Computer-assisted Sperm Analysis Research Articles

Preservation of Simmental bull sperm at 0°C in Tris dilution: effect of dilution ratio and long-distance transport.

This study aimed to assess the impact of the dilution ratio of Tris diluent, storage at 0°C, and long-distance transportation on the spermatozoa of Simmental cattle. It also validated the feasibility of the regional distribution of fresh semen. In experiment 1, semen was diluted at four dilution ratios (1:6, 1:9, 1:12, and 1:15) to determine the optimal dilution ratio of Tris diluent. In experiment 2, we assessed sperm viability, progressive motility (objectively assessed by computer-assisted sperm analyzer), and acrosome intactness in Tris dilutions kept at constant 0°C for 1, 3, 6, 9, and 12 days. We compared them to Tianshan livestock dilutions (Commercial diluent). In experiment 3, semen was diluted using Tris diluent, and sperm quality was measured before and after long-distance transport. Artificial insemination of 177 Simmental heifers compared to 156 using Tianshan Livestock dilution. The outcomes demonstrated that 1:9 was the ideal Tris diluent dilution ratio. The sperm viability, Progressive Motility, and acrosome integrity of both Tris and Tianshan dilutions preserved at 0°C gradually decreased over time. sperm viability was above 50% for both dilutions on d 9, with a flat rate of decline. The decrease in acrosome integrity rate was faster for Tianshan livestock dilutions than for Tris dilutions when stored at 0°C for 1 to 6 days. There was no significant difference (p>0.05) in sperm viability between semen preserved in Tris diluent after long-distance transportation and semen preserved in resting condition. The conception rates for Tris dilution and Tianshan livestock dilution were 49.15% and 46.15% respectively, with no significant difference (p>0.05). This shows that Tris diluent is a good long-term protectant. It has been observed that fresh semen can be successfully preserved for long-distance transport when stored under 0°C conditions. Additionally, it is feasible to distribute semen regionally.

Effect of platinum nanoparticles on rabbit spermatozoa motility and viability

Male reproductive health is becoming a frequently asked question nowadays. Spermatozoa can be highly sensitive to various substances found in the environment. With the accretive usage of nanoparticles in technology and medicine, nanoparticles may also pose as potential environmental contaminants. Platinum nanoparticles are increasingly used in medicine, mainly for cancer treatment. The aim of this study was to investigate the effect of platinum nanoparticles on the motility and viability of rabbit spermatozoa in-vitro. Rabbit semen (n = 5) was cultured with solutions with different concentrations of platinum nanoparticles (control group or CON = only 0.9% NaCl; experimental groups D = 62.5 µg/ml, E = 31.25 µg/ml, F = 15.63 µg/ml, G = 7.81 µg/ml, H = 3.91 µg/ml, I = 1.95 µg/ml, J = 0.98 µg/ml, K = 0.49 µg/ml, and L = 0.24 µg/ml platinum nanoparticles) at time intervals of 0, 2, 4, 6, 9 and 24 hours. Selected parameters were analysed by computer-assisted sperm analyzer (CASA) technology – total motility (MOT, %), progressive motility (PRO, %), curvilinear velocity (VCL, µm/s) and viability (metabolic activity) of spermatozoa using the MTT test. Platinum nanoparticles negatively affected spermatozoa motility, progressive motility, and curvilinear velocity at almost all assessed time intervals, mainly at the highest used concentrations. The initial time interval shows a slight but statistically significant increase in MOT and PRO. Spermatozoa viability was not significantly affected, according to the results of this experiment. It was shown that the effect of platinum nanoparticles is time and dose-dependent. However, further analyses are needed to draw complete conclusions.

Treatment with cholesterol just after thawing maintains the fertility of bull sperm.

Freezing and thawing diminish sperm motility and fertility by disrupting the cholesterol balance in sperm plasma and organelle membranes. The aim of this study was to elucidate the mechanisms through which exogeneous cholesterol treatment enhances the quality of frozen-thawed bull sperm. The incorporation of cholesterol was investigated using boron-dipyrromethene (BODIPY)-cholesterol, and BODIPY signals were detected not only in the plasma membrane but also in the midpiece region immediately after thawing. The positive signal of cholesterol in the midpiece region was inhibited by a scavenger receptor class B Type I (SR-BI) inhibitor, block lipid transport 1 (BLT-1). To comprehend the role of exogenous cholesterol in the functions of the plasma membrane, propidium iodide (PI)/Annexin V and peanut agglutinin lectin (PNA) staining were performed. The results showed that treatment with exogenous cholesterol increased the number of acrosome-intact sperm and decreased the number of sperm with damage to the plasma membrane. Moreover, since BODIPY signals were also observed in the midpiece region, mitochondrial function was evaluated using a flux analyzer and a flow cytometer with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) staining, revealing an increase in the number of sperm with high-mitochondrial activity and oxygen consumption. Finally, to assess sperm fertility, computer-assisted sperm analysis (CASA) and IVF were carried out. Sperm velocities and fertilization rates in IVF were significantly enhanced by the addition of cholesterol just after thawing. Thus, the treatment with cholesterol after thawing protected the plasma membrane from the stress of thawing and maintained mitochondrial function, thereby preserving the fertilization ability of frozen-thawed bull sperm for conventional IVF and artificial insemination (AI). Therefore, the application of cholesterol just after thawing is a promising option for improving the fertility of frozen-thawed sperm.

