Head morphometric changes in cryopreserved ram spermatozoa are related to sexual maturity

The importance of understanding the sperm changes after the cryopreservation process has been emphasized in human and veterinary andrology. In previous studies, we have shown that the morphometric characteristics assessed by computer-assisted analysis following the freeze-thawing process revealed differences in terms of dimension and shape between individuals that may be related to bio-physiologic factors such as sexual maturity. The purpose of this study was to determine if there are differences associated with cryoresistence and sperm head morphometric dimensions in individuals with different sexual maturity ratings (SMRs; 12, 30 and 96 months of age). Ejaculates from nine normospermic fertile rams with different SMRs were analyzed in an attempt to quantify the morphometric dimensions and the shape of sperm heads from each group after the cryopreservation process. The mean values of sperm concentration among individuals with different SMRs were significantly different (P < 0.01). Cryopreservation substantially reduced sperm motility and plasma membrane integrity irrespective of SMR assessed, with young animals being the most affected (P < 0.01). Sperm quality at thawing for all sperm parameters evaluated was significantly higher for old individuals than for middle-aged or young individuals (P < 0.01). There were no significant differences in the sperm head dimension or shape among middle-aged and old individuals (P > 0.05). However, significant differences were detected in area, perimeter and width (lower values) and length, ellipticity and elongation (higher values) in old or middle-aged individuals compared with young individuals (P < 0.01). In conclusion, this study confirms that ram age is related to sperm morphometric dimensions, and sperm size and shape may affect spermatozoa survival, being good indicators of freezability. Therefore, the present study provides information on the morphometric maturation of ram sperm and supports the idea that the dimensions of spermatozoa may be taken as an approximate indication of its relative maturity.     

Objective assessment of goat sperm head size by computer-assisted sperm morphometry analysis (ASMA)

The aim of the present study was to identify different objective categories of sperm head size evaluation, using both morphometric and statistical analyses. For this purpose, semen samples (n = 16) were collected from 4 Florida male goats and assessed using a computer-assisted sperm morphometric analysis (ASMA) to obtain sperm head size measurements. The sperm heads were grouped into categories according to 25th and 75th percentiles (the lower and upper quartile, respectively) of their area values. Thereafter, a discriminant analysis was implemented on all the sperm morphometric parameters assessed, to obtain a classification matrix for sperm head size. Sperm heads by this method were classified into 3 categories depending on their size (small, medium and large), with a globally correct assignment of 95.5%. Moreover, significant differences (p < 0.001) were recorded between individual animals for all the sperm head morphometric parameters assessed. In conclusion, by using both statistical and morphometric analyses, it was possible to recognize the three categories of sperm head size. It is expected that research could be useful in defining the relationship between sperm head size measurements and actual fertility data.     

Distinct evolutionary patterns of morphometric sperm traits in passerine birds

The striking diversity of sperm shape across the animal kingdom is still poorly understood. Postcopulatory sexual selection is an important factor driving the evolution of sperm size and shape. Interestingly, morphometric sperm traits, such as the length of the head, midpiece and flagellum, exhibit a strong positive phenotypic correlation across species. Here we used recently developed comparative methods to investigate how such phenotypic correlations between morphometric sperm traits may evolve. We compare allometric relationships and evolutionary trajectories of three morphometric sperm traits (length of head, midpiece and flagellum) in passerine birds. We show that these traits exhibit strong phenotypic correlations but that allometry varies across families. In addition, the evolutionary trajectories of the midpiece and flagellum are similar while the trajectory for head length differs. We discuss our findings in the light of three scenarios accounting for correlated trait evolution: (i) genetic correlation; (ii) concerted response to selection acting simultaneously on different traits; and (iii) phenotypic correlation between traits driven by mechanistic constraints owing to selection on sperm performance. Our results suggest that concerted response to selection is the most likely explanation for the phenotypic correlation between morphometric sperm traits.     

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.     

Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen

Both the study and the relationship between sperm design and sperm function have been a target of several researchers. In our study we have evaluated the relationship between the morphometry of sperm head and midpiece as well as the relationship between morphometry of these two spermatic components and sperm motion characteristics in the boar. Analysis of regression (lineal and multiple) and principal components analysis were used for the study of these relationships. Semen samples from five Iberian boars were taken for analysis. Analysis of morphometry was assessed by CASMA system and motility by CASA system. Sperm midpiece showed a significant relationship (positive or negative, depending on the morphometric parameter evaluated) with sperm head. VSL, LIN, STR, BCF and VAP showed a significant relationship with several head and midpiece morphometric parameters. Finally, through the analysis of multiple lineal regression we obtained several statistical models that predict STR, LIN, VCL, ALH, BCF, PC1 and PC2 (the last two variables have been obtained from a principal components analysis) as a function of one, two or three morphometric parameters. Our results suggest a co-evolution of sperm head and midpiece and in addition that sperm motion characteristics of porcine spermatozoa are influenced by morphometry of head and midpiece.     

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.     

Characterization of ram (Ovis aries) sperm head morphometry using the Sperm-Class Analyzer

Sperm morphology has been identified as a characteristic that can be useful in the prediction of fertilizing capacity. The aim of the current study was to characterize ram sperm heads morphometrically as a basis for future studies on the relationship between sperm quality and male fertility. For this purpose, ejaculates from 241 mature rams (Ovis aries) belonging to 36 different dairy herds were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer. Sperm samples, collected by artificial vagina, were diluted in phosphate buffered saline (PBS) for the analysis. A microscope slide was prepared from single-diluted fresh sperm samples. Slides were air-dried and stained with Hemacolor. A minimum of 115 sperm heads were analyzed from each male. Each sperm head was measured for four primary parameters (area, perimeter, length, width), and four derived parameters of head shape were obtained. Significant differences in sperm head morphometry were found between rams (CV for morphometric parameters ranging from 0.9 to 10.1), and there were marked differences in the sperm morphometric composition of the ejaculates. For all parameters, within-animal CVs were greater than between-animal CVs. Within-animal CVs ranged from 4.2 to 10.6, showing the high degree of sperm polymorphism present in the sheep ejaculate. Significant differences in sperm head morphometry were found between rams belonging to the different herds (i.e., origin). An important part of the variability observed on morphometric parameters was due to the male itself, with an explained variance ranging from 3.6% for regularity to 34.0% for p2a (perimeter²/[4 × π × area]). The explained variance by the herd of origin of the males ranged from 0.6% for regularity to 10.8% for area. Our results suggest that a genetic component might be responsible for the observed sperm head morphometry differences between herds.     

Morphometric characterization and classification of alpaca sperm heads using the sperm-class analyzer computer-assisted system

Sperm morphology has been identified as one characteristic which can be useful in the prediction of sperm fertility, therefore, we hope that this study aimed at establishing standardized morphological criteria might serve in future studies dealing with the search for sperm parameters which facilitate an estimation of sperm quality. For this purpose, ejaculates from fertile alpacas were used to evaluate sperm head morphometry by means of the Sperm-Class Analyzer (SCA) computer-aided image analysis system. We defined three morphological categories according to sperm head size (normal 50%, small 26%, large 24%) and five categories according to sperm head shape (normal 47%, pyriform 3%, short 20%, round 1%, long 29%). Sperm classification according to shape was performed by first morphometrically characterizing sperm heads clearly falling into each of the shape categories. Thereafter, discriminant analysis was performed on the data from these typical sperm heads and the resulting classification functions were used to categorize 2,200 spermatozoa from 11 alpacas. Classification of sperm heads by this method agreed in 88% of the cases with most of the misclassifications being due to pyriform heads classified as long heads. Morphometric values obtained from samples of 50, 100, 150, 175 and 200 sperm heads were compared. At least 150 sperm heads should be evaluated to overcome sample size influence on sperm measurements. Significant differences in sperm morphometry were found between individuals (CV for morphometric parameters ranging from 1.3 to 13.0) and there were marked differences in the sperm morphological composition of the ejaculates. Within-animal CV ranged from 4.7 to 17.8 thus showing the high degree of sperm polymorphism present in the alpaca ejaculate.     

