Comparison of different statistical approaches to evaluate morphometric sperm subpopulations in men.

This study was designed to characterize morphometric sperm subpopulations in normozoospermic men by using different statistical methods and examining their suitability to classify correctly different sperm nuclear morphologies present in human ejaculates. Ejaculates from 21 normozoospermic men were collected for the study. After semen collection and analysis, samples were prepared for morphometric determination. At least 200 spermatozoa per sample were assessed for sperm morphometry by computer-assisted sperm morphometry analysis (CASA-Morph) using fluorescence. Clustering and discriminant procedures were performed to identify sperm subpopulations from the morphometric data obtained. Clustering procedures resulted in the classification of spermatozoa into three morphometric subpopulations (large-round 30.4%, small-round 46.6%, and large-elongated 22.9%). In the second analysis, using discriminant methods, the classification was made independently of size and shape. Three morphological categories according to nuclear size (small <10.90 µm2, intermediate 10.91-13.07 µm2, and large >13.07 µm2) and four categories were defined on 400 canonical cells (100 × 4) from 10 men according to sperm nuclear shape (oval, pyriform, round, and elongated). Thereafter, the resulting classification functions were used to categorize 4200 spermatozoa from 21 men. Differences in the class distribution were observed among men from both clustering and discriminant procedures. It was concluded that the combination of CASA-Morph fluorescence-based technology with multivariate cluster or discriminant analyses provides new information on the description of different morphometric sperm subpopulations in normal individuals, and that important variations in the distribution of morphometric sperm subpopulations may exist between men, with possible functional implications.

Computer-assisted sperm morphometry fluorescence-based analysis has potential to determine progeny sex.

This study was designed to determine the ability of computer-assisted sperm morphometry analysis (CASA-Morph) with fluorescence to discriminate between spermatozoa carrying different sex chromosomes from the nuclear morphometrics generated and different statistical procedures in the bovine species. The study was divided into two experiments. The first was to study the morphometric differences between X- and Y-chromosome-bearing spermatozoa (SX and SY, respectively). Spermatozoa from eight bulls were processed to assess simultaneously the sex chromosome by FISH and sperm morphometry by fluorescence-based CASA-Morph. SX cells were larger than SY cells on average (P < 0.001) although with important differences between bulls. A simultaneous evaluation of all the measured features by discriminant analysis revealed that nuclear area and average fluorescence intensity were the variables selected by stepwise discriminant function analysis as the best discriminators between SX and SY. In the second experiment, the sperm nuclear morphometric results from CASA-Morph in nonsexed (mixed SX and SY) and sexed (SX) semen samples from four bulls were compared. FISH allowed a successful classification of spermatozoa according to their sex chromosome content. X-sexed spermatozoa displayed a larger size and fluorescence intensity than nonsexed spermatozoa (P < 0.05). We conclude that the CASA-Morph fluorescence-based method has the potential to find differences between X- and Y-chromosome-bearing spermatozoa in bovine species although more studies are needed to increase the precision of sex determination by this technique.

A comparative study of the morphometry of sperm head components in cattle, sheep, and pigs with a computer-assisted fluorescence method.

The aim of this study was to compare the sperm nuclear and acrosomal morphometry of three species of domestic artiodactyls; cattle (Bos taurus), sheep (Ovis aries), and pigs (Sus scrofa). Semen smears of twenty ejaculates from each species 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. The use of the PI/PSA combination and CASA-Morph fluorescence-based method allowed the capture, morphometric analysis, and differentiation of most sperm nuclei, acrosomes and whole heads, and the assessment of acrosomal integrity with a high precision in the three species studied. For the size of the head and nuclear area, the relationship between the three species may be summarized as bull > ram > boar. However, for the other morphometric parameters (length, width, and perimeter), there were differences in the relationships between species for sperm nuclei and whole sperm heads. Bull sperm acrosomes were clearly smaller than those in the other species studied and covered a smaller proportion of the sperm head. The acrosomal morphology, small in the bull, large and broad in the sheep, and large, long, and with a pronounced equatorial segment curve in the boar, was species-characteristic. It was concluded that there are clear variations in the size and shape of the sperm head components between the three species studied, the acrosome being the structure showing the most variability, allowing a clear distinction of the spermatozoa of each species.