Effect of tyrode albumin lactate pyruvate and modified Krebs Ringers broth media on in vitro capacitation of HD-K75 boar spermatozoa at different period of incubation.

In vitro capacitation allows for a greater understanding of the mechanisms underlying fertilization and the development of improved reproductive techniques for improving fertility rates in porcine. Tyrodes albumin lactate pyruvate (TALP) and modified Krebs Ringers Broth (m-KRB) are two medias that are commonly used in research experiments to induce capacitation in boar spermatozoa (Cañón-Beltrán et al., Theriogenology, 198, 2023 and 231; Oberlender et al., Archivos de Medicina Veterinaria, 44, 2012 and 201; Sahoo et al., International Journal of Biological Macromolecules, 241, 2023 and 124502). Moreover, understanding the morphological and functional changes in boar spermatozoa at different hours of capacitation periods might aid in the development of novel techniques for improving sperm quality and increasing the litter size. This study was carried out to investigate the effect of Tyrode albumin lactate pyruvate and modified Krebs Ringers Broth media on in vitro capacitation of HD-K75 boar spermatozoa at three different periods of incubation. A total of 24 ejaculate from four clinically healthy, 10-12 months aged HD-K75 boars, maintained at ICAR-All India Coordinated Research Project (AICRP) on pig were selected. Semen was collected by 'Simple fist' method using a portable dummy. The semen samples having 200 mL volume, 103 × 106 spermatozoa/ml concentration and 70% initial motility were selected and split into two parts and suspended in TALP and m-KRB media, respectively, and incubated for 5 h at 37°C. Seminal parameters viz. sperm viability, plasma membrane integrity and acrosomal integrity were estimated in the samples at 0, 3 and 5 h of incubation. This study revealed that there was significant variation between media in live acrosome-reacted (p < .05) and HOST-reacted (p < .01) spermatozoa, while between capacitation periods significant (p < .01) variation was observed in hyperactivated spermatozoa, live acrosome-reacted spermatozoa, HOST-reacted spermatozoa, FITC-labelled PSA, extracellular protein and sperm cholesterol. Non-significant variation was observed in total phospholipid. TALP showed overall better consequence on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. From this study, it could be concluded that both TALP and m-KRB media were virtuous to induce capacitation in HD-K75 boar spermatozoa. TALP media, however, had a better effect on sperm viability, plasma membrane and acrosomal integrity of boar spermatozoa. Out of the three different periods, 3 h capacitation period resulted in significantly (p < .01) higher incidence of sperm viability, plasma membrane and acrosomal integrity in HD-K75 boar spermatozoa.

Effects of antioxidant Bis-carboxyethyl germanium sesquioxide added to bovine semen cryopreservation medium on in vitro assessed morphofunctional sperm parameters.

This study investigated the impact of various Ge132 (Bis-carboxyethyl germanium sesquioxide) concentrations on frozen bovine semen. Ejaculates from three bulls were pooled and divided into six groups, each one with different Ge132 concentrations (0, 500, and 1000 µg/mL) and each group was incubated in different conditions (33°C for 30 min (D: D0, D500, and D1000), and the other was immediately cooled to 4°C (R: R0-control; R500 and R1000)). Thawed semen was evaluated for sperm characteristics by CASA and flow cytometer. Results showed better motility in the immediate cooling group without Ge132 compared with high Ge132 concentrations. Values for total motility dropped after 5 and 60 min in groups with high Ge132 levels and some control groups. Linearity increased with 1000 µg/mL Ge132, while straightness differed between moments in multiple groups. Membrane integrity was higher in a control group and certain Ge132 groups. Lower O2 - generation occurred without Ge132. After oxidative stress induction, lipid peroxidation intensity increased with arachidonic acid, but D1000 had lower peroxidation than R0. Overall, Ge132 appears to have provided protection against PLM when subjected to oxidative stress, since even at high concentrations it maintained sperm metabolism.

Seminal plasma removal for medium-term preservation of ram sperm at 5 °C.

