Beat Cross Frequency Research Articles

Biogenic and risk elements in wild boar testes and relation to spermatozoa motility

Environmental pollution results in serious health hazards to animals, reflected in biogenic and risk element concentrations in animal tissues. The objective of this study was to examinate concentration of selected elements in testes, and epididymal spermatozoa motility of wild boars. Wild boars were hunted in region Žuhračka - Levice, Slovak Republic. Testes were removed postmortem, spermatozoa were collected from cauda epididymis and assessed by CASA system. Elements concentrations were measured by ICP and by CV-AAS. Spermatozoa motility was 44.29% and progressive motility 18.47%. Concentration of elements in testes was in following order: K > Na > Mg > Ca > Fe > Zn > Al > Cu > Se > Mn > As > Cr > Pb > Mo > Sr > Ni > Ba > Cd > Li > Hg. The most notable correlations indicate association between Se and total spermatozoa motility, as well as with progressive motility, furthermore between As and velocity curved line and beat cross frequency. A high positive significant correlation was found between mercury and beat cross frequency. The data may serve as a fine control indicator to detect potentially toxic elements accumulated from polluted environment that can affect reproduction of wild animals.

Combination of Exogenous Spermidine and Phosphocreatine Efficiently Improved the Quality and Antioxidant Capacity of Cryopreserved Boar Sperm and Reduced Apoptosis-Like Changes.

The low resistance of boar sperm to cryopreservation dictates that addition antioxidants and energetic substances to the diluent to improve sperm quality is necessary. This study evaluated the effect of spermidine and phosphocreatine in combination on the quality, antioxidant capacity, and antiapoptotic-like changes capacity of cryopreserved boar sperm based on previous reports. The results showed that the combined application of spermidine and phosphocreatine significantly enhanced the motility, average path velocity, straight-line velocity, curvilinear velocity, beat cross frequency, acrosome integrity, plasma membrane integrity, mitochondrial activity, and DNA integrity compared with the control group (p < 0.05). In addition, the combined application of spermidine and phosphocreatine significantly enhanced the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity, and catalase activity while significantly decreasing malondialdehyde content and hydrogen peroxide content (p < 0.05). Western Blot analysis further showed that spermidine and phosphocreatine significantly decreased the expression of CASP3 and BAX and significantly enhanced the expression of BCL2 (p < 0.05); therefore, the combination of spermidine and phosphocreatine has potentially positive implications for improving the quality of cryopreserved boar sperm.

Effect of Thawing Procedure and Thermo-Resistance Test on Sperm Motility and Kinematics Patterns in Two Bovine Breeds.

This investigation aimed to analyze the effect that thawing time and temperature in combination with a termo-resistance test had on straws from dairy bulls used for artificial insemination (AI) on semen motility and kinematic variables measured with CASA systems. Eight animals of Holstein and Jersey breeds were used, and nine frozen-thawed semen doses per animal were analyzed for each breed. Three temperatures (35, 37, and 40 °C) and three thawing times (35, 40, and 45 s) were evaluated using a factorial design. Motility and kinematic patterns were analyzed using CASA-mot (Computer-Assisted Semen Analysis of motility) technology at different post-thawing times (0.5, 1, and 2 h). Sperm motility in Jersey bulls was higher (p < 0.05) than in Holstein ones (64.52 ± 1.45% and 53.10 ± 1.40%, respectively). The same effect was seen with progressive motility among the two breeds (Jersey: 45.29 ± 1.00%; Holstein: 36.30 ± 0.98%, p < 0.05). The Jersey breed presented higher values (p < 0.05) of curvilinear velocity (VCL), rectilinear velocity (VSL), average velocity (VAP), linearity on forward progression (LIN), and wobble (WOB). The Holstein breed showed a lower mean value (p < 0.05) of the beat-cross frequency (BCF) compared to the Jersey breed, thus suggesting an effect on VCL and VAP. During the post-thaw period, a gradual increase in VCL was observed at 2 h. VSL and VAP showed a decrease (p < 0.05) as the post-thaw period was prolonged. The study showed differences in sperm quality between Holstein and Jersey breeds, influenced by cryopreservation, thawing, and post-thawing incubation. Thawing at 37 °C for 30 s was considered optimal in relation to sperm motility. In addition, a decrease in sperm quality was observed as post-thawing time increased.

