Optimizing CASA Data: Transforming Sperm Coordinates into Long Format for Enhanced Machine Learning Analysis v2

Do parameters of seminal quality correlate with the results of on-farm inseminations in rabbits?

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).

BackgroundSperm motility parameters, which can be measured objectively and repeatedly by a computer-assisted sperm analysis (CASA) system, are important indicators of sperm quality. However, the sperm motility parameters assessed by a CASA system can be affected by various factors, including instrument components and settings, sperm preparation or analysis procedures. To date, no standardized protocol is available that would permit to assess sperm kinetic characteristics in passerine birds and this lack precludes any comparison of sperm swimming ability and sperm quality across species.MethodsIn this study, we chose the Tree Sparrow (Passer montanus) as the object to evaluate sperm motility parameters, including sperm motility, sperm velocity and sperm movement trajectory, at different analysis time, temperatures and pH using the WLJY-9000 CASA system.ResultsSperm motility parameters remained statistically unchanged at 1‒9 min. Progressive motility was similar at 38 °C and 40 °C, but a greater percentage of slow progressive sperm was detected at 38 °C compared to 40 °C and 42 °C. Additionally, progressive motility was lower and immotility was higher at 42 °C than 38 °C and/or 40 °C (close to the body temperature of the Tree Sparrow). The percentages of rapid progressive sperm, progressive sperm and immotile sperm were statistically similar at pH 7.0, 7.5 and 8.0 with the exception of lower percentage of progressive sperm at pH 7.0 compared to pH 7.5. In addition, slower sperm velocity and worse sperm movement trajectory were found at pH 6.0 and 9.0 than those at pH 8.0, 7.5 or 7.0.ConclusionsOur study indicates that the ideal conditions for sperm motility parameters assessment in Tree Sparrow are obtained between 1 and 9 min after dilution, an environment at body temperature (40 °C) and a pH around 7.5‒8.0. The results of this study provide a reference for the evaluation of sperm characteristics and sperm quality using a CASA system in passerine birds.

Simple SummaryA reliable sperm motility exam is important for semen analysis and breeding soundness examination. Different parameters can affect the Computer Assisted Sperm Analysis (CASA) motility results. Today, new high-resolution cameras and different chambers are introduced to CASA systems, and protocol optimization is required to render the estimation results for donkey sperm. The objective of this study is the optimization of the conditions used for donkey semen motility analysis with CASA-Mot by defining the optimum frame rate for different chamber types. Additionally, to study the effect of different chamber types, chamber field and sperm dilution on the sperm kinematic parameters with higher frame rates are examined.In order to optimize the donkey sperm motility analysis by the CASA (Computer Assisted Sperm Analysis)-Mot system, twelve ejaculates were collected from six jackasses. Capillary loaded chamber (CLC), ISAS®D4C depths 10 and 20 µm, ISAS®D4C Leja 20 and drop displacement chamber (DDC), Spermtrack® (Spk) depths 10 and 20 µm were used. Sperm kinematic variables were evaluated using each chamber and a high-resolution camera capable of capturing a maximum of 500 frames/second (fps). The optimum frame rate (OFR) (defined according to curvilinear velocity—VCL) was dependent on chamber type. The highest OFR obtained was 278.46 fps by Spk20. Values for VCL, straight-line velocity (VSL), straightness (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were high in DDC and 10 µm depth. In both DDC 10 and 20 µm, the sperm velocities (VCL, VSL, VAP) and ALH values decreased significantly from the centre to the edges, while Wobble and BCF increased. No defined behavior was observed along the CLC. However, all the kinematic variables had a higher value in a highly concentrated sample, in both chamber types. In conclusion, analyzing a minimum of nine fields at 250 fps from the centre to the edges in Spk10 chamber using a dilution of 30 × 106 sperm/mL offers the best choice for donkey computerised sperm motility analysis.

