May Bin-Jumah

Mohamed E Abd El-Hack

Sameh A Abdelnour

Yasmeen A Hendy

Hager A Ghanem

Sara A Alsafy

Asmaa F Khafaga

Ahmed E Noreldin

Hazem Shaheen

Dalia Samak

Maha A Momenah

Ahmed A Allam

Abdullah A Alkahtane

Saad Alkahtani

Mohamed M Abdel-Daim

Lotfi Aleya
email: lotfi.aleya@univ-fcomte.fr

Potential use of chromium to combat thermal stress in animals: A review

Keywords: Heat stress, chromium, growth, thermoregulation, heat shock proteins

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Heat stress (HS) occurs when the heat produced by animals surpasses their capacity to dissipate the extra heat into their surrounding environment [START_REF] Alagawany | Heat stress: effects on productive and reproductive performance of quail[END_REF][START_REF] El-Kholy | Use of acetylsalicylic acid as an allostatic modulator in the diets of growing Japanese quails exposed to heat stress[END_REF][START_REF] Farghly | Ameliorating deleterious effects of heat stress on growing Muscovy ducklings using feed withdrawal and cold water[END_REF]. There are two main categories of HS: chronic and acute. Because chronic HS comes from high temperatures over a long period, acclimatization to the environment is possible. Acute HS, on the other hand, comes from rapid increase in the environmental temperature over a short period [START_REF] Alagawany | Heat stress: effects on productive and reproductive performance of quail[END_REF]. HS elicits many physiological consequences. Its impacts include increased body and rectal temperatures, reduced feed consumption, impaired immunity, alteration of blood electrolyte balance and pH, poor feed utilization, depressed cellular energy bioavailability, impaired reproductive functions and impaired endocrine functions [START_REF] Loyau | Cyclic variations in incubation conditions induce adaptive responses to later heat exposure in chickens: a review[END_REF].

HS alters lipid, protein and carbohydrate metabolism. It also alters homeorhetic adaptations, which are largely caused by increased circulating insulin [START_REF] Baumgard | Effects of heat stress on postabsorptive metabolism and energetics[END_REF]. Hence, identification of nutritional approaches to ameliorate the effects of HS on health and production has become of interest recently ( [START_REF] Khafaga | The adaptogenic anti-ageing potential of resveratrol against heat stress-mediated liver injury in aged rats: Role of HSP70 and NF-kB signalling[END_REF]. Alternation in utilization and substrate uptake is also a result of HS, but this phenomenon is not yet fully understood.

When temperatures are high, metabolic adaptations occur to increase chances of survival. For example, feed intake in Holstein cattle during summer was found to result in lower insulin concentrations in summer than in winter and spring [START_REF] Abdelnour | Stress biomarkers and proteomics alteration to thermal stress in ruminants: A review[END_REF]. Likewise, plasma insulin was lower in heat-stressed lactating sows compared to a control group, although glucose concentration was the same [START_REF] De Braganca | Effects of low feed intake and hot environment on plasma profiles of glucose, nonesterified fatty acids, insulin, glucagon, and IGF-I in lactating sows[END_REF]. In addition, [START_REF] Sevi | Effects of ventilation regimen on the welfare and performance of lactating ewes in summer[END_REF] reported that plasma non-esterified fatty acids (NEFA) increased in sheep but declined in lactating cows due to heat exposure [START_REF] Itoh | Insulin and glucagon secretion in lactating cows during heat exposure[END_REF][START_REF] Shwartz | Effects of a supplemental yeast culture on heat-stressed lactating Holstein cows[END_REF][START_REF] Wheelock | Effects of heat stress on energetic metabolism in lactating Holstein cows[END_REF]. Under stress conditions, Cr requirements are higher due to increased urinary excretion [START_REF] Chang | Supplemental chromium for stressed and growing feeder calves[END_REF][START_REF] Pechova | Chromium as an essential nutrient: A review[END_REF][START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF].

Chromium (a low-molecular-weight binding substance) activates insulin receptors and mobilizes the glucose transporter type 4 (GLUT 4) from the inside of the cell to the surface, allowing glucose to enter the cell [START_REF] Vincent | Is the Pharmacological Mode of Action of Chromium (III) as a Second Messenger?[END_REF]. Dietary Cr supplementation (0.5 to 1.5 mg/kg of dry matter (DM)) to heat-stressed buffalo calves improved heat tolerance, immune function, and insulin effectiveness. However, it had no effect on feed intake, growth rate, tri-iodothyronine (T3) or thyroxine (T4) concentrations (Kumar et al. 2015a).

The effects of Cr supplementation on production were previously reported for the cases of heat-stressed cows [START_REF] Soltan | Effect of dietary chromium supplementation on productive and reproductive performance of early lactating dairy cows under heat stress[END_REF]) and normal cows [START_REF] Sadri | Chromium supplementation and substitution of barley grain with corn: Effects on performance and lactation in periparturient dairy cows[END_REF]). However, the information concerning the influences of Cr on growth rate in heat-stressed calves is not consistent (Kumar et al. 2015a;[START_REF] Sadri | Chromium supplementation and substitution of barley grain with corn: Effects on performance and lactation in periparturient dairy cows[END_REF][START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF]. These inconsistencies could be attributed to the chemical form of Cr used (organic vs inorganic, yeast vs chelated Cr) [START_REF] Kegley | Effect of shipping and chromium supplementation on performance, immune response, and disease resistance of steers[END_REF], the environmental conditions (normal vs heat-load conditions), Cr dosage, and method of Cr delivery (starter vs liquid).

