Computer-assisted Semen Analysis System Research Articles

Post-thaw quality of boar spermatozoa is affected by ejaculate fractions and extenders.

The aim of this study was to investigate the effect of different extenders on the post-thaw (PT) quality of sperm originating from the sperm-rich fraction (SRF) and post-sperm-rich fraction (PSRF) of boar ejaculate. Motility and velocity parameters, analyzed using a computer-assisted semen analysis (CASA) system, and membrane integrity parameters were markedly higher in frozen-thawed (FT) spermatozoa of the SRF in both the Belstville Thawing Solution (BTS) and Androhep Plus (AHP) extenders, irrespective of the post-thaw (PT) storage time. Furthermore, reduced cryo-survival was more marked in FT spermatozoa of the PSRF in both extenders following storage for 60 min. It was found that the SRF-stored samples in the AHP extender for 60 min exhibited significantly higher percentages of spermatozoa with total motility, mitochondrial function and acrosome integrity than those stored in the BTS extender. The findings of this study confirm that components of the ejaculate fractions and extender have varying effects on the cryo-survival of boar spermatozoa.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. N/A. This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Prevalence of Chlamydophila spp. and Canid herpesvirus-1 in Polish dogs.

Chlamydophila spp. affect Leydig and Sertoli cells by dysregulating spermatogenesis, inducing apoptosis and sperm DNA fragmentation, as well as benign prostate hyperplasia. Canid herpes virus 1 (CHV-1) infection in male dogs is manifested by lesions on the base of the penis and foreskin. There is a lack of information on the influence of these microorganisms on the quality of canine semen. Seroprevalence of Chlamydophila spp. (55%-61%) and CHV-1 (22%-81%) in Europe is high. The prevalence of Chlamydophila spp. and CHV-1 has been evaluated using polymerase chain reaction (PCR) only in Sweden and Croatia, respectively. No positive samples were detected in either case. The aim of this study was to evaluate the epidemiological situation in Polish male dogs (PMDs) to provide a solution to limit the spread of these microorganisms using assisted reproduction techniques or elimination from the reproduction of CHV-1 carriers. In addition, we assessed the semen quality of Chlamydophila spp. carriers and CHV-1 carriers. Cotton swabs were collected from prepuce or semen from each dog (n = 130). Real-time PCR for Chlamydophila spp. and CHV-1, as well as semen analysis, was performed using the computer-assisted semen analysis system. To the best of our knowledge, this is the first report of Chlamydophila spp. infection in PMD confirmed by real-time PCR. All parameters, except progressive movement in Chlamydophila semen carriers, were normal. The average velocity values for a dog with Chlamydia are detailed. No CHV-1 was detected. The results achieved should be verified on the basis of a larger number of studies. However, the high prevalence of these pathogens in the PMD population has not been established.

Protocolo de congelamento one-step para sêmen suíno utilizando diferentes crioprotetores

ABSTRACT: The present study evaluated the cryoprotectant efficacy of dimethylacetamide (DMA) and ethylene glycol in a one-step protocol to freeze boar sperm. The sperm-rich portion of the ejaculates from two boars were collected once a week, for 10 weeks. After collection, the ejaculates were diluted (1:1; v/v) in the cooling extender. After determining their spermatozoa concentration, the ejaculates were pooled with the same number of spermatozoa from each boar and stabilized at 20°C for 120 min. Distinct cryoprotectants were added to the cooling extender at 20 °C, at different concentrations, composing six treatments: 1.25% and 2.5% glycerol (control); 1.25% and 2.5% ethylene glycol; 2.5% and 5.0% DMA. The samples were stored in 0.25 mL straws, containing 35 × 106 spermatozoa. After 90 min at 20 °C, the straws were submitted to a cooling curve until 5 °C (0.3 to 0.5 °C/min) and kept at 5°C for 60 min. Freezing was conducted by placing the straws horizontally 5 cm above the liquid nitrogen for 10 min, followed by immersion on liquid nitrogen. After thawing at 37 °C for 30 seconds, sperm quality was evaluated through a computer-assisted semen analysis system and flow cytometry. Sperm motility was greater (P< 0.05) in treatments with 5.0% and 2.5% DMA (22.2 ± 2.6% and 20.0 ± 2.8%, respectively) than in treatment with 2.5% ethylene glycol (8.2 ± 1.0%). The integrity of the plasma membrane (P = 0.08) and mitochondrial membrane potential (P = 0.27) was similar among the treatments. The treatment with 2.5% ethylene glycol was the least efficient to maintain intact acrosome membrane (P< 0.01). Some kinetics parameters (DAP, DCL, DSL, VAP, VCL, VSL e ALH) were positively affected by 5.0% DMA. The one-step freezing protocol resulted in unsatisfactory boar sperm motility after thawing, regardless of the cryoprotectant.

