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Summary. Domestic cats experiencing a natural or FSH-induced oestrus were studied. Mated cats produced fewer (P < 0·01) unfertilized oocytes and more (P < 0·01) morulato blastocyst-stage embryos of better quality after a natural oestrus than after FSH treatment. Serum oestradiol- 17β concentrations were lower (P < 0·05) and progesterone levels rose earlier (P < 0·05) in the induced oestrus compared to the natural oestrus group. Morula/blastocyst-stage embryos from both groups transferred to 15 FSH/hCG-treated recipients produced 3 pregnancies and 2 live-born litters (1 from a natural oestrus donor and 1 from an FSH-treated donor). These results indicate that fertilization rates and embryo quality in domestic cats appear to be compromised by the FSH treatment, probably because of altered oestradiol-17β and progesterone concentrations.

Keywords: domestic cat; embryo recovery; oestrus; FSH; oestradiol-17β; progesterone.

Electroejaculates from eight snow leopards were used to determine how the motility of spermatozoa was influenced by (i) type of media (Ham's F10, PBS, human tubal fluid or RPMI-1640); (ii) holding temperature (23°C versus 37°C); (iii) washing of spermatozoa and (iv) a sperm metabolic enhancer, pentoxifylline. The duration of sperm motility was assessed by evaluating samples in each treatment every hour for 6 h and a sperm motility index (a value combining percentage sperm motility and rate of forward progression) calculated. Spermatozoa from the Ham's F10, PBS and PBS plus pentoxifylline treatments were also co-incubated with zona-intact, domestic cat eggs that were fixed and evaluated for spermatozoa bound to the zona pellucida, penetrating the outer and inner layers of the zona pellucida and within the perivitelline space. During the 6 h co-incubation, the sperm motility index in PBS with pentoxifylline was greater (P < 0.05) than in PBS alone which, in turn, was greater (P < 0.05) than in the other three test media. Washing the spermatozoa enhanced (P < 0.05) motility in both PBS and PBS plus pentoxifylline relative to unwashed samples, but there was no effect (P > 0.05) of holding temperature. Pentoxifylline supplementation enhanced (P < 0.05) the proportion of cat eggs with bound, but not penetrated, snow leopard spermatozoa. Only one to three of the 120 total cat eggs/treatment group had snow leopard spermatozoa in the inner layer of the zona pellucida, and there were no spermatozoa in the perivitelline space. Failure of sperm penetration did not appear related to an inadequacy in the culture system because nine of 27 snow leopard eggs co-incubated in PBS with conspecific spermatozoa cleaved in vitro and nine of the 18 unfertilized eggs contained spermatozoa in the perivitelline space. The snow leopard appears unique among felid species in that (i) sperm viability is highly sensitive to type of culture medium, (ii) sperm motility and function in vitro appear enhanced by a simple rather than complex culture medium and (iii) there is a mechanism that prevents these spermatozoa from fully penetrating heterologous, salt-stored, domestic cat eggs.

Summary. Laparoscopic intrauterine artificial insemination (AI) of electroejaculated spermatozoa was used to compare embryo development and conception rates in domestic cats inseminated either before or after ovulation. Females were given a single (100 iu) injection of pregnant mares' serum gonadotrophin (PMSG) followed by either 75 or 100 iu human chorionic gonadotrophin (hCG) 80 h later. Cats were anaesthetized (injectable ketamine HCl/acepromazine plus gaseous halothane) 25–50 h after administration of hCG for laparoscopic assessment of ovarian activity and for transabdominal AI into the proximal aspect of the uterine lumen. At the time of AI, 23 cats were pre-ovulatory (25–33 h after hCG injection) and 30 were post-ovulatory (31–50 h after hCG injection). Pre-ovulatory females produced 10·5 ± 1·1 follicles and no corpora lutea compared with 1·9 ± 0·5 follicles and 7·5 ± 0·9 corpora lutea for the post-ovulatory group (P < 0·05). Six days later, the ovaries of nine pre-ovulatory and 12 post-ovulatory females were re-examined and the reproductive tracts flushed. On this day, pre-ovulatory cats produced fewer corpora lutea (2·8 ± 1·5; P < 0·05) and embryos (0·4 ± 0·3; P < 0·05) than post-ovulatory females (18·9 ± 3·3 corpora lutea; 4·6 ± 1·2 embryos). Two of the 14 cats (14·3%) inseminated before ovulation and not flushed became pregnant compared with 9 of 18 cats (50·0%) inseminated after ovulation and up to 41 h after hCG injection (P < 0·05). These results indicate that ovulation in cats is compromised by pre-ovulatory ketamine HCl/acepromazine/halothane or laparoscopy or by both and that electroejaculated spermatozoa deposited by laparoscopy in utero, after ovulation, result in a relatively high incidence of pregnancy. Because ovulation usually occurs 25–27 h after injection of hCG, the lifespan for fertilization of the ovulated ovum appears to be at least 14 h in vivo in cats.

