Bull Semen Research Articles

Preserving of the bovine chilled semen viability

The aim of the work is to assess the biological usefulness of bull semen when cooled to 5 °C and stored for a period of time.Sperm cooling is less traumatic for cells than deep freezing. The fertilizing capacity of chilled sperm is higher than cryopreserved sperm, but it persists for several days, which limits its use. The study was conducted using native sperm from black-and-white (n = 6) and Ayrshire bulls (n = 3). Two diluents were used in the experiment: OptiXcell commercial diluent (IMV) (France) was used as a control, and an experimental diluent based on Tris was developed as an experiment. There was no significant difference in overall and progressive mobility between the studied diluents. In most cases, the spermatozoa were alive for 10 days. If we take into account the progressive mobility of 40% as the minimum permissible for artificial insemination, on average, the studied bulls had it during storage for 120 hours. At the same time, there were individual ejaculates that had progressive mobility (40% and higher) even after 168 hours of storage. There were no significant differences in membrane safety when diluted with the studied diluents. When stored for 72 hours, there was practically no decrease in the number of intact cells when using an experimental diluent. The preparation and application of an experimental diluent are economically more profitable than using a Western analogue — OptiXcell (IMV). At the same time, the diluent developed by the authors is not inferior in characteristics (qualitative indicators of spermatozoa), and even surpasses the foreign one.

Production and Evaluation of Frozen Semen of Sahiwal Bull Using Two Different Diluters and Protocols

The experiment was designed to evaluate the effects of diluters and freezing protocols on frozen semen production of Sahiwal breeding bulls. Imported 100% Sahiwal breeding bulls were then reared in the American dairy limited (ADL). Semen samples are processed and frozen once a week in the ADL. Spermatozoa with various motility patterns were collected; these included post-thawing, progressive, fast, slow, circular, and local. Computer Assisted Semen Analyzer (CASA) was used to measure the straight-line velocity (VSL), curvilinear velocity (VCL) and linearity (LIN) parameters. Two diluters (tris egg-yolk and tris egg-yolk aqua) and freezing protocols (slow freezing and fast freezing) were used for frozen semen production of these bulls. The significantly highest (P<0.05) number of post-thawing total, progressive and progressive fast motility (78.42±1.88, 73.50±1.90 and 59.68±2.10%), VCL (115.31±3.82µm/s) and lowest slow, circular and local motility (13.44±1.40, 0.57±0.08 and 4.92±0.41%), LIN (0.39±0.01%) was found in diluter 1 and freezing protocol 2. On the other hand, highest number of circular motility (0.77±0.18%) and VSL (44.83±1.17µm/s) was found in diluter 1 and freezing protocol 1 whereas highest number of slow and local motility (18.84±1.23 and 7.46±0.57%) and LIN (0.61±0.20%) and lowest progressive fast motility (48.73±2.28%), VCL (98.90±3.42µm/s), VSL (39.76±1.33µm/s) was found in diluter 2 freezing protocol 2 and also lowest post-thawing and progressive motility (73.44±1.94 and 66.51±2.02%) was observed in diluter 2 freezing protocol 1. The study reveals that diluter 1(one step egg yolk based) and freezing protocol 1 (5 steps) and 2 (2 steps) seems to be an efficient method for frozen semen production.

Pyrroroquinoline Quinone (PQQ) Improves the Quality of Holstein Bull Semen during Cryopreservation.

Cryopreserved semen is extensively utilized in the artificial insemination (AI) of domestic animals; however, suboptimal conception rates due to oxidative damage following AI continue to pose a challenge. The present study investigated the effects of Pyrroroquinoline Quinone (PQQ), a novel antioxidant, on the semen quality of Holstein bulls during cryopreservation, as well as its potential molecular mechanisms. Semen samples were diluted with varying concentrations of PQQ (0, 50 μmol/L, 100 μmol/L, 150 μmol/L) prior to cryopreservation. Following the freeze-thaw process, a comprehensive evaluation was conducted to assess sperm motility, plasma membrane integrity, acrosome integrity, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and adenosine triphosphate (ATP). Western blot analysis was employed to examine the levels of proteins including PGAM2, CAPZB, CAT, SOD1, and GPX1. Notably, the inclusion of 100 μmol/L PQQ significantly enhanced sperm motility, membrane integrity, and acrosome integrity post freeze-thawing (p < 0.05). Furthermore, the group treated with 100 μmol/L PQQ exhibited reduced levels of MDA and ROS (p < 0.05), while ATP levels were significantly elevated (p < 0.05). Interestingly, treatment with 100 μmol/L PQQ resulted in decreased consumption of PGAM2, CAPZB, CAT, SOD1, and GPX1 proteins in sperm after freeze-thawing, compared to the control group (p < 0.05). These findings indicate that PQQ treatment enhances the quality of bull semen, mitigates oxidative stress damage, and ultimately improves the efficacy of sperm cryopreservation.

