Morphometry and subpopulation structure of Holstein bull spermatozoa: variations in ejaculates and cryopreservation straws

Abstract

Sperm quality is evaluated for the calculation of sperm dosage in artificial reproductive programs. The most common parameter used is motility, but morphology has a higher potential as a predictor of genetic quality. Morphometry calculations from CASA-Morph technology improve morphological evaluation and allow mathematical approaches to the problem. Semen from 28 Holstein bulls was collected by artificial vagina, and several ejaculates were studied. After general evaluation, samples were diluted, packaged in 0.25 ml straws, and stored in liquid nitrogen. Two straws per sample were thawed, and slides were processed and stained with Diff-Quik. Samples were analyzed by a CASA-Morph system for eight morphometric parameters. In addition to the "classical" statistical approach, based on variance analysis (revealing differences between animals, ejaculates, and straws), principal component (PC) analysis showed that the variables were grouped into PC1, related to size, and PC2 to shape. Subpopulation structure analysis showed four groups, namely, big, small, short, and narrow from their dominant characteristics, representing 31.0%, 27.3%, 24.1%, and 17.7% of the total population, respectively. The distributions varied between animals and ejaculates, but between straws, there were no differences in only four animals. This modern approach of considering an ejaculate sperm population as divided into subpopulations reflecting quantifiable parameters generated by CASA-Morph systems technology opens a new view on sperm function. This is the first study applying this approach to evaluate different ejaculates and straws from the same individual. More work must be done to improve seminal dose calculations in assisted reproductive programs.

Figure 1

Distribution of subpopulations according to their PC values.

Figure 2

Distribution of subpopulations (SP) per ejaculate in some representative animals. SP1 (black), SP2 (dark gray), SP3 (light gray), and SP4 (white) bars. Animal 1 presents differences for subpopulations SP1, 3 and 4; Animal 3 for SP1 and 3, but the first three ejaculates present equivalent number of cells for each SP; Animal 5 for SP1 and 3, but the distribution of SP in each ejaculate was different; Animal 9 for SP1, 2 and 3; Animal 16 for SP3 and 4; and Animal 25 showed no differences among ejaculates.