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The objective of the present work was to determine the optimal supplemental chromium (Cr) concentration for alleviating the detrimental effects of heat stress on the growth performance of broilers using the literature data. A total of 53 observations from 22 experiments that assessed the growth performance of broilers fed various doses of Cr under heat stress were collected. The control groups received no supplemental Cr, whereas supplemental Cr concentrations ranged from 200 µg/ kg to 2,000 µg/kg of diet. The sources of Cr were Cr chloride (n= 12), Cr-amino acid chelate (n= 14), and Cr picolinate (n= 27). The relative change (Δ %) of average daily gain (ADG) between broilers fed a Cr-supplemented diet and those fed a control diet was calculated. To compare ΔADG among Cr sources, the source was considered a fixed variable, while the experiment and the supplemental Cr concentration were considered random variables. The ΔADG was not different among the sources. Polynomial contrast analysis indicated that ΔADG increased quadratically (p<0.05) as the dietary Cr concentration increased. The optimum supplemental Cr concentration was estimated using a one-slope broken-line model with the fixed variable of supplemental Cr and a random variable of experiment based on the NLMIXED procedure of SAS. The optimum supplemental Cr concentration to maximize ΔADG in broilers under heat stress was 687 µg/kg (SE= 137, R 2 = 0.70, and p<0.001). Taken together, the optimum Cr supplemental concentration in broiler diets to alleviate the detrimental effects of heat stress on body weight gain is 687 µg/kg, regardless of the source of Cr.


INTRODUCTION
Trivalent chromium (Cr) is an essential micronutrient for broilers (Huang et al., 2016), and Cr is known to regulate glucose levels by potentiating the actions of insulin, thereby regulating the metabolism of lipids, proteins, and carbohydrates toward anabolic processes (Brooks et al., 2016;Feng et al., 2021a). One of the major stressors in broiler production systems is heat stress caused by high environmental temperatures particularly in tropical regions of the world, which can adversely affect growth performance (Samanta et al., 2008;Norain et al., 2013), carcass traits (Toghyani et al., 2012;Huang et al., 2016), blood biochemical parameters (Moeini et al., 2011;Akbari & Torki, 2014), disease susceptibility (Hamidi et al., 2016;Hajializadeh et al., 2017), or a combination of these responses. In addition, heat stress has been known to have a negative impact on growth performance in laying hens (Kim et al., 2021a;Kim et al., 2022a) and pigs (Kim et al., 2009;Serviento et al., 2020).
Dietary supplementation of Cr has been reported to alleviate the detrimental effects of heat stress on growth performance in poultry by mitigating stressinduced immunosuppression (Norain et al., 2013;Jahanian & Rasouli, 2015). However, in some studies, supplemental Cr had no significant effects on growth performance in heat-stressed broilers (Habibian et al., 2013). Additionally, the effects of the source of Cr on alleviating efficacy were inconclusive in previous studies (Toghyani et al., 2012;Sahin et al., 2017). Furthermore, the optimal supplemental Cr concentration for alleviating the detrimental effects of heat stress on growth performance in broilers has been inconsistent among studies (Sahin et al., 2002;Toghyani et al., 2012;Ebrahimzadeh et al., 2013). The effects of the source and concentration of supplemental Cr have not always been reproducible, and individual studies may have false-positive results (type I errors) or may fail to detect small effects (false-negative, type II errors). A meta-analysis can combine growth performance data from multiple studies on the same topic, increase the precision of estimating the effects of supplemental Cr, and clarify the discrepancies among the literature (Lee, 2018). Therefore, the objectives of this meta-analysis were to analyze the effects of Cr sources on the growth performance of heat-stressed broilers and to determine the optimum supplemental Cr concentration for alleviating the detrimental influence of heat stress on the growth performance of broilers.

Data Collection and Processing
A dataset was generated by conducting an extensive search of online databases using the following keywords: broiler chicken, chromium, growth performance, heat stress, and poultry. The studies identified through the literature search were then manually screened based on their title and experimental information. The collected data included the ingredient and analyzed the composition of diets, ambient temperature, sources of Cr, supplemental Cr concentrations, experimental duration, and growth performance (Table 1). Growth performance included average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F). In cases where G:F was reported as a feed conversion ratio, the values were converted to G:F. A total of 53 observations from 22 experiments in 16 literature sources that empirically evaluated the growth performance of broilers under heat stress were collected. Data for carcass traits and serum metabolites were also collected, and all carcass trait data were converted as a percentage of body weight. The broilers were given ad libitum access to feed and water throughout the experiments. The ambient temperature ranged from 32 °C to 36 °C (34.1 ± 1.3 °C), and the experimental duration ranged from 21 to 49 days (37.8 ± 9.7 days). The dietary treatments within each experiment were divided into control and treatment groups, with the control group receiving no supplemental Cr, whereas the treatment group received supplemental Cr concentrations ranging from 200 µg/ kg to 2,000 µg/kg of diet. The nutritional values and environmental conditions in the control and treatment groups were identical within each experiment.
The relative change of measurements in broilers fed a Cr-supplemented diet compared to the control group was calculated using the following equations: Actual difference= measurement of treatment groupmeasurement of control group, Relative change (Δmeasurement %)= actual difference/ measurement of control group × 100 A total of 53 observations for relative growth performance were used for further statistical analyses ( Table 1). The sources of Cr evaluated were Cr chloride (n= 12), Cr-amino acid chelate (n= 14), and Cr picolinate (n= 27).

