38 Protective effect of glutathione concentrations on cryopreserved Kolbroek boar sperm
L. D. Sehlabela A B , M. L. Mphaphathi A , T. R. Netshirovha C and T. L. Nedambale A BA Agricultural Research Council, Animal Production, Germplasm Conservation and Reproductive Biotechnologies, Pretoria, RSA
B Tshwane University of Technology, Department of Animal Sciences, Pretoria, RSA
C University of South Africa, Department of Agriculture and Animal Health, Pretoria, RSA
Reproduction, Fertility and Development 35(2) 145-145 https://doi.org/10.1071/RDv35n2Ab38
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
During the process of boar semen cryopreservation, damaged sperm cells, impaired sperm structure, and energy metabolism changes occur due to oxidative damage. This affects the plasma membrane function and malondialdehyde (MDA) content of sperm. As an endogenous antioxidant in animals, glutathione (GSH) can significantly improve the quality of frozen-thawed boar sperm when added to the freezing diluent. The aim of the study was to determine the effect of glutathione (0, 1, 5, and 10 mM) supplementation in boar sperm-freezing extender on plasma membrane and MDA content of frozen-thawed Kolbroek sperm. A total number of 18 ejaculates (6 ejaculates per boar) were collected from three boars on the same day, twice per week. The semen samples were pooled and divided into four equal fractions and centrifuged at 800 × g for 10 min. The sperm pellets were re-extended at a ratio of 1:1 with Fraction A: control (egg yolk 20% + BTS 80%), 1 mM (egg yolk 20% + BTS 78% + GSH 2%), 5 mM (egg yolk 20% + BTS 70% + GSH 10%), and 10 mM (egg yolk 20% + BTS 60% + GSH 20%) extender. After cooling at 5°C for 60 min, Fraction B extender: control (egg yolk 20% + BTS 72 % + glycerol 8%), 1 mM (egg yolk 20% + BTS 70% + glycerol 8% +GSH 2%), 5 mM (egg yolk 20% + BTS 62% + 8% glycerol + GSH 10%), and 10 mM (egg yolk 20% + BTS 52% + glycerol 8% + GSH 20%) were added into the semen samples at a ratio of 1:2. All straws (0.5 mL) were cryopreserved in parallel using an automatic controlled-rate freezer at −5°C min−1 from +4°C to −10°C; −40°C min−1 from −10°C to −100°C; −20°C min−1 from −100°C to −140°C. The frozen straws of the semen samples were then plunged directly into the LN2 tank (−196°C) for storage. After at least two days of storage, the frozen semen straws were thawed at 37°C for 10 s on air and 1 min. After thawing (37°C), the sperm plasma membrane was assessed microscopically with the use of hyper-osmotic swelling test (HOST). The MDA content was determined with the aid of spectrophotometer measured in nmol/mL. Data were analysed using one-way analysis of variance. Treatment means were compared using least significant difference t-test. The sperm membrane with the least membrane damage by HOST was recorded from 1 mM (65.0 ± 12.2%), followed by 5 mM (63.5 ± 9.1%), then 10 mM (50.3 ± 4.9%) of GSH. The group treated with 5 nmol/L (0.065) showed the lowest MDA content in all groups (P < 0.05); however, there was a significant difference in 1 mmol/L compared to the control group and 10 nmol/L on plasm membrane permeability. In conclusion, 1 mM was the optimum concentration of GSH to be added to the freezing extender for cryopreserving semen from Kolbroek boars.
The authors acknowledge the Department of Agriculture, Land Reform and Rural Development and the Agricultural Research Council for financial assistance.
