal phase (day 8 to 12) of the donor's estrus cycle and white use of prostaglandins (PGF) to synchronize the estrus of the donors and the recipients. Alternatively, treatment may be induced on day 16 or 17 (day 0 = estrus) of the donor's natural estrous cycle, or with progesterone administration (which mimics a progesterone phase). Ultrasonography and palpation of the ovaries per rectum have been shown to have similar accuracy for determination of the number of follicles (in estrus fase) or CL (at the time of embryo recovery). However, the number of anovulatory follicles can be counted more accurately [1, 4, 33, 34].

### **4.3 Artificial insemination/mounting**

Donors should be artificially inseminated twice with a 10–12 hour interval, beginning 6 hours after the occurrence of oestrus, to ensure the time interval in which ovulation occurs. Depending on the quality of the frozen/sexed semen, a dose with a higher sperm concentration, even a double dose, can be used for each insemination. Ultrasonography is helpful in assessing the potential superovulatory response on the day prior to ovulation or at the time of AI.

### **4.4 Collection of embryos and classification**

The donor animal is kept in a standing position in a trevis. The first step in the non-surgical recovery of embryos is to determine the numbers of corpus luteum in the ovary [21]. This step is important to rule out that the superovulation response in the animal; if less or no CL is found-indicates the poor response of superovulationflushing not to be done in such animals. The donor was given an epidural anesthesia, then a wash and disinfection of the ano-vulvar region (**Figure 4**).

### **Figure 4.**

*Recovery of bovine embryos by flushing method. A. Scheme of catheter placement and reservoir-uterus-filter fluid flow, ultrasound images with catheter guidance through the cervix and visualization of uterine lavage, see white arrow. B. Inserting the stylus through the cervical lumen, C. inflating the balloon from the catheter and obtaining the dam at the top of the uterine horn, D. flow of flushing fluid and recovery of embryos.*

A two-way round tip balloon catheter (Fr. size 16 to 24) with a tul inflatable balloon is used. Once the instruments has been made ready (two/tree-way catheter), the vulvar lips are parted and the catheter with stylet is inserted into the vagina and advanced towards the lumen of the cervix. It is further advanced to the horn of the uterus until the balloon is situated at the base of the uterine horn. By blowing air, a dam is created with the tip of the uterine horn, there are located the embryonic formations between days 5–8 after ovulation. The amount of air used depends upon the size of the uterus. Basically, there are two methods of embryo collection [35]: the continuous or interrupted flow, closed-circuit system and the interrupted-syringe technique. The most commonly used medium for embryo recovery is Dulbecco's phosphate buffered saline (PBS), but there are many others ready to use (Euroflush, Vigro). Uterine horn is flushed with 30–60 ml of media and repeated until 300–800 ml of media is used up. The same process is repeated for the other horn as well [36].

Embryos are found with a 10 X magnification stereoscope after filtering the collection/washing medium through a pore filter with a diameter between 50 and 70 μm. The identified embryos are usually transferred as soon as possible, sometimes if desired it is possible to keep the embryos in that environment for a few hours at room temperature. It is also possible to cool the bovine embryos in storage medium and store them in the refrigerator for 2 or 3 days. Most often, embryos can be frozen for use at a later date.

### *Embryo Transfer DOI: http://dx.doi.org/10.5772/intechopen.99683*

A good response and an appropriate recovery rate results in getting a 4–5 embryos are recovered with each flush. This can lead to 50 freezable embryos per donor per year resulting in the birth of 30 calves after the transfer of the embryo to a recipient [24].

After the fecundation, the single-celled embryo now called the zygote undergoes rapid mitotic divisions (cell number increases, cytoplasm remaining same) called cleavage [37]. Bovine embryo descends into the uterus around day 4.5 days (estrus day 0) [38]. According to the standards, embryos are recovered from six to eight days after the onset of estrus (day 0). Embryos can be recovered even earlier from four days when the embryos arrive from the salpinx in the uterus, but before day 6 the recovery rates are lower than on days 6–8 (**Table 1**).

However, embryos can also be recovered on days 9–14, although they leave the pellucid area on days 9–10, making them more difficult to identify and isolate from cellular detritus and more susceptible to infection [39].

Identification and evaluation of embryos is one of the most important and delicate stage, the practitioner needs experience to get used to the procedure. Embryo quality and poor handling techniques can directly affect pregnancy rates. A step-by-step procedure for looking for embryos is presented in the content of this section.

Evaluation of the embryo in the uterine effluent is based on identification of several morphologic features of the embryo using light microscopy. These methods are subjective and depend on experience. The embryo is spherical and is composed of blastomeres surrounded by a gelatin-like shell and zona pellucid (**Figure 5**).

Embryos recovered 5 to 8 days after estrus are classified morphologically into the following groups, based on their stage of development. Proper evaluation requires rolling of the embryos along the bottom of the dish.

*Morula.* The blastomeres are round and not tightly joined together. Individual blastomeres are difficult to identify with each other. The blastomere cell mass of the embryo occupies most of the perivitellin space.

*Compact morula (tight morula)*. The shape and appearance of a tight mill is similar to a golf ball, in that the outer edge is slightly wavy (curly) given due to compaction. Individual blastomeres grow and become indistinguishable. The cells on the surface of the mass have a polygonal shape. The embryonic cell mass occupies 60–70% of the perivitellin space.

*Early blastocyst.* A tiny transparent (clear) space that contains fluid is visible. This area is the beginning of the blastocele (cavity). The embryo occupies 70–80% of the perivitelline space [6, 14, 37].

*Blastocyst.* The blastocele cavity becomes prominent and represents more than 70% of the embryo's volume. Inside, two groups of cells are separated and differentiated. They can be clearly recognized as a trophoblastic layer below the pellucid area and the darker cell mass occupying part of the embryo. The perivitellin space is still visible, but it is very small.


