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

Asian Journal of Andrology 2016年第6期18卷 858-862页

作者： Pilar Santolaria Alfredo Pauciullo Miguel A Silvestre Sandra Vicente-Fiel Leyre Villanova Alain Pinton Juan Viruel Ester Sales Jesus L Yaniz TECNOGAM research group Environmental Sciences Institute (IUCA) Department of Animal Production and Food Sciences Higher Polytechnic School University of Zaragoza Ctra. Cuarte SIN 22071 Huesca Spain Department of Agriculture Forest and Food Sciences University of Torino Largo Paolo Braccini 2 10095 Grugliasco TO Italy Department of Functional Biology and Physical Anthropology University of Valencia 469100 Burjassot Valencia Spain UMR 1388 INRA-ENVT INPT Animal Cytogenetis GenPhySE National veterinary school of Toulouse 23 Chemin des Capelles 31076 Toulouse Cedex 3 France Mediterranean Institute of marine and terrestrial Biodiversity and Ecology (IMBE) University of Aix Marseille Chemin de la Batterie des Lions 13007 Marseille France TECNOGAM research group Environmental Sciences Institute (IUCA) Department of Agricultural and Environmental Sciences Higher Polytechnic School University of Zaragoza Ctra. Cuarte S/N 22071 Huesca Spain

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.

关键词： bull computer-assisted sperm morphometry analysis sexing sperm morphometry

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Normozoospermic versus teratozoospermic domestic cats： differential testicular volume, sperm morphometry, and subpopulation structure during epididymal maturation

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Asian Journal of Andrology 2016年第6期18卷 871-878页

作者： Miguel Angel Gutierrez-Reinoso Manuel Garcia-Herreros National Secretariat of Higher Education Science Technology and Innovation (SENESCYT) Quito Ecuador

Teratozoospermia （〈40% morphologically normal spermatozoa/ejaculate） is a frequent phenomenon in feline species. This research was carried out to study the possible differences in testicular volume, differential sperm morphometric traits, and potential differences regarding the sperm subpopulational structure during epididymal sperm maturation in teratozoospermic feline donors. Epididymal sperm samples were collected from the caput （R1）, corpus （R2）, and cauda （R3） epididymidis in two donor groups （N： normozoospermic; T： teratozoospermic）. Aliquots were assessed for concentration, viability, motility, and acrosomal integrity. sperm morphometric descriptors from CASA-Morph analysis were analyzed by the Principal Component Analysis （PCA） and clustering analyses. Irrespective of the group analyzed, PCA revealed two Principal Components （PCs） for each epididymal region explaining more than the 93% of the variance. Surprisingly, the number of subpopulations remained constant in regions R1-R2-R3 irrespective of the donor group analyzed. However, the distribution of these subpopulations was found to be structurally different and strongly influenced by the epididymal region and the donor group. In conclusion, testicular morphometry and the sperm subpopulation structure were different in N and T donors. The alterations in subpopulations during epididymal maturation could be used as a potential clinical indicator of teratozoospermic individuals since an important influence of teratozoospermia on sperm subpopulation structure has been demonstrated.

关键词： epididymis feline sperm morphometry sperm subpopulations teratozoospermia testicular volume

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

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Asian Journal of Andrology 2020年第6期22卷 578-582页

作者： Inmaculada Palacin Pilar Santolaria Carlos Alquezar-Baeta Carles Soler Miguel A Silvestre Jesus Yaniz BIOFITER Research Group Environmental Sciences Institute(IUCA)Department of Animal Production and Food SciencesUniversity of ZaragozaHuesca 22071Spain Department of Computer Science University of ZaragozaZaragoza 50018Spain Department of Cellular Biology Functional Biology and Physical AnthropologyUniversity of ValenciaBurjassot 46100Spain

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(Pspermatozoa with an intact acrosome,those with a damaged plasma membrane had a larger sperm head and acrosome than spermatozoa with an intact plasma membrane(Psperm 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.

关键词： Bos taurus fluoresce nee microscopy image analysis sperm morphometry sperm quality

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Puma （Puma concolor） epididymal sperm morphometry

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Asian Journal of Andrology 2016年第6期18卷 879-881页

作者： Hernan Cucho Virgilio Alarcon Cesar Ordonez Enrique Ampuero Aydee Meza Caries Solere Zootechny School National University of San Atonio Abad del Cusco Postal code 921 Cusco Peru Functional Biology and Physical Anthropology Department University of Valencia C/. Dr Moliner 50 46100 Burjassot Valencia Spain Proiser R+D S.L. Scientific Park of the University of Valencia 46980 Paterna Spain

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.

关键词： principal component analysis puma sperm morphometry subpopulation wild animal

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Dog sperm head morphometry： its diversity and .=volution

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Asian Journal of Andrology 2017年第2期19卷 149-153页

作者： Caries Soler Ana Alambiaga Maria A Marti Almudena Garcia-Molina Anthony Valverde Jesus Contell Marcos Campos Department of Functional Biology and Physical Anthropology University of Valencia 46100 Burjassot Spain R+D Department Proiser R+D Scientific Park University of Valencia 46980 Paterna Spain 3Technological Institute of Costa Rica San Carlos Campus School of Agronomy 223-21001 Alajuela Costa Rica Department of Medicine and Animal Surgery University CardenaI-Herrera-CEU 46113 Moncada Valencia Spain Global Veterinaria (Reprovalcan) 46500 Sagunto Valencia Spain Clinica Veterinaria Sangueso 46009 Valencia Spain

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.

