Cryostress induces fragmentation and alters the abundance of sperm transcripts associated with fertilizing competence and reproductive processes in buffalo.

The study aimed to assess the influence of cryostress on RNA integrity and functional significance in sperm fertilizing ability. The fresh and post-thawed buffalo sperm (n = 6 each) samples were evaluated for their functional attributes, and sperm total RNA was subjected to transcriptome sequencing followed by validation using real-time PCR and dot blot. Overall, 6911 genes had an expression of FPKM > 1, and among these 431 genes were abundantly expressed (FPKM > 20) in buffalo sperm. These abundantly expressed genes regulate reproductive functions such as sperm motility (TEKT2, SPEM1, and PRM3, FDR = 1.10E-08), fertilization (EQTN, PLCZ1, and SPESP1, FDR = 7.25E-06) and the developmental process involved in reproduction (SPACA1, TNP1, and YBX2, FDR = 7.21E-06). Cryopreservation significantly (p < 0.05) affected the structural and functional membrane integrities of sperm. The expression levels of transcripts that regulate the metabolic activities and fertility-related functions were compromised during cryopreservation. Interestingly, cryostress induces the expression of genes involved (p < 0.05) in chemokine signaling (CX3CL1, CCL20, and CXCR4), G-protein coupled receptor binding (ADRB1, EDN1, and BRS3), translation (RPS28, MRPL28, and RPL18A), oxidative phosphorylation (ND1, ND2, and COX2), response to reactive oxygen species (GLRX2, HYAL2, and EDN1), and immune responses (CX3CL1, CCL26, and TBXA2R). These precociously expressed genes during cryopreservation alter the signaling mechanisms that govern sperm functional competence and can impact fertilization and early embryonic development.

Deciphering the complexity of sperm transcriptome reveals genes governing functional membrane and acrosome integrities potentially influence fertility.

Deciphering sperm transcriptome is the key to understanding the molecular mechanisms governing peri-fertilization, embryonic development, and pregnancy establishment. This study aimed to profile sperm transcriptome to identify signature transcripts regulating male fertility. Semen samples were collected from 47 bulls with varied fertility rates. The sperm total RNA was isolated (n = 8) and subjected to transcriptome sequencing. Based on the expression pattern obtained from RNA profiling, the bulls were grouped (p = 0.03) into high-fertile and sub-fertile, and signature transcripts controlling sperm functions and fertility were identified. The results were validated using the OMIM database, qPCR, and sperm function tests. The sperm contains 1100 to 1700 intact transcripts, of which BCL2L11 and CAPZA3 were abundant and associated (p < 0.05) with spermatogenesis and post-embryonic organ morphogenesis. The upregulated genes in the acrosome integrity and functional membrane integrity groups had a close association with the fertility rate. The biological functions of these upregulated genes (p < 0.05) in the high-fertile bulls were associated with spermatogenesis (AFF4 and BRIP1), sperm motility (AK6 and ATP6V1G3), capacitation and zona binding (AGFG1), embryo development (TCF7 and AKIRIN2), and placental development (KRT19). The transcripts involved in pathways regulating embryonic development such as translation (EEF1B2 and MTIF3, p = 8.87E-05) and nonsense-mediated decay (RPL23 and RPL7A, p = 5.01E-27) were upregulated in high-fertile bulls. The identified transcripts may significantly impact oocyte function, embryogenesis, trophectoderm development, and pregnancy establishment. In addition, the study also reveals that the genes governing sperm functional membrane integrity and acrosome integrity have a prospective effect on male fertility.

Elucidating the processes and pathways enriched in buffalo sperm proteome in regulating semen quality.

