Copper Super-Dosing Improves Performance of Heat-Stressed Broiler Chickens through Modulation of Expression of Proinflammatory Cytokine Genes

Continuous exposure to high ambient temperatures brings about a number of oxidative damages in chickens. Copper (Cu), an active component of a number of antioxidative defence components, should arrest these changes to take place although that may not be possible under the standard dosing regimen followed by the industry. To ascertain the optimum dose response that may be beneficial in sustaining the performance of chickens under heat stress (HS), broiler chickens (n = 400) were exposed to high ambient temperature (between 27.2°C and 35.3°C) during 1–35 d. Copper (Cu) as Cu proteinate (Cu-P) at concentrations of 37.5, 75, 112.5, and 150 mg/kg was supplemented to the diet. The negative control (NC) diet did not contain any supplemental Cu. Increasing dietary Cu improved (P < 0.001) body weight, feed intake, and conversion ratio. Serum concentrations of total cholesterol at 21 d (P = 0.009), HDL cholesterol at 35 d (P = 0.008), LDL cholesterol at 21 d (P = 0.015), and triacylglycerol at both 21 d (P = 0.033) and 35 d (P = 0.001) decreased as Cu in the diet increased. As Cu in the diet increased, hemoglobin increased (P = 0.003) at 21 d, and the heterophil to lymphocyte ratio decreased both at 21 d (P = 0.047) and 35 d (P = 0.001). Superoxide dismutase and glutathione peroxidase activities increased when dietary Cu increased to 150 mg/kg (P < 0.01). Liver Cu at 35 d increased linearly with the dose of Cu in the diet (P = 0.0001). Selected bacteria were enumerated in the digesta to ascertain if Cu super-dosing affected their population in any way in the absence of any enteric challenge. Escherichia coli and total Salmonella numbers decreased (P = 0.0001), and total Lactobacillus increased (P = 0.0001) proportionately with dietary Cu. Interleukin-6 and tumour necrosis factor-α gene expression increased linearly (P = 0.0001) as Cu in the diet increased though the response plateaued at 112.5 mg/kg. It was concluded from the present experiment that during conditions of impending HS, dietary supplementation of 112.5 to 150 mg Cu/kg diet as Cu-P may be a novel strategy to alleviate the negative effects of HS without involving any apparent risk of Cu toxicity.


