Effect of semen dilution rate and dimethyl acetamide levels on post-thaw motility and fertility parameters of rooster sperm

This study evaluated the impact of pre-freezing semen dilution rate and dimethyl acetamide (DMA) concentration on the post-thaw motility and fertility of cryopreserved rooster sperm. Rooster ejaculates were diluted with a standard EK extender to achieve low (LSC; 1 × 10⁹ sperm/mL) and high (HSC; 2 × 10⁹ sperm/mL) sperm concentrations. Each dilution group was further treated with three DMA concentrations (3%, 6%, or 9%) before cryopreservation. Post-thaw sperm motility traits were obtained by computer-assisted sperm analysis (CASA), and fertility features were evaluated through artificial insemination in hens. The current results showed that HSC significantly improved total motility (TM), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), and beat cross frequency (BCF), but reduced linearity (LIN) and straightness (STR) compared to LSC. DMA concentration had a quadratic effect on motility, with 6% yielding the highest progressive motility (PM), straight line velocity (VSL), and BCF. Fertility outcomes revealed that HSC resulted in higher fertilization rates, while neither DMA concentrations nor their interaction with dilution rates exerted significant effects on fertility traits. VCL, ALH, and BCF showed positive correlations with pipping-chicks rates, whereas STR, LIN, and WOB displayed negative correlations. These findings underscore the critical interplay between dilution rate and cryoprotectant concentration and provide practical guidance for developing more reliable cryopreservation protocols that can be applied under field conditions to enhance fertility management in poultry production.

Computer-assisted sperm class analyser (CASA) analysis of avian semen following cryopreservation indicates that their semen motility and viability parameters become compromised, due in part to oxidative stress. To mimic these observations we have treated cockerel semen with an oxidative stress inducing agent, namely hydrogen peroxide (H2O2) and monitored the motility, kinematic and viability parameters over time. Briefly, five healthy and fertile South African Venda cockerels were selected and their semen was collected using the abdominal massage technique. The semen was then treated with H2O2 at 0 &microM, 5 &microM, 50 &microM and 200 &microM concentrations for 0, 3, 16 and 24 hrs. The semen motility, kinematic and viability parameters were then determined using the CASA system while the viability was determined using the SYBR-14/PI staining. The Pearson’s correlation coefficient was determined to test the relationships between the levels of induced oxidative stress, period of exposure to oxidative stress inducing agent and the motility plus kinematic parameters. Our data revealed that in raw cockerel semen, there was high and positive correlations between total motility (TM), progressive motility (PM), rapid (RAP), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) while the kinematic parameters LIN, STR, WOB, ALH and BCF had low or negative correlations with them. Furthermore, TM, PM, RAP, VCL and VSL remained highly and positively correlated with the induced oxidative stress and also, linearity (LIN), straightness (STR), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) remained negatively correlated with the induced oxidative stress, after 3 hrs. After 24 hrs, TM, PM, RAP, VCL, VSL, VAP and ALH, became negatively correlated with the induced oxidative stress while LIN, STR, WOB and BCF became positively correlated with the induced oxidative stress. Conversely, when the H2O2 concentration used was correlated with motility and kinematic parameters over time, TM, PM, RAP, VCL, VSL, VAP became negatively correlated with oxidative stress while LIN, STR, WOB, ALH and BCF show negative or low correlations with the induced oxidative stress. This data indicates that LIN, STR, WOB, BCF and to some extend ALH, reveal the least correlations with the induced oxidative stress under persistent oxidative stress conditions in cockerel semen. In conclusion, cockerel semen, like buck semen, does not easily succumb to oxidative stress since the raw semen correlations of CASA analysed parameters are comparable to these observed after 3 hrs of H2O2 treatment. In addition, the oxidative stress levels tolerated by cockerel semen should not 5 &microM H2O2 oxidative stress levels. Lastly, lack of correlation between LIN, STR, WOB, BCF and ALH and induced oxidative stress can be used in cockerel semen to show intolerable cryopreservation conditions.

