Computer-aided Sperm Analysis Research Articles

A combination of biomarkers for predicting stallion sperm fertility.

Equine breeding would benefit greatly from reliable biomarkers of stallion or ejaculate fertility. The aim of the study was to investigate how several in vitro sperm characteristics correlate with fertility after artificial insemination, to explore the potential to build a fertility prediction model for stallions. Cooled insemination doses (3-5 per stallion) were obtained from various studs. Sperm membrane integrity, acrosome integrity, chromatin integrity, mitochondrial membrane potential, and reactive oxygen species production were evaluated by flow cytometry 24-30h after semen collection, and sperm motility was assessed by computer aided sperm analysis. Calcein violet was used to differentiate viable spermatozoa. Per season pregnancy rates for these stallions were available the following year. Positive correlations were found between pregnancy rate and straightness (r = 0.43, p ≤ 0.001), as well as pregnancy rate and the proportion of living hydrogen peroxide positive spermatozoa (r = 0.32, p ≤ 0.05). There were negative correlations between pregnancy rate and amplitude of lateral head displacement (r = -0.26, p ≤ 0.05), and between pregnancy rate and the mean fluorescence of dead superoxide positive spermatozoa (r = -0.46, p < 0.001). Principal component analysis indicated that motility, membrane integrity, DNA fragmentation, and reactive oxygen species production were associated with pregnancy rate. Therefore, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates. However, data from more individuals would be required to construct a model for fertility prediction.

Forkhead-associated phosphopeptide binding domain 1 (FHAD1) deficiency impaired murine sperm motility.

Genetic knockout-based studies conducted in mice provide a powerful means of assessing the significance of a gene for fertility. Forkhead-associated phosphopeptide binding domain 1 (FHAD1) contains a conserved FHA domain, that is present in many proteins with phospho-threonine reader activity. How FHAD1 functions in male fertility, however, remains uncertain. Fhad1-/- mice were generated by CRISPR/Cas9-mediated knockout, after which qPCR was used to evaluate changes in gene expression, with subsequent analyses of spermatogenesis and fertility. The testis phenotypes were also examined using immunofluorescence and histological staining, while sperm concentrations and motility were quantified via computer-aided sperm analysis. Cellular apoptosis was assessed using a TUNEL staining assay. The Fhad1-/-mice did not exhibit any abnormal changes in fertility or testicular morphology compared to wild-type littermates. Histological analyses confirmed that the testicular morphology of both Fhad1-/-and Fhad1+/+ mice was normal, with both exhibiting intact seminiferous tubules. Relative to Fhad1+/+ mice, however, Fhad1-/-did exhibit reductions in the total and progressive motility of epididymal sperm. Analyses of meiotic division in Fhad1-/-mice also revealed higher levels of apoptotic death during the first wave of spermatogenesis. The findings suggest that FHAD1 is involved in both meiosis and the modulation of sperm motility.

Seminal Oxidative Stress and Sperm DNA Fragmentation in Men from Couples with Infertility or Unexplained Recurrent Pregnancy Loss

(1) Background: This case–control study examined whether men from couples with unexplained recurrent pregnancy loss (RPL) or infertility exhibited higher seminal oxidative stress (OS) and sperm DNA fragmentation (SDF) compared to fertile controls. (2) Methods: The study included 30 participants from each group: unexplained RPL, unexplained infertility, and proven fertility. Data were collected at Aalborg University Hospital tertiary RPL and fertility treatment clinics (Aalborg, Denmark), excluding couples with mixed conditions for homogeneity. Semen samples were analyzed using computer-aided sperm analysis (CASA) for concentration, motility, and morphology. SDF was assessed via a CASA-based sperm chromatin dispersion test. OS was measured as static oxidation-reduction potential (sORP). (3) Results: The results showed no significant OS differences between groups. The RPL group had significantly lower SDF levels than the control group. A significant positive correlation between SDF and OS was observed in the infertility group. Overall, this study did not find significant differences in OS levels between men from couples with unexplained RPL or infertility and fertile controls, while SDF levels were lower in the RPL group compared to controls. (4) Conclusion: In conclusion, despite the existing literature suggesting that OS and SDF are negative prognostic factors, our findings suggest they may not be reliable diagnostic markers for RPL and infertility.