Rotation of liquid-preserved artificial insemination doses on roller benches affects sperm quality during storage in stallions.

Appropriate stallion semen handling is of great importance in equine artificial insemination (AI) industry. Optimal treatment of AI doses is aiming for best sperm preservation by excluding strong environmental influences and adding media that favour sperm survival. One method widely used in stallion sperm handling is the rotation of liquid-preserved semen samples on roller benches during storage. As previous studies in boars give rise to the fact that rotation should not be considered beneficial for spermatozoa anymore, the present study investigated the influence of continuous rotation of diluted stallion AI portions on sperm quality. Ejaculates (n = 15) were collected at a German AI centre and diluted with the two extenders EquiPlus and Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL. Afterwards, samples were placed separately on roller benches at 5°C in the dark, obtaining a rotation frequency of 5 revolutions per minute (rpm) and 36 rpm for four consecutive days following a split-sample design. Both groups were analysed daily in comparison to a control group (0 rpm) with an extended spectrum of spermatological methods including computer-assisted sperm analysis and flow cytometry. Statistical analyses were based on the calculation of generalized linear mixed models for each sperm parameter. The research revealed a decrease in sperm quality parameters of rotated samples compared to non-rotated control groups, visible in total sperm motility (p < .001), decreased thermo-resistance (p < .01) and a drop in pH (p < .001). Interestingly, no differences (p > .05) were detected between rotation frequencies of 5 and 36 rpm. We conclude that the fertilizing capacity of stallion semen was negatively affected by rotation during storage in vitro, irrespective of the rotation frequency. Further studies need to investigate whether field fertility in horses is similarly affected by semen rotation on roller benches in vivo.

Low-density lipoproteins, resveratrol and quercetin as alternative additives to improve boar semen cooling.

Low-density lipoproteins (LDL), quercetin (Q) and resveratrol (R) have been used for sperm preservation to improve sperm quality in different species. To evaluate the effects of LDL, Q and R during the cooling of boar semen. Fifteen boar semen samples were diluted in a BTS extender supplemented with the treatments: LDL at 6%, Q at 10 μM (Q10), 30 μM (Q30) and 50 μM (Q50), or R at 10 μM (R10), 30 μM (R30) and 50 μM. A control without supplementation was included. The semen was stored by cooling at 16°C for 96 h. Every 24 h, sperm motility and kinetics were evaluated using a computer-assisted sperm analyzer (IVOS). At 24 and 96 h of cooling, functional membrane integrity and mitochondrial membrane potential (ΔΨM) of sperm were evaluated by the hypoosmotic swelling test (HOST) an flow cytometry with JC-1 probe, respectively, LDL improved progressive motility of sperm during cooling. Likewise, LDL increased average path velocity (VAP) and straight-line velocity (VSL) and/or curvilinear velocity (VCL) during the first 48 h of cooling. The use of Q between 10 and 30 μM caused a reduction in total motility, progressive motility and amplitude of the lateral head displacement during the entire cooling period, as well as a decrease in VAP, VSL and VCL at 96 h of cooling. LDL, Q10, Q30 and Q50 modulated mitochondrial activity by reducing high-ΔΨM sperm at 0 and 96 h of cooling. During the cooling of the boar semen prior to artificial insemination, the parameters of sperm quality that could influence fertility decrease; however, the inclusion of antioxidants and additives that protect the plasma membrane, such as LDL, could mitigate the damaging effects on spermatozoa. It is concluded that LDL can improve the motility and kinetics of boar semen during cooling while it could modulating the sperm's mitochondrial activity. On the contrary, Q could alter the motility and kinetics of boar sperm during the cooling period.

Bovine sperm HSP-70 molecules: a potential cryo-tolerance marker associated with semen quality and fertility rate.