**Table 1.** *Day of collection of embryo.*

**Figure 5.** *Schematic diagram of a transferable embryo (expanded blastocyst phase).*

*Expanding or expanded blastocyst.* There is no more perivitelline space between the trophoblastic cell layer and the interior of the area. The area of the pellucida stretches becomes thinner as the blastocyst expands. A small, well-compacted internal cell mass is observed positioned in one part of the embryo. The color of the embryo becomes pale to clear and is due to the large amount of fluid present inside.

*Collapsed blastocyst.* A perivitelline space can be identified together with a very thin pellucid area. The blastocyst may be partially collapsed, with a smaller blastocyst cavity, or completely collapsed and have the appearance of a compact morula.

*Hatched blastocyst.* After a continuous growth, the blastocyst expands to rupture and the embryo escapes from the pellucid area. From this moment the embryos pass into the gastrulation phase. The hatched blastocysts can be spherical with a welldefined blastocyst or they can be collapsed, similar to cellular detritus. Identifying embryos at this stage is especially difficult for the inexperienced operator.

When blastocysts/gastrules without areas or hatching are collected, there is a higher risk of damage due to handling. In addition, hatched blastocysts are sticky and can adhere to micropipette handling tubes. Therefore, the use of embryonic filters is not recommended when there is a suspicion that hatched embryos will be recovered (> day 7.5).

Embryos are then classified according to quality based on morphologic appearance. Excellent/good, fair, and poor quality embryos are considered transferable into recipients. Excellent or good quality embryos (Code 1) are freezable (**Figure 5**).

### *4.4.1 Codes for embryo quality*

**Code 1:** *Excellent or good*. The mass of the embryo is symmetrical and spherical with individual blastomeres (cells) they are uniform in size, color and density. The embryo was in accordance with the expected stage of development (collection day).

### *Embryo Transfer DOI: http://dx.doi.org/10.5772/intechopen.99683*

The irregularities are usually minor and more than 85% of the cellular material should be a compact and intact embryonic mass. This is based on the observation of the percentage of embryonic cells represented by the extruded material in the perivitelline space. The pellucid area should be smooth and smooth and could adhere to a micro-plate or a straw.

**Code 2:** *Fair*. Some irregularities can be observed in the general shape of the embryo mass or in the size, color and density of individual cells. At least 50% of the cellular material must be an intact, viable mass of embryos.

**Code 3:** *Poor*. Some major irregularities in the shape of the embryo mass, or the size, color and density of blastomeres, are identified. At least 25% of the cellular material should be like an intact, viable mass of embryos.

**Code 4:** *Dead or degenerating*. Degenerate embryos, oocytes, or I-cell embryos are nonviable.

Embryos of fair quality can be transferred fresh, if the receptors are available and synchronized. The category of good and excellent quality embryos have a high probability of surviving cryopreservation. The EITS considers that the export of embryos of poor and fair quality is inadequate [40]. The assessment of bovine embryos has recently been revised and is constantly improving [41], but the IETS manual has the most comprehensive library of embryonic images useful to practitioners.

**Loading the Straw.** Immediately before direct transfer, the embryos are individually aspirated into sterile 0.25 ml French straw. The embryo is usually loaded from the support vessel into the straw using a 1 ml syringe attached to the end of the straw stopper. First, a 3 cm medium column is aspirated into the straw, then a 0.5 cm air column is aspirated, then a 3 cm medium column containing the embryo, followed by another air bubble.

**Selection and preparation of receptor.** The recipient should be non-pregnant, cyclic (minimum of two normal cycles), should have CL on at least one of the ovaries. The embryo stage should match the uterine age of the recipient for a successful pregnancy to occur. While perfect synchrony is desirable, recipients that are 1 day out of phase can be considered acceptable; this means that a 7-day embryo can be transferred into a recipient who showed heat 6–8 days earlier. The lower quality embryo is more sensitive to asynchrony. The recipient should not have any pathological condition which can hinder its pregnancy. Pregnancy rates following embryo transfer are best when the recipient is in estrus from 36 hours before to 12 hours after the donor [42].

Synchronous recipients can be produced in three ways:


**Embryo transfer** to the recipient can be done surgical or non-surgically. However non-surgical is more ethical to use. The recipient is secured in a Travis and the vulvar area is cleaned. As the animal is in the luteal phase. Epidural anesthesia is induced to prevent straining and defecation. The insertion of the tip of the instrument into the desired uterine horn should be done quickly, and smoothly. Trauma to the delicate endometrium causes bleeding, and blood (complement in the serum) is embryocidal. Ruminants embryos are transferred to the uterine horn and the same procedure as A.I. is followed except that in ET embryos are deposited deep in the horn ipsilateral to CL [44].

Pregnancy rates for IVP embryos were lower in commercial embryo transfer programs than for in vivo embryos [45].

Pregnancy rates are 10% lower in frozen embryos than the fresh ones [37]. Using heifers as recipients, there have been reports that in some 10% of such animals (heifers) it is difficult, if not impossible, to carry out ET via the cervix.

Any kind of stress to the recipient should be avoided. Any other routine treatments scheduled (eg antiparasitic) must take place at least 3 weeks before the transfer; also changes in the feeding regime should be prohibited for 3–4 weeks before and after embryo transfer. Beneficiaries must be accommodated where they can be easily and quietly handled on the day of transfer [23]. Any stressors should be removed.

### **5. Embryonic mortality**

It is said that about 25–40% of embryonic losses are produced in the first few days after transfer to the cow [46, 47]. It has been observed that most of these females return to heat at an interval after 20–22 days, presenting a complete and normal sexual cycle [48]; Therefore, it is believed that embryonic mortality (EM) could occur between days 7 and 17, the period from embryo transfer (ET) until it settles at maternal recognition of pregnancy [49]. In a lower proportion, but just as important, is the pregnancy losses that occur between days 28 and 98, after the transfer and the percentages between 7% and 33% have been reported [50].

