Efeito da adição do ácido palmítico e da vitamina E ao diluidor Tris-gema na criopreservação de sêmen canino / Effect of addition of palmitic acid and Vitamin E to the Tris-egg yolk diluter in canine semen cryopreservation

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. 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. 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 100µM 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.(AU)

Suplementação do ácido oleico no dilente seminal sobre o potencial mitocondrial de espermatozoides caprinos criopreservados / Supplementation of oleic acid to the seminal diluter on the mitochondrial potential of cryopreserved caprine sperm

The research aimed to evaluate the antioxidant effect of supplementation of 0.5μM, 5μM and 50μM of oleic acid to the TRIS-yolk extender on the mitochondrial potential (MIT) during the cryopreservation of goat sperm. For that, four Anglo-nubian goats were used, in which five samples / animal were collected, using artificial vagina. After evaluating the swirling and motility of the ejaculates, the pool was made, then diluted in TRIS-Gem and divided according to the treatments. After processing, the samples were packaged in 0.25mL straws and cryopreserved using the TK 3000® machine. After a minimum of 5 days of storage in a cryogenic cylinder, thawing was performed to assess the MIT of goat sperm after cryopreservation, using the lipophilic cationic fluorochrome JC-1. The data were submitted to analysis of variance (ANOVA), using the general linear models procedure (Proc GLM), and the Duncan test was used to compare the averages, with a 5% probability. The analyzes were performed using the Statistical Analysis System program (SAS Institute Inc, 2013). Thus, it was observed that the concentrations of 0.5μM and 5μM of oleic acid maintained the mitochondrial potential similar to the control, differing (p<0.05) only the concentration of 50μM. It can be concluded that 0.5μM and 5μM oleic acid are able to maintain the mitochondrial potential, prolonging the viability of cryopreserved goat sperm.

Influência de um ácido graxo adicionado ao diluente seminal sobre a integridade da membrana plasmática de espermatozoides caprinos criopreservados / Influence of a fatty acid added to the seminal extender on the integrity of the plasmatic membrane of cryopreserved caprine sperm

The objective of this study was to evaluate the antioxidant effects of supplementing different concentrations (0.5μM, 5μM and 50μM) of polyunsaturated fatty acid, arachidonic acid to the TRIS-yolk diluter on the integrity of the plasma membrane during the cryopreservation of goat sperm. For this purpose, four Anglo-nubian goats were used, in which five samples / animal were collected, using artificial vagina. After evaluating the swirling and motility of the ejaculates, the pool was made, then diluted in TRIS-Gem and divided according to the treatments. After processing, the samples were packaged in 0.25mL straws and cryopreserved using the TK3000® machine. Defrosting occurred after at least 5 days of storage in a cryogenic cylinder. Then, the integrity of the plasma membrane of goat sperm post cryopreservation was carried out, using the double staining method, where carboxyfluorescein diacetate (DCF) and propidium iodide (IP) were used. The data were analyzed and the results of the researched variable were subjected to analysis of variance (ANOVA) using the general linear models procedure (Proc GLM) and the Duncan test was used to compare the means, with a 5% probability. The analyzes were performed using the Statistical Analysis System program (SAS Institute Inc, 2013). After analysis, it was observed that the control group had the best percentage, and differed significantly (p<0.05) from the treatment with 50μM of arachidonic acid. It was concluded that the 50μM arachidonic acid concentration is not effective to maintain the integrity of the plasma membrane, and to minimize the oxidative stress of cryopreservation.

Análises de membrana plasmática, acrossoma e mitocôndria de espermatozoides ovino após criopreservação com adição do ácido fólico no meio Tris-gema / Plasma membrane, acrosome and mitochondria analysis of sheep sperm after cryopreservation with addition of folic acid in the tris-yolk medium

