Cryopreservation of European eel (Anguilla anguilla) spermatozoa: effect of dilution ratio, foetal bovine serum supplementation, and cryoprotectants

The main aim of the present study was to investigate the effect of sperm freezing medium dilution ratio (1:1, 1:2, and 1:5 v/v), two cryoprotectants: dimethyl sulphoxide (Me(2)SO) and methanol (MeOH), and the addition of foetal bovine serum (FBS) on the cryopreservation of European eel sperm. The effect of these factors was evaluated comparing post-thawing viability with fluorescent staining (Hoechst bisbenzimide 33258) and the spermatozoa head morphometry, determined with computer-assisted morphology analysis (ASMA). The 1:5 (v/v) dilution ratio resulted in a lower viability in comparison with 1:1 and 1:2 (52.8+/-2.3% vs. 67.4+/-2.3% and 65.1+/-2.3%, respectively, p=0.0001), but without effects on the head morphology. Although the viability was not significantly different between Me(2)SO and MeOH (60.4+/-1.9 vs. 63.2+/-1.9%, respectively, p=0.305), a decrease of spermatozoa head area and perimeter was found when spermatozoa were frozen with methanol (6.19+/-0.01 vs. 6.36+/-0.01 microm(2) and 17.28+/-0.05 vs. 17.49+/-0.05 microm, for area and perimeter and MeOH and Me(2)SO, respectively, p=0.0001). Finally, a higher viability (75.1+/-1.7 vs. 48.5+/-1.7, with or without FBS, respectively, p=0.0001) and higher spermatozoa head size (6.40+/-0.01 vs. 6.15+/-0.01microm(2) and 17.88+/-0.05 vs. 16.89+/-0.05 microm, for area and perimeter, with or without FBS, respectively, p=0.0001) were found when cells were frozen-thawed in freezing media supplemented with FBS. Based on the above findings, dilution ratios lower than 1:5 (v/v) and the addition of serum improved the viability results after cryopreservation. Future studies are required in order to understand the spermatozoa membrane interchange mechanisms in response to the changes in spermatozoa head size caused by cryoprotectants and freezing media supplements.     

Morphometric characterization of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa using computer-assisted spermatozoa analysis (ASMA)

As part of a larger study on sperm quality and cryopreservation methods, the present study characterized the head morphometry of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa, using both scanning electron microscopy (SEM) and computer‐assisted morphology analysis (ASMA). The latter method has been used rarely in fish and this is its first application on sharpsnout sea bream and gilthead sea bream spermatozoa. Results obtained using SEM are expensive and time‐consuming, while ASMA provides a faster and automated evaluation of morphometric parameters of spermatozoa head. For sharpsnout sea bream spermatozoa, similar head measurement values were obtained using both ASMA and SEM, having a mean ± standard error length of 2.57 ± 0.01 μm vs 2.54 ± 0.02 μm, width of 2.22 ± 0.02 μm vs 2.26 ± 0.04 μm, surface area of 4.44 ± 0.02 μm² vs 4.50 ± 0.04 μm² and perimeter of 7.70 ± 0.02 μm vs 7.73 ± 0.04 μm using ASMA and SEM, respectively. Although gilthead sea bream spermatozoa were found to be smaller than those of sharpsnout sea bream, spermatozoal head morphometry parameters were also found to be similar regardless of evaluation method, having a mean head length of 1.97 ± 0.01 μm vs 1.94 ± 0.02 μm, head width of 1.80 ± 0.01 μm vs 1.78 ± 0.02 μm, surface area of 3.16 ± 0.03 μm² vs 3.18 ± 0.06 μm² and perimeter of 6.52 ± 0.04 μm vs 6.56 ± 0.08 μm using ASMA and SEM, respectively. The results demonstrate that ASMA can be considered as a reliable technique for spermatozoal morphology analysis, and can be a useful tool for studies on fish spermatozoa, providing quick and objective results.     

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.     

