Nonsurgical collection and nonsurgical transfer of preimplantation embryos in the domestic rabbit (Oryctolagus cuniculus) and domestic ferret (Mustela putorius furo).

The objective of this study was to develop nonsurgical methods of embryo collection and transfer in domestic rabbits (Oryctolagus cuniculus) and domestic ferrets (Mustela putorius furo) to serve as models for use in mammals in which surgical procedures are the usual means for applying embryo transfer technology. Specially designed transcervical catheters were used together with a fibre optic endoscope to visualize and then catheterize the rabbit and ferret cervices. Five consecutive transcervical uterine flushes in each of eight superovulated female rabbits 78-89 h after an ovulatory injection of LH resulted in the retrieval of 187 embryos, for an average of 23 embryos per rabbit. A total of 116 embryos were nonsurgically transferred to the uteri of ten recipients, and resulted in 23 young (20%). Eight rabbits (80%) produced young with an average litter size of 2.88 (range 1-7). Ten consecutive transcervical uterine flushes in each of 37 female ferrets 145-178 h after an ovulatory injection of hCG resulted in the retrieval of 324 embryos, an average of 8.76 embryos per ferret. A total of 251 embryos from 27 donors were nonsurgically transferred to the uteri of 31 recipients, and resulted in 65 young (26%). Twenty-eight of the recipients (90%) were initially pregnant, as indicated by postpartum necropsies, and twenty-two ferrets (71%) produced young. The average litter size was 2.95 (range 1-7). This is the first report of live births resulting from the nonsurgical collection of embryos from a donor followed by nonsurgical transfer of those same embryos to a synchronous recipient. The methods reported here can serve as models for use in other mammals in which direct visualization and manipulation of the cervix are not possible, and will be particularly useful in endangered species.

Use of Hoechst 33342 stain to evaluate live fresh and frozen bull sperm by computer-assisted analysis.

The objective of this research was to investigate possible procedures for evaluating living bull sperm stained with Hoechst 33342 while in a simple medium and in commonly used complex egg yolk-glycerol-Tris (EYGT) and whole milk-glycerol (WMG) extenders. The two semen extenders provide good cryoprotection, but the latter one virtually obscures the sperm. To evaluate sperm motion characteristics when static nonsperm particles are present, a new Hamilton Thorne epifluorescent optical system (UV) with a strobe light was developed for potential use with DNA-stained sperm. This system permitted examination for the first time of sperm motion characteristics in milk. In Experiment 1 (four bull semen replicates with five dye concentrations and three incubation times), 2.5 microg/ml of Hoechst 33342 stained live and dead sperm sufficiently in a modified Tyrode's solution to measure all sperm characteristics without depressing motility, which was validated by using phase-contrast to analyze stained and unstained controls. In Experiments 2a and 2b, each using semen from four bulls with a 5 x 5 factorial arrangement, it was determined that 40 to 60 microg/ml of dye in EYGT or WMG, with UV illumination for 20 minutes, was optimal. There was no detrimental effect on sperm motility. In Experiment 3, analyses of two ejaculates, from each of eight bulls, confirmed that motion characteristics of sperm in EYGT and WMG were not depressed when the sperm were stained with Hoechst 33342. These experiments demonstrate that the dye concentrations and exposure times developed for use with the new epifluorescent optics facilitate evaluating bull sperm frozen in particle-filled whole milk and should be useful for sperm evaluation of a variety of species when nonsperm particulate matter may otherwise interfere.

Effect of amino acids and alpha-amanitin on the development of rabbit embryos in modified protein-free KSOM with HEPES.

Four experiments were conducted to test the effects of Eagle's non-essential amino acids (NEAA) and essential amino acids (EAA), glycine, and the RNA polymerase inhibitor, alpha-amanitin, on the development of preimplantation rabbit embryos in modified protein-free KSOM medium. Embryos were distributed randomly into different treatments and cultured in 5% O2:5% CO2:90% N2. In experiment 1, 100% of the embryos became blastocysts in the medium with Eagle's 1X NEAA and 0.5X EAA, but 100% stopped development at the morula stage in KSOM without amino acids. These morulae failed to develop further when transferred to amino acid supplemented medium after 72 hr of culture. Glycine alone in modified KSOM (experiment 2) was ineffective in supporting development of 8-16-cell stage embryos past the morula stage. In experiment 3, the addition of 1X NEAA and 0.5X EAA at 0, 12, 24, 36, and 48 hr of culture resulted, respectively, in 57, 65, 65, 44, and 14% blastocysts on Day 3 (P < 0.05) and 86, 77, 77, 78, and 69% on Day 5 (P > 0.05). Omission of Eagle's amino acids until 48 hr clearly delayed embryo development. In experiment 4, when alpha-amanitin (20 microM) was added to the medium containing Eagle's amino acids after 0, 12, 24, 36, and 48 hr of culture most embryos cleaved only once or twice after adding the alpha-amanitin. Without the inhibitor, 94% of the zygotes developed into blastocysts. These results indicate that modified KSOM or KSOM plus glycine could not support rabbit embryo development past the morula stage, but this block was overcome by adding Eagle's amino acids. An exogenous source of amino acids was not critical for embryo development during the first 24 hr of culture, but was required after that for development to equal controls. Addition of alpha-amanitin at multiple pre-blastocyst stages limited further embryo development to one or two cleavage divisions, with no blastocyst development.

