Effects of repeated freeze-thawing of bovine epididymal sperm on vitro embryo development

Abstract

Successfully refreezing thawed semen could have important benefits to future animal reproductive technologies. Being able to refreeze mammalian sperm could improve chances for fertilization when sperm are cryopreserved in bulk, in limited supply, highly valuable or mistakenly thawed. This study examined the use of Biladyl® (BIL) and modified Tyrode s lactose medium (MTL) containing 20% fetal bovine serum (FBS) and ethylene glycol for refreezing bovine epididymal sperm for embryo production by in vitro fertilization (IVF) procedures. Sperm collected by retrograde flushing were subjected to freeze-thaw cycle 1 using either BIL 1 or MTL 1 (50 straws/treatment). Thirty straws of each of the cryo-preserved sperm samples were thawed and motile sperm harvested by swim-up procedure before subjecting the sperm to freezing cycle 2 in the same BIL (BIL 2) and MTL (MTL 2) extenders. In vitro matured bovine oocytes were subjected to a standard IVF procedure with the sperm from freeze-thaw cycle 1 and freezethaw cycle 2. The presumptive 1,301 zygotes were cultured in modified CR1aa supplemented with 3 mg/ml of BSA and 5% FBS for up to 13 days. Embryo development was assessed for cleavage, morula and blastocyst formation, blastocyst expansion and hatching. Cleavage, morula and blastocyst rates were higher (P<0.05) in the control (BIL 1) than in the treatments (BIL 2, MTL 1 and MTL 2). Embryo expansion and/or hatching (days 7-13), however, were not different (P>0.05) in BIL 1, MTL 1 and MTL 2. However, expansion and/or hatching on the same days were lower in BIL 2 than the other treatments. It was concluded that bovine epididymal sperm can successfully undergo two freeze-thaw cycles in either Biladyl® or modified Tyrode's lactose with FBS and ethylene glycol and can be used for potential embryo production.