Computer-assisted Sperm Analysis System Research Articles

Analysis of 12 kinds of cytokines in seminal plasma by flow cytometry and their correlations with routine semen parameters

ObjectiveTo investigate the levels of 12 kinds of cytokines in seminal plasma and their correlations with routine semen parameters. MethodsThe remaining seminal plasma samples of 134 patients undergoing routine semen examination were collected for detecting cytokines. The parameters for sperm concentration, percentage of progressively motile sperm (PR), and motility were analyzed by a computer-assisted sperm analysis (CASA) system. According to the results of sperm concentration, PR and motility, 134 patients were divided into the normal routine semen parameters group, oligoasthenospermia group and azoospermia group. The levels of 12 kinds of cytokines in seminal plasma, including interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17, interferin (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α, were detected by flow cytometry. Two seminal plasma samples were detected for 10 times, respectively, to calculate the coefficients of variation (CV) of each cytokine. The linear range of each cytokine was measured using the standard, and the correlation coefficient (r) was calculated. ResultsThe r2 of 12 kinds of cytokines detected by flow cytometry were all greater than 0.99. The reproducibility of 2 seminal plasma samples showed that the CVs of all cytokines were lower than 15 % except for TNF-α in sample 1 (15.15 %). Seminal plasma IL-6 levels were negatively correlated with semen volume (P < 0.01). Seminal plasma IL-5 levels were positively correlated with sperm concentration (P < 0.01). Seminal plasma IL-8 levels were negatively correlated with sperm motility (P < 0.01). Seminal plasma IL-8, IL-17 and IL-12P70 levels were negatively correlated with sperm PR (P < 0.05). In addition to the significant negative correlation between IL-5 and IL-17 (P < 0.05), there was a significant positive correlation between the majority of other cytokines. The levels of seminal plasma IL-17 and IL-12P70 in the oligoasthenospermia group and IL-1β and IL-12P70 in the azoospermia group were significantly higher than those in the normal routine semen parameters group (P ≤ 0.05), while the levels of IL-10 in the azoospermia group were significantly lower than that in the normal routine semen parameters group (P < 0.05). ConclusionThere are certain correlations between seminal plasma cytokines and routine semen parameters and strong correlations between different seminal plasma cytokines, suggesting that the imbalance between seminal plasma cytokines may affect sperm quality. However, it still needs to be further confirmed by large samples and multi-center clinical studies and related basic researches.

Influence of incorporating L-carnitine or Moringa oleifera leaves extract into semen diluent on cryosurvival and in vitro fertilization competence of buck sperm

This study aimed at scrutinizing efficiency of incorporating L-carnitine or M. oleifera leaves extract into semen diluent on improving cryopreservation capacity and in vitro fertilization ability of buck spermatozoa. Ejaculates (n=48) were collected by an artificial vagina from six adult Damascus bucks twice weekly during the breeding season (September–October). Following initial evaluation, ejaculates of each collection session from the same bucks were pooled, diluted (1:10) with glycerolized (3 % glycerol, v/v) tris-citric acid egg yolk diluent and were split into three aliquots. The first aliquot served as control, whereas the second and third aliquots were supplemented with 4 μL/mL L-carnitine and 400 μL/mL moringa leaves extract (v/v), respectively. Thereafter, all specimens were processed for cryopreservation and were stored in liquid nitrogen (-196 °C) for 12 months before post-thaw sperm criteria were analyzed by a computer-assisted sperm analysis (CASA) system. Integrity of sperm DNA post thawing was visualized in all semen groups by fluorescence imaging, and in vitro fertilization ability of spermatozoa was also determined. Inclusion of L-carnitine or moringa leaves extract into the diluent improved (P<0.05) post-thaw sperm physical, morphofunctional and kinematic attributes, whilst maintaining (P<0.05) integrity of sperm DNA throughout the freezing and thawing cycle. Consequently, both supplemented groups yielded higher (P<0.05) in vitro fertilization rates compared to control. These results accentuate the protective roles of these antioxidants on buck sperm against consequences of cryopreservation-induced oxidative stress, hence ameliorating post-thaw sperm quality and fertilization competence. This is crucial for successful application of AI and IVF in goat selective breeding programs.

