Organic Chromium and Vitamin E Enhance Physiological Performances, Humoral and Cellular Immune Responses in Heat-stressed Broiler Chickens

Heat stress (HS) is one of the most important environmental stressors challenging poultry production in tropical and sub-tropical countries. It has detrimental effects on poultry feed intake, body weight gain, mortality, hatchability and other important traits (Rao et al., 2012). The negative effects of heat stress will increase in the coming years due to temperature rises associated with global warming. Broiler chickens are more susceptible to high temperatures because of their greater metabolic heat production. To attenuate the negative effects of heat stress in broiler production systems, different approaches are adopted including vitamin and mineral supplementation (Lin et al., 2006). The inclusion of vit-E in feed for broiler has resulted in positive effects on growth performance when supplemented at higher levels during HS (Souza et al., 2011). Vit-E is a component and natural antioxidant of cell membranes. It involved in activation of the immune system (Hashizawa et al., 2013). Cr is an essential mineral element, present in small amounts in raw ingredients in broiler diets. Organic Cr plays a crucial role in poultry nutrition, production and health in broiler chicken under HS condtion. It is required for improving productive performance in poultry due to its important functions in metabolism, growth and reduction of lipid and protein peroxidation. Supplementation of Cr may increase body weight gain, improve feed efficiency in broilers (Farag et al., 2017). The one of the most common Cr forms used as supplements to poultry and animal diets is Cr picolinate (CrPic). CrPic is an organic source of chromium that has high bioavailability. The precise role of Cr on immune functions has not been fully elucidated. Several studies showed that immunological function has been enhanced by Cr (III) and its effects seem more pronounced during stress (Kafilzadeh et al., 2012). On the other hand, HS was shown to adversely affect immune potency (Ghazi et al., 2012). Therefore, supplementation of Cr may beneficially influence the immune response in heat-stressed broiler chicken (Lee et al., 2003). Another study revealed that the supplementation of dietary Cr increases the production of antibodies against IBR (Faldyna et al., 2003) and tetanus toxins. The study investigated the effects of CrPic and vit-E on growth performance, hemato-biochemical parameters and immune responses in broiler chicken under HS condition.

The research was conducted at the Department of Physiology, Bangladesh Agricultural University, Mymensingh during the period from March 1, 2019 to April 5, 2019. All experimental protocols were approved and performed according to the approved guidelines of animal welfare and experimentation ethics committee, Bangladesh Agricultural University, Mymensingh, Bangladesh.
 
A total of 120 healthy day old Lohman broiler chicks were purchased from local hatchery. On the 13^th day of age, chicks were randomly divided into the four equal groups (n=30): A, B, C and D. Group A was considered as non-treated control, Group B, C and D were treated with CrPic (250 mg/kg feed), Vit-E (1mL/2L) drinking water and both respectively. All groups were reared at room temperature of 28 ± 3°C followed by HS condition at a temperature range of 35 ± 3°C for 6 hours daily throughout the experimental period. Broiler starter, grower and broiler finisher feed was provided according the standard broiler farming system (Table 1). CrPic and vit-E were chosen from market available poultry products. Birds received their freshly prepared daily medication in the morning hour of each day.
 

 
Measurements of body weight and feed conversion ratio (FCR)
 
The body weight of each bird was measured in a weekly basis using digital balance and total body weight gain was calculated (body weight gain= final body weight - initial body weight). FCR was determined by the formula: total feed consumed by birds divided total body weight gain. Feed consumption is the amount of feed consumed by the birds in a period of time.
 
Blood collection and hemato-biochemical analysis
 
Blood collection, serum separation and selected hematological parameters (Hb, TEC, PCV and erythrocyte indices) were performed according to standard method (Khalil et al., 2020). The biochemical tests were performed using a UV spectrophotometer T 80, PG instruments, Great Britain.
 
Measurement of antibody titers against ND vaccine by hemagglutination inhibition (HI) test
 
HI tests on serum samples were carried out according to the method described (Yadav et al., 2018). The HI titer of the Newcastle disease virus (NDV) antigen (LaSota stock virus) was adjusted by dilution to contain 4 units of hemagglutination activity. HI titer was determined as the highest dilution of serum samples that inhibited NDV agglutination of chicken RBCs.
 
Determination of cell-mediated immune (CMI) response
 
The CMI response was assessed using the cutaneous basophil hypersensitivity (CBH) response to phyto hemagglutinin (PHA-P) (Sikandar et al., 2017). Briefly, on day 32, six healthy broilers per treatment were intradermal injected between the third and fourth digits of the right foot with 100 μg of PHA-P in 0.10 ml of sterile PBS solution. The left foot of each bird was similarly injected with 0.10 ml PBS solution to serve as control. The thickness of the skin was measured with a veriner caliper just before and 12h, 24 h and 72 h following the injection.
 
Statistical analysis
 
The hematological and biochemical parameters of broilers corresponding to Cr and vit-E supplementation are compared by performing analysis of variance using Graph Pad Prism 8.