Ethylene glycol monomethyl ether effects on health and reproduction in male rabbits.

Male Dutch rabbits were weighed and randomly assigned within each weight group to five groups of six animals each (plus one more in the highest dose group). They received 0, 12.5, 25.0, 37.5, or 50.0 mg of ethylene glycol monomethyl ether (EGME) per kg of body weight in the drinking water 5 d/week for 12 weeks. Feed and water consumption were monitored daily and body weight weekly. All animals consumed the water and feed, maintained body weight, and were in good health throughout the experiment. Semen was collected twice weekly for 12 weeks, and 96% of the ejaculates were obtained. By weeks 6 and 9, most males in groups receiving 50.0 or 37.5 mg of EGME per kg were oligospermic. Only minor changes in other characteristics of sperm obtained from treated animals were found, as measured by computer-assisted sperm analysis. Fertility of the males still producing sufficient sperm during week 12 to use for insemination was tested with 96 does producing 2839 oocytes, and fertility of treated males (41%) was not lower (P > 0.05) than 47% in controls. At necropsy, all vital organs were grossly normal, with no notable histopathology. However, the groups of animals receiving 37.5 and 50 mg of EGME per kg of body weight produced fewer sperm and had smaller testes than controls (P < 0.05). Although all rabbits appeared grossly normal, there was a marked disruption of spermatogenesis as ingestion of EGME increased above 25 mg/kg of body weight. Rabbit testes appear to be more sensitive to EGME than testes of rats or mice.

Relationship of semen quality, number of sperm inseminated, and fertility in rabbits.

The relationship between the total number of sperm inseminated, semen quality, and fertility in rabbits was investigated, using fractionated or unfractionated semen and different diluting fluids. Semen was from Dutch-belted males collected twice weekly with an artificial vagina. All does were superovulated except in Experiment 3. In Experiment 1, sperm were fractionated on discontinuous 4% and 10% bovine serum albumin columns. Sperm from each portion of the gradient, along with unfractionated controls, were diluted to give 0.25 x 10(6), 0.5 x 10(6), 1.0 x 10(6), and 2.0 x 10(6) total sperm per insemination. In Experiment 2, sperm were diluted with Dulbecco's phosphate-buffered saline to provide 0.10 x 10(6), 0.50 x 10(6), and 1.0 x 10(6) total sperm per insemination, with minimal processing time. In Experiment 3, does were allowed to kindle after inseminating 0.1 x 10(6) or 1.0 x 10(6) sperm. In Experiment 4, sperm were diluted with TALP buffer: seminal plasma 1:1 to 0.025 x 10(6), 0.05 x 10(6), and 0.10 x 10(6) total sperm per insemination. Over 2,800 embryos or unfertilized oocytes were obtained either 24 or 48 hours after insemination to measure fertility. Sperm numbers required for normal fertility were 0.50 x 10(6) in Experiment 1 and only 0.05 x 10(6) in Experiment 4. This reduction presumably was due primarily to reduced processing time and diluent change. Litter size was normal with 0.1 x 10(6) sperm (Experiment 3). In Experiment 4, computer-assisted sperm analysis (HTM 2030 system; Beverly, Massachusetts) was adapted to successfully screen out some of the "interfering" granules in rabbit semen.(ABSTRACT TRUNCATED AT 250 WORDS)

Method for obtaining bovine zygotes produced in vivo.

A superovulatory and surgical protocol was developed for recovery of bovine zygotes. Holstein cows and heifers were given follicle-stimulating hormone and cloprostenol to induce superovulation. Surgical cannulation and lavage of the uterine tube was performed 40 to 48 hours after the start of standing estrus. In general, cows had more corpora hemorrhagica than did heifers, but a higher percentage (P less than 0.05) of ova recovered from cows were infertile. Several heifers were subjected to the procedure twice, and embryo recovery rates were equivalent both times.

Bovine 1-2-cell embryo development using a simple medium in three oviduct epithelial cell coculture systems.