P-052 Machine learning-based sperm motility grading model

Abstract Study question How to construct a machine learning-based assessment system for sperm motility and pick up the superior sperm with high motility accurately and efficiently? Summary answer Machine learning algorithms can be trained to build a grading model for the classification and grading of sperm motility. What is known already There are many sperm quality parameters for male fertility evaluation such as sperm concentration, viability, morphology, pH, and color of the semen. But, many studies agree that motility is the main parameter for sperm quality evaluation. Currently, there are mainly two methods for sperm motility determination. The traditional way relies on manual observation under a microscope, but this is subjective and time-consuming. Though there are some tracking errors in computer assisted sperm analysis (CASA) system, which help to acquire more motion parameters such as curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP). Study design, size, duration 3 000 sperm samples from clinical laboratory were divided to asthenospermia (AS) group and healthy control (HC) group based on their progressive motility. Sperm motility parameters were measured by CASA system. Participants/materials, setting, methods Nine variables of sperm motility including VCL, VSL, VAP, amplitude of lateral head displacement (ALH), mean angular displacement (MAD), linearity (LIN), straightness (STR), beat-cross frequency (BCF), fractal dimension (D) were collected for ML model training. The R software was used to build several classifiers on the samples investigated, including Random Forest, Adaptive Boosting (Ad Boost), Gradient Boosting, Support Vector Machine map (SVM), kNN (k-Nearest Neighbours), and Naïve Bayes. Main results and the role of chance Motility parameters differ between the two groups with a significant difference in VAP, VSL, VCL, and ALH. In order to classify the heterogeneous dataset in an unbiased manner, we performed a cross-validation procedure of training learning algorithms using six folds (equal parts) of the dataset, each time using a separate fold as the test dataset and the remaining folds as the training dataset. We initially examined whether the classifier could be reliably predicted using a subset of the sperm characterization’s 9 parameters (VCL, VSL, VAP, ALH, MAD, LIN, STR, BCF, D). We purposefully excluded additional characteristics that may be linearly related to increasing motility. We found four learning algorithms to have high accuracy, recall, precision, and F1 score. The support Vector Machine map performed best in classifying the sperm with high motility. Limitations, reasons for caution There may be some bias in data for sperm motility parameters. Specifically, different data collected from different laboratories or acquired from different equipment which may lead to a degradation in the performance of the machine learning model. Wider implications of the findings The analysis of large amounts of case data by machine learning algorithms can help clinician better understand the sperm motility characteristics of different patients so that they can develop a personalized treatment plan for each patient. Trial registration number not applicable

P-041 Impact of cigarette smoking on level of sperm reactive oxygen species in normozoospermic men

Abstract Study question Does cigarette smoking influence the levels of sperm reactive oxygen species (ROS) in men with normal semen routine parameters? Summary answer Cigarettes impact the sperm ROS of normozoospermic smokers, whose sperm ROS level is elevated when they smoke equal to or more than 10 cigarettes daily. What is known already Increasing evidence indicates that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated ROS levels in semen. Cigarette smoking leads to an imbalance between ROS and antioxidants in the semen of smokers, which may negatively affect semen quality and male fertility. However, few studies concern whether cigarette smoking would influence the sperm ROS levels in normozoospermic men. Study design, size, duration Data of semen analysis from January 2021 to December 2023 were involved. Men with normal semen routine parameters, including sperm concentration, total sperm number, sperm progressive motility, total motility and normal forms of sperm morphology, were included in the study. Sperm ROS levels of normozoospermic men were retrospectively analyzed. Men with varicocele and leucocytospermia were excluded. Participants/materials, setting, methods Sperm count and motility were analyzed by the computer-assisted sperm analysis (CASA) system, while sperm morphology was examined after the Diff-Quik rapid staining procedure. Sperm ROS was measured by flow cytometry using the probe DCFH-DA. Sperm ROS levels of smokers and non-smokers were compared by the Mann-Whitney U test. The Kruskal-Wallis H test was used for the comparison of sperm ROS levels among the control group and smoking subgroups. Main results and the role of chance A total of 2946 subjects were involved in the present study. The included subjects were divided into smokers (n = 730) and non-smokers (n = 2216) according to the clinical records. And the included smokers were subsequently divided into three subgroups according to the daily cigarettes taken, which were group A (1∼9 cigarettes/day, n = 184), group B (1∼19 cigarettes/day, n = 315), and group C (&amp;gt;20 cigarettes/day, n = 231). No statistically significant differences in age or BMI were found between smokers and non-smokers with normozoospermia. The sperm ROS level of the smoker was higher than that of the non-smoker [14.53% (14.84%) vs. 13.07% (12.09%), p = 0.001]. Compared to the control group (non-smoker), the sperm ROS levels of group B [13.07% (12.09%) vs. 15.44% (14.28%), p = 0.002] and group C [13.07% (12.09%) vs. 14.80% (14.31%)., p = 0.042] were significantly elevated. ROS levels in the control group and group A were comparable [13.07% (12.09%) vs. 13.46% (15.74%)., p = 0.391]. Limitations, reasons for caution It is a single-center study and the above results were from a retrospective analysis. Wider implications of the findings The above results indicate that cigarette smoking, although it would not deteriorate semen routine parameters, may increase the ROS level in sperm. Elevated ROS in sperm may lead to an imbalance of redox in sperm, which may contribute to male infertility. Trial registration number not applicable