Seminal oxidative stress index can be used as a marker in the prediction of bull semen cryotolerance

In this study, it was aimed to evaluate the effect of seminal plasma activity levels of enzymatic antioxidants such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), non-enzymatic antioxidant alpha-tocopherol (Vit-E), and also total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) on post-thaw sperm quality. As well as it was aimed to investigate the possibility of the use of OSI as a marker for the estimation of bull semen freezability. For this study, 72 ejaculates were collected from 6 bulls and separated into two aliquots. The first one was centrifuged to separate seminal plasma. The latter one was cryopreserved and stored in liquid nitrogen until analysis. Post-thaw semen quality was examined in two groups (good-freezable semen (GFS) and poor-freezable semen (PFS)) through cluster analyses based on post-thaw total motility and plasma membrane and acrosome integrity. As a result of the analyses, seminal TAS, CAT, and Vit-E values were higher (P<0.05) in the GFS group, while TOS and OSI values were higher (P<0.01) in the PFS group. We also performed an ROC curve analysis to determine whether the seminal OSI value could be used to predict semen freezeability. The area under curve (AUC) value was found as 0,70 (P=0.006). In conclusion, it has been revealed that the seminal plasma antioxidant content is responsible for the freezability of semen, and the OSI value, which can be determined by performing TAS and TOS analyses instead of looking for separate antioxidant enzymes, can be used as a marker for the estimation of post-thaw semen quality at artificial insemination centers.

Polymorphism detection and characterization of sperm cells chromatin remodeling associated genes in Murrah buffalo.

Seasonal variations significantly impact buffalo bull semen production and quality, particularly during the summer months. Understanding the genetic basis of these changes is important for managing bull fertility and improving sperm quality. The present study focused on characterizing and identifying polymorphisms in chromatin remodeling genes, protamines (PRMs) and Transition Nuclear Proteins (TNPs) in Murrah buffalo bulls with varying semen quality due to seasonal effects. Our findings revealed none of the coding region variation in PRM1, PRM2, TNP1, and TNP2, these genes are highly conserved in buffalo. Two intronic variants were identified, including G16C in PRM1 intron 1 and intronic SNP in PRM2 intron 1(G96A). The complete CDS of consensus sequence of bubaline PRM1 was 86.3% identical and 94.1% similar to the bovine PRM1. Whereas the complete CDS of consensus sequence of bubaline TNP2 was 78.2% identical and 91.0% similar to bovine TNP2. Further, no statistically significant differences in the fold change of TNP1, TNP2, PRM1, and PRM2 levels between the hot summer SNA and SA groups and the winter SNA and SA groups This study represents the first comprehensive report on the characterization of bubaline PRM1 (complete CDS), PRM2 (partial CDS), TNP1 (partial CDS), and TNP2 (complete CDS) genes in buffalo sperm cells. Results of the study, clearly indicate that the genes associated with protamine (PRM1 and TNP2) are highly conserved in Bubalus bubalis. Understanding these genetic underpinnings can have implications for improving buffalo bull fertility and semen quality.

Comparative study of Tris Turmeric, Tris Turmeric Dimethyl Sulfoxide and Tris Turmeric Ethylene glycol extenders on the cryosurvivability, sperm resistance, in- vivo fertility and antioxidant status in buffalo bull semen

