Sperm Integrity Research Articles

Boar-to-Boar Variations in Quality Characteristics of Sperm from Different Ejaculates Following Freezing–Thawing

The main objective of this study was to investigate boar-to-boar variations in the quality characteristics of sperm from the sperm-rich fractions (SRFs) and whole ejaculates (WEs) following freezing–thawing. Several sperm attributes, such as motility patterns analyzed by the computer-assisted sperm analysis (CASA) system, mitochondrial function, membrane integrity, and DNA fragmentation were used to compare the cryo-survival of sperm from SRFs and WEs from boars with good and poor semen freezability (GSF and PSF, respectively). In this study, boars with post-thaw total motility (TMOT) more than 30% (>30%) were classified as having GSF, while those with post-thaw TMOT less than 30% (<30%) were classified as having PSF. Principal component analysis 1 (PCA1), which is the main component of the sample variation, explained approximately 75% of the variance between the GSF and PSF groups, reaffirming the reliability of post-thaw TMOT as a reliable criterion used to classify the animals. Most of the post-thaw sperm parameters of the SRFs and WEs were positively correlated. Furthermore, scatter plot analyses show stronger relationships between the analyzed post-thaw parameters of the frozen–thawed (FT) sperm of SRFs than those of WEs. Individual boar variations or the sperm source had marked effects on the quality characteristics of FT sperm. The higher TMOT, velocity straight line (VSL), and velocity average path (VAP) of FT sperm were more enhanced in the SRFs compared with the WEs of the PSF group. Furthermore, the mitochondrial function, membrane integrity, and DNA fragmentation of FT sperm were markedly higher in the SRFs than in the WEs, particularly for the poor freezability boars. We suggest that the freezability potential of sperm of the GSF group does not differ significantly between the SRFs and WEs, reaffirming that boar variability is an important factor that affects the cryo-survival of sperm.

Graphene oxide and silymarin combination: A novel approach to improving post-cryopreservation quality of ram sperm.

Graphene oxide (GO) has been extensively studied for its diverse biomedical applications, including drug delivery, imaging, and tissue engineering. Silymarin, as a flavonoid complex derived from the milk thistle plant, has recently shown potential health benefits, particularly concerning reproductive health. This study aims to evaluate the effects of GO and silymarin supplementation, both individually and in combination, on the characteristics of frozen-thawed ram sperm. Semen samples were collected using standard artificial insemination (AI) techniques with an artificial vagina. The collected semen was evaluated and cryopreserved in a tris-based extender containing varying concentrations of silymarin and GO (0, 10, or 20 μg/mL) or their combination. Post-thaw assessments evaluated sperm motility, viability, morphological abnormalities, DNA integrity, membrane integrity, malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, and total antioxidant capacity (TAC). Our findings revealed that the combination of 20 μg/mL silymarin and 20 μg/mL GO significantly enhanced total motility, viability, membrane integrity, and DNA integrity of sperm. Additionally, this treatment effectively reduced morphological abnormalities and MDA levels post-thawing. Notably, SOD and TAC activities were improved following the freeze-thaw compared to other treatment groups. In conclusion, the combination of silymarin and GO significantly improves the quality of frozen-thawed ram sperm by enhancing sperm parameters while reducing oxidative stress markers. The results suggest their potential as effective additives in cryopreservation protocols, providing a promising avenue for improving reproductive outcomes in rams and potentially other livestock species.

Effect of reproductive senescence on DNA integrity of epididymal sperm in dogs

Effect of reproductive senescence on DNA integrity of epididymal sperm in dogs

Seminal Plasma-Derived Exosome Preserves the Quality Parameters of the Post-Thaw Semen of Bulls with Low Freezeability.

