Effect of Chromium Picolinate, Alone or in Combination with Vitamin C or Formic Acid on Growth, Carcass Traits, Immune and Blood Parameters of Broilers under Heat Stress

Heat stress is one of the most important environmental stress inducing factors that challenge commercial broilersaround the world, especially in tropical and subtropical regions, due to the economic losses related to reduced production performance and increased mortality. There are scientific evidences for the destructive effects of heat stress on growth performance (Chougule et al., 2018), carcass characteristics (Huang et al., 2016), blood biochemical (Ding et al., 2020) and hematological parameters (Ribeiro et al., 2018).

It is well known that activation of the hypothalamus- pituitary-adrenal (HPA) axis during stress increases the level of corticosterone in blood plasma of the birds (Lu et al., 2019), which facilitates the increase of infections by suppressing the immune system (Hirakawa et al., 2020). Chromium plays an important role in increasing the metabolism of nutrients such as carbohydrates, lipids, proteins, and nucleic acids by activating the enzymes associated with their metabolic pathways (Haldar et al., 2009). In fact, the major physiological role of chromium is to improve glucose tolerance (Hamidi et al., 2016), which increases the susceptibility of tissue receptors to insulin (Ezzat et al., 2017). Corticosterone secretion reduces the sensitivity of body cells to insulin during the stress (Sirirat et al., 2012), and in this case chromium improves bird performance by lowering blood corticosterone concentrations (Toghyani et al., 2006; Dalólio et al., 2018). It is illustrated that heat stress increases the mobilization of chromium from tissues, increases urinary excretion, and reduces the retention of chromium, which results in a deficiency of chromium in the body (Sahin et al., 2002), thus, the need for chromium increases in such cases. Chromium picolinate is a combination of a low-toxicity trivalent form of chromium combined with picolonic acid (Hamidi et al., 2016). The use of chromium picolinate supplement as a nutritional strategy in the diet of broilers under heat stress can improve growth performance (Toghyani et al., 2006), and carcass yield (Sahin et al., 2003), reduce depot fat (Kulkarni et al., 2018), improve immune organ weights (Lu et al., 2019) and blood parameters (Toghyani et al., 2006).

Vitamin C (ascorbic acid) is an unnecessary vitamin in poultry diets; as birds have the enzyme gluconolactone oxidase in their kidneys, which synthesize vitamin C from glucose (Khan and Sardar, 2005). However, during heat stress the endogenous vitamin C is not sufficient to meet the needs of birds (Abidian and Khatoon, 2013). On the other hand, vitamin C existsin high concentration in immune cells, which undergoes a rapid depletion under stress (Sorice et al., 2014). Vitamin C is not a part of the metabolic pathways; however, it is an essential factor for many enzymatic reactions involved in collagen carnitine, catecholamine, and tyrosine biosynthesis (Shakeri et al., 2020). Supplementation of vitamin C in the diet of broilers under heat stress reduces the synthesis and secretion of corticosteroids and decreases the plasma corticosterone concentrations by inhibiting the key enzymes involved in the corticosterone biosynthesis pathway (21-hydroxylase and 11-β-hydroxylase). It has been reported that under stress conditions, vitamin C supplementation can increase productivity, immune response, resistancetodiseaseand viability of broiler chickens (Whitehead and Keller, 2003).

Methanoic acid, known as formic acid, is the simplest carboxylic acid. One of the main applications of formic acid is to add it to feeds due to antibacterial activity against pathogens (Milillo et al., 2011) to prevent their subsequent colony formation in the digestive system of birds (Ricke et al., 2020). Moreover, the positive effects of formic acid have been previously reported on broiler body weight gain (Panda et al., 2009), feed intake, and feed efficiency (Kim et al., 2015), depot fat (Panda et al., 2009), cellular and humoral immunity (Ragaa and Korany, 2016), relative weight of immune organs (Ghazalah et al., 2011), and mortality (Brzoska et al., 2013) under normal condition. However, based on our knowledge, there is limited information about the effect of supplemental formic acidon broiler performance under heat stress. Therefore, there is room to study whether dietary inclusion of chromium picolinate with an acifieror vitamin C can show any additive effect to eliminate the adverse effects of heat stress. Hence, the aim of the current study was to compare the influence of above mentioned additives on growth, immunity and blood parameters of broiler chickens under heat stress.