This study aimed to investigate if washing ram sperm from seminal plasma (SP) could be an effective tool to extend sperm lifespan in medium-term preservation in liquid form to optimize ovine artificial insemination protocols. To this end, in Experiment 1 SP was added to a sperm model without previous contact with this substance (ram epididymal sperm) at the beginning or the end of a 48-hour preservation protocol at 5 °C (n = 13). Sperm motility and kinetic parameters and sperm functionality in terms of sperm viability, apoptosis, mitochondrial activity and reacted acrosomes were assessed after 6 h of storage at 15 °C (standard liquid preservation method) and 24 and 48 h at 5 °C. Extended sperm showed better results after 48 h when stored in the absence than in the presence of SP in most sperm quality parameters. Moreover, the final SP supplementation of this experimental group resulted in the highest sperm motility and kinetic parameters, viability and mitochondrial activity. These results suggested that initial SP deprivation could be beneficial in a medium-term ram sperm preservation protocol in liquid form, as well as a final supplementation. Therefore, we conducted Experiment 2 to evaluate the effect of SP removal from freshly ejaculated ram semen under the same storage conditions as in Experiment 1 (n = 12). Surprisingly, SP withdrawal impaired sperm functionality, leading to increased apoptosis and decreased mitochondrial activity after 24 and 48 h at 5 °C. Conversely, SP supplementation at the end of the preservation protocol of the ejaculate processed as usual had a positive effect on sperm quality and fertility. To summarize, SP absence was beneficial for a medium-term preservation protocol (up to 48 h at 5 °C) of ram epididymal sperm, but the same preservation protocol for ram ejaculated sperm revealed a possible failure of the SP removal method in avoiding the sperm-SP interaction effect. Meanwhile, SP supplementation of ram semen at the end of the preservation protocol increased in vitro sperm quality and fertility after artificial insemination.

Comparison of computer-assisted sperm analysis and smartphone-applied sperm analysis for evaluation of frozen-thawed bull semen.

This study aims to compare the efficacy of computer-assisted sperm analysis (CASA) and smartphone-applied sperm analysis (SASA) in assessing the quality of frozen-thawed bull semen. A total of 75 straws (n = 75) semen samples were used from different production batches of five Holstein bulls. The semen analyses were conducted in three groups: Group I (CASA-37°C), semen samples were evaluated using the CASA system at 37°C (n = 25); Group II (SASA-25°C), semen samples were assessed using the SASA system at a temperature of room heat (25°C) (n = 25); and Group III (SASA-37°C), semen samples were evaluated using the SASA system at 37°C (n = 25). The frozen-thawed bull semen samples were analysed in terms of total motility (TM), progressive motility (PM), immotile, velocity average path (VAP), velocity curve linear (VCL), velocity straight line (VSL) and sperm concentration. There was no significant difference between the groups in terms of spermatozoa concentration (p > .05). However, significant differences among the groups were observed for total motile spermatozoa values (p < .001). Values of progressive motile spermatozoa were lower in Group I and Group II compared to Group III (p < .001). The immotile spermatozoa values were significant between the groups (p < .001) and were found to be proportional to total motile spermatozoa values. Additionally, the VAP, VCL and VSL values were comparable between Group II and Group III, but lower when compared to Group I. In conclusion, the results of the study demonstrate that the Sperm Cell™ system can accurately analyse the concentration of frozen-thawed bull semen. The analyses performed at room temperature indicate a parallelism between the PM value and CASA results. However, it is thought that SASA devices require a series of standardization studies in different semen extenders, different sample concentrations and different animal species, analogous to the standardization evolution process of CASA devices in semen analysis.

Post-cooling sperm processing can rescue sperm quality of cooled-stored stallion semen.

Poor sperm quality in cooled-shipped semen has been related to subpar fertility in horses. Therefore, this study aimed to evaluate the ability of post-cooling sperm processing to improve sperm parameters of cooled-stored stallion semen for artificial insemination. For all experiments, ejaculates were collected, processed, and diluted in skimmed milk-based (SM) medium and stored at 5 °C/24h. In all experiments an aliquot of unprocessed cooled semen was used as a control. In the first experiment (Exp 1.), cooled-stored semen from 16 stallions (n = 32) was processed by SpermFilter or centrifugation (600×g/10min) and resuspended in an egg yolk-based freezing medium containing permeating cryoprotectants (EY-C) for cryopreservation. Sperm recovery and motility parameters were immediately assessed after sperm resuspension in both groups and compared with unprocessed (Unp) samples. In Exp 2., cooled semen samples from six stallions (n = 18) were processed using SpermFilter and resuspended in SM or EY-C. Motility parameters and plasma membrane integrity were assessed in all groups (Unp, SM, and EY-C). In Exp 3, cooled semen from four stallions (n = 20) was processed by SpermFilter, resuspended in SM, EY-C, or egg yolk-based medium without cryoprotectants (EY-nC); and submitted to a thermoresistance test (37 °C/3h). Motility parameters, plasma membrane integrity and stability, mitochondrial membrane potential, mitochondrial superoxide generation, and DNA fragmentation index were evaluated in all groups. Finally, in Exp 4, 39 estrous cycles of 11 mares were inseminated with unprocessed (n = 6) cooled-stored semen or semen cooled at 5 °C/24h and then processed by SpermFilter and resuspended in SM (n = 5), EY-C (n = 11), EY-nC (n = 11), or centrifuged and resuspended in EY-C (n = 6). Overall, semen processing and resuspension in EY mediums (EY-C and EY-nC) improved sperm parameters compared with those of unprocessed semen (P < 0.05). Centrifugation (91 ± 5 %) recovered more sperm than SpermFilter (84 ± 9 %; P < 0.05). Sperm resuspended in EY-nC maintained better sperm parameters throughout the thermoresistance test than those in the other groups (P < 0.05). The fertility rates were similar between all groups (P > 0.05). In conclusion, processing and resuspension in EY medium can improve sperm parameters in post-cooled-stored stallion semen.