Bi-allelic variants in DNAH3 cause male infertility with asthenoteratozoospermia in humans and mice.

Are there other pathogenic genes for asthenoteratozoospermia (AT)? DNAH3 is a novel candidate gene for AT in humans and mice. AT is a major cause of male infertility. Several genes underlying AT have been reported; however, the genetic aetiology remains unknown in a majority of affected men. A total of 432 patients with AT were recruited in this study. DNAH3 mutations were identified by whole-exome sequencing (WES). Dnah3 knockout mice were generated using the genome editing tool. The morphology and motility of sperm from Dnah3 knockout mice were investigated. The entire study was conducted over 3 years. WES was performed on 432 infertile patients with AT. In addition, two lines of Dnah3 knockout mice were generated. Haematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunostaining, and computer-aided sperm analysis (CASA) were performed to investigate the morphology and motility of the spermatozoa. ICSI was used to overcome the infertility of one patient and of the Dnah3 knockout mice. DNAH3 biallelic variants were identified in three patients from three unrelated families. H&E staining revealed various morphological abnormalities in the flagella of sperm from the patients, and TEM and immunostaining further showed the loss of the central pair of microtubules, a dislocated mitochondrial sheath and fibrous sheath, as well as a partial absence of the inner dynein arms. In addition, the two Dnah3 knockout mouse lines demonstrated AT. One patient and the Dnah3 knockout mice showed good treatment outcomes after ICSI. N/A. This is a preliminary report suggesting that defects in DNAH3 can lead to asthenoteratozoospermia in humans and mice. The pathogenic mechanism needs to be further examined in a future study. Our findings show that DNAH3 is a novel candidate gene for AT in humans and mice and provide crucial insights into the biological underpinnings of this disorder. The findings may also be beneficial for counselling affected individuals. This work was supported by grants from National Natural Science Foundation of China (82201773, 82101961, 82171608, 32322017, 82071697, and 81971447), National Key Research and Development Program of China (2022YFC2702604), Scientific Research Foundation of the Health Committee of Hunan Province (B202301039323, B202301039518), Hunan Provincial Natural Science Foundation (2023JJ30716), the Medical Innovation Project of Fujian Province (2020-CXB-051), the Science and Technology Project of Fujian Province (2023D017), China Postdoctoral Science Foundation (2022M711119), and Guilin technology project for people's benefit (20180106-4-7). The authors declare no competing interests.

STUDY REGARDING EFFECT OF TRISS-BASED AND CANIPLUS EXTENDERS ON SEVERAL SPERM PARAMETERS IN MEDIUM-LARGE BREED OF DOGS

"The study was carried out in different breeds of dogs owned by breeders in the city of Cluj Napoca. Mostly medium-large breeds were used and all the participating animals have been in good health during the time of acquiring the samples. The purpose of the study was to make a comparison between our own Tris-based extender and the commercial extender CANIPLUS CHILL in accordance with semen parameters with particular regards to motility, progressive motility, and length of survival of the spermatozoa. The evaluation has been done using the Computer Aided Sperm Analysis (CASA) system at the Faculty of Veterinary Medicine of Cluj-Napoca. We have found that in medium large size breeds the commercia CaniPlus extender have shown better results on the majority of the parameters over the Tris-based extender and has the ability to preserve the integrity is spermatozoa more efficiently over time compared to Tris extender. Furthermore we identified an improvement in all parameters when comparing samples on the day of collection between large breed dogs and medium-large breed dogs in both extender types. Additionally, we had results shown increase values of most parameter in tris extender when comparing it with CaniPlus extender in medium-large size breeds on the day of collection."

