Transfer of cryopreserved equine embryos to progestin‐treated ovariectomised mares

Abstract

SummaryGood to excellent quality embryos between 160 and 980 μm in diameter were recovered 6.5 to 7 days after ovulation and washed in Dulbecco's phosphate‐buffered saline plus 10 per cent foetal calf serum (PBS). Embryos were frozen in 0.5 ml French straws and seeded at −6°C. Embryos of 200 μm diameter or less were placed in 10 per cent glycerol in PBS and cooled from −6 to −30°C at 0.3°C/min, then to −33°C at 0.1°C/min. They were then plunged into liquid nitrogen. Embryos larger than 200 μm were frozen either 1) in 10 per cent glycerol plus 8.6 per cent sucrose with cooling at 0.5°C/min from −6 to 25°C followed by plunging, or 2) in 15 per cent glycerol plus 15 per cent sucrose and placed between gel‐containing cold packs in a household freezer at — 25°C for 30 mins followed by plunging. Embryos were thawed for 20 sees in a 37°C water bath or held in air for 20 sees prior to thawing in a 37°C water bath for 20 sees. Glycerol from embryos frozen in the household freezer was removed in steps of 12, 9, 6, 3 and 0 per cent glycerol, all with 10 per cent sucrose, 6 mins/step. Glycerol was removed similarly for other treatments but the first two steps were omitted. Embryos were evaluated morphologically (1 = excellent, 5 = severely degenerate) prior to transfer to the uterus of ovariectomised mares between 24th November and 9th January. Mares were given 0.044 mg/kg bodyweight altrenogest for at least six days. Four pregnancies were established after transferring eight embryos 200 μm or less, demonstrating that progestin‐treated, ovariectomised mares can serve as recipients for frozen embryos during the non‐breeding season. However, only two of 24 larger embryos resulted in pregnancy. No difference was detected in the two methods of cooling, but post‐thaw embryo quality was markedly improved (p<0.01) by holding straws in air for 20 sees prior to placing them in a water bath.