136 GameteGuard in maturation medium improves bovine embryo quality and quantity

Abstract

The number of invitro-produced (IVP) bovine embryos generated in the United States increased 11% from 2017 to 2018, demonstrating a demand for new technology to rapidly and efficiently improve herd genetics. Unfortunately, embryo production on a per oocyte basis remains low, limiting the number of high-quality commercial embryos available for transfer or freezing. Reactive oxygen species (ROS) are a major contributor to low embryo production rates of invitro gametes and are especially problematic in maturation media. This is due to high numbers of mitochondria in the bovine oocyte producing ROS exacerbated by the invitro environment. GameteGuard® is an organic plant-based additive that provides protection against reactive oxygen species. The aims of this research were to evaluate the effects of GameteGuard addition to invitro bovine embryo production media to (1) mitigate the impact of ROS on oocyte maturation and competence, and (2) increase the production of high-quality embryos in our IVP system. Four rounds of invitro embryo production (Colorado State University protocol and media) with or without maturation medium supplemented with GameteGuard were performed (n=1175 total oocytes). Oocytes were aspirated from abattoir ovaries; those with homogeneous ooplasm and multiple layers of even cumulus cells were matured using control (n=585) or GameteGuard-supplemented (n=590) maturation medium. Oocytes were fertilized with frozen/thawed semen from proven IVF bulls (n=4) and then cultured in a two-step media system to Day 7. Blastocysts were evaluated on Day 7 according to IETS embryo grading standards. A subset of 80 blastocysts were fixed and stained after grading using an In Situ Cell Death Detection Kit (Fluorescein; Millipore Sigma) and Hoechst 33342 (Invitrogen) as apoptotic and counter-stains, respectively. Stained blastocysts were assessed by immunofluorescence microscopy to obtain the percentage of apoptotic cells and total cell count. Because multiple bulls were used, a linear mixed model was required for data analysis. Treatment was considered the fixed effect and bulls as a random effect to determine the impact of the treatment on stage and quality of blastocysts. Cleavage rates were not different (P>0.5) but GameteGuard supplementation of maturation medium resulted in a 22% increase in grade-1 embryos per total blastocysts produced (P<0.01; n=79/106 control vs. n=120/137 GameteGuard grade 1 blastocysts). Additionally, the percent of grade 2 and 3 embryos per total blastocysts decreased, (P<0.01; 27%, n=27/106 control and 11%, n=14/137 GameteGuard grade 2 and 3 blastocysts), suggesting that supplementation with GameteGuard promoted development of better-quality embryos overall. The percentage of apoptotic cells was also significantly higher in control blastocysts compared with treated blastocysts (P<0.02; 11.4%, n=38 control and 12.8%, n=42 GameteGuard). GameteGuard supplementation of the maturation medium significantly increased the production of high-quality IVP bovine embryos, thereby providing potential marketable gain for embryo producers.