Sperm head phenotype and male fertility in ram semen

Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter²/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males' fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level.     

Morphometric changes in goat sperm heads induced by cryopreservation

Two experiments were designed to evaluate the effect of cryopreservation on morphometric characteristics of the goat sperm head. To address this question, we evaluated the size of the sperm head in fresh control cells, post-cooling cells after equilibration with the glycerol preservation solution, and post-thawing cells. Assessment was by automated morphometric sperm head analysis (ASMA) using phase-contrast microscopy without staining. In the first experiment, ASMA was performed on heterospermic pooled samples (fresh, post-cooling after equilibration with the glycerol preservation solution and post-thawing): length, width, area and perimeter were measured. In the second experiment, sperm viability was assessed by Hoechst staining and head morphometry was carried out as before, simultaneously during the cryopreservation process, and the head size was identified for both live and dead spermatozoa. The data were analysed by principal component analysis (PCA). The purpose of PCA is to derive a small number of linear combinations (principal components) from a set of variables (length, width, area and perimeter), that retain as much of the information in the original variables as possible. The main findings that have emerged from this study are that (i) a simple procedure has been developed for measuring spermatozoa heads without staining, which minimises the possibility that sperm head dimensions were influenced by procedural artefacts; (ii) the dimensions of goat sperm heads after cryopreservation in skimmed milk-glucose medium were smaller than in fresh sperm, but this was due to the equilibration phase with the cryoprotectant and not to the cryopreservation process itself; and (iii) dead spermatozoa showed smaller heads than live sperm, consequent upon the loss of membrane function. No differences were observed between post-cooling cells after equilibration with the glycerol preservation solution and post-thawing spermatozoa and only minor osmotic differences between them and fresh sperm were observed.     

The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability

The Sperm Class Analyzer was used to investigate the effect of freeze-thawing procedure on Florida buck sperm head morphometry, and to relate possible changes in sperm head dimensions to cryopreservation success. Semen samples (n=76) were frozen with tris and milk-based extenders and thawed. Sperm quality samples (motility, morphology, acrosome), and sperm head morphometric values (length, width, area, perimeter, ellipticity) were compared between fresh and frozen-thawed samples. Sperm freezability was judged according to the sperm quality parameters assessed. Fertility data was obtained after artificial insemination with cryopreserved semen. Cryopreservation success was different between freezing methods. Sperm head dimensions were significantly (p<0.05) smaller in cryopreserved tris and milk spermatozoa respectively than in those of the fresh samples. The sperm head morphometric parameters that had changed after cryopreservation were lower in suitable semen samples after thawing and with successful pregnancies after artificial insemination. These data suggest that changes in sperm head morphometry might reflect spermatozoa injury occurred during cryopreservation.     

Comparison of three staining techniques for the morphometric study of rainbow trout (Oncorhynchus mykiss) spermatozoa

This study was designed to compare the performance of the kits Diff-Quick, Hemacolor and Spermac for staining the spermatozoa of rainbow trout. Automated sperm morphology analysis (ASMA) was performed using two image analysis programs to determine the sperm measurements: head size (length, width, area and perimeter), shape (ellipticity, rugosity, elongation and regularity) and tail length. Diff-Quick was found to be the best procedure for staining the trout spermatozoa. The use of this method rendered the highest number of cells correctly analyzed, and provided good colour intensity and contrast of the sperm head. No differences among the methods were detected in terms of tail length measurements. Mean values established using Diff-Quick for the main morphometric variables were: head length 2.93+/-0.13 microm; head width 2.33+/-0.15 microm and tail length 34.16+/-1.66 microm. Based on these findings, we recommend the Diff-Quick staining kit for its accurate and reproducible morphometric results. Notwithstanding, when analyzing the sperm tail of the rainbow trout, the Spermac method offers improved contrast.     