The critical nature of the period and the phenomenon of recognition and survival of the embryo at the maternal uterine endometrium during implantation requires a very careful synchronization between the transferred embryo and the recipient. Thus, the importance of both the biochemistry of the uterine environment and the signals of the embryo that generates the recognition and implantation is highlighted [51, 52]. These embryonic signals must be released at the time and concentration necessary to maintain CL morphology and maintenance of function, thus generating a continuous production of P4. Progesterone levels play an essential role in maintaining the embryotrophic environment and supporting the normal development of the concept (the embryo and all adjacent cell structures) [48].

In connection with the influence of P4 (progesterone) on certain events related to pregnancy maintenance from the early stages and the ability of PGF2α to trigger luteolysis, a number of hormonal strategies for maintaining pregnancy have been researched, developed and supported [53, 54]. These strategies tend to be based on the increased efficiency and secretion capacity of P4 by CL: secretion must occur in a timely manner, thus ensuring a suitable uterine environment for the development of the embryo transferred to the recipient bovine female. All these strategies aim to increase the load rate in ET programs [48].

### *Embryo Transfer DOI: http://dx.doi.org/10.5772/intechopen.99683*

In order to prevent the mortality of the transferred embryos, and the loss of the pregnancies during embryo transfer sessions, two main objectives are considered: - Maintaining the corpus luteum function, even inducing a new one; and Inhibition of the appearance/secretion of luteolytic factor. All procedures apply to female embryo recipients.

In the first case, it is recommended to administer a treatment with Gn-RH, more precisely HCG to develop and support the luteal tissue, or even to form another CL (by causing ovulation of the follicle, if any). In the second case, the administration of non-steroidal anti-inflammatory drugs is considered, which is said to block the synthesis of PGF.

### **6. Embryo production biosecurity and contamination risks**

The procedures for embryo production, in MOET programs, include several steps where contamination with pathogen agents may occur. For instance, the first source of potential contamination comes from the donor itself. Before ovulation, an oocyte could be contaminated by its contact with a given pathogen shed in granulosa cells or follicular fluid during infection (viremia or bacteremia). For example, in bovines, viruses were detected in follicular fluid a few days after experimental exposure to bovine viral diarrhea virus [55]. Hence, the recovery of cumulus–oocyte complexes at this moment might lead to production of contaminated embryos [6, 37].

*Disease Risk Management.* Success in embryo production by either MOET or IVEP relies on the capacity to correctly perform all technical steps, eliminating or reducing factors recognized to have negative effects. It is essential to select donors and recipients with good general health and adequate nutrition. In addition to those issues, considering that the first source of potential contamination comes from the donor itself, an important measure is to select these females, taking into account their sanitary status. When incorporating animals into the flock, their health status should be checked before and quarantine should be respected. Vaccination and deworming must be employed as prescribed, depending on the location and system of production, but always before their use as donors. Testing should be conducted for some infectious diseases, and those positive should be culled. All technicians in direct contact with the animals must be careful and well trained to ensure familiarity with and safety in the procedures. The technique must be aseptic and all labware sterile. The equipment should be cleaned and all devices that are in contact with the animals should be sterilized before reuse. Clothing should be completely cleaned before reuse [56].

In general, in IVEP, the risk of potential hazards associated with oocyte collection from slaughterhouses are higher than those collected by laparoscopic ovum pickup. Consequently, when using these ovaries, it is important to determine their origin, particularly whether ovaries came from a herd depopulated for any health cause [57]. Care must be taken in the transportation of this material to the laboratory to avoid any external contamination. For media preparation and gamete or embryo manipulation or culture, all biological products should be avoided. These reagents could be replaced by those derived from plant origin or amino acids. When cell culture is preferred for IVEP, the use of controlled cell lines, confirmed to be pathogen-free, is recommended. From a sanitary point of view, safer strategies include the use of chemically defined media that do not contain serum or somatic cells [57].

For MOET, pathogens could be present in the female genital tract and can adhere to either oocytes before fertilization or embryos before collection. Intact zona pellucida is a natural barrier to penetration of pathogen into the oocyte or prehatching embryos. However, some pathogens may adhere to the zona pellucida of oocytes and embryos; thus, the zona pellucida represents a vector for disease transmission to recipients and to embryos after hatching (once transferred). For IVEP, the magnitude of this risk may vary according to the source of ovaries or oocytes that are being used: either from laparoscopic ovum pickup when the donor health status is well known or from the slaughterhouse [57].

Follicular aspiration by laparoscopy, instead of transvaginally, practically eliminates the chance of contamination by microorganisms being carried into the follicle from the vagina via the collection needle, as has been reported in humans [58].

On the other hand, ovaries collected from slaughterhouses provide an inexpensive and abundant source of oocytes, which is usually helpful for research projects and cloning. However, considering that these ovaries are generally transported in containers together, the presence of just a few ovaries from infected animals could represent a potential source of contamination. Other general sources of possible contamination involve the presence of environmental pathogens associated with the technician, slaughterhouse, equipment for laparoscopic ovum pickup or embryo collection, or even in the laboratory, such as glassware, culture dishes, media, and incubators. Regarding media, it is known that any biological product such as fetal calf serum and bovine serum albumin used in the recovery, culture, and cryopreservation of oocytes, sperm, and embryos may constitute a risk of contamination [6, 37, 55–58].

The semen used in a ET protocol (MOET or IVEP program) must be collected from males managed under appropriate sanitary protocols that ensure their good health status. Although AI represents a useful tool for disease control when best practices are applied, a further source of risk in an embryo production program is the semen. Numerous viral, bacterial, and parasite agents may be present in semen, which may adhere to the surface of spermatozoa or they could be present in the seminal fluid or in the semen extender.

In general, the studies are in agreement when the sanitary procedures recommended by IETS are correctly implemented. The risk of disease transmission from donor to recipient and to offspring for most pathogens is negligible or, at least, is much lower than that associated with live animals. These facts confirm that embryo transfer represents a safe strategy for global genetics trade and a valuable tool for the control and eradication of several diseases in small ruminants [59, 60].