Freezability is the ability of sperm to maintain its vitality and quality from various stress during the cryopreservation process, which is very important for the success of fertilization in AI programs. Heat shock proteins (HSPs) are unique proteins induced in response to various stress, including excess reactive oxygen species (ROS) and oxidative damage to intracellular enzymes that can harm cells. This study aimed to analyze the potential of HSP-70 molecules in bovine sperm as a marker of freezability or cryo-tolerance, as well as its association with semen quality and fertility rate. The classification of bulls is based on freezability (good freezability/GF and poor freezability/PF), which is obtained from the value of post-thaw viability using the SYBR-14/PI-flow cytometry. Semen quality assessed included sperm motility and kinetics (computer-assisted sperm analyses), plasma membrane integrity (HOS test), acrosome integrity (FITC-PNA), mitochondrial membrane (JC-1), and DNA damage (Halomax kit). The bull fertility rate assessment was analyzed based on the first service conception rate of each bull derived from data on the success of artificial insemination contained in the Indonesian-integrated National Animal Health Information System (iSIKHNAS). Gene expression levels of HSP-70 bovine sperm were performed using the RT-qPCR method. The protein abundance of HSP-70 bovine sperm was determined using the enzyme immunoassay (EIA) method. Bovine sperm HSP-70 molecules, at the gene and protein level, showed a higher abundance in GF (p < 0.05) than in PF bulls. The percentage of each parameter of frozen-thawed sperm quality was significantly higher in GF (p < 0.05) than in PF bulls. The HSP-70 molecules at the gene and protein levels were significantly positively correlated (p < 0.01) with the fertility rate. Furthermore, HSP-70 molecules were negatively associated (p < 0.01) with low mitochondrial membrane potential and sperm DNA damage and positively correlated (p < 0.01) with other frozen-thawed sperm quality parameters. The overall quality of frozen-thawed sperm was closely related (p < 0.01) to the fertility rate. We may conclude that HSP-70 molecules in bovine sperm at the gene and protein level have the potential to be developed as a marker for cryo-tolerance or freezability, which may be utilized as a predictor of fertility and frozen-thawed sperm quality in bulls.

Motility Subpopulations with Distinct Motility Characteristics Using Swim-Up-Selected Sperm Cells from Norwegian Red Bulls: Effects of Freezing-Thawing and Between-Bull Variation.

Discrete subpopulations of motile sperm cells have been found for several species and are implicated to be important for sperm functionality. The aim of this present study was to examine the motile subpopulations in swim-up-selected bull spermatozoa and the relationship between subpopulations in fresh and frozen-thawed sperm cells. In experiment 1, swim-up (SWUP)-selected and non-selected (control) sperm cells were analyzed using a Computer-Assisted Sperm Analyzer (CASA). In experiment 2, the semen from nine bulls was cryopreserved and analyzed using CASA both before and after freezing and after incubation at physiological temperatures. The SWUP population had a higher proportion of total motility, progressivity, and velocity compared to the control (p < 0.05). Likewise, both incubation over time and cryopreservation affected motility and motility parameters (p < 0.05). The population of rapid progressive (RapidP) sperm cells dominated the SWUP fraction and was higher than in the control samples (p < 0.05). Furthermore, RapidP was also the main part of fresh semen, but decreased significantly over time during incubation and due to cryopreservation. In conclusion, RapidP was the main population in SWUP-selected spermatozoa and seems to be an important subpopulation contributing to the differences between treatments and in response to the freezing of sperm cells.

Evaluation of sperm and hormonal assessments in Wagyu, Nellore, and Angus bulls.

Wagyu bulls are known to have a highly exacerbated libido, as shown by the intense sexual interest of young calves. Therefore we believe that Wagyu male animals have specialized Sertoli and Leydig cells that are directly involved with the sexual precocity in this breed as mature bulls have a small scrotal circumference. This study aimed to evaluate whether there were differences in the hormone and sperm characteristics of Wagyu bulls compared with the same characteristics of subspecies Bos indicus and Bos taurus sires. Frozen-thawed semen from Wagyu, Nellore, and Angus sires were analyzed for sperm kinetics (computer-assisted sperm analysis), plasma membrane integrity, chromatin integrity, acrosome status, mitochondrial activity, lipid peroxidation and hormone [luteinizing hormone (LH) and testosterone] serum concentration. The results showed that Wagyu had lower total motility and an increased number of sperm with no motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from those breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation or hormone (LH and testosterone) concentrations. We concluded that Wagyu sires had lower total motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from these breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation, or hormone (LH and testosterone) concentrations.