Exploration of semen quality analyzed by casa-mot systems of brahman bulls infected with BLV and BHV-1

Enzootic bovine leukosis virus (BLV) and bovine herpesvirus 1 (BHV-1) are very important infectious agents for the livestock industry worldwide. The present study aimed to explore the association between natural exposure to BLV and BHV-1 with sperm quality analyzed by Computer-Assisted Semen Analysis (CASA) systems. Ten sexually mature Brahman bulls, with sanitary status BLV+/BHV-1+ (n = 2), BLV−/BHV-1+ (n = 6) and BLV-/BHV-1- (n = 2) were evaluated twice, 30 days apart. Results showed that sanitary status of each bull was not associated with semen quality. It was found that the quality of the semen from the second collection was better due to the interruption of sexual rest. The evidence thus revealed that a bull infected with BLV generated good-quality contaminated semen and, therefore, that it is essential to detect contaminated seminal samples to prevent the spread of BLV. A multivariate analysis showed the presence of four sperm subpopulations in Brahman bulls that differ significantly in their kinematic patterns and with respect to sanitary status (P < 0.05), indicating that infection-free and seronegative bulls present the best kinematic parameters, which improved discrimination of sperm quality according to sanitary status. Overall, the analyses indicate that the seropositive-infected bulls with BLV and BHV-1 should be excluded from beef cattle farms.

Effect of nutraceutical supplementation on semen quality in stallions.

The use of reproductive biotechnologies in equine practice has shown that some stallions are subfertile, so ways to improve fertility have been sought. This study aimed to evaluate the effect of nutraceutical supplementation on improving semen quality in Quarter Horse stallions. Semen from six Quarter Horse stallions was assessed for 4 months every 20days using the computer-assisted semen analysis system. They were evaluated for 60days before supplementation; then, the same stallions were re-evaluated for 60days with nutraceutical supplementation (30g/day). Volume showed no significant difference (p>0.05) with nutraceuticals. Sperm concentration (10x6 ) was significantly higher with supplementation (339.4±17.5 sperm/mL) than without supplementation (224.6±19.9). Sperm abnormalities (%) were significantly (p<0.05) lower with supplementation (14.3±0.6) than without supplementation (19.1±0.4). Sperm kinematic parameters, total motility (TM), progressive motility (PM), rectilinear velocity (VSL), the velocity of the trajectory (VAP) and curvilinear velocity (VCL), were significantly better with supplementation (p<0.05). Based on the results, it is concluded that nutraceutical supplementation improved semen parameters in Quarter Horse stallions.

Effect of Inorganic Mercury on Semen Quality, Embryo and Larval Development of Bocachico Prochilodus magdalenae

The study aimed to evaluate the effect of inorganic mercury (iHg) on the sperm quality, embryos, and larvae of Prochilodus magdalenae. Two experiments were carried out. In experiment 1, semen was activated with contaminated water at different iHg concentrations (0.0, 25, 50, 100 µg/L) and sperm kinetics were analyzed with a computer-assisted semen analysis (CASA) system. In the second trial, 2 g of oocytes were fertilized with 50 µL of milt, activated with contaminated water at different iHg concentrations (0.0, 25, 50, 100 µg/L), and maintained that way during the first hour of incubation. Samples were taken every 3–4 h until hatching to identify embryo deformations. Fertilization and hatching rates were estimated. The survival of newly hatched larvae (1 day) and larvae eight days post-hatching (dph) was also assessed. The results show that the quality parameters of semen treated with contaminated water at different iHg concentrations (25, 50, 100 µg/L) were altered and showed significant differences (p &lt; 0.05) from the control group (0 µg/L). Total motility decreased (9.8–13.2%) and the amount of immotile sperm increased (3-fold) relative to values in the control group. A significant increase in oocyte diameter and a decrease in fertilization and hatching rates were observed with the higher iHg concentrations (50–100 µg/L). Embryo deformations (&gt;40%) such as scoliosis, lordosis, and microcephaly were observed, as well as premature hatchings. The larval survival at 1 dph was reduced between 25% (25 µg/L) and 97.2% (100 µg/L) relative to that in the control group. The results suggest that bocachico semen, embryos, and larvae are sensitive to contamination with iHg from 25 µg/L.

