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  • Author: S. J. O'Brien x
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L. A. Johnston, A. M. Donoghue, S. J. O' Brien and D. E. Wildt

Summary. The influence of culture temperature and gas atmosphere on in-vitro fertilization and embryo development was examined in the domestic cat. In Exp. 1, eggs were fertilized and cultured in 5% CO2 in air at 37, 38 or 39°C. Experiment 2 evaluated the effects of 5% CO2 in air; 5% CO2, 5% O2 and 90% N2; and 10% CO2 in air. Fertilization (cleavage) and development to the morula/blastocyst stage were not influenced (P > 0·05) by variations in temperature and gas composition. Despite changing these culture conditions, egg cleavage averaged ∼75% and >80% of the 2-cell embryos proceeded to morulae in vitro. However, the partial in-vitro morula-to-blastocyst developmental block normally observed in this species was not removed.

Keywords: cat; fertilization; embryo; in-vitro culture; incubation; temperature; gas atmosphere

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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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D. E. Wildt, M. Bush, S. J. O'Brien, N. D. Murray, A. Taylor and J. A. Marshall Graves

Summary. Spermic electroejaculates (range in motile sperm/ejaculate, 0·50–122·9 × 106; mean ± s.e.m., 38·6 ± 4·9) were recovered from 47 of 48 adult koalas captured from 3 wild populations in Australia. Semen was characterized by (i) a high density of globular bodies, which prevented the estimation of sperm motility without dilution; (ii) a brownish colour; and (iii) an acidic pH. Spermatozoa were categorized on the basis of 10 head forms, most cells being a curved or hooked shape. The koala populations differed in sperm concentration and motility ratings, but not in testes size, testosterone production or proportions of spermatozoa with various head shapes. These data confirm that free-living koalas normally produce spermatozoa with a high incidence of structural heterogeneity almost solely confined to the head region; and demonstrate the utility and safety of conventional gamete and endocrine studies, approaches which will be useful for determining the impact of genetic isolation and venereal disease on species fertility.

Keywords: koala; marsupial; semen; electroejaculation; spermatozoa; testis; testosterone

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F K Hollinshead, G Evans, K M Evans, S L Catt, W M C Maxwell and J K O'Brien

The characteristics and functional capacity of ram spermatozoa frozen–thawed prior to and after flow cytometric sorting was assessed after incubation (37 °C; 6 h), in vitro fertilisation (IVF), and transfer of fresh and vitrified in vitro produced embryos. Frozen-thawed spermatozoa from two rams were allocated to four treatment groups: (i) non-sorted (Control); (ii) sorted (FS); (iii) sorted then re-frozen (FSF) and (iv) re-frozen control (FCF). Frozen-thawed samples were separated into X- and Y-chromosome bearing spermatozoa using a high-speed sperm sorter after density gradient centrifugation (X: 88 ± 1.5% and Y: 87 ± 1.1% purity). After 6 h incubation (37 °C), the percentage of motile spermatozoa was higher (P < 0.001) for FS (84 ± 2.0%) compared with all other treatments (Control: 36 ± 3.3%, FSF: 28 ± 3.1%, FCF: 20 ± 2.0%). In a sperm migration test greater numbers of FS spermatozoa penetrated 5 mm into the artificial cervical mucus compared with spermatozoa from all other treatments (152 ± 39.4 vs 31 ± 9.2 spermatozoa respectively; P < 0.05). Fertilisation and cleavage rates were higher (P < 0.05) for in vitro matured oocytes inseminated with Control compared with FSF spermatozoa. However, the Day 7 blastocyst development rate was higher for oocytes inseminated with FSF (62.2%) than FS and Control spermatozoa (52.7 and 50.0%; P < 0.05). The number of ewes pregnant (Day 60), lambing and the in vivo embryo survival rate was greater (P < 0.01) after the transfer of fresh embryos rather than vitrified embryos derived from X- and Y-spermatozoa (67.6, 64.7 and 41.2% vs 29.6, 25.9 and 14.8% respectively). Twenty-six of the 30 (86.7%) lambs derived from sex-sorted spermatozoa were of the correct sex. These results demonstrate that frozen–thawed ram spermatozoa can be sex-sorted for immediate or future use after re-cryopreservation and, in conjunction with IVF and embryo transfer, can be used to efficiently produce offspring of pre-determined sex.

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J. L. Brown, M. Bush, C. Packer, A. E. Pusey, S. L. Monfort, S. J. O'Brien, D. L. Janssen and D. E Wildt

