Computer-assisted sperm morphometry fluorescence-based analysis has potential to determine progeny sex

This study was designed to determine the ability of computer-assisted sperm morphometry analysis （CASA-Morph） with fluorescence to discriminate between spermatozoa carrying different sex chromosomes from the nuclear morphometrics generated and different statistical procedures in the bovine species. The study was divided into two experiments. The first was to study the morphometric differences between X- and Y-chromosome-bearing spermatozoa （SX and SY, respectively）. Spermatozoa from eight bulls were processed to assess simultaneously the sex chromosome by FISH and sperm morphometry by fluorescence-based CASA-Morph. SX cells were larger than SY cells on average （P 〈 0.001） although with important differences between bulls. A simultaneous evaluation of all the measured features by discriminant analysis revealed that nuclear area and average fluorescence intensity were the variables selected by stepwise discriminant function analysis as the best discriminators between SX and SY. In the second experiment, the sperm nuclear morphometric results from CASA-Morph in nonsexed （mixed SX and SY） and sexed （SX） semen samples from four bulls were compared. FISH allowed a successful classification of spermatozoa according to their sex chromosome content. X-sexed spermatozoa displayed a larger size and fluorescence intensity than nonsexed spermatozoa （P 〈 0.05）. We conclude that the CASA-Morph fluorescence-based method has the potential to find differences between X- and Y-chromosome-bearing spermatozoa in bovine species although more studies are needed to increase the precision of sex determination by this technique.

A comparative study of the morphometry of sperm head components in cattle, sheep, and pigs with a computer-assisted fluorescence method

The aim of this study was to compare the sperm nuclear and acrosomal morphometry of three species of domestic artiodactyls; cattle （Bos taurus）, sheep （Ovis aries）, and pigs （Sus scrofa）. Semen smears of twenty ejaculates from each species were fixed and labeled with a propidium iodide-Pisum sativum agglutinin （PI/PSA） combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed. The use of the PI/PSA combination and CASA-Morph fluorescence-based method allowed the capture, morphometric analysis, and differentiation of most sperm nuclei, acrosomes and whole heads, and the assessment of acrosomal integrity with a high precision in the three species studied. For the size of the head and nuclear area, the relationship between the three species may be summarized as bull 〉 ram 〉 boar. However, for the other morphometric parameters （length, width, and perimeter）, there were differences in the relationships between species for sperm nuclei and whole sperm heads. Bull sperm acrosomes were clearly smaller than those in the other species studied and covered a smaller proportion of the sperm head. The acrosomal morphology, small in the bull, large and broad in the sheep, and large, long, and with a pronounced equatorial segment curve in the boar, was species-characteristic. It was concluded that there are clear variations in the size and shape of the sperm head components between the three species studied, the acrosome being the structure showing the most variability, allowing a clear distinction of the spermatozoa of each species.

Influence of several uropathogenic microorganisms on human sperm motility parameters in vitro

Aim: The effects of certain uropathogenic microorganisms (Neisseria gonorrhoeae, Staphylococcus aureus, Staphylococcus epidermidis and Mycobacterium tuberculosis) on human sperm motility characteristics were studied in vitro. Methods: In 10 healthy fertile men, ejaculates were aseptically obtained by masturbation and With a swim-up technique, a sperm suspension of high motility and purity was obtained. Several uropathogenic bacteria were obtained from outpatients with genitourinary tract infections. The sperm suspension was incubated with the pathogens at a bacteria: sperm ratio of 50:1 at 37℃. The sperm mobility parameters were estimated with a computerassisted sperm analyzer (CASA) provided with a multiple-exposure photography system (Madi Corp., Zhejiang, China). Measurements were carried out at 0, 2 and 4 hours of incubation. Results: Staphylococcus aureus significantly decreased the sperm motility and viability, but Staphylococcus epidermidis, Mycobacterium tuberculosis and Neisseria gonorrhoeae did not. Conclusion: Staphylococcus aureus has an inhibitory effect on human sperm motility in vitro.

