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.

Hormonal treatments for increasing the oocyte and embryo production in an OPU-IVP system.

The objective was to enhance the inherent developmental ability of bovine oocytes retrieved by ultrasound-guided transvaginal aspiration. Various hormonal regimes were utilized to produce partially matured oocytes in vivo, in order to improve embryo development following IVF. In the first experiment, a two-by-two factorial design was used with FSH (multiple versus single dose) and im administration of LH (yes versus no) 6h prior to OPU. In all protocols (which lasted for nine consecutive weeks), ovarian stimulation was performed in the presence of a CIDR. One FSH administration was adequate for ovarian stimulation (9.33+/-0.7 and 10.14+/-0.7 follicles per cow per OPU session); however, multiple injections increased (P<0.05) follicular response (12.97+/-0.7 and 13.97+/-0.7). In the second experiment, a two-by-two factorial design was used to compare the effects, during ovarian stimulation, of the presence or absence of CIDR, and iv treatment with LH 6h prior to OPU (yes versus no), on oocyte competence (judged by blastocyst development rates following IVF). Presence of CIDR during superstimulation had no effect on the follicular response. Administration of LH 6h prior to OPU increased (P<0.05) the oocytes of higher morphological grades, and in the absence of a CIDR, improved (P<0.05) blastocyst development rate. Treatment with LH, 6h prior to OPU without the use of CIDR during ovarian stimulation, resulted in 2.89+/-0.4 blastocysts per cow per OPU session as compared to 1.56+/-0.4, 1.56+/-0.4 and 1.33+/-0.4 for all other groups. In conclusion, compared to single administration, multiple FSH administration increased (P<0.05) available follicles for aspiration. Moreover, when ovarian stimulation in the absence of CIDR was followed by administration of LH 6h prior to OPU, it increased (P<0.05) the number of blastocysts per OPU session.

Prolonging bovine sperm-oocyte incubation in modified medium 199 improves embryo development rate and the viability of vitrified blastocysts.

This study was conducted to compare the efficacy of four in vitro fertilization (IVF) media: Bracket and Oliphant's medium (BO), modified medium 199 (IVF-M199), modified Tyrode's medium (MTM), and modified KSOM (m-KSOM) on fertilization efficiency and blastocyst formation rate. In addition, we wanted to investigate the benefit of prolonging the IVF period (from 6 to 18 h) using the two most effective IVF media determined in our initial experiment; subsequently, blastocyst viability was assessed following vitrification. A higher incidence of polyspermic fertilization was observed in the MTM (6%) and in BO, in both the 6 and 18 h (7% and 11%, respectively) groups, than in the m-KSOM (1%) or in the IVF-M199 6 or 18 h (1 and 3%, respectively) groups. Cleavage rates were similar in BO, IVF-M199, and MTM 48 h post-fertilization; however, the lowest cleavage rate was observed for m-KSOM. A greater proportion of zygotes developed into 8-cell embryos in IVF-M199 than in other IVF media. Subsequently, a greater proportion of blastocyst formation and hatching was achieved in IVF-M199 (40% and 79%, respectively) or BO (35% and 74%, respectively) than in m-KSOM (18% and 58%, respectively) or MTM (22% and 66%, respectively). Prolonging IVF to 18 h did not alter cleavage rates; however, the highest rate of overall blastocyst formation was achieved in the IVF-M199 18 h (49%), rather than in the BO 18 h (20%) group. Vitrified/thawed blastocysts from IVF-M199 groups re-expanded and developed better, as compared to the BO 18 h group, and hatching rate and total cell number in IVF-M199 18 h group was comparable to the control groups (non-vitrified). Vitrification reduced survival compared to controls. In conclusion, IVF-M199 was successfully used for IVF, compared favorably to BO medium, and offered the advantage of an extended IVF period for up to 18 h that requires only one-half a dose of semen, and resulted in better quality blastocysts that endured vitrification with a hatching rate comparable to that of control groups.

Comparison of different transvaginal ovum pick-up protocols to optimise oocyte retrieval and embryo production over a 10-week period in cows.

The objective was to develop a simple and effective ovum pick-up (OPU) protocol for cows, optimised for oocyte harvest and subsequent in vitro embryo production (IVP). Five protocols differing in collection frequency, dominant follicle removal (DFR) and FSH stimulation were tested on groups of three cows each, over an interval of 10 consecutive weeks. Performance was evaluated on per OPU session, per week and pooled (3 cowsx10weeks) basis. Among the non-stimulated groups, on a per cow per session basis, once- or twice-weekly OPU had no effect on the mean (+/- S.E.M.) number of follicles aspirated, oocytes retrieved and blastocysts produced (0.6+/-0.8 and 0.7 +/- 0.7, respectively). However, DFR 72 h prior to OPU almost doubled blastocyst production (1.2 +/- 1.3). In stimulated groups, FSH treatment (80 mg IM and 120 mg SC) was given once weekly prior to OPU. Treatment with FSH, followed by twice-weekly OPU, failed to show any synergistic effect of FSH and increased aspiration frequency. When FSH was given 36 h after DFR, followed by OPU 48 h later, more (P < 0.05) follicles (16.0 +/- 5.0), oocytes (10.6 +/- 4.5) and embryos (2.1 +/- 1.2) were obtained during each session, but not on a weekly basis. Pooled results over 10 weeks showed an overall improved performance for the treatment groups with twice-weekly OPU sessions, due to double the number of OPU sessions performed. However, the protocol that consisted of DFR, FSH treatment and a subsequent single OPU per week, was the most productive and cost-effective, with potential commercial appeal.