To Study the Effect of Meteorological Variables on Oxidative Stress Parameters in Balck Bengal Goat

Harsh environments create stressful conditions to animals raised in extreme climatic areas, and as a result, animals develop various adaptive mechanisms that enable them to survive under conditions of extreme heat or extreme cold. The present study entitled, “The effect of Meteorological Variables on Oxidative Stress in Black Bengal Goat” was conducted on twelve adult apparently healthy Black Bengal Goats. The does were investigated for their antioxidant’s profiles during autumn (Oct to Nov) and winter (Dec to Jan) seasons. Meteorological parameters like dry bulb temperature and relative humidity were recorded daily for two times at 6 am and 3 pm for estimation of THI throughout the study period. In the present study, the mean THI found during autumn and winter season was 70.45±0.60 and 57.58±0.91 respectively while the mean THI for last one years was 72.09±0.38 during autumn and 62.14±0.28 during winter season. All the antioxidants that studied (SOD, LPO and GPx) showed significant (p<0.05) variations during autumn and winter season. The SOD (IU/ml), LPO (nmol of MDA/ml of packed cells) level was observed to be higher during winter season than the autumn season whereas GPx (IU/ml) level was found lower during winter. Thus, the SOD and LPO was inversely proportional to THI and GPx was directly proportional to the THI. The study revealed that some of the parameters studied were visibly drifted from normal values as a result of climate stress during winter season. The results also highlighted drastic variations in the values of some of the antioxidants in animals. The information obtained is useful in understanding the adaptive physiology of the Black Bengal goats during environmental stress. The data generated may also help to distinguish and discriminate healthy animals from stressed ones under special physiological status of goats.


INTRODUCTION
The term "Climate change" can be described as long-term imbalance of customary weather conditions such as temperature, radiation, wind and rainfall characteristics of a particular region. The changes definitely have an enormous impact on health and biology of both human and animal kingdom. Out of various breeds of goat in India Black Bengal goat is observed to be highly tolerant to changing weather condition. The breed is found to be confined in north eastern India mostly in west bengal, jharkhand, bihar and orissa regions. The importance of goat in the rural economy is evidenced by its unparalleled economic traits, ability to get acclimatized under diversified agro-climatic condition, unfastidious type in choosing the available forage, high fertility and short generation interval. The breed occupies a unique position since they are used as a multipurpose animal for production of milk, meat and fiber besides hides and skin. The Black Bengal Goats adapt poorly to cold climates but relatively more adapted to areas of high temperatures (Shelton & spiller, 1977). Goats appear to have superior adaptation to arid tropics because of its ability to conserve water, travel well, graze selectively and take willingly a wide Variety of vegetation (Meferlane & Howard, 1972;Maloity & Taylor 1971& Merrill et al., 1965. Adaptation of animals for ecological stress makes them better survivors for which the metabolic strength of blood is highly important. The seasonal rythms reflect the endogenous adaptive mechanism to react in advance to the regular environmental changes associated with the seasons (Piccione et al., 2009). Goats adapted to a harsh environment, perform better than other domesticated ruminants (Shkolnik & Silanikove, 1981;King, 1983;& Devendra, 1990). The range of thermo neutrality for goats is an environment having an air temperature of 13-27ºC, relative humidity of 60-70 per cent (Mishra & Puneet, 2009). The temperature below and above this range makes animal stressful and reduces productivity. The breed has developed various adaptive mechanisms that allow their survival at very high as well as very low temperatures (Al-Tamimi, 2007). In the case of short-day breeders, the reports are inadequate to depict the role of free radicals in the regulation of immunity. Various kinds of stress viz., heat and cold stress leads to the production of reactive oxygen species (ROS) such as superoxide, peroxide, hydroxyl radical and singlet oxygen species. Reactive oxygen species damage, biomolecules such as DNA/RNA, proteins and lipid peroxidation of membranes and disruption of normal cell metabolism (Rehman, 1984). Superoxide dismutase (SOD), Catalase (CAT), Lipid peroxidase (LPO) and glutathione peroxidase (GPX) are important antioxidant enzymes. Antioxidants are the substances that significantly delay or inhibit oxidation of a substrate. The production of free radicals and their neutralization by antioxidants is a normal body process. Ambient stress can reduce antioxidant activity of blood (Harmon et al., 1997) resulting in oxidative stress. Antioxidants enzymes protect the cell against cellular oxidants and prevent their accumulation. Excessive production of reactive oxygen species depletes such antioxidants and induces oxidative stress which suppresses the immune system and reduces productivity of animal (Kumar et al., 2011a).

