Heterologous Seminal Plasma Reduces the Intracellular Calcium and Sperm Viability of Cryopreserved Stallion Spermatozoa.

Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from good quality semen producing stallions at the rate of 20% and 30%, respectively, whereas the third aliquot remained as control (0% SP) and incubated at 37°C for 30 minutes. Sperm membrane integrity, mitochondrial membrane potential (MMP), mitochondrial superoxide (mtROS) generation, and intracellular calcium status were assessed at different time intervals during incubation by flow cytometry. It was observed that the dead sperm population increased (p < 0.01) during incubation in both the 20% and 30% SP-supplemented groups. However, no significant changes were observed in MMP in both the control and treatment groups at different time intervals. Interestingly, it was found that sperm mtROS production increased (p < 0.01) during incubation in the SP-supplemented groups compared with the control group. The proportion of live spermatozoa with high intracellular calcium was reduced (p < 0.01) during incubation in the SP-incubated groups. Collectively, heterologous SP addition could not repair the damages caused by the cryopreservation and further resulted in deterioration of semen quality as observed in our study by reducing viability, increasing reactive oxygen species (ROS) production possibly due to high proportion of dead cells, or some factors (yet to be identified) that are inducive of oxidative stress in stallion spermatozoa.

Sperm proteomic landscape is altered in breeding bulls with greater sperm DNA fragmentation index.

Although, it is well understood that sperm DNA damage is associated with infertility, the molecular details of how damaged sperm DNA affects fertility are not fully elucidated. Since sperm proteins play an important role in fertilization and post-fertilization events, the present study aimed to identify the sperm proteomic alterations in bulls with high sperm DNA Fragmentation Index (DFI%). Semen from Holstein-Friesian crossbred breeding bulls (n = 50) was subjected to Sperm Chromatin Structure Assay. Based on DFI%, bulls were classified into either high- (HDFI; n = 6), or low-DFI (LDFI; n = 6) and their spermatozoa were subjected to high throughput proteomic analysis. Liquid chromatography and mass spectrometry analysis identified 4567 proteins in bull spermatozoa. A total of 2660 proteins were found common to both the groups, while 1193 and 714 proteins were unique to HDFI and LDFI group, respectively. A total of 265 proteins were up regulated and 262 proteins were down regulated in HDFI group. It was found that proteins involved in capacitation [heparin binding (molecular function), ERK1 and ERK2 cascade (biological process), PI3K-Akt signalling (pathway), Jak-STAT signalling (pathway)], spermatogenesis [TLR signalling (pathway), gamete generation (biological process)] and DNA repair mechanism (biological process) were significantly altered in the bulls with high DFI%.

Genome-Wide Single-Nucleotide Polymorphism-Based Genomic Diversity and Runs of Homozygosity for Selection Signatures in Equine Breeds.

The horse, one of the most domesticated animals, has been used for several purposes, like transportation, hunting, in sport, or for agriculture-related works. Kathiawari, Marwari, Manipuri, Zanskari, Bhutia, Spiti, and Thoroughbred are the main breeds of horses, particularly due to their agroclimatic adaptation and role in any kind of strong physical activity, and these characteristics are majorly governed by genetic factors. The genetic diversity and phylogenetic relationship of these Indian equine breeds using microsatellite markers have been reported, but further studies exploring the SNP diversity and runs of homozygosity revealing the selection signature of breeds are still warranted. In our study, the identification of genes that play a vital role in muscle development is performed through SNP detection via the whole-genome sequencing approach. A total of 96 samples, categorized under seven breeds, and 620,721 SNPs were considered to ascertain the ROH patterns amongst all the seven breeds. Over 5444 ROH islands were mined, and the maximum number of ROHs was found to be present in Zanskari, while Thoroughbred was confined to the lowest number of ROHs. Gene enrichment of these ROH islands produced 6757 functional genes, with AGPAT1, CLEC4, and CFAP20 as important gene families. However, QTL annotation revealed that the maximum QTLs were associated with Wither's height trait ontology that falls under the growth trait in all seven breeds. An Equine SNP marker database (EqSNPDb) was developed to catalogue ROHs for all these equine breeds for the flexible and easy chromosome-wise retrieval of ROH along with the genotype details of all the SNPs. Such a study can reveal breed divergence in different climatic and ecological conditions.