关键词： Canis familiaris cluster analysis diversity sperm morphometry

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

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Asian Journal of Andrology 2016年第6期18卷 840-843页

作者： Jesus L Yaniz Sara Capistros Sandra Vicente-Fiel Carlos O Hidalgo Pilar Santolaria TECNOGAM research group Environmental Sciences Institute (IUCA) Department of Animal Production and Food Sciences University of Zaragoza Huesca Spain Animal Genetics and Reproduction Area Regional Agrifood Research and Development Service (SERIDA) 33394 Deva Gijan Spain

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.

关键词： artiodactyls computer-assisted sperm morphometry analysis fluorescence microscopy image analysis sperm morphometry

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Bull spermatozoa selected by thermotaxis exhibit high DNA integrity,specific head morphometry,and improve ICSI outcome

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Journal of Animal Science and Biotechnology 2023年第3期14卷 1001-1011页

作者： Sara Ruiz-Diaz Rosane Mazzarella Paula Navarrete-Lopez Raul Fernandez-Gonzalez Celia de Frutos Maria Maroto Claudia Cucala Paula Beltran-Brena Marta Lombo Dimitrios Rizos Alfonso Gutierrez-Adan Department of Animal Reproduction INIA-CSIC28040 MadridSpain Mistral Fertility Clinics S.L Clinica TambreCalle Tambre828002 MadridSpain

Background sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation(SDF)in mice,humans,and stallions.This study aimed to analyse if bull spermatozoa could be selected by thermotaxis and to assess their quality in terms of SDF as well as determine the presence of a specific sperm subpopulation based on sperm morphometry and assess their fertilizing capacity by ICSI.Methods We used frozen-thawed sperm from 6 bulls and sperm selection by thermotaxis was performed with TALP medium supplemented with 25 mmol/L of HEPES and 5 mmol/L of caffeine.In these conditions,sperm selection was achieved,obtaining a net thermotaxis of 3.6%.Subsequently,we analysed the SDF of the migrated and not-migrated spermatozoa using the neutral COMET assay,and we evaluated the size of the sperm head using Hemacolor■ staining with Motic Images Plus 3 software.Additionally,migrated and not-migrated spermatozoa by thermotaxis were used to fertilize bovine in vitro matured(IVM)oocytes by ICSI,a very inefficient procedure in cattle that is only successful when the oocyte is artificially activated.Results The results showed lower SDF(χ^(2),Pspermatozoa migrated in comparison to those not-migrated.The distribution of sperm subpopulations structure varied between groups,highlighting cluster 2,characterized by spermatozoa with small head size,and high ellipticity and elongated heads,as the most abundant in the thermotaxis migrated group.When performed ICSI(without oocyte artificial activation)with the thermotactic sperm,the blastocyst rate was 32.2%±9.3%in the group microinjected with the thermotactic spermatozoa vs.8.3%±7.8%in the group of not-migrated sperm(χ^(2),Psperm selection by thermotaxis has a much higher DNA integrity,small and elongated head size param

关键词： ART Bovine sperm morphometry sperm selection Thermotaxis

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Current status and potential of morphometric sperm analysis

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Asian Journal of Andrology 2016年第6期18卷 863-870页

作者： Alejandro Maroto-Morales Olga Garcia-Alvarez Manuel Ramon Felipe Martinez-Pastor M Rocio Fernandez-Santos A Josefa Soler Jose Julian Garde SaBio IREC (CSIC - UCLM -JCCM) Albacete Spain Biomedical Center Medical Faculty in Pilsen Charles University in Prague Pilsen Czech Republic Regional Center of Animal Selection and Reproduction (CERSYRA) JCCM Valdepenas Spain 4Institute for Animal Health and Cattle Development University of Leon LeOn Spain Department of Molecular Biology University of Leon Leon Spain Faculty of Pharmacy (UCLM) Albacete Spain

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.

关键词： computer-assisted sperm morphometric analysis mammals sperm function sperm morphometry

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

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Asian Journal of Andrology 2016年第6期18卷 889-894页

作者： Manuel Garcia-Herreros National Secretariat of Higher Education Science Technology and Innovation (SENESCYT) Quito Ecuador

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.

关键词： Gallus domesticus Numida meleaEris principal component analysis sperm morphometry sperm subpopulations

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

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Asian Journal of Andrology 2017年第2期19卷 154-159页

作者： Caries Soler Jesus Contell Lorena Bori Maria Sancho Almudena Garcia-Molina Anthony Valverde Jan Segarvall Department of Functional Biology and Physical Anthropology University of Valencia 46100 Burjassot Spain R+D Department Proiser R+D Scientific Park University of Valencia 46980 Paterna Spain Technological Institute of Costa Rica San Carlos Campus School of Agronomy 223-21001 Alajuela Costa Rica Finnish Fur Breeders Association ProFur 65101 Vaasa Finland

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.

关键词： integration of motility and morphology principal component analysis sperm morphometry subpopulation

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