Effect of vitamin C and acetylsalicylic acid supplementation on some hematological value, heat shock protein 70 concentration and growth hormone level in broiler exposed to heat stress

1.      Yalcin S, Settar P, Özkan S, Cahaner A. Comparative evaluation of three commercial broiler stocks in hot versus temperate climates. Poult Sci. 1997;76(7):921-929. https://doi.org/10.1093/ps/76.7.921

2.      Ahmadu S, Mohammed AA, Buhari H, Auwal A. An overview of vitamin C as an antistress in poultry. Malays J Vet Res. 2016; 7:9-22.

3.      Rivera RE, Christensen VL, Edens FW, Wineland MJ. Influence of selenium on heat shock protein 70 expressions in heat-stressed turkey embryos (Meleagris gallopavo). Comp Biochem Physiol. 2005;142(4):427-432. 10.1016/j.cbpa.2005.09.006

4.      Krebs RA, Bettencourt BR. Evolution of thermotolerance and variation in the heat shock protein, Hsp70. Amer Zool. 1999; 39:910-919. 10.1093/icb/39.6.910

5.      Gharib HBA, El-Menawey MA, Attalla AA, Stino FKR. Response of commercial layers to housing at different cage densities and heat stress conditions. Egyptian J Anim Prod. 2005; 42:47-70.‏

6.      Mazzi CM, Ferro MIT, Coelho AAD, Savino JVM, Macari M, Ferro JA, Givisiez PEN, Giachetto PF, Silva MM, Dionello NJL. Effect of heat exposure on the thermoregulatory responses of selected naked neck chickens. Braz J Vet Res Anim Sci. 2002;54(1):35-44. 10.1590/50102-09352002000100006

7.      Wu D, Zhang M, Lu Y, Tang S, Kemper N, Hartung J, Bao E. Aspirin-induced heat stress resistance in chicken myocardial cells can be suppressed by BAPTA-AM in vitro. Cell Stress Chaperones. 2016; 21:817-827.

8.      Truong A, Sayago AM, Kutteh WH, Ker W. Sub-chorionic hematomas are increased in early pregnancy in women taking low-dose aspirin. Fertil Steril. 2016; 105:1241-1246. 10.1016/j.fertnstert.2016.01.009

9.      Wu D, Xu J, Song E, Tang S, Zhang X, Kemper N, Hartung J, Bao E. Acetylsalicylic acid protected against heat stress damage in chicken myocardial cells and may associate with induced Hsp27 expression. Cell Stress Chaperones. 2015; 20:687-696.

10.   Alagawany M, Farag MR, El-Hack MA, Dhama K, Fowler J. Use of acetylsalicylic acid as a feed additive in poultry nutrition. World's Poult Sci J. 2017;73(3):633-642. 10.1017/S0043933917000253

11.   Sahin K, Küçük O, Sahin N. Effects of dietary chromium picolinate supplementation on performance and plasma concentrations of insulin and corticosterone in laying hens under low ambient temperature. J Anim Physiol Anim Nutr. 2001; 85:142-147. 10.1046/j.1439-0396.2001.00314.x

12.   Blaha J, Kroesna K. Effect of vitamin and electrolytes supplements on broiler performance, slaughter value and chemical composition of meat during the heat stress. Agri Trop Subtrop. 1998; 30:103-113.

13.   National Research Council (NRC). Nutrient requirements of poultry 9^th rev. ed National Academy Press, Washington, DC.

14.   Tamzil MH, Haryani NK, Indarsih B. Reduced Expression of Heat Shock Protein (HSP) 70 Gene by ascorbic acid supplementation in broiler chickens exposed to transportation stress to maintain the quality of meat and hematological parameters. Inter J Poult Sci. 2016;432-441. 10.3923/ijps.2016.432.441

15.   Shoukary RD, Madeha HA, Mootaz AM. Differential leucocyte count and total colony count changes in the heat-stressed broiler. J Adv Vet Res. 2015;5(1):21-28.

