Computer-assisted Sperm Analysis System Research Articles

Using polynomial modeling for calculation of sperm quality parameters in CASA

Measurement of sperm velocity parameters takes an important place in sperm analysis. Today, computer-assisted sperm analysis (CASA) systems are used to detect motility measurements such as VCL (Curvilinear Velocity), VSL (Linear Velocity) and VAP (Average Path Velocity). The path length of sperm cells is necessary to calculate the motility parameters and it is calculated using video processing. However, this path length is obtained by the discrete-time processing of video frames, which can lead to unrealistic results. In CASA systems, the frame rate of the videos is increased to obtain the natural path length, but in this case, it is necessary to increase the hardware cost as well. Polynomial modeling applied to measure the natural path length in discrete-time structures can solve this problem. In this study, Lagrange interpolation which is an effective polynomial modeling method was used to obtain the natural path length and velocity parameters of the sperm. The results of the study showed that, when applied the polynomial modeling to calculate VCL and VAP parameters, it was found that especially in the low frame rate, more effective data were obtained than the classical method. As a result of this study, it is recommended to use polynomial modeling in the sperm velocity calculations of CASA systems, although there is an increase in calculation time.

Evaluation of ubiquitination and sumoylation of acrosin inhibitor during in vitro capacitation of porcine sperm

The main objective of this study was to investigate the expression of acrosin inhibitor (AI), ubiquitin (Ub), and small ubiquitin-related modifier 1 (SUMO1) proteins during in vitro capacitation of pig sperm. Duroc pig sperm was divided into fresh sperm and capacitation treatment groups. Protein expression was evaluated using computer-assisted sperm analysis (CASA) systems, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and immunofluorescence. The results showed that the expression of AI (30 kDa) incapacitated sperm was significantly lower than that in fresh sperm (P < 0.05), and that the levels of ubiquitinated and SUMO1-ylated proteins in capacitated sperm were significantly higher than those in fresh sperm (P < 0.05). Immunofluorescence results showed that AI, Ub, and SUMO1 were located in the acrosome region of the fresh and capacitated sperm heads. After capacitation, the fluorescence intensity of AI and SUMO1 decreased, while that of Ub increased. The protein band at 30 kDa represented the AI-Ub-SUMO1 complex, indicating that this complex was involved in sperm capacitation. Furthermore, SUMO1 increased the stability of AI at 30 kDa, preventing its complete decomposition, while at 46 kDa, in the absence of SUMO1, AI is bound to ubiquitin, and was completely degraded.

Computer-assisted sperm analysis (CASA) in veterinary science: A review

Computer-assisted sperm analysis (CASA) allows an assessment of sperm motion and morphology more accurately and objectively than by subjective evaluation. Although, CASA instruments have improved significantly during last 40 years especially in terms of software, image capture and computer settings, little has changed regarding processes for analyzing sperm motion attributes. The main problem is related to validation, consistency and optimization of equipment and procedures. Differences among CASA systems denote problems of objective analysis of results between different semen processing units. If validated, CASA systems can provide a great tool to objectively compare sperm motility and morphology. Sperm motility is one of the indicators most evaluated before and after cryopreservation vis-à-vis quality and fertilizing ability. Researchers have determined a possible relationship of CASA outputs with bull fertility in vivo; however, a clear association has not yet been confirmed. Most CASA measures depend upon concentration, sample volume, type of extender, duration of analysis and thawing temperature. For each attribute, CASA software should provide outputs based on a range rather than means or medians for transformed data. The current review describes development, validation requirements, limitations and future expansions associated with CASA technology.

Application of artificial seminal plasma to short-term storage of a large volume of common carp (Cyprinus carpio) sperm for two weeks under controlled conditions