Sperm carries a reservoir of proteins regulating the molecular functions to attain functional competence. Semen samples collected from buffalo bulls were assessed for sperm functional attributes (n = 11) and proteome profiling (n = 6). Sperm proteins were extracted and profiled by employing LC-MS/MS. Overall, the buffalo sperm contained 1365 proteins, of which 458 were common between the groups. The unique proteins were 477 and 430 in good and poor quality semen, respectively. In the whole proteome of buffalo sperm, sexual reproduction with phosphatidylethanolamine-binding protein1 (PEBP1), fetuin-B (FETUB) and acrosin (ACR) was the most enriched (p = 8.44E-19) biological process, also with thermogenesis (p = 0.003), oocyte meiosis (p = 0.007) and vascular smooth muscle contraction (p = 0.009) apart from metabolic pathways. In good quality semen, mesenchyme migration (p = 1.24E-07) and morphogenesis (p = 0.001) were abundant biological processes. In good quality semen, the fluid shear stress (p = 0.01) and, in poor quality semen, valine, leucine and isoleucine degradation (p = 3.8E-05) pathways were enriched. In good quality semen, 7 proteins were significantly (p < 0.05) upregulated and 33 proteins were significantly (p < 0.05) downregulated. On validating the abundantly expressed sperm proteins, serine protease inhibitor Kazal-type 2-like (SPINK2; 2.17-fold) and neddylin (NEDD8; 1.13-fold) were upregulated and YBX2 was downregulated (0.41-fold) in good quality semen as compared with poor quality semen (1-fold). The present findings revealed the importance of sperm proteins in oocyte maturation, fertilization process and early embryonic development. The variations in the proteomic composition can be used as potential markers for the selection of breeding bulls.

Establishment of bioinformatics pipeline for deciphering the biological complexities of fragmented sperm transcriptome.

Despite the development of several tools for the analysis of the transcriptome data, non-availability of a standard pipeline for analyzing the low quality and fragmented mRNA samples pose a major challenge to the computational molecular biologist for effective interpretation of the data. Hence the present study aimed to establish a bioinformatics pipeline for analyzing the biologically fragmented sperm RNA. Sperm transcriptome data (2 x 75 PE sequencing) generated from bulls (n = 8) of high-fertile (n = 4) and low-fertile (n = 4) classified based on the fertility rate (41.52 ± 1.07 vs 36.04 ± 1.04%) were analyzed with different bioinformatics tools for alignment, quantitation, and differential gene expression studies. TopHat2 was effectual compared to HISAT2 and STAR for sperm mRNA due to the higher exonic (6% vs 2%) mapping percentage and quantitating the low expressed genes. TopHat2 also had significantly strong correlation with STAR (0.871, p = 0.05) and HISAT2 (0.933, p = 0.01). TopHat2 and Cufflinks combo quantitated the number of genes higher than the other combinations. Among the tools (Cuffdiff, DESeq, DESeq2, edgeR, and limma) used for the differential gene expression analysis, edgeR and limma identified the largest number of significantly differentially expressed genes (p < 0.05) with biological relevance. The concordance analysis concurred that edgeR had an edge over the other tools. It also identified a higher number (9.5%) of fertility-related genes to be differentially expressed between the two groups. The present study established that TopHat2, Cufflinks, and edgeR as a suitable pipeline for the analysis of fragmented mRNA from bovine spermatozoa.

IGF-1 treatment during in vitro maturation improves developmental potential of ovine oocytes through the regulation of PI3K/Akt and apoptosis signaling.

This study aimed to assess the effect of the insulin-like grow factor 1 (IGF-1) treatment during in vitro maturation on the gene expression and developmental ability of ovine oocytes. Ovine cumulus-oocyte complexes (COC) were matured in vitro without (control) or with the supplementation of IGF-1 (100 ng/ml) and then subjected to in vitro fertilization and culture. The rate of oocyte maturation and embryo development was recorded and expression of the selected genes (involved in the PI3K/Akt and apoptosis signaling) was assessed in the matured oocytes. The IGF-1 treatment significantly (p < .05) improved the oocyte maturation rate (%) as compared to the control (81.5 ± 2.40 vs. 73.6 ± 0.94). Similarly, as compared to the control, the IGF-1 treatment significantly (p < .05) improved the rate (%) of cleavage (54.7 ± 1.58 vs. 67.2 ± 3.65) and the formation of 4-8 cell embryos (30.7 ± 2.89 vs. 44.1 ± 4.01) and morula (20.7 ± 2.08 vs. 32.8 ± 2.78). The IGF-1 treatment significantly (p < .05) upregulated the expression of IGF1R, PI3KR1, AKT1 and BCL2 and downregulated the expression of GSK3ß, FOXO3 and CASP9 in the matured oocytes. In conclusion, the IGF-1 treatment significantly improved the developmental competence of ovine oocytes through the regulation of the PI3K/Akt and apoptosis signaling.

