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.     

Computer assisted morphometric analysis of ram sperm heads: evaluation of different fixative techniques

The recent development of automated systems for morphometric sperm head analysis has provided a series of objective parameters which have facilitated the standardization of morphological semen evaluation. This current work attempts to establish the optimum fixing conditions for the morphometric characterization of ram spermatozoa. Ejaculates were obtained from 5 Merino rams used for periodic collection of semen and were diluted at 1:50 with TEST medium. Air-dried smears were fixed either in ethanol-ether (1:1), 50% methanol, 2% glutaraldehyde or SUZA fixative, in which case the smear was pretreated with chloramine. The samples were then stained with commercial kit Hemacolor. Once the preparations had been mounted, they were analyzed with the Sperm Class Analyzer automatic sperm morphometry analysis system (ASMA). The minimum number of sperm cells analyzed per sample was 100. The parameters evaluated were the area, perimeter, length, width, shape factor and mass. The results showed significant differences in sperm head dimensions between the 4 fixation techniques, with the lowest values for all parameters corresponding to the SUZA fixative, followed by glutaraldehyde, methanol, and finally ethanol-ether. In addition, there were significant variations between animals. It can, therefore, be concluded that the working protocol must be defined when performing morphometric analysis of ram semen and that the results obtained under different conditions of fixation cannot be entirely extrapolated. Equally, the high variability among individuals suggests that, in a species like the ram with a low index of teratozoospermia, there is a need for a revision of the classic definition of normality, which should include morphometric data.     

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.     

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.     

Morphometric classification of Spanish thoroughbred stallion sperm heads

This work used semen samples collected from 12 stallions and assessed for sperm morphometry by the Sperm Class Analyzer (SCA) computer-assisted system. A discriminant analysis was performed on the morphometric data from that sperm to obtain a classification matrix for sperm head shape. Thereafter, we defined six types of sperm head shape. Classification of sperm head by this method obtained a globally correct assignment of 90.1%. Moreover, significant differences (p<0.05) were found between animals for all the sperm head morphometric parameters assessed.     

Computer automated sperm head morphometry analysis (ASMA) of goat spermatozoa

The development of computer automated sperm morphometry analysis (ASMA) allows for the objective analysis of sperm head dimensions. A number of studies have been performed to optimize the efficiency of these systems when analyzing spermatozoa from a variety of species. In this study, frozen semen from 10 fertile goat bucks was thawed and prepared on slides for morphometric analysis to evaluate technical variation and to standardize ASMA procedures for goat spermatozoa. Methods of staining, the number of spermatozoa necessary to sample and optimal microscopic magnification were assessed. Staining for 20 min in hematoxylin (HEM) was found to be optimal. The most efficient method of analyzing goat sperm morphometry was to evaluate 100 sperm cells at x20 objective magnification. Using these techniques, a sample could be analyzed in approximately 2 min. The system properly recognized and digitized spermatozoa 96% of the time with a target recognition error rate of less than 1%. The morphometric measurements of sperm heads for all 10 bucks were the following: length = 7.69microm, width = 3.80microm, width/length ratio = 0.5, area = 22.82microm and perimeter = 20.15microm. The mean coefficients of variation (CV) for all bucks ranged from 3.4% for length to 5.8% for area. Standardized sample preparation techniques and analysis were found to improve the efficiency of ASMA.     

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.     

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.     

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.     

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.     

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.     

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.     

Comparison between the toluidine blue stain and the Feulgen reaction for evaluation of rabbit sperm chromatin condensation and their relationship with sperm morphology

Sperm chromatin alteration is an important feature that can affect fertility of the male rabbit. This study compared toluidine blue staining with Feulgen reaction (as methods for evaluating chromatin alteration) and investigated the relationship between sperm morphology and chromatin alteration. Seven hundred rabbit ejaculates of animals with unknown fertility were used. Primary and secondary morphological sperm abnormalities were evaluated in semen smears with phase-contrast microscopy. Chromatin alterations were evaluated in semen smears stained with toluidine blue (pH 4.0 and 5.0) and with the Feulgen reaction. While the three methods were equally efficacious for identification of chromatin alterations, toluidine blue staining was more appropriate to characterize the intensity of chromatin alterations. The correlation between primary sperm defects and chromatin alteration was high and positive, suggesting that sperm chromatin structure affected sperm head morphology. The correlation between secondary sperm defects and chromatin alteration was also positive, but lower. The final chromatin compaction occurs in the epididymus, where secondary sperm defects originate. Therefore, the causes of secondary sperm defects could also intervene with final chromatin compaction. In summary, the toluidine blue stain was an effective means of evaluating the sperm chromatin alteration in rabbit spermatozoa.     