Several research papers described the influences of Cr supplementation to heat-stressed dairy calves on feed intake, growth rate, general health status, and blood metabolites. However, the way in which Cr affects the feed intake is not clear. To clarify this mechanism, investigation of ingestion behavior in calves supplemented with Cr is needed.

There are no review articles summarizing the beneficial impacts of Cr on heat-stressed poultry. Therefore, the aim of the present paper is to highlight the consequences of Cr on thermoregulatory responses, growth rate, metabolites of blood, health status, oxidative biomarkers, and immune indices in different heat-stressed ruminants.

Heat stress in animals

Heat stress has several direct and indirect effects on dairy animal health: it can disrupt normal metabolism as well as affect the physiological, hormonal, and immune status of animals (Abd El-Hack et al. 2019;[START_REF] Saeed | Heat stress management in poultry farms: A comprehensive overview[END_REF]. Incremental increases in environmental temperature exert direct adverse impact on the hypothalamus appetite center, with subsequent reduction in feed intake [START_REF] Baile | Control of feed intake and regulation of energy balance in ruminants[END_REF]. In lactating cows, feed intake starts to decrease at climatic temperatures of 25-26°C, and it may decline more rapidly at 30°C and drop by approximately 40% at 40°C [START_REF] Rhoads | Nutritional interventions to alleviate the negative consequences of heat stress[END_REF]. The purpose of this reduction in feed intake is to decrease production of heat in hot environments [START_REF] Kadzere | Heat stress in lactating dairy cows: A review[END_REF]. As a result, animals may develop negative energy balance (NEB), with subsequent negative effect on body weight and body condition scores [START_REF] Lacetera | Body condition score, metabolic status and milk production of early lactating dairy cows exposed to warm environment[END_REF]. Additionally, HS may affect the production and composition of milk in dairy animals [START_REF] Bouraoui | The relationship of temperature-humidity index with milk production of dairy cows in a Mediterranean climate[END_REF][START_REF] West | Effects of heat-stress on production in dairy cattle[END_REF][START_REF] Spiers | Use of physiological parameters to predict milk yield and feed intake in heat-stressed dairy cows[END_REF][START_REF] Upadhyay | Impact of climate change on reproductive functions of cattle and buffalo[END_REF][START_REF] Wheelock | Effects of heat stress on energetic metabolism in lactating Holstein cows[END_REF]. [START_REF] Berman | Estimates of heat stress relief needs for Holstein dairy cows[END_REF] concluded that the stress-response systems in dairy cattle could be affected negatively in climatic temperatures above 35°C. Heatstressed dairy cows developed reduced feed intake and NEB, which is subsequently responsible for milk synthesis decline [START_REF] Wheelock | Effects of heat stress on energetic metabolism in lactating Holstein cows[END_REF].

Moreover, HS often alters the physiological mechanisms of the rumen, with resultant increases in health problems and metabolic disorders [START_REF] Nardone | Effect of climate changes on animal production and sustainability of livestock systems[END_REF][START_REF] Soriani | Rumination time during the summer season and its relationships with metabolic conditions and milk production[END_REF]. [START_REF] Nonaka | Effects of high environmental temperatures on physiological and nutritional status of prepubertal Holstein heifers[END_REF] concluded that heat-stressed animal suffer from diminished acetate production and from increased propionate and butyrate production due to alterations in rumen function. As a result, stressed animals consume less roughage and undergo alteration of rumen pH and microbial population [START_REF] Hall | Heat stress alters ruminal fermentation and digesta characteristics, and behavior in lactating dairy cattle[END_REF], diminished rumination and rumen motility [START_REF] Nardone | Effect of climate changes on animal production and sustainability of livestock systems[END_REF][START_REF] Soriani | Rumination time during the summer season and its relationships with metabolic conditions and milk production[END_REF], decreased salivary production, and less dry-matter intake (DMI) [START_REF] Nardone | Effect of climate changes on animal production and sustainability of livestock systems[END_REF][START_REF] Soriani | Rumination time during the summer season and its relationships with metabolic conditions and milk production[END_REF]. Also, HS resulted in decreased animal growth, carcass quality and fertility, and in increased mortality rate and care costs [START_REF] Baumgard | Effects of heat stress on postabsorptive metabolism and energetics[END_REF][START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF].

It is therefore necessary to develop protocols to overcome the negative impacts of HS.

