57 Effect of Seminal Plasma Removal on Sperm Characteristics and Mitochondrial Membrane Following Cryopreservation of South African Indigenous Buck Semen

Abstract

The removal of seminal plasma has a significant effect on semen characteristics. The aim of the study was to evaluate the effect of seminal plasma removal on sperm characteristics following semen dilution with Triladyl® or Bioxcell® extenders during cryopreservation. Semen samples were collected from 6 matured South African indigenous bucks for a period of 8 weeks by means of electro-ejaculation. Semen samples were pooled and then divided into 4 aliquots (Triladyl® -washed and non-washed or Bioxcell® -washed and non-washed) and diluted (1:4 vol/vol). Assessment of sperm motility characteristics was done by computer-aided sperm analysis (CASA) technology. Evaluation of mitochondria membrane integrity was done using JC-1 staining solution. Triladyl® and Bioxcell® washed semen sample groups were centrifuged at 1500 × g for 10 min, and seminal plasma was separated from sperm pellet using 1-mL plastic pipettes. After dilution of all semen sample groups, they were cooled by placing tubes into water (25°C) and then immediately placed in a 5°C fridge for equilibration for 2 h. At the end of equilibration period, all aliquot semen sample (final dilution concentration of 150 × 106 sperm/mL) groups were loaded into straws (0.25 mL) per treatment groups and placed horizontally 5 cm above the liquid nitrogen vapour for 10 min. At the end of freezing process, all semen straws per group were plunged directly into liquid nitrogen (−196°C) and stored until thawed. Frozen-thawed semen samples per treatment were analysed for sperm motility characteristics by CASA. JC-1 staining solution was also used during evaluation of mitochondria membrane integrity of frozen-thawed semen samples per treatment. Significant differences (P < 0.05) among mean values of semen parameters were determined by Tukey’s method. Sperm total motility rate of non-washed semen in Bioxcell® (85.0 ± 3.4) and Triladyl® (73.9 ± 13.8) was significantly reduced (P < 0.05) by cryopreservation compared with fresh (98.9 ± 1.2) semen sample. There was greater (P < 0.05) sperm mitochondrial membrane damage due to cryopreservation in non-washed semen group extended with Bioxcell® (50.2 ± 20.1) compared with semen extended with Triladyl® (31.3 ± 26.8) and fresh (16.6 ± 14.2) semen sample. Sperm total motility rate in Bioxcell® (68.2 ± 13.5) and Triladyl® (63.1 ± 15.1) groups on the non-washed semen were significantly reduced (P < 0.05) following cryopreservation, compared with fresh (98.3 ± 2.7) semen sample. The percentage of sperm with damaged mitochondria membrane in washed semen was significantly reduced (P < 0.05) in Triladyl® (21.6 ± 16.8) group compared with Bioxcell® (34.7 ± 14.9) group and fresh (35.0 ± 20.8) semen sample. In conclusion, seminal plasma removal reduced sperm motility rate in both extenders (Bioxcell® and Triladyl®) following post-thaw. In addition, sperm mitochondria membrane damage was higher in non-washed semen samples extended with Bioxcell® extender.