*2.1.1 Follicular dynamics*

Ovarian follicular development is a complex dynamic process, characterized by marked proliferation and differentiation of follicular cells, providing an optimal environment for oocyte maturation and preparation for fertilization after ovulation [9]. Among the recruited follicles in each follicular wave, dominance takes place and one follicle of the cohort acquires the ability to continue growing while others undergo atresia. The regulation of each wave and follicular selection involves interactions between specific circulating gonadotropins and intrafollicular factors, ensuring that each follicle is properly stimulated to grow or regress at any stage of development [8]. From an experimental point of view, the occurrence of a wave is defined as follicular growth or simultaneous emergence of a variable number of follicles below 6–13 mm in diameter [10, 11]. In the mare, these follicular waves are classified depending on their ability to develop the dominant follicle (primary waves) or, in contrast, generate only small follicles (smaller waves). Thus, the main waves or greater originate several follicles subordinate and a dominant follicle, while smaller waves, the follicles are not larger than 30 mm in diameter and then regress [12, 13].

During each cycle produces 1 or 2 major follicular waves, differentiated according to time of onset at primary and secondary. The primary major wave occurs near the middle of the diestrus, in which the dominant follicle ovulates at the end or near the end of estrus. The largest wave precedes the previous secondary and emerges during late estrus or early diestrus. There are two anovulatory follicular waves followed by an ovulatory surge during the estrous cycle [14, 15].

**5**

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy…*

Steroidogenesis in the ovaries involves both theca and granulosa cells. The antral follicles acquire receptors for follicle-stimulating (FSH) and luteinizing (LH) hormones in the membranes of the granular cells and theca, respectively. Cholesterol passes through theca cell plasma membrane attached to a lipoprotein, is stored in cytoplasmic vacuoles, and is transported to the outer membrane of the mitochondria. The LH is released in a pulsating form from the anterior pituitary gland and binds to its receptor in the theca cell membrane, mobilizing cholesterol. Inside theca cells, the StAR protein helps transfer cholesterol to the internal mitochondrial membrane, where the cytochrome P450 (CYP) enzyme system divides cholesterol into pregnenalone (P5), and subsequently, P5 becomes to androstenedione (A4). The A4 produced in theca cells is transported through the basal membrane to the granulose cells. There FSH supports the steroidogenic pathway and converts A4

Increased concentrations of estrogen stimulate the secretion of LH, which in turn induces greater estrogen synthesis. This progressive increase in estrogen also promotes the onset of LH receptors in granulosa cells, which facilitates the transition from the antral stage to the preovulatory stage, when the oocyte reaches the final stage of maturation. At 6 days after the emergence of major follicular wave deviation occurs. This event relates to the growth rate difference of the preovulatory follicle size (22.5 mm) compared to the subordinate follicles (19 mm) [12, 13, 17]. Deviation is related to inhibin secretion [12] and i*nsulin-like growth factor-1* (IGF-1) [13, 17]. Specifically, inhibin reduces FSH secretion, making it impossible to continue the development of the subordinate follicle*.* However, the dominant follicle continues to grow at a constant rate of 2.3 mm per day until reaching a size of 40 mm in response to the increased sensitivity to FSH. As has been mentioned, at this stage of development, granulosa cells also develop receptors for LH required for

As has been demonstrated in different horse breeds such as Quarter Horse, Arabian, Thoroughbred, and Spanish Purebred, the maximum diameter of the ovulatory follicle usually varies between 40 and 45 mm [19], although the range may be higher (30–70 mm) [7, 20]. Moreover, size differences were established concerning the breeding season or the presence of multiple ovulations. Thus, the follicles reach a size 5–8 mm higher in spring than in summer or autumn and are 4–9 mm lower in

The highest concentrations of estrogen secreted by the granulosa cells of the preovulatory follicle also induce the appearance of typical behavioral manifestations of estrus. Estrogens are also responsible for reproductive changes that ensure the reception, transport of sperm and oocyte fertilization [4, 6]*.* After the preovulatory LH surge, ovulation occurs spontaneously 24–48 h before the end of the follicular phase. The ovulatory process brings rapid evacuation of the oocyte and follicular fluid after follicular rupture at ovulation fossa. Once completed, E2 concentrations return to basal levels and at the same time completing the oestrus

The diestrus or luteal phase begins at the time of ovulation with the formation of CL, which is responsible for the synthesis of P4. Unlike the follicular phase, the insensitivity of the corpus luteum (CL) photoperiod makes the length of this period more constant. Most research estimates an average duration of 14–15 days but can be more durable in mid-summer (16 days) than in spring or autumn

final oocyte maturation and ovulation after the LH surge [18].

multiple ovulations compared to the simple [20, 21].

behavior in mares [11, 22–24].

