arthurs-veterinary-reproduction-and-obstetrics-8th-edition (2).pdf

1 Endogenous and exogenous control of ovarian cyclicity In nature, it is the general rule that animals breed tive activity. Provided the environmental influ- once annually and parturition occurs in the ences are favourable at this time, then once the s...

1 Endogenous and exogenous control of ovarian cyclicity In nature, it is the general rule that animals breed tive activity. Provided the environmental influ- once annually and parturition occurs in the ences are favourable at this time, then once the spring, the time most favourable to the progeny in ‘biological clock’ is started it will continue for as that they grow up during the period of increasing long as the environment remains favourable. In light and warmth, and also at the time when food none of our domestic species is there a physiolo- for the mother is most abundant to ensure ade- gical change comparable with the menopause of quate lactation. Under the conditions of feeding women. and housing provided by domestication the Amongst non-seasonal polycyclic animals, such breeding season tends to be lengthened, and some as the cow and sow, the recurring cyclic activity is of our species, particularly the cattle, may breed interrupted by pregnancy, lactation and patho- at any time during the year; all domesticated logical conditions. In those species which are sea- animals, however, show a constant tendency to sonally polycyclic, the mare, ewe, doe (or nanny) revert to the natural breeding season. goat and cat, or monocyclic like the bitch, there For an animal to breed, it must be mated and are periods of sexual quiescence or anoestrus. hence must attract the male and be sexually When the female reaches puberty the genital receptive (in heat or in oestrus). All domestic organs increase in size. During the prepubertal species show recurring periods of sexual receptiv- period the growth of the genital organs is very ity, or oestrous cycles, which are associated with similar to that of other organ systems, but at the ripening in the ovaries of one or more puberty their growth rate is accelerated, a point Graafian follicles and culminate in the shedding well illustrated in the gilt, where the mean length of one or more ova. If a fertile mating occurs then of the uterine horns is increased by 58%, the pregnancy may ensue. 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 PUBERTY AND THE ONSET OF CYCLIC domestic species reach the age of puberty at the ACTIVITY following times: mare: 1–2 years The young female animal shows no evidence of cow: 7–18 months recurring or cyclic periods of sexual receptivity. ewe: 6–15 months The onset of such changes when the female doe or nanny goat: 4–8 months becomes sexually mature and able to reproduce is sow: 6–8 months referred to as puberty. Amongst females of the bitch: 6–20 months domestic species, puberty precedes the develop- queen cat: 7–12 months ment of physical maturity and, although they become capable of reproducing, their efficiency, The changes that occur at puberty depend particularly with respect to their fecundity, has directly upon the activity of the ovaries, which have not reached its maximum. two functions: the production of the female The initiation of puberty is largely a function of gametes and the synthesis of hormones. Let us con- the animal’s age and maturity since the female is sider the changes that occur in the ovary of the born with a genetic potential for cyclic reproduc- young heifer calf. At birth, each ovary may contain 3 1 NORMAL OESTROUS CYCLES up to 150 000 primary or primordial follicles; activate the LH surge necessary for final follicular each consists of an oocyte surrounded by a single maturation and ovulation. layer of epithelial cells, but there are no thecal Experimental evidence in prepubertal ewe lambs cells. Soon after birth, the ovaries start to develop has shown that ovarian follicles are capable of and produce growing follicles which consist of an responding to exogenous gonadotrophin stimula- oocyte with two or more layers of granulosa cells tion, and the pituitary is capable of secreting LH at and a basement membrane. The stimulus for the a frequency to stimulate ovulation. The failure of development of these follicles is intra-ovarian, and the prepubertal ewe lamb to undergo ovulation and until the heifer reaches the age of puberty they exhibit oestrus is due to the high threshold for the will develop only to the stage where they have a positive-feedback effect of oestradiol, and thus there theca interna and then start to undergo atresia. is no LH surge. At puberty, the threshold is lowered, Further development of these follicles to produce thus allowing the pituitary to respond.This is some- mature Graafian or antral follicles, of which there times referred to as the ‘gonadostat’ theory. are about 200 growing follicles at puberty in Other factors are also involved.The frequency of the heifer, is dependent upon the stimulus of LH secretion is dependent upon gonadotrophin- gonadotrophic hormones. Despite the absence of releasing hormone (GnRH) from the hypothal- oestrous cycles, there is follicular growth as has amus, which is controlled by an area in the been shown using transrectal ultrasonography hypothalamus referred to as the neural GnRH in calves from 2 weeks of age. It was seen that pulse generator. Age-related changes in brain mor- there were follicular waves in response to follicle- phology and neuronal cytoarchitecture may also be stimulating hormone (FSH) secretion that were important, since extrapolation from studies per- similar to those of the adult, and that individual formed in rats, for example, has shown an increase follicular development was characterised by grow- in the number of GnRH cells with spine-like pro- ing, static and regressing phases (Adams, 1994). cesses on the soma and dendrites. In addition, the The sheep has been used extensively for studying inhibitory effect of opioid peptides on LH secretion many of the mechanisms involved in the initiation is reduced with age, which may provide a neuro- of puberty; however, it must be stressed that sea- chemical explanation for the changes in pituitary sonality will exert an overriding influence in this sensitivity to oestradiol feedback that occur at species (see below). The onset of puberty is sig- puberty (Bhanot and Wilkinson, 1983; Wray and nalled by either the occurrence of the first oestrus Hoffman-Small, 1986). or the first ovulation; in the ewe lamb these do not The reason for the ‘silent’ first oestrus of the occur simultaneously because the first ovulation is pubertal animal is believed to be because the not preceded by behavioural oestrus. A similar central nervous system requires to be primed with response is seen in sexually mature ewes at the progesterone before it will respond and the animal onset of the normal breeding season. will show behavioural signs of heat.The first ovula- The hormone that is primarily responsible for tory cycle has been shown to be short in pubertal the onset of ovarian activity, and hence puberty, is heifers (7.7 +/– 0.2 days), and the first corpus luteinising hormone (LH). In adult ewes during luteum (CL) not only has a shorter than normal life the normal breeding season, basal LH concen- span but is also smaller in size. One explanation for trations increase together with the LH pulse this is that the dominant follicle, from which the frequency to one per hour during the period of first ovulation arises, had already entered the static maximum follicular growth. This results in the phase of growth. The subsequent interovulatory development of follicles to the preovulatory stage, interval was normal (Adams 1999). and their secretion of oestradiol, which activates the LH surge causing ovulation and corpus External factors influencing the time of luteum formation. In the prepubertal ewe lamb, onset of puberty LH pulses occur at similar amplitudes but much lower frequencies (one every 2–3 hours). As a The time of onset of puberty is determined by consequence, follicular growth is insufficient to the individual’s genotype, with smaller breeds of 4  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 animal tending to be slightly more precocious. activity of the reproductive