Lateral Head Displacement Research Articles

Effect of natural astaxanthin on sperm quality and mitochondrial function of breeder rooster semen cryopreservation

Cryopreservation causes higher reactive oxygen species (ROS) concentrations, leading to oxidative stress and lipid peroxidation damaging sperm, and using antioxidants can improve semen quality after freeze-thaw. Natural astaxanthin (ASTA) can be inserted into cell membranes and its antioxidant properties are stronger than other antioxidants. We aimed to investigate the effects of ASTA supplementation in the Beltsville Poultry Semen Extender (BPSE) on post-thaw rooster semen quality and to explore the potential mechanism of rooster semen quality change. The qualifying semen ejaculates collected from 30 adult male Jinghong No. 1 laying hen breeder roosters (65 wk old) were pooled, divided into four aliquots, and diluted with BPSE having different levels of ASTA (0, 0.5, 1, or 2 μg/mL). Treated semen was cryopreserved and kept in liquid nitrogen. The entire experiment was replicated three times independently. Sperm viability, motility, curvilinear velocity, amplitude of lateral head displacement, straightness, plasma membrane integrity, and acrosome integrity were observed to be highest (P < 0.05) with 1 μg/mL ASTA at freeze-thawing. Higher (P < 0.05) antioxidant enzyme (CAT-like, SOD) activities and free radical (·OH, O2.-) scavenging ability, less ROS and malondialdehyde (MDA) concentrations were recorded with the addition of appropriate concentrations of ASTA compared to control. In addition, the levels of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP), and lactate dehydrogenase (LDH) in the 1 μg/mL ASTA group improved compared to the control group, and decreased the amount of AIF protein level but increased the Bcl-2 protein level (P < 0:05). Collectively, these results demonstrate that adding ASTA in the BPSE promoted rooster freeze-thaw sperm quality, which may be related to reducing ROS levels, protecting the antioxidant defense system, preventing lipid peroxidation, improving mitochondrial structural and functional integrity, and inhibiting sperm apoptosis.

Impact of salinity and body size on sperm motility in three California smelt species

Three smelt species in California, USA—the endangered delta smelt Hypomesus transpacificus and longfin smelt Spirinchus thaleichthys and the introduced wakasagi H. nipponensis—experience ecological pressures, including habitat alterations, temperature changes, and fluctuating salinity levels that can affect their survival and reproductive success. The present study investigates how salinity during the spawning process influences sperm motility by analyzing 13 sperm motility traits across three salinity levels (0.4, 5, and 10 ppt). Using Principal Component Analysis, we found that the first two principal components accounted for over 65 % of the total variance. Consequently, two predominant traits from each principal component were selected to evaluate sperm motility. Specifically, the amplitude of lateral head displacement (ALH), linearity coefficient (LIN), curvilinear velocity (VCL), progressive motility (PM), and wobble coefficient (WOB) predominantly contributed to these components for both longfin smelt and wakasagi. Results showed that the salinity significantly impacts these traits, except for ALH in delta smelt and longfin smelt, and WOB and LIN in wakasagi. In addition, we found the salinity and temperature difference between the activating water and extender interact differently across species. In delta smelt, interactions between the activating water and extender temperatures significantly affected most traits, whereas in wakasagi, only the interaction between activating water salinity and extender temperature showed significant effects. No significant influence from these interactions was observed in longfin smelt. Additionally, the impact of body size (e.g., fork length) on sperm motility varies across species, with a significant influence observed in longfin smelt, while no effect was detected in delta smelt and wakasagi. Future research should focus on exploring the physiological mechanisms behind the observed effects of salinity, temperature, and body size on sperm motility, as well as examining these interactions in other species to develop broader conservation strategies under changing environmental conditions.

S-adenosylmethionine and S-adenosyl-L-homocysteine metabolism is involved in the sperm motility and in vitro fertility rate in mouse

Increased fragmentation of sperm DNA has been implicated in male infertility. Folate deficiency results in impaired methionine synthesis, depletion of S-adenosylmethionine (SAM) levels, an increase in S-adenosyl-l-homocysteine (SAH) levels, and increased DNA fragmentation. Disruption of the dynamic balance between SAM and SAH may also contribute, although the details of this process are not yet fully understood. We investigated the localization of SAM, SAH, and S-adenosylhomocysteine hydrolase (SAHH), and whether SAM/SAH metabolism contributes to sperm motility and fertilization rate. SAM, SAH, and SAHH levels were assessed in the acrosome, midpiece, and tail of spermatozoa. Chemical inhibition of SAM/SAH metabolism and extracellular SAH significantly decreased the straight-line velocity (VSL), curvilinear velocity (VCL), and amplitude lateral head displacement (ALH) of sperm cells, which were thus unable to swim forward and perform oscillatory movements in place. This significantly reduced the fertilization rate. Therefore, the disruption of the SAM/SAH balance may contribute to male infertility.

