Impact of Cryopreservation on Motile Subpopulations and Tyrosine-Phosphorylated Regions of Ram Spermatozoa during Capacitating Conditions

The heterogeneous nature of ejaculates highlights the relevance of studying the behavior of different sperm subpopulations. Changes in sperm motility and the increase in tyrosine phosphorylation are key events that usually occur during capacitation and can be modified by the cryopreservation process. However, the relationship between both events remains poorly defined throughout capacitation in the different sperm subpopulations. Fresh and frozen-thawed spermatozoa were incubated in capacitating (CAP) and non-capacitating (NC) media up to 240 min. Sperm kinematics, tyrosine phosphorylation and mitochondrial activity were measured by the CASA system and imaging flow cytometry. Four motile sperm subpopulations (SP) were identified in fresh and frozen-thawed ram semen after the cluster analysis. Incubation under CAP conditions over time led to greater changes in the percentage of spermatozoa included in each subpopulation compared to NC conditions, being different between fresh and frozen-thawed spermatozoa. The SP1, characterized by slow spermatozoa, progressively increased after 15 min in frozen-thawed samples incubated in both media but not in fresh ones. The SP4, characterized by fast and non-linear spermatozoa, showed a marked increase during CAP, but not under NC conditions, occurring more rapidly in frozen-thawed spermatozoa. This subpopulation (SP4) was also the only one positively and strongly correlated with mitochondrial activity and all phosphorylated sperm regions during capacitation, either in fresh or frozen-thawed samples. Our results indicated that in vitro capacitation induced significant changes in the distribution of motile sperm subpopulations, being affected by cryopreservation. Notwithstanding, the subpopulation which probably represents hyperactivated-like spermatozoa (SP4) also increased in frozen-thawed samples, occurring faster and simultaneously to the increment of mitochondrial activity and tyrosine phosphorylation of different sperm regions.

Comparative Proteomic Analysis of Young and Adult Bull (Bos taurus) Cryopreserved Semen

The age of the bull is widely accepted to influence the production of sperm, affecting the amount and quality of produced semen, which in turn impacts the results of cryopreservation. However, the exact influence of the maturation process on cryopreserved sperm, as well as the underlying molecular mechanisms of this process, are not fully understood. The goal of this study was to evaluate changes in the proteome of thawed semen (spermatozoa and supernatant) collected from young and adult bulls (n = 6) using the 2D-DIGE approach. The quality of semen was assessed using a CASA system and flow cytometry. We found no significant age-related variation in semen quality, with the exception of the average path velocity of sperm movement, which was higher in adult bulls. Proteomic analysis indicated 15 spermatozoa proteins and 10 supernatant proteins with significant age-related changes. Our results suggest that semen from adult bulls is better equipped with proteins related to energy production, protection of spermatozoa against oxidative stress and fertilizing ability. Proteins increased in abundance in young bull spermatozoa were connected to the cytoskeleton and its development, which strongly suggests that developmental processes are still in progress. In conclusion, our results provide novel insight into the mechanism of the development of the male reproductive system of cattle.

P–105 Clinical validation of mojo AISA, an artificial intelligence robotic CASA system

