Impact of Heat Stress on Oocyte Developmental Competence and Pre-Implantation Embryo Viability in Cattle.

Rectal and vaginal temperatures are utilised in both in vivo and in vitro models to study the effects of heat stress on oocyte competence and embryo viability in cattle. However, uterine temperature increases by only 0.5 °C in heat-stressed cows, significantly lower than simulated increases in in vitro models. Temperature variations within oviducts and ovarian follicles during heat stress are poorly understood or unavailable, and evidence is lacking that oocytes and pre-implantation embryos experience mild (40 °C) or severe (41 °C) heat stress inside the ovarian follicle and the oviduct and uterus, respectively. Gathering detailed temperature data from the reproductive tract and follicles is crucial to accurately assess oocyte competence and embryo viability under realistic heat stress conditions. Potential harm from heat stress on oocytes and embryos may result from reduced nutrient availability (e.g., diminished blood flow to the reproductive tract) or other unidentified mechanisms affecting tissue function rather than direct thermal effects. Refining in vivo stress models in cattle is essential to accurately identify animals truly experiencing heat stress, rather than assuming heat stress exposure as done in most studies. This will improve model reliability and aid in the selection of heat-tolerant animals.

Evaluation of blastocyst re-expansion, quality in relation to storage temperature, and sexing using blastocoel fluid after manual perforation with a hand-held needle involving in vivo produced equine embryos.

The present study was designed to evaluate equine blastocyst re-expansion rate, quality, and sex following perforation of the blastocoel, collection of blastocoel fluid (BF), and PCR amplification of free DNA. Experiment 1 tested the feasibility of the BF sample collection with a hand-held, small-gauged needle (26g) and subsequent PCR amplification of the TSP-Y gene for males and AMEL-Y gene for males and AMEL-X gene for females. Experiment 2 tested the application of the technique. Equine embryos were collected via uterine flushes 8d after ovulation. Thereafter, embryos (n = 19) were initially assessed and transferred to a 50 µL droplet of holding medium in which the blastocoel was manually perforated as in Experiment 1. Within 1 min of detecting a diameter decrease or collapse, the entire volume of each droplet of medium was collected and stored at -20 °C until PCR. In Experiment 1, amplification of the TSP-Y gene was positive for males at 60% (9/15) and negative for females at 40% (6/15). In Experiment 2, a total of 42 embryos were randomly assigned to a collapsed embryo (CE) or intact embryo (IE) groups and stored at room temperature (RT, 25 °C) or cold temperature (CT, 5 °C) for 24h as follows: 1) CERT, n = 11; 2) CECT n = 11; 3) IERT, n = 10; and 4) IECT, n = 10. After 24h, embryo diameter and quality were reassessed. For all collapsed embryos (n = 19), blastocoel fluid was subjected to double PCR amplification of the TSPY gene with blood from adult male and female horses as controls. Positive gene amplification indicated 57.9% (11/19) of embryos were male and negative amplification indicated 31.6% (6/19) of embryos were female. Relative to the least diameter (0%) after perforation of collapsed embryos or fullest diameter (100%) of intact embryos at T0, percentage change in diameter and quality Grade 1 or 2 embryos after 24h of storage for all groups were, respectively: 31.2% and 54% for CERT group, 28.2% and 0% for CECT group, 25.9% and 100% for IERT group, 4.3% and 80% for IECT group, respectively. Thus, needle-induced leakage and collapse of the blastocoel at T0 resulted in a high rate of blastocyst re-expansion (69%) with many embryos (54%) achieving good quality at T24 with potential for transfer as either male or female embryos. For both collapsed and intact embryos, it was observed that storage for 24h at room temperature (25 °C) was associated with improved embryo growth and morphological quality compared to storage at cold temperature (5 °C).

The use of resveratrol decreases liquid-extend boar semen fertility, even in concentrations that do not alter semen quality.

In vitro and in vivo assays were conducted to investigate the effects of trans-resveratrol (RVT) on liquid-extended boar semen during 72 h of storage at 17 °C. Thirty-six ejaculates were collected from six boars, evaluated, and extended. RVT was then added at the indicated treatment concentration (0, 0.01, 0.1 or 1 mM), and the ejaculates were cooled to 17 °C and evaluated at 0, 24, 48, and 72 h. Samples were evaluated for sperm motility, kinetics, plasma and acrosome integrity, mitochondrial membrane potential, anion superoxide levels, lipoperoxidation, and antioxidant enzyme activity. In the follow-up experiment, twenty-eight gilts were fixed-time inseminated with 0 or 0.01 mM RVT liquid-extended boar semen. After five days, they were slaughtered, and their reproductive tracts were recovered. The embryos were collected, and the pregnancy, fertility, and viable embryo rates were calculated. In the in vitro assays, total motility, plasma and acrosome membrane integrity, mitochondrial membrane potential, anion superoxide levels, and lipoperoxidation did not change at any of the evaluation times with the use of RVT up to 0.01 mM. RVT decreased SOD activity without changes in GPx. RVT used at 1 mM showed harmful effects for almost all evaluated parameters. For the in vivo assay, the same pregnancy and fertility rates were observed for both groups, while the viable embryo rate was three-fold lower in the 0.01 mM group than in the 0 mM group. The results showed a dichotomous effect of RVT; a low concentration was not harmful in vitro but was catastrophic for embryo viability.

Biopsy of bovine embryos produced in vivo and in vitro does not affect pregnancy rates.

Assisted reproductive techniques have significantly contributed to animal breeding programs. Similarly, genomics has provided important information and tools to improve the accuracy of selection. However, the greatest benefits of those tools can only be expected when they are combined, allowing animals to be selected accurately early in life. Therefore, obtaining DNA samples from embryos without compromising their viability is essential for the consolidation of preimplantation genomic selection. We aimed to evaluate the effect on the gestation rate of conducting a biopsy of in vivo (VV) and in vitro-produced (IVP) bovine embryos. The VV and IVP embryos were distributed into two groups: VV-B (biopsied embryos; n = 380) and VV-C (intact embryos-controls; n = 229) and IVP-B (biopsied embryos; n = 91) and IVP-C (intact embryos-controls; n = 227), respectively. After biopsy, embryos from both groups VV-B and IVP-B were cultured for an additional 3 hours before being transferred to synchronized recipients. To evaluate the quality of the DNA obtained in the biopsies, this was used to determine the sex of embryos by polymerase chain reaction. No effect (P > 0.05) of the biopsy was observed for any of the treatments, the pregnancy rate at D 60 post-transfer being similar for VV-B: 206/380 (54.21%) and VV-C: 128/229 (55.89%) and for IVP-B: 24/91 (26.37%) and IVP-C: 45/227 (19.82%). Also, no effect (P > 0.05) of the embryo's stage of development was detected on percentage of pregnant recipients when in vitro embryos were transferred. From the biopsies analyzed, about 90% had the sex determined, confirming that DNA was there and it was efficiently amplified. The results indicated that biopsy does not affect the viability of IVV and IVP bovine embryos and can be used in commercial programs to associate assisted reproductive technologies with genomic selection.