Hydroxytyrosol Ameliorates Busulfan-Induced Oligozoospermia in a Mouse Model

Objective Hydroxytyrosol (HT), a polyphenolic component identified in olive oil and leaves, represents one of the most extensively investigated natural phenol due to its diverse beneficial biological properties. Recent studies have shown that HT exhibits protective effects on reproductive function and can improve sperm quality. However, the precise effect and underlying mechanism of HT on oligozoospermia remain unclear. This study aims to investigate the effect and mechanism of HT across varying concentrations on oligozoospermia. Methods Oligozoospermia was induced in ICR male mice via intraperitoneal injection of busulfan (BUS, 30 mg/kg). The mice were randomly categorized into five groups as Normal, BUS, HT 10 mg/kg, HT 30 mg/kg and HT 50 mg/kg. The efficacy of HT on oligozoospermia was assessed through sperm concentration and motility using a computer-assisted sperm analysis system. Testicular and epididymal histology was examined via hematoxylin-eosin staining, while cytoskeletal integrity was evaluated by immunofluorescence. Hormone levels, along with reactive oxygen species (ROS) and malondialdehyde (MDA) were examined by ELISA kits. Differentially expressed genes were identified through RNA sequencing, and the expression levels of key genes were validated by qPCR. Results The results indicated that HT (50 mg/kg) significantly increased sperm concentration, promoted morphological recovery of testicular tissue, heightened the levels of serum testosterone, follicle-stimulating hormone, luteinizing hormone, as well as decreased the accumulation of ROS and MDA in mice with oligozoospermia. RNA sequencing analysis and RT-qPCR results revealed that HT could restore spermatogenesis and ameliorate oligozoospermia by up-regulating genes related to cilium organization, microtubule-based movement, and spermatid differentiation and development. Conclusions HT demonstrates therapeutic efficacy against busulfan-induced oligozoospermia. The mechanism of HT on oligozoospermia may be related to its influence on cilium and microtubule-based movement, along with the differentiation and development of spermatids. HT shows potential as a prospective therapeutic intervention for the treatment of oligozoospermia.

Effects of MnTBAP on Porcine Semen Cryopreservation and Capacitation.

Antioxidants protect cellular function and structure by neutralizing the oxidative stress caused by increased reactive oxygen species (ROS) during sperm freezing. Studies on cryopreservation using various antioxidants have demonstrated encouraging results. Many studies have used antioxidants to increase the efficiency of sperm freezing and to improve the success rate of artificial insemination and pregnancy. Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) is a newly synthesized antioxidant with positive effects on sperm morphology and capacitation in humans, rams, and stallions. In this study, porcine semen was treated with 0, 50, 100, and 150 μM of MnTBAP based on a Tris-egg-yolk extender and frozen to determine whether MnTBAP can assist the status of sperm during cryopreservation. First, motility was assessed using the computer-assisted sperm analysis (CASA) system, with the 100 μM treatment group showing the highest motile rate (66.8%) compared with that of the other groups (control, 51.1%; 50 μM and 150 μM, 59.6%); therefore, the remaining analyses were conducted comparing the two groups (control vs. 100 μM group; p < 0.01). Second, fluorescence staining was applied to examine the control and 100 μM groups using fluorescence microscopy. The viability (41.7% vs. 62.4%) and the acrosome integrity (77.9% vs. 86.4%) differed significantly (p < 0.05). In addition, the mitochondrial membrane potential (MMP) was 46.5% vs. 51.9%; the fragmentation rate, estimated using the Sperm-sus-Halomax kit, was 63.4% vs. 57.4%; and the detected caspase activity was 30.1% vs. 22.9%. These tended to be higher in the treated group but did not differ significantly. Third, measurements using FACSLyric revealed that the 100 μM treatment group exhibited a state of elevated normal lipid arrangement within the plasma membrane and diminished levels of apoptosis and ROS (p < 0.01). We assessed the expression of genes relevant to antioxidant effectiveness using real-time RT-qPCR. Our findings indicated significant alterations in the expression levels of various mRNA species, with the exception of NOX5 (p < 0.05). Finally, the straws were dissolved and used to treat matured denuded oocytes to investigate the effect on fertilization and embryo development in vitro. The cleavage rate was (77.6% vs. 84.1%), and the blastocyst rate was 9.7% vs. 11.4% (p < 0.05). In conclusion, these results suggest that MnTBAP positively affected sperm freeze-thawing, improving the fertilization capacity, and leading to increased embryo development.

Ultra-processed food consumption and semen quality parameters in the Led-Fertyl study.