For this experiment, a total of 160 one-day-old male broiler chicks (Ross 308) were obtained from a local hatchery and raised on deep litter pens until 28 days of age, then weighed and randomly assigned to 4 dietary treatments with 4 replicates and 10 birds each with similar group weights. The experimental treatments were (1) basal diet without any supplement (control), (2) basal diet + 400 mg/kg chromium picolinate, (3) basal diet + 400 mg/kg chromium picolinate + 240 mg/kg vitamin C, and (4) basal diet +400 mg/kg chromium picolinate + 0.5% formic acid. The basal diet based on corn and soybean meal was formulated to meet or exceed the nutrients requirements recommended by Ross 308 production manual for starter (1 to 14 days of age), grower (15 to 28 days of age), and finisher (29 to 42 days of age) periods (Table I). The birds in all experimental groups had free access to water and feed for 23 h per day. The room temperature on the first day was set at 32˚C and gradually decreased to approximately 22 ˚C and then remained constant until 28 days of age. For application of heat stress, during 29 to 42 days of age the room temperature was raised to 34˚C for 8 h every day. Feed intake and body weight gain of birds per each replicate were recorded through the expemerimental period and then feed conversion ratio was calculated. The study was approved by the Animal Ethics Committee of the University of Mohaghegh Ardabili.

At the end of the experiment, two birds per replicate, each with a body weight close to the average body weight of each replicate were slaughtered after 4 h starvation. The feather removal and cutting into different parts were then performed manually. The different parts included carcass, breast, thigh, wing, neck,internal organs (gizzard, liver, heart, pancreas, and abdominal fat), and lymphoid organs (the bursa of Fabricius, thymus, and spleen) were weighed with a 0.001 g precision digital scale and expressed as the percent of live body weight.

To measure the biochemical and hematological parameters of blood, the samples of about 5 ml were taken from wing vein of two birds of each replicate on day 42 of the experiment. The samples were then poured into two test tubes. One tube containing EDTA anticoagulant was used to measure haemoglobin (Hb), red blood cells (RBC), white blood cells (WBC), and packed cell volume (PCV). RBC and WBC were counted using NAT solution by a hemocytometer. Hb and PCV levels were measured using cyanmethemoglobin and microhematocrit methods, respectively. Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) were estimated using the following common methods:

MCV (fL) = PCV × ×1000 / RBC

MCH (pg) = Hb×10 / RBC

MCHC (mmol/l or %) = Hb / PCV

1Supplied per kg of diet: 3600000 IU vitamin A, 800000 IUvitamin D3, 7200 IU vitamin E, 710 mg vitamin B1, 2640 mg vitamin B2, 1176 mg vitamin B6, 400 mg vitamin B9, 6 mg vitamin B12, 800 mg vitamin K3, 3920 mg pantothenic acid, 12,000 mg niacin, 40 mg biotin and 200,000 mg choline chloride. 2Supplied per kg of diet: 40,000 mg manganese,20,000 mg iron, 33,900 mg zinc, 4,000 mg copper, 400 mg iodine, and 80 mg selenium.

The blood serum was used to measure blood biochemical parameters including total protein, albumin, glucose, cholesterol, triglyceride, HDL-cholesterol, LDL-cholesterol, and uric acid. Blood biochemical parameters were measured using the enzymatic method with Randox-Ransod commercial kits and auto-analyzer spectrophotometer. The levels of triiodothyronine (T3) and thyroxine (T4) were measured using ELISA kits, according to the recommended instructions, with the help of an ELISA reader device.

The collected data were statistically analyzed using the General Linear Model procedure of SAS software. Duncan’s multiple range test was used to compare the significant difference (P<0.05) between the means. The statistical model of the design was as follows:

Yij= µ +Ti + Eij

Where Yij represents each observation in the experiment, µ indicates the total mean, Ti represents the effect of the i-th experimental treatment, and Eij represents the experimental error.

The effect of chromium picolinate with or without formic acid and vitamin C on birds growth performance during heat stress (29 to 42 days of age), and the whole experimental (0 to 42 days of age) periods are presented in Table II. The chickens consuming the chromium picolinate supplement alone or in combination with either vitamin C or formic acid had significantly (P<0.05) higher daily weight gain and feed intake compared with the control group. The birds on chromium picolinate plus vitamin C had similar daily weight gain and feed intake with those on chromium picolinate alone, whereas the chickens fed with formic acid and chromium picolinate had significantly (P<0.05) lower daily weight gain and feed intake than those receiving chromium picolinate alone. Moreover, the chickens fed with chromium picolinate alone or in combination with vitamin C showed the best feed conversion ratio and was followed by the group on chromium picolinate and formic acid (P<0.05).