Current scenario and challenges ahead in application of spermatogonial stem cell technology in livestock.

PURPOSE: Spermatogonial stem cells (SSCs) are the source for the mature male gamete. SSC technology in humans is mainly focusing on preserving fertility in cancer patients. Whereas in livestock, it is used for mining the factors associated with male fertility. The review discusses the present status of SSC biology, methodologies developed for in vitro culture, and challenges ahead in establishing SSC technology for the propagation of superior germplasm with special reference to livestock. METHOD: Published literatures from PubMed and Google Scholar on topics of SSCs isolation, purification, characterization, short and long-term culture of SSCs, stemness maintenance, epigenetic modifications of SSCs, growth factors, and SSC cryopreservation and transplantation were used for the study. RESULT: The fine-tuning of SSC isolation and culture conditions with special reference to feeder cells, growth factors, and additives need to be refined for livestock. An insight into the molecular mechanisms involved in maintaining stemness and proliferation of SSCs could facilitate the dissemination of superior germplasm through transplantation and transgenesis. The epigenetic influence on the composition and expression of the biomolecules during in vitro differentiation of cultured cells is essential for sustaining fertility. The development of surrogate males through gene-editing will be historic achievement for the foothold of the SSCs technology. CONCLUSION: Detailed studies on the species-specific factors regulating the stemness and differentiation of the SSCs are required for the development of a long-term culture system and in vitro spermatogenesis in livestock. Epigenetic changes in the SSCs during in vitro culture have to be elucidated for the successful application of SSCs for improving the productivity of the animals.

Sub-fertility in crossbred bulls: deciphering testicular level transcriptomic alterations between zebu (Bos indicus) and crossbred (Bos taurus x Bos indicus) bulls.

BACKGROUND: The incidence of poor semen quality and sub-fertility/infertility is higher in crossbred as compared to Zebu males. Several attempts have been made to understand the possible reasons for higher incidence of fertility problems in crossbred males, at sperm phenotype, proteome and genome level but with variable results. Since the quality of the ejaculated spermatozoa is determined by the testicular environment, assessing the testicular transcriptome between these breeds would help in identifying the possible mechanisms associated with infertility in crossbred bulls. However, such information is not available. We performed global transcriptomic profiling of testicular tissue from crossbred and Zebu bulls using Agilent Bos taurus GXP 8X60k AMADID: 29411 array. To the best of our knowledge, this is the first study comparing the testicular mRNAs between crossbred and Zebu bulls. RESULTS: Out of the 14,419 transcripts detected in bovine testis, 1466 were differentially expressed between crossbred and Zebu bulls, in which 1038 were upregulated and 428 were downregulated in crossbred bulls. PI4KB and DPY19L2 genes, reported to be involved in sperm capacitation and acrosome formation respectively, were among the top 10 downregulated transcripts in crossbred testis. Genes involved in ubiquitination and proteolysis were upregulated, while genes involved in cell proliferation, stem cell differentiation, stem cell population maintenance, steroidogenesis, WNT signalling, protein localization to plasma membrane, endocannabinoid signalling, heparin binding, cAMP metabolism and GABA receptor activity were downregulated in crossbred testis. Among the 10 genes validated using qPCR, expression of CCNYL, SOX2, MSMB, SPATA7, TNP1, TNP2 and CRISP2 followed the same trend as observed in microarray analysis with SPATA7 being significantly downregulated and transition proteins (TNP1, TNP2) being significantly upregulated in crossbred bulls. CONCLUSIONS: Abundant proteolysis by ubiquitination and downregulation of WNT signaling, cell proliferation, differentiation and steroidogenesis might be associated with higher incidence of poor semen quality and/or sub-fertility/infertility in crossbred bulls as compared to Zebu bulls. Downregulation of SPATA7 (Spermatogenesis Associated 7) and upregulation of transition proteins (TNP1 and TNP2) in crossbred bull testis might be associated with impaired spermatogenesis processes including improper chromatin compaction in crossbred bulls.

Immuno-histological mapping and functional association of seminal proteins in testis and excurrent ducts with sperm function in buffalo.