system. There is folli- However, this inherent timing is influenced by a cular growth and regression of the corpus luteum number of external factors. of the previous cycle (in polycyclic species). The Nutrition. There is good evidence that in most uterus enlarges very slightly; the endometrium domestic species, the age of puberty is closely becomes congested and oedematous, and its related to body weight; therefore, it is not surpris- glands show evidence of increased secretory activ- ing that nutrition is an important factor. Animals ity. The vaginal mucosa becomes hyperaemic; the that are well fed with good growth rates reach number of cell layers of the epithelium starts to puberty before those that are poorly fed with slow increase, and the superficial layers become corn- growth rates. However, unless the animal is ified. The bitch shows external evidence of pro- severely malnourished, the onset of cyclical activ- oestrus with vulval oedema, hyperaemia and a ity will eventually occur. sanguineous vulval discharge. Season of the year. In those species which are Oestrus. The period of acceptance of the male. seasonal breeders, such as the ewe, mare and The onset and end of the phase are the only accu- queen cat, the age at which puberty occurs will be rately measurable points in the oestrous cycle, and influenced by the effect of season of the year. For hence are used as the baseline for determining instance, a filly born early in the year, i.e. January cycle length.The animal usually seeks out the male or February, may have her first oestrus in the May and ‘stands’ for him to mate her. The uterine, cer- or June of the following year, i.e. when she is 16 or vical and vaginal glands secrete increased amounts 17 months old. A filly foal born late in the year, of mucus; the vaginal epithelium and endometrium July or August, may not have her first oestrus until become hyperaemic and congested; the cervix is she is 21 or 22 months old. The same is true of relaxed. ewes which, depending upon the time of year at Ovulation occurs during this phase of the cycle which they are born, may reach puberty as early as in all domestic species with the exception of the 6 months or as late as 18 months old. cow, where it occurs about 12 hours after the end Proximity of the male. Studies in sheep and pigs of oestrus. Ovulation is a spontaneous process in have shown that exposure to the male of the species all domestic species with the exception of the cat, will advance the timing of the onset of puberty.This rabbit and camel, in which it is induced by the act so-called ‘ram or boar effect’ is probably mediated of coitus. by pheromonal and other sensory cues influencing During pro-oestrus and oestrus there is follicu- hypothalamic GnRH secretion. lar growth in the absence of functional corpora Climate. Anthropomorphic extrapolation has lutea, the main ovarian hormones produced being assumed that animals living in the tropics reach oestrogens. Pro-oestrus and oestrus are frequently puberty at an earlier age than those in temperate referred to collectively as the follicular phase of climates. Studies carried out in Zambia have the cycle. shown that in cattle this is not true. Metoestrus. The phase succeeding oestrus. The Disease. Any disease which can influence the granulosa cells of the ovulated follicle give rise to growth rate, either directly or because of interfer- lutein cells which are responsible for the form- ence with feeding and utilisation of nutrients, will ation of the corpus luteum.There is a reduction in delay the onset of puberty. the amount of secretion from the uterine, cervical and vaginal glands. Dioestrus. The period of the corpus luteum. THE OESTROUS CYCLE AND ITS The uterine glands undergo hyperplasia and PHASES hypertrophy, the cervix becomes constricted and the secretions of the genital tract are scant Traditionally, the oestrous cycle is divided into a and sticky; the vaginal mucosa becomes pale. number of phases. The corpus luteum is fully functional during Pro-oestrus. The phase immediately preceding this phase, and is secreting large amounts of oestrus. It is characterised by a marked increase in progesterone. 5 1 NORMAL OESTROUS CYCLES The period of the oestrous cycle when there is a and prolactin-releasing and inhibitory hormones. functional corpus luteum is sometimes referred to There is some suggestion that melatonin may not as the luteal phase of the cycle, to differentiate it act directly upon the hypothalamus/anterior pitu- from the follicular phase. itary, but indirectly via the other pineal peptide Since in most of our domestic species oestrus is hormones. the only readily identifiable phase of the oestrous Melatonin drives the reproductive response of cycle, there is some merit, in polyoestrous species, the ewe to inductive photoperiods (Bittman et al., in dividing the cycle into oestrus and interoestrus, 1983). Rhythmic administration of melatonin to the latter including pro-oestrus, metoestrus and adult ewes exerts a similar effect to increased dioestrus. Another alternative division can be into hours of darkness by inducing the onset of the follicular and luteal phases. breeding season (Arendt et al., 1983) and causes Anoestrus. The prolonged period of sexual rest changes in prolactin concentrations in the plasma during which the genital system is mainly quies- that are similar to those following exposure to cent. Follicular development is minimal; the short days (Kennaway et al., 1983). corpora lutea, although identifiable, have regressed In sheep, an intact pineal gland is required for a and are non-functional. Secretions are scanty and normal photoperiodic response to altered daylight tenacious, the cervix is constricted, and the vaginal patterns; however, other seasonal environmental mucosa is pale. cues are important, since pinealectomised ewes still show seasonal breeding (Lincoln, 1985). The mare is a seasonal breeder, but is ‘switched Natural regulation of cyclical activity on’ by increasing day length. The pineal gland is Regulation of cyclical activity in the female is a involved, since if it is removed the mare does not complex process. With the development of new show a normal response to changes in photo- techniques, particularly those of hormone assays, period. In intact mares, melatonin concentrations and the application of new molecular biological increase during hours of darkness (Grubaugh et techniques, there is a continual advance in the al., 1982). There is some evidence that foals are knowledge and understanding of the mechanisms conditioned at an early age and develop a pattern involved. Although much of the early work was of melatonin secretion from about 7 weeks of age done on laboratory animals – notably the rat and (Kilmer et al., 1982). guinea pig – there is now much more information The hypothalamus is responsible for the control about domestic species, although there are still of release of gonadotrophins from the anterior areas, particularly in the bitch, which are not fully pituitary by the action of specific releasing and understood. inhibitory substances. These are secreted by the Regulation of cyclical activity is mainly under hypothalamic neurons, and are carried from the control of the hypothalamic–pituitary–ovarian the median eminence of the hypothalamus by the axis. At one end of this axis there is the influence hypothalamic–hypophyseal portal system. In 1971 of the extrahypothalamic areas – the cerebral the molecular structure of porcine GnRH was cortex, thalamus and mid-brain – and the role determined (Matsuo et al., 1971) as being a deca- played by stimuli such as light, olfaction and peptide, and subsequently synthesised (Geiger touch (Ellendorff, 1978), whilst at the other end is et al., 1971). Opinion is divided as to whether the influence of the uterus upon the ovary. GnRH is responsible in vivo for the release of both The pineal gland appears to have an important FSH and LH (Lamming et al., 1979), although role in controlling reproduction in seasonal breed- the injection of GnRH stimulates the release of ing species and also in the timing of puberty by both FSH and LH in domestic species. As yet, no influencing the release of FSH, LH and prolactin. specific inhibitory factor such as that for prolactin Although much of the interest