Sperm Morphometric Subpopulations Research Articles

The contribution of different sperm parameters to better explain ram semen cryoresistance

In order to determine an effective procedure for explaining ram sperm cryoresistance and develop a new model for breeders classification, a retrospective study was conducted using sperm analysis data obtained over two consecutive years from a total of 82 sessions of ram semen cryopreservation. In each session, fresh ejaculates from eight males were collected via artificial vagina, pooled and frozen in liquid nitrogen vapors. After thawing, a total of 19,084 sperm tracks and 11,319 morphometric measurements were analysed. Clustering analyses were applied to establish motile and morphometric sperm subpopulations. Additionally, plasma and acrosome membrane integrity, as well mitochondrial activity using flow cytometry immediately after sperm thawing and following hypoosmotic shock test (HOST) was assessed. To develop a Ram Sperm Cryoresistance Index, Principal Component Analyses (PCA) using 22 variables were conducted. In the first PCA, the parameters that best explain cryoresistance include total motility (TM), motile subpopulation 2 (motSP2, which groups slow, very linear spermatozoa with low lateral head displacement), morphometric subpopulation 1 (morphSP1, grouping spermatozoa with the smallest head size and lowest shape values), sperm plasma membrane integrity immediately after thawing and following hypoosmotic shock test. These parameters collectively account for 77.34 % of the accumulated variance. To emphasize their importance, a second PCA was performed, revealing significant higher weighting coefficients for the quantity (TM) and quality (motSP2) of sperm movement after thawing, compared to the head size and shape of the thawed sperm (morphSP1). Furthermore, HOST Viability played a more decisive role than what was observed under isotonic conditions.

Predictive Capacity of Boar Sperm Morphometry and Morphometric Sub-Populations on Reproductive Success after Artificial Insemination.

Simple SummaryThe efficiency of swine production measured as litter size influences the profitability of the pig industry. Furthermore, sow fertility potential depends in part on the boar semen quality and reproductive efficiency. The objective of this study is to compare boar sperm head size and morphometric features of shape to evaluate their relationships with reproductive success after artificial insemination (AI). A morphometric analysis of boar ejaculate reveals morphometrically separate sub-populations. The differences between sub-populations are displayed for sperm head size. In addition, sperm clustering into sub-populations did not have a predictive capacity on litter size variables. Nevertheless, the morphometric variables of the sperm may have a predictive, albeit reduced, capacity regarding litter size variables. The results of this study therefore open up possibilities for future assessments of fertility. The aim of the study was to compare the morphometric features of sperm head size and shape from the Pietrain line and the Duroc × Pietrain boar crossbred terminal lines, and to evaluate their relationship with reproductive success after artificial insemination of sows produced from crossbreeding the York, Landrace and Pietrain breeds. Semen samples were collected from 11 sexually mature boars. Only ejaculates with greater than 70% motility rate and <15% of abnormal sperm were used for artificial inseminations (AI) and included in the study. Samples were analyzed using an ISAS®v1 computer-assisted sperm analysis system for eight morphometric parameters of head shape and size (CASA-Morph). Sub-populations of morphometric ejaculates were characterized using multivariate procedures, such as principal component (PC) analysis and clustering methods (k-means model). Four different ejaculate sub-populations were identified from two PCs that involved the head shape and size of the spermatozoa. The discriminant ability of the different morphometric sperm variables to predict sow litter size was analyzed using a receiver operating characteristics (ROC) curve analysis. Sperm head length, ellipticity, elongation, and regularity showed significant predictive capacity on litter size (0.59, 0.59, 0.60, and 0.56 area under curve (AUC), respectively). The morphometric sperm sub-populations were not related to sow litter size.

Identification of sperm morphometric subpopulations in cooled-stored canine sperm and its relation with sperm DNA integrity.

