Effect of Probiotic and Amla Powder Supplementation on Metabolic Profile and Milk Production of Summer Stressed Buffaloes Residing in Heavy Metal Polluted Areas of Ludhiana

Heavy metal contamination is one of the alarming environmental hazards in Ludhiana, the industrial hub of Punjab, India as evidenced from their presence detected in soil, water and fodder (Singh et al., 2013). Elevated heavy metal status of the cattle living in Buddha Nallah area of Ludhiana district in Punjab (Dhaliwal and Chhabra 2016) could be owing to the presence of heavy metals in surface water of Buddha Nallah and its seepage in the adjacent groundwater aquifers.
       
Some of the heavy metals like arsenic (As), cadmium (Cd), lead (Pb), mercury (Hg) are persistent, accumulative and are not metabolized in environment; thus exerting  toxic and harmful effects with challenging consequences concerned with environmental, evolutionary and ecological issues (Jaishankar et al., 2014). Heavy metals and metalloids, at the cellular level, lead to the production of reactive oxygen metabolites, elicit oxidative stress, cause DNA damage, affect membrane function, nutrient assimilation and disturb protein function/activity (Tamas et al., 2014). Even at low levels, heavy metals can upset the normal body function by playing as endocrine disruptors, production of reactive oxygen metabolites, hindering of essential functional groups or displacing the essential metal ions from biomolecules, leading to loss or inhibition of various enzyme activities and modification of metabolism (Lavicoli et al., 2009) resulting in physiological or biochemical alterations in the animal. The toxic metals hinder antioxidant enzymes and exhaust intracellular glutathione due to the pro-oxidative properties of metals and such changes may even override the adaptive potential of the animals. Arsenic toxicity can disrupt hepatic function due to cross-linking of enzymes (Patlolla et al., 2012). The higher concentrations of hexavalent Cr in blood may cause blood cell damage leading to functional damage of liver and kidney (Dartsch et al., 1998). Nickel exposure through contaminated water leads to dermatitis, allergy of skin and oral epithelium damage (Jacob et al., 2015). The heavy metal overload in tissues may also affect glycolysis, protein and lipid profile which in turn may induce damage to blood composition, kidneys, lungs, liver and reduction in energy levels (Javed et al., 2017).  The antioxidant defense has an important role in the protection of organisms against metal-induced oxidative stress. Oxidative stress is a unified concept for the assessment of metabolic status of buffaloes affected by heavy metal exposure and it may be significant in the presence of heavy metals when there are an insufficient amount of antioxidants to defend against the growing amount of free radicals. Living systems most often interact with a cocktail of heavy metals in the environment which may disrupt the metabolism of trace elements and antioxidants in the cattle (Arslan et al., 2011).
       
Summer season is especially stressful for livestock production probably due to depletion in antioxidant reserve and increases in summer-induced oxidative stress (Chabukdhara et al., 2014). The first goal of the livestock production is the delivery of safe foods for human consumption taking into account the welfare of the animal. One way is to use specific feed additives or dietary raw materials to favorably affect animal performance and welfare, particularly through the modulation of the gut microbiota which plays a critical role in maintaining host health.
       
Probiotics e.g. Saccharomyces cerevisiae improve resistance to pathogenic bacteria colonization and enhance host mucosal immunity; thus resulting in reduced pathogen load, improved animal health and a reduced risk of food-borne pathogens in foods. Feeding Saccharomyces cerevisiae may have a dosage-independent beneficial effect in supporting the physiologic adaptation after parturition, resulting in higher milk production and lower milk somatic cell count (Zaworski et al., 2014). The feeding of live yeast in dairy cattle helps to improve the rumen function leading to greater milk yield and milk protein (Rossow et al., 2017). Probiotic supplementation improves the vital health parameters, erythrocytic lipid peroxidation and hematology in buffalo calves during summer season (Singh et al., 2018).
       
