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L. A. Johnston, D. L. Armstrong, and J. L. Brown

The annual reproductive cycle of the male snow leopard (Panthera uncia) was characterized by evaluating seminal and endocrine traits monthly. Testicular volume was greatest (P < 0.05) during the winter months when the quality of ejaculate was optimal. Ejaculate volume, total sperm concentration ml−1, motile sperm concentration per ejaculate, sperm morphology and sperm motility index were lowest during the summer and autumn months compared with the winter and spring. Peripheral LH, FSH and testosterone concentrations were also lowest during the summer months, increasing during the autumn just before the increase in semen quality, and were maximal during the winter months. There was a direct relationship (P < 0.01) between: (1) testosterone and testicular volume, total sperm concentration ml−1, motile sperm concentration per ejaculate and ejaculate volume, and (2) LH and testicular volume and motile sperm concentration per ejaculate. In summary, although spermatozoa were recovered throughout the year, optimal gamete quality was observed during the winter and spring. Although previous studies in felids have demonstrated seasonal effects on either seminal or endocrine traits, this is the first study to demonstrate a distinct effect of season on both pituitary and testicular function.

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In the pseudopregnant rat, deciduomata have been generally produced by surgical or chemical trauma of the uterus. The optimal administration time for these stimuli was found to be Day 4 of pseudopregnancy, and maximal decidual responses were obtained 5 days later (De Feo, 1963a). However, we have observed that deciduomata developed spontaneously in the absence of uterine trauma, and attempted to determine its frequency.

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Wistar-strain, female rats obtained from the Royal Hart colony, and weighing approximately 250 g, were used. They were housed in a controlled illumination environment of 14 hr light and 10 hr darkness, and were maintained on unlimited Purina Chow and water. During oestrus, the animals were made pseudopregnant by cervical stimulation with a glass rod or matings with vasectomized males. Day 1 of pseudopregnancy

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J. L. Brown, K. L. Goodrowe, L. G. Simmons, D. L. Armstrong, and D. E. Wildt

Summary. Frequent blood samples were collected to study hormonal responses to GnRH in male and female leopards and tigers. Animals were anaesthetized with ketamine–HCl and blood samples were collected every 5 min for 15 min before and 160 min after i.v. administration of GnRH (1 μg/kg body weight) or saline. No differences in serum cortisol concentrations were observed between sexes within species, but mean cortisol was 2-fold greater in leopards than tigers. GnRH induced a rapid rise in LH in all animals (18·3 ± 0·9 min to peak). Net LH peak height above pretreatment levels was 3-fold greater in males than conspecific females and was also greater in tigers than leopards. Serum FSH increased after GnRH, although the magnitude of response was less than that observed for LH. Basal LH and FSH and GnRH-stimulated FSH concentrations were not influenced by sex or species. Serum testosterone increased within 30–40 min after GnRH in 3/3 leopard and 1/3 tiger males. Basal testosterone was 3-fold greater in tiger than leopard males. LH pulses (1–2 pulses/3 h) were detected in 60% of saline-treated animals, suggesting pulsatile gonadotrophin secretion; however, in males concomitant testosterone pulses were not observed. These results indicate that there are marked sex and species differences in basal and GnRH-stimulated hormonal responses between felids of the genus Panthera which may be related to differences in adrenal activity.

Keywords: GnRH, leopard, tiger, LH, cortisol, testosterone

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L. Robertson, H. L. Brown, H. J. Staines, and G. J. Wishart

The inner perivitelline layer, separated from laid chicken eggs, was investigated as readily available material for studying the spermatozoa–egg interaction in vitro. This layer was found to have a similar response to hydrolysis by spermatozoa as the inner perivitelline layer from ovulated and follicular ova, in terms of the numbers of points of hydrolysis made by spermatozoa during a 5 min incubation at 40°C. Initiation of hydrolysis of the inner perivitelline layer was found to occur within 2.5 min, after which the size, but not the number of holes, increased with time. The frequency of the points of hydrolysis per unit area of the inner perivitelline layer was positively correlated with the concentration of spermatozoa in the incubation medium. The perivitelline hydrolysis assay was able to detect more damaged spermatozoa in samples that had been either stored at 5°C or cryopreserved in liquid N2 than did other tests of sperm quality, which are known to overestimate the fertilizing ability of stored avian semen.

