In ejaculates, viable spermatozoa of normal morphology are sometimes a very low percentage ofthe total cell population. This Application Sheet presents a detailed protocol for the recovery of ahighly viable fraction of bovine spermatozoa for use in fertilization. 

The recommended strategyinvolves, in the first instance, adjustment of the density of a semen sample to approx. 1.17 g/ml.Ideally, two lower density solutions are then layered on top, so that the viable semen of normalmorphology band at the interface between theses two layers. 

These cells are thus completely separatedfrom both the non-viable cells (and any soluble material released from partially broken cells), which allremain in the load zone, and any morphologically abnormal cells that band at the top of the least denselayer. 

This result is depicted in Figure 1.Iodixanol is the gradient solute of choice: firstly, all of the solutions are easily prepared by dilutionof OptiPrep™ with any buffered saline or a special diluent formulated for the maintenance of spermviability, while Nycodenz® solutions must be prepared from Nycodenz® powder.

Secondly, to raisethe density of the ejaculate to approx 1.170 g/ml it is necessary to mix it with a high-density medium(usually >1.26 g/ml). Nycodenz® solutions are hyperosmotic above ρ = 1.16 g/ml, thus the seminalfluid would also become hyperosmotic. Consequently with Nycodenz® the semen has to be loaded at alower density in the middle, or top of the gradient. 

This is not the case with iodixanol; OptiPrep™ or adense solution prepared from OptiPrep™ and the chosen diluent, can be added to a raw ejaculatewithout increasing its osmolality (see Notes 1 and 2)