Mitigating Heat Stress By Dietary Manipulation of Dried Pulp of Tamarind (Tamarindus Indica L.) In Broilers Ration: Growth Dynamics, Lipids Prole, Redox Balance, Cellular And Humoral Immune Response

A total of 700 day old broiler chicks (Arbor Acres) were weighed and allocated to 28 oor pens (25 birds per pen). Five diets were prepared and allocated in a completely randomized design consisting of a control (basal diet) and four levels of dried Tamarind seeds pulp (DTSP) powder at the rate of 5 (DTSP-5), 7.5 (DTSP-7.5), 10 (DTSP-10) and 12.5 (DTSP-12.5) g/kg of feed for 35 days under heat stress condition. Body weight and feed conversion ratio (FCR) increased signicantly (P<0.05) in the DTSP fed broilers compared to the control. Blood concentration of paraoxonase-1 (PON1) was signicantly (P<0.05) higher in the DTSP-10 and DTSP-12.5 compared to the control. However, antibody titre against New Castle disease (ND) was signicantly (P<0.05) higher in DTSP-7, DTSP-10 and DTSP-12.5 compared to the control. Blood cholesterol and triglyceride were signicantly (P<0.05) lower in DTSP-7, DTSP-10 and DTSP4-12.5 compared to the control. However, blood HDL was signicantly (P<005) higher in DTSP-10 and DTSP-12.5 compared to the control. Blood LDL was signicantly (P<0.05) lower in DTSP-7, DTSP-10 and DTSP-12.5 compared to the control. Signicantly (P<0.05) higher total leucocytes count (TLC) was found in DTSP-12.5, however, heterophils were signicantly (P<0.05) lower in the same group. In contrast, lymphocytes increased signicantly (P<0.05) in DTSP-7, DTSP-10 and DTSP-12.5 while monocytes was signicantly (P<0.05) decreased in the same group. In conclusion, DTSP-5 supplementation improved growth, however, redox balance, cholesterol prole and cellular and humoral immunity increased in DTSP-5, DTSP-7.5 and DTSP-12.5 in broilers exposed to heat stress.


Introduction
Though there is no universal de nition of heat stress, however, it can be de ned as environmental temperature beyond physiological temperature of the body, which results in adverse consequences and ultimately reduces the growth performance of the broiler chickens (Khan et al. 2012). In poultry production, heat stress occurs when the ambient temperature exceeds 25°C. The heat exposure may be from several days to several weeks and the temperature may go as high as 50°C (Kpomasse et al. 2021). Research studies have reported the deleterious effects of heat stress on growth, livability, carcass quality, physiological, and biochemical changes in broilers (Khan et  has strong carminative effect (Cabuk et al. 2003). Dried Tamarind seeds pulp (DTSP) is an excellent source of polyphenol such as oligomeric anthrocyanidins, anthrocyanidin and tannins (Pumthong 1999).
Further, Gu et al. (2003) reported that DTSP contains phenolic antioxidants such as epicatechin, 3,4dihydroxyphenyl acetate, methyl 3,4-dihydroxybenzoate, 40-dihydroxyacetophenone and 2-hydroxy-30. The extract of Tamarind pulp has also exhibited antioxidant potential in vitro (Tsuda et al. 1993). The pulp of this fruit is though edible, it is still considered as an industrial waste in countries where it is grown abundantly (Galvao et al. 2019). The dry matter of pulp generated during processing of this fruit is about 50 to 65% and comprised of modest amount of crude protein (74.8 g/kg), neutral detergent bre (609g/kg), non-berous carbohydrate (268 g/kg) in addition to lignin (185 g/kg) and tannin (75 g/kg) as reported Souza et al. 2018). Despite a number of valuable phytoconstituents and properties of Tamarind pulp, there is scant scienti c information on its application in broiler production and health under heat stress. Therefore, the aim of the present study was to evaluate the inclusion of different levels of DTSP on the production performance, cellular and humoral immunity, lipids pro le and antioxidant status in broiler exposed to heat stress.

