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R. J. ABLIN, P. GUINAN and I. M. BUSH

Summary.

Inoculation of female rabbits with `coagulo-prostatic fluid' (CPF), a secretory-specific autoantigen of male accessory sexual glands, permitted the identification by the methods of gel precipitation and passive haemagglutination of tissue- and species-specific antibodies to CPF. Subsequent mating of females with high titres of circulating antibodies to CPF at the time of coitus resulted in impairment of reproduction in three of four animals studied.

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JoGayle Howard, M. Bush, V. de Vos and D. E. Wildt

Summary. A regimented electroejaculation protocol (120 electrical stimulations; 10-30 V) was used to collect semen and characterize ejaculate quality from 9 adult, free-ranging African elephants under anaesthesia. Eight of the 9 ejaculates contained high concentrations of progressively motile spermatozoa. The overall mean ejaculate volume, sperm concentration/ml ejaculate, sperm motility, sperm status and ejaculate pH were 93·3 ml, 2408·6 × 106 spermatozoa/ml, 70%, 3·9 and 7·4, respectively. A high percentage (mean 77·5%) of spermatozoa within each ejaculate was morphologically normal. Of the aberrant spermatozoa, 72% had a cytoplasmic droplet defect. When sperm viability was tested in vitro at 37°C, sperm motility rating declined by at least half of the initial assessment within 3·5 h of semen collection. Generally, spermatozoa maintained motility in vitro for < 6 h. Serum testosterone ranged from 1·4 to 8·2 ng/ml in 4 males evaluated in the morning (07:30–08:00 h). In 4 of the 5 bulls assessed in the afternoon (15:00–18:00 h), testosterone levels were <0·9 ng/ml. The remaining bull, evaluated at 16:00 h, had exceptionally high testosterone concentrations (peak 25·6 ng/ml) and a preputial discharge potentially indicative of 'musth'. The present study demonstrates that high quality semen can be collected consistently from the African elephant and that striking differences exist in serum testosterone amongst free-ranging males which may be due, in part, to a diurnal rhythm.

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M. Chaudhuri, D. G. Kleiman, D. E. Wildt, M. Bush, E. S. Frank and R. B. Thau

Summary. Urinary concentrations of conjugated oestrone and pregnanediol-3-glucuronide were measured during and after spontaneous and induced oestrus and during pregnancy. Behavioural oestrus was preceded by a rise in oestrone values from < 10 ng/mg creatinine (Cr) to peaks of 45 ng/mg Cr. Maximal lordotic response and mating activity coincided with the decline in oestrone levels. After presumed ovulation, urinary pregnanediol glucuronide concentrations increased from <5 to 15–30 ng/mg Cr. Further increases in this steroid (to 60–80 ng/mg Cr) occurred 114 days after mating, presumably coincident with implantation. These high levels of pregnanediol glucuronide were maintained for 3 weeks, began to decline 1 week before parturition and fell to a nadir (<5 ng/mg Cr) immediately after delivery. When FSH was administered i.m. for 5 days, urinary oestrone values rose markedly and were maximal (580 ng/mg Cr) on Day 7. Mating first occurred on Day 20 and 500 i.u. hCG were given i.m. Urinary pregnanediol glucuronide levels during the next 5 months were similar to those in the previous year during pregnancy with values rising 105–108 days after mating. However, no birth occurred. These results support the suggestion that pandas exhibit delayed implantation and demonstrate that the panda is responsive to exogenous gonadotrophins.