PSVI-18 Association between residual feed intake and reproductive-related traits in young bulls

Abstract Enhancement of feed efficiency (FE) along with reproductive performance is critical to maximize profitability of beef production systems. Residual feed intake (RFI) is a measure of FE, assessing differences between expected and actual feed intake. Published data suggest a negative impact of RFI on reproductive performance. The objective of this experiment was to establish the relationship between RFI and reproductive parameters related with puberty in young bulls. Growing, Brangus bulls (n = 58; 10 to 12 mo of age) were tested for FE over 56 d and ranked as low RFI (LRFI) and high RFI (HRFI). Scrotal circumference (SC) was measured at the greatest diameter of the testicles by the same operator using a scrotal measuring tape, and semen samples were collected by electroejaculation at the beginning and at the end of the test. Fresh semen was visually evaluated (VE) for visual motility (VM), morphology [major defects (MAD)], minor defects (MID), total defects (TD)], plasma membrane integrity (PMI) via Eosin-nigrosin stain and sperm concentration (Con). Sperm kinematics were objectively determined simultaneously via computer-assisted sperm analysis (CASA-IVOS II System, Hamilton Thorne, Beverly, MA) within 6 h of collection in a medium designed specifically for holding sperm. Statistical analysis included repeated measures to assess interactions between Group (LRFI, HRFI) and Time (0 and 56 d of RFI measurement). Results are presented as mean ± SEM in Table 1. The main effect of Group did not affect CASA derived traits, while MAD and TD were affected among VE traits. A Time effect was observed in the amplitude of lateral head displacement (ALH), beat cross frequency (BCF), curvilinear velocity (VCL) and linearity (LIN). A tendency was indicated for total motility (TM), and average path velocity (VAP) among CASA derived traits, while all VE traits were affected by Time except for visual motility (VM). Finally, an interaction effect (Group x Time) was observed in LIN and plasma membrane integrity (PMI). In conclusion, age at puberty as determined by VM, Con, and SC (Group: P = 0.12, Day: P &amp;lt; 0.001, Group x Day: P = 0.79) was not associated with RFI. Overall, fertility-related measurements were not influenced by RFI, with time emerging as the primary influencing factor across evaluated parameters. Evaluation of reproductive parameters following d 56 FE testing may be a useful determinant for identifying differences in seminal characteristics of young bulls.

Pre- and Post-Thaw Addition of L-Carnitine and Pyruvate: Effect on Stallion Sperm Parameters.

The addition of antioxidants to cryopreservation media reportedly improves sperm post-thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L-carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post-thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA-glucose-based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk-based extender with 20% egg yolk and 5% dimethylformamide (milk control); (3) Extender1 supplemented with 50 mM L-carnitine and 10 mM pyruvate (EDTA-carnitine-pyruvate); and (4) Extender 2 supplemented with 50 mM L-carnitine and 10 mM pyruvate (milk-carnitine-pyruvate). In Experiment 2, 50 mM L-carnitine and 10 mM pyruvate were added post-thaw to samples cryopreserved with extenders 1 and 2 (EDTA control and milk control). Sperm kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability were evaluated after thawing. No significant differences (p > 0.05) were observed for most of the kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability of spermatozoa, between the samples frozen in the presence or absence of L-carnitine and pyruvate, nor between the samples after the post-thaw addition of these components. A higher (p < 0.05) mean velocity and higher (p < 0.05) amplitude of lateral head displacement were observed in the samples frozen in the milk-based extender with the addition of L-carnitine and pyruvate after thawing. The addition of 50 mM L-carnitine and 10 mM pyruvate, either to the freezing extenders or after thawing, was not deleterious for sperm; however, it did not improve equine sperm motility, viability, acrosome and DNA integrity, nor decrease membrane lipid peroxidation after thawing.

Flow cytometry evaluation of Dalagh ram post-thaw semen incorporated with curcumin-loaded niosomal nanocarriers

