Amplitude Of Lateral Head Displacement Research Articles

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.

A combination of biomarkers for predicting stallion sperm fertility.

Equine breeding would benefit greatly from reliable biomarkers of stallion or ejaculate fertility. The aim of the study was to investigate how several in vitro sperm characteristics correlate with fertility after artificial insemination, to explore the potential to build a fertility prediction model for stallions. Cooled insemination doses (3-5 per stallion) were obtained from various studs. Sperm membrane integrity, acrosome integrity, chromatin integrity, mitochondrial membrane potential, and reactive oxygen species production were evaluated by flow cytometry 24-30h after semen collection, and sperm motility was assessed by computer aided sperm analysis. Calcein violet was used to differentiate viable spermatozoa. Per season pregnancy rates for these stallions were available the following year. Positive correlations were found between pregnancy rate and straightness (r = 0.43, p ≤ 0.001), as well as pregnancy rate and the proportion of living hydrogen peroxide positive spermatozoa (r = 0.32, p ≤ 0.05). There were negative correlations between pregnancy rate and amplitude of lateral head displacement (r = -0.26, p ≤ 0.05), and between pregnancy rate and the mean fluorescence of dead superoxide positive spermatozoa (r = -0.46, p < 0.001). Principal component analysis indicated that motility, membrane integrity, DNA fragmentation, and reactive oxygen species production were associated with pregnancy rate. Therefore, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates. However, data from more individuals would be required to construct a model for fertility prediction.

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.

Protein factors of capacitation and decapacitation in canine seminal plasma

The aim of the present study was to determine the proteins in canine seminal plasma with direct effect on specific sperm parameters during fertilisation. The influence of seminal plasma proteins was focused on three processes: capacitation, hyperactivation and decapacitation. Size exclusion chromatographic fractional separation of proteins from seminal plasma based on their molecular weights was performed. In addition, two-dimensional electrophoresis of two seminal protein fractions was done. The effect of protein molecules on the motion and kinetic parameters of the male gametes in in vitro conditions was monitored by computer-assisted sperm analysis. The activity of alkaline phosphatase, creatine kinase and alanine aminotransferase in whole seminal plasma and each chromatography fraction was measured spectrophotometrically using the BA-88A semi-automatic chemistry analyzer. The protein fractions with different molecular weights affected the in vitro motility and kinetic characteristics (curvilinear velocity, straightness, linearity and amplitude of lateral head displacement) of spermatozoa, leading to hyperactivation or initiating decapacitation kinetics changes. The detailed study of the seminal plasma proteome would add fundamental information about the processes associated with physiological changes occurring in spermatozoa before fertilisation. The study of the canine seminal plasma proteome could add relevant information about its effects on the fertilising ability of the male gametes and the changes occurring in them before fertilisation.

Effects of semen quality on pregnancy rate in artificially inseminated dairy cows