The freeze-thaw process leads to structural and functional damage due to excessive accumulation of reactive oxygen species (ROS). The addition of exogenous antioxidants to sperm diluents is of great importance to overcome oxidative damage during freezing. The purpose of this study was to explore the effects of three diluents Tris Turmeric, Tris Turmeric Dimethyl Sulfoxide and Tris Turmeric Ethylene glycol on the cold survival ability of buffalo sperm. Semen was collected from five local adult male buffalo breeds. A base diluent of Tris-citric acid-fructose (TCF) was prepared, adding 20% whole egg yolk (TCFY). The Tris extender without turmeric, without DMSO, and without EG was kept as a control. Other extenders are Tris containing turmeric TT (100 ml/5 ml Tris), Tris containing turmeric dimethyl sulfoxide TTD (100 ml/5 ml Tris + 1.5% DMSO) and Tris containing turmeric and ethylene glycol TTE EG (100 ml/5 ml Tris + 1.5% EG). Semen samples were added and a pure sperm concentration of 60 × 106/ml was achieved. Frozen buffalo sperm after thawing showed significant improvements in all research parameters of the three breeding samples compared to the control. Tris Turmeric Ethylene was the type that best improved sperm survival under frozen conditions, followed by Tris Turmeric and Tris Turmeric Dimethyl Sulfoxide compared to the control. A significant decrease in sperm motility after thawing was evident as usage time increased in all expanders. There was a significant increase in total antioxidant content (TAC) and insignificant change in malondialdehyde (MDA) of the diluent used compared to the control. Conception rate (CR) was higher in Tris Turmeric Ethylene glycol (65.2%), followed by Tris Turmeric (60.3%) and Tris Turmeric Dimethyl Sulfoxide (55.9%) compared to the control (36, 7%). It can be concluded that Tris Turmeric Ethylene Glycol is considered the best agent for improving cold survival and sperm fertility, followed by Tris Turmeric and Tris Turmeric Dimethyl Sulfoxide.

Effect of Skim Nanoparticles on the Motility and Kinematics of Simmental Cattle Frozen Semen

Nanotechnology has positively impacted the development of extenders used in the cryopreservation of spermatozoa in livestock. The inclusion of nano skim in the extender is expected to preserve semen quality after thawing. This study aimed to determine the optimal concentration of nano skim. Various concentrations of nano skim (0%, 1.66%, 3.33%, 5%, 6.66%, 8.33%, and 10%) with the addition of 10% nano egg yolk in each extender were compared. Fresh semen samples were collected from three three-year-old Simmental bulls using an artificial vagina. The study parameters included motility (total motility, progressive motility) and kinematics (velocity curve linear (VCL), velocity straight linear (VSL), velocity average pathway (VAP), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and wobble (WOB). A completely randomized design was employed, and data were analyzed using analysis of variance (ANOVA). Fresh semen dilution results indicated that only the control group (0%) and the nano skim group with concentrations of 6.66% and 8.33% were eligible for further processing into frozen semen. The results showed no significant difference between the three treatment groups on sperm motility and kinematics of Simmental cattle after thawing (p0.05). The control group exhibited the highest percentage of total motility, VCL, VAP, and ALH values, while the 6.66% nano skim group outperformed the 8.33% nano skim group. The highest percentage of progressive motility and VSL value was observed in the 6.66% nano-skim group compared to the 8.33% nano-skim and control groups. The highest values of BCF, WOB, LIN, and STR was observed in the nano-skim group at 8.33% compared to the nano-skim group at 6.66% and control. The study concluded that 6.66% nano skim was the optimal concentration to maintain the quality of frozen semen of Simmental cattle.

PSLBI-12 Screening and isolation of Fusobacterium necrophorum and Fusobacterium varium from seminal, and vagino-uterine microbiota of healthy cattle and sheep

