59 Incubation of frozen/thawed bovine embryos

Abstract

High-quality, properly developed bovine embryos are crucial to the embryo transfer industry and enable cryopreservation; however, ∼30% of invivo embryos do not develop to the appropriate stage and are discarded. Currently, frozen/thawed embryos are transferred directly into a recipient, and the quality of embryos post-thaw is seldom evaluated. The objective of this experiment was to evaluate frozen/thawed bovine embryos immediately post-thaw, and again after incubation in different environments. Frozen/thawed bovine embryos (n=30/treatment) processed for direct transfer were thawed (30°C water; 30s) and graded and staged in holding medium. Embryos were then placed into either holding medium, phosphate-buffered saline supplemented with 15% fetal bovine serum (v/v) and antibiotic/antimycotic (2μL mL−1) (PBS+FBS), or a commercially available invitro culture (IVC) medium for ∼18h. Embryos in holding medium and PBS+FBS were loaded into 0.25-mL straws with the appropriate medium, and the straws were sealed and submerged in 38.5°C water. The IVC embryos were placed individually into 25-μL culture drops on tissue-coated 60-mm plastic Petri dishes overlaid with mineral oil and incubated (18h) at 38.5°C in 5% CO2 and air at 100% humidity. Embryos were then collected, regraded, and staged, and comparisons among groups were analysed via the Kruskal–Wallis H-test. Quality score of all embryos decreased by at least one-third post-thaw; however, the developmental stage was unaffected by the freeze/thaw cycle. Following incubation, all embryos suffered a significant (P<0.05) decrease in embryo quality but the IVC group demonstrated less (P<0.05) of a decline in resulting quality (Table 1). The IVC group demonstrated significant development (P<0.05) during incubation compared to the Holding and PBS+FBS groups (Table 1) indicating that on average, viability was maintained during IVC. Regardless of group, the zona pellucida was damaged during the freeze/thaw process in 31.1% of embryos. These data illuminated embryo damage after cryopreservation, and demonstrated that short-term invitro incubation of frozen/thawed embryos (IVC) facilitated continued development and may be a practical mechanism to salvage poor quality or developmentally suppressed embryos. Future research will focus on salvaging fresh embryos that are classified as degenerate and may prove useful in the bovine embryo industry, and for cattle producers alike, by ultimately increasing the number of transferable embryos that would otherwise be discarded. Table 1. Descriptive statistics (mean±s.e.m.) of bovine embryos pre-freeze and post-thaw and incubation Item Media Pre-freeze Post-thaw Post-incubation Grade Holding 1.4±0.1 1.7±0.1a 3.3±0.1b,x PBS+FBS 1.2±0.1 1.6±0.1a 3.1±0.1b,x IVC1 1.2±0.1 1.6±0.1a 2.5±0.2b,y Stage Holding 4.0±0.1 4.0±0.1 3.9±0.1x PBS+FBS 4.7±0.2 4.4±0.2 4.5±0.2y IVC 4.3±0.1 4.3±0.1a 5.8±0.2b,z a,b, x–zDifferent superscripts within row (a, b) and column (x, y, z) indicate a significant difference (P<0.05, Kruskal-Wallis H-test). 1Invitro culture medium.