Various publications [57] describe the possibility of transmitting diseases and the management of prevention procedures. The procedures for managing these risks have been described in the *OIE Terrestrial Code* [61], which explicitly refers to the specifications published in the IETS manual. These procedures are included in national regulations for the transfer of embryos between herds.

Adherence to these procedures ensures that embryo transfers contribute to improving the animal health of a population by controlling the movement of genetic material between herds. The basic concept behind these regulations is the official validation of embryo transfer teams. This approval is a very important method of veterinary regulations, as they are usually based more on animals coordinated in protocols and their products. However, in this case, the safety of embryo transfer procedures is based on the correct ethics and technique of the head of the embryo transfer team [62].

*Embryo Transfer DOI: http://dx.doi.org/10.5772/intechopen.99683*

The criteria used by national veterinary services for the approval of embryo transfer teams are based on the *Terrestrial Code*. For example, in Chapter 4.7. it is stated that: "the embryo collection team is a group of competent technicians-operators, including at least one veterinarian, who carry out the production, collection, processing and storage of embryos".

It is recommended that the following conditions be met:

	- checking the health of the embryo donor
	- implementation of appropriate disease control measures when handling or operating donors
	- disinfection and hygiene procedures;

e.the embryo collection team should have adequate equipment for:


These facilities do not necessarily have to be in the same location;


### **7. Conclusion**

Embryotransfer *in vivo*, (IVD by MOET) is a procedure that can significantly increase the amount of offspring a genetically significant ruminants can carry. This it is a multistep procedure involving superovulation, synchronization of donor and

recipient, insemination of donor, collection, isolation, evaluation, genetic testing freezing and transfer of embryo. This is the shortest path to genetic progress on economic efficiency in large and small ruminant farms.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Ștefan Gregore Ciornei Department Clinics, Biotechnology of Reproduction, Faculty of Veterinary Medicine, Iasi University of Life Sciences (IULS), Iasi, Romania

\*Address all correspondence to: stefan\_ciornei@yahoo.com

© 2021 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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### **Chapter 4**

## Doppler Ultrasound in the Reproduction of Mares

*Camila Silva Costa Ferreira and Rita de Cássia Lima Morais*

### **Abstract**

Doppler ultrasonographic (US) is a method that provides real-time information on vascular architecture and hemodynamic aspects of blood vessels. It can determine the presence, direction, and speed of blood flow, being subdivided into the categories of color Doppler (color flow and power flow) and pulsed Doppler. The objective of this chapter was to compile data from several studies addressing the use of US Doppler correlated with pathophysiological phenomena of equine reproduction. Initially we decided to describe the technique, advantages, and disadvantages of each Doppler mode. Then the applicability of US Doppler in mares related to equine reproduction. Thus, within this chapter, you will find the form of use and descriptions of studies carried out on vascular perfusion of the follicular dynamics, the corpus luteum, the uterine segments, which we have divided into post-insemination evaluation, endometritis diagnosis and pregnancy diagnosis. So, we hope that this chapter will expand the knowledge about US Doppler and increase the number of veterinarians who will introduce the technique into their practical routine.

**Keywords:** equine, diagnosis, Doppler ultrasound, reproduction

### **1. Introduction**

In ultrasonographic (US), images of the body are obtained from the reflection or scattering of a pulsed high frequency sound beam that is sent by a mobile transducer to examine the body [1]. Each time the sound beam encounters acoustic interfaces in its path, there are changes in the density or elasticity of the medium, where the fraction of sound energy is reflected or scattered. This can happen on the walls of an organ or even along a tissue with a heterogeneous structure. The retro-scattered wave (or "echo") is detected and processed by the device, which will assign a gray scale according to the amplitude of the signal returned. Therefore, an ultrasound image corresponds to a 2D map of the tissue's acoustic reflectivity. The body can also be investigated in Doppler mode to obtain flow information, widely applied in the analysis of the circulatory system [2].

In the 1980s, the renowned researcher Dr. O.J. Ginther stated that since the introduction of transrectal palpation, ultrasound diagnosis has been the most profound technological advance in the field of research and the reproductive clinic of large animals [3]. In the late 1990s, studies were started using US Doppler to determine

physiological and pathological changes in the mare's reproductive tract [4]. Over the past decades, the use of US has reached great dimensions, not only in research centers, but also commercially in livestock activities, having made great improvements in the clinical diagnosis and reproductive efficiency of large animals [5].

In this context, the objective of this chapter was to compile data from several studies addressing the use of US Doppler correlated with pathophysiological phenomena of equine reproduction.

### **2. Doppler ultrasonographic**

US Doppler is a method that provides real-time information on vascular architecture and hemodynamic aspects of blood vessels. It can determine the presence, direction and speed of blood flow, being subdivided into the categories of color Doppler (color flow and power flor) and pulsed Doppler [6].

In color flow Doppler US (UCF) is considered the classic examination of the color mode within US Doppler, which allows non-invasive assessment of the presence, direction, speed, and quality of blood flow. Two distinct colors are used, usually variations of red tones for positive flows and blue for negative flows representing the vascular blood perfusion of a structure, where the colored pixels indicate the direction of the blood cells in relation to the transducer [7–9].

In power flow Doppler US (UPF) is a variation of the UCF and has been used as the test of choice to evaluate the vascularization of the uterus and ovaries of mares and components of the testicular bag of the stallions [7, 9–15]. It is observed with this technique an increase of the sensitivity of display of the blood flow inside the tissue of 3 to 5 times, in comparison with UCF. The higher sensitivity allows the evaluation of vessels with a small diameter or slow flow that does not appear in a conventional color flow because of incompatible speed ranges and Doppler angles. [16]. Blood flow is generally characterized homogeneously by orange tones and the speed of flow is indicated in both modes by the intensity of the tones, the lighter the color the faster the flow and the darker the color the slower the flow [17]. In this way, the advantages of UPF are greater sensitivity to weak flow; effects of the Doppler angle on the Doppler frequency are ignored; and aliasing does not affect the display of colors [9].