The relationship between ram sperm head morphometry and fertility depends on the procedures of acquisition and analysis used

Sperm head morphometry is a parameter in the evaluation of semen that has been associated with fertility in two ways: comparing morphometric measures between predefined groups of fertility; or analyzing morphometric data by multivariate techniques to identify cell populations. We analyzed the morphometry of ram sperm head by three procedures and checked its relationship with male fertility. A Computer-Aided Sperm Morphometric Assessment procedure (CASMA), an image analysis software (NIS-Elements) in combination with an optical microscope (MO-NIS) and this image analysis software in combination with a scanning electron microscope (SEM-NIS) were used. Eight morphometric parameters were assessed: length, width, area, perimeter, ellipticity, form factor, elongation and regularity. We observed significant differences between the morphometric data of sperm head obtained with three study procedures. The CASMA procedure shows the highest values for all parameters and the SEM-NIS procedure the lowest. The analysis of a semen sample, when only the mean of morphometric parameters is used to describe the cell population, is too limited to interpret their fertilizing capacity. It is essential to analyze the complex structure of the samples by defining subpopulations by multivariate methods. With few exceptions, the means of each morphometric parameter differ between the three subpopulations analyzed in each procedure. Only the subpopulations obtained with the MO-NIS procedure showed a significant correlation with male fertility. In short, it is necessary to establish an instrumental standard for the analysis of sperm morphometry to obtain reliable results and we believe that the MO-NIS system presents these basic requirements.     

Effect of cetrorelix on sperm morphology during migration through the epididymis in the cynomolgus macaque (Macaca fascicularis)

The importance of the cynomolgus monkey as a model for human reproductive medicine prompted this examination of epididymal sperm morphology. Computer-aided sperm morphological analysis was used for the first time to provide morphometric data on sperm heads as they traversed the epididymal duct of Macaca fascicularis. The duct was divided into six regions, starting close to the testis (proximal) and ending close to the vas deferens (distal). To determine the androgen-dependence of the changes, one group of animals received a GnRH-antagonist (Cetrorelix, Asta Medica, Frankfurt, Germany) to induce testicular regression and lower epididymal androgens, while a control group received only vehicle. Epididymides were removed 16 and 25 days after treatment, and sperm heads were analysed by a computer-assisted morphometric analyser. Cluster analysis revealed swollen sperm head cells in proximal regions 1 and 2 of the epididymis, but fewer such forms distally. Normal head shapes became the majority in region 4 and these underwent a gradual but statistically significant decrease in size (area, perimeter, length, width) and shape as they reached the distal regions. In the animals given Cetrorelix, sperm with swollen heads were found more distally than in the controls, although they were also never present in the distal cauda (region 6). Normal heads still became predominant in region 4 after 16 days treatment, and in region 6 after 25 days. The normal forms in the cauda epididymidis of treated animals were significantly larger than cells from control animals. We conclude that epididymal sperm maturation in the monkey is characterised by both a loss of sensitivity to distortion on air-drying, and by a decrease in sperm head size. The former, but not the latter, is attained by sperm in androgen-deficient epididymides from GnRH-antagonist-treated monkeys.     

Automated sperm morphometry and morphology analysis of canine semen by the Hamilton-Thorne analyser

