Blood biochemical profile of four fast-growing broiler strains under high ambient temperature

The present study was carried out to evaluate the effect of optimum and high ambient temperatures on biochemical parameter of four broiler strains. Broiler chicks (n = 242) of four different commercial strains (Ross, Hubbard, Cobb and Arber Acer) on day 15 were divided into two groups: thermo-neutral zone (TN) group and high ambient temperature zone (HAT) group. Chicks in TN group were housed at constant room temperature (25 °C ± 2 C° and RH 65 ± 5%) while chicks in HAT group were kept under HAT of summer. Chicks in each group were further divided into four subgroups, i.e., TN-Ross, TN-Hubbard, TN-Cobb, TN-Arber Acer and HAT-Ross, HAT-Hubbard, HAT-Cobb, HAT-Arber Acer. Each subgroup was further subdivided into four replicates having ten chicks per replicate. Blood was collected on day 21 and 42. Mean serum aspartate aminotransferase (AST), alanine amino transaminase (ALT), alkaline phosphates (ALP), glucose, cholesterol, triglyceride, and low-density lipoprotein (LDL) were significantly higher (P < 0.05) in HAT group, while total serum protein and high-density lipoprotein (HDL) were significantly higher (P < 0.05) in TN group. In TN zone group, significantly (P < 0.05) lower AST, ALT, glucose, cholesterol, triglyceride, and LDL and significantly (P < 0.05) higher HDL and total protein were recorded for Cobb and Hubbard strains. In HAT zone group, significantly (P < 0.05) lower AST, ALT, glucose, cholesterol, triglyceride, and LDL and significantly (P < 0.05) higher HDL and total protein were recorded for Ross and Arber Acer strains. The findings of the present study suggested that Ross and Arber Acer strains were more tolerant to summer HAT of tropical areas than Cobb and Hubbard, while Cobb and Hubbard strains were more effective in TN environment.


Introduction
High environmental temperature during summer months of the year is also one of the most serious climate problems of tropical and subtropical countries of world, which exerts deleterious effects on performance of broiler chickens [1][2][3]. High ambient temperature and relative humidity (RH) are considered to be the most crucial factors leading to the reduction in bird's performance [3,4].
Blood plays very important role in carrying nutrient, metabolic waste, and hormonal transmission [5][6][7]. The blood biochemical profile would reflect the physiological state of the body [7]. The serum biochemical profile provides valuable information about health and immune status of animals [8]. High temperature increases serum aspartate aminotransferase and alanine amino transaminase [9][10][11], glucose [12][13][14], cholesterol [15,16], and triglyceride in broiler chicks [17]. Although the responses to heat stress differ between chickens of different genetic backgrounds [18,19]. Strains selected for rapid growth are significantly less resistant to heat stress than strains having slow growth rate [20,21]. Little is known about the biochemical profile of different broiler strains during heat stress; therefore, the present research study was designed to investigate the effect of high environmental temperature on biochemical profile of four different broiler strains.

Materials and methods
The study was approved by ethical committee on welfare and rights of animals, Faculty of Animal Husbandry and Veterinary Sciences, the University of Agriculture, Peshawar, Pakistan.

Climatic data
House temperature was recorded continuously during the experimental period. Temperature was recorded after every four hours in the center of the house at 08:00 am, 12:00 pm, 4:00 pm, 8:00 pm, 12:00 am, and 4:00 am, and then minimum and maximum of each day were averaged by week (Table 1).

Experimental design and birds' husbandry
The experiment was conducted in a completely randomized design with two factors, i.e., two temperature zones with four different broiler strains. A total of 240-day-old broiler chicks of four commercial broiler strains (Ross, Hubbard, Cobb, and Arber Acres) of the same age and size were used for the study. In first 2 weeks, chicks were placed in brooding room and then on day 15, chicks were divided into two groups: thermo-neutral zone (TN) group and high ambient temperature zone (HAT) group. Chicks in TN group were housed at constant room temperature (25°C ± 2 C°) and relative humidity (RH, 65 ± 5%) while chicks in HAT group were kept at HAT. Both the groups were kept in two different rooms, which were identical in terms of size, construction materials, and equipments. The temperature was recorded after every four hours in the center of the house, and minimum and maximum temperature of each day were averaged per week. Chicks in each group were designated as subgroup TN-Ross, TN-Hubbard, TN-Cobb, TN-Arber Acer and HAT-Ross, HAT-Hubbard, HAT-Cobb, HAT-Arber Acer. Each subgroup was further subdivided into four replicates having ten chicks per replicate. Feed and water were provided ad libitum. During first 21 days, chicks were provided with starter ration containing 22% crude protein (CP) and 3000 kcal/kg metabolizable energy (ME). During last three weeks, chicks were provided with finisher ration containing 20% CP and 3200 kcal/kg ME ( Table 2). All birds of four strains were vaccinated against infectious diseases.