has been in the has been identified for gonadotrophins. action of the indoleamine melatonin, there is Specific neurotransmitter substances are increasing interest in the other pineal peptide hor- involved in the regulation of the release of pitu- mones, namely arginine vasotocin, gonadotrophin itary hormones. The role of three monoamines 6  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 has now been fairly well established (Kordon, 1978). Noradrenaline stimulates the release of Extrahypothalamic centres Pineal FSH and LH; the inhibition of the conversion of dopamine to noradrenaline blocks the ‘oestradiol- Melatonin and other pineal induced’ release of LH, which is responsible for peptides ovulation; whilst serotonin inhibits the basal Hypothalamic centres secretion of LH and regulates other neurosecre- Surge Episodic/ tonic tory systems. Dopamine also has an important e os GnRH uc role in the control of prolactin release. Gl Progesterone/oestrogens There is good evidence that in domestic species Anterior GH the secretion of FSH and LH is controlled by pituitary Oestrogens two functionally separate, but superimposable, Liver systems. These are the episodic/tonic system, FSH/LH Insulin GH which is responsible for the continuous basal secretion of gonadotrophin and stimulates the Oestrogens growth of both germinal and endocrine compo- IGF nents of the ovary, and the surge system, which 's controls the short-lived massive secretion of Ovary gonadotrophin, particularly LH, responsible for Insulin Pancreas ovulation. There are two hypothalamic centres Oestrogens/ that are involved in controlling these two systems PGF2α progesterone (Figure 1.1). With the exception of the cat, rabbit and camel, Oxytocin all domestic species are spontaneous ovulators. Uterus However, in these three species ovulation is induced by the stimulation of sensory receptors in Fig. 1.1 Endocrine control of cyclical reproductive the vagina and cervix at coitus. This initiates a activity. ——, stimulation; - - -, inhibition; PGF2α, neuro-endocrine reflex ultimately resulting in the prostaglandin2α; IGFs, insulin-like growth factors; GH, growth hormone. (Adapted from Lamming et al., 1979.) activation of GnRH neurons in the surge centre and release of a surge of LH. Not only does the anterior pituitary have a be isolated from follicular fluid. It has also been direct effect upon ovarian functions by stimulat- isolated from the testis and seminal plasma (see ing folliculogenesis, follicular maturation, ovula- Chapter 29). Inhibin and oestradiol act in concert tion and corpus luteum formation, but the ovary in suppressing FSH secretion. Inhibin, which is has an effect upon the hypothalamus and anterior produced by all antral follicles, has a longer half- pituitary.This is mediated by oestradiol, produced life, and sets the overall level of negative feedback, by the maturing follicle, and by progesterone, pro- whereas oestradiol, which is produced only by duced by the corpus luteum. The episodic/tonic those antral follicles that have the potential for hypothalamic release centre is influenced by the ovulation, is responsible for the day-to-day fluctu- negative-feedback effect of oestradiol and pro- ations (Baird et al., 1991). Two other peptide hor- gesterone. Low levels of progesterone also have a mones have been isolated from ovarian follicular modulating influence on this centre, which fluid; these have been designated activin, which appears to be particularly important in ruminants stimulates, and follistatin, which suppresses, FSH (Lamming et al., 1979). In the cow, ewe and sow secretion.Their roles in controlling and regulating (and probably in other domestic species) FSH follicular growth are not known. secretion is also controlled by a number of The positive-feedback effect of oestradiol on ovarian-derived peptide hormones. The first that hypothalamic–pituitary function is well demon- has been characterised, inhibin, is produced by strated in farm animals, since the preovulatory the granulosa cells of large antral follicles, and can surge of oestradiol stimulates the release of LH, 7 1 NORMAL OESTROUS CYCLES which is so necessary for the process of ovulation that only FSH can induce early follicular growth, and corpus luteum formation. The response of so that when the granulosa cells have matured the anterior pituitary to GnRH is influenced by and are able to respond to endogenous LH, the the levels of ovarian steroids so that there is formation of a fully developed vesicular follicle increased responsiveness shortly after the level of occurs. Large amounts of a peptide similar to the progesterone declines and that of oestradiol rises hormone inhibin, produced by the Sertoli cells of (Lamming et al., 1979). There are probably self- the testis, have been found in bovine and porcine regulatory mechanisms controlling gonadotrophin follicular fluid and granulosa cells. This hormone secretion acting locally within the anterior pitu- probably selectively inhibits FSH release from the itary and hypothalamus. anterior pituitary but it may also have a local role Tonic release of gonadotrophins, especially LH, in controlling ovarian function; it has been shown does not occur at a steady rate but in a pulsatile to inhibit the binding of FSH to granulosa cells in fashion in response to a similar release of GnRH the cow (Sato et al., 1982). Recently, the roles of from the hypothalamus. The negative feedback of insulin-like growth factors (IGFs), notably IGF-1, progesterone is mediated via a reduction in pulse and their associated binding proteins have been frequency of gonadotrophin release, whereas shown to exert an influence on ovarian function at oestradiol exerts its effect via a reduced pulse the level of the granulosa, thecal and luteal cells, amplitude. The onset of cyclical activity after par- probably by acting synergistically with gonado- turition (see Chapter 7), at puberty or at the start trophins (Lucy et al., 1999). Furthermore, there is of the breeding season is associated with increased also good evidence in the cow that growth hor- pulse frequency of tonic gonadotrophin secretion. mone (GH) also has a role in regulating ovarian When the ram is placed in contact with ewes function either directly or by stimulating the syn- before the start of the breeding season, there is thesis and secretion of IGF-1 by the liver (Lucy increased frequency of pulsatile LH release, which et al., 1999). These findings are of considerable stimulates the onset of cyclical activity (Karsch practical interest, because it is likely that they may 1984). be important in mediating the effects of nutrition Progesterone appears to play a critically im- on reproduction. portant role in the inhibition of the tonic mode of Throughout the oestrous cycle, during preg- LH secretion in the ewe (Karsch et al., 1978). nancy and other reproductive stages, there is Progesterone is thus the main regulatory hormone dynamic follicular activity with growth and which controls the oestrous cycle of the sheep and atresia; only about 1% of antral follicles sub- probably of other species too. Thus when the con- sequently ovulate. There appear to be two differ- centration of progesterone in the circulation falls, ent patterns of follicular growth in mammals associated with the regression of the corpus (Fortune, 1994). Thus in cattle, sheep and horses luteum, there is release of LH from the anterior the development of antral follicles to sizes close to pituitary. The rise in LH triggers the secretion of that at ovulation occurs throughout the oestrous oestradiol; this sudden rise stimulates the surge cycle including the luteal phase, whereas in the centre for the LH release and, as a result of this pig and rat the development of preovulatory-size sudden increase, ovulation of the mature follicle follicles only occurs in the follicular phases of the occurs (Karsch et al., 1978). cycle in the absence of a functional corpus luteum In some species, notably the cow (see Figure (CL). Follicular development occurs in stages, the 1.28 later), there is also a concomitant surge in following terminology for which is now generally FSH; although its significance is unclear it may accepted (Webb et al., 1999): be part of the ‘ovulation-inducing’ hormone complex. For this reason