Diyala Agricultural Sciences Vol (14) No 1, 2022: 47-53 Pakiza Hamira Wasman ISSN: 2073-9524 eISSN: 2310-8746 74 Effect of L-Threonine Supplementation to Diet on Some Productive and Physiological of Traits Broiler Chickens Under Heat Stress Conditions Pakiza Hamira Wasman Salahaddin University, College of Agricultural Engineering Science, Animal Resources Department, Erbil, Iraq Corresponding author: Pakiza.wasman@su.edu.krd Article history: Received: 23 January 2022 Accepted: 9 May 2022 Published: 30 June 2022 Abstract This experiment was conducted in Erbil, Iraq, from July 7 to August 17, 2021, for determine the effects of L-Threonine supplementation on several productive and physiological traits Under heat stress conditions (36.04 ± 2 °C), A total of 200 broiler chicks were randomly distributed into five treatments: T1 control treatment, T2, T3, T4 and T5 standard diet with L-Threonine 250, 500, 750 and 1000 mg/kg, respectively. The results showed that the addition of L-Threonine to the ration improved broiler production performance under heat stress conditions such as body weight it increased significantly (P≤0.05) in T3 and T5 and reached 2187 and 2235 g, respectively compared to 2145 g in the T1 and reduce H/L ratio in T4 and T5 compared with T1 and did not affect effect (P≥0.05) on physiological traits such as red and white blood cell count, hematocrit, cholesterol, glucose, total protein, aspartate aminotransferase and alanine aminotransferase activities. Keywords: L-Threonine, physiological traits, broilers, heat stress https://dx.doi.org/10.52951/dasj.22140106 This article is open-access under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/) Introduction Tropic hot weather during summer is one of the significant factors that negatively on broiler performance (Dalolio et al., 2021). Broiler chickens eat less and spend more time drinking water, panting, and resting around the waterer and other cool locations in the pen when the heat stresses them. Also, the broiler production performance was affected negatively (Wasti et al., 2020). Furthermore, heat-stressed broilers have shown an increase in abdominal fat and a loss in carcass protein due to heat stress (Mohamed et al., 2019). It also increases the need for nutrients and amino acids such as methionine, lysine and Threonine. So The growth rates of broiler under heat stress conditions depend on a sufficient supply of amino acids to produce proteins involved in various physiological functions, such as signal transduction, hormone signaling, cell structure, and antioxidant systems (Maharjan, 2020). Therefore, nutritional and management therapies can be introduced to minimize the heat stress due to high environmental temperatures in broiler production. L-Threonine “is often the third limiting amino acid, and it is important for gut health, morphology, and function, as well as the optimal utilization of total sulfur amino acids and lysine, immunity, carcass traits, structural protein synthesis, antibody, uric acid, and pancreatic enzymes, intestinal barrier maintenance, and mucin synthesis. The intestinal mucosa is protected by the mucus layer, which contains mucins, and glycoproteins that need Threonine for production” (Al-Hayani, 2017; Al-Sagan et al., 2018). Estalkhzir et al., (2013) found that supplementing broiler diets with threonine improved body weight, feed conversion, and dressing percentage. According to Rezaeipour et. al., (2014) reported that mailto:Pakiza.wasman@su.edu.krd http://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0002-1407-1649 Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 47-53 74 “threonine supplementation for the first 42 days improved Feed Conversion in broiler” and based on NRC (1994) requirement for threonine is 0.48% of the diet. More research is needed to quantify amino acid needs under heat stress conditions, This experiment aimed to study the effects of L-Threonine Supplementation on some productive and physiological traits broiler (Ross 308) Under Heat Stress Conditions. Materials and methods Ethic Approval The study proposal presented was accepted by the Scientific Ethical Committee of the College of Veterinary Medicine, University of Diyala. Experimental design This experiment was conducted in (The sako field) in (Gurato/Mergasor/Erbil/Iraq) from the period (5 / 7 / 2021 to 17 / 8 / 2021) and aimed to determine the effects of the use L-Threonine in the broiler as on some productive