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.

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.

Spermiogram, Kinetics, Flow Cytometric Characteristics and DNA Damage Degree in Boar Ejaculates: Summarization and Clustering.

Boar semen analysis includes sperm motility, concentration, morphology and other more complex analyses such as membrane integrity, DNA damage and seminal plasma components. This study aims to summarize these numerous data by linear combinations of them, to classify ejaculates in several categories (clusters) and to investigate the potential differences among clusters on fertility and prolificacy. Young Pietrain boars (23 ± 3.6 months) were investigated: ten boars from the Nucléus genetic line (group 1: 90 ejaculates weekly) and five boars from the Batallé genetic line (group 2: 30 ejaculates weekly). Computer-assisted semen analysis (CASA) examined motility. Sperm viability, acrosome reaction, early apoptosis, mitochondrial activity and DNA damage were studied by flow cytometry analysis. SPSS v.26 software was used to perform principal component analysis (PCA) and clustering. Three principal components (PC1: speed; PC2: linear path; PC3: DNA damage) were detected and four clusters identified in both groups. Clusters also differed significantly in several variables not included in these PCs (group 1: beat cross frequency and poly (ADP-ribose) polymerase; group 2: cathepsin B, abnormal forms, mitochondrial activity and high DNA stainability). PCA and clustering achieved adequate description of these ejaculates, but no differences among clusters were found for fertility or prolificacy, probably because the minimum sperm requirements had been met.

The efficacy and functional consequences of interactions between human spermatozoa and seminal fluid extracellular vesicles.

Seminal fluid extracellular vesicles (SFEVs) have previously been shown to interact with spermatozoa and influence their fertilisation capacity. Here, we sought to extend these studies by exploring the functional consequences of SFEV interactions with human spermatozoa. SFEVs were isolated from the seminal fluid of normozoospermic donors prior to assessing the kinetics of sperm-SFEV binding in vitro, as well as the effects of these interactions on sperm capacitation, acrosomal exocytosis, and motility profile. Biotin-labelled SFEV proteins were transferred primarily to the flagellum of spermatozoa within minutes of co-incubation, although additional foci of SFEV biotinylated proteins also labelled the mid-piece and head domain. Functional analyses of high-quality spermatozoa collected following liquefaction revealed that SFEVs did not influence sperm motility during incubation at pH 5, yet SFEVs induced subtle increases in total and progressive motility in sperm incubated with SFEVs at pH 7. Additional investigation of sperm motility kinematic parameters revealed that SFEVs significantly decreased beat cross frequency and increased distance straight line, linearity, straightness, straight line velocity, and wobble. SFEVs did not influence sperm capacitation status or the ability of sperm to undergo acrosomal exocytosis. Functional assessment of both high- and low-quality spermatozoa collected prior to liquefaction showed limited SFEV influence, with these vesicles inducing only subtle decreases in beat cross frequency in spermatozoa of both groups. These findings raise the prospect that, aside from subtle effects on sperm motility, the encapsulated SFEV cargo may be destined for physiological targets other than the male germline, notably the female reproductive tract. A male's influence over the biological processes of pregnancy extends beyond the provision of sperm. Molecular signals present in the ejaculate can influence the likelihood of pregnancy and healthy pregnancy progression, but the identity and function of these signals remain unclear. In this study, we wanted to understand if nano-sized particles present in the male ejaculate, called seminal fluid extracellular vesicles, can assist sperm in traversing the female reproductive tract to access the egg. To explore this, we isolated seminal fluid extracellular vesicles from human semen and incubated them with sperm. Our data showed that seminal fluid extracellular vesicles act to transfer molecular information to sperm, but this resulted in only subtle changes to the movement of sperm.

