Computer-assisted Sperm Analysis Parameters Research Articles

Homozygous ACTL9 mutations cause irregular mitochondrial sheath arrangement and abnormal flagellum assembly in spermatozoa and male infertility.

To identify novel variants in ACTL9 and new phenotypes responsible for male infertility. Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing (WES). Computer-assisted sperm analysis (CASA) was used to test the motility of spermatozoa. The ultrastructure of flagella and the mitochondrial sheath were assessed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Immunostaining was used to validate the localization and expression of ACTL9 and ACTL7A. An Actl9-mutated mouse model was used to validate the phenotypes by CASA and TEM. We identified novel homozygous variants in ACTL9 in two independent Chinese families. Spermatozoa with ACTL9 mutations showed decreased CASA parameters and a higher proportion of spermatozoa with abnormal morphology, exhibiting coiled flagella and a thickened midpiece. The spermatozoa were characterized by chaotic or irregular '9+2' structures and irregular mitochondrial sheath arrangements in the flagellum. Actl9 knock-in mice also showed abnormal CASA parameters and irregular '9+2' structures in flagella. Our study expands the mutation spectrum and phenotypic spectrum of ACTL9.

Enhancing evaluation of bull fertility through multivariate analysis of sperm

Computer-assisted sperm analysis (CASA) has become the predominant tool for assessing bull semen in artificial insemination programs. Despite such popularity CASA's ability to predict fertility has been limited, especially when emphasis is based upon single motion characteristics. Our hypothesis is that numerical sets of CASA measures provide a more effective method to differentiate the potential fertilization capacity of bulls and that bulls can be clustered based upon sets of CASA measures. Therefore, we used CASA to evaluate frozen-thawed semen samples from 307 Holstein and 152 Jersey bulls sourced from USDA-ARS's National Animal Germplasm Program gene bank. Sperm was evaluated immediately after thawing and 30 min later. We evaluated sperm kinetic and morphometric means and variances to capture the structure of CASA data in relation to various sources of variation. These data were subjected to univariate and multivariate statistical methods to investigate animal and management factors affecting sperm characteristics measured by CASA. Clustering with K-means identified 4 clusters of bulls based upon each cluster's set of CASA parameters after thawing. There was little overlap among clusters for sets of CASA measures. At the extremes, bull cluster 1 (BC1, n = 180) and BC3 (n = 101) had different sire conception rates (SCR) -0.07 vs -1.29, respectively and sets of CASA measures. Interestingly, BC2 had CASA measures that could be perceived as negative, e.g., cell size at 8.18mm2 vs 6.37mm2 for BC4 and total motility of 29.7% vs 48.7% for BC3, but SCR for BC2 was higher (-0.79) than BC3 (-1.29). Despite such discrepancies for some BC2 CASA values it appears the potentially negative effects were offset by the levels of other CASA values. Our findings suggest improved approaches for using CASA could lie in evaluating multiple CASA measures as sets within specific numerical ranges rather than as independent measures.

PSII-7 Is Scrotal Ultrasound a Useful Tool in Ram Breeding Soundness examination?

