Effect of Variable Levels of Metabolisable Energy and Metabolisable Protein On Growth, Immunity and Health Performance in Male Murrah Buffalo Calves (Bubalus

In calve nutrition; malnutrition is still the one of the limiting factor in developing countries (Olafadehan and Adewumi 2009). Poor nutrition and imbalance nutrition supply primarily effects health and production of the animal which rendered the animal to reach its maximum genetic potential level. To understand the effects of different level of dietary energy and protein on health and immunity of the ruminant, the present study was considered to investigate the effects of different levels of ME and MP on body weight changes, dry matter intake, feed conversation eciency and various immunity and health parameters. For growth trial, Twenty four calves were divided into four groups with six animals (n = 6) in each group. All animals were fed individually and for feeding four types of concentrates were prepared; Treatment 1 (control, in accordance with ICAR 2013 feeding standard), Treatment 2 (T2, 30% extra metabolisable energy above ICAR 2013 feeding standard), Treatment 3 (30% extra metabolisable protein above ICAR 2013 feeding standard) and T4 (30% extra metabolisable protein and 30% extra metabolisable energy). The growth trial was conducted for 12 months and during this trial period dry matter intake was measured daily, body weight measured fortnightly and blood was collected on monthly interval for evaluating blood immunity parameters. There was no signicant alteration in average body weight changes when compared between groups. The overall mean of total DMI (kg) for T1, T2, T3 and T4 groups were 6.86 ± 0.49, 6.49 ± 0.49, 6.66 ± 0.51 and 6.68 ± 0.48 respectively. There was no signicant variation in GPx, SOD, catalase, cortisol and IgG due to different levels of ME and MP. The study outcomes states providing higher energy and protein did not caused any stress and any alteration in various health parameters in Murrah buffalo calves during growth period.


Introduction
The buffaloes are considered as the main dairy animals in India, contributing 51% to the total milk production of the country (BAHFS, 2015). Diversity of the buffaloes in India is represented by 13 registered and well de ned breeds' along with non-descript populations; Murrah is the prominent buffalo breed in the country followed by Surti, Mehesana and Jafarabadi breeds (Vohra et. al., 2016). In tropical countries like India, buffaloes showed better e ciency in utilization of nutrients over cattle and had comparatively higher resistance to disease (Paul et. al., 2003). In calve nutrition; malnutrition is still the one of the limiting in developing countries (Olafadehan and Adewumi 2009). Apart from genetics, undernutrition and imbalance feeding are the major causes of lower growth rate (Bhatti et. al., 2007).
As breeding bull sires the whole herd so it is considered as half of the herd but generally bull's nutrition is neglected. The nutrition of the buffalo bulls has important in uence on the quantity and quality of the semen. Under-nutrition which includes mainly de ciency of major and minor nutrients is the most common cause of poor growth rate.
One of the major dietary nutrients which in uence the e ciency of other nutrients in feed is dietary energy which also effects growth and production by the animal (Hosseini et. al., 2008). The other most important dietary nutrient effecting reproductive performances of bulls is the available digestible protein in the diet. The feed with de ciency in protein and energy causes rapid weight loss which in turn in uences the productive life of bull (Mecham et. al.,1963). Therefore, the optimum energy and protein levels for better growth and better health of young buffalo male needs to be critically understood, so that male bull productive life can be increased.
Keeping in view, the bene ts of feeding higher energy and protein on growth, production and immunity from early age and during growth trial and this study was undertaken to determine the effects of feed energy and protein levels on body weight changes, total dry matter intake, health and immunity performance of growing Murrah buffalo calves.

Material And Methods
The study was carried out at Livestock Research Centre of National Dairy Research Institute (N.D.R.I.), Karnal, Haryana, India. Twenty four male Murrah calves were selected and distributed randomly into four groups (6 animals each) according to their body weight so that average body weight of each group was similar at the time of starting of experiment. Four types of concentrates were prepared; Treatment 1 (control, in accordance with ICAR 2013 feeding standard), Treatment 2 (T2, 30% extra metabolisable energy above ICAR 2013 feeding standard), Treatment 3 (30% extra metabolisable protein above ICAR 2013 feeding standard) and T4 (30% extra metabolisable protein and 30% extra metabolisable energy).
Concentrate mixture, green fodder (sugargraze) and dry roughage (wheat straw) were supplied at the ratio of 40: 40: 20 (on dry matter basis) in all three types of TMRs. Blood samples were collected at monthly interval during experiment period to elicit the effect of higher energy and protein level.
DM intake was recorded daily by subtracting the residual DM from the quantity of DM offered. To estimate body weight, the animals were weighed before feeding and watering in the morning on two consecutive days at the start of experimental feeding and thereafter at fortnightly intervals during the entire experimental period. Blood samples (8-10 ml) were collected in sterile heparinised vacutainer tubes (vacuette, Greiner bio-one, GmbH, Austria) from jugular vein puncture, causing least stress to the animal.
Blood samples were taken on monthly intervals. Immediately after collection, tubes were kept in ice and transported to the laboratory for further processing.