Introduction
High ambient temperature (HT) causes acute (a short and rapid increase in temperature) or chronic (extended exposure to moderately high temperature) heat stress (HS) and retards the performance of modern fast-growing broiler chickens leading to signifcant economic losses [1].Te intensive genetic selection for rapid growth has imparted higher metabolic rates in these birds and has made them less heat tolerant [2].Oxidative damage of intestinal mucosa [3,4] and change in small intestinal microbiota composition occur upon exposure to acute or even chronic HT, which not only impairs the general performance traits but also negatively afects the immune system in various ways, thus making chickens vulnerable to impending enteric and systemic diseases [5].Chickens, like all other avian species, do not possess any functional heat dissipation mechanism except panting, and hence, when humidity in the environment increases concomitantly with temperature, the bird's core body temperature may exceed the upper critical limit to cause mortality [1,2].Climate-controlled houses play a crucial role in keeping the microenvironment surrounding the birds comfortable and conducive to optimum production and are one of the physical strategies employed worldwide to prevent HS-related mortality in broiler chickens.However, subtle physiological changes are inevitable even in environmentally controlled houses in areas experiencing high ambient temperature and humidity.Tese physiological changes may have negative impacts at the tissue and cellular levels, which may be sufcient to bring down the performance indices.Many dietary strategies, which are likely to mitigate the negative efects of HS by modulating the cellular and molecular functionality in commercially reared chickens, are being explored in recent times when owing to global warming, and maintaining the rearing temperature of a chicken-producing house within the thermoneutral zone has become a challenge.Trace minerals are one of the candidates which, when applied judiciously in the dietary regimen, may provide a solution to this problem amongst the trace minerals.Te scope of copper (Cu) in sustaining the performance of chickens exposed to HS seems quite promising although this scope has not been hitherto researched to a great extent.
Dietary Cu requirement for broiler chickens varies from 8 mg/kg [6] to 16 mg/kg [7].Adding Cu at higher concentrations in broiler diets for growth promotion has been reported [8].Interestingly, such "more than the requirements" applications of Cu have not been extrapolated to study its efects in ameliorating HS.As an essential component of cytochrome oxidase, lysyl oxidase, superoxide dismutase, ceruloplasmin, and metallothionein, Cu maintains the redox balance and helps to achieve the targeted growth performances in poultry [9].At an inclusion level of 150 to 200 mg/ kg diet, Cu reportedly resulted in superior body weight (BW) and feed conversion ratio (FCR) in broiler chickens [10].Tis supplementation strategy, which may be termed Cu superdosing, should be a useful tool in sustaining the performance of domestic fowl, especially that of broiler chickens, under conditions of HS since Cu is an active component of the redox system of the body and considerable oxidative damages take place when a biological system gets exposed to elevated ambient temperature for a prolonged period.However, selecting the inclusion level of Cu should be performed carefully to prevent any possible toxicity since, as it will be discussed in more details in this study in a later section, Cu in higher inclusion levels may get accumulated in hepatic tissues and initiate oxidative damages by production of free radicals.Tis efect may vary with the source of Cu and hence is highly variable, and so far, no data are available with regard to Cu presented as yeast protein chelates.
In the present experiment, broiler chickens were exposed to naturally occurring cyclic HS from 1 to 35 d and were supplemented with Cu at graded levels up to 150 mg/kg diet as a yeast protein-Cu chelate (Cu-P).Te experimental conditions simulated the practical situations often encountered by broiler operations, especially in tropical and subtropical countries, and paired controls for the heatstressed groups needed to be improved.However, the basic objective of the experiment was to ascertain the efect of HT in broiler chickens when diets were near-defcient in terms of Cu and whether Cu super-dosing could alleviate the likely negative efects of HS.Tis design did not necessarily warrant heat-stressed paired control.Te efects of Cu super-dosing on the expression of certain key immunemodulatory genes were also ascertained to assess if this strategy could help broiler chickens combat any impending diseases during exposure to HS.

Experimental Design.
Te 35 d study was conducted with a fock of male broiler chickens (n � 400) fed a basal diet supplemented with a trace mineral premix devoid of Cu (negative control, NC).Te NC diet was supplemented with Cu-P at varying levels to achieve the fnal Cu concentrations of 37.5, 75, 112.5, and 150 mg/kg, which corresponded to 250 (Cu-P 250), 500 (Cu-P 500), 750 (Cu-P 750), and 1000 (Cu-P 1000) mg/kg of the Cu-P in the diet.Te customized Cu-P was manufactured in a commercial trace mineral production facility (Zeus Biotech P Ltd., Hebbal, Mysuru, India).Te diets were adequate concerning all nutrients except Cu (Table 1) and did not contain any antibiotic growth promoter or probiotic.A phytase (Buttiauxella phytase with a declared activity of 5000 FTU/g, Danisco Animal Nutrition, Marlborough, UK) was used with its full matrix in all the diets.Diclazuril (0.5%) was added to prevent a possible coccidiosis outbreak.A yeast protein complex (similar to the one used to produce the Cu proteinate) was added to all the diets at the rate of 1 g/kg, and an equivalent amount of this was replaced when Cu-P was added to the diet.Tis was performed to ensure that the results obtained in the treatment groups were due to the efects of Cu only and not due to the yeast protein present in the Cu-P.