The aims of this study were to (i) identify different morphometric subpopulations in cooled-stored canine sperm and their patterns of distribution during cool-storage for up to 240hr and (ii) determine whether or not morphometric sperm subpopulations (sP) are related to sperm DNA integrity. For that purpose, morphometric parameters were analysed by computer-assisted sperm analysis (CASA) and sperm DNA fragmentation (sDFi) using the sperm Halomax test. Four morphometric sperm heads subpopulations were identified: sP1 (large and rounded), sP2 (large and elongated), sP3 (small and rounded) and sP4 (small and elongated). sP1 was the most predominant subpopulation for up to 72hr and thereafter sP3 increased progressively. sDFi increased after 48hr of cool-storage. Although sP3 showed a positive correlation with sDFi, and both increased over time, it could not be ensured that only the sperm with fragmented DNA are accumulated in sP3. In conclusion, sP3 and DNA fragmentation increased progressively during cool-storage, becoming possible indicators of sperm damage. However, it cannot be concluded that sP3 only contains sperm with fragmented DNA.

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.

Predictive capacity of sperm quality parameters and sperm subpopulations on field fertility after artificial insemination in sheep

This study was designed to evaluate the relevance of several sperm quality parameters and sperm population structure on the reproductive performance after cervical artificial insemination (AI) in sheep. One hundred and thirty-nine ejaculates from 56 adult rams were collected using an artificial vagina, processed for sperm quality assessment and used to perform 1319 AI. Analyses of sperm motility by computer-assisted sperm analysis (CASA), sperm nuclear morphometry by computer-assisted sperm morphometry analysis (CASMA), membrane integrity by acridine orange-propidium iodide combination and sperm DNA fragmentation using the sperm chromatin dispersion test (SCD) were performed. Clustering procedures using the sperm kinematic and morphometric data resulted in the classification of spermatozoa into three kinematic and three morphometric sperm subpopulations. Logistic regression procedures were used, including fertility at AI as the dependent variable (measured by lambing, 0 or 1) and farm, year, month of AI, female parity, female lambing-treatment interval, ram, AI technician and sperm quality parameters (including sperm subpopulations) as independent factors. Sperm quality variables remaining in the logistic regression model were viability and VCL. Fertility increased for each one-unit increase in viability (by a factor of 1.01) and in VCL (by a factor of 1.02). Multiple linear regression analyses were also performed to analyze the factors possibly influencing ejaculate fertility (N=139). The analysis yielded a significant (P<0.05) relationship between sperm viability and ejaculate fertility. The discriminant ability of the different semen variables to predict field fertility was analyzed using receiver operating characteristic (ROC) curve analysis. Sperm viability and VCL showed significant, albeit limited, predictive capacity on field fertility (0.57 and 0.54 Area Under Curve, respectively). The distribution of spermatozoa in the different subpopulations was not related to fertility.

Sperm population structure in high and low field fertility rams

The aim of the present study was to investigate whether differences in field fertility of rams are reflected in differences in sperm morphometric and kinematic population structures. The association between sperm morphometric and kinematic subpopulations was also investigated. Ejaculates from 8 adult rams, 4 with high and 4 with low field fertility, were collected weekly using an artificial vagina over 6 consecutive weeks. Analyses of sperm motility using computer-assisted sperm analysis (CASA) and sperm nuclear morphometry using computer-assisted sperm morphometry-fluorescence were performed. Clustering procedures using the kinematic and morphometric data from high and low field fertility rams resulted in the classification of spermatozoa in three kinematic and three morphometric sperm subpopulations. The distribution of subpopulations between rams of high and low field fertility was significantly different (P<0.05), with higher percentages of spermatozoa exhibiting fast and linear movements and those with large and long nuclei in the high fertility group. However, these subpopulations were not correlated. Logistic regression analyses were also performed to evaluate the relative utility of sperm subpopulations to classify rams in high and low field fertility. Total progressive sperm motility and the proportion of large and long spermatozoa were identified as the most consistent indicators of fertility. It was concluded that high and low fertility rams had clear differences in morphometric and kinematic sperm subpopulations, and that the most consistent indicators of fertility were the total progressive motility and the proportion of spermatozoa with large and long head present in the ejaculate.

