Bovine Spermatozoa Research Articles

The effect of adjusting settings within a Computer-Assisted Sperm Analysis (CASA) system on bovine sperm motility and morphology results.

Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data.

Sperm DNA 5-methyl cytosine and RNA N6-methyladenosine methylation are differently affected during periods of body weight losses and body weight gain of young and mature breeding bulls.

Aiming to characterize the effects of nutritional status on epigenetic markers, such as DNA 5-methyl cytosine (mC) methylation and RNA N6-methyladenosine (m6A) methylation, of bovine sperm, 12 Angus × Hereford crossbred breeding bulls were submitted to nutritional changes for a period of 180 d: no change in body weight (BW) (phase 1 = 12 d), BW loss (phase 2 = 78 d), and BW gain (phase 3 = 90 d) in a repeated measures design. Animals were fed Beardless wheat (Triticum aestivum) hay and mineral mix. Statistical analyses were performed using SAS 9.4 (SAS Inst., Cary, NC). Higher levels of RNA m6A (P = 0.004) and DNA methylation (P = 0.007) of spermatic cells were observed at phase 2 compared with phase 1. In phase 3, sperm RNA m6A methylation levels continued to be higher (P = 0.004), whereas the DNA of sperm cells was similar (P = 0.426) compared with phase 1. Growing bulls had a tendency (P = 0.109) of higher RNA m6A methylation levels than mature bulls. Phase 2 altered scrotal circumference (P < 0.001), sperm volume (P = 0.007), sperm total motility (P = 0.004), sperm progressive motility (P = 0.004), total sperm count (P = 0.049), normal sperm (P < 0.001), abnormal sperm (P < 0.001), primary sperm defects (P = 0.039), and secondary sperm defects (P < 0.001). In phase 3, bulls had scrotal circumference, sperm volume, sperm motility, sperm progressive motility, total sperm count, normal and abnormal spermatozoa, and primary and secondary spermatozoa defects similar to phase 1 (P > 0.05). Serum concentrations of insulin-like growth factor-1 and leptin decreased during phase 2 (P = 0.010), while no differences (P > 0.05) were detected between phases 3 and 1; growing bulls tended (P = 0.102) to present higher leptin levels than mature bulls. Specific for mature bulls, DNA methylation was positively correlated with leptin concentration (0.569, P = 0.021), whereas for young bulls, DNA methylation was positively correlated with abnormal spermatozoa (0.824, P = 0.006), primary spermatozoa defect (0.711, P = 0.032), and secondary spermatozoa defect (0.661, P = 0.052) and negatively correlated with normal spermatozoa (-0.824, P = 0.006), total sperm count (-0.702, P = 0.035), and sperm concentration (-0.846, P = 0.004). There was no significant correlation (P > 0.05) between RNA m6A and hormones and semen traits. In conclusion, the nutritional status of breeding bulls alters epigenetic markers, such as DNA methylation and RNA m6A methylation, in sperm, and the impact of change seems to be age dependent. These markers may serve as biomarkers of sperm quality and fertility of bulls in the future. Detrimental effects on sperm production and seminal quality are observed at periods and places when and where environmental and nutritional limitations are a year-round reality and may carry hidden players that may influence a lifetime of underperformance.

The addition of crocin in the freezing medium extender improves post-thaw semen quality.