Introduction: Sperm cryopreservation is a useful storage technique in artificial insemination. Nanoparticles and nanovesicles such as exosomes are widely used in sperm cryopreservation procedures to alleviate cold-induced injury inflicted during sperm freezing. Objective: The objective of the present study was to examine the impact of varying concentrations of exosomes derived from seminal plasma added to a freezing extender on the quality of post-thawed bull sperm. Methods: Five Holstein bulls were chosen based on their samples having less than 30% progressive motility. After exosome extraction, semen samples from bulls (n = 5) with progressive sperm motility ≤30% were collected, diluted with different exosome concentrations (0, 25, 50, and 100 μg/mL), and aspirated into 0.5 mL straws. After the freeze-thaw process, sperm total and progressive motility, viability, morphology, plasma membrane integrity, mitochondrial activity, and apoptosis status were assessed. Furthermore, the expression levels of annexin (ANX1), dystrophy-associated Fer-1-like protein (DYSF), fibronectin 1 (FN1), and reactive oxygen species modulator 1 (ROMO1) were evaluated via real-time polymerase chain reaction (PCR). Results: Adding different concentrations of exosomes (25, 50, and 150 μg/mL) significantly increased the progressive motility, viability, and membrane integrity of sperm compared with the control group (p < 0.05). For the apoptosis index, treatment with 100 μg/mL exosomes significantly increased the percentage of live cells (p < 0.05), while the percentage of necrotic cells decreased significantly (p < 0.05) compared with 25 μg/mL exosome. The results of quantitative PCR showed that the expression levels of ANX1 were significantly (p < 0.05) upregulated at 50 μg/mL exosome, and the expression of ROMO1, FN1, and DYSF were downregulated upon treatment with different exosome concentrations. Conclusions: In conclusion, supplementing the freezing diluent with exosome-derived seminal plasma could preserve the quality parameters of the post-thaw semen of the bull with low freezeability and could be used as a helpful method for reproductive programs.

Regulatory role of exosomal proteins MIF and PEBP1 in sperm storage in black rockfish (Sebastes schlegelii) ovary

Sperm entry into the female reproductive tract triggers the production of exosomes that play a vital role in regulating sperm storage, release, and viability. Exosomal proteins are considered to be key factors influencing these processes. In this study, we focused on macrophage migration inhibitory factor (MIF) and phosphatidylethanolamine-binding protein 1 (PEBP1) to investigate their amino acid sequence identification, expression patterns, and functional roles in post-mating ovarian tissues of black rockfish (Sebastes schlegelii). Phylogenetic analysis revealed that MIF and PEBP1 cluster separately across species, with distinct groupings for fish and mammals, underscoring their evolutionary conservation and essential biological roles. We observed significant upregulation of mif and pebp1 gene expression during various stages of post-mating sperm storage, which was reflected in corresponding increases in protein levels. Exosomes were successfully isolated from the ovarian fluid of black rockfish, with particle sizes ranging from 40 to 220 nm, and exhibiting typical exosomal morphological characteristics. More importantly, the presence of MIF and PEBP1 was confirmed in these exosomes. Both proteins were localized in ovarian follicular and epithelial cells. Functional analysis revealed that recombinant Sebastes schlegelii MIF (rSsMIF) and PEBP1 (rSsPEBP1) were capable of transferring into sperm after incubation, suggesting their binding interaction. Both proteins effectively preserved sperm integrity, including intact heads and undamaged flagella. After 24 h of incubation with rSsMIF and rSsPEBP1, sperm viability was maintained at 88 % and 85 %, respectively, compared to only 58 % in the control group. Additionally, sperm motility was also significantly improved following treatment with both proteins, with the proportion of motile sperm increasing by 14 % and 20 %, respectively. Moreover, the present study found that the regulation of sperm viability and motility by two types of exosomal proteins may accompanied by intracellular reactive oxygen species (ROS) and calcium ions. The levels of ROS in sperm decreased to varying extents after incubation with the two proteins, whereas intracellular calcium ion concentrations increased. These findings provide new insights into the roles of MIF and PEBP1 in sperm storage and fertility, suggesting potential applications in reproductive biotechnology.

Paternal Contributions to Recurrent Pregnancy Loss: Mechanisms, Biomarkers, and Therapeutic Approaches.

