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Int.J.Curr.Microbiol.App.Sci (2020) 9(3): 2464-2471 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 3 (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.903.282 Assessment of Oxidative Stress by Trace Elements in Pregnant Kankrej Cows Jinnal Patel*, Sandhya S. Chaudhary, Abid Dadawala and Vishal Patel Department of Animal Physiology & Biochemistry, College of Veterinary Science & Animal Husbandry, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar -385 506, Gujarat, India *Corresponding author ABSTRACT Keywords Antioxidant trace elements, Copper, Iron, Oxidative stress, pregnant Kankrej cow, Zinc Article Info Accepted: 20 February 2020 Available Online: 10 March 2020 The present study was carried out with the objectives to study oxidative stress in periparturient cows by estimation of the levels of antioxidant trace elements copper, zinc and iron in plasma at 30, 15 days before parturition, on the day of parturition, 15 and 30 days after parturition. During study significantly higher levels of zinc (P<0.05) and iron (P<0.01) was observed between control and parturited animals. This indicates higher oxidative stress on the day of parturition. Zinc and iron decreased significantly from 30 days before parturition and attained lowest levels on the day of parturition. However, the level increased gradually thereafter up to 30 days after parturition. Significant increase in copper level was observed on the day of parturition, thereafter the levels decreased. Copper is a constituent of SOD enzyme and levels of SOD also increased on the day of parturition. The present study indicates some kind of role of the antioxidant trace elements, zinc and iron in protecting cows from oxidative stress as their levels significantly decreased during pregnancy and parturition. Introduction A living creature has to survive under the environment created as a result of interactions of internal and external factors. There are many physiological processes namely parturition, milk production, preterm labor, lactation which create a stressful condition in the animal body. As it is very well known that during the various biochemical metabolic processes nascent oxygen is released and its level is regulated by various antioxidative agents of the body. Deficiency of any such antioxidative agent will favour the prevailing of the nascent oxygen which will be damaging the cell’s metabolic processes or cell itself and produce 2464 Int.J.Curr.Microbiol.App.Sci (2020) 9(3): 2464-2471 reactive oxygen species (ROS), such as hydroxyl radicals, superoxide anion, and hydrogen peroxides. The balance between the rate at which oxidative damage is induced (input) and the rate at which it is efficiently repaired and removed (output) determines the level of oxidative stress. The rate at which damage is caused is determined by how fast the ROS are generated and then inactivated by endogenous defense agents called antioxidants. In a healthy animal, ROS and antioxidants remain in balance, when the balance is disrupted towards an over abundance of ROS, oxidative stress occurs. Oxidative stress can also lead to the modification of important physiological and metabolic functions. This damage can affect a specific molecule or the entire organism and leads to peroxidative damage of lipids and other macromolecules, with consequent alteration of cell membranes and other cellular components (Toyokuni, 1999). Trace minerals, such as iron (in catalase), copper, zinc, manganese (in superoxide dismutase) and selenium (in glutathione peroxidase), are essential to the structure and function of many of these antioxidant enzymes (Kleczkowski et al., 2003). Oxidative stress plays a role in multiple physiological processes from oocyte maturation to fertilization, embryo development during pregnancy, in normal parturition and in initiation of preterm labor (Agrawal et al., 2005). Pregnancy is a physiological state accompanied by a high energy demand for many bodily functions and an increased oxygen requirement. Because of the increased intake and utilization of oxygen augmented level of oxidative stress would be expected (Gitto et al., 2002). Looking to the immense importance of trace elements in pregnancy we determined the level of endogenous antioxidant minerals viz. zinc, copper and iron