Animal Reproduction, PDF

In nature, it is the general rule that animals breed once annually and parturition occurs in the spring, the time most favourable to the progeny in that they grow up during the period of increasing light and warmth, and also at the time when food for the mother is most abundant to ensure ade- quate...

In nature, it is the general rule that animals breed once annually and parturition occurs in the spring, the time most favourable to the progeny in that they grow up during the period of increasing light and warmth, and also at the time when food for the mother is most abundant to ensure ade- quate lactation. Under the conditions of feeding and housing provided by domestication the breeding season tends to be lengthened, and some of our species, particularly the cattle, may breed at any time during the year; all domesticated animals, however, show a constant tendency to revert to the natural breeding season. For an animal to breed, it must be mated and hence must attract the male and be sexually receptive (in heat or in oestrus). All domestic species show recurring periods of sexual receptiv- ity, or oestrous cycles, which are associated with the ripening in the ovaries of one or more Graafian follicles and culminate in the shedding of one or more ova. If a fertile mating occurs then pregnancy may ensue. PUBERTY AND THE ONSET OF CYCLIC ACTIVITY The young female animal shows no evidence of recurring or cyclic periods of sexual receptivity. The onset of such changes when the female becomes sexually mature and able to reproduce is referred to as puberty. Amongst females of the domestic species, puberty precedes the develop- ment of physical maturity and, although they become capable of reproducing, their efficiency, particularly with respect to their fecundity, has not reached its maximum. The initiation of puberty is largely a function of the animal's age and maturity since the female is born with a genetic potential for cyclic reproduc- tive activity. Provided the environmental influ- ences are favourable at this time, then once the 'biological clock' is started it will continue for as long as the environment remains favourable. In none of our domestic species is there a physiolo- gical change comparable with the menopause of women. Amongst non-seasonal polycyclic animals, such as the cow and sow, the recurring cyclic activity is interrupted by pregnancy, lactation and patho- logical conditions. In those species which are sea- sonally polycyclic, the mare, ewe, doe (or nanny) goat and cat, or monocyclic like the bitch, there are periods of sexual quiescence or anoestrus. When the female reaches puberty the genital organs increase in size. During the prepubertal period the growth of the genital organs is very similar to that of other organ systems, but at puberty their growth rate is accelerated, a point well illustrated in the gilt, where the mean length of the uterine horns is increased by 58%, the mean weight of the uterus by 72% and the mean weight of the ovaries by 32% between 169 and 186 days of age (Lasley, 1968). Females of domestic species reach the age of puberty at the following times: mare: cow:7--18months ewe: doe or nanny goat: 4--8 months sow: bitch:6--20months queen cat: The changes that occur at puberty depend directly upon the activity of the ovaries, which have two functions: the production of the female gametes and the synthesis of hormones. Let us con- sider the changes that occur in the ovary of the young heifer calf. At birth, each ovary may contain 1--2 years 6--15 months 6--8 months 7--12 months 3 1 NORMAL OESTROUS CYCLES up to 150 000 primary or primordial follicles; each consists of an oocyte surrounded by a single layer of epithelial cells, but there are no thecal cells. Soon after birth, the ovaries start to develop and produce growing follicles which consist of an oocyte with two or more layers of granulosa cells and a basement membrane. The stimulus for the development of these follicles is intra-ovarian, and until the heifer reaches the age of puberty they will develop only to the stage where they have a theca interna and then start to undergo atresia. Further development of these follicles to produce mature Graafian or antral follicles, of which there are about 200 growing follicles at puberty in the heifer, is dependent upon the stimulus of gonadotrophic hormones. Despite the absence of oestrous cycles, there is follicular growth as has been shown using transrectal ultrasonography in calves from 2 weeks of age. It was seen that there were follicular waves in response to follicle- stimulating hormone (FSH) secretion that were similar to those of the adult, and that individual follicular development was characterised by grow- ing, static and regressing phases (Adams, 1994). The sheep has been used extensively for studying many of the mechanisms involved in the initiation of puberty; however, it must be stressed that sea- sonality will exert an overriding influence in this species (see below). The onset of puberty is sig- nalled by either the occurrence of the first oestrus or the first ovulation; in the ewe lamb these do not occur simultaneously because the first ovulation is not preceded by behavioural oestrus. A similar response is seen in sexually mature ewes at the onset of the normal breeding season. The hormone that is primarily responsible for the onset of ovarian activity, and hence puberty, is luteinising hormone (LH). In adult ewes during the normal breeding season, basal LH concen- trations increase together with the LH pulse frequency to one per hour during the period of maximum follicular growth. This results in the development of follicles to the preovulatory stage, and their secretion of oestradiol, which activates the LH surge causing ovulation and corpus luteum formation. In the prepubertal ewe lamb, LH pulses occur at similar amplitudes but much lower frequencies (one every 2--3 hours). As a consequence, follicular growth is insufficient to activate the LH surge necessary for final follicular maturation and ovulation. Experimental evidence in prepubertal ewe lambs has shown that ovarian follicles are capable of responding to exogenous gonadotrophin stimula- tion, and the pituitary is capable of secreting LH at a frequency to stimulate ovulation. The failure of the prepubertal ewe lamb to undergo ovulation and exhibit oestrus is due to the high threshold for the positive-feedback effect of oestradiol, and thus there is no LH surge. At puberty, the threshold is lowered, thus allowing the pituitary to respond.This is some- times referred to as the 'gonadostat' theory. Other factors are also involved.The frequency of LH secretion is dependent upon gonadotrophin- releasing hormone (GnRH) from the hypothal- amus, which is controlled by an area in the hypothalamus referred to as the neural GnRH pulse generator. Age-related changes in brain mor- phology and neuronal cytoarchitecture may also be important, since extrapolation from studies per- formed in rats, for example, has shown an increase in the number of GnRH cells with spine-like pro- cesses on the soma and dendrites. In addition, the inhibitory effect of opioid peptides on LH secretion is reduced with age, which may provide a neuro- chemical explanation for the changes in pituitary sensitivity to oestradiol feedback that occur at puberty (Bhanot and Wilkinson, 1983; Wray and Hoffman-Small, 1986). The reason for the 'silent' first oestrus of the pubertal animal is believed to be because the central nervous system requires to be primed with progesterone before it will respond and the animal will show behavioural signs of heat.The first ovula- tory cycle has been shown to be short in pubertal heifers (7.7 +/-- 0.2 days), and the first corpus luteum (CL) not only has a shorter than normal life span but is also smaller in size. One explanation for this is that the dominant follicle, from which the first ovulation arises, had already entered the static phase of growth. The subsequent interovulatory interval was normal (Adams 1999). External factors influencing the time of onset of puberty The time of onset of puberty is determined by the individual's genotype, with smaller breeds of 4 animal tending to be slightly more precocious. However, this inherent timing is influenced by a number of external factors. Nutrition. There is good evidence that in most domestic species, the age of puberty is closely related to body weight; therefore, it is not surpris- ing that nutrition is an important factor. Animals that are well fed with good growth rates reach puberty before those that are poorly fed with slow growth rates. However, unless the animal is severely malnourished, the onset of cyclical activ- ity will eventually occur. Season of the year. In those species which are seasonal breeders, such as the ewe, mare and queen cat, the age at which puberty occurs will be influenced by the effect of season of the year. For instance, a filly born early in the year, i.e. January or February, may have her first oestrus in the May or June of the following year, i.e. when she is 16 or 17 months old. A filly foal born late in the year, July or August, may not have her first oestrus until she is 21 or 22 months old. The same is true of ewes which, depending upon the time of year at which they are born, may reach puberty as early as 6 months or as late as 18 months old. Proximity of the male. Studies in sheep and pigs have shown that exposure to the male of the species will advance the timing of the onset of puberty.This so-called 'ram or boar effect' is probably mediated by pheromonal and other sensory cues influencing hypothalamic GnRH secretion. Climate. Anthropomorphic extrapolation has assumed that animals living in the tropics reach puberty at an earlier age than those in temperate climates. Studies carried out in Zambia have shown that in cattle this is not true. Disease. Any disease which can influence the growth rate, either directly or because of interfer- ence with feeding and utilisation of nutrients, will delay the onset of puberty. THE OESTROUS CYCLE AND ITS PHASES Traditionally, the oestrous cycle is divided into a number of phases. Pro-oestrus. The phase immediately preceding oestrus. It is characterised by a marked increase in activity of the reproductive system. There is folli- cular growth and regression of the corpus luteum of the previous cycle (in polycyclic species). The uterus enlarges very slightly; the endometrium becomes congested and oedematous, and its glands show evidence of increased secretory activ- ity. The vaginal mucosa becomes hyperaemic; the number of cell layers of the epithelium starts to increase, and the superficial layers become corn- ified. The bitch shows external evidence of pro- oestrus with vulval oedema, hyperaemia and a sanguineous vulval discharge. Oestrus. The period of acceptance of the male. The onset and end of the phase are the only accu- rately measurable points in the oestrous cycle, and hence are used as the baseline for determining cycle length.The animal usually seeks out the male and 'stands' for him to mate her. The uterine, cer- vical and vaginal glands secrete increased amounts of mucus; the vaginal epithelium and endometrium become hyperaemic and congested; the cervix is relaxed. Ovulation occurs during this phase of the cycle in all domestic species with the exception of the cow, where it occurs about 12 hours after the end of oestrus. Ovulation is a spontaneous process in all domestic species with the exception of the cat, rabbit and camel, in which it is induced by the act of coitus. During pro-oestrus and oestrus there is follicu- lar growth in the absence of functional corpora lutea, the main ovarian hormones produced being oestrogens. Pro-oestrus and oestrus are frequently referred to collectively as the follicular phase of the cycle. Metoestrus. The phase succeeding oestrus. The granulosa cells of the ovulated follicle give rise to lutein cells which are responsible for the form- ation of the corpus luteum.There is a reduction in the amount of secretion from the uterine, cervical and vaginal glands. Dioestrus. The period of the corpus luteum. The uterine glands undergo hyperplasia and hypertrophy, the cervix becomes constricted and the secretions of the genital tract are scant and sticky; the vaginal mucosa becomes pale. The corpus luteum is fully functional during this phase, and is secreting large amounts of progesterone. ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 5 1 NORMAL OESTROUS CYCLES The period of the oestrous cycle when there is a functional corpus luteum is sometimes referred to as the luteal phase of the cycle, to differentiate it from the follicular phase. Since in most of our domestic species oestrus is the only readily identifiable phase of the oestrous cycle, there is some merit, in polyoestrous species, in dividing the cycle into oestrus and interoestrus, the latter including pro-oestrus, metoestrus and dioestrus. Another alternative division can be into follicular and luteal phases. Anoestrus. The prolonged period of sexual rest during which the genital system is mainly quies- cent. Follicular development is minimal; the corpora lutea, although identifiable, have regressed and are non-functional. Secretions are scanty and tenacious, the cervix is constricted, and the vaginal mucosa is pale. Natural regulation of cyclical activity Regulation of cyclical activity in the female is a complex process. With the development of new techniques, particularly those of hormone assays, and the application of new molecular biological techniques, there is a continual advance in the knowledge and understanding of the mechanisms involved. Although much of the early work was done on laboratory animals -- notably the rat and guinea pig -- there is now much more information about domestic species, although there are still areas, particularly in the bitch, which are not fully understood. Regulation of cyclical activity is mainly under the control of the hypothalamic--pituitary--ovarian axis. At one end of this axis there is the influence of the extrahypothalamic areas -- the cerebral cortex, thalamus and -- and the role played by stimuli such as touch (Ellendorff, 1978), whilst at the other end is the influence of the uterus upon the ovary. The pineal gland appears to have an important role in controlling reproduction in seasonal breed- ing species and also in the timing of puberty by influencing the release of FSH, LH and prolactin. Although much of the interest has been in the action of the indoleamine melatonin, there is increasing interest in the other pineal peptide hor- mones, namely arginine vasotocin, gonadotrophin and prolactin-releasing and inhibitory hormones. There is some suggestion that melatonin may not act directly upon the hypothalamus/anterior pitu- itary, but indirectly via the other pineal peptide hormones. Melatonin drives the reproductive response of the ewe to inductive photoperiods (Bittman et al., 1983). Rhythmic administration of melatonin to adult ewes exerts a similar effect to increased hours of darkness by inducing the onset of the breeding season (Arendt et al., 1983) and causes changes in prolactin concentrations in the plasma that are similar to those following exposure to short days (Kennaway et al., 1983). In sheep, an intact pineal gland is required for a normal photoperiodic response to altered daylight patterns; however, other seasonal environmental cues are important, since pinealectomised ewes still show seasonal breeding (Lincoln, 1985). The mare is a seasonal breeder, but is 'switched on' by increasing day length. The pineal gland is involved, since if it is removed the mare does not show a normal response to changes in photo- period. In intact mares, melatonin concentrations increase during hours of darkness (Grubaugh et al., 1982). There is some evidence that foals are conditioned at an early age and develop a pattern of melatonin secretion from about 7 weeks of age (Kilmer et al., 1982). The hypothalamus is responsible for the control of release of gonadotrophins from the anterior pituitary by the action of specific releasing and inhibitory substances. These are secreted by the hypothalamic neurons, and are carried from the median eminence of the hypothalamus by the hypothalamic--hypophyseal portal system. In 1971 the molecular structure of porcine GnRH was determined (Matsuo et al., 1971) as being a deca- peptide, and subsequently synthesised (Geiger et al., 1971). Opinion is divided as to whether GnRH is responsible in vivo for the release of both FSH and LH (Lamming et al., 1979), although the injection of GnRH stimulates the release of both FSH and LH in domestic species. As yet, no specific inhibitory factor such as that for prolactin has been identified for gonadotrophins. Specific neurotransmitter substances are involved in the regulation of the release of pitu- itary hormones. The role of three monoamines mid-brain light, olfaction and 6 has now been fairly well established (Kordon, 1978). Noradrenaline stimulates the release of FSH and LH; the inhibition of the conversion of dopamine to noradrenaline blocks the 'oestradiol- induced' release of LH, which is responsible for ovulation; whilst serotonin inhibits the basal secretion of LH and regulates other neurosecre- tory systems. Dopamine also has an important role in the control of prolactin release. There is good evidence that in domestic species the secretion of FSH and LH is controlled by two functionally separate, but superimposable, systems. These are the episodic/tonic system, which is responsible for the continuous basal secretion of gonadotrophin and stimulates the growth of both germinal and endocrine compo- nents of the ovary, and the surge system, which controls the short-lived massive secretion of gonadotrophin, particularly LH, responsible for ovulation. There are two hypothalamic centres that are involved in controlling these two systems (Figure 1.1). With the exception of the cat, rabbit and camel, all domestic species are spontaneous ovulators. However, in these three species ovulation is induced by the stimulation of sensory receptors in the vagina and cervix at coitus. This initiates a neuro-endocrine reflex ultimately resulting in the activation of GnRH neurons in the surge centre and release of a surge of LH. Not only does the anterior pituitary have a direct effect upon ovarian functions by stimulat- ing folliculogenesis, follicular maturation, ovula- tion and corpus luteum formation, but the ovary has an effect upon the hypothalamus and anterior pituitary.This is mediated by oestradiol, produced by the maturing follicle, and by progesterone, pro- duced by the corpus luteum. The episodic/tonic hypothalamic release centre is influenced by the negative-feedback effect of oestradiol and pro- gesterone. Low levels of progesterone also have a modulating influence on this centre, which appears to be particularly important in ruminants (Lamming et al., 1979). In the cow, ewe and sow (and probably in other domestic species) FSH secretion is also controlled by a number of ovarian-derived peptide hormones. The first that has been characterised, inhibin, is produced by the granulosa cells of large antral follicles, and can Fig. 1.1 Endocrine control of cyclical reproductive activity. ------, stimulation; - - -, inhibition; PGF2α, prostaglandin2α; IGFs, insulin-like growth factors; GH, growth hormone. (Adapted from Lamming et al., 1979.) be isolated from follicular fluid. It has also been isolated from the testis and seminal plasma (see Chapter 29). Inhibin and oestradiol act in concert in suppressing FSH secretion. Inhibin, which is produced by all antral follicles, has a longer half- life, and sets the overall level of negative feedback, whereas oestradiol, which is produced only by those antral follicles that have the potential for ovulation, is responsible for the day-to-day fluctu- ations (Baird et al., 1991). Two other peptide hor- mones have been isolated from ovarian follicular fluid; these have been designated activin, which stimulates, and follistatin, which suppresses, FSH secretion. Their roles in controlling and regulating follicular growth are not known. The positive-feedback effect of oestradiol on hypothalamic--pituitary function is well demon- strated in farm animals, since the preovulatory surge of oestradiol stimulates the release of LH, ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 Extrahypothalamic centres GH Hypothalamic centres peptides Surge Episodic/ tonic Anterior pituitary Oestrogens Ovary PGF2α Oxytocin Insulin Oestrogens/ progesterone Uterus Pineal Melatonin and other pineal Glucose Liver Oestrogens GH Insulin FSH/LH GnRH Progesterone/oestrogens IGF\'s Pancreas 7 1 NORMAL OESTROUS CYCLES which is so necessary for the process of ovulation and corpus luteum formation. The response of the anterior pituitary to GnRH is influenced by the levels of ovarian steroids so that there is increased responsiveness shortly after the level of progesterone declines and that of oestradiol rises (Lamming et al., 1979). There are probably self- regulatory mechanisms controlling gonadotrophin secretion acting locally within the anterior pitu- itary and hypothalamus. Tonic release of gonadotrophins, especially LH, does not occur at a steady rate but in a pulsatile fashion in response to a similar release of GnRH from the hypothalamus. The negative feedback of progesterone is mediated via a reduction in pulse frequency of gonadotrophin release, whereas oestradiol exerts its effect via a reduced pulse amplitude. The onset of cyclical activity after par- turition (see Chapter 7), at puberty or at the start of the breeding season is associated with increased pulse frequency of tonic gonadotrophin secretion. When the ram is placed in contact with ewes before the start of the breeding season, there is increased frequency of pulsatile LH release, which stimulates the onset of cyclical activity (Karsch 1984). Progesterone appears to play a critically im- portant role in the inhibition of the tonic mode of LH secretion in the ewe (Karsch et al., 1978). Progesterone is thus the main regulatory hormone which controls the oestrous cycle of the sheep and probably of other species too. Thus when the con- centration of progesterone in the circulation falls, associated with the regression of the corpus luteum, there is release of LH from the anterior pituitary. The rise in LH triggers the secretion of oestradiol; this sudden rise stimulates the surge centre for the LH release and, as a result of this sudden increase, ovulation of the mature follicle occurs (Karsch et al., 1978). In some species, notably the cow (see Figure 1.28 later), there is also a concomitant surge in FSH; although its significance is unclear it may be part of the 'ovulation-inducing' hormone complex. For this reason it is probably incorrect to assign a separate and specific physiological role for the two pituitary gonadotrophins. Thus, although ovulation and steroidogenesis can be initiated by both FSH and LH, it would appear that only FSH can induce early follicular growth, so that when the granulosa cells have matured and are able to respond to endogenous LH, the formation of a fully developed vesicular follicle occurs. Large amounts of a peptide similar to the hormone inhibin, produced by the Sertoli cells of the testis, have been found in bovine and porcine follicular fluid and granulosa cells. This hormone probably selectively inhibits FSH release from the anterior pituitary but it may also have a local role in controlling ovarian function; it has been shown to inhibit the binding of FSH to granulosa cells in the cow (Sato et al., 1982). Recently, the roles of insulin-like growth factors (IGFs), notably IGF-1, and their associated binding proteins have been shown to exert an influence on ovarian function at the level of the granulosa, thecal and luteal cells, probably by acting synergistically with gonado- trophins (Lucy et al., 1999). Furthermore, there is also good evidence in the cow that growth hor- mone (GH) also has a role in regulating ovarian function either directly or by stimulating the syn- thesis and secretion of IGF-1 by the liver (Lucy et al., 1999). These findings are of considerable practical interest, because it is likely that they may be important in mediating the effects of nutrition on reproduction. Throughout the oestrous cycle, during preg- nancy and other reproductive stages, there is dynamic follicular activity with growth and atresia; only about 1% of antral follicles sub- sequently ovulate. There appear to be two differ- ent patterns of follicular growth in mammals (Fortune, 1994). Thus in cattle, sheep and horses the development of antral follicles to sizes close to that at ovulation occurs