Optimization of cryopreservation media to attenuate oxidative damage on post-thaw spermatozoa is crucial. This study aimed to assess the antioxidant functionality of curcumin-loaded niosomal nanocarriers (CurLNN) on the quality of cryopreserved ram spermatozoa. Semen samples (n=16) of four Dalagh rams were extended with different concentrations (10 or 20 μL) of curcumin (Cur) or CurLNN and then cryopreserved using a standard protocol. The findings indicated that sperm cells exposed to curcumin, specifically CurLNN at a concentration of 20 μL, displayed increased levels of total and progressive motility, greater lateral head displacement and linearity values, improved plasma membrane function, and reduced abnormality (P < 0.05). Viability and plasma membrane functionality increased by supplementing cryopreservation medium with Cur20 and CurLNN (P < 0.05). A significant decline (P < 0.05) in malondialdehyde (MDA) and reactive oxygen species (ROS) concentrations was observed by supplementing cryopreservation medium with both curcumin and its nano-sized. There was a significant (P < 0.05) increase in the percentage of spermatozoa with mitochondrial activity when the cryopreservation medium was incorporated with CurLNN. Spermatozoa treated with CurLNN demonstrated higher viability and lower late apoptosis (P < 0.05) compared with other groups. There were no differences among groups in terms of the percentage of necrotic and early apoptotic spermatozoa. In conclusion, the results of this study demonstrated that nano-sized curcumin, particularly at a 20 µL concentration, had a higher antioxidant potential, leading to improved sperm functional quality by mitigating oxidative damage during cryopreservation.

Effect of cooling rates and equilibration times on post-thaw sperm quality of Kail rams

Context The conflicting findings regarding the impact of equilibration time on post-thawed sperm quality underscore the need for further research to evaluate the impact of equilibration time and cooling rate on post-thaw sperm quality of ram semen. Aims The current study aimed to assess the combined impact of cooling rates and pre-freezing equilibration times on post-thaw sperm quality in Kail ram semen (n = 5). Methods Semen collection was performed using an artificial vagina at 42°C. The pooled semen was divided into equal aliquots and subjected to either slow cooling (SC, −0.27°C/min) or moderate cooling (MC, −0.36°C/min) rates, transitioning from 37°C to 4°C. Equilibration times of 0, 4, 8, and 12 h were employed before freezing. Key results Semen samples undergoing the SC rate and equilibrated for 4 h exhibited higher (P &lt; 0.05) percentages of progressive motile (PM), rapid progressive (RP), and medium progressive (MP) sperm compared with the MC rate. However, total motility remained unaffected by the cooling rate (P &lt; 0.05). Semen equilibrated for 4 h demonstrated higher (P &lt; 0.05) percentages of PM and RP sperm, as well as improved kinematics (curvilinear velocity, average path velocity, and straight-line velocity) compared with other equilibration times. Nevertheless, equilibration time had no (P &gt; 0.05) impact on the amplitude of the lateral head displacement for semen samples subjected to the MC rate. Notably, the cooling rate did not affect post-thaw sperm kinematics, plasma membrane integrity, or live-sperm percentage (P &gt; 0.05). Semen samples equilibrated for 4, 8, and 12 h exhibited a higher (P &lt; 0.05) percentage of sperm with intact plasma membrane and viability than did those equilibrated for 0 h. Conclusions In conclusion, slow cooling rate and a 4 h equilibration period were shown to be optimal for preserving post-thaw sperm quality in Kail rams. Implications The findings highlighted the combined effect of cooling rate and equilibration time on post-thaw sperm quality for optimising sperm cryopreservation protocols in the context of ram semen.

Effect of Mitoquinone on sperm quality of cryopreserved stallion semen

This study aimed to investigate the effect of mitochondria-targeted antioxidants (Mitoquinone, MitoQ) on the quality of frozen-thawed stallion semen. Semen samples collected from three fertile stallions aged 10 - 13 years, were filtered, centrifuged in a skimmed milk-based extender, and diluted to a final concentration of 50 × 106 sperm/mL in freezing medium. Diluted semen was divided into five experimental groups supplemented with MitoQ at concentrations of 0 (control), 25, 50, 100, and 200 nM and then subjected to freezing after cooling and equilibration. After thawing, semen was evaluated for motility and kinetics at different time points. Sperm viability, plasma membrane, acrosome, DNA integrity, mitochondrial membrane potential, apoptosis, and intracellular reactive oxygen species (ROS) concentrations were evaluated. The results revealed that MitoQ at concentrations of 25, 50, and 100 nM improved (P< 0.01) the total sperm motility after 30 minutes of incubation. In addition, 25 nM MitoQ improved the sperm amplitude of lateral head displacement values (P< 0.01) after 30 minutes of incubation. Conversely, negative effects on sperm motility, kinetics, and viability were observed with the highest tested concentration of MitoQ (200 nM). The various concentrations of MitoQ did not affect the plasma membrane, acrosome, and DNA integrity, or the mitochondrial membrane potential and intracellular ROS concentrations. In conclusion, supplementation of MitoQ during cryopreservation, had a mild positive effect on sperm motility and kinetics especially at a concentration of 25 nM, while the highest concentration (200nM) has a detrimental effect on motility and viability parameters of frozen-thawed stallion sperm.

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.

