How important is the cooling rate in cryopreservation of (bull) semen, and what is its relation to thawing rate and glycerol concentration

The rate of cooling is known to be an important factor for freezing and thawing of bull semen. In a recent experiment we have studied the effects of the cooling rates in different parts of the cooling and freezing protocol using a central composite design multifactor approach, including also a number of medium parameters (to be reported later). We did not include thawing rate in this design, but ventured to test the effect of thawing rate in a separate multifactorial experiment, including the main factors Cooling rate, Thawing rate, and Glycerol concentration. Bull semen from five bulls was frozen in standard Tris-egg yolk medium containing 0.4, 0.6, or 0.8 M glycerol using cooling rates of 1, 10, or 100 degrees C/min. The thawing rates were 2400, 100, or 10 degrees C/min. Sperm motility was assessed by microscopy and sperm integrity was assessed by flow cytometry after staining with Sybr 14 and propidium iodide. The factor Bull had a significant effect, but in a qualitative sense, the overall response to the factors Cooling rate, Thawing rate and Glycerol concentration was similar for all bulls. There was a significant three factor interaction between Glycerol concentration, Cooling rate, and Thawing rate, but the effect of Glycerol concentration was less important than those of the other factors. A decrease of thawing rate led to decreased sperm survival at all combinations of cooling rates and glycerol concentrations studied. This is in agreement with earlier studies (Rodriguez et al., 1975; Robbins et al, 1976) and indicates that the use of the highest thawing rate as a fixed value in further optimisation studies is justified.