Differential distribution of sperm subpopulations and incidence of pleiomorphisms in ejaculates of captive howling monkeys (Alouatta caraya)

The aim of this study was to develop an objective method to determine the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates of howling monkeys (Alouatta caraya) by using a combination of computerized analysis system (ASMA) and principal component analysis (PCA) methods. Ejaculates were collected by electroejaculation methods on a regular basis from five individuals maintained under identical captive environmental, nutritional, and management conditions. Each sperm head was measured for dimensional parameters (Area [A, (square micrometers)], Perimeter [P, (micrometers)], Length [L, (micrometers)], and Width [W, (micrometers)]) and shape-derived parameters (Ellipticity [(L/W)], Elongation [(L - W)/(L + W)], and Rugosity [(4лA/P (2))]). PCA revealed two principal components explaining more than the 96 % of the variance. Clustering methods and discriminant analyzes were performed and seven separate subpopulations were identified. There were differences (P < 0.001) in the distribution of the seven subpopulations as well as in the incidence of abnormal pleiomorphisms (58.6 %, 49.8 %, 35.1 %, 66.4 %, and 55.1 %, P < 0.05) among the five donors tested. Our results indicated that differences among individuals related to the incidence of pleiomorphisms, and sperm subpopulational structure was not related to the captivity conditions or the sperm collection method, since all individuals were studied under identical conditions. In conclusion, the combination of ASMA and PCA is a useful clinical diagnostic resource for detecting deficiencies in sperm morphology and sperm subpopulations in A. caraya ejaculates that could be used in ex situ conservation programs of threatened species in Alouatta genus or even other endangered neotropical primate species.

A comparative study of sperm morphometric subpopulations in cattle, goat, sheep and pigs using a computer-assisted fluorescence method (CASMA-F)

This study was designed to compare the sperm nuclear morphometric subpopulations of four species of domestic artiodactyls (cattle, sheep, goat and pigs). Samples from 20 males of each species were collected. After semen collection, sperm concentration and motility were measured and samples prepared for morphometric determinations. Smears were fixed with 2% glutaraldehyde, stained with Hoechst 33342 and photographed. At least 200 spermatozoa per sample were processed using the Image J analysis open software. Clustering procedures were performed to identify sperm subpopulations using the morphometric data obtained from each species. Results of the present study show that, applying the computer-assisted sperm morphometry analyisis-fluorescence (CASMA-F) technology and multivariate cluster analyses, it was possible to determine the subpopulations of spermatozoa with different morphometric characteristics in the four species studied. Bulls and boars had two clearly differentiated size categories: large and small. However, the final sperm subpopulations were four in the bull (large-round, large-elongated, small-round, and small-elongated) and only three in the boar (large, small-elongated and small-round). In small ruminant species, three sperm nuclei size categories were established: large, average sized and small. Two of these subpopulations were also elongated in goat bucks, with three subpopulations (large-round, small-elongated and average size-elongated). In the ram three morphometric subpopulations were also obtained (large, small and average size-round), but none was elongated. When comparing among species, sperm subpopulations were smaller in the buck and less elliptical and elongated in the ram than those in the other species studied. Male variability was identified in the distribution of sperm subpopulations described in the four species studied. It was concluded that the combination of CASMA-F technology with multivariate cluster analyses allow the study of morphometric sperm subpopulations and that there are important variations in the subpopulations among the four species studied.

Identification of sperm head subpopulations with defined pleiomorphic characteristics in ejaculates of captive Goeldi's monkeys (Callimico goeldii)

The aim of this study was to evaluate the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates from the vulnerable Goeldi's monkey (Callimico goeldii) by using a combination of computerized analysis system and Principal Component Analysis (PCA) methods. Each sperm head was measured for four primary spermatozoal head dimensional parameters (area [A (μm2)], perimeter [P (μm)], length [L (μm)] and width [W (μm)]) and three head shape derived parameters (ellipticity [(L/W)], elongation [(L−W)/(L+W)] and rugosity [(4πA/P2)]). Six separate subpopulations (SPs) were identified: SP1, constituted by very large, narrow and very elliptical spermatozoa (A=16.85±1.56μm2, W=2.75±0.42μm and ellipticity=2.16±0.24); SP2, characterized by average sized, short, wide and round spermatozoa (A=15.00±1.92μm2, L=5.06±0.49μm, W=3.51±0.31μm and ellipticity=1.44±0.15); SP3, represented by small, wide and slightly round spermatozoa (A=14.95±1.75μm2, W=3.47±0.29μm and ellipticity=1.48±0.14); SP4 included very small, short and very round spermatozoa (A=14.15±2.38μm2, L=4.90±0.57μm and elongation=0.18±0.05); SP5 consisted of average sized and slightly elliptical spermatozoa (A=15.14±1.72μm2 and ellipticity=1.49±0.14); and SP6 included large and round spermatozoa (A=16.30±1.62μm2 and elongation=0.19±0.04). There were differences in the sperm subpopulation distribution (P<0.001) among the five donors analyzed. In conclusion, the results of the current study confirmed that the use of computer sperm analysis methods combined with PCA cluster analyses are useful methods to identify, classify, and characterize different sperm head morphometric subpopulations in neotropical primates. Broadening our knowledge of C. goeldii sperm morphometric abnormalities as well as developing reliable techniques for sperm evaluation may be essential for ex situ conservation of this threatened species.

