Puberty in Females and Estrous Cycle

Puberty is the ability of the animal to produce gamete, i.e. ovum in females. The onset of puberty results from integrated sequences of biological events that lead to progres­sive maturation of sexual characteristics to attain full repro­ductive capacity.

22.2.1 Factors Affecting Puberty

The attainment of puberty depends on the maturation of the hypothalamic-pituitary-ovarian axis, and the HPO axis matu­ration requires adequate body weight and age. Thus, the factors which can alter the maturation of the hypothalamic- pituitary-ovarian axis are the genetics of the animals, nutritional level, energy metabolism, appropriate body size, body weight environment, and photoperiod.

Generally, females attain puberty earlier than males of the same species, with minor exceptions. In the male rabbit, the onset of puberty is earlier than in males.

22.2.1.1 Breed, Age, and Body Weight

The functionality of the HPO axis is highly related to the adequate somatic growth of the animal. Hence, age and body weight play a significant role in achieving reproductive effi­ciency. However, it is species specific and varies between breeds of a particular species (Table 22.8). The age of puberty is moderately heritable in all species. In cattle and buffaloes, it ranges between 0.16-0.57 and 0.24, respectively. In gen­eral, the puberty occurs in bovine species at 11 months (male 7-18 and female 9-24); in ovine, 7 months (male 6-9 and female 4-14); in porcine, 7 months (male 5-8 and female 5-7); in equine 14 months (male 10-24 and female 12-19); canine 9 months (male 5-12 and female 6-24) and feline 9 months (male 8-10 and female 4-12). Usually, puberty occurs when an animal reaches 55-60% of its adult body weight in beef cattle, 30-40% in dairy cattle, 60-65% in buffalo, 40-65% in sheep, 40-60% in goat, 40-50% in pig, 60-65% in horse, and 40-60% in the dog.

22.2.1.2 Nutrition

Plane of nutrition, particularly protein and energy and cal­cium and phosphorus in the ration, directly influence the HPO axis. Lack of optimum level of nutrition inhibits ovarian follicular development and steroidogenesis and reduces the synthesis and release of GnRH, FSH, LH, and GH. Several endocrine factors link energy metabolism and reproduction. Leptin, produced from adipose tissue, skeletal muscle, stom­ach, mammary tissue, placenta and pituitary, activates the GnRH pulse generator in heifers, rodents, and other non-ruminants to activate the HPO axis.

Fig. 22.6 Control of gonadotropin-releasing hormone secretion and commencement of puberty in cattle. The upper part of the graph shows the estrogenic control on GnRH secretion of (gonadotropin­releasing hormone) in the hypothalamus with the occurrence of puberty, and the lower part of the figure illustrates the secretory pattern of GnRH. During pre-pubertal periods, the tonic GnRH centre comprises an arcuate nucleus (ARC) with kisspeptin (KNDy = kisspeptin/neurokinin B/dynorphin) neurones is under the negative control of estrogen released from the non-ovulated follicles of the ovary during folliculogenesis process. It facilitates the secretion of low-frequency GnRH from the GnRH neurone (negative feedback of estrogen, depicted by a dotted curved arrow). After puberty, the tonic centre becomes non-sensitive to estrogen feedback, and GnRH is secreted at a high frequency (shown as a bold-faced curve-linear graph in the lower part of the figure). At the same time, the surge centre comprising POA/AVPV (pre-optic area/anteroventral periventricular nucleus) Kisspeptin neurones is sensitised with the estrogens (positive feedback of estrogens, depicted with a bold-face curved arrow) at the onset of puberty. It causes sudden spikes (surges) of GnRH secretion from the GnRH neurone (shown in the lower part of the figure). The positive feedback of estrogens causes activation of the hypothalamic- pituitary-ovarian axis and the profuse release of estrogen results in ovulation. The events of pulsatile release of GnRH inform the tonic and surge centres before and after puberty and are presented at a scale of 20 days on the X-axis of the graph, considering one estrous cycle of cattle.

and it increases food intake and storage of energy as fat and reduces anxiety and stress.

22.2.1.3 Environment

Season, particularly the season of birth, and photoperiods are two major factors that modulate puberty and sexual maturity.

22.2.1.3.1 SeasonofBirth

The young born during autumn and winter attains puberty earlier than the young born during spring. It is due to the long photoperiod and fodder availability in autumn and winter, favouring growth. The high environmental temperature in summer reduces the activity of gonads. Sheep are usually bred in autumn, and young are born in spring. Lambs born in spring and autumn will attain puberty earlier than summer- born lambs. The autumn and winter (September to February) are the buffalo’s breeding season, and the calving season is spring (July to November). Puberty occurs earlier in spring- born buffalo calves than in their summer-born mates. Horse favours long-day period for breeding and foaling as well. In the horse, the frequency of FSH and LH pulses is more in early spring and decreases gradually and becomes lowest during winter.

22.2.1.3.2 Photoperiod

Photoperiod means daylight exposure, affecting puberty onset in seasonal breeders. Long-day photoperiod consists of a maximum of 16 h of day length, and short-day

Table 22.8 Physiological age and body weight at puberty of various breeds of domestic animals

Source: Data compiled from various sources

photoperiod denotes a maximum of 8 h of daylight exposure.

Seasonal breeders are mostly found in the temperate zone (60-70° N/S), where photoperiod alters the reproduction in animals. The effect of photoperiod on reproduction is less evident in the domestic animals closer to the equator, where the daylight and dark phases are almost equal.

22.2.1.3.3 Seasonal Breeder

A seasonal breeder shows reproductive activity during a particular season and maintains reproductive quiescence dur­ing the rest of the year. Seasonal breeders are generally short- day and long-day breeders (Table 22.9). In short-day breeders, decreasing day length (in autumn and winter) influences the onset of estrus (sheep and goat). In contrast, increasing day length during summer and spring stimulates estrus in long-day breeders (mares). Generally, the long-day breeders give birth within the same breeding season or same season of the following year. Seasonal breeding is a kind of adaptive strategy for the survival of the offspring and mothers. The changes in the food resources available that control the animal’s energy expenditure plays a central role in reproductive seasonality through integrated endocrine and genetic mechanisms. Therefore, apart from photoperiod and melatonin, the seasonal breeding is controlled by neuropep­tide Y (NPY), Kisspeptin, GnIH, gonadotropins, estrogen, GH, IGF-I, leptin, and thyroid hormones that are related to the energy metabolism of an animal.

Table 22.9 Seasonal breeders with their breeding season

Cattle, pigs, and rabbits are the non-seasonal breeders. But, ovulation rate, conception rate, and litter size are less in summer in pigs. Parity one is more susceptible to repro­ductive infertility in the sow. There are another group of mammals competent to be fertile at any time or can resume fertility at a short period in a favourable environment, called opportunistic breeders. Human is an example of opportunis­tic breeder and can mate throughout the year. Golden spiny mouse temporary halts its reproduction in high salinity in drying desert; it can resume fertility during rainfall. Small rodents and tree kangaroo are also opportunistic breeders. The dog considers a seasonal breeder, but the exact breeding season(s) are difficult to determine. They show breeding activity after 6 months. The rainy season is one of the pre­ferred breeding seasons for the dog may be due to higher humidity and less environmental temperature that favours the pheromone signals for the activation of sexual response.

