Transvaginal ultrasound-guided oocyte retrieval in cattle: State-of-the-art and its impact on the in vitro fertilization embryo production outcome.

Transvaginal ultrasound-guided oocyte retrieval (commonly called OPU) and in vitro embryo production (IVP) in cattle has shown significant progress in recent years, in part, as a result of a better understanding of the full potential of these tools by end users. The combination of OPU and IVP (OPU-IVP) has been successfully and widely commercially used worldwide. The main advantages are a greater number of embryos and pregnancies per unit of time, faster genetic progress due to donor quick turn around and more elite sires mating combinations, larger spectrum of female age (calves, prepuberal, heifer, cow) and condition (open, pregnant) from which to retrieve oocytes, a reduced number of sperm (even sexed) required to fertilize the oocytes, among other benefits. OPU-IVP requires significant less donor preparation in comparison to conventional embryo transfer (<50% of usual FSH injections needed) to the extent of no stimulating hormones (FSH) are necessary. Donor synchronization, stimulation, OPU technique, oocyte competence, embryo performance, and its impact on cryopreservation and pregnancy are discussed.

Recent progress in bovine in vitro-derived embryo cryotolerance: Impact of in vitro culture systems, advances in cryopreservation and future considerations.

Cryopreservation of in vitro-derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high-merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra-low temperature storage method in order to maximize donor ovum pickup (OPU) turn-over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation, and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled-rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in-straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology.

Recent advances in bovine sperm cryopreservation techniques with a focus on sperm post-thaw quality optimization.

Sperm cryopreservation facilitates the storage and transport of germplasm for its use in artificial insemination (AI) and other advanced reproductive technologies. The cryopreservation process can damage sperm and compromise functionality. Several cryobiological studies have found that the physical and biological factors that affect sperm survival at low temperatures during the cryopreservation process often involve the integrity of sperm membrane. In this review, the behaviour of the sperm membrane against cooling, cold shock, ice crystal formation, oxidative stress, osmotic changes, reorganization of the lipid bilayer and addition of cryoprotective agents (CPA) is discussed. In addition, the phenomenon of reactive oxygen species (ROS) and its relationship with the cryopreservation process is also described. Semen cryopreservation techniques have progressed slowly in past years, and the current performance, measured as post-thawed survival, is not very different compared to past decades. Recent advances in understanding the structure of the cell membrane, its function and metabolism have driven to new conservation systems, including lyophilization and vitrification. However, none of these technologies is commercially available, although its future appears very promising.

Effect of spermatozoa motility hyperactivation factors and gamete coincubation duration on in vitro bovine embryo development using flow cytometrically sorted spermatozoa.

The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30min before 18h fertilisation with hyperactivating factors, namely 10mM caffeine (CA), 5mM theophylline (TH), 10mM caffeine and 5mM theophylline (CA+TH); and untreated spermatozoa (control). In Experiment 2, matured B. taurus oocytes were fertilised using a short (8h) or standard (18h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA+TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8h (3%) to 18h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode's albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte-sperm coincubation time (8h) resulted in similar overall embryo performance rates compared with the prolonged (18h) interval.

Comparison of different fertilisation media for an in vitro maturation?fertilisation?culture system using flow-cytometrically sorted X chromosome-bearing spermatozoa for bovine embryo production.

High demand exists among commercial cattle producers for in vitro-derived bovine embryos fertilised with female sex-sorted spermatozoa from high-value breeding stock. The aim of this study was to evaluate three fertilisation media, namely M199, synthetic oviductal fluid (SOF) and Tyrode's albumin-lactate-pyruvate (TALP), on IVF performance using female sex-sorted spermatozoa. In all, 1143, 1220 and 1041 cumulus-oocyte complexes were fertilised in M199, SOF and TALP, respectively. There were significant differences among fertilisation media (P < 0.05) in cleavage rate (M199 = 57%, SOF = 71% and TALP = 72%), blastocyst formation (M199 = 9%, SOF = 20% and TALP = 19%), proportion of Grade 1 blastocysts (M199 = 15%, SOF = 52% and TALP = 51%), proportion of Grade 3 blastocysts (M199 = 58%, SOF = 21% and TALP = 20%) and hatching rates (M199 = 29%, SOF = 60% and TALP = 65%). The inner cell mass (ICM) and trophectoderm (TE) cells of Day 7 blastocysts were also affected by the fertilisation medium. Embryos derived from SOF and TALP fertilisation media had higher numbers of ICM, TE and total cells than those fertilised in M199. In conclusion, fertilisation media affected cleavage rate, as well as subsequent embryo development, quality and hatching ability. SOF and TALP fertilisation media produced significantly more embryos of higher quality than M199.

Comparison of Gonadotropin-Releasing Hormone versus Estrogen-Based Fixed-Time Artificial Insemination Protocols in Grazing Bos taurus Suckled Beef Cows.

Fixed-timed artificial insemination (FTAI) protocols for beef cattle in South America are primarily based on estradiol esters and intravaginal progesterone-releasing devices (IVPD). The objective of this study was to determine the optimal gonadotropin-releasing hormone (GnRH)-based protocol as an alternative to the use of estrogen-based protocols in grazing Bos taurus suckling beef cows. All cows received an IVPD on the day of protocol initiation and prostaglandin F2α (PG) plus equine chorionic gonadotropin (eCG) treatments at the time of IVPD removal. In Experiment 1, cows (n = 235) were randomly assigned to one of four treatments: (i) 7-day estradiol = 2 mg of estradiol benzoate (EB) at IVPD insertion on Day 9 and 1 mg of estradiol cypionate (ECP) at IVPD removal on Day 2; (ii) 7-day GnRH = 10 µg of GnRH at IVPD insertion on Day 10, IVPD removal on Day 3 and GnRH at FTAI; (iii) 7 & 7 estradiol = PG at IVPD insertion on Day 16, EB on Day 9 and ECP at IVPD removal on Day 2; (iv) 7 & 7 GnRH = PG at IVPD insertion on Day 17, GnRH on Day 10, IVPD removal on Day 3 and GnRH at FTAI. In Experiment 2, cows (n = 462) were randomly assigned to one of four treatments: (i) 6-day estradiol = EB at IVPD insertion on Day 9, IVPD removal on Day 3 and GnRH at FTAI; (ii) 7-day estradiol; (iii) 7-day GnRH; (iv) 7 & 7 GnRH. In Experiment 1, plasma progesterone concentrations and percentage of cows with a corpus luteum (CL) at IVPD removal, and pregnancy per AI (P/AI) were greater for cows subjected to GnRH-based protocols compared with cows subjected to estrogen-based protocols (p < 0.01). In Experiment 2, cows subjected to the 7 & 7 GnRH protocol had the greatest P/AI (p < 0.01). In summary, GnRH-based FTAI protocols resulted in similar or greater P/AI compared to estrogen-based FTAI protocols in grazing postpartum Bos taurus suckled beef cows. The greatest P/AI was attained with the 7 & 7 GnRH protocol.