Evaluation of plasma minerals visa-vis physiological status and seasonal variation in goats of Shuhama Alusteng area of Kashmir valley

Mineral disorders can have a great impact on animal performance and to draw optimum production the deficiency gap needs to be abridged. Regular assessment of livestock vis-a-vis physiological demands and/or geo-climatic variations is an important management strategy to overcome severe economic losses. A study was undertaken to evaluate the plasma mineral profile of goats in Shuhama Alusteng area of Kashmir valley, aimed to set a platform for formulation of areaand species-specific mineral supplement(s). A total of 114 blood samples from goats belonging to different physiological states were collected in four different seasons of the year. The plasma macro-minerals like Ca, P and Mg were measured using standard kits, while micro-minerals like Cu, Zn and Fe were measured using atomic absorption spectrophotometer (AAS). Young stock revealed significantly higher Ca, but lower Mg status. Also, Ca level was lower throughout the year except autumn, whereas Mg was lower in winter and spring seasons. Cu and Zn concentrations, though adequate in all the categories, were near the critical values in spring and summer. Fe was adequate but could be provided during spring. Fe concentration observed was adequatewell above the critical concentration irrespective of the physiological status or season of the year. The study also revealed a good percentage of samples deficient in one or the other mineral throughout the year, suggesting Ca, Mg, Cu and Zn supplementation during specific periods. Further, formulating a mineral supplement(s) demands larger sampling size for confirmation of these findings besides the impact study of agro-geo-climatic conditions of the area on animal mineral status.

(Ben Salem and Smith, 2008).This livestock-based economy contributes about 4.36% to the total Gross domestic product (GDP) of India and 24.72% to GDP-Agriculture, despite being seriously affected by various diseases.In J&K-India, 75% population is rural with agriculture as the main stay and livestock-rearing as the subsidiary one.As per 18 th Livestock Census of 2007, the goat population in J&K is about 2.07 million out of the total 10.99 million livestock strength.The agriculture and allied sectors contribute about 38% to the state GDP of which 11% is contributed by livestock sector (DES, 2007).Goats supply precious animal proteins of high biological value in the form of meat, milk, plus essential minerals and fat-borne vitamins.Their increased contribution to animal production is justified by the presence of 94% of the total 674 million world population being found in the developing countries characterized by inadequate food supply and the need for increased food security for the poor (Devendra, 1999).The largest meat consumption in India is chevon/mutton with a share of 46.3% in rural areas, 49.2% in urban areas, and 47.4% nationally.The per capita consumption of meat (chicken, mutton and chevon) in India's Kashmir valley is among the highest in the country (Gandhi and Zhou, 2010).India is currently the largest producer of milk in the world, goats contributing about 3.58%.
Mineral disorders range from acute deficiency or toxicity, characterized by well marked clinical signs and pathological changes, to mild and transient conditions too difficult to diagnose and expressed as a vague unthriftiness or unsatisfactory growth and production (Vargas and McDowell, 1997).Macro-minerals Ca, P, Mg etc play a key role in skeletal development, muscle functions, and transmission of nerve impulse.Inadequacy in diet mostly causes muscular weakness, while mild to moderate deficiency causes sub-minimal productivity and increased susceptibility to other metabolic as well as infectious diseases (NRC, 2001).Trace elements (Cu, Zn, Fe etc) are required for synthesis of many proteins and activation of vast array of enzyme systems (Ceylan et al., 2008).Their concentration must usually be maintained within quite narrow limits, if the functional and structural integrity of the tissues is to be safeguarded and the growth, health and productivity of animals to be maintained at optimum levels.Their supplementation above predicted requirements during stressful conditions mitigates cellular oxidative damage and increases disease resistance (Gressley, 2009).Levels of requirements as well as thresholds of deficiency and toxicity vary with age, sex, production level, activity level, season, and pasture availability, presence of antagonists e.g.Cu and Mo etc, species and genetic strain of the animal (Guyot et al., 2009;Tashi et al., 2005).Significant species differences have been reported for Cu, I, Mo and As (Haenlein, 1991).
For maintenance of normal health and sustained efficient production of livestock, it is necessary to ensure adequate dietary intake of essential nutrients (Hosnedlava et al., 2007).Large number of livestock around the world thrives on mineral deficient diets (McDowell et al., 1993).Continued ingestion of such diets culminates in mineral imbalances in the body leading to physiological and pathological consequences (Prasad and Gowda, 2005).Mostly pastures are considered adequate in nutritive value (energy and protein) to sustain mature animals, the quality of grasses is often inadequate for growing animals whose physiological demands are higher (Pastrana et al., 1991).Mineral deficiencies that affect livestock at pasture include those of macro and micro minerals (Khan et al., 2005).Signs of mineral disorders are often non-specific and in cases of marginal deficiencies may go unnoticed.Further, the interpretation of such signs is even difficult in presence of multiple mineral deficiencies or if complicated by gastrointestinal parasitic burden (Suttle and Jones, 1989).Mineral supplementation is a least cost input for livestock improvement.Nevertheless, supplements should be used only when requirements cannot be met within the available feed, and only when local conditions dictate.
In Kashmir, goats are mainly maintained on grazing with little or no mineral supplementation.Hence, the study was undertaken to assess mineral status of goats in Shuhama Alusteng area of Kashmir valley so as to initiate the work for formulation of area specific mineral supplements and to devise the supplementation strategy for ensuring optimum production performance and prevention of health disorders.