We previously demonstrated that artificial seminal plasma (ASP), developed for common carp (Cyprinus carpio), enabled considerable improvement of the fertilizing capacity of sperm in this species. Here, for the first time, the effectiveness of using ASP (ASP: 2 mM CaCl2, 1 mM MgSO4, 40 mM KCl, 100 mM NaCl, 20 mM Tris; pH 7.5 and 310 mOsm kg−1) for short-term storage of tenfold diluted sperm for up to two weeks at 10 °C was evaluated. Additionally, the fertilizing capacity of stored sperm was investigated. Sperm were stored in a volume of 5 ml in containers equipped with ventilation plugs enabling gas exchange of the stored sperm. During the study, the quality of the sperm was verified using the computer-assisted sperm analysis (CASA) system after 1, 2, 5, 9, 11 and 14 days of storage. After 14 days of common carp sperm storage in ASP sperm motility (MOT, %), the progressive motile sperm (PRG, %) and curvilinear velocity of sperm (VCL, μm s−1) were confirmed to be 55%, 25% and 120 μm s−1, respectively. At the same time, in undiluted sperm (Control), we did not observe sperm motility. Only until the 2nd day of storage in undiluted samples motility and sperm kinetic parameters remained high level (84%, 34% and 144 μm s−1 for MOT, PRG and VCL, respectively). The fertilization of eggs and hatching rate after 14 days of sperm storage in ASP were 80% and 40%, respectively, and did not differ significantly from the fertilization and hatching rate determined after fresh sperm was used for fertilization. In summary, for the first time, the fertilization capacity of common carp sperm after 14 days of short-term storage under ASP conditions was confirmed, indicating the possibility of using previously collected and diluted ASP sperm in reproduction at a time convenient for the breeder. Thus, it is possible to reduce the stressful and time-consuming handling of fish during breeding under controlled conditions, which has an impact on the welfare of spawners.

P–123 How to develop accurate Computer Assisted Sperm Analysis (CASA) AI in the absence of protocol standardization and abundance of human error when performing semen analyses?

Abstract Study question How can an automation &amp; artificial intelligent tools be developed to perform according to WHO recommendations? Summary answer Developing CASA performs at &amp;lt; 20% error margin requires AI trained with high quality datasets and a robotic system adheres to WHO guidelines. What is known already A survey of 40 andrology laboratories, in 22 countries, revealed that &amp;gt; 90% had nonconformities in correct use of equipment, standardisation of protocols and quality control, leading to a lack of compliance to WHO protocols. Conventional CASA systems can standardize analysis, but controversy has occurred due to differences between manual and automated analyses stemming from: 1) all cells in a semen sample are detected including debris; 2) protocol variation when compared to top-notch manual analysis. The first point can be addressed by AI. The second point can be addressed by robotics designed to adhere to WHO guidelines. Study design, size, duration A mojo AISA (AI-powered semen analysis) system was placed in four clinical laboratories mentioned above capturing images of over 300 samples, one million images were generated over a course of 2 years. Mojo AISA’s AI was trained on data collected from the four clinics using robotic system is developed according to WHO guidelines. Participants/materials, setting, methods For an AI to detect sperm accurately, sperm samples were captured using mojo AISA smart microscopy and then the extracted sperm images expertly annotated. To evaluate the system-ability for semen analysis, fresh sample were analysed for concentration and motility by a manual operator and compared to a mojo AISA test. Main results and the role of chance To train the sperm detection AI, representative sperm images were carefully captured using mojo AISA and processed according to the following criteria: the number of images and videos to train and to test the model: 50,000 spermatozoon head and tails with various variations the variety of images: data used to train the AI has to be representative of the population that will undergo the analysis: 1) wide concentration ranges from 0 to 300 M/ml, 2) high and low density of debris and cells, 3) Presence of slight aggregations careful and precise annotation: expert andrology scientists annotated sperm images and identify objects to exclude, such as debris in seminal plasma, Mojo AISA is an attempt strictly build CASA AI system to WHO-guidelines. The marriage of AI and robotics automation has shown a promising results to mimic humans when measuring a semen sample and attempt to obtain results comparable to the manual analysis. mojo AISA’s performance improved three-fold (from 0,85 to 0,95 Pearson sperm count correlation and from &amp;gt;100% means relative error to 25% mean relative error). Limitations, reasons for caution Lack of standardization for semen analysis laboratory process globally is a bottleneck towards building a robust multi-center study, on-site CASA testing and generating an actionable data pool for studying the causes behind male fertility declineWider implications of the findings: Key learnings for parties advancing developing AI based on images and videos for application in the fertility space. Trial registration number Not applicable

Cryobank of Mediterranean Brown Trout Semen: Evaluation of the Use of Frozen Semen up to Six Hours Post-Collection