Current status and potential of morphometric sperm analysis

The spermatozoon is the most diverse cell type known and this diversity is considered to reflect differences in sperm function. How the diversity in sperm morphology arose during speciation and what role the different specializations play in sperm function, however, remain incompletely characterized. This work reviews the hypotheses proposed to explain sperm morphological evolution, with a focus on some aspects of sperm morphometric evaluation; the ability of morphometrics to predict sperm cryoresistance and male fertility is also discussed. For this, the evaluation of patterns of change of sperm head morphometry throughout a process, instead of the study of the morphometric characteristics of the sperm head at different stages, allows a better identification of the males with different sperm cryoconservation ability. These new approaches, together with more studies employing a greater number of individuals, are needed to obtain novel results concerning the role of sperm morphometry on sperm function. Future studies should aim at understanding the causes of sperm design diversity and the mechanisms that generate them, giving increased attention to other sperm structures besides the sperm head. The implementation of scientific and technological advances could benefit the simultaneous examination of sperm phenotype and sperm function, demonstrating that sperm morphometry could be a useful tool for sperm assessment.

Sperm speed is associated with sex bias of siblings in a human population

Recent studies investigating possible causes of male subfertility have largely focused on how lifestyle or environmental factors impact on the process of spermatogenesis, Markedly, fewer studies have investigated those risk factors that result in reduced sperm quality, such as poor sperm motility. The speed at which sperm swim is a major predictor of fertility and is extremely variable in human populations. It has been hypothesized that offspring sex may be adaptively manipulated to maximize the offspring＇s reproductive fitness （e.g., parents with genes for good male fertility traits, such as high sperm speed, would produce primarily sons and fewer daughters because the offspring will inherit advantageous male fertility genes）. Conversely, parents with poor male fertility genes would produce primarily daughters, We tested whether there was an association between how fast a man＇s sperm swam and the sex bias of his siblings in a sample of men attending clinic for fertility investigations with their partner and with a wide range of semen characteristics, including sperm speed. We found that the sex bias of a man＇s siblings is associated with his sperm speed; men with female-biased siblings had significantly slower sperm （judged using computer-assisted sperm analysis （CASA）） than men from male-biased sibships. This observation suggests family composition is an important factor that needs to be considered in future eDidemiological and clinical studies of human fertility,

Morphometric and kinematic sperm subpopulations in split ejaculates of normozoospermic men

This study was designed to analyze the sperm kinematic and morphometric subpopulations in the different fractions of the ejaculate in normozoospermic men. Ejaculates from eight normozoospermic men were collected by masturbation in three fractions after 3-5 days of sexual abstinence. Analyses of sperm motility by computer-assisted sperm analysis （CASA-Mot）, and of sperm morphometry by computer-assisted sperm morphometry analysis （CASA-Morph） using fluorescence were performed. Clustering and discriminant procedures were performed to identify sperm subpopulations in the kinematic and morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three kinematic subpopulations （slow with low ALH [35.6% of all motile spermatozoa], with circular trajectories [32.0%], and rapid with high ALH [32.4%]）, and three morphometric subpopulations （large-round [33.9% of all spermatozoa], elongated [32.0%], and small [34.10%]）. The distribution of kinematic sperm subpopulations was different among ejaculate fractions （P 〈 0.001）, with higher percentages of spermatozoa exhibiting slow movements with low ALH in the second and third portions, and with a more homogeneous distribution of kinematic sperm subpopulations in the first portion. The distribution of morphometric sperm subpopulations was also different among ejaculate fractions （P〈 0.001）, with more elongated spermatozoa in the first, and of small spermatozoa in the third, portion. It is concluded that important variations in the distribution of kinematic and morphometric sperm subpopulations exist between ejaculate fractions, with possible functional implications.

Comparison of different statistical approaches to evaluate morphometric sperm subpopulations in men