Background and Objectives: Recurrent pregnancy loss (RPL) affects numerous couples worldwide and has traditionally been attributed mainly to maternal factors. However, recent evidence highlights significant paternal influences on pregnancy viability and outcomes. This review aims to comprehensively examine male contributions to pregnancy loss, focusing on underlying mechanisms, novel biomarkers, and integrated strategies for improved reproductive success. Materials and Methods: A comprehensive narrative review was conducted by searching databases including PubMed and Embase for the literature published from January 2004 to October 2024. Studies focusing on paternal influences in RPL-encompassing oxidative stress, genetic and epigenetic mechanisms, health conditions, lifestyle factors, environmental exposures, and advancements in sperm proteomics-were included. Inclusion criteria were peer-reviewed articles in English that directly addressed paternal factors in RPL; studies not meeting these criteria were excluded. Results: The review identified that paternal factors such as advanced age, metabolic and cardiovascular health issues, chronic diseases, lifestyle habits (e.g., smoking, alcohol consumption, poor diet), and environmental exposures significantly affect sperm integrity through mechanisms like oxidative stress, DNA fragmentation, and epigenetic alterations. Advanced paternal age and poor health conditions are associated with increased risks of miscarriage and adverse pregnancy outcomes. Novel sperm proteomic biomarkers have been identified, offering potential for enhanced diagnostics and personalized interventions. Integrated approaches involving multidisciplinary assessments, preventive strategies, and genetic counseling are essential for effectively addressing RPL. Conclusions: Integrating paternal factors into clinical evaluations is crucial for effectively addressing recurrent pregnancy loss. Recognizing and modifying paternal risk factors through lifestyle changes, medical interventions, and environmental management can improve pregnancy outcomes. The findings underscore the need for incorporating paternal assessments into standard care and highlight the importance of future research focusing on standardizing diagnostic protocols, expanding studies on paternal contributions, and integrating proteomic biomarkers into clinical practice to facilitate personalized treatment strategies.

Association of NOX5 Expression with Sperm Activity and Motility in Pathospermic Infertile Men.

The newest NOX isoform, NOX5, has been found in mammalian spermatozoa. Many physiological and pathological situations in spermatozoa are mediated by reactive oxygen species (ROS). NOX5 is the main source of ROS in spermatozoa. Our purpose was to investigate the changes in NOX5 expression and the effect of NOX5 expression on sperm motility, chromatin integrity, and oxidative status in oligoasthenozoospermic compared to normozoospermic men. Semen samples were collected from 30 normozoospermic (NS) and 30 oligoasthenozoospermic (OAS) men. NOX5 protein expression in sperm samples was evaluated by immunohistochemistry and western blot. Oxidative stress status was evaluated by total antioxidant capacity (TAC), total oxidant capacity (TOC), and oxidative stress index (OSI) parameters. Chromatin integrity in spermatozoa was evaluated by toluidine blue staining. NOX5 expression levels were significantly higher in OAS group than in NS group (p<0.001). In addition, chromatin integrity was significantly higher in the OAS group in comparison to NS group (p<0.001). TAC levels were higher in the NS group, but OSI and TOC levels were significantly higher in OAS group (p<0.001). It was found that NOX5 protein expression was positively correlated with oxidative stress and chromatin integrity and negatively correlated with motility (p<0.01). These results suggest that overexpression of NOX5 may be the source of excessive ROS production and oxidative stress injuries in oligoasthenozoospermic men. Considering that NOX5 expression is positively correlated with oxidative stress and chromatin integrity but negatively correlated with motility, it can be considered a biomarker to be used in assisted reproductive procedures.

Ameliorative impacts of carvacrol on DNA integrity, oxidative stress, and sperm quality in asthenozoospermic infertile individuals during cryopreservation.

Nowadays, the cryopreservation process can produce reactive oxygen species (ROS), which cause damage to the motility, cell membrane, and DNA integrity of sperm. This study is aimed at evaluating the impact of carvacrol, as a powerful antioxidant, on sperm features and improving oxidative stress throughout the cryopreservation procedure. In this prospective study, semen samples from 25 patients with asthenozoospermia were separated into three groups: fresh, freezing, and freezing + carvacrol (a dose of 100µM). The subsequent parameters were evaluated using standard methods in all three groups: sperm motility according to WHO criteria, sperm morphology using Papanicolaou staining, sperm viability with eosin-nigrosin staining, DNA integrity with acridine orange staining, levels of antioxidant enzymes (catalase, glutathione, and superoxide dismutase), total antioxidant capacity (TAC), and malondialdehyde (MDA) using ELISA. Also, DNA fragmentation was analyzed by the SDFA kit, and mitochondrial membrane potential (MMP) was assessed by rhodamine staining. Average sperm viability, motility, mitochondrial membrane potentiality, integrity of sperm membrane, and antioxidant enzyme levels are meaningfully reduced in the freezing group in contrast to the control group. The freezing group showed a meaningful rise in the mean MDA levels and DNA fragmentation compared to the control group. In the freezing group supplemented with carvacrol, a meaningful rise could be visible in mean percentages of viability, motility, and antioxidant enzyme levels, whereas mean levels of MDA and DNA fragmentation meaningfully declined in contrast to the freezing group. The results demonstrate that carvacrol, a potent antioxidant, offers significant protection against the loss generated by the freeze-thaw procedure, thereby improving sperm quality.