Phyto-compounds acting as antioxidants could be a solution in ameliorating these adverse effects because they are readily available often at low cost. Amla or Indian gooseberry (Emblica officinalis) is an important dietary source of vitamin C, amino acids, minerals along with phenolic compounds, tannins, phenyllembelic acids, phenyllemblin, cuccuminoides, rutin and emblicol (Yokozawa et al., 2007). Amla is a haematanic and lipolytic tonic which strengthens the liver, helping it in eliminating toxins from the body and categorized as one of the strongest rejuvenant in Indian pharmacopeia. The supplementation of amla powder in summer stressed buffaloes can maintain the antioxidant status and ameliorate heat stress in Murrah buffaloes under field condition (Lakhani et al., 2015; 2017). Keeping in view the above facts, the present study was designed to monitor the influence of probiotic (Saccharomyces cerevisiae strain¹⁰²⁶) and amla powder (Embilica officinalis) supplementation on metabolic and productive status of summer stressed lactating buffaloes in relation to environmental heavy metal exposure.

Location of study area
 
The dairy farm alongside of Buddha Nallah on Haibowal Road (Haibowal) with drinking water heavy metal levels above the permissible limits (FSSAI, 2010) viz. Chromium-0.05 µg/ml.
       
Nickel-0.02 µg/ml; Arsenic-0.05 µg/ml and Lead-0.05 µg/ml. Heavy metals affected area was selected through a survey in Ludhiana district, Punjab, India, where the drinking water heavy metals concentration was above the maximum permissible blood level of chromium (>0.05 µg/ml), nickel (>0.02 µg/ml), arsenic (>0.05 µg/ml) and lead (>0.05 µg/ml) were used to decide the exposure group.
       
The buffaloes were maintained by their owner and provided with standard diet and ad libitum water.
 
Experimental plan
 
Twenty summer stressed lactating Murrah buffaloes of the same age group, close parity and in early lactation from the exposed area were divided into two groups:
 
 
       
Saccharomyces cerevisiae¹⁰²⁶ used in this study was procured from Alltech Biotechnology Pvt. Ltd. Bangalore, India and Amla powder from Himalaya Drug Company, Bangalore, India.
 
Temperature humidity index
 
Temperature and relative humidity were recorded inside the shed with the help of thermo hygrometer (Lakhani et al., 2016). Temperature humidity index (THI) of the animal shed was calculated using the formula: Where,
Ta   = Average ambient temperature in °C.
RH  = Average relative humidity.
               
Monthly recording of temperature, relative humidity and temperature humidity index (THI) of the experimental buffalo shed are as under:
 


Sampling schedule
 
Whole blood and milk samples of the experimental buffaloes were collected aseptically in the morning hours from both control and treatment group on the day of starting of supplementation (i.e. day 0) and thereafter on days 15 and 30.
       
The blood samples (10 mL) were collected in heparinized vials by jugular venipuncture. Two milliliters of whole blood was kept at -20°C for quantitation of heavy metals, trace minerals and reduced glutathione. Rest of blood was centrifuged at 2500 rpm for 10 minutes. Plasma was separated and erythrocyte pellet was used for preparation of hemolysate. The RBC hemolysate was prepared as per Yeotikar et al., (2019).
       
The lymphocytes peripheral blood mononuclear cells (PBMC), were separated by density gradient centrifugation (400×g, 30 minutes at 22°C) with the help of a density gradient media (Ficoll-Paque^TM PREMIUM, density = 1.084 g/mL) as per the instructions of the manufacturer. Lymphocytes from the interface were washed twice with phosphate-buffered saline (PBS) of pH 7.4, resuspended in PBS and counted in the hemocytometer. Lymphocytes (1×10⁶ cells/ml) of control and treatment groups were ruptured by ultrasonication (Misonix; Ultrasonic Liquid Processor) using 0.6 ml of PBS and then centrifuged for 10 min (10000×g at 4°C). The supernatant was used for assay of markers of oxidative stress viz. intracellular reactive oxygen species and thiobarbituric acid reactive substances according to Lebel et al., (1992) and Fraga et al., (1988) respectively.
       