throughout the oestrous cycle including the luteal phase, whereas in the pig and rat the development of preovulatory-size follicles only occurs in the follicular phases of the cycle in the absence of a functional corpus luteum (CL). Follicular development occurs in stages, the following terminology for which is now generally accepted (Webb et al., 1999): recruitment -- gonadotrophin stimulation of a pool of rapidly growing follicles selection -- a process whereby one or more of the recruited follicles are selected to develop further 8 ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 dominance -- the mechanism whereby one (the dominant follicle), or several follicles, undergo rapid development in an environment where the growth and development of other follicles is suppressed. The pattern of follicular dynamics has been summarised, particularly in ruminant species, by Adams (1999), and it is appropriate to quote this as follows: '(1) follicles grow in a wave-like fashion; (2) periodic surges in circulating FSH are associated with follicular wave emergence; (3) selection of a dominant follicle involves the decline in FSH and acquisition of LH responsive- ness; (4) periodic anovulatory follicular waves continue to emerge until the occurrence of an LH surge; (5) within species, there is a positive rela- tionship between the duration of the oestrous cycle and the number of follicular waves; (6) prog- esterone is suppressive to LH secretion and the growth of the dominant follicle; (7) the duration of the interwave interval is a function of follicular dominance, and is negatively correlated with cir- culating FSH; (8) follicular dominance in all species is more pronounced during the first and last follicular waves of the oestrous cycle; (9) preg- nancy, the prepubertal period and seasonal anoestrus are characterised by regular, periodic surges of FSH and emergence of anovulatory follicular waves.' The CL is rapidly formed from the Graafian fol- licle after ovulation primarily from the granulosa and the thecal cells; in the ewe, for example, its mass increases 20-fold over 12 days (Reynolds and Redmer, 1999). For some time it was assumed that, once formed, it remained a relatively static structure but now it has been shown that when it is functionally mature there is rapid cellular turnover, although there is little change in size.The fully formed CL consists of a number of different cell types: the steroid-secreting large and small luteal cells, fibroblasts, smooth muscle, pericytes and endothelial cells. It has the greatest blood supply per unit tissue of any organ (Reynolds and Redmer, 1999). In the ewe, based on volume, the large luteal cells comprise 25--35%, the small luteal cells 12--18% and vascular elements 11% (Rodgers et al., 1984). Although the CL develops as a result of ovulation, in some species, notably the bitch, there are early signs of luteinisation of the follicle before it has ovulated. The stimulus for the formation and maintenance of the CL prob- ably varies within species.The hormones which are most likely to be involved are prolactin and LH, but there is some evidence that they are involved together, perhaps in association with FSH. Although all three hormones are probably involved in the induction of luteinisation of granulosa cells, the available evidence suggests that FSH is prob- ably not required for the maintenance of luteal function. The difference between species is well illustrated by the observation that LH will prolong luteal function in the sow, but prolactin will not (Denamur et al., 1966; Anderson et al., 1967). However, in the ewe prolactin appears to be more important as a luteotrophic agent, since LH will exert an effect only if infused from day 10 to day 12 of the oestrous cycle. The role of prolactin in the control of repro- duction in many domestic species is still largely speculative, and in many cases it is only possible to extrapolate from studies in the traditional lab- oratory species. Unlike other anterior pituitary hormones that require hypothalamic stimulation, it appears that prolactin secretion is spontaneous, and that it is largely controlled by inhibition by hypothalamically-derived prolactin inhibitory factor (PIF), which is believed to be dopamine. There is some evidence to suggest that dopamine may have a dual role as a stimulant of prolactin secretion, rather like a prolactin-releasing factor (PRF). Much interest has been directed towards the role of certain endogenous peptides with opioid activity such as β-endorphin and met-enkephalin. These substances have been found in high con- centrations in hypothalamic--hypophyseal portal blood. The administration of exogenous opioid peptides inhibits the secretion of FSH and LH whilst stimulating the secretion of prolactin. If an opiate antagonist such as naloxone is infused, there is an increase in mean concentrations of gonadotrophins in the plasma and the frequency of episodic gonadotrophin secretion. The effect of opioids appears to be influenced by the steroid environment of the animal; for example, in ewes, naloxone increased the mean plasma concen- tration of LH and the episodic frequency in a 9 1 NORMAL OESTROUS CYCLES high-progesterone environment. However, in ovariectomised ewes or those with oestradiol implants, naloxone had no effect (Brooks et al., 1986). It is possible that the negative feedback of progesterone on LH release (see below) may be mediated via opioids (Brooks et al., 1986). The presence of a functional CL, by virtue of its production of progesterone, inhibits the return to oestrus by exerting a negative feedback effect upon the anterior pituitary; this is most obvious during pregnancy (see Chapter 3). In the normal, non-pregnant female, oestrus and ovulation occur at fairly regular intervals; the main control of this cyclical activity would appear to be the CL.There is also evidence that the CL also exerts a positive intra-ovarian effect by increasing the number of small antral follicles in that ovary (Pierson and Ginther, 1987). Although it has been known for nearly 80 years that in certain species of animal the uterus influ- ences ovarian function (Loeb, 1923) the mech- anism has been fully understood only in recent years. It has been demonstrated that in many species removal of part or all of the uterus will result in the prolongation of the life span of the CL (du Mesnil du Buisson, 1961; Rowson and Moor, 1967); these species include the cow, mare, ewe, goat and sow. In the human, dog and cat the normal life span of the CL is unaltered in the absence of the uterus. In the cow, ewe and goat the 'luteolytic' action of the uterine horn is directed exclusively to the CL on the adjacent ovary (Ginther, 1974). Thus, if one of the uterine horns is surgically removed on the side adjacent to the ovary with a CL then the latter will persist. If the contralateral horn is removed, then the CL will regress at the normal time. It appears that in these species the luteolytic substance is transported directly from the uterus to the ovary. In the ewe it has been shown experimentally that the most likely route for transport of the substance is the middle uterine vein, since when all other struc- tures between the ovary and uterus are severed there is still normal regression of the CL (Baird and Land, 1973). In the mare no local effect can be demon- strated, since if the ovary is transplanted outside the pelvic cavity, luteal regression still occurs (Ginther and First, 1971). It is generally assumed that in this species the luteolysin is transported throughout the systemic circulation. In the pig the luteolytic substance is trans- ported locally (du Mesnil du Buisson, 1961) but not exclusively to the adjacent ovary. It has been shown that, following surgical ablation of parts of the uterine horns, provided at least the cranial quarter of the uterine horn is left, regression of the CLs occurs in both ovaries. If more than three-quarters of the horn is excised, then regres- sion of the CLs occurs only in the ovary adjacent to the intact horn. In the bitch, the mechanisms of control of the life span of the CLs are not fully understood, and in this species even in the absence of pregnancy there is always a prolonged luteal phase traditionally called metoestrus. Although the importance of the middle uterine vein in the transfer of the luteolytic substance has been demonstrated, the mechanisms whereby the luteolytic substance passes to the ovary have not been conclusively shown in all species, although they have been fairly well evaluated in the ewe and cow. In the former species, it appears that the close proximity of the ovarian artery and utero- ovarian vein is important, particularly since at their points of approximation the walls of the two vessels are thinnest; there is no anastomosis (Coudert et al., 1974). This allows the leakage of the luteolytic substance from the uterine vein into the ovarian artery and thus to the ovary, by a form of counter-current exchange through the walls of the vessels. It has been suggested (Ginther, 1974) that the variation in the response to partial or total hysterectomy in different species is probably due to differences in the relationships between the vasculature of the uterus and ovaries. It was not until 1969 that the substance responsible for luteolysis was identified, when the duration of pseudo-pregnancy in the rat was shortened by the injection of prostaglandin F2α (PGF2α). This same substance has subsequently been shown to have potent luteolytic activity in the ewe, doe, cow, sow and mare. Although it has been proved only in ruminants and the guinea pig that it is the natural luteolysin, it is likely that it is also true for the other species listed. PGF2α is a derivative of the unsaturated hydroxy acids linolenic and arachidonic acids. It derived its name because it was first isolated from 10 fresh semen and it was assumed to be produced in the prostate gland. It is synthesised in the endometrium of a number of species (Horton and Poyser, 1976), and in the ewe it has been demon- strated in increasing amounts at and around the time of luteal regression (Bland et al., 1971). Luteal regression can be viewed from two aspects. Firstly, functional regression is rapid, so that the secretion of progesterone declines rapidly. Secondly, as regards structural regression when the CL is reduced in size, the latter process takes longer than the former. In ruminants, luteal regression is caused by episodic release of PGF2α from the uterus at intervals of about 6 hours.This is induced by oxytocin secreted by the CL; thus, each episode of PGF2α release is accompanied by an episode of oxytocin release. Furthermore, PGF2α stimulates further secretion of oxytocin from the ovary. It has been postulated that the abundant, non-steroido- genic endothelial cells of the CL may mediate the actions of PGF2α, and that its physical demise is due to the action of invading macrophages which may secrete cytokines, such as tumour necrosis factor (TNF)α (Meidan et al., 1999). The sensitivity of the uterus to oxytocin is determined by the concentration of endometrial oxytocin receptors. At the time of luteal regression in sheep they rise approximately 500-fold (Flint et al., 1992). Their concentration is determined by the effects of progesterone and oestradiol. Thus, the high concentrations of progesterone which occur after the formation of the CL reduce the number of receptors, so that in the normal oestrous cycle of the ewe they start to increase from about day 12. Exogenous oestradiol causes premature induction of oxytocin receptors, result- ing in premature luteolysis (Flint et al., 1992). In non-ruminant species, much less is known about the mechanisms of luteolysis. The CL becomes more sensitive to the leuteo- lytic effect of PGF2α as it ages. The early CL is unresponsive to PGF2α. THE MARE Cyclic periodicity Fillies are often seen in oestrus during their second spring and summer (when they are year- lings), but under natural conditions it is unusual for them to foal until they are over 3 years old. The mare is normally a seasonal breeder, with cyclic activity occurring from spring to autumn; during the winter she will normally become anoe- strous. However, it has been observed that some mares, especially those of native pony breeds, will cycle regularly throughout the year. This tendency can be enhanced if the