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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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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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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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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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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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S. L. Monfort, C. Wemmer, T. H. Kepler, M. Bush, J. L. Brown and D. E. Wildt

Summary. Direct radioimmunoassays (RIA) for urinary oestrone conjugates and pregnanediol-3α-glucuronide (PdG) were used to study ovarian activity patterns and pregnancy in Eld's deer. In 2 does, urinary metabolite patterns were compared to temporal patterns of plasma LH, oestradiol-17β and progesterone. Preovulatory LH peaks occurred coincident with behavioural oestrus, and plasma progesterone secretion paralleled PdG excretion. Although plasma oestradiol-17β levels fluctuated between 5 and 10 μg/ml throughout the oestrous cycle, no preovulatory oestrogen surge was observed. Based on PdG excretion, non-conception oestrous cycles averaged 21·5 ± 2·1 days (±s.e.m., n = 65); however, 2 of 13 does exhibited prolonged oestrous cycles (30·1 ± 4·4 days; range 14–62 days, n = 14) characterized by sustained PdG excretion. Excluding these 2 females, the mean oestrous cycle was 18·5 ± 0·3 days (range 14·23 days, n = 51). Behavioural oestrus (12·24 h duration) was observed in 42 of 65 cycles (64·6%), and always corresponded with intercyclic troughs in PdG excretion (2·5 days duration). Mean gestation duration (n = 10) was 33·5 ± 0·4 weeks. PdG concentrations increased (P < 0·05) by Week –32 (3rd week of gestation), plateaued between Weeks −31 and −25, increased (P < 0·05) markedly by Week –22 and then rose steadily until parturition, declining (P < 0·05) rapidly thereafter. Mean excretion of oestrone conjugates remained low until Week −30, increased (P < 0·05) steadily to Week −24 (P < 0·05) and then returned to baseline by Week −17. Increased (P < 0·05) oestrone conjugates concentrations were detected again by Week −4 followed by a rapid increase to peak pregnancy levels by Week −1, declining (P < 0·05) precipitously after parturition. The results confirm that the Eld's deer is seasonally polyoestrous with onset (January–March) and cessation (August–October) of regular, cyclic ovarian activity coinciding with increasing and decreasing daylengths, respectively. Urinary PdG excretion accurately reflects cyclic ovarian activity and markedly elevated concentrations of this metabolite provide an accurate index of pregnancy. The simultaneous monitoring of oestrone conjugates appears useful for estimating the stage of pregnancy and predicting parturition onset.

Keywords: Eld's deer; oestrogen; progesterone; urinary steroids; LH; seasonality

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J. L. Brown, D. E. Wildt, L. G. Phillips, J. Seidensticker, S. B. U. Fernando, S. Miththapala and K. L. Goodrowe

Summary. In Study 1, semen was collected using a standardized electroejaculation procedure. Males (N = 8) produced ejaculates with a high incidence of sperm abnormalities (77 ± 3·3%). After electroejaculation under anaesthesia, serum cortisol concentrations increased (P < 0·05), while testosterone concentrations decreased (P < 0·05) and LH and FSH concentrations were unchanged (P > 0·05) over a 2-h bleeding period. In Study 2, male and female leopards were bled at 5-min intervals for 3 h and given (i.v.): (1) saline (N = 2/sex); (2) GnRH (1 μg/kg body weight) 30 min after the onset of sampling (N = 5/sex); or (3) ACTH (250 μg) at 30 min followed by GnRH 1 h later (N = 5/sex). Basal concentrations of serum LH, FSH and cortisol were comparable (P > 0·05) between male and female leopards. After GnRH, peak LH concentrations were 2-fold greater (P < 0·05) in males than females while FSH responses were similar. In males, testosterone concentrations increased 2–3-fold following GnRH. After ACTH, serum cortisol concentrations doubled within 15 min in both sexes. Administration of ACTH 1 h before GnRH did not affect GnRH-induced LH or FSH release (P > 0·05); however, testosterone secretion was only 30% of that observed after GnRH alone (P < 0·05). We conclude that (1) the high incidence of sperm abnormalities in the leopards of Sri Lanka may be related to parallel findings of genetic homozygosity; and (2) decreases in basal and GnRH-stimulated testosterone secretion were related to increases in serum cortisol after electroejaculation or ACTH and were not associated with changes in pituitary gonadotrophin secretion.

Keywords: leopard; spermatozoa; GnRH; ACTH; LH; FSH; cortisol; testosterone