The extent of vascularization of the color US Doppler can be estimated subjectively through the percentage of pixels (colored signs) of a tissue or objectively by counting the colored pixels via software [10].

In spectral Doppler US mode, the artery blood flow wanted is found and then the cursor ("gate") is positioned in the lumen of the artery to evaluate the sample volume [18]. The volume evaluations of the samples are reflected in graphs, called spectrum, which represent the speed of the blood flow of the artery in question for several times within the cardiac cycle or of an individual arterial pulse [19]. The brightness of the spectral trace, represented in the gray scale, is also used to represent the amplitude of each frequency component, indicating the amount of blood cells that pass at a particular speed [6]. Changes in the evaluated vessel may suggest changes in the spectral tracing and this fact may be indicative of physiological, systemic changes or of some disease present at the site [20].

In the quantitative analysis of the spectral Doppler US tracing, most devices have automatic configuration to automatically calculate the average of the displacement frequency or speed. The maximum point reached in the spectrum is called the systolic peak velocity (PSV) and the minimum point in the wave morphology is the value of

the final diastolic velocity (EDV). The medium flow can be calculated by multiplying the average speed by the vessel area [9, 20].

Hemodynamic indices, such as systole-diastole ratio, resistivity index (RI) and pulsatility index (PI), allow the comparison of flow during systole and diastole. RI and PI have a negative correlation with the vascular perfusion of the tissue irrigated by the artery in question, that is, the lower the RI and PI, the greater the vascular perfusion in the tissue supplied by that vessel [10]. The changes in these indexes help in the identification of stenosis, thrombosis and changes in vascular resistivity and parenchyma dysfunctions, or in the characterization of disease malignancy [6].

### **3. Use in mares reproduction**

### **3.1 Follicular dinamic**

The UCF has the potential to predict the follicular state (ovulatory or anovulatory future) of the dominant follicles during the transition period. During the anovulatory transition season, vascular changes in the walls of the future anovulatory follicle and of a future dominant ovulatory follicle were studied from 25 mm in 7 days after the follicle was 30 mm. The blood flow area was smaller for anovulatory follicles of dominant size than for ovulatory follicles at the time when blood flow determinations started at 25 mm [21, 22]. There is a hypothesis for anovulation that involves hormones and follicular angiogenesis during the transition period [23]. In this regard, pre-ovulatory vascular changes were compared between the first and the next ovulation of the year in 40 pony mares for 6 days before ovulation [24]. Although the area of blood flow from the follicle increased the day of ovulation in both groups, the results demonstrated that follicle vascularization and LH peak were attenuated before the first ovulation of the year, with no indication that estradiol was involved in the differences between the first and the last ovulation.

Regarding the blood flow of the preovulatory follicle, recent studies have shown a daily increase in vascularization of the dominant follicle wall as it matures and approaches the day of ovulation [22, 24–26]. However, on the day of ovulation, a few hours before ovulation, an abrupt decrease in blood perfusion was detected in the wall of the preovulatory follicle [9, 25].

Vascular perfusion is related to the peripheral area of the follicle and its diameter [19]. Therefore, research shows that a higher pregnancy rate is associated with a greater blood flow in the preovulatory follicle (POF). Because a higher percentage of Doppler signals was observed in the follicular wall and a reduction in the Doppler indices of RI and PI in the ovarian vessels in mares that impregnated in comparison to mares that did not impregnate [27].

Another study demonstrated that larger, well-vascularized POF produce larger corpus luteum (CL), with more blood flow and a higher systemic concentration of progesterone (P4), which can lead to a better uterine environment for the establishment of pregnancy. In addition, the repetition of the POF diameter value in individuals during spring and autumn may be estimate the best breeding time during the transition period. The lower blood flow of CL observed during the last estrous cycle of the reproductive season is another important finding of the study, which may clarify the luteal insufficiency of the transition period [28].

The morphology and vascularization of anovulatory hemorrhagic follicles (HAF) in mares and the endocrinology immediately prior to the formation of HAF were

studied in control and HAF groups [29]. The day of ovulation and the first day of HAF formation, as indicated by the turbidity of the follicular fluid, were defined as Day 0. The frequency of discrete ultrasound indicators in imminent ovulation gray scale and the diameter of the follicle on Day −1 did not differed between the groups with future ovulation and HAF. However, the circumference of the follicle wall of future HAF had more signs of color Doppler than in control mares [25].

### **3.2 Corpus luteum**

The UCF can be effective in identifying mares that fail or regress CL, before any decrease in P4 circulation, decrease in CL area, echogenicity changes in B-mode image or when uterine/cervical tone becomes apparent [30].

A daily assessment of CL between the day of ovulation and the eighth day after ovulation was performed by Romano et al. [15]. The authors observed that the CL area was weakly correlated with luteal vascular perfusion and plasma P4 concentrations. However, a positive correlation was observed between luteal vascular perfusion and plasma P4 concentrations. Furthermore, the number of colored pixels and the total pixel intensity were positively correlated with vascular perfusion and the plasma P4 concentration.

From a daily analysis, via UPF of the CL, a transient increase in the total luteal area was observed during the first days after ovulation, demonstrating that old mares had a greater luteal area than young mares between D2 and D8 and in D18-D19 (p < 0.05). However, old mares have a late increase in luteal vascularization during the first gestational days (p < 0.05). However, the CL of young and old mares showed similar and constantly high vascularization from D14. It has also been observed that the progressive increase in plasma P4 concentrations observed up to D8 was followed by a gradual decrease until to intermediate levels of P4. Thus, concluding that the newly formed CL of old mares underwent a compensatory structural remodeling to guarantee the local blood supply and the continuous output of P4 during early pregnancy to maintain it without suffering from the age effect [11].

To check if there could be a difference between the vascularization of the CL between the possible days for embryo transfer in the recipient mares, it was observed that the CL classified as adequate for the embryo transfer procedure by US mode B were also those classified with the better vascularization by UPF maintaining the pattern of proportional growth of the size of the CL with the concentration of P4 [31].