Study question Can a CASA system based on Artificial Intelligence perform as well as manual semen assessment, within the WHO error margins? Summary answer The AI-based CASA systems that mimic high quality assessments show great potential for reducing clinical workloads while increasing treatment efficacy. What is known already The field of male-factor fertility investigation is still lacking an automated semen analysis system that can be widely clinically adopted. By leveraging state-of-the-art robotics and Artificial Intelligence (AI), it was possible to build mojo AISA which is an AI and robotic platform designed according to WHO recommendation for semen analysis. This system is based on AI software with a unique convolutional neural network (CNN) that detects and measures sperm concentration and motility while ruling out unwanted cells and debris in raw samples. Study design, size, duration This study presents and validates the mojo AISA device. A total of 60 patient samples at ANOVA Karolinska University Hospital were collected and results from manual assessment were compared to mojo AISA for concentration and motility. Semen samples were assessed manually (WHO 2010) and concurrently with Mojo AISA. Manual measurements ranged from 1–206M/ml. This study lasted from May 2020 to December 2020 following informed consent and ethics committee practices of ANOVA. Participants/materials, setting, methods Sample preparation protocol for mojo AISA consisted of placing two 10ul drops and covering with two 22x22mm coverslip. Manual assessment followed ANOVA EQA procedures akin to the WHO. A CNN was trained using videos captured with mojo AISA as input data. Images were annotated to form a validation set by which the AI was trained. To account for sampling error, videos of Hamilton Thorne Accubeads+ were captured using mojo AISA and the mojo counting chambers. Main results and the role of chance Comparing the concentration measured by mojo AISA with the known value for each microbead, results are in agreement of 86%, within the confidence interval of the microbeads. The mean relative error was 6.7% and maximum error was 11%. Therefore, Accubeads+ validation proved no observational error regarding the use of mojo AISA microscope. As for comparing mojo AISA to manual assessment for concentration, Pearson (Spearman) correlation was 0.95 (0.97). The mean relative error was 24.8% and maximum relative error was 71.1%, where 90% of samples were below 50% error. By looking at the concentration range between 10 and 20 M/ml, mojo AISA displayed a mean error of 18.5%. For motility, as comparing mojo AISA to manual assessment, a result of 35.4% mean relative error was obtained. To conclude, mojo’s robotic solution shows promise for clinical practice as the AI continues to improve. In 6 months, sperm concentration correlation improved by 3-fold. Next, the AI will be further clinically trained for low concentration. Limitations, reasons for caution mojo AISA requires further development, especially for very low concentration ranges, below 5M/ml, due to high sensibility to false positive detections. The same applies to post-vasectomy samples. Additionally, the necessity to compute the motility of each sperm scales poorly with high concentration generating a poor experience for high volume clinics. Wider implications of the findings: Automation is crucial in several industries. It enables fertility clinics & andrologists to standardize male factor infertility measurements (if paired with widespread standardization of protocols for automation) while enabling them to put more focus on demanding activities of their profession and removes human biases of inter-laboratory performance. Trial registration number Not applicable

Sperm filtration as an alternative technique for seminal plasma separation in boars

The present study aimed to evaluate the filtration for separating seminal plasma of boars’ ejaculate by means of sperm viability and the occurrence of hyperactivation and lipid peroxidation in fresh semen and after cooling for up to 96 hours. The ejaculate of eight healthy boars of different breeds was collected and the gelatinous portion was separated and discarded. In the laboratory, the semen was fractioned into three aliquots (groups G1, G2 and G3) as follows: G1: semen with plasma diluted in BTS (TOTAL BTS); G2: semen centrifuged at 600G/10’ (BTS CEN); and G3: semen filtered with the Sperm-filter® following dilution of the retained cells with BTS (BTS FIL). The analyses were performed at three moments: with fresh samples (D0) and after 48 (D2) and 96 hours (D4) of cooling at 17ºC. The kinetic evaluation was performed using the CASA system, which provided data for the classification of sperm hyperactivity. For lipid stress analysis, the TBARS (thiobarbituric acid reactive substance) test was performed. The variance analysis test was conducted to compare the results between the groups and moments analyzed. The results showed better total motility values (%) for G1 at D0 (67.9, P= 0.001), D2 (36.6, P= 0.004) and D4 (26.1, P= 0.003). The occurrence of hyperactivity was observed in G2 and G3 at moments D2 and D4. In addition, TBARS showed higher peroxidation levels for G1 at D0 (8.1 mM MDA/ml, P= 0.01), D2 (7.4 mM MDA/ml, P= 0.02), and D4 (6.41mMol MDA/ml, P= 0.008) when compared to G2 and G3. Since the filtration method did not demonstrate any damage to the sperm viability, the study concluded that sperm filtration is an accessible and valid tool to replace centrifugation.