Cryopreservation of black seabream (Acanthopagrus schlegelii) sperm

In recent years, biotechnology has had a significant impact on the aquaculture industry, particularly in the field of breeding. Molecular selection breeding has emerged as a novel approach to breeding. Reducing the cost of genetic information for individuals with desirable traits after breeding has become an important research direction. Cryopreservation technology allows bypassing time and space constraints in genetic breeding, simplifying broodstock management. This study presents a detailed cryopreservation method for black seabream sperm, evaluating extender type, glucose concentration, cryoprotectant type and concentration, sperm-dilution ratio, and cooling protocols. Sperm motility parameters were analyzed using computer-assisted sperm analysis (CASA) before and after two days of freezing. This involved using an RS solution with a glucose concentration of 15 g/L and adding a 5% final concentration of EG as the sperm cryoprotectant. After mixing the sperm and solution at a ratio of 1:2, we subjected it to 5 min fumigation at 5 cm above the liquid nitrogen surface before plunging it into the nitrogen. Sperm motility reached 85.46 ± 7.32% after two days. Various enzymatic activities showed changes over 20 days post-cryopreservation. This improved cryopreservation protocol for black seabream sperm is beneficial for genetic breeding and reproduction and provides reference for studying the cryodamage mechanisms of black seabream sperm.

Identification of potential indicators for testis quality in swamp eels (Monopterus albus) through metabolomics analysis

The swamp eel (Monopterus albus) is a commonly cultured freshwater fish. Selection and cultivation of fecund broodstock is crucial for efficient artificial reproduction in this species. However, there are currently no guidelines for selecting high-quality males in M. albus. This study quantitatively investigated the sperm motility of male swamp eels during the breeding season by using computer-assisted sperm analysis system. Variability in the proportion and number of motile sperm was observed between individual males. The testes were grouped into high-quality and low-quality according to sperm quality. Morphometric parameters of the two groups were analyzed. We found that high-quality testes were longer (150 ​± ​27 ​mm verses 127 ​± ​19 ​mm) than low-quality testes. Besides, the proportion of testes with a dark appearance was slightly higher (58.3% verses 48.1%) in the high-quality group than in the low-quality group. Metabolomic analysis was conducted to compare the metabolite profiles of high-quality and low-quality testes. Glycerophospholipids, organic acids, glycerolipids, amino acids, sphingolipids, and nucleotides were the most abundant metabolites in the testes of swamp eels. Moreover, 135 differential metabolites were identified. Several nucleotide derivatives, including 5′-deoxyadenosine, 2′-deoxyadenosine, and ribosyl adenosine, were the most significantly enriched metabolites in high-quality testes. These metabolites are potential indicators for the testis quality in M. albus. Altogether, our work provides systematic and quantitative data about the physiological and biochemical characteristics of M. albus testis, which provides an evidential basis for the selection of high-fecundity parents to improve artificial reproduction efficiency of M. albus.

Genome-Wide Identification of Aqp Family Related to Spermatogenesis in Turbot (Scophthalmus maximus).

The development and maturation of sperm entails intricate metabolic processes involving water molecules, amino acids, hormones, and various substances. Among these processes, the role of aquaporins (aqps) in the testis is crucial. Turbot (Scophthalmus maximus) is a significant marine flatfish species in China; however, natural egg laying in females is not feasible under cultured conditions. Consequently, artificial insemination becomes necessary, requiring the retrieval of sperm and eggs through artificial methods. In this study, we combined genomic, transcriptomics, RT-qPCR, computer-assisted sperm analysis (CASA), and immunohistochemistry to investigate the involvement of the aqp family in spermatogenesis in turbot. Through genomic data analysis, we identified 16 aqps genes dispersed across 13 chromosomes, each exhibiting the characteristic major intrinsic protein (MIP) domain associated with AQPs. The results from RNA-seq and RT-qPCR analysis revealed prominent expression of aqp4, 10, and 12 during the proliferative stage, whereas aqp1 showed primary expression during the mature stage. aqp11 displayed high expression levels during both MSII and MSV stages, potentially contributing significantly to the proliferation and maturation of male germ cells. Conversely, aqp8 showed elevated expression levels during the MSIII, MSIII-IV, and MSIV stages, suggesting its direct involvement in spermiogenesis. Immunohistochemical analysis unveiled the predominant localization of AQP1 protein in male germ cells rather than Sertoli cells, specifically concentrated in the head of sperm within cysts. Furthermore, a noteworthy decline in sperm motility was observed when sperm were subjected to treatment with either the AQP1-specific inhibitor (HgCl2) or the AQP1 antibody. However, no direct correlation was found between the expression of Smaqp1 and sperm quality. Overall, these findings provide new insights into the involvement of aqps in teleost spermatogenesis. Moreover, they hold potential for improving techniques related to sperm activation and cryopreservation, offering valuable knowledge for future advancements in this field.