Semen quality of progeny-tested breeding bulls maintained at the National Artificial Insemination Centre, Arusha, Tanzania

The success or failure of artificial insemination (AI) depends on the quality of semen used. This study was conducted to investigate the effect of breed, age, collection period, and ejaculate number on semen quality collected from 15 breeding bulls available at the centre with different ages (4 Friesian, 4 Ayrshire, 3 Jersey, 2 Simmental and 2 Boran breeds) maintained at National Artificial Insemination Center (NAIC), Arusha, Tanzania. A total of 600 ejaculates were collected for four weeks per month consecutively from January to May 2022, weekly per bull and twice a day; morning (6.00 - 9.00 AM) and evening (3.00 - 6.00 PM) sessions). The ejaculates were first examined visually for volume, color, foreign bodies, and consistency whereas sperm concentration (SPC), progressive motility (PM) and total motility (TM) were assessed using Computer Assisted Semen Analysis System (CASA). Viability/vitality (SPVI) and morphology (SPNR) of sperms were analyzed microscopically after staining with Eosin-Nigrosin. Data were subjected to SAS program and results showed that ejaculate volume, progressive motility, total motility, sperm viability, and morphology were significantly (P&lt;0.05) affected by age and collection period. Simmental, Friesian, and Ayrshire bulls produced more voluminous semen than Jersey and Boran bulls. Jersey bulls had higher (P&lt;0.05) sperm concentrations and live spermatozoa as compared to other bulls. Boran and Jersey's bulls exhibited higher PM and TM than the other bulls. The period of semen collection (morning versus evening) negatively affected semen volume, total motility, and semen viability in which morning harvests performed better than evening collections. Middle aged bulls (3-5 years) produced semen of higher quality compared to other groups (&lt; 3 years and &gt; 5 years old bulls). In conclusion, age, breed, period of semen production and ejaculate number revealed significant effect on bovine semen characteristics.

Effects of Extenders Supplementation with Gum Arabic and Antioxidants on Ram Spermatozoa Quality after Cryopreservation

Semen cryopreservation is very important in animal agriculture to maximize the number of daughters of genetically superior males and to distribute the cryopreserved semen of good males all over the world. However, the freezing process generates some damage to sperm that reduce their fertilizing ability after thawing. Moreover, egg yolk, which is the most common animal-origin cryoprotectant used in semen dilution, is considered a source of biosecurity risk. In the current study, we aimed to compare the replacement of egg yolk in the extender by gum arabic (5%) along with supplementation with antioxidant cysteine or ascorbic acid on semen quality and freezability in Noemi rams in vitro. Semen from six rams were collected with an artificial vagina two times per week. Semen evaluation parameters such as color, volume, pH, general motility, percentage motility, concentration and cell viability ratio were assessed. Spermatozoa motility and concentration were estimated with the computer-assisted semen analysis system. The semen samples were frozen using a Tris extender containing either 15% egg yolk or 5% gum arabic. For antioxidant-supplemented extenders, cysteine or ascorbic acid was dissolved at concentrations of 0.10, 0.50 or 1.0 mM in egg yolk or gum arabic extender. The semen from each ejaculate of each ram were resuspended with a specific extender with glycerol (5%); the final volume after dilution was 1 mL semen to 4 mL extender. The samples were then cooled to 4 °C for 120 min, loaded into 0.5 mL straws and frozen in liquid nitrogen for 7 days. Supplementation of gum arabic or egg yolk extenders for ram semen with antioxidants such as cysteine or ascorbic acid has beneficial effects on semen quality after cold storage or cryopreservation. However, supplementation of a 5% gum arabic extender with cysteine at 0.5 or 1 mM concentration or ascorbic acid at 0.5 mM concentration improved the quality of spermatozoa postcryopreservation. It could be concluded that gum arabic is a good alternative for egg yolk in Noemi ram semen extenders. Antioxidants are necessary to support the addition of gum arabic to the extender to help the ram spermatozoa to survive freezing-thawing and oxidative stresses.

