Effect of Dilution Media and Incubation Time on Motility, Kinetic and Morphometric Parameters of Canine Spermatozoa Using CASA System

The present study aimed to evaluate the effect of two dilution media and five incubation times at 37°C on the kinetic and morphometric parameters of canine spermatozoa (spz) using computer-assisted sperm analysis (CASA). To do so, 14 ejaculates were collected from six dogs, and initially assessed (microscopy for motility and photometer for concentration). Each ejaculate was divided into two aliquots and diluted in two different solutions (PBS 'phosphate-buffered saline' and a commercial buffer solution 'EBB: Easy Buffer B') at a concentration of 25 million spz/mL then incubated at 37°C for five different times (T0, 10, 20, 30 and 40 min after collection) before being analysed by Hamilton-Thorne IVOS II CASA system. The analyser automatically generated two motility percentages, eight kinetic parameters and six morphometric parameters. The results showed that the sperm analysis by HT-IVOS II immediately after collection without prior incubation (T0), presented a decrease in the percentages of motility and kinetic parameters. On the other hand, incubation at 37°C for 20 min did not show any significant difference compared to T0 + 10 min. The spz retained their motility, kinetic and morphometric parameters in a similar way to the reference time (10 min). On the contrary, the incubation times T0 + 30 min and T0 + 40 min showed a highly significant difference compared to T0 and T0 + 10 min. The percentages of total and progressive motility and the kinetic parameters dropped considerably, so these times (30 and 40 min) are unsuitable for the incubation of canine spz before analysis by the HT-IVOS II system. To conclude, incubation at T0 + 20 min seems to be a good alternative to T0 + 10 min and PBS medium tends to be an acceptable substitute of EBB medium; this trend requires further exploration to be confirmed.