Serum biochemical profile
Blood samples were collected on day 21 and day 42 from randomly selected birds (one bird per replicate) for biochemical analysis. Blood was collected from the wing vein with the help of disposable syringe using 22 gauge needles. Blood samples were centrifuged at 4000 rpm for 10 min for serum separation. Serum samples were analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), glucose, cholesterol, triglyceride, total protein, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) using autoanalyzer (Microlab 300 semiautomated biochemical analyzer) and its accompanying kits. AST and

Statistical analysis
Data were subjected to two-way analysis of variance with temperatures and strain as the main effects. The following model was used: where l = population mean, bj = treatment effect; treatment comprised of TN and HAT zones, ak = The strain effect (four commercial broiler strains Ross, Hubbard, Cobb, and Arber Acres), cl = The strain by temperature zone interaction, eijklm = The random error associated with kth chicks and jth treatment in experimental unit.
Least significant test was used to compare the differences among treatment means. All statistical analysis was carried out using statistical package of SAS [23].

Results
Mean serum AST and ALT of four different broiler strains reared in TN and HAT zones are presented in Table 3. Temperature zones significantly (P \ 0.05) affected serum AST and ALT in different broiler strains in all recorded stages. On day 21 in TN zone, significantly (P \ 0.05) lower AST and ALT were found in Cobb and Hubbard, while in HAT zone, no significant (P \ 0.05) difference was recorded in mean serum AST level. On day 42, in TN zone, significantly (P \ 0.05) lower AST level was recorded in Hubbard and was followed by Cobb, Arber Acer, and Ross, while in HAT zone, significantly (P \ 0.05) lower AST was found in Arber Acer followed by Ross. Similarly, on day 42 in TN zone, significantly (P \ 0.05) lower and the same ALT level was recorded in Ross, Hubbard, and Cobb. Mean ALT level of Arber Acer was not different from Ross, Hubbard, and Cobb on day 42. In HAT zone on day 42, significantly (P \ 0.05) lower and the same serum ALT level was recorded in Ross and Arber Acer.
Mean serum ALP and protein of four different broiler strains reared in TN zone and HAT zone are presented in Table 4. Temperature zone significantly (P \ 0.05) affected serum ALP and protein in different broiler strains at all recorded stages. On day 21 in TN zone, significantly (P \ 0.05) lower and the same serum ALP was found in Cobb and Hubbard, while in HAT zone, significantly lower (P \ 0.05) and the same ALP level was recorded in Ross and Arber Acer followed by Cobb and Hubbard. On day 42 in TN zone, no significant differences were recorded in mean serum ALP levels of different broiler strains. On day 42 in HAT zone, significantly lower (P \ 0.05) ALP was recorded in Arber Acer as compared to all other strains. In TN zone, significantly (P \ 0.05) higher and the same serum protein was recorded for Hubbard and Cobb, while in HAT zone, significantly (P \ 0.05) higher and the same serum protein was recorded for Ross and Arber Acer on day 21. On day 42 in HAT zone, significantly higher (P \ 0.05) serum protein was found in Ross followed by Arber Acer, while the lowest and the same serum protein levels were recorded for Hubbard and Cobb.
Mean serum glucose of four different broiler strains reared in TN and HAT zone is presented in Table 5. Temperature zone significantly (P \ 0.05) affected serum glucose in different broiler strains at all recorded stages. On day 21 in TN zone, significantly (P \ 0.05) lower and the same serum glucose level was found in Cobb and Hubbard followed by Arber Acer and Ross, while in HAT zone, significantly lower glucose was recorded in Arber Acer followed by Ross. Significantly (P \ 0.05) higher and the same glucose level was recorded in Hubbard and Cobb on day 21 in HAT zone. On day 42 in TN zone, significantly (P \ 0.05) lower serum glucose level was recorded in Hubbard and Cobb. On day 42 in HAT zone, significantly (P \ 0.05) lower serum glucose was found in Ross followed by Arber Acer, while significantly (P \ 0.05) higher and the same serum glucose levels were recorded in Cobb and Hubbard.
Mean serum cholesterol and triglycerides of four different broiler strains reared in TN zone and HAT zone are presented in Table 6. Temperature zone significantly (P \ 0.05) affected serum cholesterol and triglycerides level in different broiler strains at all recorded stages. On day 21 in TN zone, significantly (P \ 0.05) lower serum cholesterol was found in Hubbard followed by Cobb, while in HAT zone, significantly lower and the same serum On day 21 in TN zone, significantly (P \ 0.05) lower serum triglyceride was found in Hubbard and Cobb, while higher and the same triglyceride level was recorded in Ross and Arber Acer. On day 21, in HAT zone, significantly (P \ 0.05) lower and the same triglyceride was recorded in Ross and Arber Acer. On day 42, in TN zone, no significant difference was recorded in serum triglyceride levels among the strains. In HAT zone on day 42, significantly lower (P \ 0.05) serum triglyceride was found in Ross followed by Arber Acer, Hubbard, and Cobb.
Mean serum LDL and HDL of four different broiler strains reared in TN and HAT zones are presented in Table 7. Temperature zone significantly (P \ 0.05) affected LDL in different broiler strains on all recorded stages. On day 21 in TN zone, significantly (P \ 0.05) lower LDL was found in Hubbard followed by Cobb, Ross, and Arber Acer. Mean LDL level was the same in Cobb, Ross, and Arber Acer in TN group on day 21. In HAT zone on day 21, significantly (P \ 0.05) low and the same LDL was recorded in Ross and Arber Acer. On day 42 in TN zone, no significant difference was found in mean LDL, while in HAT zone, significantly (P \ 0.05) lower LDL was found in Ross followed by Arber Acer. On day 21 in TN zone, significantly (P \ 0.05) higher HDL was found in Hubbard followed by Cobb while the lowest HDL level was recorded for Ross and Arber Acer. On day 21, in HAT zone, significantly (P \ 0.05) higher serum HDL was found in Ross, while HDL level was lower and the same in all other strains. On day 42 in TN zone, significantly (P \ 0.05) higher and the same serum HDL was found in Cobb and Hubbard. On day 42, in HAT zone, significantly (P \ 0.05) higher serum HDL was recorded in Ross followed by Arber Acer, Cobb, and Hubbard.

