Several extenders to preserve fertilizing capacity of preserved canine semen have been successfully tested, but further studies are requisite to improve its quality. Effect of supplementation of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione (GSH) to Tris-citric acid-fructose-egg yolk extender on Labrador dog sperm survival during storage at 4° was evaluated. Different concentrations per antioxidant i.e. SOD (50-300 IU/ml), GPX (1.5-2.5 IU/ml), CAT (100400 µg/ml) and GSH (2.5-10 µM/ml) were evaluated to look for an optimum dose. Semen was analyzed for motility, viability and plasma membrane integrity (PMI) after every 24 hrs till 72 hrs of preservation. Semen was also analyzed for acrosome integrity (AI) at 0, 72 hrs and lipid peroxidation at 72 hrs of storage. Values for motility, viability, PMI, AI were significantly (p<0.05) higher in the presence of 200IU/ ml SOD, 2 IU/ml GPX, 200 µg/ml CAT and 7.5 µM/ml GSH compared to control and other tested doses. MDA concentration was non-significantly (p>0.05) less in the presence of all doses of SOD and 2.0 IU GPX compared to control at 72 hrs of preservation. MDA concentration in the presence of 200 µg/ml catalase and 7.5 µM/ml GSH was non-significantly (p>0.05) higher than control. It was concluded that SOD, catalase, GPX and GSH at a concentration of 200 IU, 2.0 IU, 200µg and 7.5 µM per ml were optimum concentrations to be supplemented to the extender for positive effect. Supplementation of antioxidants could improve sperm attributes by maintaining proportionate level of oxidative stress during preservation of Labrador dog semen at 4° for 72 hrs.
Effect of supplementation of different doses of antioxidants was observed on semen during preservation at 4°C.
SOD, catalase, GPX and GSH at a concentration of 200 IU, 2.0 IU, 200µg and 7.5 µM per ml were optimum concentrations to be supplemented to the extender for positive effect
Artificial insemination (AI) with fresh semen in canines presents pregnancy rates almost similar to natural mating. The use of chilled or cryopreserved semen leads to the reduction in fertility rate (
ROS concentration is controlled by antioxidants of the seminal fluid and inside the sperm cell, thereby avoiding ROS overload and preserving semen quality (Michael
Antioxidants were purchased from Sigma-Aldrich Company. Study was conducted in two phases. Effect of supplementation of SOD, GPx, CAT and GSH to TEY buffer was observed on Labrador dog semen stored at 4ºC from 0-72 hrs. Different concentrations per antioxidant i.e. SOD (50-300 IU/ml), GPx (1.5-2.5 IU/ml), CAT (100-400 µg/ml) and GSH (2.5-10 µM/ml) were evaluated to look for an optimum dose.
All the procedures were approved by the CPCSEA, New Delhi vide F. No 25-19-2018-CPCSEA, dated 22/11/2018. Six Labrador dogs were maintained in individual pens. They were daily fed 500 g cooked feed twice daily and water provided ad libitum. Dogs were given regular exercise in the form of walking / running for one hour daily in the morning and evening. Dogs were dewormed and vaccinated for rabies, CDV, CAV2, CPV, CPI and CAV1. Semen was collected by message method twice a week.
Tris - citric acid-fructose buffer (Tris, 3.08 g; citric acid, 1.78 g; fructose, 1.25 g /100 ml, pH 7.2, gentamycin, 5 mg) was prepared and autoclaved at 15 lb for 15 min. Buffer was filtered through 0.2-micron membrane filter. Egg yolk and gentamycin at a concentration of 15 % and 50µg/ml were added to the buffer on the day of experiment, again filtered through 0.2-micron membrane filter and kept at 37°C (TEY extender).
Semen of dogs exhibiting >70 % motility was pooled for each trial and divided into required aliquots. To each aliquot SOD (50,100,200 and 300 IU/ml), GPX (1.5, 2.0, 2.5 µM/ml), CAT (100, 200, 300, 400 IU/ml) and GSH (2.5, 5.0, 7.5 and 10 µM/ml) were added and incubated at 37°C for 10 min. To each aliquot, extender was added and semen-extender mixture was centrifuged at 960 g for 3 min. Loose pellet was re-suspended in extender containing respective concentration of the antioxidant to get a final sperm concentration between 150-200 spermatozoa / ml. Sperm suspension was again equilibrated at 37oC for 10 min and evaluated for motility, viability, membrane and acrosome integrity. Samples in tubes were shifted to a container containing warm water (37oC) and placed in a cooling cabinet at 4ºC. Semen was analyzed for motility, viability and membrane integrity after every 24 hrs till 72 hrs of storage. Semen was also analyzed for acrosome integrity at 0, 72 hrs and lipid peroxidation at 72 hrs of storage.