This study was designed to characterize morphometric sperm subpopulations in normozoospermic men by using different statistical methods and examining their suitability to classify correctly different sperm nuclear morphologies present in human ejaculates. Ejaculates from 21 normozoospermic men were collected for the study. After semen collection and analysis, samples were prepared for morphometric determination. At least 200 spermatozoa per sample were assessed for sperm morphometry by computer-assisted sperm morphometry analysis （CASA-Morph） using fluorescence. Clustering and discriminant procedures were performed to identify sperm subpopulations from the morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three morphometric subpopulations （large-round 30.4%, small-round 46.6%, and large-elongated 22.9%）. In the second analysis, using discriminant methods, the classification was made independently of size and shape. Three morphological categories according to nuclear size （small 〈10.90 μm^2, intermediate 10.91-13.07 μm^2, and large 〉13.07 μm^2） and four categories were defined on 400 canonical cells （100 × 4） from 10 men according to sperm nuclear shape （oval, pyriform, round, and elongated）. Thereafter, the resulting classification functions were used to categorize 4200 spermatozoa from 21 men. Differences in the class distribution were observed among men from both clustering and discriminant procedures. It was concluded that the combination of CASA-Morph fluorescence-based technology with multivariate cluster or discriminant analyses provides new information on the description of different morphometric sperm subpopulations in normal individuals, and that important variations in the distribution of morphometric sperm subpopulations may exist between men, with possible functional implications.

用传统方法与计算机辅助精液分析软件CRISMAS分析166名丹麦青年男性精液样本的结果比较

The aim of the present study was to compare assessments of sperm concentration and sperm motility analysed by conventional semen analysis with those obtained by computer-assisted semen analysis （CASA） （Copenhagen Rigshospitalet Image House Sperm Motility Analysis System （CRISMAS） 4.6 software） using semen samples from 166 young Danish men. The CRISMAS software identifies sperm concentration and classifies spermatozoa into three motility categories. To enable comparison of the two methods, the four motility stages obtained by conventional semen analysis were, based on their velocity classifications, divided into three stages, comparable to the three CRISMAS motility categories： rapidly progressive （A）, slowly progressive （B） and non-progressive （C＋ D）. Differences between the two methods were large for all investigated parameters （P〈0.001）. CRISMAS overestimated sperm concentration and the proportion of rapidly progressive spermatozoa and, consequently, underestimated the percentages of slowly progressive and non-progressive spermatozoa, compared to the conventional method. To investigate whether results drifted according to time of semen analysis, results were pooled into quarters according to date of semen analysis. CRISMAS motility results appeared more stable over time compared to the conventional analysis; however, neither method showed any trends. Apparently, CRISMAS CASA results and results from the conventional method were not comparable with respect to sperm concentration and motility analysis. This needs to be accounted for in clinics using this software and in studies of determinants of these semen characteristics.

Dog sperm head morphometry： its diversity and .=volution

Dogs have been under strong artificial selection as a consequence of their relationship with man. Differences between breeds are evident that could be reflected in seminal characteristics. The present study was to evaluate differences in sperm head morphometry between seven well-defined breeds of dog： the British Bulldog, Chihuahua, German Shepherd, Labrador Retriever, Spanish Mastiff, Staffordshire Terrier, and Valencian Rat Hunting dog. Semen samples were obtained by masturbation and smears stained with Diff-Quik. Morphometric analysis （CASA-Morph） produced four size and four shape parameters. Length, Ellipticity, and Elongation showed higher differences between breeds. MANOVA revealed differences among all breeds. Considering the whole dataset, principal component analysis （PCA） showed that PC1 was related to head shape and PC2 to size. Procluster analysis showed the British Bulldog to be the most isolated breed, followed by the German Shepherd. The PCA breed by breed showed the Chihuahua, Labrador Retriever, Spanish Mastiff, and Staffordshire Terrier to have PC1 related to shape and PC2 to size, whereas the British Bulldog, Valencia Rat Hunting dog, and German Shepherd had PC1 related to size and PC2 to shape. The dendrogram for cluster groupings and the distance between them showed the British Bulldog to be separated from the rest of the breeds. Future work on dog semen must take into account the large differences in the breeds＇ sperm characteristics. The results provide a base for future work on phylogenetic and evolutionary studies of dogs, based on their seminal characteristics.