Keywords: giant panda; reproductive cycle; ovulation induction; oestrone; pregnanediol

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S. L. Monfort, K. D. Dahl, N. M. Czekala, L. Stevens, M. Bush and D. E. Wildt

Summary. Urinary excretion of oestrone conjugates, pregnanediol-3α-glucuronide (PdG) and 20α-hydroxypregn-4-en-3-one were measured from 8 weeks before oestrus to 2 weeks post partum and bioactive FSH was monitored during the periovulatory interval in a female giant panda. A biphasic urinary bioactive FSH excretory profile appeared to indicate a broad (∼ 10 day) follicular phase followed by a sharp preovulatory bioactive FSH surge coincident with an acute increase in urinary oestrone conjugates and behavioural oestrus. Weekly concentrations of urinary oestrone conjugates and PdG increased (P < 0·001) by Week 9 of gestation with 20α-hydroxypregn-4-en-3-one levels increasing 10–30-fold (P < 0·001) between Weeks 11 and 14. These observations indicate that the monoestrous giant panda does not appear to require a prolonged period of endogenous FSH release or multiple FSH peaks for ovarian priming and follicle selection to proceed normally. Furthermore, the delayed rise in urinary steroid excretion during the second half of gestation in the giant panda supports the concept of delayed implantation while the estimation of steroid conjugates in urine offers a non-invasive approach for monitoring pregnancy status in this endangered species.

Keywords: giant panda; oestrogen; progesterone; FSH; delayed implantation

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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. G. Howard, M. Bush, C. Morton, F. Morton, K. Wentzel and D. E. Wildt

Summary. A study was conducted to determine an optimum technique for semen cryopreservation and the biological competence of frozen–thawed ferret spermatozoa. Fifty-two fresh electroejaculates from 4 males were evaluated for sperm percentage motility, forward progressive motility, motility index (SMI) and acrosomal integrity. To determine the optimum temperature for maintaining sperm motility in vitro and the influence of glycerol on sperm motility, seminal aliquants were diluted in TEST diluent (containing either 0 or 4% glycerol) and maintained at 25° or 37°C. For cryopreservation, semen was diluted in each of 3 cryodiluents (TEST, PDV, BF5F), cooled for 30 min at 5°C and pelleted on solid CO2 or frozen in 0·25 ml straws (20°C/min to −100°C). Following thawing, SMI and acrosomal integrity were determined. Ten females with maximum vulval swelling were given 90 i.u. human chorionic gonadotrophin and laparoscopically inseminated in utero with spermatozoa previously frozen using the optimum diluent and freeze–thaw method. The maintenance temperature of 25°C was superior (P < 0·05) to 37°C for sustaining sperm motility, and glycerol did not influence (P > 0·05) motility for up to 11 h of culture. After thawing, motile spermatozoa were recovered in all treatment groups, but sperm motility and normal acrosomal ratings were highest using the PDV diluent, the pelleting method and thawing at 37°C (P < 0·05). Seven of the 10 ferrets (70%) inseminated with spermatozoa frozen by this approach became pregnant and produced 31 kits (mean litter size 4·4; range 1–9 kits). These results illustrate the sensitivity of ferret sperm motility and acrosomal integrity to different cryopreservation conditions; and demonstrate the biological competence of frozen–thawed ferret spermatozoa.

Keywords: ferret; sperm; semen cryopreservation; artificial insemination

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D. E. Wildt, M. Bush, C. Morton, F. Morton and J. G. Howard

Summary. Five domestic ferrets previously maintained for 12 weeks under a 16L:8D photoperiod were electroejaculated weekly for 15–65 weeks while continuing to be exposed to the prolonged light cycle. Two ferrets sustained spermatogenesis for 20 and 26 weeks, while sperm production in the remaining males either was sporadic or decreased, remained depressed and then increased to peak levels observed in other males. Regardless of the temporal spermatogenesis patterns within males, the number of electroejaculated spermatozoa with residual cytoplasmic droplets or abnormal acrosomes increased in all ferrets over time. Diluted ejaculates meeting artificial insemination criteria were deposited intravaginally or by transabdominal laparoscopy into the uterine horns of females treated 0 or 24 h earlier with 90 i.u. hCG. Vaginal insemination was ineffective (0 pregnancies in 10 attempts), but 17/24 ferrets (70·8%) inseminated laparoscopically became pregnant and delivered live young (mean litter size, 5·2 kits). Number of motile spermatozoa deposited in utero (1·6–10·0 × 106 cells), presence of glycerol in the sperm dilution medium (0 versus 4%) and time of hCG administration (0 versus 24 h before insemination) had no effect on pregnancy results or litter size.