**2.2 Luteal phase**

(13 days) [5, 6].

*DOI: http://dx.doi.org/10.5772/intechopen.90387*

into E2 [16].

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy… DOI: http://dx.doi.org/10.5772/intechopen.90387*

Steroidogenesis in the ovaries involves both theca and granulosa cells. The antral follicles acquire receptors for follicle-stimulating (FSH) and luteinizing (LH) hormones in the membranes of the granular cells and theca, respectively. Cholesterol passes through theca cell plasma membrane attached to a lipoprotein, is stored in cytoplasmic vacuoles, and is transported to the outer membrane of the mitochondria. The LH is released in a pulsating form from the anterior pituitary gland and binds to its receptor in the theca cell membrane, mobilizing cholesterol. Inside theca cells, the StAR protein helps transfer cholesterol to the internal mitochondrial membrane, where the cytochrome P450 (CYP) enzyme system divides cholesterol into pregnenalone (P5), and subsequently, P5 becomes to androstenedione (A4). The A4 produced in theca cells is transported through the basal membrane to the granulose cells. There FSH supports the steroidogenic pathway and converts A4 into E2 [16].

Increased concentrations of estrogen stimulate the secretion of LH, which in turn induces greater estrogen synthesis. This progressive increase in estrogen also promotes the onset of LH receptors in granulosa cells, which facilitates the transition from the antral stage to the preovulatory stage, when the oocyte reaches the final stage of maturation. At 6 days after the emergence of major follicular wave deviation occurs. This event relates to the growth rate difference of the preovulatory follicle size (22.5 mm) compared to the subordinate follicles (19 mm) [12, 13, 17]. Deviation is related to inhibin secretion [12] and i*nsulin-like growth factor-1* (IGF-1) [13, 17]. Specifically, inhibin reduces FSH secretion, making it impossible to continue the development of the subordinate follicle*.* However, the dominant follicle continues to grow at a constant rate of 2.3 mm per day until reaching a size of 40 mm in response to the increased sensitivity to FSH. As has been mentioned, at this stage of development, granulosa cells also develop receptors for LH required for final oocyte maturation and ovulation after the LH surge [18].

As has been demonstrated in different horse breeds such as Quarter Horse, Arabian, Thoroughbred, and Spanish Purebred, the maximum diameter of the ovulatory follicle usually varies between 40 and 45 mm [19], although the range may be higher (30–70 mm) [7, 20]. Moreover, size differences were established concerning the breeding season or the presence of multiple ovulations. Thus, the follicles reach a size 5–8 mm higher in spring than in summer or autumn and are 4–9 mm lower in multiple ovulations compared to the simple [20, 21].

The highest concentrations of estrogen secreted by the granulosa cells of the preovulatory follicle also induce the appearance of typical behavioral manifestations of estrus. Estrogens are also responsible for reproductive changes that ensure the reception, transport of sperm and oocyte fertilization [4, 6]*.* After the preovulatory LH surge, ovulation occurs spontaneously 24–48 h before the end of the follicular phase. The ovulatory process brings rapid evacuation of the oocyte and follicular fluid after follicular rupture at ovulation fossa. Once completed, E2 concentrations return to basal levels and at the same time completing the oestrus behavior in mares [11, 22–24].

#### **2.2 Luteal phase**

The diestrus or luteal phase begins at the time of ovulation with the formation of CL, which is responsible for the synthesis of P4. Unlike the follicular phase, the insensitivity of the corpus luteum (CL) photoperiod makes the length of this period more constant. Most research estimates an average duration of 14–15 days but can be more durable in mid-summer (16 days) than in spring or autumn (13 days) [5, 6].

*Animal Reproduction in Veterinary Medicine*

mones in mediating pathological processes.

the levels of oestradiol (E2) and P4 [6, 7].

of sperm and finally culminates with ovulation [5, 6, 8].