L-carnitine supplementation in conventional slow and ultra-rapid freezing media improves motility, membrane integrity, and fertilizing ability of dog epididymal sperm

This study aimed to assess the impact of L-carnitine (LC) supplementation in conventional-slow (CS) and ultra-rapid (UR) freezing media on post-thaw quality and fertilizing ability of dog epididymal spermatozoa. Sperm samples were collected from 60 epididymides obtained from 30 adult orchiectomized dogs via retrograde flushing. Twenty pooled sperm samples were then created (3 epididymal samples/pool). Four treatments were established according to the freezing method (CS and UR) and LC supplementation (5 and 0 mM [control, Co]): CS-LC5, CS-Co, UR-LC5, and UR-Co. The CS freezing involved exposing 0.25 mL straw to liquid nitrogen vapors (LN2), while UR freezing submerged 30-µL drops of sperm samples directly into LN2. Sperm kinematics, membrane integrity, and fertilizing ability (by heterologous in vitro fertilization using bovine oocytes) were evaluated for all treatments. Post-thaw results revealed that the CS freezing treatments resulted in significantly higher values (P < 0.05) of curvilinear and average-path velocities, and beat-cross frequency compared to the UR freezing treatments, regardless of LC supplementation. The CS-LC5 and UR-LC5 treatments cryoprotected the sperm by increasing (P < 0.05) the percentage of ‘live-sperm/intact-acrosome’ compared to their controls treatments CS-Co and UR-Co. Regarding fertilizing ability, the CS-LC5 treatment yielded a higher percentage (P < 0.05) of pronuclei formation compared to both UR treatments. The UR-LC5 treatment, however, obtained greater percentage (P < 0.05) than their control UR-Co. In conclusion, supplementation with L-carnitine in conventional-slow and ultra-rapid freezing improved sperm motility, plasma, and acrosome membranes integrity and fertilizing ability of dog epididymal spermatozoa.

Optimizing the post-thaw quality of cryopreserved masu salmon (Oncorhynchus masou) sperm: Evaluating the effects of antioxidant-supplemented extender

The oxidative damage induced by cryopreservation significantly contributes to the overall decline in the quality of cryopreserved sperm. This underscores the necessity for exploring mitigation measures, such as supplementing the freezing media with antioxidants, aimed at enhancing sperm quality post-thaw. In this study, we determined the effects of adding five antioxidants [Vitamin C, taurine, trolox, Butylhydroxytoluene (BHT), and uric acid] to a glucose–methanol extender at three concentrations—low, intermediate, and high—on the motility, viability, concentration, and fertilization rates of the sperm of masu salmon (Oncorhynchus masou), which is an economically important fish resource in Northeast Asia. Our research reveals various sperm quality parameters of the same species react differently to different antioxidants or even different concentrations of the same antioxidants, which adds another layer of complexity to the selection criteria for an appropriate antioxidant for supplementation beyond the specific specificity of antioxidants. Moreover, the effects of the antioxidants were often nonlinear and paradoxical, highlighting the importance of applying accurate dosage. Compared with the control group (no antioxidant supplementation), only uric acid and taurine supplementation at intermediate concentrations (5 mM) significantly improved the post-thaw sperm viability and percentage of motile sperm. Meanwhile, BHT supplementation generally improved the following kinematic traits of sperm: straight-line velocity (VSL), linearity, average path velocity, and curvilinear velocity (VCL). Similarly, 5-mM taurine supplementation also significantly improved sperm VSL, VCL, and beat cross frequency. In contrast, 10-mM Vitamin C supplementation (high concentration) adversely affected multiple parameters, leading to a decline in fertilization capacity. Based on these findings, the selection criteria for antioxidants should go beyond species specificity and include sperm quality parameters the breeder considers crucial for predicting post-thaw sperm fertility, as well as the ways in which particular antioxidants influence these parameters. We recommend 5-mM taurine supplementation for masu salmon sperm cryopreservation because of its positive effects on sperm viability, concentration, and some kinematic parameters.