Abstract Fusobacteria necrophorum is an important opportunistic bacterial pathogen associated with liver abscesses, foot rot, necrotic laryngitis and endometritis in both dairy and beef cattle. Recently, we identified F. necrophorum as one of the taxa in the uterine microbiota that are positively associated with pregnancy outcome in beef cows, and that Fusobacterium was identified as the most dominant genus in the bovine seminal microbiota. These findings indicate that Fusobacterium spp. may have a role in reproductive health and cattle fertility. Our objectives were to detect and isolate the F. necrophorum subsp necrophorum (FNN), F. necrophorum subsp. funduliforme (FNF) and F. varium (FV) in seminal, vaginal and uterine microbial ecosystems as well as fetal samples by culture and real-time PCR (qPCR) methods. The qPCR was performed on the DNA (n = 215) extracted from bull semen (n = 37), ram semen (n = 100), and vaginal (n = 25) and uterine swabs (n = 32), caruncles (n = 9), allantoic fluid (n = 6), and amniotic fluid (n = 6) from pregnant beef cows. The qPCR targeted the hgdA gene, and leukotoxin promoter regions (lktA-n and lktA-f) to detect and quantify FV, FNN and FNF, respectively. Cryopreserved samples (n = 174; 57 bull semen, 10 ram semen, 38 vaginal, and 47 uterine swabs) were cultured to detect and isolate FNN, FBF and FV by direct plating and after culturing in anaerobic peptone-yeast extract broth containing sodium lactate broth with josamycin, vancomycin and norfloxacin for selective enrichment of FN and FV. Also, 28 samples of 6-mo-old bovine fetuses caruncles, allantoic and amniotic fluids, and intestinal tissue) were screened for Fusobacterium species. By qPCR, FNF was detected in 75.7% of bull semen, and 8.0% of vaginal swabs as well as 33.3% of both allantoic fluid and caruncles, but was undetected in ram semen, uterine swabs, and amniotic fluids. Subspecies necrophorum was identified in 37.8% of bull semen, 3% of ram semen, 8.0% of vaginal and 15.6% of uterine 55.6 % of caruncles, 50% of allantoic fluid samples, but was not detected in amniotic fluid samples. Meanwhile FV was only detected in vaginal, uterine swabs and caruncles at 4%, 6.3%, and 11.1%, respectively. The culture method detected FNF in 63.2% of bull semen and 5.3% of vaginal samples, while FNN was identified in 1.8% of bull semen and 2.1% of uterine samples. F. varium was not isolated from any of the samples. Overall, FNF was the most prevalent subspecies present in the seminal microbiome of bulls. Identification of FNN subspecies and FV in seminal, vaginal, and uterine microbiota of healthy cattle, as well as in healthy calf fetus-associated samples suggests potential transfer from bulls to cows and then on to offspring, and potential involvement of the Fusobacterium spp. in cattle fertility.

PSXIII-14 Effect of melatonin supplementation during in vitro embryo culture on cleavage rates following in vitro maturation and fertilization of bovine oocytes

Abstract In vitro embryo production (IVEP) is an important advanced reproductive technology and has great potential in enhancing livestock production. However, in vitro culture (IVC) of embryos exposes them to stress resulting in compromising quality and efficiency of embryo production. Melatonin has been reported in several studies to reduce oxidative stress and improve the efficiency of IVEP. Our objective was to evaluate the effect of melatonin supplementation in IVC media on cleavage rates of bovine embryos. Bovine ovaries (n = 22) were obtained from local abattoir with ovarian weight of 7.29 ± 3.31 g, and size of 3.65 ± 0.69 cm. Cumulus-oocyte complexes (COCs) were recovered from 2 to 8 mm size follicles by aspiration using an 18-gauge needle attached with a 10 mL syringe. COCs were matured, fertilized, and cultured in vitro using IVF Bioscience bovine media and their protocols. Oocytes were in vitro matured for 23 h at 38.8°C, 5.5% CO2 in a humidified atmosphere and assessed for maturation by assessing COC expansion. Fully expanded and partially expanded COCs were fertilized for 20 h in a 4-well dish with frozen/thawed bull semen. Following IVF, the cumulus cells were removed by vertexing, and the presumptive zygotes were cultured in vitro with and without 1 µM melatonin in 4-well dishes in triplicates under the oil for embryo development at 38.8°C and 5.5% CO2. Cleavage rates were determined on d 3 of culture. The percentage of cleaved embryos was calculated as (number of zygotes cleaved/total number of fertilized oocytes) x100. Data were analyzed by student’s unpaired t- test with Welch’s correction using GraphPad Prism software. The cleavage rate was greater in melatonin supplemented group (54.17 ± 11.24%) than the control (49.83 ± 7.36%) but the difference was not significant (P &amp;gt; 0.05). In conclusion, under our experimental conditions, melatonin supplementation during embryo culture, following IVM/IVF of the bovine oocytes, did not significantly improve cleavage rates.