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R. Qvist, L. F. Blackwell, H. Bourne, and J. B. Brown

Summary. An in-vitro culture system was developed in which primary mouse follicles from 12–16-day-old mice grew to the preovulatory stage. The important determinants of growth in culture were the inclusion of stroma with the primary follicles, the age of the mouse, the presence of FSH and LH, the use of culture dishes with a hydrophobic membrane and the use of post-menopausal human serum to supply growth factors. During culture the pieces of ovarian tissue containing the primary follicles coalesced to form characteristic spherical clusters. The cultured follicles appeared to be normal as determined by the appearance and organization of the granulosa cells, the appearance of the antrum and the accompanying steroidogenesis, but the ova had not resumed meiosis. The results show that the growth of mouse follicles starting from the primary stage is critically dependent on adequate concentrations of FSH.

Keywords: FSH; mouse; culture; follicle; in vitro

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R. J. Wordinger, D. Brown, E. Atkins, and F. L. Jackson

Summary. Pregnant mice were injected subcutaneously with diethylstilboestrol (DES: 10 μg/kg body weight in 0·1 ml corn oil) or corn oil alone on Day 15 or 16 of gestation (Day 1 = day of copulatory plug) and allowed to give birth. Female progeny from control and DES-exposed animals were superovulated with exogenous gonadotrophins at 6–8 weeks of age. In-vivo results indicated that the total number of ovulated ova, 2-cell embryos and blastocysts were significantly increased in DES-exposed progeny but that there was a decline in developmental potential from the ovulated ova stage to the blastocyst stage in these animals. However, there was no significant difference in the in-vitro development of 2-cell embryos to the blastocyst stage between control and DES-exposed animals. These results indicate that the ovaries of mice exposed in utero to DES are capable of responding to exogenous gonadotrophins and that second generation progeny have the potential for normal development to the early post-blastocyst stage of embryogenesis. The in-vivo decline in developmental potential may be attributable to reproductive tract abnormalities rather than ova/embryo defects.

Keywords: diethylstilboestrol; mouse; embryo; in vitro; development

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M. C. Schiewe, T. A. Fitz, J. L. Brown, L. D. Stuart, and D. E. Wildt

Summary. Ewes were treated with exogenous follicle-stimulating hormone (FSH) and oestrus was synchronized using either a dual prostaglandin F-2α (PGF-2α) injection regimen or pessaries impregnated with medroxy progesterone acetate (MAP). Natural cycling ewes served as controls. After oestrus or AI (Day 0), corpora lutea (CL) were enucleated surgically from the left and right ovaries on Days 3 and 6, respectively. The incidence of premature luteolysis was related (P < 0·05) to PGF-2α treatment and occurred in 7 of 8 ewes compared with 0 of 4 controls and 1 of 8 MAP-exposed females. Sheep with regressing CL had lower circulating and intraluteal progesterone concentrations and fewer total and small dissociated luteal cells on Day 3 than gonadotrophin-treated counterparts with normal CL. Progesterone concentration in the serum and luteal tissue was higher (P < 0·05) in gonadotrophin-treated ewes with normal CL than in the controls; but luteinizing hormone (LH) receptors/cell were not different on Days 3 and 6. There were no apparent differences in the temporal patterns of circulating oestradiol-17β, FSH and LH. High progesterone in gonadotrophin-treated ewes with normal CL coincided with an increase in total luteal mass and numbers of cells, which were primarily reflected in more small luteal cells than in control ewes. Gonadotrophin-treated ewes with regressing CL on Day 3 tended (P < 0·10) to have fewer small luteal cells and fewer (P < 0·05) low-affinity PGF-2α binding sites than sheep with normal CL. By Day 6, luteal integrity and cell viability was absent in ewes with prematurely regressed CL. These data demonstrate that (i) the incidence of premature luteal regression is highly correlated with the use of PGF-2α; (ii) this abnormal luteal tissue is functionally competent for 2–3 days after ovulation, but deteriorates rapidly thereafter and (iii) luteal-dysfunctioning ewes experience a reduction in numbers of small luteal cells without a significant change in luteal mass by Day 3 and, overall, have fewer low-affinity PGF-2α binding sites.