**2.1 Follicular phase**

*2.1.1 Follicular dynamics*

Since an understanding of endocrinology in equine species is useful when considering hormone treatment of cyclic and pregnant mares, this chapter considers a basic review and applications of this information in clinical therapeutic situations. For this reason, this chapter aims to provide an overview of the endocrine changes that occur in the mare in response to gestation and to discuss the key role of hor-

**2. Neuroendocrine control of the estrus cycle in cycling mares**

The estrous cycle is defined as the interval of time between two consecutive ovulations. The approximate length varies between 18 and 22 days, considering on average a period of 21 days [4, 5]. The current nomenclature stipulates that the estrous cycle consists of two differentiated stages: estrus or follicular phase and diestrus or luteal phase. These phases are characterized by internal modifications of the sexual organs and glandular system as well as behavioral alterations based on

Estrus, heat or follicular phase is characterized by the presence of follicles at different stages of development, and the simultaneous increase in the secretion of E2. It has a duration of about 5–7 days, with a variability of 3–9 days related to the season. Thus, estrus is extended in autumn (7–10 days) and is shortened considerably, in late spring and early summer (4–5 days). During this period the mare is sexually receptive to the stallion genital tract and is ready to receive and transport

Ovarian follicular development is a complex dynamic process, characterized by marked proliferation and differentiation of follicular cells, providing an optimal environment for oocyte maturation and preparation for fertilization after ovulation [9]. Among the recruited follicles in each follicular wave, dominance takes place and one follicle of the cohort acquires the ability to continue growing while others undergo atresia. The regulation of each wave and follicular selection involves interactions between specific circulating gonadotropins and intrafollicular factors, ensuring that each follicle is properly stimulated to grow or regress at any stage of development [8]. From an experimental point of view, the occurrence of a wave is defined as follicular growth or simultaneous emergence of a variable number of follicles below 6–13 mm in diameter [10, 11]. In the mare, these follicular waves are classified depending on their ability to develop the dominant follicle (primary waves) or, in contrast, generate only small follicles (smaller waves). Thus, the main waves or greater originate several follicles subordinate and a dominant follicle, while smaller waves, the follicles are not larger than 30 mm in diameter and then

During each cycle produces 1 or 2 major follicular waves, differentiated according to time of onset at primary and secondary. The primary major wave occurs near the middle of the diestrus, in which the dominant follicle ovulates at the end or near the end of estrus. The largest wave precedes the previous secondary and emerges during late estrus or early diestrus. There are two anovulatory follicular waves fol-

lowed by an ovulatory surge during the estrous cycle [14, 15].

**4**

regress [12, 13].

## *2.2.1 Formation of corpus luteum*

The disorganization of the follicular wall after ovulation allows blood vessels and fibroblasts invade the follicular cavity. Luteinization involves structural and functional changes in granulosa and theca cells. These are the same cells that initially produced E2 and become into luteal cells that produce P4. P4 remains high from day 5 post-ovulation until the end of the diestrus and exerts specific functions related to the preparation of the endometrium to accept and maintain pregnancy, endometrial gland development and inhibition of myometrial contractility [24].

Have been described two types of CL regarding the presence or absence of central blood clot. In a high percentage of cases (50–70%) in place of ovulation, a core clot develops surrounded by luteal tissue. This type of condition is defined as a corpus hemorrhagic. The cavity begins to fill with blood, fibrin, and transudate for the first 24 h, reaching the maximum size at 3 days. Around day 5 post-ovulation CLs that develop a central cavity usually, have a significantly higher size (32.8 mm) to those without it (26.0 mm). The ratio of the maximum diameter of the CL is 65–80% compared to pre-ovulatory follicle size and has an outer wall thickness of 4–7 mm corresponding to the portion of luteinized tissue. As happens with the size, texture also changes depending on the type of CL. The CL that develops the central cavity is denser than those that lack it, in which the structure is more spongy [25]. Usually, the ratio of non-luteal luteal tissue of the corpus hemorrhagic is minimal during the early diestrus and maximum in halfway of diestrus. These events are associated with the gradual decrease of fluid as a result of the production and organization of connective tissue associated with the clotting mechanism [26, 27]. Notably, the formation of one type or another of CL is a random event. The morphology luteal repeatability is not always observed in subsequent ovulation [26–28].

Furthermore, continuous P4 levels during diestrus reduce the frequency and intensity of gonadotrophin-releasing factor (GnRH) pulses by a feedback mechanism. However, because the pulses of FSH are higher than those of LH, a new follicular wave is developed during this period. In the absence of pregnancy, the end luteal phase culminates with the lysis of CL induced by the PGF2α of endometrial origin and decreased concentrations of P4 [5, 6]. Luteal regression involves several structural and functional events characterized by decreased vascularization, an increase of connective tissue, hyalinization, atrophy and fibrosis [29].