Effects of extender filtration and egg yolk concentration on canine semen cryopreservation.

The semen cooling and freezing extenders commonly contain the chicken egg yolk (EY) as the main sperm cryoprotectant. Besides its advantages, the EY has large lipoprotein granules that cause several physical and biological interferences. The previous studies have proposed several methods to resolve the problems with the EY-based semen extenders, including mechanical agitation, EY fractionation, replacing the EY with purified EY LDL, and ultrasonication. In the current research, we aimed to evaluate the syringe filtration (220 nm) of an EY-based canine semen freezing extender as a simple and cheap method to remove the EY granules. We also studied the possibility of re-aggregation of EY granules after cooling, freeze/thawing, and lyophilization/rehydration of the filtered extenders. Additionally, we compared the effects of the filtration on lipid profile, turbidity, EY particle size distribution, and osmolality of the EY-based extenders. Next, we examined the effects of filtered extenders containing different levels of EY (5%, 10%, 15%, 20%, and 25%) versus the control extender (20% EY, unfiltered) on post-thaw sperm quality traits. We collected the semen samples from seven clinically healthy mixed-breed adult dogs and pooled them for sperm freezing procedures. Samplings were repeated at least five times, independently. Our results indicated that the syringe filtration could remove the large EY particles and reduce the extender turbidity without affecting the lipid profile of the whole extender solution. The filtered extender supplemented with 25% (v/v) EY led to the best post-thaw canine spermatozoa quality markers. The frozen-thawed spermatozoa evaluations included motility parameters (computer-assisted semen analysis system), membrane and acrosome integrity (hypo-osmotic swelling test, chlortetracycline binding assay), DNA fragmentation (sperm chromatin dispersion assay), membrane lipid peroxidation (MDA levels), apoptosis (Annexin V/propidium iodide assay), and fertility-associated sperm mRNA transcript abundance (protamine 2 and 3). In conclusion, the syringe filtration of the EY-based semen extenders was a simple and cheap method that could effectively remove large EY lipoprotein granules and possibly prevent EY-origin bacterial contamination of the final extender solution. The EY at 25% (v/v) concentration in the filtered extenders resulted in the highest canine spermatozoa cryo-tolerance.

A Method of Digital Washing of Fresh Semen Samples

Background and Aim: Semen analysis is of great importance in the evaluation and treatment of male reproductive pathology [1]. Semen analysis may be performed on fresh/unwashed or washed semen samples. The semen washing process eliminates undesirable cells and particles from a fresh specimen. Computer-Assisted Semen Analysis (CASA) systems have shown to work extremely well, particularly on washed debris-free semen samples [2]. However, analyzing fresh samples could be problematic, especially in the presence of non-sperm cells.

The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa

BackgroundThe wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry.ResultsPercoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis.ConclusionsWhile creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

P-038 Comparison of three methods of semen analysis: A novel at-home sperm test kit, a computer-assisted assessment and an embryologist

