Evaluation of follicular growth and tissue viability in vitrified/warmed domestic dog ovaries after in vitro culture.

Cryopreservation of gametes is an important tool to preserve fertility, but for most species, including domestic dogs, data regarding ovarian tissue cryopreservation are limited. We aimed to evaluate the follicular and tissue viability and follicular growth after in vitro culture of domestic dog ovarian cortical slices cryopreserved by vitrification. Ovarian cortex was obtained from ten pairs of ovaries from domestic dogs using two methods (A and B), one for each ovary from the same bitch. At least four slices for each method were obtained from each ovary, one was processed for histology and the other three were vitrified. When the vitrified slices were warmed, one slice from each method was processed for histology and the remaining two slices were cultured in vitro for 7 days, after which they were processed for histological evaluation. Density of follicles in fresh samples was similar for both methods. For Method A, density of secondary follicles decreased, while the density of primordial follicles was maintained throughout the process. For Method B, density of primary follicles decreased after 7 days of incubation, but density of secondary follicles increased, confirming follicular growth in Method B. Overall, there were no differences between Methods A and B in follicular integrity after incubation. Fresh samples showed better arterial, venous and follicle preservation, followed by vitrified-warmed samples, but no differences were observed between methods. In conclusion, the methodology used to isolate the ovarian cortex may affect tissue and follicle viability as well as follicular development during in vitro culture.

Predominant suppression of follicle-stimulating hormone ß-immunoreactivity after long-term treatment of intact and castrate adult male rats with the gonadotrophin-releasing hormone agonist deslorelin.

Gonadotrophin-releasing hormone (GnRH) agonists are used to treat gonadal steroid-dependent disorders in humans and to contracept animals. These agonists are considered to work by desensitising gonadotrophs to GnRH, thereby suppressing follicle-stimulating hormone (FSH) and luteinising hormone (LH) secretion. It is not known whether changes occur in the cellular composition of the pituitary gland after chronic GnRH agonist exposure. Adult male Sprague-Dawley rats were treated with a sham, deslorelin, or deslorelin plus testosterone implant for 41.0 ± 0.6 days. In a second experiment, rats were castrated and treated with deslorelin and/or testosterone. Pituitary sections were labelled immunocytochemically for FSHß and LHß, or gonadotrophin α subunit (αGSU). Deslorelin suppressed testis weight by two-thirds and reduced plasma FSH and LH in intact rats. Deslorelin decreased the percentage of gonadotrophs, although the effect was specific to the FSHß-immunoreactive (-ir) cells. Testosterone did not reverse the deslorelin-induced reduction in the overall gonadotroph population. However, in the presence of testosterone, the proportion of gonadotrophs that was FSHß-ir increased in the remaining gonadotrophs. There was no effect of treatment on the total LHß-ir cell population, although the loss of FSHß in bi-hormonal cells increased the proportion of mono-hormonal LHß-ir gonadotrophs. The castration-induced plasma LH and FSH increases were suppressed by deslorelin, testosterone or both. Castration increased both LH-ir and FSH-ir without increasing the overall gonadotroph population, thus increasing the proportion of bi-hormonal cells. Deslorelin suppressed these increases. Testosterone increased FSH-ir in deslorelin-treated castrate rats. Deslorelin did not affect αGSU immunoreactivity, suggesting that the gonadotroph population per se is not eliminated by deslorelin, although the ability of gonadotrophs to synthesise FSHß is compromised. We hypothesise that the FSH dominant suppression may be central to the long-term contraceptive efficacy of deslorelin in the male.

Diagnostic value of transcervical endometrial biopsies in domestic dogs compared with full-thickness uterine sections.

Transcervical endometrial biopsy is a useful tool for obtaining information about uterine health in some species. The clinical application of information gained from histopathological interpretations of endometrial biopsies in the bitch has not been validated. We hypothesized that transcervical endometrial biopsy samples would be as diagnostic as full-thickness uterine sections in identifying cystic endometrial hyperplasia (CEH), inflammation and periglandular fibrosis. Endometrial biopsies were obtained from 20 female adult dogs. Vaginal swabs, gross appearance of the vulva and vaginal tract, and serum progesterone values were used to determine the stage of the oestrous cycle at the time of sampling. The uteri were removed between 1 and 6 days after the biopsy procedure, and full-thickness sections were collected from each uterine horn and ovary and processed for histopathology. Two pathologists, blinded to the origin of each sample, compared full-thickness sections from the excised uteri to the biopsy samples collected via the transcervical technique. Pathologic features noted included: CEH, inflammation and periglandular fibrosis. Pathological diagnoses obtained from the biopsy sections were compared with those obtained from the full-thickness sections, as well as comparing diagnoses between the two pathologists, using McNemar's test. Of the 59 total biopsy samples obtained, 54 were considered diagnostic. All stages of the canine oestrous cycle were represented (anoestrus, proestrus, oestrus and dioestrus). Pyometra was not noted in any of the transcervical biopsy sections, but was noted in many of the full-thickness sections collected from dogs in dioestrus, suggesting either that biopsy is not a sensitive indicator of pyometra or that the procedure may induce pyometra in dioestrous dogs. Transcervical endometrial biopsy showed similar sensitivity as full-thickness sections in detecting CEH, inflammation and fibrosis. No differences in describing lesions were detected between pathologists.