Primary structure determination and physicochemical characterization of DSP-3, a phosphatidylcholine binding glycoprotein of donkey seminal plasma.

Major proteins of the seminal plasma in a variety of mammals such as bovine PDC-109, equine HSP-1/2, and donkey DSP-1 contain fibronectin type-II (FnII) domains and are referred to as FnII family proteins. To further our understanding on these proteins, we carried out detailed studies on DSP-3, another FnII protein of donkey seminal plasma. High-resolution mass-spectrometric studies revealed that DSP-3 contains 106 amino acid residues and is heterogeneously glycosylated with multiple acetylations on the glycans. Interestingly, high homology was observed between DSP-1 and HSP-1 (118 identical residues) than between DSP-1 and DSP-3 (72 identical residues). Circular dichroism (CD) spectroscopic and differential scanning calorimetric (DSC) studies showed that DSP-3 unfolds at ~45 °C and binding of phosphorylcholine (PrC) - the head group moiety of choline phospholipids - increases the thermal stability. Analysis of DSC data suggested that unlike PDC-109 and DSP-1, which exist as mixtures of polydisperse oligomers, DSP-3 most likely exists as a monomer. Ligand binding studies monitoring changes in protein intrinsic fluorescence indicated that DSP-3 binds lyso-phosphatidylcholine (Ka = 1.08 × 105 M-1) with ~80-fold higher affinity than PrC (Ka = 1.39 × 103 M-1). Binding of DSP-3 to erythrocytes leads to membrane perturbation, suggesting that its binding to sperm plasma membrane could be physiologically significant.

Freezability and Fertility Rates of Stallion Semen Supplemented With Trehalose in Lactose Extender.

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. One of the reason for this diminished quality is osmotic stress that spermatozoa experiences during freezing and thawing process. The aim of the present study was to evaluate the cryoprotective effect of trehalose on stallion sperm quality and field fertility rates subjected to cooling and freeze-thawing process. Semen samples were collected from six Marwari breed stallions, divided into three different treatments in a final concentration of 150 × 106 sperm/mL by using Lactose based extender containing 0, 50, and 150 mM of trehalose then subjected to cryopreservation after equilibration. Sperm motility, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, DNA integrity and oxidative stress related parameters of the stallion spermatozoa were analyzed at fresh, prefreeze and post thaw stages. Thirty (30) reproductively healthy mares were inseminated with frozen-thawed semen either supplemented with (treatment) or without (control) trehalose to evaluate the field fertility. Results of the current study indicated that, the extender containing 50 mM trehalose has enhanced the functional plasma membrane, acrosomal, DNA integrities and augmented the mitochondrial membrane potential. Trehalose supplementation to the semen extender not only ameliorated the semen quality parameters, but also protected the stallion sperm from oxidative stress by reducing the levels of reactive oxygen species (ROS) and lipid peroxidation (LPO). The inclusion of 50 mM trehalose in semen extender resulted in significantly (P < .05) increased post-thaw progressive motility and viability compared to the control group. Mares inseminated with frozen-thawed semen supplemented with 50 mM trehalose tended to have better pregnancy rates than controls (non-significant [P < .05]) although a larger fertility trial is required to determine if this effect reaches the level of significance. In conclusion, addition of 50 mM trehalose yielded in better quality stallion semen after cooling and post-thawing in terms of reducing the oxidative stress and enhancing the motility, integrities of acrosome, plasma membrane, mitochondrial potential and DNA.

High-throughput proteomic characterization of seminal plasma from bulls with contrasting semen quality.