Pituitary responses to gonadotrophin-releasing hormone (GnRH) and prolactin and steroid secretory profiles were examined in two populations of adult, female lions in the Serengeti (one outbred in the Serengeti Plains and one inbred in the Ngorongoro Crater) to determine whether reductions in genetic variability adversely affected endocrine function. GnRH-induced gonadotrophin secretion was also examined after adrenocorticotrophic hormone (ACTH) treatment to determine whether acute increases in serum cortisol altered pituitary function. Anaesthetized lions were administered (i) saline i.v. after 10 and 100 min of blood sampling; (ii) saline at 10 min and GnRH (1 μg kg−1 body weight) after 100 min; or (iii) ACTH (3 μg kg−1) at 10 min and GnRH after 100 min of sampling. Basal serum cortisol and basal and GnRH-induced gonadotrophin secretion were similar (P > 0.05) between females of the Ngorongoro Crater and Serengeti Plains. After ACTH, serum cortisol increased two-to threefold over baseline values and the response was unaffected (P > 0.05) by location. ACTH-induced increases in serum cortisol had no effect on subsequent basal or GnRH-stimulated luteinizing hormone (LH) or follicle-stimulating hormone (FSH) secretion. Overall mean serum progesterone concentrations ranged from 0.2 to 5.4 ng ml−1 with the exception of four females (two in the Serengeti and two in the Crater; progesterone range, 18.4–46.5 ng ml−1) that were presumed pregnant (three of these females were observed nursing cubs several weeks later). There were no differences (P > 0.05) between Serengeti and Crater lions in mean serum progesterone, oestradiol or prolactin concentrations, and hormone secretion was not influenced (P > 0.05) by GnRH or ACTH treatment. Although Ngorongoro Crater lions have decreased genetic variability, the reproductive–endocrine system of females appears functionally normal compared with outbred counterparts living in the Serengeti Plains. Furthermore, the acute rise in serum cortisol after ACTH administration in lions fails to alter subsequent GnRH-induced gonadotrophin release, suggesting that short-term changes in adrenal activity do not markedly affect pituitary responsiveness in this species.

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J. L. Brown, M. Bush, C. Packer, A. E. Pusey, S. L. Monfort, S. J. O'Brien, D. L. Janssen and D. E. Wildt

Summary. Pituitary–gonadal function was examined in male lions free-ranging in the Serengeti Plains or geographically isolated in the Ngorongoro Crater of Tanzania. Lions were classified by age as adult (6·1–9·8 years), young adult (3·3–4·5 years) or prepubertal (1·4–1·6 years, Serengeti Plains only). Each animal was anaesthetized and then bled at 5-min intervals for 100 min before and 140 min after i.v. administration of saline or GnRH (1 μg/kg body weight). Basal serum LH and FSH concentrations were similar (P > 0·05) among age classes and between locations. In Serengeti Plains lions, net LH peak concentrations after GnRH were ∼ 25% greater (P < 0·05) in prepubertal than in either adult or young adult animals. GnRH-stimulated LH release was similar (P > 0·05) between adult and young adult lions, and these responses were similar (P > 0·05) to those measured in Ngorongoro Crater lions. Basal and GnRH-stimulated testosterone secretion was higher (P < 0·05) in adult than in young adult lions and lowest (P < 0·05) in prepubertal lions. Age-class differences in testosterone production were related directly to the concentrations of LH receptors in the testis (P < 0·05). Basal and GnRH-stimulated testosterone secretion and gonadotrophin receptor concentrations within age classes were similar (P > 0·05) between lions of the Serengeti Plains and Ngorongoro Crater. Lower motility and higher percentages of structurally abnormal spermatozoa were observed in electroejaculates of young adult compared to adult Serengeti Plains males (P < 0·05) and were associated with decreased steroidogenic activity. In contrast, there were no age-related differences in ejaculate characteristics of Ngorongoro Crater lions. Seminal quality in the Crater population was poor in adult and young adult animals and was unrelated to alterations in pituitary or testicular function. In summary, only seminal quality in adult male lions was affected by location, whereas age significantly affected both basal and GnRH-stimulated testosterone secretion and seminal quality (Serengeti Plains only) in sexually mature males. The striking seminal/endocrine differences among pride (breeding) males of different ages raises questions about the impact of age on individual reproductive performance in this species.

Keywords: lion; GnRH; LH; testosterone; receptors; testis; spermatozoa

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T R Robeck, K J Steinman, M Greenwell, K Ramirez, W Van Bonn, M Yoshioka, E Katsumata, L Dalton, S Osborn and J K O'Brien

The reproductive physiology of the Pacific white-sided dolphin, Lagenorhynchus obliquidens, was characterized to facilitate the development of artificial insemination (AI) using cryopreserved spermatozoa. Specific objectives were to: 1) describe reproductive seasonality of the Pacific white sided dolphins; 2) describe urinary LH and ovarian steroid metabolites during the estrous cycle; 3) correlate LH and ovarian steroidal metabolite patterns to ultrasound-monitored follicular growth and ovulation; and 4) assess the efficacy of synchronizing estrus, sperm collection/cryopreservation, and intrauterine insemination. Ovulations (64%, n=37) and conceptions (83%, n=18) occurred from August to October. Peak mean serum testosterone (24 ng/ml), cross-sectional testicular area (41.6 cm2), and sperm concentration (144.3×107 sperm/ml) occurred in July, August, and September respectively. Spermatozoa were only found in ejaculates from July to October. Estrous cycles (n=22) were 31 d long and were comprised of a 10 d follicular and 21 d luteal phase. Ovulation occurred 31.2 h after the onset of the LH surge and 19.3 h after the LH peak. Follicular diameter and circumference within 12 h of ovulation were 1.52 and 4.66 cm respectively. Estrus synchronization attempts with altrenogest resulted in 17 (22%) ovulatory cycles with ovulation occurring 21 d post-altrenogest. Ten AI attempts using cryopreserved semen resulted in five pregnancies (50%). The mean gestation length was 356 days (range 348–367). These data provide new information on the Pacific white-sided dolphin's reproductive physiology and collectively enabled the first application of AI in this species.