Abstract Varicocele, a condition of abnormally dilated veins within the pampiniform plexus, is associated with reduced male fertility in several species but its frequency and impact in rams is not well documented. Semen was collected and pampiniform plexus ultrasound performed in early December during breeding soundness examinations (BSE) of mature Composite-IV rams over a three-year study. A portion of rams were evaluated in multiple years and data from a total of 129 collections on 90 rams were available for analyses. Semen parameters were estimated by computer-assisted sperm analysis (CASA). Imaging software was used to quantify the cross-sectional area of five vessels per testis and average areas of the left testis (AAL), right testis (AAR), and both testes (AAT) were calculated. The correlation between AAL and AAR measured on the same ram within a year was moderate and positive (0.51; P < 0.001). However, correlations between AAL, AAR, and AAT measured on the same ram in consecutive years were not different from zero. Average vessel area was analyzed with fixed effects of testis side (AAL or AAR), year (2020, 2021, or 2022), ram age (2, 3, or ≥ 4 yr-old) and a random effect of ram. Across years, AAL was 23 to 25% greater than AAR and 16 to 18% greater in ≥ 4-yr-old than younger rams (P < 0.01). Unilateral varicocele (UV) was diagnosed when AAL/AAR or AAR/AAL ratio exceeded 1.5 in a ram and, for rams sampled in multiple years, those diagnosed in one year were considered affected for all subsequent years. Within year, prevalence of UV ranged from 16 to 36%. Scrotal circumference (SC) and CASA parameters were analyzed with fixed effects of year, ram age, and UV status and a random effect of ram. Within 2- and 3-yr-olds, sperm from UV rams had lower motility than unaffected rams (42.1 ± 6.7% vs 62.2 ± 4.1% and 47.8 ± 7.4% vs 70.1 ± 4.7%, respectively; P ≤ 0.02) but no differences were observed within 4-yr-old rams. Across ages, sperm from UV rams had a lower percentage of normal than unaffected rams (91.5 ± 1.1% vs 94.3 ± 0.74%; P = 0.03). Sperm concentration, progressive motility, and SC were unaffected by UV status. Future studies will evaluate the effect UV status on siring capacity. Ultrasound methodologies previously refined to diagnose varicocele in boars helped identify sub-fertile rams that would not have been classified by routine BSE procedures. However, these methodologies would benefit from concurrent machine learning and software development to ensure imaging consistency across technicians and streamline procedures for real-time decision making. USDA is an equal opportunity provider and employer.

P-720 Biallelic variants in IQCN cause sperm flagella assembly defects and male infertility

Abstract Study question What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? Summary answer Deficiency in IQCN is responsible for sperm flagella assembly defects and abnormal computer-assisted sperm analysis (CASA) parameters, which result in male infertility. What is known already Manchette is a transient structure that is involved in the shaping of the spermatid nucleus and protein transport of flagella. Most recently, our group reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN led to total fertilization failure (TFF) and defective acrosome structure phenotypes. However, the related function of IQCN in sperm flagella assembly is still unknown. Study design, size, duration Fifty infertile males were recruited from the Reproductive and Genetic Hospital of China International Trust Investment Corporation (CITIC)-Xiangya from January 2014 to October 2022. Participants/materials, setting, methods Genomic DNA was extracted from the peripheral blood samples of the affected individuals for whole-exome sequencing. The ultrastructure of the flagella was detected by transmission electron microscopy (TEM). CASA was used to test the parameters of sperm motility. An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPER-Cas9 technology. Immunoprecipitation (IP) followed by liquid chromatography‒mass spectrometry was used to select the differentially expressed IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. Main results and the role of chance Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our infertile cohort. The sperm from the affected individuals showed irregular “9 + 2” structure of sperm flagella, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. The CASA parameters in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. The partial of peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or the chaotic arrangement of DMTs were observed in the principal piece and end piece of sperm flagella. The hyperactivation ability of sperm from Iqcn-/- male mice was decreased than sperm from Iqcn+/+ male mice. The CDC42 and IFT protein families are hub proteins that interact with IQCN and regulate flagella assembly during spermiogenesis. Deficiency in IQCN causes the loss of binding with CDC42 and IFT74, which leads to the absence or abnormal localization of CDC42 and IFT74 in mouse and human spermatozoa. Limitations, reasons for caution More cases are needed to demonstrate the relationship between the IQCN variations and the phenotypes. Wider implications of the findings Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, thus providing a genetic marker for sperm motility deficiency and male infertility. Trial registration number N.A.

Biallelic variants in IQCN cause sperm flagellar assembly defects and male infertility.