Effect of Age on the Body Parameters, Scrotum, Seminal Characteristics, Spermatozoa Integrity and Sexual Behavior in Ganjam Buck

Background: Bucks raised for breeding purposes receive no special attention and are raised at random without following the correct selection process. Besides, the fertility of the male bucks is never addressed. Goat breeders have noted a sharp decline in the reproductive efficiency of Ganjam goats, associated with a lengthened kidding interval, fewer successful pregnancies and decreased twining frequency. The measurement of male fertility parameters is crucial for estimating the breeding performance of any species since males contribute 50% of the reproductive efficiency. Methods: Six Ganjam bucks of age 1.5(B1), 2(B2), 2.5(B3), 3.5(B4), 4(B5) and 4.5(B6) years procured after selection on the basis of their body morphometry and sexual behaviour were maintained under optimum management practice. They were trained for semen collection. For study on effect age on the semen qualities, they were classified into two age groups and ejaculates were collected during two phases. Bucks of age below 3 were considered under Age group I and bucks older than 3 years were assigned Age group II. Twelve ejaculates from each buck were collected, six ejaculates between the month of July to August and six ejaculates during October to November. The total of 72 ejaculates was evaluated for the study of seminal characteristics. Besides, the external body parameters, scrotal parameters and sexual behaviour of the bucks were also recorded before each semen collection. Result: The bucks of age group II were found to be having significantly higher (p less than 0.01) body weight, mean body length and average value of chest girth than age group I bucks in the present study. The mean value of scrotal width of the bucks of age group II was significantly higher (p less than 0.01) as compared to that of age group I. The mean value of abnormalities of the bucks of age group II was significantly higher (p less than 0.01) than that of age group I. The mean value of acrosome integrity and MBRT of the bucks of age group II was significantly higher (p less than 0.05) to that of age group I. The present study will be helpful in the selection of Ganjam buck at appropriate age for breeding purpose basing on their body parameters, scrotum, seminal characteristics, spermatozoa integrity and sexual behavior.

Effect of silver nanoparticles on donkey sperm parameters and ultrastructure.

The aim of this study was to determine the effect of silver nanoparticles (AgNPs) on donkey sperm parameters and ultrastructure. AgNPs were synthesized, purified and resuspended in the extender. Nine frozen-thawed donkey sperm samples were exposed to different concentrations of AgNPs (0, 1.25, 2.5, 5, 12.5, 25 and 50 μg/mL). Sperm parameters: total (TMOT, %) and progressive (PMOT, %) sperm motility, plasma (LIVE, %) and acrosomal membrane integrity (AIS, %), and sperm morphology (MORF, %) were evaluated immediately after AgNPs exposure (T0) and after 2 h of incubation (T2). The interaction beween AgNPs and spermatozoa was visualized by transmission electron microscopy (TEM). At T0, sperm motility and AIS were reduced (p < .05) when using concentrations ≥50 and ≥25 μg/mL, respectively. At T2, sperm motility and LIVE were significantly decreased (p < .05) in concentrations ≥25 and ≥50 μg/mL, respectively. TEM analysis revealed nanoparticle adhesion to the acrosomal region of the plasma membrane. In conclusion, AgNPs at concentrations ≥25 μg/mL impair motility, acrosome and plasma membrane integrity of donkey sperm, which may be mediated by adhesion to the acrosomal region of the sperm surface membrane.