Lead and calcium crosstalk tempted acrosome damage and hyperpolarization of spermatozoa: signaling and ultra-structural evidences

BackgroundExposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 μg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h.ResultsLead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria.ConclusionsPb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.Graphical

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

Effect of resveratrol supplementation in conventional slow and ultra-rapid freezing media on the quality and fertility of bull sperm

The study investigated the impact of resveratrol (RES) on bull sperm cryopreservation employing conventional slow (CS) and ultra-rapid (UR) freezing methods on sperm quality and in vitro fertility. Twenty-four ejaculates from four bulls were divided into four groups based on the cryopreservation method and RES addition: CS-RES (n = 80), CS-Co (n = 80), UR-RES (n = 24), and UR-Co (n = 24). The CS freezing involved exposing sperm straws with 5% glycerol to liquid nitrogen (LN2) vapors, while UR freezing submerged sperm drops with 100 mM sucrose directly into LN2. Overall, sperm kinematic parameters and integrity of plasma and acrosome membranes significantly decreased (P < 0.001) after cryopreservation. Post-thaw values of motilities (total [TM] and progressive [PSM]), velocities (curvilinear and straight-line), beat cross frequency (BCF), and sperm with intact plasma membrane/intact acrosome (PI-/PNA-) were higher (P < 0.05) with CS-RES and CS-Co treatments compared to UR-RES and UR-Co treatments. CS-RES treatment resulted in greater percentages (P < 0.05) of TM, PSM, PI-/PNA-, and fertility (blastocyst rate) than their control, CS-Co; while UR-RES showed higher BCF values (P < 0.05) than its control, UR-Co. Additionally, UR-RES treatment exhibited lower oxidative stress percentages than UR-Co (P < 0.05). This study presents the following conclusions: (1) the CS freezing resulted in better cryosurvival of bull sperm than UR freezing; (2) the RES supplementation to CS freezing medium improved sperm motility, membrane integrity, and fertility; and (3) despite low cryosurvival sperm and fertility, the RES addition to ultra-rapid freezing medium reduced oxidative stress.

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.

Gallic acid supplementation partially ameliorates reproductive aging in rooster breeders by improving semen quality, sperm kinetics, hormones, and antioxidant status

Aging leads to decreased fertility in roosters, which is likely due to increased oxidative stress. This study evaluated the antioxidant effects of gallic acid (GA) supplementation on sperm quality and fertility of aged roosters. This study evaluated whether gallic acid (GA) supplementation can mitigate age-related fertility decline. Roosters were randomly assigned to: control, 100 mg/kg GA, or 200 mg/kg GA. Semen parameters, sperm kinetics, hormone levels, fertility rate, and hatchability were assessed. GA increased semen concentration, membrane integrity and viability while decreasing defects versus control (P<0.01). Testosterone was higher in GA groups (P<0.01) without affecting gonadotropins. Furthermore, 200 mg/kg GA optimized motility, velocity, linearity, and beat cross frequency versus control and 100 mg/kg GA (P<0.01). Fertility and hatchability were higher in both GA groups. In conclusion, GA supplementation in aged roosters improves sperm quality, antioxidant status, testosterone, and fertility outcomes, likely by mitigating oxidative stress. The 200 mg/kg dose elicited optimal effects on motion parameters.

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.

L-carnitine enhances the kinematics and protects the sperm membranes of chilled and frozen-thawed Peruvian Paso horse spermatozoa

l-carnitine (LC) transports fatty acids to the mitochondria for energy production, reducing lipid availability for peroxidation through β-oxidation. This research examines the effect of LC supplementation to two skimmed milk-based extenders on the cryosurvival of chilled (5°C) and frozen-thawed Peruvian Paso horse spermatozoa .An initial experiment determined the optimal LC concentration (0, 1, 5, 10, 25, and 50 mM) when added to INRA-96® and UHT (skimmed milk + 6% egg yolk) extenders, using nine ejaculates from three stallions chilled for up to 96 h. Subsequently, the effect of 25 mM LC supplementation (the optimal concentration) on chilling (INRA-96) and freezing (INRA-Freeze®) extenders was evaluated using eight pooled samples from sixteen ejaculates (2 ejaculates/pool) from four stallions. Results indicated that all LC concentrations produced significantly higher values (P<0.05) for kinematic variables (total [TM] and progressive motilities, curvilinear [VCL] and straight-line [VSL] velocity, and beat-cross frequency [BCF]), and the integrity of plasma/acrosome membranes (IPIA) compared to non-supplemented chilled sperm samples for up to 96 h with both extenders. Moreover, the use of 25 mM LC was more efficient (P<0.05) in preserving the post-chilled values of velocity, BCF, and IPIA for the long term than lower LC concentrations (1–10 mM). Post-thaw values of total motility, the amplitude of lateral head displacement (ALH), and IPIA were significantly improved (P<0.05) when INRA-Freeze extender was supplemented with 25 mM LC. In conclusion, supplementation of l-carnitine to skimmed milk-based extenders enhanced kinematic variables and protected the membrane integrity in chilled and frozen-thawed Peruvian Paso horse spermatozoa.