What is the effect of defects in the manchette protein IQ motif-containing N (IQCN) on sperm flagellar assembly? Deficiency in IQCN causes sperm flagellar assembly defects and male infertility. The manchette is a transient structure that is involved in the shaping of the human spermatid nucleus and protein transport within flagella. Our group recently reported that the manchette protein IQCN is essential for fertilization. Variants in IQCN lead to total fertilization failure and defective acrosome structure phenotypes. However, the function of IQCN in sperm flagellar assembly is still unknown. Fifty men with infertility were recruited from a university-affiliated center from January 2014 to October 2022. Genomic DNA was extracted from the peripheral blood samples of all 50 individuals for whole-exome sequencing. The ultrastructure of the spermatozoa was assessed by transmission electron microscopy. Computer-assisted sperm analysis (CASA) was used to test the parameters of curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP). An Iqcn knockout (Iqcn-/-) mouse model was generated by CRISPR-Cas9 technology to evaluate sperm motility and the ultrastructure of the flagellum. Hyperactivation and sperm fertilizing ability were assessed in a mouse model. Immunoprecipitation followed by liquid chromatography-mass spectrometry was used to detect IQCN-binding proteins. Immunofluorescence was used to validate the localization of IQCN-binding proteins. Biallelic variants in IQCN (c.3913A>T and c.3040A>G; c.2453_2454del) were identified in our cohort of infertile men. The sperm from the affected individuals showed an irregular '9 + 2' structure of the flagellum, which resulted in abnormal CASA parameters. Similar phenotypes were observed in Iqcn-/- male mice. VSL, VCL, and VAP in the sperm of Iqcn-/- male mice were significantly lower than those in Iqcn+/+ male mice. Partial peripheral doublet microtubules (DMTs) and outer dense fibers (ODFs) were absent, or a chaotic arrangement of DMTs was observed in the principal piece and end piece of the sperm flagellum. Hyperactivation and IVF ability were impaired in Iqcn-/- male mice. In addition, we investigated the causes of motility defects and identified IQCN-binding proteins including CDC42 and the intraflagellar transport protein families that regulate flagellar assembly during spermiogenesis. More cases are needed to demonstrate the relation between IQCN variants and phenotypes. Our findings expand the genetic and phenotypic spectrum of IQCN variants in causing male infertility, providing a genetic marker for sperm motility deficiency and male infertility. This work was supported by the National Natural Science Foundation of China (81974230 and 82202053), the Changsha Municipal Natural Science Foundation (kq2202072), the Hunan Provincial Natural Science Foundation (2022JJ40658), and the Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-Xiangya (YNXM-202114 and YNXM-202201). No conflicts of interest were declared. N/A.

Toxicity and deleterious impacts of selenium nanoparticles at supranutritional and imbalance levels on male goldfish (Carassius auratus) sperm

BackgroundSelenium has a major role in male reproduction and antioxidative mechanisms. Although deficiency of this element can result in damages to the body's organs, this metalloid can induce deleterious effects in organisms by causing oxidative stress. This study assessed the spermatotoxicity of selenium nanoparticles (SeNPs) in goldfish (Carassius auratus) based on genotoxicity, antioxidant status, sperm quality, and histopathology. MethodsThe fish with an average weight of 70 g (n = 288) were divided into four experimental groups (three replicates) and fed three times a day with SeNPs at different levels of 0, 0.1, 0.5, and 1 mg kg diet for 30 and 60 days. ResultsAfter 30 and 60 days of feeding trial, compared to the control group, spermatocrit percentage markedly decreased at 1 mg kg SeNPs on day 30 as well as at 0.5 and 1 mg kg on day 60 (p < 0.05). Computer-assisted sperm analysis parameters especially VCL, VSL, and VAP decreased in response to SeNPs (p < 0.05). Percentage of fast speed progressive sperm cells was highest in fish fed with 0.1 mg kg SeNPs following the dietary experiment and significantly reduced in a SeNPs dose-dependent manner (p < 0.05). In addition, the levels of Malondialdehyde and Glutathione peroxidase were significantly elevated in seminal plasma of all SeNPs-treated groups (p < 0.05). On day 60, DNA damage of sperm was greatly increased at 1 mg kg SeNPs (p < 0.05). Moreover, the highest percentage of spermatocyte and spermatid were observed at the highest dose of SeNPs while the highest percentage of spermatozoa was recorded at the lowest and moderate SeNPs doses. ConclusionThese findings suggested that non-optimal doses of SeNPs could reduce sperm quality, induce oxidative stress, and DNA damage in sperm, and disrupt testis development.