As shown in Table III, supplementation of different dietary treatments had no significant effect on relative weights of carcass, breast, thigh, wing, neck, gizzard, liver, heart, and pancreas compared with the control. However, all treatments significantly (P<0.05) decreased relative weight of abdominalfat of the birds. The effect was more pronounced by chromium picolinate alone or in combination with vitamin C.

Table IV shows the effect of the supplements on immune organ weights (the bursa of Fabricius, thymus, and spleen) of birds at 42 days of age. All supplements significantly (P<0.05) increased the relative weights of bursa of Fabricius and thymus in comparison with the control.The relative weight of spleen was also significantly (P<0.05) increased by the treatments and the birds on chromium picolinate alone or in combination with vitamin C had the heaviest spleen.

Table II. The effects of dietary treatments on boriler chickens growth performance under heat stress during the experimental periods.

SEM, Standard error of means. a,bMeans in the same row with different letters are significantly different (P<0.05).

Table III. The effects of dietary treatments on carcass traits (%, as the percent of live body weight) of boriler chickens under heat stress on day 42.

SEM, Standard error of means. a,bMeans in the same row with different letters are significantly different (P<0.05).

Table IV. The effects of dietary treatments on relative weight of immune organs ((%, as the percent of live body weight) of boriler chickens under heat stress on day 42.

SEM, Standard error of means. a,bMeans in the same row with different letters are significantly different (P<0.05).

Table V. The effects of dietary treatments on blood biochemical parameters of boriler chickens under heat stress on day 42.

SEM, Standard error of means. a,bMeans in the same row with different letters are significantly different (P<0.05).

SEM, Standard error of means. a,bMeans in the same row with different letters are significantly different (P<0.05).

The concentration of serum metabolites of broilers as affected by dietary treatments are presented in Table V. The supplements caused a significant increase in the concentration of total protein (P<0.05). The level of albumin was increased by chromium picolinate alone or in combination with vitamin C (P<0.05). A significant decrease was observed in the concentration of glucose, total cholesterol, and LDL cholesterolas the influence of the dietary treatments (P<0.05). There were no significant effect of the treatments on triglyceride, HDL cholesterol, and uric acid levels of the birds serum.

According to the results shown in Table VI, all dietary supplements significantly (P<0.05) decreased blood MCV, heterophils percent and heterophil to lymphocyte ratio and decreased MCHC percent and WBC counts compared with the control. The percentage of blood lymphocytes was significantly (P<0.05) increased by chromium picolinate and chromium picolinate plus vitamin C. The other hematological parameters including RBC, Hb, PCV, MCH, T3 and T4 were not significantely affected by the treatments (P>0.05).

It is demonstrated that heat stress leads to reduced feed intake, growth performance and feed efficiencyin broiler chickens (Hu et al., 2019). Weight loss is not only due to less feed intake, but also due to the direct effect of ambient temperature on the physiology and metabolism of broilers (Geraert et al., 1996).

The improvement observed in growth response of broilers under heat stress in this study (Table II) is consistent with previous studies which report the similar results by adding chromium picolinate supplement alone (Hamidi et al., 2016; Chougule et al., 2018; Hridoy et al., 2021) or in combination with vitamin C (Sahin et al., 2003) to the diet. The increased daily feed intake during heat stress (29 to 42 days of age) and the whole experimental periodis in line with earlier reports indicating increased appetite and feed intake by chromium picolinate supplement alone (Toghyani et al., 2006; Hamidi et al., 2016; Sahin et al., 2017; Chougule et al., 2018) or in combination with vitamin C (Sahin et al., 2003).

There is contradictory results on chromium picolinate effect on birds feed conversion ratio under heat stress. In keeping with our finding, some studies have been reported improved feed conversion ratio by adding chromium picolinate alone (Samanta et al., 2008; Ezzat et al., 2017; Chougule et al., 2018; Hamidi et al., 2019) or in combination with vitamin C (Sahin et al., 2003) or with vitamin E (Hridoy et al., 2021), whereas the others have not shown any improvement by adding chromium picolinate alone (Toghyani et al., 2006; Tawfeek et al., 2014; Huang et al., 2016) under heat stress or with vitamin C (Haq et al., 2018) under normal temperature conditions. Inconsistency in the results obtained by different studies might be explained by the supplemental level of chromium picolinate and other variables such as bird age, health status, and most importantly, the type (acute or chronic) and duration of stress and etc.

