Strong evidences suggest that many different sperm subpopulations co-exist within the mammalian ejaculate. These subpopulations have been identified in a number of species; however, to the best of our knowledge, no data exist regarding the existence of sperm subpopulations within the canine ejaculate. Ejaculates were obtained by masturbation from four mongrels and processed using a standard freezing protocol. Motility data were analysed before and after cryopreservation using a computer assisted sperm analysis (CASA) system ISAS. On raw data, a principal component analysis (PCA) was performed to reduce the number of motility descriptors to a few informative variables, and then a K-means cluster procedure was performed and then a regression analysis to validate the clusters obtained in the second analysis. ANOVAs and chi-squared analyses were used to compare clusters and males. PCA revealed that two principal components represented more that the 88% of the variance with eigenvalues of 3.25 and 3.02, respectively. The clustering and discriminant analysis using curvilinear velocity and linear velocity as variables revealed the existence of 11 sperm subpopulations--four of them characterized by high velocities, two by medium values and five by low velocities. After freezing-thawing, nine subpopulations were found--four of high velocities, two of medium and three of low velocities. It is concluded that freezing-thawing not only impairs sperm motility but also produces changes in the sperm subpopulation structure in the canine ejaculate, that the evaluation of the sperm structure subpopulations is a better indicator of semen quality and freezeability than the use of mean values, and that two sperm motility quality indexes can be used to resume of the variables obtained from the CASA analysis.
It is widely accepted that sperm morphology is a strong indicator of semen quality. As the sperm head mainly comprises the sperm DNA, it is have been proposed that subtle changes in sperm morphology may be related to abnormal DNA content. Semen from four mongrel dogs was used to investigate DNA quality by means of the sperm chromatin structure assay (SCSA), and for computerized sperm morphometry (CASMA). Each sperm head was measured for nine primary parameters [head area (A), head perimeter (P), head length (L), head width (W), 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, 4piA/P(2); FUN3, (L - W)/(L + W); FUN4, piLW/4A]. Significant differences were found in all CASMA-derived parameters among dogs (p < 0.001). Linear regression models including sperm head shape factors 1, 3 and 4 predicted the extent of DNA denaturation (p < 0.001). We conclude that the CASMA analysis can be considered a powerful tool to improve the spermiogram.
Sperm morphometric indexes obtained after principal component analysis were used to investigate its value as diagnostic tests for freezability. Semen from six dogs was frozen-thawed following a standard protocol. Before freezing, computer-assisted analysis of sperm morphometry (CASMA) was performed. The principal component analysis (a statistical technique for simplifying a dataset, by reducing multidimensional datasets to lower dimensions for analysis) performed in the data obtained after the CASMA analysis gave four morphometric indexes. After thawing, ejaculates were evaluated for early changes in sperm membranes using the combination of two fluorescent probes, YO-PRO-1 and ethidium homodimer and flow cytometry. Significant differences in the percentages of intact membranes post-thaw were observed among dogs (p < 0.01). Significant non-parametric correlations were found between index 3 and the percentage of intact membranes after thawing (R = 0.432 p < 0.05). Receiving operating system curves demonstrate a good diagnostic value for indexes 2 and 3 in the prediction of freezability, with areas under the curve of 0.798 and 0.786, respectively.
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