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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%., Competing Interests: Declaration of competing interest All authors hereby declare that there is no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

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 × 10 6 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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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 (K a  = 1.08 × 10 5  M -1 ) with ~80-fold higher affinity than PrC (K a  = 1.39 × 10 3  M -1 ). Binding of DSP-3 to erythrocytes leads to membrane perturbation, suggesting that its binding to sperm plasma membrane could be physiologically significant., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Musti J. Swamy reports financial support was provided by India Ministry of Science & Technology, Department of Science and Technology. Musti J. Swamy reports financial support was provided by University of Hyderabad., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The present study was conducted to evaluate the functional membrane integrity of cryopreserved spermatozoa from stallions belonging to two contrasting motility groups (high and low motility groups). A dual staining method comprising of carboxy fluoroscein diacetate (CFDA) and propidium iodide (PI) was used for this purpose. Stallions were classified into either high (post thaw progressive motility e"35%) or low (post thaw progressive motility <25%) motility groups. The proportion of membrane intact spermatozoa was significantly (P<0.05) higher in high motility group. Interestingly, it was observed that the proportion of moribund population was significantly (P<0.05) higher in low motility group as compared to high motility group. It was inferred that higher proportion of moribund spermatozoa might be one of the important contributing factors for poor sperm motility in stallions belonging to low motile group. [ABSTRACT FROM AUTHOR]

The study was conducted with the objective to evaluate the post thaw semen qualities of Poitou donkey by addition of α-tocopherol, Pentoxifylline and Taurine in semen extender. Semen was divided in four different treatments having final concentration of 150 x 106 sperm/ml with use of cryoprotectant (5% DMF) containing without supplement (control) and following supplements with concentration: á-tocopherol (1 mM), Pentoxifylline (3.5 mM) and Taurine (25 mM), all stored in liquid nitrogen for 24 hrs. All samples after thawing process were maintained at 37°C, were analyzed and performed. Spermatozoa motility, live sperm count, host reactive, acrosomal integrity and DNA integrity were recorded and performed for each additive and also for an untreated control. In between the overall treatments, Pentoxifylline (T2) and Taurine (T3) revealed the higher (P<0.01) post thaw sperm motility (53.78±0.87 and 52.28±0.90%) compared to α-tocopherol-T1 (50.15±0.80%) and control (39.65±0.52%) while, T2 and T3 differed non-significantly. Between the treatments, T2 and T3 revealed the higher (P<0.01) post thawed live sperm count (60.62±0.82and 59.46±0.84%) compared to T1 (57.12±0.77%) and control (45.43±0.56%). In between the treatments, T2 and T3 revealed the higher (P<0.01) values of post thaw host reactive sperms (43.28±0.77 and 42.18±0.84%) compared to T1 (40.21±0.72%) and control (30.15±0.40%). Whereas, T2 and T3 differed non-significantly. Between the treatments, T2 and T3 revealed the higher (P<0.01) post thaw Acrosomal integrity (87.03±0.52and 87.03±0.45%) compared to T1 (84.62±0.34%) and control (80.34±0.24%) while T2 and T3 differed non-significantly. In between the treatments (T2 and T3) revealed the higher (P<0.01) DNA integrity (88.28±0.37 and 87.50±0.28%) compared to both treatment T1 (84.75±0.26%) and control (81.03±0.37%) while T2 and T3 differed non-significantly. In above study Pentoxifylline was found to improve sperm motility, live sperm count, host reactive, acrosomal integrity and DNA integrity and may also enhance the quality of frozen equine semen by increasing the fertility rate probably. [ABSTRACT FROM AUTHOR]