Chronic heat stress suppresses the activity of the appetite center in the hypothalamus and reduces feed intake, by affecting the ambient temperature receptors and the transmission of nerve impulses to the hypothalamus (Marai et al., 2007). Reducing feed intake leads to a reduction in consumption of nutrients used for growth, which is part of the bird’s physiological adaptation to heat stress (Lara and Rostagno, 2013), so that fewer nutrients are available to the bird for activity and enzymatic synthesis, hormone production, and heat regulation (Attia et al., 2017). In addition, stress increases the mobilization of chromium from tissues and increases urinary excretion, and decreases its retention in the body (Sahin et al., 2002). Thus, the need for chromium increases in such cases. In current study, observation of increased feed intake by supplemental chromium picolinatecompared with the control may be because of providing high demand of birds for chromium during heat stress (Sahin et al., 2010). On the other hand, heat stress increases the concentration of corticosterone in the blood (Zhao et al., 2009) and decreases the sensitivity of body cells to the insulin (Sirirat et al., 2012). Chromium supplement reduces blood glucose and increases appetite (Chougule et al., 2018) by increasing the sensitivity of tissue receptors to insulin and increasing glucose uptake by cells (Ezzat et al., 2017). As a result, increasing feed intake by providing more glucose, amino acids and other nutrients to muscle tissues and cells and increased protein retention can lead to improved weight gain (Hamidi et al., 2016), as seen in this study (Table II).

Heat stress has an adverse effect on serum concentrations of vitamins such as vitamin C (Kucuk et al., 2003). It has been reported that vitamin C supplement improves the absorption and retention of chromiumin the body (Ahmed et al., 2005; Dalólio et al., 2018), thereby improving physiological functions of broilers under heat stress (Sahin et al., 2003; Haq et al., 2016). However, in the present study there was no synergy between vitamin C and chromium picolinate. Accordingly, the synergy of chromium picolinate and vitamin C leads to improved feed intake, better nutrient digestibility, and feed efficiency in this group, as previously reported by Sahin et al. (2003).

In keeping with our results (Table III), no significant effect of chromium picolinate supplementation on relative weight of broiler carcass (Tawfeek et al., 2014; Zheng et al., 2016), breast (Xiao et al., 2017; Kulkarni et al., 2018; Ding et al., 2020), and thigh (Xiao et al., 2017; Kulkarni et al., 2018) has been reportedunder heat stress. However, anincrease in relative carcass and breast weights by adding chromium picolinatehas also been illustrated under heat stress conditions (Samanta et al., 2008; Ding et al., 2020). Unlike our findings, the increase of hot and cold carcass weights by combination of chromium picolinateand vitamin C under heat stress (Sahin et al., 2003) and carcass and breast weights by chromium yeast with vitamin C in normal conditions (Haq et al., 2018) has been reported.No significant effect of chromium picolinate supplement has been previously recorded on relative weights of thigh, wing, gizzard, liver, heart, and pancreasunder heat stress (Toghyani et al., 2006; Samanta et al., 2008; Kulkarni et al., 2018), which is consistent with our results. However, Sahin et al. (2003) has been reported increased liver, heart, and gizzard weights as the result of chromium picolinate supplementation to the diet of heat stressed broilers.There are contrary reports on the effect of chromium picolinate on the liver weight of broilers under heat stress. Lien et al. (1999) reported that supplemental chromium picolinate at the levels of 1600 and 3200 µg/kg had no effect on liver weight, while at 800 µg/kg level increased the organ weight. An increase in the liver weigh has been also observed by Sahin et al. (2002) in broilers fed with chromium picolinate. Furthermore, no effect of chromium picolinate supplementation has been shown on liver weight of broilers under heat stress (Toghyani et al., 2006; Tawfeek et al., 2014).

