Maintaining canine sperm function and osmolyte content with multistep freezing protocol and different cryoprotective...

Area of Science:

• Reproductive Biology
• Cryobiology
• Veterinary Science

Background:

• Cryopreservation of canine semen causes osmotic stress, reducing sperm quality and osmolyte content.
• Conventional single-step cryoprotectant protocols negatively impact sperm motility, morphology, and viability.

Purpose of the Study:

• To evaluate a multistep cryoprotectant loading/dilution protocol for canine spermatozoa.
• To compare the efficacy of glycerol versus ethylene glycol in a multistep protocol.
• To identify osmolytes involved in regulating osmotic stress during canine sperm cryopreservation.

Main Methods:

• A multistep protocol involving serial cryoprotectant addition in four steps over 90 seconds was developed.
• The multistep protocol was compared against a conventional single-step protocol for canine semen cryopreservation.
• Sperm progressive motility, membrane integrity, morphology, and osmolyte content (carnitine, glutamate) were assessed post-thaw.

Main Results:

• The multistep protocol significantly improved post-thaw progressive motility (23.3% vs. 12.5%) and membrane integrity (66.5% vs. 49.5%) compared to the single-step method.
• Multistep cryopreservation minimized the loss of crucial osmolytes, carnitine and glutamate, in canine spermatozoa.
• Glycerol, when used in the multistep protocol, demonstrated superior outcomes in maintaining sperm quality over ethylene glycol.

Conclusions:

• A multistep cryopreservation protocol effectively mitigates osmotic stress in canine spermatozoa.
• This improved protocol enhances frozen-thawed canine sperm morphology, motility, and osmolyte content.
• The multistep protocol using glycerol is recommended for superior cryopreservation of canine semen.