An objective quality indicator for evaluating filtering of microscopic video in CASA system

Computer-Aided Sperm Analysis (CASA) has received great attention in the diagnosis of male reproductive health for its objectivity and accuracy. But its accuracy may be greatly affected by algorithms used in microscopic video preprocessing, especially algorithms for microscopic videos filtering. Owing to the Peak Signal to Noise Ratio (PSNR) and Structural Similarity (SSIM) indicators are not suitable for microscopic images, objective indicators for evaluating filtering of microscopic videos are scarce at present, leading to filtering algorithms are chosen arbitrarily. In addition, it is critical to establish quality control indicators for CASA from the perspective of image processing, but they are rarely mentioned for the reason that algorithms adopted in image processing aren’t public. To reduce influences of filtering on diagnostic results, it is imperative to establish an objective quality control indicator for evaluating filtering of microscopic video from the perspective of image preprocessing. The principle of formatting sperm tracking trajectory and the rules of grading trajectory continuity were introduced firstly. Then, impacts of filtering algorithms on the continuity of sperm trajectory were investigated and the relation between the continuity of sperm trajectory and the fluctuation of sperm segmentation thresholds was further explored. Finally, an objective indicator for appraising the quality of filtering for microscopic video was established based on SADTM (Sum of Absolute Difference between Threshold and the Mean).The proposed indicator can objectively evaluate the quality of microscopic video filtering and provide a guideline for algorithm selection, laying foundation for quality control for CASA.

Relationship between long non-coding RNA MALAT1 and HOTAIR expression with sperm parameters, DNA and malondialdehyde levels in male infertility

BackgroundSperm quality is a complex index used to evaluate the fertility potential of men. The long non-coding RNA (lncRNA) MALAT1 participate in sperm development and HOTAIR have critical roles in the regulation of oxidative stress responses. This study aimed to evaluate the relationship of lncRNA MALAT1 and HOTAIR expression with sperm parameters, DNA fragmentation and malondialdehyde (MDA)levels in sperm fertility. MethodsIn this experimental study, semen samples (n = 30 fertile, n = 30 infertile) men were collected and evaluated for sperm parameters by computer-aided sperm analysis(CASA). Sperm DNA integrity quality was assessed by the Acridine orange(AO) test. MDA levels were determined by the Thiobarbituric acid reaction method. The expression of MALAT1 and HOTAIR was detected by RT-PCR. ResultsWe observed a decreased level of MALAT1and HOTAIR expression in the infertile men (p < 0.001). The relative expression level of MALAT1and HOTAIR showed a positive correlation with motility and morphology (p < 0.001). Subsequently, we found the DNA damage and MDA levels was negatively correlated with expression level of genes of sperm (p < 0.001). ConclusionIn this study the low expression of MALATI and HOTAIR resulted in the high level of MDA, DNA damage, and reduced motility of sperm. This study suggests the therapeutic opportunities in respect to MALATI and HOTAIR expression in the sperm function.

Ensembling noisy segmentation masks of blurred sperm images

Background:Sperm tail morphology and motility have been demonstrated to be important factors in determining sperm quality for in vitro fertilization. However, many existing computer-aided sperm analysis systems leave the sperm tail out of the analysis, as detecting a few tail pixels is challenging. Moreover, some publicly available datasets for classifying morphological defects contain images limited only to the sperm head. This study focuses on the segmentation of full sperm, which consists of the head and tail parts, and appear alone and in groups. Methods:We re-purpose the Feature Pyramid Network to ensemble an input image with multiple masks from state-of-the-art segmentation algorithms using a scale-specific cross-attention module. We normalize homogeneous backgrounds for improved training. The low field depth of microscopes blurs the images, easily confusing human raters in discerning minuscule sperm from large backgrounds. We thus propose evaluation protocols for scoring segmentation models trained on imbalanced data and noisy ground truth. Results:The neural ensembling of noisy segmentation masks outperforms all single, state-of-the-art segmentation algorithms in full sperm segmentation. Human raters agree more on the head than tail masks. The algorithms also segment the head better than the tail. Conclusions:The extensive evaluation of state-of-the-art segmentation algorithms shows that full sperm segmentation is challenging. We release the SegSperm dataset of images from Intracytoplasmic Sperm Injection procedures to spur further progress on full sperm segmentation with noisy and imbalanced ground truth. The dataset is publicly available at https://doi.org/10.34808/6wm7-1159.