De Vasconcelos Azevedo et al. [32] evaluated the vascularization and function of CL of recipient mares of the Mangalarga Marchador embryo at the time of embryo transfer through UCF. As a result, they noticed that the mares that were pregnant showed a correlation with the increase in the vascularization of the CL, as well subjective methods as objective methods, and the plasma concentration of P4.

### **3.3 Uterine evaluation**

The hemodynamic evaluation of the uterus can be done by spectral data collected from the uterine arteries and their ramifications [33], or by subjective or objective assessments of the blood flow of the endometrium that provide data regarding local and specific changes in the evaluated area [8, 34].

The first investigations relating US Doppler to equine uterine physiology were carried out by Stolla and Bollwein [35], Mayer et al. [36] and Bollwein et al. [33], who obtained a cyclic pattern in uterine blood flow. Bollwein et al. [33] performed the

### *Doppler Ultrasound in the Reproduction of Mares DOI: http://dx.doi.org/10.5772/intechopen.98951*

measurement of the RI of the uterine artery on different days of the equine estrous cycle. They are verifying that there was no difference between the values of the left and right arteries. Not showing a correlation of the values found in the flow with the presence or absence of follicles or luteum corpus ipsilateral to the evaluated horn, indicating that the circulation is distributed equally to the two horns of the uterus. The authors also found that the average RI for all evaluated days was higher when observed in multiparous mares than when observed in nulliparous mares. This work also found significantly higher RI values on days 0 and 10 of the estrous cycle compared to days 5, 15 and 20 (considering D0 the day of ovulation). The authors correlated the lower RI value observed on day 5 to the possibility of an increase in blood supply to the uterus in this initial luteal phase due to the moment of embryo entry into the uterus.

Subsequent studies by Bollwein et al. [37], confirmed these statements, in addition to noting that the lowest uterine PI values were record during the initial diestrus, in a stage, when they found a peak in uterine tonus in mares. They speculated, therefore, that the tone and contractility of the uterus do not seem to be regulated by the uterine blood supply in cyclic mares.

Ferreira et al. [31] evaluated the flow of the uterine artery of recipient's mares on the day of embryo transfer. It was observed that in animals where the RI of the dorsal branches of the uterine arteries close to 1.0 are proportional to mares with greater vascularization of the corpus luteum and a high plasma P4 concentration. Being able to use these indices to select mares with greater aptitude for the development of the embryo when comparing two similar mares clinically by conventional ultrasound exams.

Other studies correlating the uterine flow of normal mares with subfertility characterized by biopsy endometrial were performed by Stolla and Bollwein [35] and Blaich et al. [38]. The authors show that in all mares there was an increase in vascular resistance in the pre-ovulatory phase and in 8 mares the peak occurred 8 days after ovulation. The blood flow impedance decreased from D1 after ovulation until it reached its lowest level during a luteal phase in all mares. They also observed that there was a gradual increase in vascular resistance in the initial follicular phase and that cyclical changes occurred in all mares with varying amplitudes. As they compared the flows according to the endometrial classifications, they observed that, for all mares with histological category IIb and III, there was an increase in the PI and average RI throughout the estrous cycle, when compared with the mares in the group that had normal fertility.

### *3.3.1 Uterine evaluation after artificial insemination*

Although adequate blood flow is essential for the normal functioning of the reproductive system [9], there are few in vivo studies that describe the uterine hemodynamics of non-pregnant mares [34, 39].

Changes in blood flow velocity verified by spectral Doppler US of the dorsal uterine arteries after reproduction, suggest that there is an increase in endometrial blood flow during semen transport and uterine clearance [40]. However, currently, the evaluation of the Doppler indices of the mesometrial arteries and the vascular perfusion of the uterine tissue during the pre- and post-reproductive periods and in mares with endometritis have been little researched.

Bollwein et al. [41] also evaluated, during three estrous cycles, the vascular perfusion of the uterine artery using the mean maximum velocity spectral Doppler index (MVM). The authors related the quantification of flow to the effect of the infusion

of semen extender, seminal plasma, or pure semen. In response to the dilution infusion, no effect on uterine blood flow was observed. Controversial results have been reported after one hour of infusion of seminal plasma or pure semen, where an increase in VMM values was observed in both uterine arteries. Therefore, the authors concluded that the increase in endometrial perfusion in these groups may be associated with inflammation and vasodilator components present in the seminal plasma.

Ferreira et al. [8] observed a transient increase in uterine vascular perfusion without mesometrial changes in the PI during the first 8 hours after artificial insemination. However, in the research by Ferreira et al. [42, 43], where PI Doppler measurements of the mesometrial arteries and UPF of the organ in relation to the semen effect were used, changes in the blood flow velocity of the uterine arteries were observed only in the first hour after the infusion of crude semen.

Ferreira et al. [42, 43] also evaluated uterine vascular perfusion before and after artificial insemination correlating with the age of the mares and the presence/ absence of endometrial degenerative processes. There were no differences in perfusion between the horns, however, they showed in the organ, an early and transient increase in the blood flow of the uterus in response to artificial insemination in all mares. However, the increase in mesometrial arterial resistance was strongly associated with severe endometrial degenerative changes after AI, regardless of age.

In horses, the AI procedure can be performed by depositing the semen in the body of the uterus or at the apex of the uterus horn to use a reduced inseminating dose. In order to assess the uterine inflammatory response to the different semen deposition sites, Araújo [44] analyzed the uterine perfusion by means of spectral Doppler US of the dorsal uterine arteries and with subjective UCF in the uterus. The values of RI and PI identified after AI were similar on the contralateral and ipsilateral sides of the mesometrial and dorsal uterine arteries. Regarding the evaluation time, a difference was observed where, both the RI and the PI, were lower in the moment before ovulation and AI and higher values were recorded in 24 h after. As for the analysis of organ perfusion by score, this did not show any difference within each experimental group regarding the evaluation time. Regarding the place of insemination, the color scores showed significant differences between the two experimental groups, with a predominance of scores 1.0 and 2.0 in the group of mares inseminated at the apex of the uterine horn, and the score 3.0 in the group inseminated in the body of the uterus.