The region-specific expression of seminal proteins in testis and excurrent duct system determines the quality and function of the spermatozoa. In the present study, localization and expression of some of the seminal proteins such as insulin-like growth factor receptor 1ß (IGF-1Rß), phosphatidylethanolamine-binding protein 4 (PEBP4), α-tubulin and tissue factor pathway inhibitor 2 (TFPI2) were carried out in testis, excurrent duct system and spermatozoa of buffalo. IGF-1Rß was localized in the cells of the seminiferous tubules of the testis, except in primary spermatocytes. The PEBP4 was localized only in the elongated spermatid, whereas α-tubulin and TFPI2 proteins were localized in all cells of the seminiferous tubule including spermatocyte. In the buffalo spermatozoa, IGF-1Rß, PEBP4, α-tubulin and TFPI2 were localized in the acrosome region, the post-acrosomal region till the tail end, post-acrosome to the entire tail region and the equatorial region, respectively. The study indicates that IGF-1R, α-tubulin and PEBP4 proteins regulate spermatogenesis, whereas TFPI2 may be involved during the zona binding process of the buffalo spermatozoa.

Immune regulatory molecules as modifiers of semen and fertility: A review.

Declining fertility rates in both human and animals is a cause for concern. While many of the infertility cases are due to known causes, idiopathic infertility is reported in 30% of the infertile couples. In such cases, 18% of the infertile males carry antisperm antibodies (ASAs). Such data are lacking in livestock, wherein 20-30% of the animals are being culled due to low fertility. In males, the blood-testis barrier (BTB) and biomolecules in the semen provide an immuno-tolerant microenvironment for spermatozoa as they traverse the immunologic milieu of both the male and female reproductive tracts. For example, insults from environmental contaminants, infections and inflammatory conditions are likely to impact the immune privilege state of the testis and fertility. The female mucosal immune system can recognize allogenic spermatozoa-specific proteins affecting sperm kinematics and sperm-zona binding leading to immune infertility. Elucidating the functions and pathways of the immune regulatory molecules associated with fertilization are prerequisites for understanding their impact on fertility. An insight into biomolecules associated with spermatozoal immune tolerance may generate inputs to develop diagnostic tools and modulate fertility. High-throughput sequencing technologies coupled with bioinformatics analyses provides a path forward to define the array of molecules influencing pregnancy outcome. This review discusses the seminal immune regulatory molecules from their origin in the testis until they traverse the uterine environment enabling fertilization and embryonic development. Well-designed experiments and the identification of biomarkers may provide a pathway to understand the finer details of reproductive immunology that will afford personalized therapies.

Cryoprotective role of organic Zn and Cu supplementation in goats (Capra hircus) diet.

The current study focused on cryopreservation and assessment of characters of post-thaw semen of indigenous Osmanabadi bucks maintained with standard diet, supplemented with different concentrations of organic zinc (Zn), copper (Cu) or in combination, for a period of 180 days. The different doses of organic Zn and Cu were fed per kg DM basis, Zn groups (low: Zn20, medium: Zn40 and high: Zn60), Cu groups: (low: Cu12.5, medium: Cu25 and high: Cu37.5) and combination of Zn + Cu groups (low: Zn20 + Cu12.5, medium: Zn40 + Cu25 and high: Zn60 + Cu37.5) respectively. The control group bucks were maintained mainly on the basal diet without any additional mineral supplementation. Two hundred and forty (240) semen samples were collected from 40 bucks aged 11 months, through electro ejaculator method, processed and analysed for sperm quality parameters both at pre freeze and post-thaw stage. The semen samples were diluted in Tris egg yolk extender, cooled and equilibrated for 4 h at 5 °C, cryopreserved using programmable freezer (PLANER Kryo 360-1.7) and stored at -196 °C. The organic trace minerals (Zn, Cu and Zn + Cu) protected the spermatozoa against the cryoinjury and maintained higher post-thaw semen parameters except in high Zn group. Additional feeding of organic Cu and Zn to bucks had a protective role and resulted in higher sperm liveability, plasma membrane and acrosome integrities, motility and velocity and reduced oxidative stress in supplemented goats (P < 0.05).

Novel insights into the role of cell-free seminal mRNAs on semen quality and cryotolerance of spermatozoa in bulls (Bos taurus).

