Demand for animal products is increasing with changes in consumer tastes and expanding markets; particularly in developing countries where affluence is spreading. So there is challenge for animal scientist to meet the demand of people with limited feed resources. Many chemical feed additives have been used to improve the productivity of the animals but they have drawbacks of residue and health hazards to consumers. So natural feed supplements have to search upon. Azolla, an aquatic fern supplementation in the ration been reported to improve the performance of chicks (Yadav and Chhipa, 2016); bucks (Kumar et al., 2016) and buffalo calves (Indira et al., 2009). Consequently, spirulina is emerging as a potential candidate to fulfill these criteria. Spirulina (Arthrospira platensis) is a blue-green microalgae, a source of protein of high biological value, vitamins, minerals and essential fatty acids, along with a number of bioactive compounds. Its dietary supplementation has been found to improve the animal health and productivity (Holman and Malau-Aduli, 2012). Spirulina supplementation has shown to reduce oxidative stress and enhance humoral and cell-mediated immune functions in human being. Spirulina is widely available in the market as food supplement for both humans and animals. Keeping the above facts in view, the present study was conducted to explore the potential of Spirulina platensis to improve immunity and reducing oxidative stress in Barbari goats.

Materials and Methods

Eighteen male Barbari goats were divided into three groups, control (Group 1) and treatment groups (Group 2 and Group 3), consisting of six animals in each group in a completely randomized design (CRD). All the goats were kept under uniform management conditions by housing them in well ventilated sheds. All the goats were fed with complete pellet feed having Bengal gram straw and concentrate mixture in 60:40. In case of control group no supplementation of Spirulina platensis (SP) was done, while in treatment groups of goats Spirulina was supplemented @ 0.25% (Group 2) and 0.50% (Group 3) of DMI respectively. An experimental feeding on three groups of goats was carried out for 90 days. Blood was collected before feeding from each animal of all three groups at 90^th days of experimental feeding by puncturing the jugular vein with the help of a clean sterilized needle. Blood was collected in acid citrate dextrose buffer (@ 1.5 ml/10 ml of blood) in 2 ml eppendorf tube on ice. Blood samples collected in acid citrate dextrose (ACD) buffer for assays of oxidative stress indices were centrifuged in refrigerated condition at 2000 rpm for 20 min to harvest the erythrocytes. Buffy coat was removed and erythrocytes were washed thrice with ice cold phosphate buffer saline (PBS) solution. Packed erythrocyte was used for estimation of reduced glutathione (GSH). Erythrocytic suspension was prepared by mixing equal volume of erythrocytes and normal saline solution (NSS). This suspension was used for haemoglobin estimation. Exactly, 0.5 ml of erythrocytic suspension was mixed with 4.5 ml of stabilizing solution [0.5025 g EDTA (2.7 mM, pH-7.0) and β-Mercaptoethanol (0.7 mM) 24.55 µL in 500 ml distilled water] to form haemolysate. Haemolysate and RBC suspension were kept at -70^oC and used for antioxidants assay. Catalase was estimated in the RBC haemolysate as per the method of Cohen et al. (1970). Superoxide dismutase (SOD) activity of RBC haemolysate samples was measured using Nitro blue tetrazolium (NBT) as a substrate after suitable dilution according to the method of Marklund and Marklund (1974), with certain modifications as suggested by Minami and Yoshikawa (1979).

The cell mediated immune (CMI) response was assessed in the goats after 90 days of experimental feeding by delayed type hypersensitivity (DTH) reaction by measuring the increase in skin thickness (Kim et al., 2000) in response to intra-dermal inoculation of phytohaemaglutinin-p (PHA-p). The skin on both the sides of the neck was shaved with the help of a razor and injected with 25 µg of PHA-p in 100 µL of PBS (pH 7.4) at two different sites 4 cm apart from each other on one side of neck and 100 µL of PBS at two different sites 4 cm apart on other side of the neck as a control. The skin thickness/ induration were measured at 0, 24, 48, 72 and 96 h post sensitization with the help of a vernier calliper. All the injections and skin measurements were made by the same person to minimize the variations. Immunoglobulins in plasma sample were estimated by Zinc turbidity method (Mc Ewan et al., 1970).

The data collected during study was statistically analyzed using generalized linear model (GLM) procedures and difference between treatments and periods was analyzed by using analysis of variance (ANOVA) as per Snedecor and Cochran (1989) using SPSS 1995.The difference between mean was obtained using Duncan’s test at 95% level of significance.

Results and Discussion

The effect of Spirulina platensis supplementation on erythrocytic antioxidants enzymes catalase and superoxide dismutase (SOD) activity studied during feeding trial (0, 45 and 90^th days) is presented in Table 1.