The aim of this study was to evaluate the effects of different cold-storage time intervals between collection and semen-freezing on both fresh and cryopreserved semen motility parameters and the post-thaw fertilizing ability of Mediterranean brown trout semen. The ejaculates were split into six aliquots and stored on ice from 1 to 6 h, until freezing. Fresh and post-thaw sperm motility was evaluated by a Computer-Assisted Sperm Analysis system, whilst the fertilizing ability was assessed by in vivo trials. In fresh semen, at 3 h of storage, a significant decrease of total motility, linear movement (STR, LIN) and beat cross frequency (BCF) was recorded, whilst the amplitude of lateral displacement of the spermatozoon head (ALH) underwent a significant increase. In frozen semen, no significant difference was observed for all the motility parameters evaluated, except for the total motility between 1 and 6 h of storage and the duration of sperm movement between 1 and 5 h. Cold-storage time did not significantly affect the percentage of live embryos following the use of frozen semen. In conclusion, our results showed that, if necessary, the Mediterranean brown trout semen can be frozen even until 6 h post-collection without losing its fertilizing ability.

Obesity Causes Abrupt Changes in the Testicular Microbiota and Sperm Motility of Zebrafish.

BackgroundObesity is a recognized risk factor for low fertility and is becoming increasingly prevalent in many countries around the world. Obesity changes intestinal microbiota composition, causes inflammation of various organs, and also reduces sperm quality. Several microorganisms are present in the testis. However, whether obesity affects the changes of testicular microbiota and whether these changes are related to reduced fertility in obese men remain to be elucidated.MethodsIn the present study, a zebrafish obesity model was established by feeding with egg yolk powder. Sperm motility was measured by the Computer Assisted Sperm Analysis system, testicular microbial communities was assessed via 16s RNA sequencing, the immune response in zebrafish testis was quantified by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and the testicular tissue structure was detected by electron microscopy and hematoxylin–eosin staining.ResultsCompared with the control group, zebrafish sperm motility was dramatically reduced, the expression of testicular proinflammatory cytokines in the testes was upregulated, and the blood–testis barrier structure was disrupted in the obese group. In addition, testicular microbiome composition was clearly altered in the obese group.ConclusionObesity alters testicular microbiota composition, and the reason behind the decreased sperm motility in obese zebrafish may be related to changes in the testicular microbial communities.

Post-Thaw Sperm Quality and Functionality in the Autochthonous Pig Breed Gochu Asturcelta.

Simple SummaryGenetic resource banks were created to preserve the genetic material of endangered, rare, valuable individuals or genetically relevant breeds. Sperm cryopreservation is a practical and widespread strategy to preserve these genetic materials. This study aimed to characterize the frozen-thawed sperm of the native pig breed Gochu Asturcelta, considering the effects of boar age and season of semen collection on post-thaw sperm quality. We found that the boar age did not have a significant effect on the sperm parameters assessed. However, the season significantly affected many of these parameters (motility, viability, acrosomal status, mitochondrial activity). In general, sperm samples collected in spring and summer showed higher quality post-thawing, the lowest in winter. Our findings demonstrated that the post-thawing sperm quality of Gochu Asturcelta was in the range of results for commercial breeds, bringing a good prospect for the use of assisted reproductive technologies in this local breed.Genetic resource banks (GRB) preserve the genetic material of endangered, valuable individuals or genetically relevant breeds. Semen cryopreservation is a crucial technique to reach these goals. Thus, we aimed to assess the sperm parameters of semen doses from the native pig breed Gochu Asturcelta stored at the GRB of Principado de Asturias (GRB-PA, Gijón, Spain), focusing on intrinsic and extrinsic (boar, season) factors. Two straws per boar (n = 18, 8–71 months of age) were thawed, pooled, and assessed after 30 and 150 min at 37 °C by CASA (computer-assisted sperm analysis system; motility and kinematic parameters) and flow cytometry (viability, acrosomal status, mitochondrial activity, apoptosis, reactive oxygen species, and chromatin status). The effects of age, incubation, and season on post-thawing quality were determined using linear mixed-effects models. Parameters were on the range for commercial boar breeds, with chromatin status (SCSA: fragmentation and immaturity) being excellent. Incubation decreased sperm quality and functionality. The boar age did not have a significant effect (p > 0.05), but the between-boar variability was significant (p < 0.001). The season significantly affected many parameters (motility, kinematics, viability, acrosomal status, mitochondrial activity), especially after 150 min of incubation. In general, samples collected in spring and summer showed higher quality post-thawing, the lowest in winter. In conclusion, the sperm doses from the Gochu Asturcelta breed stored at the GRB-PA showed excellent chromatin status and acceptable characteristics after thawing. Therefore, boar and seasonal variability in this autochthonous breed could be relevant for cryobank management.

