Vitrification and direct warming in iso-osmotic holding medium of equine in vitro embryos supports competitive pregnancy rates

Improved efficiency of ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) resulted to a steep increase in the number of in vitro produced (IVP) equine embryos worldwide. These IVP blastocysts are routinely cryopreserved and both slow freezing and vitrification result in high pregnancy rates. The use of minimal volume cryodevices and fast, multistep warming protocols can be challenging for veterinary practitioners receiving vitrified embryos, but direct warming in embryo holding medium supports embryo viability (Canesin et al. Theriogenology. 2020;151:151-158). The aim of this study was to compare pregnancy rates in a clinical OPU-ICSI program using a three-step vitrification/warming protocol vs. a commercial vitrification kit and direct warming in embryo holding medium. Embryos were produced in autumn and winter of 2021-2022 by OPU-ICSI (Papas et al. Animals. 2021;11:2004). Day of vitrification (7-12 post ICSI) was similar for both vitrification groups. Blastocysts were vitrified in a minimal volume on a Cryolock (Irvine Scientific) following two methods: (1) a three-step protocol (n=21): holding in basal vitrification medium (BVM; DMEM-F12 with 20% FCS), vitrification in BVM with 1.5M ethylene glycol (5 min), followed by BVM with 7M ethylene glycol and 0.6M galactose (40 s), and warming in warming medium (WM; DPBS with 0.1% glucose,36 mg/L pyruvate, and 0.4% BSA) with 0.3M sucrose (1 min), followed by WM with 0.15M sucrose (5 min) and holding in WM (5 min), all performed at 38.2°C (Choi and Hinrichs. Theriogenology. 2017; 87:48-54); (2) direct warming (n=21): vitrification at 23°C with a commercial VIT-Kit (Minitube, Germany) in VS1 (5 min), VS2 (5 min) and VS3 (40 s), containing increasing concentrations of glycerol and/or ethylene glycol, based on Eldridge-Panuska et al. (Theriogenology. 2005; 63:1308-1319), and direct warming at 38°C for 5 min in 4 ml of Emcare holding medium (Spervital, The Netherlands). For both protocols, warmed embryos were washed four times in Emcare and transferred immediately to a day 4 recipient mare. The effect of vitrification method on pregnancy rate at 14 days and 25 days was determined using binomial logistic regression analyses. Pregnancy rates at 14 and 25 days were respectively 66.7% and 47.6% for the three-step method and 76.2% and 52.4% for the direct warming. No difference was found between the two methods (p=0.496 and p=0.882, respectively). In conclusion, vitrification and warming of IVP blastocysts using commercial media and direct warming supports pregnancy and provides an easy method for embryo warming and transfer by equine veterinary practitioners.