Seminal plasma proteins are the major extrinsic factors that can modulate the sperm quality and functions. The present study was carried out to compare the proteomic profiles of seminal plasma from breeding bulls producing good and poor quality semen in an effort to understand the possible proteins associated with semen quality. A total of 910 and 715 proteins were detected in the seminal plasma of poor and good quality semen producing bulls, respectively. A total of 705 proteins were common to both the groups, in which 380 proteins were upregulated and 89 proteins were downregulated in the seminal plasma of poor quality semen, while 236 proteins were co-expressed. The proteins negatively influencing sperm functions such as CCL2, UQCRC2, and SAA1 were among the top ten upregulated proteins in the seminal plasma of poor quality semen. Proteins having a positive role in sperm functions (NGF, EEF1A2, COL1A2, IZUMO4, PRSS1, COL1A1, WFDC2) were among the top ten downregulated proteins in the seminal plasma of poor quality semen. The upregulation of oxidation-reduction process-related proteins, histone proteins (HIST3H2A, H2AFJ, H2AFZ, H2AFX, HIST2H2AB, H2AFV, HIST1H2AC, HIST2H2AC, LOC104975684, LOC524236, LOC614970, LOC529277), and ubiquinol-cytochrome-c reductase proteins (UQCRB, UQCRFS1, UQCRQ, UQCRC1, UQCRC2) indicate deranged oxidation-reduction equilibrium, chromatin condensation and spermatogenesis in poor quality semen producing bulls. The expression of proteins essential for motile cilium (CCDC114, CFAP206, TEKT4), chromatin integrity (PRM2), gamete fusion (IZUMO4, EQTN), hyperactivation, tyrosine phosphorylation, and capacitation [PI3K-Akt signalling pathway-related proteins (COL1A1, COL2A1, COL1A2, SPP1, PDGFA, NGF)] were down regulated in poor quality semen producing bulls. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03474-6.

Cryopreservation process alters the expression of genes involved in pathways associated with the fertility of bull spermatozoa.

In bovines, cryopreserved semen is used for artificial insemination; however, the fertility of cryopreserved semen is far lower than that of fresh semen. Although cryopreservation alters sperm phenotypic characteristics, its effect on sperm molecular health is not thoroughly understood. The present study applied next-generation sequencing to investigate the effect of cryopreservation on the sperm transcriptomic composition of bull spermatozoa. While freshly ejaculated bull spermatozoa showed 14,280 transcripts, cryopreserved spermatozoa showed only 12,375 transcripts. Comparative analysis revealed that 241 genes were upregulated, 662 genes were downregulated, and 215 genes showed neutral expression in cryopreserved spermatozoa compared to fresh spermatozoa. Gene ontology analysis indicated that the dysregulated transcripts were involved in nucleic acid binding, transcription-specific activity, and protein kinase binding involving protein autophosphorylation, ventricular septum morphogenesis, and organ development. Moreover, the dysregulated genes in cryopreserved spermatozoa were involved in pathways associated with glycogen metabolism, MAPK signalling, embryonic organ morphogenesis, ectodermal placode formation, and regulation of protein auto-phosphorylation. These findings suggest that the cryopreservation process induced alterations in the abundance of sperm transcripts related to potential fertility-associated functions and pathways, which might partly explain the reduced fertility observed with cryopreserved bull spermatozoa.

Genom-wide analysis identifies single nucleotide polymorphism variations and altered pathways associated with poor semen quality in breeding bulls.

The reason for poor semen quality among the breeding bulls is not well understood. In the present study, we performed high-throughput RNAseq analysis of spermatozoa to identify the SNPs present in good and poor-quality semen-producing Holstein Friesian breeding bulls. A total of 21,360 and 44,650 SNPs were identified in good and poor-quality semen with a minimum read depth of 20, among which 4780 and 8710 novel variants were observed in good and poor-quality semen, respectively. Greater SNPs and indels variations were observed in poor compared to good-quality semen. In poor-quality semen, SNP variations were observed in ZNF280B, SLC26A2, DMXL1, OR52A1, MACROD2 and REV1 genes, which are associated with regulation of spermatogenesis, post-testicular maturation, Cl- channel activity, V-ATPase-mediated intracellular vesicle acidification, a mono-ADP-ribosyl hydrolase and ATR-Chk1 checkpoint activation. GO analysis of filtered genes with significant variations between good and poor-quality semen showed enrichment in important pathways related to semen quality such as MAPK signalling pathway, Akt signalling pathway, focal adhesion, cAMP signalling pathway, and Rap1 signalling pathway. Network analysis of filtered genes in poor-quality semen showed variations in pathways of purine metabolism, pyrimidine metabolism, prolactin signalling pathway and RNA cap-binding complex. It is inferred that SNP in genes involved in maintaining sperm functions could be the reason for poor-quality semen production in bulls, and the identified SNPs hold potential to be used as biomarkers for semen quality in bulls.