How age, captivity and cryopreservation affect sperm quality and reproductive efficiency in precocious Atlantic salmon (Salmo salar L. 1758)

In order to restore an extinct population in a river basin, enough biological material for restocking purposes needs to be produced which requires male semen of good quality but also in large quantities. For sperm production in Atlantic salmon, it is possible to rely on (i) early matured males or (ii) sperm cryopreservation. However, the quality of both, fresh and cryopreserved spermatozoa can be influenced by biological factors such as the age of the fish or factors linked to their environment (captive vs. wild fish). this study investigates the impact of these factors to ensure good reproductive success. For this purpose, spermatozoa viability (SYBR-14/PI) and mitochondrial membrane potential (JC-1) were evaluated with flow cytometry staining techniques. Motility as well as velocity and trajectory parameters were evaluated using the Computer Assisted Sperm Analysis system (CASA). DNA integrity was assessed with the help of the Comet assay. After assisted reproduction, the reproductive success was determined at two developmental stages (i.e. eyed stage and hatching). For the first time, spermatozoa quality and reproductive success were compared between adult males and early parr of different ages and origins. Spermatozoa quality and reproductive success of fresh semen was similar for anadromous males and early parr regardless of their age and origin. This observation further supports the use of highly available young parr from captivity for reproduction purposes. Cryopreservation on the other hand, affected spermatozoa quality differently in the different conditions tested. The mitochondrial potential and motility of parr from captivity was more severely affected by cryopreservation, which directly translated into reduced reproductive success. This is the first report of decreased cryoresistance of spermatozoa of precocious salmon induced by captivity. It is therefore recommended that only the semen of anadromous males should be cryopreserved.

Beneficial Influence of Soybean Lecithin Nanoparticles on Rooster Frozen-Thawed Semen Quality and Fertility.

Simple SummarySoy lecithin (SL) can be used in to prevent spermatozoa cryodamage during cryopreservation by mitigating the efflux of cholesterol or phospholipids, thus reducing the formation of intracellular ice crystals. SL nanoparticles (nano-SL) have a smaller particle size and higher solubilizing capacity as compared with those that have not undergone nanotreatment. Thus, they allow for a better interaction or coating of sperm, decreasing cold shock injury during freezing–thawing processes. The objective of this study was to determine the optimal concentration of nano-SL. In order to achieve this, we assessed the quality of frozen–thawed semen in vitro and in vivo. We found that a nano-SL dosage of 1.0% in the semen extender had an affirmative influence on post-thawing quality in roosters, improving various parameters related to sperm motion, protecting the membrane and acrosome integrities, increasing mitochondrial activity and antioxidant capacity, and reducing the oxidative stress caused by the cryopreservation process. Moreover, enrichment of 1.0% nano-SL in the semen extender improved the fertilizing capacity of rooster sperm after artificial insemination. The present study aimed to investigate the impact of different concentrations (0%, 0.5%, 1.0%, 1.5%, and 2.0%) of nano-soybean lecithin (SL) in the extender on sperm quality, sperm motion characteristics, and fertility outcomes of post-thawed rooster semen. Adult Ross broiler breeder roosters (n = 20) were subjected to semen collections twice a week for three weeks. At each collection, semen samples were pooled and allocated into five treatments corresponding to different nano-SL concentrations (control, SL0.5, SL1.0, SL1.5, and SL2.0). Sperm parameters, including motility (collected using a computer-assisted sperm analysis system), plasma membrane and acrosome integrities, and mitochondrial activity were assessed. Sperm malondialdehyde (MDA) and antioxidant activities (total antioxidant capacity (TAC); superoxide dismutase (SOD); glutathione peroxidase (GPx)) were evaluated. The fertility and hatchability obtained with frozen–thawed rooster semen supplemented with the optimum nano-SL concentration were assessed after artificial insemination. The results showed that the addition of 1% nano-SL into the extender led to a higher semen motility in roosters, improved plasma membrane and acrosome integrities, and higher mitochondrial activity of post-thawed rooster semen in comparison to controls (p < 0.05). The MDA levels in the SL0.5 and SL1.0 groups were lower than the other groups (p < 0.05). TAC activities in SL0.5, SL1.0, and SL1.5 groups were significantly higher than those in the other groups (p < 0.05). It was observed that the concentration of SOD was higher in the SL1.0 group than in the other groups (p < 0.05). The activity of GPx was not influenced in any of the cases (p > 0.05). Moreover, the percentages of fertility and hatchability in the SL1.0 group were higher (56.36% and 58.06%) than those in the control group (42.72% and 40.43%). In summary, the addition of nano-SL to the extenders enhanced the post-thawed semen quality and fertility of roosters by reducing the level of oxidative stress. The optimum nano-SL concentration was 1.0%. These results may be beneficial for improving the efficacy of semen cryopreservation procedures in poultry breeding.