Semen cryopreservation is arguably the most important method or technique contributing to the advancement of modern animal production. However, the quality of sperm after thawing is still highly variable. The addition of antioxidant compounds to the freezing medium has been used customarily to counteract the harmful effects of Reactive Oxygen Species (ROS) that are produced during the freeze/thaw process. Crocin, a potent antioxidant, improves the fertilizing capacity of spermatozoa. In this study, we evaluated the potential of crocin (0, 0.5 and 1mM) as an extender additive to diminish the damaging effects of cryopreservation on bovine spermatozoa. Post-thaw semen quality was assessed by means of motility, viability and lipid peroxidation (LPO). We further investigated the effect of crocin supplementation upon freezing on sperm quality parameters during their incubation at 37°C for up to 2hr. Overall, the data assessment indicates that crocin facilitated a general improvement of the quality of freeze/thawed spermatozoa, under the present experimental conditions. Crocin (1mM) maintained a higher percentage of alive spermatozoa with intact acrosome with rapid and progressive motility, compared to the control extender. Moreover, the spermatozoa cryopreserved in the presence of crocin exhibited higher values in CASA kinematic parameters (VCL, VSL, VAP, ALH) immediately after thawing. Furthermore, the positive effect of crocin on motility parameters was also sustained over a period of 2hr incubation at 37°C. This effect of crocin may be attributed to the observed inhibition of LPO during the incubation period. Thus, the results indicate that the addition of crocin (especially at a final concentration of 1mM) in the freezing extender medium may benefit the preservation of the quality parameters of spermatozoa that are compromised by the freeze/thaw heat shock and the stress during handling for IVF or artificial insemination.

113&#x2003;The effects of bisphenols on cryopreserved bovine spermatozoa in vitro.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

The effect of IBMX and prolactin on functional status of cryopreserved bull spermatozoa

Purpose: investigate the effect of IBMX (activator of protein phosphorylation) and prolactin (PRL) on the functional state of cryopreserved bovine spermatozoa using inhibitory analysis.Materials and methods. Frozen-thawed semen samples from 60 black-and-white bulls was used in the experiments. For capacitation, cells were incubated in Sp-TALP medium supplemented with 6 mg/ml bovine serum albumin and various compounds: an inductor of capacitation (IBMX at concentrations of 1 μM, 10 μM, 50 μM, 100 μM), hormone (PRL at concentrations of 1 ng, 10 ng, 50 ng, 100 ng) and inhibitors of protein kinases C (Ro 31-8220 at a concentration of 10 ng/ml) and protein kinase A (H-89 at a concentration of 10 μM). The incubation was carried out at 38°C in an atmosphere of 5% CO2, 98% humidity for 4 hours. The functional status of the cells was determined by the chlortetracycline test.Results. It was shown that IBMX at all experimental concentrations did not affect the post-ejaculatory maturation (capacitation and acrosome reaction) of spermatozoa, while all concentrations of PRL (1-100 ng/ml) promoted the acrosome reaction in capacitated cells. In the presence of a protein kinase A inhibitor, there was a decrease in number of capacitated and an increase in number of acrosome-reactive spermatozoa under the action of IBMX at a concentration of 100 μM and no changes under the action of a protein kinase C inhibitor. Also, in case of protein kinase C inhibition the PRL-related stimulation of the acrosome reaction was canceled, while the usage of H-89 did not affect the functional status of spermatozoa, mediated by PRL. Thus, the influence of IBMX and PRL on the processes of post-ejaculatory maturation in thawed bovine spermatozoa was studied using the inhibitory analysis.Conclusion. At the capacital stage, all studied IBMX concentrations did not affect the ratio of deconved cells with various functional status. Prode also contributed to the passage of the acrosomous reaction in the rolled spermatozoa after defrosting. Inhibition of protein kinase A when incubating cells with IBMX has mediated the processes of acrosomal exocytosis in ripped cells and did not affect this process under the action of the PRR, while the protein kinase inhibitor C changed the ratio of cells with various functional status in the direction of increasing the percentage of cells at the rate of occasion I did not participate in intracellular action provided IBMX on deconved cells.