Identification and functional prediction of miRNAs that regulate ROS levels in dielectric barrier discharge plasma-treated boar spermatozoa.

Dielectric barrier discharge (DBD) plasma regulates the levels of reactive oxygen species (ROS), which are critical for sperm quality. MicroRNAs (miRNAs) are non-coding single-stranded RNA molecules encoded by endogenous genes, which regulate post-transcriptional gene expression in animals. At present, it is unknown whether DBD plasma can regulate sperm ROS levels through miRNAs. To further understand the regulatory mechanism of DBD plasma on sperm ROS levels, miRNAs in fresh boar spermatozoa were detected using Illumina deep sequencing technology. We found that 25 known miRNAs and 50 novel miRNAs were significantly upregulated, and 14 known miRNAs and 74 novel miRNAs were significantly downregulated in DBD plasma-treated spermatozoa. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that target genes of differentially expressed miRNAs were involved in many activities and pathways associated with antioxidants. We verified that DBD plasma significantly increased boar sperm quality and reduced ROS levels. These results suggest that DBD plasma can improve sperm quality by regulating ROS levels via miRNAs. Our findings provide a potential strategy to improve sperm quality through miRNA-targeted regulation of ROS, which helps to increase male reproduction and protect cryopreserved semen in clinical practice.

Flavone and 3-hydroxyflavone supplementation in cryopreservation medium protects canine sperm against apoptosis and lipid peroxidation.

Cryopreservation is a pivotal technique in safeguarding genetic material across diverse species, despite its inherent challenges linked to induced spermatozoa damage, notably apoptosis and lipid peroxidation (LPO). Given the insufficient antioxidant defense of spermatozoa against LPO, there is a rising interest in integrating additional additives into extenders to ameliorate mammalian semen quality. Among these additives, flavonoids have garnered considerable attention due to their potent antioxidative properties. Hence, our study aimed to assess the efficacy of flavone (FL) and 3-hydroxyflavone (3-OH = ) supplementation in the cryopreservation medium to protect canine sperm against the damaging impacts of freezing and ensure the preservation of their reproductive potential. Semen was collected from five Beagle stud dogs and then pooled. Then, the sample was divided into 7 groups, each treated with 1) 0 mM, 2) 0.1 mM FL, 3) 0.2 mM FL, 4) 0.4 mM FL, 5) 0.1 mM 3-OH = , 6) 0.2 mM 3-OH = , 7) 0.4 mM 3-OH = . Semen samples were subjected to cryopreservation in French straws and glycerol as a cryoprotectant. In the frozen thawed semen, sperm motility parameters by CASA system and sperm membrane integrity, acrosome status, mitochondrial activity, DNA fragmentation, early apoptosis with capacitation, and LPO were assessed using flow cytometry just after thawing (0 h) and 4 h post thaw. Results reveal significant increase in the proportion of live spermatozoa with undamaged acrosomes in the FL 0.1 and 3-OH = 0.2 groups at 0 h post thaw. At this time point, 3-OH = 0.1 significantly reduced the DNA fragmentation index (DFI) compared to the FL 0.1 and 0.2 groups. However, after the next 4 h, 3-OH = 0.4 exhibited the lowest (P < 0.05) DFI compared to FL 0.2 and 3-OH = 0.1. Additionally, 3-OH = 0.4 showed the highest (P < 0.05) proportion of non apoptotic and non capacitated spermatozoa compared to FL 0.1 0 h post-thaw. Simultaneously, the same group demonstrated significant reduction in apoptotic and capacitated sperm cells, at 0 h and 4 h post-thaw. Moreover, 3-OH = at 0.1 (0 h and 4 h) and 0.2 mM (4 h) significantly enhances the proportion of live sperm without LPO post thaw. Whitin the FL groups, only 0.4 FL significantly increased the percentage of live sperm without LPO. No significant effect of the tested substances was observed on sperm motility, cell membrane integrity, or mitochondrial activity. These findings highlight the promising role of flavone and 3-hydroxyflavone in enhancing sperm resilience during cryopreservation, suggesting their protective function against acrosome damages, capacitation, apoptosis and lipid peroxidation.