Effect of quercetin, L-ergothioneine and H89 on sperm motility and kinematic pattern, plasma membrane functionality and in vitro heterologous fertilizing capacity of cryopreserved equine semen

Semen cryopreservation causes extensive chemical and physical damage to sperm structure, which generates premature aging and reduces viability and fertility of spermatozoa. The addition of antioxidants to freezing extenders can reduce the oxidative damage caused by excessive generation of reactive oxygen species (ROS), and the premature aging could be reduced by adding an enzyme inhibitor that prevents an anticipated capacitation. The aim of this study was to evaluate the in vitro effect of quercetin (Q), L-ergothioneine (E) and H89 addition to cryopreserved equine spermatozoa. Six experimental groups were stablished: control, Q, E, H89, H89Q and H89E. The analyzed parameters were sperm motility and kinematic using computer assisted sperm analysis (CASA), plasma membrane functionality with the hypoosmotic swelling test (HOST) and fertilizing capability with in vitro heterologous fertilization. Quercetin reduced curvilinear velocity (VCL) and increased beat-cross frequency (BCF), while its combination with H89 (H89Q) reduced total motility, progressive motility, VCL and hyperactive sperm (HA). Likewise, H89 and its combination with E (H89E) decreased VCL and amplitude of lateral head displacement (ALH). No significant differences were observed among treatments for membrane functionality and fertilizing capacity of sperm. In conclusion H89 in combination with Q and E reduced sperm motility or some kinematic parameters. However, they did not influence plasma membrane functionality and in vitro fertilizing capacity of frozen-thawed equine semen.

Effect of Microfluidic and Density gradient techniques to select Frozen-thawed Epididymal Sperm from Bighorn rams.

Gamete preservation of wild species is a priority in conservation programs. However, field techniques to recover sperm from deceased animals may impair its final quality.The cryopreservation process also has its challenges, and the minimal required quality parameters may differ depending on the intended use of the semen.Often, postmortem harvested and cryopreserved sperm may need to undergo a selection process to improve the final quality of the sample. Certain sperm selection techniques that employ centrifugal forces can increase the risk of damaging the cells. In contrast, microfluidics uses hydrostatic pressure and capillary forces, avoiding the need for costly equipment and minimizing cell damage.This study aims to compare sperm motion parameters, morphology, and acrosome integrity of cryopreserved-thawed epididymal bighorn ram sperm using two commercial selection techniques, VetCount Harvester® device (MFD) and BovipureTM (NidaCon International AB, Möndal, Sweden) density gradient centrifugation (DGC).Sperm parameters associated with quality were, progressive motile sperms (PMS), straight velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), linearity (LIN), straightness (STR), beat cross frequency (BCF), amplitude of lateral head deviation (ALH) and plasma membrane integrity (PMI). Computer Assisted Sperm Analyzer was used for the semen analysis. Eosin/nigrosine and Spermac stains were used to assess morphology and acrosome integrity, respectively. Two veterinarians counted the total number of morphologically normal sperm with complete acrosome.Mean sperm concentration was higher (P < 0.01) before filtration (129.56 ±53.9 ×106/ml) than DCG (68.42 ±29.18 ×106/ml) and MFD (52.94 ±28.76 ×106/ml). PMS was higher (P <0.01) after selection using the DGC (79.70 ± 11.11) and MFD (81.23 ±5.64) compared to the no sperm-selected controls (45.36 ±14.67). Sperm kinetics parameters (VCL, VSL, VAP, BCF, LIN, and STR) were higher in the DGC (P<0.01). However, ALH (P<0.01) was significantly higher in the MFD and Control. Morphological normal sperm, acrosome integrity, and plasma membrane integrity were higher (P<0.01) in samples processed with DGC and MFD.In conclusion, microfluidics and single-layer centrifugation improved motion parameters, viability, and proportion of morphologically normal sperm with intact acrosomes from frozen-thawed sperm harvested from Bighorn epididymis. The VetCount Harvester® microfluidic device was comparable to DGC and could be an alternative option for sperm selection. Further research is needed to explore the effect of selected sperm using this technique on artificial insemination, re-cryopreservation, and fertility outcomes.