Bird Husbandry, Diet Preparation, and Measurement of Performance Traits.
Te experimental fock of one-day-old chickens (Vencobb 430 Y, Venkateshwara Hatcheries, Pune, India) was distributed in one of the fve treatments following a completely randomized design.Te chicks were weighed immediately after arrival and placed on a litter of fresh wood shavings and chopped paddy straw in individual pens (1.2 m × 1.2 m).A single pen constituted a replicate, and 8 such replicate pens were in a treatment group (10 chicks in a replicate and 80 chicks per treatment).Te lighting period involved 23 h light during the frst 7 days, followed by 20 h a day during the remainder.Te experimental diets were produced by mixing all ingredients, excluding the Cu-P.Tis mix was then divided into 5 equal parts, and a premix (2 g/ kg) containing Cu-P at variable quantities and fnely ground maize (as fller) was added to each of these parts to achieve Each kg contained vitamin A 13.5 MIU, vitamin D3 4.5 MIU, vitamin E 60 g, vitamin K3 3.5 g, vitamin B1 3.5 g, vitamin B2 8.0 g, vitamin B6 3.5 g, vitamin B 12 0.02 g, biotin 0.145 g, pantothenic acid 14.5 g, folic acid 2.25 g, niacin 60 g; 4 Buttiauxella phytase with a declared activity of 5000 FTU/g; (Transasia Bio-Medicals Ltd., India) in a semiautomated blood biochemistry analyser (RT-9100, Rayto Life and Analytical Sciences Co., Ltd., Shenzhen, China).Blood samples from the left brachial veins were collected in glass tubes containing EDTA as an anticoagulant.Te tubes were handled with care to prevent damage to the blood cells.After blood sampling, a cellulose swab was pressed onto the wound until the bleeding stopped.Blood samples were brought to a laboratory near the broiler house for diferential leukocyte counts.Blood smears were performed under dustfree and almost sterile conditions in the laboratory to achieve high-quality smears.Using a glass rod, one drop of blood was placed on a grease-free microscopic slide.Te drop was spread on the glass slide by the wedge smear technique.Te smears were air-dried, stained with Giemsa, and observed under the 40x objective of a trinocular microscope (Coslab 32 LED, Coslab India, Ambala, India).For the diferential count, 200-400 leucocytes were determined.Te H/L ratios were calculated by dividing the relative heterophil counts by the relative lymphocyte counts.Another aliquot of the fresh whole blood collected from the left brachial vein was used for the estimation of hemoglobin (Hb) by azidemethaemoglobin method in the blood analyser using a commercially available Hb kit (BeneSphera Hemoglobin Kit, Pennsylvania, USA).

Bacteria Enumeration in Ileal Digesta
. At the end of the feeding trial, one bird from each of the replicate pens (n = 8 birds from a group) was selected randomly and slaughtered humanely (by inserting air emboli in the brachial vein).Te small intestine was removed, and the tissue debris was washed of with sterile phosphate bufer saline (PBS).Ligatures were put on the Meckel's diverticulum and on the ileocecal junction with the help of sterile twines to prevent the mixing of caecal ileal contents.After repeated washing with sterile PBS, the ileum was separated, and the digesta present therein was emptied into autoclaved polystyrene tubes by applying gentle digital pressure.Te tubes were stored at 4 °C and, within 48 h, were cultured in specifc media for enumeration of total Salmonella (HiTouch ™ Complementary DNA (cDNA) was synthesised from the said RNA using a cDNA reverse transcription kit (Quan-tiTect Reverse Transcription kit, Qiagen Inc., Hilden, Germany) and stored at −20 °C.Te primer sets for the target genes and the housekeeping gene, beta-actin, were designed using the NCBI GenBank sequences (Table 2).Real-time PCR was performed in a 96-well microplates' Bio-Rad CFX-96 real-time PCR instrument (Bio-Rad Laboratories Inc., Hercules, California, USA) under the following conditions: 95 °C for 3 minutes for initial denaturation, followed by 40 cycles of 95 °C for 30 seconds, and at 60 °C for 1 minute.Gene expression analysis was performed by the 2 −ΔΔCt method [14] in which the absolute and relative abundance of a particular immune modulatory gene was expressed as the cycle threshold (Ct) of the target genes and the fold chain increment (2 −ΔΔCt ) in the expression of the target gene in the treatment groups (in this case, the groups supplemented with 37.5 to 150 mg Cu/kg diet) relative to the untreated control group (in this case, the receiving no supplemental Cu).Te latter was assigned a unit value of 1 (or 100%), and the values obtained with the other treatment groups were expressed relative to this unit value.Te mean of qRT-PCR for a single biological replicate was the average of three technical replicates, and the mean was used as a single data point for calculation.

Expression Analysis of Immune Modulatory Genes.
Expression of the genes (Table 5) coding for IL-6 and TNF-α, expressed in terms of Ct target gene, increased linearly (P � 0.0001) as the dose of Cu increased in diet (Table 6) indicating a signifcantly high abundance of both the genes at Cu inclusion levels of 750-1000 mg/kg diet.Te abundance of the said genes (ΔCt) relative to the NC group also increased with the dose of Cu in diet (P � 0.0001 linear, P � 0.001 quadratic) for both IL-6 and TNF-α.Te Ct values for the beta-actin gene, which served as the housekeeping gene, for both IL-6 and TNF-α remained unafected by dietary Cu level (P > 0.05) and in both the analyses, which ran independently, and the Ct values of the beta-actin gene remained close to each other.