Comparison of Semen Diluted Basis (Tris-Egg Yolk) with Diluted Commercial Semen (Andromed®) in Frozen Goat Semen on Sperm Quality by CASA

The process of producing frozen goat semen involves a preparation of semen diluents, which is complicated. Since it involves a rapid temperature reduction, the quality of the sperm deteriorates. This study aimed to determine the comparison of semen diluted basis (Tris-egg yolk) with diluted commercial semen (Andromed®) in frozen goat semen on sperm quality by Computer aided sperm analysis (CASA) after thawing at 0, 1 and 3h. by using 1 Goat (Boer) semen. The semen was collected by means of an artificial vagina diluted with both types of semen diluent. Then the semen was preserved by a freezing method. The results showed that the preliminary assessment of fresh semen quality was good and suitable for use in frozen semen production. Additionally, it was found that frozen goat semen diluted basis (Tris-egg yolk) and diluted commercial semen (Andromed®) had no significant difference in motility, progressive motility, VAP, VSL, VCL, ALH, BCF (HZ), STR (%) and LIN (%) at 0h. However, at 1h, the motility and the progressive motility of Andromed® were 87.20±2.71% and 46.98±6.03%, while those of Tris-egg yolk were 68.55±11.5% and 34.20±4.42% respectively which these values were significantly different (P&lt;0.05). At 3h, the motility and the progressive motility of Andromed® were 73.45±3.04% and 31.58±6.63%, while those of Tris-egg yolk were 10.43±1.63% and 1.37±0.06% respectively which these values were significantly different (P&lt;0.01). In addition, at 1 and 3h, the VAP, VSL, VCL, ALH, and STR values of Andromed® diluted sperm were higher than those of Tris-egg yolk. From this study, it was found that commercial semen diluent (Andromed®) helps to maintain the quality of frozen goat semen and reduce the steps of semen diluent preparation. It can also be applied in farms or agencies.

Dietary Moringa oleifera leaves to male rainbow trout (Oncorhynchus mykiss) broodstock: Effects on sperm quality and reproductive performance

Moringa oleifera leaves (MOL), a superfood rich in protein and vitamins, is native to the Indian subcontinent. Moringa were added to the broodstock trout diet to examine their influence on the reproductive performance of male broodstock rainbow trout (Oncorhynchus mykiss). Females were fed with a commercial broodstock trout diet without any inclusion of moringa leaves. In 12 circular fiberglass tanks, 36 male rainbow trout aged 3+ (1786.36 ± 35.36 g; 56.08 ± 0.94 cm) were divided into four groups (n = 9) (control, MOL-1, MOL-2, and MOL-3) with three parallels each. Fourteen weeks of feeding trials were performed where powdered moringa leaf was used in the diets at various levels: 0% (control), 4% (MOL-1), 8% (MOL-2), and 16% (MOL-3). Sperm quality parameters and fertilization success were investigated. Sperm motility parameters were examined at 10.5 °C using a computer-aided sperm analysis (CASA) system, and all samples were analyzed on the same day. Sperm volume and seminal plasma osmolality did not differ significantly across groups (p > 0.05). Spermatozoa concentration (6.71 ± 0.25, 7.54 ± 0.31, 8.44 ± 0.55, and 7.66 ± 0.35 × 109 cells ml−1 in control, MOL-1, MOL-2, and MOL-3 groups, respectively), spermatocrit ratio (18 ± 2.55%, 25 ± 3.16%, 38 ± 6.44%, and 28 ± 5.15%), and motility duration differed significantly (p < 0.05) among groups. Total motility percentage did not change, but progressive motility (74.72 ± 5.94%, 88.52 ± 4.35%, 89.36 ± 1.76%, and 90.47 ± 2.67%) was considerably higher in all three moringa groups than in control one (p < 0.05). Average Path Velocity (VAP), Straight Line Velocity (VSL), and Curvilinear Velocity (VCL) significantly differ between control and treatment groups (p < 0.05). The fertilization rate in the MOL-2 group was similar to the control and MOL-3 groups, while significant differences were observed from the eyeing stage, with the highest result observed in the MOL-2 group (p < 0.05). Hatching rates were also highest in the MOL-2 group (81.65 ± 0.49%), followed by MOL-3 (79.08 ± 1.27%) and MOL-1 (74.40 ± 2.32%), with the control group having the lowest hatching rate (74.24 ± 0.98%) (p < 0.05). Progressive motility, VAP, VSL, and VCL have significantly enhanced in the moringa-treated groups than the control one, hence boosting the fertilization success. The present study revealed that moringa leaves in the diet improve the sperm quality parameters and enhance the reproductive performance in broodstock male rainbow trout (Oncorhynchus mykiss). Therefore, 8% moringa leaf can be recommended as a supplement diet for male rainbow trout broodstock.