it is probably incorrect recruitment – gonadotrophin stimulation of a to assign a separate and specific physiological role pool of rapidly growing follicles for the two pituitary gonadotrophins. Thus, selection – a process whereby one or more of although ovulation and steroidogenesis can be the recruited follicles are selected to develop initiated by both FSH and LH, it would appear further 8  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 dominance – the mechanism whereby one (the the bitch, there are early signs of luteinisation of dominant follicle), or several follicles, undergo the follicle before it has ovulated. The stimulus for rapid development in an environment where the formation and maintenance of the CL prob- the growth and development of other follicles ably varies within species.The hormones which are is suppressed. most likely to be involved are prolactin and LH, but there is some evidence that they are involved The pattern of follicular dynamics has been together, perhaps in association with FSH. summarised, particularly in ruminant species, by Although all three hormones are probably involved Adams (1999), and it is appropriate to quote this in the induction of luteinisation of granulosa cells, as follows: ‘(1) follicles grow in a wave-like the available evidence suggests that FSH is prob- fashion; (2) periodic surges in circulating FSH are ably not required for the maintenance of luteal associated with follicular wave emergence; (3) function. The difference between species is well selection of a dominant follicle involves the illustrated by the observation that LH will prolong decline in FSH and acquisition of LH responsive- luteal function in the sow, but prolactin will not ness; (4) periodic anovulatory follicular waves (Denamur et al., 1966; Anderson et al., 1967). continue to emerge until the occurrence of an LH However, in the ewe prolactin appears to be more surge; (5) within species, there is a positive rela- important as a luteotrophic agent, since LH will tionship between the duration of the oestrous exert an effect only if infused from day 10 to day cycle and the number of follicular waves; (6) prog- 12 of the oestrous cycle. esterone is suppressive to LH secretion and the The role of prolactin in the control of repro- growth of the dominant follicle; (7) the duration duction in many domestic species is still largely of the interwave interval is a function of follicular speculative, and in many cases it is only possible dominance, and is negatively correlated with cir- to extrapolate from studies in the traditional lab- culating FSH; (8) follicular dominance in all oratory species. Unlike other anterior pituitary species is more pronounced during the first and hormones that require hypothalamic stimulation, last follicular waves of the oestrous cycle; (9) preg- it appears that prolactin secretion is spontaneous, nancy, the prepubertal period and seasonal and that it is largely controlled by inhibition by anoestrus are characterised by regular, periodic hypothalamically-derived prolactin inhibitory surges of FSH and emergence of anovulatory factor (PIF), which is believed to be dopamine. follicular waves.’ There is some evidence to suggest that dopamine The CL is rapidly formed from the Graafian fol- may have a dual role as a stimulant of prolactin licle after ovulation primarily from the granulosa secretion, rather like a prolactin-releasing factor and the thecal cells; in the ewe, for example, its (PRF). mass increases 20-fold over 12 days (Reynolds and Much interest has been directed towards the Redmer, 1999). For some time it was assumed role of certain endogenous peptides with opioid that, once formed, it remained a relatively static activity such as β-endorphin and met-enkephalin. structure but now it has been shown that when it These substances have been found in high con- is functionally mature there is rapid cellular centrations in hypothalamic–hypophyseal portal turnover, although there is little change in size.The blood. The administration of exogenous opioid fully formed CL consists of a number of different peptides inhibits the secretion of FSH and LH cell types: the steroid-secreting large and small whilst stimulating the secretion of prolactin. If an luteal cells, fibroblasts, smooth muscle, pericytes opiate antagonist such as naloxone is infused, and endothelial cells. It has the greatest blood there is an increase in mean concentrations of supply per unit tissue of any organ (Reynolds and gonadotrophins in the plasma and the frequency Redmer, 1999). In the ewe, based on volume, the of episodic gonadotrophin secretion. The effect of large luteal cells comprise 25–35%, the small opioids appears to be influenced by the steroid luteal cells 12–18% and vascular elements 11% environment of the animal; for example, in ewes, (Rodgers et al., 1984). Although the CL develops naloxone increased the mean plasma concen- as a result of ovulation, in some species, notably tration of LH and the episodic frequency in a 9 1 NORMAL OESTROUS CYCLES high-progesterone environment. However, in that in this species the luteolysin is transported ovariectomised ewes or those with oestradiol throughout the systemic circulation. implants, naloxone had no effect (Brooks et al., In the pig the luteolytic substance is trans- 1986). It is possible that the negative feedback of ported locally (du Mesnil du Buisson, 1961) but progesterone on LH release (see below) may be not exclusively to the adjacent ovary. It has been mediated via opioids (Brooks et al., 1986). shown that, following surgical ablation of parts of The presence of a functional CL, by virtue of its the uterine horns, provided at least the cranial production of progesterone, inhibits the return to quarter of the uterine horn is left, regression of oestrus by exerting a negative feedback effect the CLs occurs in both ovaries. If more than upon the anterior pituitary; this is most obvious three-quarters of the horn is excised, then regres- during pregnancy (see Chapter 3). In the normal, sion of the CLs occurs only in the ovary adjacent non-pregnant female, oestrus and ovulation occur to the intact horn. In the bitch, the mechanisms of at fairly regular intervals; the main control of this control of the life span of the CLs are not fully cyclical activity would appear to be the CL. There understood, and in this species even in the is also evidence that the CL also exerts a positive absence of pregnancy there is always a prolonged intra-ovarian effect by increasing the number of luteal phase traditionally called metoestrus. small antral follicles in that ovary (Pierson and Although the importance of the middle uterine Ginther, 1987). vein in the transfer of the luteolytic substance has Although it has been known for nearly 80 years been demonstrated, the mechanisms whereby the that in certain species of animal the uterus influ- luteolytic substance passes to the ovary have not ences ovarian function (Loeb, 1923) the mech- been conclusively shown in all species, although anism has been fully understood only in recent they have been fairly well evaluated in the ewe and years. cow. In the former species, it appears that the It has been demonstrated that in many species close proximity of the ovarian artery and utero- removal of part or all of the uterus will result in the ovarian vein is important, particularly since at prolongation of the life span of the CL (du Mesnil their points of approximation the walls of the two du Buisson, 1961; Rowson and Moor, 1967); these vessels are thinnest; there is no anastomosis species include the cow, mare, ewe, goat and sow. (Coudert et al., 1974). This allows the leakage of In the human, dog and cat the normal life span of the luteolytic substance from the uterine vein into the CL is unaltered in the absence of the uterus. In the ovarian artery and thus to the ovary, by a form the cow, ewe and goat the ‘luteolytic’ action of the of counter-current exchange through the walls of uterine horn is directed exclusively to the CL on the vessels. It has been suggested (Ginther, 1974) the adjacent ovary (Ginther, 1974). Thus, if one of that the variation in the response to partial or total the uterine horns is surgically removed on the side hysterectomy in different species is probably due