Sperm quality is often evaluated through computer-assisted semen analysis (CASA) and is an indicator of boar fertility. The aim of this research was to study the relationship between CASA motility parameters and fertility results in pigs. Insemination records and semen parameters from a total of 45,532 ejaculates collected over a 3-yr period were used. The statistical model for analysis of fertility data from these inseminations included factors related to sow productivity. The boar- and semen-related variance (direct boar effect) were corrected for the effects of individual boar, genetic line of the boar, age of the boar, days between ejaculations, number of sperm cells in an ejaculate, number of sperm cells in an insemination dose, and AI station. The remaining variance was analyzed if semen motility parameters had a significant effect. This analysis revealed significant (P < 0.05) effects of progressive motility, velocity curvilinear, and beat cross frequency on farrowing rate (FR). Total motility, velocity average path, velocity straight line, and amplitude of lateral head displacement affected (P < 0.05) total number of piglets born (TNB). Boar- and semen-related parameters explained 5.3% of the variation in FR and 5.9% of the variation in TNB. Motility parameters, measured by CASA, explained 9% of the boar- and semen-related variation in FR and 10% of the boar- and semen-related variation in TNB. Individual boar and genetic line of the boar affected (P < 0.0001) the variation in FR and TNB. No differences (P > 0.05) were observed between effects of AI stations on fertility outcome, underscoring the objectivity of the CASA system used. Motility parameters can be measured with CASA to assess sperm motility in an objective manner. On the basis of the motility pattern, CASA enables one to discriminate between the fertilizing capacity of ejaculates, although this depends on the genetic line of the boar used in AI stations.

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.

African medicinal plant like soursop (Annona muricate L.) within annonaceae are known for their biological, therapeutic, and pharmacological properties with little or no toxicity. The use of such plant requires good knowledge of the toxicity dosage, purity, suitable extraction solvent and adverse effects. The leaves, seeds, fruits, barks, and roots of African medicinal plants have been used for various nutraceuticals and functional effects according to African folk medicine. The aim of this study is to evaluate the semen quality, oxidative activity and spermatozoa kinematics of rooster semen in soursop juice extender. About 30 roosters were used for the in vitro analysis. Semen was collected twice a week for 2 weeks through dorsal-abdominal massage technique. The evaluation was done hourly until semen quality declined at the 5th-hour. The pooled semen was allotted to seven treatments of semen extenders as undiluted semen, dextrose saline, 10% soursop juice extender, 20% soursop juice extender, 30% soursop juice extender, 40% soursop juice extender, and 50% soursop juice extender for the study. The percentage motility, progressive motility, nonprogressive motility, curvilinear velocity, average path velocity, straight line velocity, linearity, straightness, amplitude of lateral head, beat cross frequency and wobble were analyzed using computer aided sperm analysis. Oxidative status (antioxidant activity and lipid peroxidation) was determined by assay. Result of rooster semen at room temperature and after 1-hour dilution showed that percentage motility, nonprogressive motility, and average path velocity were significantly (P < 0.05) reduced by different soursop juice extenders compared to undiluted semen. After 2-hour dilution of rooster semen, nonprogressive motility, average path velocity, curvilinear velocity, straight line velocity, wobble, liveability and amplitude of lateral head parameters were significantly (P < 0.05) increased by different soursop juice extenders compared to undiluted semen. Antioxidant activity and lipid peroxidation in both room temperature and after 5-hour dilution were affected by different soursop juice extenders in rooster semen. In conclusion, supplementation of soursop juices as an extender to rooster undiluted semen played an improvement role on spermatozoa fertility and oxidative status during processing or preserving ejaculates for insemination.