Study questionHow does computer-assisted semen analysis (CASA) (Lenshooke, LOGIXX) and at-home sperm testing (ExSeed) compare with manual methodology assessment of male fertility based on WHO criteria (5thEdition); a three-method comparison study.Summary answerAll methods showed good agreement for concentration and limited agreement for motility highlighting the need for further development of alternatives to manual assessment.What is known alreadySeveral studies have shown good correlation between CASA systems and laboratory-based manual semen analysis, but only a few have carried out a three-way comparison study. One of which showed positive correlation with motility and concentration between a smartphone-based semen analysis and laboratory-based CASA system and positive correlation between concentration and motility between the smartphone-based CASA system and microscopic-based results. The at-home sperm test kit may play a role in motivating infertile males to visit clinics for early diagnosis and also reducing the need for multiple visits to the fertility clinic for repeat semen analyses.Study design, size, durationFifty patients (between 29 and 56 years) attending a fertility clinic were included in the study between September 2021 to December 2021. Semen samples were split into 3 aliquots and evaluated using manual semen assessment (MSA) according to the WHO 5th Edition (2010) guidelines, the ExSeed Home Sperm Test (HST) and the LensHooke® X1 PRO Semen Quality Analyzer (CASA).Participants/materials, setting, methodsThe semen samples were collected from fifty participants at CARE Fertility Tunbridge Wells. The samples were placed in an incubator at 37°C for 30 minutes to liquefy. After liquefaction, sperm concentration, total motility, total motile sperm count (TMSC) and normal morphology were evaluated. Spearman’s Rank correlations (>0.7) and Chi-squared tests were used and the p-value < 0.05 was considered as statistically significant.Main results and the role of chanceAll variables were highly significantly (p < 0.0002) positively correlated between all 3 methods.The greatest correlations were obtained for sperm concentration (CASA/HST: r = 0.826; MSA/HST: r = 0.870; MSA/CASA: r = 0.871) and TMSC (MSA/CASA: p = 0.792; CASA/HST: r = 0.800; MSA/HST: r = 0.854). Correlations for motility were markedly lower (MSA/HST: r = 0.611; CASA/HST: r = 0.717; MSA/LCASA: r = 0.750). The lowest correlation was found for morphology (MSA/CASA: r = 0.500). The HST device does not determine morphology.As compared to MSA, using the HST device agreement for normal or low sperm concentration (≥15 × 106/mL or < 15 × 106/mL respectively) was identified in 84.3% of the cases, whereas 94.0% with CASA. The agreement between CASA and HST was 82.0%. For total motility ≥ or < 40%, the agreements were 68.0% (CASA/HST), 56.9% (MSA/HST) and 82.0% (MSA/CASA), respectively.For the identification of patients with normal morphology >4%, the agreement between MSA and CASA was 30%.Limitations, reasons for cautionThe small sample size was 50 cases. The home testing device does not assess morphology. Furthermore, it is difficult to ascertain whether a consumer would carry out the analysis with this device with the same accuracy as an embryologist.Wider implications of the findingsThe need for repeated semen analyses, the effect of the COVID-19 pandemic and the discomfort some patients feel in a clinical setting, necessitate the need for evaluation of novel semen analysis approaches. These emerging technologies have potential to be more patient friendly, convenient and efficient than standard semen assessment methods.Trial registration numbernot applicable

Evaluating the potential of improving sperm quality traits in farmed Arctic charr (Salvelinus alpinus) using selective breeding

Arctic charr (Salvelinus alpinus) is a high-value species for the Nordic aquaculture. The highly variable reproductive performance that is commonly observed in commercial farms is hindering the expansion of the Arctic charr industry in Sweden. Traits related to sperm motility (total motility; curvilinear velocity; average path velocity; straight-line velocity) and concentration can play a pivotal role in male fertility. Selective breeding practices could offer solutions and contribute to improving male fertility. The current study aimed to investigate the magnitude of genetic variance for sperm quality traits in a selectively bred population of Arctic charr from Sweden and evaluate the possibility of their improvement through selection. Sperm motility and concentration were recorded using a computer-assisted semen analysis (CASA) system and a NucleoCounter, respectively, in over 400 males from year-class 2017. Double digest restriction-site associated DNA sequencing (ddRAD-seq) was applied in a subset of the recorded animals (n = 329), resulting in the detection of over 5000 single nucleotide polymorphisms (SNPs). Moderate heritability estimates were obtained for the recorded semen traits using both pedigree (0.21–0.32; SE 0.09) and genomic (0.23–0.26; SE 0.09) relationship matrices. A genome-wide association study (GWAS) detected a single SNP significantly associated (P < 1e-05) with total sperm motility on chromosome LG7 in relatively close proximity (500 Kb) to PTPN11 a gene previously associated with sperm quality traits in mammals. Moreover, weighted single-step genomic best linear unbiased prediction (WssGBLUP) pinpointed genomic regions explaining more than 3 % of the additive genetic variance for both the motility traits and the sperm concentration. Finally, the efficiency of genomic prediction was tested using a 3-fold cross-validation scheme. Higher prediction accuracy for total motility and velocities (both curvilinear and average path) was obtained using genomic information (0.26–0.29, SE 0.03–0.06) compared to pedigree (0.20–0.28, SE 0.04–0.07), while for sperm concentration a pedigree-based model (0.22 SE 0.03) was more efficient than the genomic model (0.14 SE 0.04). Overall, our results indicate that the recorded sperm quality traits are heritable, and could be improved through selective breeding practices.