16.   Rahman I, Kode A, Biswas SK. Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc. 2007; 1:3159-3165.10.1038/nprot.2006.378

17.   Steel R GD, Torrie JH, Dickey DA. Principles and procedures of statistics: A Biometrical Approach. 3^rd ed. New York: McGraw-Hill Book Co.; 1997.350-386p 10.4236/blr.2014.5424.

18.   Mozo J, Emre Y, Bouillaud F, Ricquier D, Criscuolo F. Thermoregulation: what role for UCPs in mammals and birds? Biosci Rep. 2005;25(3-4):227-249.https://doi.org/10.1007/s10540-005-2887-4

19.   King YT, Lin CS, Lin JH, Lee WC. Whole-body-hyperthermia-induced thermotolerance is associated with the induction of heat shock protein 70 in mice. J Exp Biol. 2002; 205:273-278.

20.   Abele D, Heise K, Pörtner HO, Puntarulo S. Temperature-dependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam Mya arenaria. J Exp Biol. 2002; 205:1831-1841.

21.   Hassan AA, Al-Ma'atheedi MS. Effect of some antioxidants on blood picture and antioxidants status in roosters exposed to oxidative stress. Iraqi J Vet Sci. 2012;26(2):55-61.  http://dx.doi.org/10.33899/ijvs.2012.67441

22.   Jassim HM, Hassan AA. Changes in some blood parameters in lactating female rats and their pups exposed to lead: Effects of vitamins C and E. Iraqi J Vet Sci. 2011;25(1):1-7.

23.   Bryszewska M, Zavodnik IB, Niekurzale A, Szosland K. Oxidative processes in red blood cells from normal and diabetic individuals. Biochem Mol Biol Int. 1995; 37:345-354.

24.   Arbos KA, Claro LM, Borges L, Santos CA, Weffort AM. Human erythrocytes as a system for evaluating the antioxidant capacity of vegetable extracts. Nutr Res. 2008; 28:457-463. https://doi.org/10.1016/j.nutres.2008.04.004

25.   Sivilotti ML. Oxidant stress and hemolysis of the human erythrocyte. Toxicol Rev. 2004; 23:169-188. 10.1016/j.nutres.2008.04.004

26.   Pandey KB, Rizvi SI. Biomarkers of oxidative stress in red blood cells. Biomed Papers. 2011;155(2):131-136. 10.5507/bp.2011.027

27.   Abdullah AM. Effect of acetylsalicylic acid (ASA) in drinking water on productive performance and blood characteristic of layer hens during heat stress. Inter J Poult Sci. 2010;9(4):382-385.

28.   Tras BF, Inal AL, Bas V, Ahunok M, Elmas E, Yazar. Effects of continuous supplementations of ascorbic acid, aspirin, vitamin E and selenium on some hematological parameters and serum superoxide dismutase level in broiler chickens. Poult Sci. 2000; 41:664-666. 10.1080/00071660020009225

29.   Usman BA, Mani AU, Muyiwa OB. Effects of dietary treatments of ascorbic acid on the blood parameters, egg production and quality in quail (Coturnix coturnix japonica) subjected to heat stress. Inter J Poult Sci. 2008;7(4):344-349.

30.   Kim JH, Mamuad LL, Yang CJ, Kim SH, Ha JK, Lee WS, Lee SS. Hemato-biochemical and cortisol profile of Holstein growing-calves supplemented with vitamin C during the summer season. Asian-Australasian J Amin Sci. 2012;25(3):361. 10.5713%2Fajas.2011.11438

31.   Adenkola AY, Angani MT. Ascorbic acid supplementation effect on hematology and oxidative stress parameters of broiler chicken during the hot-dry season in southern Guinea Savannah. J Poult Res. 2017;14(1):28-33.

32.   Altan O, Altan A, Cabuk M, Bayraktar H. Effects of heat stress on some blood parameters in broilers. Turk J Vet Anim Sci. 2000; 24:145-148.

