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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

Induction of maturation and spermiation in the male European eel: assessment of sperm quality throughout treatment

Weekly injections of human chorionic gonadotropin (hCG; 1·5 IU g⁻¹ W ) induced spermiation in 87–100% farmed male European eels Anguilla anguilla over a 10-week period, producing 3 ml (100 g)⁻¹ milt volume, at 1˙4×10⁹ spermatozoa ml⁻¹ sperm mobility peaked in ninth week of treatment with 97% of males with motile sperm. Gonadotropin-treated males showed earlier but similar morphological changes to control males. Whereas higher sperm density was obtained 6 h after hCG administration, higher percentage of motile cells and longer sperm mobility were observed 24 h after the treatment indicating that this is the optimum time to obtain good sperm after the hormonal induction. Several monounsaturated fatty acids in sperm increased significantly from week 5 to 9 of treatment (when highest volume, density and mobility of milt were recorded), whereas eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and total n-3 polyunsaturated fatty acids (PUFAs) were significantly reduced. Fatty acid levels returned to initial values after 13 weeks of treatment. These results are in agreement with the reduction of n-3 PUFAs recently observed in the ovary of female Japanese eel during artificial maturation, and an attempt is made to suggest a physiological explanation of its possible relation with sperm quality.     

Morphometry characterisation of European eel spermatozoa with computer-assisted spermatozoa analysis and scanning electron microscopy

The aim of the present study was to characterise European eel spermatozoa morphometrically, as a basis for future studies on the morphological effects of methods for sperm cryopreservation and sperm quality. This characterisation was carried out measuring several spermatozoa morphology parameters (head length, width, area and perimeter) by scanning electron microscopy (SEM), in comparison with measurements developed in European eel spermatozoa with computer-assisted morphology analysis (ASMA). Spermatozoa head morphology showed differences in width (1.15+/-0.01 microm versus 1.12+/-0.01 microm), perimeter (14.68+/-0.13 microm versus 13.72+/-0.19 microm) and area (5.36+/-0.06 microm2 versus 1.12+/-0.01 microm2) for ASMA and SEM, respectively. When head length was evaluated, significant differences were found, being higher for SEM methodology (5.09+/-0.04 microm versus 4.29+/-0.03 microm). The curved and elongated spermatozoa head in eels means a problem for the ASMA system (Sperm Class Analyser), Morfo Version 1.1, Imagesp, Barcelona, Spain), causing an error in the length measurements. However, similar results were obtained by both techniques when spermatozoa head length was considered as the greater length between two points within the object (4.29+/-0.03 microm versus 4.31+/-0.04 microm for ASMA and SEM, respectively). In conclusion, this is one of the first applications of ASMA in fish and the first in this species, and confirms this system as a useful tool with wide applications in future fish spermatozoa studies. Width, perimeter and area could be used as parameters for the spermatozoa morphology evaluation, whereas the length requires a new programming of the Imagesp software.     

L’analyse automatisée de la morphologie du spermatozoïde

Examination of sperm morphology is one factor of evaluation of sperm function, but it can also be considered as a biomarker of testicular function. All publications showed a high variability in observed results, in relation with different methods of staining slides and classifying sperm morphology, and a large subjectivity in the visual assessment. Automated sperm morphology analysis (ASMA) have the potential to provide more objective, accurate, and precise morphometric measurements of spermatozoa. Standardisation of the methods of slides preparation is first essential. Analysis of the sperm head morphometry appears the more accessible for the ASMA and could give selective parameters in the evaluation of fertility, in complement with motion sperm analysis. In the other hand automated analysis of all sperm abnormalities appears illusory with actual instruments, because the midpiece or the flagellum is a little structure weakly stained, and thus difficult to be identified by the computer. Until more rigorous and consistent definitions of sperm features can be developped, in relation with testicular function, the pronostic value of existing sperm abnormalities classifications is limited.     