Selection based on the Breeding Soundness Evaluation is associated with the improvement of the reproductive quality of young Nellore bulls.

Breeding soundness evaluation (BSE) is the best methodology to estimate the fertility potential of future bulls and performing indirect selection for their fertility. However, the outcome of the BSE is influenced by several factors, including genetics, environment, and BSE guidelines. Herein, in this retrospective study, our first aim was to characterize the reasons for failure in 46,566 BSE from 2-year-old beef Bos indicus bulls (Nellore) born from 1997 to 2018. Our second aim was to determine whether or not BSE was associated with reproductive potential improvement of the bulls over the years. Due to changes in the BSE criteria, we used the same dataset, but only bulls born from 2002 to 2018 were included resulting in 35,856 BSE. For the second aim, the effect of the year and farm were included in the model of the multivariate logistic regression. We also determined if the main reasons for BSE failure decreased over time. Bulls were classified as approved (satisfactory potential breeders and qualified for natural breeding service) and not approved (deferred and unsatisfactory potential breeders). The reasons for BSE failure in Nellore bulls were poor semen quality (53.1 %) and physical defects (46.9 %), with the main physical defect being testis abnormalities (19.7 %). The overall percentage of bulls approved each year was 87.1 %, with no improvement over the years of study. However, the percentage of approved bulls at the first BSE increased over the years (P < 0.05). This increase was evident by a reduction in the difference between the overall percentage of the bulls approved vs the percentage of bulls approved at the first BSE. Furthermore, there was an increase in the percentage of bulls classified as satisfactory potential breeders in the BSE and an evident decrease in the percentage of bulls qualified only for natural breeding service (P < 0.05). In addition, an increase of the scrotal circumference (SC) of the herd was found (P < 0.05). These results indicate the overall quality of the bulls improved over the years. To associate and identify the main sperm abnormalities, 3461 not approved bulls were clustered. The most frequent defects were strongly coiled tail spermatozoa, proximal droplets, and acrosomal defects. Overall, there was no change in the frequency of bulls not approved by the sperm morphology nor the frequency of the main sperm abnormalities over the years. Nevertheless, the frequency of the defects remained very low, implying they were controlled. Additionally, abnormalities in the testis decreased over the years and was associated with the increase in the SC of the herd and a decrease of culled bulls due to low SC. In conclusion, this study demonstrates that there is an association between implementation and use of BSE with improvements in the reproductive quality of future generation bulls.

The contribution of different sperm parameters to better explain ram semen cryoresistance.

In order to determine an effective procedure for explaining ram sperm cryoresistance and develop a new model for breeders classification, a retrospective study was conducted using sperm analysis data obtained over two consecutive years from a total of 82 sessions of ram semen cryopreservation. In each session, fresh ejaculates from eight males were collected via artificial vagina, pooled and frozen in liquid nitrogen vapors. After thawing, a total of 19,084 sperm tracks and 11,319 morphometric measurements were analysed. Clustering analyses were applied to establish motile and morphometric sperm subpopulations. Additionally, plasma and acrosome membrane integrity, as well mitochondrial activity using flow cytometry immediately after sperm thawing and following hypoosmotic shock test (HOST) was assessed. To develop a Ram Sperm Cryoresistance Index, Principal Component Analyses (PCA) using 22 variables were conducted. In the first PCA, the parameters that best explain cryoresistance include total motility (TM), motile subpopulation 2 (motSP2, which groups slow, very linear spermatozoa with low lateral head displacement), morphometric subpopulation 1 (morphSP1, grouping spermatozoa with the smallest head size and lowest shape values), sperm plasma membrane integrity immediately after thawing and following hypoosmotic shock test. These parameters collectively account for 77.34 % of the accumulated variance. To emphasize their importance, a second PCA was performed, revealing significant higher weighting coefficients for the quantity (TM) and quality (motSP2) of sperm movement after thawing, compared to the head size and shape of the thawed sperm (morphSP1). Furthermore, HOST Viability played a more decisive role than what was observed under isotonic conditions.