BENEFICIALL EFFECT OF THE EUROPEAN RASPBERRY (RUBUS IADEUS) EXTRACT ON THE ACTIVITY AND OXIDATIVE PROFILE OF BOVINE SPERMATOZOA

Berries belong to the best dietary sources of bioactive compounds. These fruits contains bioactive compounds such as antioxidants (phenolic compounds and fruit colorants), vitamins (ascorbic acids) and minerals. This study aimed to estimate the effect of three concentrations (75 µg/mL, 150 µg/mL and 300 µg/mL) of the European raspberry (Rubus iadeus) on the selected quality parameters of bovine spermatozoa after 0h, 2h, and 24h of in vitro culture. Sperm motility was evaluated using the Computer-assisted sperm analysis (CASA) system. The mitochondrial activity was detected by the metabolic (MTT) assay. The determination of ROS (Reactive Oxygen Species) quantity was observed through the luminol method. The protein and lipid oxidation were evaluated using spectrophotometric assays. The experiment showed that the highest applied concentration of European raspberry extract at 2h (p&lt;0.05) and all concentration after 24h (p&lt;0.05; p&lt;0.001) exhibited motility-promoting effects. We observed the highest increase of mitochondrial activity using 300 µg/mL by 2h (p&lt;0.05) and using 150 µg/mL (p&lt;0.05) and 300 µg/mL (p&lt;0.01) after 24h. Exposure to this extract led to decreasing levels of ROS generation at 2h of incubation (p&lt;0.05) with the highest dose as well as after 24h using 150 µg/mL (p&lt;0.01) and 300 µg/mL (p&lt;0.001) applied doses. The protein oxidation levels showed decreasing rates at 2h (p&lt;0.05; 300 µg/mL) and at time of 24h (p&lt;0.01; 150 µg/mL and p&lt;0.001; 300 µg/mL). All selected raspberry concentration prevented oxidative degeneration of lipids during 2h (p&lt;0.05) and after 24h (p&lt;0.05; p&lt;0.001). Our results indicate, that European raspberry extract has beneficial effect on the measured parameters of the bovine spermatozoa during in vitro incubation but depending on time and applied dose.

Increase of Dna Fragmentation Evaluated Through The Alkaline Comet Is Concomitant With A Decrease In The Quality of Frozen-Thawed Bovine Sperm

Increase of Dna Fragmentation Evaluated Through The Alkaline Comet Is Concomitant With A Decrease In The Quality of Frozen-Thawed Bovine Sperm

Optimization of Bovine Sperm Cryopreservation Using Iterative Optimization and Machine Learning

Optimization of Bovine Sperm Cryopreservation Using Iterative Optimization and Machine Learning

Staphylococcus-Induced Bacteriospermia In Vitro: Consequences on the Bovine Spermatozoa Quality, Extracellular Calcium and Magnesium Content.

Bacterial contamination of bovine ejaculates intended for artificial insemination may be reflected in a significant economic loss due to unsuccessful fertilization as well as health issues of the recipients. The Staphylococcus genus represents a large part of bacteriocenosis of bovine ejaculates. Therefore, this study aims to get a closer look on the effects of Staphylococcus-induced bacteriospermia under in vitro conditions on bovine sperm quality. Prior to inducing bacteriospermia, spermatozoa were separated from each ejaculate using Percoll® Plus gradient medium in order to limit the effects only to the selected bacterial species. Seven Staphylococcus species previously isolated from bovine semen were used for our experiments at a turbidity of 0.5 McFarland (equivalent to 1.5 × 108 colony-forming units per mL). The contaminated semen samples were incubated at 37 °C and at times of 0, 2, and 4 h, motility, mitochondrial membrane potential, reactive oxygen species (ROS) generation, sperm DNA fragmentation, and magnesium (Mg) and calcium (Ca) extracellular concentration were analyzed and compared with the control group (uncontaminated). The results showed no significant changes at the initial measurement. However, significant adverse effects were observed after 2 h and 4 h of incubation. Most notably, the presence of S. aureus, S. warneri, S. kloosii, and S. cohnii caused a significantly increased ROS production, leading to sperm DNA fragmentation, changes in the mitochondrial membrane potential, and a decreased sperm motility. Furthermore, the presence of Staphylococcus species led to lower extracellular concentrations of Mg and Ca. In conclusion, the overgrowth of Staphylococcus bacteria in bovine semen may contribute to oxidative stress resulting in sperm DNA fragmentation, altered mitochondrial membrane potential, and diminished sperm motility.