Discussion
Te thermoneutral zones for broiler chickens are 28∼34, 25∼31, 22∼28, 20∼25, 18∼24, and 18∼24 °C for each of the frst six weeks of age, respectively [15].So, it may be assumed that the birds in this experiment were under HS from 14 d of age onwards.Tis exposure to HS negatively afected overall BW and FCR, especially towards the terminal phase, and this happened irrespective of the level of Cu supplemented to the diets.Te concentration of Cu in the NC diet was close to 8 mg/kg, which matches the NRC [6] recommended level.However, the data obtained in this experiment show that when the level of supplemental Cu as Cu-P was elevated to 37.5 mg/kg diet, the BW of the birds at the time of harvest increased by almost 4% over that in the NC group.In other words, a minimum of an additional 37.5 mg/kg Cu in the diet was required to reverse the detrimental efects of HS on performance, and it was possible even to achieve even a dose-dependent linear improvement in performance traits when the concentration of added Cu in the diet was increased up to 150 mg/kg diet.Te present investigation adds to the existing data pool that dietary Cu super-dosing (as Veterinary Medicine International high as 125 to 150 mg Cu/kg diet) may improve BW, FCR [16][17][18], and nutrient digestibility [19] in adults as well as young chickens alike.Te present data have corroborated the fndings of El-Kassas et al. [20], who reported that dietary supplementation of Cu nanoparticles ameliorated the depression in BW and ADG observed in Ross and Cobb broiler chickens exposed to HS apparently due to a decreased FI [21] and suboptimal thyroid functioning [22] along with overt participation of corticosterone [23].In this experiment, FI was largely similar across the treatments up to 21 d.Considering growth response as a function of FI, it may be assumed that dietary Cu concentration up to 8 mg/kg was insufcient to support normal growth and FCR during this period in the presence of moderate HS, which is plausible because Cu plays an important role in growth promotion [24], and energy digestibility [25].Te apparent Cu inadequacy in the NC group of birds failed to support the growth performance in the said group of birds because the negative efects of the dietary Cu inadequacy were exacerbated as the birds approached maturity in the presence of HS.Te concentration of blood metabolites in poultry may change upon exposure to HS [26,27] due to oxidative damage in tissues [28,29].Acute HS reportedly elevated glucose and cholesterol in the serum of broiler breeders [27], while, in broiler chickens, a 23.9 to 37 °C cyclic HS for four weeks increased glucose and lowered triglyceride and total cholesterol [26].However, any such specifc trend was absent in the present study indicating the subtle efect of HS and Cu super-dosing on the serum metabolites.It is      Veterinary Medicine International important that both cholesterol and triacylglycerol decreased despite an elevated FI in the Cu-supplemented groups.It is plausible that superdosing of Cu improved utilization of nutrients at the tissue level, which, in turn, increased the cholesterol and triacylglycerol uptake from circulation into the tissue level, and this explains the comparatively better performance in the Cu supplemented groups even in the presence of HS.Te activities of SOD, GPX, and MDA in serum were not afected by dietary Cu concentrations both at 21 d and 35 d, unlike other fndings [27], and hence, it became apparent that Cu super-dosing in this experiment did not infuence the oxidative properties of the experimental chickens.Circulatory oxidative enzyme activities vary a lot depending on the temperature and latency of heat exposure [4,21,27], and the present data did not give any clue about the efects of Cu super-dosing on oxidative damages in the experimental birds.Te dose-dependent increase in liver Cu concentration agreed with earlier fndings obtained with Cu sulfate, tribasic Cu chloride [17,30], and Cu hydroxychloride [31] in broiler chickens.Te available literature shows a strong correlation between the source of Cu and its assimilation rate in tissues [32], and abruptly increasing dietary Cu concentration may increase Cu concentration in excreta, thus raising serious environmental concerns.Te increase in Hb due to Cu super-dosing was reported earlier [20] and suggests that the Cu from the yeast protein chelate was well assimilated.An elevated Hb might be correlated with a greater nutrient turnover rate, and it may be assumed that despite HS, Cu super-dosing sustained the pool of nutrients that bolstered the performance of the birds.
Te H : L ratio, a widening of which is suggestive of a stressed condition in poultry [33], further indicated that Cu super-dosing ameliorated the HS in this experiment, with narrower ratios observed in all the Cu-supplemented groups.Considering H : L ratios of 0.2, 0.5, and 0.8 being indicative of low, optimal, and high degrees of stress in poultry, respectively [34], the stress was on the extremely higher side in the NC group, which came down to the level of "optimal" with 150 mg/kg Cu in the diet.