Nutritional intervention is considered to be an economical method for increasing production in summer [START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF]. Indeed, dietary chromium enhances cellular glucose uptake [START_REF] Chen | Chromium activates glucose transporter 4 trafficking and enhances insulin-stimulated glucose transport in 3T3-L1 adipocytes via a cholesterol-dependent mechanism[END_REF] by increasing the binding of insulin to its extracellular receptors (NRC, 1994;[START_REF] Vincent | Is the Pharmacological Mode of Action of Chromium (III) as a Second Messenger?[END_REF]. In addition, studies showed that average daily feed intake (ADFI), reproduction, immune response and growth performance are improved by Cr supplementation (Burton et al., 1993b;[START_REF] Hayirli | Effect of chromium supplementation on production and metabolic parameters in periparturient dairy cows[END_REF][START_REF] Hung | Dietary nano-chromium tripicolinate increases feed intake and decreases plasma cortisol in finisher gilts during summer[END_REF][START_REF] Lindemann | Dietary chromium picolinate additions improve gain:feed and carcass characteristics in growing-finishing pigs and increase litter size in reproducing sows[END_REF][START_REF] Sadri | Chromium supplementation and substitution of barley grain with corn: Effects on performance and lactation in periparturient dairy cows[END_REF][START_REF] Sales | Effects of dietary chromium supplementation on performance, carcass characteristics, and meat quality of growing-finishing swine: a meta-analysis[END_REF][START_REF] Yasui | Effects of chromium propionate supplementation during the periparturient period and early lactation on metabolism, performance, and cytological endometritis in dairy cows[END_REF]. The adverse effect of HS in ruminants and their body reactions against it are summarized in Figure 1. Similarly, the adverse effect of HS on animal performance, production and reproduction is summarized in Figure 2.

General properties of chromium and sources

Trivalent Cr (Cr 3+) has an effect on rodents (rats and mice), raising their sensitivity to insulin [START_REF] Vincent | Is chromium pharmacologically relevant? Journal of trace elements in medicine and biology: organ of the Society for Minerals and Trace Elements[END_REF]. Despite the fact that, according to a report published by the European Food Safety Authority, there is no evidence that Cr is an essential trace element for either animals or humans [START_REF] Vincent | Is chromium pharmacologically relevant? Journal of trace elements in medicine and biology: organ of the Society for Minerals and Trace Elements[END_REF], Cr has for decades been thought to be an essential nutrient, and current studies show that high pharmacological and nutritional doses should be maintained as a beneficial element [START_REF] Hua | Molecular mechanisms of chromium in alleviating insulin resistance[END_REF][START_REF] Vincent | The bioinorganic chemistry of chromium(III)[END_REF][START_REF] Vincent | New Evidence against Chromium as an Essential Trace Element[END_REF][START_REF] Ibrahim | Pathological Evaluation of The Effect of Zinc Oxide Nanoparticles on Chromium-Induced Reproductive Toxicity in Male Albino Rats[END_REF].

Farm animals, humans and laboratory animals have been the subject of studies of Cr as a dietary supplement. These studies showed that stress resulted in Cr deficiency (when Cr is considered essential) (e.g. see [START_REF] Lukaski | Chromium supplementation and resistance training: effects on body composition, strength, and trace element status of men[END_REF] and in increased loss of Cr through urinary excretion.

Cr supplementation also overcomes the effects of stress stemming from animal shipping and meat quality improvement. The reviews made by the (NRC, 1994) in the mid-1990s and by [START_REF] Lindemann | Dietary chromium picolinate additions improve gain:feed and carcass characteristics in growing-finishing pigs and increase litter size in reproducing sows[END_REF] are the most comprehensive reviews about the use of Cr supplementation for farm animals. No conclusions about the need for Cr supplementation in the diets of sheep, fish, rats, horses, and rabbits have been drawn or stated by the Animal Research Committee. Nor have any recommendations been made about Cr supplementation for poultry, cattle and swine, even though Cr does have beneficial impacts on heat-stressed cattle and pig leanness and on the effectiveness of reproduction (NRC, 1994).

Mode of action of chromium in animal nutrition

Chromium chloride, chromium polynicotinate, and chromium picolinate (CrP, Figure 3) are considered to be nutritional supplements and micronutrients. Several investigations concluded that chromodulin is a naturally occurring biomolecule that binds to trivalent Cr and could explain the action of Cr in metabolism of lipids and carbohydrates [START_REF] Davis | Isolation and characterization of a biologically active chromium oligopeptide from bovine liver[END_REF]. Chromodulin is able to stimulate insulin-dependent glucose metabolism in adipocytes [START_REF] Yamamoto | Purification and properties of biologically active chromium complex from bovine colostrum[END_REF]. Moreover, it could activate the membrane phosphotyrosine phosphatase in adipocyte membranes [START_REF] Davis | The biologically active form of chromium may activate a membrane phosphotyrosine phosphatase (PTP)[END_REF]. Supplemented levels of Cr are expected to induce little alteration on body weight and body composition. However, the administration of high levels of Cr could potentially lead to altered Cr status, with subsequent alteration in metabolisms [START_REF] John | Vincent Recent advances in the nutritional biochemistry of trivalent chromium[END_REF].

In addition, CrP has the potential to control levels of blood sugar in diabetes, control blood pressure levels, and reduce cholesterol. Cr acts as a cofactor for insulin; therefore, it activates insulin receptor kinase and increases the number of insulin receptors. CrP attenuates weight gain, but the mechanism for this is unknown [START_REF] Anton | Effects of chromium picolinate on food intake and satiety[END_REF]. It may have an action through its influence on the balance of energy to either decrease feed intake or increase energy expenditure.

Cr affects reproduction by enlarging the seminal vesicles, increasing prostate weight and reducing testis weight [START_REF] Marouani | Effects of hexavalent chromium on reproductive functions of male adult rats[END_REF]. It also affects weight loss by decreasing fat percentage and increasing lean body mass [START_REF] Anderson | Effects of chromium on body composition and weight loss[END_REF].