Composition of the ration and management:
The does were provided green fodder, routine grazing (daily for four to six hours) with balanced ration. Animals were maintained on a standard ration at the rate of 250 gm/animal/day with green fodder and the water was provided ad-libitum. The animals were dewormed with anthelmintic fembendazole @ 10mg/kg of body weight 15 days prior to the start of experiment.

Experimental Design:
The selected twelve animals were maintained at normal animal husbandry practices. The animals were kept in Instructional Farm of Small Ruminants; RVC. The experiment was conducted during two seasons i.e., autumn (Oct-Nov) and winter (Dec-Jan).

Blood Sampling:
The blood samples with anticoagulant (EDTA) in vials were collected from jugular vein of each animal on 0 th day of experiment and thereafter on every 15 th day up to 120 days for haematological examination. The collected blood samples were taken immediately to the laboratory in ice box.  Fig 1). The mean ambient temperature was found to be significantly lower (p<0.01) on day 60th during autumn. Similarly, during winter season, the mean ambient temperature recorded on 15 th day was found to be significantly (p<0.01) higher as compared to that was on day 30 th and 45 th . The mean ambient temperature was found to be 6.91±0.62, 3.55±0.60, 3.89±0.40 and 5.16±0.88 on 15 th day, 30th, 45th and 60 th respectively. Significantly lower level of ambient temperature (p<0.01) was observed on day 60 as compared to day 15, 30 and 45 during autumn. The difference in ambient temperature between autumn and winter was found to be highly significant on each fortnight, it was higher during autumn whereas it was lower than the thermal comfort zone (12-24) during winter, and it was ≤ 5 which creates cold stress in animals.

Relative humidity (%)
Table-2 suggests that the difference between Relative humidity was non-significant between Autumn and Winter at all the fortnights except for that at day 15 th the t-value (p critical) in that case was 0.04 (p<0.05) and thus the relative humidity was significantly higher in Autumn (69±0.33) than in winter (66.  Statistically, non-significant differences were observed in average relative humidity (%) on different fortnights during autumn as well as during winter. There was a gradual fall in relative humidity during winter whereas a rise was observed on day 30 during autumn followed by a gradual fall on day 45 and 60. In our finding the average relative humidity during winter was higher than the findings of Rathwa et al. (2017), because of heavy rainfall in this region (tropical humid).  Fig 3) THI value of autumn was higher than winter at all the four fortnightly intervals.  In the present study, significant differences were observed within season as well as between seasons. Significantly lower level of THI (p<0.01) was observed on day 60 during autumn whereas significantly lower level of THI (p<0.01) was observed on day 30 and 45 as compared to day 60 and 45. The THI was significantly higher during autumn as compared to winter is in consistent with the findings of Rathwa et al. (2017) who also reported a higher THI during summer.

Oxidative Stress Parameter: 2.7.1 Lipid peroxidation (LPO)
Lipid peroxidation in tissue homogenate was estimated in terms of malondialdehyde (MDA) production by the modified method of Stock and Dormandy (1971) as described by Jain (1988). 0.5 ml of tissue homogenate was suspended in 0.5 ml of PBS. To this 0.5 ml of 30 % TCA was added. Tubes were vortexes and allowed to stand on ice for at least 2 h. Tubes were centrifuged at 2000 rpm for 15 minutes. 1 ml of the supernatant was transferred into another tube and to this 0.075 ml of 0.1 M EDTA and 0.25 ml of 0.67 % thiobarbituric acid in 0.05 M NaOH was added. It was mixed and the tubes were kept in a boiling water bath for 15 min and then allowed to cool at room temperature. Absorbance was read at 532 nm. The amount of lipid peroxidation is expressed in nanomoles of MDA formed per mg of protein. After allowing it to stand for 5 min to avoid crust formation on top of the supernatant, it was centrifuged for 20 min at 2000 rpm at room temperature. Two ml of supernatant was mixed with 2.5 ml of Tris buffer (1M; pH 8.0 and 0.2 ml of 5, 5dithiobis-2-nitrobenzoic acid reagent. The absorbance was read immediately at 412 nm in spectrophotometer. The GSH content has been expressed as mM/ml blood or mM/g tissue by using a molar extinction coefficient of 13.6 103 M -1 cm -1 .