Vitrification of oocytes from endangered Mexican gray wolves (Canis lupus baileyi).

Careful genetic management, including cryopreservation of genetic material, is central to conservation of the endangered Mexican gray wolf. We tested a technique, previously used to vitrify human and domestic animal oocytes, on oocytes from domestic dogs as a model and from the endangered Mexican wolf. This method provided a way to conserve oocytes from genetically valuable older female Mexican wolves as an alternative to embryos for preserving female genes. Oocytes were aspirated from ovaries of 36 female dogs in December and March (0 to 65 oocytes per female) and from six female wolves (4 to 73 per female) during their physiologic breeding season, or following stimulation with the GnRH agonist deslorelin. Oocytes from dogs were pooled; half were immediately tested for viability and the remainder vitrified, then warmed and tested for viability. All oocytes were vitrified by being moved through media of increasing cryoprotectant concentration, placed on Cryotops, and plunged into liquid nitrogen. There was no difference in viability (propidium iodide staining) between fresh and vitrified, warmed dog oocytes (65.7 and 61.0%, respectively, P = 0.27). Oocyte viability after warming was similarly assessed in a subset of wolves (4 to 15 oocytes from each of three females; total 29 oocytes). Of these, 57.1% of the post-thaw intact oocytes were viable, which was 41.4% of all oocytes warmed. These were the first oocytes from a canid or an endangered species demonstrated to have maintained viability after vitrification and warming. Furthermore, our results demonstrated that vitrification of oocytes with the Cryotop technique was an option for preserving female gametes from Mexican wolves for future use in captive breeding programs, although in vitro embryo production techniques must first be developed in canids for this technique to be used.

Influence of cooling rates and addition of Equex pasta on cooled and frozen-thawed semen of generic gray (Canis lupus) and Mexican gray wolves (C. l. baileyi).

A current priority for the preservation of the endangered Mexican gray wolf (Canis lupus baileyi) is the development of a sperm-based genome resource bank for subsequent use in artificial insemination. To optimize the quality of cryopreserved sperm, the procedures involved in processing semen before and during freezing need to be improved. The aim of this study were to examine the effects of: (i) different cooling periods before freezing and (ii) addition of Equex pasta (Minitüb, Tübingen, Germany) on the characteristics of sperm from the generic gray wolf and the Mexican gray wolf after cooling and cryopreservation. For Mexican wolf sperm, cooling for 0.5 and 1.0 h had a less detrimental effect on cell morphology than cooling for 2.5 h, whereas the slower cooling rate (2.5 h) had a less detrimental effect on functional parameters and seemed to cause less damage to plasma membrane and acrosome integrity than 0.5 and 1.0 h. For the generic gray wolf, cooling semen for 2.5 h had less detrimental effect on plasma membrane integrity and viability; together with the 0.5 h cooling time, it yielded the highest percentages of intact acrosomes. As previously shown in the domestic dog, Equex pasta had no beneficial effect on sperm characteristics in either wolf species.

Monitoring pregnancy in babirusa (Babyrousa babyrussa) with transabdominal ultrasonography.

This case report documents the application of transabdominal ultrasonography for detecting pregnancy and monitoring fetal development in an unanesthetized, unrestrained babirusa (Babyrousa babyrussa). A 4-yr-old multiparous female babirusa was trained to accept monitoring via transabdominal ultrasonography on a weekly basis postcoitus. Uterine changes associated with pregnancy were first detected at 30 days gestation. Three fetuses were first visualized on day 38, and two of the three fetuses were detectable until term. One fetus failed to develop and appeared to be resorbed between days 80 and 94 of the 161-day pregnancy. The growth of the remaining two fetuses was monitored via cranial measurements taken during the weekly scans throughout the pregnancy. By monitoring a subsequent pregnancy via ultrasound in the same female it was possible to confirm her tendency towards partial resorption, with earliest detection of uterine changes on day 32 and apparent resorption of one of three fetuses at approximately the midpoint of gestation. Two rectal scans of a different female under anesthesia at 28 and 50 days gestation showed stages of development comparable to those visualized transabdominally. However, the transabdominal scans produced better images of the reproductive tract, perhaps because of the close proximity of the pregnant uterus to the abdominal wall.