Is ultra-processed food (UPF) consumption associated with semen quality parameters? Higher UPF consumption was inversely associated with total sperm count, sperm concentration, and total motility in men of reproductive age. The consumption of UPF, which has been rising during the last decades, has been demonstrated to be positively associated with several chronic diseases such as diabetes or cardiovascular diseases. However, the scientific evidence on its potential impact on semen quality remains notably limited. A cross-sectional analysis was conducted using data from 200 healthy men (mean age 28.4 ± 5.5 years) enrolled in the Led-Fertyl (Lifestyle and Environmental Determinants of Seminogram and Other Male Fertility-Related Parameters) study between February 2021 and April 2023. UPF consumption (% of energy from UPF) was estimated according to the NOVA classification system using a validated 143-item semi-quantitative food frequency questionnaire. Total sperm count, sperm concentration, sperm vitality, total motility, progressive motility, and normal sperm forms were set as the main outcomes. Microscopic parameters were analyzed using a phase-contrast microscope and a computer-assisted sperm analysis (CASA) system. Semen samples were collected and tested according to World Health Organization 2010 standards. Multivariable linear regression models were fitted to estimate the associations between UPF tertile and semen quality parameters. Sperm concentration (β: -1.42 × 106 spz./ml; 95% CI: -2.72 to -0.12) and motility (β: -7.83%; 95% CI: -15.16 to -0.51) were lower in participants in the highest tertile of UPF compared to the lowest. A similar association was observed for sperm count when UPF was analyzed per 10% increment of energy from UPF consumption (β: -1.50 × 106 spz.; 95% CI: -2.83 to -0.17). Theoretically replacing 10% of energy from UPF consumption with 10% of energy from unprocessed or minimally processed food consumption was associated with a higher total sperm count, sperm concentration, total motility, progressive motility, and normal sperm forms. Cross-sectional studies do not permit the drawing of causal inferences. Measurement errors and reporting bias cannot be entirely ruled out. This work suggests that consumption of UPF may have an impact on certain semen quality parameters. Furthermore, opting for unprocessed or minimally processed foods instead of UPFs could potentially benefit semen quality. If these results are replicated in future epidemiological studies with different long-term designs, these novel findings could provide valuable insights for updating or even designing preventive and interventional programs to address infertility among men of reproductive age. This study was supported by the Spanish government's official funding agency for biomedical research, ISCIII, through the Fondo de Investigación para la Salud (FIS), the European Union ERDF/ESF, 'A way to make Europe'/'Investing in your future' [PI21/01447], and the Diputació de Tarragona (2021/11-No.Exp. 8004330008-2021-0022642). J.S.-S. gratefully acknowledges the financial support of ICREA under the ICREA Academia program. C.V.-H. received a predoctoral grant from the Generalitat de Catalunya (2022 FI_B100108). M.Á.M. was supported by the Sara Borrell postdoctoral fellowship (CD21/00045-Instituto de Salud Carlos III (ISCIII)). M.F.d.l.P. was supported by a predoctoral grant from the Rovira i Virgili University and Diputació de Tarragona (2020-PMF-PIPF-8). All authors have no conflict of interest to declare. N/A.

PLCζ, WBP2NL and TNF-α expression in spermatozoa is associated with stallion fertility and seminal quality?

This study aims to investigate the gene expression of sperm-borne phospholipase C zeta (PLCζ), WW domain-binding protein 2N-Terminal Like (WBP2NL), and Tumor necrosis factor (TNF-α), as a negative control, in spermatozoa and their relationship with fertility and seminal quality in stallions. Ejaculates from 40 Criollo stallions were used, whose fertility was assessed on the basis of their pregnancy rate per cycle in at least two breeding seasons. Pregnancy rates ranged from 20% to 90% and were used to divide the stallions into two groups: High rates (≥ 50%) (n = 25), and Low rates (< 50%) (n = 15). A computer-assisted sperm analysis system - (CASA) analyzed semen after collection. Also were evaluated the physical and functional integrity of the plasmatic membrane and sperm morphology alterations. All stallions expressed PLCζ, WBP2NL, and TNF-α. PLCζ positively correlates with conception rate, total motility (TM), progressive motility (PM), plasmatic membrane functionality, and integrity. A simple linear regression was detected between pregnancy rate and PLCζ expression (P = 0.003), TM (P < 0.001) and PM (P < 0.001). PLCζ gene expression was higher (P = 0,012) in the High rates group than in the Low group. WBP2NL and TNF-α did not correlate with seminal quality and stallion's fertility. It was concluded that PLCζ gene expression in the spermatozoa might be used as a biomarker of fertility and seminal quality in stallions. Parameters of sperm kinetics also showed, positive correlation between TM, PM and pregnancy rate.

Enhancing Sperm Motility Parameters in Patients with Asthenospermia: A Combined Approach of Acupuncture at Fusiguan Point and Tamoxifen Citrate Tablets.

