99 COMPARISON OF IN VITRO DEVELOPMENT FOLLOWING CRYOPRESERVATION OF MEISHAN AND WHITE CROSS SWINE EMBRYOS

Abstract

Development of improved protocols for cryopreservation of zona pellucida-intact porcine embryos could greatly impact the swine industry. Our aim was to investigate in vitro development following cryopreservation of embryos from Chinese Meishan (M) and occidental white cross (WC) breeds using a modified protocol described previously (Misumi K et al. 2003 Theriogenology 60, 253–260). First-parity M sows (n = 11) and WC gilts (n = 13) were observed for estrus every 12 h and inseminated at 12 and 24 h after estrous onset within breed using semen from 2 different boars. Females were sacrificed between Days 4.5 and 6 after estrus and embryos were collected using Beltsville embryo culture medium (BECM). Compact morula (CM) or blastocyst stage embryos from each female within breed were randomly allocated either directly into the culture system to serve as controls (68 M and 48 WC embryos) or to undergo cryopreservation. A total of 101 M and 78 WC embryos were cryopreserved using the following protocol: (1) 5 min in BECM + 10% ethylene glycol (EG); (2) 5 min in BECM + 10% EG + 0.27 M sucrose + 1% polyethylene glycol (PEG); and (3) 30 to 45 s in BECM + 40% EG + 0.36 M sucrose + 2% PEG. In the last solution, 5 to 10 embryos in a 5- to 10-μL microdrop attached to a fine glass pipette were exposed to the vapor phase of liquid nitrogen (LN₂) for 15 s and then plunged into LN₂. The pipette tip was broken and the tip and associated frozen microdrop were placed inside an LN₂-submerged 2-mL cryotube containing a hole in the lid for 1 h. Next, embryos were thawed using a 4-step (5 min each) procedure: (1) BECM + 5% EG + 0.57 M sucrose; (2) BECM + 2.5% EG + 0.29 M sucrose; (3) BECM + 0.3 M sucrose; and (4) BECM alone. All procedures were performed with solutions maintained at 37°C. Cryopreserved and control embryos were cultured in 50 μL drops of modified Whitten's medium + 1.5% BSA under oil at 37°C in a 5% CO₂ in air environment and scored daily for development. For embryos undergoing cryopreservation, retrieval rates from cryovials were 92% and 96% for M and WC, respectively. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than for WC (44%; P < 0.001; χ²-test). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than for frozen embryos from both breeds. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98% and 95%, respectively), but more M embryos developed to the hatched blastocyst stage (22% for M v. 9% for WC; P < 0.05). Our results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos.

Funding for M. Montagner was provided by CAPES, Brazil.