in plasma of pre and post parturient cows. Materials and Methods The present study was carried out in Department of Animal Physiology and Biochemistry and in collaboration with Department of Animal Nutrition, College of Veterinary Science and Animal Husbandry, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar. The samples from twelve healthy Kankrej cows were taken from Livestock Research Station, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar. The animals were kept under loose system of housing, in well-ventilated hygienic sheds and fed on standard ration including green fodder. Twelve animals were divided into two groups of six animals each. Group–I (control group) comprised of six healthy non-pregnant Kankrej cows in their second or third lactation. Group-II (experimental or group) comprised of six Kankrej cows. periparturient periparturient Approximately 10 ml of blood was collected aseptically in heparinized vials by puncturing the jugular vein of animals under study. Blood was collected only once at diestrous stage from Group I animals. Blood samples were collected at approximately 30, 15 days before parturition, on the day of parturition, 15 and 30 days after parturition from Group II animals. The plasma samples were separated from whole blood by centrifugation at 3000 rpm for 15 minutes, and stored at -200C for further analysis. Biochemical analyses of plasma samples were carried out for trace elements. 2465 Int.J.Curr.Microbiol.App.Sci (2020) 9(3): 2464-2471 Trace elements 0.2 ml of blood plasma samples were digested with 1.8 ml of triple acid mixture (concentrated sulphuric acid: perchloric acid: nitric acid in the ratio of 1:2:1) till it becomes colorless. After digestion the final volume was made to 10 ml with triple glass distilled water. Copper, zinc and iron concentration from the digested samples were determined by atomic absorption spectrophotometer (Model AAS 4141, Electronic Corporation of India Ltd. make) method as described by (Oser, 1979). Statistical analysis The significance of difference between means for different biochemical parameters were tested by applying paired t-test for dependant samples and unpaired t-test for independent samples assuming equal variances (Snedecor and Cochran, 1994). parturition, 15 and 30 days after parturition (1.06 ± 0.06, 1.05 ± 0.07, 1.19 ± 0.07 and 1.23 ± 0.06 ppm, respectively) of the experiment. A decreasing trend in levels was observed from 30 day before parturition, while reverse trend was observed after the day of parturition. Copper Control group with periparturient group Copper concentration in blood plasma of nonpregnant Kankrej cows was observed to be 1.19± 0.01 ppm, and the same has been presented in Table. Results and Discussion Highly significant differences (p<0.01) were observed for copper level between nonpregnant group with periparturient group at 30, 15 day prepartum and 30 day postpartum. Non-significant differences were observed between non- pregnant group with periparturient group on the day of parturition and 15 day after parturition. Zinc Periparturient group Control group with periparturient group The result indicated that the plasma copper concentration was highly significantly higher on the day of parturition (1.17 ± 0.03 ppm) compared to 30 and 15 days before parturition, 15 and 30 days after parturition (0.62 ± 0.03, 0.71 ± 0.03, 1.13 ± 0.03 and 0.82 ± 0.03 ppm, respectively) of the experiment. The mean concentration of zinc in blood plasma of non-pregnant Kankrej cows was 1.24 ± 0.02 ppm, while that in periparturient cows at 30 and 15 days before parturition, on the day of parturition, 15 and 30 days after parturition were 1.06 ± 0.06, 1.05 ± 0.07, 1.02± 0.07 1.19 ± 0.07 and 1.23 ± 0.06 ppm, respectively and the same has been presented in Table. Iron Control group with periparturient group Periparturient group The results indicate that the plasma zinc concentration was significantly lowest on the day of parturition (1.02 ± 0.07 ppm) compared to 30 and 15 days before The average level of iron in blood plasma of non-pregnant Kankrej cows was 2.00 ± 0.08 ppm. The mean concentration of iron in blood plasma of control and periparturient group is given in Table. 