Consistent with our results, the reduction of abdominalfathas been previously reported by adding chromium picolinate supplement to the diets of broilers under heat stress (Samanta et al., 2008; Huang et al., 2016; Kulkarni et al., 2018). A similar observation has also been seen by supplemental chromium yeast with vitamin C under normal temperature conditions (Ahmed et al., 2005; Haq et al., 2018). One probable reason for this observation may be due to the effect of chromium oninhibition of fat synthesis or fat mobilization, or both (Suksombat and Kanchanatawee, 2005). Furthermore, it has been revealed that chromium enhances cellular utilization of glucose by stimulating insulin action (Vincent, 2000). According to Mertz (1993), glucose is converted to lipid and stored in adipose tissue at low levels of insulin. Chen et al. (2018) reported that the activity of fatty acid synthase, acetyl coenzyme A carboxylase, hormone-sensitive lipase, and lipoprotein lipase, which are essential enzymes in the metabolism, transport and storage of fatty acids, arereduced by chromium picolinate supplement. Thus, induced lower deposition of fat by chromium supplementation might be occurred by increased utilization of glucose and fatty acids and inhibition of fat biosynthesis.

On the other hand, heat stress increases the level of blood corticosterone by activating the hypothalamic-pituitary-adrenal axis (Zhao et al., 2009). Chromium picolinate, however, reduces the level of plasma corticosteroids (Dalólio et al., 2018) by inhibiting key enzymes involved in the corticosteroid biosynthesis pathway (21-hydroxylase and 11-β-hydroxylase) (Alhassani and Alshukri, 2016), thereby causing are duction in the synthesis and secretion of corticosterone in broilers under heat stress, which in turn resulted in decreased catabolism and increased protein synthesis in muscle tissue and reduced abdominal fat. This can improve meat quality of under heat stressed broilers.

The bursa of Fabricius, thymus, and spleen are the important organs of the immune systems in broilers. It is shown that heat stress reduces the weight of these organs (Hirakawa et al., 2020), may be due to the lowered feed intake and subsequent fewer nutrients providing for their proper growth (Bartlett and Smith, 2003). All supplements increased the birds feed intake (Table II) and led to high relative weight of the bursa of Fabricius, thymus and spleen (Table IV). However, the effect of chromium picolinate with formic acid on both feed intake and spleen relative weight was less than chromium picolinate alone or in combination with vitamin C. Lu et al. (2019) reported that different levels of chromium picolinate supplement (0.4, 0.8, 1.6, or 3.2 mg/kg) increased immune organ weights of the broilers under heat stress. Hamidi et al. (2016) also reported an increase in thymus and spleen weights by adding chromium picolinate supplement to the diet of heat stressed broilers. However, inconsistent with our results, Toghyani et al. (2007) did not report any significant effect of chromium picolinate on immune organs weights. In addition to the effect of feed intake on immune organs, the influence of corticosterone also should be noted, as it is shown that corticosterone causes the weight loss of lymphoid organs (Haq et al., 2018). Therefore, the supplementation of chromium picolinatein the current study may has been caused high immune organ weights by affecting on this hormone as mentioned above.

Biochemical parameters provide valuable information about the health and wellbeing status of animals. Heat stress in broilers leads to some changes in serum biochemical parameters such as reducing total protein, albumin, and HDL- cholesterol, and increasing glucose, total cholesteol, triglyceride, and LDL-cholesterollevels (Ding et al., 2020). Supplementaion of chromium picolinate, alone or in combination with vitamin C, icreased the levels of total protein and albumin in the blood (Table V). A similar observation has been previously reported by adding chromium picolinatealone (Ezzat et al., 2017; Chougule et al., 2018; Hamidi et al., 2019) or in combination with vitamin C to the diet under heat stress (Sahin et al., 2003), or by adding chromium yeast plus vitamin C in the diet of broilers under normal temperature conditions (Haq et al., 2018). However, there is a report indicating no significant effect of chromium picolinate on the concentration of blood total protein and albuminunder heat stress (Hridoy et al., 2021). It is shown that high ambient temperatures reduces protein synthesis and increases protein catabolism in the body (Gous and Morris, 2005). The chromium supplement enhances glucose uptake by the cells due to stimulating insulin action (Vincent, 2000). Indeed, increasing serum insulin concentration regulates protein metabolism by increasing cellular amino acid uptake and protein synthesis (Colgan, 1993). Therefore, the rate of protein anabolism is higher than the rate of protein catabolism (Sahin et al., 2002). On the other hand, vitamin C also limits the catabolism of lipids and proteins by lowering corticosterone levels (Kucuk et al., 2003).