Simple SummarySemen quality in ostriches is still being studied to optimize the reproductive performance of these birds in a commercial farming environment. Various methods such as mass sperm motility scoring and computer-aided sperm analysis (CASA) technology have been employed successfully to evaluate semen quality characteristics such as sperm motility. The relationship between these methods in assessing sperm motility in ostriches is unknown. This study evaluated the relationship between the CASA and the mass sperm motility scoring method when analyzing ostrich sperm motility. Positive significant correlations between these methods for sperm motility traits were recorded, suggesting that farmers could utilize the affordable mass sperm motility scoring to screen ostrich males for sperm quality.Semen analyses have gained momentum in various livestock industries. However, in farmed ostriches, semen analysis is still in its experimental stage, and males are not screened for sperm quality before breeding. This study investigated the correlations between computer-aided sperm analysis (CASA) technology and the traditional, yet affordable, mass sperm motility score. Semen was collected from nine South African Black ostrich males (mean age ± SD: 5.25 ± 1.21 years), using the dummy female method for 5 consecutive days monthly, for 8 months. Mass sperm motility scores were recorded on a scale of 1–5 (1: little to no sperm movement; 5: rapid sperm movement). The CASA traits recorded were: total motility (MOT), progressive motility (PMOT), curve–linear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), linearity (LIN), straightness (STR), wobble (WOB), and beat-cross frequency (BCF). The results revealed positive correlations between mass sperm motility and PMOT, MOT, VCL, and VAP ranging from 0.34 to 0.59 (p < 0.0001). In contrast, negative correlations were recorded between mass sperm motility and LIN, STR, and BCF, with correlations ranging from −0.20 to −0.39 (p < 0.0001). VSL, ALH, and WOB were not correlated to mass sperm motility (p > 0.05). Ostrich farmers may thus be able to evaluate sperm motility reliably and potentially select breeding males by using the affordable mass sperm motility scoring method. Determining the correlation between these methods and fertility after artificial insemination or natural mating is however needed.

The present study was undertaken to identify the differences in sperm kinematics between buffalo bulls with different fertility ratings. Murrah buffalo bulls (n=9) that were routinely used for breeding purpose under progeny testing programme were utilized for the study. Bull fertility was determined based on in vivo fertility trials and the conception rates (CR) were adjusted for different non-genetic parameters. Based on the adjusted CR, bulls were classified into high, medium and low fertile group. Frozen semen samples of these bulls were obtained and sperm kinematic parameters were assessed using a computer assisted sperm analyzer. The kinematic parameters analyzed included the curvilinear velocity (VCL), the linear velocity (VSL), the average path velocity (VAP), the amplitude of lateral head displacement (ALH), the linearity (LIN), the straightness coefficient (STR) and the beat cross frequency (BCF). In high fertile bulls, the proportion of motile spermatozoa was higher (p&lt;0.001) than the medium and low fertile bulls. The VAP and VCL of sperm motion were significantly higher (P&lt;0.05) in high fertile bulls compared to either medium or low fertile bulls. The VSL was significantly lower in low fertile bulls (P&lt;0.005) compared to either high or medium fertile bulls. Spermatozoa from high fertile bulls had significantly higher (P&lt;0.05) BCF, STR, ALH and LIN compared to either medium or low fertile bulls. Buffalo bull fertility was significantly and positively correlated with sperm motility, VAP, VSL, VCL and ALH.

Effects of melatonin implants on the reproductive performance and endocrine function of camel (Camelus dromedarius) bulls during the non-breeding and subsequent breeding seasons

Motility patterns of spermatozoa are an indicator of their fertilizing capacity. Reduced glutathione (GSH) has been reported to induce positive effects on the biological quality of the frozen-thawed spermatozoa. The aim of this study was to investigate the effects of GSH addition to semen extender based on the following kinetic parameters: continuous line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF), total motility (tMOT) and progressive motility (pMOT), and to appoint them as indicators of its presence. A soybean-based extender was used for dilution of the semen samples, fractioned in two parts, one containing GSH (5 mMol/ml) and second without GSH. The ejaculates (n=48) were collected from two rams (January - May, 2013) which were classified in two groups according to the used extender: Group 1 (with GSH, n=24) and Group 2 (without GSH, n=24), and then frozen in liquid nitrogen on -196°C degrees in a programmable freezer. Assessment of the samples has been performed post-thawing (30 sec. at 37°C) on CASA equipment, acquiring kinetic parameters. Results showed that only VSL and BCF have a statistically significant difference between group1 and group 2 (102.98±15.13 vs. 88.47±20.63, t=2.77, p&lt;0.01 and 32.01±2.68 vs. 89.47±2.92, t=3.13, p&lt;0.01, respectively). The summary of the investigation concludes that none of the kinetic parameters could be appointed as indicators that confer the positive effect of GSH as an additive to soy-bean semen extender.