A comparative study of two methods to determine acrosome integrity of frozen-thawed boar sperm

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P&amp;gt;0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P&amp;gt;0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.

A new open-source hardware device to measure vertical sperm motility and concentration.

Sperm motility and concentration are commonly evaluated parameters in semen analysis. Those parameters are assessed objectively with commercial instrumentation such as computer-assisted sperm analysis systems (CASA) and hemocytometer. In CASA systems, sperm motility is assessed in the horizontal plane imposed by the stage of the microscope. Thus, there is lack of measurement of the vertical velocity of sperm. The female reproductive tract is a tridimensional space which the sperm traverse to reach the ovum, and there is a need for instruments measuring parameters more relevant to this real-world situation. In this report we describe the design, construction and use of an open-source hardware (OSH) device for evaluation of the vertical velocity of sperm, called UPSPERM. This device was also used to measure sperm concentration, and agreement with hemocytometer was evaluated. Bland-Altman analysis shows good agreement between these two methods of sperm counting. As a first application of UPSPERM, we evaluated the changes in boar sperm motility at distinct pH values between 7.0 and 8.0. The UPSPERM results showed that the vertical velocity of sperm was highest at pH7.6 and 7.8. We propose that our UPSPERM offers a reliable and affordable option for obtaining measurements of vertical velocity and sperm concentration.

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 CVs 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.

Simplified sperm testing devices: a possible tool to overcome lack of accessibility and inconsistency in male factor infertility diagnosis. An opportunity for low- and middle- income countries.

Manual semen assessment (MSA) is a key component in a male's fertility assessment. Clinicians rely on it to make diagnostic and treatment decisions. When performed manually, this routine laboratory test is prone to variability due to human intervention which can lead to misdiagnosis and consequently over- or under- treatment. For standardisation, continuous training, quality control (QC) programs and pricy Computer-Assisted Sperm Analysis (CASA) systems have been proposed, yet, without resolving intra- and inter-laboratory variability. In response, promising simplified sperm testing devices, able to provide cost-effective point-of-care male infertility diagnosis are prospected as a plausible solution to resolve variability and increase access to sperm testing. A throughout literature research for semen testing, sperm analysis, smart-phone assisted semen analysis, 'at-home' semen testing, male infertility, infertility in developing countries, infertility in low- and middle-income countries (LMIC) and quantitative sperm analysis was performed. A total of 14 articles, specific to 'at-home' simplified sperm assessment, were included to treat the core subject. Continuous training and consistent QC, are sine qua none conditions to achieve accurate and comparable MSA. Compliance does not rule-out variability, nevertheless. Emerging simplified sperm assessment devices are an actual alternative to resolve the lack of standardisation and accessibility to sperm analysis. YO ® , SEEM ® , and ExSeed ® are commercially available, user-friendly smartphone-based devices which can accurately measure volume, sperm concentration (millions/ml) and total motile sperm count. More broadly, by cost-effectiveness, availability, accuracy and convenient application, these devices could effectively select patients for first-line artificial reproduction treatments such as intrauterine insemination. Accuracy and cost-effectiveness make smart-phone based sperm testing devices a practical and realistic solution to overcome variability in MSA. Importantly, these tools represent an actual opportunity to standardise and improve male subfertility diagnosis and treatment, especially in LMIC. However, before clinical application is possible, guidelines, further testing with special attention on accuracy in washed sperm, availability, cost-benefit and reliability are required.

