Effect of Low Protein Diet Supplemented with Protease Enzyme and Sodium Bisulphate in Litter on Carcass, Biochemical, Respiratory Tract Lesions and Immunity Status of Birds during Winter Season

The quality of litter is a single major factor in deciding the emission of various harmful gases especially ammonia. Therefore a study was conducted to determine the effect of low protein diet supplemented with protease enzyme and litter amended with sodium bisulphate on carcass parameters, serum biochemical, respiratory tract lesions and immune status of birds during winter season. The experiment was conducted for six week on 240, day old broiler chicks which were randomly distributed into four treatment groups. One bearing control group (Tc) had no dietary and litter amendments and the other three included, litter amendment with sodium bisulphate (Ts), dietary amendment with low protein supplemented with protease enzyme (Tp) and both dietary and litter amendment (Tsp), each having 60 birds in three replications of 20 number. Blood sample for serum biochemical parameters were collected at 3rd and 6th week of age and at the end of experiment, four birds from each treatment were randomly slaughtered for carcass trait measurement and respiratory tract lesions. The results revealed a higher dressing and breast percentage in Ts group as compared to other treatment groups. Total protein level was also significantly higher in Ts and control group. The histopathology revealed that the gross as well as microscopic lesions revealed a better health status of chicks in the treatment groups Ts and Tsp than those in control and Tp group. The immune status was also better in the litter treated groups. Thus it can be concluded that the treatment of litter with sodium bisulphate had significant influence on carcass, serum biochemical, respiratory and foot pad lesions and immune status of birds.

In the modern scenario, poultry farming is gaining strength with fast pace of development to attain mega commercial agro-industrial business in India. Due to changing demands of people, rapid urbanization and population growth, the broiler industry has been the most profitable agro-economic enterprise. Broilers are generally reared in deep litter system of rearing. The broiler house environment is a reflection of overall efficiency of grows out operation. The bedding material used in deep litter system absorbs moisture in order to keep litter dry and ensures a comfortable environment for birds. Wet litter results in high ammonia production which negatively affects productive performance of broilers (Ritz, 2006).
Keeping litter dry is a critical part of management of any poultry farm because bird spends most of their lifetime in close contact with the bedding material. Litter quality has a major effect on health and performance of birds. Dry litter helps to control the ammonia level, provide a healthy flock environment, reduces accusation due to hock, foot pad burns and breast blisters. Uric acid and nitrogen in the litter is converted to ammonium (NH4+) by the action of microbes in litter. This ammonium is converted to ammonia depending upon the moisture content, temperature and acidity of litter. Ammonia production is favored by high temperature and high pH (Ritz et al., 2004). Researchers suggested that high ammonia Proch et al. concentration (>25ppm) can impair performance and immunity of bird and increase susceptibility to respiratory tract infections (Kristensen and Wathes, 2000;Nagaraja et al., 1983). Increased levels of ammonia from litter cause severe irritation to respiratory tract and skin of birds resulting in pododermatitis and breast blister. There are several methods to decrease the nitrogen content in litter and ammonia produced from litter. The main methods include dietary amendment, litter amendment, adequate ventilation, use of acidifier in poultry litter and use of enzymes. Many scientists have reported that the most potent method to inhibit ammonia production in a poultry house is chemical litter amendment. Many litter additives such as alum, clay, FeSO 4 , NaHSO 4 have been used to reduce litter pH, reduce ammonia volatilization and inhibit microbial load on litter. Sodium bisulphate, a dry crystalline acidifier is widely used by the broiler industry to control ammonia levels and to provide a comfortable environment to birds. It is readily soluble in water and has a pH <1. Sodium bisulphate decreases ammonia volatilization by lowering the litter pH, interacting with the uric acid and by lowering the microbial population that produces ammonia. The excretion of nitrogen in litter can also be reduced by supplementation of various enzymes in a low protein diet. The low protein diet supplemented with proteolytic enzymes like protease helps in effective utilization of available protein present in the diet. In view of above facts and figures, the present study was planned to estimate the effect of sodium bisulphate in litter and protease enzyme in a low protein diet on carcass parameters, serum biochemical, respiratory tract lesions and immune status of birds during winter season.

MATERIALS AND METHODS
The whole experiment was carried out as per the code of practices approved by Institute of Animal Ethics Committee GADVASU, Ludhiana, Punjab-141004, India (Permission no: GADVSAU/2016/IAEC/32/15).

Research Methodology
The experiment was conducted at poultry research farm of department of Livestock Production Management, Guru Angad Dev Veterinary and Animal Science University, Ludhiana. All preparation in the poultry house was made much before the arrival of chicks. After removal of all equipments and old litter, the house was thoroughly cleaned, washed and disinfected. Around two inches layer of litter of rice husk was spread all over the floor. Sodium bisulphate @ 25gm/sq.ft was mixed in the litter of treatment groups Ts and Tsp. The brooders were kept in the centre of each pen to ensure uniform heating and were switched on 24 hours before the arrival of chicks to obtain the required temperature. Day old, 240 commercial VENCOBB-400 broiler chicks with similar body weight range and average group weight were randomly divided into four groups, each having 60 birds in 3 replications of 20 birds each. In the experiment (Table 1), four various management strategies viz; litter manipulation with sodium bisulphate (T S ), dietary manipulation with low CP and protease supplementation (T P ) and a combination of both litter manipulation with sodium bisulphate and dietary manipulation with low CP and protease supplementation (T SP ), was tested and compared with control group without implication of any litter or nutritional strategy (T C ) during December 2016 to January 2017.   (Silva, 2007). The ingredient composition of the diet given to different treatment group is presented in Table 2 prepared as per ICAR (2013) schedule. All other management and rearing practices were similar for all the groups.