Assessment of an iPad-based Sperm Motility Analyzer for Determination of Canine Sperm Motility

The objective of this study was to evaluate the repeatability and accuracy of canine sperm motility (total and progressive) assessment with a tablet-based Canine iSperm ® instrument compared to computer-assisted sperm analysis (CASA). The experiment used fresh and frozen/thawed canine semen samples for comparisons of semen analysis parameters (concentration, total motility, and progressive motility) between a CASA system, iSperm ®, and NucleoCounter ® SP-100 ™ (concentration) instruments. Spearman’s Rho correlational analysis was used to identify significant associations between motility assessment methods. Significant positive correlations were found between CASA assessment and iSperm ® for both progressive and total motility measurements. We also determined the coefficient of variation (CV) for repeatability of sample analysis for iSperm ® and CASA for fresh sperm, wherein each sample was assessed 10 times on both devices. For fresh and frozen-thawed samples, concentration assessment by iSperm ® showed high variability (CV= 19.9 ± 1.5%). For iSperm ® assessment of total and progressive motility, the CV’s were 6.3 ± 0.5% and 10.7 ± 0.8%, respectively. The results indicate that the iSperm ® application offers an accurate and alternative measurement of motility to traditional CASA analysis, though caution should be taken when assessing concentration due to the high CV observed in this study.

Sperm kinematic subpopulations of the American crocodile (Crocodylus acutus)

There has been very limited use of computer assisted semen analysis (CASA) to evaluate reptile sperm. The aim of this study was to examine sperm kinematic variables in American crocodile (Crocodylus acutus) semen samples and to assess whether sperm subpopulations could be characterized. Eight ejaculates (two ejaculates/male) from four sexually mature captive crocodiles were obtained. An ISAS®v1 CASA-Mot system, with an image acquisition rate of 50 Hz, and ISAS®D4C20 counting chambers were used for sperm analyses. The percentages of motile and progressively motile spermatozoa did not differ among animals (P > 0.05) but there was a significant animal effect with regards to kinematic variables (P < 0.05). Principal component (PC) analysis revealed that kinematic variables grouped into three components: PC1, related to velocity; PC2 to progressiveness and PC3 to oscillation. Subpopulation structure analysis identified four groups (P < 0.05), which represented, on average, 9.8%, 32.1%, 26.8%, and 31.3% of the total sperm population. Males differed in the proportion of sperm in each of the kinematic subpopulations. This new approach for the analysis of reptile sperm kinematic subpopulations, reflecting quantifiable parameters generated by CASA system technology, opens up possibilities for future assessments of crocodile sperm and will be useful in the future development of assisted reproduction for these species.

Evaluation of porcine semen quality by portable and desktop CASA systems – Short communication

When using artificial insemination in porcine reproduction, one of the most important requirements is the suitable quality of semen regarding its total motility (TM) and progressive motility (PM). Computer-assisted sperm analysis (CASA) is an appropriate method to analyse the quality of semen. Recently a portable instrument has been developed to help specialists in their everyday field work. In our study, semen quality was measured simultaneously by the portable device (Ongo) and a laboratory CASA system (Microptic) to compare TM and PM values obtained by these appliances at a concentration of 50 × 106 spermatozoa/mL. Agreement between measurements was evaluated with a Bland-Altman plot. Strong correlation was found between the investigated instruments for all the three parameters, i.e. sperm concentration, TM and PM. However, a few measurements fell outside the defined range of acceptance.

Time and Dose-Dependent Effects of Viscum Album Quercus on Rabbit Spermatozoa Motility and Viability in Vitro

The target of this study was to evaluate the effect of extract of the European mistletoe – Viscum album quercus L. on spermatozoa motility and viability in vitro. The CASA system was used to determine the spermatozoa motility parameters at different time intervals (0, 1, 2 and 3 h) and spermatozoa viability was determined in five different doses of Viscum album quercus L [10 (QA), 6.6 (QB), 3.3 (QC), 2.5 (QD) and 2 (QE) mg/ml]. Results in experimental groups detected a significant deterioration on rabbit spermatozoa after 1, 2 and 3 hours, compared to the control. The initial total spermatozoa motility showed increased value for all doses of Viscum album quercus in comparison to control. After in vitro culture a dose–dependent decrease (QA: reduction of 69.7 %, QB: reduction of 40.9 %) was found. For the progressive spermatozoa most significant decrease (86.8 % for QA vs. 48.5 % for QB) was detected compared to the control after 3 hours of culture. Spermatozoa viability (MTT test) was decreased in all experiment groups at the end of experiment, but the differences were not significant. Significant alterations of membrane integrity were found in groups with the highest Viscum album quercus concentration (QA, QB), but acrosome integrity showed no significant changes. Results suggest negative dose– and time–dependent effect of Viscum album quercus at higher doses on spermatozoa motility and viability parameters in vitro.