Materials And Methods
This experiment was carried out under the care and use of experimental animals committee, The University of Agriculture Peshawar.

Preparation of DTSP powder
Fresh fruit of Tamarind was obtained from Tamarind tree, washed with cleaned water and shade-dried for two weeks at room temperature. The pulps were ground into ne powder. Animals and experimental designs A total of 700 day old broiler chicks (Arbor Acres) were weighed and allocated to 28 oor pens (25 birds per pen) bedded with wood shavings. Each pen measured 160 ×240 cm, equipped with round pen feeders and drinkers. Birds had free access to feed through feeders and drinkers throughout the study. Birds were vaccinated against the infectious diseases including New Castle disease according to the standard schedule. The basal food composition is given in Table 1. Birds were kept under uorescent lighting for 23 h for the rst week and then a 20 light: 4 dark light cycle. Birds were exposed to a cyclic heat stress cycle as shown in Table 2.  Experimental protocols and samplings Five diets were prepared and allocated in a completely randomized design consisting of a control (basal diet) and four levels of DTSP powder at the rate of 5 (DTSP-5), 7.5 (DTSP-7.5), 10 (DTSP-10) and 12.5 (DTSP-12.5) g/kg feed for 35 days including rst week as adaptation period. Feed intake, body weight gain and feed conversion ratio were measured per pen basis on a weekly interval and the average of each parameter for the whole period was recorded. On day 35, three birds per pen were slaughtered and 3ml of blood samples were taken into two separate tubes, one with EDTA and another without EDTA. Blood without EDAT was centrifuged (3000 rpm for 10 minutes) and preserved at -80°C. Blood with EDTA was stored at -4°C until analysis. Serum melanodialdehyde (MDA) and paraoxonase (PON-1) For the determination of lipid peroxidation in the serum samples, thiobarbituric acid (TBA) reaction was used as described by Ohkawa et al. (1979). The colour produced by the reaction of TBA and MDA was measured spectrophotometrically at 532nm. Blood PON-1 was determined by the method of Mackness et al. (1991) using phenol as a substrate. The working reagent was consisted of 1mM phenylacetate and calcium chloride and 20mM Tris HCl buffer. Change in the absorbance was recorded for ve minutes at 270 nm after diluting the sample in 1:3 with buffer before mixing with the substrate. Cellular and humoral immunity Total leucocytes (TLC) and differential leucocytes were carried on blood with EDTA by the method described by Chand et al. (2016). Humoral response against the ND virus was carried out using haemagglutination inhibition test.

Blood lipid pro le
Blood lipid pro le of the control and treatment groups was determined with the help of commercial kits (Biocheck, UK) using chemistry analyzer.

Statistical analysis
Analysis of data was done by software (Statistix 8.1) by using Completely Randomized Design.
Signi cant difference among the groups was computed using Tukey test at the level of 5% probability.

Results
The ndings of feed intake, weight gain and FCR in broilers exposed to heat stress in response to different levels of DTSP is given in Table 2. The feed intake data did not differ between the control and the treatment groups. Body weight increased signi cantly (P < 0.05) in the DTSP fed broilers compared to the control. Similarly, FCR of the DTSP supplemented birds was signi cantly (P < 0.05) higher compared to the control broilers.
The effect of different levels of DTSP on MDA, PON1 and ND titre on broilers during heat stress is given in Table 3. The value of MDA did not change between the control and treatment groups. Blood concentration of PON1 was signi cantly (P < 0.05) higher in the DTSP-10 and DTSP-12.5 compared to the control. However, ND titre was signi cantly (P < 0.05) higher in DTSP-7, DTSP-10 and DTSP-12.5 compared to the control. The ndings of the effect of DTSP in broilers during heat stress on the blood cholesterol pro le are given in Table 4. The results indicated that blood cholesterol and triglyceride were signi cantly (P < 0.05) lower in DTSP-7, DTSP-10 and DTSP-12.5 compared to the control. However, blood HDL was signi cantly (P < 005) higher in DTSP-10 and DTSP-12.5 compared to the control. Blood LDL was signi cantly (P < 0.05) lower in DTSP-7, DTSP-10 and DTSP-12.5 compared to the control. The ndings of total blood leucocytes count and differential leucocytes count in broilers during heat stress is given in Table 5. Signi cantly (P < 0.05) higher TLC was found in DTSP-12.5 compared to the control and rest of the treatment groups, however, heterophil concentration was signi cantly (P < 0.05) lower in the same group compared to the control and the other treatment groups. However, lymphocytes increased signi cantly (P < 0.05) in DTSP-7, DTSP-10 and DTSP-12.5 while monocytes signi cantly (P < 0.05) decreased in the same group.

