Fertilizing ability of Pinzgau bull sperm in vitro after cryostorage

Abstract

The aim of the study was to evaluate fertilizing ability of Pinzgau bull sperm after different periods of cryostorage. The sperm samples from 16 bulls were arranged into the three groups according to cryostorage period as follows: less than 7 years (group 1), 7 to 13 years (group 2) and 14 or more years (group 3). Straws with frozen sperm doses were thawed in a water bath and the motile fraction of sperm was selected by modified swim-up method. Motile spermatozoa (2 x 106 .ml-1) were co-incubated with matured bovine oocytes in fertilization drops (IVF-TALP medium) under mineral oil at 39 °C in presence of 25 μg/ml heparin for 20 hours. Totally, 739 oocytes were used in the in vitro fertilization test. Following fertilization the presumptive zygotes were stained with DAPI nuclear stain and the status of chromatin was examined under fluorescence microscope. Penetration of the sperm head into the ooplasm of oocyte and fertilization (formation of pronuclei) were evaluated. No significant difference in fertilizing ability among the experimental groups was found. The rates of penetrated plus fertilized eggs for group 1, 2 and 3 were 79.31, 76.40 and 76.14 %, respectively. Significantly higher pronuclear formation rate was observed in the group 1, where 72.9 % of eggs had two visible pronuclei at 20 hours following incubation with sperm compared to 65.72 and 47.21 % in groups 2 and 3, respectively. Also, 4 % of fertilized eggs from the group 1 reached syngamy of both pronuclei, probably due to a faster formation of pronuclei, whilst no syngamy was observed in groups 2 and 3. In conclusion, period of cryostorage had no direct influence on penetrating and fertilizing ability of Pinzgau bull sperm doses. However, it is assumed that cryostorage length can affect the speed of pronucleus formation.