To explore the effect of acupuncture at Fuguan point combined with tamoxifen citrate tablet on sperm motility parameters. A total of 115 individuals with asthenospermia were categorized based on different treatment regimens: 53 patients in the control group (receiving tamoxifen citrate tablets) and 62 patients in the observation group (undergoing acupoint acupuncture in conjunction with tamoxifen citrate tablets). Both groups underwent a 3-month treatment period. The computer-assisted sperm analysis system was employed to measure various motility parameters of human sperm, including sperm motility rate, average path velocity (VAP), lateral swing amplitude (ALH), percentage of class a sperm, and percentage of class a + b sperm. Prior to treatment, no statistically significant differences were observed between the two groups in terms of sperm motility rate, VAP, ALH, percentage of class a sperm, and percentage of class a + b sperm (p > 0.05). Following treatment, both groups exhibited significant enhancements in sperm motility rate, VAP, ALH, percentage of class a sperm, and percentage of class a + b sperm compared to pretreatment levels (p < 0.05). Furthermore, all measured indicators in the observation group demonstrated significantly superior improvements than those of the control group, with the differences proving statistically significant (p < 0.05). The combination of acupuncture at Fusiguan point and tamoxifen citrate tablets exerts a notably positive effect on sperm motility in individuals diagnosed with asthenospermia.

Ursolic acid attenuates oligospermia in busulfan-induced mice by promoting motor proteins.

Oligospermia is one of the most common reasons for male infertility which is troubling numerous couples of child-bearing age. This investigation scrutinizes the implications and mechanistic underpinnings of ursolic acid's effect on busulfan-induced oligospermia in mouse models. A singular intraperitoneal injection of busulfan at a dosage of 30 mg/kg induced oligospermia. Two weeks subsequent to this induction, mice were subjected to various dosages of ursolic acid (10, 30, and 50 mg/kg body weight, respectively) on a daily basis for four consecutive weeks. Following this treatment period, a meticulous analysis of epididymal sperm parameters, encompassing concentration and motility, was conducted using a computer-assisted sperm analysis system. The histopathology of the mice testes was performed utilizing hematoxylin and eosin staining, and the cytoskeleton regeneration of the testicular tissues was analyzed via immunofluorescent staining. Serum hormone levels, including testosterone, luteinizing hormone, and follicle-stimulating hormone, as well as reactive oxygen species levels (inclusive of reactive oxygen species and malondialdehyde), were gauged employing specific enzyme-linked immunosorbent assay kits. Differentially expressed genes of testicular mRNA between the oligospermia-induced group and the various ursolic acid treatment groups were identified through RNA sequencing analysis. The results revealed that a dosage of 50 mg/kg ursolic acid treatment could increase the concentration of epididymal sperm in oligospermia mice, promote the recovery of testicular morphology, regulate hormone levels and ameliorate oxidative damage. The mechanism research results indicated that ursolic acid increased the expression level of genes related to motor proteins in oligospermia mice.

Specific setup and methodology for computer-assisted sperm analysis (CASA) in evaluating elasmobranch sperm

1.One-third of elasmobranch species are threatened with extinction, and the precise assessment of their sperm parameters and quality is one stage in the development of reproductive technology for animal conservation.2.Computer-Assisted Sperm Analysis (CASA) is the gold standard for sperm analysis; however, there were not one setup established for elasmobranchs.3.This study presents the description of a specific setup developed for elasmobranch semen analysis using the CASA system.4.In addition, it provides a gold-standard technique protocol for elasmobranch sperm quality assessment.5.The described setup presented an accuracy of more than 90 percent in fourteen distinct species of freshwater and marine sharks, stingrays, and rays, failing only when there were two or more bundled sperm.

Comparative study on the gamete quality, artificial propagation and larval development of common goldfish, shubunkin, black moor, and oranda variants of goldfish (Carassius auratus)

Due to the origin and diversity of goldfish, it is a suitable model organism to investigate the effect of artificial selection on reproduction and larval development. In the present study, four, well-known variants of goldfish (common goldfish, shubunkin, black moor, oranda) with considerable phenotypical differences were compared within the framework of gamete quality assessment, propagation trial, larvae rearing and morphology assessment. In our first experiment, sperm concentration, sperm volume per fish (sperm volume) and volume of sperm per kg of body weight of males did not differ significantly between the variants. Concerning the measured Computer-Assisted Sperm Analysis (CASA) system parameters, shubunkin and oranda had significantly higher progressive motility (pMOT, %) than black moor. Tendencies in female gamete production were observed in the second experiment, despite not all cases displaying significant differences. For pseudo-gonadosomatic index (PGSI, %), higher values were recorded for the globular-bodied goldfish, while the contrary was seen for the number of eggs per gram. Out of the four variants, a significantly higher fertilization rate was determined for oranda, while a significantly lower hatch rate was calculated for black moor. In the third experiment, a significant difference in the body length of freshly hatched larvae was observed between common goldfish and shubunkin during larvae rearing. However, at 3 days post-hatching (dph), shubunkin and oranda had a significant difference in this parameter. At 10 dph, common goldfish and shubunkin had significantly lower body weights than black moor and oranda. Moreover, developmental variances between common goldfish and black moor were observed in larvae morphology. At the end of larvae rearing, a significantly higher survival rate was recorded for common goldfish than for shubunkin and oranda. Even though not all parameters show considerable differences between the variants, black moor is far behind the others in terms of hatching rate and morphological abnormalities.Thousands of years of artificial selection have had a noticeable mark on the reproductive capacity and larval development of goldfish variants studied. Our results may contribute to further research on the impact of artificial selection on reproductive biology.

