Flow cytometric and microscopic evaluation of post-thawed ram semen cryopreserved in chemically defined home-made or commercial extenders
M. Emamverdi A , M. Zhandi A D , A. Zare Shahneh A , M. Sharafi A , A. Akhlaghi B , M. Khodaei Motlagh C , F. Dadkhah A and N. Dadashpour Davachi A
+ Author Affiliations
- Author Affiliations
A Department of Animal Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.
B Department of Animal Science, College of Agriculture, Shiraz University, Shiraz, Iran.
C Department of Animal Science, Faculty of Agriculture, Arak University, Arak, Iran.
D Corresponding author. Email: mzhandi@ut.ac.ir
Animal Production Science 55(4) 551-558 https://doi.org/10.1071/AN13215
Submitted: 26 May 2013 Accepted: 27 November 2013 Published: 14 January 2014
Abstract
The present study was designed to determine the effect of three different extenders on ram sperm quality during a freeze–thawing procedure using flow cytometric and microscopic evaluations. Several in vitro qualitative analyses of post-thawed sperm parameters including motility and velocity parameters, plasma membrane functionality, total abnormality, capacitation status, acrosome integrity, mitochondrial activity and apoptosis features were considered. In the breeding season, seven ejaculates from each Zandi ram were collected routinely twice a week. Following semen collection, samples were pooled and equally divided into three aliquots. Each aliquot was diluted and frozen with one of the following extenders: (1) Tris-based extender containing 1.5% (w/v) soybean lecithin (TSL), as a chemically defined extender, (2) Bioxcell, a commercial soybean lecithin-based extender, and (3) Tris-based extender containing 20% (v/v) egg yolk (TEY). The results of the present study indicated no differences in total [TSL (55.8 ± 2.02%) vs TEY (50.2 ± 2.02%; P < 0.05)] and progressive motility of spermatozoa [TSL (26.2 ± 1.36%) vs Bioxcell (22.4 ± 1.36%; P < 0.05)]. Semen freezing by means of TSL resulted in a higher percentage of live spermatozoa (39.42 ± 1.81%) compared with TEY (29.17 ± 1.81%; P < 0.05), and a higher percentage of functional plasma membrane (50.8 ± 192%) compared with TEY (44 ± 1.92%) and Bioxcell (38.8 ± 1.92%; P < 0.05). The effect of extenders on sperm capacitation status showed that the percentage of post-thawed capacitated spermatozoa was higher in TEY (61.9 ± 1.48%) compared with that in TSL (56.6 ± 1.48%; P < 0.05). The evaluation of post-thawed spermatozoa indicated that the percentage of live spermatozoa with active mitochondria was higher in TSL (53.05 ± 2.31%) compared with Bioxcell (45.92 ± 2.31; P < 0.05) and the percentage of intact acrosome spermatozoa was higher in TSL (84.55 ± 2.51%) compared with TEY (74.91 ± 2.51%; P < 0.05). The use of TSL and Bioxcell extenders reduced the percentage of apoptotic spermatozoa (40.82 ± 2.07% and 42.22 ± 2.07%, respectively), compared with TEY (51.34 ± 2.07%; P < 0.05). Post-thawing dead spermatozoa were increased when semen was frozen by Bioxcell (25.69 ± 1.28%). The results of this study showed that TSL extender may provide stabile milieu and conditions for ram sperm cryopreservation compared with Bioxcell and TEY extenders. Whether TSL extender can improve the artificial insemination results remains, however, an open question.
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