37 The comparison of different freezing methods and thawing temperatures on Windsnyer boar semen quality
M. A. Thema A B , M. L. Mphaphathi B C , M. R. Ledwaba A B and T. L. Nedambale A BA Tshwane University of Technology, Department of Animal Science, Pretoria, Republic of South Africa
B Agricultural Research Council, Animal Production, Germplasm, Conservation, Reproductive and Biotechnologies, Irene, Republic of South Africa
C Department of Animal, Wildlife and Grassland Sciences, University of the Free State, Bloemfontein, Republic of South Africa
Reproduction, Fertility and Development 34(2) 253-254 https://doi.org/10.1071/RDv34n2Ab37
Published: 7 December 2021
© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
The present study aimed to evaluate the effect of different freezing methods (slow freezing and liquid nitrogen vapour) and thawing temperatures (5, 18, 37, and 40°C) on post-thawed sperm motility and morphology of Windsnyer boar semen. Total of 18 ejaculates (6 replication/boar) were collected from three Windsnyer boars of proven fertility with the use of hand-gloved technique method, twice per week. Semen was extended with Beltsville thawing solution (BTS), held at 18°C for 3 h, and centrifuged. Following centrifugation, seminal plasma was discarded, and semen precipitate was resuspended with fraction A (20% egg yolk and BTS) and cooled at 5°C for 1 h. Cooled semen was further diluted with fraction B (20% egg yolk, BTS, and 16% glycerol) and loaded into straws. Semen straws were equally allocated for liquid nitrogen (LN2) vapour and slow-freezing (controlled rated) methods. During LN2 vapour method, semen straws were placed 3 cm above the LN2 level for 20 min in a polystyrene box. During controlled rated freezing method, semen straws were transferred to the controlled rated freezer set at −3°C min−1 + 5°C to −5°C; −30°C min−1 from −5°C to −15°C and −50°C min−1 from −15°C to −60°C. Frozen semen straws were plunged immediately into the LN2 tank (−196°C) for storage and further analysis. Thawing was accomplished by immersing the semen straws in warm (37°C) water for 1 min or 40°C for 30 s or 18°C for 3 min and 5°C for 5 min. Sperm morphology and motility percentages were evaluated following thawing. Sperm motility was evaluated with the use of Sperm Class Analyser® system (Microptic). The nigrosine-eosin staining solution was mixed with semen inside the Eppendorf tube, smeared across the microscope glass slide, and air-dried at room temperature. Sperm morphology was evaluated at 100× magnification under the microscope. A total of 200 sperm per slide/treatment was evaluated for sperm morphology. Data were analysed using one-way ANOVA. The sperm total motility (93.9%) and progressive motility (47.7) were recorded on raw/fresh semen. Semen straws frozen with LN2 vapour recorded highest post-thaw sperm total motility and progressive motility at 37°C (13.9; 8.9%), 5°C (8.9; 6.0%), 18°C (17.4; 7.9%), and 40°C (26.8; 11.1%) compared with the controlled rated freezing treatment (P < 0.05). A difference was found between post-thawed average normal live sperm percentages of LN2 vapour (5–40°C; 48.3%) and controlled-rated (5–40°C; 32.1%; P < 0.05). The results obtained indicated that thawing temperature influenced motile and live sperm percentages (P < 0.05). The values of the live, motile, and velocity sperm parameters were higher in the semen frozen with the aid of LN2 vapour compared with the controlled rated freezer (P < 0.05). In conclusion, Windsnyer boar semen thawed at 40°C using LN2 vapour method resulted in better sperm quality.