Extracellular vesicles in porcine seminal plasma maintain sperm function by reducing Lyso-PC

Seminal plasma (SP) exosomes are membrane vesicles with 30−120 nm diameter, which have been shown to regulate the physiological processes in sperm cells. However, the mechanism through which SP extracellular vesicles (EVs) maintain sperm function remains unclear. To explore the mechanism of EVs in sperm preservation, different EV concentrations (EV-0, EV-1, EV-4, EV-8, EV-16) were added to semen liquid preservation solution. Sperm motility was detected via computer-assisted sperm analysis systems, plasma membrane integrity was assessed using SYBR-14 and propidium iodide staining, and total antioxidant capacity and lipid oxidation levels were evaluated after 0, 2, 4, 6 days of liquid-storage at 17 °C. Differences in sperm metabolites between the EV-0 and EV-8 groups stored for 4 days, were analyzed using untargeted metabolomics [liquid chromatography with tandem mass spectrometry (LC-MS/MS)]. Moreover, mitochondrial reactive oxygen species (mtROS) levels were assessed when sperm incubated with EV-0 and EV-8 during 0, 2, 4, 6 days, and embryonic development were assessed when sperm incubated with EV-0 and EV-8 stored for 4 days at 17 °C. Our data showed that compared to EV-0, sperm motility and plasma membrane integrity, antioxidant capacity, lipid oxidation levels and cleavage rates were significantly increased in the EV-8 group (P < 0.05), lysophosphatidylcholine (Lyso-PC) metabolisma and mtROS levels were significantly reduced in the EV-8 group (P < 0.05). In conclusion, SP EVs inhibit the premature increase in Lyso-PC, exert antioxidant effects on mtROS, and preserve sperm's ability to fuse with oocytes.

Trends in sperm quality by computer-assisted sperm analysis of 49,189 men during 2015-2021 in a fertility center from China.

Sperm quality, including semen volume, sperm count, concentration, and total and progressive motility (collectively, "semen parameters"), has declined in the recent decades. Computer-assisted sperm analysis (CASA) provides sperm kinematic parameters, and the temporal trends of which remain unclear. Our objective is to examine the temporal trend of both semen parameters and kinematic parameters in Shanghai, China, in the recent years. This retrospective study analyzed semen parameters and kinematic parameters of 49,819 men attending our reproductive center by using CASA during 2015-2021. The total sample was divided into two groups: samples that surpassed the WHO guideline (2010) low reference limits ("above reference limit" group, ARL; n = 24,575) and samples that did not ("below reference limit" group, BRL; n = 24,614). One-way analysis of variance, Kruskal-Wallis test, independent samples t-test, and covariance analysis were used to assess the differences among groups. Year, age, and abstinence time were included in the multiple linear regression model of the ARL group to adjust the confounders and depict the trends in sperm quality. Among all the total sample and the ARL and BRL groups, the age of subjects increased in recent years. Semen volume and sperm count showed declined tendency with years in the total sample, the ARL and BRL groups, and the subgroup of age or abstinence time, whereas sperm velocities showed increased tendency with years on the contrary. The multiple linear regression model of the ARL group, adjusting for age and abstinence time, confirmed these trends. Semen volume (β1= -0.162; CI: -0.172, -0.152), sperm count (β1= -9.97; CI: -10.813, -9.128), sperm concentration (β1 = -0.535; CI: -0.772, -0.299), motility (β1 = -1.751; CI: -1.830, -1.672), and progressive motility (β1 = -1.12; CI: -0.201, -0.145) decreased with year, whereas curvilinear line velocity (VCL) (β1=3.058; CI: 2.912, 3.203), straight line velocity (VSL) (β1=2.075; CI: 1.990, 2.161), and average path velocity (VAP) (β1=2.305; CI: 2.224, 2.386) increased over time (all p < 0.001). In addition, VCL, VSL, and VAP significantly declined with age and abstinence time. The semen parameters declined, whereas the kinematic parameters increased over the recent years. We propose that, although sperm count and motility declined over time, sperm motion velocity increased, suggesting a possible compensatory mechanism of male fertility.

Exposure to silver and titanium dioxide nanoparticles at supra-environmental concentrations decreased sperm motility and affected spermatozoa subpopulations in gilthead seabream, Sparus aurata.

