S. Chaubal

Optimizing IVF with sexed sperm in cattle.

Sperm-sorting by flow cytometry separates X-sperm from Y-sperm with an accuracy as high as 90% or more. This technology offers farmers and the livestock industry the potential to nearly double productivity, by producing the desired sex to optimize breeding programs. Sorting speed and fertility variation of sorted sperm, however, remain limiting factors for widespread application, particularly in traditional AI programs. Alternatively, in vitro fertilization is a feasible and efficient means to increase the fertilization efficiency of sex-sorted sperm in cattle. Procedures to increase fertilization rate and improve embryo quality include optimizing heparin concentrations for semen of each bull, reducing fertilization drop size to increase sperm concentration, use of fructose instead of glucose in culture media, and use of vitrification protocols with extremely rapid cooling and warming rates.

Oocyte Source and Hormonal Stimulation for In Vitro Fertilization Using Sexed Spermatozoa in Cattle

The aim of this study was to investigate the efficiency of in vitro embryo production in cattle utilizing sexed sperm from two bulls and oocytes recovered by OPU. Twenty donor animals were employed in eight OPU replicates: the first four OPU trials were conducted on animals without hormone treatment, and the last four were run on the same animals, following FSH subcutaneous and intramuscular administration. A higher rate of blastocyst development was recorded in stimulated, as compared to nonstimulated animals, (25.2% versus 12.8%, P = .001). Ocytes derived from slaughterhouse (SH) ovaries were also fertilized with sperm from the same bulls. Overall, non-sexed sperm used with oocytes derived from SH ovaries was significantly more efficient for blastocyst development than was sexed sperm with these same SH derived oocytes and sexed sperm with stimulated donor oocytes (39.8% versus 25.0% and 25.2%, P = .001). In conclusion, the use of sexed sperm with OPU-derived oocytes resulted in a significantly higher blastocyst development when donors were hormonally stimulated; furthermore, the level of efficiency achieved was comparable to that attained when the same sexed sperm was tested on oocytes derived from SH ovaries.

Beneficial Effect of Young Oocytes for Rabbit Somatic Cell Nuclear Transfer

This study was designed to examine the effect of the age of rabbit oocytes on the developmental potential of cloned embryos. The metaphase II oocytes used for nuclear transfer (NT) were collected at 10, 12, 14, and 16 h post-hCG injection (hpi). The total number of oocytes collected per donor (21.4-23.7) at 12 to 16 hpi was similar, but significantly higher than that collected at 10 hpi (16.2). Additionally, a significant improvement in blastocyst development was achieved with embryos generated by electrically mediated cell fusion (56.0%), compared to those from nuclear injection (13.1 %) (Experiment 1). Markedly higher blastocyst development (45.8-54.5%) was also achieved with oocytes collected at 10-12 hpi than from those collected 14-16 hpi (8.3-14.3%) (Experiment 2). In Experiment 3, the blastocyst rates of NT embryos derived from oocytes harvested 12 hpi (39.2-42.8 %) were significantly higher than from those collected at 16 hpi (6.8-8.4 %) (p < 0.05), regardless of the donor cell age. Kinase activity assays showed variable changes of activity in rabbit oocytes over the period of 10-16 hpi; however, there was no correlation with preimplantational development (blastocyst rate vs. MPF, R = 0.326; blastocyst rate vs. MAPK, R = -0.131). Embryo transfer of NT embryos utilizing 12 hpi oocytes resulted in one full-term but stillborn, and one live cloned rabbit; thus, an efficiency of 1.7 % (n = 117) (Experiment 4). These results demonstrated that NT utilizing relatively young rabbit oocytes, harvested at 10-12 h after hCG injection, was beneficial for the development of NT embryos.