Supplementing semen extenders with a combination of phosphorus and vitamin B12 Improves post-thawed cryopreserved rooster semen quality.

Semen cryopreservation is an important technique for preserving the genetic material of numerous species. However, frozen semen is highly susceptible to sperm DNA damage and reduced motility, resulting in decreased fertility. The standard method for cryopreservation and several approaches have not been elucidated. This study aimed to determine the effects of supplementing rooster semen extender with a combination of phosphorus and vitamin B12 on cryopreserved semen quality. Semen was collected weekly via dorso-abdominal massage from 57 Burmese × Vietnam-crossbred Thai native roosters aged 1-3 years. In total, 139 semen samples were collected, pooled, and diluted to 200 million sperm per dose. The pooled sample was divided into six experimental groups: a control group (0.00%) diluted with modified Beltville Poultry Semen Extender (BPSE) and five treatment groups diluted with modified BPSE supplemented with phosphorus and vitamin B12 at concentrations 0.02, 0.04, 0.06, 0.08, and 0.10%, respectively. The semen samples were frozen and evaluated at 0, 15, and 30 min after thawing. Sperm kinematic parameters were determined using a computer-assisted sperm analysis system. Sperm quality was evaluated by measuring sperm viability, mitochondrial activity, acrosome integrity, and plasma membrane integrity. Statistical analyses were performed using a general linear mixed model (MIXED) in SAS. Factors in the statistical model were experimental groups, time after thawing, and interaction between experimental groups and time after thawing. Total and progressive motilities were greater in semen supplemented with 0.04% phosphorus and vitamin B12 compared with those in the control (p < 0.05). At 15 min post-thawing, VCL, VAP, and HPA in the 0.04% phosphorus and vitamin B12 supplementation group was greater than that in the control (p < 0.05). Phosphorus and vitamin B12 supplementation did not affect sperm kinematics at 0 and 30 min after thawing (p > 0.05). All the sperm parameters that were tested for the 0.04% phosphorus and vitamin B12 supplementation group in modified BPSE were the highest at all the timepoints after thawing. Thus, supplementing frozen semen extender with 0.04% phosphorus and vitamin B12 increased sperm motility, sperm kinematic parameters, and sperm quality.

Starvation-induced autophagy modulates spermatogenesis and sperm quality in Nile tilapia

Spermatogenesis is a finely regulated process that involves the interaction of several cellular mechanisms to ensure the proper development and maturation of germ cells. This study assessed autophagy contribution and its relation to apoptosis in fish spermatogenesis during starvation. To that end, Nile tilapia males were subjected to 0 (control), 7, 14, 21, and 28 days of starvation to induce autophagy. Testes samples were obtained for analyses of spermatogenesis by histology, electron microscopy, immunohistochemistry, and western blotting. Sperm quality was assessed using a computer-assisted sperm analysis (CASA) system. Data indicated a significant reduction in gonadosomatic index, seminiferous tubule area, and spermatozoa proportion in fish subject to starvation compared to the control group. Immunoblotting revealed a reduction of Bcl2 and Beclin 1 associated with increased Bax and Caspase-3, mainly after 21 and 28 days of starvation. LC3 and P62 indicated reduced autophagic flux in these starvation times. Immunolabeling for autophagic and apoptotic proteins occurred in all development stages of the germ cells, but protein expression varied throughout starvation. Beclin 1 and Cathepsin D decreased while Bax and Caspase-3 increased in spermatocytes, spermatids, and spermatozoa after 21 and 28 days. Autophagic and lysosomal proteins colocalization indicated the fusion of autophagosomes with lysosomes and lysosomal degradation in spermatogenic cells. The CASA system indicated reduced sperm motility and velocity in animals subjected to 21 and 28 days of starvation. Altogether, the data support autophagy acting at different spermatogenesis stages in Nile tilapia, with decreased autophagy and increased apoptosis after 21 and 28 days of starvation, which results in a decrease in the spermatozoa number and sperm quality.