Stress stimulates the hypothalamus to produce corticotropin, which stimulates the pituitary to produce the adrenocorticotropic hormone, which in turn stimulates the adrenal cortex to release corticosterone. Corticoids depress the immune system and reduce the concentration of serum protein. They also raise blood glucose levels and decrease the utilization of glucose, thus functioning as antagonists to Cr, which influence the release of corticosteroids. The role of Cr 3+ against cellular glucose uptake is summarized in Figure 4.

Impacts of chromium in heat-stressed animals

The potential beneficial impact for dietary supplementation of Cr during HS could be obtained through several pathways. The main findings related to Cr supplementation in heatstressed animals are summarized in Table 1 and discussed in the following sections.

1 Thermoregulatory responses

Thermoregulatory response is considered to be the first indicator of HS. HS is associated with increased respiration, pulse rate and body temperature in homothermic animals. A dietary supplement of 0.05 mg of Cr-methionine/kg of body weight significantly reduced the respiration rate (RR) in heat-stressed dairy calves (Mousavi et al. 2019b). Cr (0.05 mg/kg of body weight) had the same effect but had no effect on RT (Kargar et al., 2018b). In heat-stressed calves, Crmethionine at 0.05 mg/kg of body weight reduced diarrhea to -0.9 d, pneumonia to -0.7 d and medication to -1.5 d before weaning (Kargar et al., 2018b;Mousavi et al., 2019a).

Most studies indicated that Cr decreases RT to overcome the effects of HS (Kargar et al., 2018b). Mousavi et al. (2019a) found no effect of Cr-methionine on diarrhea, pneumonia, medication days or RT in heat-stressed calves. Also, Kumar et al. (2015a) found no effect of 0.5, 1 or 1.5 mg of inorganic Cr/kg on pulse rate (PR), RR or RT in heat-stressed buffalo calves. An-Qiang et al. ( 2009) measured the values of RR and RT for cows undergoing Cr supplementation (3.6, 7.2 or 10.8-milligram chromium/head per day) and cows without any supplementation and found no differences. According to the previous results, Cr has no impact on somatic cell count and or on RT, PR or RR in lactating cows (Qi et al. 2018b). A separate study indicated that Cr propionate (200 ppb) has no effect on RR, skin temperature (ST) or body temperature (BT) in heat-stressed pigs [START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF].

Growth performance

Thermal stress has been reported to decrease feed intake, weight gain and effectiveness of feed intake [START_REF] Abdelnour | Stress biomarkers and proteomics alteration to thermal stress in ruminants: A review[END_REF]. Separate studies corroborated that heat-load results in decreased feed intake and growth performance and that indicated that, in turn, it results in decreased energy expenditure for tissue synthesis [START_REF] Johnson | Effects of in utero heat stress on postnatal body composition in pigs: II. Finishing phase[END_REF][START_REF] Kerr | Influences of dietary protein level, amino acid supplementation and environmental temperature on performance, body composition, organ weights and total heat production of growing pigs[END_REF][START_REF] Bellego | Effect of high temperature and low-protein diets on the performance of growing-finishing pigs[END_REF][START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF]. To counter this, Cr supplementation can result in increased feed intake in heat-stressed animals and hence growth performance, as shown by several studies [START_REF] Hayirli | Effect of chromium supplementation on production and metabolic parameters in periparturient dairy cows[END_REF][START_REF] Hung | Dietary nano-chromium tripicolinate increases feed intake and decreases plasma cortisol in finisher gilts during summer[END_REF][START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF][START_REF] Sahin | Effects of dietary chromium and ascorbic acid supplementation on digestion of nutrients, serum antioxidant status, and mineral concentrations in laying hens reared at a low ambient temperature[END_REF].

Likewise, Cr yeast increased feed intake in heat-stressed lactating dairy cows compared with a control group (21.24 and 19.56 kg/day, respectively) [START_REF] Alsaiady | Effect of chelated chromium supplementation on lactation performance and blood parameters of Holstein cows under heat stress[END_REF][START_REF] Hayirli | Effect of chromium supplementation on production and metabolic parameters in periparturient dairy cows[END_REF].