Relationship of sperm plasma membrane andacrosomal integrities with sperm morphometry inBos taurus

To date,sperm morphometric studies have assessed whole sperm populations without considering sperm function.The aim of this study was to evaluate the relati on ship of sperm membrane and acrosomal integrity with sperm morphometry in liquid semen samples collected from bulls.To this end,sperm morphometry was performed on cryopreserved semen samples from 16 bulls by a combination of fluorescent dyes,including Hoechst 33343,carboxyfluorescein diacetate,and propidium iodide.This allowed discrimination of different subpopulations on the basis of sperm membrane and acrosomal integrity and analysis of the morphometries of the sperm head,nucleus,and acrosome using a specific plug-in module created on ImageJ.Acrosomal integrity was related to sperm morphometry as the heads of spermatozoa with a damaged acrosome were significantly smaller than those with a normal acrosome(P<0.001).In the case of spermatozoa with an intact acrosome,those with a damaged plasma membrane had a larger sperm head and acrosome than spermatozoa with an intact plasma membrane(P<0.001).No significant differences in the sperm head size were observed between sperm subpopulations without an acrosome or in the nuclear sperm morphometry of the different subpopulations.There was a positive correlation between the sperm motility values of the samples and the morphometric parameters for in tact spermatozoa.These correlations were particularly strong for the morphometric parameters of the sperm acrosome.We conclude that there are clear differences in the sperm morphometry depending on the status of the sperm membrane and acrosome and this should be considered when performing this kind of analysis.

Puma （Puma concolor） epididymal sperm morphometry

The Andean puma （Puma concolor） has not been widely studied, particularly in reference to its semen characteristics. The aim of the present study was to define the morphometry of puma sperm heads and classify their subpopulations by cluster analysis. Samples were recovered postmortem from two epididymides from one animal and prepared for morphological observation after staining with the Hemacolor kit. Morphometric data were obtained from 581 spermatozoa using a CASA-Morph system, rendering 13 morphometric parameters. The principal component （PC） analysis was performed followed by cluster analysis for the establishment of subpopulations. Two PC components were obtained, the first related to size and the second to shape. Three subpopulations were observed, corresponding to elongated and intermediate-size sperm heads and acrosomes, to large heads with large acrosomes, and to small heads with short acrosomes. In conclusion, puma spermatozoa showed no uniform sperm morphology but three clear subpopulations. These results should be used for future work in the establishment of an adequate germplasm bank of this species.

Sperm kinematic, head morphometric and kinetic- morphometric subpopulations in the blue fox （Alopex lagopus）

This work provides information on the blue fox ejaculated sperm quality needed for seminal dose calculations. Twenty semen samples, obtained by masturbation, were analyzed for kinematic and morphometric parameters by using CASA-Mot and CASA-Morph system and principal component （PC） analysis. For motility, eight kinematic parameters were evaluated, which were reduced to PC1, related to linear variables, and PC2, related to oscillatory movement. The whole population was divided into three independent subpopulations： SP1, fast cells with linear movement; SP2, slow cells and nonoscillatory motility; and SP3, medium speed cells and oscillatory movement. In almost all cases, the subpopulation distribution by animal was significantly different. Head morphology analysis generated four size and four shape parameters, which were reduced to PC 1, related to size, and PC2, related to shape of the cells. Three morphometric subpopulations existed. SPI： large oval cells; SP2： medium size elongated cells; and SP3. small and short cells. The subpopulation distribution differed between animals. Combining the kinematic and morphometric datasets produced PC1, related to morphometric parameters, and PC2, related to kinematics, which generated four sperm subpopulations - SPI.. high oscillatory motility, large and short heads; SP2; medium velocity with small and short heads; SP3. slow motion small and elongated cells; and SP4. high linear speed and large elongated cells. Subpopulation distribution was different in all animals. The establishment of sperm subpopulations from kinematic, morphometric, and combined variables not only improves the well-defined fox semen characteristics and offers a good conceptual basis for fertility and sperm preservation techniques in this species, but also opens the door to use this approach in other species, included humans.

