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...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.展开更多
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 sp...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.展开更多
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...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.展开更多
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 ...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.展开更多
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 ...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.展开更多
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 stal...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),P<0.001,13.3%reduction,n=8)and lower head size parameters(length and width,P<0.01;and perimeter and area,P<0.001;n=4)in those spermatozoa 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),P<0.05).Conclusion Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity,small and elongated head size parameters,and different sperm subpopulation structure than the not-selected spermatozoa.Additionally,we evidenced that thermotactic spermatozoa improve ICSI success rates.展开更多
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. ...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.展开更多
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 eva...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.展开更多
This work evaluates sperm head morphometric characteristics in adolescents from 12 to 18 years of age, and the effect of varicocele. Volunteers between 150 and 224 months of age （mean 191, n = 87）, who had reached o...This work evaluates sperm head morphometric characteristics in adolescents from 12 to 18 years of age, and the effect of varicocele. Volunteers between 150 and 224 months of age （mean 191, n = 87）, who had reached oigarche by 12 years old, were recruited in the area of Barranquilla, Colombia. Morphometric analysis of sperm heads was performed with principal component （PC） and discriminant analysis, Combining seminal fluid and sperm parameters provided five PCs： two related to sperm morphometry, one to sperm motility, and two to seminal fluid components. Discriminant analysis on the morphometric results of varicocele and nonvaricocele groups did not provide a useful classification matrix. Of the semen-related PCs, the most explanatory （40%） was related to sperm motility. Two PCs, including sperm head elongation and size, were sufficient to evaluate sperm morphometric characteristics. Most of the morphometric variables were correlated with age, with an increase in size and decrease in the elongation of the sperm head. For head size, the entire sperm population could be divided into two morphometric subpopulations, SP1 and SP2, which did not change during adolescence. In general, for varicocele individuals, SP1 had larger and more elongated sperm heads than SP2, which had smaller and more elongated heads than in nonvaricocele men. In summary, sperm head morphometry assessed by CASA-Morph and multivariate cluster analysis provides a better comprehension of the ejaculate structure and possibly sperm function. Morphometric analysis provides much more information than data obtained from conventional semen analysis.展开更多
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 ma...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.展开更多
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...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.展开更多
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 mor...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.展开更多
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 dis...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.展开更多
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 ...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.展开更多
文摘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.
文摘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.
基金This work was supported by the Spanish Ministry of Economy and Finance(MINECO)(grant AGL2017-85030-R)the European Territorial Cooperation Operational Program-Spain,France,and Andorra Area 2014-20(Program DietaPYR2 EFA144/16)and the DGA-FSE(grant A07_17R).We would like to acknowledge the use of the Research Support Service-SAI,University of Zaragoza.
文摘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.
文摘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.
文摘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.
基金funded by the Spanish Ministry of Science and Innovation-MCIN (RTI2018-093548-B-100 and PID202M225070B-100 to A.Gutierrez-Adan and PID2019-1l1641 RB-100 to D.Rizos,funded by MCIN/AEI/10.13039/501100011033/and European Union"NextGeneration EU"/PRTR)supported by a Juan de la Cierva postdoctoral contract (FJC2019-040385-1)from the MCIN+1 种基金supported by a"Doctorados Industriales2018"fellowship of Comunidad de Madrid (IND2018/BIO-9610)supported by FPI scholarships from the MCIN (PRE2020-094452 and PRE2019-088813 respectively)。
文摘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),P<0.001,13.3%reduction,n=8)and lower head size parameters(length and width,P<0.01;and perimeter and area,P<0.001;n=4)in those spermatozoa 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),P<0.05).Conclusion Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity,small and elongated head size parameters,and different sperm subpopulation structure than the not-selected spermatozoa.Additionally,we evidenced that thermotactic spermatozoa improve ICSI success rates.
文摘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.
文摘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.
文摘This work evaluates sperm head morphometric characteristics in adolescents from 12 to 18 years of age, and the effect of varicocele. Volunteers between 150 and 224 months of age （mean 191, n = 87）, who had reached oigarche by 12 years old, were recruited in the area of Barranquilla, Colombia. Morphometric analysis of sperm heads was performed with principal component （PC） and discriminant analysis, Combining seminal fluid and sperm parameters provided five PCs： two related to sperm morphometry, one to sperm motility, and two to seminal fluid components. Discriminant analysis on the morphometric results of varicocele and nonvaricocele groups did not provide a useful classification matrix. Of the semen-related PCs, the most explanatory （40%） was related to sperm motility. Two PCs, including sperm head elongation and size, were sufficient to evaluate sperm morphometric characteristics. Most of the morphometric variables were correlated with age, with an increase in size and decrease in the elongation of the sperm head. For head size, the entire sperm population could be divided into two morphometric subpopulations, SP1 and SP2, which did not change during adolescence. In general, for varicocele individuals, SP1 had larger and more elongated sperm heads than SP2, which had smaller and more elongated heads than in nonvaricocele men. In summary, sperm head morphometry assessed by CASA-Morph and multivariate cluster analysis provides a better comprehension of the ejaculate structure and possibly sperm function. Morphometric analysis provides much more information than data obtained from conventional semen analysis.
文摘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.
文摘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.
文摘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.
文摘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.
文摘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.
