Organic Zinc and Copper Supplementation-Associated Changes in Gene Expression and Protein Profiles in Buck Spermatozoa.

Mineral supplementation has greater impact on male reproduction; however, the mechanism of action has not been studied in detail. The present study was aimed to deal with the lacuna in mechanism of action of mineral supplementation on improvement in sperm characteristics. A group of 40 bucks (aged 5 months) were assigned to 10 groups (4 in each group) based on their body weight and fed with concentrate mixture: basal roughage (minimal diet) in equal proportion to all the bucks. Among the 10 groups, one was considered as control, without any additional mineral supplementation, and the remaining 9 were treatment groups (3 groups each in Zn, Cu, and Zn + Cu). In treatment groups, organic Zn was fed in three different doses as 20, 40, and 60 mg/kg DM; organic Cu was fed in three different doses as 12.5, 25, and 37.5 mg/kg DM; and organic Zn + Cu was combinedly supplied as 20 + 12.5, 40 + 25, and 60 + 37.5 based on their mg/kg DM for 8 months period. The neat semen samples were processed for spermatozoal gene (stress- NOS3, HSP70, HIF1A; fertility- MTF1, MTA1, TIMP2, TNFa, and EGFR) expression studies through qRT-PCR and protein profile changes through single- and two-dimensional gel electrophoresis. Significantly, the stress-responsive genes were downregulated, and fertility-related genes were upregulated in treatment groups. A significant correlation had been noticed among the genes studied: HIF1A with MTA1 (P < 0.05) and MTF1 with EGFR, TIMP2, TNFa, and NOS3 (P < 0.01) respectively. The organic Zn and Cu feeding modulated the expression of stress- and fertility-related genes and protein abundance, thereby improved the sperm characteristics.

Organic Zn and Cu supplementation imprints on seminal plasma mineral, biochemical/ antioxidant activities and its relationship to spermatozoal characteristics in bucks.

This experiment was conducted to study the effect of mineral supplementation on seminal plasma minerals level, biochemical constituents and total antioxidant capacity of Osmanabadi bucks. The study comprised of forty healthy bucks, aged five months were randomly assigned to ten groups (n = 4 per group). The control group was fed with a basal diet without any additional mineral supplementation. In addition to basal diet, treatment bucks were supplemented with three graded doses of organic Zinc (Zn) as 20, 40 and 60 mg/kg dry matter (DM); organic Copper (Cu) as 12.5, 25, 37.5 mg/ kg DM and combination of Zn + Cu as Zn20+Cu12.5, Zn40+Cu25, Zn60+Cu37.5 mg /kg DM basis respectively. Minerals were supplemented for 8 months and the separated seminal plasma used for analysis of minerals, biochemical profile, total antioxidant capacity (TAC), lipid peroxidation (LPO), and protein carbonylation (PC). In treatment groups, significantly lower LPO and PC were observed, except Zn60 and Zn60+Cu37.5, where higher malondialdehyde (MDA) (P < 0.05) formed. The TAC was relatively higher (P < 0.05) in Zn20, Zn40, Cu12.5 and Zn60+Cu37.5 than control. The minerals and biochemical parameters were significantly altered and positive relationship was observed among them. From this study, it was concluded that supplemented minerals changed the seminal plasma minerals profile (Zn- 7-13; Cu- 0.5-1.9 mg/L), reduced the stress (LPO and PC of control Vs treatment as 0.3 Vs 0.1 nmol/ml and 25.7 Vs 4.3 nmol protein carbonyl/mg protein), which improved the sperm quality in Zn40, all Cu treatments and Zn60+Cu37.5 groups respectively.

Transcriptomic Profiling of Buffalo Spermatozoa Reveals Dysregulation of Functionally Relevant mRNAs in Low-Fertile Bulls.

Although, it is known that spermatozoa harbor a variety of RNAs that may influence embryonic development, little is understood about sperm transcriptomic differences in relation to fertility, especially in buffaloes. In the present study, we compared the differences in sperm functional attributes and transcriptomic profile between high- and low-fertile buffalo bulls. Sperm membrane and acrosomal integrity were lower (P < 0.05), while protamine deficiency and lipid peroxidation were higher (P < 0.05) in low- compared to high-fertile bulls. Transcriptomic analysis using mRNA microarray technology detected a total of 51,282 transcripts in buffalo spermatozoa, of which 4,050 transcripts were differentially expressed, and 709 transcripts were found to be significantly dysregulated (P < 0.05 and fold change >1) between high- and low-fertile bulls. Majority of the dysregulated transcripts were related to binding activity, transcription, translation, and metabolic processes with primary localization in the cell nucleus, nucleoplasm, and in cytosol. Pathways related to MAPK signaling, ribosome pathway, and oxidative phosphorylation were dysregulated in low-fertile bull spermatozoa. Using bioinformatics analysis, we observed that several genes related to sperm functional attributes were significantly downregulated in low-fertile bull spermatozoa. Validation of the results of microarray analysis was carried out using real-time qPCR expression analysis of selected genes (YBX1, ORAI3, and TFAP2C). The relative expression of these genes followed the same trend in both the techniques. Collectively, this is the first study to report the transcriptomic profile of buffalo spermatozoa and to demonstrate the dysregulation of functionally relevant transcripts in low-fertile bull spermatozoa. The results of the present study open up new avenues for understanding the etiology for poor fertility in buffalo bulls and to identify fertility biomarkers.

Impact of environmental contaminants on reproductive health of male domestic ruminants: a review.