33.   Huff GR, Huff WE, Balog JM, Rath NC, Anthony NB, Nestor KE. Stress response differences and disease susceptibility reflected by heterophil to lymphocyte ratio in Turkeys selected for increased body weight. Poult Sci. 2005; 84:709-717. 10.1093/ps/84.5.709

34.   Altan Ö, Pabuçcuoğlu A, Altan A, Konyalioğlu S, Bayraktar H. Effect of heat stress on oxidative stress, lipid peroxidation and some stress parameters in broilers. British Poult Sci. 2003;44(4):545-550. https://doi.org/10.1080/00071660310001618334

35.   Langsdorf LJ, Zydney AL. Effect of uremia on the membrane transport characteristics of red blood cells. Blood. 1993;81(3):820-827.

36.   Maurice DV, Lightsey SF, Abudabos A, Toler JE. Factors affecting ascorbic acid biosynthesis in chickens: III. Effect of dietary fluoride on l‐gluconolactone oxidase activity and tissue ascorbic acid (ASA) concentration. J Anim Physiol Anim Nutrit. 2002;86(11‐12):383-388. 10.1046/j.1439-0396.2002.00402.x

37.   Holik V. Management of laying hens to minimize heat stress. Lohmann Inform. 2009;44(1):16-29.

38.   Abou El-Soud SB, Ebeid TA, Eid YZ. Physiological and antioxidative effects of dietary acetylsalicylic acid in laying Japanese quail (Coturnix japonica) under high ambient temperature. J Poult Sci. 2006;43(3):255-265.

39.   Maak S, Melesse A, Schmidt R, Schneider F, Von Lengerken G. Effect of long-term heat exposure on peripheral concentrations of heat shock protein 70 (Hsp70) and hormones in laying hens with different genotypes. British Poult Sci. 2003;44(1):133-138.‏10.1080/0007166031000085319

40.   Abdulwahid AJ, Hassan AA. Effect of zinc sulphate and cadmium chloride on heat shock protein 70 and some sex hormones in adult male rabbits. Iraqi J Vet Sci. 2019;32:29-35 10.33899/ijvs.2019.153874

41.   Gong WJ, Golic KG. Loss of Hsp70 in Drosophila is pleiotropic, with effects on thermotolerance, recovery from heat shock and neurodegeneration. Gen. 2006;172(1):275-286.

42.   Surai PF. Antioxidant systems in poultry biology: Heat shock proteins. J Sci. 2015;5(12):1188-1222.

43.   AL-Ma’atheedi MS, Hassan AA. Effect of vitamin c and selenium on some physiological and reproductive characters in adult roosters exposed to oxidative stress induced by hydrogen peroxide. Iraqi J Vet Med. 2012;36(1):32-42.

44.   Lin H, Decuypere E, Buyse J. Acute heat stress induces oxidative stress in broiler chickens. Comp Biochem Physiol A Mol Integr Physiol. 2006;144(1):11-17.‏10.1016/j.cbpa.2006.01.032

45.   Droge, W. Free radicals in the physiological control of cell function. Physiol Rev. 2002;82:47-95. ‏doi.org/10.1152/physrev.00018.2001

46.   Mashaly MM, Hendricks 3rd GL, Kalama MA, Gehad AE, Abbas AO, Patterson PH. Effect of heat stress on production parameters and immune responses of commercial laying hens. Poult Sci. 2004;83(6):889-894. 10.1093/ps/83.6.889

Hassan A A, Asem R A, Al-Chalabi A S. Assessment the level of some heavy metal in vital body fluids and blood parameters in lactating Awassi ewes. Iraqi J Vet Sci. 2019:33:297-301. DOI: 10.33899/ijvs.2019.162908

Discussion

Endothermic animals (including mammals and birds) are able to control their body temperature by the regulation of their basal metabolic rate (thermoregulation). The significance of endothermy is enhanced enzymatic activities and a continuous body temperature that allows animals to be active at different external temperatures (18). Body thermoregulation plays an important role in tissue protection against oxidative stress which enhanced by elevation of body temperature, oxidative stress took place as a result of activation of ROS production in mitochondrial matrix through respiratory chain process (19). In cells, the respiratory chain is responsible for reactive oxygen species (ROS) production, mainly by complexes I and III. When the electrochemical gradient increases, the activity of the respiratory chain diminishes, raising the half-life of ubisemiquinone (20). Thus, the ROS production is dependent on the mitochondrial membrane potential and alteration in body temperature will lead to alteration in response capacity of the cells to produce ROS. Heat stress plays an important role in tissue oxidation due to enhance the phosphorylation of ADP into ATP takes place within the mitochondrial inner membrane respiratory chain via reoxidize the reduced cofactors (NADH, FADH2) produced by the various cellular metabolic processes (18).