Pellet cryopreservation for chicken semen: Effects of sperm working concentration, cryoprotectant concentration, and equilibration time during in vitro processing

The aim of this study was to use computer-assisted sperm analysis (CASA) to examine changes in motion parameters of rat spermatozoa incubated under culture conditions that support IVF. Rat cauda epididymal spermatozoa were evaluated in six replicate experiments, at 0 and 4h of incubation. CASA was conducted at 60 Hz on digital 1s tracks ( approximately 100 spermatozoa/rat). Mean values of CASA parameters that describe the vigour of spermatozoa [curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF)] increased, while those indicating progressiveness [straight line velocity (VSL), linearity (LIN) and straightness (STR)] decreased between 0 and 4 h. Visual inspection of sperm tracks after 4 h of incubation revealed classical hyperactivation patterns. Bivariate models were evaluated to objectively define the subpopulation of hyperactivated (HA) spermatozoa. Of all models considered, ALH and LIN, VCL and LIN, BCF and LIN, VCL and BCF, and VCL and ALH showed significant changes in the percentage of HA spermatozoa after the 4 h incubation period. The efficacy of detecting HA spermatozoa was evaluated using sperm tracks that were visually classified as HA or progressive. VCL and LIN provided the most accurate prediction of HA spermatozoa. It was concluded that analysis of CASA data using bivariate models could be used to detect and monitor hyperactivation in rat spermatozoa.

The present study was undertaken to assess the motility and velocity parameters of sperm of freezable and nonfreezable ejaculates by computer-assisted sperm analyser (CASA) such as Hamilton-Thorne Semen Analyser IVOS 11 in mithun semen. Fifty ejaculates (twenty-five ejaculates each for freezable and nonfreezable semen ejaculates) were collected from ten matured mithun bulls. CASA parameters, motility parameters such as forward progressive motility (FPM) (%), nonprogressive motility (NPM) (%), total motility (TM) (%), and static sperms (SM) (%); velocity parameters such as curvilinear velocity (VCL) (μm/sec), straight line velocity (VSL) (μm/sec), average path velocity (VAP) (μm/sec), linearity (LIN) (%), straightness (STR) (%), wobble (WOB) (%), amplitude of lateral head displacement (ALH) (μm), and beat/cross-frequency (BCF) (Hz) were measured by CASA analyser. The result revealed that these parameters varied significantly (P<0.05) between the freezable and nonfreezable ejaculates and freezable ejaculates have significantly (P<0.05) higher value than nonfreezable ejaculates. It was concluded that most of the CASA parameters were significantly lower in nonfreezable ejaculates than in freezable ejaculates in mithun and confirmed that the CASA was effective for a quick and objective analysis of motility and velocity parameters in mithun semen.

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p<0.05). There was no difference (p>0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.

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

Impact of age, clinical conditions, and lifestyle on routine semen parameters and sperm kinematics

The aim of the study was to determine the sperm motility parameters in wild Atlantic salmon and sea trout to define criteria important for selection of milt for controlled fertilisation. Parameters for these species were determined in the fish migrating into north-western rivers of Poland at spawning time. Eight motility parameters percentage of motile sperm (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight line velocity (VSL), linearity (LIN), straightness (STR), amplitude of lateral head displacement (ALH), beat cross frequency (BCF) and motility duration were subjected to computer-assisted sperm analysis (CASA). Milt of most individuals studied representing both salmon and trout showed spermatozoa density of 12–22 × 109 ml−1 and a high percentage of motile sperm (>70%). In general, spermatozoa swim progressively with slightly curved trajectories (mean STR = 70%, LIN = 65%) and velocity VCL of 180 μm s−1 (salmon) and 190 μm s−1 (trout), at 10 s post-activation. Such sperm is easily accessible in the wild populations of salmon and sea trout and is recommended for use in reproduction trials. The spermatozoa of sea trout seem to show a greater tendency to follow curvilinear trajectories than those of salmon, both in the beginning and the final phase of motion. In the first phase of motility, the values and time dependencies of the motility parameters were similar in both species. In the end phase of movement differences in LIN and BCF time dependencies were found in the samples representing the two species. In salmon the linearity and beat cross frequency remained stable in this phase, contrary to the patterns in sea trout for which LIN decreased while BCF increased in the end period of movement. Durations of movement were similar in both species (ranges of 20–40 s).