Environmental contaminants are gaining more attention in the livestock sector lately due to their harmful effects on productivity and fertility of livestock. Recent research indicates that many domestic ruminants are becoming subfertile/infertile due to confounding reasons associated with management. Contaminants like metals, metalloids, herbicides, pesticides, insecticides, chemicals, or natural contaminants are present everywhere in day to day life and are becoming a threat to the livestock. Studies on a broad-spectrum of animals suggest that high doses of acute or low doses of chronic exposure to the contaminants lead to disruption of multi-organs/systems including reproductive function. The lowered reproductive efficiency in animals is attributed to the endocrine disruptor activities of the environmental contaminants on the gonads, affecting gametogenesis and steroidogenesis. In vitro studies on testicular cells and the semen suggest that spermatozoa are more susceptible to damage by environmental contaminants. The quality of the semen happens to be a critical factor in the livestock industry. Contaminants affecting gametogenesis and steroidogenesis may lead to devastating consequences to the livestock reproduction, and thus the production. However, there is a lack of collective data on the effect of such environmental contaminants on the fertility of male domestic ruminants. This review discusses the studies related to the impact of environmental contaminants on male fertility in large (bull and buffalo) and small (sheep and goat) ruminants by focusing on the underlying molecular interactions between the contaminants and gonads.

Ellagic and ferulic acids protect arsenic-induced male reproductive toxicity via regulating Nfe2l2, Ppargc1a and StAR expressions in testis.

Arsenic (As) - induced oxidative stress causes male reproductive toxicity apart from its other generalized systemic effects. Some phytochemicals through their antioxidant properties might help to overcome such toxic effects. The aim of the study was to elucidate the protective role of the selected phytochemicals, ellagic and ferulic acids against the As-induced reproductive toxicity. Forty two healthy male Swiss albino mice were randomly assigned to six groups (each @ n = 7). Group A served as the control, while group B received 200 ppm of As through drinking water. The group C and D mice were administered Per os (P.O) with 50 mg/kg BW of ellagic and ferulic acids, respectively on alternate days. Group E or F received 50 mg of ellagic or ferulic acid + 200 ppm of As for forty days. Ellagic and/ ferulic acid significantly reduced the accumulation of As, protein carbonylation (PC), lipid peroxidation (LPO) in addition to altering the antioxidant enzymes (CAT and SOD) activities, reduced glutathione (GSH) and total antioxidant capacity (TAC) in the testicular tissues. A significantly (p < 0.05) altered sperm functions (viability, functional membrane integrity, Δψm and sperm kinematics like total motility, rapid, progressive motile and type-A (STR > 80%, ALH > 2.5 µm) and testicular damage induced by the As were ameliorated (p < 0.05) by the phytochemical treatments. These phytochemicals due to their antioxidant activities were found to attenuate the As-induced oxidative stress, testicular damage, and sperm abnormalities via regulating the expressions of Nfe2l2, StAR and Ppargc1a. The study revealed that ellagic and ferulic acids might be potential therapeutic options to protect the male reproductive system from As-poisoning.

Protective role of epigallocatechin-3-gallate on arsenic induced testicular toxicity in Swiss albino mice.

Arsenic, often referred to as the king of poisons is carcinogenic in humans and animals. It affects multiorgan systems including reproduction. The present study was undertaken to explore the protective role of green tea compound, epigallocatechin-3-gallate (EGCG) on arsenic induced testicular toxicity in Swiss albino mice. Thirty two adult male mice were randomly assigned to four groups (n=8). Group I served as control without test chemical. The group II received arsenic (200ppm) through drinking water, group III received only EGCG (20mg/kgb.wt., intraperitoneally, alternate days) and group IV was administered arsenic+EGCG for 40days. Factorial experimental design was employed to assess the treatment effect. The EGCG restored arsenic induced decrements in epididymal sperm concentration, kinematic attributes (total motility, rapid, progressive motile, fast progressive, VSL, VAP, VCL, BCF, LIN, WOB, STR and Type A), structutal membrane integrity, functional membrane integrity and mitochondrial membrane potential. As evidenced by the histoarchitectural studies, the EGCG reversed the deleterious effects of arsenic on testicular malondialdehyde (p<0.05) levels, reduced glutathione, antioxidative enzymes and spermatogenesis. Overall, the results suggest that EGCG reduces the testicular oxidative stress induced by arsenic poisoning and thereby protect the reproductive system.

Impact of arsenic(V) on testicular oxidative stress and sperm functional attributes in Swiss albino mice.

The arsenic (As) is a multi system effector including reproduction. The present study examined the association of graded doses of As(V) on testicular microenvironment and sperm function in mice. Thirty-six adult male mice were randomly assigned to six groups (n = 6). Group A served as control without test chemical. The groups B, C, D, E, and F were administered graded doses of 10, 25, 50, 100, and 200 ppm As(V), respectively, through drinking water for 40 days. A dose-dependant significant (P < 0.05) decrements were observed in epididymal sperm kinematic attributes (progressive motility, rapid, fast progressive, VCL, VSL, VAP, LIN, STR, WOB and TYPE A (STR >80 %, ALH 2.5 µm) by CASA), viability, plasma membrane functional integrity, and mitochondrial membrane potential which were associated with insignificant decrease in serum testosterone levels. The histoarchitectural studies of testes showed progressive loss of spermatozoa concentration in the seminiferous tubules as the As(V) dose increased. The mice exposed to As(V) had an increase in the As accumulation, protein carbonylation, and lipid peroxidation levels associated with alterations in SOD, CAT, and GST activities in the testes. In conclusion, higher doses of As(V) (more than 50 ppm) were found to be testicular toxicants which impaired semen quality by inducing oxidative stress in the testicular microenvironment.