Dietary supplementing South African indigenous rams with flaxseed oil and ascorbic acid improves cryopreserved semen quality and in vitro fertility.

The purpose of this study was to evaluate how ascorbic acid with dietary flaxseed oil affects the quality and fertility of cryopreserved ram sperm in South African indigenous rams. Treatment diets were supplemented 60 days before semen collection to afford proper spermatogenesis, adaptation to the feed formulated and fed throughout the study. Semen was collected with the use of artificial vagina following dietary supplementation with five treatment diets (neg. cont. - negative control, pos. cont. - positive control, FLO - 5% Flaxseed oil, ASA - 4% Ascorbic acid, and FLO + ASA). Semen was then extended using tris-based extender and cryopreserved using the programmable freezer (CBS Freezer 2100 series, Laboratory consumables & chemical suppliers, America). Ovaries were collected from a neighbouring slaughter house and conveyed to the lab in 0.9% saline at 37 °C. Data (sperm parameters and in vitro fertility) was then exposed to the GLM (General Linear Model) in Minitab 17. Pearson's correlation coefficient was utilized to investigate the relationship between cryopreserved sperm quality and in vitro fertility. The student Least Significant Difference Test was used to separate the treatment means, and differences were accepted when the p-value was less than 0.05. The FLO + ASA group had higher (p < 0.05) progressive (36.33 ± 1.87), total (88.24 ± 2.24), rapid motility (27.52 ± 1.74), intact plasma membrane (75.67 ± 2.08), total fertilization (65.98 ± 7.39), and total cleavage (66.19 ± 6.50) when compared to other treatment groups. Total fertilization rate had a medium significant (p < 0.001) medium correlation with the progressive motility (r2 = 0.435), total motility (r2 = 0.447) and rapid motility (r2 = 0.409). In conclusion, dietary flaxseed and ascorbic acid (FLO + ASA) improves cryopreserved semen quality, in vitro fertilization rate, and the total cleavage rate. Noteworthy, the progressive, total and rapid motility play a crucial in the in vitro fertilization rate.

Dietary supplementation with ginseng extract enhances testicular function, semen preservation, and fertility rate of mature and aging Thai native roosters.

The decrease in fertility in aging roosters is related to the reduced quality of ejaculated sperm. This study aimed to investigate the effect of dietary supplementation with ginseng extract at various concentrations (0-150 mg/kg) on testicular function, semen preservation, and fertility at different stages of sexual maturity (mature and aging roosters) in Thai native roosters. Pradu Hang Dum roosters at 32 (mature; n = 24) and 75 (aging; n = 24) weeks of age were fed diets with non-supplemented or supplemented ginseng extracts (50, 100, and 150 mg/kg) until the end of the experiment. In experiment 1, fresh semen samples were examined for the quality parameters of semen volume, sperm concentration, sperm motility, sperm viability, lipid peroxidation, and enzymatic activities. In experiment 2, semen was preserved at 5 °C for up to 48 h, and the semen quality and fertility potential were determined. In experiment 3, testicular function and testosterone concentrations were evaluated. The results showed that ginseng extract supplementation in the diets of both mature and aging roosters at 50 and 100 mg/kg improved fresh semen quality (P < 0.05). A decrease in malondialdehyde levels in fresh semen was observed with increasing enzyme activities. In mature roosters, the progressive motility of cold-stored semen and fertility rates were higher in the G50 and G100 groups compared to the control and G150 groups after 24 h of storage (P < 0.05). In aging roosters, the highest significant differences in progressive motility, viability, and fertility rates were observed in the G50 and G100 groups at all storage times (P < 0.01). These improvements might be attributed to good testicular function in spermatogenesis, as revealed by the results of histological examination and testosterone concentrations. However, higher doses of ginseng extract supplementation negatively affected sperm quality. In summary, the recommended dose of ginseng extract supplementation in diets is 50 mg/kg. Fertility results indicated that insemination with semen preserved for 24 h was satisfactory in both mature and aging roosters.

Cr (VI)-induced ribosomal DNA copy number variation is associated with semen quality impairment: Evidence from human to animal study.