Bull Sperm Tracking and Machine Learning-Based Motility Classification

Sperm motility measurement using computer assisted sperm analysis (CASA) has been widely accepted as a substitute for manual measurement but still faces several challenges. In the tracking phase, tracking errors caused by detection failure often occur when measuring fresh bull semen. Tracking errors occur for two reasons: (1) the sperm move very fast, which makes them appear blurry, and (2) partial occlusion, which frequently occurs. This study proposes the mean angle of sperm motion and Tracking-Grid to predict the position of the sperm that failed to be detected. The Tracking-Grid has also been found useful in tracking fast-moving sperm. The proposed methods reduce identity switch (ID-switch) and achieve a multi-object tracking overall accuracy (MOTAL) of 73.2. The MOTAL result exhibits 5% less ID-switch and is 15.6 MOTAL points higher than state-of-the-art simple online and real-time tracking with a deep association metric (Deep SORT). The speed achieved is 41.18 frames per second (fps), which is 1.8 times faster than Deep SORT. In sperm motility classification, most researchers use one or several CASA parameters with a static threshold value. Such a method is effective for motile-progressive sperm classification but is less reliable for identifying non-motile-progressive sperm such as vibrating and floating sperm. This study proposes a machine learning-based motility classifier using a support vector machine with three CASA parameters: curvilinear velocity (VCL), straight-line velocity (VSL), and linearity (LIN), which we call the bull sperm progressive motility classifier (BSPMCsvm3casa). Experimental results show that BSPMCsvm3casa’s mean accuracy is 92.08%, which is 2.51–9.67 points higher than other classification methods.

A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird's Nest on the quality of chilled Arabian stallion semen.

The aim of this study was to evaluate the effects of adding different concentrations of edible bird’s nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, E-Z mixin® treated semen had significantly higher TM and PM than EquiPlus® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.

Effects of chilled storage and pH of activating solution on different motility parameters in burbot (Lota lota) sperm

The effects of a simple saline solution prepared using two different pH (4.4 and 8.5) on sperm motility in burbot were investigated. Results were recorded during a 96-hour chilled storage (4°C) in 24-hour intervals. Measurements were focused on the detailed characteristics of motility using 12 parameters obtained from the Computer-assisted Sperm Analysis (CASA). Significantly higher progressive motility (pMOT), distance average path (DAP), distance curved line, distance straight line (DSL), average path velocity (VAP), curvilinear velocity, straight line velocity, and beat cross frequency (BCF) were observed with the activating solution buffered at pH 8.5 in comparison with pH 4.4. Already after 24 h a significant reduction was measured in pMOT (0 h: 49 ± 24%, 24 h: 12 ± 7%). Similar decreasing tendency was recorded only after 72 h in DAP (0 h: 26 ± 4 µm/s, 72 h: 19 ± 9 µm/s), DSL (0 h: 21 ± 5 µm/s, 72 h: 17 ± 8 µm/s), VAP (0 h: 59 ± 9 µm/s, 72 h: 43 ± 21 µm/s), and BCF (0 h: 28 ± 2 Hz, 72 h: 18 ± 10 Hz). The response of different investigated CASA parameters to different treatments varied in our experiments. According to our studies, numerous burbot sperm motility parameters are sensitive to chilled storage and to low pH of the activating solution. Our results could support the effective sperm quality assessment and successful artificial propagation process in burbot.