Keywords: ferret; semen; electroejaculation; spermatozoa; artificial insemination; hCG

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C. C. Platz Jr, D. E. Wildt, J. G. Howard and M. Bush

Summary. Semen was collected by a standardized electroejaculation procedure from a giant panda on 4 occasions. Ejaculate volume, sperm count and % sperm motility were 2·3–3·6 ml, 62–562 × 106 spermatozoa/ml and 45–85% respectively. The results, although limited to a single male, suggested a seasonal influence on ejaculate and gonadal parameters with improved ejaculate volume, sperm motility and increased testicular size in the season proximate to the female's oestrous period. Frozen–thawed spermatozoa were motile with no apparent abnormalities induced by the freezing procedure.

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L. Munson, J. L. Brown, M. Bush, C. Packer, D. Janssen, S. M. Reiziss and D. E. Wildt

Reduced genetic variability is known to adversely affect ejaculate quality in inbred lions (Panthera leo) physically isolated in the Ngorongoro Crater compared with outbred lions inhabiting the adjacent Serengeti Plains in East Africa. This study compared the histomorphology of testicular biopsies from these two lion populations. Ngorongoro Crater lions had fewer (P <0.05) seminiferous tubules with spermiogenesis and fewer (P <0.05) spermatids per seminiferous tubular cross-section than Serengeti Plains lions, although seminiferous tubular diameter did not differ (P > 0.05) between populations. Interstitial areas were greater (P <0.05) in Crater than in Plains lions, but no qualitative differences were evident, suggesting that proportionately less testicular area was occupied by seminiferous tubules in Crater lions. None of the lions in either population had evidence of testicular degeneration. Overall results suggest that inbred Crater lions have reduced spermiogenesis and less total seminiferous tubular area per testis. These data further support the premise that genetic homogeneity compromises reproductive traits in free-living, male African

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J. G. Howard, M. Bush, V. de Vos, M. C. Schiewe, V. G. Pursel and D. E. Wildt

Summary. Electroejaculates from free-ranging, African elephants were frozen to test various seminal diluents, freezing methods and thawing media on post-thaw sperm viability and structural integrity. In Study I, each ejaculate was tested with each of 7 cryoprotective diluents. After cooling to 5°C and equilibration on ice (4°C) for 120 min, each aliquant was pellet frozen on solid CO2, stored in liquid nitrogen and thawed (37°C) in saline or tissue culture solution. Amongst all diluents, post-thaw sperm motility, motility duration in vitro (37°C) and acrosomal integrity were greatest (P < 0·05) when diluent BF5F was used. Thawing medium had no effect on results. In Study II, the optimal diluent from Study I (BF5F) was compared with the diluent SGI. Results were not affected by a 90- or a 150-min cooling–equilibration interval in an electronic cooler (5°C); however, post-thaw sperm motility rating and duration of motility in vitro were greater (P <0·01) with the pellet than the straw container freezing method. When the pelleting method was used, diluents BF5F and SGI provided comparable cryoprotection. Duration of post-thaw motility was enhanced 2-fold and up to 12h by maintaining thawed semen at 21 rather than 37°C (P <0·05) All diluents provided some protection on acrosomal integrity, but the overall proportion of intact acrosomes after thawing was markedly less in Study II, apparently as a result of the slower initial cooling rate (∼1·5°C/min) compared to that of Study I (∼6·5°C/min). This study demonstrates the feasibility of cryopreserving semen from free-ranging African elephants and indicates that spermatozoa must effectively survive freezing when the BF5F or SGI diluent is used in conjunction with the pelleting method.