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.     

Sperm morphology and fertility of progeny-tested AI dairy bulls in Sweden

Use of bull semen with high levels of sperm abnormalities, reflecting genital dysfunction, is not recommended for artificial insemination (AI) since it would most likely lead to subfertility. Sperm quality, including sperm morphology, may deteriorate with increasing age of the bull thus becoming a source of concern when using older, progeny-tested AI bull sires. Although a relationship between sperm morphology and fertility after AI in progeny-tested bull sires has been reported, it is yet unclear which sperm abnormalities are most critical. This constituted the core aim of a 22-month long retrospective study in proven (aged 60-84 months at the start of the study) AI sires of the Swedish Red (SR, n=8) and Swedish Holstein (SLB, n=4) breeds where their semen (107 freezing batches in total, built by a single ejaculate (n=3) or pooling two consecutive ejaculates (n=104) collected at 1-3 months interval), were subjected to detailed morphological examinations on wet- and dry, stained smears. Attention was paid to between- and within-bull variations with regard to presence and level of sperm abnormalities. Sperm morphology differed significantly between sires and ejaculates, with 6/12 sires having ejaculates containing >10% of morphologically deviating sperm head shapes, a commonly used threshold for young AI bulls in Sweden. However, with the exception of pear-shaped or narrow-at-the-base anomalies, the mean values for individual defects were always within the limits expected for a normal bull sire, and were therefore considered acceptable. The percentage of morphologically normal spermatozoa was positively related to fertility, whose output differed significantly among bulls. Among sperm abnormalities, the proportion of morphologically deviating sperm head shapes were negatively correlated with fertility, pear-shaped sperm heads in particular. In conclusion, the relationship between sperm morphology and fertility after AI calls for frequent (2-3 months interval) detailed assessments of sperm morphology in AI stud bull sires.     

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.     

The effects of cryopreservation on the morphometric dimensions of caprine sperm heads

Cryopreserved semen has been utilised in the artificial insemination of livestock species for over 40 years, even though the detrimental effects of cryopreservation on sperm function and fertility are well documented. In the present study, computer-automated sperm-head morphometry was used to determine if goat sperm-head morphometry was affected by freezing and thawing. A microscope slide was prepared from single semen samples, collected by artificial vagina, from 10 sexually active Saanen bucks. The remainder of each sample was frozen in a tris-citrate-yolk extender. After thawing, semen smears were prepared on microscope slides. All slides were stained in haematoxylin and mean sperm-head measurements of length, width, width/length, area and perimeter were determined for each slide by computer aided sperm morphometry analysis. The effects of sperm freezing on sperm-head dimensions within and among all bucks were determined. No significant (P > 0.10) freezing effect was found between fresh semen and postthaw samples for length (7.00 μm vs 7.13 μm), width (3.77 μm vs 3.87 μm), width/length (0.54 μm vs 0.54 μm), area (19.67 μm² vs 20.57 μm²) and perimeter (18.62 μm vs 18.83 μm) when analysed across all bucks. Significant differences (P < 0.05) were however found within three bucks for area, perimeter, length and width, with the percentage increase in measurements being significantly greater than in the remaining bucks. The variability of the morphometric dimensions were not affected by freezing. The results indicate that semen freezing did not affect the overall dimensions of sperm heads across the entire population of bucks sampled. However, since sperm-head dimensions from three bucks were affected, changes in sperm-head morphometry may be indicative of spermatozoa of the semen from individuals to successfully freeze. Because the overall mean sperm-head dimensions acquired from frozen/thawed semen were not different from those of fresh semen, previously reported measurements of goat sperm heads are probably reflective of fresh semen. More importantly, retrospective studies of sperm-head morphometry and fertility may now be performed utilising extensive breeding records from frozen semen.     

Studies on camel semen. I. Electroejaculation and some aspects of semen characteristics

A method for collection of camel semen by electroejaculation has been described for the first time. The volume of the ejaculates obtained was slightly lower than that obtained using an artificial vagina. The sperm count was considerably higher, but other parameters including pH, percentage of live sperm and sperm dimensions were comparable to those of ejaculates collected by the artificial vagina. The camel spermatozoon is generally smaller than those of other animals. The head is somewhat elliptical in shape and slightly tapering at the base. Mean sperm dimensions for the head, middle piece and tail in μm were 5.5, 6.9 and 35.6 respectively; total sperm length was 48.0 μm.     

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.