Effects of egg yolk and soybean lecithin on sperm quality determined by computer-assisted sperm analysis and confocal laser scanning microscope in chilled canine sperm

Abstract

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.

Keywords: canine; egg yolk; extender; soybean; sperm.

Figure 1

Canine spermatozoa stained with the fluorescent combination of H324, PI, FITC‐PSA and JC‐1 under a confocal laser scanning microscope (600 ×  magnification). (a) Intact plasma and acrosome membrane, and high mitochondrial membrane potential. (b) Intact plasma membrane, damaged acrosome membrane and high mitochondrial membrane potential. (c) Damaged plasma membrane, intact acrosome membrane and high mitochondrial membrane potential. (d) Intact plasma membrane, damaged acrosome membrane and low mitochondrial membrane potential. (e) Damaged plasma membrane, intact acrosome membrane and low mitochondrial membrane potential. (f) Damaged plasma and acrosome membrane, and low mitochondrial membrane potential.