The study aimed to evaluate the quality of fresh and frozen-thawed semen of five adult Holstein-Friesian crossbred bulls and the pregnancy rate of cows inseminated with frozen semen of those bulls. The fresh semen of breeding bulls collected for artificial insemination (AI) programme in the field was of good quality with volume (6.7 ± 0.2 ml - 8.9 ± 0.5 ml), concentration (904.2 ± 56.4 million/ml), mass activity (3.3 ± 0.2-3.6 ± 0.2), total motility (77.0 ± 1.1% – 92.1 ± 0.6%), progressive motility (67.0 ± 1.2% – 87.4 ± 0.6%) and semen viability (73.0 ± 0.6% to 85.4 ± 0.7%). The computer assisted sperm analysis (CASA) results showed that diluted pre-freezing semen had good sperm total motility (50.1 ± 3.8 % to 59.0 ± 4.7%), progressive motility (30.0 ± 1.2%-39.0 ± 1.6%), the velocity traits of straight linear velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP) of sperm ranged from 48.0 ± 1.3 - 71.3 ± 0.7 μm/s,118.1 ± 2.8-181.3 ± 10.9 μm/s and 68.4 ± 2.5 to 91.0 ± 2.9 μm/s, respectively. Bull 1 showed significantly higher VSL (71.3 ± 0.7 μm/s), VCL (181.3 ± 10.9 μm/s) and VAP (91.0 ± 2.9 μm/s) compared to others. Viability of frozen-thawed semen was lower in Bull 5 (73.0 ± 1.71%) compared to others. Although in frozen-thawed semen these parameters declined, the semen was sufficiently good to be used in AI in the field. The overall pregnancy rate using frozen semen was 55.6% and the highest pregnancy rate (62%) was in cows that were inseminated with frozen semen of Bull 1, but the differences between bulls was not significant. The pregnancy rate had positive correlation with sperm count, total motility, progressive motility, VCL, VSL, VAP, amplitude of lateral head displacement (ALH), beat cross frequency (BCF), linearity (LIN), straightness (STR), sperm viability. In artificially inseminated cows, the intensity of oestrus of cows, timing of AI, site of semen deposition and season had a significant effect on pregnancy rate. In conclusion, the fresh and frozen-thawed semen of breeding bulls supplied in North-East Bangladesh for AI programme were good quality. Heat detection and insemination timing need to be improved to increase the pregnancy rate.&#x0D; Bangl. vet. 2022. Vol. 39, No. 1-2, 1–15

Discriminant Analysis and Data Mining CHAID Decision Tree as Tools to Evaluate the Buffering Effect of Hydroxytyrosol on Reactive Oxygen Species in Rooster Sperm Cryopreservation.

Sperm cryopreservation is effective in safeguarding genetic biodiversity in avian species. However, during this process, spermatozoa are very susceptible to plasma membrane peroxidation in the presence of high concentrations of reactive oxygen species (ROS). To mitigate this effect, the addition of exogenous antioxidants, such as hydroxytyrosol (3,4-dihydroxyphenylethanol; HT), an antioxidant derived from olive oil, to the cryopreservation sperm diluent, could be useful. To verify this, a cryopreservation diluent was supplemented with different concentrations (0 μg/mL, 50 μg/mL, 100 μg/mL, and 150 μg/mL) of HT. For this, semen was collected in 10 replicates from 16 roosters of the Utrerana avian breed, and a pool was prepared with the optimum quality ejaculates in each replicate. After cryopreservation, spermatozoa were thawed and different in vitro semen quality parameters were evaluated. A discriminant canonical analysis (DCA) was carried out and revealed that total motility (TM; Lambda = 0.301, F = 26,173), hypo-osmotic swelling test (HOST; Lambda = 0.338, F = 22,065), and amplitude of lateral head displacement (ALH, Lambda = 0.442; F = 14,180) were the variables with the highest discriminant power. Finally, a chi-squared automatic interaction detection (CHAID) decision tree (DT) was performed excluding fresh semen samples and ROS was found to be the most valuable variable to discriminate between the different established freezing groups. Samples in the absence of HT or with low concentrations of this antioxidant showed less desirable ROS values in cryopreserved rooster semen. The present study could lead to the improvement of cryopreservation techniques for the genetic material of local poultry breeds and optimize the conservation programs of endangered native avian breeds.