Integrated multi-omics analyses reveals molecules governing sperm metabolism potentially influence bull fertility.

Bull fertility is of paramount importance in bovine industry because semen from a single bull is used to breed several thousands of cows; however, so far, no reliable test is available for bull fertility prediction. In the present study, spermatozoa from high- and low-fertility bulls were subjected to high-throughput transcriptomic, proteomic and metabolomic analysis. Using an integrated multi-omics approach the molecular differences between high- and low-fertility bulls were identified. We identified a total of 18,068 transcripts, 5041 proteins and 3704 metabolites in bull spermatozoa, of which the expression of 4766 transcripts, 785 proteins and 33 metabolites were dysregulated between high- and low-fertility bulls. At transcript level, several genes involved in oxidative phosphorylation pathway were found to be downregulated, while at protein level genes involved in metabolic pathways were significantly downregulated in low-fertility bulls. We found that metabolites involved in Taurine and hypotaurine metabolism were significantly downregulated in low-fertility bulls. Integrated multi-omics analysis revealed the interaction of dysregulated transcripts, proteins and metabolites in major metabolic pathways, including Butanoate metabolism, Glycolysis and gluconeogenesis, Methionine and cysteine metabolism, Phosphatidyl inositol phosphate, pyrimidine metabolism and saturated fatty acid beta oxidation. These findings collectively indicate that molecules governing sperm metabolism potentially influence bull fertility.

High throughput deep proteomic analysis of seminal plasma from stallions with contrasting semen quality.

Seminal plasma proteins and pathways associated with sperm motility have not been elucidated in stallions. Therefore, in the current study, using the high throughput LC/MS-MS approach, we profiled stallion seminal plasma proteins and identified the proteins and pathways associated with sperm motility. Seminal plasma from six stallions producing semen with contrasting sperm motility (n = 3 each high-and low-motile group) was utilized for proteomic analysis. We identified a total of 1687 proteins in stallion seminal plasma, of which 1627 and 1496 proteins were expressed in high- (HM) and low- motile (LM) sperm of stallions, respectively. A total number of 1436 proteins were co-expressed in both the groups; 191 (11%) and 60 (3.5%) proteins were exclusively detected in HM and LM groups, respectively. A total of 220 proteins were upregulated (>1-fold change) and 386 proteins were downregulated in SP from LM group stallions as compared to HM group stallions, while 830 proteins were neutrally expressed in both the groups. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed dysregulation of the important proteins related to mitochondrial function, acrosome, and sperm cytoskeleton in the seminal plasma of stallions producing ejaculates with low sperm motility. High abundance of peroxiredoxins and low abundance of seminal Chaperonin Containing TCP1 Complex (CCT) complex and Annexins indicate dysregulated oxidative metabolism, which might be the underlying etiology for poor sperm motility in LM group stallions. In conclusion, the current study identified the seminal plasma proteomic alterations associated with poor sperm motility in stallions; the results indicate that poor sperm motility in stallions could be associated with altered expression of seminal plasma proteins involved in oxidative metabolism.

Melatonin and canthaxanthin enhances sperm viability and protect ram spermatozoa from oxidative stress during liquid storage at 4°C.