22.2.2 Estrous Cycle

The rhythmic sexual behavioural pattern exhibited by the female animals after the attainment of puberty is called the estrous cycle. The sign of sexual receptivity is called estrus or heat, which denotes the initiation of the cycle. Estrous cycle can also be defined as the duration between two successive estruses. The estrous cycle is classified into four distinct phases: estrus, metestrus, diestrus and proestrus. During the proestrus and estrus, follicular development or generation of follicular wave(s) occur, followed by ovulation. Hence, these two phases are collectively called the follicular phase. The luteal phase is characterised by the formation of the corpus luteum and its lysis. The luteal phase consists of metestrus and diestrus.

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Estrus: Etymology

The name estrus was derived from the Greek word ‘oistros’. In ancient Greece (1690-1700), the oistros was a gadfly, a biting fly under the genus Oestrus that hurt animals and forced them to react. The reaction of the animals attacked by such flies is similar to the behaviour during the estrus. In 1850, the word oistros was used as the Latin word ‘oestrus’; later, in 1890 as ‘estrus’.

The events and duration of these phases (Fig. 22.7) are species-specific (Table 22.10 and 22.11). The events of the estrous cycle are regulated mainly by two gonadotropins (FSH and LH) and ovarian sex steroids (estrogens and pro­gesterone) hormones that change dramatically during different phases of estrous cycle (Fig. 22.8). The animals showing a regular estrous cycle are called cyclic animals. Pregnancy causes physiological cessation of the cycle, and it resumes after the parturition as postpartum estrus. The failure to express the estrous cycle without any physiological reasons (such as pregnancy) is termed ‘anestrus’ (Fig. 22.7). In the majority of the cases, anestrus is temporary due to some pathological reasons, but prolonged anestrus leads to infertility, and persistent infertility leads to sterility.

22.2.2.1 Classification of Estrous Cycle

Estrous cycle can classify in several ways. Based on the occurrence of the estrous cycle, animals can be classified into monoestrous, polyoestrous, and seasonally polyestrous. Monoestrous animals exhibit only one estrous cycle per year. The true monoestrous animals are foxes, wolves, and bears. Animals like cows and sows show frequent, periodic estrous cycles throughout the year are called polyestrous animals. Animals that exhibit periodic estrous cycles only during a particular season are called seasonally polyestrous animals. They can be classified further into short-day breeders and long-day breeders. Animals that come in estrus during the decreasing day length are called short-day breeders, for example, buffaloes, deers, and ewes of non-tropical regions. The animals that show estrus during the increasing day length are called long-day breeders (mare and queen). Based on ovarian activity, the estrous cycle can be classified into regu­lar, spontaneous, and induced ovulatory estrous cycles. In the animals showing a regular estrous cycle, ovulation and for­mation of the corpus luteum (CL) occur regularly during a cycle. The corpus luteum formation is independent of mating, for example, cow, sheep, mare, and sow. In the spontaneous ovulatory cycle, ovulation occurs spontaneously, but CL will not be functional until mating, for example, rat and mouse. In the induced ovulatory cycle, ovulation and CL formation depends on whether mating has occurred or not, for example cat, rabbit, mink, and camel. There are sensory neurones in the female genital tract of induced ovulators which are sensitised during copulation and stimulate the hypothalamus for LH surge, which causes ovulation and subsequent CL formation. The classification of the estrous cycle based on ovarian activity is presented in Table 22.10.

22.2.2.2 Estrus

It is a well-defined period characterised by sexual receptivity or acceptance of males. The most distinguishable feature of this phase is behavioural change. The estrus begins with the first acceptance and ends with the last acceptance of the male. A large and matured Graafian follicle protrudes above the ovarian surface. Oviducts are tonic with mature epithelia and active cilia. Contractions of the oviduct occur that facilitate sperm transportation after insemination. The fimbriated end of the oviduct comes close affinity to the Graafian follicle to

Fig. 22.7 Various phases of cow’s estrous cycle. Figure depicts the four distinct phases of estrous cycle with associated ovarian changes. The cycle is initiated with estrus, followed by metestrus, diestrus, and proestrus (boldface circle). The estrus is characterised by pre-ovula- tory or Graafian follicle’s presence, and ovulation is occurred during metestrus in the cow; corpus luteum is formed in diestrus and

regressed in proestrus. Pregnancy leads to prolonged diestrus, and the cycle resumption occurs from the proestrus after the termination of pregnancy (straight arrow). The pseudo pregnancy also causes prolonged anestrus (dotted arrow), and the cycle is restored from proes- trus after the termination of pseudopregnancy

Table 22.10 Characteristic pattern of various types of estrous cycle

capture the ova during ovulation. The secretion of oviductal fluid increases. The uterus appears tonic and becomes turgid. There is increased blood supply to the uterus. Uterine muco­sal growth is evident, and there is increased mucous secre­tion. Vaginal mucosa becomes pale pink due to increased vascularity. In dogs and cats, thickening of vaginal mucosa and desquamation of cornified epithelial cells occurs. The cervix is relaxed and edematous. Stringy mucous hangs from the vulva. There is increased neutrophilic infiltration into the uterine lumen, and the stroma becomes edematous. Glandular ducts secrete a thin serous fluid which flushes the tract and assists sperm transportation. In most domestic species, ovu­lation occurs during estrus, but in cows, ovulation takes place 12 h after the end of estrus. In induced ovulators, the estrus may be prolonged up to 7-10 days in the absence of males.

During estrus, the level of LH, as well as estrogens, is increased gradually. It causes the selection of the dominant follicle and its maturation. LH surge occurs at the end of this phase with the profuse synthesis of estrogens favouring ovu­lation. Secretion of FSH is also reduced due to high estrogen levels, which favours follicular maturation. The synthesis of PGF2α has also occurred at the time of ovulation. The mor­phological alterations of the female reproductive tract are manifested mainly by the estrogens with progesterone priming. The absence of progesterone priming results in poor estrus manifestation (silent estrus), as seen in the first estrus after puberty and the first postpartum estrus. Silent estrus is common in cow, ewe, and sow and less frequent in mare and bitch; as progesterone starts secreting from theca externa of the Graafian follicle before estrus in these animals. The behavioural signs of estrus are also manifested by estrogens acting over the pre-optic area, ventromedial hypo­thalamus (VMH), amygdale, midbrain, and pituitary. The sexual receptivity is manifested through estrogen receptor α at the ventromedial hypothalamus (VMH), the centre for sexual receptivity. Progesterone has an inhibitory role on

Table 22.11 Duration of estrous cycle and its various phases

Source: Data collected from various sources

^a Estrus is considered for the standing estrus period

^b Repeated mating is preferred in the natural breeding programme in polytocous species

^c For successful ovulation 3-4 mating is required within 24 h for ovulation

^d After formation of active corpus luteum in pseudopregnancy stage

^e Other than the intravaginal route, intraperitoneal route of insemination can be used in guinea pig as there is an opening of the ventral part of the ovarian bursa

^f Up to ovulation

sexual receptivity in all induced ovulators (rabbit, ferret), guinea pig, and hamster as it antagonises the estrogenic effects on the CNS. Some neuronal networks involved in the expression of sexual receptivity are GnRH neurons (at the medial pre-optic area), noradrenergic neurons (at the medial pre-optic area and the VMH-secreting norepineph­rine), and dopaminergic neurons (at ventral tegmental areas and the substantia nigra of midbrain-secreting dopamine).