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

To assess the impact of aging on routine semen and computer-assisted sperm analysis (CASA) motility parameters according to the current World Health Organization guidelines; and to evaluate the effect of obesity and lifestyle (alcohol consumption, cigarette smoking) in older men's semen. Blind cross-sectional study. Research laboratory and andrology and reproduction laboratory. A population of 11,706 men. None. Semen analysis: routine (semen volume, sperm concentration and count, motility, vitality, morphology, hypo-osmotic swelling test, round and peroxidase-positive cell concentration) and CASA (straight-line velocity, curvilinear velocity, average path velocity, linearity, straightness, beat cross frequency, wobble, amplitude of lateral head displacement, and mean angular displacement) parameters; and body mass index. A negative correlation was found between age and routine semen parameters: volume, sperm count, motility, vitality, total motile spermatozoa and normal-motile spermatozoa, round cell concentration, and hypo-osmotic swelling test values. Several CASA variables (straight-line velocity, curvilinear velocity, average path velocity, beat cross frequency, amplitude of lateral head displacement, and mean angular displacement) were also negatively affected. Using 40years as a cut-off value, significant differences in most parameters correlated to age. In a selected subpopulation of men unexposed to known fertility-compromising factors, the same evaluations were performed, finding some parameters still decreased. Although obesity exerted a significant deleterious effect on older patients' semen quality, alcohol consumption and cigarette smoking mildly affected it. Male aging, with the contribution of unhealthy conditions, are paramount effectors of sperm quality deterioration.

The effects of environmentally relevant concentrations of aldrin and methoxychlor on the testes and sperm of male Clarias gariepinus (Burchell, 1822) after short-term exposure.

The organochlorine pesticides aldrin (0.14μg/L) and methoxychlor (0.23μg/L) were both present in the Albasini Dam, Limpopo Province, South Africa, during a field survey in 2014. The use of aldrin has been banned in the USA since 1987 and restricted in South Africa since 1992. The use of methoxychlor, however, remains undefined with little information available about its registration in South Africa despite being banned in Europe (2002) and USA (2003). The aim of this study was to determine the potential effects of environmentally relevant concentrations of aldrin and methoxychlor on the reproductive system of male catfish, Clarias gariepinus. Males were exposed for 96h to the two pesticides under controlled laboratory conditions. Following exposure, each fish was weighed and measured, and a necropsy performed to determine any macroscopic abnormalities and the general health of the fish. The fish were killed and dissected and the testes removed, weighed and measured to determine the gonadosomatic index (GSI). The right testis of each fish was sectioned for histopathological assessment and to calculate the testes index (IT). The left testis was used for computer-assisted sperm analysis (CASA). The histopathological assessment of the testes showed histopathological changes such as of melano-macrophage centres (MMCs) and vacuolation of spermatogonia and spermatocytes. However, the classification of these changes indicated that the testes tissue structure was normal with slight histological changes. No statistically significant differences (p > 0.05) were found in the CASA parameters between exposure groups. The results of this study showed that the environmentally relevant concentrations of aldrin and methoxychlor did not have a negative effect on the motility of the mature sperm, but adverse effects were noted in the early stages of spermatogenesis, indicating possible effects over longer exposure periods.

Effects of zearalenone, α-zearalenol, and genistein on boar sperm motility in vitro

Genistein (GEN) and zearalenone (ZEA), environmental oestrogens commonly present in feedstuff for pigs, are known for their effects on reproductive functions. The aim was to verify the in vitro effects of 0.5–20 µM concentrations of GEN, ZEA and its metabolite α-zearalenol (α-ZOL) on pig sperm motility. A dose-dependent increase of the immotile sperm amount against fast and medium-fast sperm clusters was observed with all three oestrogens from the lowest concentrations tested. Individual CASA (computer-assisted sperm analysis) parameters of motile sperms seemed to be less sensitive indicators. This should be considered especially in toxicological research on a sperm model. Background of inconsistencies in to date-published papers is discussed. The results shift the effective concentrations of ZEA, α-ZOL, and GEN to values achievable in vivo and raises the questions of risk assessment of these compounds in pig reproduction.