Performance evaluation of sperm concentration, motility, and morphological analysis for GSA-810 series of sperm quality analysis system.

The performance evaluation of each computer-assisted sperm analysis (CASA) system may provide a basis for the interpretation of clinical results and further improvement of the CASA system. The accuracy of the GSA-810 CASA system was evaluated by detecting latex bead quality control products. The precision of sperm concentration, morphology, and percentages of progressively motile sperm (PR) were evaluated by coefficient of variation (CV). Three samples with sperm concentration of about 100 × 106 /mL were diluted to evaluate the linear range. The detection values of latex beads were within the range of target values. The CVs of sperm concentration and PR were significantly and negatively correlated with sperm concentration (r = -0.561, p = 0.001) and PR value (r = -0.621, p < 0.001), respectively. The R2 values of the linear range of sperm concentration were ≥0.99. There was no significant difference in sperm motility and PR within 1-10 min at 36.5°C ± 0.5°C. The coincidence rates of sperm morphology and sperm head morphology for 36 semen samples analyzed by the GSA-810 system and manual method were 99.40% and 99.67%, respectively. The CVs of the percentage of sperm with abnormal morphology and percentage of sperm with abnormal head morphology were less than 5%. The GSA-810 system can accurately analyze normal semen samples, but the repeatability of the results is poor for oligozoospermia and asthenozoospermia samples. The future CASA system for analyzing sperm morphology should focus on recognizing the middle and tail segments of a spermatozoon.

Improving the quality of chilled semen from Thai native chicken using phosphorus and vitamin B12 supplementation in semen extender

This study aimed to determine phosphorus and vitamin B12 supplementation effect in semen extender on the quality and fertility ability of chilled Thai native rooster semen. Eighty-four ejaculates of semen from 26 Thai native roosters (Burmese x Vietnam crossbreed) were included. Semen was collected by applying dorsal–abdominal massage once a week, pooled, diluted to 500 million sperms per dose, and divided into six groups. The semen samples used for control group were diluted with modified Beltsville poultry semen extender (BPSE). For the treatment groups 2–6: semen samples were diluted with modified BPSE and enriched with phosphorus and vitamin B12 (Octafos® Octa Memorial Co., Ltd., Bangkok, Thailand) at concentrations 0.02%, 0.04%, 0.06%, 0.08%, and 0.10%. Semen fertility ability was tested in six replications by inseminating layer hens. Thirty-six Thai native hens were randomly assigned to three groups (control, 0.04%, and 0.08%) of 12 hens and were inseminated with a dose of 0.2 mL on collecting day. Sperm motion characteristics (i.e., sperm motility, sperm progressive motility, and sperm kinetic parameters) were measured using a computer-assisted sperm analysis system (SCA®, Proiser S.L., Valencia, Spain). Sperm viability, mitochondrial activity, acrosome integrity, plasma membrane integrity, and malondialdehyde (MDA) concentration were also evaluated. The sperm motion characteristics were the highest in the 0.04% supplementation group on all days of collection, especially the VCL and VAP (P < 0.05). The viability, mitochondrial activity, plasma membrane and acrosome integrity of spermatozoa were greater in the 0.04% supplementation group than in the control groups (P < 0.05). The 0.04% supplementation group had the lowest MDA concentration in all days of collection. The 0.04% supplementation group were higher both fertility (66.59% vs. 48.50%: P < 0.05) and hatching rates (58.80% vs. 43.18%: P < 0.05) than in the control group. In conclusion, 0.04% phosphorus and vitamin B12 concentrations supplementation in semen extender improved rooster semen quality and fertility in chilled rooster semen.

Granulocyte-Macrophage Colony-Stimulating Factor Cytokine Addition After the Freeze-Thawing Process Improves Human Sperm Motility and Vitality in Asthenoteratozoospermia Patients.