22.2.2.2.1 Behavioural Estrus

The estrus is characterised by the appearance of a series of visible psychological behaviour under the influence of estrogens. The most predominant sign of estrus is standing to be mounted. The animals under estrus remain standing and allow other (cow or bull) animals to be mounted with the elevation of hindquarters, called lordosis reflex. It is also called standing heat and may continue up to 18-19 h in a cow (the total estrus phase may be up to 20-30 h). The female on standing heat may allow a maximum of 50 times mount­ing with a few seconds duration in a cow. The other primary signs include bellowing, red and swollen vulva, and clear cervical mucus that appears as viscous elastic strands hanging from the vulva and sometimes spread over the tail, flanks, and perineal region. Upon microscopic examination, the cervical mucus shows characteristics ‘ferning pattern’ due to high chloride content. In some cows and mostly in heifers, bloody mucus may discharge 1-3 h after the estrus. It is called metestrus bleeding. This sign confirms that the animal was in estrus. The metestrus bleeding is due to dripping from the uterine blood vessels caused by excess estrogens during estrus. The secondary signs of estrus are restlessness, sniffing the genitalia, licking the vulva of other animals, lip curling, frequent micturition, rubbing the chin on the back or rump of the other animal (chin-resting), loss of appetite and reduced milk yield. The characteristic female sexual behaviour manifests only during the estrous cycle’s estrus phase in all mammalian species, except humans. This distinguishable external expression helps identify the reproductive state of the animals, and hence, the period of estrus is considered day 1 (the day of initiation) of the cycle.

22.2.2.2.1.1 The Ideal Time for Insemination

The time of ovulation is species specific (Table 22.11); hence, the timing of insemination also varies between spe­cies. The ideal time of insemination can predict based on several factors, such as the duration of standing heat, ovula­tion time, the viability of ovum and spermatozoa, and the duration taken by the sperm to achieve fertilising capability

Table 22.12 The duration of different events to predict the ideal time of insemination in cow

Fig. 22.8 Relative concentration of various female reproductive hormones in different phases of estrous cycle in cow. Figure shows the relative changes of the concentrations of luteinising hormone (LH), follicle-stimulating hormone (FSH), estrogen and progesterone (Y- axis) during different phases of the estrous cycle of the cow (X-axis). Different phases of the cycle, viz. estrus (0-1 day), metestrus (1-4 days), diestrus (4-19 days), and proestrus (19-21 days), are illustrated above with the hormonal profile (line arrow). The cycle initiated at estrus with the LH surge. The concentration of estrogen also rises at the initiation of estrus and continues up to the entire estrus

phase. The progesterone level gradually increases during the metestrus, and the concentrations of other hormones decrease. In diestrus, the progesterone level peaked on day ten and sustained up to day 17, followed by a sudden fall to reach a base level after day 19. The concentration of FSH and estrogens follow characteristics of wavy patterns during diestrus. Proestrus (day 20-21) is characterised by a higher level of FSH and estrogen. The proestrus and estrus consider follicular phase, and metestrus and diestrus are called the luteal phase; marked at the top of the figure and demarketed with a line arrow

in the female reproductive tract (capacitation). The average duration of such events in cattle is presented in Table 22.12.

Considering the above facts, the a.m./p.m. or p.m./a.m. rule is practised in artificial insemination in cows. A cow exhibited in standing heat in the morning should inseminate in the afternoon of that day. A cow shown in standing heat in the afternoon or evening should be bred the following morning.

22.2.2.2.2 Heat Detection Techniques

Appropriate detection of estrus or heat is the essential prereq­uisite for a successful breeding programme. Visual observation is the best method to detect the standing heat, but it is of limited use during silent heat and repeat breeding animals. There are several aids to detect the standing heat in animals, such as (1) Teaser bull: Vasectomised bulls are routinely used to detect heat in farms with an efficiency of 84%. But sexually transmitted diseases are the major demerits of this method. (2) Milk progesterone analysis. During proestrus and estrus, the level of progesterone will decrease. In cows, low progesterone levels may continue for nearly 6 days. But, it is not an ideal tool for detecting the standing heat. (3) Mount detector device is an electronic gadget that can detect mounting and is very useful for identifying the postpartum estrus. (4) Animal wise action lists can sometimes assist in detecting the heat in the herd and encaged animals. (5) Videography of the animals’ behaviour is more accurate, but it is costly. (6) Vaginal electrical resistance (ER) probe can be used to detect heat, where the resistance value is minimum during standing estrus. The maintenance cost of the device and transmission of diseases are the major demerits of this tool. (7) Vaginal cytology is extensively used to detect heat in laboratory animals. (8) Periodic evaluation of estrogens and progester­one metabolites in urine and faeces are used to detect heat in wild animals during the breeding season, followed by confir­mation through the cytological test.

22.2.2.2.3 Postpartum Estrus

The immediate estrus with subsequent occurrence of ovula­tion and formation of corpus luteum after parturition is called postpartum estrus. The uterus returns to its normal tone with proper myometrial activity to support the successive gestations. Generally, the postpartum estrus is anovulatory, and the animal may not be able to fertilise after insemination. During the postpartum period, the progesterone level is minimised due to luteolysis. The regressed corpus luteum transformed into scar tissue. Follicular wave is initiated under the influence of FSH. Lactation, particularly in high yielding animals, causes increased prolactin secretion, which inhibits gonadotropin release and delays the occurrence of postpartum estrus (lactational anestrus), whereas weaning advances the period. The occurrence of postpartum estrus varies between species. It is nearly 45-60 days in cow, 30-90 days in buffalo, 33-90 days (depending upon season and latitudinal location) in doe and ewe, 6-15 days in mare, 90 days or more in bitch, 45-60 days in queen (domestic cat),

3- 18 h in rat, 2-14 h in guinea pig, 14-24 h in the mouse.

22.2.2.3 Metestrus

It is the transition period between ovulation and the formation of CL. It is a poorly developed period. In cows, ovulation occurs in the first part of this phase. It is mainly under the influence of progesterone produced by CL; hence, this period is considered under the luteal phase. Progesterone inhibits FSH secretion and restricts the further growth of Graafian follicles. The level of LH remains at a moderate level with declining estrogens. In cows, the growth of endometrial glands begins, and capillary haemorrhage occurs that leads to menstrual bleeding. The secretion of mucus is decreased. The uterus becomes less tonic, and the superficial epithelium becomes hypertrophic and pseudostratified cells are modified to tall columnar epithelium cells. Uterine contraction is reduced. In most animals, fertilisation takes place in this phase.