Keywords: ovulation; anaesthesia; artificial insemination; laparoscopy; cat

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

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.

Summary. Eight female pumas were treated i.m. with 1000 (N = 5) or 2000 (N = 3) i.u. PMSG followed 84 h later by 800 i.u. hCG. Eggs were recovered 24–26 h after hCG from ovarian follicles by using laparoscopy and transabdominal aspiration. Mature eggs were inseminated in vitro 4–6 h later whereas immature eggs were cultured for 24 h and then inseminated. Electroejaculates from 3 pumas were diluted with mKRB before insemination to evaluate the influence of sperm concentration on fertilization. Seven of 8 pumas responded with follicle development, and 140 eggs were recovered from 145 follicles (96·6%; 77 mature, 43 immature, 20 degenerate eggs; mean ± s.e.m., 20·0 ± 5·9 eggs/female). Overall fertilization rate was 43·5% (total eggs fertilized = 40) despite using inseminates containing 82·99% pleiomorphic spermatozoa. Of the 36 immature oocytes matured in vitro and inseminated, 12 were fertilized even though 50% of the inseminating spermatozoa contained an acrosomal defect. Fertilization rate of mature oocytes collected from follicles appeared unrelated (P > 0·05) to PMSG dose or number of spermatozoa/inseminate. This study demonstrates that a high proportion of follicular eggs can be recovered laparoscopically from adult pumas treated with PMSG and hCG. These gametes are capable of being fertilized in vitro (immediately or after maturation in vitro) even with low quality semen with a high incidence of sperm pleiomorphisms.

Keywords: in vitro fertilization; puma; oocyte maturation; teratospermia

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

Summary. Pituitary, gonadal and adrenal activity were compared in free-living, adult African buffalo bulls during the breeding and nonbreeding seasons. Frequent blood samples were collected for 2 h from anaesthetized bulls treated intravenously with saline, gonadotrophin-releasing hormone (GnRH, 200 μg), human chorionic gonadotrophin (hCG, 10 000 i.u.) or adrenocorticotrophic hormone (ACTH, 1·5 mg). Electroejaculates also were collected from anaesthetized bulls during the breeding and nonbreeding seasons. Pretreatment testosterone concentrations among bulls varied more during the breeding (0·17–23·0 ng/ml) than the nonbreeding (0·15–2·21 ng/ml) season. The variation within the breeding season was attributed to 8 of 25 bulls producing higher (P < 0·05) serum testosterone (High-T; 16·28 ± 2·03 ng/ml) and testicular LH receptor (1·53 ± 0·22 fmol/mg testis) concentrations compared with their seasonal counterparts (Low-T; 0·95 ± 0·26 ng/ml; 0·38 ± 0·04 fmol/mg) or with all bulls during the nonbreeding season (0·90 ± 0·27 ng/ml; 0·31 ± 0·04 fmol/mg). The magnitude of GnRH- and hCG-induced increases in serum testosterone was similar (P > 0·05) between Low-T bulls and bulls during the nonbreeding season. In the High-T animals treated with GnRH or hCG, serum testosterone did not increase, suggesting that secretion was already maximal. Peak serum LH concentrations after GnRH were greater (P < 0·05) in bulls during the nonbreeding than the breeding season; FSH responses were similar (P > 0·05). ACTH treatment did not increase serum cortisol concentrations above the 2-fold increase measured in bulls treated with saline, hCG and GnRH (P > 0·05). Ejaculate volume, sperm motility and the proportion of morphologically normal spermatozoa were greater (P < 0·05) during the breeding than in the nonbreeding season, but the total number of spermatozoa/ejaculate was similar (P > 0·05). We suggest that season effects endocrine function and seminal quality in free-living, male African buffalo. During the breeding season some, but not all, adult bulls produce high circulating concentrations of testosterone associated with increased testicular LH receptor binding. These findings suggest that this species may experience selective gonadal suppression, perhaps related to differences in social status.