Computer-assisted sperm morphometry has the potential to eliminate several drawbacks inherent to the current methods of sperm morphology evaluation, and allows for the identification of subtle sperm characteristics which cannot be detected by visual evaluation. In the present study, the Metrix Oval Head Morphology software implemented in the Hamilton-Thorne CEROS (version 12.1; HTR 12.1 Metrix) computer-aided semen analyser was evaluated for canine sperm morphometry and morphology analysis. Comparison of sperm morphometric measurements of 200 spermatozoa from pooled semen samples (n = 4) at 40x and 60x demonstrated a more accurate identification of the sperm head boundaries at a magnification level 60x. Dilution of pooled semen samples (n = 4) to a sperm concentration of 50 x 10(6) ml(-1) allowed for a correct evaluation of the sperm cell dimensions whereas 100 x 10(6) and 200 x 10(6) ml(-1) resulted in a higher percentage of rejected spermatozoa due to overlapping. No differences in morphometric dimensions were found when 100 or 200 spermatozoa were evaluated for each of 15 dogs. The mean morphometric parameters of canine spermatozoa, based on the fresh ejaculates of 23 dogs, were: major 6.65 +/- 0.20 microm; minor 3.88 +/- 0.14 microm; area 20.66 +/- 1.04 microm2; elongation 58.64 +/- 2.58 %; perimeter 17.57 +/- 0.43 microm and tail length 48.93 +/- 10.16 microm. Large variations in morphometric dimensions were detected among individual dogs. After cryopreservation, significantly lower morphometric dimensions were obtained for all the evaluated sperm samples (n = 12). Finally, a correlation of 0.82 (P < 0.05) was established for the percentage of normal spermatozoa assessed by subjective evaluation and by the HTR 12.1 Metrix (n = 39 semen samples). In conclusion, dilution of the semen samples to approximately 50 x 10(6) spermatozoa/ml and an objective lens magnification of 60x, analysing at least 100 spermatozoa, are the technical settings proposed to obtain reliable and objective sperm morphometric measurements by the HTR 12.1 Metrix in canine.     

Computer-assisted sperm head morphometry analysis (ASMA) of cryopreserved ram spermatozoa

Normal sperm morphology has been shown to be indicative of male fertility; however, subjective methods of assessing morphology are highly variable. Computer-assisted sperm morphometry analysis (ASMA) has been developed for the objective analysis of sperm head dimensions. Developing applicable protocols for sperm head morphometry analysis increases the efficiency of these systems. The objective of the current study was to develop accurate methods for employing ASMA of ram sperm heads. Staining methods, optimal sperm sample numbers microscopic magnification and sampling variation within and between technicians were assessed. Frozen semen from 10 fertile rams was thawed and prepared on slides for morphometric analysis. Staining spermatozoa with hematoxylin and rose bengal stains yielded the best results. Ram sperm head morphometry was accurately evaluated on at least 100 spermatozoa at x 40 objective magnification. Using these techniques, a sample could be analyzed in approximately 3 min. No significant differences in sperm head measurements were detected between 2 technicians. The system properly recognized and digitized ram spermatozoa 95% of the time. The morphometric measurements of sperm heads for all rams were as follows: length = 8.08 microns, width = 4.80 microns, width:length ratio = 0.59, area = 29.13 micron 2 and perimeter = 23.93 microns. The mean within analysis coefficients of variation for all individual analyses and parameters ranged from 4.8% for length to 6.0% for area. The variation between replicate analysis was 2.4% or less for both technicians. When applying proper sample preparation and analysis procedures no differences in measurements or variation were observed between the 2 system operators.     

Study of nuclear and acrosomal sperm morphometry in ram using a computer-assisted sperm morphometry analysis fluorescence (CASMA-F) method

The aim of this study was to develop a new method that allows morphometric assessment of the sperm nucleus and acrosome in the ram using fluorescence microscopy and free software. The study was divided into three experiments. In the first experiment, semen smears from 20 ejaculates 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 using the ImageJ program. The computer-assisted sperm morphometry analysis fluorescence (CASMA-F) method used allowed the differentiation, capture, and morphometric analysis of most sperm nuclei, acrosomes, and whole heads with high precision and the assessment of the acrosomal status. In the second experiment, sperm nuclear morphometry by CASMA-F was compared by staining with the PI/PSA combination and staining with Hoechst 33342 as in previous studies. Similar results were obtained using both methods. In the third experiment, CASMA-F with PI/PSA was compared with a more conventional CASMA method (semen smears stained with Hemacolor (HEM) and processed with the ISAS commercial software, HEM). Spermatozoa displayed a bigger size when processed with CASMA-F than with HEM method in all primary sperm head morphometric parameters, but results using both methods were correlated. It was concluded that the CASMA-F method allows the simultaneous assessment of sperm nucleus, acrosome, and head in the ram.     