ANALYSIS OF THE QUALITY OF SEXED BULL SEMEN BY COMPUTER-ASSISTED SEMEN ANALYSIS-CASA

The quality of the ejaculate is determined by examination of the macroscopic (volume, density, color, smell) and microscopic properties of the sperm (concentration of spermatozoa in 1 ml and the total number of spermatozoa in the ejaculate, morphology, vitality, mobility of spermatozoa and integrity of the acrosomal membrane). Computer assisted semen analysis (CASA) is an automated system that measures sperm motility, kinetics and concentration. Some systems have modifications for evaluating the morphological characteristics of spermatozoa. The assessment of morphological characteristics of spermatozoa can be performed by microscopic examination (presence of normal and pathological spermatozoa, integrity of sperm cell membrane and acrosome abnormalities). The Blom method, as the method of the staining of the sperm smear with eosin and nigrosine, determines the vitality of the semen. In modern cattle breeding, sexed bull semen (semen from which desired gender of calf will be delivered) is used more and more. The analysis of sexed semen, and especially the comparison of its quality with conventional semen, is an increasingly common requirement on the cattle production market. The aim of the work was to examine the quality of 15 samples of sexed bull semen, originating from imports. The tests included determination of concentration and kinetics using the CASA system, as well as assessment of vitality and morphological characteristics by Blom staining. The average sperm concentration in 15 samples was 19.61x106/ml of ejaculate. The total motility was 39.42%, while the number of immobile spermatozoa in the samples was 60.58%. The number of morphologically normal spermatozoa varied from 2.91x106 to 6.11x106, and the percentage of pathological spermatozoa from 8% to 28%. Taking into account the results obtained and the absence of standards related to sexed semen in the currently valid Regulation on the manner of marking of sperm, the manner of keeping records on sperm production, as well as on the conditions that must be met by animal sperm in terms of quality (Official Gazette of the Republic of Serbia, 38/14), it is necessary to carry out more frequent analyzes of sexed semen using the CASA method in order to obtain values that would become an integral part of the Regulation and thereby facilitate the manipulation of sexed semen on the market.

In vitro effect of ferrous sulphate on bovine spermatozoa motility parameters, viability and Annexin V-labeled membrane changes.

The aim of this study was to assess the dose- and time-dependent in vitro effects of ferrous sulphate (FeSO4.7H2O) on the motility parameters, viability, structural and functional activity of bovine spermatozoa. Spermatozoa motility parameters were determined after exposure to concentrations (3.90, 7.80, 15.60, 31.20, 62.50, 125, 250, 500 and 1000 μM) of FeSO4.7H2O using the SpermVisionTM CASA (Computer Assisted Semen Analyzer) system in different time periods. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, and the Annexin V-Fluos was applied to detect the membrane integrity of spermatozoa. The initial spermatozoa motility showed increased average values at all experimental concentrations compared to the control group (culture medium without FeSO4.7H2O). After 2 h, FeSO4.7H2O stimulated the overall percentage of spermatozoa motility at the concentrations of ≤ 125 μM. However, experimental administration of 250 μM of FeSO4.7H2O significantly (P < 0.001) decreased the spermatozoa motility but had no negative effect on the cell viability (P < 0.05) (Time 2 h). The lowest viability was noted after the addition of ≥ 500 μM of FeSO4.7H2O (P < 0.001). The concentrations of ≤ 62.50 μM of FeSO4.7H2O markedly stimulated (P < 0.001) spermatozoa activity after 24 h of exposure, while at high concentrations of ≥ 500 μM of FeSO4.7H2O the overall percentage of spermatozoa motility was significantly inhibited (P < 0.001) and it elicited cytotoxic action. Fluorescence analysis confirmed that spermatozoa incubated with higher concentrations (≥ 500 μM) of FeSO4.7H2O displayed apoptotic changes, as detected in head membrane (acrosomal part) and mitochondrial portion of spermatozoa. Moreover, the highest concentration and the longest time of exposure (1000 μM of FeSO4.7H2O; Time 6 h) induced even necrotic alterations to spermatozoa. These results suggest that high concentrations of FeSO4.7H2O are able to induce toxic effects on the structure and function of spermatozoa, while low concentrations may have the positive effect on the fertilization potential of spermatozoa.

