Zinc oxide and selenium nanoparticles can improve semen quality and heat shock protein expression in cryopreserved goat (Capra hircus) spermatozoa.

BACKGROUND: Reactive oxygen species (ROS) are strongly linked with oxidative stress (OS) generated during the process of sperm cryopreservation. Indeed, cellular damage from ROS has been implicated during sperm cryopreservation which causes deterioration in sperm quality and antioxidant nanoparticles (NPs) have been successful in preventing such damage. The interaction of NPs with sperm cells has been less frequently explored in farm animals. OBJECTIVE: The present study explored the effect of NP supplementation on sperm ultrastructure, potential interaction with sperm membrane (plasma and acrosome membrane), heat shock protein (HSP) gene expression levels and sperm quality in cryopreserved buck semen. MATERIALS AND METHODS: Thirty-two (32) ejaculates were collected from four (4) adult male bucks and then diluted in Tris- citric acid- fructose- egg yolk (TCFY) extender containing the Zinc-oxide (ZnO) and Selenium (Se) NP treatments (T0: Control; TZn: 0.1 mg/mL ZnO NPs and TSe: 1 µg/mL Se NPs) after initial evaluation. Diluted semen was packed in 0.25 mL French mini straws and then stored in liquid nitrogen (LN2). Sperm parameters, lipid peroxidation (LPO) profile, sperm head morphology ultrastructural classification under transmission electron microscope (TEM), potential interaction of NPs with sperm membrane and expression of HSP genes were evaluated in the different treatment groups. RESULTS: We found a significant (p < 0.05) increase in the percentage of spermatozoa with intact plasma membrane, and intact acrosome in the ZnO (0.1 mg/mL) and Se (1 µg/mL) NP supplemented groups in comparison to the frozen control group. TEM assessment revealed no internalization of both ZnO and Se NPs into the sperm structure. Few occasional contacts of ZnO NPs with the sperm membrane and a few agglomerates of Se NPs around the area of damaged membranes were visualized. HSP70 and HSP90 mRNA levels were significantly (p < 0.001) higher in the NP supplemented groups in comparison to the control. HSP70 and HSP90 mRNA levels had a strong positive association with sperm motility and a weak to moderate association with other sperm parameters. CONCLUSIONS: Current findings indicated that ZnO NPs are more potent than Se NPs in ameliorating peroxidative damages during sperm cryopreservation, increases semen quality parameters possibly by increasing the expression levels of HSP genes in buck semen. Furthermore, NP supplementation may have a potential role in preserving sperm head ultrastructure by acting as an antioxidant and reducing OS during various degrees of cellular insults, which needs to be further explored.

In Vitro and In Vivo Studies on the Efficacy of Zinc-Oxide and Selenium Nanoparticle in Cryopreserved Goat (Capra hircus) Spermatozoa.

Different nanoparticles (NPs) are currently being investigated for their potential role as cryoprotectant during semen cryopreservation in several mammalian species. It may be possible to improve semen quality following cryopreservation by supplementation of NPs in the freezing extenders. The present study was carried out in semen collected from four (4) Assam Hill Goat bucks (10 ejaculates per buck) to investigate the effect of supplementing zinc oxide (ZnO) and selenium (Se) NPs in Tris-citric acid-fructose yolk (TCFY) extender on in vitro sperm quality and in vivo fertility rate after freeze-thawing. The size morphology and zeta potential of ZnO and Se NPs were evaluated prior to its incorporation in the freezing extender. Qualified semen samples (> 70% progressive motility) were divided into five (5) aliquots and then diluted in TCFY extender containing ZnO and Se NP supplementation at different concentrations (T0, control; T1, 0.1 mg/mL ZnO NPs; T2, 0.5 mg/mL ZnO NPs; T3, 0.5 µg/mL Se NPs; and T4, 1 µg/mL Se NPs). Diluted semen was packed in 0.25 mL straws and then stored in liquid nitrogen. After thawing, post-thaw in vitro sperm attributes were evaluated. Finally, the effect of NPs on in vivo fertility rate was checked in heat-synched does (n = 70) by artificial insemination (AI) using straws that showed superior results during the in vitro study. Results showed that ZnO and Se NPs were poly-crystalline in nature with particle size below 100 nm (nm). The evaluated post-thaw sperm in vitro attributes were significantly (p < 0.001) higher in T1 in comparison to T0. The antioxidant enzyme activities were significantly (p < 0.001) higher in T1. Lipid peroxidation (LPO) profile was significantly (p < 0.001) lower in T1. Sperm motility and mitochondrial membrane potential (MMP) had a highly significant (r = 0.580, p < 0.05) association in T1. No significant (p > 0.05) differences in pregnancy rates were recorded after AI in the different treatments. In conclusion, extender supplemented with 0.1 mg/mL ZnO NPs improved post-thaw semen quality of goat spermatozoa consequently by increasing activities of endogenous antioxidant enzymes thereby lowering LPO levels. However, improved in vitro outcomes might not correspond to improved field fertility outcomes.

Polymorphism and phylogenetic analysis of FecB and FecG genes in native sheep of Meghalaya, India.

The present study was conducted to identify the polymorphism of two major fecundity genes, viz. FecB and FecG, in indigenous sheep of Meghalaya and to assess phylogenetic relationship with 15 breeds of sheep and to estimate the sequence distances between them. Blood samples were collected from 50 adult ewes and PCR-RFLP was performed to detect the polymorphism of these genes. Further digestion of FecB and FecG was done with restriction enzymes AvaII and DdeI, respectively. In the case of FecB gene, two genotypes, viz. AA and AB, were identified where AA genotype yielded one fragment (190 bp) and AB genotype yielded two fragments (160 and 30 bp). The frequencies of A and B alleles were calculated as 0.64 and 0.36 and those of AA and AB genotypes as 0.280 and 0.720 respectively, whereas FecG gene was found to be monomorphic, with only a single genotype designated as AB genotype. Measure of relatedness among Indian and exotic sheep in terms of both the fecundity genes threw light on the evolutionary origin of different sheep breeds.

Allelic variability of estrogen receptor (ESR) gene and its effect on litter traits of Doom pigs.

The present study was conducted to identify the polymorphism of estrogen receptor (ESR) gene and its biological association with litter traits (litter size at birth, litter size at weaning, litter weight at birth and litter weight at weaning) of Doom pigs native to Assam. A total of 50 adult pigs (12 males and 38 females) chosen randomly from three different herds (Herd I, Herd II, and Herd III) were utilized in the present study. Detection of polymorphism of ESR gene was done by means of PCR-RFLP method. The amplified PCR product was digested with Pvu II restriction enzyme. PCR-RFLP analysis of ESR gene revealed polymorphic banding pattern. Two genotypes viz. AA and AB were identified. AA genotype yielded one fragment (120 bp) and AB genotype yielded three fragments (120, 65, and 55 bp). BB genotype was not found in the population under study. The frequencies of A and B alleles were found to be 0.650 and 0.350, respectively, and the genotypic frequencies of ESR gene were found to be 0.300 and 0.700 for AA and AB genotypes, respectively. There was no significant (P > 0.05) effect of ESR genotype on litter traits and the population under study was not in Hardy-Weinberg Equilibrium for ESR gene. Clustal W Multiple alignment of partial sequence of ESR gene revealed single nucleotide changes at 33, 65, 70, 83, and 92nd nucleotide positions. The presence of Pvu II polymorphism and identification of single nucleotide variation of ESR gene opens interesting prospects for improvement of litter traits in Doom pig through selective breeding program, especially based on marker-assisted selection.