adjacent to the ovary with a CL then the latter will to differences in the relationships between the persist. If the contralateral horn is removed, then vasculature of the uterus and ovaries. the CL will regress at the normal time. It appears It was not until 1969 that the substance that in these species the luteolytic substance is responsible for luteolysis was identified, when the transported directly from the uterus to the ovary. In duration of pseudo-pregnancy in the rat was the ewe it has been shown experimentally that the shortened by the injection of prostaglandin F2α most likely route for transport of the substance is (PGF2α). This same substance has subsequently the middle uterine vein, since when all other struc- been shown to have potent luteolytic activity in tures between the ovary and uterus are severed the ewe, doe, cow, sow and mare. Although it has there is still normal regression of the CL (Baird and been proved only in ruminants and the guinea pig Land, 1973). that it is the natural luteolysin, it is likely that it is In the mare no local effect can be demon- also true for the other species listed. strated, since if the ovary is transplanted outside PGF2α is a derivative of the unsaturated the pelvic cavity, luteal regression still occurs hydroxy acids linolenic and arachidonic acids. It (Ginther and First, 1971). It is generally assumed derived its name because it was first isolated from 10  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 fresh semen and it was assumed to be produced in lings), but under natural conditions it is unusual the prostate gland. It is synthesised in the for them to foal until they are over 3 years old. endometrium of a number of species (Horton and The mare is normally a seasonal breeder, with Poyser, 1976), and in the ewe it has been demon- cyclic activity occurring from spring to autumn; strated in increasing amounts at and around the during the winter she will normally become anoe- time of luteal regression (Bland et al., 1971). strous. However, it has been observed that some Luteal regression can be viewed from two mares, especially those of native pony breeds, will aspects. Firstly, functional regression is rapid, so cycle regularly throughout the year.This tendency that the secretion of progesterone declines rapidly. can be enhanced if the mares are housed and Secondly, as regards structural regression when the given supplementary food when the weather is CL is reduced in size, the latter process takes longer cold and inclement, and if additional lighting is than the former. In ruminants, luteal regression is provided when the hours of daylight are short. caused by episodic release of PGF2α from the Horse breeding has been influenced by the uterus at intervals of about 6 hours.This is induced demands of thoroughbred racing, because in the by oxytocin secreted by the CL; thus, each episode northern hemisphere foals are aged from 1 January, of PGF2α release is accompanied by an episode of irrespective of their actual birth date. As a result, oxytocin release. Furthermore, PGF2α stimulates the breeding season for mares has been, for over a further secretion of oxytocin from the ovary. It has century, determined by the authorities as running been postulated that the abundant, non-steroido- from 15 February to 1 July. Since the natural genic endothelial cells of the CL may mediate the breeding season does not commence until about actions of PGF2α, and that its physical demise is the middle of April, and maximum ovarian activity due to the action of invading macrophages which is not reached until July, it is obvious that a large may secrete cytokines, such as tumour necrosis number of thoroughbred mares are bred at a time factor (TNF)α (Meidan et al., 1999). when their fertility is suboptimal (see Chapter 26). The sensitivity of the uterus to oxytocin is The winter anoestrus is followed by a period of determined by the concentration of endometrial transition to regular cyclic activity. During this oxytocin receptors. At the time of luteal regression transition, the duration of oestrus may be irregu- in sheep they rise approximately 500-fold (Flint lar or very long, sometimes more than a month. et al., 1992). Their concentration is determined The manifestations of heat during the transitional by the effects of progesterone and oestradiol. phase are often atypical and make it difficult for Thus, the high concentrations of progesterone the observer to be certain of the mare’s reproduc- which occur after the formation of the CL reduce tive status. Also, before the first ovulation, there is the number of receptors, so that in the normal poor correlation between sexual behaviour and oestrous cycle of the ewe they start to increase ovarian activity; it is common for the early heats to from about day 12. Exogenous oestradiol causes be unaccompanied by the presence of palpable premature induction of oxytocin receptors, result- follicles, and some long spring heats are anovula- ing in premature luteolysis (Flint et al., 1992). tory. However, once ovulation has occurred, In non-ruminant species, much less is known regular cycles usually follow. about the mechanisms of luteolysis. The average length of the equine cycle is 20–23 The CL becomes more sensitive to the leuteo- days; the cycles are longer in spring and shortest lytic effect of PGF2α as it ages. The early CL is from June to September. Typically, oestrus lasts 6 unresponsive to PGF2α. days and dioestrus 15 days. Ovulation occurs on the penultimate or last day of heat, and this rela- THE MARE 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 Cyclic periodicity manual rupture of the ripe follicle resulted in ter- Fillies are often seen in oestrus during their mination of oestrus within 24 hours. The diameter second spring and summer (when they are year- of the ripe follicle is 3–7 cm. During the last day 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 Fig. 1.3 Ovaries of a 9-year-old farm mare in dioestrus. subsequent ones. Corpus luteum in right ovary, orange in colour; pleats During oestrus, a single egg is usually released, loose. 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 Fig. 1.4 Ovaries of a 4-year-old shire mare in dioestrus. 27% of double ovulations, and Arthur (1958) Corpus luteum in left ovary, brownish-red in colour; found an overall frequency of 18.5%, with a pleats distinct; central cavity containing blood clot. Right ovary contains a follicle filled with blood. 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 Fig. 1.5 Ovaries of a 6-year-old farm mare in dioestrus. protrusions of corpora lutea be seen, but because A corpus luteum in each ovary, orange-yellow in colour; of the curvature of the ovary and the presence of pleats distinct. 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. 1.6 Ovaries of a 6-year-old hunter mare in dioestrus. Corpus luteum in right ovary, pale yellow in colour; pleats distinct. Central cavity. 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 Fig. 1.2 The ovaries of a 5-year-old farm mare in oestrus. Specimens obtained in May. Regressing corpus a size of 1–3 cm. By the first day of oestrus one luteum in left ovary, bright yellow ochre in colour. follicle is generally considerably larger than the 12  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 remainder, having a diameter of 2.5–3.5 cm. next 24 hours. Quite frequently the mare shows During oestrus, this follicle matures and ruptures evidence of discomfort when the ovary is palpated when it has attained a diameter of 3–7 cm. After soon after ovulation. Unless sequential transrectal ovulation, the other follicles regress, until, during palpation or ultrasonic examinations are per- the first 4–9 days of the ensuing dioestrus, no fol- formed, it is sometimes possible to confuse a licles larger than 1 cm are likely to be present. mature follicle with the early corpus haemorrhag- Several hours before ovulation the ripe follicle icum, since before ovulation the follicular antrum becomes much less tense. The collapsed follicle is is filled with follicular fluid and then soon after recognised by an indentation on the ovarian ovulation it becomes filled with blood. For this surface; there is usually some haemorrhage into reason mares are sometimes incorrectly diagnosed the