22.2.2.4 Diestrus

It is the most prolonged phase of the estrous cycle. The corpus luteum matures and secrets progesterone. Several small follicles of successive wave(s) will appear in the ovary. Due to the effect of progesterone, there is increased hypertrophy of endometrial glands, and the uterine muscles become flaccid. The basal cells of the endometrial glands become active and secrete thick uterine milk into the lumen of the uterus. The cervix is closed, and vaginal mucus is scanty. The vaginal mucous membrane becomes pale. Estro­gen priming is essential to get the optimum effect of proges­terone during diestrus as the estradiol activates progesterone receptors in the endometrium.

On the other hand, the progesterone antagonises estradiol and causes negative feedback on the GnRH for the secretion of FSH. Thus, the estrogen and FSH level is minimised with the increasing level of progesterone (Fig. 22.8). In cows, the corpus luteum attains its maximum size within 7-8 days after ovulation, i.e. within the first 4 days of the diestrus and sustains up to day 16. If the animal becomes pregnant, the peak size of the corpus luteum will persist throughout preg­nancy to provide progesterone. The corpus luteum regresses in non-pregnant females by PGF2α at the end of this phase, and the progesterone level is dropped suddenly.

22.2.2.5 Proestrus

It is an ill-defined period that usually lasts for 2 days in domestic animals. Growth of the follicles occurs under the influence of FSH with increased production of estradiol. Estrogens cause the formation of the antrum and also cause endometrial hypertrophy. Estrogens also stimulate the expression of pre-ovulatory sexual behaviour in some animals like rats. Increased vascularity of uterine mucosa causes hyperplasia of endometrial cells. The level of proges­terone is minimum during this phase. The ovaries are characterised by remnants of CL (corpus albicans) of the earlier cycles. There is increased growth of oviductal cells and cilia. The vaginal epithelium thickens due to increased vascularity and is cornified in dogs and cats. The LH level rises at the end of the proestrus.

The primary follicles are primarily involved in the wave. The size of the follicles is species specific. Ovary and the entire reproductive tract become hyperaemic. The uterus and vagina are distended. Non-cornified nucleated epithelial cells are available on vaginal cytology. This is the first phase of the follicular stage.

22.2.2.6 Anestrus

Anestrus is the period of complete reproductive incompe­tence due to ovarian inactivity when the animals fail to express estrus behaviour. When estrus behaviour is less intense, it is called sub-estrus. It is the period between the diestrus of a cycle and the proestrus of the next cycle. During anestrus, the temporary inactivation of the HPO axis during anestrus restricts follicular development and ovulation. The secretion of estrogen is decreased and results in weak estrus behaviour. The inactivation of the HPO axis is due to reduc­ing GnRH secretions from the GnRH pulse generator. Anes­trus is of two types. In anovulatory anestrus, follicle maturation is affected due to reduced FSH, and no ovulation occurs due to lack of LH. In persistent luteal anestrus, the CL does not regress, and the progesterone secreted from this CL inhibits FSH secretion. The ovary of anestrus animals becomes oval and somewhat flattened in shape with large numbers of small follicles, and the endometrium becomes atrophic with dense stroma and small endometrial glands.

In bitch, two types of persistent anestrus are seen, primary persistent anestrus and secondary persistent anestrus. Anes­trus without a previous estrous cycle is called primary persis­tent anestrus, whereas secondary persistent anestrus occurs after the first estrous cycle. The primary persistent anestrus develops due to genetic and chromosomal disorders, autoim­mune diseases (oophoritis), ovarian aplasia, ovariectomy, or ovariohysterectomy at a young age and progesterone- releasing ovarian cysts. Management problems, like over­exercise and nutritional deficiency, also act as predisposing factors for primary persistent anestrus in bitches. The regular interval between two estruses in bitch is about 4-10 months; hence, if a bitch fails to return heat after a gap of 10— 18 months from its previous estrus. It can be designated as secondary persistent anestrus and seen in an aged animal with an irregular estrous cycle. Infections in the reproductive tract, metabolic diseases, endocrine disturbances (Cushing’s dis­ease, dysfunction of the thyroid gland), systemic diseases, and altering reproductive hormonal balance may cause sec­ondary persistent anestrus. Luteal cyst and the ovarian tumour may cause both primary and secondary anestrus in bitch.

22.2.2.6.1 Cause of Anestrus

Anestrus may occur due to several reasons (Table 22.13). Physiological anestrus occurs due to pregnancy, lactation, and breeding seasons. Sometimes congenital defects, nutritional deficiencies, and stress factors also suppress the HPO axis and cause anestrus. The anestrus period may also vary between species, breed, parity, and age.

22.2.2.6.1.1 CysticOvarian Diseases

Cyst is a fluid-filled structure. The follicular and luteal cyst may develop in the ovary, particularly in cattle. A follicular cyst is formed within unovulated follicle, caused due to lack of LH surge required for ovulation. Prolonged exposure to phyto-estrogen and administration of estrogen and progester­one may reduce the optimum LH level for ovulation, causing the development of a follicular cyst. The pathological state builds up the cystic ovarian disease (COD). A thin layer of theca cells covers a follicular cyst. The theca cells continu­ously produce progesterone. But it does not have the receptors for PGF2α. Hence, luteolysis is not occurred, caus­ing anestrus. Animals having follicular cyst with anestrus may exhibit constant estrus (heat) due to estrogen produced by granulosa cells. When the cyst develops in the corpus luteum, it is called a luteal cyst. The luteal cyst contains a thick luteal cell layer, which will produce continuous proges­terone and causes anestrus. The thick layer of luteal cells can respond to exogenous PGF2α.

22.2.2.6.2 Management of Anestrus

The anestrus can be treated after removing the underline causes with proper management and specific hormonal ther­apy. PGF2α is frequently used to treat luteal cysts. First, FSH and LH are applied to induce ovulation and CL formation for the follicular cysts. Then, PGF2α is administered 7 days apart to induce luteolysis. Estrus can be induced using hCG and eCG in wild cats (like Amur leopard cat) during the non-breeding season. The presence of males can increase ovulation rate and reduce the period of anestrus in the non-breeding season of seasonal breeders, like goats and sheep, along with gonadotropin administration. In bitches, serotonin-antagonists and dopamine-agonists can reduce the anestrus period by increasing FSH levels and decreasing the prolactin levels in lactating animals.

Table 22.13 Factors causing anestrus in animals

22.2.2.6.3 Silent Heat

The lack of behavioural estrus symptoms is termed silent heat. It is completely different from anestrus as reproductive organs undergo normal cyclic changes in silent heat. The predominant cause of silent heat is the lack of estrogen receptors. The silent heat is commonly seen in the animals experiencing the first estrus due to the absence of progester­one priming. Silent heat can also occur during the first post­partum estrus. Seasonal breeders also exhibit silent heat at the beginning of every breeding season. Persisted silent heat may cause anestrus due to insufficient estrogen production, nutritional deficiency, and various pathological conditions. In persisted silent heat, ovulation may occur.

