136 CLOSED SYSTEM OF GEL-LOADING-TIP VITRIFICATION FOR IN VITRO-PRODUCED BOVINE BLASTOCYSTS BEFORE AND AFTER BIOPSY
K. Tominaga A B , F. Iwaki A , E. Yamaguchi B , Y.-M. Dou C , I. Kijihana C and S. Hochi DA Hyogo Prefectural Institute for Agriculture, Forestry and Fisheries, Kasai, Hyogo, Japan
B Awaji Agricultural Research Center, Minami-Awaji, Hyogo, Japan
C Nippon Medical and Chemical Instruments Co., Ltd., Osaka, Japan
D Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, Japan
Reproduction, Fertility and Development 19(1) 185-186 https://doi.org/10.1071/RDv19n1Ab136
Submitted: 12 October 2006 Accepted: 12 October 2006 Published: 12 December 2006
Abstract
Ultra-rapid cooling by direct plunging of gel-loading-tip (GL-tip) into liquid nitrogen (LN2) contributed to the high post-warm survival rates of in-vitro-produced (IVP) bovine embryos (Tominaga and Hamada 2001 J. Reprod. Dev. 47, 267–273). Since GL-tip vitrification and other ultra-rapid vitrification methods have potential risk of microbiological contamination in the LN2, the current study was undertaken to develop a closed system of GL-tip vitrification for IVP bovine blastocysts before and after biopsy. Day 7 blastocysts were produced in vitro (Tominaga et al. 2000 Theriogenology 53, 1669–1680), and biopsied for sex determination (Tominaga and Hamada 2004 Theriogenology 61, 1181–1191). The blastocysts were exposed first to 10% ethylene glycol (EG) +10% DMSO in TCM-199/20% FCS for 2 min at 37°C, and then placed in vitrification medium with 20% EG + 20% DMSO + 0.6 M sucrose in TCM 199/20% FCS for 30 s at 37°C. Three to 4 blastocysts in 0.6 µL of the vitrification medium were aspirated into a GL-tip (NK-tip; Nippon Medical & Chemical Instruments Co., Ltd., Osaka, Japan). Immediately before being plunged into LN2, the tip of the GL-tip was sealed by dipping into 100% glycerol at 4°C, and the opposite open end of the GL-tip was sealed by inserting a holding stick under LN2 vapor. After warming in 0.25 M sucrose solution at 37°C, the glycerol-seal was removed from the tip by gentle shaking. The blastocysts diluted in a two-step manner were cultured up to 72 h for survival assay. Within the 72 h of culture, intact embryos that initiated hatching and biopsied embryos that re-expanded were considered as surviving. Two more biopsied blastocysts derived from ovum pickup, IVF, and IVC were vitrified–warmed in the closed system, and then transferred to 2 recipients. The results indicate no significant difference in the post-thaw survival rates of either intact or biopsied blastocysts in both closed and open systems (Table 1). One recipient became pregnant and delivered a female calf. Gestation period (287 days) and birth weight (22 kg) were within the normal range of the Tajima strain of Japanese Black cattle. We reached the conclusion that the closed system of GL-tip vitrification was simple and highly effective for IVP bovine blastocysts before and after biopsy.
|