The aim of the present study was to ascertain the effectiveness of seminal plasma mRNAs as markers to assess the reproductive performance of bulls. Semen samples (33 ejaculates) from 11 bulls were evaluated for sperm kinematic and functional parameters. Total RNA was isolated from cell-free seminal (cfs) using TRIzol LS reagent and the concentration of cfs-RNA was 24.4±2.3µgmL-1 seminal plasma. The cfs-RNA was fragmented to a size of 25-500bp. Of the cfs-mRNAs screened using real time PCR, expression of protamine 1 (PRM1) was positively (P<0.05) associated with the mitochondrial membrane potential of raw semen, whereas expression of Fas Ligand (FASLG) was negatively (P<0.05) associated with sperm velocity, membrane integrity and chromatin distribution in post-thaw semen samples. The percentage of Type A spermatozoa (amplitude of lateral movement of head >2.5µm and straightness >85%) in raw semen was positively (P<0.05) associated with bone morphogenetic protein 2 (BMP2), ubiquitin conjugating enzyme E2D3 (UBE2D3), tumour-associated necrotic factor-associated death domain (TRADD) and caspase-3 (CASP3) expression. Nerve growth factor (NGF) expression was positively (P<0.05) associated with the maintenance of post-thaw functional membrane integrity in spermatozoa and could be used to assess the cryotolerance of bull semen. In conclusion, the expression of cfs mRNAs can be used to assess the reproductive performance of males and to predict the sensitivity of spermatozoa to cryoinjury.

IGF1 stabilizes sperm membrane proteins to reduce cryoinjury and maintain post-thaw sperm motility in buffalo (Bubalus bubalis) spermatozoa.

Insulin like growth factor 1 (IGF1) in the seminal plasma is reported to improve sperm motility by reducing oxidative stress. The present study was conducted to assess the effect of addition of IGF1 on sperm function and protein composition during cryopreservation process. Semen samples were collected from six Murrah buffaloes (2 ejaculates from each animal) and diluted (80 million/ml) in tris egg yolk extender and divided into control, T1, T2 and T3, groups supplemented with 0, 50, 100 and 150 ng of IGF1/mL, respectively. The semen was filled in straws (250 µL) and straws from each group were divided into two batches. One batch was processed for freezing and another batch was incubated at 4 °C for 4 h. The sperm kinematic and functional parameters were studied in both the batches. A significant (P < 0.05) positive effect of IGF1 was observed on functional membrane integrity (%) during incubation at 4 °C for 4 h in T3 as compared to control group. The spermatozoa (%) positive for structural membrane integrity, mitochondrial membrane potential and the metabolic activity in post-thaw semen were significantly (P < 0.05) high in T3 than the control group. The acrosomal integrity was significantly (P < 0.05) higher in T2 group as compared to control. The proteins (kDa) of 17.3 with pI 4.2 (calmodulin), 11.3 with pI 6.5 (dermcidin) and 18.1 with pI 5.5 (sperm acrosome membrane associated protein3) were protected in IGF1 group. The study suggests that IGF1 can be added to the extender for improving cryosurvial of buffalo spermatozoa.

Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus).

Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at -80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy+TRIzol and PureLink+TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800-900 ng/30-40 × 10(6)) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50-2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa.

Relationship between seminal plasma tuberoinfundibular peptide of 39 residues and sperm functional attributes in buffalo (Bubalus bubalis).

The buffalo seminal plasma protein profile and its relationship with sperm quality have not been studied in detail. Thus, the aim of the present study was to profile buffalo seminal plasma proteins and to assess the relationship between differentially expressed proteins and sperm characteristics. Semen samples (n = 44) were collected from 11 Murrah buffalo bulls (four ejaculates from each animal) and seminal plasma protein profiling was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionisation time-of-flight analysis of one of the differentially expressed proteins, namely the 11-12 kDa protein, identified it as tuberoinfundibular peptide of 39 residues (TIP39). Western blot analysis confirmed the presence of TIP39, with TIP39 expression in seminal plasma varying among bulls. Based on TIP39 levels, bulls were classified into two groups, those with high and low protein. The percentages of spermatozoa positive for mitochondrial membrane potential test, chromatin distribution test, synthetic media sperm penetrability test and acrosomal integrity test were significantly (P < 0.05) high in the high protein group. The present study is the first to demonstrate the presence of TIP39 in buffalo seminal plasma and the positive effect of TIP39 on the functional parameters and fertilising ability of spermatozoa.