Effect of prenatal perfluoroheptanoic acid exposure on spermatogenesis in offspring mice

BackgroundPerfluoroheptanoic acid (PFHpA), a persistent organic pollutant widespread in the environment, is suspected as an environmental endocrine disruptor for its disturbance effect on hormone homeostasis and reproductive development. Whereas the effect of intrauterine PFHpA exposure during gestation on spermatogenesis of male offspring mice is still unknown. ObjectiveThis study aimed to explore the effect of prenatal PFHpA exposure on the reproductive development of male offspring mice and the role of N6-methyladenosine (m6A) during the process. MethodsFifty-six C57BL/6 pregnant mice were randomly divided into 4 groups. During the gestation period, the pregnant mice were exposed to 0, 0.0015, 0.015, and 0.15 mg/kg bw/d PFHpA from gestational day 1 (GD1) to GD16 by oral gavage. The male offspring mice were sacrificed by spinal dislocation at 7 weeks old. The body weight, testicular weight, and brain weight were weighed, and the intra-testicular testosterone was detected. The sperm qualities were analyzed with computer-aided sperm analysis (CASA). The testicular tissues were taken to analyze the pathological changes and examine the global m6A RNA methylation levels. Quantitative real-time PCR (qRT-PCR) was adopted to figure out the mRNA expression levels of m6A-related enzymes in testicular tissues of different PFHpA treated groups. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was applied to further explore the m6A RNA methylation at a whole-genome scale. ResultsCompared with the control group, no significant differences were observed in body weight, testicular weight, testicular coefficient, and the visceral-brain ratio of testicular tissue in the PFHpA treated groups. And no significant change was observed in intra-testicular testosterone among the four groups. CASA results showed a decrease of sperm count, sperm concentration, and total cell count, as well as an increase of sperm progressive cells’ head area after prenatal PFHpA exposure (P < 0.05). Hematoxylin and eosin staining of pathological sections showed seminiferous tubules morphological change, disorder arrangement of seminiferous epithelium, and reduction of spermatogenic cells in the PFHpA treated groups. PFHpA significantly decreased global levels of m6A RNA methylation in testicular tissue (P < 0.05). Besides, qRT-PCR results showed significant alteration of the mRNA expression levels of seven m6A-related enzymes (Mettl3, Mettl5, Mettl14, Pcif1, Wtap, Hnrnpa2b1, and Hnrnpc) in the PFHpA treated groups (P < 0.05). MeRIP-seq results showed a correlation between prenatal PFHpA exposure and activation and binding of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Cnga3 and Mpzl3 showed differential expression in the enrichment subcategories or pathways. ConclusionsExposure to PFHpA during the gestation period would adversely affect the development of seminiferous tubules and testicular m6A RNA methylation in offspring mice, which subsequently interferes with spermatogenesis and leads to reproductive toxicity.

VISEM-Tracking, a human spermatozoa tracking dataset

A manual assessment of sperm motility requires microscopy observation, which is challenging due to the fast-moving spermatozoa in the field of view. To obtain correct results, manual evaluation requires extensive training. Therefore, computer-aided sperm analysis (CASA) has become increasingly used in clinics. Despite this, more data is needed to train supervised machine learning approaches in order to improve accuracy and reliability in the assessment of sperm motility and kinematics. In this regard, we provide a dataset called VISEM-Tracking with 20 video recordings of 30 seconds (comprising 29,196 frames) of wet semen preparations with manually annotated bounding-box coordinates and a set of sperm characteristics analyzed by experts in the domain. In addition to the annotated data, we provide unlabeled video clips for easy-to-use access and analysis of the data via methods such as self- or unsupervised learning. As part of this paper, we present baseline sperm detection performances using the YOLOv5 deep learning (DL) model trained on the VISEM-Tracking dataset. As a result, we show that the dataset can be used to train complex DL models to analyze spermatozoa.