Effects of CatSper Stimulation and Inhibition by Progesterone and NNC on Human Sperm

Calcium ion enters the sperm through a specific calcium channel, CatSper. This voltage-sensitive channel is stimulated by intracellular alkalization and progesterone. This study is aimed at investigating the effects of CatSper inhibition or stimulation on sperm motility, viability, and sperm function regulators such as mitochondrial membrane potential (MMP), ATP, and reactive oxygen species (ROS) production. This study was performed on 30 semen samples of fertile volunteers, referred to Shiraz Fertility Center. The semen samples were diluted to 20 × 10 6 sperm/mL. The samples were divided randomly into control, solvent, progesterone (10 μM), NNC (2 μM), and NNC+progesterone groups. Sperm kinematics, viability, MMP, ATP content, and the amount of ROS production were assessed using VT-SPERM3.1, eosin staining, JC1 flow cytometry, bioluminescence, and chemiluminescence methods, respectively. Sperm viability and total and progressive motility were significantly decreased in the NNC and NNC+progesterone groups. The amplitude of lateral head displacement (ALH) and curvilinear velocity (VCL) was reduced in the NNC-containing groups. These parameters did not change in the progesterone group. ROS production by viable spermatozoa in the NNC and NNC+progesterone groups was significantly higher than the controls. MMP and ATP content did not show any significant difference with controls in none of the experimental groups. NNC inhibits the CatSper and reduces human sperm motility and viability. These harmful NNC effects were not due to their impact on MMP or ATP production but are likely because of intracellular calcium reduction and higher ROS production. Progesterone at 10 μM concentration had no significant effect and may not be a considerable stimulator for CatSper.

P-090 The in vitro motility characteristics of human sperm collected by microfluidic device

Abstract Study question Do the in vitro motility characteristics of sperm collected using a microfluidic device (ZyMot) differ from those of sperm collected by density gradient centrifugation (DGC)? Summary answer ZyMot-collected sperm had longer-lasting motility than those collected by DGC, but the head beat-cross frequency was inferior due to the absence of washing in vitro. What is known already ZyMot (DxNow, Inc., USA) can sort highly motile sperm with low reactive oxygen species (ROS), and DNA damage, without centrifugation. In IUI cycles, although the total number of ZyMot collected sperm was lower than that of DGC, it was reported that the pregnancy rate of ZyMot cycles was higher than that of DGC cycles. Washing sperm with a medium is essential for in vitro capacitation; however, the protocol for microfluidic devices lacks this procedure. The aim of this study was to clarify the motility characteristics of ZyMot-collected sperm. We also examined how washing ZyMot-collected sperm affected motility. Study design, size, duration The specimens were provided by patients who underwent semen analysis between October 2022 and January 2023, with written consent. We used 12 samples with a semen volume of 2.5 ml or more, or a motile sperm concentration of 4 million/ml or more. The same donor samples were divided into three groups (DGC, ZyMot, and ZyMot with wash). Dunnett’s test was performed with the DGC group as the control group. Participants/materials, setting, methods In the DGC group, 2-layer DGC (400G, 20 min) was performed, followed by washing (400G, 5 min). In the ZyMot group, ZyMot was used according to the manufacturer’s protocol. In the ZyMot with wash group, ZyMot-collected sperm was centrifugally washed (300G, 5 min). All samples were cultured at 30 °C until motility analysis. Sperm motility was analyzed using a computer-assisted sperm analysis system (SMAS) after 0, 4, and 24 h of semen preparation. Main results and the role of chance There was no significant difference in the motility rate of the two ZyMot groups compared to that of the DGC group at 0 and 4h after sperm preparation. On the other hand, the motility of the ZyMot group tended to be higher (53.3%, p = 0.0787), and the motility of the ZyMot with wash group was significantly higher (85.4%, p &amp;lt; 0.001) than that of the DGC group (33.5%), after 24h of sperm preparation. The straight-line velocity, curvilinear velocity, and average speed of the ZyMot and ZyMot with wash group did not differ from those of the DGC group at 0 and 4h. All speed parameters of the ZyMot with wash group were significantly higher than those of the DGC group, after 24h of culturing. Regarding sperm head movement, there was no significant difference in the amplitude of lateral head displacement between the two ZyMot groups compared to that of the DGC group at 0 and 4h of culture. In contrast, the sperm head beat-cross frequency of the ZyMot group was lower than that of the DGC group (9.18±0.31 Hz vs 8.71±0.49 Hz, p = 0.0409). Both head movement parameters of the ZyMot with wash group were significantly higher than those of the DGC group. Limitations, reasons for caution We chose semen samples with a high number of motile sperm because we divided the sperm into three experimental groups. Therefore, it is unclear whether our findings are applicable to patients with poor semen parameters. Wider implications of the findings ZyMot may be advantageous for IUI cycles because ZyMot-collected sperm maintains sustainable motility for extended periods. Alternately, ZyMot may be unfavorable compared to DGC in conventional IVF cycles due to inferior sperm head movement, which is a marker of capacitation. However, this disadvantage can be overcome by washing. Trial registration number not applicable