Effects of Butylated Hydroxytoluene and Sorbitol as Diluent Components on Structural and Surface Ultrastructural Changes of Gaga Chicken Sperm During Cryopreservation

The Gaga chicken is an indigenous Indonesian breed that is important to preserve using semen cryopreservation technology. The study was conducted to determine the effect of adding sorbitol and butylated hydroxytoluene (BHT) in the diluent on the structural and surface ultrastructure of cryopreserved Gaga chicken sperm during cryopreservation /frozen storage. The study aimed to assess how adding sorbitol and butylated hydroxytoluene (BHT) to the diluent affects the structure and surface ultrastructure of cryopreserved Gaga chicken sperm. A completely randomized design was employed with four treatments and 10 replications including egg yolk-lactate ringer diluent (EYLR) as the control group, EYLR diluent with 3 mM BHT, EYLR diluent with 2% sorbitol, and EYLR diluent with both 3 mM BHT and 2% sorbitol. Semen was collected using a massage technique from 4 male chickens aged approximately 10 months, pooled semen was diluted, packaged in 0.25 mL straws, equilibrated for 2 hours at 5 °C, pre-freeze for 10 minutes, frozen for 24 hours, and thawed for 30 seconds at 37 °C. The parameters evaluated were sperm plasma membrane integrity, acrosome integrity, DNA damage, mitochondrial functionality, and surface ultrastructure. The results showed that the treatment had a significant effect on plasma membrane integrity and post-thawing mitochondrial functionality compared to the control, but no effect was observed on acrosome integrity or DNA damage. The results showed that the combination treatment of BHT with sorbitol had a significant effect on plasma membrane integrity and post-thawing mitochondrial function, but did not affect acrosome integrity or DNA damage when compared to the control group. Ultrastructural observations indicated that cryopreservation caused damage to the head, middle, and tail of the sperm in the control groups. However, these changes were prevented by the diluent containing a combination of BHT and sorbitol. The addition of both components (BHT 3 mM + sorbitol 2%) effectively maintained plasma membrane integrity, mitochondrial functionality, and surface ultrastructure of Gaga chicken sperm during cryopreservation. Keywords: Butylated hydroxytoluene, Chicken sperm, Cryopreservation, Sorbito, Structure, Sperm ultrastructure

Exploring the Effects of Nitric Oxide and Reactive Oxygen Species on Buffalo Sperm Quality: A Comprehensive Review

Buffalo reproduction plays a vital role in the livestock industry, where sperm quality is a critical determinant of successful fertilization and overall fertility. Nitric oxide (NO) and reactive oxygen species (ROS) are key regulators of various physiological processes, including sperm function. However, their dual role in modulating sperm quality is complex, as both excessive and insufficient levels can lead to detrimental effects. This review explores the intricate balance between NO, ROS and sperm quality in buffalo, emphasizing the molecular mechanisms underlying these interactions. NO is a signaling molecule involved in the regulation of sperm motility, capacitation and acrosome reaction, but its overproduction can result in nitrosative stress, impairing sperm function. Similarly, ROS, at physiological levels, are essential for sperm capacitation and acrosome reaction, yet an imbalance towards oxidative stress can lead to lipid peroxidation, DNA fragmentation and reduced sperm viability. This review also delves into the role of exogenous NO donors, such as sodium nitroprusside (SNP), in modulating NO and ROS levels, and their consequent impact on sperm quality. The dual effects of NO and ROS on buffalo sperm highlight the importance of maintaining an optimal redox balance to preserve sperm integrity and functionality. In conclusion, understanding the molecular pathways through which NO and ROS influence sperm quality is crucial for developing targeted strategies to enhance reproductive outcomes in buffalo. Future research should focus on identifying biomarkers of oxidative stress and evaluating the potential of antioxidant therapies to mitigate the adverse effects of NO and ROS, ultimately improving buffalo fertility.

Exploratory Metabolomics and Lipidomics Profiling Contributes to Understanding How Curcumin Improves Quality of Goat Semen Stored at 16 °C in Tropical Areas.