Sperm preparedness and adaptation to osmotic and pH stressors relate to functional competence of sperm in Bos taurus

The adaptive ability of sperm in the female reproductive tract micromilieu signifies the successful fertilization process. The study aimed to analyze the preparedness of sperm to the prevailing osmotic and pH stressors in the female reproductive tract. Fresh bovine sperm were incubated in 290 (isosmotic-control), 355 (hyperosmotic-uterus and oviduct), and 420 (hyperosmotic-control) mOsm/kg and each with pH of 6.8 (uterus) and 7.4 (oviduct). During incubation, the changes in sperm functional attributes were studied. Sperm kinematics and head area decreased significantly (p < 0.05) immediately upon exposure to hyperosmotic stress at both pH. Proportion of sperm capacitated (%) in 355 mOsm/kg at 1 and 2 h of incubation were significantly (p < 0.05) higher than those in 290 mOsm media. The magnitude and duration of recovery of sperm progressive motility in 355 mOsm with pH 7.4 was correlated with the ejaculate rejection rate (R2 = 0.7). Using this information, the bulls were divided into good (n = 5) and poor (n = 5) osmo-adapters. The osmo-responsive genes such as NFAT5, HSP90AB1, SLC9C1, ADAM1B and GAPDH were upregulated (p < 0.05) in the sperm of good osmo-adapters. The study suggests that sperm are prepared for the osmotic and pH challenges in the female reproductive tract and the osmoadaptive ability is associated with ejaculate quality in bulls.

Sperm chromatin condensation as an in vivo fertility biomarker in bulls: a flow cytometry approach

BackgroundGenetic selection in cattle has been directed to increase milk production. This, coupled to the fact that the vast majority of bovine artificial inseminations (AI) are performed using cryopreserved sperm, have led to a reduction of fertility rates over the years. Thus, seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency. In humans, sperm chromatin condensation evaluated through chromomycin A3 (CMA3) has recently been purported to be a powerful biomarker for sperm functional status and male infertility. The objectives of the present study were: a) to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability, and b) to test whether this parameter could be used as a predictor of in vivo fertility in bulls. The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males. Reproductive outcomes of each sire were determined by non-return rates, which were used to classify bulls into two groups (highly fertile and subfertile).ResultsChromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry. Sperm quality parameters (morphology, viability, total and progressive motility) were also assessed. Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility. Sperm morphology, viability and total motility presented an area under the ROC curve (AUC) of 0.54, 0.64 and 0.68, respectively (P > 0.05), and thus were not able to discriminate between fertile and subfertile individuals. Alternatively, while the percentage of progressively motile sperm showed a significant predictive value, with an AUC of 0.73 (P = 0.05), CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls. Specifically, the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility, with an AUC of 0.78 (P = 0.02).ConclusionsChromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility, with a potential ability to maximising the efficiency of dairy breeding industry.

Mammalian sperm hyperactivation regulates navigation via physical boundaries and promotes pseudo-chemotaxis.