Enhancing evaluation of bull fertility through multivariate analysis of sperm

Computer-assisted sperm analysis (CASA) has become the predominant tool for assessing bull semen in artificial insemination programs. Despite such popularity CASA's ability to predict fertility has been limited, especially when emphasis is based upon single motion characteristics. Our hypothesis is that numerical sets of CASA measures provide a more effective method to differentiate the potential fertilization capacity of bulls and that bulls can be clustered based upon sets of CASA measures. Therefore, we used CASA to evaluate frozen-thawed semen samples from 307 Holstein and 152 Jersey bulls sourced from USDA-ARS's National Animal Germplasm Program gene bank. Sperm was evaluated immediately after thawing and 30 min later. We evaluated sperm kinetic and morphometric means and variances to capture the structure of CASA data in relation to various sources of variation. These data were subjected to univariate and multivariate statistical methods to investigate animal and management factors affecting sperm characteristics measured by CASA. Clustering with K-means identified 4 clusters of bulls based upon each cluster's set of CASA parameters after thawing. There was little overlap among clusters for sets of CASA measures. At the extremes, bull cluster 1 (BC1, n = 180) and BC3 (n = 101) had different sire conception rates (SCR) -0.07 vs -1.29, respectively and sets of CASA measures. Interestingly, BC2 had CASA measures that could be perceived as negative, e.g., cell size at 8.18mm2 vs 6.37mm2 for BC4 and total motility of 29.7% vs 48.7% for BC3, but SCR for BC2 was higher (-0.79) than BC3 (-1.29). Despite such discrepancies for some BC2 CASA values it appears the potentially negative effects were offset by the levels of other CASA values. Our findings suggest improved approaches for using CASA could lie in evaluating multiple CASA measures as sets within specific numerical ranges rather than as independent measures.

Long-term cryopreservation of bull semen as a main strategy for the ex situ conservation of endangered Polish Red cattle

Abstract Regular verification of the quality of cryopreserved semen derived from native cattle is one of the tasks performed at the bank as recommended by the Food and Agriculture Organization. The purpose of the present study was to evaluate the quality of semen from PR bulls stored for 40, 50 and 60 years in the BMB using standard evaluation parameters such as sperm motility as well as structural-functional parameters such as plasma membrane integrity, transmembrane mitochondrial potential and sperm chromatin damage. Semen pellets from 27 PR bulls (3 ejaculates/bull) were tested. The data were analysed by one-way and two-way ANOVA, and the significance of the difference (P≤ 0.01) between the means was determined using Duncan’s test. Our study results revealed that the long-term storage of semen had no effect on sperm characteristics after thawing. However, statistically significant differences (P≤0.01) in sperm plasma membrane integrity and transmembrane mitochondrial potential, following storage in liquid nitrogen were noted between bulls at all time points. However, there were no significant differences (P&gt;0.01) in sperm chromatin damage between breeds or between different storage times, and the degree of DNA fragmentation ranged from 0.4 to 0.8%.

Fetal sex effects on maternal health can now be tested via randomization: A first-in-class illustration in cows on glucoregulatory outcomes

BackgroundThe maternal-offspring relationship, such as whether fetal sex influences maternal health, is essential to explore to advance prenatal and maternal health. While associations exist between fetal sex and maternal health outcomes, it is unclear whether these reflect a causal relationship. ObjectiveTo demonstrate that fetal sex can be randomly assigned to test the causal effect of fetal sex on maternal outcomes. MethodsHolstein dairy cows were stratified and randomized using sealed opaque envelopes to be artificially inseminated with either X- or Y-sorted bull semen until 40 cows became pregnant. Monthly body weight measurements were recorded, and an intravenous glucose tolerance test was performed 30 days before the expected calving day. The primary outcome was insulin area under the curve (AUC), and secondary outcomes were clearance rate, half-life, and AUC for glucose, insulin, and non-esterified fatty acid concentrations. An intention-to-treat (ITT) approach using multiple imputation was employed for primary analysis, and an as-treated (AT) approach was used for secondary analysis. ResultsWe demonstrated that we could successfully randomize the assignment of fetal sex to dams and test for causal effects of fetal sex on glucoregulatory outcomes using dairy cows as a model. Insulin AUC was not statistically different between groups (ITT p = 0.857, AT p = 0.874), and other outcomes were also not statistically different (p > 0.05). ConclusionWe demonstrated that causal effects of fetal sex on maternal outcomes can be causally tested in dairy cows. Our study did not provide statistical evidence to support an effect of fetal sex on maternal glucose-related outcomes.

Validated DNA isolation method ensuring successful long-read sequencing of cattle semen genome.