One of the intriguing qualitative parameters worth mentioning here is the reduction in the panting rate, especially towards the later age, in the Cu-supplemented birds.Panting and polypnea started from the 3rd week and occurred in all the dietary groups during the daytime when the temperature was at its peak, and the birds reverted to normal breathing activities when the room temperature was towards its lower side.Te relative humidity of the experimental house was not too high, facilitating a quick reversal of the birds to normal breathing.Te severity of panting increased further towards the terminal part of the experiment.Qualitatively, the reversal from panting to normal breathing occurred to a greater extent in the birds receiving Cu-P at higher doses, although owing to the visual nature of the observation, it was not possible to conclude about the exact inclusion level that resulted in reduced panting.
Exposure to HS brings about a profound shift in the neuroendocrine cascade in chickens, starting with the autonomic nervous system inducing tachycardia, panting, and enhanced blood fow towards the periphery to facilitate maximum heat loss to maintain body temperature [35].Panting has far-reaching consequences.Owing to compromised splanchnic circulation, the muscle glycogen breaks down, and the capacity of muscles to store energy reduces [36,37], thus disrupting the functioning of the electron transport chain.Te electrons leak out from the mitochondria, generating reactive oxygen species [3,38].Cu is a pro-oxidant and scavenges the free radicals [39], being an integral part of the Cu-zinc superoxide dismutase [40], which is one of the main components of the antioxidant defence mechanism.It is claimed that Cu improves the efciency of the electron transport system [41] and increases the antioxidant activities in fsh [42].In heat-stressed broiler chickens, dietary Cu supplementation as Cu-oxide nanoparticles decreased degenerative changes in hepatic tissues and modulated mRNA transcripts for heat shock protein, superoxide dismutase, and glutathione peroxidase [43].In the present study, no oxidative stress biomarkers were affected by HS or level of Cu in diet, which is intriguing.With impending HS-induced oxidative damages, an upregulation in the expression of the nuclear factor (erythroid-derived 2) like −2 gene takes place that further triggers a variety of downstream target genes [44] which, in turn, through a series of rate-limiting reactions maintain glutathione and superoxide dismutase homeostasis [45].In the present experiment, there was a numerical increment in the SOD at both 21 and 35 d, which suggests that the total antioxidant capacity increased by Cu super-dosing, although that of MDA is suggestive of little or no lipid peroxidation.
It should be noted that most stressor stimuli operate through an elevated corticosterone activity [22], again strongly controlled by the circadian rhythm [46].Blood samples for the biomarker assays were collected in the morning when the room temperature was not still high enough to induce an overt HS reaction.As a result, the corticosterone-mediated stress responses possibly did not become strong enough to induce discernible changes in the circulatory concentrations of the biomarkers discussed previously.
Te data related to the absolute number of bacteria in digesta suggested that there was an apparent shift in the numbers of bacterial species in the ileal digesta following Cu supplementation, which is in corroboration with the hypothesis that states that the growth-promoting efect of Cu is mediated through a modulated growth hormone action and a changed gastrointestinal microbiota [31].An altered bacterial composition of the digesta with fewer potential pathogens such as E. coli and Salmonella decreases birds' susceptibility to diseases and reduces the recruitment of lymphocytes in protecting the intestinal tissues, which, in turn, might improve nutrient absorption [47].Te change in gut microbiota composition may be a direct efect of HS or may be a result of the altered immune profle in which the neuroendocrine behaviour shifts more towards corticosterone secretion during the periods of HS [48,49], and it was apparent that due to graded level of Cu supplementation, the numbers of the potential pathogens decreased, and those of Lactobacillus increased.Inhibition of bacterial growth under Veterinary Medicine International the infuence of Cu is highly dependent on the availability of free Cu in the media, and it is assumed that Cu salts with higher solubility should have greater bactericidal efcacy [50,51], and the changes in bacterial numbers observed in the present study indicated that Cu from Cu-P was probably sufciently available to bring about the desired changes in the ileal contents.
In human models, IL-6 serves as a pro-infammatory gene during conditions of chronic infammation, while during acute infammation, it performs an antiinfammatory action [52,53].Presumably, HS in the experimental birds might have disrupted the integrity of the mucosal barrier [54], which, in turn, facilitated the paracellular transport of endotoxins into the bloodstream leading to the activation of the innate immune system and systemic infammation [36] and increased the expression of the IL-6 gene in liver coding for the proinfammatory cytokine.It is possible that the upregulation in the expression of the IL-6 gene in the liver due to Cu super-dosing happened under the infuence of ceruloplasmin, which was expected to increase dose-dependent with Cu level in diet [37].
