*2.3.2 Follicle stimulating hormone*

*Animal Reproduction in Veterinary Medicine*

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].

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

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

**2.3 Neuroendocrine control of the estrus cycle**

factors have during the estral cycle in the mare.

*2.3.1 Gonadotrophin releasing factor*

*2.2.1 Formation of corpus luteum*

**6**

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 ovulation, maintaining increased during diestrus [31].

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 the development of immature follicles [4].

#### *2.3.3 Luteinizing hormone*

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 luteal phase [8, 13, 22].

#### *2.3.4 Estradiol-17β*

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 the preovulatory release of LH. After ovulation, E2 levels begin to decrease, reaching basal levels at day 5 post-ovulation [13, 19].

Although estrogen levels are directly related to the degree of ovarian activity, sexual receptivity and reproductive tract changes [4, 6, 13, 31, 35] there is no evidence of a direct relationship between the intensity of endometrial edema and E2 concentration. This situation is much clearer on P4. Swelling occurs when P4 levels are <1 ng/ml, so this hormone could be responsible in principle on the intensity of edema, among other behavioral and morphological changes of the cervix and uterus [35]. However, at the time of ovulation inverse correlations are established between E2 and FSH levels associated with the negative feedback effect of inhibin, as previously referred [31].

#### *2.3.5 Progesterone*

The steroidogenic activity of P4 depends on the action of LH on theca cells. As noted above, levels of P4 are <1 ng/ml during estrus [19, 36]. After ovulation, it increases progressively and significantly to the 5th or 6th day, with values similar to those of pregnant mares during the first 14 days of gestation. At this time the CL is fully functional and P4 levels remain high until day 9 [35, 37], consistent with the maximum diameter reached by the CL [7, 20, 35, 37]. However, peripheral concentrations of P4 are highly variable between mares. This variability is associated with secretory capacity CL and hormonal catabolic rate. Perhaps this fact may explain the differences in P4 levels between different breeds during the first 5 days of the luteal period*,* despite the similarity in length of estrous cycles. Among other factors related to variations in levels of P4 highlights the number of ovulations. In fact, double ovulations induce higher concentrations of P4 compared to simple ones [35].

P4 inhibits the secretion and pulsatile release of GnRH and LH but does not modify the pattern of FSH [7, 13, 15]. This event, unlike what happens in other species, enabling a new wave of follicular growth and in some cases the presence of ovulation during diestrus related to high levels of this hormone [18, 22, 38]. After lysis of the CL at the end of diestrus, P4 is drastically reduced to levels <1 ng/ml, a fact which promotes the mare returns to estrus [19, 36].