### *3.3.2 Endometritis' diagnosis*

A research to measure uterine blood perfusion using UCF (subjective and objective) was carried out by Sá et al. [45] to investigate changes in vascularization of uterine segments of mares after intrauterine inoculation of E. coli. The authors found significant differences in the blood flow evaluated before inoculation of the bacteria (M0) and 24 h after inoculation (M1), where blood perfusion in M1 showed almost twice the M0 in the three uterine segments evaluated, but no significant differences were found in evaluations carried out between the follow-ups. In four animals evaluated, endometrial cytology and mode B ultrasound examinations were not sufficient to detect uterine infection, however, through UCF, it was possible to verify a significant increase in uterine vascularization in these mares compared to the values before inoculation.

In a second experiment carried out by Sá et al. [45], the evaluation by the UCF was performed 24 h after the intrauterine infusion of E. coli. Half of the mares with

### *Doppler Ultrasound in the Reproduction of Mares DOI: http://dx.doi.org/10.5772/intechopen.98951*

uterus inoculated by bacteria were subjected to a treatment consisting of a uterine lavage using 50 mL of the Fitoclean® phytotherapy solution (Organnact Saúde Animal, Brazil) diluted in 950 mL of Ringer with Lactate and subsequent intrauterine infusion with 40 mL phytotherapy solution Fitoclean® diluted in 60 mL of Ringer with Lactate. In the control group, an intrauterine wash was performed with Ringer's serum with pure lactate, with subsequent infusion of 100 mL of the same substance. UCF was performed before treatment (A), 24 h (B) and 48 h (C) after treatment. The authors reported that uterine perfusion was greater at time A than at time B and C, but the decrease in uterine bacterial load was not verified. The authors assume that the decrease in vascularization in the post-treatment groups can be attributed to the vasoconstriction caused by the reaction of components with anti-inflammatory properties present in the product, which may have caused the decrease in local perfusion even with the presence of microorganisms.

A third research was carried out in the study by Sá et al. [45]. The mares in which pathogenic agents were identified in the samples collected 10 days after the end of the second mentioned experiment, were subjected to antibiotic therapy by intrauterine infusion of 100 mL Gentamicin (Gentrin® Uterine Infusion, Ourofino Saúde Animal, São Paulo, Brazil) for three days, according to the sensitivity shown in the antibiogram. UCF was performed seven days after treatment, comparing the tests performed before E. coli pre-inoculation, post-treatment with Fitoclean® and one week after antibiotic therapy (M0, M1 and M2, respectively). The authors observed a reduction in vascularization in the group treated with Fitoclean® after antibiotic therapy, however, blood perfusion in this group was still greater than M0, even with antibiotics being able to eliminate the bacteria present. Based on the results obtained in this study, it was possible to identify acute endometritis through the UCF, but the vascular perfusion identified did not correlate with the uterine laboratory tests performed.

Abdelnaby et al. [46] performed spectral Doppler ultrasonography of the dorsal uterine arteries and UCF of the uterus of mares with and without endometritis, correlating the data with the impact of the pathology on the oxidative and hormonal state. The results revealed a significant increase in the metabolites of estradiol, malondialdehyde and nitric oxide associated with a significant decrease in progesterone and total antioxidant capacity in the endometritis group. The uterine blood flow analyzed by the UCF showed a significant increase in the endometritis group, while the spectral mode showed a significant increase in the PSV and TAMV indices and in the blood flow area rate accompanied by a significant decrease in the PI and RI Doppler indices. In addition, the elevated uterine blood perfusion was correlated with the accumulation of fluid inside the uterus, with a marked difference between the uterine horns in relation to the size of the UCF staining area, which may be due to the marked increase in fluid accumulation in the right in endometritis group.

Morais [13] evaluated, through UPF, the intensity of colored pixels (IPC) of the uterine segments (body and horns) during estrus before and after uterine treatment with DMSO. From the evaluations it was noted that mares with negative cytology (despite positive culture test) had less blood flow than mares with positive cytology. IPC was reduced in mares that became pregnant. In the mares that remained empty, the CPI remained high. Thus, UPF can be used as an auxiliary diagnostic method in some cases of equine endometritis. In pregnant mares, blood flow in the endometrium and RI decreased. Empty mares blood flow in the endometrium and RI remained high or increased.

### **3.4 Doppler ultrasonography in pregnancy diagnosis**

Changes in the hemodynamics of the equine reproductive tract during early and late pregnancy have already been described several times in the literature. Bollwein et al. [40] to compare the vascularization of the right and left uterine horns of cyclical mares and in initial pregnancies, observed that on days 11 and 15 to 29 of gestation, mean values of blood flow velocity were higher and RI lower in both horns uterine of pregnant mares compared to empty mares.

Subsequently, Bollwein et al. [47] investigated the blood flow in both uterine arteries (ipsilateral and contralateral to the fetus) every four weeks, from the second week until the moment of delivery. A highly significant regression was observed in the RI averages according to the week of gestation, reaching, at the end of the evaluations, values lower than half of those initially recorded (0.89 ± 0:01 to 0.39 ± 0:03). The volume of blood flow in the ipsilateral and contralateral uterine horns increased significantly according to the week from the middle of gestation.

In other research, it was shown that the transient changes in vascular perfusion accompany the mobility and the fixation of the embryonic vesicle [34]. Pregnant and non-pregnant mares have similar and low endometrial vascularity in the first eight days after ovulation. However, from D11 there was a gradual increase in the volume of blood flow in both uterine horns during the embryonic mobility phase and a higher speed in blood flow in the uterine horn of fixation of the conceptus, in relation to the opposite side.

