Automated sperm morphometry and morphology analysis of canine semen by the Hamilton-Thorne analyser

Abstract

Computer-assisted sperm morphometry has the potential to eliminate several drawbacks inherent to the current methods of sperm morphology evaluation, and allows for the identification of subtle sperm characteristics which cannot be detected by visual evaluation. In the present study, the Metrix Oval Head Morphology software implemented in the Hamilton-Thorne CEROS (version 12.1; HTR 12.1 Metrix) computer-aided semen analyser was evaluated for canine sperm morphometry and morphology analysis. Comparison of sperm morphometric measurements of 200 spermatozoa from pooled semen samples ( n=4) at 40× and 60× demonstrated a more accurate identification of the sperm head boundaries at a magnification level 60×. Dilution of pooled semen samples ( n=4) to a sperm concentration of 50×10 6 ml −1 allowed for a correct evaluation of the sperm cell dimensions whereas 100×10 6 and 200×10 6 ml −1 resulted in a higher percentage of rejected spermatozoa due to overlapping. No differences in morphometric dimensions were found when 100 or 200 spermatozoa were evaluated for each of 15 dogs. The mean morphometric parameters of canine spermatozoa, based on the fresh ejaculates of 23 dogs, were: major 6.65±0.20 μm; minor 3.88±0.14 μm; area 20.66±1.04 μm 2; elongation 58.64±2.58%; perimeter 17.57±0.43 μm and tail length 48.93±10.16 μm. Large variations in morphometric dimensions were detected among individual dogs. After cryopreservation, significantly lower morphometric dimensions were obtained for all the evaluated sperm samples ( n=12). Finally, a correlation of 0.82 ( P<0.05) was established for the percentage of normal spermatozoa assessed by subjective evaluation and by the HTR 12.1 Metrix ( n=39 semen samples). In conclusion, dilution of the semen samples to approximately 50×10 6 spermatozoa/ml and an objective lens magnification of 60×, analysing at least 100 spermatozoa, are the technical settings proposed to obtain reliable and objective sperm morphometric measurements by the HTR 12.1 Metrix in canine.