Date of Award
Doctor of Philosophy (PhD)
A total of 374 non-surgically recovered bovine embryos (EMB) was used to evaluate in vitro culture systems for micromanipulated (MICRO) EMB and to assess EMB development following various MICRO procedures. In Experiment I, viability at 12, 24, 36, 48, 60 and 72 h of culture of 39 demi-embryos (DE) in Ham's F-10 with 10% FCS (HF-10) was 43, 41, 33, 28, 21 and 18% compared with 69, 69, 69, 67, 62 and 62% for DE cocultured with bovine uterine fibroblasts (BUF) in HF-10. These results demonstrate the BUF coculture system's superiority to HF-10 for culturing bovine DE. Experiment II was conducted to assess quartering blastocyst stage EMB. In Trt (A) 15 EMB were MICRO into quarter embryos (QE). IN Trt (B) 15 EMB were bisected into DE, cultured for 2 to 4 h and split into QE. Intact EMB (IE) in Trt (C) served as non-MICRO controls. Fifty-four QE produced in Trt (A) and 34 QE produced in Trt (B) were cocultured with BUF in HF-10 as were IE in Trt (C). Eight QE (14.8%) in Trt (A) and 6 QE (17.6%) in Trt (B) re-expanded into blastocysts while 10 (66.6%) of the IE in Trt (C) were viable in culture. These data suggest that blastocyst stage EMB cannot be split into viable QE using this technique. The effect of puncturing the zona pellucida (ZP) on in vitro blastocyst hatching was evaluated in Experiment III. Seventy-five blastocysts were equally allotted across three Trt. Using MICRO techniques the ZP of EMB were punctured at the ICM (Trt A) or apex of trophoblast (Trt B) while EMB in Trt (C) were non-MICRO controls. All EMB were cocultured with BUF cells in HF-10. Time at which hatching was initiated and size of hatched blastocysts were similar across Trts. EMB in Trt (A) and (B) completed hatching earlier (P < .05) than control EMB (48.9 (+OR-) 5.8, 43.5 (+OR-) 4.4 and 61.1 (+OR-) 4.8 h, respectively). One EMB in Trt (A) and (B) split into half EMB via hatching through the ZP puncture. In Experiment VI, pregnancy rates for non-surgically transferred IE (n = 91) and DE (n = 123) were 46.2% and 37.4%, respectively. Pregnancy rates for DE transferred with and without a ZP were 39.7% and 34.0%, respectively. MICRO of EMB can be conducted while maintaining acceptable pregnancy rates and replacing DE in a ZP is not a prerequisite for successful non-surgical transfer.
Voelkel, Steven A., "In Vitro Culture and Non-Surgical Transfer of Micromanipulated Bovine Embryos." (1985). LSU Historical Dissertations and Theses. 4078.