The effect of sperm concentration in the ejaculate on morphological traits of bull spermatozoa

Experiments were performed on 75 ejaculates obtained from 19 bulls representing different cattle breeds used at the Masovian Centre for Animal Breeding and Reproduction in ?owicz. Fresh ejaculates were measured in respect to their volume and sperm count in the ejaculates was determined. The ejaculates were classified based on the criterion of sperm concentration and divided into five groups. Sperm morphometric measurements were taken from each bull and assessment of semen morphology was done on the basis of examination under a microscope using preparations made from fresh ejaculates. For each slide, morphometric measurements were taken of 15 randomly selected spermatozoa characterised by normal morphology and well visible under the microscope. Additionally, in each preparation morphometry of 500 spermatozoa was evaluated, numbers of spermatozoa with normal morphology and morphological abnormalities were recorded and these were categorized into spermatozoa with major and minor defects. An insignificant correlation was observed between the sperm concentration in the ejaculate and morphological traits, dimensions and shapes of bull spermatozoa. The less concentrated ejaculates contained spermatozoa with a slightly larger head circumference and a more elongated head shape in comparison with the spermatozoa in the more concentrated ejaculates. The highest frequency of morphologically malformed spermatozoa, both in the case of primary and secondary alterations, was observed in ejaculates with sperm concentration of no more than 1000 x 10(3)/mm3.     

Sexual selection on protamine and transition nuclear protein expression in mouse species

Post-copulatory sexual selection in the form of sperm competition is known to influence the evolution of male reproductive proteins in mammals. The relationship between sperm competition and regulatory evolution, however, remains to be explored. Protamines and transition nuclear proteins are involved in the condensation of sperm chromatin and are expected to affect the shape of the sperm head. A hydrodynamically efficient head allows for fast swimming velocity and, therefore, more competitive sperm. Previous comparative studies in rodents have documented a significant association between the level of sperm competition (as measured by relative testes mass) and DNA sequence evolution in both the coding and promoter sequences of protamine 2. Here, we investigate the influence of sexual selection on protamine and transition nuclear protein mRNA expression in the testes of eight mouse species that differ widely in levels of sperm competition. We also examined the relationship between relative gene expression levels and sperm head shape, assessed using geometric morphometrics. We found that species with higher levels of sperm competition express less protamine 2 in relation to protamine 1 and transition nuclear proteins. Moreover, there was a significant association between relative protamine 2 expression and sperm head shape. Reduction in the relative abundance of protamine 2 may increase the competitive ability of sperm in mice, possibly by affecting sperm head shape. Changes in gene regulatory sequences thus seem to be the basis of the evolutionary response to sexual selection in these proteins.     

Quantitative changes in sperm head morphology during passage through the male excurrent duct system of the rabbit

A fine adjustment of sperm head size and shape occurs during maturation and storage within the male excurrent duct of the rabbit. This remodelling, as judged by morphometric values of area, perimeter, length, width, and shape factors, takes place mostly in passage from the seminiferous tubules of the testis to the distal caput of the epididymis. The dimensions of sperm heads from the distal corpus of the epididymis break the general tendency toward a reduction in size and more elliptical shapes. A period of transport and storage within the epididymal cauda and vas deferens follows in which there are no further changes in sperm head morphometry. It can be concluded that the period immediately following sperm release from the testis is crucial to the final morphological maturation of spermatozoa. Moreover, the fact that changes are detected in the appearance of sperm heads at successive stages of sperm maturation suggests that the dimensions of a particular epididymal spermatozoon may be taken as an approximate indication of its relative maturity. Mol. Reprod. Dev. 51:203–209, 1998. © 1998 Wiley-Liss, Inc.