Dimethyl sulfoxide and glycerol as cryoprotectant agents of stallion semen: effects on blastocyst rates following intracytoplasmic sperm injection of IVM equine oocytes*
Nancy L. Cook
A B E , Keith R. Masterson B , David Battaglia B , Rick Beck C and Elizabeth S. Metcalf B D
+ Author Affiliations
- Author Affiliations
A Advanced Equine Reproduction, 1123 Fredensborg Cyn Rd, Solvang, CA 93463, USA.
B Andrology Division, Department of Obstetrics and Gynecology, Oregon Health and Science University School of Medicine, 3303 SW Bond Ave, CH10F, Portland, OR 97239, USA.
C In-Foal, Inc., 39185 Diamond Valley Rd, Hemet, CA 92543, USA.
D Honahlee, PC, 14005 SW Tooze Rd, Sherwood, OR 97239, USA.
E Corresponding author. Email: nlcook4aer@gmail.com
Reproduction, Fertility and Development 32(3) 253-258 https://doi.org/10.1071/RD19266
Submitted: 9 July 2019 Accepted: 16 October 2019 Published: 9 December 2019
Abstract
Numerous variables affect in vitro blastocyst development following intracytoplasmic sperm injection (ICSI). The paternal factor is affected by initial semen quality, processing techniques and final selection of individual spermatozoon for injection. This study investigated whether there was an effect of sperm cryoprotectant agent (CPA) on equine in vitro blastocyst production, and reviews recent developments examining how processing equine semen affects ICSI outcomes. Single ejaculates from five stallions were collected and processed in a freezing extender containing either 1 M dimethyl sulfoxide (DMSO) or 3.5% glycerol. Immature equine oocytes were obtained from ovarian follicles of mares during diestrus by transvaginal aspiration (n = 128). After in vitro maturation, MII oocytes (n = 90) were fertilised by ICSI with thawed stallion spermatozoa (n = 45 in both the DMSO and glycerol groups). The embryo cleavage rate was greater in the DMSO than glycerol group (73.3% vs 46.7% respectively; P = 0.0098), but the blastocyst development rate per fertilised oocyte was similar between the two groups (28.9% vs 15.6% respectively; P = 0.128), as was the blastocyst production rate per cleaved embryo (39.4% vs 33.3% respectively; P = 0.653). In this study, cryopreservation of equine spermatozoa in 1 M DMSO was correlated with significantly higher cleavage rates in IVM oocytes fertilised by ICSI compared with spermatozoa cryopreserved using 3.5% glycerol. Although not statistically significant in this small number of stallions, increased blastocyst production and individual stallion variability was observed among CPA treatments. This warrants further critical examination of cryoprotectants used in equine sperm subpopulations used for ICSI in a larger number of stallions.
Additional keywords: assisted reproductive technology, cleavage, IVF.
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