MATERIALS AND METHODS
The study area lies between 34.23°N 74.78°E and is characterized by sub-humid temperate climate with mean annual rainfall of 744 mm and mean annual temperature of 13.4°C.In the study area, the livestock rearing is a subsidiary occupation with almost no inputs and use of technology.The animals, selected randomly, had no history of deworming/vaccination.Jugular venous blood collected in heparinized vials using 18 gauge needles from goats belonging to different physiological states viz.kids/weaners (aged 1 to 6 months), adults and in different seasons of the year as shown in Table 1, was centrifuged at 2000 rpm for 15 min to harvest plasma, stored at -20°C, and subsequently processed and analyzed for minerals.

Calcium
Plasma calcium was estimated by O-Cresolphthalein Complexone (OCPC) endpoint assay (Ca Test Kit supplied by Span Diagnostics Ltd.India).20 µl of plasma samples were taken in labeled test tubes followed by 1000 µl of working Ca reagent.Standard was prepared in triplicate with 20 µl of Ca standard in test tubes mixed with 1000 µl of working Ca reagent.Test tube containing 1000 µl working Ca reagent was used as reagent blank.After mixing the reagents, test tubes were incubated at 37°C or room temperature (15 to 30°C) for 5 min.Analyzer programmed (578 nm) as per assay conditions and absorbance of standards followed by plasma samples was taken against blank.

Phosphorus
Plasma inorganic phosphorus (P i ) was estimated by UV Molybdate, endpoint assay (P Test Kit supplied by Span Diagnostics Ltd.India).10 µl of plasma samples were taken in labeled test tubes to which 1000 µl of Reagent 1 was added and mixed well.Test tubes containing 10 µl of P i standard and 1000 µl of Reagent 1 were taken in triplicate as standard.Reagent blank was prepared by taking 1000 µl of Reagent 1 in the test tube.All test tubes containing test samples, standards and blank were mixed properly by shaking and incubated at 37°C for 5 min.Analyzer was programmed as per assay parameters (340 nm) and blanked with reagent blank.The absorbance of the standard and plasma samples was taken against blank.

Magnesium
Plasma Mg was estimated by Calmagite method (Mg Kit supplied by Crest Biosystems, India).To labeled test tubes, 0.01 ml of plasma samples were added followed by 0.5 ml of buffer reagent (L 1 ), and 0.5 ml of color reagent (L 2 ).Standard was prepared in triplicate which contained 0.5 ml of L 1 reagent, 0.5 ml of L 2 reagent and 0.01 ml of Mg standard.Test tube containing L 1 and L 2 reagents (0.5 ml each) plus 0.01 ml distilled water was used as reagent blank.The contents in test tubes were mixed well by shaking and incubated at 25°C for 5 min.Absorbance of standard and the samples was recorded against blank at 510 nm.