and physiological traits of broiler chickens Under Heat Stress Conditions. A total of 200 one-day old unsexed (Ross-308) broilers chicks were randomly distributed into five treatments and each treatment was further subdivided into fourth replicates:  T1: a standard diet without L-Threonine (control treatment)  T2: a standard diet with L-Threonine 250 mg/kg  T3: a standard diet with L-Threonine 500 mg/kg  T4: a standard diet with L-Threonine 750 mg/kg  T5: a standard diet with L-Threonine 1000 mg/kg The chicks of each treatment were housed in Full Environmental Control Houses and all of the experimental diets were designed to suit the nutritional needs of broiler chicks according to the NRC (1994) (See table 1). Feeds and water were supplied ad-libitum, the temperature and humidity were measured inside the breeding farms by using electronic thermometers distributed in different parts of the farm. Table 1. Chemical analysis of the basal die Ingredients starter (1-10days) Grower (11-24days) Finisher (25-35days) Protein % 22.50 20 18 Energy (Kcal/kg) 3069 3185 3250 Fat% 4.45 5.61 5.95 Lysine % 1.33 1.21 1.21 Methionine % 0.64 0.56 0.50 Ca % 0.98 0.95 0.90 P % 0.62 0.58 0.54 The preventive vaccination program was against the most common infectious diseases such as Newcastle disease and infectious bronchitis as shown in Table 2. Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 47-53 74 Table 2. Vaccination program of broiler chicks during the experimental period Age (days) Vaccine Route of vaccination 1-3 Newcastle disease + Infectious Bronchitis eye drop 10-12 Infectious bronchitis eye drop 16-17 Hantavirus Pulmonary Syndrome Sub cut.1/2 cc 22 Infectious bronchitis drinking water 28 Newcastle disease drinking water Productive traits Live body weight (LBW): live body weight of each replicate and treatment was individually measured at the one day old and weekly in the morning by weighing the broilers of one replicate individually as follows (Miah et al., 2022): LBW (g) = sum of broiler weights per replicate/number of broiler per replicate Body weight gain (BWG): The BWG was calculated using the following equation according to (Miah et al., 2022): BWG = LBW2- LBW1/ number of bird Feed intake (FI): The weekly feed intake for each repeater was calculated at the end of each week of the experiment by weighing a certain amount of diet provided to the birds for each repeater during the week and removing from it the weight of the remaining diet at the end of the week (Miah et al., 2022). Feed conversion ratio (FCR): FCR was measured weekly as Kg of FI / Kg of BWG (Miah et al., 2022). Physiological traits Five ml of blood was collected from five birds (male) from each experimental treatment at 42 days of age, blood count parameters were determined: Red blood cell (RBC), weight blood cell (WBC), Hemoglobin (Hb), Hematocrit (PCV) and H/L ratio according to Archer (1965) and Natt and Herick (1952). Then another portion of the blood was centrifuged for ten minutes at 1000 rpm. All blood serum biochemical features, including cholesterol, glucose, total protein, albumin and globulin, uric acid, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, were measured photometrically using commercially available testing kits. Statistical analysis The data were statistically analyzed by the General Linear Models procedure of the SPSS software (SPSS, 2011). Statistically significant differences observed among treatments were separated using Duncan’s multiple range test with 5% probability level. Results and Discussion Table 3 shows the impact of threonine supplementation on body weight (BW), body weight gain (BWG), and other production traits in the broiler chickens. It can be seen from the table 3 that threonine treatments have a significant effect (p≤0.05) on BW when compared to the control treatment. In T3 and T5, it reached BW 2187 and 2235 g, respectively, compared to 2145 in T1 and T5. The current study also found that broiler BWG was considerably greater (p≤0.05) in T3 and T5 than in T1, T2, and T4, but had no effect on FI or FCR. Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 47-53 05 Table 3. Effect of L- threonine supplementation on productive performance in broiler chickens (Mean±SE) FCR FI (g/ bird ) BWG(g/ bird) FBW (g) IBW (g) Treatment 1.51 ± 0.01 3187 ± 68.28 2104 ± 45.92 b 2145 ± 22.73 b 41.89 ± 0.35 T1 1.52 ± 0.08 3173 ± 79.01 2086 ± 37.17b 2128 ± 21.37 b 41.23 ± 0.27 T2 1.47 ± 0.06 3172 ± 61.22 2145 ± 16.23 ab 2187 ± 10.15 ab 41.69 ± 0.13 T3 1.51 ± 0.03 3179 ± 98.75 2103 ± 94.22 b 2144 ± 47.97 b 41.30 ± 0.21 T4 1.44 ± 0.03 3177 ± 13.22 2194 ± 43 a 2235 ± 22.94 a 41.29 ± 0.29 T5 NS NS * * NS P-value  T1 control treatment, T2, T3, T4 and T5 standard diet with L-Threonine 250, 500, 750 and 1000 mg/kg respectively  The different letters within the same column indicate a significant difference between the means according to Duncan's test  * means that there is a significant effect of treatment and NS means non-significant effect of treatment in the ANOVA table. Table 4 shows that using L-Threonine supplement has no significant effect (P ≥ 0.05) on the broiler red and white blood cells (RBC and WBC) compared to T1 (control treatment), In comparison to T1 and the remainder of the L-Threonine treatments, the H/L ratio was much lower (P ≤ 0.05) in T4 and T5, indicating that heat stress was less of an effect. Table 4. Effect of L- threonine supplementation on physiological performance in broiler chickens (Mean±SE) H/L WBC (*10 3 /mm 3 ) RBC (*10 6 /mm 3 ) Treatment 0.34 ± 0.01 a 21.75 ± 0.17 2.11 ± 0.04 T1 0.31 ± 0.00 ab 21.90 ± 0.36 2.12 ± 0.04 T2 0.32 ± 0.02 ab 22.09 ± 0.13 2.10 ± 0.07 T3 0.28 ± 0.00 b 21.38 ± 0.17 2.17 ± 0.05 T4 0.27 ± 0.01 b 22.03 ± 0.14 2.07 ± 0.11 T5 * NS NS P-value  T1 control treatment, T2, T3, T4 and T5 standard diet with L-Threonine 250, 500, 750 and 1000 mg/kg respectively.  The different letters within the same column indicate a significant difference between the means according to Duncan's test .  * means that there is a significant effect of treatment and NS means non-significant effect of treatment in the ANOVA table. As shown in tables 5 and 6, the addition of L- threonine did not affect on the biochemical traits of blood serum (Glucose, Total protein, Cholesterol and Uric acid) or the activity of liver enzymes (AST and ALT) when compared to the control treatment T1. Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 47-53 05 Table 5. Effect of L- threonine supplementation on biochemical traits of blood serum in broiler chickens (Mean±SE) Uric acid (mg/dl) Cholesterol (mg/dl) Total protein (mg/dl) Glucose (mg/dl) Treatment 7.12 ± 0.27 143.78 ± 5.30 2.23 ± 0.13 182.3 ± 5.9 T1 6.40 ± 0.22 149.34 ± 11.64 2.48 ± 0.09 191.42 ± 7.2 T2 6.78 ± 0.14 147.53 ± 7.59 2.56 ± 0.10 190.9 ± 6.6 T3 6.24 ± 0.52 147.74 ± 1.09 2.66 ± 0.18 191.2 ± 4.6 T4 6.54 ± 0.10 145.56 ± 2.87 2.79 ± 0.10 187.8 ± 10.6 T5 NS NS NS NS P-value  T1 control treatment, T2, T3, T4 and T5 standard diet with L-Threonine 250, 500, 750 and 1000 mg /kg respectively and NS means non-significant effect of treatment in the ANOVA table. Table 6. Effect of L- threonine on the activity of liver enzymes in broiler chickens (Mean±SE) ALT (U/L) AST (U/L) Treatment 13.68 ± 0.46 b 117.50 ± 3.97 T1 13.19 ± 1.01 b 112.83 ± 1.53 T2 12.44 ± 0.70 b 110.51 ± 1.04 T3 11.85 ± 1.07 a 110.53 ± 0.82 T4 11.69 ± 0.21 a 112.18 ± 1.27 T5 NS NS P-value  T1 control treatment, T2, T3, T4 and T5 standard diet with L-Threonine 250, 500, 750 and 1000 mg /kg respectively  NS means non-significant effect of treatment in the ANOVA table. The digestive system is lined with mucous membranes, and threonine is required for mucous membrane function, which includes adjusting the pace at which food travels through the digestive tract for optimum nutrient absorption and maintaining appropriate production of digestive enzymes (Dalolio et al., 2021), also the L -threonine maintain appropriate secretion of digestive enzymes which leads to increased feed digestibility and improved production traites (BW and BWG) (Rao et. al., 2011 and Johannsen et. al., 2012) . According to Rezaeipour et al,. (2012), increasing dietary L-threonine improved morphological aspects of the small intestine, resulting in greater development rate, whereas birds fed threonine-deficient diets exhibited decreased nutrient absorption in the digestive tract, "and Boosts in the number of heterophils are an essential measure of immune system function, and threonine increases globulin levels in blood and lymphocytes. (Azzam et. al., 2011). Conclusion Our findings show that L-Threonine supplementation can improve broiler production performance such as live body weight and body weight gain, and reduce the negative effects of heat stress. Still, it has no effect on feed intake, feed conversion ratio, blood serum biochemical traits, or liver enzyme activity. Conflict of interests The author's name appears below. He declares that he has no financial or non- financial interests (such as personal or professional ties, affiliations, expertise, or beliefs) in the subject matter or materials covered in this manuscript. Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 47-53 05 Acknowledgments Thank you to the Saco field manager and his team for assisting with the study. References Al-Hayani W.K., (2017). Effect of threonine supplementation on broiler chicken productivity traits. International Journal of Poultry Science, 16(4):160- 168. Al-Sagan A.A., Khalil S.I. and Smith M.P., (2018). The Benefit of L-Threonine Supplementation on Growth Performance, Carcass Characteristics, Intestinal Morphology and Litter Quality of Broilers. Brazilian J. Poul. Sci., 20(4): 753-758. Archer, R. K., (1965). ComParative Clinical Hematology. Oxford Black Well Scientific Publications. Azzam M.M.M., X.Y. Dong, P. Xie, C. Wang and X.T. Zou, (2011). The effect of supplemental L-threonine on laying performance, serum free amino acids and immune function of laying hens under high-temperature and high- humidity environmental climates. J. Applied Poult. Res., 20: 361-370. Dalolio, F.S., Albiono L.F.T., DA Silva J.N., Fireman A.K.A.T., Junior A.M.B, M. Busanello, Junior V.R., (2021). Dietary chromium-methionine supplementation and broiler (22–43 days) responses during heat stress. 1. Growth performance and carcass yield, metabolisable energy and serum biochemistry. Anim. Prod. Sci. 61, 586- 595. Estalkhzir, F.M., S. Khojasteh and M. Jafari, (2013). The effect of different levels of threonine on performance and carcass characteristics of broiler chickens. J. Novel Applied Sci., 2: 382-386. Johannsen, D. L., Galgani, J. E., Johannsen, N. M., Zhang, Z., Covington, J. D., and Ravussin, E. (2012). Effect of short- term thyroxine administration on energy metabolism and mitochondrial efficiency in humans. PLoS One, 7(7), e40837. https://doi.org/10.1371/journal.pone.00 40837 Kidd M.T and Tillman P.B. (2016). Key principles concerning dietary amino acid responses in broilers. Animal Feed Science and Technology, 221:314-322. Maharjan, P., Garret M., Katie H., Justina C., Antonio B., Jordan W., Nawin S., Antonio K. , Nadia Y. , Victor N. , Judy E., and Craig C. (2020). Effect of digestible amino acids to energy ratios on performance and yield of two broiler lines housed in different grow- out environmental temperatures . Poult. Sci., 99: 6884-6898. Miah M.Y., Saha S., Koiri N., Mahbub A., Ashraful Islam M. and Channarayapatna G., (2022). Effects of dietary methionine and threonine on growth performance, carcass traits and blood metabolites of broilers in a hot environment. Europ. Poult. Sci., 86:1- 13. Mohamed, A.S.A., Lozovskiy A.R., Ali A.M.A., (2019). Strategies to combat the deleterious impacts of heat stress through feed restrictions and dietary supplementation (vitamins, minerals) in broilers. J. Indones. Trop. Anim. Agric. 44, 155-166. Natt M.P. and Herick C.A, (1952). A new blood diluent for counting the erythrocytes and leucocytes of the chicken. Poultry Science, 31:735-738. NRC., (1994). Nutrient Requirements of Poultry. 9th Edn., National Academy Press, Washington, DC., USA., ISBN- 13: 9780309048927, Pages: 155. https://doi.org/10.1371/journal.pone.0040837 https://doi.org/10.1371/journal.pone.0040837 Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 47-53 05 Rao, S.V.R., M.V.L.N. Raju, A.K. Panda, N.S. Poonam, O.K. Moorthy, T. Srilatha and G.S. Sunder, (2011). Performance, carcass variables and immune responses in commercial broiler chicks fed graded concentrations of threonine in diet containing sub-optimal levels of protein. Anim. Feed Sci. Technol., 169: 218-223. Rezaeipour, V. and S. Gazani, (2014). Effects of feed form and feed particle size with dietary L-threonine supplementation on performance, carcass characteristics and blood biochemical parameters of broiler chickens. J. Anim. Sci. Technol., Vol. 56. DOI: 10.1186/2055-0391-56-20 Rezaeipour, V., H. Fononi and M. Irani, (2012). Effects of dietary L-threonine and Saccharomyces cerevisiae on performance, intestinal morphology and immune response of broiler chickens. South Afr. J. Anim. Sci., 42: 266-273. SPSS, (2011). Statistical Package for Social Science version 20 for window LEAD Technologies. Inc. USA . Wasti, S., Sah N., Mishra B., (2020). Impact of heat stress on poultry health and performances, and potential mitigation strategies. Animals 10, 1266.