Efficacy of the GnRH analogue deslorelin for suppression of oestrous cycles in cats.

The aim of this study was to develop a method for long-term but reversible inhibition of oestrous cycles in female cats by downregulation of GnRH receptors with deslorelin released from a long-acting implant. In a blind study with mature cats (n = 20), a 6 mg deslorelin implant was administered s.c. to ten cats and a placebo implant was administered to ten cats. Occurrence of oestrus and general health were observed daily, and individual faecal samples were collected at 3 day intervals for 14 months and analysed for oestradiol content. All the placebo-treated queens continued to undergo normal oestrous cycles during the study. Oestrus was accompanied by peaks in oestradiol concentrations of > or = 20 ng g-1 faeces. Treatment with deslorelin initially stimulated oestradiol release, which accompanied treatment-induced ovulations. Thereafter, oestradiol concentrations decreased to 1-10 ng g-1 faeces and remained low for extended periods. Observations of small increases in oestradiol concentrations in one cat led to a second treatment with 6 mg deslorelin in five cats on day 155 after first treatment. Faecal oestradiol concentrations remained < 20 ng g-1 faeces in the five single treatment cats for 8.0, 8.5, 11.0 and 14.0 (two cats) months. Cats receiving two implants had the first oestradiol peak > 20 ng g-1 faeces after treatment at 7.5, 11.0 (two cats), 11.5 and 14.0 months. After 14 months, two cats had returned to normal cyclic activity, two had irregular small oestrogen peaks and six showed no cyclic activity. For months 2-5, 6-10 and 11-14, oestrogen values in treated cats were significantly different from control values (P < 0.001, 0.05 and 0.02, respectively). Differences in oestrogen concentration between control cats and cats that were treated twice were significant (P < 0.001) during months 6-10 only. The general health of treated cats was unchanged throughout the study. These results confirm that deslorelin can effectively suppress ovarian activity in domestic cats, but that the duration of suppression varies among individuals.

Control of reproduction and sex related behaviour in exotic wild carnivores with the GnRH analogue deslorelin: preliminary observations.

The GnRH analogue deslorelin, in long-acting implants, was used in an attempt to temporarily control reproduction or aggression in wild carnivores in southern Africa and the USA. In the southern African study, 6 mg deslorelin was administered to cheetahs (eight females, four males), one female leopard and wild dogs (six females, one male) housed in groups, and 12 mg deslorelin was administered to two lionesses. None of the animals became pregnant after deslorelin administration apart from one wild dog that was mated at the initial treatment-induced oestrus. Two wild dogs and one lioness came into oestrus 12 and 18 months after deslorelin administration, respectively, thus demonstrating that the anti-fertility effects of deslorelin are reversible. Two lionesses and four cheetahs underwent oestrus without allowing mating 2-14 days after treatment. Simultaneous administration of progestins to three bitches and one lioness did not suppress oestrus. Male cheetahs had no spermatozoa on day 82 after treatment and did not impregnate two untreated females. Of three untreated female wild dogs housed with treated males, only the first female to enter oestrus (21 days after deslorelin administration) became pregnant. One month after treatment, plasma testosterone concentrations of male dogs were at basal values. In the USA study, three male sea otters that had been treated with 6 mg deslorelin ceased antagonistic behaviour and blood testosterone concentrations and size of the testes were still sharply reduced 24 months after treatment. Male red (n = 7) and grey (n = 5) wolves received 6 mg deslorelin in December 1998 but no effects on seasonal spermatogenesis and behaviour were observed. In a black-footed cat, sperm production, libido and aggressiveness decreased in response to treatment with 3 mg deslorelin and penile spines were not observed within 3 months after treatment, but were observed again 4-6 months later. Treatment of female red (n = 5) and grey (n = 5) wolves with deslorelin in December 1999 triggered preseason oestrus and mating, which were followed by one abortion and one successful pregnancy. Contraception was achieved in female Fennec foxes (n = 7) and two lionesses, which was observed in the foxes by an absence of increases in faecal progesterone concentrations. In two male bush dogs, administration of 3 mg deslorelin once or twice was insufficient to suppress reproductive function or behaviour.