P-065 HyperSperm, a new sperm preparation method for IVF/ICSI improves sperm capacitation while maintaining a high safety profile

Abstract Study question Does HyperSperm improve sperm capacitation without affecting sperm quality? Summary answer HyperSperm enhances sperm hyperactivated motility, a hallmark of capacitation, without compromising sperm viability, acrosome integrity, or DNA fragmentation. What is known already HyperSperm is a novel semen preparation method for IVF/ICSI based on mimicking effective capacitation that occurs in the female tract. HyperSperm, which consists of 3 media used sequentially to treat sperm before insemination, gave rise to more high-quality blastocysts compared to current standard of care in a pilot split oocyte study in 10 couples. Study design, size, duration This prospective in vitro study was conducted between July 2023 and January 2024 in an academic department. A consecutive cohort of 15 normozoospermic individuals were recruited and included. Each participant provided a semen sample by masturbation after written informed consent was obtained on the day of the collection. Participants/materials, setting, methods Semen samples from normozoospermic individuals (n = 15) were divided into two halves. After density gradient centrifugation, Control half was incubated in modified HTF (Human Tubal Fluid) for 2-4 hours, while the other half underwent HyperSperm treatment. The following parameters were assessed: sperm viability (eosin exclusion); motility and kinematic parameters (CASA); acrosome integrity (fluorescein-labeled Pisum sativum lectin staining); and DNA fragmentation by TUNEL. Differences between groups were evaluated by paired T-test. Significant differences were considered when p &amp;lt; 0.05. Main results and the role of chance Following treatments, sperm viability (90±3% Control vs. 91±3% HyperSperm) and motility (87±2% Control vs. 89±3% HyperSperm) were similar between groups (p &amp;gt; 0.05). Notably, HyperSperm induced a significant hyperactivation increase (9±2% Control vs. 22±3% HyperSperm), along with associated kinematic parameters (i.e., curvilinear velocity and lateral head displacement) (p &amp;lt; 0.05). Furthermore, HyperSperm treatment did not promote motility decrease over time. Finally, there were no differences in the percentage of sperm with intact acrosome (91±2% Control vs. 90±2% HyperSperm) and DNA fragmentation (3±1% Control vs. 3±1% HyperSperm) between groups (p &amp;gt; 0.05). Altogether, HyperSperm has shown to significantly improve sperm capacitation parameters while maintaining a high safety profile in the treated sample. Limitations, reasons for caution The study is limited to normozoospermic samples, and further research should validate these findings across broader male populations undergoing IVF/ICSI. Wider implications of the findings HyperSperm has demonstrated an increase in the production of high-quality blastocysts via IVF, that would reduce barriers to treatment access through higher cumulative success rates and lower treatment repetitions. The observed increase in hyperactivation combined with the safety profile of HyperSperm supports its potential for future adoption in reproductive clinics. Trial registration number not applicable

O-019 Outcomes and correlations between traditional Intracytoplasmic Sperm Injection (ICSI) method and a new AI-enhanced system

Abstract Study question In the context of ICSI, how does the Sperm Tracer Deep Neural Network analysis compare to the embryologist’s sperm selection technique? Summary answer Sperm Tracer AI system significantly improves fertilization rates, offering a standardized, objective approach to sperm selection, but nuanced correlations highlight complexities in sperm parameters. What is known already Traditional sperm selection during ICSI relies on subjective, manual methods, introducing variability and affecting pregnancy success. In response, AI applications, aim to enhance objectivity, potentially improving pregnancy rates. Study design, size, duration Between September and December 2023, the study examined 26 fresh ICSI cycles in a total of 268 sibling oocytes. Men aged 18 years and above with 34.4 mean women’s age. The Sperm Tracer group underwent single embryo culture, while the Embryologists' group underwent group embryo culture. The study analyzes statistical differences in fertilization rates, day 3 and day 5 embryo development. The study duration aligns with the analyzed cycles. Participants/materials, setting, methods The study compared real-time AI-assisted sperm evaluation with the Sperm Tracer system to the traditional ICSI method by embryologists. Correlation analysis was performed for the following kinematic parameters: Amplitude of Lateral Head displacement (ALH), Dance (DNC), Linearity (LIN), Straightness (STR), Wobble (WOB), Curvilinear Velocity (VCL), Linear Velocity (VSL) and Average Path Velocity (VAP). Statistical analysis involved Odds Ratios (OR) with 95% confidence intervals, Mann-Whitney U tests and correlation analyses by Pearson’s coefficient. Main results and the role of chance The Sperm Tracer system exhibited a noteworthy enhancement in fertilization rates (OR: 0.46, 95% Confidence Intervals (CI) [0.23, 0.89], p = 0.0224) but no significant differences in cleavage stages, blastocyst formation, or the production of good-quality embryos compared to conventional methods. For correlation analysis used two groups of sperm population. Group A comprises spermatozoa subjected to ICSI with the Sperm Tracer system, resulting in unsuccessful oocyte fertilization and Group B consists of spermatozoa that achieved successful fertilization. In Group A, correlation analysis identified significant negative associations between DNC and linear parameters (LIN, STR, WOB) (p &amp;lt; 0.001), highlighting the clinical relevance in sperm motility. Group B displayed a distinct negative correlation between DNC and LIN (p = 0.03941), indicating potential effects on sperm linear velocity. LIN in Group A exhibited strong negative correlations with WOB (p = 0.0001), moderate negative correlations with STR (p = 0.0028), and VCL (p = 0.0773). Moreover, ALH analysis in Group A unveiled robust negative correlations with LIN, STR, and WOB, suggesting clinical significance in sperm motility. These intricate findings underscore the Sperm Tracer system's primary advantage in optimizing fertilization rates, offering valuable insights for refining sperm selection strategies in assisted reproduction. Limitations, reasons for caution The study faces limitations due to a small sample, impacting generalizability. Divergence in culture embryo strategies introduces bias, necessitating validation in larger studies. The system includes morphology analysis, but no data analysis has been conducted. Exploring the correlation between morphology, considering embryo culture variations, and motility could provide valuable insights. Wider implications of the findings This study highlights AI applications like Sperm Tracer, showcasing their potential to improve assisted reproduction outcomes. Recognizing AI's impact on IVF success is vital for adoption. Integrating AI into clinic protocols may boost IVF efficiency, requiring training for embryologists to ensure effective utilization. Trial registration number Not applicable