Influence of staining and sampling procedures on goat sperm morphometry using the Sperm Class Analyzer

Computer-assisted sperm morphometry analysis (ASMA) has improved the assessment of sperm morphology, but the results depend on the use of adequate sampling and staining procedures of spermatozoa from individual species. In this study, the Sperm Class Analyzer ASMA system was used for the morphometric analysis of goat sperm heads. Semen samples, obtained from four bucks, were used to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris' Haematoxylin) on the accuracy of image processing and sperm morphometry, the effect of the number of cells analysed and the repeatability of the method. These experiments were performed to obtain objective, accurate and reliable sperm morphometric measurements of goat spermatozoa. Diff-Quik and Harris' Haematoxylin were significantly (p<0.05) more accurate than Hemacolor. However, Diff-Quik obtained the highest proportion of correctly analysed sperm heads (86.06%) and the lowest coefficients of variation on the image processing and morphometric measurements. The staining methods affected significantly the sperm dimensions (p<0.001) with increased values from Diff-Quik than Hemacolor and Harris' Haematoxylin, respectively (Diff-Quik>Hemacolor>Harris' Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. The repeatability of results obtained was very high since no differences were found when measuring the same sperm on multiple attempts. In conclusion, to obtain objective, accurate and repeatable sperm morphometric measurements by the Sperm Class Analyzer system in goats, the analysis of 100 spermatozoa from slides which have been previously stained with Diff-Quik is recommended.     

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.     

Effect of Hoechst 33342 on stallion spermatozoa incubated in KMT or Tyrodes modified INRA96

The only known means of effectively separating populations of X and Y bearing sperms is the Beltsville sexing technology. The technology implies that each individual sperm is interrogated for DNA content, measuring the intensity of the fluorescence after staining the spermatozoa with Hoechst 33342. Because there are no data regarding the effect of the staining on stallion sperm, ejaculates were incubated up to 90 min in presence of 0, 4.5, 9, 22.5, 31.5, 45, 54, 67.5, 76.5 and 90 μM of Hoechst 33342, in two media, KMT or INRA-Tyrodes. After 40 and 90 min of incubation, motility (CASA) and membrane integrity (flow cytometry after YoPro-1/Eth staining) were evaluated. In KMT extender sperm motility significantly decreased after 45 min of incubation when sperm were incubated in the presence of concentrations of Hoechst of 45 μM or greater (P<0.05). When incubated in modified INRA96, stallion spermatozoa tolerated greater concentrations of Hoechst, because sperm motility only decreased when incubated in presence of 90 μM (P<0.05) and membrane integrity was not affected. After 90 min of incubation the same effect was observed, but in this case at concentrations over 45 μM the percentage of total motile sperm was also reduced although only in samples incubated in KMT. To produce this effect in samples incubated in Tyrodes modified INRA 96, Hoechst had to be present at concentrations over 67.5 μM. Apparently, the detrimental effect of Hoechst to stallion spermatozoa varies depending on the media, and INRA modified extender may be an alternative to KMT.     

Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation?

Previous studies have shown sperm quality post-cryopreservation differs depending on the fraction of the seminal plasma boar spermatozoa are fortuitously contained in. As such, spermatozoa contained in the first 10 mL of the sperm-rich fraction (portion I) have better sustained handling procedures (extension, handling and freezing/thawing) than those contained in the ulterior part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction, portion II). However, those studies were performed using pooled samples. In the present study, individual ejaculates were used. Split ejaculates (portions I and II) from five boars were frozen and thawed using a conventional freezing protocol, followed by computer-assisted motility and morphology analysis (CASA and ASMA, respectively), as well as an Annexin-V assay for spermatozoa from each boar and ejaculate portion. Significant differences between portions were observed in all ASMA-derived variables, except in one boar. Also significant differences were observed between boars and ejaculate portions in sperm quality post-thaw. We identified, however, boars showing best results of motility and sperm membrane integrity post-thaw in portion I, while in other boar the best results was observed in portion II. It is concluded that the identification of the ejaculate portion more suitable to sustain cryopreservation in each individual boar may be a readily applicable and easy technique to diminish variation in sperm freezability among boars.     

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.     

Automated sperm head morphology analyzer for open-source software

Sperm head morphology has been identified as a characteristic that can be used to predict a male's semen quality. In the present study, we have developed an automated sperm head morphology analysis (ASMA) plug-in for open-source ImageJ software (http://rsbweb.nih.gov/ij/). We describe the plug-in's functionality, and confirm its validity for sperm head morphology analysis using fish sperm. Sperm head morphological measurements (length and width) made with the ASMA plug-in did not differ from manual measurements. Using the plug-in to measure sperm head-shaped objects of known size, the associated plug-in error rate was < 0.5%. Brightness and contrast ratios influenced sperm head measurements, suggesting the need for standardized protocols. This plug-in was effective at measuring elliptical (i.e., Atlantic cod) as well as slightly irregular (i.e., Chinook salmon) shaped sperm heads. In conclusion, our ASMA plug-in represents a versatile alternative to costly sperm morphology software.     