Feed efficiency and average daily gain increased in the case of calves in their growth stage fed Cr at a level of 4 mg/head [START_REF] Chang | Supplemental chromium for stressed and growing feeder calves[END_REF]. This result is due to the increase in total levels of immunoglobulins in serum [START_REF] Burton | Supplemental chromium: its benefits to the bovine immune system[END_REF][START_REF] Burton | Effects of supplemental chromium on production of cytokines by mitogen-stimulated bovine peripheral blood mononuclear cells[END_REF][START_REF] Chang | Supplemental chromium for stressed and growing feeder calves[END_REF]. Moreover, the supplementation of 0.05 mg Cr/kg of body weight of Cr-methionine in cows' diets increased feed efficiency and DMI [START_REF] Arthington | Supplemental dietary chromium does not influence ACTH, cortisol, or immune responses in young calves inoculated with bovine herpesvirus-1[END_REF]. Supplementation of 3.6, 7.2 and 10.8 mg chromium/head/day to heat-stressed dairy cows resulted in a significant increase in DMI (p < 0.05) according to [START_REF] Zhi-Sheng | Effect of Chromium Picolinate Supplementation on Early Lactation Performance, Rectal Temperatures, Respiration Rates and Plasma Biochemical Response of Holstein Cows under Heat Stress[END_REF]. Mousavi et al. (2019a) reported that supplementation of Cr to dairy calves under HS resulted in increases in DMI (P = 0.002) and in average daily gain (ADG) (p= 0.02) but had no effect on effectiveness of feed (P = 0.93) or on body weight (p = 0.14). On the other hand, Kargar et al. (2018b) found that dietary supplementation of chromium improved the intake of feed and growth rate in dairy Holstein calves exposed to high environmental temperature (83.3 units). Also, the addition of Cr at a level of 0.05 mg/kg of body weight to a milk substitute or to diets for cattle during the pre-and postweaning periods led to increases, at the end of the experiment, in feed intake, DM intake and average body weight gain, by 16.4%, 11.85% and 7.07% respectively. Cr supplementation improves growth rate and body weight by enhancing the absorption and digestion of nutrients [START_REF] Ghorbani | Performance and metabolic responses of Holstein calves to supplemental chromium in colostrum and milk[END_REF]Kargar et al., 2018a). Moreover, in finisher gilts exposed to high temperatures, the application of nano-chromium tripicolinate (400 ppb) resulted in a reduction in cortisol level by 25% and an increase in feed intake by 6% [START_REF] Hung | Dietary nano-chromium tripicolinate increases feed intake and decreases plasma cortisol in finisher gilts during summer[END_REF]. [START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF] discovered that final body weight increased in heat-stressed finishing pigs when fed with 200 ppb of chromium propionate.

Rumination activity was reported to decrease as a result of environmental heat load [START_REF] Soriani | Rumination time during the summer season and its relationships with metabolic conditions and milk production[END_REF]. Decreased rumen motility as a result of a slowing in the fractional digesta passage rate has led to a decrease in feed intake [START_REF] Nikkhah | Eating time modulations of physiology and health: life lessons from human and ruminant models[END_REF]. Although the mechanism by which Cr affects rumination patterns is unclear, the insulin-potentiating pathway may be involved. In contrast with previous results, Cr supplementation given over 120 days to Murrah buffalo calves had no significant effect on feed conversion ratio (FCR), ADG or intake of DM (Kumar et al., 2015a). Similar results recorded by Qi et al. (2018a), [START_REF] Subiyatno | Metabolite and hormonal responses to glucose or propionate infusions in periparturient dairy cows supplemented with chromium[END_REF] showed that Cr has no influence on DM intake. Also, the results of tests performed under normal conditions by [START_REF] Bunting | Effects of dietary inclusion of chromium propionate and calcium propionate on glucose disposal and gastrointestinal development in dairy calves[END_REF] and [START_REF] Ghorbani | Performance and metabolic responses of Holstein calves to supplemental chromium in colostrum and milk[END_REF] showed that Cr has no influence on feed intake or total DMI. The timing of supplementation of Cr to the diet (pre-partum or immediately post-partum) had an impact on the response.

Overall, the addition of Cr yeast to the diet has been observed to attenuate the negative impacts of high environmental temperatures and result in a reduction in feed intake and efficiency. Cr requirements for calves increase under a variety of stress conditions [START_REF] Chang | Supplemental chromium for stressed and growing feeder calves[END_REF][START_REF] Pechova | Chromium as an essential nutrient: A review[END_REF]. Such variations are due to changes in the concentration of supplementation and the method of adding Cr to milk only or colostrum plus milk. Cr requirements as a whole rise during thermal heat load; this is a result of increased Cr urinary excretion, which in turn results in diminished feed intake (Kargar et al., 2018b;[START_REF] Pantelic | The impact of the chromium supplementation on insulin signalling pathway in different tissues and milk yield in dairy cows[END_REF][START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF]. In short, to attenuate the impacts of HS, dietary Cr should be used to improve both the performance of animals and their welfare (Kargar et al., 2018b).

Milk yield and composition

Adding (4 g/head/day) of dietary chelated chromium to the diet of heat-stressed dairy cows has no effect on milk fats, protein, lactose, or the non-fat percentage of solids [START_REF] Alsaiady | Effect of chelated chromium supplementation on lactation performance and blood parameters of Holstein cows under heat stress[END_REF]. A significant rise (p < 0.05) in milk protein, fat and lactose levels by 1.043%, 1.066% and 1.020% respectively was recorded by [START_REF] Nikkhah | Eating time modulations of physiology and health: life lessons from human and ruminant models[END_REF] when heat-stressed dairy cows were supplied with 0.05 mg Cr/kg. Moderate levels of Cr resulted in an increase in the ingestion of feed, DM effectiveness and a decline in insulin levels, leading to a significant augmentation in milk secretion as a result of increased mammary nutrient flow [START_REF] Winkelman | The effects of increasing doses of 2 preparations of longacting insulin on short-term plasma profiles of glucose and insulin in lactating dairy cows[END_REF].