Carcass parameters
On 42 nd day, 4 birds in each treatment were randomly sacrificed for the recording of carcass yield, cut-up parts using the standard procedures (Ricard and Rouvier, 1967).

Biochemical parameters
At the age of 3 rd and 6 th weeks, four birds from each treatment were randomly selected for collection of blood samples. The serum biochemical parameters evaluated were SGOT, SGPT, LDH, Creatinine, BUN, total protein and blood glucose by using BPC BioSed Srl kits (Roma, Italy).

Histopathological evaluation
All tissues samples viz. trachea, lungs, liver and kidney were collected and fixed in 10% neutral buffered formalin solution at the time of sacrifice. After fixation tissue samples were given overnight washings under tap water. Then, dehydration of samples was done through ascending grades of alcohol (70%, 80%, 90%, and absolute alcohol) followed by clearing with acetone and benzene. Tissues were embedded in paraffin wax (Leica Microsystem, Paraplast tissue embedding medium, 56°C for further processing) and 4-5μ thick sections were cut. The paraffin sections were stained with routine hematoxylin technique (Luna, 1968). Slides were examined by BX6I Research Photomicrograph Microscope System of Olympus Corporation, USA. The tissues were screened for the presence of any histopathological changes in the organ system.

Immune status
On day 7 th and 14 th post vaccination, four birds from each treatment were randomly picked up to obtain blood sample. Two ml of blood was drawn by cardiac puncture and was allowed to clot at room temperature. The serum thus collected was used to evaluate the antibody titre. The antibody specific for New Castle Virus (NDV) was detected in sera of chicks by means of haemagglutination inhibition (HI) test.

Foot Pad Score
All the birds in each treatment were examined from 4 th week at weekly interval to record the occurrence of foot pad lesion if any.

Statistical analysis
The collected data from the experiment was subjected to statistical analysis using Software Package for Social Sciences (SPSS, version 20.0) by analysis of variance (Snedecor and Cochran, 1980) to test the difference among treatments. The treatment means were compared by Duncan's Multiple Range Test (Duncan, 1995) at 5% level of significance (p ≤ 0.05).

RESULTS AND DISCUSSION
The data on carcass yield and cut-up part of the meat in various treatment groups have been presented in Table 3. The data revealed that dressing percentage of different treatments varied from 61 to 65%. The Ts group had significantly (p ≤ 0.05) higher dressing percentage followed closely by Tsp group then by Tc and Tp group. The yield of breast meat was also significantly (p ≤ 0.05) higher in Ts group as compared to control group. The abdominal fat % although non significant but was numerically higher in control group as compared to other treatment groups. This might be due to the amendment of litter with sodium bisulphate because it has antibacterial and bacteriostatic properties. Similar findings were found by Karimi et al. (2011) and Younis et al. (2016) who stated that dressing percentage and giblet percentage is more in litter treated groups as compared to control group. Enzyme activities of SGOT and SGPT are an indirect assessment for the feed consumed and these activities are used as an index for liver tissue integrity. The data for all biochemical parameters is presented in Table 4 and revealed that during initial phases of growth (3 rd week), the level of SGPT was significantly (p ≤ 0.05) higher in Ts and control group as compared to Tp and Tsp group. Total protein level was also higher in Ts and control group. However in later stages of growth (6 th week), SGPT level was numerically higher in Ts group as compared to all other treatment groups. The total protein level was significantly (p ≤ 0.05) higher in Ts and control group as compared to Tp and Tsp. These findings are in concurrent with the findings of Soliman and Hassan (2017) who reported that there occurs significant improvement in biochemical parameters of the birds which were reared in litter treated groups.
The changes in the histopathology of trachea, lungs and kidney of broiler chicks is shown in Fig. 1, 2 and 3. Tezrich et al. (1998) also reported significantly betterment of respiratory lesion in sodium bisulphate treated litter chicks than that of untreated groups. The reduction in respiratory tract lesions among broilers raised on litter treated groups was associated with reduction in atmospheric ammonia concentration.
The antibody titre data for immune status of the birds is presented in Table 5.    These data revealed that at 7 th day post vaccination, the antibody titre of New Castle Disease (NDV) in Ts and Tsp groups had significant (p ≤ 0.05) higher titre as compared to Tc and Tp group. On 14 th day post vaccination, both litter treated groups Ts and Tsp had significantly (p ≤ 0.05) higher value than the control and Tp group indicating poor immune response in the later group. The results are in agreement with the findings of Sahoo et al. (2015) who reported that antibody titre of treated litter group was higher as compared to untreated litter group. As reported by Abbasi et al. (2014) diets with different CP and threonine levels had no marked effect on antibody titers against NDV.
The foot pad score numerically had lower values in litter amended groups i.e. Ts and Tsp as compared to Tp and control group (Table 6). No incidence of leg abnormality was observed in the control as well as treatment group. There was zero breast blister score in the control as well as treatment group. The cake formation in the litter was more in Tp and control group as compared to Ts and Tsp group.
The results are in agreement with the findings of Nagraj et al. (2007) who reported that sodium bisulphate as a litter amendment numerically reduced the incidence of pododermatitis by 10 or more percentage points.

CONCLUSION
Addition of sodium bisulphate in litter had a significant (p ≤ 0.05) effect on dressing and breast percentage of the birds. Low protein diet with protease supplementation had no significant effect on carcass parameters. There was no change in serum biochemical levels except SGPT and total protein levels probably by improving nutrient digestibility, mainly protein. The immune status of the birds reared in litter treated groups had better immunity status as compared to untreated litter group. Moreover, the foot pad score was also better in the litter amended groups. So, the use of sodium bisulphate in litter is beneficial to maintain the litter quality to have a profitable broiler production.