Marine pollution by nanoparticles (NPs) can be reprotoxic for fish and disturb successful reproduction of wild populations. In gilthead seabream (Sparus aurata), a mild effect on sperm motility was observed after exposure to high concentrations of silver NPs. Considering the great heterogeneity traits within a sperm sample, it is possible that NPs affect spermatozoa accordingly, modulating subpopulation profile. Thus, this work aimed to analyse NP effects in sperm motility in general and considering spermatozoa population structure, using a subpopulation approach. Seabream sperm samples from mature males were exposed for 1 h to increasing concentrations of titanium dioxide (1, 10, 100, 1000 and 10,000 μg L-1) and silver (0.25, 25 and 250 μg L-1) NPs, including Ag NP and Ag+, dissolved in a non-activating medium (0.9 % NaCl). Concentrations chosen include realistic (10-100 and 0.25 μg L-1, respectively, for TiO2 and Ag) and supra-environmental values. The mean particle diameter was determined as 19.34 ± 6.72 and 21.50 ± 8.27 nm in the stock suspension, respectively, for titanium dioxide and silver. After the ex vivo exposure, sperm motility parameters were determined using computer-assisted sperm analysis, and sperm subpopulations were later identified using a two-step cluster analysis. Results revealed a significant reduction in total motility after exposure to the 2 highest concentrations of titanium dioxide NPs, while curvilinear and straight-line velocities were not altered. Exposure to silver NPs (Ag NP and Ag+) lowered significantly total and progressive motilities at all concentrations, while curvilinear and straight-line velocities were significantly lower only at the highest concentration. Sperm subpopulations were also affected by the exposure to both titanium dioxide and silver NPs. In both cases, the highest levels of NPs triggered a decrease in the percentage of fast sperm subpopulations (38.2% in TiO2 1000 μg L-1, 34.8.% in Ag NP 250 μg L-1, and 45.0% in Ag+ 250 μg L-1 vs 53.4% in the control), while an increase on slow sperm subpopulations. A reprotoxic effect was proven for both NPs, but only at supra-environmental concentrations.

Evaluation of the effects of the recommended oral dose of diflubenzuron on bovine sperm and oocyte quality using CASA and OPU-IVEP.

The aim of this study was to evaluate potential effects of diflubenzuron on the production and quality of gametes, and on in vitro embryo production (IVEP) outcomes, in cattle. Two experiments were performed, the first to evaluate effects on semen, and the second on cumulus-oocyte complexes (COC) and on IVEP. Nelore (Bos taurus indicus) bulls (n = 14) or heifers (n = 16) were allocated into control (CG) or treatment (DIF) groups. All groups received a mineral mix supplement added (DIF) or not (CG) with diflubenzuron (30 mg/head/day), during 8 weeks. Animals were weighed and blood samples were collected throughout the experimental period. Every other week, bulls were subjected to semen collection and heifers to transvaginal ultrasound-guided follicle aspiration sessions. Semen underwent physical and morphological evaluation, and samples were stored for further computer-assisted sperm analysis. The COC recovered were evaluated according to morphology and those classified as viable were sent to an IVEP laboratory. Diflubenzuron had no effect (P > 0.05) on average body weight or in any blood hematological or biochemical endpoints, regardless of gender. In experiment 1, there was no difference (P > 0.05) between DIF and CG groups for sperm concentration, morphology, or kinetics. In experiment 2, there was also no effect of diflubenzuron on the number of total, viable, or grade I oocytes, as well as on cleavage or blastocyst rates (P > 0.05). In summary, the oral administration of diflubenzuron, within the recommended dose, has no short-term negative effects on sperm production and quality or on oocyte yield and developmental potential in vitro, in cattle.

A Modified-Herringbone Micromixer for Assessing Zebrafish Sperm (MAGS).

Sperm motility analysis of aquatic model species is important yet challenging due to the small sample volume, the necessity to activate with water, and the short duration of motility. To achieve standardization of sperm activation, microfluidic mixers have shown improved reproducibility over activation by hand, but challenges remain in optimizing and simplifying the use of these microdevices for greater adoption. The device described herein incorporates a novel micromixer geometry that aligns two sperm inlet streams with modified herringbone structures that split and recombine the sample at a 1:6 dilution with water to achieve rapid and consistent initiation of motility. The polydimethylsiloxane (PDMS) chip can be operated in a positive or negative pressure configuration, allowing a simple micropipettor to draw samples into the chip and rapidly stop the flow. The device was optimized to not only activate zebrafish sperm but also enables practical use with standard computer-assisted sperm analysis (CASA) systems. The micromixer geometry could be modified for other aquatic species with differing cell sizes and adopted for an open hardware approach using 3D resin printing where users could revise, fabricate, and share designs to improve standardization and reproducibility across laboratories and repositories.