Sperm head morphometry in ejaculates of adult marmosets (Callithrix jacchus): A model for studying sperm subpopulations and among-donor variations

In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P < 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies.

Seasonal dynamics of sperm morphometric subpopulations and its association with sperm quality parameters in ram ejaculates

Sperm morphologic assessment is considered an irreplaceable part of standard laboratory routine analyses in the diagnosis of male fertility. Thus, in an attempt to quantify the effects of season on sperm morphology and its functional significance in relation to sperm quality parameters, sperm head morphometric traits were analyzed by using an objective computerized analysis combined with principal components analysis (PCA) cluster analysis to establish the relationship between the distribution of the subpopulations found and sperm quality in each season. There were slight variations on sperm motility and sperm membrane integrity indexes (P > 0.05). However, the mean values for sperm concentration substantially changed among seasons in all individuals studied (P < 0.01). There were significant differences in sperm morphometric parameters (P < 0.01) as well as in the distribution of morphometric subpopulations between seasons (P < 0.001). In conclusion, this study confirmed that there was an important seasonal effect on sperm morphometric traits. In addition, the distribution of these subpopulations seems to be related to the season studied and the ejaculate quality which would be a very important indicator of sperm function. The substantial information derived from these morphometric subpopulations has provided new knowledge which can be used in future studies using sperm morphometry as a seasonal indicator in ram ejaculates.

Morphometrically-distinct sperm subpopulations defined by a multistep statistical procedure in Ram ejaculates: intra- and interindividual variation

The existence of sperm subpopulations within the mammalian ejaculate has now been widely recognized. However, to the best of our knowledge, no data exist regarding the existence of sperm morphometric subpopulations within the ovine ejaculate. Computer assisted sperm morphometry analysis (ASMA) data and clustering methods were used in this study to identify sperm-head subpopulations in ram semen. Two experiments were carried out. In Experiment 1, ejaculates from 226 mature rams of the Manchega breed belonging to 36 different herds were used. A minimum of 100 sperm heads were analyzed from each male and eight morphometric characteristics for each individual sperm were recorded. Subpopulation analysis was performed in sequential steps: variable group analysis and correlation analysis to select which morphometric characteristics to use in cluster analyses; nonhierarchical clustering analysis using sperm head length and p2a (also known as roundness) shape factor as initial classificatory variables; and hierarchical clustering analysis to obtain the final number of clusters. The clustering analyses, based on 26 306 individual cells, revealed the existence of four sperm subpopulations (SP1, SP2, SP3 and SP4) with different morphometric characteristics. Significant differences in the proportion of spermatozoa in the SP1 and SP3 were found between rams belonging to different herds. In Experiment 2, the intra- and intermale variability on the distribution of sperm subpopulations was assessed. Three ejaculates from each of 21 rams were collected and the same multistep clustering analysis was performed. For all subpopulations defined, the intermale variability resulted in high values, being the intramale variability much lower. This fact would allow the use of sperm head morphometry to characterize a male and might provide valuable information to asses its fertility. In conclusion, our results show that using computer assisted sperm morphometry analysis and multivariate cluster analyses, four sperm subpopulations with different head phenotype were identified in ram ejaculates.

