Use of Relief Contrast(®) objective to improve sperm morphometric analysis by Isas(®) casa system in the ram

The aim of this study was to develop a new method for morphometric assessment of the sperm head and acrosome in the ram. Ejaculates from 10 adult males were collected using an artificial vagina. For each ejaculate, 10 semen smears were prepared, air-dried and divided (in pairs) into the following five treatment groups: (i) washed in distilled water and allowed to dry without further processing (DRY); (ii) fixed in 50% methanol (MET); (iii) fixed in 2% glutaraldehyde (GLUT); (iv) fixed and stained with Hemacolor(®) (HEM) and (v) fixed and stained with SpermBlue(®) (SB). The prepared slides were examined with a 40 × Relief Contrast(®) objective (RC) and processed with ISAS(®) commercial software. The use of RC optics increased the contrast between acrosome and sperm head, allowing capture and morphometric analysis by ISAS of sperm heads and the acrosome, even in non-stained samples. MET and GLUT groups resulted in a lower number of acceptable, that is, correctly delineated, sperm heads than those in the SB, and SB and HEM groups, respectively (p < 0.05). The higher proportion of sperm discarded in MET and GLUT groups may be explained by a higher presence of artefacts. For the majority of the primary morphometric parameters of the sperm head and the acrosomal area, the relationship between treatments was the following: GLUT> HEM≥ MET≥ SB> DRY. When studying the proportion of the sperm head covered by the acrosome, the relation between treatments was: MET> DRY = GLUT = SB> HEM. It was concluded that the new method for sperm morphometric assessment allows the simultaneous assessment of sperm head and acrosome in the ram by the first time, even in unprocessed semen smears.