follicle, and the coagulum hardens within the as having failed to ovulate. For the next 3 days the (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) 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. (c) luteinising mass can be felt as a resilient focus, but monly occupied by a variable amount of dark- later it tends to have the same texture as the brown fibrin. The cyclical CL begins to regress at remainder of the ovary. In pony mares, however, of about the 12th day of the cycle, when there is a par- known history from daily examinations, Allen allel fall in the blood progesterone concentration. (1974) reports that it is possible to follow the From this day onwards the events previously growth of the CL by palpation because in ponies it described recur. Ovulation, with the subsequent forms a relatively large body in a small ovary. The formation of a CL, does not always occur; the folli- CL attains maximum size at 4–5 days, but it does cle may regress or sometimes undergo luteinisation not protrude from the ovarian surface. On section (see Figure 1.10(b)). of the ovary it is brown and later yellow and of a tri- B-mode ultrasound imaging with a rectal trans- angular or conical shape, with the narrower end ducer has been used to visualise follicles (see impinging on the ovulation fossa. Its centre is com- Figures 1.7–1.12). This is particularly useful in 14  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 (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) (a) as a large anechoic (black) area. (a) (b) Fig. 1.10 (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. detecting the possibility of twin ovulations and could be used to predict the time of ovulation. also in determing the timing of ovulation. Ginther The same author has used this technique to assess (1986) observed that in the preovulatory period corpora lutea. He identified differences in the there was a change in the shape of the follicle and echogenic properties of the CL depending upon a thickening of the follicular wall, which, together the persistence of the corpus haemorrhagicum; with the assessment of the size of the follicle, this he identified in about 50%. 15 1 NORMAL OESTROUS CYCLES (a) (b) 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). (c) During winter anoestrus, both ovaries are typi- licles. During oestrus the ovary of the thorough- cally small and bean-shaped, common dimensions bred mare may contain two or even three follicles, being 6 cm from pole to pole, 4 cm from the hilus each of 4–7 cm, and these, with other subsidiary to the free border and 3 cm from side to side. Not follicles, combine to give it a huge size. During uncommonly, however, in early spring or late dioestrus, however, with an active CL and only autumn, the anoestrous ovaries are of medium or atretic follicles the ovary may be little larger than large size and knobbly due to the content of in anoestrus. numerous follicles of 1–1.5 cm diameter. During By visual examination of the vagina and the the cycle, there are large variations in the ovarian cervix using an illuminated speculum, it is possible size depending on the number and size of the fol- 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 be seen lying on the vaginal floor, with its folds vagina are pale pink, while mucus is scanty and oedematous; the vaginal walls are glistening with sticky. During oestrus, there is a gradual increase clear lubricant mucus. After ovulation there is a in the vascularity of the genital tract and relaxation gradual reversion to the dioestrous appearance. of the cervix with dilatation of the os. As oestrus During anoestrus, as in pregnancy, both the advances and ovulation time approaches, the vagina and cervix are blanched; the cervix is cervix becomes very relaxed and its protrusion can 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 Fig. 1.13 Exposure of the clitoris (ct) in response to teasing. 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 (Evans and Irvine, 1975). The pattern of LH mare is accustomed to the procedure and that, secretion is also unusual in this species since there because of the interval between the end of the last is no sudden surge of this hormone but a gradual oestrus and the start of the next, she is teased increase and persistence of elevated levels for 5–6 15–16 days after the end of the last oestrus. days on either side of ovulation. Oestrogens in the peripheral circulation reach peak values during oestrus whilst concentrations of progestrone and Endocrine changes during the oestrous other progestrogens follow closely the physical cycle changes of the CL. The trends in endocrine changes are shown in Figure 1.14.The secretion of FSH is biphasic with THE COW surges at approximately 10–12 day intervals. One surge occurs just after ovulation, with a second Cyclic periodicity surge in mid- to late dioestrus approximately 10 days before the next ovulation. It has been sug- Under conditions of domestication, normal and gested that this increase in FSH secretion, which well-cared-for cattle are polyoestrous throughout is unique to the mare, is responsible for priming the year. The age at first oestrus, or puberty, is the development of a new generation of follicles, affected by nutrition and season of birth, and one of which will ovulate at the next oestrus ranges from 7 to 18 months, with an average of 10 18  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 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 Fig. 1.14 Trends in hormonal concentrations in the peripheral circulation of the mare during the oestrous during the hours of night, perhaps when the cycle. animals are least disturbed (Williamson et al., 1972; Esslemont and Bryant, 1976). months. A small proportion of heifers do not ovu- Ovulation is spontaneous, and occurs on late at the first heat, and in a majority of young average 12 hours after the end of oestrus. cattle the oestrus associated with the first ovula- tion is ‘silent’ (Morrow et al., 1969). Poor feeding Signs of oestrus and calfhood disease delay puberty. Once puberty has been reached, cyclical activity should persist, Where artificial insemination is used, the accurate except during pregnancy, for 3–6 weeks after detection of oestrus by the herdsperson is para- calving, during high milk yield (especially if there mount in ensuring optimum fertility. Poor detec- is some evidence of dietary insufficiency), and tion is probably the most important reason with a number of pathological conditions (see affecting delayed breeding (Wood, 1976), whilst Chapter 22). Some cows and heifers also fail to in the USA Barr (1975) has calculated that in 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 114 46 307 128 Duration of oestrus (hours) 11.3 +/– 6.9 13.9 +/– 6.1 7.3 +/– 7.2 7.8 +/– 5.4 No. of standing events 18.8 +/– 12.8 30.4 +/– 17.3 7.2 +/– 7.2 9.6 +/– 7.4 19 1 NORMAL OESTROUS CYCLES Ohio dairy herds dairy farmers appeared to be Sometimes there are signs of a vulval discharge losing twice as many days due to failure to detect of transparent mucus whose elasticity causes it to heat as to conception failures. hang in complete clear strands from the vulva to There are great variations amongst individual the ground; it also adheres to the tail and flanks. cattle in the intensity of heat signs; the manifest- The vulva may be slightly swollen and congested, ations tend to be more marked in heifers than in and there is a small elevation of temperature. The cows. However, it is generally agreed that the most tail may be slightly raised.The hair of the tail-head reliable criterion that a cow or heifer is in oestrus is often ruffled and the skin sometimes becomes is that she will stand to be mounted by another excoriated due to mounting by other cows. For (Williamson et al., 1972; Esslemont and Bryant, the same reason, the rear of the animal may be 1974; Foote, 1975). soiled with mud. At range, the oestrous cow may The oestrous animal is restless and more active; wander from the herd, and if isolated there will be Kiddy (1977), using pedometers, found that there bellowing. When she is put with a bull, the two was an average increase in activity of 393% at this animals lick each other and the cow often mounts time. More recently, Lewis and Newman (1984) the bull before standing to