The glass tubes for milk collection were soaked in 20% Nitric acid solution for 24 hours and rinsed twice with the deionized water (Belete et al., 2014). The fresh milk samples (50 mL) were collected in clean sterilized screw capped glass tubes during the morning milking time from each buffalo. While milking, the udder of the individual buffalo was washed with the distilled water. Immediately after collection, milk samples were transported to the laboratory in ice packs and stored at -20°C until further analysis (Enb et al., 2009). The milk samples were used for quantification of heavy metals, trace minerals and milk components viz; milk fat, SNF, protein, ash and lactose per cent. The quantity of milk was measured twice a day and total quantity was taken as milk yield (kg) per day. 
 
Processing of blood and milk samples for heavy metals
 
All glass wares were first cleaned with 10% HNO₃ solution followed by washing with the distilled water and then sterilized in the hot air oven at 160°C for 60 minutes. To 1 ml of whole blood samples, added 15 ml of a tri-acid mixture (10:4:1 HNO₃, H₂SO₄ and HClO₄) and left overnight. Then these mixtures were digested on a hot plate at 250°C until a transparent solution and volume reduced to 1-2 mL. After cooling, the digested samples were filtered using Whatman filter paper no. 42 and the volume of filtrate was made up to 10 ml with double glass distilled water (Allen et al., 1986).
       
Milk samples were digested by the method described by Ahmad et al., (2017) with slight modifications. To 10 mL of milk sample, added 12 mL of 1:3 H₂O₂ (30%) and HNO₃ (65%) in acid prewashed teflon vessels. After standing overnight, samples were heated on a hot plate until their volume reduced to 2 mL. After cooling, the digested samples were filtered using Whatman No. 42 filter paper and the filtrate diluted to 20 mL with double glass distilled water.
       
Blood and milk levels of Cr, Ni, As and Pb, erythrocytic malondialdehyde (MDA), superoxide dismutase (SOD) activity, the levels of reduced glutathione (GSH), vitamins C and E were analyzed as described by Yeotikar et al., (2019). The concentration of glucose, total cholesterol, triglycerides, total protein, albumin, creatinine, urea and the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AKP), gamma-glutamyl transferase (GGT) and creatine kinase (CK) in plasma were determined using BPC Biosed kits (SRL, Rome Italy) on the fully automatic biochemical analyzer (Global 240 BPC Biosed).
       
The amla powder procured from Himalaya drug Co., Bangalore was analyzed for the physicochemical parameters. Pharmacognostic authentication of the amla powder was done by the Ayush Center, Department of Veterinary Pharmacology and Toxicology, GADVASU, Ludhiana. Quality of amla powder was consistent to the Ayurvedic Pharmacopoeia of India (API), Department of Health and Family Welfare, Government of India as well as to the Veterinary Monograph of Indian Pharmacopoeia (Indian Pharmacopoeia Commission 2010). The dose of Amla powder (86 mg/kg b.w.) was decided as per the API.
       
In a pestle and mortar, 5 g amla powder was finely triturated in 2% m-phosphoric acid solution and then filtered through a Whatman filter paper no. 1 and made up the total volume to 100 mL. The alcoholic extract of amla powder was prepared by the method described by Shukla et al., (2009). Five grams of amla powder was extracted in 50 ml of dichloromethane: methanol (1:1) mixture. The extract was evaporated to dryness in a water bath at 40°C. After the solvent was evaporated the extract was weighed and dissolved in ethanol (10 mg/ml) and used as alcoholic extract of amla powder. The extract of the amla powder was prepared for assessment of its antioxidant property, vitamins (E ans C as per Yeotikar et al., 2018), minerals by acid digestion and atomic absorption spectrophotometer, DPPH radical scavenging activity (Blois 1958) and total antioxidant activity  and tannins (Sadasivam and Manickam 1997), total phenolic compounds (Sadasivam and Manickam 1997) of the amla powder. The biochemical analysis, heavy metal and mineral element concentrations of amla powder have been presented in Table 1.
 