Epididymal DIS3 exosome ribonuclease is not necessary for mouse sperm maturation or fertility

DIS3 is an RNA exosome associated ribonuclease that degrades a wide range of transcripts that can be essential for cell survival and development. The proximal region of the mouse epididymis (initial segment and caput) plays a pivotal role in sperm transport and maturation required for male fertility. However, whether DIS3 ribonuclease mediates RNA decay in proximal epididymides remains unclear. Herein, we established a conditional knockout mouse line by crossing a floxed Dis3 allele with Lcn9-cre mice in which the recombinase is expressed in the principal cells of initial segment as early as post-natal day 17. Morphological and histological analyses, immunofluorescence, computer-aided sperm analysis and fertility were used for functional analyses. We document that DIS3 deficiency in the initial segment had no effect on male fertility. Dis3 cKO males had normal spermatogenesis and initial segment development. In cauda epididymides of Dis3 cKO mice, sperm abundance, morphology, motility, and the frequency of acrosome exocytosis were comparable to controls. Collectively, our genetic model demonstrates that loss of DIS3 in the initial segment of the epididymis is not essential for sperm maturation, motility, or male fertility.

Serum interleukin-38 and -41 levels as candidate biomarkers in male infertility

BackgroundInterleukin (IL)-38 and IL-41 are novel cytokines, but their role in male infertility (MI) is unknown. The purpose of this study was to measure the levels of serum IL-38 and IL-41 in patients with MI and correlate these levels with semen indexes. MethodsEighty-two patients with MI and 45 healthy controls (HC) were recruited for this study. Semen parameters were detected using computer-aided sperm analysis, Papanicolaou staining, ELISA, flow cytometry, peroxidase staining and enzyme methods. Serum IL-38 and IL-41 levels were determined by ELISA. ResultsSerum IL-38 levels were decreased (P < 0.01) in patients with MI compared with HC. Serum IL-41 levels were significantly higher in patients with MI than in HC (P < 0.0001). In patients with MI, serum IL-38 levels were positively correlated with semen white blood cell counts (r = 0.29, P = 0.009), and there was a positive correlation between semen white blood cell counts and sperm concentration (r = 0.28, P = 0.0100) and seminal plasma elastase (r = 0.67, P < 0.0001). Receiver operating characteristic curve analysis showed that the area under the curve of IL-38 for diagnosing MI was 0.5637 (P > 0.05), and the area under the curve of IL-41 for diagnosing MI was 0.7646 (P < 0.0001). ConclusionsSerum IL-38 levels were significantly lower, and serum IL-41 levels were higher in patients with MI. These results suggest that IL-38 and IL-41 may be novel biomarkers for the diagnosis of MI.

The Loss-Function of KNL1 Causes Oligospermia and Asthenospermia in Mice by Affecting the Assembly and Separation of the Spindle through Flow Cytometry and Immunofluorescence

KNL1 (kinetochore scaffold 1) has attracted much attention as one of the assembly elements of the outer kinetochore, and the functions of its different domains have been gradually revealed, most of which are associated with cancers, but few links have been made between KNL1 and male fertility. Here, we first linked KNL1 to male reproductive health and the loss-function of KNL1 resulted in oligospermia and asthenospermia in mice (an 86.5% decrease in total sperm number and an 82.4% increase in static sperm number, respectively) through CASA (computer-aided sperm analysis). Moreover, we introduced an ingenious method to pinpoint the abnormal stage in the spermatogenic cycle using flow cytometry combined with immunofluorescence. Results showed that 49.5% haploid sperm was reduced and 53.2% diploid sperm was increased after the function of KNL1 was lost. Spermatocytes arrest was identified at the meiotic prophase I of spermatogenesis, which was induced by the abnormal assembly and separation of the spindle. In conclusion, we established an association between KNL1 and male fertility, providing a guide for future genetic counseling regarding oligospermia and asthenospermia, and a powerful method for further exploring spermatogenic dysfunction by utilizing flow cytometry and immunofluorescence.