Study on sperm motility and velocity parameters of freshly collected mithun semen through computer-assisted sperm analyser (CASA)

The objective of this study was to evaluate the quality of mithun semen by computer assisted sperm analysis (CASA). Ejaculates (50) were analysed by CASA. Semen motility was evaluated for kinetics parameters. The percentage of total motile and progressive motile spermatozoa were determined. Several velocities parameters were also determined viz., average path velocity (VAP, μm/s), track speed (VCL,μm/s), progressive velocity (VSL, μm/s), lateral amplitude (ALH), beat frequency (BCF, Hz), straightness (%), elongation (%), linearity (%), and area (μm sq). The result revealed a strong positive correlation between various CASA parameters (PMOTVAP, PMOT- VSL, VAP-VSL, VAP-ALH, VSL-ALH, STR-LIN).The present study confirmed the usefulness of CASA for a quick and objective analysis of sperm concentration, motility and other velocity parameters.

102 Affect of Ram Semen Extenders and Supplements on Computer Assisted Sperm Analysis Parameters

102 Affect of Ram Semen Extenders and Supplements on Computer Assisted Sperm Analysis Parameters

Use of computer-assisted sperm analysis and flow cytometry to detect seasonal variations of bovine semen quality

Seasonal fluctuations of climate are considered a major factor affecting spermatogenesis and semen quality in the bovine. Our study aimed to investigate the effect of season on functional parameters of frozen-thawed bovine semen using computer-assisted sperm analysis (CASA) and flow cytometry. For this purpose, 86 ejaculates were collected from five mature Holstein-Friesian bulls kept under subtropical conditions during summer (August to September; n = 43) and winter (December to January; n = 43) months. Semen was diluted with a Tris-egg yolk-based extender and frozen at −196 °C. Computer-assisted sperm analysis was performed immediately after thawing (0h) and after 3 hours of incubation (3h) to evaluate the percentage (%) of total motile, progressively motile, and rapidly motile sperm. In addition, the average path, curvilinear, and straight-line velocities as well as the amplitude of lateral head displacement of sperm were determined. The percentages of sperm with intact plasma membrane and acrosome (PMAI, %), with high mitochondrial membrane potential (HMMP, %), with low intracellular Ca+2 levels (LOW-Ca+2, %), and with high DNA fragmentation index (DFI%, %) were flow cytometrically determined at 0 and 3h. The survival rate of sperm under hypotonic conditions (HYPO-SURV, %) and the percentage of sperm with inducible acrosome reaction (IAR, %) were assessed using flow cytometry at 0 and 3h, respectively. The fixed effect of season (winter vs. summer) on the quality parameters of sperm was explored by applying linear mixed-effects models. The results showed an improvement of all CASA parameters, except for the straight-line velocity (P > 0.05) in winter compared with summer for both unincubated and incubated sperm (P < 0.01 in all cases). Ejaculates collected in summer had lower values of IAR (P < 0.001) as well as PMAI, HMMP, and LOW-Ca+2 at 0 and 3h (P < 0.01 in all cases). On the contrary, HYPO-SURV and DFI% (at 0 and 3h) were not affected by season (P > 0.05 in all cases). Concluding, the employment of CASA and flow cytometry revealed season-related alterations in the functional status of cryopreserved bovine sperm, which suggest an adverse effect of summer heat stress on motility, plasma membrane and acrosome integrity, inducibility of acrosome reaction, mitochondrial function and intracellular Ca+2 content, but not on the DNA integrity of sperm after freezing–thawing.

Spermatozoa Motility Parameters of the Pumpkinseed (Lepomis gibbosus) in a Standardized Solution and Ambient Water.

