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Sperm survival kinetics in different types of bull semen: progressive motility, plasma membrane integrity, acrosomal status and reactive oxygen species generation

Mushtaq Ahmad A B , Nasim Ahmad B , Amjad Riaz B and Muhammad Anzar A C D

A Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7S 1K9, Canada.
B Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan.
C Agriculture and Agri-Food Canada, Saskatoon Research Center, Saskatoon, SK S7N 0X2, Canada.
D Corresponding author. Emails: muhammad.anzar@agr.gc.ca; muhammad.anzar@usask.ca

Reproduction, Fertility and Development - http://dx.doi.org/10.1071/RD13400
Submitted: 4 June 2013  Accepted: 19 January 2014   Published online: 28 February 2014


 
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Abstract

This study was designed to compare the kinetics of sperm survival in different types of bull semen. Fresh ejaculates from four bulls were pooled, diluted in Tris-citric acid-egg yolk-glycerol extender, cooled to 4°C, frozen in LN2 and thawed at 37°C. Fresh, diluted, cooled and frozen–thawed semen were incubated at 37°C, and evaluated at 0, 2, 4, 6, 12 and 24 h after the beginning of incubation. In Experiment 1, progressive sperm motility, normal acrosomes and plasma membrane integrity and asymmetry were determined. In Experiment 2, generation of superoxide anion (O2) along with plasma membrane permeability and generation of hydrogen peroxide (H2O2) along with plasma membrane integrity were assessed. In Experiment 1, frozen–thawed semen had shorter survival times for progressive sperm motility, and spermatozoa with intact plasma membranes and acrosomes (IPM-IACR) as compared with other types of semen (P < 0.05). Fresh spermatozoa underwent a necrotic pathway, diluted and cooled spermatozoa underwent an apoptosis-like pathway and frozen–thawed spermatozoa underwent both necrotic and apoptosis-like pathways. In Experiment 2, spermatozoa in all four types of semen exhibited O2•– generation and increased plasma membrane permeability, and became necrotic without H2O2 generation during incubation (P < 0.05). In conclusion, frozen–thawed semen had shorter sperm longevity, which has important implications relating to the timing of artificial insemination. Different types of semen followed different death pathways. During incubation, spermatozoa in all types of semen generated O2•–, which increased the permeability and compromised the integrity of the plasma membrane.

Additional keywords: cryopreservation, hydrogen peroxide, membrane asymmetry, membrane permeability, superoxide anion.


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