Folic acid is closely linked to cobalamin, which is a carrier of hydroxymethyl and ant groups. The objective of this work was to evaluate the effect of adding folic acid to the TRIS-yolk diluter and possible effect on the membrane, mitochondria and acrosome of sheep sperm after thawing the semen. There were six sheep and seven collections of each animal in sessions between 48 and 72 hours. After collection and analysis, the semen samples were mixed and submitted to the formation of a pool. Diluted in TRIS-yolk medium and divided into 3 groups; Group control; Group 2: 10,000µM of folic acid and in Group 3: 5000µM of folic acid. Subsequently, the semen samples were packaged in 0.25mL straws and processed in a semen freezing machine. After a few days, the semen was thawed and evaluated: plasma membrane integrity, acrosome function and integrity. They were analyzed using an analysis of variance (ANOVA) followed by the Newman-Keuls test, using SAS. The additionof a micronutrient with potential antioxidant action in the semen indicates that it can reduce the action of free radicals that alter the plasma membrane and sperm DNA. The evaluation of the plasma membrane integrity, mitochondrial activity and the acrosome integrity of post-thawed sperm were not significantly different between the groups evaluated. Thus, it is concluded that the addition of folic acid in concentrations of 5000µM and 10000µM to the TRIS-yolk seminal diluter does not significantly influence the variables evaluated in this experiment.

Influência de um ácido graxo adicionado ao diluente seminal sobre a integridade da membrana plasmática de espermatozoides caprinos criopreservados / Influence of a fatty acid added to the seminal extender on the integrity of the plasmatic membrane of cryopreserved caprine sperm

The objective of this study was to evaluate the antioxidant effects of supplementing different concentrations (0.5μM, 5μM and 50μM) of polyunsaturated fatty acid, arachidonic acid to the TRIS-yolk diluter on the integrity of the plasma membrane during the cryopreservation of goat sperm. For this purpose, four Anglo-nubian goats were used, in which five samples / animal were collected, using artificial vagina. After evaluating the swirling and motility of the ejaculates, the pool was made, then diluted in TRIS-Gem and divided according to the treatments. After processing, the samples were packaged in 0.25mL straws and cryopreserved using the TK3000® machine. Defrosting occurred after at least 5 days of storage in a cryogenic cylinder. Then, the integrity of the plasma membrane of goat sperm post cryopreservation was carried out, using the double staining method, where carboxyfluorescein diacetate (DCF) and propidium iodide (IP) were used. The data were analyzed and the results of the researched variable were subjected to analysis of variance (ANOVA) using the general linear models procedure (Proc GLM) and the Duncan test was used to compare the means, with a 5% probability. The analyzes were performed using the Statistical Analysis System program (SAS Institute Inc, 2013). After analysis, it was observed that the control group had the best percentage, and differed significantly (p<0.05) from the treatment with 50μM of arachidonic acid. It was concluded that the 50μM arachidonic acid concentration is not effective to maintain the integrity of the plasma membrane, and to minimize the oxidative stress of cryopreservation.(AU)

Suplementação do ácido oleico no dilente seminal sobre o potencial mitocondrial de espermatozoides caprinos criopreservados / Supplementation of oleic acid to the seminal diluter on the mitochondrial potential of cryopreserved caprine sperm

The research aimed to evaluate the antioxidant effect of supplementation of 0.5μM, 5μM and 50μM of oleic acid to the TRIS-yolk extender on the mitochondrial potential (MIT) during the cryopreservation of goat sperm. For that, four Anglo-nubian goats were used, in which five samples / animal were collected, using artificial vagina. After evaluating the swirling and motility of the ejaculates, the pool was made, then diluted in TRIS-Gem and divided according to the treatments. After processing, the samples were packaged in 0.25mL straws and cryopreserved using the TK 3000® machine. After a minimum of 5 days of storage in a cryogenic cylinder, thawing was performed to assess the MIT of goat sperm after cryopreservation, using the lipophilic cationic fluorochrome JC-1. The data were submitted to analysis of variance (ANOVA), using the general linear models procedure (Proc GLM), and the Duncan test was used to compare the averages, with a 5% probability. The analyzes were performed using the Statistical Analysis System program (SAS Institute Inc, 2013). Thus, it was observed that the concentrations of 0.5μM and 5μM of oleic acid maintained the mitochondrial potential similar to the control, differing (p<0.05) only the concentration of 50μM. It can be concluded that 0.5μM and 5μM oleic acid are able to maintain the mitochondrial potential, prolonging the viability of cryopreserved goat sperm.(AU)