The present study revealed that heat stress exposure induced a significant reduction in RBCs, Hb concentration, and PCV accompanied by an elevation in MCV. These results, are similar to the previous findings, detailed that exposure to oxidative stress on the 6^th week of adult rooster caused a significant decrease in Hb and PCV values as compared with the control group (21), other researchers documented that oxidative stress causes a significant increase in MCV (22). The etiology of that result is may be due to that the RBCs are highly sensitive to oxidative injury because of its high level of oxygen contents. Moreover, the hemoglobin is a potent initiator of the oxidative stress process development in the RBCs (23). So, the RBCs are considered as the main cells that influenced by oxidative stress (24). Hemoglobin reduction due to heat stress might be due to the influence oxygen binding protein as an antioxidant mechanism and gradually converted to methemoglobin (metHb), so MetHb is unable to bind or transport oxygen (25,26). Under normal circumstances, the concentration of metHb in RBCs is preserved at less than 1% of the whole hemoglobin (26). Hemoglobin oxidation also initiates the development of disulfide cross-links between globin chains and deforming the hemoglobin structure that leads to Heinz bodies’ formation (26). This accumulation of membrane-bound denatured protein (Heinz bodies) may be eliminated through reticuloendothelial macrophages system, but more significant cellular changes results in RBC hemolysis (25).

Vitamin C addition in drinking water to stressed broiler lead to a significant rise in RBCs count associated with decreased MCV. Whereas giving of acetylsalicylic acid improve RBC and PCV, associated with reduction MCV, MCH, and MCHC. Our results oppose with (27) who documented no significant influence on red blood cell, packed cell volume and hemoglobin were noticed when acetylsalicylic acid additive to heat stress layers. The current findings disagree with Tras et al. (28), who found that the hematological values of vitamin C, acetylsalicylic acid treated groups, had no effect on the serum superoxide dismutase liver, glutathione peroxidase, and catalase-like antioxidant enzymes because of the nature of the non-steroidal anti-inflammatory reagents as an antioxidant agent. Furthermore, Usman et al. (29) found that there was no changes in RBC, HB, PCV, MCV, MCHC values in the quails under the heat stress at 38 ºC supplemented to the vitamin C. Ultimately similar to (30) who report in his work that supplementation of vitamin C during summer season improves the hematological values. Vitamin C may be attributed to its capability to preserve the integrity of the erythrocyte membrane (31) and to its antioxidant capacity by scavenging the free radicals from the cellular environment leading to high malondialdehyde formation (31). Current results found that administration of vitamin C simultaneously with heat stress had no effect on hematological values. The etiology of this effect, is that the vitamin C plays a major role as antioxidant cellular compounds which has the ability to neutralize free radicals when poultry are undergoing stress (14).