Ovulatory cycles and anovulatory periods in the addax (Addax nasomaculatus).

Changes in serum oestradiol and progesterone were measured to study their dynamics during ovulatory cycles in six female addax, an endangered antelope. Blood was collected three times per week, during chute restraint, for 3 months (November to February) before introduction of a male, and continued until pregnancy was diagnosed with ultrasound. Serum was analysed by enzymeimmunoassay. Mean luteal phase, interluteal phase, and cycle durations were 22.7 +/- 2.0, 8.78 +/- 0.5 and 32.3 +/- 1.7 days, respectively. Ultrasonography revealed coiled uterine horns and maximum follicle and corpus luteum diameters of 15 and 27 mm, respectively. Each female experienced an anovulatory period, during which oestradiol continued to fluctuate, but progesterone remained below 2 ng ml-1. These periods ranged from 39 to 131 days and were not synchronous; ovulatory cycles resumed spontaneously in all females. All four females placed with a male conceived. Because addax give birth all year round, they are not considered seasonal breeders. The sporadic periods of anovulation that occurred during the winter months of this study suggest a possible seasonal effect. However, systematic sampling has not been conducted during summer and early autumn and will be necessary to address this question.

The influence of social and endocrine factors on urine-marking by captive wolves (Canis lupus).

Although serum hormones varied seasonally in all adult animals, only dominant male and female wolves urine-marked. Serum testosterone and urine-marking rates, which increased during the fall/winter breeding season, were positively correlated in both male and female dominant wolves. Estradiol, which increased in conjunction with proestrus and estrus, was not correlated with female urine-marking. These findings suggest that hormonal influence on urine-marking in the wolf is modulated by social factors and contrast with those for both domestic dogs and coyotes, two other members of the genus Canis.

Development of spermatozoa in the rhea.

We have examined the ultrastructural changes that take place during spermiogenesis in the rhea. Spermatozoa are characterized by a curved head and a midpiece. A thin rod extends from the anterior tip of the spermatozoon through the center of the nucleus. A 3-mu-long distal centriole occupies the entire midpiece. The principal piece is characterized by a small fibrous sheath and tiny dense fibers that are only observed in the region of the principal piece, which is immediately behind the annulus. During development a circular manchette surrounds the nucleus of young spermatids. Later the microtubules of the circular manchette become reorganized into a longitudinal manchette. A long distal and short proximal centriole are observed in early round spermatids. The distal centriole becomes associated with the plasma membrane. Later the proximal centriole is observed in association with the nucleus. The area around the centriole pair then accumulates dense material, which is associated with either the centrioles or the circular manchette. The longitudinal manchette forms and then disappears and mitochondria subsequently associate with the distal centriole. The long centriole of the rhea enables this species to develop a midpiece similar to the midpiece of mammalian sperm without the complex intercellular movements that characterize mammalian spermiogenesis.

Nuclear shaping in spermatids of the Thai leaf frog Megophrys montana.

Transmission electron microscopy of Thai leaf frog testis revealed a unique pattern of spermatid nuclear morphogenesis. Chromatin condenses into a continuous cylindrical coil within a roughly spherical nucleus. Later the nuclear membrane conforms to the contours of the uncoiling nuclear contents. In the mature sperm, the long, tapering nucleus is helically shaped. This developmental sequence occurs in the absence of a microtubular manchette, raising questions about the role of this structure in nuclear shaping in spermatozoa of other species.

Pinealectomy or superior cervical ganglionectomy do not alter reproduction in the wolf (Canis lupus).

Twelve wolves (6 male and 6 female) were used to study the role of the pineal in photoperiodic mediation of seasonal reproduction. Eight wolves were pinealectomized (PNX) or sham-pinealectomized (S-PNX) at 5 mo of age, and 4 were superior cervical ganglionectomized (SCGX) at 16 mo of age (2 males and 2 females per treatment). All attained puberty at the species-typical time, during their second breeding season, except 2 SCGX males that did not survive. Reproductive cycles of an additional male that was SCGX as an adult and the PNX and S-PNX wolves, followed for a minimum of 3 yr, did not differ from each other or from those of unoperated colony wolves on measures of serum testosterone and luteinizing hormone for males, or of serum estradiol and progesterone for females. Nor was the range of dates for ovulation different for treated vs. untreated females. Surgical transection of the olfactory tracts of 1 male and 1 female PNX wolf, inducing anosmia to control for the possibility of pheromonally synchronized cycles, also failed to alter the seasonality of these reproductive parameters. These results do not conform to the model of pineal mediation of sexual cycles for photoperiod-sensitive species. In spite of evidence for photoperiod influence, the wolf apparently relies on a system other than the pineal for seasonal control of reproduction.