Predictive Capacity of Boar Sperm Morphometry and Morphometric Sub-Populations on Reproductive Success after Artificial Insemination.

Simple SummaryThe efficiency of swine production measured as litter size influences the profitability of the pig industry. Furthermore, sow fertility potential depends in part on the boar semen quality and reproductive efficiency. The objective of this study is to compare boar sperm head size and morphometric features of shape to evaluate their relationships with reproductive success after artificial insemination (AI). A morphometric analysis of boar ejaculate reveals morphometrically separate sub-populations. The differences between sub-populations are displayed for sperm head size. In addition, sperm clustering into sub-populations did not have a predictive capacity on litter size variables. Nevertheless, the morphometric variables of the sperm may have a predictive, albeit reduced, capacity regarding litter size variables. The results of this study therefore open up possibilities for future assessments of fertility. The aim of the study was to compare the morphometric features of sperm head size and shape from the Pietrain line and the Duroc × Pietrain boar crossbred terminal lines, and to evaluate their relationship with reproductive success after artificial insemination of sows produced from crossbreeding the York, Landrace and Pietrain breeds. Semen samples were collected from 11 sexually mature boars. Only ejaculates with greater than 70% motility rate and <15% of abnormal sperm were used for artificial inseminations (AI) and included in the study. Samples were analyzed using an ISAS®v1 computer-assisted sperm analysis system for eight morphometric parameters of head shape and size (CASA-Morph). Sub-populations of morphometric ejaculates were characterized using multivariate procedures, such as principal component (PC) analysis and clustering methods (k-means model). Four different ejaculate sub-populations were identified from two PCs that involved the head shape and size of the spermatozoa. The discriminant ability of the different morphometric sperm variables to predict sow litter size was analyzed using a receiver operating characteristics (ROC) curve analysis. Sperm head length, ellipticity, elongation, and regularity showed significant predictive capacity on litter size (0.59, 0.59, 0.60, and 0.56 area under curve (AUC), respectively). The morphometric sperm sub-populations were not related to sow litter size.

Retinoic Acid Induced Protein 14 (Rai14) is dispensable for mouse spermatogenesis.

BackgroundRetinoic Acid Induced Protein 14 (Rai14) is an evolutionarily conserved gene that is highly expressed in the testis. Previous experiments have reported that small interfering RNA (siRNA)-mediated gene knockdown (KD) of Rai14 in rat testis disrupted spermatid polarity and transport. Of note, a gene knockout (KO) model is considered the “gold standard” for in vivo assessment of crucial gene functions. Herein, we used CRISPR/Cas9-based gene editing to investigate the in vivo role of Rai14 in mouse testis.MethodsSperm concentration and motility were assayed using a computer-assisted sperm analysis (CASA) system. Histological and immunofluorescence (IF) staining and transmission electron microscopy (TEM) were used to visualize the effects of Rai14 KO in the testes and epididymides. Terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) was used to determine apoptotic cells. Gene transcript levels were calculated by real-time quantitative PCR.ResultsRai14 KO in mice depicted normal fertility and complete spermatogenesis, which is in sharp contrast with the results reported previously in a Rai14 KD rat model. Sperm parameters and cellular apoptosis did not appear to differ between wild-type (WT) and KO group. Mechanistically, in contrast to the well-known role of Rai14 in modulating the dynamics of F-actin at the ectoplasmic specialization (ES) junction in the testis, morphological changes of ES junction exhibited no differences between Rai14 KO and WT testes. Moreover, the F-actin surrounded at the ES junction was also comparable between the two groups.ConclusionIn summary, our study demonstrates that Rai14 is dispensable for mouse spermatogenesis and fertility. Although the results of this study were negative, the phenotypic information obtained herein provide an enhanced understanding of the role of Rai14 in the testis, and researchers may refer to these results to avoid conducting redundant experiments.