Obtaining high-quality DNA suitable for long-read sequencing can be difficult for many types of tissues and cells, and it is a key step in current genomic studies. The challenge is even greater when it comes to isolating genomic DNA from mammalian spermatozoa, as DNA is tightly packed into a cell with a robust membrane rich in disulfide bonds. Here we describe a method for isolating high molecular weight DNA from Bovine commercial semen straws. This protocol includes a cleaning step to remove diluents and preservatives used for the long-term storage of the semen, which may affect long read sequencing. It is based on a simple salting-out method and avoid the use of spin columns, strong mixing or intensive centrifugation, in order to limit DNA fragmentation. However, we have adapted this protocol to facilitate the disruption of cell membranes and disulfide bonds with strong chaotropic and reducing agents. The average size of the fragments produced was approximately 49 kb, ranging from 25 to 85 kb, according to the femto pulse profiles.This method was used to isolate DNA from semen straws, more than 80 of them were successfully sequenced using the Continuous Long-Read (CLR) sequencing mode on the PacBio SequelII platform to study genome diversity and notably to detect large structural variations within genomes.

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.

Effect of cryopreservation and semen extender on extracellular vesicles isolated from bull semen.

Semen cryopreservation is the most popular practice for semen production for artificial insemination and in vitro fertilization in cattle. The Seminal plasma contains extracellular vesicles (spEVs) which modulate sperm viability and function during oocyte fecundation. The study of spEVs in frozen-thawed semen doses may yield novel indicators for predicting bull fertility, but the presence of the semen extender may hinder molecular profiling of spEVs. The aim of this study was to provide extensive characterization of EVs isolated from seminal plasma before and after the cryopreservation process and the addition of a commercial animal protein-free semen extender to understand the potential influence of EVs originating from the extender in hindering the use of spEVs derived biomarkers for assessment of bull fertility. EVs were isolated from the seminal plasma (with or without the extender), from the cryopreserved straw devoid of spermatozoa, and from the extender using two different methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), and characterized for their structure and composition. Physical characterization of EVs showed that size and particle numbers were related to the method of isolation. spEVs were larger but less abundant (UC: 168.9 nm, n = 2.68 × 109; SEC: 197.0 nm, n = 6.42 × 109) compared to extender EVs (UC: 129.0 nm, n = 2.68 × 1011; SEC: 161.8 nm, n = 6.47 × 1011). Western blotting analysis (WB) confirmed the presence of typical EV markers in spEVS: the membrane bound CD9 (25 kDa) and the luminal markers Alix (96 kDa) and TSG101 (48 KDa). Although Transmission Electron Microscopy confirmed the presence of a lipid bilayer structure in all preparations, no specific EV markers were detected in the vesicles isolated from extender when the Single Molecule Array (SiMoa) was used. A total of 724 Bos taurus miRNAs were identified in at least one preparation. The percentage of miRNAs identified in EVs from the extender (0.05%-0.49% of the total reads) was lower than in the preparation containing spEVs (10.56%-63.69% of the total reads). Edge-R identified a total of 111 DE-miRNAs between EVs isolated from the extender by two methods. Among them, 11 DE-miRNAs (bta-miR-11980, bta-miR-11987, bta-miR-12057, bta-miR-1246, bta-miR-125b, bta-miR-181b, bta-miR-2340, bta-miR-2358, bta-miR-2478, bta-miR-2898, and bta-miR-345-3p) were also abundant in EVs isolated from seminal plasma preparations with extender. This study clearly demonstrates that the presence of the extender does not prevent the characterization of spEVs in cryopreserved semen. However, the molecular profiling of spEVs can be influenced by the isolation method used and by the presence of some miRNAs from the extender. Therefore, in such studies, it is advisable to characterize both spEVs and the vesicles isolated from the extender.

Metagenomic identification of bull semen microbiota in different seasons

A seasonal effect on sperm quality parameters was observed previously. Although identification of the bull semen microbiota by 16S rRNA sequencing was performed previously, it has not been carried out in commercial semen samples from different seasons, and its connection with sperm quality parameters has not been evaluated yet. The objectives in this study were; (i) to evaluate diversity of bull semen microbiota and sperm quality parameters in different seasons, and (ii) to find if specific bacteria were associated with seasonal differences in specific sperm quality parameters. Bull semen microbiota was identified in 54 commercial bull semen samples from 3 seasons (winter, spring, summer). Sperm quality was analysed by Computer Assisted Sperm Analyses (CASA) and Flow Cytometry (FC). From 28 phyla in all samples, six phyla were identified in samples from all seasons, with observed seasonal differences in their distribution. At genus level, 388 genera were identified, of which 22 genera had a relative abundance over 1 % and showed seasonal differences in bacterial diversity, and 9 bacteria genera were present in all seasons. Differences between spring and summer (P < 0.05) were observed for live hydrogen peroxide positive sperm cells. A trend towards significance (0.10 >P > 0.05) was observed for some CASA kinematics (VCL and LIN) and FC parameters (High respiratory activity, and live hydrogen peroxide positive sperm cells) between seasons. Nevertheless, associations between sperm quality parameters and specific bacteria were observed in spring.