Reactive oxygen species (ROS) exert a vital role in sperm quality during semen preservation, where excessive ROS leads to oxidative damage and undermines sperm integrity. Curcumin, a botanical extract, is capable of neutralizing ROS and enhancing the activity of antioxidant enzymes. This study was aimed at evaluating the effects of curcumin on sperm viability, acrosome integrity, and antioxidant levels, as well as metabolomic and lipidomic profiles. The results demonstrated that curcumin at 25 µmol/L significantly enhanced sperm motility, plasma membrane, and acrosome integrity, elevated the levels of antioxidant enzymes (T-AOC, CAT, SOD), and decreased ROS production (p < 0.05). Metabolomic analysis identified 93 distinct metabolites that showed significant differences between the control and curcumin-treated groups. KEGG pathways emphasized the participation of these metabolites in key metabolic processes such as the citric acid cycle, cholesterol metabolism, and fatty acid metabolism. Curcumin treatment brought about notable variations in lipid profiles, including increased levels of phosphatidylcholine, acylcarnitine, and triglyceride over the storage time, suggesting enhanced lipid anabolic activity. Overall, the supplementation of curcumin at 25 µmol/L effectively mitigates oxidative stress and prolongs the viability of semen storage at 16 °C by modulating specific metabolic and lipid profiles.

The Effect of Metallic Nanoparticles Supplementation in Semen Extender on Post-thaw Quality and Fertilizing Ability of Egyptian Buffalo (Bubalus bubalis) Spermatozoa.

Nanomaterials offer several promising prospects in the field of farm animal reproduction, encompassing a broad range of applications such as transgenesis and the precise delivery of substances to sperm cells, antimicrobial, antioxidants properties as well as their potent role in improving cryopreservation methods. The aim of the present study is to explore the effect of supplementing the semen extender with 10µg/mL nano gold (Au-NPs10), 10µg/mL nano silver (Ag-NPs10), 1µg/mL nano selenium (Se-NPs1), and 100µg/mL nano zinc oxide (ZnO-NPs100) on sperm characteristics and kinematics parameters, acrosome integrity, oxidative biomarkers, morphological and apoptosis-like changes of frozen-thawed buffalo bull sperm, and, ultimately, their fertilizing capacity. The results revealed that all aforementioned nano materials significantly improved viability, progressive motility, membrane integrity, acrosome integrity, and kinematic parameters as well as apoptosis-like changes of post-thawed buffalo bull sperm compared to the control (p < 0.05). No discernible effects were observed on sperm ultrastructure morphology measures as a response to the addition of these metallic nanoparticles to the extender. The values of caspase 3 significantly decreased by 64.22, 45.99, 75.59, and 49.39% in Au-NPs10, Ag-NPs10, Se-NPs1, and ZnO-NPs100treated groups, respectively, compared to the control. The addition of 100µg ZnO-NPs to the extender significantly decreased the total count of bacteria, fungi, and yeast compared to the control (p < 0.05). The AuNPs10 and SeNPs1 treated groups showed lower content of malondialdehyde, hydrogen peroxide, and nitric oxide concentrations and higher values of total antioxidant capacity of post-thawed extended semen (p < 0.05). Pregnancy rates increased by 17.5, 20, and 30% in buffalo cows inseminated with sperm treated with ZnO-NPs100, Se-NPs1, and Au-NPs10, respectively, compared to the control group. The present results indicate that the freezing extender supplemented with metallic nanoparticles can be an effective strategy to enhance the cryotolerance and fertility potential of buffalo bull sperm.

The quality and characteristics of bovine sperm are compromised by Toxoplasma gondii antigens, impacting in in vitro bull fertility

Studies in various species have demonstrated different results on the effects of T. gondii infection on sperm quality. It has also been demonstrated that in some stages of the disease, there is elimination of cellular debris or even the intact parasite in the semen. The present work aimed to evaluate the in vitro effects of the presence of soluble T. gondii antigens in bovine semen on sperm integrity. The spermatozoa were treated with T. gondii antigens in double serial dilutions classified as high, medium and low doses (8, 4, 2 µg/ml) in "TALP-Sperm” and “TALP-Fert" media. The results showed that T. gondii antigens affect sperm motility and mitochondrial activity, and cause changes in sperm chromatin integrity, as well as damage to the sperm membrane and acrosome. Finally, spermatozoa treated with T. gondii antigens were evaluated in the in vitro production of embryos (IVEP). The use of semen contaminated with antigens in IVEP routines did not lead to a decrease in the fertilization of oocytes, as sperm undergo selection before in vitro fertilization, which eliminates the most altered sperm. However, early embryonic development was affected, probably by structural changes that were not eliminated in the selection process. The results demonstrated that the presence of soluble T. gondii antigens in bovine semen alters sperm integrity and vital characteristics for the fertilization process and embryonic development and therefore causes fertility problems in males.