Discussion
Temperature zone significantly affected serum AST, ALT, ALP, and protein concentration in different broiler strains at all recorded stages. The increase in serum biochemical parameters could possibly be linked to the higher production of reactive oxygen species (ROS) at cellular level. Free radicals affect blood serum metabolites [22,24]. The increased serum ALT level may be due to the decreased antioxidative enzymes and ROS at the cellular level [21].
Decreased activity of antioxidative enzymes alters the balance between the production of ROS and the antioxidant system, which affects serum metabolites [21]. Under heat stress conditions, alteration in serum enzymes activity occurs [11,25]. Serum ALT level is measured clinically as a part of diagnostic liver function tests to determine liver health status, and its activities are generally increased with muscle or liver damage [26,27]. Our findings are in agreement to the results of Kohn and Nyska [28] who reported increased AST and serum ALT level for broilers exposed to high temperature (42-45°C). Our results are in line with the findings of another study, which reported increased serum activities of AST and ALT at high ambient in broiler chicks [29].
Mean serum protein in different broiler strains reared in TN and HAT zones was significantly affected by temperature zone. Significantly lower serum protein was recorded in HAT zone. Oxidative stress increases production of ROS [30][31][32][33] resulting in denaturation of biomolecules such as nucleic acid, protein, and enzymes. The reduction in serum protein may be linked to the increased oxidative stress caused by HAT, which could be partially due to the reduced intake of protein and deficiency of essential amino acids [34]. Reduced digestibility of proteins at HAT may also be responsible for low serum protein level [35]. Our results are in agreement with previous results of Faisal et al. [36] who reported that HAT decreases serum protein in Japanese quails.
Temperature zone significantly affected mean serum glucose levels of four different broiler strains at all recorded stages. Significantly higher serum glucose level was recorded at HAT. The increased blood glucose could be due to the endocrine activity of adrenal hormones, which accelerate gluconeogenesis in the cell [37,38]. Contrary to the findings of the resent study, Sands et al. [39] reported High serum cholesterol levels at HAT may be caused by hyperactivity of the adrenal gland, which produces adrenocorticotropic hormones [38] for which cholesterol acts as a precursor. Our findings are in agreement with the results of Sohail et al. [10] who reported that serum cholesterol in broiler was higher under HAT than TN zone. Gursu et al. [17] found that heat stress (34°C) increases serum concentrations of cholesterol. The increase in serum triglyceride may be due to the overproduction of glucocorticoids and corticosterone at HAT. High ambient temperature stimulates the release of corticosterone from the adrenal glands and increases plasma concentrations of glucocorticoids in birds [27]. Glucocorticoids stimulate triglyceride synthesis in the liver by enzyme phosphatidate phosphodydrolase [41]. Our findings are in agreement with the results of Imik et al. [42] who found that serum triglyceride, LDL, and HDL were higher in broilers reared in HAT zone compared to birds reared in TN zone.
It is concluded from the present study that interaction of broiler strains and temperature zones affected the serum biochemistry. Ross and Arber Acer strains were more tolerant to summer high ambient temperature of tropical areas than Cobb and Hubbard.