P-052 Machine learning-based sperm motility grading model

Abstract Study question How to construct a machine learning-based assessment system for sperm motility and pick up the superior sperm with high motility accurately and efficiently? Summary answer Machine learning algorithms can be trained to build a grading model for the classification and grading of sperm motility. What is known already There are many sperm quality parameters for male fertility evaluation such as sperm concentration, viability, morphology, pH, and color of the semen. But, many studies agree that motility is the main parameter for sperm quality evaluation. Currently, there are mainly two methods for sperm motility determination. The traditional way relies on manual observation under a microscope, but this is subjective and time-consuming. Though there are some tracking errors in computer assisted sperm analysis (CASA) system, which help to acquire more motion parameters such as curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP). Study design, size, duration 3 000 sperm samples from clinical laboratory were divided to asthenospermia (AS) group and healthy control (HC) group based on their progressive motility. Sperm motility parameters were measured by CASA system. Participants/materials, setting, methods Nine variables of sperm motility including VCL, VSL, VAP, amplitude of lateral head displacement (ALH), mean angular displacement (MAD), linearity (LIN), straightness (STR), beat-cross frequency (BCF), fractal dimension (D) were collected for ML model training. The R software was used to build several classifiers on the samples investigated, including Random Forest, Adaptive Boosting (Ad Boost), Gradient Boosting, Support Vector Machine map (SVM), kNN (k-Nearest Neighbours), and Naïve Bayes. Main results and the role of chance Motility parameters differ between the two groups with a significant difference in VAP, VSL, VCL, and ALH. In order to classify the heterogeneous dataset in an unbiased manner, we performed a cross-validation procedure of training learning algorithms using six folds (equal parts) of the dataset, each time using a separate fold as the test dataset and the remaining folds as the training dataset. We initially examined whether the classifier could be reliably predicted using a subset of the sperm characterization’s 9 parameters (VCL, VSL, VAP, ALH, MAD, LIN, STR, BCF, D). We purposefully excluded additional characteristics that may be linearly related to increasing motility. We found four learning algorithms to have high accuracy, recall, precision, and F1 score. The support Vector Machine map performed best in classifying the sperm with high motility. Limitations, reasons for caution There may be some bias in data for sperm motility parameters. Specifically, different data collected from different laboratories or acquired from different equipment which may lead to a degradation in the performance of the machine learning model. Wider implications of the findings The analysis of large amounts of case data by machine learning algorithms can help clinician better understand the sperm motility characteristics of different patients so that they can develop a personalized treatment plan for each patient. Trial registration number not applicable

Cryopreservation alters buffalo sperm kinematics and mitochondrial parameters, acrosome and intra-cellular calcium status