The sunfish are classified as an invasive species after their introduction into a new habitat. Milt quality and spermatozoa movement in several sunfish were described. The aim of this study was to determine the basic milt characteristics and spermatozoa motility parameters in pumpkinseed males after using ambient water and a standardized solution to examine the parameters in mimicked natural conditions and in controlled conditions allowing interspecies data comparisons. Nine spermatozoa motility parameters were traced using computer-assisted sperm analysis (CASA) after activation with 30 mM NaCl and ambient water at 20°C. Spermatozoa motility parameters on activation, except for ALH, differed after using saline solution and ambient water. The CASA parameters were higher in the first medium. On the other hand, spermatozoa motility was high in both media, with an average of 89.6 and 85.2% in saline solution and ambient water, respectively. In the media, initial spermatozoa motility was characterized by a mean curvilinear velocity of 122.4 and 108.5 μm s-1, and a linearity of 79.7 and 67.1%, respectively. The active motility phase in the saline solution was approx. 8 min was longer than that measured in ambient water (mean 3 min). The pumpkinseed spermatozoa reveal good motility parameters, especially the high percentage of motile sperm, linearity, long duration of spermatozoa movement, as well as good velocity. It can be assumed that apart from reproductive strategy, spermatozoa motility parameters are valuable features of the male reproductive biology which may contribute to reproductive success.

Effects of preincubation of eggs and activation medium on the percentage of eyed embryos in ide (Leuciscus idus), an externally fertilizing fish

Standardization of fertilization protocols is crucial for improving reproductive techniques for externally fertilizing fish in captive breeding. Therefore, the objectives of this study were to determine the effects of preincubation of eggs and activation medium on the percentage of eyed embryos for ide (Leuciscus idus). Pooled eggs from five females were preincubated in three different activating media for 0, 30, 60, 90, and 120 seconds and then fertilized by pooled sperm from five males. At the eyed-egg stage, the percentage of viable embryos was later calculated. Results showed that preincubation time was significant for the freshwater activation medium (P < 0.001), such that the percentage of eyed embryos declined across the preincubation time gradient. Additionally, there was an effect on the percentage of eyed embryos when eggs were incubated with Woynarovich solution (P < 0.001), such that a decline was detected at 90 seconds, whereas no effect was detected for the saline water medium. Activating medium had a significant effect on the percentage of eyed embryos for each preincubation time (P < 0.05). More precisely, freshwater produced the lowest percentage of eyed embryos at all preincubation times (ranged from 1.9% at 120 seconds to 43.6% at 0 seconds), whereas saline water and Woynarovich solution produced the highest percentage of eyed embryos at 0 seconds and 30 seconds before incubation. Woynarovich solution produced the highest percentage of eyed embryos at 60 seconds (65.26%), whereas saline water produced the highest percentage at 90 seconds (68.37%). No difference was detected between saline water and Woynarovich solution at 120 seconds. Examination of sperm traits showed no impact of activating medium on computer assisted sperm analysis parameters. Together, these results suggest that saline water or Woynarovich solution improve fertilization rate in ide during IVF; thus, these media are useful for standardizing fertilization protocols and controlled reproduction for this species.

Cryopreservation of Siberian sturgeon (Acipenser baerii , Brandt, 1869) and sterlet (Acipenser ruthenus , Linnaeus, 1758) semen and its influence on sperm motility parameters assessed using a computer-assisted sperm analysis (CASA) system