Rapid reversible immobilization of feral stallions using etorphine hydrochloride, xylazine hydrochloride and atropine sulfate.

Forty-eight newly captured free-ranging feral stallions (Equus caballus) from two different locations and six captive stallions were immobilized using combinations of etorphine hydrochloride, xylazine hydrochloride and atropine sulfate with or without acepromazine. Six animals were immobilized twice, 1 mo apart. The drugs were administered either intramuscularly (n = 13) or intravenously (n = 44). Mean immobilization time (+/- SE) after intravenous (i.v.) injection of etorphine, xylazine and atropine was 55 +/- 4 sec (range 20 to 185 sec) compared to 708 +/- 131 sec (range 390 to 1,140 sec) for intramuscular (i.m.) injection. Immobilization was reversed with i.v. administration of 3 to 11 mg diprenorphine hydrochloride and 16 to 24 mg yohimbine hydrochloride. Average time from administration to standing and walking was 86 +/- 7 sec (n = 55). Reversal of etorphine-induced immobilization with an amount of diprenorphine equal to the etorphine and administered i.v. was as effective as a 2:1 ratio of diprenorphine to etorphine. Acepromazine had no effect on induction time, but decreased relaxation after immobilization and prolonged ataxia after reversal of the etorphine and xylazine. Eight free-ranging horses were immobilized in 708 +/- 132 sec by darting with 5.5 mg etorphine, 1,300 mg xylazine and 15 mg atropine from a helicopter. Three animals died during the study: one immediately after reversal of an i.v. administration, one from a broken neck during induction from darting, and one was found a week later at the site of darting.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure of spermatozoa of the white-naped crane.

Spermatozoa of the white-naped crane were examined with the light and electron microscopes. The relatively simple spermatozoa have a small acrosome, a short midpiece, and a flagellum lacking both dense fibers and fibrous sheath. Crane spermatozoa are unique in that the degree of condensation varies among spermatozoa, but in no spermatozoon does chromatin become highly compacted. Incomplete condensation of chromatin may be responsible in part for the variety of sperm head shapes, including elongate, curved, 'S'-shaped, droplet and spherical forms. Crane sperm may provide a system to better understand the mechanism and function of chromatin condensation in spermatozoa.

Sexual behavior of mares.

Behavior during the estrous phase of the ovulatory cycle of the mare is analogous in most ways to that of estrous females of other species. Proceptive behaviors bring the mare into the proximity of the male and attract his attention. Positioning facilitates mounting, intromission, and ejaculation. Estrous signs appear to be more intense in the few days prior to ovulation than during the transition periods that separate the recurring estrous and diestrous phases. Sexual behavior is absent during diestrus. Detection of estrus in mares is problematic in that it requires the presence (or at least facsimile acoustic or tactile stimuli) or a stallion. Unexplained conditions such as silent or subestrus make reliable determinations even more difficult. The choice of methods for distinguishing estrous from nonestrous mares depends, among other factors, upon characteristics of individual mares, as well as the number of mares to be evaluated. Detection of true estrus--that is, sexual behavior that is associated with follicular growth and ovulation--is confounded by the occurrence of estrus-like behavior independent of gonadal stimulation. Seasonally anovulatory and ovariectomized mares may regularly solicit and accept copulation despite low or even no circulating levels of ovarian steroids. Instead, endocrine support of sexual behavior in these mares appears to be provided by adrenal cortical androgens and/or estrogens. A smaller percentage of pregnant mares may also show some signs of estrus but are not likely to be receptive to mating. The ability of the mare to dissociate sexual behavior from ovulation is unique among infraprimate species studied. The display of estrous behavior and willingness to copulate, even at times when conception is impossible, may have functional significance in promoting band cohesiveness in wild and feral populations. Even without copulation, continued motivation to remain in proximity to the band stallion may contribute to the stability of the social unit outside the ovulatory season.

Effect of anosmia on reproduction in male and female wolves (Canis lupus).