BACKGROUND: Little is known about the effects of different seasons on the cryopreservation success of buffalo sperm in terms of kinematics and sperm functional parameters. OBJECTIVE: To study the effect of three seasons (winter, comfort and summer) and cryopreservation on sperm kinematics and functional properties in buffalo bulls. MATERIALS AND METHODS: Semen ejaculates (n = 90) collected during three seasons i.e. winter (n = 30), comfort (n = 30), summer (n = 30) were evaluated for sperm kinematics and functional properties. RESULTS: Sperm kinematics with respect to total (TM), progressive (PM) and rapid motility (RM) was higher (P&lt;0.05) in fresh sperm compared to sperm that had been frozen-thawed. Similarly, all kinematic parameters [viz. average path velocity (VAP), straight linear velocity (VSL), curvilinear velocity (VCL), beats cross frequency (BCF), lateral head displacement (ALH), linearity (LIN) and straightness (STR)] were higher (P&lt;0.01) at the fresh stage. With respect to season, frozen-thawed semen TM (57.67 ± 115%), PM (50.2 ± 1.15%) and RM (51.6 ± 1.19%) were higher (P&lt;0.01) when using sperm collected during winter. The stage of cryopreservation (i.e., equilibration and freeze-thawing) also showed significant effects (P&lt;0.01) on mitochondrial superoxide positive status (MSPS), mitochondrial membrane potential (MMP), acrosome status and intra-cellular calcium status. CONCLUSION: The season of sperm collection and cryopreservation have significant effects on buffalo bull sperm kinematics and functional properties.

Freezing Hu ram semen: influence of different penetrating cryoprotectants and egg yolk level on the post-thaw quality of sperm.

The Hu sheep is a renowned breed known for its high reproductive rate. It is in estrus all year round, and its breeding population is gradually expanding. However, the current techniques for cryopreserving semen have limited effectiveness, which hinders the continuous development of this species. The purpose of this study is to explore the effects of different penetrating cryoprotectants (CPAs) and egg yolk (EY) concentrations on the cryopreservation of Hu ram semen to determine the most effective combination. In this study, the effects of glycerol (GLY), ethylene glycol (EG), dimethylacetamide, dimethyl sulfoxide, different proportions of GLY and EG, EY on sperm quality after thawing were investigated by detecting sperm total motility (TM), progressive motility (PM), straight-line velocity, curvilinear velocity, average path velocity, amplitude of lateral head displacement, wobble movement coefficient, average motion degree, functional integrity (plasma membrane integrity, acrosome integrity) and reactive oxygen species (ROS) level. When GLY and EG were added together, compared to other concentration groups, 6% GLY significantly (p<0.05) increased TM, PM, plasma membrane integrity, and acrosome integrity of thawed sperm. Additionally, it significantly (p<0.05) decreased the ROS level of sperm. In this study, the TM, PM, and membrane integrity of the 6% EG were significantly (p<0.05) higher than those of the control, 1% GLY+5% EG and 6% GLY+6% EG groups. Compared to other concentration groups, 20% EY significantly (p<0.05) improved the TM, PM, and plasma membrane integrity of thawed sperm. However, the integrity of the acrosome increased with the higher concentration of EY. In conclusion, the post-thawed Hu ram semen diluted with a diluent containing 6% GLY and 20% EY exhibited higher quality compared to the other groups.

The effect of age and bracing on vehicle occupant responses during sled-simulated evasive swerving

Objective Bracing can reduce adult occupants’ out-of-position postures, but it is unclear if this finding can be extended to child occupants. We investigated the effect of bracing, and age on the motion of vehicle occupants of different ages during sled-simulated pre-crash maneuvers. Methods Forty seatbelt restrained subjects (9–40 y.o.) experienced sled simulated evasive swerving maneuvers during a brace condition, where subjects actively brace before the maneuver onset, and an unbraced condition. A 3D motion capture system, electromyography (EMG), and seatbelt load cells captured head and trunk kinematics, muscle activation, and seat belt reaction forces, respectively. The effects of age and bracing on peak lateral head and trunk displacement were examined with Mixed Effects Model (p ≤ 0.05). Results In the braced condition, all subjects had reduced normalized peak head (0.09 ± 0.05) and trunk (0.11 ± 0.05) displacements compared to the unbraced condition (Head: 0.14 ± 0.06, Trunk: 0.17 ± 0.07) (p < 0.05). For the into-the-belt direction in the unbraced condition, children showed the smallest peak head and trunk displacement of all age groups (p < 0.01) while teens had the greatest peak head and trunk displacement compared to all other age groups (p < 0.02). Similar trends were found in the out-of-the-belt direction in the unbraced condition (p < 0.01). In the unbraced condition, all occupants showed greater shoulder seat belt reaction loads than the braced condition (unbraced 1.6–2.7 N/kg, braced 1.0–1.4 N/kg). Children also showed greater neck muscle activation (50–64% MVIC) compared to other muscles and older occupants regardless of the bracing condition or direction of motion. Conclusion Bracing reduced out-of-position postures across all ages. In the unbraced condition, children move less than teens and adults due to greater activation in the neck muscles. In the unbraced condition, teens showed the greatest motion, potentially due to their developing neuromotor control transitioning to a more mature stage. Across all age groups in the unbraced condition, occupants relied more heavily on the seat belt to maintain their position rather than their muscles to brace, suggesting the importance of good seatbelt-torso interaction in unbraced occupants.