Sperm morphometric subpopulations are differentially distributed in rams with different maturity age in cryopreserved ejaculates

It is widely accepted that sperm morphology is a good indicator of fertility and it has been proposed that sperm quality may be related to subtle changes in sperm head morphology. However, a precise estimation of the morphology of ram sperm would be very useful to improve reproductive success in ovine. Computer-assisted morphometric analysis and clustering analysis have been important tools to study sperm subpopulations in domestic animals. However, to the best of our knowledge, no data exist studing morphometric differences regarding to sperm subpopulations within the ovine ejaculate. The aim of this study was to test the presence and distribution of sperm morphometric subpopulations in cryopreserved ejaculates from yearling and mature rams using an objective method by computer analysis system and to establish the relationship between the distribution of the subpopulations found and sperm quality in each individual ram. Principal component analysis revealed that three principal components for yearlings and four components for mature rams that represented more than 84% of the cumulative variance in both cases. After cluster analysis, three sperm morphometric subpopulations for yearlings (CLY) and four for mature (CLM) rams were identified with defined sperm dimensions and shapes. CLY1 included big, round and short sperm (37%), CLY2 included average size and slightly elliptical and elongated sperm (48%), CLY3 included small, long, elliptical and elongated sperm cells (15%). CLM1 consisted of average size and moderate elliptical and elongated (26%), CLM2 consisted of small, long, elliptical and elongated (31%), CLM3 consisted of small and round (32%) and CLM4 included big, short and round (8%) spermatozoa respectively. There were significant differences in the distribution of the three subpopulations (P < 0.001) as well as in the sperm concentration, total motility (%), sperm viability (%) and the overall (P < 0.05) in the ejaculates among the four yearling rams tested. Same results were found for the four subpopulations and the different sperm quality parameters in the ejaculates among the four mature rams tested. In conclusion, cryopreserved ram semen showed a specific structure with regard to sperm morphometric subpopulations. In addition, the distribution of these subpopulations seems to be related to stud maturity age and the ejaculate quality which would be a very important indicator of sperm function. Thus, analysis of sperm morphometric subpopulation structure together with functional tests could provide valuable information to assess the cryoresistence of ram spermatozoa.

Identification of Sperm‐Head Morphometric Subpopulations in Iberian Red Deer Epididymal Sperm Samples

Computer-assisted sperm morphometry analysis (CASMA) was used in this study to identify sperm morphometric subpopulations in Iberian red deer epididymal sperm samples. Epididymal sperm samples were collected from 37 mature stags and were divided. One portion was diluted in a Tris-citrate-egg yolk medium. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours using a conventional protocol. After thawing, sperm smears were prepared as described for extended samples. All slides were air-dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 145 sperm-heads were analyzed from each sample by means of the Sperm-Class Analyser, and the mean measurements recorded. Each sperm-head was measured for four primary sperm-head parameters, and five parameters of head shape. All sperm morphometric parameters evaluated were placed in a statistical database and a multivariate cluster analysis was performed. The clustering analyses, based on 10 867 individual spermatozoa, revealed the existence of three subpopulations (SP(1), SP(2), SP(3)) of spermatozoa with different morphometric characteristics (p < 0.001). The proportion of spermatozoa present in any of the three subpopulations remained constant (p > 0.05) through the cryopreservation process. Pre-freeze and post-thaw sperm quality was in vitro evaluated by microscopic assessments of individual sperm motility and of plasma membrane and acrosome integrities. In conclusion, our results show that applying the CASMA techniques and multivariate cluster analyses, it was possible to determine that three subtle subpopulations of spermatozoa with different morphometric characteristics coexist in red deer semen.

Identification of sperm morphometric subpopulations in the canine ejaculate: do they reflect different subpopulations in sperm chromatin integrity?