22.2.3 Factors Affecting Estrous Cycle

22.2.3.1 Species and Breed

Species variations in the lengths of estrous cycles among species (Table 22.11) are determined primarily by the dura­tion of the luteal phase. Large mammals have a longer luteal phase than animals with small body stature. The evolutionary advantages of having shorter luteal phases in small mammals are short gestation periods, absence of lactational anestrus, and early attainment of puberty in young. Small mammals are susceptible to predation, and a lengthy non-pregnant cycle is not advantageous. The animals with a longer estrous cycle are prone to extinction. The duration of cycle length and occurrence of estrus cycle varied between breeds of certain species. In sheep, more duration with fewer cycles in a season is seen in the Santa Ines breed than Romney Marsh breed. Generally, the breeds of the tropical region, Dorsets and the fine-wool breeds like Rambouillet and Merino have short anestrus than the other breeds like Hampshire, Suffolk, and Border Leicester. The breeding season starts in Rambouillet breed around July, whereas Landrace initiates their breeding during September. A short estrous cycle and proestrus often occur in German shepherds than Pointers in dogs. German shepherd breeds also exhibit long estrus.

22.2.3.2 Seasons and Photoperiod

The photoperiod, latitude, and season cause HPO axis devia­tion and seasonal reproductive activity in certain species. Estrus duration is increased during summer with less mount­ing activity in cattle. Total daily light exposure is important to control the onset of estrous cycle. The duration of seasonal anestrus in long-day breeders may reduce by increasing pho­toperiod duration with artificial light. In sheep and goats, decreased day length influences the onset of the estrous cycle. In mares, increased total daily hours of light hasten the onset of estrous cycle. Rapid alterations in the exposure to artificial light can extend the cyclic activity in short-day breeders. Melatonin implantation also has a similar effect.

22.2.3.2.1 Temperature

The secondary sign of estrus is displayed more in the early morning and late evening due to favourable ambient temperatures ranging between 75 and 85 °F in the cow. Excessive heat during summer directly reduces reproductive efficiency. In cattle, the estrous cycle length can be increased up to 25 days during summer compared to 20-22 days in cool weather. The reduced reproductive activity around summer is due to reduced thyroid activity, high progesterone from gonadal and non-gonadal (adrenal gland) sources, oxidative stress, and reduced LH secretion. Heat stress also affects follicular development with diminished aromatase activity and lower production of 17β-estradiol (Fig. 22.9). Reduced estradiol and poor LH concentration cause silent heat around summer. The poor quality oocytes with reduced follicular fluid and less viable granulosa cells produced under thermal stress may affect fertilisation rates. It is designated as summer sterility usually occurs in high yielding cows and buffaloes of the tropical and sub-subtropical region.

In sow, summer stress (beyond 35-40 °C ambient temper­ature) negatively modulates the HPO axis causing less ovu­lation rate, reduced litter size, increased anestrus, and duration of postpartum estrous occurrence. The effect of high ambient temperature and heat stress on reproductive cycles can be minimised by increasing antioxidant levels, hormonal manipulation, and nutritional and housing manage­ment. In rats, cold exposure enhances progesterone levels and diminishes estrogen levels. In an extremely cold environ­ment, LH level increases and results in impaired ovarian function with irregular estrous cycle, decreased ovulation rate, and even induced follicular cysts.

22.2.3.3 Nutrition

Nutritional deficiency impairs the secretions of gonadotropic hormones from the pituitary gland. Negative energy balance has a detrimental impact on the reproductive activity of dairy animals. Insufficient energy intake during the growth period results in delayed onset of estrous and reduced fertility in females. Malnutrition also causes poor estrus manifestations and inadequate follicular maturation. Dietary fat, calcium salt, and long-chain fatty acids often influence the follicular growth under energy-deficient conditions, particularly in lac­tating bovines and swine. Feeding phytoestrogens like clover (red clover), barley and oat grains, alfalfa, and some growing legumes (mature and dry legumes have less estrogen) may cause delayed estrus.

Fig. 22.9 Effect of malnutrition on the female reproductive system. Figure shows the effect of heat stress cause to insufficient hormone secretion. Constant scarcity of sufficient nutrition leads to prolonging energy deficiency. The consequence of it results in various hormonal insufficiency and changes in the metabolic or systemic status of the animal. It affects the cellular and physiological function of the female reproductive system directly with an increased calving interval, poor pregnancy rate, and repeat breeding

22.2.3.3.1 Nutritional Flushing

Increasing nutrient intake suddenly from sub-optimum to the optimum level to improve body condition before and during the breeding time to augment ovulation and conception are called flushing. It is generally practised in ewes but can be applied to sows, does or even cows. Flush-feeding positively impacts the GnRH pulse generator and hypophyseal sensitiv­ity to GnRH. Thus, flushing induced gonadotropin release increases the follicular wave and ovulation rate. Nutritional flushing also influences the release of some metabolic hormones and growth factors like insulin, growth hormone, IGF-I, and leptin, which affect the HPO axis and stimulate gonadotropin release. Flushing is generally practised during pre-estrus, 1-2 weeks earlier than estrus during breeding time. Flushing is more beneficial for lean animals than fatty animals. Flushing is helpful in sows, as feed intake is gener­ally reduced in post-weaning conditions.

22.2.3.4 Lactation

Suckling anestrus is common in high yielding cattle and buffaloes due to its high prolactin and oxytocin. Both these two hormones have a negative impact on the HPO axis. In hyperprolactinemia, dopamine secretion is increased, which inhibits the GnRH release from the hypothalamus and subse­quently FSH and LH release from the anterior pituitary. Prolactin suppresses the estrogen-induced LH release, and oxytocin suppresses the GnRH pulse generator. All these factors cause impaired estrus manifestation and ovulation. Poor follicular development leads to low estradiol production. Hence, the occurrence of silent heat is increased. Some high yielding animals also experienced negative energy balance during the lactation period, which adversely affects the HPO axis’s functionality.

22.2.3.4.1 Interaction with the Opposite Sex

The presence of males can influence the occurrence of estrus through bio-stimulation. The bio-stimulation is mediated mainly through pheromones. Flehmen response is a common phenomenon in a wide range of ungulate mammals. The other examples of bio-stimulation are the bull effect in cattle, ram effect in sheep, self-enurination in goats, Vandenbergh effect in mice and boar, Whitten effect in mice, and Bruce effect in rodents.

22.2.4 Distinguishable Attributes of Estrous Cycle in Various Species

22.2.4.1 Cattle

The cow is a non-seasonal polyestrous animal, and the dura­tion of estrous cycle is 19-21 days. Different attributes of the estrous cycle in cows are discussed in different sections as a model animal.

22.2.4.2 Buffalo

Buffaloes are known as shy breeders due to their less intense estrus behaviour. The occurrence of silent heat is very com­mon in buffaloes, particularly during summer, due to heat stress. Buffaloes exhibit estrus behaviour from evening to night; hence, estrus detection is challenging for buffaloes. The major causes of silent estrus in buffaloes are the small ovarian size and less primordial follicles (20,000 vs 150,000 in cattle) that produce a small quantity of estrogen (17- β-estradiol). The follicular atresia is also more in buffaloes than in cattle with fewer antral follicles. The vasectomised teaser can detect standing estrus; milk progesterone estima­tion and ultrasonography are used to confirm the estrous cyclic stages.