Assessment of sperm motility in livestock: Perspectives based on sperm swimming conditions in vivo

Evaluation of sperm motility is well-established in farm animals for quickly selecting ejaculates for semen processing into insemination doses and for evaluating the quality of preserved semen. Likewise, sperm motility is a fundamental parameter used by spermatologists in basic and applied science. Motility is commonly assessed using computer-assisted semen analysis (CASA). Recent increases in computational power, as well as utilization of mobile CASA systems and open-source CASA programs, broaden the possibilities for motility evaluation. Despite this technological progress, the potential of computer-generated motility data to assess male fertility remains challenging and may be limited. Relevance for fertility assessment could be improved if measurement conditions would more closely mimic the in vivo situation. Hence, this review is focused on the current trends of automated semen assessment in livestock and explores perspectives for future use with respect to the physiological and physical conditions encountered by sperm in the female reproductive tract. Validation of current CASA systems with more complex, microfluidic-based devices mimicking the female reproductive tract environment could improve the value of sperm kinematic data for assessing the fertilizing capacity of semen samples, not only for application in livestock but also for use in conducting assisted reproduction techniques in other species.

Effects of L-carnitine combined with pancreatic kininogenase on thioredoxin 2, thioredoxin reductase 1, and sperm quality in patients with oligoasthenospermia.

BackgroundTo study the effects of L-carnitine (LC) combined with pancreatic kininogenase on thioredoxin 2 (Trx 2), thioredoxin reductase 1 (TrxR 1), and sperm quality in patients with oligoasthenospermia.MethodsA total of 300 male infertility patients with oligoasthenospermia who were treated in the andrology clinic of our hospital from December 2019 to December 2020 were randomly divided into an LC group and combined treatment group, and 50 males with normal semen were selected as a control group. The computer-assisted semen analysis system (CASA) was used to detect the total number, vitality, and forward motility of the sperm before and after treatment, and sperm morphology was detected by the Diff-Quik method of the sperm staining kit. Sperm chromatin dispersion (SCD) method was used to detect sperm DNA fragments, and Western-blot was used to detect the protein expression of Trx 2 and TrxR 1.ResultsThere were no significant differences in sperm density, motility rate, forward motile sperm rate, and DNA fragmentation rate in oligoasthenospermia patients before treatment (P>0.05). However, after 1 month of treatment, the sperm density, motility rate, and forward motile sperm rate were all higher than before treatment (P<0.05), while the DNA fragmentation rate was lower than before treatment. At the same time, each index of semen in the combination group was higher than that in the LC group (P<0.05), and the total effective rate in the combination group was significantly higher than in the LC group (P<0.01). The expression of Trx2 protein in oligoasthenospermia patients was significantly increased (P<0.05), while the expression of TrxR1 protein was significantly decreased (P<0.05). After 3 months of treatment, the expression of Trx2 protein was significantly decreased (P<0.05), while the expression of TrxR1 protein was significantly increased (P<0.05).ConclusionsThe results suggest Trx 2 and TrxR 1 may be candidate protein markers for oligoasthenospermia. LC combined with pancreatic kininogenase in the treatment of male oligoasthenospermia can effectively promote sperm maturation, enhance sperm motility, and improve semen quality, which has high application value.