Insulin growth factor (IGF) activity, which has functional and structural homology with insulin receptors, is supported through the improvement of milk production, which also mimics the action of bovine somatotropin (bST) [START_REF] Alsaiady | Effect of chelated chromium supplementation on lactation performance and blood parameters of Holstein cows under heat stress[END_REF]. Likewise, milk production increased, resulting in a decline in lipolysis. In a separate study, increase in glucose uptake, which leads to a rise in DMI (NRC, 1994), resulted from receiving 10 mg/day of Cr from 21 days pre-partum to 35 postpartum [START_REF] Mcnamara | Adipose tissue metabolism and production responses to calcium propionate and chromium propionate[END_REF]. Additionally, milk yield was found to increase (p=0.013) after the supplementation of chromium picolinate to the diet of heatstressed cows [START_REF] Zhi-Sheng | Effect of Chromium Picolinate Supplementation on Early Lactation Performance, Rectal Temperatures, Respiration Rates and Plasma Biochemical Response of Holstein Cows under Heat Stress[END_REF]. On the other hand, An-Qiang et al. ( 2009) reported that dietary Cr had no significant effect on milk components (protein, fat and lactose) in dairy cows under high temperature (79.61). The beneficial effect of chromium on glucose and NEFA metabolism in dairy cattle is illustrated in Figure 5.

Blood variables

Cr yeast (4 mg/day) fed to lactating cows failed to show any effect on the blood serum levels of hemagglutination (HA), cholesterol, triglycerides, urea, IGF-1, BHBA, albumen, and glucose [START_REF] Mirzaei | Chromium improves production and alters metabolism of early lactation cows in summer[END_REF][START_REF] Soltan | Effect of dietary chromium supplementation on productive and reproductive performance of early lactating dairy cows under heat stress[END_REF] but did show a drop in total blood and protein concentration [START_REF] Soltan | Effect of dietary chromium supplementation on productive and reproductive performance of early lactating dairy cows under heat stress[END_REF]. Serum, insulin and NEFA reduced significantly when 0.05 mg Cr/kg of body weight of Cr yeast was supplemented [START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF]. Recently, in dairy farm calves suffering from HS, dietary supplementation with 0.05 mg of Cr-methionine/kg of body weight had no significant influence on glucose, albumin, A/G ratio, triglyceride cholesterol, β-Hydroxybutyrate (βHBA) or blood urea nitrogen (BUN). Nevertheless, the serum concentration of globulin tended to be higher in calves supplemented with Cr +3 during the experimentation period (Mousavi et al., 2019a).

Calves supplied with chromium at a level of 0.05 mg/kg body weight tended to have higher serum concentrations of glucose and higher ratios of insulin to glucose measured at the end of the experiment (Kargar et al. 2018b). The same results were recorded by [START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF] and [START_REF] Ghorbani | Performance and metabolic responses of Holstein calves to supplemental chromium in colostrum and milk[END_REF]. Similarly, [START_REF] Jin | Effects of dietary supplementation of active dried yeast on fecal methanogenic archaea diversity in dairy cows[END_REF] suggested that dietary supplementation of chromium picolinate (3.5 mg of Cr/cow daily) to lactating cows under HS conditions did not exhibit any changes in glucose, BUN, cholesterol or creatinine. [START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF] reported similar results as he demonstrated that heat-stressed finishing pigs supplied with 200 ppb of chromium propionate did not show any shift in most of the blood parameters except for neutrophils. Heat-stressed pigs treated with chromium propionate supplementation (200 ppb) showed an increase in blood neutrophils by 37% compared to the control group [START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF]. Adding chromium (p < 0.01) was found to raise albumin/globulin ratio as well as cholesterol level [START_REF] Soltan | Effect of dietary chromium supplementation on productive and reproductive performance of early lactating dairy cows under heat stress[END_REF]. Enhanced insulin action allows glucose to enter the cell and can repress lipolysis. Reduced mobilization of fats was reported to decrease dependence on body storage and lessen NEFA [START_REF] Yang | Effects of chromium supplementation on early lactation performance of Holstein cows[END_REF]. Reduced NEFA raises DMI through the reversed lipostatic mechanism: as the levels of NEFA increase, the intake of food will decrease [START_REF] Forbes | Voluntary food intake and diet selection in farm animals: Second edition[END_REF][START_REF] Mcnamara | Adipose tissue metabolism and production responses to calcium propionate and chromium propionate[END_REF]. Milk production was found to increase when insulin action is improved as a result of the reduction in NEFA and insulin levels despite fixed glucose levels [START_REF] Hayirli | Effect of chromium supplementation on production and metabolic parameters in periparturient dairy cows[END_REF]. [START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF] and [START_REF] Ghorbani | Performance and metabolic responses of Holstein calves to supplemental chromium in colostrum and milk[END_REF] reported similar results. Similarity, [START_REF] Jin | Effects of dietary supplementation of active dried yeast on fecal methanogenic archaea diversity in dairy cows[END_REF] suggested that dietary supplementation of 3.5 mg of chromium picolinate daily to lactating cows under heat-stress conditions did not exhibit any changes in cholesterol, glucose, BUN or creatinine levels. In finisher gilts reared under summer conditions, plasma glucose, insulin and NEFA levels in serum did not change in chromium tripicolinate nanoparticles-treated pigs (400 ppb) compared to those in the control group [START_REF] Hung | Dietary nano-chromium tripicolinate increases feed intake and decreases plasma cortisol in finisher gilts during summer[END_REF]. [START_REF] Bareille | Modulation of the feeding response of lactating dairy cows to peripheral insulin administration with or without a glucose supply[END_REF] found evidence indicating that insulin can affect rumination by increasing the motility of reticulum and rumen through enhancing the entry of glucose into the cells. They also reported an increase in rumination time when glucose was infused intravenously (2.75 mmol/kg) to dairy cows. However, supplemental Cr did not affect blood insulin concentration at weaning or rumination time during the pre-weaning period in their study, despite the fact that rumination patterns changed (decreased rumination frequency and increased rumination duration and interval). Therefore, the relationship between blood insulin concentration and rumination patterns needs to be investigated further.

