How to process semen from difficult stallions

Selection of stallions is performed by phenotypic assessments, such as the conformation of the animal and their athletic performance,⁹ unlike bovines where reproductive parameters are assessed in bulls before becoming a commercial sire. Another factor that affects sperm quality is age, and often owners decide to freeze semen from stallions that have advanced age and may be subfertile. Also is knowed that some breeds as Spanish and Mangalarga stallions are less resistant to semen preservation.

Consequently, several techniques have been developed to increase the sperm quality of stallions. Stallions whose semen does not resist the centrifugation process, the use of such techniques as SpermFilter, and cushioned centrifugation may be beneficial. For stallions whose semen has a low resistance to cryopreservation, the use of extenders with specific cryoprotectants, such as amines, in general improve sperm cryosurvival and fertility.

When the initial quality of semen is poor, sperm selection using commercially available density gradients, such as EquiPure (Nidacon) or Androcoll-E (Minitube) can be performed before cryopreservation.These gradients select the sperm that exhibit better progressive motility, cell integrity, and no morphologic defects . Another alternative is to select sperm after thawing. For this procedure, the contents of four straws are gently layered on top of 2 mL of Equipure, centrifuged at 300 to 400 G for 20 minutes, and the pellet resuspended with the freezing extender. Based in our experience this protocol improves the motility and the fertility of frozen semen of some stallions. It is postulated that the removal of bad quality or dead sperm that can generate oxidative reagents can improve the fertility.

Cushion centrifugation (CC) is an alternative technique for removing SP from semen. This method aims to maximize the sperm recovery using a high G force (1000 G/ 30 minuts). Another possibility for removal of seminal plasma is to use a filtering method composed of a synthetic hydrophilic membrane (Spermfilter , Botupharma Brasil) ) that retains the sperm cells and allows for the passage of SP , concentrating the sperm cells with less damage and also less cell compactation. Recently publications have been shown an increase on fertility of cooled semen that was reprocessed by Spermfilter after 24 hours of refrigeration.

Methylformamide has been used as the preferential cryoprotector for Stallion semen it causes less osmotic damage to sperm than glycerol because of the lower molecular weight and viscosity.⁷ For stallions with semen that has satisfactory freezability (“good freezers”), the use of extenders containing dimethylformamide or methylformamide may not result in a significant increase in post thaw sperm motility; however, it does increase the fertility of the frozen semen. In stallions whose semen has low resistance to cryopreservation (“bad freezers”), the use of extenders containing dimethylformamide or methylformamide provides a significant improvement in sperm motility and fertility compared with extenders containing only glycerol. The use of a combination of cryoprotectants affords better protection to sperm compared with the use of single agents. Several equine reproduction centers in Europe, the United States, and Brazil have preferentially used a commercial extender (BotuCrio), which includes a combination of methylformamide and glycerol.

For semen cooling and transportation, the use of the new formulation of extenders containing casein and cholesterol have been show to increase the cooling ability and also membrane integrity. Casein is a milk protein that avoid the binding of seminal plasma proteins that usually destabilize the sperm membrane inducing damage and precocious capacitation. Cholesterol increases membrane fluidity improving the ability of the sperm cell to resist to osmotic stress.

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