OBJECTIVES: This study aimed to analyze the possible role of rDNA copy number variation in the association between hexavalent chromium [Cr (VI)] exposure and semen quality in semen donors and further confirm this association in mice. METHODS: In this cross-sectional study, whole blood and semen samples were collected from 155 semen donors in the Zhejiang Human Sperm Bank from January 1st to April 31st, 2021. Adult C57BL/6 J male mice were treated with different doses of Cr (VI) (0, 10, or 15 mg/kg b.w./day). Semen quality, including semen volume, total spermatozoa count, sperm concentration, progressive motility, and total motility, were analyzed according to the WHO laboratory manual. Cr concentration was detected using inductively coupled plasma mass spectrometry. The rDNA copy number was measured using qPCR. RESULTS: In semen donors, whole blood Cr concentration was negatively associated with semen concentration and total sperm counts. Semen 5 S and 45 S rDNA copy numbers were negatively associated with whole blood Cr concentration and whole blood 5.8 S rDNA copy number was negatively associated with semen Cr concentration. In mice, Cr (VI) damaged testicular tissue, decreased semen quality, and caused rDNA copy number variation. Semen quality was related to the rDNA copy number in whole blood, testicular tissue, and semen samples in mice. CONCLUSION: Cr (VI) was associated with decreased semen quality in semen donors and mice. Our findings suggest an in-depth analysis of the role of the rDNA copy number variation in the Cr (VI)-induced impairment of semen quality.

Outcomes of dietary alpha-lipoic acid on testicular vascularization, steroid hormones, and seminal quality in aged Baladi bucks.

BACKGROUND: Senescence is accompanied by a progressive decrease in male reproductive performance, mainly due to oxidative stress and endothelial dysfunction. Alpha lipoic acid (ALA) is a potent antioxidant, that diffuses freely in aqueous and lipid phases, possessing anti-inflammatory and anti-apoptotic properties. This study aimed to examine the effects of supplemental dietary ALA on testicular hemodynamics (TH), circulating hormones, and semen quality in aged goats. Twelve Baladi bucks were divided into two groups (n = 6 each); the first fed a basic ration and served as a control group (CON), while the second received the basic ration supplemented with 600 mg ALA/ kg daily for consecutive eight weeks (ALA). RESULTS: There were improvements in testicular blood flow in the ALA group evidenced by a lower resistance index (RI) and pulsatility index (PI) concurrent with higher pampiniform-colored areas/pixel (W3-W6). There were increases in testicular volume and decreases in echogenicity (W3-W5; ALA vs. CON). Compared to the CON, ALA-bucks had higher serum concentrations of testosterone, estradiol, and nitric oxide (W3-W5). There were enhancements in semen traits (progressive motility, viability, morphology, and concentration, alanine aminotransferase enzyme) and oxidative biomarkers (catalase, total antioxidant capacity, and malondialdehyde). CONCLUSIONS: ALA dietary supplementation (600 mg/kg diet) improved aged bucks' reproductive performance by enhancing the testicular volume, testicular hemodynamics, sex steroids, and semen quality.

Asian elephant (Elephas maximus) seminal plasma: establishing the proteome and effect on spermatozoa when added to cryomedium.

Context The removal or supplementation of ejaculates with seminal plasma (SP) can affect cryotolerance and post-thaw survival of spermatozoa in many species. In the Asian elephant (Elephas maximus ), elucidation of the SP proteome and investigation of how it affects spermatozoa may enable improvement of cryopreservation protocols. Aims Herein, we characterise the Asian elephant SP proteome and investigate the impacts of SP on sperm cryotolerance in the presence of conspecific or heterospecific SP. Methods Proteomic analysis of Asian elephant SP was performed using mass spectrometry on nine samples from three individuals. In a separate study, SP was removed from six ejaculates and spermatozoa were resuspended in Tris extender supplemented with: no seminal plasma (NOSP), conspecific SP from ejaculates exhibiting 'good' (GSP, >60%) or mixed sperm total motility (MSP), or horse SP (HSP). Samples underwent cryopreservation, and sperm parameters were compared prior to cryopreservation and after thawing (0 and 2h). Key results Mass spectrometry identified 155 proteins from an array of families. Significant differences were observed in post-thaw sperm quality between SP treatments: high concentrations of MSP (25%, v/v) displayed greater average path and straight-line velocity immediately after thawing (P P P Conclusions and implications These preliminary findings suggest the potential of SP to enhance the cryosurvival of Asian elephant spermatozoa, with HSP showing particularly promising results compared to conspecific SP (GSP). Further research into the specific effects of Asian elephant SP proteins is warranted.

Improving semen quality of rams fed with ration containing protected maggot oil.