Optimized addition of nitric oxide compounds in semen extender improves post-thaw seminal attributes of Murrah buffaloes.

Semen dilution and cryopreservation alter the homogeneity of seminal plasma, resulting in a non-physiological redox milieu and consequently poor sperm functionality. Considering the concentration-specific bimodal action of nitric oxide (NO) in the regulation of sperm functions, cryopreservation media supplemented with optimized concentrations can improve the semen attributes. The present study aimed to evaluate the effect of adding an optimized concentration of sodium nitroprusside (SNP) and N-nitro-L-arginine methyl ester (L-NAME) in an extender on in vitro semen quality. An aliquot of semen samples (n = 32) from Murrah buffalo bulls (n = 8) was divided into control (C) and treatment (T-I: SNP in extender at 1µmol/L; T-II: L-NAME in extender at 10µmol/L). Fresh semen quality parameters showed no significant difference at 0h except for the structural integrity in the T-II group. Post-thaw semen quality parameters and sperm kinematics using computer-aided sperm analysis (CASA) revealed significantly higher (p < 0.05) cryoresistance in the treatment groups. Viability, acrosome integrity, and membrane integrity were significantly higher (p < 0.05) in both treatment groups; however, the results were pervasive in T-II. Lower abnormal spermatozoa were observed in both T-I and T-II. SNP supplementation led to a significant rise (p < 0.05) in NO, whereas L-NAME reduced the NO concentration in post-thawed samples, which was directly correlated with different sperm functionality and associated biomarkers viz. total antioxidant capacity (TAC) and thiobarbituric acid reactive substance (TBARS). It was concluded that the cryopreservation media supplemented with SNP and L-NAME at 1µmol/L and 10µmol/L, respectively, lower the cryo-damage and improve post-thaw seminal attributes.

A novel approach to sperm selection: Nanoparticle-based purification improves quality of Angora cryopreserved buck’s semen

The Aim: The aim of the study was to investigate the applicability of an up-to-date method, sperm selection with nanoparticles at different temperatures (37ºC, 21ºC, and 4ºC), on Angora buck semen. Material and Methods: Semen samples were collected from 3 Angora bucks with 7 repetitions and each sample was diluted and divided into 4 groups. Nanopurification procedure was applied by using a nanoparticle dose of 10 μg/ml to the three groups at 37ºC, 21ºC, and 4ºC while in the control group samples were cryopreserved without nanopurificiation treatment. Post-thaw motility and morphology parameters of the sperm samples were analyzed by using computer-aided sperm analysis (CASA) and fluorescein microscopy.Results: After nanopurification at 37ºC total motility, linearity (LIN) (P&lt;0.05), curvilinear velocity (VCL), straight-line velocity (VSL), and acrosome integrity parameters (P&lt;0.001) were higher than the control group while the semen samples nanopurified at 21ºC were superior to the control group in terms of LIN, straightness (STR) (P&lt;0.05), VCL, VSL, average path velocity (VAP), wobble and acrosome integrity (P&lt;0.001). There was no significant difference between the control and the 4ºC group.Conclusion: In conclusion, nanopurification procedure on buck semen was useful in terms of parameters that affect fertility and the method can be performed at 21ºC, as well as 37ºC, to adapt the procedure to field conditions;