Antioxidants are used to minimize oxidative stress during liquid semen storage. The main aim of current study was to elucidate effect of supplementing melatonin and canthaxanthin in Tris-based extender could enhance seminal quality of ram at 4°C up to 72 h. A total of 48 ejaculates were collected from breeding Magra rams (n = 8) and were preliminarily subjected for various macroscopic and microscopic semen evaluation tests. These ejaculates were pooled and divided into three equal aliquots. Two aliquots were diluted (1:10) using extender encompassing final concentration of 1mM melatonin and 25 µM canthaxanthin and stored at 4°C. Third aliquot with extender only was kept as control. Structural and functional seminal changes were observed at different time points of preservation. Results revealed that mean values for progressive sperm motility, viability and total antioxidant capacity were significantly higher (p < 0.05) in melatonin group while hypo-osmotic swelling test was significantly (p < 0.05) higher in canthaxanthin group. Total sperm abnormalities and malondialdehyde levels were significantly (p < 0.05) lower in both treatment groups indicating their antioxidant efficacy in protection of spermatozoa from oxidative stress. Results of study indicated that supplementation of these antioxidants to ram semen could be used to enhance storage life of liquid semen at 4°C up to 72 h.

Applications of Genome Editing Tools in Stem Cells Towards Regenerative Medicine: An Update.

Precise and site-specific genome editing through application of emerging and modern gene engineering techniques, namely zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/ Cas9) have swiftly progressed the application and use of the stem cell technology in the sphere of in-vitro disease modelling and regenerative medicine. Genome editing tools facilitate the manipulation of genes in various types of cells with target-specific nucleases. These tools aid in elucidating the genetics and etiology behind different diseases and have immense promise as novel therapeutics for correcting the genetic mutations, making alterations, and curing diseases permanently, which are not responding and resistant to traditional therapies. These genome engineering tools have evolved in the field of biomedical research and have also been shown to have a significant improvement in clinical trials. However, their widespread use in the research revealed potential safety issues, which need to be addressed before implementing such techniques for clinical purposes. Significant and valiant attempts need to be made in order to surpass those hurdles. The current review outlines the advancements of several genome engineering tools and describes suitable strategies for their application towards regenerative medicine.

Purification, molecular characterization and ligand binding properties of the major donkey seminal plasma protein DSP-1.

Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the protein is heterogeneously glycosylated and also contains multiple acetylations, occurring in the attached glycans. Structural and thermal stability studies on DSP-1 employing CD spectroscopy and differential scanning calorimetry showed that the protein unfolds at ~43 °C and binding to phosphorylcholine (PrC) - the head group moiety of choline phospholipids - increases its thermal stability. Intrinsic fluorescence titrations revealed that DSP-1 recognizes lyso-phosphatidylcholine with over 100-fold higher affinity than PrC. Further, interaction of DSP-1 with erythrocytes, a model cell membrane, revealed that DSP-1 binding is mediated by a specific interaction with choline phospholipids and results in membrane perturbation, suggesting that binding of this protein to sperm plasma membrane could be physiologically significant.

Epididymosomes: A potential male fertility influencer.

During transit and storage in epididymis, spermatozoa undergo final maturation, acquire motility, functional competence and the ability to fertilise an oocyte. Epididymal secretions contain a complex biochemical milieu of diverse inorganic ions, proteins, metabolites and other molecules. Since it is believed that spermatozoa are translationally silent, proteins appearing in them are thought to be synthesised elsewhere, including epididymis, and then incorporated to the cells. One of the important mechanisms suggested to be involved in transfer of epididymal secretions to spermatozoa is through exosomes called epididymosomes. Epididymosomes released from the epididymal epithelium contain proteins, noncoding RNAs and distinct set of lipids that are transferred to spermatozoa while they pass through the different epididymal regions. Owing to the importance of these molecules for sperm maturation and fertilising ability, research on epididymosomes has gained increasing attention during the last decade. This review is focused on epididymosomes, with emphasis on recent advances in the understanding of mechanisms of epididymosomal cargo transfer to spermatozoa and potential roles of epididymosomes in sperm function and beyond. Possibilities of utilising the molecular signatures of epididymosomes as a tool for male fertility assessment are also discussed.

Cholesterol Loaded Cyclodextrin Supplementation Enhances the Cholesterol-to-Phospholipid Ratio and Diminishes Oxidative Stress in Jack Spermatozoa During Cryopreservation.