22.2.4.3 Ewe

Sheep are the seasonally polyestrous short-day breeder. The estrous cycle length varies from 16 to 17 days, depending on the breed. The duration of breeding seasons is also diverse. Breeds like Leceister, Scottish Blackface, Texel, and Shet­land have a short breeding season (less than 4 months). The breeds of the medium breeding season (4-6 months) are Hampshire, Suffolk, and Charollais. The long breeding sea­son (6-8 months) is seen in Rambouillet, Dorset, Finn, and Romanov. The tropical breeds of sheep (Nellore, Bellary, Malpura, Yankasa, and Uda) are generally considered non-seasonal breeders. Progesterone priming is important for follicular wave and sexual receptivity in ewe; hence, the silent estrus usually occurs of the first cycle in each breeding season. Estrus symptoms are less prominent in sheep than in other domestic animals like cattle, pigs, etc. The presence of ram (ram effect), even with the teaser and androgen-treated whether, can be used to detect estrus. In general, the induc­tion of estrus and ovulation are seen within 48 h of the ram effect. But, the first estrus induced by the ram effect is of shorter duration (with 5-6 days of diestrus). It is followed by a true estrus, seen around 25 days after the ram effect. This process is effectively used to synchronise estrus in ewes. Some animals exhibit long estrus duration with sexual recep­tivity and high ovulation rate due to high gonadotropin. Flushing before breeding also creates a higher ovulation rate and increases litter size in ewes.

22.2.4.4 Doe

The reproductive character of goats depends upon the habitat. The breeds of the temperate region are seasonal breeders. Autumn and winter are their breeding seasons. The breeds of the tropical area are non-seasonal breeders. The tropical breeds of goats exhibit prolonged anestrus with reduced litter size, probably due to malnutrition. Flushing, manipulation of photoperiod and hormonal treatment can reduce the duration of anestrus in seasonal breeders. The does exhibit a short estrous cycle followed by a regular cycle. The short estrous cycle is related with less ovulation rate. In tropical regions, does are often exhibited a short estrous cycle during high rainfall. In does, generally, four or more follicular waves are seen in one cycle, and ovulation occurs from the follicle(s) of the last wave. The ovulation rate is usually 1-3 eggs per doe, with a maximum of 5 depending upon the breed and man­agement of the animal. Application of exogenous progester­one is generally used to synchronise the estrous, and eCG and FSH or hCG are used for superovulation in does. The corpus luteum maintained its peak luteal activity from day 7 to 10 of the cycle and regressed from day 11 in non-pregnancy. In pregnancy, luteal progesterone is the primary source of pro­gesterone to maintain the gestation.

22.2.4.5 Sow

The onset of puberty occurs in most pig breeds at 5-7 months. The pig is a non-seasonal polyestrous polytocous animal. Photoperiod has little or no influence on the estrous cycle, but long darkness exposure may delay the estrus. Standing estrus is coincides with ovulation and generally seen in the middle of the estrus phase and continues up to 8-36 h. Duration of standing estrus is usually more in sows than gilts. Typical behavioural estrus symptoms are restlessness, decreased appetite, increased vocalisation, pricked ears, and swollen and reddish or pinkish coloured vulva. Back­pressure test (BPT) is used to detect estrus in the sow to see the standing reflex during the heat. The sow gets immobilised for 10-15 s after applying back pressure on both sides. The onset of estrus can be accelerated by the presence of boar (Vandenbergh effect, details in sex pheromone). The applica­tion of boar saliva is often used to synchronise the estrus in the sow. The boar presence also stimulates oxytocin secretion in the sow to facilitate sperm transport after the insemination. Hormonal manipulation with the combinations of PMSG and hCG to the gilt, aged 7 months or more, can also accelerate the onset of puberty. But, the results are questionable as cyclic gilt would not respond appropriately due to suppres­sion of progesterone by PMSG (when the gilt is in the luteal phase) or FSH, LH, and estrogen will reach too high upon PMSG treatment (when the gilt is in follicular phase). Multi­ple breeding during the standing estrus period can enhance the litter size. The visual symptoms of ovulation are the reddening of the vulva before ovulation, which subsides after ovulation. Hence, artificial insemination should practice when the reddening of the vulva is subsided. About 15-30 follicles can ovulate from both ovaries in one cycle, depending upon mainly the breed and nutritional status. Gilts can be bred on the second or third cycle to achieve better litter size. Exposure to unfamiliar boar during preg­nancy may cause abortion, compared with the Bruce effect in mice. Lactational anestrus is very common after farrowing in the sow. Nutritional factor influences the duration of postpar­tum estrus. Early weaning at 21 days can reduce the period of postpartum anestrus with the appearance of estrus within

4- 7 days. Proper nutrition during the postpartum period is essential for uterine involution and the growth of the piglets. Hence, the low weaning weight of piglets during the first lactation causes reduced litter size in second (or subsequent) lactations. It is called second litter syndrome.

22.2.4.6 Mare

Mare is a seasonally polyestrous animal. In non-tropical regions, summer is the breeding time; autumn considers a transition time and winter is characterised by anestrus in mare. In the northern hemisphere, the duration of estrus is shorter than in winter due to rapid follicular development under favourable photoperiod. But the duration of diestrus does not alter due to season. Mares exhibit prolonged estrus of 4-8 days, comprising 20-30% of the total duration of estrous cycle. Sometimes a period (1-2 days) of decreased behavioural signs is seen in the mares during the estrus stage, known as split estrus or slit heat. It is because follicles of one follicular wave fail to develop fully, and a second follicle develops to secret estrogen leading to the second strong phase of estrus behaviour. Normal estrus behaviours of mares include squealing at other horses and frequent urina­tion with small amounts. After approaching the stallion, estrus-mares lowered the pelvis and spread her hind limbs. The mare exposes her perineal region with the elevation of the tail. The rhythmic eversion of the clitoris, called clitoral winking, is also documented in estrus mares.

In some cases, unique facial expressions are seen characterised by relaxed facial muscles and lowered head with ears turned to the side. The characteristics of Flehmen’s response are also common in mares. Mares exhibit estrus behaviour during the anovulatory period due to estrogenic steroids of adrenal gland origin and unique among other ungulates. Another distinguishing feature of the mare estrous cycle is the postpartum estrus called ‘foal heat’. It is generally seen during the first week after birth, and the mare can be conceived at this time. But, breeding is usually preferred 2-­3 months after the foaling. The ovulation can occur before LH surge in the mare; hence, progesterone starts to release from the primary corpus luteum.

22.2.4.7 Rat

The commencement of puberty and appearance of estrous cycle were seen around the 33rd day after birth in female rats. The beginning of estrous cycle is indicated by vaginal open­ing. High estradiol concentration associated with vaginal unfolding was the main reason for sexual receptivity and first ovulation. From the onset of sexual maturity up to the age of 12 months, the mean cycle length in the female rat is 4 days. In some individuals, 6-day is seen. The estrous cycle in rats was characterised by four stages: proestrus, estrus, metestrus, and diestrus. Sometimes the cycle may consist of five phases having two metestrus, metestrus-I (15-18 h) and metestrus-II (6 h) (Table 22.11). Ovulation occurs from the beginning of proestrus to the end of estrus. A persistent estrus with constant sexual receptivity was seen in females aged

6- 18 months.

