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Short Communication · Volume 54, Issue 5, 19 July 2022 · Open Access

Effect of Glutathione and L-Cysteine Supplementation on the Quality of Frozen Beetal Buck Spermatozoa

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Affiliations & Notes ⌄
  1. 1Animal Reproduction and Genetics Program, Animal Sciences Institute, National Agricultural Research Centre, PARC, Islamabad 44000 * Corresponding author: hammad.fayyax@gmail.com
Article Info ⌄
Journal:
Pakistan Journal of Zoology
Published:
19 July 2022
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From the article

  • This is the very first study to evaluate the combined effect of GLU and CYS on the post-thaw quality of buck spermatozoa.
  • In this study, supplementation of two antioxidants in different combinations significantly improved the sperm quality in terms of motility, PMI, and livability.
  • Our results indicated that the optimum combination of GLU and CYS in freezing extender can protect buck sperm from cryoinjuries.
  • In conclusion, this study exhibited that glutathione and L-cysteine at the concentration of 1mM and 10mM may be the optimum combination in extender to ameliorate cryopreservation of Beetal buck spermatozoa.

Selected from the article's own text.

Abstract

This study was designed to assess the cryosurvival of beetal buck spermatozoa with the supplementation of Glutathione (GLU) and L-cysteine (CYS) in the extender. In the first experiment, semen samples from each of the five mature bucks were pooled and diluted in Tris-citric acid (TCA) extender containing Glutathione (0.5mM, 1mM), L-cysteine (5mM CYS, 10mM CYS), and control. In experiment 2, semen samples (n=5) were diluted in TCA extender containing differential doses of combined antioxidants (0.5 mM GLU+ 5mM CYS, 0.5mM + 10mM CYS, 1mM GLU + 5mM CYS, 1mM GLU + 10mM CYS and Control). At post-thawing, progressive motility, plasma membrane integrity (PMI), and livability of buck spermatozoa were higher (P< 0.05) with 1mM GLU and 10mM CYS as compared to control. In the second experiment, post-thaw progressive motility, PMI, and livability were higher (P< 0.05) with 1mM GLU + 10mM CYS and 1mM GLU + 5mM CYS compared to other doses of GLU+CYS and control. We concluded that optimized supplementation of Glutathione and L-cysteine in TCA extender ameliorated frozen-thawed quality of beetal buck spermatozoa.

Materials and methods

Semen from Five healthy beetal bucks each buck was collected twice a week (for 6 weeks) using an artificial vagina at 42°C. Collected samples with more than 60% motility and 1.5 × 109 sperm concentration per mL were pooled and subdivided into five aliquots. Each aliquot was diluted with tris-citric extender [Tris 3.07 g/100mL, citric acid 1.64 g/100mL, fructose 1.26 g/100mL, 1000 IU/mL streptomycin sulfate, 5 mL of glycerol (v/v); 15mL of egg yolk (v/v)] containing different concentrations of glutathione (0.5mM GLU, 1mM GLU) and L-cysteine (5mM CYS, 10mM CYS) alone and in combination viz. 0.5mM GLU+5mM CYS, 0.5mM GLU+10mM CYS, 1mM GLU+5mM CYS, 1mM GLU+10mM CYS.

For freezing, extended sperm suspensions were cooled from 37°C gradually to 4°C in 90 min and equilibrated at 4°C for 2 h in the cold cabinet and packaged in 0.54 mL French straws (50 million sperm/straw). Packaged straws were placed horizontally on racks in cold cabinet then held at liquid nitrogen vapors 4 cm higher than the level of liquid nitrogen for 10 min followed by immersing in liquid nitrogen at -196°C for storage.

For post-thaw sperm evaluation assays, motility, plasma membrane integrity (PMI), and livability were considered as criteria. Motility analysis was performed according to Gillan et al. (2008). PMI of spermatozoa was analyzed by hypo-osmotic swelling (HOS) assay as described by Correa and Zavos (1994). Post-thaw livability was assessed by eosin–nigrosine staining. A total of 200 spermatozoa were counted in different fields of the phase-contrast microscope. Spermatozoa gaining partial or complete stain were recorded non-viable, and spermatozoa with unstained heads were considered as viable.

All data were expressed as mean with the standard error of the mean (SEM) among different treatment groups. One-way analysis of variance (ANOVA) was performed for data analysis and the Tukey test as post-hoc were performed on SPSS (version 20.0). The probability level of P < 0.05 was considered significant.

Results

Table I shows the effects of differential concentrations of GLU and CYS (combined or separate) on post-thaw progressive motility, PMI, and livability. All sperm parameters were higher (P<0.05) in 1mM GLU, 5mM CYS and 10mM CYS groups than control. The highest sperm parameters were observed in the 10mM CYS group. The highest motility was observed in 1mM GLU + 10mM CYS and 1mM GLU + 5mM CYS groups. A similar trend was seen in the case of livability and PMI, in which high concentration groups exhibited significantly higher results as compared to control and other groups.

Discussion

The plethora of ROS during the freeze-thaw process affects sperm quality parameters, compromising its survival, declining its internal antioxidant defense system, and fertilization potential. Frozen sperm is more prone to lipid peroxidation as compared to fresh sperm.

This is the very first study to evaluate the combined effect of GLU and CYS on the post-thaw quality of buck spermatozoa. In this study, supplementation of two antioxidants in different combinations significantly improved the sperm quality in terms of motility, PMI, and livability. Our results are in agreement with the study conducted in bovine semen . However, these findings contradicted the studies conducted in ram and buck in which inclusion ratios of GLU were high (5mM and 10mM). This may be due to mitochondrial DNA degradation is associated with high GLU concentration . Also, it has been studied that the high levels of antioxidants negatively affect plasma membrane functionality . Therefore, improving the effects of GLU on PM and other parameters may be due to its low inclusions which affected the functionality of the plasma membrane. Results of our study agreed with the findings of Zhandi et al. (2015) who reported that low concentrations of GLU in freezing extender improved the frozen-thawed sperm qualities of buck.

Cysteine is a well-known ROS scavenger. In this study addition of CYS in extender also positively affected motility, plasma membrane integrity, and livability agreeing with the studies performed in bull ram , and buck .

However, this is the first study on the combined effect of two related antioxidants tested on frozen-thawed buck sperm quality. Our findings were consistent with the same study in ram. This discrepancy may be due to the difference in antioxidant concentrations used and semen characteristics of the two species. Our results indicated that the optimum combination of GLU and CYS in freezing extender can protect buck sperm from cryoinjuries.

In conclusion, this study exhibited that glutathione and L-cysteine at the concentration of 1mM and 10mM may be the optimum combination in extender to ameliorate cryopreservation of Beetal buck spermatozoa.

Statement of conflict of interest

The authors have declared no conflict of interests.

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