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&amp;gt;T and c.3040A&amp;gt;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.

P-075 Artificial intelligence for sperm analysis: A KNIME-Based automated analysis of sperm morphology

Abstract Study question To evaluate an automated sperm morphology assessment method using the KNIME Analytics Platform compared to manual sperm morphology analysis performed as per WHO guidelines. Summary answer Our method emphasizes how artificial intelligence technologies have the potential to foster standardization of sperm morphology assessment with comparable precision and reliability. What is known already Manual sperm morphology assessment is considered the most difficult parameter to standardize due to its subjective nature, strongly linked to the operator's level of expertise. Indeed, there is a high degree of inter and intra-laboratory variability. Manual examination is time-consuming and laborious. There were many attempts to automate sperm morphology analysis, especially with CASA (Computer Assisted Sperm Analysis) systems, but their performance is still disputable. One of the difficulties in this field of study is the lack of publicly available datasets. Besides, the available databases are only focused on the sperm head morphology. Study design, size, duration A total of 37 semen samples from men attending our laboratory for infertility investigation were included, over a period of one year. For each sample, semen smears were fixed and stained by the Spermoscan® kit. Participants/materials, setting, methods A total of 1000 images of individual spermatozoa were obtained using the MMC® CASA system. The number of images per sample depended on its quality. Three experts have classified these spermatozoa according to modified David classification for sperm morphology. The results were then processed and an algorithm created using teh KNIME Analytics Platform, trained and tested to classify spermatozoa. This workflow uses CNN (convolutional neural network) to perform image classification on our dataset. Main results and the role of chance Of the 1000 images analyzed, we counted : 116 Normal sperm morphology, 67 abnormal post-acrosomal region, 128 abnormal acrosomes, 8 elongated heads, 6 thin heads,10 microcephalic,7 multiple heads, 27 coiled tails, 7 cytoplasmic residues, 17 angulated tails, 6 short tails, 4 multiple tails, and 697 associated abnormalities. For each image, a notebook file containing sperm abnormalities as assessed by the three experts was created, in addition to sperm head, mid-piece and tail dimensions obtained by the CASA system. Our dataset was randomly partitioned into 2 groups: 80% of data formed the training set, and The remaining 20% formed the fully independent test set. The best performance with the KNIME-Based algorithm was achieved for post-acrosomal abnormalities (97 % True positive rate), and the worst for multiple tail abnormalities (69 % True positive rate). Our machine learning model classifies sperm morphology at high accuracy (99.5 %). The overall process occurs in less than 10 seconds. Limitations, reasons for caution Our database included various head, midpiece and tail anomalies. However, the number of images in each category is unequal due to the limited occurrence of some morphological abnormalities. Thus, it is important to increase the number of images in these categories to obtain better results. Wider implications of the findings Our goal is to expand our modified David’s classification-based database. We aim to improve the performance of our model, and to put it at the service of learning and routine use in laboratories. Trial registration number not applicable

O-073 Delta-9-THC acts on the calcium channel CatSper and alters human sperm function

Abstract Study question Do the main psychoactive phytocannabinoid delta-9-tetrahydrocannabinol (THC) and its non-psychoactive analog cannabidiol (CBD) affect human sperm function? Summary answer THC reduces the ligand-dependent activation of sperm-specific Ca2+ channel CatSper and affects human sperm functions in vitro. What is known already Marijuana (Cannabis sativa) is one of the most commonly used recreational drugs worldwide. Although the impact of phytocannabinoids on reproductive health has been investigated, there is no evidence of a direct effect of THC on CatSper function. Study design, size, duration We studied the effects of the main psychoactive phytocannabinoid, THC, its non-psychoactive analog, CBD, as well as their major metabolites on Ca2+ influx via CatSper in human spermatozoa. THC and CBD were selected to further evaluate their action on progesterone-, prostaglandin-, and pH-induced activation of human CatSper. The effects of THC and CBD on hyperactivation, progressive motility in viscous media, and acrosomal exocytosis were also assessed. Participants/materials, setting, methods The impact of phytocannabinoids on CatSper activity was investigated on sperm samples from healthy volunteers using kinetic Ca2+ fluorimetry. Motility assessment was performed using Computer-Assisted Sperm Analysis (CASA). Sperm penetration into viscous media was assessed using a modified Kremer test. Acrosomal exocytosis was evaluated by flow cytometry using Pisum sativum agglutinin-stained spermatozoa. Main results and the role of chance Both THC and CBD suppress natural ligand-induced calcium influx via CatSper. In particular, THC inhibits progesterone-induced Ca2+ influx via CatSper at pharmacologically relevant concentrations in a non-competitive manner and reduces the pH-induced activation of CatSper. In addition, THC impaired sperm hyperactivation and penetration into viscous media and induced spontaneous acrosomal exocytosis in vitro. Limitations, reasons for caution This is an in vitro study. Future studies are needed to test the physiological relevance in vivo and whether THC can disrupt human sperm function. Wider implications of the findings The action of THC on CatSper in human sperm might impair the fertilization process. Healthcare providers, especially fertility clinicians, should be aware of the potentially negative effects of cannabis consumption on sperm physiology Trial registration number not applicable