Mammalian sperm migration within the complex and dynamic environment of the female reproductive tract toward the fertilization site requires navigational mechanisms, through which sperm respond to the tract environment and maintain the appropriate swimming behavior. In the oviduct (fallopian tube), sperm undergo a process called "hyperactivation," which involves switching from a nearly symmetrical, low-amplitude, and flagellar beating pattern to an asymmetrical, high-amplitude beating pattern that is required for fertilization in vivo. Here, exploring bovine sperm motion in high-aspect ratio microfluidic reservoirs as well as theoretical and computational modeling, we demonstrate that sperm hyperactivation, in response to pharmacological agonists, modulates sperm-sidewall interactions and thus navigation via physical boundaries. Prior to hyperactivation, sperm remained swimming along the sidewalls of the reservoirs; however, once hyperactivation caused the intrinsic curvature of sperm to exceed a critical value, swimming along the sidewalls was reduced. We further studied the effect of noise in the intrinsic curvature near the critical value and found that these nonthermal fluctuations yielded an interesting "Run-Stop" motion on the sidewall. Finally, we observed that hyperactivation produced a "pseudo-chemotaxis" behavior, in that sperm stayed longer within microfluidic chambers containing higher concentrations of hyperactivation agonists.

PSX-A-7 Late-Breaking: Correlation of mitochondrial membrane potential and rough motility scores in cryopreserved bovine spermatozoa

Abstract Spermatozoon motility is an important factor in successful artificial reproductive technologies. Successful reproduction requires properly developed spermatozoa with adequate forward, progressive motility that allows for transport through the female reproductive tract. Motility is driven by production of ATP; however, cryopreservation is known to have damaging effects on spermatozoa. Mitochondria utilize oxidative phosphorylation to synthesize ATP through an electrochemical proton motive force that is composed primarily of mitochondrial membrane potential (Δψm). The mitochondrial membrane potential can be measured using a fluorescent, carbocyanine JC-1 dye (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide). The objective of this study was to examine correlations between the mitochondrial membrane potential and motility score. Cryopreserved semen samples from three bulls were donated to Tarleton State University for research purposes. Samples were thawed, placed under a phase contrast microscope, and analyzed by three individuals with varying levels of training for rough motility. These samples were then prepared with JC-1 dye according to manufacturer’s instructions. Two microliters of dye were added to a stock preparation of each sample. Samples were examined on a FACS Calibur flow cytometer at University of Texas Southwestern Core in Dallas, TX. Flow cytometry analysis was performed using FlowJo V10.7. Statistical analysis was performed using SAS v9.4. The Pearson correlation coefficient showed a strong, positive correlation (r = 0.90) between the mitochondrial membrane potential and motility (P = 0.28), thus indicating as the mitochondrial membrane potential increases, so does the rough motility score. These data represent a subset of a population that demonstrates the need of further research on the ability for spermatozoa to produce ATP and the correlation in forward, progressive motility. This research can provide a foundation in which future researchers may develop an assay that allows for testing of mitochondrial membrane potential by producers to select bulls with greater breeding potential.

Assessment of Sperm Binding Capacity in the Tubal Reservoir Using a Bovine Ex Vivo Oviduct Culture and Fluorescence Microscopy.

Sperm binding within the oviductal sperm reservoir plays an important role for reproductive success by enabling sperm survival and maintaining fertilizing capacity. To date, numerous in vitro technologies have been established to measure sperm binding capacity to cultured oviductal cells or oviductal explants. However, these methods do not accurately represent the microenvironment and complex multi-molecular nature of the oviduct. In this paper, we describe a novel protocol for assessing sperm binding capacity in the tubal sperm reservoir using an ex vivo oviduct culture in the bovine model. This protocol includes the staining of frozen-thawed bovine spermatozoa with the DNA-binding dye Hoechst 33342, the co-incubation of stained sperm in closed segments of the oviduct and the visualization and quantification of bound spermatozoa by fluorescence microscopy. By generating overlays of multiple Z-stacks of randomly selected regions of interest (ROIs), spermatozoa bound in the sperm reservoir can be visualized and quantified within the 3D arrangement of the oviductal folds. This method, which is applicable to multiple species, can be used to assess individual sperm binding capacity in males for prognostic purposes as well as to assess the impact of diseases and medications on the formation of the sperm reservoir in the oviduct in humans and animals.

In vitro effect of ferrous sulphate on bovine spermatozoa motility parameters, viability and Annexin V-labeled membrane changes.

