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A. M. Donoghue, M. R. Bakst, D. R. Holsberger, and D. J. Donoghue

A progressive decline in fertility over the course of egg production may be observed when turkey hens are inseminated weekly with semen stored for 24 h. In vitro storage of spermatozoa before insemination results in lower fertilization, possibly because fewer spermatozoa survive selection and storage in the hen's sperm storage tubules in vivo; alternatively, stored spermatozoa may be as capable of reaching the egg as are fresh spermatozoa, but unable to penetrate and fertilize the egg normally. The objective of this study was to determine whether this decline in fertility is a result of fewer spermatozoa reaching the egg after insemination with spermatozoa stored in vitro. Hens were inseminated weekly over the first 12 weeks of egg production with either fresh semen (n = 30 hens) or semen stored for 24 h (n = 30 hens). A total of 301 eggs was evaluated by determining the density distribution of spermatozoa embedded in the outer perivitelline layer. For the 12 weeks of egg production, the fertility of hens inseminated with fresh semen remained greater than 94%. Conversely, the percentage fertility of eggs from hens inseminated with stored semen in weeks 1–3 was greater than 94% but thereafter fertility averaged 86%. There was no difference in hatchability of fertile eggs between the two treatments over all weeks combined, and weekly throughout the study (P > 0.05). The mean number of spermatozoa in the perivitelline layer was higher (P < 0.001) when hens were inseminated with fresh (12.1 ± 1.3 spermatozoa per 5.5 mm2 membrane) versus stored semen (2.5 ± 0.3 spermatozoa per 5.5 mm2 membrane) over all weeks combined, and weekly throughout the study (P < 0.05). As a result of storage for 24 h, fewer spermatozoa are stored in the sperm storage tubules and, consequently, fewer spermatozoa are present at the site of fertilization, thus contributing to the depressed fertility.

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J. G. Howard, M. A. Barone, A. M. Donoghue, and D. E. Wildt

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

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A. M. Donoghue, A. P. Byers, L. A. Johnston, D. L. Armstrong, and D. E. Wildt

The ovarian response to equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG), the effect of timing of ovulation relative to hCG injection and the use of laparoscopic intrauterine artificial insemination (AI) were examined in two subspecies of tiger (Panthera tigris). Adult female tigers were subjected to the same eCG/hCG treatment followed by laparoscopy under xylazine/diazapam/ketamine HCl anaesthesia at 39–42 h (Group I, n = 9), 46–49 h (Group II, n = 5) or 51–55 h (Group III, n = 5) after hCG. Six of these females, observed to be postovulatory at the time of laparoscopy (Group II, n = 3; Group III, n = 3), were subjected to intrauterine AI. The number of preovulatory follicles observed on the ovaries of Group I females was twofold greater (P < 0.05) than the number observed on ovaries of females in Group II and III. Fewer (P < 0.05) corpora lutea were observed on ovaries of Group I females (1.3 ± 0.6) compared with the number of corpora lutea in Group II and III (combined average, 7.8 ± 0.8 corpora lutea per female). Only one of ten females in Groups II and III failed to ovulate by the time of laparoscopy. Four Group I females never ovulated, based on a laparoscopic re-evaluation 4 weeks later. One female inseminated 46 h after hCG (Group II) became pregnant and delivered a healthy cub after a normal gestation. There were no apparent differences between subspecies in response to the same ovulation induction protocol. Results demonstrate the importance of the relationship between exogenous gonadotrophin treatment and onset of anaesthesia for laparoscopic examination and AI in tigers. Data clearly indicate that anaesthesia/laparoscopy conducted too early (39–42 h after hCG) compromises the number of females and proportion of follicles ovulating. In contrast, ovulation success is high if anaesthesia/laparoscopy is performed after this time, and intrauterine insemination can result in healthy young.

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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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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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T. L. Roth, J. G. Howard, A. M. Donoghue, W. F. Swanson, and D. E. Wildt

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.

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A. M. Donoghue, L. A. Johnston, U. S. Seal, D. L. Armstrong, L. G. Simmons, T. Gross, R. L. Tilson, and D. E. Wildt

Summary. Electroejaculates from tigers were collected and half was used fresh to inseminate tiger eggs in vitro and domestic cat eggs stored in a hypertonic salt solution. The remainder was pelleted, frozen in a solution of 20% egg yolk, 11% lactose and 4% glycerol, thawed and cultured with tiger and domestic cat eggs. The motility index ((sperm % motility) + (status rating × 20))/2 for thawed spermatozoa was about 86% of that in fresh aliquots. Of the 49 tiger oocytes inseminated in vitro with fresh spermatozoa, 34 (69·4%) cleaved, compared with 33 of 47 oocytes (70·2%) cultured with thawed spermatozoa (P > 0·05). Embryos generated by either sperm treatment could develop in vitro to the 16-cell or morula stage. Fresh and thawed tiger spermatozoa were equally capable (P > 0·05) of binding and penetrating the outer and inner zona pellucida of domestic cat eggs. These results demonstrate the ability of frozen–thawed tiger spermatozoa to (i) penetrate homologous and heterologous eggs and (ii) result in conspecific, advanced development of preimplantation embryos in vitro.

Keywords: tiger; spermatozoa; cryopreservation; in vitro fertilization; embryo