 
Statistical analysis
 
The differences between the mean values of the parameters belonging to the control group and supplemented group in heavy metal exposed area during summer season were analyzed for significant differences using two way ANOVA with interaction and the group means were compared by post hoc test of Fischer’s least significant difference (LSD).

Heavy metal and trace mineral level
 
The blood and milk concentrations (mean±SE) of heavy metals Cr, Ni, As and Pb (Table 2) and trace minerals Zn, Fe, Cu, Mn and Co (Table 3) in the buffaloes of the treatment group were not affected by probiotic and amla supplementation on 15^th and 30^th days of supplementation in comparison to control group. Their mean concentrations in raw milk were lower than permissible limits as described earlier by Yeotikar et al., (2018). Saccharomyces cerevisiae are able to bind the toxin and remove, inactivate or reduce the availability of the heavy metals which contaminate foods and feed (Zoghi et al., 2014). Yeast in combination with lactic acid bacteria can remove heavy metals, which might be due to complex binding of probiotic to contaminants by the cell walls (Allam et al., 2015). The probiotics inhibit absorption of heavy metal by protecting the intestinal barrier and can prevent the adverse health effects in humans and animals (Zhai et al., 2016). The mean levels of Zn, Fe, Mn and Co in the milk of buffaloes of treatment group were significantly (P<0.01) higher on both 15^th and 30^th day of supplementation in comparison to the control group. Kowalik et al., (2016) found the significant retention of Cu and Zn in the rams fed with Saccharomyces cerevisiae.
 

 

 
Oxidative stress markers
 
The mean values of erythrocytic malondialdehyde in the buffaloes of the treatment group showed significant (P<0.05) decrease whereas the mean values of erythrocytic SOD activity, whole blood reduced glutathione, plasma vitamins C and E in the buffaloes of the treatment group showed significant (P<0.05) increase as compared to the control group at 15^th and 30^th day of supplementation (Table 4) which indicates that the amla powder supplementation has a positive effect in relieving the stress on the animals. Similar results were reported by Lakhani et al., (2016). A significant increase in GSH, Vitamin C and E levels by supplementation with amla powder and probiotic to a level comparable with those found in the control group of animals suggested the antioxidant role of amla powder (Shukla et al., 2009). Amla inhibits the lipid peroxidation induced by heavy metal toxicity (Thilakchand et al., 2013).
 

       
Lymphocytic TBARS and intracellular ROS (Table 4) were decreased significantly (P<0.01) on 15^th day and 30^th day after supplementation, in comparison to the control group.  Amla contains vitamin C which is a chain blocker of lipid peroxidation and similar results were reported by Tanaka et al., (2007), Kumar et al., (2011a) in heat stressed buffaloes and dairy cows.
 
Blood biochemical constituents
 
The mean values of plasma glucose, total cholesterol, globulin, creatinine and BUN in the buffaloes of the treatment group (Table 5) were significantly (P<0.05) lowered and the mean values of plasma total protein, albumin, A: G ratio and BUN: Creatinine ratio in the buffaloes of the treatment group were significantly (P<0.05) increased in comparison to the control group at 15^th and 30^th day of supplementation. As per Kowalik et al., (2016), the rams fed with yeast showed significant decrease in blood levels of total cholesterol and triglycerides which could be due to the changes in rumen fermentation. However, concentrations of plasma total protein, albumin, A: G ratio and BUN: creatinine ratio were significantly (P<0.05) increased by feeding Saccharomyces cerevisiae and amla powder. Feeding live yeast to dairy cows during the hot season lowered plasma values of urea N whereas values of plasma glucose, triglyceride, total cholesterol and total protein were not influenced; the feeding of probiotic live yeast might have improved protein utilization which reduces the plasma levels of urea in mid lactating dairy cows. Hansen et al., (2017) noticed reduced plasma levels of urea and increased levels of albumin in yeast culture supplemented buffaloes as supplementation of Saccharomyces cerevisiae supports protein conversion efficiency and energy in the buffaloes. Supplementation of Yea Sacc1026 in buffalo calves during summer season was effective in ameliorating the adverse effects of summer stress on animal physiological parameters (Singh et al., 2018).
 