The cryopreservation-thawing process of spermatozoa cells has negative impacts on their structure, function, and fertility parameters, which are known as cryoinjury. Asthenozoospermia patients are more susceptible to cryoinjury. Granulocyte-macrophage colony-stimulating factor (GM-CSF) increases sperm glucose uptake via the induction of glucose transporters, resulting in increased sperm motility. This study aimed to investigate the efficiency of GM-CSF supplementation of the cryopreservation media for semen samples of asthenoteratozoospermia patients. The study was carried out on 20 semen samples from infertile men referred to diagnosing semen analysis. To avoid subjective bias, two main sperm motility parameters, including velocity along the curvilinear path and velocity along the straight-line path were considered by the computer-assisted sperm analysis system. Afterward, each semen sample was divided into three equal aliquots and randomly assigned to one of the following groups: group I (control, freezing media only), group II (+GM-CSF, freezing medium supplemented with 2 μL/mL GM-CSF), or group III (GM-CSF added after thawing and washing). Following semen thawing, standard parameters, mitochondrial membrane potential (MMP), and the DNA Fragmentation Index were analyzed. Total sperm motility (progressive and non-progressive) improved significantly in group III samples after a 30-minute incubation with GM-CSF compared with the control group (26.5% ± 3.1% vs. 17.51% ± 2.59%). However, no differences in progressive motility or sperm morphology were found among the three thawed samples. The percentage of vitality was significantly higher in group III compared with the other two groups (28.38% ± 3.4% vs. 22.4% ± 3.08% and 22.14% ± 2.77%, respectively) (p < 0.05). JC-1 levels (a marker of MMP) were not significantly different between the examined groups (44.95% ± 8.26% vs. 36.61% ± 6.95% vs. 46.67% ± 7.7%, for control, group II, and group III, respectively) (p > 0.05). GM-CSF may be advantageous as an additive after freezing, improving total motility and viability after 30 minutes of post-thaw incubation; however, when supplied to the freezing media before cryopreservation, it is unable to protect against cryoinjury.

The growth performance of pond-reared common carp (Cyprinus carpio) larvae propagated using cryopreserved sperm.

The aim of our study was to determine the efficacy of utilizing cryopreserved common carp sperm (in comparison to fresh sperm) for propagation at a Hungarian aquaculture facility. The sperm was frozen in 5mL straws using an extender method that was previously tested in common carp. Sperm motility was monitored using a computer-assisted sperm analysis system. The hatching and malformation rates among the specimens were recorded before the stocking of larvae in both groups. The growth (body weight, total length) and survival rates of the fish were measured during the pre-nursing (from May to June: between 1 and 26days post hatching) and grow-out periods (from June to October: between 26 and 105days post hatching) of the same year. The fresh sperm, which was collected and pooled prior to fertilization, showed high MOT (97%), pMOT (92%), VCL (106µms-1), LIN (75%), and ALH (1.84µm). Prior to the fertilization trial of the cryopreserved sperm, low MOT (34%), pMOT (14%), and VCL (61µms-1) values were observed in frozen-thawed sperm. A significantly higher hatching rate was measured in the fresh sperm group (87%) when compared to the cryopreserved sperm group (42%). No significant difference in the overall malformation rate was observed in larvae originating from either the fresh or frozen sperm. A significant difference between the two test groups was observed in the incidence of deformed tails (fresh: 20%, cryopreserved: 55%). Except for one sampling period, no significant difference in the body weight and total length of the fish larvae was found between the two groups throughout the pre-nursing and grow-out periods. A significantly higher larvae survival rate was noted in the fresh sperm (72%) as compared to the cryopreserved group (43%) by the end of the pre-nursing stage. However, no significant difference in survival rate was observed for the cryopreserved sperm (96%) in comparison to the fresh sperm (95%) by the end of the grow-out stage. The results of this study showed, for the first time in large-scale pond culturing, an equal growth and viability in larvae propagated from cryopreserved sperm when compared to fresh sperm (despite the limited available rearing ponds provided by the commercial company).

Can in vitro embryo production be estimated from semen variables in Senepol breed by using artificial intelligence?

Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

Seasonal and age-related changes in sperm quality of farmed arctic charr (Salvelinus alpinus)

BackgroundSubstantial variation in male fertility is regularly observed in farmed Arctic charr. However, detailed investigations of its fluctuation during a reproductive season and across years are lacking. Furthermore, information about the effect of underlying genetic factors influencing sperm quality is scarce. The current study focused on seasonal and age-related factors that may affect sperm quality characteristics in males reared in natural and delayed photoperiods. Animals were sampled three times for two consecutive years, and sperm quality parameters were recorded using a computer-assisted sperm analysis (CASA) system. Thereafter, high-throughput sequencing technologies were applied, aiming to identify genomic regions related to the variation of sperm quality throughout the reproductive season.ResultsAn across-season variation in the recorded sperm quality parameters was evident. Overall, 29% and 42% of males from the natural and delayed spawning groups had a highly variable total progressive motility. Males at four years of age showed significantly higher sperm motility and velocities during the early October and November recordings compared to the following year when the same animals were five years of age. On the other hand, the opposite was observed regarding sperm concentration during the last sampling. A genome-wide FST scan detected SNP differentiation among males with high and low variability in total progressive motility (PM) on eight chromosomes (FST > 0.17), Genome wide windows with the highest FST contained SNPs in proximity (within 250 kb up- and downstream distance) to 16 genes with sperm quality biological functions in mammalian species.ConclusionOur findings provide a detailed view of seasonal, age-related, and genetic effects on sperm quality and can be used to guide decisions on broodstock selection and hatchery management.