Cryopreservation of goat spermatozoa in the Mekong Delta region of Vietnam: evaluating cryoprotectant effectiveness

Conserving and improving the quality of breeds is crucial for enhancing small ruminant production in Vietnam. To support this purpose, cryopreservation was offered as a promising method for semen preservation and maintaining genetic diversity. Moreover, many small-scale farmers in Mekong Delta rely on goats for both income and nutrition, making them an important part of farming activities. This study aimed to develop a sperm cryopreservation protocol and identify the optimal cryoprotectant for goat sperm in the Mekong Delta region of Vietnam. Semen samples were diluted with TCG-EggYolk freezing medium containing cryoprotectant agent (CPA) in a 1:1 ratio. The samples were then placed in 0.5 mL French straws, cooled to 15 °C and 5 °C, and subjected to nitrogen vapor before being immersed in liquid nitrogen at -196 °C. Experiment 1 utilized Glycerol as the CPA in concentrations of 0 %, 5 %, 8 %, and 11 %. Experiment 2 employed dimethyl sulfoxide (DMSO) as the CPA with the same concentrations. Thawed samples were evaluated for sperm quality. Results indicated that in Experiment 1, increasing the Glycerol concentration improved the overall motility, viability, and membrane integrity of sperm. The best outcomes were achieved with 8 % Glycerol, resulting in progressive motility of 57.5 % (P&lt;0.05). In Experiment 2, the extender containing 8 % DMSO demonstrated significantly better performance in terms of overall motility, viability, and membrane integrity compared to 5 % or 11 % DMSO. Progressive motility was 53.5 % (P&lt;0.05). In conclusion, the study determined that using 8 % Glycerol or 8 % DMSO is the optimal choice for freezing goat sperm in the Mekong Delta region of Vietnam.

Fertility after photodynamic inactivation of bacteria in extended boar semen.

Antimicrobial resistance is an increasing challenge in semen preservation of breeding animals, especially in the porcine species. Bacteria are a natural component of semen, and their growth should be inhibited to protect sperm fertilizing capacity and the female's health. In pig breeding, where semen is routinely stored at 17°C in the liquid state, alternatives to conventional antibiotics are urgently needed. Photodynamic inactivation (PDI) of bacteria is a well-established tool in medicine and the food industry but this technology has not been widely adopted in semen preservation. The specific challenge in this setting is to selectively inactivate bacteria while maintaining sperm integrity and functionality. The aim of this study was to test the principle of PDI in liquid stored boar semen using the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) and a white light LED-setup. In the first step, photophysical experiments comprising singlet oxygen phosphorescence kinetics of TMPyP and determination of the photosensitizer triplet time revealed a sufficiently high production of reactive singlet oxygen in the Androstar Premium semen extender, whereas seminal plasma acted as strong quencher. In vitro experiments with extended boar semen showed that the established PDI protocol preserves sperm motility, membrane integrity, DNA integrity, and mitochondrial activity while efficiently reducing the bacteria below the detection limit. A proof-of-concept insemination study confirmed the in vivo fertility of semen after photodynamic treatment. In conclusion, using the PDI approach, an innovative tool was established that efficiently controls bacteria growth in extended boar and maintains sperm fertility. This could be a promising contribution to the One Health concept with the potential to reduce antimicrobial resistance in animal husbandry.