This study aimed to investigate the effects of refrigeration and cryopreservation on the sperm kinetic and morpho-functional parameters of 5/8 Girolando bulls. Eleven ejaculates from each bull were collected, diluted, and divided into two portions: one portion was subjected to refrigeration at 5ºC for 24 and 48 hours, while the second portion underwent cryopreservation(-196ºC). Post-thawing, various kinetic parameters were analyzed using the Computer Assisted Sperm Analysis (CASA) system. These parameters included total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness coefficient (STR), wobble coefficient (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Additionally, plasma membrane integrity (PMI), acrosomal membrane integrity (AMI), mitochondrial membrane potential (MMP), and chromatin condensation (CC) were examined for morpho-functional analyses. The results indicated that kinetic parameters (TM, PM, VCL, VSL, VAP) and morpho-functional analyses (PMI, MMP) differed significantly (P &lt; 0.05) between cryopreserved and refrigerated semen, as well as other parameters such as LIN, ALH, and BCF across all groups. The acrosomal membrane integrity showed no differences, but CC varied significantly among all groups. In conclusion, semen from 5/8 Girolando bulls can be preserved for up to 48 hours at 5ºC, providing an alternative for the short-term use of genetic material from this breed.

Rat epididymal sperm motion changes induced by ethylene glycol monoethyl ether, sulfasalazine, and 2,5-hexandione

Oxidative stress has been identified as a major cause of low seminal fertility. Among the components of stallion seminal plasma, some enzymatic and non-enzymatic antioxidants have been identified, which protect sperm from injurious effects of reactive oxygen species (ROS); however, the characterisation of these components is still in preliminary stages, as well as their relationship with freezability. The aim of this study was to evaluate the effect of some components of seminal plasma (SP) on stallion semen freezability. Semen of 30 Colombian Creole horses, and a total of 60 ejaculates, were collected. Semen was centrifuged to recover the SP. It was lyophilized and some components were assayed: total protein concentration (TP) by Bradford assay, CRISP3 protein concentration by ELISA, vitamin C (CVIT), vitamin E (EVIT) and vitamin A (AVIT), by HPLC; content of iron (Fe), copper (Cu), magnesium (Mg) and Zinc (Zn) by atomic absorption spectroscopy flame. Semen was supplemented with 10% stallion lyophilized SP and cryopreservation was performed. Post-thaw, total motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF), were assessed using a computer-assisted sperm analysis (SCA®, Microptic SL, Barcelona, Spain). Sperm viability (SV) was determined by the Live/Dead kit (Molecular Probes Inc., Eugene, OR, USA). Normal sperm morphology (NM) was performed by the supravital technique and plasmatic membrane integrity (MI) was evaluated by the hypo-osmotic test. For statistical analysis, completely randomised mixed models were fitted. Levels according to the concentration of components of SP (high, medium, and low) were established. Comparisons of the means between levels were done with Tukey’s test. The significance level used for all assessments was P &lt; 0.05. Means for TP of 0.35 mg BSA/g, CRISP3 of 55.22 ng/mg, CVIT of 2.66 mg/g, EVIT of 72.36 µg/g, AVIT of 37.37 µg/g, Fe of 17.37 mg/kg, Cu of 33.64 mg/kg, Mg of 109.08 mg/kg, and Zn of 0.49 g/100 g of SP were found. We found that a high level of CRISP3, AVIT, Cu, and Fe had higher results for post-thaw TM, PM, NM; medium levels of TP and Mg showed higher post-thaw TM, PM, NM, and MI; and lower levels of Zn had better results for post-thaw TM, PM, VCL, and VAP. In contrast, high and medium levels of CVIT had a deleterious effect on post-thaw TM, PM, SV, NM, and MI. We concluded that there is a relationship between concentrations of seminal plasma components and stallion semen freezability.