Identification of hub genes and their expression profiling for predicting buffalo (Bubalus bubalis) semen quality and fertility.

Sperm transcriptomics provide insights into subtle differences in sperm fertilization competence. For predicting the success of complex traits like male fertility, identification of hub genes involved in various sperm functions are essential. The bulls from the transcriptome profiled samples (n = 21), were grouped into good and poor progressive motility (PM), acrosome integrity (AI), functional membrane integrity (FMI) and fertility rate (FR) groups. The up-regulated genes identified in each group were 87, 470, 1715 and 36, respectively. Gene networks were constructed using up- and down-regulated genes from each group. The top clusters from the upregulated gene networks of the PM, AI, FMI and FR groups were involved in tyrosine kinase (FDR = 1.61E-11), apoptosis (FDR = 1.65E-8), translation (FDR = 2.2E-16) and ribosomal pathway (FDR = 1.98E-21), respectively. From the clusters, the hub genes were identified and validated in a fresh set of semen samples (n = 12) using RT-qPCR. Importantly, the genes (fold change) RPL36AL (14.99) in AI, EIF5A (54.32) in FMI, and RPLP0 (8.55) and RPS28 (13.42) in FR were significantly (p < 0.05) up-regulated. The study suggests that the expression levels of MAPK3 (PM), RPL36AL + RPS27A or RPL36AL + EXT2 (AI), RPL36AL or RPS27A (FMI) and RPS18 + RPS28 (FR) are potential markers for diagnosing the semen quality and fertility status of bulls which can be used for the breeding program.

Relationship among seminal antigenicity, antioxidant status and metabolically active sperm from Holstein-Friesian (Bos taurus) bulls.

Sperm antigenicity has been implicated as a regulatory factor for acquiring fertilizing competence in the female reproductive tract. Overt immune response against the sperm proteins leads to idiopathic infertility. Hence, the aim of the study was to evaluate the influence of the auto-antigenic potential of sperm on the antioxidant status, metabolic activities and reactive oxygen species (ROS) in bovine. Semen from Holstein-Friesian bulls (n = 15) was collected and classified into higher (HA, n = 8) and lower (LA, n = 7) antigenic groups based on micro-titer agglutination assay. The neat semen was subjected to the evaluation of bacterial load, leukocyte count, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lipid peroxidation (LPO) levels. Antioxidant activities in seminal plasma and intracellular ROS levels in the post-thawed sperm were estimated. The number of leukocytes was lower (p < .05) in the HA than the LA semen. The percentage of metabolically active sperm was higher (p < .05) in HA than the LA group. The activities of total non-enzymatic antioxidant, superoxide dismutase (SOD) and catalase (CAT) were higher (p < .05) while glutathione peroxidase activity was lower (p < .05) in the seminal plasma of LA group. The LPO levels of neat sperm and the percentage sperm positive for intracellular ROS in the cryopreserved sample were lower (p < .05) in the HA group. Auto-antigenic levels were positively correlated with the percentage of metabolically active sperm (r = 0.73, p < .01). However, the seminal auto-antigenicity was negatively (p < .05) correlated with the levels of SOD (r=-0.66), CAT (r=-0.72), LPO (r=-0.602) and intracellular ROS (r=-0.835). The findings were represented in graphical abstract. It is inferred that the higher auto-antigenic levels protect the quality of bovine semen by promoting sperm metabolism and lowering ROS and LPO levels.

Dietary n-3 PUFA augments pre-ovulatory follicle turnover and prolificacy in well-fed ewes.