The protective effects of antioxidants against endogenous and exogenous oxidative stress on bull sperm.

Oxidative stress, caused by both endogenous and exogenous factors, affects sperm function by damaging morphology and reducing metabolic activity, leading to reduced fertilization ability. The purpose of this study was to investigate the effects of oxidative stress on bull sperm and to evaluate the efficacy of targeted antioxidants in mitigating these detrimental effects. Fresh bull semen samples were subjected to hydrogen peroxide (H2O2) and antimycin treatments to induce oxidative stress, and the antioxidants PQQ, ergothioneine, and vitamin C were applied to counteract the induced stress. Sperm motility, viability, and reactive oxygen species (ROS) levels in the cytoplasm and mitochondria of sperm were assessed using computer-assisted sperm analysis (CASA) and flow cytometry. The treatment with H2O2 rapidly decreased sperm viability, and antimycin-induced mitochondrial ROS mainly decreased sperm motility; PQQ and vitamin C effectively reduced mitochondrial ROS, while ergothioneine and vitamin C reduced cytosolic ROS. In frozen-thawed sperm, oxidative stress was elevated in both cytoplasm and mitochondria, and all three antioxidants improved sperm motility by inhibiting ROS production. Furthermore, the localization of oxidized lipids (4-hydroxynonenal) in sperm was detected using immunofluorescence, indicating that oxidative stress affects the head and midpiece of sperm. These findings highlight the potential of targeted antioxidants to mitigate the detrimental effects of oxidative stress on bull sperm and provide valuable insights to improve semen quality and optimize the use of antioxidants in artificial insemination.

ИЗУЧЕНИЕ БИОЛОГИЧЕСКОЙ ПОЛНОЦЕННОСТИ СПЕРМАТОЗОИДОВ В СПЕРМЕ С БАКТЕРИАЛЬНОЙ ОБСЕМЕНЕННОСТЬЮ

The aim of the study is to investigate the relationship between bacterial contamination of bull semen and the biological value of spermatozoa, as well as reproductive performance. The object of the study was Holstein bulls (n = 17). The material for the study was frozen-thawed semen of bulls in the amount of 61 samples. Microbiological studies of sperm doses were carried out using standard nutrient media in accordance with GOST 32222-2013. The total number of microorganisms was calculated by the number of grown colony-forming units (CFU/ml) in 1 cm3, with subsequent determination of their morphological pro-perties. Sperm motility and morphology were determined using the Argus CASA program. The state of DNA in spermatozoa was studied by the acridine orange test (AO test) using a fluorescence microscope. The study revealed a high positive correlation (r = +0.940**) between the number of microorganisms in sperm and pathology in individual sperm segments. Analysis of variance confirmed a statistically significant effect of the number of microorganisms on sperm morphology F = 123.2 (p=0.000). The correlation relationship between the fragmentation index and the content of microorganisms has a high value (r = +0.965**). Sperm motility negatively correlates (r = –0.768**) with the number of microorganisms (CFU/ml). In the presence of Mycoplasma spp in the samples, the number of cows with fruitful insemination was 35.5 % due to reduced motility, morphology and high fragmentation of sperm DNA (58 %). Sperm motility in samples containing Mycoplasma spp averages 24.5 %, while in samples without these microorganisms it is 48.9 %. Thus, it can be assumed that the presence of Mycoplasma spp in samples and high bacterial contamination rates lead to to lower reproductive performance in bulls.