Estimation of micro-minerals
For estimation of trace elements the plasma samples were digested and subsequently analyzed as per the standard procedures (Kolmer et al., 1951;Sharma et al., 2003).To 3 ml of sample in digestion tubes an equal volume of concentrated HNO 3 was added and mixed well.The tubes were kept overnight at room temperature followed by low heat (70 to 80°C) digestion until the volume of the samples reduced to 1 ml.To this, 3 ml of double acid mixture (HNO 3 and HClO 4 in 3:1 ratio) was added and low heat digestion continued until the digested samples became watery clear and emitted white fumes.Heating was continued until the volume of the samples got reduced to ~0.5 ml.Final volume of the filtrate was made 10 ml with triple distilled de-ionized water after warming the solution.During digestion of plasma samples simultaneous digestion of reagent blank was also undertaken and final volume of 10 ml stored to have the blank.Atomic absorption spectrophotometer (AAS-Model No ECIL 4141) was used for estimation of trace minerals.At least 3 standards of known concentration were used for calibration and then the unknown test samples were analyzed.Sample analysis was done by attached computer and concentration of mineral samples was expressed in parts per million (ppm).

Statistical analysis
Collected data were analyzed for mean, standard error and analysis of variance (ANOVA) by using SPSS software (version 16).

RESULTS
The overall plasma mineral concentration in goats belonging to different physiological states and in different seasons, plus the percentage of analyzed samples deficient in minerals is presented in Tables 2 and 3.The overall Ca concentration observed was below the critical value and significantly lower (P < 0.05) in pregnant and lactating does, bucks and dry goats as compared to kids.The Ca concentration in spring, summer and winter was significantly lower (P < 0.05) than the autumn season.The percent samples observed deficient in Ca were in the order of pregnant > bucks > lactating > dry > kids/weaners, and summer > spring > winter > autumn.The P i concentration noticed was adequate, but significantly lower (P < 0.05) in lactating does and dry goats as compared to bucks.In autumn and spring, the P i concentration was significantly lower (P < 0.05) than winter and summer seasons.The percent samples observed P i deficient were in the order of lactating > dry > pregnant > kids/weaners > bucks, and autumn > spring > summer > winter.The Mg concentration recorded was significantly lower (P < 0.05) in kids as compared to dry goats, and significantly lower (P < 0.05) in winter compared to the autumn season.The percent samples observed Mg deficient were in the order of lactating > kids/weaners > bucks > pregnant > dry, and spring > winter > autumn > summer.
The plasma Cu and Zn concentrations found were adequate in all the goats throughout the year except spring and summer seasons in which it was near to critical concentration.The Cu concentration observed was significantly lower (P < 0.05) in kids and bucks as compared to dry goats.Also, the Cu concentration recorded was significantly lower (P < 0.05) in spring but higher in winter season.The percent samples observed Cu deficient were in the order of bucks > kids/weaners >  pregnant > lactating > dry, and summer > spring > winter/autumn.The Zn concentration noticed was higher, but not significantly, in dry goats and pregnant does as compared to kids.Moreover, the Zn concentration recorded was significantly lower (P < 0.05) in spring but higher in winter season.The percent samples observed Zn deficient were in the order of kids/weaners > lactating > pregnant > dry > bucks, and spring > summer > autumn > winter.The plasma Fe concentration recorded was adequate in all goats and all throughout the year, but significantly higher (P < 0.05) in pregnant does as compared to kids and dry goats.Also, it was significantly lower (P < 0.05) in spring and summer as compared to autumn and winter seasons.The percent samples observed Fe deficient were in the order of lactating > dry > bucks > kids/weaners > pregnant, and spring > autumn > summer > winter.