Chromium supplementation had little impact on insulin and blood glucose levels for both control and heat-stressed calves [START_REF] Ghorbani | Performance and metabolic responses of Holstein calves to supplemental chromium in colostrum and milk[END_REF][START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF]. Kumar et al. (2015a) reported a reduction in blood insulin concentration in buffalo calves under HS when 1.0 and 1.5 mg Cr/kg of DMI was supplemented, but not in the case of 0.5 mg Cr/kg. Consistent with these results, [START_REF] Bunting | Effects of dietary inclusion of chromium propionate and calcium propionate on glucose disposal and gastrointestinal development in dairy calves[END_REF] found that blood glucose decreased and insulin levels increased when 0.5 mg of Cr/kg of DMI was supplemented to dairy calves. Chromium supplementation increased both insulin concentrations and the ratio of insulin to glucose, suggesting that high levels of insulin were needed in order to make blood free from glucose (weak insulin sensitivity) and that resistance to insulin increased over that time period [START_REF] Bunting | Effects of dietary inclusion of chromium propionate and calcium propionate on glucose disposal and gastrointestinal development in dairy calves[END_REF].

Hormones

After 0.05 mg of Cr-methionine/kg of body weight was given in supplementation to dairy calves exposed to HS, Mousavi et al. (2019a) observed no notable effect on thyroxin (T3, T4, T3/T4 ratio), and cortisol hormone activities. However, they also reported that calves fed with Cr recorded high insulin levels and insulin-to-glucose ratios compared to those in the control group.

Similarly, Kargar et al. (2018b) reported that chromium supplementation (0.05 mg/kg of body weight) had no influence on serum levels of T3, T4 or cortisol when measured at pre-and postweaning. Calves fed .04 mg Cr/kg of body weight showed no difference in T3 or T4 blood concentration.

The same results were observed in buffalo calves that received 0.5, 1.0, and 1.5 mg of Cr/kg of DMI (Kumar et al., 2015a). However, Cr supplementation of 0.03 mg of Cr/kg of body weight increased blood T4 concentration and reduced T3-to-T4 ratio in dairy calves [START_REF] Ghorbani | Performance and metabolic responses of Holstein calves to supplemental chromium in colostrum and milk[END_REF]. On the other hand, it was reported that raising Cr doses (from 0 to 0.02 and 0.04 mg Cr/kg of body weight) resulted in a quadratic decline in serum T4, whereas blood T3 decreased only with the higher Cr dose supplementation in dairy calves [START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF].

Antioxidants

Cr supplementations do not influence malondialdehyde (MDA) or glutathione peroxidase (GPx) serum levels, but the concentration of superoxide dismutase (SOD) tended to increase in calves receiving Cr-methionine at levels of 0.05 mg Cr/kg of body weight at weaning. Furthermore, serum activities of catalase in calves fed Cr-methionine was higher after weaning (p < 0.001) [START_REF] Jin | Effects of dietary supplementation of active dried yeast on fecal methanogenic archaea diversity in dairy cows[END_REF]. Moreover, Mousavi et al. (2019a) revealed a significant reduction of creatine kinase as a response to yeast-chromium (Cr yeast, 8 mg/d) dietary supplementation to lactating heat-stressed cows; this result may be linked to application of different types of animals, suitable doses of chromium, and its chemical forms.

On the other hand, (Qi et al., 2018a) reported that supplementation of chromium picolinate (3.5 mg of Cr/cow daily) under hot conditions did not have an effect on antioxidant activities such as serum total antioxidant capacity (T-AOC), SOD activity, or MDA levels of lactating cows (Qi et al. 2018a). Qi et al. (2018a) moreover suggested that Cr can inhibit inflammation in lactating cows by promoting the release of Hsp72, facilitating the production of IL-10, and inhibiting the degradation of IκBα under HS conditions. Kumar et al. (2015a) observed that 1mg Cr/kg DM had positive impacts on both cellmediated and humeral immunity in heat-stressed buffalo calves. Kumar et al. (2015a) investigated different levels: 0.5, 1.0 and 1.5 mg of inorganic Cr/kg DM of the diet of buffalo calves under high environmental temperature conditions: buffalo calves receiving 1.5 mg/kg DM of Cr (p < 0.01) showed the highest immune status values and plasma Cr concentration during the stages of the experiment. It was found that buffalo calves receiving 1.5 mg/kg of DM of Cr exhibited a boost in the proliferation of B or T lymphocyte values (+16.50%), the phagocytic activity of neutrophils (13.72%), fluorescence recovery after use of the photobleaching (FRAP) technique (27.88%) and immunoglobulin of plasma (13.14%) compared with the control buffalo calves (Kumar et al., 2015a). The negative influence of HS on cows can be reduced through improved immunological activities under the effect of chromium supplementation. A significant enhancement in lymphocytes proliferation and blastogenesis in inorganic Cr given as supplementation to periparturient buffaloes was reported by several researchers (Burton et al., 1993a;[START_REF] Chang | Supplemental chromium for stressed and growing feeder calves[END_REF]Kumar et al., 2015b).