Morphometric differences in sperm head dimensions of fertile and subfertile stallions

Gross morphological evaluation of stallion spermatozoa is of clinical value in assessing male fertility in the horse. While of value, methods of subjective sperm classification yield highly variable results. Recent development of computer-assisted sperm morphometry analysis (ASMA) technology has allowed for the objective analysis of sperm head morphometry. In the current study, ASMA was employed to determine morphometric differences in sperm head dimensions between fertile and subfertile stallions. At least 200 spermatozoa from each of 10 fertile and 10 subfertile stallions were analyzed by a commercial ASMA instrument. The mean measurements for length, width, area, perimeter, and width/length for each stallion were recorded and group means compared by a two-sample t-test. The mean measurements for length, area and perimeter were significantly larger in the subfertile than the fertile group (5.77 microm vs 5.33 microm, 12.66 microm vs 11.37 microm and 14.59 microm vs 13.64 microm, respectively). The width of sperm heads from stallions in the subfertile group also tended to be larger than those of fertile stallions. The data suggest that differences in the dimensions of sperm heads may exist between fertile and subfertile stallions.     

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.     

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.     

Computer automated morphometric analysis of bull sperm heads

Morphological type classification of spermatozoa is an important component of the modern semen evaluation; however, current methods of analysis are subjective and highly variable between technicians. To reduce the subjectivity and thus variability of sperm morphology assessment, computer automated sperm head morphology analysis (ASMA) has been developed. Previous studies have shown the importance of standardizing ASMA procedures to optimize accuracy. The objective of this study was to standardize ASMA procedures for evaluating bull sperm heads. Semen from 10 fertile bulls was used to standardize procedures for optimal analysis of bull spermatozoa. Sample preparation methods, sperm staining methods and microscopic magnifications were compared. Semen samples that were diluted to a standard concentration of 200 x 10(6) sperm/ml were more efficiently analyzed than raw samples. A modified GZIN staining procedure, incorporating rose bengal as an acrosomal stain, was used for accurate ASMA at a magnification of x 60. The mean morphometric measurements for all bulls were the area (27.30 microM), perimeter (25.36 microM), length(8.65 microM), width(4.40 microM) and width/length (0.50). Within the analyses, coefficients of variation ranged from 3.45% for length to 8.52% for area. The ASMA system correctly digitized sperm heads 97% of the time. Results of this study indicate that bull sperm heads can be accurately analyzed using current standard procedures of ASMA technology.     

Differences in boar sperm head shape and dimensions recorded by computer-assisted sperm morphometry are not related to chromatin integrity

Although sperm head shape and relative dimensions are considered reliable indicators of sperm quality, their quantification is most often operator-driven, e.g., subjective. Artificial insemination semen doses from 35 mature stud boars of known fertility and belonging to three breeds and two hybrid breeds (Duroc, Large White, Landrace, respectively, Yorker and Risco) were used in this study. Sperm samples were extended to 100x10(6) cells per mL and 10microL of the sperm suspension used to made smears which, stained, were examined using phase contrast microscopy interfaced with an automated sperm morphology analyzer (ASMA, ISAS). Each sperm head was measured for four primary parameters [area (A) microm(2), perimeter (P) mum, length (L) microm, width (W) microm], and four derived parameters of head shape [(L/W), (4piA/P(2)), ((L-W)/(L+W)), (piLW/4A)]. Definition of head size was statistically performed. The threshold for each class was established on the basis of the area values, considering the 25th percentile as small and the 75th percentile as large spermatozoa. In a second step, sperm head shape was determined as normal, elliptic, abnormal (rugose) contour, long or irregular and percentiles set as above to define spermatozoa with normal values for each shape parameter. Significant differences were found among breeds in the size of morphologically normal spermatozoa, which were significantly larger and more elliptic (P<0.001) in the Duroc breed. Sperm chromatin integrity was studied using the SCSA-assay, with significant differences observed in the degree of fragmentation intensity (DFI) although this value was consistently low in all animals studied. The hereby-validated ASMA was able to determine significant differences in sperm shape and dimensions among breeds, which were not accompanied by deviations in chromatin structure neither within nor between fertile AI-boars.     

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.     

