Elsevier

Small Ruminant Research

Volume 190, September 2020, 106169
Small Ruminant Research

Supplementation of soybean lecithin-based cryopreservation medium with glutathione: Fertility and flow cytometry study of ram frozen-thawed semen

https://doi.org/10.1016/j.smallrumres.2020.106169Get rights and content

Highlights

  • The effects of reduced glutathione was evaluated as a cryo-additive for ram semen cryopreservation.

  • Using 4 mM reduced glutathione improved quality indicators of ram semen during cryopreservation.

  • Lipid peroxidation was reduced using 4 mM reduced glutathione in soybean lecithin-based extender.

Abstract

This study was aimed to evaluate the beneficial effects of reduced glutathione antioxidant in soybean lecithin-based cryopreservation medium on fertility and quality parameters of ram semen. In experiment 1, semen samples were collected from five rams and diluted in the freezing extender contained various concentrations of glutathione as follows: extender without glutathione (G0), extender containing 0.5 mM (G0.5), 1 mM (G1), 2 mM (G2), 4 mM (G4) and 8 mM (G8) glutathione. Motility, membrane functionality, morphology, mitochondrial activity, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation and reactive oxygen species (ROS) concentration were assessed after thawing. In experiment 2, pregnancy, parturition, lambing and twining rates were evaluated after artificial insemination with frozen-thawed semen. In results, G4 showed higher (P ≤ 0.05) total and progressive motility, membrane functionality, acrosome integrity, and lower (P ≤ 0.05) lipid peroxidation and ROS concentration compared to the other groups. Mitochondrial activity and viability were higher (P ≤ 0.05) and late apoptotic-like changes were lower (P ≤ 0.05) in G2 and G4 compared to the other groups. DNA fragmentation and late apoptotic-like changes were higher (P ≤ 0.05) in G8 compared to the other groups. No significant difference was observed among groups in cases of morphology and reproductive performance. In conclusion, supplementation of cryopreservation medium with 4 mM reduced glutathione improves the quality parameters of ram spermatozoa but it was not reflected to the reproductive performance.

Introduction

Cryopreservation is an effective technique to preserve spermatozoa for a long time. However, freeze-thaw processes produce physical and chemical stresses on spermatozoa that reduces sperm viability and fertility (Evans, 1991). Plasma membrane of spermatozoa being rich in polyunsaturated fatty acids is susceptible to oxidative damage during freeze-thaw process (Alvarez and Storey, 1983). To overcome this problem, supplementation of cryopreservation medium with different antioxidants was investigated in different species such as glutathione in the bulk (Salmani et al., 2013), alpha-tocopherol in bull (Motemani et al., 2017) and CoQ10 in rooster (Masoudi et al., 2018).

Soybean lecithin (SL) is an extracellular cryoprotectant, which has recently been successfully applied for semen freezing in bull (Aires, 2003), human (Reed et al., 2009), buffalo (Akhter et al., 2012), bulk (Salmani et al., 2013) and ram (Masoudi et al., 2019a). Supplementation of extender containing SL with glutathione as an antioxidant could be helpful to preserve ram post-thawed semen quality. Glutathione is the major non-protein thiol compound in animal cells, which participates in a number of cellular functions such as amino acids transportation, synthesis of DNA and protein, reduction of disulfide bonds and protection against oxidative stress. It has been displayed that the sulfhydryl groups of reduced glutathione confer protection against cell damage by oxidants, free radicals and electrophiles (Agarwal et al., 2006).

This study was aimed to investigate the effect of different concentrations of reduced glutathione in SL-based extender on ram post-thaw semen quality in sheep.

Section snippets

Chemicals and ethics

All chemicals were purchased from Merck (Darmstadt, Germany) and Sigma (St. Louis, MO, USA) companies. This study was approved by Research Ethics Committees of Animal Science Research Institute of Iran (ASRI-2018−0661).

Semen collection and extender preparation

Semen samples were collected via artificial vagina twice a week from five mature Zell rams (3–4-years, 70−75 kg) in six replicates twice a week during breading season [Tehran, Iran (35.68 °N, 51.38 °E), average day temperature: 32 °C, day length: 13 h, humidity: 30 %]. Samples

Motility characteristics, membrane functionality and morphology

The effects of different concentrations of glutathione on motility characteristics of ram frozen-thawed semen is presented in Table 1. Sperm total motility and progressive motility in G4 were greater than other groups. On the other hand, G8 showed the least total motility and progressive motility compared to other groups. For VAP, VSL, VCL, ALH, BCF and LIN, no significance difference was observed among the different groups.

Fig. 1 shows the effects of different concentrations of glutathione on

Discussion

The purpose of this study was to investigate the effects of different concentrations of reduced glutathione in soybean lecithin-based extender on quality parameters and fertility of frozen-thawed ram spermatozoa.

Previous studies presented freezing process decreased semen glutathione concentration in bull (80 %) (Bilodeau et al., 2000), boar (32 %) (Gadea et al., 2004) and human (63 %) (Gadea et al., 2011), while supplementation of freezing media via glutathione enhanced the semen quality in

Conclusion

In conclusion, supplementation of ram plant-based cryopreservation medium with reduced glutathione preserved quality parameters of frozen-thawed semen in this study. Therefore, reduced glutathione could be considered as a suitable cryo-additive for cryopreservation of ram semen in SL-based freezing media.

Declaration of Competing Interest

The authors have no conflicts of interest to disclose.

Acknowledgement

The authors would like to acknowledge the financial support of Iran National Science Foundation under grant number of 95007081.

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