2466 Int.J.Curr.Microbiol.App.Sci (2020) 9(3): 2464-2471 Periparturient group The plasma iron concentration at 30, 15 days before parturition, on the day of parturition, 15 and 30 day after parturition were 1.69 ± 0.05, 1.66 ± 0.05, 1.62 ± 0.05, 1.77 ± 0.11 and 1.99 ± 0.10 ppm, respectively of the experiment. Iron levels were lower in prepartum animals than postpartum. The lowest concentration of iron was observed on the day of parturition. These micronutrients are cellular antioxidants, preventing peroxidative damage, in the cytoplasm (trace elements), and are essential for a well-functioning immune system (Weiss 2002). The variation observed in the blood content of minerals is largely in response to physiological changes that account during the periparturient period in dairy cows. Trace minerals, such as iron (in catalase), copper, zinc, manganese (in superoxide dismutase) and selenium (in glutathione peroxidase), are essential to the structure and function of many of the antioxidant enzymes (Kleczkowski et al., 2003) and reduces oxidative stress. Zinc Control group with periparturient group Significantly higher level of zinc was found between non-pregnant cows and 15 day prepartum cows, between non-pregnant cows and recently parturited cows. On the day of parturition zinc levels were lowest. However non-significant differences were observed between non- pregnant group with 30 day prepartum, 15 and 30 day postpartum. Zinc has a critical role in repair and maintenance of uterine lining following parturition and early return to normal reproductive function and oestrus. Zinc has also been shown to increase plasma betacarotene, which has been directly correlated to improved conception rate and early embryonic development. Our findings are in agreement with Pavlata et al., (2005), Akhtar et al., (2009), Cahit et al., (1999). However Dhami et al., (2003) found significantly lower zinc level in lactating animals than in cyclic and pregnant heifers. However, nonsignificant difference was observed for zinc level by Asif et al., (1996) and Gosai, N.M., (1998). Periparturient group Plasma zinc concentration was highly significantly lower on the day of parturition as compared different days of intervals during pre and post parturient period. There were highly significant differences (p<0.01) among all the five zinc levels measured on different days before and after parturition except significant differences were observed between 30 day before parturition with 15 day after parturition, 30 day before parturition with 30 day after parturition. Similar results were observed by Dufty et al., (1977), Goff and Stable (1990), Meglia et al., (2001, 2004), Mehere et al., (2002), Jacob et al., (2003), Pankajkumar and Sharma (2005) and Ram Pravesh (2006). Non-significant difference was observed for zinc level by Asif et al., (1996). Cahit et al., (1999) found significantly higher concentration of zinc in early pregnant cows than in non-pregnant cows and concluded that zinc has an important role in early pregnancy in cows from the observed higher levels in early period of pregnant cows than non- pregnant cows. Rapid need for zinc in synthesis of colostrum may explain why Zn concentration is 22 % lower in blood of cows on day of calving (Kincaid, 2008). Zinc, is responsible for normal functions of certain antioxidant enzymes. The blood concentration of zinc decreased dramatically at calving mainly in 2467 Int.J.Curr.Microbiol.App.Sci (2020) 9(3): 2464-2471 response to colostrum formation (Goff and Stabel, 1990), and to redistribution to other tissues such as, liver. The peripartum in dairy cows is considered a stressful period, and stress can induce synthesis of metallothionein, a protein associated with zinc metabolism, making zinc less available (Spears et al., 1991; Xin et al., 1993). Copper Control group with periparturient group Highly significant differences (p<0.01) were observed for copper level between nonpregnant group with periparturient group at 30, 15 day prepartum and 30 day postpartum. However, non-significant differences were observed between non- pregnant group with periparturient group on the day of parturition and 15 day after parturition. Increased copper concentration during parturition may be required to trigger the endocrine glands related to the physiology of initiation of labor pain and process of parturition. Copper is an important cofactor of superoxide dismutase, an enzyme, which protects cells from the pro