The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5, 2, and 3 mg/mL CLC to obtain 120 × 106 sperm/mL spermatozoa concentration. The semen was cryopreserved using customized freezing protocols. Evaluation of seminal parameters, the C:P ratio, and the oxidative status of jack spermatozoa was analyzed at all stages of cryopreservation. The oxidative status in the jack semen was evaluated by measuring malondialdehyde, glutathione and total antioxidant capacity levels. The results indicated that the mean percent values for various seminal quality parameters and the oxidative parameters were found to be significantly higher (P < .05) in CLC-treated groups with the highest values for 2 mg of CLC/120 × 106 spermatozoa. In conclusion, the present study revealed that the supplementation of CLC before cryopreservation has significantly reduced the oxidative stress and also increased the C:P ratio during semen cryopreservation process. Furthermore, a reduction in lipid peroxidation levels, reduced damage to the sperm plasma and acrosome membranes and improvement in the post-thaw sperm integrity as well as stability were recorded.

Ameliorative Effect of Ascorbic Acid and Glutathione in Combating the Cryoinjuries During Cryopreservation of Exotic Jack Semen.

The present study was designed to study the adverse effects of cryopreservation and evaluation of the cryoprotective effect of reduced glutathione (GSH) and ascorbic acid (AA) supplementation on exotic jack semen in combination or alone. For this, 24 semen samples from four adult and fertile jacks were collected via artificial vagina using an estrus jenny as dummy. After semen collection, the semen was evaluated for various qualitative and quantitative parameters in fresh, cooled, and frozen-thawed semen. The semen pellet was extended with the freezing extender containing either AA (0.9 g/L), GSH (2.5 mM), or combination of both (AA 0.9 g/L + GSH 2.5 mM), and another aliquot was kept as control without adding the antioxidants. The jack semen underwent cryodamage, which was evident by the observation of significant (P < .05) decline in the seminal quantitative parameters at various stages of cryopreservation process. Prefreeze and postthaw semen evaluation revealed that the values of plasma membrane, acrosome integrity, and chromatin integrity were found to be significantly higher (P < .01) in the group of samples supplemented with the combination (0.9 g/L AA +2.5 mM GSH) than AA- and GSH-alone or control groups. Supplementation of antioxidants to the freezing extender improved jack prefreeze and postthaw semen quality with the superiority of GSH over AA alone. From the present study, it was inferred that, exotic jack spermatozoa are susceptible to injuries because of cryopreservation, but these cryo-induced damage can be ameliorated significantly (P < .05) with the use of antioxidants and contribute to the improvement of semen cryopreservation procedures.

Biochemical components of seminal plasma and their correlation to the fresh seminal characteristics in Marwari stallions and Poitou jacks.

AIM: To investigate various biochemical components of seminal plasma in Marwari stallions and Poitou Jacks and to find out their correlation with that of the seminal characteristics. MATERIALS AND METHODS: In this study, semen was collected from six Marwari stallions and six Poitou jacks aged from 4 to 6 years and with known fertility status. The semen collection from the stallions were collected during the breeding season, i.e., between the months of April and June. From the collected semen ejaculates, we estimated the values of some biochemical components, viz., total protein content, total lipid content, and enzymes such as glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), alkaline phosphatase (ALP), acid phosphatase (ACP), and lactate dehydrogenase (LDH) as well as concentrations of glucose, cholesterol, total calcium (Ca), and phosphorus (P) and correlations among different seminal parameters were statistically examined using the Pearson correlation coefficient. RESULTS: In this study, we found positive correlations between semen volume as well as sperm concentration and GOT, GPT, ALP and ACP for both the group stallions. Significant correlation between motility and glucose, GOT and GPT could be an indication for their role metabolism and protection against free radicals to the spermatozoa. CONCLUSION: Based on the results, it is concluded that there is a positive correlation between some biochemical values such as glucose, Ca, ALP, and LDH and seminal parameters which play a key role in capacitation and onward movement of the spermatozoa.