The aim of this study was to assess the dose- and time-dependent in vitro effects of ferrous sulphate (FeSO4.7H2O) on the motility parameters, viability, structural and functional activity of bovine spermatozoa. Spermatozoa motility parameters were determined after exposure to concentrations (3.90, 7.80, 15.60, 31.20, 62.50, 125, 250, 500 and 1000 μM) of FeSO4.7H2O using the SpermVisionTM CASA (Computer Assisted Semen Analyzer) system in different time periods. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, and the Annexin V-Fluos was applied to detect the membrane integrity of spermatozoa. The initial spermatozoa motility showed increased average values at all experimental concentrations compared to the control group (culture medium without FeSO4.7H2O). After 2 h, FeSO4.7H2O stimulated the overall percentage of spermatozoa motility at the concentrations of ≤ 125 μM. However, experimental administration of 250 μM of FeSO4.7H2O significantly (P < 0.001) decreased the spermatozoa motility but had no negative effect on the cell viability (P < 0.05) (Time 2 h). The lowest viability was noted after the addition of ≥ 500 μM of FeSO4.7H2O (P < 0.001). The concentrations of ≤ 62.50 μM of FeSO4.7H2O markedly stimulated (P < 0.001) spermatozoa activity after 24 h of exposure, while at high concentrations of ≥ 500 μM of FeSO4.7H2O the overall percentage of spermatozoa motility was significantly inhibited (P < 0.001) and it elicited cytotoxic action. Fluorescence analysis confirmed that spermatozoa incubated with higher concentrations (≥ 500 μM) of FeSO4.7H2O displayed apoptotic changes, as detected in head membrane (acrosomal part) and mitochondrial portion of spermatozoa. Moreover, the highest concentration and the longest time of exposure (1000 μM of FeSO4.7H2O; Time 6 h) induced even necrotic alterations to spermatozoa. These results suggest that high concentrations of FeSO4.7H2O are able to induce toxic effects on the structure and function of spermatozoa, while low concentrations may have the positive effect on the fertilization potential of spermatozoa.

Effects from disruption of mitochondrial electron transport chain function on bull sperm motility

Sperm mitochondrial function is essential for normal physiology and fertility, but the importance of mitochondrial activity to support specific sperm functions, such as motility, varies between species. It was previously believed that mitochondrial function was not necessary for bull sperm motility [1]; however, this theory is contradicted by recently reported findings that the upper fraction of bull sperm swim-up preparations had both high motility and elevated mitochondrial oxygen consumption rates [2]. The objective of this study was to investigate the relationship between mitochondrial function and motility in bull sperm. We hypothesized that sperm motility would be positively correlated with mitochondrial oxygen consumption (MITOX) but unaffected by pharmacological inhibition of electron transport chain (ETC) activity. This was accomplished by monitoring both mitochondrial oxygen consumption and motility parameters in the presence of mitochondrial effector drug treatments. Duplicate ejaculates were collected by electroejaculation from Black Angus bulls (n = 4). Oxygen consumption, as % air saturation (pO2; oxygen partial pressure), over time was monitored in the presence of 5 drug treatments: vehicle control, FCCP, Antimycin (ANTI), Oligomycin (Oligo), and FCCP + Oligomycin (FCCP + OLIGO). Duplicate aliquots were prepared for concurrent motility assessment by computer-assisted sperm analysis (CASA) at 6 and 30 min post-treatment (t6 and t30). The impact of treatments on pO2 (in % air saturation) over time were assessed by generalized linear mixed effects modeling (GLMM) which was also used to test for differences in average motility across treatment conditions and time points (t6 and t30). Pearson product-moment correlation was used to investigate relationships between oxygen consumption and motility parameters. Overall, pO2 differed over time between treatment conditions (p < 0.0001). When compared to the vehicle treatment, ANTI and OLIGO significantly inhibited oxygen consumption (p < 0.05, adjusted), and FCCP stimulated a marked increase in oxygen consumption. No significant differences in motility over time were observed between treatments, so comparison of motility parameters between treatment conditions was performed with pooled timepoints. Motility parameters were only observed to differ significantly from the vehicle with ANTI Treatment, for which significant decreases in numerous parameters, including total motility (p = 0.007), progressive motility (p = 0.01), DAP (p = 0.01), VAP (p = 0.01) and VSL (p = 0.02) were identified. For the vehicle treatment, correlational analysis did not reveal any significant correlations between pO2 and any motility parameters at t6; however, several significant correlations were identified at t30. Mean pO2 was negatively correlated with local motility (p < 0.01) and positively correlated with DCL, DAP, and VCL (p < 0.05). Results from this study suggest that bovine sperm motility is impacted by mitochondrial functionality, with ETC inhibition by ANTI causing significant reduction in motility parameters. This study also demonstrates the use of a new technology for the assessment of bovine sperm mitochondrial function. This modality for evaluation of bull sperm mitochondrial function will inform future efforts to understand bull sperm function and fertility and aid investigations into toxicological agents.