Table 1: Effect of Spirulina platensis supplementation on erythrocytic antioxidant profile of different groups of goats

Means bearing different superscripts (a,b) differ significantly; Means bearing different superscripts (A and B) in a row differ significantly Means bearing different superscripts (X and Y) in a column differ significantlyy at (P<0.05)

The catalase activity (U/mg Hb) was 47.01, 45.66 and 44.39 in Group 1, 2 and 3 respectively at 0 day which changed to 45.49, 63.86 and 61.74 at 90 days of experimental feeding. There was significant (P<0.05) increase in catalase activity in the Spirulina supplemented group (Group 2 and Group 3) as compared to control group (Group1). The catalase activity (U/mg Hb) was significantly (P< 0.05) higher in Group 2 (63.86) that was statistically similar to Group 3 (61.74) and lowest for Group 1 (45.49) after 90 days of Spirulina supplementation. Similar trend was reported for superoxide dismutase activity. The superoxide dismutase activity (U/mg Hb) was 49.43, 55.36 and 58.67 in Group 1, 2 and 3 respectively at 0 day which changed to 49.91, 65.34 and 67.72 at 90 days of experimental feeding. There was significant (P<0.05) increase in superoxide dismutase activity in the Spirulina supplemented group (Group 2 and Group 3) as compared to control group (Group 1). The mean superoxide dismutase activity (U/mg Hb) was highest for Group 3 (63.19) that was statistically similar to Group 2 (60.35) and was lowest for Group 1 (49.67). The findings are in agreement with Abdel-Daim. (2014) who reported that buck fed concentrates with Spirulina platensis (SP) had significantly higher levels SOD concentration compared to the control group. Treatment with SP offered protection through attenuation of lipid peroxidation and decreased production of free-radical derivatives, as evident from the decreased levels of serum MDA, and normalization of GSH and SOD levels. Reddy et al. (2004) also suggested that Spirulina supplementation resulted in significantly higher activities of superoxide dismutase and catalase in the erythrocytes with concomitant increase in reduced tripeptide glutathione content in broiler chickens. The antioxidative effect Spirulina is related to several active ingredients, notably phycocyanin, polysaccharides, α-tocopherol and β-carotene that have potent antioxidant activities working, individually or synergy, directly on free-radicals (Riss et al., 2007) The effect of Spirulina platensis supplementation on total immunoglobulin concentration in blood serum studied during feeding trial (0, 45^th days and 90^th days) is presented in Table 2.

Table 2: Effect of Spirulina platensis supplementation on total immunoglobulin concentrations (mg/ml)

Means bearing different superscripts (a to d) differ significantly; Means bearing different superscripts (X to Z) in a column differ significantlyy at (P<0.05)

Mean total immunoglobulin concentration (mg/ml) changed from 36.62 to 36.64 in Group 1; 36.44 to 46.29 in Group 2 and 32.84 to 50.34 in Group 3 in 0 to 90 days of experimental feeding. There was significant increase in total immunoglobulin concentration in the Spirulina supplemented group (Group 2 and Group 3) as compared to control group (Group1). Qureshi et al. (1996) reported that no differences were observed in anti-sheep red blood cells antibodies during primary response. However, during secondary response K-strain chicks in all Spirulina dietary groups had higher total anti SRBC titers with 10,000 ppm SP group being highest versus the o ppm group. In broiler chicks IgG concentration was increased in 10,000 ppm SP group over the control. Qureshi et al. (1996) fed increasing levels of SP to chicken poults and evaluated immunological performance after injection of sheep red blood cells. No differences in total Ig were observed 7 days after the first injection. However, 7 days after a second injection, higher total Ig concentrations were observed in all poults fed SP. This suggests that the immune enhancement response is not immediate it means that to reach the final conclusion need longer duration of feeding trial. Hayashi et al. (1994) reported that in the primary immune response to sheep red blood cells (SRBC), significant increase of splenic PFCs per 10⁶ cells was observed in mice fed Spirulina diet (SP-10 and SP-20), as compared to control group. However, HA titers observed in SP-10 and SP-20 was almost the same as the control group. While, immunoglobulin G (IgG)-antibody production in the secondary immune response hardly effected. The skin thickness (mm) measured at 0, 12, 24, 48, 72 and 96 h post sensitization with the help of a vernier caliper is presented in Fig. 1.

