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