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Morphometry and subpopulation structure of Holstein bull spermatozoa： variations in ejaculates and cryopreservation straws

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Asian Journal of Andrology 2016年第6期18卷 851-857页

作者： Anthony Valverde Hector Arenan Maria Sancho jesus Contell jesus yaniz Alejandro Fernandez Caries Soler Technological Institute of Costa Rica San Carlos Campus S Xenetica Fontao Fontao-Esperante 27210 Lugo Spain R+D Department Proiser R+D Scientific Park University of Valencia 46980 Paterna Spain Department of Animal Production and Food Science Politechnical School of Huesca University of Zaragoza 22071 Huesca Spain Department of Functional Biology and Physical Anthropology University of Valencia 46100 Burjassot Spain

Sperm quality is evaluated for the calculation of sperm dosage in artificial reproductive programs. The most common parameter used is motility, but morphology has a higher potential as a predictor of genetic quality. Morphometry calculations from CASA-Morph technology improve morphological evaluation and allow mathematical approaches to the problem. Semen from 28 Holstein bulls was collected by artificial vagina, and several ejaculates were studied. After general evaluation, samples were diluted, packaged in 0.25 ml straws, and stored in liquid nitrogen. Two straws per sample were thawed, and slides were processed and stained with Diff-Quik. Samples were analyzed by a CASA-Morph system for eight morphometric parameters. In addition to the ＂classical＂ statistical approach, based on variance analysis （revealing differences between animals, ejaculates, and straws）, principal component （PC） analysis showed that the variables were grouped into PC1, related to size, and PC2 to shape. Subpopulation structure analysis showed four groups, namely, big, small, short, and narrow from their dominant characteristics, representing 31.0%, 27.3%, 24.1%, and 17.7% of the total population, respectively. The distributions varied between animals and ejaculates, but between straws, there were no differences in only four animals. This modern approach of considering an ejaculate sperm population as divided into subpopulations reflecting quantifiable parameters generated by CASA-Morph systems technology opens a new view on sperm function. This is the first study applying this approach to evaluate different ejaculates and straws from the same individual. More work must be done to improve seminal dose calculations in assisted reproductive programs.

关键词： cryopreservation Holstein bull sperm morphometry subpopulations

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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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Morphometric and kinematic sperm subpopulations in split ejaculates of normozoospermic men

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Asian Journal of Andrology 2016年第6期18卷 831-834页

作者： Pilar Santolaria Caries Soler Pilar Recreo Teresa Carretero Araceli Bono Jose M Berne jesus L yaniz TECNOGAM research group Environmental Sciences Institute (IUCA) Department of Animal Production and Food Sciences University of Zaragoza Huesca Spain Department of Functionat 8iotogy and Physical Anthropology University of Valencia 46100 Burjassot Valencia Spain R+D Department Proiser R+D Scientific Park University of Valencia 46980 Paterna Valencia Spain TECNOGAM research group Environmental Sciences Institute (IUCA) Departament of Human Anatomy and Histolosy Faculty of Health and Sport Sciences of Huesca PLaza Universidad 3 22002 Huesca Spain TECNOGAM research group Environmental Sciences Institute (IUCA) Hospital General San Jorge de Huesca Area de Urologia Av. Martinez de Velasco 36 22004 Huesca Spain

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.

关键词： man sperm morphometry by computer-assisted sperm morphometry analysis sperm motility by computer-assisted spermanalysis sperm subpopulations split ejaculate

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

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Asian Journal of Andrology 2016年第6期18卷 819-823页

作者： jesus L yaniz Sandra Vicente-Fiel Caries Soler Pilar Recreo Teresa Carretero Araceli Bono Jose M Berne Pilar Santolaria TECNOGAM research group Environmental Sciences Institute (IUCA) Department of Animal Production and Food Sciences Higher Polytechnic School University of Zaragoza Ctra. Cuarte S/N 22071 Huesca Spain Department of Functional Biology and Physical Anthropology University of Valencia 46100 Burjassot Valencia Spain R+D Department Proiser R+D Scientific Park University of Valencia 46980 Paterna Valencia Spain TECNOGAM research group Environmental Sciences Institute (IUCA) Departament of Human Anatomy and Histology Faculty of Health and Sport Sciences of Huesca Plaza Universidad 3 22002 Huesca Spain TECNOGAM research group Environmental Sciences Institute (IUCA) Hospital General San Jorge de Huesca Area de Urologla Av. Martfnez de Velasco 36 22004 Huesca Spain

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.

关键词： computer-assisted sperm morphometry analysis man sperm subpopulations

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

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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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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 *** 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 *** 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 *** 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 *** 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 *** was a positive correlation between the sperm motility values of the samples and the morphometric parameters for in tact *** correlations were particularly strong for the morphometric parameters of the sperm *** 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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