The relationship between sperm quality parameters and stallion fertility

There are stallions with apparently good sperm quality that achieve low pregnancy rates when their semen is used for artificial insemination (AI). Thus, additional methods of evaluating sperm quality that result in better methods of predicting stallion fertility, are essential. Semen samples were obtained in April-June from 8 stallions, 4 trotters and 4 warmblood sport horses, which were sampled Monday, Wednesday and Friday each week. From each stallion, 1-4 samples were obtained, and evaluated after cold storage for 24 hours. The samples were evaluated for kinematic variables using CASA, and for membrane integrity, mitochondrial membrane potential (MMP), production of reactive oxygen species, acrosome integrity and chromatin integrity using flow cytometry. Relationships with fertility, calculated as number of pregnant mares/number of inseminated mares for samples used for artificial insemination after transport in cold storage, were evaluated by linear regression, following log-transformation of variables found not to be normally distributed. The following sperm variables were found to have a positive relationship with fertility: Straightness (r2=0.86, p= 0.0009), Viable with High mitosoxred/Low MMP (r2=0.70, p= 0.01), Viable non acrosome reacted (r2=0.68, p= 0.01), Viable by calceinviolet (r2=0.67, p= 0.01), Viable non H2O2-producing (r2=0.66, p= 0.01), Viable with High mitosoxred/High MMP (r2=0.65, p= 0.02), Viable non superoxide-producing (r2=0.60, p= 0.02), Linearity (r2=0.56, p= 0.03). The only variable found to have a negative relationship with fertility was Amplitude of lateral head displacement (r2=0.63, p= 0.02). The other 26 measured variables did not show a significant relationship with fertility. The results of the study shows that both kinematic variables as well as flow cytometric analysis can be valuable for relating fertility with sperm quality in vitro. The flow cytometry results indicate that analysis of the production of different reactive oxygen species can be valuable when predicting stallion fertility. Acknowledgments: Special thanks to Menhammar, Västerbo, Markebäck and Stockholm Seminstation for providing stallion semen. This work was supported by funding from FORMAS (2018-01083; awarded to AJ). The work was also supported by the Swedish University of Agricultural Sciences via the Cells for Life Platform.

The influence of L-proline and fulvic acid on oxidative stress and semen quality of buffalo bull semen following cryopreservation.

This study investigates the effects of cryopreservation and supplementation of Azeri water buffalo's semen with proline (Lp) and fulvic acid (FA). Therefore, this study aimed to assess motility parameters, sperm viability, oxidative stress parameters, and DNA damage to detect the optimum concentrations of Lp and FA for buffalo semen cryopreservation. Thirty semen samples of three buffalo bulls were diluted in Tris-egg yolk extender and divided into 12 equal groups including control (C), Lp-10, Lp-20, Lp-40, Lp-60, Lp-80 (containing 10, 20, 40, 60, 80mM L-proline, respectively), FA-0.2, FA-0.5, FA-0.8, FA-1.1, FA-1.4 and FA-1.7 (containing 0.2%, 0.5%, 0.8%, 1.1%, 1.4% and 1.7% fulvic acid, respectively). The velocity parameters, TM and PM were improved by FA-1.7, FA-1.4, Lp-40 and Lp-60 groups compared to the C group but no significant difference was found regarding the amplitude of lateral head displacement and straightness compared to the control groups. The percentage of sperm viability and PMF were increased by FA-1.7, FA-1.4, FA-1.1, Lp-40 and Lp-60 groups compared to C group, while in terms of sperm DNA damage FA-1.7, FA-1.4, FA-1.1, Lp-10, Lp-20, Lp-40 and Lp-60 groups showed better results compared to C group. The results also showed that FA-1.7, FA-1.4, FA-1.1, Lp-20, Lp-40 and Lp-60 groups could improve TAC, SOD, GSH and decrease MDA levels. Also, FA-1.7, FA-1.4, Lp-20 and Lp-40 groups could improve GPx levels but just FA-1.7, and Lp-40 groups could improve CAT levels compared to C group. Thus, it can be concluded that L-proline and fulvic acid supplementations can improve the quality parameters of post-thawed buffalo bull semen.