Assessment of antioxidant status
Antioxidant enzymes (superoxide dismutase (SOD), catalase and glutathione peroxidase (GP X )) were estimated using RBC hemolysate within three days after collection of blood.

Preparation of blood lysate
Erythrocytes and erythrocyte membranes are more vulnerable to lipid peroxidation due to constant exposure to high oxygen tension and richness in polyunsaturated fatty acid, respectively. Besides this, the erythrocytes contain several defense mechanisms against oxidative stress which include both enzymatic and non-enzymatic antioxidants. The simplicity, availability and ease of isolation make erythrocytes as an excellent model for in vivo studies on cellular antioxidant response to oxidative stress under clinical conditions and preparation of hemolysate is an initial step in the analysis of different oxidative stress markers.

Principle
Red blood cells when placed in a hypotonic (with a lower solute concentration than that is found in the cells) solution, it rapidly moves into the cells by osmosis, causing the cells to swell and burst releasing hemoglobin in the surroundings. This phenomenon is known as hemolysis. This fresh haemolysate was further used for estimation of various enzymatic and non-enzymatic antioxidants. Normal saline (0.9%): for this 4.5 g NaCl (sodium chloride) was dissolved in DDW to make the volume up to 500 mL.

Procedure
Blood was collected in ACD (at 1.5 mL/10 mL of blood) in 2.0 mL eppendrof tube.
Sample was thoroughly mixed with the anticoagulant ACD.
Samples were centrifuged in refrigerated conditions (4-6°C) at 2500 rpm for 20 mm. Supernatant (plasma) and buffy coat was carefully removed.
1 mL of ice-cold normal saline was added to packed RBC in each tube which was gently mixed by up and down movements 2-4 times.
Samples were again centrifuged at 4-6°C for 20 min. at 2500 rpm.
Above steps were repeated thrice. 0.5 mL of packed erythrocytes were taken and an equal quantity of normal saline was added to form RBC suspension.
0.2 mL of RBC suspension was taken in a separate 2 mL vial and 1.8 mL of ice-cold EDTA stabilizing solution was added to make haemolysate.
If further dilution was required then EDTA stabilising solution was used.
Haemolysate was analysed within three days.

Statistical Analysis
Analysis of data was done by SPSS (22.0) software using ANOVA (turkey-test). Mean and standard error were calculated and comparisons between and within groups were made.

Body weight changes
The data pertaining to change in body weight was measured fortnightly and the values are presented in respectively. Body weight at last fortnight and overall mean of body weight indicates numerically higher body weight achieved in T4 group however change is not signi cantly higher. The mean body weight did not varied signi cantly in any of the fortnight or in the overall mean value. .49 ± 0.43 and 9.01 ± 0.44. The DMI intake was gradually increased throughout the trial which is normal as body weight and requirement increased with progress in age and trial. There were no signi cant changes among the different group in any of the fortnight as far as total DMI was concern. The overall mean DMI (kg/d) for T1, T2, T3 and T4 group were 6.86 ± 0.49, 6.49 ± 0.49, 6.66 ± 0.51 and 6.68 ± 0.48 respectively which did not showed any signi cant alteration in between the different groups.     The mean body weight changes did not varied signi cantly in any of the fortnight or in the overall mean value. The result may be attributed to higher energy and protein provided in treatment rations is not su cient to cause gain in body weight and this extra energy and protein is either not utilized or aiding in some other production parameters. Mahmoudzadeh et. al. (2007) fed three levels of energy and two levels of proteins in 15 months male buffalo calves and results of this study showed no nal body weight difference among treatment which was in support to results of this study. Patil (2013) fed 10% higher and 20% higher energy than control (NRC, 2001) to Murrah buffalo calves and also observed no alteration in body weight changes in different groups.

Dry matter intake and Feed conversation ratio
There was no change in total dry matter intake and feed conversion ratio during the feeding trial of the study. This could be possibly due to increase in ME and MP in ration did not affect feed intake and as there was also no change in total body weight gain, therefore no alteration was observed in feed  Tauqir et. al. (2011) fed three levels of CP and two levels of ME, result of this study DMI had not changed signi cantly when CP was fed at low (11.85%) and medium (14.25%) level

Blood antioxidant and immunity parameters
Parameters like blood GPx, SOD, catalase and IgG were not altered during the trial indicating no type of stress and no variation in health and immunity status of animals. GPx plays key role in antioxidant defence system by decreasing H 2 O 2 and other peroxides whereas SOD also catalyses peroxide radical and functions as rst intracellular defense against free radicals. Values did not vary during the study indicated no harmful effects of providing higher energy and protein on health and immunity parameters.
From the present study it can be concluded that feeding 30% higher ME and MP did not aided in body weight changes and in dry matter intake. Higher ME amd MP also did not caused any variation in stress and immunity parameters of Murrah buffalo. Therefore upto 30% extra ME and MP could be fed in growing Murrah buffalo calves without causing any stress though it did not increased dry matter intake and body weight changes. Declarations