Cryoprotectors (ACPs) are substances added in seminal extenders with function to reduce damage to spermatozoa during freezing. However, this practice is a process that leads to injury and / or cell death that may decrease fertilization. The objective of this study was to evaluate the association of ACPS Glycerol (G) and ethylene glycol (E) with the Tris-egg yolk extender (TG) on the parameters of integrity and functionality of cryopreserved canine sperm cells. Ten canine male ejaculates were diluted in 3 treatments: 5% G (T1), and 5% (T2) and G+ E (final concentration of 2.5% +2.5%) (T3). The ejaculates were evaluated, readily diluted, packaging in 0.25 ml straws and cryopreserved in seminal freezing machine. Post-thawed samples were evaluated for sperm motility (SM), acrosome integrity (AI), membrane integrity (MI), DNA integrity (DI), mitochondrial functionality (MF), rate of penetration with heterologous porcine oocytes (RP) and number of sperm /penetrated oocytes (NS). The SM was evaluated under a light microscope and the analyzes of AI, MI, DI and MF were assessed with a fluorescence microscope (400X) with specific fluorophores dyes for each analysis. Fertilization was performed at 750 μl drop of M2 HEPES containing 0.4% BSA plus 250 μl of sêmen, the oocytes were subsequently recovered and stained with Hoechst 33342 (10μg/ml) and submitted to the evaluation of RP and NS on fluorescence microscope (400X). No statistical difference was observed between treatments for SM, MI, DI and FM. Regarding IA the T2 differ (P <0.05) from the treatments 1 and 3 (24.8 ± 18.3, 50.2 ± 25.4, 38.1 ± 27.5, respectively). The best results for RP were T3 (9.8 ± 9.3) and T2 (8.5 ± 1.51) that were statistically different from T1 (1.3 ± 1.3). In the analysis of NS the T3 showed the best results (0.9 ± 0.8), showing statistical difference of T1 (0.2 ± 0.2) and T2 (0.13 ± 0.13). Therefore the combination of glycerol and ethylene glycol is an effective alternative for cryopreservation of canine semen.
Rizzoto, G., Moreira, F., Varela Jr, A.s., Dode, M., Nobre, M.o., Corcini, C.d. (2014). Effect of the addition of glycerol and ethylene glycol in association upon spermatic viability parameters in the cryopreservation of canine semen. PUBVET, 271, 1-14 [10.22256/pubvet.v8n22.1808].
Effect of the addition of glycerol and ethylene glycol in association upon spermatic viability parameters in the cryopreservation of canine semen
Rizzoto G;
2014
Abstract
Cryoprotectors (ACPs) are substances added in seminal extenders with function to reduce damage to spermatozoa during freezing. However, this practice is a process that leads to injury and / or cell death that may decrease fertilization. The objective of this study was to evaluate the association of ACPS Glycerol (G) and ethylene glycol (E) with the Tris-egg yolk extender (TG) on the parameters of integrity and functionality of cryopreserved canine sperm cells. Ten canine male ejaculates were diluted in 3 treatments: 5% G (T1), and 5% (T2) and G+ E (final concentration of 2.5% +2.5%) (T3). The ejaculates were evaluated, readily diluted, packaging in 0.25 ml straws and cryopreserved in seminal freezing machine. Post-thawed samples were evaluated for sperm motility (SM), acrosome integrity (AI), membrane integrity (MI), DNA integrity (DI), mitochondrial functionality (MF), rate of penetration with heterologous porcine oocytes (RP) and number of sperm /penetrated oocytes (NS). The SM was evaluated under a light microscope and the analyzes of AI, MI, DI and MF were assessed with a fluorescence microscope (400X) with specific fluorophores dyes for each analysis. Fertilization was performed at 750 μl drop of M2 HEPES containing 0.4% BSA plus 250 μl of sêmen, the oocytes were subsequently recovered and stained with Hoechst 33342 (10μg/ml) and submitted to the evaluation of RP and NS on fluorescence microscope (400X). No statistical difference was observed between treatments for SM, MI, DI and FM. Regarding IA the T2 differ (P <0.05) from the treatments 1 and 3 (24.8 ± 18.3, 50.2 ± 25.4, 38.1 ± 27.5, respectively). The best results for RP were T3 (9.8 ± 9.3) and T2 (8.5 ± 1.51) that were statistically different from T1 (1.3 ± 1.3). In the analysis of NS the T3 showed the best results (0.9 ± 0.8), showing statistical difference of T1 (0.2 ± 0.2) and T2 (0.13 ± 0.13). Therefore the combination of glycerol and ethylene glycol is an effective alternative for cryopreservation of canine semen.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
