The effects of different cryoprotectants on fertility parameters for stallion epididymal sperm — ASN Events
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  3. The effects of different cryoprotectants on fertility parameters for stallion epididymal sperm

The effects of different cryoprotectants on fertility parameters for stallion epididymal sperm (#408)

Lee HA Morris 1 , Jody A Blomfield 1 , Cleo Hogne-Beattie 1
  1. EquiBreed NZ Ltd, Te Awamutu, NZ, New Zealand

This study investigated the effects of two cryoprotectants, glycerol (GLY) and dimethylformamide (DMF,) and the effects of post-thaw dilution with a protein-free modified Tyrode’s media (SW3, JEL Media, NZ), on  fertility parameters of frozen-thawed stallion epididymal sperm.

Epididymal sperm were recovered from 2yo thoroughbred colts (n=5). Each sample was divided equally into one of 4 different cryoprotectant treatment groups (GLY 2.5%, DMF 2.5%, DMF 5%, or combination GLY2.5%: DMF 2.5%) and frozen in 0.5ml straws in liquid Nitrogen vapour. After thawing, the effects of the different cryoprotectants and addition of SW3 media (1:1, v/v) on motility, morphology, acrosome status, plasma membrane integrity, mitochondrial status, DNA and chromatin integrity and in vivo fertility were evaluated.

The sperm motility was assessed microscopically at 200x magnification and morphology was evaluated at 1000x magnification under oil using phase contrast microscopy after eosin-nigrosin staining. Acrosome status after staining with FITC-PNA was assessed at 1000x magnification using fluorescent microscopy (wavelength 450-490nm). Sperm plasma membrane integrity was assessed using the hypo-osmotic swelling test. The mitochondrial status, DNA and chromatin integrity were evaluated, respectively, using the MTT assay, the alkaline comet assay and the acridine orange chromatin integrity test. Mares were inseminated with 200 x106 epididymal sperm frozen with either GLY2.5% (n = 10) or the combination GLY:DMF (n = 10) at 4 hours prior to ovulation.

Dilution of the frozen-thawed samples with SW3 media significantly improved the motility of GLY2.5% (p=0.02) treated sperm compared to the other treatment groups. No other differences were observed after dilution with SW3 media. Pregnancy rates in the GLY:DMF treatment group (50%) were higher (χ2, p = 0.05) than in the GLY treatment group (10%). There were no significant differences between cryoprotectant treatments on sperm motility, morphology, plasma membrane integrity, acrosome status, mitochondria or DNA.

Funded by New Zealand Equine Research Foundation.