Single vs. Group In Vitro Culture of Bovine Embryos: Research in Progress
Austin Byrd and John Gibbons
Texas Tech University, School of Veterinary Medicine
Amarillo, TX 79106
Introduction:
Producing viable embryos and reducing the time between generations is an important, valuable, fast-moving, and trending topic amongst many progressive cattle producers and relies heavily upon effective Assisted Reproductive Techniques (ART’s). In vitro fertilization (IVF) is becoming one of the more popular ARTs as it allows for quicker generation turnover, multiple sires to be used with a single oocyte collection, and more embryos / calves in a given time period. Further, IVF might be chosen over conventional ARTs such as superovulation and embryo transfer because the conventional approaches produce few embryos per collection and the costs are significant. However, the number of oocytes recovered per transvaginal-follicular aspiration session is variable and thus the number of oocytes ultimately placed into culture may be low. The focus of this preliminary study was to evaluate the percentage of ova that cleave and develop to the blastocyst stage when cultured in single vs. group environments.
Materials and Methods:
Ovaries were collected from cattle at a local slaughterhouse and transported to the lab in 25° C sterile saline within six hours of collection. Follicles between 5 and 10 mm were aspirated into 50 ml conical tubes and the follicular fluid and debris were allowed to stand for >10 minutes. The precipitant was aspirated and placed in a petri dish with warm Phosphate-Buffered Saline (PBS) to search for oocytes. Selected oocytes were washed 3 times (modified PBS) and placed (10 per drop) into 50 mL drops of in vitro maturation media for 20-22 hours. In vitro maturation (and fertilization and culture) was conducted in a 5% CO2 (and air) environment at 39.0° C using commercially available in vitro maturation, fertilization, and culture medias.
Matured oocytes were washed 3 times and randomly placed in wells of fertilization media (500 mL) in groups of 50 oocytes per well. Oocytes were then co-incubated (18 hours) with Percoll-separated sperm cells (500,000 / mL) from frozen / thawed semen of a well characterized bull.
Presumptive zygotes were washed 3 times and randomly placed in in vitro culture media in one of three groups: individually, groups of five, or groups of ten presumptive zygotes. Volume of the culture media was based on the number of presumptive zygotes (5 mL per zygote). On Day 7 of culture, embryos were evaluated to determine percent cleavage (> than 1 cell) and development to the blastocyst stage.
Results:
On Day 7 of culture, embryo cleavage (> 1 cell) was similar among groups; however, the development to the blastocyst stage was dependent upon the number of ova in the culture drop (5 mL of media per ova; Figure 1). Although not statistically different (P<0.10), this “helper effect” was evident among the embryos cultured individually, in groups of 5, and in groups of 10.
Discussion:
These data suggested that there was an apparent “helper effect” which was related to the number of presumptive zygotes in the in vitro culture environment. Future research will explore the identity of this “helper effect” which may lead to custom designed culture media dependent upon the number of ova in the culture cohort. In upcoming research, embryos will undergo daily examination to better determine when after cleavage developmental arrest occurs. Perhaps the most important physiological event during that timeframe between embryo cleavage and development to the blastocyst stage is the transition from maternal to embryonic control of the genome. Factors that regulate this transition have not been fully elucidated but may be related to the “helper effect” demonstrated by in vitro culture of bovine ova in large groups.