174 EMBRYO TRANSFER OF VITRIFIED IVF EMBRYOS IN CATTLE: PREGNANCY COMPARISON AFTER SINGLE AND DOUBLE TRANSFER
F.L. Du A , A. Dinnyes A , L.Y. Sung A , J. Xu B , S. Jiang A , C.X. Tian A and X. Yang AA Department of Animal Science, Center for Regenerative Biology, University of Connecticut, Storrs, CT, USA. email: fdu@canr.uconn.edu;
B Evergen Biotechnologies Inc., Storrs, CT, USA.
Reproduction, Fertility and Development 16(2) 209-209 https://doi.org/10.1071/RDv16n1Ab174
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
Abstract
Advancement in vitrification of in vitro-produced bovine embryos will benifit the cattle breeding and production industry. The objective was to evaluate whether bilateral (double) embryo transfers (ET) can improve pregnancy rate compared to ipsilateral (single) transfers. Bovine cumulus-oocyte complexes collected from slaughterhouse ovaries were matured for 20–22 h, and subsequently subjected to a standard Brackett and Oliphant in vitro fertilization (IVF). Six hours after IVF, embryos denuded of cumulus were cultured in defined CR1 medium supplemented with essential and non-essential amino acids (CR1aa), plus 6 mg mL−1 BSA for 2 days at 39°C under 5% CO2, 5% O2 and 90% N2, and then cultured in CR1aa medium supplemented with 7.5% FBS for a further 5 days on bovine cumulus monolayers. Expanded blastocysts with tighter compaction of the inner cell mass (quality 1) were selected on Day 7 for cryopreservation via modified solid surface vitrification (Dinnyes et al., 2000 Biol. Reprod. 513–8). Vitrification solution contained HEPES-buffered TCM199 supplemented with 20% FBS, ethylene glycol and dimethylsulphoxide. A droplet of 1–2 μL vitrification solution containing 4–5 blastocysts was dropped directly onto a cooled surface within 30 s after 3-min incubation in equilibration solution. Prior to ET, embryos were warmed and subsequently washed several times in 0.25 M sucrose rehydration solution and M199 + 7.5% FBS medium. The warmed embryos from initial trials were cultured for 2 to 72 h to evaluate their viability after vitrification. During ET trails, vitrified embryos were loaded into transfer straws (one embryo per straw) after warming. The treatments were as following, (1) single transfers, one embryo was transferred into the horn ipslateral to CL; (2) double transfers, one embryo was transferred by non-surgical means into each uterine horn of a synchronous recipient on Day 7. ET trails were conducted in both the USA (double transfers) and China (single v. double transfers). Pregnancy was determined by palpation per rectum around Day 70 after transfer. The data were compared by Student’s t-test. The survival rate of vitrified IVF embryos reached as high as 91.4% (n = 256) 2 h post-warming, and hatching rate was 70.7% (n = 154) 72 h after culture in vitro, respectively. The data (Table 1) show that double transfers resulted in a significantly higher pregnancy rate than did single transfers (P < 0.05). With double transfers, a higher pregnancy rate was achieved in the USA than in China (76.2% v. 45.6%, P = 0.079). This study confirms that double embryo transfers can improve the pregnancy outcome after ET, perhaps because bilateral placement of embryos may increase embryonic signals to the maternal environment. Further evaluation of gestation length, single/twin conception and calving difficulty is under investigation.
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