The reduction of serum glucoselevelin this trial (Table V) was in keeping with perivious studies by adding chromium picolinatealonealone (Sahin et al., 2017; Chougule et al., 2018; Hamidi et al., 2019) or in combination with vitamin C (Sahin et al., 2003; Saracila et al., 2021) to the diet of broilers under heat stress. It has been reported that chromium, as an active glucose tolerance factor (GTF), increases the susceptibility of tissue receptors to insulin and thus increases glucose uptake by the cells (Ezzat et al., 2017) and decreses its level in the blood.It is possible that chromium and vitamin C supplement also reduced blood glucose levels by activating insulin, thereby increasing insulin uptake into tissues (Khukhodziinai et al., 2021).

According to the results, all supplements reduced total and LDL cholesterol levels (P<0.05) with no effectonthe concentration of triglyceride or HDL cholesterol. Consistent with our results, several studies have shown that the use of chromium picolinate supplements in the diet of broilers under heat stress reduces serum cholesterol (Sahin et al., 2017; Chougule et al., 2018; Hridoy et al., 2021). Also, a reduction in total cholesterol concentrations in broilers blood have been reported by the addition of chromium picolinate plus vitamin C (Sahin et al., 2003) or chromium picolinate plus vitamin E under heat stress (Hridoy et al., 2021). Inconsistent with our results, Saracila et al. (2021) reported no decrease of serum total cholesterol by adding chromium picolinate (200g / kg) with vitamin C (0.25 g / kg) to the diet of broilers under heat stress. Similar to our results, no changes in triglyceride or HDL cholesterol levels was observed by supplementing chromium picolinate (200 or 400 µg/kg) (Sands and Smith, 2002) or in triglyceride level by adding chromium picolinate (200 µg/kg) plus vitamin C (0.25 g/kg) to the diet of heat stressed broilers (Saracila et al., 2021). Haq et al. (2018) also reported that supplementation of chromium yeast with vitamin C did not affect the concentration of HDL cholesterol of broilers under normal temperature conditions. Moreover, a decrease in triglyceride and an increase in HDL cholesterol concentration of broilers blood have been reported by chromium picolinate under heat stress (Sahin et al., 2002; Samanta et al., 2008; Tawfeek et al., 2014), which is in opposite to our findings.

The increase oftotal cholesterol and triglyceride levelsin the blood of broilers under heat stress might be due to the higher levels of stress hormones that stimulate lipolysis (Hajati et al., 2018), or due to the reduced feed intake, which causes the bird to increase lipid metabolism or lipolysis to meet its energy needs (Rashidi et al., 2010). The reduction in serum total cholesterol and triglyceride concentrations can be explained by the stimulatory effect of organic chromium on insulin secretion, there by increasing glucose uptake to oxidize or convert to fatty acids for storing as triglyceride in adipose tissue. It has been reported that chromium increases the activity of lipoprotein lipase and lecithin-cholesterol acyltransferase, which in turn accelerates esterification and excretion of cholesterol, stimulates HDL synthesis, and increases LDL receptors in the liver, thereby reducing blood LDL content, and at the same time, increasing HDL levels (Brindley and Salter, 1991). On the other hand, vitamin C reduces the concentration of cholesterol in the liver and serum by accelerating the conversion of cholesterol to bile acids. Because cholesterol is transported in the blood by lipoprotein complexes (VLDL, LDL, and HDL), the concentrations of cholesterol and lipoprotein are positively correlated (Linne and Ringsrud, 1999). As asimultaneous reduction in total cholesterol and LDL cholesterol levels was observed in this study (Table V). However, differences in the results of different studies may be related to chromium sources and levels, stress conditions, and disease incidence (Huang et al., 2016).

Consistent with our results, Samanta et al. (2008) and Sathyabama et al. (2017) reported that chromium picolinate supplement had no significant effect on serum uric acid concentration in broilers under heat stress compared with the control.

