Differences in embryo weight and chemical composition, and their implications for cryopreservation survival

Abstract

OBJECTIVE: Cryopreservation is considered a standard of care in ART; however, great variability still exists in embryo survival rates. The process assumes that water is highly conserved across embryos of the same species. Therefore, any dramatic shift in the embryos' chemical nature might lead to failure of the cryopreservation process. The objective of the present study was to determine if embryos from the same species have significantly different chemical composition. Embryo weight and lipid content were compared between Jersey cattle, a breed with significant body fat content and considered difficult to cryopreserve, and beef cattle breeds with known high success rates for cryopreservation. DESIGN: A laboratory based study of cryopreservation using cattle embryos of various breeds. MATERIALS AND METHODS: Frozen day 6 cattle blastocysts of Jersey and beef breeds were thawed using standard procedures, and equilibrated into P-1 medium. Specific gravities were then determined by “dropping” the embryos through two solutions (50 embryos /solution/breed) of known osmolarity and timing their decent over a standard distance. The system had been previously standardized using glass spheres of known size and weight. Additionally, ten embryos of both Jersey and beef cattle (20 embryos total) were thawed, equilibrated into P-1 and then fixed for electron microscopy (EM) using standard techniques. Random sections were then examined using a Hitachi H-7650 TEM (1500X). Resulting micrographs were then examined to quantify the lipid content of the embryo. Data between groups were analyzed by student's T test using SPSS. RESULTS: Specific gravity data suggest the beef cattle embryos to be 20-27% heavier than Jersey embryos (P < .001). EM data suggest these differences are due to differing lipid content; with Jersey embryos having approximately 36% lipid/unit area as compared to only 8% in beef cattle embryos (P < .01); thus, explaining the Jersey embryos lighter weight. CONCLUSIONS: The data suggest that subtle differences in embryo weight can be quantified using a simple specific gravity technique. In the present study, animals with a known higher body fat content were shown to have embryos of lighter weight. Electronic microscopy appeared to confirm these embryos had higher lipid content. These data would appear to suggest that “standard” cryopreservation protocols might not be effective with all embryos and selective cohorts might require modification of the cryoprotectants.