These studies were designed to develop a coculture system using a simple medium to promote development of 1-cell bovine embryos through the 8-16-cell stage to morula and blastocyst stages. Monolayers for coculture were prepared from bovine oviduct epithelial cells (BOEC). In vivo-fertilized 1-2-cell embryos and ova (384) were surgically collected from superovulated cows. In Experiment 1, embryos cocultured in a simple glucose-free and serum-free medium (CZB) developed with superior scores of embryo quality than embryos cocultured in Ham's F-10 with serum, and a greater percentage developed past 8-16 cells than embryos cocultured in CMRL-1066 with serum (p less than 0.05). In Experiment 2, embryos cocultured with fresh BOEC monolayers averaged more (p less than 0.05) cells than did embryos in coculture with frozen-thawed BOEC monolayers or in BOEC-conditioned medium. Without glucose in the simple medium for the first 48 h of culture, more embryos blastulated (p less than 0.01) by Day 5.5 of culture (Day 6.5 of donor's estrous cycle) than embryos in the same medium with glucose present throughout. In Experiment 3, more embryos tended to hatch in BOEC coculture (p less than 0.10) than in conditioned medium. These results show that a chemically simple medium with fresh BOEC monolayers can provide a significant benefit for coculture of early bovine embryos.

Development and survival after transfer of cow embryos cultured from 1-2-cells to morulae or blastocysts in rabbit oviducts or in a simple medium with bovine oviduct epithelial cells.

This study compares development of bovine 1-2-cell embryos in bovine oviduct epithelial cell co-culture (Group EC) with a glucose- and serum-free simple medium (CZB), or after surgical transfer to ligated oviducts of rabbits (Group RO). Embryos were surgically collected from superovulated donor cows 40-48 h after the beginning of oestrus and randomly distributed between the two groups. Embryos were cultured or incubated for 5 days. In Exp. 1, embryo quality scores and total numbers of cells in the two groups were compared. In Exp. 2, pairs of similarly treated morulae were transferred to each of 10 or 12 recipients in the Groups RO and EC, respectively. Total cell counts per embryo in both groups averaged 52 (P greater than 0.05), and the in-vitro culture system was equivalent to the rabbit oviducts in promoting embryo development for all characteristics measured. Embryo survival, as determined by ultrasound between Days 39 and 43 after oestrus, in 13 ideal recipients was 57% for embryos in Group EC and 58% for embryos Group RO. None of the 9 less desirable recipients was pregnant for either group. These results establish that cattle zygotes can develop to morulae in culture with bovine oviduct epithelial cells in a simple medium and can produce normal pregnancy rates.

Fertility of fresh and frozen rabbit semen inseminated at different times is indicative of male differences in capacitation time.

Some reports indicate that sperm from different males differ in capacitation time, and other reports suggest that freezing sperm may affect their capacitation time. These two variables were specifically studied in rabbits in a fertility trial with 96 does inseminated with approximately 1.6 million motile fresh or frozen sperm from three different bucks at 15, 10, 5, and 0 h before expected ovulation. Fresh semen averaged 84% live (unstained) sperm and 88% had normal acrosomes; corresponding values for frozen sperm were 44% and 54%. On the basis of does that became pregnant, average litter size with fresh semen was 5.5 and with frozen semen was 4.8 (p greater than 0.05), but overall, does bred with frozen semen produced fewer young (p less than 0.05). On the basis of total does and total semen, average litter size from insemination at 15, 10, 5, and 0 h was 2.8, 4.2, 3.8, and 1.7, and average litter size for the three bucks was 4.0, 1.8, and 3.6. There was no interaction of type of semen (fresh or frozen) with the other variables in the model (p greater than 0.05). Bucks and time of insemination affected both the proportion of does that were pregnant and litter size (p less than 0.01). A major interaction between buck and time of insemination (p less than 0.01) was due apparently to both differential sperm survival and probable capacitation time among bucks. This major interaction should be considered in designing in vitro and in vivo fertility studies, and for selecting males for use in artificial insemination.

Measurement of semen quality, fertility, and reproductive hormones to assess dibromochloropropane (DBCP) effects in live rabbits.

Thirty-six sexually mature Dutch rabbits were divided into six equal groups to receive in the drinking water 5 days/week for 10 weeks 0, 0.94, 1.88, 3.75, 7.50, and 15.00 mg of DBCP/kg body wt. General health, body weight, semen quality (four ejaculates per male per week), and libido were measured throughout. Fertility, blood follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were measured the last week and cauda epididymal sperm were examined at sacrifice. There was no effect of DBCP on general health or body weight. There was considerable variation in ejaculate volume, percentage motile sperm, and sperm concentration per milliliter within groups and among weeks. However, between the first 2 weeks and the last 2 weeks of the experiment sperm output had increased by 19% in the three lower DBCP groups and decreased by 16% in the three higher DBCP groups (p less than 0.01). The proportion of sperm with abnormal tails also increased as DBCP dosage increased. Fertility was unaffected. FSH was elevated (p less than 0.01) in the group receiving 15 mg/kg of DBCP, which is consistent with the impairment of spermatogenesis. Libido, LH, and testosterone levels were not affected. Sperm morphology was the most sensitive indicator of a DBCP effect in the live animal, being affected at a daily oral intake greater than or equal to 1.88 mg DBCP/kg body wt.