Cryopreservation of canine sperm using egg yolk and soy bean based extenders

•Dog sperm cryopreservation in vegetal protein based extender (LP1℗) is evaluated.•Freezing spermatozoa in LP1℗ adversely affected sperm function and acrosome integrity.•TGC addition after semen collection improved acrosome integrity when frozen in LP1.

Animal protein-based extenders are widely used despite being a potential source of bacterial or mycoplasma contamination. Its replacement with vegetal protein-based extenders could represent an interesting alternative for dog sperm cryopreservation. This technique could be further improved by the addition of Tris-Glucose-Citric acid (TGC) that could physically protect the spermatozoa and improve its homeostasis. The aim of this study was to evaluate a cryopreservation protocol for dog spermatozoa using a soybean-based extender (LP1℗) as well as the effects of the addition of (TGC) immediately after the semen collection. Eleven ejaculates from purebred adult dogs were collected, centrifuged in the absence or presence of TGC and processed as fresh or cryopreserved spermatozoa with: egg yolk-based extender (CaniPRO) or LP1℗. Freezing the spermatozoa in LP1℗ reduced the amplitude of the lateral head displacement, the percentage of spermatozoa that showed the intact acrosome and the mitochondrial function (P < 0.05). These samples also showed a trend towards increased percentage of apoptotic spermatozoa (P < 0.05). The addition of TGC before centrifugation did not improve the seminal parameters and adversely affected motility (P < 0.05) in the spermatozoa cryopreserved in CaniPRO. However, TGC did not affect motility and increased (P < 0.05) the percentage of intact acrosomes in the spermatozoa cryopreserved in LP1℗, reaching similar values than those cryopreserved in CaniPRO.In conclusion, LP1® plus TGC provide the same level of protection to dog spermatozoa cryopreservation than the egg yolk based extender CaniPRO when comparing standard post-thaw sperm quality parameters.