A state of pseudopregnancy of 10-14 days’ duration was seen in aged females who failed to breed after persistent estrus. The rat estrous cycle is characterised by frequent and abrupt changes in the concentration of hormones. The FSH, LH, and prolactin levels increase in proestrus’s afternoon, and a surge usually occurs in the late afternoon. The estrous cycle in rats is influenced by light and age, nutrition, dark and light cycle, temperature, stress and social relationships. The female rodents cycle occurs more regularly when males are present in the room (i.e. the Whitten effect) although mice are more sensitive to this phenomenon than rats. Period of anes­trus has also occurred in rats. It may be due to low progester­one in the absence of corpus luteum along with high prolactin, cause of pituitary tumours or pituitary dysfunction of other reasons, which suppress LH secretion. The cyclic rats contain three or more generations of variable sets of corpora lutea. The first half of pregnancy is maintained by the progesterone secreted from the corpus luteum. In the second half, the progesterone is released from the placenta.

22.2.4.7.1 Heat Detection by Vaginal Cytology in the Rat

Rodents’ estrous cycles were characterised by histomorphological alterations in ovaries, the uterus and the vagina. There are several methods to determine the stages of the estrous cycle in rats. Vaginal cytology is one of the most reliable tools to classify estrous cycle in rats. The basic principle behind this method was the rapid alterations in the vaginal mucosa during different stages of estrous cycle under the influence of sex steroids, particularly estradiol, which facilitated the cornification of the vaginal epithelium. The desquamation of mucosal epithelium occurred under estra­diol withdrawal. The vaginal smear can obtain after inserting a cotton-tipped swab moist with physiological saline into the vagina, followed by subsequent rolling against the vaginal wall. The stains visualised exfoliated vaginal epithelial cells were 0.1% crystal violet, Wright’s Giemsa, Toluidine blue O, and a single differential stain. There were three types of cells: leucocytes, cornified epithelial cells, and nucleated epithelial cells. Their relative proportions in vaginal smear evaluated the estrous cycle stage (Table 22.14).

22.2.4.8 Guinea Pig

Female guinea pigs attain puberty at 2 months of age (55-70 days). The estrous cycle length of the guinea pig is 16-19 days having the estrus period of 6-11 h. Different phases of the estrous cycle can detect by identifying cell types of the vaginal smear. Mainly three kinds of epithelial cells are seen during different cycle phases (Fig. 22.10). The cells are the parabasal (immature or smallest squamous epi­thelium having a large nucleus compared to cytoplasm),

Fig. 22.10 Vaginal cytological changes during different phases of estrous cycle. Figure depicts the appearance of three distinct types of exfoliated vaginal epithelial cells viz. superficial cells, intermediate cells, and parabasal cells in various phases of estrous cycle

Table 22.14 Gross and microscopic features of rat reproductive organs during estrous cycle

Note'. Bold words are the major distinguishable features. S. basale stratum basale, S. spinosum stratum spinosum, S. granulosum stratum granulosum, S. corneum stratum corneum

intermediate and superficial cells, and RBC and WBC (neu­trophil). Proestrus is characterised by the presence of superfi­cial nucleated cells and RBCs. Superficial cells are the largest epithelial cells appearing as polygonal flat shapes, transpar­ent, eosinophilic, with very small and dark nucleus or enucleated. Enucleated superficial cells are prominent during the proestrus-estrus transition phase. In estrus, superficial cells with a pyknotic nucleus are dominated along with RBC infiltration. No parabasal cells are found in estrus, but their presences, together with neutrophils, are more in metes- trus. The diestrus phase is characterised by similar cell types and large and small intermediate cells. Intermediate cells are larger than parabasal cells having an almost round or oval shape with large and prominent nuclei (small intermediate) and polygonal shape with small nucleus (large intermediate). Multichromatic (multicoloured) stains like Papanicolaou stain or Pap stain are generally used in cytological staining techniques.

22.2.4.9 Mouse

Mice are polyestrous non-seasonal breeders and spontaneous ovulator. The estrous cycle of the mouse is very short,

4- 6 days. The age of maturity is 6 weeks, and the reproduc­tive cycle continues throughout the life span (2-3 years in laboratory mice). Lactational anestrus is also common in mice due to the luteotropic action of prolactin. The postpar­tum estrous period is also very less, 12-20 h. The phase of proestrus and estrus lasts nearly 1-2 days, whereas sexual receptivity lasts for about 13 h. The estrogen level gradually increases in proestrus and remains higher in the morning until ovulation and slowly reaches baseline in the afternoon. Estro­gen and progesterone levels gradually increase in diestrus within 1-2 days. Thus, ovulation generally occurs during the night due to a surge of FSH and LH. However, the FSH and LH levels remain low in estrus, metestrus, and diestrus. Ovulation occurs around 10 h after the beginning of estrus. Generally, 10-20 ova can be ovulated in a cycle.

Various cytological changes occur in the vagina, uterus, and ovary during different phases of the estrous cycle due to estrogen’s action. The visual assessment of the vagina and vulva is one of the most accepted, non-invasive and fastest techniques for estrous cycle assessment. The vaginal opening is swollen, broad, wrinkled, and reddish-pink during proes- trus, while in estrus, it is less swollen, less moist, and less pink. Metestrus is characterised by the presence of white cellular debris with a narrow vaginal opening. In diestrus, the vaginal opening is closed, and no swelling occurs. Vagi­nal cytology is also a reliable method of estrus detection in mice. The presence of numerous leukocytes, nucleated epi­thelial cells, and cornified cells is evident during different cycle phases.

The cycle continues throughout the life span after maturity (6 weeks), except during lactation due to prolactin. Hence, within about 25 days, a mouse can give birth to two succes­sive litters. Thus, mice are used as an animal model in the experimental research study. The Whitten effect can synchro­nise the estrous cycle, and puberty can be enhanced through the Vandenbergh effect. The presence of an unfamiliar male (other than a mating partner) may cause abortion due to the Bruce effect.

22.2.4.10 Bitch

The ovarian cycle of dogs occurs twice a year except for Basenji breed (cycles once a year). Bitches enter their first heat between 6-10 months, but it may occur at 18-24 months in some breeds. The proestrus begins with vaginal bleeding and ends when a bitch allows a male to mate. The duration of proestrus is about 9 days (ranges from 3-25 days). The bitches are playful and attractive compared to males but refuse to mount. In late proestrus, the bloody discharge fades and becomes transparent to straw colour. The proestrus is under the influence of estrogen synthesised from developing follicles. The estrogen level peaks in late proes- trus and then declines at the basal levels at the beginning of estrus. The progesterone concentration increases from basal levels at late proestrus and increases at the onset of estrus and ovulation. The source of progesterone is partially luteinised follicles before ovulation and developing CL. Estrus starts with the first acceptance of males and ends with refusal. The duration of estrus is about 9 days but may range from 2 to 18 days. The standing heat in bitches is characterised by a declining estrogen level and increasing progesterone level. The bitch attracts males from a distance due to pheromones. The vaginal discharge becomes straw-coloured or pink at the time of estrus. Ovulation occurs 24-48 h after the LH surge at 24-72 h. The eggs are released as primary oocytes and become fertile after 24-72 h. The mature ova have a life span of 2-4 days. The diestrus begins with the end of stand­ing heat and ends when the progesterone reaches its basal level. The unique feature of CL in bitches is that the CL remains functional whether it is pregnant or not. The luteal phase ends in a pregnant bitch after the parturition. But, in non-pregnant bitch, the CL functioned for 75-100 days and regressed due to ageing.