The effect of reclined seat-back angles on the LODC with and without a belt-positioning booster during far-side lateral-oblique impacts

Objective In frontal crashes belt-positioning boosters (BPB) may prevent submarining when the seatback is reclined. It is unclear if the BPB can also mitigate injuries in far-side lateral-oblique crashes in reclined conditions, where current restraints are less effective in reducing lateral excursion. This study aimed to understand reclined child injury risk during lateral-oblique impacts, with and without a booster seat, by using the Large Omni-Directional Child (LODC) test device. Methods The LODC was tested in nine lateral-oblique impact (80° from frontal) sled tests (target 31.3 km/h, duration 58 ms maximum, peak acceleration 21 g). Three seatback angles (25°, 45°, and 60°) with and without the BPB were compared on a production passenger seat with an integrated seatbelt. The LODC moved toward the buckle side during the far-side lateral-oblique impact. Abdominal pressure (left and right), seatbelt loads, anterior superioriliac spine (ASIS) forces, and pelvis lateral rotation were examined. The LODC head and knee excursions were extracted from a 3D-motion capture system. Results In the reclined noBPB condition, peak abdominal pressures on the buckle side reached up to 186 kPa and the ASIS forces demonstrated an early (∼ 50 ms) peak and a subsequent drop in the reclined noBPB conditions, suggesting that the belt slid into the abdomen. With the BPB, peak pelvis lateral rotation was greater than in the noBPB conditions but decreased with increasing reclined seatback angles (BPB: 31° to 36.5° vs no-BPB: −5° to −4.6°). Peak lap belt forces were greater in the reclined noBPB conditions (4.8-4.9 kN) compared to all conditions with the BPB (2.9-3.2 kN). The greatest lateral head excursion was observed in the BPB 25° condition (895 mm) and the lowest in the noBPB 45° condition (748 mm). Conclusions The BPB may prevent the lap belt intrusion into the abdomen in reclined configurations in far side lateral-oblique impacts. The greater pelvis lateral rotation and head displacement with the BPB may lead to contact with other rear-seated occupants and/or vehicle structures, although motion decreased with the BPB as the reclined seatback angle increased. This suggests that reclined seats may help reduce BPB-seated children’s lateral excursion in far-side lateral-oblique impacts.

Effects of Different Diluents and Freezing Methods on Cryopreservation of Hu Ram Semen.

The purpose of this study was to investigate the effects of different diluents and freezing methods on the quality of thawed sperm after cryopreservation and find an inexpensive and practical method for freezing Hu ram semen for use in inseminations under farm conditions. Ejaculates were collected from five Hu rams. In experiment I, ejaculates were diluted with eight different freezing diluents (basic diluents A, B, C, D, E, F, G, and H). After dilution and cooling, the samples were loaded into 0.25 mL straws and frozen using the liquid nitrogen fumigation method. In experiment II, diluent C was used as the basic diluent and the semen was frozen using liquid nitrogen fumigation and two program-controlled cooling methods. For analysis, frozen samples were evaluated in terms of motility parameters (total motility (TM), progressive motility (PM)), biokinetic characteristics (straight-line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), wobble movement coefficient (WOB), average motion degree (MAD)), reactive oxygen species (ROS) level, and membrane and acrosome integrity. In experiment I, diluent C had higher TM, PM, and acrosome and membrane integrity and lower ROS compared to other extenders (p < 0.05) except diluent A. Diluent C exhibited higher (p < 0.05) VCL, VAP, ALH, WOB, and MAD compared to diluents B, D, E, and F. In experiment II, TM and all biokinetic characteristics did not show significant differences (p > 0.05) amongst the three freezing methods. Liquid nitrogen fumigation resulted in higher (p < 0.05) PM, membrane integrity, acrosome integrity, and lower ROS level compared to the program. In conclusion, the thawed semen diluted with diluent C had higher quality compared to other diluents. The liquid nitrogen fumigation demonstrated superior semen cryopreservation effects compared to the program-controlled cooling method using diluent C.

Effect of bicarbonate and polyvinyl alcohol on in vitro capacitation and fertilization ability of cryopreserved equine spermatozoa.

Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumin lactate pyruvate medium (non-capacitating; NC) or Tyrode's albumin lactate pyruvate supplemented with bicarbonate, bicarbonate and polyvinyl-alcohol, bicarbonate and bovine serum albumin, polyvinyl-alcohol and bovine serum albumin alone. Protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels, sperm motility, and acrosome reaction percentages were evaluated. After determining the best condition media (capacitating; CAP), heterologous intracytoplasmic spermatozoa injection on pig oocytes was performed and the phospholipase C zeta sperm localization pattern was evaluated. Incubation of frozen-thawed equine spermatozoa with bicarbonate and polyvinyl-alcohol in atmospheric air for 45min induced an increase in protein kinase A-phosphorylated substrates and tyrosine phosphorylation levels compared to NC condition. Sperm incubation in bicarbonate and polyvinyl-alcohol medium showed an increase in total motility and progressive motility with respect to NC (p ≤ 0.05). Interestingly, three parameters associated with sperm hyperactivation were modulated under bicarbonate and polyvinyl-alcohol conditions. The kinematic parameters curvilinear velocity and amplitude of lateral head displacement significantly increased, while straightness significantly diminished (curvilinear velocity: bicarbonate and polyvinyl-alcohol=120.9±2.9 vs. NC=76.91±6.9µm/s) (amplitude of lateral head displacement: bicarbonate and polyvinyl-alcohol=1.15±0.02 vs. NC=0.77±0.03µm) (straightness: bicarbonate and polyvinyl-alcohol=0.76±0.01 vs. NC=0.87±0.02) (p ≤ 0.05). Moreover, the spontaneous acrosome reaction significantly increased in spermatozoa incubated in this condition. Finally, bicarbonate and polyvinyl-alcohol medium was established as CAP medium. Although no differences were found in phospholipase C zeta localization pattern in spermatozoa incubated under CAP, equine spermatozoa pre-incubated in CAP condition for 45min showed higher fertilization rates when injected into matured pig oocytes (NC: 47.6% vs. CAP 76.5%; p ≤ 0.05). These findings underscore the importance of bicarbonate and polyvinyl-alcohol in supporting critical events associated with in vitro sperm capacitation in the horse, resulting in higher oocyte activation percentages following heterologous intracytoplasmic spermatozoa injection. This protocol could have an impact on reproductive efficiency in the equine breeding industry.

Quercetin in semen extender improves frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats.

This study investigated the antioxidant effect of quercetin-treated semen on frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats. In total, 32 ejaculates from four fertile bucks were diluted in Tris-based egg yolk extender with varying levels of quercetin (0, 1, 5, 10, and 15 μM). Qualified semen samples were pooled and frozen in French straws. The results revealed that the addition of quercetin in the semen extender increased (p < 0.05) frozen-thawed sperm total motility (TM), progressive motility (PM), rapid velocity (RV), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), and amplitude of lateral head (ALH) displacement in contrast to the control group. Quercetin supplementation had no effect on beat cross frequency (BCF), straightness (STR), and linearity (LIN) (p > 0.05). Quercetin showed significantly higher (p < 0.05) plasma membrane and acrosome integrity and viability (p < 0.05) of spermatozoa in contrast to the control group. Quercetin in the semen extender significantly increased (p < 0.05) superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and total antioxidant capacity (TAC) levels while reduced (p < 0.05) the contents of total oxidant status (TOS) and malondialdehyde (MDA), which were in contrast to the control group. Ultrasound results revealed that 24 out of 30 (80%) goats were found pregnant when semen was treated with 5 μM quercetin while the control group showed 18 out of 30 (60%) animals were pregnant. Thus, the study concluded that 5 μM quercetin-treated semen was found to be efficient, showed increased antioxidant status, and reduced oxidant production, leading to improved spermatozoa quality and in-vivo fertility in goats.

Arsenic exposure causes decline in sperm motility accompanied by energy metabolism disorders in mouse testes

Arsenic (As) is a notorious environmental toxicant widely present in various natural environments. As exposure has been correlated with the decline in sperm motility. Yet, the mechanism has not been fully elucidated. Adult male C57 mice were given 0, 1, or 15 mg/L NaAsO2 for 10 weeks. The mature seminiferous tubules and sperm count were decreased in As-exposed mice. Sperm motility and several sperm motility parameters, including average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), beat-cross frequency (BCF), linearity (LIN), straightness (STR), and amplitude of lateral head displacement (ALH), were declined in As-exposed mice. RNA sequencing and transcriptomics analyses revealed that differentially expressed genes (DEGs) were mainly enriched in metabolic pathways. Untargeted metabolomics analyses indicated that energy metabolism was disrupted in As-exposed mouse testes. Gene set enrichment analysis showed that glycolysis and oxidative phosphorylation were disturbed in As-exposed mouse testes. As-induced disruption of testicular glucose metabolism and oxidative phosphorylation was further validated by RT-PCR and Western blotting. In conclusion, As exposure causes decline in sperm motility accompanied by energy metabolism disorders in mouse testes.