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K. L. Goodrowe, J. G. Howard, and D. E. Wildt

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.

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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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A. M. Donoghue, L. A. Johnston, K. L. Goodrowe, S. J. O'Brien, and D. E. Wildt

Thirty-six domestic cats received 100 iu hCG (i.m.) on day 1, 2 or 3 of a natural, behavioural oestrus. Twenty-two anoestrous cats were injected with 150 iu pregnant mares' serum gonadotrophin (PMSG; i.m.) followed 84 h later by 100 iu hCG. Twenty-four to 26 h after hCG, all cats were examined laparoscopically to determine the number of ovarian follicles and to recover follicular eggs. Mature eggs were cultured with conspecific spermatozoa and examined 30 h later for cleavage. Within the natural oestrus group, cats on day 1 produced fewer (P < 0.05) follicles and total eggs than females on day 2 or 3, and 88.9% of the day 1 eggs were degenerate or immature and unsuitable for in vitro fertilization (IVF). Although only 54.5% of the cats in the PMSG/hCG group exhibited overt oestrus, mean (± sem) numbers of follicles (9.7 ± 0.8) and oocytes recovered (8.7 ± 0.8) were at least twofold greater (P < 0.001) than those measured in the natural oestrus group (3.7 ± 0.6; 3.4 ± 0.6, respectively) or subgroups on day 2 (3.7 ± 0.4; 3.3 ± 0.4) and day 3 (5.7 ± 0.8; 5.3 ± 0.8). Overall, the proportion of eggs cleaving in vitro was similar (P > 0.05) between the natural oestrus group (48.3%) and the PMSG/hCG group (50.9%), but the latter group produced more than twice the number of embryos per donor. Embryo quality was unaffected (P > 0.05) by day of hormone treatment, and more than 80% of all two-cell embryos were rated good-to-excellent quality. In summary, there is a temporal relationship between day of sexual receptivity and follicular egg viability in the domestic cat: eggs on the first day of oestrus are not optimally responsive to an LH-like stimulus. There is also no evidence that PMSG/hCG treatment compromises egg quality or subsequent fertilizability in vitro. On the contrary, use of these gonadotrophins markedly improves overall IVF efficiency by increasing the total number of high quality embryos produced.

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A. M. Miller, M. E. Roelke, K. L. Goodrowe, J. G. Howard, and D. E. Wildt

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

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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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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

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B. C. Buckrell, C. J. Gartley, K. G. Mehren, G. J. Crawshaw, W. H. Johnson, I. K. Barker, J. Balke, C. Coghill, J. R. G. Challis, and K. L. Goodrowe

Summary. Over a 3-year period, 32 Dall's sheep (Ovis dalli dalli) embryos were transferred into 24 domestic sheep (O. aries) recipients and 4 were transferred into 2 Dall's sheep recipients. In the first year, none of the 10 O. aries recipients was diagnosed pregnant. In the following 2 years, 9 (37%) of the domestic sheep recipients were pregnant on Day 18, 8 (33%) on Day 40, 6 (25%) on Day 90 and 4 (16%) on Day 120; 1 aborted at Day 125 and another at Day 145. Pregnancies were established only in ewes that had previously been recipients of Dall's sheep embryos. The 2 remaining pregnant sheep were treated with progesterone from Day 125 until the fetuses were determined to be dead at Day 145. Both of the Dall's sheep recipients (Year 2) established pregnancies; 1 live Dall's sheep lamb was born 174 days after mating. No differences in serum progesterone, oestrone, prostaglandin F-2α metabolites or cortisol concentrations could be detected during pregnancy between recipients carrying Dall's sheep embryos, recipients receiving progesterone treatment or domestic ewes carrying domestic sheep pregnancies. Six fetuses were necropsied (1 at Day 125 and 5 at Day 145–146): all fetuses were premature and had various degrees of hydranencephaly. No significant differences were found when cotyledon numbers were compared among domestic ewes carrying Dall's sheep lambs, Dall's sheep ewes lambing naturally and domestic ewes lambing naturally. These results demonstrate that the transfer of Dall's sheep embryos to domestic ewes results in the establishment but subsequent loss of pregnancy and that these losses occur throughout gestation.

Keywords: sheep; embryo transfer; interspecies; pregnancy failure; superovulation