It has been shown already that heat stress in broilers reduces RBC (Hassan and Asim, 2020), Hb level (Ding et al., 2020), PCV percentage (Ribeiro et al., 2018), increases MCV, and decreases MCHC (Jassim and Hassan, 2011), which are part of the bird’s response to temperature regulation (Yahav, 1999). Howevere, RBC and Hb did not change in the current study due to the dietary treatments (Table VI). The findings are in line with Toghyani et al. (2006) and Hridoy et al. (2021), who also reported no effect of chromium picolinate on these parameters. However, Ezzat et al. (2017) found that the addition of chromium picolinate at the level of 1200 µg/kg to the diet of broilers under heat stress caused anincrease in RBC and Hb. It is noted that that chromium, as an antioxidant, can protect vitamin C against oxidative damage. Insulin that is activating by chromium,isalso involved in the transport of vitamin C to red blood cells (Sahin et al., 2001). Accordingly, chromium picolinate plus vitamin C can more effectively protect RBCs and Hb against oxidative damage and provide more nutrients by increasing the ability of oxygen transferring under stress conditions (Oleforuh-Okoleh et al., 2015). In the current study, the positive effect of adding vitamin C to chromium picolinate containing diet is reflected by a non-significant increase in Hb levels (P=0.057).

The percentage of PCV was not affected by the supplements (Table VI), which was consistent with the findings of Toghyani et al. (2006) and Hridoy et al. (2021). MCV, MCH, and MCHC are indicators of red blood cells, and since MCH and MCHC are calculated using Hb, PCV, or RBC values, any changes in Hb, PCV, and RBC are directly reflected in MCH and MCHC (Ding et al., 2020). Consistent with our results, Abdelhady et al. (2017) reported that chromium supplement (1400 µg/kg) in the diet of Japanese quails under heat stress reduced MCV. However, Toghyani et al. (2006) and Hridoy et al. (2021) reported that chromium picolinate supplement in the diet of broilers under heat stress did not affect MCV. It is possible that the supplements caused the reduction of MCV due to their antioxidant capacity to maintain the integrity of RBS membranes (Adekola et al., 2010). The supplements had no significant effect on MCH (P>0.05), which was in line with the finding of Toghyani et al. (2006) and Hridoy et al. (2021) and increased MCHC, that was consistent with the result of Toghyani et al. (2006). In contrast, Hridoy et al. (2021), did not find any changes in MCHC by chromium picolinate supplement. Since MCHC reflects the haemoglobin content of red blood cells, an increase in its amount may indicates increased haemoglobin production and subsequent oxygen-carrying capacity of this cells (Isroli et al., 2020). In keeping with our result, Ezzat et al. (2017) also reported that the addition of chromium picolinate supplement to the diet of broilers under heat stress caused a significant increase in WBC. It is revealed that broilers exposed to various stresses show an increase in heterophils and a decrease in lymphocytes, which leads to an increase in the H/L ratio (Aengwanich and Suttajit, 2010). Hence, the H/L ratio is a reliable indicator for avian stress (Felver-Gant et al., 2012). Gross and Siegel (1983) found that the number of heterophils in the blood of chickens increased by feeding corticosterone. According to the results, the supplements caused a significant decrease inheterophils and H/L ratio (P<0.05). Such reductions might be mediated by decreased corticosterone secretion, which induced by chromium supplement (Dalólio et al., 2018). The decrease in the H/L ratio by adding chromium (Norain et al., 2013) or vitamin C (Hassan and Asim, 2020) to the diet of broilers under heat stress has already been reported.

It has been shown that under heat stress, dietary supplementation of chromium increases the concentrations of T3 and T4 in the blood of broilers (Dalólio et al., 2018). The same observation has been reported by supplemental chromium picolinate alone (Sahin et al., 2003; Ezzat et al., 2017) or in combination with vitamin C (Sahin et al., 2003) in heat stressed broilers. Although in this study the treatments had no significant effect on T3 and T4 concentrations, the influence of chromium picolinate, alone or in combination with vitamin C on T4 tended to be increased (P= 0.064).

In conclusion, the use of dietary chromium picolinate (400 mg/kg) alone or in combination with vitamin C (240 mg/kg) ameliorates the adverse effects of heat stress by improving performance and immune status, and reducing abdominal fat of the birds. Supplementation of vitamin C (240 mg/kg) to the diet failed to show any synergistic effect with chromium picolinate, however, addition of formic acid (0.5%) worsens the influence of supplemental chromium picolinate on growth response, abdominal fat andsome blood parameters.

Not any funds were provided for this study.

The study was approved by the University of Mohaghegh Ardabili, Ardabil, Iran.

The study was approved by the ethics committees of the authors’ institutions.

The authosr have declared no conflict of interest.