mares are housed and given supplementary food when the weather is cold and inclement, and if additional lighting is provided when the hours of daylight are short. Horse breeding has been influenced by the demands of thoroughbred racing, because in the northern hemisphere foals are aged from 1 January, irrespective of their actual birth date. As a result, the breeding season for mares has been, for over a century, determined by the authorities as running from 15 February to 1 July. Since the natural breeding season does not commence until about the middle of April, and maximum ovarian activity is not reached until July, it is obvious that a large number of thoroughbred mares are bred at a time when their fertility is suboptimal (see Chapter 26). The winter anoestrus is followed by a period of transition to regular cyclic activity. During this transition, the duration of oestrus may be irregu- lar or very long, sometimes more than a month. The manifestations of heat during the transitional phase are often atypical and make it difficult for the observer to be certain of the mare's reproduc- tive status. Also, before the first ovulation, there is poor correlation between sexual behaviour and ovarian activity; it is common for the early heats to be unaccompanied by the presence of palpable follicles, and some long spring heats are anovula- tory. However, once ovulation has occurred, regular cycles usually follow. The average length of the equine cycle is 20--23 days; the cycles are longer in spring and shortest from June to September. Typically, oestrus lasts 6 days and dioestrus 15 days. Ovulation occurs on the penultimate or last day of heat, and this rela- tionship to the end of heat is fairly constant and irrespective of the duration of the cycle or the length of oestrus; Hammond (1938) found that manual rupture of the ripe follicle resulted in ter- mination of oestrus within 24 hours. The diameter of the ripe follicle is 3--7 cm. During the last day ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 11 1 NORMAL OESTROUS CYCLES before ovulation, the tension in the follicle usually subsides, and the palpable presence of a large fluc- tuating follicle is a sure sign of imminent ovulation. The onset of heat after foaling occurs on the fifth to 10th day. This foal heat is sometimes rather short, 2--4 days. It is traditional to cover a mare on the ninth day after foaling. The first two post-parturient cycles are a few days longer than subsequent ones. During oestrus, a single egg is usually released, and there is a slight preponderance of ovulations from the left ovary. Assessing the functional activ- ity of the two ovaries on the basis of post-mortem counts of CLs in 792 equine genitalia, Arthur (1958) recorded an incidence of 52.2% of ovula- tions from the left ovary. Twin ovulation com- monly occurs in mares; Burkhardt (1948), in a study of June--July slaughterhouse specimens, saw 27% of double ovulations, and Arthur (1958) found an overall frequency of 18.5%, with a summer peak of 37.5%. However, there is a strong breed influence on twin ovulation; thoroughbreds are prone to it, but pony mares rarely show it. A fascinating finding by Van Niekerk and Gernaeke (1966) was that only fertilised eggs pass into the uterus; non-fertilised eggs remain for months in the uterine tubes, where they slowly disintegrate. All equine ovulations occur from the ovulation fossa; only at the ovarian hilus may occasional protrusions of corpora lutea be seen, but because of the curvature of the ovary and the presence of the adjacent substantial fimbriae these protusions cannot be identified by rectal palpation. Day (1939) has given a clear picture of the changes which occur in the equine ovary during an oestrous cycle. Figures 1.2--1.6 are diagrams of ovaries of the mare, half natural size, at different Fig. 1.3 Ovaries of a 9-year-old farm mare in dioestrus. Corpus luteum in right ovary, orange in colour; pleats loose. stages of the oestrous cycle, whereas Figures 1.7--1.12 show examples of whole ovaries, cross- sections and B-mode ultrasound images. Just before the onset of heat, several follicles enlarge to a size of 1--3 cm. By the first day of oestrus one follicle is generally considerably larger than the Fig. 1.4 Ovaries of a 4-year-old shire mare in dioestrus. Corpus luteum in left ovary, brownish-red in colour; pleats distinct; central cavity containing blood clot. Right ovary contains a follicle filled with blood. Fig. 1.5 Ovaries of a 6-year-old farm mare in dioestrus. A corpus luteum in each ovary, orange-yellow in colour; pleats distinct. Fig. 1.2 The ovaries of a 5-year-old farm mare in oestrus. Specimens obtained in May. Regressing corpus luteum in left ovary, bright yellow ochre in colour. 12 Fig. 1.6 Ovaries of a 6-year-old hunter mare in dioestrus. Corpus luteum in right ovary, pale yellow in colour; pleats distinct. Central cavity. remainder, having a diameter of 2.5--3.5 cm. During oestrus, this follicle matures and ruptures when it has attained a diameter of 3--7 cm. After ovulation, the other follicles regress, until, during the first 4--9 days of the ensuing dioestrus, no fol- licles larger than 1 cm are likely to be present. Several hours before ovulation the ripe follicle becomes much less tense. The collapsed follicle is recognised by an indentation on the ovarian surface; there is usually some haemorrhage into the follicle, and the coagulum hardens within the next 24 hours. Quite frequently the mare shows evidence of discomfort when the ovary is palpated soon after ovulation. Unless sequential transrectal palpation or ultrasonic examinations are per- formed, it is sometimes possible to confuse a mature follicle with the early corpus haemorrhag- icum, since before ovulation the follicular antrum is filled with follicular fluid and then soon after ovulation it becomes filled with blood. For this reason mares are sometimes incorrectly diagnosed as having failed to ovulate. For the next 3 days the ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 (a) (b) (c) Fig. 1.7 Ovary from an acyclical (anoestrus) mare. (a) The ovary was hard on palpation with no evidence of follicular activity. Note the ovulation fossa (o). (b) Cross- section of the ovary. Note that there are a few small follicles (f) \< 1 cm in diameter which are contained within the ovarian matrix. (c) B-mode ultrasound image of the same ovary showing small anechoic (black) areas \< 1 cm in diameter which are follicles (f). 13 1 NORMAL OESTROUS CYCLES (a) (b) (c) Fig. 1.8 Ovary from a mare in the early follicular phase. (a) The ovary was soft on palpation with evidence of large follicles near the surface of the ovary (f). Note the ovulation fossa (o). (b) Cross-section of the ovary. Note three follicles are at least 2 cm in diameter. (c) B- mode ultrasound image of the same ovary showing one large anechoic (black) area about 3.5 cm in diameter which is a follicle (f), together with three smaller ones. luteinising mass can be felt as a resilient focus, but later it tends to have the same texture as the remainder of the ovary. In pony mares, however, of known history from daily examinations, Allen (1974) reports that it is possible to follow the growth of the CL by palpation because in ponies it forms a relatively large body in a small ovary. The CL attains maximum size at 4--5 days, but it does not protrude from the ovarian surface. On section of the ovary it is brown and later yellow and of a tri- angular or conical shape, with the narrower end impinging on the ovulation fossa. Its centre is com- monly occupied by a variable amount of dark- brown fibrin. The cyclical CL begins to regress at about the 12th day of the cycle, when there is a par- allel fall in the blood progesterone concentration. From this day onwards the events previously described recur. Ovulation, with the subsequent formation of a CL, does not always occur; the folli- cle may regress or sometimes undergo luteinisation (see Figure 1.10(b)). B-mode ultrasound imaging with a rectal trans- ducer has been used to visualise follicles (see Figures 1.7--1.12). This is particularly useful in 14 ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 (a) (b) Fig. 1.9 Ovary of a mare with a single large preovulatory follicle. (a) Section of the ovary showing a 4 cm follicle (f). (b) B-mode ultrasound image of a different ovary showing a 4--5 cm preovulatory follicle (f) as a large anechoic (black) area. \(a) B-mode ultrasound image of an ovary showing the corpus haemorrhagicum. (b) B-mode ultrasound image of a 5 cm anovulatory follicle that is undergoing luteinisation. (a) (b) Fig. 1.10 detecting the possibility of twin ovulations and also in determing the timing of ovulation. Ginther (1986) observed that in the preovulatory period there was a change in the shape of the follicle and a thickening of the follicular wall, which, together with the assessment of the size of the follicle, could be used to predict the time of ovulation. The same author has used this technique to assess corpora lutea. He identified differences in the echogenic properties of the CL depending upon the persistence of the corpus haemorrhagicum; this he identified in about 50%. 15 1 NORMAL OESTROUS CYCLES (a) (b) (c) Fig. 1.11 Ovary of a mare in early dioestrus. (a) The corpus luteum (cl), although present, could not be palpated externally, whereas a follicle (f) could be identified. Note the ovulation fossa (o). (b) Section of the same ovary. Note that the corpus luteum (cl), still with a central blood clot, impinges on the ovulation fossa (o) where ovulation occurred. Also, one large follicle (f) and several smaller ones can be identified. (c) B-mode ultrasound image of a different ovary showing the corpus luteum (cl) and follicles (f). During winter anoestrus, both ovaries are typi- cally small and bean-shaped, common dimensions being 6 cm from pole to pole, 4 cm from the hilus to the free border and 3 cm from side to side. Not uncommonly, however, in early spring or late autumn, the anoestrous ovaries are of medium or large size and knobbly due to the content of numerous follicles of 1--1.5 cm diameter. During the cycle, there are large variations in the ovarian size depending on the number and size of the fol- licles. During oestrus the ovary of the thorough- bred mare may contain two or even three follicles, each of 4--7 cm, and these, with other subsidiary follicles, combine to give it a huge size. During dioestrus, however, with an active CL and only atretic follicles the ovary may be little larger than in anoestrus. By visual examination of the vagina and the cervix using an illuminated speculum, it is possible to detect the preovulation period. In dioestrus, the 16 ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 (a) (b) (c) Fig. 1.12 Ovary of a mare in mid-dioestrus. (a) The corpus luteum (cl), although present, could not be palpated externally; there was no evidence of any follicles. Note the ovulation fossa (o). (b) Section of the same ovary. Note the corpus luteum (cl), which impinges upon the ovulation fossa (o) where ovulation has occurred. (c) B-mode ultrasound image of the same ovary showing a speckled area corresponding to the corpus luteum (cl). cervix is small, constricted and firm; it and the vagina are pale pink, while mucus is scanty and sticky. During oestrus, there is a gradual increase in the vascularity of the genital tract and relaxation of the cervix with dilatation of the os. As oestrus advances and ovulation time approaches, the cervix becomes very relaxed and its protrusion can be seen lying on the vaginal floor, with its folds oedematous; the vaginal walls are glistening with clear lubricant mucus. After ovulation there is a gradual reversion to the dioestrous appearance. During anoestrus, as in pregnancy, both the vagina and cervix are blanched; the cervix is constricted and generally turned away from the 17 1 NORMAL OESTROUS CYCLES midline, the external os being filled with tenacious mucus. On palpating the uterus per rectum, cyclic changes can be detected.With the development of the CL the uterus increases in tone and thickness, but these features diminish when the CL regresses. At oestrus there is no increase of tone. During anoestrus and for the first few days after ovulation the uterus is flaccid. During dioestrus, pregnancy and pseudopreg- nancy the cervix is identified on rectal palpation as a narrow firm