Assessing coral sperm motility

The declining reproductive viability of corals threatens their ability to adapt to changing ocean conditions. It is vital that we monitor this viability quantitatively and comparatively. Computer-assisted sperm analysis (CASA) systems offer in-depth analysis used regularly for domestic and wildlife species, but not yet for coral. This study proposes quality control procedures and CASA settings that are effective for coral sperm analysis. To resolve disparities between CASA measurements and evaluations by eye, two negative effects on motility had to be resolved, slide adhesion (procedural) and sperm dilution (biological). We showed that the addition of bovine serum albumin, or caffeine, or both to fresh sperm reduced adhesion in the CASA cassettes, improved motility and motile sperm concentration (P < 0.0001), yet these additions did not affect measurements of total sperm concentration. Diluting coral sperm reduced sperm motility (P = 0.039), especially from heat-stressed corals. We found CASA concentration counts comparable to haemocytometer and flow cytometer measures (P = 0.54). We also found that motile sperm per egg is a useful predictor of fertilisation success, using cryopreserved sperm. Standard measurements of coral reproductive characteristics inform our understanding of the impacts of climate change on reef populations; this study provides a benchmark to begin this comparative work.

Effect of Repeated Freezing and Thawing on Nguni Sperm Parameters Evaluated by Computer Assisted Sperm Analyzer System

The aims of the current study were to assess the effect of double freezing and thawing on Nguni semen parameters. Semen was collected from Nguni bulls. The Sperm Class Analyzer® system was used to evaluate sperm motility rates prior and post freezing, repeatedly. Semen samples were diluted with egg yolk citrate extender. Cooled semen samples were loaded into straws and placed inside the programmable freezer for freezing. The frozen semen straws were stored inside liquid nitrogen tank. The frozen semen straws were then thawed at 15°C (1st and 2nd freezing and thawing followed the same process) for a duration of 5 min and further assessed post-thawed at 0 and 15 min during in vitro liquid storage at 15°C. The treatment means were analyzed using Fisher’s protected t-test least. There was no significant differences recorded on raw semen sperm total motility (93.2%) and 1st frozen-thawed at 0 min (82.6%), with recovery rate of 88.6% of the sperm total motility. There was a drastic decline observed in sperm total motility during the 1st frozen-thaw at 15 min (77.6%), 2nd frozen-thaw at 0 min (31.3%) and 2nd frozen-thaw at 15 min (30.1%; P<0.05). In conclusion, the freezing and thawing process did not decrease the bull’s sperm total motility percentage during the 1st freezing and thawing process, as compared with raw semen. However, a drastic decline was observed during the 2nd freezing-thawing process, as compared with raw semen.

32 An approach to invitro oocyte maturation and fertilization in threatened Saharan Dorcas gazelle (Gazella dorcas osiris) using frozen-thawed epididymal sperm cells

The Dorcas gazelle (Gazella dorcas), in the family Bovidae, is one of the smallest species of the genus Gazella. Its population is decreasing and it is classified as vulnerable by the International Union of Conservation of Nature Red List of Threatened Species. The subspecies Saharan Dorcas gazelle (G. dorcas osiris), originating in the central-western Saharan and Sahel regions, is almost extinct in the wild because of over-hunting. Thereon, assisted reproductive techniques (ARTs) could help in its population management. Here, we performed invitro maturation (IVM) of oocytes recovered from an adult female of Saharan Dorcas gazelle (10 years old) to attempt IVF with frozen-thawed epididymal sperm cells from an adult male (9 years old) of the same subspecies, following protocols for ARTs in cattle. Epididymal sperm was obtained after postmortem examination by cannulation of vas deferens with commercial semen extender Gent A (Minitub). Motility features were assessed using a computer-assisted sperm analysis system (Proiser SL), and viability was evaluated using eosin/nigrosin staining. After collection, viability (88.3%), total (17.8%) and progressive (8.7%) motility were assessed. A total of 820.5×106 sperm cells was obtained and diluted (1:1) in a semen extender with permeable cryoprotectants (Gent B, Minitub). Then, the sample was refrigerated in straws (200×106 mL−1; 0.5mL) at 4°C for 1h and exposed to liquid nitrogen vapors (LN2) for 10min before being directly plunged into and kept on LN2. A total of 35 oocytes were recovered by slicing, 27 of which were selected for IVM (24h, 38.5°C, 5% CO2 in humidified air). Motile sperm were obtained by centrifugation of thawed semen (10min, 1000×g) by performing a density gradient (BoviPure, Nidacon). Post-thawing viability (72.9%) and total (14%) and progressive (4%) motility were evaluated just before IVF. After IVM, 17 oocytes were incubated for IVF for 20h with 1×106 thawed sperm cells mL−1 (although the first polar body extrusion was observed only in 5 of the 17 oocytes before IVF; 29.4%). Also, 7 oocytes selected for IVM were vitrified and kept for the genetic bank, and 3 were fixed. Thereafter, all oocytes that underwent IVF were incubated for invitro embryo culture (38.5°C, 5% CO2, 5% O2 in humidified air) to avoid discarding any potential zygote. Cleavage was assessed at 48h post-IVF. From those, only 2 oocytes successfully achieved IVF out of the 5 that showed first polar body extrusion (40%), although cleavage was asymmetric and embryos did not progress. The rest of oocytes (12) did not achieved IVM, IVF, or cleavage. Results suggest that optimal protocols and appropriate individualized conditions for vulnerable nondomestic ungulates are required for successful ARTs. Future efforts are needed to improve preservation strategies for the gametes/embryo conservation of endangered species. JG and MRC contributed equally to this work. JG is supported by AGAUR (2018 FI_B 00236) and MRC by FPU (Training for Academic Staff) (FPU15/06029).