DISCUSSION
The lower plasma Ca (below the critical concentration) in pregnant and lactating does could be attributed to negative Ca balance as a result of dietary imbalances of Ca and P, higher requirements due to pregnancy, and dietary interaction with other minerals (Maynard et al., 1979) plus excessive Ca secretion through milk (Asif et al., 1996).The results obtained are in agreement with the findings of Remberg et al. (1970) who reported an outflow of Ca into milk at the onset of lactation accompanied by a reduction in the plasma Ca pool.The adequate Ca level observed in kids might be due to more efficient Ca absorption in young than older animals (Ricks, 1996).Also, in growing animals net Ca retention occurs in body, while in adults the amount ingested equals that lost if metabolic requirement is met (Church and Pond, 1988).Moreover, absorption efficiency is well known to fall with age which partly relates to decline in vitamin D stores (Robert, 1989).The optimum Ca level observed in autumn season might be due to higher dietary availability of Ca during dry season than wet season plus the higher absorption efficiency in the drier months (Khan, 2003).The study revealed adequate plasma P i concentration irrespective of the physiological status of the animals and/or the season of the year.However, the lower P i concentration in lactating does could be due to increased P i excretion into milk and is in agreement with the findings of Braithwaite (1983) who noticed a marked increase in P i secretion in milk during lactation in sheep.The plasma Mg concentration figured around the critical concentration in kids/weaners compared to rest of the adult stock, and might be assigned to more rapid uptake of Mg by young than adult animals (Ahmed et al., 2000).Furthermore, exchange of radio Mg in bone was 5 to 10 times greater in young than old animals (Breibart et al., 1960).Since young animals have more water content than old animals, more water ions are adsorbed on the surface of bone crystal resulting in low Mg ions in the blood (Fontenot et al., 1989).The maximum Mg excretion through feces in winter/spring than during summer/autumn, and thus less absorption through the gastrointestinal tract could be the reason for lower plasma Mg concentration in colder months (Khan, 2003).The plasma Cu and Zn concentrations found were adequate in all the animal categories throughout the year except spring and summer seasons in which it was near to critical concentration.However, significantly higher Cu concentration in dry, lactating and pregnant goats compared to kids and bucks might be attributed to increased requirements for growth in yearling dry does, for sustained lactation in milking does, and higher progesterone level or to the increased fetal demands and utilization of maternal Cu for the development of fetal nervous system in pregnant does (Elnageeb and Adelatif, 2010).Moreover, increase in plasma Cu levels in the form of ceruloplasmin owing to increase in estrogen levels has been noticed in late pregnancy (Howell et al., 1968).The significantly lower Cu level in spring and summer compared to winter season is in agreement with the findings of Pastrana et al. (1991).The higher Zn concentration in dry goats and pregnant does might be due to increased requirements for growth in yearling dry does, increased rate of Zn accumulation in fetus and increased demands for Zn towards end of pregnancy (Elnageeb and Adelatif, 2010) in addition to higher plasma albumin levels in pregnant animals to which Zn is bound primarily (Davis, 1984).The results obtained are in agreement with the findings of Williams (1977).However, late gestation associated hemodilution may reduce serum Zn level (Masters and Fels, 1980).The significantly higher Zn concentration in winter compared to rest of the seasons is in agreement with the findings of Khan et al. (2008), but contradicts the observations made by Cermak et al. (2006) and Mtui et al. (2007).The plasma Fe concentration recorded was adequate in all physiologically varied categories and all throughout the year, hence no need of its supplementation.However, significantly higher Fe concentration observed in the pregnant stock is in agreement with the findings of Tainturier et al. (1984) who recorded relatively high Fe concentration from 3 rd to 7 th month of pregnancy compared to minimum concentration when lactation commenced.However, Asif et al. (1996) observed nonsignificant differences with respect to physiological status in plasma Fe content in cattle.The amount of Fe seems not to be dependent on the dietary Fe intake owing to its complex absorption mechanism in GIT.Fe absorption (in duodenum) is normally restricted by a mucosal block (McDowell, 1985) in which Fe in mucosal cells is released into plasma by the conversion of Fe 3+ to Fe 2+ .Moreover, high levels of certain divalent metals enhance the Fe requirements by competing for absorption site in the intestine (Underwood, 1981).The amount of Fe absorbed depends on Fe balance of the animal, hence Fe depleted animals absorb more Fe than the nondepleted ones.The significantly higher Fe concentration in winter compared to rest of the seasons is in agreement with the findings of Merkel et al. (1990) and Rojas et al. (1993).
The findings of the present study suggest that goats of all categories in the study area should be supplemented with Ca throughout the year except autumn, Mg during winter and spring, and Cu and Zn during spring and summer seasons.Also, the dosage should be recommended as per the physiological needs of an animal.Moreover, the soil-plant-animal system of the area should be evaluated with respect to minerals which would confirm these findings and allow the formulation of area specific mineral supplement(s).This study could well serve as a mere indicator and set a platform for much detailed and wider scale investigations in future where in larger sample size and other livestock species throughout the valley could be studied.

Table 1 .
Total number of plasma (Goat) samples collected from the study area.

Table 2 .
Effect of physiological status and seasonal variation on plasma minerals in goats.

Table 2 .
Contd.Means bearing different uppercase superscripts across the columns for each parameter differ significantly (P < 0.05).Means bearing different lower case superscripts across the rows for each parameter differ significantly (P < 0.05).