Spermatozoal morphologies and fructose and citric acid concentrations in agouti (Dasyprocta leporina) semen

This study was conducted to identify the levels of fructose and citric acid, and sperm morphologies in agouti (Dasyprocta leporina) semen. These parameters may be important in identifying highly fertile semen from the agouti. The objectives were: (1) to investigate spermatozoal abnormalities in agouti semen and (2) to determine the concentrations of seminal fructose and citric acid in agouti semen samples. Semen samples were collected from 16 anaesthetised male agouti by electro-ejaculation. Fructose and citric acid concentrations were 256.86+/-63.54 mg/dl and 1877+/-147 mg/dl, respectively, measured with ELISA kits. Sperm morphologies, examined using eosin-negrosin staining, showed 11 morphologies. The most abundant (68.5%) sperm morphology (M1) showed no known sperm defects. Means for head, mid piece, tail and total length of the agouti spermatozoa was 5.23+/-0.04 microm, 5.18+/-0.08 microm, 37.52+/-0.24 microm and 47.96+/-0.25 microm, respectively for M1 sperm. The means of spermatozoa head and mid piece width and semen volume were 3.26+/-0.04 microm, 0.70+/-0.02 microm and 0.47+/-0.16 ml, respectively. It was concluded that as the fructose concentration in agouti ejaculate increased the percentage of spermatozoa with known spermatozoa defects increased (r=0.506; P<0.037; n=32). It is suggested that the M1 sperm could be the most competitive spermatozoa in agouti ejaculate. In conclusion standards for identifying fertile agouti semen were established.     

Morphometry of stallion spermatozoa by computer-assisted image analysis

Metric measurements of stallion spermatozoal heads were determined for live, unfixed spermatozoa and for Feulgen-stained spermatozoa by videomicroscopy and computerized image analysis. Two ejaculates were collected from each of five stallions of normal fertility. Air-dried semen smears were Feulgen-stained, and live, unfixed spermatozoa were examined as wet-mount preparations. For Feulgen-stained spermatozoa, videoimages (x3850) were captured, and sperm heads were detected via image segmentation and particle analysis. For live, unfixed spermatozoa, phase contrast videoimages (x3850) were measured to determine width and length of the sperm head. For Feulgen-stained spermatozoa, there were significant effects (P < 0.001) of stallion and ejaculate on measured parameters of area, circumference, and the length and width of the sperm head. For live, unfixed spermatozoa, there were significant effects of stallion on length and width and of ejaculate on length of the sperm heads. There was a very poor correlation between length and width of sperm heads between Feulgen-stained and live, unfixed spermatozoa. Two indices of sperm shape (oval factor and aspect ratio) were also determined. Both aspect ratio and oval factor were significantly affected by stallion (P < 0.001); however, oval factor was not affected by ejaculate and therefore may represent a less variable determination of sperm head shape across stallions. Overall, length and width of stallion sperm heads were larger (P < 0.01) for live, unfixed spermatozoa than for Feulgen-stained spermatozoa (length: 6.3 +/- 0.4 vs 5.08 +/- 0.44; width: 3.08 +/- 0.34 vs 2.71 +/- 0.28 mum, respectively). Computerized image analysis may be useful as a means to objectively measure sperm head dimensions in the stallion and could be useful in future studies to determine associations with stallion fertility.     

Morphology and ultrastructure of Siberian sturgeon (Acipenser baerii) spermatozoa using scanning and transmission electron microscopy

BACKGROUND INFORMATION: Available data concerning the sperm morphology of teleost fishes demonstrate wide variation. In the present study, the spermatozoa of Siberian sturgeon (Acipenser baerii Brandt, 1869), a chondrostean fish, was investigated. In contrast with teleost fish, chondrostean spermatozoa have a head with a distinct acrosome, whereas other structures, such as a midpiece and a single flagellum, are present in spermatozoa of most species. RESULTS: The average length of the head including the acrosome and the midpiece was 7.01+/-0.83 microm. Ten posterolateral projections derived from the acrosome were present on a subacrosomal region, with mean lengths of 0.94+/-0.15 microm and widths of 0.93+/-0.11 microm. The nucleus consisted of electrodense homogeneous nuclear chromatin. Three intertwining endonuclear canals, bound by membranes, traversed the nucleus longitudinally from the acrosomal end to the basal nuclear fossa region. There were between three and six mitochondria, two types of centrioles (proximal and distal) in the midpiece and two vacuoles composed of lipid droplets. The flagellum (44.75+/-4.93 microm in length), originating from the centriolar apparatus, had a typical 9+2 eukaryotic flagellar organization. In addition, there was an extracellular cytoplasm canal between the cytoplasmic sheath and the flagellum. CONCLUSIONS: A principal components analysis explained the individual morphological variation fairly well. Of the total accumulated variance, 41.45% was accounted for by parameters related to the head and midpiece of the sperm and the length of the flagellum. Comparing the present study with previous studies of morphology of sturgeon spermatozoa, there were large inter- or intra-specific differences that could be valuable taxonomically.     