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

The aim of this study was to evaluate the effects of dilution rate and holding time on various motility parameters using computer-assisted sperm analysis (CASA). The semen samples were collected from three Ghezel rams. Samples were diluted in seminal plasma (SP), phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and Bioexcell. The motility parameters that computed and recorded by CASA include curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), straightness (STR), linearity (LIN), amplitude of lateral head displacement (ALH), and beat cross frequency (BCF). In all diluters, there was a decrease in the average of all three parameters of sperms movement velocity as the time passed, but density of this decrease was more intensive in SP. The average of ALH between diluters indicated a significant difference, as it was more in Bioexcell in comparison with the similar amount in SP and PBS. The average of LIN in the diluted sperms in Bioexcell was less than two other diluters in all three times. The motility parameters of the diluted sperms in Bioexcell and PBS indicated an important and considerable difference with the diluted sperms in SP. According to the gained results, the Bioexcell has greater ability in preserving motility of sperm in comparison with the other diluters but as SP is considered as physiological environment for sperm. It seems that the evaluation of the motility parameters in Bioexcell and PBS cannot be an accurate and comparable evaluation with SP.

We previously reported that single layer centrifugation (SLC) with Percoll® (GE Healthcare, Uppsala, Sweden) of fresh bovine semen resulted in improved sperm progressive motility and movement, as evidenced by computer-assisted sperm analyzer (CASA) after freezing-thawing. However, no report has been found in the literature on the use of Percoll Plus® (PP; GE Healthcare), a nontoxic colloid, for the same purpose. Thus, this study aimed to verify the effects of SLC-PP before bull sperm freezing on sperm kinematics after cryopreservation. Ejaculates were collected from 3 Nellore bulls (6 from each) using an artificial vagina. After collection, the semen was assessed and pooled, and then 1 billion spermatozoa either diluted [D; 1:2 (v/v)] in freezing extender (FE, without glycerol) or undiluted (UD) was layered on top of a 9-mL column of PP (in 15-mL centrifuge tubes) at concentrations of 70% or 90% to form the 70D, 70UD, 90D, and 90UD treatment groups. Following centrifugation for 13 min at 839 × g [except for the control (C) group], the supernatant was removed and the sperm pellet diluted to 50 × 106 sperm mL−1 in FE medium plus glycerol. Then, frozen–thawed sperm samples were analysed by CASA (MMC Sperm, St. Petersburg, Russia) for the following parameters: total motility (TM, %), progressive motility (PM, %), curvilinear velocity (VCL, µm−1), straight line velocity (VSL, µm s−1), average path velocity (VAP, µm s−1), amplitude of lateral head displacement (ALH, µm), beat cross frequency (BCF, Hz), linearity (LIN, %), and straightness (STR, %). For statistical analyses, ANOVA and Student-Newman-Keuls test were used. Data are presented as mean ± SEM with P &lt; 0.05 taken as significant. No difference was found among the groups for TM, VSL, BCF, and STR. However, the percentage of PM was higher (P &lt; 0.05) in the SLC-selected sperm samples (values ranging from 42.0 ± 7.0 to 47.4 ± 11.4) than in C (28.8 ± 5.0), and ALH was lower in 70UD (1.6 ± 0.12) and 70D (1.7 ± 0.10) than in C (1.9 ± 0.2). Moreover, 70UD (49.0 ± 1.0), 90UD (50.0 ± 3.0), and 90D (50.0 ± 4.0) displayed higher percentage of LIN (P &lt; 0.05) compared with C (45.0 ± 2.0) and 70D (48.0 ± 3.0). On the other hand, similar results were obtained for VCL (from 126.3 ± 8.0 to 130.0 ± 20.5) and VAP (from 82.7 ± 14.5 to 85.1 ± 6.9) in C, 70UD, and 70D, but these values differed (P &lt; 0.05) from those for VCL in 90UD (104.6 ± 10.3) and 90D (97.2 ± 22.0) as well as for VAP in 90UD (72.2 ± 11.0) and 90D (71.8 ± 9.6). These are the first data demonstrating favourable influences of SLC with 70% Percoll Plus® to select distinct sperm subpopulations as evidenced by enhanced PM, LIN, and ALH. Thus, SLC-PP could optimize the production of frozen bull semen by decreasing the number of sperm per insemination dose, and help to circumvent limitations associated with the poor semen quality sometimes found in bulls of high genetic merit. This research was funded by FAPESP # 2015/20986-3, MasterFertility and Tairana Artificial Insemination Station, Brazil.