A comparison of human chorionic gonadotropin and luteinizing hormone releasing hormone on the induction of spermiation and amplexus in the American toad (Anaxyrus americanus)

ABSTRACT: BACKGROUND: Captive breeding programs for endangered amphibian species often utilize exogenous hormones for species that are difficult to breed. The purpose of our study was to compare the efficacy of two different hormones at various concentrations on sperm production, quantity and quality over time in order to optimize assisted breeding. METHODS: Male American toads (Anaxyrus americanus) were divided into three separate treatment groups, with animals in each group rotated through different concentrations of luteinizing hormone releasing hormone analog (LHRH; 0.1, 1.0, 4.0 and 32 micrograms/toad), human chorionic gonadotropin (hCG; 50, 100, 200, and 300 IU), or the control over 24 hours. We evaluated the number of males that respond by producing spermic urine, the sperm concentration, percent motility, and quality of forward progression. We also evaluated the effects of hCG and LHRH on reproductive behavior as assessed by amplexus. Data were analyzed using the Generalized Estimating Equations incorporating repeated measures over time and including the main effects of treatment and time, and the treatment by time interaction. RESULTS: The hormone hCG was significantly more effective at stimulating spermiation in male Anaxyrus americanus than LHRH and showed a dose-dependent response in the number of animals producing sperm. At the most effective hCG dose (300 IU), 100 % of the male toads produced sperm, compared to only 35 % for the best LHRH dose tested (4.0 micrograms). In addition to having a greater number of responders (P < 0.05), the 300 IU hCG treatment group had a much higher average sperm concentration (P < 0.05) than the treatment group receiving 4.0 micrograms LHRH. In contrast, these two treatments did not result in significant differences in sperm motility or quality of forward progressive motility. However, more males went into amplexus when treated with LHRH vs. hCG (90 % vs. 75 %) by nine hours post-administration. CONCLUSION: There is a clear dichotomy between the two hormones' physiological responses on gamete production and stimulation of amplexus. Understanding how these two hormones influence physiology and reproductive behaviors in amphibians will have direct bearing on establishing similar breeding protocols for endangered species.     

Automatic evaluation of ram sperm morphometry

This study was designed to develop a new method based on fluorescence microscopy and image analysis for the automatic assessment of sperm morphometry and to study separately the effect of drying and fixation on the parameters of head sperm morphometry in the ram. The study was divided into two experiments. In the first experiment, ejaculates from 25 adult males were collected using an artificial vagina, diluted and divided into four sample aliquots. The first was labeled directly with Hoechst 33342 (FRESH), and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying (DRIED), and the other were fixed either with glutaraldehyde (GLUT), or with methanol (MET), and labeled with Hoechst afterward. Digital images of the fluorescence-labeled sperm were recorded with a digital camera, and sperm heads were automatically captured and analyzed using the ImageJ program. The method used allowed a fast and automatic selection of most sperm heads for a given image with high precision. There was a general trend toward significant decrease in head length, width, area and perimeter of air-dried sperm compared with fresh sperm. On average, this decrease was of 4.1% in length, 4.3% in width, 9.1% in area, and 2.8% in perimeter. Between semen smears, fixation with glutaraldehyde significantly increased head sperm dimensions. The smears fixed with glutaraldehyde method is recommended for a more practical use than with fresh samples, providing better quality images than the other methods, and because the morphometric results obtained were more similar to the FRESH group than those of the DRIED and MET. In the second experiment, ejaculates from adult males were used to compare the sperm head morphometric results obtained with the new method developed (using the GLUT treatment as reference) with a more conventional CASMA method (semen smears stained with Hemacolor and processed with the ISAS commercial software, HEM). The GLUT method allowed the analysis of 100% of sperm, whereas only 93% of sperm could be analyzed using HEM. Spermatozoa displayed a bigger size when processed with HEM than with GLUT method in all primary sperm head morphometric parameters. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters. The new method developed allows automatic determination of sperm head morphometry in a reduced time, which facilitates its use in routine semen analysis. It was concluded that the automation of sperm morphometry is feasible using fluorescence microscopy and image analysis and that the effect of drying and fixation was less important than previously stated.