Effect of Addition Palmitic Acid and Vitamin E to the Tris-Egg Yolk Diluter in Canine Semen Cryopreservation

Abstract

Background: Canine sperm is a very delicate cell that is quite susceptible to oxidative stress since the cytoplasm is restricted and features little antioxidant reserves. Furthermore, the sperm membrane has some polyunsaturated fatty acids sensitive to lipid peroxidation, which makes it important to addition antioxidant substances to the diluter aiming at decreasing such stress to the sperm cell, particularly during seminal cryopreservation. Several antioxidants have been used in this process in some domestic animal’s species, however, the use of palmitic acid has been little reported in works on cryopreservation of semen of the canine species. Hence, this study aimed to assess the effect of addition antioxidants palmitic acid and vitamin E to the Tris-egg yolk diluter on the semen quality of dogs after thawing.Materials, Methods & Results: Samples were collected from the ejaculates of 4 adult dogs, apparently healthy, of the American Pit Bull Terrier breed of kennels in the city of Teresina, PI, places where the pre-freezing procedures of the dog’s semen were performed. The samples were diluted in Tris citric acid fructose (3.28 g Tris-hydroxymethyl-aminomethane, 1.78 g citric acid monohydrate and 1.25 g D-fructose), dissolved in 100 mL distilled water, and added 20% egg yolk and 6% glycerol, at the concentration of 100x106 sptz/mL. The semen samples were divided into 3 mL aliquots to form 3 experimental groups: G1 - Only Tris-egg yolk (Control group); G2 - Tris-egg yolk + 100 µM palmitic acid; and G3 - Tris-egg yolk + 116 µM vitamin E. Semen was collected weekly over a period of little over 2 months. After thawing, thermorresistance test (TTR) was carried out at 0, 30, 60, and 90 min to assess spermatics motility and vigor, in addition to analysis of integrity of plasma membrane, acrosomal membrane and mitochondrial activity of the sperm, using fluorescent probes. These assessments were performed out at the Animal Reproduction Biotechnology Laboratory (LBRA/UFPI). In the TTR, G2 and G3 didn´t exhibit significant results for spermatics motility or vigor when compared with the control group. The palmitic acid and vitamin E also had no significant effects on the parameters of acrosomal membrane integrity or mitochondrial activity. However, sperm cryopreserved with the addition of palmitic acid exhibited significant differences for plasma membrane integrity, providing greater protection to the sperm cells in G2.Discussion: The palmitic acid is one of the most saturated fatty acids in human semen, with reports of great proportions also in the seminal plasma of dogs. Its main role is to protect the plasma membrane from external damage, improving viability and fertility of the sperm after cryopreservation. Data is scarce in the literature on the composition of fatty acids in canine semen and regarding the use of palmitic acid as a seminal antioxidant in that species, which grants further studies aiming to investigate such valuable information for canine reproduction. It is concluded that addition palmitic acid at 100 µM concentration to the Tris-egg yolk diluter was able to preserve the integrity of the plasma membrane during the process of cryopreservation of canine semen.Keywords: dog, semen, antioxidants, cryopreservation.Descritores: cão, sêmen, antioxidantes, criopreservação.