Machine Learning Approach for Muscovy Duck (Cairina moschata) Semen Quality Assessment.

This study aimed to develop a comprehensive approach for assessing fresh ejaculate from Muscovy duck (Cairina moschata) drakes to fulfil the requirements of artificial insemination in farm practices. The approach combines sperm kinetics (CASA) with non-kinetic parameters, such as vitality, enzyme activities (alkaline phosphatase (AP), creatine kinase (CK), lactate dehydrogenase (LDH), and γ-glutamyl-transferase (GGT)), and total DNA methylation as training features for a set of machine learning (ML) models designed to enhance the predictive capacity of sperm parameters. Samples were classified based on their progressive motility and DNA methylation features, exhibiting significant differences in total and progressive motility, curvilinear velocity (VCL), velocity of the average path (VAP), linear velocity (VSL), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), and live normal sperm cells in favour of fast motility ones. Additionally, there were significant differences in enzyme activities for AP and CK, with correlations to LDH and GGT levels. Although motility showed no correlation with total DNA methylation, ALH, wobble of the curvilinear trajectory (WOB), and VCL were significantly different in the newly introduced classification for "suggested good quality", where both motility and methylation were high. The performance differences observed while training various ML classifiers using different feature subsets highlight the importance of DNA methylation for achieving more accurate sample quality classification, even though there is no correlation between motility and DNA methylation. The parameters ALH, VCL, triton extracted LDH, and VAP were top-ranking for "suggested good quality" predictions by the neural network and gradient boosting models. In conclusion, integrating non-kinetic parameters into machine-learning-based sample classification offers a promising approach for selecting kinetically and morphologically superior duck sperm samples that might otherwise be hindered by a predominance of lowly methylated cells.

The effect of luteolin on spermatological parameters, apoptosis, oxidative stress rate in freezing rabbit semen.

The aim of the present study was to determine the effects of Luteolin (LUT) on semen quality, oxidative stress, apoptosis, acrosomal integrity, mitochondrial membrane potential and dead sperm ratio in rabbits. Ejaculates from six New Zealand rabbits were collected, evaluated and pooled. The pooling was divided into five groups as control (no additive) LUT 25 µM, LUT 50 µM, LUT 100 µM and LUT 200 µM and LUT added. It was then filled into a falcon tube with Tris-based extender at a final concentration of approximately 35 x 106 spermatozoa. Diluated rabbit semen samples were drawn into frozen and thawed. Frozen semen straws were thawed at 37°C in 30 seconds. According to our findings, no statistical difference was found between all doses of luteolin and the control group in the CASA (computer assisted sperm analysis) analysis performed at 4°C. However, total motility, progressive motility and rapid sperm percentage were found to be higher in the frozen and thawed rabbit semen at a dose of LUT 50 µM compared to the other groups (p⟨0.05). While amplitude of lateral head displacement (ALH) and beat cross-frequency (BCF) values were found at the lowest dose of LUT 200 µM, a statistically significant difference was observed between the other groups. When the flow cytometry results were examined, no statistical difference was found between the rate of dead sperm, acrosomal integrity, mitochondrial membrane potential and apoptosis rate. Morever, the H2 O2 percentage was found to be lower in all experimental groups compared to the control group (p⟨0.001). In conclusion, the addition of LUT in long-term storage of rabbit semen provided a protective effect for spermatozoa with its antioxidative properties against damage caused by cryopreservation.

