The reduction of spermatozoa survival time is a major problem of canine chilled sperm for artificial insemination. The aim of the study was to improve the quality of canine chilled sperm during storage time. We therefore, evaluated the effects of eight treatments with different levels of soybean lecithin concentration (1, 3 and 5%) and egg yolk (20%) in Tris‐citric‐fructose or Tris‐citric‐fructose‐mineral salts extender on chilled canine sperm quality during 10 days of storage. The sperm motility was analysed by computer‐assisted sperm analysis (CASA), whereas plasma membrane integrity, acrosome membrane integrity and mitochondrial membrane potential parameters were determined using a fluorescent staining combination of propidium iodide (PI), Hoechst 33342 (H342), fluorescein isothiocyanate‐conjugated Pisum sativum agglutinin (FITC‐PSA) and 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolyl‐carbocyanine iodide (JC‐1) by confocal laser scanning microscope. The results showed that egg yolk was found to be better than soybean lecithin in Tris‐citric‐fructose or Tris‐citric‐fructose‐mineral salts extender for maintaining the quality of chilled canine sperm within 10 days of storage (P < 0.05). Although egg yolk in Tris‐citric‐fructose extender could maintain the motility better than other extenders, egg yolk in Tris‐citric‐fructose‐mineral salts extender was the highest in intact plasma membrane, intact acrosome membrane and high mitochondrial membrane potential (P < 0.05). In contrast, the sperm quality of soybean lecithin in Tris‐citric‐fructose‐mineral salts extender was lower than that of soybean lecithin in Tris‐citric‐fructose extender, and soybean lecithin 1% was greater than soybean lecithin 3% and 5% in plasma membrane integrity, acrosome membrane integrity and mitochondrial membrane potential (P < 0.05). In conclusion, soybean lecithin cannot replace egg yolk in Tris‐citric‐fructose or Tris‐citric‐fructose‐mineral salts extenders, and egg yolk in Tris‐citric‐fructose‐mineral salts extender is superior to other extenders in chilling canine sperm.

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Background:Oxidative stress during chilled storage is a major problem withcanine sperm. To improve the quality of chilled canine sperm during storage, many synthetic antioxidants have been examined, but different outcomes were investigated depending on antioxidant properties. The bioactive compounds of essential oils fromOcimum gratissimumleaves are known as a natural antioxidant source. This study aimed to evaluate the antioxidant effects of essential oils from Ocimum gratissimumleavesas a supplement in extender on chilled canine sperm during 12 days of storage. Results:The results showed thatlow concentrations of Ocimum gratissimum essential oils (25, 50, and 100µg/mL) have beneficial effectson sperm quality, whereasOcimum gratissimumessential oils athigh levels (above 200µg/mL) have harmful effects. Specifically, the addition of 100µg/mL ofOcimum gratissimum essential oilsto the extender had the greatestbeneficial effect in improving the quality of chilled canine sperm, and had a significant difference in all sperm quality parameters except motility when compared to the control group (p<0.05). Conclusions:Ocimum gratissimum essential oilshave an impact on chilled canine sperm quality in a dose-dependent manner, and the best results areachieved with a maximum dose ofOcimum gratissimum essential oils of 100µg/mL.

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