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.     

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.     

Ultrastructure of spermatozoa of tench Tinca tinca observed by means of scanning and transmission electron microscopy

Structure of tench (Tinca tinca L.) spermatozoa was investigated by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spermatozoa of 26.1+/-3.8 microm total length possessed typical primitive simple structure, called "aqua sperm", without acrosomal head structures. It was probably the smallest spermatozoon described among cyprinid fishes. Heads were mostly composed of dense and slightly granular material, which appeared to be fairly homogeneous except for the occasional appearance of vacuoles. The midpiece remained separated from the flagellum by the cytoplasmic channel; it was cylindric/cone-shaped, 0.86+/-0.27 microm in length and 1.17+/-0.24 microm in width at proximal part. The proximal centriole was located in the "implantation fossa". The distal centriole appeared almost tangential to the nucleus and it functioned as a basal body for the flagellum. It had an orientation of 140 degrees with respect to the distal centriole. The sperm flagellum with 25.45+/-2.47 microm of total length had no any fin. The diameter of the flagellum perpendicular to the plane of the doublet of central microtubules was 173.67+/-20.45 nm and horizontal plane of the central microtubules was 200.71+/-20.45 nm. Peripheral doublets and the central doublet of microtubules measured 23.39+/-3.18 and 35.88+/-4.44 nm in width, respectively. The diameter of a microtubule was only 9.14+/-2.97 nm. A vesicle was attached to the most basal region of the flagellum and located just under plasma membrane of the flagellum.     

Ultrastructure and motility of the caudal epididymis spermatozoa from the volcano mouse (Neotomodon alstoni alstoni Merriam, 1898)

The volcano mouse Neotomodon alstoni alstoni is a genus endemic to the higher elevations of the Mexican transvolcanic belt. In the present study we examined for the first time the morphological features of the spermatozoa taken from the caudal epididymis of this species by transmission and scanning electron microscopy. Spermatozoan motility was studied in sucrose and bicarbonate solutions; vitality and morphology were observed by light microscopy. Transmission electron microscopy shows that the head of spermatozoon is asymmetric and possesses a large and curved hook. The axoneme of the spermatozoan tail is highly developed at fibers 1, 5, and 6. Absolute and relative measurements of the length of the head, the midpiece, and the rest of the tail were also obtained. N. alstoni alstoni spermatozoa were hyperactive in the presence of 290 mM sucrose and 10 and 20 nM bicarbonate solutions exhibited high motility (180-190 microm/sec), and high flagellum beating frequency (10-12 Hz). In contrast, the spermatozoa in 310 mM sucrose solution showed scarce motility (13.5 +/- 3.8 microm/sec) and low beating frequency (1.5 +/- 0.4 Hz). It is proposed that the volcano mouse spermatozoa possess some features very similar to other mammalian spermatozoa and that bicarbonate triggers caudal epididymal sperm motility of this species. J. Exp. Zool. 287:316-326, 2000.     

Motion characteristics of Murrah buffalo bull spermatozoa in various seasons and its relationship with functional integrity of the plasmallema