Effect of Different Activation Solutions and Protein Concentrations on Ide (Leuciscus idus) Sperm Motility Analysis with a CASA System.

The purpose of this study was to compare four activation solutions (AS)-Woynarovich, Lahnsteiner, Kucharczyk, and Perchec-with the addition of 0.5% bovine serum albumin (BSA) for ide (Leuciscus idus) sperm activation and analysis with a CASA system. It was found that ide sperm can be activated using each AS within a pH range of 7.4-9.0 and an osmolality range of 160-200 mOsm kg-1. The effect of Woynarovich and Perchec solutions supplemented with BSA and casein at concentrations of 0.25, 0.5, 1.0, and 2.0% were also analyzed during the experiment. These two AS without protein supplementation (pure solutions) were the controls. Woynarovich and Perchec solutions supplemented with the minimum BSA concentration (i.e., 0.25%) significantly improved sperm motility (89.05% and 86.63%, respectively) compared to the controls (20.39 and 28.48%, respectively). Similar increases were also noted in progressively motile sperm (PRG, %), the curvilinear velocity of sperm (VCL, µm s-1), and the amplitude of lateral head displacement (ALH, µm). A similar trend in CASA parameters was also noted when casein was added to Woynarovich and Perchec solutions at a concentration of 0.25%. We concluded that 0.25% doses of each of the proteins were sufficient to prevent sperm adhesion to glass slides, and they can be used in research on ide sperm motility measurements.

Impact of quercetin, carnosine, and ozone in the cryopreservation on Nellore (Bos indicus) semen.

The objective of this study was to reduce the effects of cryoinjury caused in bovine semen by cryopreservation. Ejaculates were collected from Nellore bulls and subjected to freezing in C (control), ozone (15, 30, and 60 µg mL-1 of ozone), quercetin (25, 50, and 100 µg mL-1 of quercetin), and carnosine groups (100, 200, and 300 ng mL-1 of carnosine). Samples were evaluated post-thaw (M0) and post-rapid thermoresistance test (M30) for sperm kinetics (total motility, progressive motility, curvilinear speed, linearity and amplitude of lateral head displacement) and cell structure viability (plasma membrane integrity, acrosomal integrity, mitochondrial potential, membrane fluidity, and lipid peroxidation). There were no differences (P > 0.05) between the control, quercetin, and carnosine-treated groups for the parameters evaluated at M0 and M30. In turn, supplementation with ozone resulted in lower values for sperm kinetics (P < 0.05) and lower mitochondrial potential at M30 (P < 0.05). Quercetin and carnosine at the concentrations used did not promote significant gains in frozen semen, nor did they demonstrate cytotoxicity. We expected to obtain positive results with the use of ozone. Nonetheless, the addition was harmful to the parameters of sperm kinetics, and its effect was not observed as a possible pro-antioxidant. We believe that the fact that the gas did not harm the sperm structure opens avenues for tests with lower dosages, since, by reducing its concentration, we could minimize the damage to sperm kinetics.