RESULT AND DISCUSSION
Climatic changes have range of impact on physical, mental & community health of animals. The present experiment was conducted to study the effect of meteorological variables on oxidative stress in Black Bengal Goat. Harsh environments constitute stressful conditions to animals raised in extreme climatic areas, and as a result, animals develop various adaptive mechanisms that enable them to survive under conditions of extreme heat or extreme cold.

Lipid Peroxidation (mol of MDA produced /ml)
The process of lipid peroxidation is one of oxidative conversion of polyunsaturated fatty acids known as melanodialdehyde (MDA), which is the most commonly studied, biologically relevant free radical reaction (Esterbauer et al., 1991& Celi, 2011 and considered to be the best indicators of oxidative stress (Nielsen et al., 1997& Georgieva, 2005. Lipid peroxidation level (mol of MDA) in Black Bengal goats was compared during autumn and winter. Using independent t-test, the periodical variation in the lipid peroxidation level at fortnightly interval was analyzed with one way ANOVA. The effect of 15-day interval was highly significant during both autumn as well as during winter (P<0.01). In the present study, the mean value of lipid peroxidation level (nmol of MDA/ml of packed cells) of Black Bengal Goat was 0.51±0.01, 0.54±0.008, 0.55±0.006, 0.56±0.005 in Autumn and 0.59±0.009, 0.62±0.012, 0.65±0.012, 0.67±0.011 in Winter (Table 4, Fig 4) on day 15 th , 30 th , 45 th & 60 th . The independent t test (t value P critical) shows that the effect of season on the abovementioned parameter was highly significant (P<0.01) at all the periodical intervals. While the mean value of lipid peroxidation (mol of MDA) in winter was greater than autumn at all the periodical interval mentioned.

Fig. 4: Bar diagram depicting Lipid peroxidation (nmol of MDA) of Black Bengal Goats in autumn and winter season at different periods (Mean±SE)
Increasing trend was observed in the level of LPO during winter. Significantly higher levels of LPO were observed on each fortnight during winter as compared to autumn. Which indicates stress level was increased during this period. However, the level was lower in comparision to the findings of Rathwa et al. (2017).

Superoxide dismutase (IU/ml)
Super dismutase (SOD) is the important antioxidant enzyme present in the body and provides defensive mechanism against free radical mediated oxidative damage. Antioxidant activity of SOD mediated by dismutation reaction, where SOD scavenges highly reactive superoxide radical and converts it to oxygen molecule and less reactive H 2 O 2 molecule (Fridovich, 1972). Superoxide dismutase (U) of Black Bengal Goats in Autumn and Winter season was found out at different periods (Mean±S.E) namely 15 th , 30 th , 45 th and 60 th day i.e at an interval of 15 days between the two periods. The effect was found to be highly significant between autumn and winter season (P<0.01) at all the periodical intervals on the basis of tvalues. The mean values were greater during autumn than winter in all the cases. The oneway ANOVA predicted non-significant effect during autumn and highly significant effect during winter for the variation due to 15-day interval.
The  Fig 5) on day 15 th , 30 th , 45 th and 60 th . Decreasing trend was observed in the level of SOD, which was significantly higher on day 15 and 30 as compared to day 60 during autumn. Our findings were in agreement with the findings of Rathwa et al. (2017) in sheep. The higher SOD activity in black Bengal goats during winter season was probably a response to higher superoxide generated in these animals. Superoxide dismutase catalyzes the dismutation of superoxide to oxygen and hydrogen peroxide. Thus, a higher SOD activity leads to increase in hydrogen peroxide level in the cells which is metabolized by intra cellular components by GPx which protecting the organism against deleterious effects of free radicals.  Fig 6) respectively during autumn and winter on day 15 th , 30 th , 45 th & 60 th . The level of GPx gradually decreased till day 60 th during autumn as well as during winter. Significantly lower level of GPx was observed on each fortnight during winter than autumn season. In present study decreased level of GPx was observed during winter that may be due to glutathioneassociated metabolism which is a major mechanism for cellular protection against agents which generate oxidative stress. In the present finding, the level was higher than the findings reported by Rathwa et al. (2017) in indigenous sheep.