O-257 IGF2BP3 induces apoptosis of spermatogenic cells by regulating the stability of Tcf4 mRNA in an m6A-dependent manner, thereby affecting the occurrence of azoospermia

Abstract Study question Does IGF2BP3 regulate Tcf4 mRNA stability in an N6-methyladenosine (m6A)-dependent manner, affect spermatogenesis, and lead to idiopathic non-obstructive azoospermia (iNOA)? Summary answer IGF2BP3 induces apoptosis of spermatogenic cells by regulating the stability of Tcf4 mRNA in an m6A-dependent manner, which in turn affects spermatogenesis leading to iNOA. What is known already Azoospermia is the most serious phenotype of male infertility, affecting approximately 10-15% of male infertility patients. iNOA refers to a class of non-obstructive azoospermia of unknown etiology for which there is no effective treatment. The etiology of iNOA is poorly understood, and its genetic and molecular mechanisms are poorly studied. IGF2BP3 was found to be highly expressed in tumor tissues and to be a post-transcriptional regulator in cancers. The study found that IGF2BP3 is an m6A reading protein that recognizes mRNA m6A modification and enhances mRNA stability and translation. Study design, size, duration We collected testicular tissue from 120 iNOA and 120 patients with obstructive azoospermia through prospective studies to analyze differentially expressed genes by high-throughput RNA sequencing. Participants/materials, setting, methods IGF2BP3 knockout and overexpression mouse models were constructed. Spermatogenesis was assessed by immunostaining, HE staining, spermatogenic specific gene expression, and computer-assisted sperm analysis (CASA). The stability of target mRNA was conducted by ActD. Merip-qRCR was used to detect IGF2BP3 binds to the target gene in an m6A-dependent manner; the m6A modification site of Tcf4 was predicted by SRAMP and verified by double luciferase reporting experiments. Overexpression Tcf4 to analyze its effect on spermatogenesis. Main results and the role of chance RNA-seq showed that IGF2BP3 was lowly expressed in iNOA testicular tissue which was verified by qRT-PCR and immunohistochemistry. After knocking down IGF2BP3, the spermatogenesis process is impaired, HE showed that the arrangement of spermatogenic cells in the seminiferous tubule was disordered and reduced, the spermatogonia and spermatoblasts were significantly reduced, and there were very few sperm in the seminiferous tubule. CASA showed that sperm density was significantly lower than that of the control group (p &amp;lt; 0.05). Western blot and qRT-PCR showed that spermatogenic cells-specific genes Nanos3, Sycp3, Tnp1, proliferation indexes PLZF and PCNA were all reduced compared with the control group. Overexpression of IGF2BP3 can reverse spermatogenesis in azoospermia mice; after knocking down IGF2BP3, the fertility of mice in vivo and in vitro was impaired; apoptosis of spermatogenic cells increased in testicular tissues of mice after knocking down IGF2BP3 and decreased in mouse testicular tissue after knocking down IGF2BP3. IGF2BP3 induces apoptosis of spermatogenic cells by regulating the stability of Tcf4 mRNA in an m6A-dependent manner, resulting in impaired spermatogenesis; overexpression of Tcf4 in GC1 cells knocked down IGF2BP3 reduces apoptosis and increases cell proliferation. Limitations, reasons for caution In this study, we constructed knockdown and overexpression mice models by in situ injection of IGF2BP3 lentivirus in the testes, and the results were more accurate if conditional knockout mice model was used. Wider implications of the findings We found direct clinical and in vivo animal model evidence for the causes of male infertility in patients with idiopathic non-obstructive azoospermia, and for the first time found that IGF2BP3 affects spermatogenesis by regulating the stability of target genes in an m6A-dependent manner. Trial registration number not applicable