Statistical Analysis
The 53 observations for the relative change of growth performance were analyzed using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, USA).
To compare the effects of Cr sources on the relative change of growth performance, the source was considered a fixed variable, whereas the experiment and the supplemental Cr concentration were considered random variables. The least squares means of each Cr source were calculated, and pairwise comparisons were conducted using the least significant difference method, with Tukey's adjustment if an effect was significant. To evaluate the linear and quadratic effects of supplemental Cr concentrations on the relative change of growth performance, pre-planned polynomial contrasts were used, with Cr concentrations as a fixed variable, and the experiment and the source of Cr as random variables. The least squares means of each Cr concentration were then calculated.
Due to the quadratic response of ΔADG to the increasing supplemental Cr concentrations, a brokenline analysis was conducted for ΔADG. The optimal supplemental Cr concentrations for mitigating the detrimental effects of heat stress on ΔADG in broiler chickens were estimated using a one-slope brokenline analysis, employing the NLMIXED procedure of SAS (Robbins et al., 2006). In the one-slope broken-line models, the experiment was considered a random variable. An individual Cr-supplemented diet in the treatment group was the experimental unit and statistical significance was considered at a p-value less than 0.05.
Mean, standard deviation, and 95% confidence interval were calculated for growth performance, carcass traits, and serum metabolites regardless of the sources and doses of supplemental Cr. The data were analyzed using the UNIVARIATE procedure of SAS, and significance was declared for a p-value less than 0.05 (2-tailed), when the 95% confidence interval did not include zero.

RESULTS
The ΔADG, ΔADFI, and ΔG:F were not affected by the sources of Cr (Table 2). The ΔADG increased quadratically (p<0.05) with increasing dietary Cr concentration (Table 3). The optimum supplemental Cr concentration to maximize ΔADG in broilers under heat stress was 687 µg/kg (SE= 137, R 2 = 0.70, and p<0.001) based on one-slope broken-line analysis (Figure 1).
Supplementing Cr in broiler diets under heat stress improved (p<0.05) ΔADG, ΔADFI, and ΔG:F regardless of the sources and doses of Cr (Table 4). Additionally, Cr supplementation increased (p<0.05) the relative changes of carcass yield, dressing yield, the weight of Bursa of Fabricius, thymus, and breast meat; however, it decreased (p<0.05) the weight of heart and abdominal fat. Broilers fed the Cr-supplemented diet showed greater (p<0.05) relative changes in insulin, total protein, and Cr concentrations in serum compared with those fed the control diet. Supplementation of Cr in broiler diets under heat stress led to decreased (p<0.05) relative changes of glucose and corticosterone concentrations in serum regardless of the sources and doses of Cr.