Keywords: buffalo; LH; FSH; testosterone; testis; gonadotrophin receptors; seasonality; spermatozoa

Summary. Blood, testicular biopsies and electroejaculates were collected from adult male impala, free-ranging in the Kruger National Park (Republic of South Africa), during the breeding (rut; April–May) and nonbreeding (September–October) seasons. Blood samples were collected at 5-min intervals for 120 min from anaesthetized males (n = 7 impala/group) treated intravenously with saline, gonadotrophin-releasing hormone (GnRH: 1 μg/kg body weight) or human chorionic gonadotrophin (hCG: 10 or 30 iu/kg). Semen was collected from six more animals during the breeding season and 12 animals during the nonbreeding season using a standardized electroejaculation protocol. Ejaculates obtained during the nonbreeding season were of inferior quality to those collected during the breeding season, and were characterized by lower sperm concentrations, poorer sperm motility and more morphologically abnormal sperm forms. Within season, there were no differences in testosterone secretion between the two hCG doses, and these responses were similar to those observed after GnRH, but during the rut, testosterone secretion stimulated by both GnRH and hCG was approximately nine times greater than during the nonbreeding season. This seasonal increase in testosterone production was associated with a doubling in testicular volume and concentrations of luteinizing hormone (LH) receptors. Although concentrations of testicular follicle-stimulating hormone (FSH) receptors were similar between seasons, receptor content increased during rut as a result of increased testicular volume. In contrast to testosterone secretion, basal LH and FSH secretions were unaffected by season and GnRH-induced gonadotrophin secretion was reduced during rut. These data indicate that: (i) seminal quality in free-ranging impala undergoes seasonal changes coincident with alterations in testicular steroidogenic activity, and (ii) the increased testosterone secretion observed during rut is associated with increased testicular sensitivity to LH (via increased gonadotrophin receptors) rather than to increased circulating gonadotrophin concentrations or pituitary responsiveness to GnRH.

Keywords: impala; LH; FSH; testosterone; testis; receptors

We tested the ability of several GnRH analogues to suppress pituitary–testicular activity and potentially musth in free-ranging African elephants (Loxodonta africana). In Study 1, adult bulls were given 4 or 12 mg GnRH antagonist (Detirelix) or saline i.m. on day 0 (n = 3 bulls per treatment). Animals were then recaptured on day 2 (about 48 h later) and given 300 μg GnRH i.v. to assess the ability of the antagonist to block pituitary activity. Detirelix reduced (P < 0.05) basal concentrations of serum LH and testosterone on day 2 compared with day 0, with no effect of dose. Similarly, LH and testosterone release induced by GnRH were also reduced (P < 0.05) in the Detirelix-treated bulls (50–70% reduction in peak concentrations). In Study 2, elephants were given 30 mg of a structurally similar GnRH antagonist (103-201-40; n = 6), 22.5 mg of a long-acting GnRH agonist (Lupron Depot; n = 4) or D-mannitol carrier (n = 4) i.m. on day 0. All bulls were recaptured and given GnRH on day 2 (103-201-40 treatment) or on days 2 and 20 (Lupron Depot treatment) after the initial injection. In contrast to Detirelix, 103-201-40 did not inhibit basal or GnRH-induced LH or testosterone secretion. Pituitary–testicular responses to Lupron Depot were initially stimulatory, as evidenced by increased (P < 0.05) LH and testosterone secretion on days 0 and 2. By day 20, basal LH concentrations had returned to baseline values and the response to GnRH was markedly reduced (P < 0.05), indicating that the pituitary was at least partially desensitized. Basal testosterone concentrations had also returned to baseline values by day 20 after Lupron Depot treatment. However, despite the attenuated LH response to GnRH, subsequent testosterone secretion was increased (P < 0.05) compared with controls, suggesting the testes of agonist-treated bulls had, instead, become hyper-responsive to small increases in LH secretion. These results suggest that GnRH analogues can suppress the pituitary–gonadal axis in African elephants; however, longer treatment periods, more frequent injection intervals or higher doses are probably needed to inhibit testosterone secretion completely and, thus, musth.