Using a computer assisted sperm analysis (CASA) system, sperm motility parameters were determined in semen of the Siberian sturgeon (Acipenser baerii) and sterlet (Acipenser ruthenus) prior to and after cryopreservation. Semen was collected from 15 Siberian sturgeon and 15 sterlet males, diluted 1:1 (sperm:freezing medium) in 23.4 mm sucrose, 0.25 mm KCl, 30 mm Tris (pH of 8.0) and 10% methanol, loaded in 0.25 ml straws and frozen over liquid nitrogen vapor. Post-thaw sperm motility was activated in 10 mm Tris buffer (pH 8.5), containing 20 mm NaCl and 2 mm CaCl2. A significant decrease in the percentage of motile sperm (MOT: from 41.3% to 25.3%), curvilinear velocity (VCL: from 121.9 to 112.2 μm s−1), amplitude of lateral head displacement (ALH: from 10.4 to 5.4 μm) and momentum (MOM: from 4591 to 1038 m s−1) was observed as effect of the cryopreservation process in the Siberian sturgeon semen. A significant decrease in the values of most CASA parameters, including MOT, VAP, VCL, VSL, ALH and MOM, were also noted following the cryopreservation of sterlet semen. In the sterlet, an additional decrease in VAP (from 102.4 to 80.3 μm s−1) and VSL (from 86.8 to 69.8 μm s−1) values was noted after cryopreservation when compared to fresh semen. Unfortunately, the cryopreservation of semen from both species led to a drop in CASA parameters, which are important to the successful fertilization of eggs. MOT and VCL parameters were the most affected in these species. They can be used as sensitive criteria to check changes in motility.

Biochemical factors of common carpCyprinus carpioL. 1758, seminal plasma and its relationship with sperm motility parameters

Summary Compared were the volume and quality of carp Cyprinus carpio L. obtained from semen of males not subjected to hormonal stimulation (N = 10) but injected with 0.9% NaCl in the amount of 1 ml kg−1 b.w., as well as those stimulated using Ovopel (N = 10) in the amount of 1 granule kg−1 b.w. Semen was collected from the males 24 h after injection of hormonal preparation, to determine the pH, total volume (TVS) and volume recalculated per kg of body mass, concentration and total sperm production (TSP). Differences in sperm motility parameters between fish groups were determined by a computer-assisted sperm analysis (CASA) system. Sperm motility was activated using 10 mm Tris buffer, containing 100 mm NaCl (pH 9.0, osmolality 200 mOsm kg−1). The pH, osmolality, total protein concentration and activity of acidic phosphatase (AcP), lactate dehydrogenase (LDH), s-N-acetylglucosaminidase and arylsulfatase (AS) were determined in the seminal plasma of both test groups. The stimulation of male carp using Ovopel did not significantly affect CASA parameters, but did lead to an over ten-fold increase in the values of TVS and VOS and nearly six times higher values of TSP as compared to the Control group. A significant increase in pH following stimulation with Ovopel indicates that cyclic AMP increased, thanks to which sperm acquire a capacity to move during their passage through the sperm ducts. An increase in the values of CASA parameters is a likely reflection of these changes.

Variation among Beef Bulls in the Ratio of X- to Y-Chromosome Bearing Spermatozoa

A study was conducted to evaluate variation in the ratio of X- to Y-bearing sperm of individual ejaculates and to determine any relationship between skewed sex ratio and either routine morphological evaluation or computer assisted sperm analysis (CASA). Semen was collected from bulls weekly for 6 consecutive weeks, sperm DNA was recovered and quantitative, and real-time PCR was used to determine the ratio of X- to Y-bearing sperm in each ejaculate. The overall mean of X-bearing sperm within ejaculates was 54.7% over the 6 weeks of semen collections. The percentages of X-bearing sperm were similar (P > 0.5) across all collections. Between bulls, there were differences (P < 0.05) in the mean percentage of X-bearing sperm. No significant correlations were found between CASA parameters and percentage of X-bearing sperm across bulls, so analysis was done within each bull. Different combinations of CASA and/or morphological parameters were found to correlate with the percentage of X-bearing sperm but the prediction equations were specific for individual bulls and unlikely to be of use across bulls. These results confirm that the ratio of X- to Y-bearing sperm may be skewed in some ejaculates of bull semen. Some sperm parameters measured by CASA or routine morphological evaluation were associated with semen sex ratio.