tubular structure; at oestrus it is soft and broad. A temporary pneumovagina assists in this examination (Allen, 1978). Signs of oestrus The mare becomes restless and irritable; she fre- quently adopts the micturition posture and voids urine with repeated exposure of the clitoris (Figure 1.13). When introduced to a stallion or teaser, these postures are accentuated; the mare raises the tail to one side and leans her hindquar- ters.The vulva is slightly oedematous, and there is a variable amount of mucoid discharge. A mare which is not in oestrus will usually violently oppose the advances of a stallion, and for this reason when 'trying' mares at stud it should be done over a gate, box-door or stout fence. If the mare is in oestrus the stallion usually exhibits 'flehmen'. Good stud management requires that a mare is accustomed to the procedure and that, because of the interval between the end of the last oestrus and the start of the next, she is teased 15--16 days after the end of the last oestrus. Endocrine changes during the oestrous cycle The trends in endocrine changes are shown in Figure 1.14.The secretion of FSH is biphasic with surges at approximately 10--12 day intervals. One surge occurs just after ovulation, with a second surge in mid- to late dioestrus approximately 10 days before the next ovulation. It has been sug- gested that this increase in secretion, which is unique to the mare, is responsible for priming the development of a new generation of follicles, one of which will ovulate at the next oestrus Fig. 1.13 teasing. Exposure of the clitoris (ct) in response to (Evans and Irvine, 1975). The pattern of LH secretion is also unusual in this species since there is no sudden surge of this hormone but a gradual increase and persistence of elevated levels for 5--6 days on either side of ovulation. Oestrogens in the peripheral circulation reach peak values during oestrus whilst concentrations of progestrone and other progestrogens follow closely the physical changes of the CL. THE COW Cyclic periodicity Under conditions of domestication, normal and well-cared-for cattle are polyoestrous throughout the year. The age at first oestrus, or puberty, is affected by nutrition and season of birth, and ranges from 7 to 18 months, with an average of 10 18 FSH Fig. 1.14 show overt signs of oestrus yet have normal cycli- cal activity, a condition referred to as 'silent heat' or suboestrus. This may, however, be due to failure of the herdperson to observe the signs, rather than a failure of the cow to show signs. In heifers, the average length of the oestrous cycle is 20 days, and in cows 21 days, the normal ranges being 18--22 and 18--24 days, respectively. For many years, the average duration of oestrus has tradition- ally been recognised as being about 15 hours with a wide range of 2--30 hours; however, there is good evidence that in the 'modern' Holstein and Jersey cows, as compared with the heifer, the average is much shorter, perhaps an average of 8 hours. This has been shown using a radio frequency data com- munication system called 'Heat Watch' (Nebel et al., 1997), and is summarised in Table 1.1. There are a number of factors which can influ- ence the duration: breed of animal, season of year, presence of a bull, nutrition, milk yield, lactation number and, perhaps most important, the number of cows that are in oestrus at the same time (Wishart, 1972; Esslemont and Bryant, 1974; Hurnik et al., 1975).There is also good evi- dence that more signs of oestrus are observed during the hours of night, perhaps when the animals are least disturbed (Williamson et al., 1972; Esslemont and Bryant, 1976). Ovulation is spontaneous, and occurs on average 12 hours after the end of oestrus. Signs of oestrus Where artificial insemination is used, the accurate detection of oestrus by the herdsperson is para- mount in ensuring optimum fertility. Poor detec- tion is probably the most important reason affecting delayed breeding (Wood, 1976), whilst in the USA Barr (1975) has calculated that in ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 Trends in hormonal concentrations in the peripheral circulation of the mare during the oestrous cycle. months. A small proportion of heifers do not ovu- late at the first heat, and in a majority of young cattle the oestrus associated with the first ovula- tion is 'silent' (Morrow et al., 1969). Poor feeding and calfhood disease delay puberty. Once puberty has been reached, cyclical activity should persist, except during pregnancy, for 3--6 weeks after calving, during high milk yield (especially if there is some evidence of dietary insufficiency), and with a number of pathological conditions (see Chapter 22). Some cows and heifers also fail to Table 1.1 Duration of oestrus and number of standing events (mean and standard deviation) as determined by the 'Heat Watch' oestrus detection system (from Nebel et al., 1997) Nulliparous heifers Pluriparous cows Holstein Jersey Holstein Jersey No. of animals Duration of oestrus (hours) No. of standing events 114 11.3 +/-- 6.9 18.8 +/-- 12.8 46 13.9 +/-- 6.1 30.4 +/-- 17.3 307 7.3 +/-- 7.2 7.2 +/-- 7.2 128 7.8 +/-- 5.4 9.6 +/-- 7.4 19 1 NORMAL OESTROUS CYCLES Ohio dairy herds dairy farmers appeared to be losing twice as many days due to failure to detect heat as to conception failures. There are great variations amongst individual cattle in the intensity of heat signs; the manifest- ations tend to be more marked in heifers than in cows. However, it is generally agreed that the most reliable criterion that a cow or heifer is in oestrus is that she will stand to be mounted by another (Williamson et al., 1972; Esslemont and Bryant, 1974; Foote, 1975). The oestrous animal is restless and more active; Kiddy (1977), using pedometers, found that there was an average increase in activity of 393% at this time. More recently, Lewis and Newman (1984) found that pedometer activity was about twice as great in oestrus compared with the luteal phase of the cycle. In their study, 75% of cows showed peak pedometer readings on the day of onset of oestrus whereas 25% peaked 1 day after oestrus. There tends to be grouping of sexually active indi- viduals; there is a reduction in the time spent eating, resting and ruminating, and frequently a reduction in milk yield. Reduced milk yield has been shown to be a reliable indicator of the onset of oestrus; there is usually a compensatory rebound at the next milking (Horrell et al., 1984). In this study of 73 dairy cows, it was found that if a cow produced 75% of her usual yield there was a 50% chance of her being in oestrus. On the rare occasions that it fell to 25%, oestrus was always present. As the cow approaches oestrus she tends to search for other cows in oestrus, and there is licking and sniffing of the perineum. During this period, during oestrus and just afterwards, the cow will attempt to mount other cows; quite often before she does this she will assess the receptivity of the other cows by resting her chin on the rump or loins. If the cow to be mounted is responsive and stands, she will mount and sometimes show evidence of pelvic thrusting (Esslemont and Bryant, 1974). If the cow that is mounted is not in oestrus she will walk away and frequently turn and butt the mounting cow. A positive mounting response lasts about 5 seconds (Hurnik et al., 1975); however, if both cows are in oestrus it will be increased to about 7.5 seconds. In a group of 60 cows, Esslemont and Bryant (1976) observed that 33 cows were mounted on average 56 times. Sometimes there are signs of a vulval discharge of transparent mucus whose elasticity causes it to hang in complete clear strands from the vulva to the ground; it also adheres to the tail and flanks. The vulva may be slightly swollen and congested, and there is a small elevation of temperature. The tail may be slightly raised.The hair of the tail-head is often ruffled and the skin sometimes becomes excoriated due to mounting by other cows. For the same reason, the rear of the animal may be soiled with mud. At range, the oestrous cow may wander from the herd, and if isolated there will be bellowing. When she is put with a bull, the two animals lick each other and the cow often mounts the bull before standing to be mounted by him. For a short time after service, the cow stands with raised tail and arched back, and where actual service has not been seen this posture indicates that mating has occurred. Within 2 days of service, there is an occasional yellowish-white vulval discharge of mucus con- taining neutrophil leucocytes from the uterus. At about 48 hours after heat, irrespective of service, heifers and many cows show a bright red sanguin- eous discharge, the blood coming mainly from the uterine caruncles. The body temperature of dairy cows falls about 0.5°C the day before oestrus, increases during oestrus and falls by about 0.3°C at ovulation. The vaginal temperature, of 37.74°C, was lowest on the day before oestrus, increased by 0.1°C on the day of oestrus, and increased for the next 6 days until a plateau was reached.This was followed by a gradual decline from 7 days before oestrus (Lewis and Newman, 1984). Practical detection of this is tedious; however, the use of microwave telemetry systems may enable such measurements to be made in the future (Bobbett et al., 1977). Automated methods of measuring the related increase in milk temperature in the milking parlour have also been described (Maatie, 1976; Ball, 1977). Vaginal pH also fluctuates throughout the oestrous cycle but is lowest, namely 7.32, on the day of oestrus (Lewis and Newman, 1984). Cyclic changes in the vagina The main variations are in the epithelial cells of the anterior vagina and in the secretory function of the 20 cervical glands (Hammond, 1927; Cole, 1930). During oestrus, the anterior vaginal epithelium becomes greatly thickened due to cell division and to the growth of the tall, columnar, mucus-secret- ing superficial cells. During dioestrus, these cells vary from flat to low columnar. Leucocytic in- vasion of the vaginal mucosa is maximal 2--5 days after oestrus. Copious secretion of mucus by the cervix and anterior vagina begins a day or so before heat, increases during heat and gradually diminishes to the fourth day after heat.The mucus is transparent and flows readily. Associated with these features of the cervical mucus are variations in its crystallisation patterns which can be seen when dried smears of mucus are examined microscopically. During oestrus, and for a few days afterwards, the crystals are dis- posed in a distinct aborisation pattern, while for the remainder of the cycle this pattern is absent. This phenomenon, together with the character and amount of cervical mucus, are dependent on the concentration of oestrogen. The postoestrous vaginal mucus shows floccules composed of leu- cocytes, and, as previously mentioned, blood is frequently present. Hyperaemia of the mucosae of the vagina and cervix is progressive during pro-oestrus and oestrus; the vaginal protrusion of the cervix is tumefied and relaxed, so that one or two fingers can be inserted into the cervical os. During metoestrus, there is a rapid reduction in vascular- ity, and from 3 to 5 days after heat the mucosa is pale and quiescent and the external os is con- stricted while the mucus becomes scanty, sticky and pale yellow or brown. There are also cyclic variations in vaginal thermal conductance and vaginal pH, the former rising just before oestrus (Abrams et al., 1975). When pH electrodes were placed in the cervical end of the vagina the pH fell from 7.0 to 6.72 one day before the first behav- ioural signs of oestrus, and at the start of oestrus fell again to 6.54 (Schilling and Zust, 1968). Cyclic changes in the uterus During oestrus, the uterus is congested, and the endometrium is suffused with oedematous fluid; its surface is glistening. The muscularis is physio- logically contractile so that when the uterus is pal- pated per rectum this muscular irritability, coupled with the marked vascularity, conveys a highly characteristic tonic turgidity to the pal- pating fingers; the horns feel erect and coiled.This tonicity is present the day before and the day after oestrus but is at its maximum during heat, and, with experience, the veterinarian can detect oestrus on this sign alone. Between 24 and 48 hours after oestrus the uterine caruncles show petechial