The study aimed to to evaluate the effect of feeding protected maggot oil at different levels on the ram sperm quality. The study used 15 local rams with an age of approximately 10-12 months and an initial weight of 19.99 ± 3.97 kg. The feeding rate was 4% of body weight per day. Feed was given 3 times a day, specifically in the morning (08.00 WIB), afternoon (12.00 WIB) and evening (16.00 WIB). Water was provided ad libitum. This study used 3 treatments and 5 groups as replicates. The treatments used concentrates with different levels of protected maggot oil: P0(0% protected maggot oil (control)), P1(4% protected maggot oil), and P2(8% protected maggot oil). The variables measured were nutrient consumption, blood cholesterol levels, scrotal circumference, and sperm quality. Blood cholesterol and scrotal circumference measured at the end of the experimental diet. Semen samples were collected and analysed before and at the end of the experimental diet. The data obtained were analysed using ANOVA, with further testing using Duncan's test for significant differences. The results showed that there were no significant differences in the consumption of dry matter, crude protein, crude fiber, scrotal circumference, volume, colour, pH of semen, sperm concentration, live percentage, abnormal percentage, plasma membrane, and acrosome integrity of spermatozoa. There were significantly (p < 0.05) produced higher consumption of oleic and palmitic acids in 8% protected maggot oil compared to 4% treatments, the treatments containing 4% and 8% protected maggot oil produced significantly (p < 0.05) higher consumption of lauric and myristic acids, blood cholesterol levels, and sperm motility than the control. The result indicates that protected maggot oil up to 8% in the ram diet have positive effect on improving the microscopic quality of ram sperm, i.e. increased sperm motility.

Characteristics of Bachaur bull semen.

The Bachaur is a mediumized draft purpose breed which has been recognized by ICAR-National Bureau of Animal Genetic Resources (NBAGR) Karnal, India, and presently is on the verge of extinction. Since there are no data regarding the seminal parameters of this breed, this work was performed to evaluate seminal parameters of freshly ejaculated semen. A total of three healthy breeding Bachaur bulls aged 2.5-5 years were selected for the study which were maintained under identical managemental conditions. Semen parameters of these bulls were studied across 10 ejaculates. The average scrotal circumference and testicular weight of the three bulls were 27.78 ± 1.2 cm and 400.67 ± 26.6 g, respectively. The average overall volume (mL), pH, concentration (million/mL), liveability (%), abnormality (%), HOST (%) and acrosome integrity (%) were 2.20 ± 0.19, 6.86 ± 0.06, 1245.60 ± 23.49, 85.09 ± 0.91, 4.13 ± 0.06, 81.16 ± 1.18 and 83.54 ± 1.32, respectively. The average overall mass motility of three Bachaur bulls was 3.57 ± 0.06 in 0-5 scale and individual motility averaged 84.78 ± 1.70 per cent. The volume of ejaculates in Bachaur bull seemed to be lower as compared to other exotic and Indian breeds. However, the semen parameters with regard to mass motility, liveability, abnormalities, hypo-osmotic swelling test (HOST) and acrosomal integrity seemed similar to other exotic and Indian breeds.

The sperm storage capacity in hens was correlated with the morphological differences of the oviduct and uterus-vagina junction.

The fertilization rate is an important index to evaluate the reproductive capacity of hens, which is mainly affected by semen quality, timing of artificial insemination (AI), and the ability to store sperm. A high sperm storage (SS) capacity can extend the interval, reduce the frequency, and decrease the labor costs of AI. However, relatively few studies have investigated the SS capacity of hens. Therefore, the aims of the present study were to identify factors influencing the SS capacity of Guangxi partridge chickens and to explore the impact of the sperm count in different sections of the oviduct and sperm storage tubules (SSTs), in addition to the number and surface area of SSTs on SS capacity at different time points after AI. We found that SS capacity was positively correlated to the egg production rate (P < 0.01) and body length (P < 0.05). On post-AI days 5, 10, and 15, the sperm count was higher in uterus-vagina junction (UVJ) than the magnum, isthmus, and infundibulum (P < 0.01), but gradually decreased over time. Also, the duration of SS and the sperm count of the UVJ was greater in the high SS group than the low SS group (P < 0.05). Histopathological analysis of the UVJ showed that the number and surface area of the SSTs (P < 0.01), as well as the proportion of SSTs containing sperm, were greater in the high SS group at all time points post AI (P < 0.01), while the proportion of SSTs containing sperm gradually decreased over time. Collectively, these results highlight the potential for selective breeding of SS capacity and show that SS capacity is related to laying performance and body length of Guangxi partridge hens. In addition, SS capacity was positively correlated to the surface area of SSTs and the proportion containing sperm. A greater sperm count stored in the UVJ was correlated to more sperm transported to the infundibulum and subsequent greater SS capacity of hens.