Melatonin improves rate of monospermic fertilization and early embryo development in a bovine IVF system.

The developmental competence of male and female gametes is frequently reduced under in vitro conditions, mainly due to oxidative stress during handling. The amino-acid derived hormone melatonin has emerged as a potent non-enzymatic antioxidant in many biological systems. The goal of the present study was to evaluate the effects of melatonin on post-thaw sperm quality, fertilizing ability, and embryo development and competence in vitro after in vitro fertilization. Frozen-thawed bovine spermatozoa were incubated either in the presence of 10−11 M melatonin (MT), or its solvent (ethanol; Sham-Control), or plain Tyrode’s Albumin Lactate Pyruvate medium (TALP, Control). Computer-Assisted Sperm Analysis (CASA) and flow cytometry data after 30 min, 120 min, and 180 min incubation did not reveal any significant effects of melatonin on average motility parameters, sperm subpopulation structure as determined by hierarchical cluster, or on the percentage of viable, acrosome intact sperm, or viable sperm with active mitochondria. Nevertheless, in vitro matured cumulus-oocyte-complexes fertilized with spermatozoa which had been preincubated with 10−11 M melatonin (MT-Sperm) showed higher (P < 0.01) rates of monospermic fertilization, reduced (P < 0.05) polyspermy and enhanced (P < 0.05) embryo development compared to the Control group. Moreover, the relative abundance of MAPK13 in the in vitro-derived blastocysts was greater (P < 0.05) than observed in the Control group. In conclusion, adding melatonin to the sperm-preparation protocol for bovine IVF improved proper fertilization and enhanced embryonic development and competence in vitro.

DOUBLE WASHING PROCESS DO NOT HAVE EFFECT ON SPERM TOTAL RNA CONCENTRATION IN SEMEN ASSAY SAMPLE

As highly specialized cells, sperm are well designed to fertilize with oocytes. To achieve its function, round spermatids undergo dramatic changes, spermiogenesis. Along with the elimination of cytoplasm and the shift of histone with protamine, spermatids progressively switch off transcription and translation to inactive all cellular event that are not relevant to fertilization. However, recently, many different research groups have introduced sperm RNA in rat, mouse, bovine, swine, equine, and human sperm.