MAP3K1, SPEF2 and PLCZ1 genes association with fertility and semen quality traits of AI bulls in Andhra Pradesh

Bull fertility plays an important role in improving the economic value of the herd and is measured in terms of semen quality traits, which is influenced by both genetic and non-genetic factors. Polymorphism of MAP3K1/ CviQI (rs463712269), SPEF2/HpyCH4V (rs722354121) and PLCZ1/AvaII (rs208019489) and their association with scrotal circumference, sperm motility and plasma membrane integrity, respectively were studied on 239 bulls of different breeds reared in different frozen semen bull stations. The MAP3K1/CviQ1 assay revealed that T allele was more frequent in Ongole and Murrah population. RFLP at SPEF2/ HpyCH4V locus indicated fixation of T allele in both the exotic purebred cattle (Holstein Friesian and Jersey) whereas selective advantage of C allele was observed in Murrah buffaloes. Except in Holstein Friesian crossbred, other cattle genetic groups were with CC genotype for PLCZ1/AvaII assay and the Murrah population had low genetic diversity and heterozygosity excess. The PLCZ1/ AvaII genotypes had significant influence on the plasma membrane integrity of sperm in Holstein Friesian, Jersey and Ongole bulls. The plasma membrane integrity of sperm was reported to be high in heterozygotes (CG) of Jersey and Ongole cattle. The present study, concluded the importance of PLCZ1 gene as a marker for semen quality assessment and selection in bulls.

Advancements in Understanding and Enhancing Antioxidant-Mediated Sperm Cryopreservation in Small Ruminants: Challenges and Perspectives.

Cryopreservation poses significant challenges to the preservation of sperm integrity and function, particularly in small ruminants where cryodamage is pronounced. This review explores the molecular mechanisms underlying sperm cryodamage and strategies for improving cryopreservation outcomes, with a focus on the role of antioxidants. Cryopreservation-induced alterations in proteins and RNA transcripts critical for sperm function, including motility, capacitation, fertilization, and embryo development, are discussed. Proteomic, transcriptomic, and epigenomic advancements have provided valuable insights into these mechanisms, offering potential biomarkers for predicting sperm freezability and enhancing cryopreservation strategies. Combining technologies such as mass spectrometry and flow cytometry allows for a comprehensive understanding of molecular and cellular changes induced by the freezing-thawing process. However, challenges remain in optimizing cryoprotectant formulations and antioxidant supplementation to improve post-thaw sperm fertility. Further research is needed to explore a wider range of novel cryoprotectants, antioxidants, and proteins for cryopreservation media, as well as to validate their efficacy in enhancing sperm viability and function. Additionally, investigations into the effects of cryopreservation on RNA transcripts and epigenetic factors in small ruminant species are warranted to advance our understanding of sperm preservation. Overall, this review highlights the importance of antioxidants in mitigating cryodamage and underscores the need for continued research to refine cryopreservation protocols and improve reproductive outcomes in small ruminants.

Effect of Season on the Scrotum, Seminal Characteristics, Spermatozoa Integrity and Sexual Behavior in Ganjam Buck

Background: Bucks raised for breeding purposes receive no special attention and are raised at random without following the correct selection process. Besides, the fertility of the male bucks is never addressed. Goat breeders have noted a sharp decline in the reproductive efficiency of Ganjam goats, associated with a lengthened kidding interval, fewer successful pregnancies and decreased twining frequency. The measurement of male fertility parameters is crucial for estimating the breeding performance of any species since males contribute 50% of the reproductive efficiency. Methods: A total of 120 Ganjam bucks of different age groups reared by the local farmers of Rambha, Khalikote and Chhatrapur areas of Ganjam district were used for the present study. A total of six healthier bucks of age 1.5(B1), 2(B2), 2.5(B3), 3.5(B4), 4(B5) and 4.5(B6) years were procured from the Farmers for the experimental purpose. For study on effect of season on the semen qualities, twelve ejaculates from each buck were collected, six ejaculates between the month of July to August (season 1) and six ejaculates during October to November (Season 2). Result: The scrotal length, scrotal width, scrotal circumference have highly significant positive degree of association with Flehmen’s reaction and Libido score and all the parameters exhibited highly significant negative degree of association with reaction time. Moreover, the scrotal length, scrotal width and scrotal circumference had significantly higher positive degree of association with colour, volume of the semen and negative degree of association with MBRT. Further, the sperm concentration revealed a highly significant degree of association with scrotal length, scrotal width, scrotal circumference, livability % and acrosome integrity and a significant degree of association with mass activity. Livability % of the semen samples of the six bucks revealed a highly significant degree of association with scrotal length, scrotal width, scrotal circumference, sperm concentration and acrosome integrity.