ITRAQ-based proteomic analysis of sperm reveals candidate proteins that affect the quality of spermatozoa from boars on plateaus

BackgroundTibetan pigs (TP) exhibit heritable adaptations to their hypoxic environments as a result of natural selection. However, candidate proteins that affect the sperm quality of boars on plateaus have not yet been clearly investigated.MethodsIn this study, to reveal the candidate proteins that affect the quality of spermatozoa of boars on plateaus, we analyzed the sperm quality using computer-assisted semen analysis (CASA) system and reactive oxygen species (ROS) levels. We also compared the proteomes of sperm proteomes between TP and Yorkshire pigs (YP) raised at high altitudes using the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC–MS/MS) proteomic method, and confirmed the relative expression levels of the four proteins by western blotting.ResultsThe sperm quality of the TP was superior to that of the YP on plateaus. Of the 1,555 quantified proteins, 318 differentially expressed proteins (DEPs) were identified. Gene ontology (GO) analysis revealed that the DEPs were predominantly associated with the sorbitol metabolic process, removal of superoxide radicals, cellular response to superoxide, response to superoxide and regulation of the mitotic spindle assembly. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were mainly enriched in pathways involved in the regulation of the actin cytoskeleton, glutathione metabolism, oxidative phosphorylation, and estrogen signaling. Based on the protein–protein interaction (PPI) network analysis, we identified 8 candidate proteins (FN1, EGF, HSP90B1, CFL1, GPX4, NDUFA6, VDAC2, and CP) that might play important roles and affect the sperm quality of boars on plateaus. Moreover, the relative expression levels of four proteins (CFL1, EGF, FN1, and GPX4) were confirmed by western blot analysis.ConclusionsOur study revealed 8 candidate proteins (FN1, EGF, HSP90B1, CFL1, GPX4, NDUFA6, VDAC2, and CP) that affect the sperm quality of boar on plateaus and provide a reference for further studies on improving sperm quality and the molecular breeding of boars on plateaus.

A Combined Flow Cytometric Semen Analysis and miRNA Profiling as a Tool to Discriminate Between High- and Low-Fertility Bulls.

Predicting bull fertility is one of the main challenges for the dairy breeding industry and artificial insemination (AI) centers. Semen evaluation performed in the AI center is not fully reliable to determine the level of bull fertility. Spermatozoa are rich in active miRNA. Specific sperm-borne miRNAs can be linked to fertility. The aim of our study is to propose a combined flow cytometric analysis and miRNA profiling of semen bulls with different fertility to identify markers that can be potentially used for the prediction of field fertility. Sperm functions were analyzed in frozen-thawed semen doses (CG: control group) and high-quality sperm (HQS) fraction collected from bulls with different field fertility levels (estimated relative conception rate or ERCR) by using advanced techniques, such as the computer-assisted semen analysis system, flow cytometry, and small RNA-sequencing. Fertility groups differ for total and progressive motility and in the abnormality degree of the chromatin structure (P < 0.05). A backward, stepwise, multiple regression analysis was applied to define a model with high relation between in vivo (e.g., ERCR) and in vitro (i.e., semen quality and DE-miRNA) fertility data. The analysis produced two models that accounted for more than 78% of the variation of ERCR (CG: R2 = 0.88; HQS: R2 = 0.78), identifying a suitable combination of parameters useful to predict bull fertility. The predictive equation on CG samples included eight variables: four kinetic parameters and four DNA integrity indicators. For the HQS fraction, the predictive equation included five variables: three kinetic parameters and two DNA integrity indicators. A significant relationship was observed between real and predicted fertility in CG (R2 = 0.88) and HQS fraction (R2 = 0.82). We identified 15 differentially expressed miRNAs between high- and low-fertility bulls, nine of which are known (miR-2285n, miR-378, miR-423-3p, miR-191, miR-2904, miR-378c, miR-431, miR-486, miR-2478) while the remaining are novel. The multidimensional preference analysis model partially separates bulls according to their fertility, clustering three semen quality variable groups relative to motility, DNA integrity, and viability. A positive association between field fertility, semen quality parameters, and specific miRNAs was revealed. The integrated approach could provide a model for bull selection in AI centers, increasing the reproductive efficiency of livestock.