Análises de membrana plasmática, acrossoma e mitocôndria de espermatozoides ovino após criopreservação com adição do ácido fólico no meio Tris-gema / Plasma membrane, acrosome and mitochondria analysis of sheep sperm after cryopreservation with addition of folic acid in the tris-yolk medium

Folic acid is closely linked to cobalamin, which is a carrier of hydroxymethyl and ant groups. The objective of this work was to evaluate the effect of adding folic acid to the TRIS-yolk diluter and possible effect on the membrane, mitochondria and acrosome of sheep sperm after thawing the semen. There were six sheep and seven collections of each animal in sessions between 48 and 72 hours. After collection and analysis, the semen samples were mixed and submitted to the formation of a pool. Diluted in TRIS-yolk medium and divided into 3 groups; Group control; Group 2: 10,000µM of folic acid and in Group 3: 5000µM of folic acid. Subsequently, the semen samples were packaged in 0.25mL straws and processed in a semen freezing machine. After a few days, the semen was thawed and evaluated: plasma membrane integrity, acrosome function and integrity. They were analyzed using an analysis of variance (ANOVA) followed by the Newman-Keuls test, using SAS. The additionof a micronutrient with potential antioxidant action in the semen indicates that it can reduce the action of free radicals that alter the plasma membrane and sperm DNA. The evaluation of the plasma membrane integrity, mitochondrial activity and the acrosome integrity of post-thawed sperm were not significantly different between the groups evaluated. Thus, it is concluded that the addition of folic acid in concentrations of 5000µM and 10000µM to the TRIS-yolk seminal diluter does not significantly influence the variables evaluated in this experiment.(AU)

Efeito do Inibidor do ativador do plasminogênio 1 (PAI-1) nos parâmetros cinéticos de espermatozoides criopreservados de touros Curraleiro Pé Duro / Inhibitor effect of plasminogen activator 1 (PAI-1) in the kinetic parameters of sperm cryopreserved of Curraleiro Pé-Duro bulls

This study was conducted to evaluate the effect of different plasminogen activator inhibitor 1concentrations (70 ƞg, 140 and 210 ƞg ƞg) in the kinetic parameters of sperm cryopreserved of Curraleiro FootHard bulls. Sperm kinetics were analyzed by means of a computer aided system (CASA). The variables evaluatedwere: (MT-%) (MOP-um / s), (VCL - um / s), (VSL - um / s), (VAP-um / s), (LIN -%) ( Str-%) (ALH - uM)Wobble (WOB -%) and (BCF-Hz). The cryopreserved sperm in the presence of the inhibitor of plasminogenactivator 1 in a concentration of 210 ƞg decreased velocity parameters in a straight line (VSL - um / s) andlinearity (LIN -%). In conclusion, supplementation of the Inhibitor of plasminogen activator 1 (PAI-1),cryopreservation of bovine semen does not improve the kinetic parameters as compared to the control.(AU)

Efeito do Inibidor do ativador do plasminogênio 1 (PAI-1) nos parâmetros cinéticos de espermatozoides criopreservados de touros Curraleiro Pé Duro / Inhibitor effect of plasminogen activator 1 (PAI-1) in the kinetic parameters of sperm cryopreserved of Curraleiro Pé-Duro bulls

This study was conducted to evaluate the effect of different plasminogen activator inhibitor 1concentrations (70 ƞg, 140 and 210 ƞg ƞg) in the kinetic parameters of sperm cryopreserved of Curraleiro FootHard bulls. Sperm kinetics were analyzed by means of a computer aided system (CASA). The variables evaluatedwere: (MT-%) (MOP-um / s), (VCL - um / s), (VSL - um / s), (VAP-um / s), (LIN -%) ( Str-%) (ALH - uM)Wobble (WOB -%) and (BCF-Hz). The cryopreserved sperm in the presence of the inhibitor of plasminogenactivator 1 in a concentration of 210 ƞg decreased velocity parameters in a straight line (VSL - um / s) andlinearity (LIN -%). In conclusion, supplementation of the Inhibitor of plasminogen activator 1 (PAI-1),cryopreservation of bovine semen does not improve the kinetic parameters as compared to the control.