The potential significance of antioxidants in livestock reproduction: Sperm viability and cryopreservation.

Male reproductive efficiency is primarily defined by the generation of high-quality and viable sperm cells in farm animals. However, the literature shows that male fertility has declined in recent years due various factors including heat stress, which causes the development of free radicals and reactive oxygen species (ROS) which damages sperm cells. This review aimed to examine the potential significance of antioxidants in increasing and preserving sperm quality and viability. Data used to produce this review paper came from recently published articles in peer reviewed journals. Google Scholar, Science Direct, Research Gate, Web of Science, and the Directory of Open Access Journals were used to access the data. Various studies have shown that antioxidants play acritical role in preserving the sperm quality and viability by protecting sperm cells from the potential damage from oxidative stress induced by the development of oxygen species imbalances. However, there is less information on the use of natural or synthetic antioxidants to preserve semen quality through in vivo procedures, despite its growing popularity and promising results. Hence, there is a need for researchers to explore more on this topic, especially in other livestock species than poultry.

Evaluation of the testicular artery Doppler velocimetry and its correlation with sperm defects in domestic cats.

Several studies have demonstrated the correlation between Doppler velocimetric parameters of testicular artery and semen quality in domestic species, but in felines data are scarce. This study aimed to correlate the Doppler velocimetry of the testicular artery with sperm kinetics and sperm defects, in sedated and non-sedated cats. Forty tomcats were divided into two groups: sedated (SG; n=20) with dexmedetomidine (10 µm/kg) and ketamine (12 mg/kg), and non-sedated (NSG; n=20). The animals were subjected to ultrasound Doppler velocimetry of the distal supratesticular and marginal region of the testicular artery and subsequently orchiectomized. Epididymal tail spermatozoa were recovered and analyzed using a CASA system for motility, and morphology took place. Animals of SG presented a significantly higher velocity in the marginal region of the cat's testicular artery [peak systolic velocity (PSV) 11.51 cm/s; end-diastolic velocity (EDV) 7.72 cm/s] compared to NSG (PSV 7.72 cm/s, P < 0.001; EDV 4.93 cm/s, P < 0.001). Sedated cats presented higher pulsatility and resistivity indexes than non-sedated cats. The supratesticular PSV of NSG was moderately correlated with major (rs = 0621; P < 0.001) and total sperm defects (rs = 0614; P < 0001). Doppler velocimetry was fairly correlated with minor, major, and total sperm defects. In conclusion, Doppler velocimetric evaluation emerges as an important possibility in the reproductive evaluation of tomcats, once the testicular artery hemodynamics were associated with sperm defects. However, it is advisable to carry out this evaluation in non-sedated animals. If sedation is necessary, peripheral vasoconstriction should be considered.

Enhancing cryopreserved ram sperm quality at -80 °C with Spirulina platensis and Salvia verbenaca extracts.

This study was conducted in two steps to evaluate the influence of freezing methods and natural extracts on cryopreserved ram sperm quality. Initially, the research compared the effects of two freezing methods: liquid nitrogen (LN2) versus -80 °C, on post-thawed ram semen on total and progressive motilities and velocity parameters. Experiment I revealed no significant differences (P > 0.05) between the LN2 and -80 °C freezing methods, indicating similar effects on the analyzed parameters. Experiment II aimed to examine the influence of Spirulina platensis (SP) and Salvia verbenaca (SV) extracts added to egg yolk extender on cryopreserved sperm quality, utilizing the -80 °C freezing method. Various concentrations (1.25, 3.75, 6.25 and 8.75 µg*mL-1) of acetone (Ac-SP and Ac-SV) and hexanoic (Hex-SP), as well as methanolic (MeOH-SV) extracts, were added into the extender. A thorough assessment of post-thawed sperm quality parameters, encompassing motility, velocity parameters, viability, membrane integrity, abnormality and lipid peroxidation was conducted. The outcomes demonstrated that 1.25 and 3.75 g*mL-1 of Ac-SP and Hex-SP and 1.25 µg*mL-1 of AC-SV and MeOH-SV increased the post-thawed ram sperm quality. In conclusion, this study emphasizes the antioxidant properties of SP and SV extracts, highlighting their potential to protect cryopreserved sperm cells from oxidative stress at -80 °C.