100 The evolution and premature hyperactivation of kinetic sperm subpopulations are affected by inbreeding level in Retinta cattle

Computer-assisted sperm analysis (CASA) has become a powerful tool to study sperm heterogeneity because it allows to cluster individual sperm in subpopulations (Sp) according to their kinetic parameters, which are associated with fertility and sperm quality. In cattle, inbreeding (mating between close relatives) affects sperm quality. Inbreeding estimation has become more accurate with the increasing availability of genomic methodologies, such as the detection of runs of homozygosity (ROH). Additionally, genomic inbreeding values (FROH) allow us to determine which metabolic pathways are differentially affected by this genetic trait. The aim of this study was to assess the effect of inbreeding on evolution of sperm Sp over time in cattle. Sperm samples (n=100) from 50 Retinta bulls (two replicates) were analysed in a sperm longevity experiment at time 0 (T0, after thawing), 1 (T1) and 2 (T2) h. At each time point, eight parameters were measured using a CASA system (Sperm Class Analyzer 5.4), including curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s); average path velocity (VAP, μm/s), percentage of linearity (LIN,%: VSL/VCL), percentage of straightness (STR,%: VSL/VAP), wobble coefficient (WOB,%: VAP/VCL), mean amplitude of lateral head displacement (ALH, μm) and beat-cross frequency (BCF, Hz). The presence of Sp was determined by a two-step multivariate analysis including non-hierarchical followed by hierarchical analysis in 80,154 motile sperm. Four sperm Sp were identified: Sp1 (rapid and highly progressive sperm); Sp2 (progressive sperm with intermediate speed); Sp3 (slow and non-progressive sperm); and Sp4 (fastest, hyperactive-like, non-progressive sperm). To determine the effect of inbreeding, individuals were clustered into lowly (FROH &amp;lt; 0.125, n=27) and highly (FROH &amp;gt; 0.125, n=23) inbred individuals. After thawing (T0), the percentage of sperm Sp4 was higher and showed premature hyperactivation in highly inbred animals, which was previously associated with reduced fertility (Table 1). However, highly inbred individuals showed an increased percentage of rapid (Sp1) and intermediate (Sp2) progressive sperm after T1 and T2 incubation, respectively, compared with lowly inbred bulls (P&amp;lt;0.001). These results suggest that sperm velocity and progression persist for longer in highly inbred bulls, at least after a short incubation. In conclusion, our results demonstrate that inbreeding affects sperm motility pattern and premature hyperactivation in cattle. Table 1. Percentage of subpopulations (Sp) by inbreeding group and time Time Sp1 Sp2 Sp3 Sp4 Highly inbred Lowly inbred Highly inbred Lowly inbred Highly inbred Lowly inbred Highly inbred Lowly inbred 0 33.17a 34a 32.39a 32.56a 28.07a 27.93a 6.37b 5.51a 1 31.73b 26.13a 36.75a 41.65b 23.80a 24.37a 7.72a 7.85a 2 11.73b 7.67a 53.75b 48.78a 29.68a 39.27b 4.83a 4.28a a,bDifferent superscripts by subpopulation in the same row denote significant difference (P&amp;lt;0.05).