Fig.1: Effect of Spirulina (Spirulina platensis) supplementation on cell mediated immune response against PHA-p in different groups of goats

The skin thickness (mm) was 5.16, 4.16 and 4.66 at pre injection (0 hr) which increased to 14.33, 17.16 and 18.58 at 12 hr in Group 1, Group 2 and Group 3 respectively. After 12 hr to 96 hr post inoculation a statistically significant (P<0.05) decreasing trend in skin thickness was reported with period. The skin thickness (mm) was 9.33 (24h), 8.41 (48h), 5.41 (72h) and 5.32 (96h) in control group (Group 1) while 10.16 (24h), 9.33 (48h), 5.83 (72h) and 4.7 (96h) in Group 2 and 11.58 (24h), 9.58 (48h), 7.08 (72h) and 4.90 (96h) in Group 3. Overall mean skin thickness (mm) was 9.39 in 0.5% Spirulina supplemented group, 8.56 in 0.25% Spirulina supplemented group and 7.97 in un supplemented group of goats, showing improvement in cell mediated immunity on Spirulina supplementation. Qureshi et al. (1996) reported that 10,000 ppm Spirulina group chick had higher PHP-p mediated lympho proliferation response over the 0 ppm control group.

Conclusion

From present study it can be concluded that supplementation of Spirulina @ 0.25 and 0.50 of DMI had significantly improved the antioxidant status and immunity in growing Barbari goats.

Acknowledgements

The fellowship provided to first author by Director, ICAR-Indian Veterinary Research Institute, Izatnagar and research facilities provided by Director, ICAR-Central Institute for Research on Goats, Makhdoom for this work are greatly acknowledged.

References

1. Abdel-Daim, M. M. (2014). Pharmacodynamic interaction of Spirulina platensis with erythromycin in Egyptian Baladi bucks (Capra hircus). Small Rumin. Res. 120: 234–41.
2. Cohen, G., Dembiec, D. and Marcus, J. (1970). Measurement of catalase activity in tissue extracts. Analytical Biochemistry. 34: 30–38.
3. Hayashi, O., Katoh, T. and Okuwaki, Y. (1994). Enhancement of antibody production in mice by dietary Spirulina platensis. Journal of Nutritional Science and Vitaminology .40: 431-441.
4. Holman, B. W. B. and Malau-Aduli, A. E. O. (2012). Spirulina as a livestock supplement and animal feed. of Anim. Physiology and Nutr. 97:615-623.
5. Indira, D., Rao, K.S., Suresh, J., Naidu, K.V. and Ravi, A. (2009). Azolla (Azolla pinnata) as feed supplement in buffalo calves on growth performance. Indian J. Nut. 26: 345-348.
6. Kim, H. W., Boon, P., Chewa, B. P., Wonga, T. S., Parka, J. S., Byrnea, K. M., Hayekb, M. G. and Reinhart, G. A.( 2000). Modulation of humoral and cell mediated immune responses by dietary lutein in cats. Immun. and Immunopath. 73: 331–341.
7. Kumar Ravindra, Gangwar C. Tripathi, P. and Chaudhary, U.B. Effect of azolla supplementation on semen quality, haematology and rumen metabolites in Barbari bucks. Ind. J. Small Rumin.  22: 186-189
8. Marklund, S. and Marklund, (1974). Involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. European J. of Biochemistry 47: 469-474.
9. McEwan, A. D., Fisher, E. , Selman, I.  E. and Penhale, W.  J. (1970). A turbidity test for the estimation of immune globulin levels in neonatal calf serum.  Clinica Chemica Acta. 27: 155–163.
10. Menami, M. and Yoshikawa, H. (1979). Simplified assay method of superoxide dismutase activity of clinical use. Clinica Chemica Acta. 92: 337-342.
11. Reddy, B. , Yuvaraj, N., Babitha, V., Ramnath, V., Philominia, P. T. and Sabu, M. C. (2004). Antioxidant and hypolipidemic effects of Spirulina and natural carotenoids in broiler chicken. Ind. Vet. J. 81: 383-386.
12. Riss, J., Ecord’e, K. D. and Sutra, T. (2007). Phycobiliprotein C-phycocynin from Spirulina platensis is powerfully responsible for reducing oxidative stress and NADPH oxidase expression induced by an atherogenic diet in hamsters. Agric. and Food Chem. 55: 7962- 7967.
13. Snedecor, G.W. and Cochran, W.G. (1989). Statistical Methods. 7^th The Iowa State University, Iowa, USA.
14. (1995). Statistical Packages for Social Sciences. Version 7.5. SPSS Inc., IL, USA.
15. Yadav, C.M. and Chhipa, B.G. (2016).Influence of inclusion of different levels of Azolla (Azolla pinnata) meal in the diet on the performance of Pratapdhan chicks. Indian J. Nut. 33: 350-352.