Escherichia coli membrane‐derived oxygen‐reducing enzyme system (Oxyrase) protects bubaline spermatozoa during cryopreservation

Abstract

The objective of this study was to determine the effectiveness of deoxygenation of semen extender using Escherichia coli membrane-derived oxygen scavenger (Oxyrase) on post-thaw quality of buffalo (Bubalus bubalis) spermatozoa. Sixteen semen ejaculates, four each from four bulls, were each divided into five equal fractions, diluted using Tris-egg yolk extender supplemented with different concentrations of Oxyrase (0, 0.3, 0.6, 0.9, and 1.2 U/ml), designated as treatments T1, T2, T3, T4, and T5, respectively, and cryopreserved. Immediately after thawing, Oxyrase did not improve sperm kinetics and motility; however, it improved the keeping quality (significantly lower deterioration of post-thaw sperm motility after incubation for 120 min) in T3. Further, T3 reduced (p < .05) cholesterol efflux and protected the intactness of the sperm plasma membrane. Flow cytometry with Fluo-3 AM/propidium iodide (PI) dual staining revealed the highest (p < .05) proportion of live spermatozoa with low intracellular calcium in T3. Oxyrase supplementation protected spermatozoa from premature capacitation which was confirmed by low expression of tyrosine-phosphorylated proteins (32, 75, and 80 kDa) and a relatively lower percentage of F-pattern (uncapacitated spermatozoa) in chlortetracycline assay. Importantly, the Oxyrase fortification decreased superoxide anion in a dose-dependent manner indicating reduced availability of oxygen at sperm mitochondrial level. Similarly, in Oxyrase-fortified sperm, malondialdehyde concentration, an index of lipid peroxidation, is also reduced in a dose-dependent manner. In conclusion, we demonstrate that deoxygenation of buffalo semen by Oxyrase has the potential of improving post-thaw sperm quality by overcoming the problem of cryocapacitation and oxidative damage during cryopreservation process.

Effect of mitochondria‐targeted antioxidant on the regulation of the mitochondrial function of sperm during cryopreservation

“…Superoxide anion is the principal ROS produced by mitochondria. The MitoSOX‐based assay has been used to identify sperm population that generated high superoxide after the freeze–thaw process due to the ability of MitoSOX to permeate live cells where it selectively targets mitochondria (Arjun et al, 2021; Dalal, Chandolia, Jan, et al, 2020; Patil et al, 2020). It is quickly oxidized by superoxide but not by other ROS.…”

A review on applications and toxicities of metallic nanoparticles in mammalian semen biology

“…These changes in the seminal attributes arising due to oxidative stress could be assessed by various staining procedures (Rautela et al, 2020) and computer assisted semen analysis (Singh et al, 2021). Many attempts have been done in past for the reduction of oxidative damage in the semen, namely, neutralising ROS through enzymatic, non‐enzymatic, plant‐based antioxidants or reductants (reviewed by Kumar et al, 2019; Silvestre et al, 2021; Kumar et al, 2021, 2022); or by minimising the sources like the semen radiation exposure, leucocytes and dead and defective spermatozoa to reduce the level of free radical generation (Arzondo et al, 2012; Bisla, Ramamoorthy, et al, 2020; Bisla, Rautela, et al, 2020; Bisla, Rautela, et al, 2021; Durfey et al, 2019; Durfey, Burnett, et al, 2017; Durfey, Swistek, et al, 2017; Feugang, 2017; Feugang et al, 2015; Odhiambo et al, 2014); or by using oxygen scavenger like E. coli derived enzyme oxyrase (Dalal et al, 2020; Darr et al, 2016; Dong et al, 2010; London et al, 2017; Ngou et al, 2020; Shore, 2019); or by using nitrogen gassing to reduce the oxygen tension for partially deoxygenating the extender, a source of generation of free radicals in the microenvironment of spermatozoa (Amin et al, 2018; Balamurugan et al, 2018, 2020; Bhutia et al, 2021; Katiyar et al, 2020; Kumar, Prasad, et al, 2018; Kumar, Prasad, et al, 2018; Mustapha et al, 2021; Pande et al, 2015).…”