be mounted by him. found that pedometer activity was about twice as For a short time after service, the cow stands with great in oestrus compared with the luteal phase of raised tail and arched back, and where actual the cycle. In their study, 75% of cows showed service has not been seen this posture indicates peak pedometer readings on the day of onset of that mating has occurred. oestrus whereas 25% peaked 1 day after oestrus. Within 2 days of service, there is an occasional There tends to be grouping of sexually active indi- yellowish-white vulval discharge of mucus con- viduals; there is a reduction in the time spent taining neutrophil leucocytes from the uterus. At eating, resting and ruminating, and frequently a about 48 hours after heat, irrespective of service, reduction in milk yield. Reduced milk yield has heifers and many cows show a bright red sanguin- been shown to be a reliable indicator of the onset eous discharge, the blood coming mainly from the of oestrus; there is usually a compensatory uterine caruncles. rebound at the next milking (Horrell et al., 1984). The body temperature of dairy cows falls about In this study of 73 dairy cows, it was found that if 0.5°C the day before oestrus, increases during a cow produced 75% of her usual yield there was oestrus and falls by about 0.3°C at ovulation. The a 50% chance of her being in oestrus. On the rare vaginal temperature, of 37.74°C, was lowest on the occasions that it fell to 25%, oestrus was always day before oestrus, increased by 0.1°C on the day of present. As the cow approaches oestrus she tends oestrus, and increased for the next 6 days until a to search for other cows in oestrus, and there is plateau was reached.This was followed by a gradual licking and sniffing of the perineum. During this decline from 7 days before oestrus (Lewis and period, during oestrus and just afterwards, the Newman, 1984). Practical detection of this is cow will attempt to mount other cows; quite often tedious; however, the use of microwave telemetry before she does this she will assess the receptivity systems may enable such measurements to be made of the other cows by resting her chin on the rump in the future (Bobbett et al., 1977). Automated or loins. If the cow to be mounted is responsive methods of measuring the related increase in milk and stands, she will mount and sometimes show temperature in the milking parlour have also been evidence of pelvic thrusting (Esslemont and described (Maatie, 1976; Ball, 1977). Bryant, 1974). If the cow that is mounted is not in Vaginal pH also fluctuates throughout the oestrus she will walk away and frequently turn oestrous cycle but is lowest, namely 7.32, on the and butt the mounting cow. A positive mounting day of oestrus (Lewis and Newman, 1984). response lasts about 5 seconds (Hurnik et al., 1975); however, if both cows are in oestrus it will Cyclic changes in the vagina be increased to about 7.5 seconds. In a group of 60 cows, Esslemont and Bryant (1976) observed The main variations are in the epithelial cells of the that 33 cows were mounted on average 56 times. anterior vagina and in the secretory function of the 20  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 cervical glands (Hammond, 1927; Cole, 1930). pated per rectum this muscular irritability, During oestrus, the anterior vaginal epithelium coupled with the marked vascularity, conveys a becomes greatly thickened due to cell division and highly characteristic tonic turgidity to the pal- to the growth of the tall, columnar, mucus-secret- pating fingers; the horns feel erect and coiled.This ing superficial cells. During dioestrus, these cells tonicity is present the day before and the day after vary from flat to low columnar. Leucocytic in- oestrus but is at its maximum during heat, and, vasion of the vaginal mucosa is maximal 2–5 days with experience, the veterinarian can detect after oestrus. Copious secretion of mucus by the oestrus on this sign alone. Between 24 and 48 cervix and anterior vagina begins a day or so hours after oestrus the uterine caruncles show before heat, increases during heat and gradually petechial haemorrhages, and these give rise to the diminishes to the fourth day after heat.The mucus postoestrous vaginal discharge of blood. In heifers is transparent and flows readily. there are often also associated perimetrial sub- Associated with these features of the cervical serous petechiae. During dioestrus the endo- mucus are variations in its crystallisation patterns metrium is covered by a scanty secretion from the which can be seen when dried smears of mucus uterine glands. are examined microscopically. During oestrus, and for a few days afterwards, the crystals are dis- Cyclic changes in the ovaries posed in a distinct aborisation pattern, while for the remainder of the cycle this pattern is absent. Usually one follicle ovulates and one ovum is lib- This phenomenon, together with the character erated after each heat, but twin ovulations occur and amount of cervical mucus, are dependent on in 4 or 5% of cows, and triplet ovulations more the concentration of oestrogen. The postoestrous rarely. In dairy cattle, about 60% of ovulations are vaginal mucus shows floccules composed of leu- from the right ovary, although in beef cattle the cocytes, and, as previously mentioned, blood is functional disparity between the ovaries is not frequently present. great. Hyperaemia of the mucosae of the vagina and The size and contour of the ovaries will depend cervix is progressive during pro-oestrus and on the phase of the cycle. It is best to begin by oestrus; the vaginal protrusion of the cervix is studying the organs of a mature unbred heifer. tumefied and relaxed, so that one or two fingers Post-mortem section of such ovaries will reveal can be inserted into the cervical os. During the most significant structures in them to be metoestrus, there is a rapid reduction in vascular- Graafian follicles and CLs. ity, and from 3 to 5 days after heat the mucosa is pale and quiescent and the external os is con- Follicular growth and development stricted while the mucus becomes scanty, sticky and pale yellow or brown. There are also cyclic Follicular growth and atresia throughout the cycle variations in vaginal thermal conductance and is a feature in the cow (Matton et al., 1983). In the vaginal pH, the former rising just before oestrus studies of Bane and Rajakoski (1961), two waves (Abrams et al., 1975). When pH electrodes were of growth were demonstrated, with the first wave placed in the cervical end of the vagina the pH fell beginning on the third and fourth day, and the from 7.0 to 6.72 one day before the first behav- second starting on the 12th to 14th day of the ioural signs of oestrus, and at the start of oestrus cycle. Consequently, a normal follicle of 9–13 mm fell again to 6.54 (Schilling and Zust, 1968). 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 Cyclic changes in the uterus 15th and 20th days; the ovulatory follicle is selected During oestrus, the uterus is congested, and the at about 3 days before ovulation (Pierson and endometrium is suffused with oedematous fluid; Ginther, 1988). Others have observed three waves its surface is glistening. The muscularis is physio- of follicular development in most oestrous cycles logically contractile so that when the uterus is pal- (Sirois and Fortune, 1988; Savio et al., 1990). The 21 1 NORMAL OESTROUS CYCLES most notable feature was the regularity of the quently feels flattened and soft. If such an ovary is number of waves of follicular growth per oestrous examined post-mortem it will be seen that the cycle, which probably reflected genetic or environ- surface from which ovulation has occurred is mental influences. Follicular growth is under the wrinkled and possibly bloodstained. The CL influence of FSH, with normally one follicle develops by hypertrophy and luteinisation of the obtaining dominance and subsequently ovulating. granulosa cells lining the follicle. Enlargement is The dominance does not appear to be mediated by rapid. By the 48th hour after ovulation it has the effect of inhibin but probably by some yet attained a diameter of about 1.4 cm. At this stage unknown intra-ovarian mechanism which does not the developing CL is soft, and yields on palpation. involve the