Keywords: sheep; superovulation; premature luteal regression; prostaglandin F-2α; receptor

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D. V. Brown, P. L. Senger, S. L. Stone, J. A. Froseth, and W. C. Becker

The roles of selenium (Se) and glutathione peroxidase in reproductive function are poorly understood, but it is possible that they may be important for normal reproduction in the male. In rats fed a Se-deficient diet, the testes accumulated and retained more 75Se than did other tissues 1 week after injection (Brown & Burk, 1972; Burk, Brown, Seely & Scaief, 1972). Autoradiographic studies (Brown & Burk, 1972) of spermatozoa recovered from rat epididymides have shown that 75Se is associated with the midpiece of the spermatozoon. Gould (1970) has suggested that 75Se is probably incorporated into late spermatocytes or early spermatids in the rat. Rotruck et al. (1973) demonstrated that Se is a component of glutathione peroxidase, an enzyme which has been reported to exist in dog, goat, ram and human semen (Li, 1975). The objectives of the present study were to determine whether glutathione peroxidase is present in ejaculated bovine semen and the relationship of the enzyme levels to sperm concentration and ejaculate volume.

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J. L. Brown, D. E. Wildt, N. Wielebnowski, K. L. Goodrowe, L. H. Graham, S. Wells, and J. G. Howard

Faecal oestradiol and progestogen metabolite excretion was monitored in adult, female cheetahs (Acinonyx jubatus) (n = 26) for 1–24 months. Increased faecal oestradiol excretion was associated with mating or equine chorionic gonadotrophin (eCG) administration for artificial insemination, whereas increased progestogen metabolites were observed during natural and human chorionic gonadotrophin (hCG)-induced pregnant and nonpregnant luteal phases. On the basis of oestradiol excretory patterns, duration of the oestrous cycle (mean ± sem) was 13.6 ± 1.2 days with high oestradiol concentrations lasting for 4.1 ± 0.8 days. In non-gonadotrophin-treated cheetahs, 75% showed evidence of oestrous cyclicity; however, none evaluated for 1 year or longer were continuously cyclic. Rather, cyclicity was interrupted by periods of anoestrus, often exceeding several months in duration. These inactive ovarian periods were unrelated to season and were not synchronous among females. Mean duration of gestation (breeding to parturition) was 94.2 ± 0.5 days, whereas duration of faecal progestogen metabolite excretion during the nonpregnant luteal phase was 51.2 ± 3.5 days. On the basis of progestogen metabolite evaluations, spontaneous ovulation (non-mating induced) occurred only once in two females (2 of 184 oestrous cycles; 1.1%). Peak eCG-stimulated, preovulatory oestradiol concentrations were similar to those associated with natural oestrus, whereas progestogen metabolite profiles after hCG resembled those during pregnant and nonpregnant luteal phases after natural mating. In summary, results confirm that the cheetah is polyoestrus and ovulation is almost always induced. However, new evidence suggests that many females inexplicably experience periods of anoestrus unrelated to season, while 25% of the cheetahs examined expressed no ovarian activity during the study period.

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S. L. Monfort, J. L. Brown, M. Bush, T. C. Wood, C. Wemmer, A. Vargas, L. R. Williamson, R. J. Montali, and D. E. Wildt

Blood samples, morphometric measurements and behavioural data were collected weekly for 52 weeks from six adult Eld's deer stags exposed to natural fluctuations in photoperiod (38°N latitude). Mean (± sem) serum LH concentrations reached peak values in the autumn (October), three months before FSH and testosterone concentrations reached peak values in early winter (January). Prolactin concentrations were inversely related (r = −0.733, P < 0.001) to LH and directly related to daylength, and maximal concentrations were observed during mid-summer (July) and minimal concentrations during early winter (January). The temporal pattern of circulating inhibin was positively correlated with FSH (r = 0.88, P < 0.001), but lagged behind the seasonal FSH increase by 1–3 weeks. Antler length, body weight and chest girth were maximal during pre-rut (December–January). Maximal scrotal circumference and combined testes volume were observed in mid-winter (February), whereas peak neck girth and behavioural aggression occurred 1–3 months later (March–May). On the basis of quarterly electroejaculation results, motile spermatozoa were produced in all seasons. However, the greatest number of motile spermatozoa per ejaculate was observed during the winter and spring, whereas the highest incidence of sperm pleiomorphisms (> 80%) was detected in the autumn. Histological assessments of the regressed testis (July) revealed fewer germ cells undergoing spermatogenesis and an increased incidence of degenerating and abnormal cell types. In summary, Eld's deer exhibit a circannual hypothalamic–pituitary–gonadal cycle with onset of pituitary activation occurring during the autumn and winter, whereas gonadal activity peaks during the winter and spring as daylengths are increasing. Marked circannual variations in circulating prolactin suggest that Eld's deer may use photoperiodic cues to modulate seasonal fertility; however, the existence of an endogenous seasonal rhythm operating independently of photoperiod cannot be excluded.