Sperm subpopulations in avian species： a comparative study between the rooster （Gallus domesticus） and Guinea fowl （Numida meleagris）

The main aims of this research were to study possible differences in objective morphometric sperm characteristics, establish normative sperm morphometry standards, and evaluate the presumed different subpopulation distribution of avian spermatozoa from the rooster （Gallus domesticus） and Guinea fowl （Numida meleagris） as model avian species. Seventy-two ejaculates （36 per species studied） were obtained manually, following a training period involving gently combined dorso-abdominal and lumbo.sacral massage of the birds. Ejaculates were processed for volume, sperm concentration, viability, motility, and morphology. Moreover, samples were submitted for sperm morphometric assessment using objective Computer-Assisted Semen Analysis for Morphometry （CASA-Morph） methods, with sperm morphometric descriptors evaluated by Principal Component Analysis （PCA） and multivariate clustering analyses. There were several differences observed between the avian species in values obtained for ejaculate volume and sperm concentration （P 〈 0.001）. Irrespective of species, PCA revealed two Principal Components （PCs） explaining more than 80% of the variance. In addition, the number of subpopulations differed with species （three and five subpopulations for rooster and Guinea fowl, respectively）. Moreover, the distribution of the sperm subpopulations was found to be structurally different between species. In conclusion, our findings from using CASA-Morph methods indicate pronounced sperm morphometric variation between these two avian species. Because of the strong differences observed in morphometric parameter values and their subpopulation distribution, these results suggest that application of objective analytical methods such as CASA-Morph could substantially improve the reliability of comparative studies and help establish valid normative sperm morphological values for avian species.

Deep learning methods for noisy sperm image classification from convolutional neural network to visual transformer:a comprehensive comparative study

Background With the gradual increase of infertility in the world,among which male sperm problems are the main factor for infertility,more and more couples are using computer-assisted sperm analysis(CASA)to assist in the analysis and treatment of infertility.Meanwhile,the rapid development of deep learning(DL)has led to strong results in image classification tasks.However,the classification of sperm images has not been well studied in current deep learning methods,and the sperm images are often affected by noise in practical CASA applications.The purpose of this article is to investigate the anti-noise robustness of deep learning classification methods applied on sperm images.Methods The SVIA dataset is a publicly available large-scale sperm dataset containing three subsets.In this work,we used subset-C,which provides more than 125,000 independent images of sperms and impurities,including 121,401 sperm images and 4,479 impurity images.To investigate the anti-noise robustness of deep learning classification methods applied on sperm images,we conducted a comprehensive comparative study of sperm images using many convolutional neural network(CNN)and visual transformer(VT)deep learning methods to find the deep learning model with the most stable anti-noise robustness.Results This study proved that VT had strong robustness for the classification of tiny object(sperm and impurity)image datasets under some types of conventional noise and some adversarial attacks.In particular,under the influence of Poisson noise,accuracy changed from 91.45%to 91.08%,impurity precison changed from 92.7%to 91.3%,impurity recall changed from 88.8%to 89.5%,and impurity F1-score changed 90.7%to 90.4%.Meanwhile,sperm precision changed from 90.9%to 90.5%,sperm recall changed from 92.5%to 93.8%,and sperm F1-score changed from 92.1%to 90.4%.Conclusion Sperm image classification may be strongly affected by noise in current deep learning methods;the robustness with regard to noise of VT methods based on global information is greater than that of CNN methods based on local information,indicating that the robustness with regard to noise is reflected mainly in global information.

Effect of low-dose fenvalerate on semen quality capacitation in adult mice

Background Fenvalerate （FEN） has been demonstrated to be a reproductive toxicant in humans and rodents. However,little is known about whether short-term exposure to low-dose FEN produces reproductive toxicity.Methods We administered FEN （0.009 375, 0.1875, 3.750, or 45.00 mg·kg-1d-1 by gavage for 30 days） to male ICR mice and compared reproductive toxicity parameters between groups receiving different concentrations of FEN.Reproductive toxicity was evaluated by computer-assisted semen quality analysis （CASA）, chlortetracycline （CTC） assay,and histopathology.Results The sperm morphology and testis histology of FEN-exposed mice （all doses） were similar to that in controlling mice. Exposure to FEN at a concentration of 0.1875 mg·kg-1d-1 decreased sperm path straightness （STR） and linearity （LIN） （both P〈 0.05）, but had no significant impact on average path velocity （VAP）, straight line velocity （VSL）, curvilinear velocity （VCL）, lateral amplitude （ALH）, beat cross frequency （BCF）, or progressive motility （MOT）. FEN reduced the rate of mouse sperm capacitation in a dose-dependent manner.Conclusion The present results demonstrate that exposure to low-dose FEN for 30 days reduces semen quality and sperm capacitation in adult mice.