Investigating Hereditary Disorders of Simmental Frozen Bull Semen Imported to Türkiye during 10-Year Period

This study was aimed to determine Hereditary defects of frozen Simmental bull semen imported to Turkiye between 2013 and 2022. As the source of this study, the websites of various companies that produce semen, bull catalogues, and databases of bull semen companies have been used. A total of 13 websites including Simmental semen databases and bull catalogues were investigated. The result of the study showed that 438 of 1301 Simmental bull’s frozen semen carry at least one genetic defect during 10-year period of import. The most common hereditary defects are Bovine Male subfertility (BMS) for 164 frozen bull semen, Trombopathia (TP) for 155 bull semen and Fleckvieh Haplotype 4 (FH4) for 127 bull semen respectively. Significant effect was observed between different years and the rate of carriers was significantly decreased after 2019. As for origin of bull semen, the bull semen imported from Czech Republic showed significantly the least hereditary defect rate compared with those imported from Austria, Germany and Italy.

PRESENT SCENARIO OF LIVESTOCK BREEDING AND MANAGEMENT PRACTICES IN THE NORTHERN PART OF BANGLADESH

&lt;p&gt;&lt;strong&gt;Background: &lt;/strong&gt;Bangladesh has a rich heritage of livestock rearing. The management and breeding practices differ across the country. &lt;strong&gt;Objectives:&lt;/strong&gt; To study the present scenario of breeding and management practices of livestock at northern Teesta river basin based Lalmonirhat district in Bangladesh. &lt;strong&gt;Methodology: &lt;/strong&gt;Total 100 farmers were selected using random sampling technique from 5 upazilas of Lalmonirhat district during July to December, 2022. Data were analyzed using SPSS 23.0 statistical package. &lt;strong&gt;Results: &lt;/strong&gt;Results showed that most of the farmers (65%) at the northern part of Bangladesh were middle-aged, 41% of them completed secondary education, and their occupation was mainly agriculture (55%). About 37.78% of respondents supplied roadside grass as roughage to their livestock. Maximum number of respondents (60.24%) supplied hand mixed feed as a source of concentrate whereas 39.76% of respondents used commercial feed. 96% of farmers practiced artificial insemination to inseminate their cows and heifers while in goat nearly all respondent farmers (98.41%) practiced natural mating. Among the breeding companies, 41.41% farmers preferred semen from the Bangladesh Rural Advancement Committee (BRAC), followed by Advanced Chemical Industries (ACI) (30.30%), Government (Department of Livestock Services (DLS) (22.22%), and others (6.07%). About 41.76% respondents used 50% Sahiwal - 50% local genotype bull to breed the cows whereas 23.08% respondents used 100% Sahiwal, 18.68% used 75% Holstein Friesian-25% local, 5.49% used 100% Holstein Friesian, 3.30% used 50% Holstein Friesian-50% Local and only 3.30% used 87.5% Holstein Friesian-12.5% Local genotype bull semen, respectively.&lt;strong&gt; &lt;/strong&gt;On the other hand, most of the farmers (98.41%) chosen Black Bengal breeding buck during breeding and remaining used crossbred (Black Bengal Goat- Jamunapari) to breed their does. The actual price of bull semen from Govt. (DLS) was 30 Bangladeshi Taka (BDT) on average for all the breeds whereas, the price ranged 110-200 BDT depending on cattle breeds and bull/bucks Identification (ID) in different private enterprises. Farmers had to pay on an average&lt;strong&gt; &lt;/strong&gt;181.18 BDT for DLS originated semen in addition to that BRAC Artificial Insemination (AI) workers took an average of 425.61 BDT from farmers to inseminate their cows. The average milk yield/d was 1.82±0.14, 3.35±0.40, and 2.74±0.27 liters for local, HF crossbred and Sahiwal crossbred respectively in that region. The prevalence of repeat breeding incidences was 13.74%, 14.21%, and 15.17%, for Local, Sahiwal crossbred, and Holstein Friesian crossbred genotypes respectively. The highest incidence of disease was found as Lumpy Skin Disease (14.67%) followed by Foot and Mouth Disease (FMD) (11.98%) and other parasitic and metabolic diseases. In the study area, it was found that maximum number of the respondents (95%) faced excessive feed price problem along with some other problems. &lt;strong&gt;Implications: &lt;/strong&gt;Government intervention is necessary to improve the situation of livestock production in the country. &lt;strong&gt;Conclusion:&lt;/strong&gt; This study showed the overall scenario of livestock production, breeding, and management in the northern part of Bangladesh which could be helpful for the govt., Non-governmental organizations (NGO’s) and policymakers to take realistic steps for the improvement of livestock production in the northern part of Bangladesh.&lt;/p&gt;