Significant heparin effect on bovine embryo development during sexed in vitro fertilization

The aim of this study was to investigate the effect of different heparin concentrations in the course of sexed in vitro fertilization (IVF), on bovine embryonic development and development to term following embryo transfer (ET). With a total of 9156 oocytes for IVF, sorted as well as unsorted sperm from four bulls had different heparin requirements for achieving the highest rate of development in vitro. However, when optimal heparin concentrations were used (40 to 80 µg/ml), the performance of X-sorted sperm (0.3 × 106/ml/IVF droplet) from all four bulls, as judged by blastocyst development (Bulls A, B, C, and D: 25.2, 19.7, 25.1, and 9.8%, respectively), was significantly increased, and the blastocyst rate was comparable to that observed with unsorted sperm at certain heparin concentrations within the four bulls. We determined that near-optimal blastocyst development was possible with sorted sperm from all four bulls, when a heparin concentration of 40 µg/ml was used. Pregnancy rates at d 70 post ET ranged from 39.1 to 40.3% (P > 0.05), and the calving rates ranged from 34.4 to 35.1% (P > 0.05), when heparin was used at a concentration of 10 μg/ml (n = 236), 20 μg/ml (n = 189), and 40 μg/ml (n = 305), respectively. Our study demonstrates that, although the sorted sperm of different bulls performed optimally over a range of heparin concentrations, a generally accepted heparin concentration of 40 µg/ml can be set for sexed IVF. This improvement is beneficial when sexed embryo production by ovum pickup and IVF is an essential component of genetic breeding programs.

261 DIFFERENT TRANSVAGINAL OVUM PICK-UP STRATEGIES TO OPTIMIZE THE OOCYTE RETRIEVAL AND EMBRYO PRODUCTION OVER A FIXED PERIOD OF TIME

The objective of this study was to compare the efficacy of different oocyte retrieval schemes over a period of 10 weeks. Fifteen multiparous Angus cows were randomly assigned (n = 3/group) to the following groups: 1) OPU once/week (7-day interval), 2) OPU twice/week (3- and 4-day interval, alternately), 3) Dominant follicle removal (DFR) + OPU once/week. DFR followed by OPU 72 h later, 4) DFR + FSH + OPU once/week. DFR followed 36 h later by FSH (Folltropin, Bioniche, Belleville, Ontario, Canada) (120 mg s.c. and 80 mg i.m. administered simultaneously) followed by OPU 48 h later, 5) FSH + OPU twice/week. FSH followed by OPU1 30 h later and OPU2 96 h after OPU1. The interval between OPU2 and next FSH was approximately 42 h. The follicles were aspirated using an Aloka ultrasound scanner (Wallingford, CT, USA) and a 5-MHz probe. The COCs were selected based on morphology and matured in TCM-199, supplemented with 10% FCS, 0.01 units mL-1 bFSH, 0.01 units mL-1 bLH and antibiotics. Fertilization (Day 0) was carried out with TALP-FERT medium containing capacitation factors. Frozen semen from the same bull was used (1 × 106 mL-1) throughout. After 18 h the presumptive zygotes were cultured in SOF with 5% FCS (Holm P et al., 1999 Theriogenology 52, 683–700). The embryos were evaluated based on IETS guidelines (Grades 1 and 2 selected). The data were analyzed by chi-square test and ANOVA. In all parameters, the DFR followed by FSH and subsequent OPU once/week protocol gave the best results on a per-cow-per-week as well as total (3 cows over 10 weeks) basis. Though OPU was done only once/week, this group produced more total oocytes (303) than groups where OPU was done twice/week, either with FSH (286) or without FSH (229) and also produced more total embryos on Day 8 (71 blastocysts, 23.4% of oocytes cultured) than the latter two groups (64, 22.4% and 49, 21.4%, respectively). Among the nonstimulated groups, the OPU twice/week group had more total oocytes (236) than groups with OPU once/week, either without DFR (137) or with preceding DFR (160). However, a preceding DFR seemed to have a positive effect on oocyte quality as this group had a better embryo development rate (26.9%), producing more total embryos (42). In comparison, OPU twice/week produced total 49 embryos (21.4%) and OPU once/week produced 26 (19.4%). In conclusion, DFR coupled with single-shot FSH administration can be used effectively over a period of at least 10 consecutive weeks and can increase (P < 0.05) the oocyte yield by two-fold and embryo production following IVF by two and half-fold, as compared to routine OPU-IVF done once a week. Table 1 Per cow per week performance