Silva and Ginther [29] observed, through UCF, an early vascular indicator of the future position of the embryo itself, which consisted of a colored point in the image of the endometrium close to the wall of the embryonic pole. The early indicator was detected in each mare 0.5 ± 0.1 days after fixation and 2.5 ± 0.2 days before the first visible embryo detection. The author also reports that by US Doppler we can monitor the inadequate early orientation of this embryo. Studies have shown that this type of problem is correlated with a flabby uterus and a defective embryonic dorsal invasion of the endometrium. However, the asymmetric increase in the allantoic sac can spontaneously correct the disorientation, so that the orientation for the formation of the umbilical cord is in a normal position around 12 o'clock.

Using spectral-mode US Doppler, Chen and Stolla [48] developed the uterine index (UI) to predict embryonic death in mares. The calculation used by the authors considers the following formula: UI = (RI-p - RI-np) x100, where RI-p is the RI of the uterine artery on the side of the pregnant uterine horn and RI-np is the RI of the artery uterine horn of the non-pregnant uterine horn. According to the authors, UI less than five is indicative of embryonic death evident in the next 24 hours, while mares with UI more than 10 did not present any apparent hemodynamic disorder.

Ferreira et al. [12] observed that the uterine doppler indices (RI and PI) of pregnant mares decreased progressively. Unlike other studies, this research also observed, through measurements of mesometrial RI and PI, an increase in vascular perfusion between D3 and D6 post-OV. This study was one of the triggers that helped future inquiries regarding the use of the technique as an early pregnancy diagnosis and determination of the ideal moment for embryo collection, especially when aimed at cryopreservation.

Ousey et al. [49] evaluated the blood flow of the uterine artery and other Doppler indices during pregnancy to compare placental and fetal development in young and elderly mares. However, no difference was found in the evaluated indexes. Thus, the authors stated that the similarity in the Doppler indices between the groups of

### *Doppler Ultrasound in the Reproduction of Mares DOI: http://dx.doi.org/10.5772/intechopen.98951*

elderly and young mares reflects the absence of severe pathological changes in the endometrial vascularization and glandular tissue found in the elderly mares used. However, employing other types of blood perfusion measurements, it has already been observed that mares with diffuse endometrial degeneration had reduced uterine vascular perfusion when compared to pregnant mares with unchanged endometrium. The hypothesis was then raised that severe angiosis can reduce the capacity of the vessels to adapt to the varied demands of uterine circulation [50]. In addition, endometrial pathological changes have been strongly associated with degeneration of uterine vessels in mares [51].

Ferreira et al. [42], used in their research, in addition to the analysis of uterine perfusion with UPF, a spectral analysis of the insertion of uterine arteries in the mesometrium, which is also reported to be an efficient method for the objective examination of uterine blood flow during the first 20 days gestation. With this methodology, the authors detected early and transient increases in uterine blood flow in pregnant mares, regardless of age and presence of endometrial changes. The increase in uterine blood flow in the initial pregnancy described by this and other authors, may be caused by the effects of vasoactive factors sought by pre-implanted embryos, as described in horses [34].

Another analysis carried out in the study by Ferreira et al. [42], was an observation of the negative effect of age and endometrial degeneration without uterine blood flow from mares during early pregnancy. The authors associated this apparent inability of the aged uterus to respond to vasoactive factors derived from the embryo with the progressive changes in the architecture of the uterine vascular network observed in older mares. In addition, it has already been added that age-related degenerative changes in the endometrium can affect the development of placental microcotyledons and their associated blood flow [49].

With UCF it is also possible to determine fetal equine sex between 90 and 180 days of gestation by observing fetal gonads [52]. This assessment was later confirmed by Pricking et al. [53], who obtained high rates of sex determination accuracy when associated with Us mode B, color Doppler and 3D transabdominal tomography ultrasound techniques. In addition, the application of this new 3D imaging ultrasound technology has enabled the diagnosis of gender in 18 cases in which B-mode and Doppler ultrasonography have shown dubious results.

In relation to the gestation of hybrids, pregnant donkey showed greater uterine vascularization in the uterine horn contralateral to that of embryonic fixation. And that older mares showed less blood flow in the uterine artery, as they had higher PI and RI [54]. Another difference observed was comparing the PI of the fetal umbilical cord artery, which showed a statistical difference in pregnant with donkey semen when compared to pregnant with stallion semen [55].

Nieto-Olmedo et al. [14] detected differences in uterine vascular perfusion between pregnant and non-pregnant mares early between days 7 and 8 post-ovulation, demonstrating that the UPF associated with computerized analyzes is an effective method for the early diagnosis of pregnancy. The authors observed that the area of vascular perfusion of the uterus (mm2 ) and pixel intensity increases in pregnant mares compared to mares without embryonic recovery. The technique can be used in routine clinical practice to maximize the embryo recovery rates of donor mares and to predict the diagnosis of pregnancy before embryo collection.

Relationship of uterine vascular perfusion associated with involution of the postpartum uterus was mentioned in the study by Lemes et al. [56] using the UCF mode of uterine segments. The authors observed that vascular perfusion increased in endometrial and mesometrial tissues in the first 2 to 4 days after delivery, followed by a progressive decrease until the second week postpartum. The profile of vascular perfusion in the uterus described after the first postpartum ovulation is similar to that observed during the estrous cycles and initial gestation observed previously, indicating a rapid return of the uterus to the pre-gestational uterine characteristics in the mares. In such cases, it has been reported that the rapid reduction in uterine diameter, absence of intrauterine fluid and vascular decrease in the layers of the uterus, may indicate a favorable uterine environment for the development of the embryo in the foal's heat.

### **Author details**

Camila Silva Costa Ferreira\* and Rita de Cássia Lima Morais Federal Rural University of Rio de Janeiro, Seropédica, RJ, Brazil

\*Address all correspondence to: vetcamilaferreira@gmail.com

© 2021 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.

*Doppler Ultrasound in the Reproduction of Mares DOI: http://dx.doi.org/10.5772/intechopen.98951*

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### Section 3