Motility was evaluated by wet mount and track method. Viability was assessed by staining the spermatozoa with syber green-propidium iodide stain kit (Sigma). Hypoosmotic swelling test (HOST) was performed to analyze the integrity of sperm membrane (
Malondialdehyde concentration (end product of LPO) was estimated by the method of
Significant differences (5% level) among the extenders were tested by one-way Anova using SPSS 16 program (Student version for windows, SPSS Inc. 233 South Wacker Drive, 11th floor Chicago, IL 60606-6412). Normality of the data was assessed using the ShapiroWilk test and homogeneity of variances was evaluated using the Levene test.
Treatment of fresh semen with different concentrations of SOD and GPX at 37° for 10 min did not show significant difference in sperm attributes of freshly extended semen. However, treatment with 200 µg/ml catalase and 7.5µM/ ml GSH exhibited significantly higher values for sperm attributes in freshly extended semen compared to control and other tested doses (
Preservation of semen in TEY extender supplemented with 100 and 200 IU SOD revealed significantly higher values of individual motility, viability and PMI at 24 hrs, 48 hrs and 72 hrs compared to control and 300 IU (
Effect of supplementation of superoxide dismutase (100-300 IU/ml) to TEY extender on sperm attributes during storage of Labrador dog semen at 4°C for 72 hrs
Effect of supplementation of glutathione peroxidase (1.5-2.5 IU/ml) to TEY extender on sperm attributes during storage of Labrador dog semen at 4°C for 72 hrs
The values for motility, viability and plasma membrane integrity were significantly (p<0.05) high in TEY extender supplemented with 1.5 and 2.0 IU GPX / ml compared to control and other doses at 24 hrs of preservation (
Supplementation of TEY extender with 200 µg/ml catalase resulted in significantly (p<0.05) higher motility, viability and plasma membrane integrity compared to control and other doses at 24, 48 and 72hrs of preservation (
Values for motility, viability and plasma membrane integrity were significantly (p<0.05) high in extender supplemented with 7.5 µM/ml GSH compared to control and other doses from 24 – 72 hrs of preservation (
A gradual decline in sperm attributes was noticed with increase in time of preservation irrespective of the antioxidant treatment. This observation supported previous studies on preservation of chilled dog spermatozoa in extenders (
It may be inferred that SOD, GPX, catalase and GSH acted in a dose dependent manner and 200 IU SOD, 2.0 IU GPX, 200 µg catalase and 7.5 µM GSH per ml semen were optimum doses for preservation of Labrador dog semen at 4oC. Although motility, viability, plasma membrane integrity and acrosome integrity declined gradually in the presence of optimum dose of antioxidants, but still remained > 60% at 72 hrs of preservation. Contrary to our observations, extender containing 100, 400 and 1,600 U/ ml of CAT or SOD or the combination did not enhance sperm motility, viability and acrosomal integrity during storage at 5°C (
Effect of supplementation of Catalase (µg/ml) to TEY extender on sperm attributes during storage of Labrador dog semen at 4°C for 72 hrs.
Effect of supplementation of Glutathione (mM/ml) to TEY extender on sperm attributes during storage of Labrador dog semen at 4°C for 72 hrs
Addition of GSH at a concentration of 7.5 µM/ml was an optimum dose in improving sperm attributes compared to control during storage of Labrador dog semen at 4°C. Contrary to this observation, supplementation of the extender with 5 mM GSH improved the chilled and frozen-thaw semen quality (
Among the three concentrations of GPX tested, only 2 IU/ml supplementation to the extender could improve the sperm attributes in Labrador dogs. But,
It may be inferred from these studies that the different species respond variably to the antioxidants. Moreover, influence of environment including temperature, extenders and endogenous antioxidants may also be possible reasons behind variation. Different concentration of antioxidants should be evaluated for a species under investigation as per the conditions of particular lab, as optimum dose for one species may not be effective or may be toxic for the other species due to the change in physiological condition of semen extender. We also observed higher doses of antioxidants as toxic to dog spermatozoa. Addition of 400 mM and 10 mM GSH caused negative effects on ram (
There was no significant (p<0.05) difference in MDA concentration in control and antioxidant treated samples except GPX. Rather MDA concentration was nonsignificantly high in the presence of optimum catalase and GSH concentration. The addition of catalase in semen extenders resulted in significantly (p<0.0005) lower total ROS values compared with the controls in canine (
In the dog, seminal fluid has been found to be deleterious for the
It was concluded that 200 IU SOD, 2.0 IU catalase, 200µg GPX and 7.5 µM GSH per ml were optimum concentrations to be supplemented to the extender for positive effect. Supplementation of enzymatic and nonenzymatic antioxidants could improve sperm attributes by maintaining proportionate level of oxidative stress during preservation of Labrador dog semen at 4°C for 72 hrs. Artificial insemination using liquid semen supplemented with antioxidants and exhibiting enhanced sperm attributes will definitely results in achieving higher fertility rate.
This work was funded by a Research Grant from Department of Biotechnology, Ministry of Science and Technology, New Delhi (SAN No.102/IFD/SAN/5331/2017-2018).