Correlation between in vitro sperm kinetic, oxidative stress assessments and field fertility of cryopreserved bull semen

This study assessed kinetic parameters and oxidative stress in bull sperm after post-thaw (PT) or after sperm selection by Percoll™ gradient, and thermo resistance test (SS + TRT) to identify useful indicators of field fertility. For the experiment, commercial doses of frozen semen were obtained from six Aberdeen Angus bulls. Three of the bulls were classified as high fertility and three as low fertility according to the IFert™ index provided by the international breeding company CRV Lagoa. Pooled semen samples were distributed between two treatment groups for analysis: post-thaw (PT) or sperm selection (SS) (Percoll™) and thermal resistance test (SS + TRT). The samples were evaluated using sperm kinetics (CASA) (motility %, progressive motility %, VCL µm/s, VSL µm/s, VAP µm/s, LIN %, STR % and WOB%), production of reactive oxygen species (ROS), lipid peroxidation, superoxide dismutase (SOD) enzyme activity and total antioxidant capacity. Data were analyzed using Two-Way ANOVA, considering the fertility index, the treatment used in the samples as effects, and the interaction between these factors. When a significant effect was observed, the values were compared using the Bonferroni test. A Pearson Correlation analysis was performed between the fertility indices and the sperm parameters analyzed in vitro, to evaluate the relationship between sperm quality and the fecundity rate obtained by the bulls. Sperm kinetic parameters, including total motility, progressive motile, and beat cross-frequency, were higher in low fertility compared to high fertility bulls (P &lt; 0.05). However, curvilinear velocity was greater in high fertility bulls followed by SS + TRT. Straight-line velocity, average path velocity, linearity, and beat cross-frequency beat were higher in high fertility bulls after SS + TRT. Reactive oxygen species was correlated with fertility after SS. In addition, there was a decrease in lipid peroxidation was observed only in high fertility bulls. However, lipid peroxidation and high fertility were correlated after PT and SS + TRT. The combination of in vitro sperm kinetic parameters predicted in vivo fertility more accurately than individual kinetic parameters. The lipid peroxidation of sperm is an important indicator of fertility in bulls. High fertility bulls appeared to be more susceptible to lipid peroxidation, which was only reduced in high fertility bulls, suggesting that their sperm can repair the damage induced by oxidative stress.

Effect of crocin supplementation in the extender on the quality of chilled canine semen

The aim of the study was to evaluate the effects of crocin on canine sperm quality parameters during prolonged storage at 4 °C. Ejaculates from 10 dogs were diluted in a TRIS- egg yolk extender supplemented with 0 (control group), 0.5, 1, and 2 mM crocin and stored at 4 °C. Sperm membrane functional integrity, motility, and kinetics were assessed after 3 h, 24 h, 4 days and 7 days of storage. Based on the results, the more efficient concentration of crocin (0.5 mM) was chosen to evaluate sperm intracellular ROS levels, lipid peroxidation, and DNA fragmentation vs. the control. Semen with the addition of 0.5 mM crocin with respect to the control exhibited: i) increased (P < 0.05) sperm membrane functionality at 4 and 7 days of storage; ii) higher (P < 0.05) average path (VAP), straight-line velocities (VSL), and beat cross frequency (BCF) at 4 d of storage at 4 °C; iii) decreased (P < 0.05) intracellular ROS levels after 3 and 24 h storage. No differences in lipid peroxidation and DNA fragmentation were recorded between the control and C0.5 groups at any time point. Lipid peroxidation did not increase over time, while DNA fragmentation increased (P < 0.05) in both groups after 4 days of storage. The results demonstrated that the enrichment of extender with crocin improves to a certain extent canine semen quality, particularly after 4 days of storage at 4 °C.