Scientifc papers are ample to suggest that the negative efects of HS stretch beyond a depressed BW and FCR [55] but may extend to retarded growth of the digestive tract and disrupted the integrity of the mucosal barrier as well [54].Te latter, in turn, diminishes the innate immunity which allows pathogens to bind to and damage the intestinal epithelium [56] and facilitates the paracellular transport of endotoxins into the bloodstream leading to reactivation of the innate immune system and systemic infammation [36].Te expression of the IL-6 gene, which increased as the dose of Cu increased, suggested that an oxidative damage might have taken place at the cellular level due to the HS, and this occurred irrespective of the level of Cu in the diet.As a matter of fact, several counter-current mechanisms operated on the experimental birds which not only confounded the data related to the expression of the concerned genes but at the same time also made them difcult to explain.For example, the possible oxidative stress mentioned previously might have had precipitated an innate immune response that increased the expression of the IL-6 gene which codes for the proinfammatory cytokine responsible for eliciting some adaptive immune responses and precipitates a condition termed as sickness behaviour [57].Both Cu super-dosing and exposure to HS might increase ceruloplasmin in blood [58,59] and elicit pro-infammatory and antiinfammatory responses together which could increase the expression of IL-6 gene.As discussed earlier, the H : L ratio indicated that Cu super-dosing apparently decreased the level of stress on the birds in this experiment.Terefore, rather than the proinfammatory responses, it is the ceruloplasminmediated antiinfammatory pathway that increased the expression of the IL-6 gene in this case.Tis observation was, however, contradictory to an earlier study [37] which reported that the supplementation of 16 mg Cu per kg feed decreased liver concentration of IL-6 and TNF-α.Te defciency of Cu might delay the acute phase response because Cu is linked to synthesis of acute phase proteins such as ceruloplasmin, cytokine synthesis, and the generation of reactive oxygen species during phagocytosis [37,60], and a moderate increment in the IL-6 and TNF-α activity was expected when Cu was supplemented at plethoric levels.Excessive Cu (as high as 330 mg/kg dry matter) reportedly caused pathologic changes and induced oxidative stress triggering NF-κB pathway and induced infammation through activation of proinfammatory programs [61][62][63].Tus, it may not be inappropriate to assume that the benefcial efects of Cu super-dosing were partially negated by subtle toxic efects elicited in the experimental chickens.As a matter of fact, one of the concerns in designing the present experiment was the possibility of toxicity elicited by ingestion of almost 20 times more Cu than what has been suggested by NRC [6].Dietary concentration of as high as 300 mg/kg Cu should cause growth depression Based on the current literature, Malinak et al. reported that dietary concentration of as high as 300 mg/kg Cu should cause growth depression [64] while that as high as 800 mg/kg may cause mortality [65,66].However, most of the toxicological data refer to the sulfate or oxide salts of Cu.Tribasic Cu chloride at more than 2000 mg/kg reportedly caused degenerative changes in gastrointestinal tract mucosa leading to high mortality in broiler chickens [64], but no such data are available for yeast protein chelates of Cu.Copper sulfate included at the rate of 300 to 450 mg/kg diet caused mild diarrhoea, anorexia, and reduced BW in some studies [30,67] while many authors failed produce identical toxicological efects with similar or higher inclusion level of Cu in diets of broiler chickens [32,68,69].Apart from the form in which Cu is presented, the other factor that infuences Cu toxicity in cases of excessive intake is the solubility of the Cu salt at acidic and neutral pH.Copper as tri-basic Cu chloride (TBCC) is almost insoluble at neutral pH and at an acidic pH (in the presence of 0.1% HCl), and the solubility is reported to be approximately 76% in 1 h although Cu sulfate was almost completely soluble in both the pH [70].Tis explains why more than 2000 mg/kg Cu in the form of TBCC was required to induce hepatic degeneration and gastrointestinal disorders in broiler chickens [64].No such data are available for yeast protein chelates of Cu though so the data obtained with this experiment are going to be the frst of its kind.Apparently, signs of toxicity were not evidenced in the experimental chickens in this study except for the BW gain and FCR getting plateaued beyond 112.5 mg Cu/kg diet despite a dose-dependent increase in Cu absorption up to 150 mg/kg Cu in the diet.Although, serum concentration of MDA, a key biomarker for cellular oxidative damages caused by super-dosing of Cu did not show any remarkable change with the dose of Cu in diet, considering the stalled performance indices, a subtle toxic efect beyond the inclusion level of 112.5 mg/kg could not be ruled out.