Effects of three male reproductive toxicants on rat cauda epididymal sperm motion

The manual method of semen analysis is vital in andrology laboratories. To solve the labor-intensive, time-consuming, and subjective problem, computer-assisted sperm analysis (CASA) systems have been developed. However, it is unclear whether the consistency of semen analysis results especially in male infertility diagnoses can be achieved. A total of 326 individuals were recruited from January 14, 2020, to October 22, 2020. The manual method was used as the gold standard. Pairwise comparisons were conducted between CASAs (Hamilton-Thorne CEROS II Clinical, LensHooke X1 Pro, and SQA-V Gold Sperm Quality Analyzer) and manual method. LensHooke had the best performance in concentration, while the others showed moderate performances (intraclass correlation coefficient [ICC]: CEROS-0.723, LensHooke-0.842, SQA-V-0.631). CEROS II had moderate performances in motility, and the others only had poor agreements (ICC: CEROS-0.634, LensHooke-0.417, SQA-V-0.451). Morphology analyses were not consistent with manual results (ICC: LensHooke-0.160, SQA-V-0.261). In Bland–Altman plots, CEROS II (P = .379) and SQA-V Gold (P = .218) showed consistent measurements in concentrations and total sperm count with the manual method, while the others were inconsistent. LensHooke X1 Pro (κ=0.701) and CEROS II (κ = 0.664) showed substantial performances in oligozoospermia, and SQA-V Gold had moderate agreements (κ = 0.588). In asthenozoospermia, LensHooke X1 Pro had moderate performances (κ = 0.405) and CEROS II had fair agreement (κ = 0.249), while low agreement using SQA-V Gold (κ = 0.157). In teratozoospermia, LensHooke X1 Pro (κ = 0.177) and SQA-V Gold (κ = 0.008) could not have consistent results either. When choosing treatment based on morphology, the ratio of intracytoplasmic sperm injection (ICSI) approximates 0.5 in our unit. However, the ratios were around 0.31 and 0.15 using LensHooke X1 Pro and SQA-V Gold, indicating the reduction of ICSI work in routine treatment. CASA results were not consistent with manual results, and the deviations might result in skewed in vitro fertilization/ICSI allocation in subsequent treatment. Interestingly, tested CASA systems tend to skew to conventional in vitro fertilization instead of ICSI. Although CASA technologies have been improving recently, the manual method cannot be replaced by the tested CASA systems at present and the results should be treated with caution. CASA algorithms should be improved, especially in morphology. Future strict studies should be designed to evaluate the CASA systems with both internal and external validations.

The present study investigated the effects of exogenous cyclic AMP (cAMP)-dependent protein kinase inhibitor (H89) on kinematic parameters of epididymal ram sperm in vitro. After incubation of epididymal sperm samples for 45 and 90 min in the presence of H89 (0.5, 5, and 50 μM), motion parameters were evaluated by computer-aided sperm analysis. At 50 μM concentration of H89, there was a significant decrease in the fast progressive motility (at 45 and 90 min), and also most of the kinematical parameters such as curvilinear velocity, straight line velocity, average path velocity, mean angular degree (MAD), amplitude of lateral head displacement, beat cross-frequency, linearity, and straightness but not in wobble (WOB) at 45 min. After 90 min all kinematic parameters were significantly decreased at 50 μM concentration except MAD and WOB. In conclusion, high concentrations of exogenous cAMP-dependent protein kinase inhibitor (H89) decrease the motion parameters of ram epididymal sperm.

Sperm morphological examination, computer-assisted sperm analysis (CASA) and histopathologic examination of the testis and epididymis were performed for male rats treated orally with boric acid for 3 weeks at dosage levels of 50, 150 and 500 mg/kg/day, and treated males were mated with untreated females. None of the males treated with 500 mg/kg/day could impregnate untreated females. The fertility index showed a tendency to decrease in males treated with 150 mg/kg/day. At necropsy, the pre-implantation loss rate in females mated with males treated with 150 mg/kg/day was higher than the control values. Upon epididymal sperm analysis using the CASA system, all parameters including the number of sperm and sperm motions were found to be affected in males treated with 500 mg/kg/day, and the number of sperm, percent motile, velocities and amplitude of lateral head displacement (ALH) were affected in males treated with 150 mg/kg/day. Upon sperm morphological examination, head and tail abnormalities were observed in males treated with 150 and 500 mg/kg/day. In the histopathological examination, atrophy of the seminiferous tubules and multinucleated giant cells in the testes were observed in males treated with 500 mg/kg/day.