The present study evaluated the effect of dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) on preovulatory follicle (POF) turnover, prolificacy, and endocrine and metabolic milieu in Malpura sheep. Fifty cyclic ewes with 3-3.5 body condition scores on a five-point scale were allocated equally to two groups (n = 25) following estrus synchronization and were supplemented with 0.6 mL/kg body weight of n-3 PUFA-rich fish oil (FO) or palm oil (PO) as control, for 60 d following an acclimatization period of 7 d. All ewes were mated with sexually active rams at the end of the supplementation period. On ultrasonographic ovarian scanning at the last fourth estrus, the mean number of POFs was 77.8% greater (P < 0.01) in FO ewes than in the PO ewes. The proportion of ewes with multiple ovulations two months after the beginning of supplementation was 56% in the FO group as compared to 8% in the PO group. The number of fetuses was 46% higher (P < 0.01) in the FO than in the PO ewes at d 45 of gestation. At lambing, the twinning percent in the FO ewes was three times greater than in the PO ewes (27.3 vs. 9.1%). Plasma cholesterol, estradiol, and insulin concentrations were lower (P < 0.01) in ewes fed with FO than those offered PO group at the end of the feeding period. It was concluded that the dietary supplementation of n-3 PUFA-rich FO in well-fed Malpura ewes improved the number of follicles and ovulation rate which led to an increased prolificacy, accompanied by a reduction of plasma cholesterols, estradiol, and insulin.

Evaluation of maize grain and polyunsaturated fatty acid (PUFA) as energy sources for breeding rams based on hormonal, sperm functional parameters and fertility.

The objective of the present study was to elucidate the effect of different sources of dietary energy (maize vs polyunsaturated fatty acid (PUFA) on semen functional parameters and fertility of adult rams. Eighteen adult rams were divided into two groups (maize and PUFA, n=9). The main energy source for the rams in the maize group was coarsely ground maize grain, whereas in the PUFA group it was sunflower oil (rich in 18:2 linoleic acid, an omega-6 acid). The ration was fed for a minimum period of 60 days and thereafter semen was collected for evaluation. The proportion of progressive forward motility was significantly (P<0.05) higher in the PUFA group compared with the maize group. Sperm lipid peroxidation as measured by malondialdehyde formation (µM per 1×10(9) spermatozoa) was significantly (P<0.05) higher in the PUFA group compared with the maize group. When the semen was diluted with Tris-egg yolk-citrate buffer and incubated for 24h at 4°C, the proportions of plasmalemma integrity, the sperm subpopulation positive for functional membrane and acrosomal integrities, and mitochondrial membrane potential were significantly (P<0.05) higher in PUFA-fed than in maize-fed animals. The different sources of energy did not influence the serum and seminal plasma IGF-I levels. The cleavage rate (percentage) did not differ significantly between PUFA- (45.4±4.91) and maize- (44.63±6.8) fed animals. In conclusion, PUFA feeding influenced sperm quality by altering or stabilising membrane integrity. The present study indicates that PUFA may improve semen quality but did not improve in vitro fertilisation.

Microenvironment of the male and female reproductive tracts regulate sperm fertility: Impact of viscosity, pH, and osmolality.

BACKGROUND: Terminally differentiated mammalian sperm are exposed to gradients of viscosity, pH, and osmolality both in the male and female reproductive tract during their perilous journey to quest the ovum. The complex physicochemical factors play an integral role in preparing sperm for the fertilization process. OBJECTIVES: To elucidate the influence of the reproductive tract microenvironment especially viscosity, pH, and osmolality in regulating sperm functional and fertilization competence. MATERIALS AND METHODS: The data used in this review were collected from the research papers and online databases focusing on the influence of viscosity, pH, and osmolality on sperm function. DISCUSSION: The gradients of viscosity, pH, and osmolality exist across various segments of the male and female reproductive tract. The changes in the viscosity create a physical barrier, pH aid in capacitation and hyperactivation, and the osmotic stress selects a progressive sperm subpopulation for accomplishing fertilization. The sperm function tests are developed based on the concept that the male genotype is the major contributor to the reproductive outcome. However, recent studies demonstrate the significance of sperm genotype-environment interactions that are essentially contributing to reproductive success. Hence, it is imperative to assess the impact of physicochemical stresses and the adaptive ability of the terminally differentiated sperm, which in turn would improve the outcome of the assisted reproductive technologies and male fertility assessment. CONCLUSION: Elucidating the influence of the reproductive tract microenvironment on sperm function provides newer insights into the procedures that need to be adopted for selecting fertile males for breeding, and ejaculates for the assisted reproductive technologies.