#### **2.3 Neuroendocrine control of the estrus cycle**

Physiological events that occur during the estrous cycle are regulated by the coordinated interaction of various hormones and releasing factors like GnRH, FSH, LH, E2, P4, and PGF2α, among others [22]. In this section we will describe a synthesis of the most notable changes and the physiological participation that all these factors have during the estral cycle in the mare.

#### *2.3.1 Gonadotrophin releasing factor*

The increased photoperiod during spring and summer causes decreased secretion of melatonin. This signal has a positive effect on the pulses of hypothalamic GnRH, which in turn controls the release of gonadotropins [27]. GnRH pulses produced every 45 min originate predominantly LH secretion whereas those occur every 6 h stimulate the secretion of FSH. The high-frequency pulses of GnRH (2 pulses per hour) during estrus favors an increase in LH and FSH decline, while reducing the frequency to 2 pulses per day, leads an increase of FSH and LH inhibition [30]. These endocrine events, allowing the emergence of follicular waves, E2

**7**

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy…*

synthesis, and ovulation during estrus and appearance of the CL with P4 release

Follicle-stimulating hormone describes two types of secretion patterns during the estrous cycle in the mare: uni or bimodal. The bimodal pattern occurs frequently during the spring transition period and the ovulatory season. The first peak of FHS appears between the 8th and 14th day of the cycle, the moment in which the largest follicle reached a diameter of 13 mm [18]. This initial increase precedes the beginning of the deviation and is associated with increased synthesis of inhibin by the largest follicle [8, 13, 15, 18, 31] and persists until the preovulatory follicle reaches 22 mm of diameter. The second peak of FSH begins on day 15 of the cycle and it is necessary to complete the development of the preovulatory follicle [19, 31]. Unlike the bimodal pattern, the first peak of FSH would be absent in the unimodal pattern [18]. In the latter pattern, FSH levels remain low during estrus, rise in times around

FSH is also involved in the development of the LH receptors in the preovulatory follicle [32, 33]. At the start of follicular growth, low levels of estradiol exert negative feedback on the hypothalamic-hypophysis axis (HHA) controlling the tonic or basal release of gonadotropin. This mechanism controls the follicular growth and E2 synthesis continuously preventing ovarian overstimulation. After the period of follicular growth, once the dominant follicle has been selected, the E2 and inhibin levels are significantly increased. This elevation of E2 is responsible for the characteristic changes of the genital tract and signs of heat during estrus. Furthermore, this response exerts positive feedback on the HHA, favoring the emergence of preovulatory LH surge, necessary to produce the ovulation. Additionally, the stimulatory effects of E2 on LH combined to the inhibitory action of inhibin on FSH create the ideal microenvironment for the final maturation of the oocyte, inhibiting

LH levels gradually increase from day 5 to the day of ovulation, when it reaches the maximum concentration [7, 34]. The pre-ovulatory LH surge occurs as a result of the positive feedback mechanism exerted in the adenohypophysis by E2 concentrations secreted by the granulosa cells of the preovulatory follicle. However, the peak of E2 is reached 2 days before the LH surge. During diestrus, LH is released in a pulsatile manner, with a frequency of 1.4 pulses per 24 h and for a period of 20–40 min at the central level, or 2–4 h per pulse at the peripheral level [34]. Therefore, P4 secretion is maintained by basal levels of LH. The decline of LH at the end of diestrus is a result of the combined effect of decreased estrogen positive feedback, and the resurgence of negative feedback induced by P4 on the HHA. This gonadotropin not only participates in the development and maturation of the primary follicles but also in the development and maintenance of CL during the

The ability of estrogen synthesis is dependent on the effect of FSH on granulosa cells. In the absence of P4, estrogens begin to be actively secreted by the preovulatory follicle 5–7 days before ovulation. This event coincides with the time of departure and reaches the peak 2 days before ovulation [5, 22], and will be responsible for

*DOI: http://dx.doi.org/10.5772/intechopen.90387*

ovulation, maintaining increased during diestrus [31].

the development of immature follicles [4].

*2.3.3 Luteinizing hormone*

luteal phase [8, 13, 22].

*2.3.4 Estradiol-17β*

during diestrus [24].

*2.3.2 Follicle stimulating hormone*

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy… DOI: http://dx.doi.org/10.5772/intechopen.90387*

synthesis, and ovulation during estrus and appearance of the CL with P4 release during diestrus [24].