DISCUSSION
Broiler chickens subjected to heat stress exhibit lower growth performance compared with those kept under a thermoneutral environment (Samanta et al., 2008;Norain et al., 2013). In our meta-analysis, we identified 5 studies that investigated the effects of ambient temperatures on growth performance in broilers fed a    control diet without Cr supplementation. The studies included a positive control group kept under thermoneutral conditions and a negative control group subjected to a tropical or hyperthermic environment (Sands & Smith, 1999;Samanta et al., 2008;Jahanian & Rasouli, 2015;Hajializadeh et al., 2017;Hamidi et al., 2022). The results indicated that broilers kept under a hyperthermic environment had decreased ADG by 24.0% (standard devia-tion= 7.2% data not shown), ADFI by 13.5% (3.8%), and G:F by 11.9% (10.0%) compared with those under thermoneutral conditions. The hypothalamus plays a crucial role in regulating energy balance and feed intake by manipulating the endocrine system and body temperature (Berthoud, 2002). Heat stress affects the rostral cooling center of the hypothalamus, which simulates the medial satiety center, inhibiting the lateral appetite center and resulting in reduced feed intake (Silanikove, 2000). This reduction in feed intake affects the center of satiety in the hypothalamus, resulting in a decrease in the heat increment associated with digestion and absorption of feed and nutrient metabolism. Lower G:F induced by heat stress is attributed to physiological and immunological adaptations in birds, and a part of the consumed energy is used for heat dissipation, resulting in impaired G:F (Jahanian & Rasouli, 2015). The combination of reduced feed consumption and efficiency explains the decreased ADG in broilers kept under heat stress.
The empirical studies utilized in this meta-analysis employed various feed formulations, growth phases, including starter, grower, and finisher, as well as initial body weight and experimental duration. As a result, the growth performance of broilers fed the control group and the actual difference in growth performance between broilers fed the treatment and control groups varied. In 22 experiments, ADG and ADFI of broilers fed the control group under heat stress ranged from 30.4 g/d to 60.2 g/d (SD= 8.8 g/d) and 50.9 g/d to 137.7 g/d (SD = 24.7 g/d), respectively. To combine and compare the 22 experiments, we employed the relative change, which is a unitless value calculated as the actual difference divided by the growth performance of broilers fed the control group, to minimize biases among the experiments. The use of relative changes is widely employed in meta-analyses for nutritional research in pigs and poultry (Cowieson & Roos, 2013;Kim et al., 2021b).
Sources of Cr can be categorized into inorganic sources (Cr chloride) and organic sources, including Cr nicotinate, Cr yeast, Cr-amino acid chelate, and Cr picolinate (Huang et al., 2016). In most studies, organic Cr has been added in the form of Cr-amino acid chelate or Cr picolinate (Samanta et al., 2008;Moeini et al., 2011;Ghazi et al., 2012;Toghyani et al., 2012;Habibian et al., 2013;Huang et al., 2016). Of the 4 studies employed in this meta-analysis that simultaneously compared the alleviating effects of Cr chloride and Cr-amino acid chelate on growth performance, none showed significant differences in ADFI between the 2 sources (Moeini et al., 2011;Ghazi et al., 2012;Habibian et al., 2013;Huang et al., 2016). Average daily feed intake in heat-stressed broilers fed the Cr-amino acid chelate-supplemented diet was numerically increased at -2.3%, 0.7%, 3.0%, and 4.5% (mean= 1.48%) compared with those fed the Cr chloride-supplemented diet.
The ΔADG increased as the dietary Cr concentration increased. In our study, only 2 experiments used the polynomial contrast to determine the effects of increasing supplemental Cr levels on ADG in heatstressed broilers, and the results showed that ADG increased linearly (Sahin et al., 2002) and quadratically (Samanta et al., 2008) as the doses of Cr increased. Our results agreed with a previous meta-analysis in which Cr picolinate significantly increased the ADG of broilers under heat stress but had no significant effect on ADFI and G:F (Feng et al., 2021b). Heat stress conditions can lead to immunosuppression and oxidative stress by reducing antioxidant defenses and increasing the production of free radicals, and these factors are associated with the harmful effects of heat-stressed broilers (Norain et al., 2013;Jahanian & Rasouli, 2015). Dietary supplementation of Cr has been known to alleviate the detrimental effects of heat stress on growth performance in poultry by ameliorating stress-induced immunosuppression (Norain et al., 2013;Jahanian & Rasouli, 2015). Additionally, dietary Cr supplementation increases insulin plasma concentration and regulates glucose level by potentiating the insulin, acting as an insulin cofactor and enabling proper metabolic transformations of carbohydrates, proteins, and lipids towards the anabolic side (Feng et al., 2021b). Furthermore, Cr supplementation could increase the digestion and absorption of feeds by improving intestinal morphology in stressed broilers, thereby increasing the amount of nutrients available for growth (Bunglavan et al., 2014). A combination of one or more factors by Cr supplementation ameliorated the reduced body mass gain in heat-stressed broilers.
To determine the optimal nutrient concentration, the broken-line analysis has been widely employed (Choi & Kim, 2019;Kim et al., 2022b). Our study found that the optimal supplemental Cr concentration to maximize ΔADG in broilers under heat stress was 687 µg/kg based on one-slope broken-line analysis. Spears et al. (2019) reported that Cr supplementation at 400 µg/kg and 2,000 µg/kg over the normal life span of broilers (49 days) did not adversely affect performance and mortality. Moreover, Cr supplementation at 2,000 µg/kg did not affect Cr concentrations in breast muscle and skin with adhering fat. Therefore, the optimal Cr supplemental dose of 687 µg/kg to alleviate the detrimental effects of heat stress on ADG may have no toxicity on broilers and may not present a human health concern.
Serum glucose, corticosterone, and total protein concentrations are stress indicators in broilers. The concentration of serum glucose and corticosterone has been reported to increase, whereas that of serum total protein has been reported to decrease in broilers under heat stress (Sahin et al., 2002). In the present work, Cr supplementation alleviated the changes in serum glucose, corticosterone, and total protein concentrations by heat stress based on multiple studies, indicating that Cr effectively mitigates heat stress. Chromium supplementation increases circulating insulin concentrations, regulating glucose levels by acting as an insulin cofactor and enabling proper metabolic transformations of carbohydrates, proteins, and lipids towards the anabolic side (Feng et al., 2021b). Additionally, Cr supplementation can manipulate the carcass composition to yield more lean meat, thus increasing protein accretion in broilers (Samanta et al., 2008). In the present study, Cr supplementation increased carcass and dressing yields of broilers by 1.7% and 3.2%, respectively, whereas it decreased abdominal fat weight by 15.0%. Moreover, Cr supplementation increased the weight of breast meat by 8.1%, confirming that the broilers fed the Cr-supplemented diet retain more protein and weight gain.

CONCLUSION
The growth performance of broilers fed Crsupplemented diets is not affected by the source of Cr under heat stress. The relative change of weight gain in broilers subjected to a hyperthermic environment quadratically increases as dietary Cr concentration increases. The optimal Cr supplemental concentration in broiler diets to alleviate the detrimental effects of heat stress on body weight gain is 687 µg/kg, regardless of the source of Cr.