 
Plasma enzymes
 
The mean values of plasma ALT, AST, ALP, CK and GGT in the buffaloes of the treatment group were significantly  (P<0.05) lowered as compared to control group at 15^th and 30^th day of supplementation. Preclinical studies in arsenic-induced toxicity in mice supplemented (Table 6) with amla resulted in elevated levels of antioxidant enzymes, reduced levels of serum aminotransferases and lipid peroxidases. This might be due to the action of heavy metals like arsenic on liver which causes generation of free radicals by reacting with the sulfhydryl group of enzymes and proteins (Thilakchand et al., 2013).
 

 
Milk yield and quality
 
In the present study, the mean values of milk yield in thebuffaloes of the treatment group were significantly (P<0.05) higher as compared to the control group at 15^th and 30^th day of supplementation (Fig 1). The overall significant increase in the milk yield of the supplemented buffaloes was 20.57% in comparison to the control group buffaloes. In Murrah buffaloes supplemented with Saccharomyces cerevisiae during transition period, the milk yield was significantly higher as compared to the control group (Para et al., 2018). The probiotics including Saccharomyces cerevisiae enhance the digestibility of food ingredients/nutrients, provide more energy for milk production, reduce fat mobilization and improve the milk yield in dairy cows (Yalcin et al., 2011). The increased milk yield due to feeding of Saccharomyces cerevisiae might be due to its galactopoietic effect. Feed supplement composed of Embilica officinalis and Saccharomyces cerevisiae enhanced feed digestibility by enhancing intestinal bioavailability of nutrients (Patil 2014). Saccharomyces cerevisiae reduces the detrimental effects of heat stress in dairy cows, reduces energy and nutrient losses and increases milk production by manipulating the microbial fermentation in the rumen (Wang et al., 2010).
 

       
The mean values of milk fat percent and milk ash percent in the buffaloes of the treatment group were significantly (P<0.05) higher as compared to the control whereas mean values of milk SNF, milk protein and milk lactose were unaffected at 15^th and 30^th day of supplementation (Fig 2). Results reported by Para et al., (2018) revealed similar findings in Murrah buffaloes supplemented with dietary yeast. Cattle supplemented with the herbal mixture and yeast Saccharomyces cerevisiae exhibited significant improvement in the milk fat, SNF and protein (Patil 2014). The higher milk fat content in the yeast supplemented buffaloes might be attributed to the yeast promoted population of microflora which enhances the fiber digestion and VFA production. The supplementation of yeast in the dairy cows caused no significant effect on milk fat and protein content (West and Bernard 2011). The milk protein percent of yeast treated cows showed significantly higher values than control (Bruno et al., 2009). The difference in the results of various studies might be due to the differing animal species, lactation stage, parities, a dose of the yeast and diet composition (Hansen et al., 2017). The live yeast feeding is increases the numbers of rumen cellulolytic bacteria which helps in improving the fiber digestibility in ruminants (Banadaky et al., 2013). The increased milk fat percent in the yeast supplemented buffaloes might be due to increased acetate concentrations.
 

 
Economics
 
A balance sheet indicating the economics of probiotic and amla powder supplementation in the summer stressed buffaloes of heavy metal exposed area is given in Table 7 The net profit was calculated as Rs. 246.93 and Rs. 279.97 per buffalo per day in control and supplemented animals, respectively. The overall percentage of monetary gains from buffaloes fed with probiotic and amla powder was >13.38% as compared to control.