Discussion
In the present study, feed intake did not differ between the control and treatment groups of birds under heat stress conditions; however, weight gain and FCR were signi cantly improved in the treatment groups. Similar to our study, Aengwanich et al. (2009)  In the current study, no signi cant change was observed in the serum MDA concentration in the control and treatment groups, however, PON1 concentration was increased signi cantly in DTSP-10 and DTSP-12.5 compared to the control. In some recent studies, improved serum PON1 was observed in broilers supplemented with different natural compounds such as taurine, In the present study, antibody titre against ND decreased in the control group and improved in DTSP-7.5, DTSP-10.0 and DTSP-12.5. The lymphocyte count is decreased during heat stress ) as seen in this study and hence there is a lower antibody titre against an infection. The higher antibody titre against ND in this study seems to be due to the production of greater levels of lymphocytes in response to Tamarind supplementation.
The search for novel agents regulating blood cholesterol has gained momentum over the last few decades, resulting in a signi cant number of reports on natural agents with anticholesteremic activities.
In view of the previous experiments, we evaluated the effect of DTSP on serum lipid pro le. The seed coat of Tamarind is a low-cost source of antioxidant and exhibits antioxidant activity against thiocyanate and thiobarbituric (Pazhanivelan et al. 2008). Lipids are easily susceptible to the injury of free radicals. Compounds in Tamarid have been reported to improve the lipid pro le (Martinello et al. 2006). In the current study, Tamarind pulp exhibited a signi cant reduction in lipid pro le in serum of broilers. Environmental temperature is one of the most important factors affecting the immune system of broiler chickens. Exposure of the chickens to high ambient temperature causes signi cant physiological changes including immunosuppression and high mortality rate (Khan et al. 2012). Leucocyte count has been used as an indicator of heat stress in poultry. In the current study, TLC increased signi cantly in birds fed with DTSP at the rate of 12.5 g/kg. Moreover, heterophil and monocytes count increased in the control and DTSP-5 and the same were restored at the higher doses. Lymophocytes count was also lower in the control and DTSP-5, however, it was restored at higher doses. Rise in TLC is considered to enhance the immune system with increased resistance against infectious diseases and improvement in production performance. Almost similar ndings were reported by Shinde et al. In conclusion, weight gain and FCR were improved in broilers supplemented with DTSP. PON1 was improved in DTSP-10 and DTSP-12.5 while antibody titre was greater in DTSP-7.5, DTSP-10 and DTSP-12.5 in broilers during heat stress. Except DTSP-5, total cholesterol and LDL increased in the rest of the treatment groups. Furthermore, TLC increased signi cantly in DTSP-12.5 while lymphocytes count was greater in all the treatments except DTSP-5. Similarly, heterophils and moncytes count decreased signi cantly in all the treatment groups except DTSP-5.

Declarations
Con ict of interest

Competing interest
There is no potential competing interest with this study.
Consent to participate and consent to publish All the authors have equally participated in this study and agreed to publish this work in this journal.

Authors Contribution
MNH conducted the study and analyzed the samples. NC developed the study design. RUK edited, submitted and revised the paper.

Data availability
Data is available in the thesis.