haemorrhages, and these give rise to the postoestrous vaginal discharge of blood. In heifers there are often also associated perimetrial sub- serous petechiae. During dioestrus the endo- metrium is covered by a scanty secretion from the uterine glands. Cyclic changes in the ovaries Usually one follicle ovulates and one ovum is lib- erated after each heat, but twin ovulations occur in 4 or 5% of cows, and triplet ovulations more rarely. In dairy cattle, about 60% of ovulations are from the right ovary, although in beef cattle the functional disparity between the ovaries is not great. The size and contour of the ovaries will depend on the phase of the cycle. It is best to begin by studying the organs of a mature unbred heifer. Post-mortem section of such ovaries will reveal the most significant structures in them to be Graafian follicles and CLs. Follicular growth and development Follicular growth and atresia throughout the cycle is a feature in the cow (Matton et al., 1983). In the studies of Bane and Rajakoski (1961), two waves of growth were demonstrated, with the first wave beginning on the third and fourth day, and the second starting on the 12th to 14th day of the cycle. Consequently, a normal follicle of 9--13 mm was present from the fifth to the 11th day before becoming atretic. In the second wave the ovulatory follicle developed, and was 9--13 mm between the 15th and 20th days; the ovulatory follicle is selected at about 3 days before ovulation (Pierson and Ginther, 1988). Others have observed three waves of follicular development in most oestrous cycles (Sirois and Fortune, 1988; Savio et al., 1990). The ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 21 1 NORMAL OESTROUS CYCLES most notable feature was the regularity of the number of waves of follicular growth per oestrous cycle, which probably reflected genetic or environ- mental influences. Follicular growth is under the influence of FSH, with normally one follicle obtaining dominance and subsequently ovulating. The dominance does not appear to be mediated by the effect of inhibin but probably by some yet unknown intra-ovarian mechanism which does not involve the suppression of FSH secretion. In addi- tion, other metabolic hormones such as insulin growth factor 1 (IGF-1) may also be involved in follicular growth patterns (see review by Webb et al., 1992). Thus, during dioestrus several large follicles will be found ranging in size up to 0.7--1.5 cm in diameter. These follicles do not alter the general oval contours of the ovaries but do cause some overall variation in gross ovarian size. The ease of palpating them rectally will depend upon the size, degree of protrusion and relationship with the corpus luteum. During pro-oestrus and oestrus, the follicle which is soon to rupture enlarges, and ovulation occurs when it has attained a size of at least 1.9 cm (Hammond, 1927). On rectal palpation of the ovaries during heat it is usually possible to detect the ripening follicle as a slightly bulging, smooth soft area on the surface of one of them. Ovulation may occur from any aspect of the ovarian surface, and the shape of that organ sub- sequently when the CL develops will be chiefly influenced by this site. The point of ovulation is usually in an avascular area of the follicular wall, and consequently haemorrhage is not a feature of bovine ovulation, although there is marked post- ovulatory congestion around the rupture point, and sometimes a small blood clot is present in the centre of the new CL. The corpus luteum of the oestrous cycle On rupture, the ovum is expelled through a small breach in the surface of the follicle and, conse- quent on the escape of the greater part of its fluid, the follicle collapses. If the opportunity arose for repeatedly carrying out rectal examinations during heat and for the 24 hours succeeding it, this collapse would be detected. The ovary fre- quently feels flattened and soft. If such an ovary is examined post-mortem it will be seen that the surface from which ovulation has occurred is wrinkled and possibly bloodstained. The CL develops by hypertrophy and luteinisation of the granulosa cells lining the follicle. Enlargement is rapid. By the 48th hour after ovulation it has attained a diameter of about 1.4 cm. At this stage the developing CL is soft, and yields on palpation. Its colour is dull cream, and the luteinised cells can be seen in the form of loose pleats. The CL attains its maximum size by the seventh to eighth day of dioestrus (Figure 1.15). The luteinised pleats are now relatively compact, and the body comprises a more or less homogeneous mass, yellow to orange-yellow in colour. Its shape varies; the majority are oval, but some are irregularly square or rectangular. The greatest dimension of the fully developed structure varies from 2.0 to 2.5 cm; the changes in the dimensions of the CL are shown in Figure 1.16. Its weight also varies; in the authors' series, fully developed CLs have varied from 4.1 to 7.4 g. (Similar variations also occur in the weights of the CLs of pregnancy, ranging from 3.9 to 7.5 g.) Sometimes, the centre of the yellow body is occupied by a cavity (Figures 1.17 and 1.27). This has been seen in 25% of those collected by the author. The size of the cavity varies; in the majority it is small, averaging 0.4 cm in diameter, but occasionally it is large, up to 1 cm or more. It is occupied by yellow fluid. In the case just described, there is evidence of ovula- tion by the presence of a pin-head depression in the centre of the projection from the surface of the ovary.This serves to differentiate the CL from the abnormality of the cow's ovary: luteinisation of the walls of the follicle without ovulation. Nevertheless, it is probable that this is the condi- tion which has been described in the past as cystic corpus luteum and regarded as pathological; the presence of a cavity is normal. Fig. 1.15 Ovary of cow in mid-dioestrus. (a) A mature corpus luteum (cl) with ovulation papilla could be readily palpated together with a mid-cycle follicle (f). (b) Section of the same ovary showing the solid corpus luteum (cl) and mid-cycle follicle (f). (c) B-mode ultrasound image of the same ovary showing a speckled area corresponding to the corpus luteum (cl) and the mid-cycle follicle (f). 22 ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 Fig. 1.16 The development of follicular waves (- - - -) and corpus luteum (------) during the oestrous cycle of the cow. (A--E refers to Figures 1.18--26.) Projection of the corpus luteum from the surface of the ovary As the CL enlarges, it tends to push itself out of the ovary, stretching the surface of the latter, until by the time it attains maximum development it often forms a distinct projection.The degree and type of this projection vary. In the majority it is a distinct bulge about 1 cm in diameter with a clear-cut con- striction where it joins the general contour of the ovary. In other cases it is nipple-like (Figure 1.15). In a third type the projection is indistinct but diffuse and occupies the greater part of the ovary. It would seem that the type of protrusion which develops depends on the extent of the surface of the ovary occupied by the follicle just before ovulation. Figures 1.18--1.26 show bovine ovaries\* (natural size) during the oestrous cycle. Regressing corpus luteum The CL maintains its maximum size and remains unaltered in appearance until the onset of pro- oestrus, i.e. 24 hours or so before the onset of \*Throughout the book, sketches of bovine ovaries are of a section from the attached to the free border through the poles. In those cases in which this section did not pass through the greatest dimension of the significant corpus luteum or the largest follicle, the sketch has been made as though it did so but without materially altering the size of the ovary. (a) (b) (c) 23 1 NORMAL OESTROUS CYCLES (a) (b) (c) Fig. 1.18 Fig. 1.17 Ovary of cow in mid-dioestrus. (a) A mature corpus luteum (cl) with prominent ovulation papilla and mid-cycle follicle (f). (b) Section of the same ovary showing the corpus luteum (cl) with a central lacuna which was filled with orange-yellow fluid, and the mid-cycle follicle (f). Note that the 'wall' of the corpus luteum comprises at least 5 mm thickness of luteal tissue. (c) B-mode ultrasound image of the same ovary showing a speckled area corresponding to the 'wall' of the corpus luteum (cl), the central lacuna, and also the mid-cycle follicle (f). Fig. 1.19 Ovaries of a first-calf heifer in oestrus. 1, ripe follicle; 2, regressing corpus luteum, brick-brown. Stage A in Figure 1.16. Ovaries of a first-calf heifer in oestrus. 1, ripe follicle; 2, regressing corpus luteum, bright yellow; 3, corpus albicans. Stage A in Figure 1.16. 24 Fig. 1.20 Ovaries of a nulliparous heifer just after ovulation. 1, collapsed follicle, surface wrinkled and blood-stained petechiae in wall; 2, regressing corpus luteum, bright yellow. Stage B in Figure 1.16. Fig. 1.21 Ovaries of a young cow 1 day after ovulation. 1, developing corpus luteum, pleats loose, colour pale cream, central cavity; 2, regressing corpus luteum, bright orange-yellow; centre filled by connective tissue; 3, corpus albicans. Stage B in Figure 1.16. Fig. 1.22 Ovaries of a young cow 2 days after ovulation. 1, twin corpora lutea, some haemorrhage; 2, regressing corpus luteum, bright yellow. Stage C in Figure 1.16. Fig. 1.23 Ovaries of a 4-year-old cow in early dioestrus. 1, active corpus luteum, pleats loose, colour orange- yellow, central cavity; 2, regressing corpus luteum, dense and brown; 3, corpus albicans. Stage C in Figure 1.16. heat. From this point, it undergoes rapid reduc- tion in size and changes in colour and appearance. By the middle of oestrus, its diameter is reduced to 1.5 cm and its protrusion is much smaller and less distinct, while its colour is changing to bright Fig. 1.24 Ovaries of a 6-year-old cow in early dioestrus. 1, active corpus luteum, orange-yellow, atypical protrusion; 2, regressing corpus luteum, small, shrunken, scarlet; 3, corpus albicans; 4, follicle. Stage D in Figure 1.16. yellow. (This colour contrasts strikingly with that of the active body.) Its consistency is dense, and already scar tissue invasion is commencing. By the second day of dioestrus, its size is reduced to about 1 cm and its outline is becoming irregular. ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 Fig. 1.25 Ovaries of nulliparous heifers in dioestrus. 1, corpus luteum 2, largest follicle. Stage E in Figure 1.16. 25 1 NORMAL OESTROUS CYCLES Fig. 1.26 Size of the ovaries From the foregoing account, it will be appreciated that the size of an ovary will depend chiefly on the period in the oestrous cycle at which it is exam- ined and whether or not it contains an active CL. The presence of follicles does not alter the size of an ovary to anything like the same extent. In the great majority of heifers and young cows exam- ined at any time between the sixth and 18th days of the dioestrous period, one ovary will be dis- tinctly larger than the other. The approximate dimensions of the larger one will be 3.5 cm from pole to pole, 3 cm from the attached to the free border and 2.8 cm from side to side. (All ovarian dimensions given in this book are in this order.) From some part of its surface the CL will project. The smaller ovary will have approximate dimen- sions of 2.5 by 1.5 by 1.2 cm, and it will be flat from side to side. During the first 4 or 5 days of the interoestrus phase there will be relatively little variation in size, for as yet the developing CL has not attained sufficient bulk to influence the size of the ovary significantly, while the regressing CL has lost its significant bulk. During oestrus, also, there will be little difference in size. If the ovary which contains the follicle undergoing preovul- ation enlargement also contains the regressing CL (and this is often the case), the ovary containing the two structures will be a little larger than the other, but not strikingly so. Ovaries of the multiparous cow The ovaries of the normal multiparous cow do not differ greatly from those of the heifer or first calver. They tend, however, in many cases to be larger. This increase in size is due in part to the progressive deposition of scar tissue resulting from prolonged function, and in some cases also to the presence of large numbers of small but visible follicles. Not infrequently, the ovary that does not contain a CL measures 4 by 3 by 2 cm. Nevertheless, it is generally possible in mid- dioestrus to detect the CL, for, quite apart from its protrusion, the ovary containing it is plum-like, whereas the other is distinctly flattened from side to side. On section of such ovaries, the CLs, both active and regressing, and the follicles approach- ing maturity are identical with those described for Ovaries of parous cows in dioestrus. 