The reproductive success of bovine sperm after sex-sorting: a meta-analysis

In the three decades since its inception, the sex-sorting technology has progressed significantly. However, field studies report conflicting findings regarding reproductive outcomes. Therefore, we conducted this meta-analysis of all trials published between 1999 and 2021. Non-return rates after 24 or 60 d (NRR 24/60), pregnancy, calving, abortion, and stillbirth rates were compared after AI with sex-sorted vs non-sorted sperm. Additionally, the impact of recent developments in the sex-sorting technology was assessed. Of 860 studies found, 45 studies (72 trials) provided extractable data and were included. Overall, the results of this meta-analysis provided evidence that the NRR 24/60 was diminished by 13%, pregnancy rates were reduced by 23% (25% cows, 21% heifers) and calving rates were reduced by 24% when using sex-sorted sperm. Enhancing the dosage to 4 million sex-sorted sperm/straw (including recent improvements, high vs low dose) as well as using fresh sex-sorted sperm (sorted vs non-sorted) increased pregnancy rate ratios by 7 percentage points. The refinement of the sex-sorting technology after 2015 resulted in a lowered reduction of pregnancy and calving rate of 19% and 23%, respectively. Whereas abortion rates were similar, the stillbirth of male calves was increased by 6.3%.

P–051 Differential resilience of sperm from different mammals to DNA decondensation

Abstract Study question Does sperm from different species with different protamine 1/protamine 2 ratios have different resilience to sperm decondensation? Summary answer Sperm cells from species whose DNA is condensed with both protamine 1 and protamine 2 require less time in deprotamination steps. What is known already Sperm cells present a highly particular DNA condensation that is acquired during sperm differentiation, where most part of histones are replaced by protamines. Protamines are key elements for DNA condensation and, while protamine 1 is more conserved among species, protamine 2 has evolved differentially, existing only a few species that retain the mature protein in their sperm DNA. Changes in protamine expression rates have been described to be associated to head sperm size and shape. In addition, reduced amounts of protamine 2 are related to male infertility in species in which this protein is present. Study design, size, duration Cryopreserved sperm samples were treated with lysis solutions to induce DNA decondensation and formation of sperm haloes. In these treatments, the effect of different incubation times with proteinase K added to the lysis solution upon DNA decondensation was tested by analyzing core diameter, halo diameter and the Halo/core ratio in at least 50 sperm per sample. Participants/materials, setting, methods Species included in the study were Human, Equine, Donkey, Porcine and Bovine. Sperm samples from five different individuals for each species were included in the study. DNA decondensation included three lysis steps: first, a SDS + DTT incubation for 30 minutes; second, a DTT + NaCl treatment for 30 minutes; and third, a DTT + NaCl + Proteinase K treatment with a variable time of 0, 30 or 180 minutes. Main results and the role of chance The halo/core diameter, used as a representation of the degree of DNA decondensation, for 0 minutes, 30 minutes and 180 minutes of proteinase K incubation were: 4.68±0.51, 4.32±0.51 and 4.77±0.64, respectively for human sperm; 4.15±0.41, 4.57±0.53 and 4.68±0.63, respectively for Equine sperm; 4.40±0.64, 4.00±0.37 and 4.17±0.19, respectively for donkey sperm; 1.77±0.2, 3.05±0.14 and 4.13±0.39, respectively for porcine sperm; and 2.40±0.40, 3.36±0.22 and 4.19±0.38, respectively for bovine sperm. Differences of halo/core ratio in different times were only observed in porcine and bovine sperm, where increasing degrees of DNA decondensation were found (p &amp;lt; 0.05). Therefore, these results show that while longer incubations in lysis solutions with proteinase K lead to higher DNA decondensation in porcine and bovine, they do not induce higher decondensation in human, equine and donkey. This evidence, coupled to the fact that porcine and bovine sperm present null or very low protamine 2 content, suggests that its presence might confer higher DNA decondensation susceptibility. Limitations, reasons for caution Only sperm cells with normal sperm haloes were analyzed in the present study. As multiple studies show, haloes exhibited by sperm cells with DNA damage display higher diameter, that is why they were strictly excluded in this study with the aim to elucidate the average DNA decondensation. Wider implications of the findings: Sperm DNA might have different degrees of DNA condensation, which can be associated to a higher difficulty of DNA decondensation, thus having implications in the sensitivity tests that assess sperm DNA integrity. Trial registration number Not applicable.