Effects of CrPic and vit-E on growth performances and FCR in broilers
 
The effects of organic CrPic and vit-E on live body weight, body weight gain and FCR are presented in Fig 1 and Table 2. After one week of the CrPic and vit-E supplementation, the highest body weight was recorded in group B (CrPic) followed by groups D, C and A. The values were differed significantly (p<0.05). On the 28^th day of age, the highest body weight was recorded in group B (CrPic) followed by group C and D. On the 35^th day, there was a sharp rise in body weight in the birds of treatment groups. The highest body weight was recorded in group B (CrPic) and the lowest body weight was in group A (control). All the data were statistically significant (p<0.05) among the groups. The result showed the body weights and body weight gain were increased slowly in the control group but a rise in body weight was noticed during the last three weeks in the treated groups (B, C and D). Organic CrPic had a more pronounced effect on body weight gain in HS conditions. In parallel, FCR values were significantly reduced in all treated groups (Fig 1 and Table 2). Increased body weight gain in the treated group might be due to an increased feed utilization, digestion, absorption and metabolism of supplied feed nutrient. The present observation was in line with the previous findings of Haq et al., 2016.  Both Cr and vit-E improved feed efficiency in the later phases of growth in the broiler. Under HS conditions, CrPic might play a crucial role in poultry nutrition, production and health. It enhances growth performance. Rao et al., 2012 also stated that Cr supplementation alone increases weight gains of poultry. Vit-E with drinking water was also found to be useful to improve feed intake, body weight gain and feed efficiency broilers. CrPic and vit-E singly or in combination had a significant (p<0.05) lowering effect on FCR in broiler and especially CrPic treated birds achieved minimum FCR than other groups. A similar decrease was observed by previous findings of Moeini et al., 2011; Niu et al., 2009.
 

 

 
Effects of CrPic and vit-E on hemato-biochemical parameters
 
The mean values of Hb, TEC, PCV and erythrocyte indices (MCV, MCH and MCHC) were slightly increased in the treated groups but remained within the normal range and statistically non-significant (p>0.05) (Table 3). The mean serum ALT values were varied among the groups but didn’t differ significantly. The highest AST level was recorded in group C and the lowest was in group A . The mean values of creatinine levels also varied among the groups (Table 4). The data indicated that birds fed supplemented CrPic had increased creatinine levels. The mean values of serum total protein, albumin and glucose of different treatment groups were found differed slightly among the treatment groups. Still, the values were within the normal ranges. Broilers fed dietary chromium and vit-E had increased serum cholesterol, triglyceride, LDL-c and HDL-c (Table 4). The data were statistically significant at p<0.05. It is reported that Cr supplemented birds had significantly increased hemoglobin, MCH and MCHC (Faraget_al2017). Birds fed supplemented vit-E also had slightly increased hematological values in this current study, which is supported by a previous report (Biswas et al., 2011).
 

 

 
Two important liver enzymes were varied slightly in the present research. AST values but not ALT, were increased significantly in the birds of Vit-E fed group. Dietary addition of Se and bacterial organic Se (vitamin E) significantly affects broiler serum ALT, AST, LDH activities and reduces serum creatinine level (Farag et al., 2017). The underlying mechanism of increase activity of AST in treated group need to investigate. Current study didn’t find any alternation of serum total protein, albumin and glucose level in blood although glucose levels were reduced in CrPic treated birds. it is reported (Haldar and Ghosh 2008; Tawfeek et al., 2014) that serum glucose was decreased. In contrast, total protein, albumin concentrations increased when dietary Cr and Se singly or in combination, were supplemented. TC, LDL-c and HDL-c were found increased in the sera of CrPic and Vit-E fed birds in the present study. The results are not agreed with other findings that supplementation of Cr decreased the blood cholesterol and LDL-c whereas HDL-c concentration was increase during starter and finisher phase of broilers (Aslanian et al., 2011). Under heat stress conditions, the serum concentrations of TC, TG and LDL-cholesterol were increased. Serum HDL-cholesterol decreased in broilers supplemented with selenium and vit-E (Habibian et al., 2014). It was also reported that cholesterol decreased when dietary Cr and Se singly or combined were supplemented (Tawfeek et al., 2014).
 
Effects of CrPic and vit-E on humoral and cellular immune response
 
The HI titer values were calculated in different groups of broilers (Fig 2). It was clearly found that birds of group B had the highest antibody titers against the ND vaccine compared with the control and other groups (p<0.01). These results revealed that the administration of Cr to ND vaccinated birds orally resulted in potentiation of the chicken immune response to ND vaccination under HS. Cellular immunity of broiler chickens fed CrPic and vit-E were assessed by CBH response (Table 5). The CBH response elicited by the PHA-P was evaluated by determining toe web skin thickness. Results revealed that significant skin increases were observed in PHA-P treated right toe web compared to PBS treated left foot. Supplementation of Cr and Vit-E in birds achieved a different degree of CBH responses in a time-dependent manner. The current study confirmed that Cr has immune stimulants activities that can promote recovery from immune suppression states and can enhance the innate and specific immunity. This result is in line with the findings of previous reports (Eze et al., 2014) which concluded that dietary supplementation of Cr improved the immune response to ND vaccine under HS conditions in broilers. Assessment of the cellular immunity of broiler chickens fed CrPic and vit-E by CBH response induced the immune responses of birds under HS conditions.
 

 

Supplementation of CrPic at the dose rate of 250 mg/kg feed improves the physiological performances, antibody titers against NDV and cellular immune response in heat-stressed broiler chickens.

This research work was funded by the Ministry of Science and technology, Government of the People’s Republic of Bangladesh under special allocation of the Science and Technology.

The author declares no conflict of interest.