Semen was collected from six adult (3.5-7-year-old) Murrah buffalo bulls at weekly intervals for 1 year and evaluated for routine parameters, motion characteristics, reactivity in hypoosmotic solution, and acrosomal and other morphological abnormalities of the spermatozoa. The overall motility (MOT), straight line velocity (VSL), curvilinear velocity (VCL), linearity (LIN), lateral head displacement (ALH) and average path velocity (VAP) were 66.85+/-2.79%, 26.58+/-0.24 and 107.07+/-1.47 microm/s, 26.91+/-0.01%, 11.19+/-0.09 and 61.78+/-2.79 microm/s, respectively. Significant seasonal variation was observed in sperm kinematics and hypoosmotic swelling (HOS) reactivity. Except for LIN, the mean values of sperm dynamics were higher during summer and rainy season and significantly lower in winter season. Sperm kinematics showed significant (P<0.01) positive correlation (r=0.25-0.60) with plasmallemal integrity. Ejaculates with less than 50% HOS-reactive spermatozoa had significantly lowered MOT, VSL, VCL and VAP as compared to the ejaculates with >50% HOS-positive spermatozoa. No significant difference was observed in sperm kinematics among the ejaculates having 50-70% and >70% HOS-reactive spermatozoa. The trend of motion dynamics of the spermatozoa with respect to HOS reactivity was similar in all the three seasons (summer, rainy and winter). The results indicate that ejaculates having more than 50% of HOS-reactive sperm show a higher magnitude of sperm kinematics compared to ejaculates having less than 50% HOS-positive spermatozoa.     

Fine structure and morphology of sterlet (Acipenser ruthenus L. 1758) spermatozoa and acrosin localization

Ultrastructure of sterlet Acipenser ruthenus L. 1758 sperm was examined by scanning and transmission electron microscopy, which allowed us to use various methods for visualizations of different parts of sterlet spermatozoa. Sperm cells possess a head with a distinct acrosome, a midpiece and a single flagellum surrounded by the flagellar plasma membrane. The average length of the head including the acrosome and the midpiece was estimated as 5.14+/-0.42 microm. Nine to 10 posterolateral projections were derived from the acrosome. Three inter-twining endonuclear canals bounded by membranes traversed the nucleus in its whole length from the acrosome to the implantation fossa. Acrosin was located in all the three parts (acrosome, endonuclear canals and implantation fossa). The proximal and distal centrioles located in the midpiece compacted of nine peripheral triplets of microtubules. One cut of the midpiece contained from two to six mitochondria with area of 215+/-85 nm(2) in average. The flagellum was 42.47+/-1.89 microm in length with typical eukaryotic organization of one central pair and nine peripheral pairs of microtubules. It passed through a cytoplasmic channel in the midpiece, which was formed by an invagination at the plasmalemma. The flagellum gradually developed two lateral extensions of its plasma membrane, so-called "fins". Detected morphological variation can be described by four principal component axes corresponding to groups of individual morphometric characters defined on the sperm structures. Correlations among the characters indicate that the sperms are variable in their shape rather than size. Significant variation among examined fish individuals was found only in flagellum and nucleus length. Comparison between the present and previous studies of morphology of sturgeon spermatozoa confirmed large inter- and/or intra-specific differences that could be of substantial taxonomic value.     

The effect of age on the morphometric sperm traits of domestic pigs (Sus scrofa domestica)

The experiment was conducted using 20 male domestic pigs, which were in 2 (equal-sized) age groups: under 14 months old and over 18 months old. At least 5 ejaculates from each male were taken, and in each ejaculate, morphometric measurements of 50 spermatozoa were made. The measured parameters were head area, head length and width, and flagellum length. For each ejaculate, the basic physical traits and the frequency of occurrence of developmental anomalies of the spermatozoa were examined. It was found that sperm dimensions tended to increase along with the boars' age. Considerable and statistical differences in head area and flagellum length were proved. Spermatozoa collected from older boars (above 18 months of age) had a head area larger by 0.49 microm(2)(P< or = 0.01) and a flagellum longer by 0.67 microm (P< or =0.01) than spermatozoa collected from younger boars (under 14 months of age). The differences in head length and width between the spermatozoa of the tested boars were considerably smaller and were not statistically provable. Significant correlations between the head area of spermatozoa and the head length (r = 0.56, P< or =0.05) and head width (r = 0.36, P< or =0.05) were found. However, the head length was not significantly directly correlated with its width (r = 0.18, P< or =0.05), and flagellum length was negatively correlated with spermatozoan head width (r = -0.71, P< or =0.05). Slight correlations between the morphometric traits of the sperm and the physical traits of the ejaculates (r = -0.11 to 0.16) were found, although in most cases, the correlations were not statistically provable.