Conclusions
Dietary Cu at a concentration of 8 mg/kg, as suggested by NRC [6], was insufcient to support growth and feed efciency under conditions of HT.Supplementation of 112.5 to 150 mg Cu/kg diet improved BW by 8% and FCR by almost 8 Veterinary Medicine International points in conditions of acute HS.Terefore, redefning the Cu requirement for modern broiler chickens, especially those likely to be exposed to environmental stresses, has become inevitable.Super-dosing the diet with as high as 112.5 to 150 mg Cu/kg as Cu-yeast-protein chelate may be a novel strategy to neutralize the negative efects of impending high ambient temperature without involving any apparent risk of Cu toxicity.

2. 1 .
Ethical Statement.Te protocol was reviewed by the Institutional Animal Ethics Committee of the Research Station to ensure that the stipulations laid down by the Committee for the Purpose of Control and Supervision of Experiments on Animals (Ministry of Fisheries, Animal Husbandry and Dairying, Department of Animal Husbandry and Dairying, Government of India, 2020) were adhered to.

Figure 1 :
Figure1: Maximum, minimum, and average daily temperature of the experimental room measured during the period of experimentation(1-35 d).Temperature was measured daily at hourly intervals and averaged on a 24 h basis to be plotted.Te shaded area indicates the standard ambient temperature to be maintained for optimum production (Cobb Broiler Management Guide, 2013 available from http://www.cobbvantress.com).Te mean maximum temperature was 35.27 °C (32.9-36.7 °C), and the mean minimum temperature was 27.22 °C (26.2-29.4°C).

