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.