LH and prolactin are the two luteotropic factors. The luteotropic action of prolactin is seen in the second half of the luteal phase. The uterine involution occurs during anes­trus and extends up to 4.5 months. Vaginal cytology is used to identify the estrus in bitches. The proestrus is characterised by increased cornified cells with RBC and leukocytes. In estrus, mostly cornified cells are evident in the vaginal smear. There is an abrupt change in the vaginal cells from superficial to basal cells with increased leukocyte infiltration in diestrus. The anestrus is characterised by foam cells and leukocytes. The best time for mating is between the tenth and 14th day of estrous cycle. The breeding time can also be predicted by progesterone assay (Table 22.15).

Long proestrus is one of the common disturbances in the estrous cycle of bitch due to a lack of estrogenic peak during proestrus with higher progesterone levels. Split estrus is also another problem in bitch. In split estrus, the proestrus starts due to incomplete luteolysis (in chronic premature luteolysis or hypothyroidism), when the progesterone level falls

Table 22.15 Prediction of breeding time based on progesterone level

Ideal mating time is around 15-24 ng (48-75 nmol)

Two matings, 48 h apart

slightly. It results in the initiation of proestrus, but proper follicular development will not achieve due to progesterone. Thus, proestrus is prolonged, followed by either failure to estrus or exhibiting a short estrus but no ovulation. Vaginal bleeding and vulvar swelling during this time may be con­fused with estrus. After about a month, the normal cycle will start. This incidence is called split estrus, wherein the first phase is a pseudo or short non-ovulatory estrus followed by a true estrus. This incidence is mostly common in young experiencing the first estrus in each breeding season but can be seen in adults.

22.2.4.11 Cat (Queen)

The onset of puberty varies from 3 to 12 months of age. The Burmese cats mature earlier than the Persian and free-ranging cats. Environmental factors influence the onset of puberty, particularly in the regions away from the equator. Cats are seasonally polyestrous animals. Short-haired breeds come to cycle throughout the year. Increasing day length makes the females cycle all round the year. Being an induced ovulatory, queens have three possibilities of estrous cycle

1. Proestrus, estrus (non-bred), inter-estrus

2. Proestrus, estrus (sterile mating), diestrus, anestrus

3. Proestrus, estrus (fertile mating), pregnancy

Proestrus is a short phase of 1-3 days characterised by rubbing against objects by the queens, followed by rolling with purring, rhythmic opening and closing of claws, squirming and scratching. The queens exhibit a monotone howling (heat cry) for 3 min at a time during proestrus and estrus. Queens are spraying urine and sebaceous secretions to attract males. The estrus behaviour includes crunching lordo­sis and a copulatory stance. Tails are laterally displaced, and serosanguinous discharge from the vulva is observed. The estrus lasts for 4-6 days, and the sexual receptivity is seen on the 3rd and 4th. The queens do not ovulate unless mating occurs. Ovulation can be induced by stimulation with male penile spines or artificially by glass rods (several insertions of 10 s for 5-10 min after 48 h). The period between two successive estrous cycles of a cyclic queen is called the inter-estrus phase. It is 2-17 (average 10) days. The copula­tion before estrus will not induce ovulation, and hormonal applications such as eCG (for multiple follicular developments) and hCG (for ovulation) can be applied to augment the ovulation process. The heat symptoms will subside within 1-2 days after ovulation in the absence of fertilisation and the diestrus period continues for 35-40 days, termed pseudopregnancy. If the queen gets pregnant, she will not return to estrus till the seasonal peak or the following year. About 10% of pregnant queens display estrus behaviour at the 3-6th week of gestation. In the absence of tomcats, females are in estrus for 10-14 days, and the estrous cycle is 29 days. If the breeding does not occur, metestrus is an inter- estrus period between two successive estruses.

22.2.4 Menstrual Cycle

The term ‘menstrual cycle’ is derived from the Latin word ‘mensis’, meaning months, and the cycle lasts for approxi­mately 28 days. The periodic discharge of blood and other substances (sloughed lining of endometrial cells of the uterus and mucus), including an unfertilised ovum from the female genital tract of human and non-human primates (monkey and ape), is called menstruation. The repetition of such events from one menstruation to the next is called the menstrual cycle. It is generally a cycle of 28 days, with 21-35 days. It starts after puberty, with the development of the HPO axis, at the age of 10-11 years in girls with irregular cycle duration, whereas regular cyclicity develops after 17 years after maturity.

22.2.5.1 Phases of the Menstruation Cycle

The menstrual cycle can broadly classify into pre-ovulatory and post-ovulatory phases. The pre-ovulatory phase divides into bleeding (menstruation, day 1-day 4) and the proliferative phase (day 5-day 14). The post-ovulatory phase is also called secretory phage (day 14-day 28). The first day of bleeding is considered day 1 of the cycle. The normal amount of blood discharged during menstruation is about 4-40 mL of arterial origin, and the blood is unclotted due to fibrinolysin content. Ovulation generally occurs on days 12-14, and the ovum can be viable up to 24 h after ovulation. The changes in the ovaries, endometrium, and endocrine alterations during different menstrual cycle days are summarised in Table 22.16.

Table 22.16 Characteristics of the menstruation cycle

Know More..............

Mittelschmerz

The word Mittelschmerz comes from the German words ‘middle’ and ‘pain’. It means the pelvic and lower abdominal pain that occurs during ovulation halfway through the menstrual cycle. Hence, it is also called ovulation pain. Pain is generally one-sided, corresponding with the ovary involved in ovulation. It is a physiological process different from the abdominal pain related to some endometriosis and inflammation due to sexually transmitted infections.

22.2.5.2 Menstruation Cycle and Estrous Cycle

Other than the duration of the cycle and its occurrence, the estrous cycle and menstrual cycle have some striking differences. The major difference is in sexual receptivity. The animals are sexually receptive only during the estrus phase of the cycle, whereas females are receptive throughout the cycle in the menstrual cycle. The corpus luteum in the menstruation cycle can synthesise a small quantity of estro­gen, but CL in the estrous cycle cannot produce estrogen. Sometimes, menstruation may be confused with metestrus bleeding in cows, but these two are different phenomena. Menstrual bleeding is due to the imbalance between estrogen and progesterone regulates the endometrial lining. The met- estrus bleeding in cows is due to high estrogen levels during estrus, leading to blood leakage from vessels on the surface of the uterus. The metestrus bleeding can be called pseudomenstruation and is seen in bitches during proestrus and estrus.

22.3