suppression of FSH secretion. In addi- Its colour is dull cream, and the luteinised cells tion, other metabolic hormones such as insulin can be seen in the form of loose pleats. The CL growth factor 1 (IGF-1) may also be involved in attains its maximum size by the seventh to eighth follicular growth patterns (see review by Webb day of dioestrus (Figure 1.15). The luteinised et al., 1992). pleats are now relatively compact, and the body Thus, during dioestrus several large follicles comprises a more or less homogeneous mass, will be found ranging in size up to 0.7–1.5 cm in yellow to orange-yellow in colour. Its shape varies; diameter. These follicles do not alter the general the majority are oval, but some are irregularly oval contours of the ovaries but do cause some square or rectangular. The greatest dimension of overall variation in gross ovarian size. The ease of the fully developed structure varies from 2.0 to palpating them rectally will depend upon the size, 2.5 cm; the changes in the dimensions of the CL degree of protrusion and relationship with the are shown in Figure 1.16. Its weight also varies; in corpus luteum. the authors’ series, fully developed CLs have During pro-oestrus and oestrus, the follicle varied from 4.1 to 7.4 g. (Similar variations also which is soon to rupture enlarges, and ovulation occur in the weights of the CLs of pregnancy, occurs when it has attained a size of at least ranging from 3.9 to 7.5 g.) Sometimes, the centre 1.9 cm (Hammond, 1927). On rectal palpation of of the yellow body is occupied by a cavity (Figures the ovaries during heat it is usually possible to 1.17 and 1.27). This has been seen in 25% of detect the ripening follicle as a slightly bulging, those collected by the author. The size of the smooth soft area on the surface of one of them. cavity varies; in the majority it is small, averaging Ovulation may occur from any aspect of the 0.4 cm in diameter, but occasionally it is large, up ovarian surface, and the shape of that organ sub- to 1 cm or more. It is occupied by yellow fluid. In sequently when the CL develops will be chiefly the case just described, there is evidence of ovula- influenced by this site. The point of ovulation is tion by the presence of a pin-head depression in usually in an avascular area of the follicular wall, the centre of the projection from the surface of and consequently haemorrhage is not a feature of the ovary. This serves to differentiate the CL from bovine ovulation, although there is marked post- the abnormality of the cow’s ovary: luteinisation ovulatory congestion around the rupture point, of the walls of the follicle without ovulation. and sometimes a small blood clot is present in the Nevertheless, it is probable that this is the condi- centre of the new CL. tion which has been described in the past as cystic corpus luteum and regarded as pathological; the presence of a cavity is normal. 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- Fig. 1.15 Ovary of cow in mid-dioestrus. (a) A mature quent on the escape of the greater part of its fluid, corpus luteum (cl) with ovulation papilla could be readily the follicle collapses. If the opportunity arose for palpated together with a mid-cycle follicle (f). (b) Section repeatedly carrying out rectal examinations of the same ovary showing the solid corpus luteum (cl) and mid-cycle follicle (f). (c) B-mode ultrasound image of during heat and for the 24 hours succeeding it, the same ovary showing a speckled area corresponding this collapse would be detected. The ovary fre- to the corpus luteum (cl) and the mid-cycle follicle (f). 22  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 (a) 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.) (b) 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 (c) 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. 23 1 NORMAL OESTROUS CYCLES (a) (b) 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 (c) follicle (f). Fig. 1.18 Ovaries of a first-calf heifer in oestrus. 1, ripe Fig. 1.19 Ovaries of a first-calf heifer in oestrus. 1, ripe follicle; 2, regressing corpus luteum, bright yellow; 3, follicle; 2, regressing corpus luteum, brick-brown. Stage corpus albicans. Stage A in Figure 1.16. A in Figure 1.16. 24  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 Fig. 1.24 Ovaries of a 6-year-old cow in early dioestrus. Fig. 1.20 Ovaries of a nulliparous heifer just after 1, active corpus luteum, orange-yellow, atypical ovulation. 1, collapsed follicle, surface wrinkled and protrusion; 2, regressing corpus luteum, small, shrunken, blood-stained petechiae in wall; 2, regressing corpus scarlet; 3, corpus albicans; 4, follicle. Stage D in Figure luteum, bright yellow. Stage B in Figure 1.16. 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 Fig. 1.25 Ovaries of nulliparous heifers in dioestrus. and brown; 3, corpus albicans. Stage C in Figure 1.16. 1, corpus luteum 2, largest follicle. Stage E in Figure 1.16. heat. From this point, it undergoes rapid reduc- yellow. (This colour contrasts strikingly with that tion in size and changes in colour and appearance. of the active body.) Its consistency is dense, and By the middle of oestrus, its diameter is reduced already scar tissue invasion is commencing. By the to 1.5 cm and its protrusion is much smaller and second day of dioestrus, its size is reduced to less distinct, while its colour is changing to bright about 1 cm and its outline is becoming irregular. 25 1 NORMAL OESTROUS CYCLES 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 Fig. 1.26 Ovaries of parous cows in dioestrus. 1, corpus from prolonged function, and in some cases also luteum; 2, largest follicle; 3, corpus albicans. Stage E in Figure 1.16. 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. By this time its colour is changing to brown. By Nevertheless, it is generally possible in mid- the middle of dioestrus, it has shrunk to a size of dioestrus to detect the CL, for, quite apart from about 0.5 cm, and its surface protrusion is little its protrusion, the ovary containing it is plum-like, larger than a pin-head. As it gets older its colour whereas the other is distinctly flattened from side tends to change to red or scarlet. Small red rem- to side. On section of such ovaries, the CLs, both nants of corpora lutea tend to persist for several active and regressing, and the follicles approach- months. ing maturity are identical with those described for 26  ENDOGENOUS AND EXOGENOUS CONTROL OF OVARIAN CYCLICITY 1 the heifer. There is, however, an additional struc- pation) and the appearance (as determined by sec- ture to be recognised: old scarred CLs of previous tioning after slaughter) of the ovaries and their pregnancies. They generally show as a white, contents. The advent of transrectal B-mode real- pin-head-sized projection on the surface of the time grey scale ultrasound imaging, particularly ovary, and on section are found to comprise using a 7.5 MHz transducer, has enabled detailed, mainly scar tissue. They are irregular in outline, accurate sequential examination of the ovaries to with a maximum dimension of about 0.5 cm. be made without adversely affecting the cow’s Their colour is white (corpus albicans) or brown- health or fertility. The principles of the technique ish-white. The CL of pregnancy does not atrophy are described in Chapter 3, and for a detailed after parturition as quickly as does that of the description of the echogenic appearance of the oestrous cycle after it has ceased to function. It is ovaries, readers are advised to consult Pierson and an appreciable structure for several weeks after Ginther (1984) and Boyd and Omran (1991). parturition, brown in colour and about 1 cm in The following normal structures can be identi- diameter. It becomes progressively invaded by fied (see Figures 1.15 and 1.17): the ovarian scar tissue and remains throughout the cow’s life. stroma, antral follicles, CLs and ovarian blood On post-mortem the presence of the corpus albi- vessels. In addition, pathological structures such cans serves to distinguish the cow from the heifer as ovarian cysts can be seen (see Chapter 22).The and in the former a count of the corpora albi- ovarian stroma has a mottled echotexture. The cantia gives the number of calves borne. antral follicles are readily identif