Sperm parameters in the Great Dane: Influence of age on semen quality

Not all sires have sperm suitable for chilled or frozen storage, and success in artificial insemination (AI) varies highly among individual dogs and breeds. Fertilizing potential is further complicated as sperm quality declines with the aging process. Due to the rapidity of aging and senescence in large breed dogs, associated health and fertility changes may be observed over a shorter period, though this period remains undefined for any breed. Working with a population of purebred Great Danes (GD), our aims were (1) to characterize the distribution of a series of sperm parameters, (2) to distinguish sources of variation in sperm quality within this rapidly aging breed, and (3) to identify changes in sperm quality that may accompany aging. Ejaculates collected from young, middle-aged, and senior Great Dane dogs (n = 50) were evaluated for semen volume, total sperm number and viability, and reactive oxygen species (ROS), in addition to sperm morphology and kinematic parameters. Total testicular volume was also determined using ultrasonography. Testicular volume was not a predictor of sperm production in the GD, however, significant differences between coat colors were identified. Age was negatively associated with total motility, progressive motility, and amplitude of lateral head displacement (ALH) (p < .05). We identified significant relationships between GD male age and TM, PM, and immotility with −9.9%, −9.0%, and +8.3% change per year of age, respectively, which support the anecdotal reports of decline of the fertility with the advance of age in this breed. Sperm of younger GD dogs aged 12 ≤ x < 24 months had significantly higher TM, PM, ALH, and nonlinear motility (p < .05) than older dogs (x ≥ 48 months). High ROS levels were positively associated with TM and PM, average pathway distance (DAP) and straight line distance (DSL), average pathway velocity (VAP), straight line velocity (VSL), and the presence of hairpin tails (p < .05). While age and ROS have significant influences on sperm parameters in the GD, the influence of selection for breed specific phenotypes could help explain the functional significance of the diversity among GD males.

Evaluation of metabolomic profiling and oxidative stress markers of bull seminal plasma and their relationship with sperm motility before and after thawing

Mechanisms underlying differentiation of good or poor freezability ejaculates in bull are still poorly understood. This study aims at investigating oxidative stress parameters and metabolomic profiling of seminal plasma (SP) in relation to bull semen motility before and after cryopreservation. Semen samples were collected by artificial vagina from 20 bulls aged from 10 to 12 mo. An aliquot of semen was centrifuged to obtain SP (stored at -80°C), another was diluted 1:1 with a pre-warmed extender and cryopreserved. Motility assessment (CASA) was performed on fresh and thawed semen (0 and 3 h after thawing). Semen samples were divided into good freezers (GF) and bad freezers (BF) groups according to thawed semen motility, with cutoff values as follows: TM ≥ 60% and PM ≥ 30%. SP was used for oxidative stress markers analysis by spectroscopy techniques and metabolomic profile by nuclear magnetic resonance (NMR). The relationship between NMR spectra and semen quality was evaluated by the PCA/PLS-DA analysis. General linear models and mixed linear models were used to evaluate the relationship between seminal plasma oxidative stress parameters and semen quality. A clear separation between GF and BF ejaculates was observed on the first two principal components of the variance (PC1 vs PC2) of NMR spectra of SP. The increase of advanced oxidative protein products (AOPP) and thiols concentrations on SP were significantly related with higher straightness (STR), beat cross frequency (BCF) and linearity (LIN) and the decrease of amplitude of lateral head displacement (ALH) of fresh semen. Thiols and AOPP concentrations on SP were positively related to semen freezability. In addition, higher AOPP concentration was significantly related to higher TM, PM and LIN of thawed spermatozoa, whereas higher levels of thiols in SP concentration were positively related to the increment of sperm PM after thawing. These findings suggested that SP composition can modulate bull semen freezabilty. Further analyses could elucidate which metabolites and oxidative stress parameters could be used as biomarkers of good semen quality after cryopreservation.