Cornichon protein CNIH4 is not essential for mice gametogenesis and fertility

BackgroundCornichon is a functionally conserved transmembrane protein family that generally acts as a cargo-sorting receptor and cycles between the ER and the Golgi. Four Cornichon family members (CNIH1-4) have been identified. The key residues responsible for CNIH1-3 to bind to AMPA receptors are not conserved in CNIH4. Additionally, the function of CNIH1-3 in GPCR signaling is less established, while more established in case of CNIH4 protein that interact with GPCR and control their exportation. Many GPCRs are known for their essential roles in male and female gonad development. But whether CNIH4 plays a role in gametogenesis remains unknown. DesignMice carrying the Cnih4 knockout allele (Cnih4tm1a−/−) were generated by insertion of a LacZ reporter and a polyadenylation site after exon 1. Western blot, Immunofluorescence, computer-aided sperm analysis and other methods were used in the functional analysis. ResultsWe identified that both Cnih4tm1a−/− male and female mice have normal fertility. Though, the sperm count, morphology, and motility of Cnih4tm1a−/− mice were slightly impaired compared to those of wild-type mice, the testes to body weight ratio and testicular histology were similar to those in control mice. Histological examination of Cnih4tm1a−/− ovaries detected follicles from primordial to antral stages and the numbers of follicles at each stage were also comparable to wild-type controls. Normal fertility was noticed after six-month fertility tests. That was likely due to the compensatory role of Chin3, which significantly upregulated in the Cnih4tm1a−/− mice to preserve the fertility role. ConclusionDespite CNIH4 showing enriched expression in mouse germ cells, our genetic knockout studies demonstrated that CNIH4 is not essential for gametogenesis and fertility in mice although with a slight reduction in count, motility and morphology of sperm in male mice.

Dimethylacetamide - an alternative to glycerol as cryoprotectant of Malabari buck semen

The key role of a cryoprotectant is to minimize the chemical and physical stress which occurs during cooling, freezing and thawing of semen. The difference between the cryoprotectant (CPA) occurs in their permeability coefficient and the structural model of the cryogenic agent. The beneficial effect of dimethylacetamide (DMA) as a cryoprotectant especially for sperms had been observed in several studies. The aim of the study was to study the cryoprotective effect of DMA in freezing the Malabari buck semen compared to glycerol. Ten ejaculates were taken from fourMalabaribucks . After preliminary evaluation sample split technique was followed with Tris based extender containing glycerol (6.7 per cent) as cryoprotectant (control) and Trisextender containing DMA (3 per cent) as cryoprotectant (treatment group). The semen straws (0.25mL) after filling were subjected for equilibration and manual freezing. Sperm kinetics was studied using computer-aided sperm analyzer. Pre-freeze and post-thaw evaluation included sperm viability, sperm abnormality, hypo osmotic test, acrosome integrity test and DNA fragmentation. Results indicated that inclusion of 6.7 per cent glycerol had significantly higher (p&lt;0.05) post-thaw values than DMA. From our study we conclude that 6.7 per cent glycerol was better than 3 per cent DMA in cryopreservation of Malabari buck semen.

Effect of High Viscosity on Energy Metabolism and Kinematics of Spermatozoa from Three Mouse Species Incubated under Capacitating Conditions

In order to sustain motility and prepare for fertilization, sperm require energy. The characterization of sperm ATP production and usage in mouse species revealed substantial differences in metabolic pathways that can be differentially affected by capacitation. Moreover, spermatozoa encounter different environments with varying viscoelastic properties in the female reproductive tract. Here, we examine whether viscosity affects sperm ATP levels and kinematics during capacitation in vitro. Sperm from three mouse species (Mus musculus, M. spretus, M. spicilegus) were incubated under capacitating conditions in a modified Tyrode's medium containing bicarbonate, glucose, pyruvate, lactate, and bovine serum albumin (mT-BH) or in a bicarbonate-free medium as a non-capacitating control. Viscosity was increased with the inclusion of polyvinylpyrrolidone. ATP was measured with a bioluminescence kit, and kinematics were examined with a computer-aided sperm analysis system. In M. musculus sperm, ATP declined during capacitation, but no differences were found between non-capacitating and capacitating sperm. In contrast, in M. spretus and M. spicilegus, ATP levels decreased in capacitating sperm. Increasing viscosity in the medium did not modify the timing or proportion of cells undergoing capacitation but did result in additional time- and concentration-dependent decreases in ATP in M. spretus and M. spicilegus under capacitating conditions. Additionally, increased viscosity altered both velocity and trajectory descriptors. The limited impact of capacitation and higher viscosity on M. musculus sperm ATP and kinematics could be related to the low intensity of postcopulatory sexual selection in this species. Responses seen in the other two species could be linked to the ability of their sperm to perform better under enhanced selective pressures.