Data of current study of the broiler exposure to the heat stress caused significant decline in the lymphocyte accompanied with a significant elevation in Heterophils as a result of the heterophils/lymphocyte ratio greater than normal, whereas giving of vitamin C in drinking water caused significant increase in percentage of lymphocyte and decreased in percentage of Heterophils which cause H/L ratio decreased. However, the administration of acetylsalicylic acid with drinking water did not affect the percentage of the DLC but decrease in H/L ratio. Leucocyte response has been linked to heat stress due to its relation to circulating ACTH hormone which effect on corticosterone levels through decreasing its serum level leading to decreasing white blood cell count (32). The high temperature in hot weather is enough to cause increased body temperature as well as changes circulating leucocyte components in chickens, in addition, to that, pervious results observed that vitamin C avoided the releasing of leucocytes into peripheral bloodstream from their body reservoir, presumably because of its inhibition function in the secretion of corticosteroids during stress (31). Another previous work reported that after 2 hours of heat stress exposure, chickens showed higher heterophils correlated with declining lymphocytes (33). Consequently, the percentage of heterophils /lymphocytes increased owing to heat stress and is considered as an indicator of physiological stress in harmony with earlier research (34). The H/L ratio was expressed in birds as a stress index (35). Adenkola et al. (31) reported that vitamin C supplementation reduces the negative effects of heat stress in birds. Therefore, it is advisable to add vitamin C during the hot-dry season to reduce heat stress effect. Normally, vitamin C, a synthesis in the fowl's kidneys, is concerned with expelling surplus free radicals from the body, yet are either drained or collected under pressure condition; in this way presenting cells to their ruinous impacts (36). Another work by (37) have suggested various methods by using electrolytes and vitamins to improve the harmful impacts of heat stress on birds.

In our study, the administration of acetylsalicylic acid declines the H/L ratio and this finding agrees with (27). This decrease could be due to the action of acetylsalicylic acid on reducing the release of serum corticosterone in the summer season (38). The result of the present study revealed a significant elevation in Hsp70 in heat stressed broiler. Our findings are in agreement with other researchers who found a significant rise in Hsp70 level relative to the control group in laying hens when exposed to mild heat stress (39). This outcome was relating to other investigation which uncovers that administration of male rabbits to the cadmium chloride as oxidative stress in tap water daily for 6^th weeks cause significant increase in serum Hsp70 concentration in compassion with control group (40). The cause of the rise in serum Hsp70 level may be due to an increase in the expression of Hsp70 genes in broiler's liver during the altered ambient temperature (14), and the heat shock protein 70 gene is very susceptible to changes in the environment (41). The results of the research show that enhanced expression of Hsp70 in chicken tissue is one of the most significant protective reactions to prevention the detrimental changes in protein structure and functions owing to multiple stresses (42).

The result of the present study demonstrated that exposure to heat stress reduces the body weight and liver weight, this result agrees with (43) who reported that six weeks of oxidative stress induced by hydrogen peroxide supplementation with drinking water caused a significant reduction in the body weight of adult white Leghorn male chicken. Lin et al. (44), the researcher found that elevated body temperatures could lead to metabolic changes involving oxidative stress induction and the liver is more susceptible to oxidative stress in broilers during acute exposure to heat stress. Oxidative stress should be considered as part of broiler stress response to heat stress exposure (44). Under stressful environmental circumstances, enhanced concentrations of ROS happen as the bird's body tries to keep its heat homeostasis. As a result, the body begins to produce heat shock proteins in an attempt to safeguard itself from ROS deleterious cellular effects (45). Heat stress reduced feed consumption and activation of the hypothalamus pituitary hypophysial axis and decreased feed consumption is consequent to detrimental impacts of heat stress leading to reduced body weight, feed effectiveness, egg production and quality (46). Current research has shown no significant change in serum glutathione concentration between groups, which is not agreement with Hassan et al. (47) who found that serum glutathione concentration increased in Awassi ewes during the spring season compared to those lactating their lambs in hot dry season. This finding also disagree with prior research that found the administration of vitamin C to adult rooster with hydrogen perioxide resulted in a significant increase in liver glutathione levels relative to hydrogen peroxide group (43). In the present research, the cause of uninfluenced growth hormone and glutathione concentration maybe due to the length of the experiment or the dose of vitamin C and acetylsalicylic acid were not sufficient to trigger modifications in growth hormone and glutathione.

Conclusion

From this research, it is found that vitamin C and acetylsalicylic acid lowered the adverse effect of heat stress so suggested to offer vitamin C and acetylsalicylic acid during the warm season.

Acknowledgements

The searcher would like to appreciate the College of Veterinary Medicine, University of Mosul for offering all the equipment to finish the current job.

Conflict of interest

The researchers note that there is really no conflict of interest.