A statistical approach using sequentially principal component analysis (PCA) clustering and discriminant analysis was developed to disclose morphometric sperm subpopulations. In addition, we used a similar approach to disclose subpopulations of spermatozoa with different degrees of DNA fragmentation. It is widely accepted that sperm morphology is a strong indicator of semen quality and since the sperm head mainly comprises the sperm DNA, it has been proposed that subtle changes in sperm head morphology may be related to abnormal DNA content. Semen from four mongrel dogs (five replicates per dog) were used to investigate DNA quality by means of the sperm chromatin structure assay (SCSA), and for computerized sperm morphometry (ASMA). Each sperm head was measured for nine primary parameters: head area (A), head perimeter (P), head length (L), head width (W), acrosome area (%), midpiece width (w), midpiece area (a), distance (d) between the major axes of the head and midpiece, angle (theta) of divergence of the midpiece from the head axis; and four parameters of head shape: FUN1 (L/W), FUN2 (4pi A/P2), FUN3 ((L - W)/(L + W)) and FUN 4 (pi LW/4A). The data matrix consisted of 2361 observations, (morphometric analysis on individual spermatozoa) and 63,815 observations for the DNA integrity. The PCA analysis revealed five variables with Eigen values over 1, representing more than 79% of the cumulative variance. The morphometric data revealed five sperm subpopulations, while the DNA data gave six subpopulations of spermatozoa with different DNA integrity. Significant differences were found in the percentage of spermatozoa falling in each cluster among dogs (p < 0.05). Linear regression models including sperm head shape factors 2, 3 and 4 predicted the amount of denatured DNA within each individual spermatozoon (p < 0.001). We conclude that the ASMA analysis can be considered a powerful tool to improve the spermiogram.

OC3 Morphometry Characterisation of Catalan Donkey Spermatozoa and Identification of Sperm Morphometric Subpopulations

This study determined morphometric patterns of Catalan donkey spermatozoa. For this purpose, 32 ejaculates were collected from seven Catalan donkeys. Semen smears were stained with Harris’ Hematoxilin stain and analyzed with an ASMA system. VARCLUS and FASTCLUS procedures were applied to determine the most significant morphology parameters and the morphologic subpopulations respectively. Mean values of head morphometry were: length (LH) 5.45 ± 0.53 μm, width (WH) 2.68 ± 0.21 μm, area (AH) 11.56 ± 1.32 μm2, percentage of reacted acrosomes 45.2 ± 6.95%, FUN1 (LH/AH) 2.0 ± 0.21, FUN3 (4piS/PH2) 0.3 ± 0.04. Mean values of midpiece were: width (WMP) 1.2 ± 1.08 μm, area (AMP) 3.2 ± 0.99 μm2, angle between head and midpiece (AHMP) 8.8 ± 17.46 degrees. Four different sperm morphometric subpopulations were identified. Subpopulation 1 (2.18% of spermatozoa) showed a WMP of 2.66 μm, an AMP of 39.96 μm2. Subpopulation 2 (72.78% of spermatozoa) showed a WMP of 1.00 μm, an AMP of 4.01 μm2. Subpopulation 3 (24.24% of spermatozoa) showed a WMP of 1.36 μm, an AMP of 14.11 μm2. Finally, subpopulation 4 (0.94% of spermatozoa) showed a WMP of 7.70 μm, an AMP of 171.53 μm2. It is noteworthy that four morphologic subpopulations were identified suggesting a possible relationship between these and the four motility subpopulations previously described (Miró et al. 2005, Theriogenology 63: 706–1716).

Identification of Sperm Morphometric Subpopulations in Two Different Portions of the Boar Ejaculate and Its Relation to Postthaw Quality

A statistical approach using sequentially principal component analysis (PCA), clustering, and discriminant analyses was developed to identify sperm morphometric subpopulations in well-defined portions of the fresh boar ejaculate. Semen was obtained as 2 portions (the first 10 mL of the sperm-rich fraction and the rest of the ejaculate, respectively) and frozen using a conventional protocol. Before freezing, an aliquot was used for computer-assisted sperm morphometry analysis (ASMA). Postthaw quality was evaluated using computer-assisted sperm analysis (CASA), and an annexin-V/PI assay evaluated sperm membranes. The PCA revealed that 3 variables represented more than 78% of the cumulative variance in sperm subpopulations. The clustering and discriminant analyses, based on 5780 individual spermatozoa, revealed the existence of 4 sperm subpopulations. The relative percentage of these subpopulations varied between boar and ejaculate portions. Linear regression models based on measured morphometric characteristics could account for up to 36% of the percentage of intact sperm membranes postthaw. The ASMA protocol used in our study was useful to detect subtle morphometric differences between spermatozoa, and the combination of this analysis with a multivariate statistical procedure gave new information on the biological characteristics of boar ejaculates that is not given by conventional sperm analysis.