1, corpus luteum; 2, largest follicle; 3, corpus albicans. Stage E in Figure 1.16. By this time its colour is changing to brown. By the middle of dioestrus, it has shrunk to a size of about 0.5 cm, and its surface protrusion is little larger than a pin-head. As it gets older its colour tends to change to red or scarlet. Small red rem- nants of corpora lutea tend to persist for several months. 26 the heifer. There is, however, an additional struc- ture to be recognised: old scarred CLs of previous pregnancies. They generally show as a white, pin-head-sized projection on the surface of the ovary, and on section are found to comprise mainly scar tissue. They are irregular in outline, with a maximum dimension of about 0.5 cm. Their colour is white (corpus albicans) or brown- ish-white. The CL of pregnancy does not atrophy after parturition as quickly as does that of the oestrous cycle after it has ceased to function. It is an appreciable structure for several weeks after parturition, brown in colour and about 1 cm in diameter. It becomes progressively invaded by scar tissue and remains throughout the cow's life. On post-mortem the presence of the corpus albi- cans serves to distinguish the cow from the heifer and in the former a count of the corpora albi- cantia gives the number of calves borne. The fully developed corpus luteum is present by the seventh day and persists unchanged until the onset of pro-oestrus at the 19th or 20th day. Figure 1.27 shows exceptional corpora lutea. Ultrasonic appearance of the ovaries In the previous sections of this chapter there are descriptions of the texture (as determined by pal- pation) and the appearance (as determined by sec- tioning after slaughter) of the ovaries and their contents. The advent of transrectal B-mode real- time grey scale ultrasound imaging, particularly using a 7.5 MHz transducer, has enabled detailed, accurate sequential examination of the ovaries to be made without adversely affecting the cow's health or fertility. The principles of the technique are described in Chapter 3, and for a detailed description of the echogenic appearance of the ovaries, readers are advised to consult Pierson and Ginther (1984) and Boyd and Omran (1991). The following normal structures can be identi- fied (see Figures 1.15 and 1.17): the ovarian stroma, antral follicles, CLs and ovarian blood vessels. In addition, pathological structures such as ovarian cysts can be seen (see Chapter 22).The ovarian stroma has a mottled echotexture. The antral follicles are readily identifiable as anechoic (black) structures of variable size, with a clearly defined line of demarcation between the follicular wall and antrum. Follicles will not always be regular and spherical in shape. CLs have a well- defined border and a mottled echogenic appear- ance which is less echogenic than the ovarian stroma; the presence of a fluid-filled lacuna can be readily identified as a dark, non-echogenic area in the centre. Differentiation between developing and regressing CLs can be difficult. Ovarian blood vessels, which can be confused with antral follicles, are black, non-echogenic structures. Movement of the transducer will usually demon- strate their elongated appearance. Endocrine changes during the oestrous cycle The trends in concentrations of reproductive hor- mones in the peripheral circulation are illustrated schematically in Figure 1.28; it is important to stress that hormones are secreted in a pulsatile manner and fluctuate considerably. An effective description is given by Peters (1985a,b). Just before the onset of behavioural oestrus, there is a sudden rise in plasma oestrogens, particularly oestradiol. Peak values occur at the beginning of oestrus with a subsequent decline to basal concentrations at the time of ovulation. During the rest of the cycle, there are fluctuations in concentrations, although there ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 Fig. 1.27 Examples of vacuolated or cavitated bovine corpora lutea, showing single (1) and sometimes multiple (2) cavities. 27 1 NORMAL OESTROUS CYCLES 200 100 0 20 10 0 200 100 0 6 3 0 0 21 Ovulation Ovulation Days of oestrous cycle Progesterone Total oestrogens LH (ng/ml) FSH (ng/ml) (ng/ml) (pg/ml) Oestrus Oestrus Fig. 1.28 induced by restraint for venepuncture is sufficient to cause a significant rise. THE EWE The sexual season of most breeds of sheep in Britain is from October to February, during which time there are 8--10 recurrent oestrous cycles. The stimulus for the annual onset of sexual activity is declining length of daylight. The extent of the breeding season diminishes with increase of lati- tude; thus at the equator ewes may breed at any time of year, whereas in regions of high latitude -- in both northern and southern hemispheres -- the breeding season is restricted and distinct, with a prolonged phase of anoestrus after parturition.The breed of ewe also influences the duration of the breeding season; for example, in Dorset Horns it is distinctly longer than in other breeds -- whereby three crops of lambs can be obtained in 2 years -- but in hill breeds, like the Welsh Mountain and Scottish Blackface, it is shorter. Local breeds of central Europe and the Merino in Australia may not show an annual anoestrus. Also, in 'ordinary' breeds in Britain, like the Clun Forest, which nor- mally show a distinct seasonal activity, isolated instances of successful mating may occur in every month of the year (Lees, 1969). Ewe-lambs and yearling ewes have shorter breeding seasons than older ewes. The seasonal onset of sexual activity can be advanced by artifical manipulation of the photoperiod and by the use of hormonal agents (described later in this chapter). The average duration of oestrus in mature ewes of British breeds is about 30 hours, and is at least 10 hours less in immature ewes. In Merinos, heat may last 48 hours. Ovulation occurs towards the end of oestrus, and the length of the oestrous cycle averages 17 days. Signs of oestrus and mating behaviour Oestrous ewes are restless. They seek the ram, and together form a following 'harem'. The ram 'tries' members of this group for receptivity by pawing with a forefoot, by rubbing his head along the ewe's side and by nipping her wool. A non- receptive ewe trots away, but when in full heat she Trends in hormone concentrations in the peripheral circulation of the cow during the oestrous cycle, with three follicular waves. is a discrete peak around day 6 of the cycle (Glencross et al., 1973) which may be related to the first wave of follicular growth (Ireland and Roche, 1983).The pre-oestrus rise in oestrogens stimulates the surge of LH from the anterior pituitary which is necessary for follicular maturation, ovulation and CL formation. A secondary less discrete peak has been demonstrated 24 hours after the ovulatory surge of gonadotrophin (Dobson, 1978). The changes in progesterone concentrations mimic closely the physical changes of the CL. In a number of cows there is evidence of a delay in progesterone production or secretion by the CL (Lamming et al., 1979) which does not appear to affect the fertility of the individual adversely. Peak values are reached by days 7 and 8 after ovulation, and decline fairly quickly from day 18. When progesterone values fall to fairly low basal levels the removal of the anterior pituitary block allows the sudden release of gonadotrophins. Prolactin values are frequently difficult to obtain since stress 28 stands, waggles her tail and moves it laterally. The vulva is slightly swollen and congested, and there is often a slight discharge of clear mucus.The ram mounts and makes a series of probing pelvic thrusts and then dismounts. After variable inter- vals further mounts occur before intromission is achieved, and this is characterised by a deep pelvic thrust. An essential feature of successful coitus in the Najdi and Awassi breeds of the Middle East is the lateral displacement by the ram of the fat tail of the ewe. Rams of other breeds are unable to move the tail sufficiently to perform intromission. More ovine matings occur during early morn- ing and evening. When several rams run with a flock, a hierarchy is established and the dominant male attracts the largest harem, but, despite this, a majority of ewes mate with more than one ram. Also, ewes show a preference for rams of their own breed or for a ram of their particular group if that group is mixed with other groups of differ- ent origin (Lees and Weatherhead, 1970). The number of services received by an oestrous ewe averages a little above four. Rams may serve from eight to 38 ewes in a day. Changes in the ovaries The ovaries of the ewe are smaller than those of the cow, and their shape is nearer the spherical. During anoestrus their size is approximately 1.3 by 1.1 by 0.8 cm, and the largest follicles present vary from 0.2 to 0.6 cm. Studies of folliculogene- sis are more difficult than in the cow or mare because transrectal ultrasonic sequential access to the ovaries is much more difficult. However, using either ultrasonography or laparoscopy evidence suggests that the ewe is similar to the cow, usually with three or four follicular waves in each oestrous cycle; if there are three then there are two during the luteal and one during the follicular phases (Noel et al., 1993; Leyva et al., 1995). There is less certainty about the mechanisms involved in the emergence of the dominant follicle(s). Even during anoestrus, folliculogenesis occurs with fol- licles reaching the size of those frequently present during cyclical activity (Bartlewski et al., 1995). At the onset of oestrus, one or more follicles will have attained a size of 1 cm. Their walls are thin and transparent and the liquor folliculi appears purple in colour. Grant (1934) has observed that rupture of the follicle is preceded by the elevation of a small papilla above the general surface; ovulation occurs through rupture of this papilla about 24 hours after the onset of heat.The development of the CL is rapid, being linear from day 2 to day 12 after ovulation (Jablonka-Shariff et al., 1993); by the fifth day of dioestrus it is 0.6 cm in diameter, and it attains its maximum size of 0.9 cm when it has a central cavity (Roux, 1936). As the dioestrous period advances, its colour changes from blood red to pale pink. Its size remains constant until the onset of the next oestrus, when regression is rapid and the colour changes, first to yellow and later to brownish yellow. The luteolytic mechanism is similar to that in the cow where at the end of dioestrus, due to the effect of oestradiol and progesterone, there is an increase in the number of uterine oxytocin receptors, and at the same time luteal oxytocin stimulates uterine PGF2α secretion and vice versa (Mann and Lamming, 1995). The first CLs formed after the first ovulations at the beginning of the breeding season have a shorter life span than subsequent ones. In twin ovulations the CLs may occupy the same or opposite ovaries. During pregnancy, the CL is 0.7--0.9 cm in diameter. Its colour is pale pink, but the central cavity has dis- appeared, having become filled by white tissue. Ovulation with CL formation, but without signs of heat, may occur during the so-called anoestrous period -- spurious ovulation (Grant, 1934). As to the number of ova shed at oestrus, genetic and nutritional factors play a part. Hill sheep in the UK generally have one lamb, but if they are transferred to lowland pastures where herbage is rich (before the onset of the breeding season), twins become common. With lowland breeds the general expect- ancy is an average of 1.5 lambs per ewe. Roux (1936) in South Africa has noted that age is also a factor in the incidence of twinning. It attains its maximum when ewes are 5--6 years old, after which it remains constant. Primiparous ewes are very much less likely to have twins than pluriparous ones. Ewes of the Border Leicester and Lleyn breeds are the most prolific of British sheep, and commonly bear triplets. The Finnish Landrace and Cambridge breeds normally produce two to four lambs per pregnancy. ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 29 1 NORMAL OESTROUS CYCLES Endocrine changes during the oestrous cycle The endocrine changes are shown in Figure 1.29. Just before the onset of oestrus there is a rise in oestrogens in the peripheral circulation, particu- larly oestradiol-17β. This is followed by a sudden surge of LH which reaches a peak about 14 hours before ovulation; coincidental with this peak is a rise in FSH. There is also a second FSH peak 2 days after ovulation. Progesterone concentrations follow closely the physical changes of the corpora lutea, but max- imum values are lower than those of the cow, i.e. 2.5--4 ng/ml. Prolactin fluctuates throughout the oestrous cycle; however, concentrations rise during oestrus and ovulation, presumably reflecting the role of this hormone in the formation of the CLs. THE DOE (NANNY) GOAT The breeding season in Britain is from August to February with the greatest activity in the months of October, November and December. Near the equator, there is no evidence of seasonality but continuous cyclic activity. The doe is poly- oestrous, with an interoestrus interval of 20--21 days, although it is rather irregular at the begin- ning of the breeding season. The duration of oestrus is 30--40 hours, with ovulation occurring 12--36 hours after the onset. The detection of heat in a doe is difficult in the absence of a male goat.The vulva shows some evi- dence of oedema and hyperaemia; the tail is wagged from side to side and up and down (the most reliable sign according to Llewelyn et al., 1993); the doe is restless and more vocal, has a reduced appetite and milk yield, and shows mounting behaviour. The presence of the phero- mones from the male goat, which can be trans- ferred from the scent gland on to a cloth, will often intensify the signs. The ovaries are of variable shape, depending upon the structures which are present, the longest dimension being about 2.2 cm. The largest fol- licles reach a maximum size of about 1.2 cm in diameter, and when they protrude from the surface often have a bluish tinge. There are few studies on follicular dynamics, however Ginther and Kot (1994), using ultrasonography, reported that there were four waves of growth, with ovula- tion occurring during the fourth wave. The corpora lutea are pink in colour. The endocrine changes measured in the peri- pheral circulation are very similar to those of the ewe. THE SOW Puberty in gilts is reached at about 7 months, but diet, breed (including the degree of in-breeding) and season of birth influence its onset. At the first oestrus the number of ovulations is low, but it increases thereafter so that if mating is delayed until the third heat a larger litter will result. The cross-breeding of in-bred lines increases the ovu- 90 60 30 0 30 20 10 0 100 50 0 3 2 1 0 0 17 Ovulation Ovulation Days of oestrous cycle Progesterone Total oestrogens LH (ng/ml) FSH (ng/ml) (ng/ml) (pg/ml) Oestrus Oestrus Fig. 1.29 Trends in hormone concentrations in the peripheral circulation of the ewe during the oestrous cycle. lation rate, as does the provision of a high-energy 30 diet for 11--14 days before the expected oestrus; continuing such a diet after mating, however, increases embryonic loss. Fecundity is best from the fourth to seventh gestations. Although the domestic sow is generally consid- ered to be polyoestrous, the wild pig is a seasonal breeder, the main period being late autumn with a second peak of activity in April (Claus and Weiler, 1985). There is some evidence of the in- fluence of photoperiod on reproduction in the domestic sow; for example, anoestrus is more pre- valent in summer, and to a lesser extent February and March, whilst the ovulation rate is lower in summer. Claus and Weiler (1985) found that by reducing the day length artificially from May to August they were able to decrease the interval from weaning to oestrus from 23.6 to 5.7 days. For the most part, the recurrent reproductive cycles of 21 days are interrupted by pregnancy and lactation. The average length of oestrus is 53 hours, with spontaneous ovulation occurring between 38 and 42 hours from the beginning (Signoret, 1971). During lactation, the physical stimuli of suckling suppresses cyclical activity, but many sows show an anovulatory oestrus 2 days after farrowing. When weaning occurs at 5 or 6 weeks, oestrus can be expected in 4--6 days. Earlier weaning results in a slightly longer time interval. Signs of oestrus Beginning 3 days before oestrus, the vulva becomes progressively swollen and congested; these features persist throughout oestrus and gradually subside during the 3 days afterwards. Restlessness is an unfailing sign of the approach of heat, and a peculiar repetitive grunt is emitted. With other sows the pro-oestrous animal sniffs their vulvae and may try to ride them, or will be the recipient of such attentions. The boar is sought, and in his presence the pro-oestrous female noses his testicles and flanks and may mount him but will refuse to be mounted. At the height of oestrus the sow assumes a stationary, rigid attitude with her ears cocked, and she appears to be quite oblivious to her environment. She generally remains still during coitus, which lasts an average of 5--7 minutes, but when mating with heavy boars gilts may become fidgety. Burger (1952) demonstrated that oestrus could readily be determined by firmly pressing the loin of the sow with the palms of both hands; the oestrous sow will stand motionless with cocked ears whereas sows not in heat will object to this approach. The same immobilisation response can be elicited if the attendant sits astride the sow, and it can also be obtained in the absence of a boar by reproduc- ing the voice or odour of the boar. The substance responsible for boar odour has been identified as 5α-andost-16-ene-3-one and it is secreted by the salivary glands. In the form of an aerosol it can be sprayed in the vicinity of sows to promote the standing reaction of oestrus. 'Silent heats' occur in about 2% of porcine cycles. Cyclic changes in the ovaries The ovaries of the mature cyclical sow are rel- atively large and mulberry-like, the surface lobul- ation being due to the elevations of the large follicles and CLs, which, when mature, attain diameters of 0.8--1 cm and 1--1.3 cm, respectively. It is much more difficult to study follicular dynamics in the sow compared with the cow and mare; firstly, there are the problems of identifying an individual follicle amongst a large number, and secondly, because until the recent development of small transrectal B-mode ultrasound transducer probes, it has been almost impossible to study changes sequentially in the same animal. It has been shown that, except during the follicular phase of the cycle, there is continuous prolifer- ation and atresia of follicles so that there is gener- ally a pool of about 50 between 2 and 5 mm in diameter. Between about day 14 and day 16 of the cycle, there is recruitment of follicles, probably under the influence of gonadotrophin stimulation induced because of the decline in progesterone and the withdrawal of the negative feedback effect. A substantial number of these follicles are destined for ovulation, although it is not possible to identify the pre-ovulatory population until day 21--22 of the oestrous cycle. The growth of selected pre-ovulatory follicles during the follicu- lar phase is associated with rapid atresia of small follicles, and a block to their replacement in the proliferating pool, thus indicating some intra- ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 31 1 NORMAL OESTROUS CYCLES ovarian control mechanism (Foxcroft and Hunter, 1985). The precise nature of the substances is not fully understood, although various substances have been proposed: steroids, growth factors (epi- dermal/transforming, fibroblast and insulin-like growth factors) and their related binding proteins, and other regulatory substances such as 'follicle regulatory protein' (Hunter and Picton, 1999). The ripe follicle is sea-shell pink colour with a fine network of surface blood vessels and a very trans- parent focus which indicates the site of imminent ovulation. Haemorrhagic follicles are common. After ovulation there remain a considerable number of follicles of about 0.4 cm, some of which gradually enlarge to 0.9 cm by the succeed- ing day 18. Immediately after ovulation, the ruptured fol- licle is represented by a congested depression, but the accumulation of blood clot soon gives it a conical shape. By day 3, its cavity is filled with dark red blood clot, which by day 6 is replaced by a connective tissue plug or by a slightly yellow fluid; clots may persist up to day 12 and fluid up to day 18. The CLs attain their maximum size at 12--15 days, after which they gradually regress to the next oestrus.They are dark red up to day 3 but then change to, and remain, wine red until day 15. As the CLs regress between days 15 and 18 the colour changes rapidly from wine red to yellow, creamy yellow or buff. The mechanism of luteo- lysis is not fully understood in the sow, for although PGF2α is secreted by the uterus as early as 12--16 days of the cycle, which is earlier than in other species (Bazer et al., 1982); the CLs are unresponsive to exogenous PGF2α until about 12--23 days after ovulation. During the process of luteolysis, the CLs are invaded by macrophages which produce tumour necrosis factor (TNF); this substance in association with PGF2α probably causes luteolysis (Wuttke et al., 1995). In addi- tion, there is also a suggestion that TNF inhibits oestradiol production, thereby removing a luteotrophic source. There is further rapid regression of the CLs at the next oestrus, but throughout the succeeding dioestrus the CLs remain as distinct entities; after this they regress sharply to grey pin-head foci. When, therefore, a cyclic sow is slaughtered in the first half of dioestrus the ovaries may show the wine-coloured CLs of the current cycle, also the smaller pale CLs of the previous cycle and the grey specks of the third-generation CLs. During the luteal phases of the cycle, oestrogens are very luteotrophic; as a result it is possible to prolong the life span of the CLs for several weeks, result- ing in a pseudopregnancy (Dzuik, 1977). Endocrine changes during the oestrous cycle The endocrine changes are shown in Figure 1.30. Oestrogens in the peripheral circulation start to rise at the time that the CLs begin to regress, reaching a peak about 48 hours before the onset of oestrus. The ovulatory LH peak occurs at the beginning of oestrus and 8--15 hours after the oestrogen peak; values remain low and fluctuate throughout the rest of the cycle. FSH concentra- tions vary considerably; however, there appears to 8 4 0 8 4 0 60 30 0 30 20 10 0 0 21 Ovulation Ovulation Days of oestrous cycle Oestrus Oestrus Progesterone Total oestrogens LH (ng/ml) FSH (ng/ml) (ng/ml) (pg/ml) 32 Fig. 1.30 Trends in hormone concentrations in the peripheral circulation of the sow during the oestrous cycle. be some pattern of secretion. Brinkley et al. (1973) demonstrated two surges, one concurrent withtheLHpeakandalargeroneonday2or3 of the cycle; Van de Wiel et al. (1981) found a similar pattern, the peak coinciding with the minimum value of oestradiol. As with other species, the progesterone concentrations closely follow the physical changes of the CLs. For the first 8 days after ovulation there is a good correla- tion between progesterone levels and the number of CLs; however, by 12 days it is less obvious (Dzuik, 1977). Two prolactin surges have been identified (Brinkley et al., 1973; Van de Wiel et al., 1981), the first one concomitant with the pre-ovulatory LH and oestrogen surges and a second during oestrus. THE BITCH Reproductive activity in the bitch differs from the polycyclic pattern of other species in that there are no frequent, recurring periods of heat. All bitches have a prolonged period of anoestrus or sexual quiescence between successive heats irrespective of whether they have been pregnant or not; this pattern has been described as monocyclic (Jöchle and Andersen, 1977). The average interval between successive oestrous periods is 7 months, but it is variable, and there is some evidence that the breed of th