oxidative influence of free radicals (Kleczkowski, 2003). Our findings are an agreement with Pavlata et al., (2005), Akhtar et al., (2009). However non-significant difference was reported by Asif et al., (1996), Gosai et al., (1999) and Cahit et al., (1999) for copper concentration. Dhami et al., (2003) found low copper in parturient and lactating cows compared to young calves, pregnant heifers and repeat breeding cows. Periparturient group Significantly higher level of plasma copper was found on the day of parturition as compared to 30, 15 days before parturition, 15 and 30 days after parturition. Copper is constituent of SOD enzyme and level of SOD also increased on the day of parturition. Pregnancy is a copper dependant process and the body demands additional copper from conception onwards. Reduction in zinc and copper availability in the early postpartum period (Muehlenbein et al., 2001) of dairy cows might explain the reduction of SOD activity (Michiels et al., 1994). The level of copper during parturition was higher than pre parturition or early lactation. This might be due to conservation of copper by increasing absorption and preventing losses via excretory pathways in order to accommodate high demands of fetus. At parturition, increased circulatory levels of estrogen stimulate ceruloplasmin synthesis by the liver and thereby resulting in increased concentration of copper. Our finding were similar to those observed by Meglia et al., (2001), Mehere et al., (2002), Jacob et al., (2003). Pankajkumar and Sharma (2005), Asif et al., (1996), Cahit et al., (1999) and Ram Pravesh (2006) found non-significant difference for Cu level, contradictory to our findings. Devraj (1982) reported copper level was low up to 3 day postpartum which rose in subsequent stages. Iron Control group with periparturient group The average level of iron in blood plasma of non-pregnant Kankrej cows was significantly higher than 30, 15 day pre-partum. Highly significant difference were observed between control animals and on the day of parturition, while non-significant differences were observed between non- pregnant group with 15 and 30 day post-partum group. Iron being an integral part of cytochrome oxidase/peroxidase and catalase enzymes, is responsible for biological oxidation. Our findings are an agreement with Akhtar et al., 2468 Int.J.Curr.Microbiol.App.Sci (2020) 9(3): 2464-2471 (2009), Setia et al., (1994). The nonsignificant difference was found by Asif et al., (1996), for iron concentration. The opposite result was observed by Dhami et al., (2003). Table.1 Mean and standard error of zinc, copper and iron in blood plasma of Kankrej cows Group Group-I Group-II 1. 2. 3. 4. 5. Animal Zinc (ppm) Copper (ppm) Iron (ppm) Non-pregnant 1.24 ±0.021 Kankrej cows (Control) Peri parturient cows 1.19 ±0.011 2.00 ±0.081 30 day before 1.06 ±0.061,a parturition 15 day before 1.05 ±0.071*,a**,b parturition On the day of 1.02 ±0.071*,a**,b**,c parturition 15 day after 1.19 ±0.071,a*,b**,c**,d parturition 30 day after 1.23±0.061,a*,b**,c**,d** parturition 0.62 ±0.031**,a 1.69 ±0.051*,a 0.71±0.031**,a**,b 1.66 ±0.051*,a**,b 1.17 ±0.031,a**,b**,c 1.62 ±0.051**,a**,b**,c 1.13±0.031,a**,b**,c**,d 1.77 ±0.111,a,b,c,d 0.82±0.031**,a**,b**,c**,d** 1.99±0.101,a**,b**,c**,d** Means bearing same numerical superscript within a column did not differ significantly (P≤0.05). Means having same alphabetical superscript within a column did not differ significantly (P≤0.05) from each other for Group-II. *=significant (P<0.05) and ** =significant (P<0.01) Periparturient group Present study revealed that the plasma iron was significantly lower on the day of parturition as compared to different days of intervals during pre and post parturient period. Moreover, highly significant differences (p<0.01) for iron concentration were observed between all periparturient groups except between 30 day prepartum with 15 days postpartum , 15 day prepartum with 15 days postpartum and on the day of parturition with 15 days postpartum differences were non-significant. Similar results were observed by Jacob et al., (2003) and Ram Pravesh (2006). Pankajkumar and Sharma (2005) found adequate level of iron in pregnant cattle of specific area. Asif et al., (1996) found non- significant difference for Fe level. 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