Ide (Leuciscus idus) sperm short-term storage: Effects of different extenders and dilution ratios

In cyprinids, Tyrode’s (TLP) and Volckaert’s (VRT) solutions are the most frequently used extenders for short-term sperm storage. The effectiveness of TLP and VRT on ide (Leuciscus idus) sperm short-term storage was analyzed using a × 4 (sperm:extender) dilution ratio over 48 h. A × 4 (1:3) dilution ratio was compared to a × 10 (1:9) dilution ratio for ide sperm storage using TLP supplemented with antibiotics and was tested for a 14-day period. Sperm motility (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity of sperm (VCL, µm s-1), movement linearity (LIN, %), beat cross frequency (BCF, Hz), and the amplitude of lateral head displacement (ALH, µm) were verified using a CASA system. After 48 h, most CASA parameters were significantly higher in TLP compared to VRT. The dilution ratio also had a significant impact (P < 0.0001) on the efficiency of ide sperm short-term storage over 14 d in comparison to undiluted sperm (control). Ide sperm diluted with TLP supplemented with penicillin/streptomycin and stored short term, regardless of the dilution ratio used, retained motility and fertilization capacity over 14 d at 4 °C. The highest embryo survival rates of 70% and 73% were noted using sperm diluted with TLP at × 4 and × 10 dilution ratios compared to 48% for raw sperm that was not stored short term.

Assessment of an open-access CASA software for bovine and buffalo sperm motility analysis

The aim of this study was to verify the reliability of an open access CASA software (BGM) to evaluate the sperm motility of cattle and buffalo, comparing motility and kinematic parameters to those of a commercial one (HTM). Thirty frozen-thawed samples for each species were analyzed with both HTM and BGM, after 1 h of incubation at 37 °C. Sperm viability and mitochondrial membrane potential (MMP) were evaluated through flow cytometric analysis. Agreement of all motility variables between the two systems was assessed. Correlation analysis was performed to identify relationships between motion parameters and sperm viability and MMP. Bland Altman analysis showed good agreement between methods for all motility parameters except for curvilinear velocity (VCL) in cattle, and for average path (VAP), VCL and (amplitude of lateral head displacement) ALH in buffalo, that showed a proportional bias (P > 0.05). In both systems, positive correlation between both viability and high MMP and total and progressive motility of cattle spermatozoa were found; viability and the sperm with high MMP were positive correlated only with VAP, straight-line (VSL), VCL and ALH evaluated with HTM system. Different results were found for buffalo sperm motility parameters, since viability had positive correlations and mitochondrial activity negative ones. Results suggested that motility assessment performed by these two systems are comparable. The discrepancy of VCL, VAP, and ALH could be due to the difference in the algorithms between software. The open-access CASA plug-in is a reliable alternative to the expensive commercial CASA system for sperm motility assessment in cattle and buffalo.

Relationship between quality parameters and fertilizing ability of cryopreserved sexed bull sperm

Abstract The aim of the experiment was to assess the correlation between sperm quality parameters and field fertility after AI with sex-sorted (X-bearing) bull semen. A total of 32 ejaculates from 26 Holstein-Friesian bulls were analyzed to assess sperm motility parameters (CASA), viability (SYBR-14/PI), apoptotic-like changes (YO-PRO-1/PI), chromatin structure (SCSA), and ATP content. In order to determine sperm fertilizing ability, 816 heifers and 727 cows were inseminated. Ultrasound diagnosis of pregnancy was performed on day 35 after insemination. For each ejaculate, the percentage of pregnant females was calculated separately. The results revealed that the pregnancy rate ranged from 20.0 to 85.7% for heifers and from 7.7 to 66.7% for cows. On the basis of the pregnancy rate (PR) obtained, the ejaculates were divided into 3 groups: high PR (about 25% of ejaculates), medium PR (about 50% of ejaculates) and low PR (about 25% of ejaculates). Significant differences were detected for amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) between high- and low-fertility ejaculates in heifers. Pearson’s correlation analysis showed a significant relationship between the BCF and the pregnancy rate for heifers (r = 0.53, P&lt;0.01) and there was a trend towards significance for ALH (r = −0.37, P = 0.07). There was no relationship between the sperm quality parameters and pregnancy rate of cows. In conclusion, the present study identified markers of sexed bull sperm function that were related to the fertility of inseminated heifers.