Table 1 :
Composition (g/kg) and nutritive values (g/kg as fed, unless stated otherwise) of the basal diet.
Te quantity of the maize was added in the above premix as fller was adjusted from the fnal formulation.Te diets were 1 Contained 490.5 g crude protein per kg; 2 presented in the form of a protein chelate and contained (g/kg) iron(33.8),manganese (65.9), zinc (64.0), selenium (0.67), chromium (1.8), and iodine, as potassium iodide (4.28); 3 [13]ere homogenized and dried at 80 °C for 24 h in a hot air oven.Te dried tissue samples were digested with concentrated nitric acid (HNO 3 ) at 180 °C.Te digested samples were diluted with 1M HNO 3 and used for analysis of Cu in a fame atomic absorption spectrophotometer[13].
Salmonella Count Flexi Plate FL-023, for selective isolation and enumeration of Salmonella typhi and other Salmonella species; Hi Media Laboratories, Mumbai, India), Escherichia coli (Luria Bertani Agar, Miller or Miller Luria Bertani Agar, M1151, Hi Media Laboratories, Mumbai, India), and Lactobacillus spp.(LactobacillusMRSAgar, M641, Hi Media Laboratories, Mumbai, India).2.7.Copper in the Liver.Te whole liver from the slaughtered birds was removed after evisceration and washed with PBS to remove the tissue debris and blood.Tissue samples (approximately 5 of 35.3 °C.Te mean minimum temperature was 27.2 °C (26.2 to 29.4 °C).Te mean temperature during 1-35 d was 29.7 °C.Apart from a transient dip during 21-26 d, the maximum and minimum temperatures remained fairly constant.Te relative humidity of the house (data not shown) varied in a day depending on the time of measurement, with relative humidity as high as 91% being recorded during the early morning.Relative humidity decreased in the daytime and went as low as 35%.During the overall period of 1-35 d, the average relative humidity of the experimental room was 65% (48-77%).Liveability was similar across the groups.Te mortality recorded during the study period was accidental, and necropsy examination did not reveal any pathognomonic lesion that could correlate with over-dosing of Cu in the dead birds.ratio in the NC group was observed compared to the treated groups at 21 and 35 d (P < 0.05).Amongst the Cu-supplemented groups, the ratio was narrower, with 150 mg Cu/kg diet at 21 d and 112.5 mg Cu/kg diet at 35 d (Table4).Te concentration of Cu in the liver (Table4) at 35 d increased linearly with the dose of Cu (P � 0.0001).3.5.Bacteria in IlealDigesta.Te number of E. coli in ileal digesta decreased linearly (P � 0.0001), while that of Salmonella decreased quadratically (P � 0.0001) with the dose of Cu in the diet.Lactobacillus number increased when Cu was supplemented to the NC diet (P � 0.0001, linear and quadratic).
mean and pooled standard error of the mean.Probability values of P < 0.05 were considered statistically signifcant, and means were separated by Tukey's B test.Orthogonal polynomial contrasts were applied to determine the linearity of dose-response for Cu levels in the diet.°C with an average 3.3.Performance Traits.Compared to the NC group, added Cu increased (Table 3) BW at 21 d (linear P � 0.0001, quadratic P � 0.013) and 35 d (linear P � 0.0001), ADG during 1-21 d (linear P � 0.0001, quadratic P � 0.013), 22-35 d (linear P � 0.029) and 1-35 d (linear P � 0.0001), FI during 22-35 d (linear P � 0.0001) and 1-35 d (linear P � 0.0001), and FCR during 1-21 d (linear P � 0.0001, quadratic P � 0.004) and 1-35 d (linear P � 0.0001, quadratic P � 0.007).at 35 d.Triacylglycerol was higher in the NC group at 21 d and 35 d, and Cu supplementation decreased it linearly at 21 d (P � 0.033) and quadratically at 35 d (P � 0.001).Hb increased (linear P � 0.003) at 21 d, and heterophil decreased at 21 d (linear P � 0.018) and 35 d (linear and quadratic P � 0.001), while lymphocyte count did not change (P > 0.05) as dietary Cu levels increased.As a result, a wider H : L
Temperature was measured daily at hourly intervals and averaged on a 24 h basis to be plotted.Te shaded area indicates the standard ambient temperature to be maintained for optimum production (Cobb Broiler Management Guide, 2013 available from http://www.cobbvantress.com).Te mean maximum temperature was 35.27 °C (32.9-36.7 °C), and the mean minimum temperature was 27.22 °C (26.2-29.4°C).

Table 3 :
Performance traits of broiler chickens supplemented with incremental doses of copper (as yeast protein) during 1-35 d 1 .

Table 4 :
Serum analytes and haematological parameters at 21 and 35 d in broiler chickens supplemented with incremental doses of copper as (as yeast protein)1.
1Tere were 8 replications/treatment with n � 10 birds/replication.Samples were collected from a single bird selected randomly from a replicate.Means with dissimilar superscript in a row varied signifcantly.

Table 5 :
Relative expression of genes coding for interleukin-6 and tumour necrosis factor alpha at 35 d in hepatic tissues of broiler chickens supplemented with incremental doses of copper (as yeast protein)1.Tere were 8 replications/treatment with n � 10 birds/replication.Samples were collected from a single bird selected randomly from a replicate.Means with dissimilar superscript in a row varied signifcantly.

Table 6 :
Selected bacteria numbers (log 10 colony-forming units/g digesta) at 35 d of age in ileal digesta of broiler chickens supplemented with incremental doses of copper (as yeast protein).
1Tere were 8 replications/treatment with n � 10 birds/replication.Samples were collected from a single bird selected randomly from a replicate.Means with dissimilar superscript in a row varied signifcantly.