Anosmia was produced in two female and three male wolves by transection of the olfactory peduncle and was confirmed by their inability to detect meat, urine, feces, anal-gland secretions, and fish emulsion. All operated animals continued to investigate the environment with their noses, to interact normally with other pack members, and to feed at levels which maintained presurgical body weights. No effect was found on reproductive physiology (females: estradiol or progesterone concentrations, ovulation, pregnancy or parturition; males: testosterone, testicular recrudescence or sperm numbers, motility or maturation). One anosmic female became dominant and although she urine-marked with a flexed leg, the rate was lower than typical for dominant females and perhaps contributed to her failure to pair-bond with the dominant male. One anosmic male raised-leg-urinated while competing for pack dominance and when kenneled away from other males. Precopulatory, copulatory, and maternal behavior were observed for one anosmic female and appeared normal. However, neither male that was sexually naive before surgery showed interest in proestrous or estrous females. The possibility that secondary degeneration of brain regions mediating sexual behavior was responsible for the failure of these males to respond was not supported. Not only was the lack of male sexual response the only serious deficit following transection, but the male which was sexually experienced prior to surgery did copulate successfully during his second postoperative breeding season despite continued anosmia. Chemosensory priming from female urine during the protracted proestrous phase, as well as urinary and vaginal odors during estrus, appear to be critical for induction of full sexual potency in sexually naive males. The importance of urine and vaginal secretions in the sexual response of experienced males is uncertain.

The effect of estradiol and progesterone on the sexual behavior of ovariectomized mares.

Daily treatment (5 days) with estradiol resulted in increased levels (p less than 0.05) of proceptive behavior in ovariectomized as compared to control mares (N = 8 per treatment) within 4 hr of injection and for the 4 subsequent days. Ejaculations occurred more often (p less than 0.05) in estrogen-treated mares on days 2-5, but the number of precopulatory investigations by the stallions was not altered. Progesterone treatment resulted in an absence of sexual behavior except in one mare on Day 1. Control mares exhibited varying levels of sexual interest. The concurrent administration of estradiol and progesterone produced a biphasic effect on proximity-related behaviors. Proximity behaviors were initially (Day 1) greater and subsequently less in the group treated with both hormones than in the group treated with estradiol alone. Injections of free estradiol resulted in a shorter latency to effect for two measures of proceptivity than did injections of estradiol benzoate. A dose response test for progesterone showed no effect with 0, 1, or 10 mg, but 100 mg was inhibitory (p less than 0.05). These results demonstrated that within 4 hr estradiol stimulated, while progesterone inhibited estrous behavior in ovariectomized mares and that concurrent administration of estradiol and progesterone produced a biphasic effect, first enhancing, then suppressing some aspects of the estrous response. The relatively short latency to action of all treatments and the biphasic effect of concurrent estradiol and progesterone may be attributable to low level, endogenous hormones.

Assessment of the sexual behavior of pregnant mares.

Pregnant mares (N = 12) were observed with a stallion from early gestation until parturition for sexual behavior. Observations were conducted for 20 min per day for 5 days each month from September until July. No mares exhibited full estrus and no intromissions or ejaculations occurred at any time during the study. Social interactions such as male approach and female/female mutual grooming occurred with greater mean frequency or duration in some months of spring and fall than in winter months. (P less than 0.05). None of the recorded behaviors differed by month of gestation. In a comparison of the behavior of diestrous and pregnant mares in harem groups, no estrous behavior occurred and no differences in social interactions were found. Although progesterone may be sufficient to cause the absence of sexual behavior in diestrous mares, another estrous-inhibiting substance may be present during pregnancy at times of high estrogen and low progesterone.

Changes in plasma cortisol concentrations during the ovulatory cycle of the mare.

Daily blood samples from four mares were assayed for cortisol through a total of eight ovulatory cycles. Mean cortisol concentrations on days -14, -13, -10, -9 and -8 before ovulation (dioestrus) were greater than on days -5 to -1 (oestrus). The highest mean (+/- S.E.M) value of cortisol occurred on day -10 (260 +/-28 nmol/l) and the lowest on day -2 (142 +/- 14 nmol/l). A single episode on a day in late dioestrus characterized the maximum cortisol value per cycle for five of eight cycles. Extraction of plasma samples with petroleum ether or chromatography before assay, to eliminate interference from progesterone and its metabolites, did not alter the pattern of high dioestrous and low oestrous cortisol concentrations. Maximum follicular diameter at ovulation was negatively correlated with mean cortisol concentration for that cycle. These results indicate that in the mare the adrenals secrete cortisol more actively during dioestrus than during oestrus and suggest that a decline in cortisol values at oestrus may favour full follicular growth and ovulation.