Immunity

It was reported that beef calves given supplementation of 15 mg/kg DM of Cr) had a slight improvement in cell-mediated immunity. Supplementation of Cr may have improved immune status by increasing immunoglobulin (IgM) and decreasing blood cortisol levels in buffalo calves compared to those in the control group, although an increase in cortisol level after exposure to high temperatures was also observed [START_REF] Abdelnour | Stress biomarkers and proteomics alteration to thermal stress in ruminants: A review[END_REF]Kumar et al., 2015b). This led to depression in immunity by preventing proliferation of lymphocytes, the activation of lymphocytes factors, and the production of T-cell growth factors [START_REF] Munck | Physiological functions of glucocorticoids in stress and their relation to pharmacological actions[END_REF][START_REF] Roth | Effect of glucocorticoids on the bovine immune system[END_REF]. [START_REF] Wang | Effects of chromium nanocomposite supplementation on blood metabolites, endocrine parameters and immune traits in finishing pigs[END_REF] recorded that supplementation of 200 ppb Nano-Cr/kg in pigs increased the serum levels of IgG and IgM by 20.6% and 13.2% respectively. Likewise, serum total levels of Ig and IgM increased after supplementation of Cr yeast to the diets of stressed calves (Burton et al., 1993a). On the other hand, [START_REF] Kegley | Immune response, glucose metabolism, and performance of stressed feeder calves fed inorganic or organic chromium[END_REF] found that Cr nicotinate and Cr yeast supplementation for stressed calves had no impact on total levels of IgM.

To develop immunoglobin concentration and the proliferation of lymphocytes, advanced antioxidants are needed for the production of immunoglobulin [START_REF] Bach | Nitrogen metabolism of early lactation cows fed diets with two different levels of protein and different amino acid profiles[END_REF].

Heat shock proteins (HSPs)

Heat-stressed lactating Holstein cows with a chromium diet (Cr yeast, 8 mg/d) were found to have a significant elevation (p<0.05) of blood HSP mRNA expression on d 44 of exposure, and Kumar et al. (2015b) found that the addition of 1.5 mg of Cr/kg to the diet of heat-stressed buffalo calves decreased Hsp70 significantly. Similarly, Qi et al. (2018a) confirmed that the supplementation of 3.5 mg Cr/kg daily to the diets of heat-stressed lactating cows increased Hsp72 significantly (11.68 ppb) compared to those that did not receive Cr (6.21 ppb).

Conclusion and future perspectives

The rapidly changing climate, global warming, and accompanying rapid growth in population size are sounding the alarm for the development of critical strategies to manage livestock productivity and reproducibility. As shown in this current review, HS has an effect on the nutritional metabolic and antioxidant status of livestock. When exposed to HS conditions, animals maintain thermoregulation and homeostasis via metabolic and physiological adjustments that can have a negative influence on growth performance. Feed intake is lowered, and growth rates consequently decrease. Alterations in nutrient partitioning and utilization as well as in immune and metabolic functions have also been reported. Cr restores productivity, performance, nutrient digestibility, antioxidant profile and immune status while decreasing lipid peroxidation, cholesterol and fat content in animals after exposure to HS. Therefore, dietary supplementation with Cr may be recommended as a nutritional strategy to modulate the negative effects of HS in broilers.

Proper nutritional management of livestock so that animals maintain energy balance could have a positive effect on reproductive status and resulting embryos, future progeny and production. More in-depth studies using genomic and metabolomic technologies are needed to clearly investigate the genomic and epigenetic mechanism of action of HS on livestock development. Most of the literature discussing the effect of HS and/or NEB on livestock health is based on in vivo chromium effects. Because of its marked role in poultry performance and production, Cr could be considered as one of the very essential components in poultry feed during times of HS. Furthermore, the effect of Cr diet interaction with livestock health status during HS is a new area that should be considered for future research. Another future consideration is proper attention on breeding programs, for selection of HS-tolerant breeds, instead of only selection for high-production breeds. • Cr-methionine (0.05 mg /kg BW0.75). • Cr (0.05 mg/kg BW0.75).

Acknowledgment

• Cr-methionine at 0.05 mg/kg BW0.75 -

• Significantly reduced the Respiration rate (RR).

• Reduced the RR but has no effect on rectal temperature (RT).

• Reduced the days with diarrhea (-0.9 d), pneumonia (-0.7 d) and total medication days (-1.5 d) before weaning in heat stressed calves.

• No effect of Cr methionine on diarrhea, pneumonia, medication days and RT in heat stressed calves.

• (Mousavi et al., 2019b) • (Kargar et al., 2018b) • (Mousavi et al., 2019a) • (Mousavi et al., 2019a) Buffalo calves.

• Cr (0.5, 1 or 1.5 mg inorganic Cr/kg

• No effect on PR, RR and RT in heated buffalo calves.

• (Kumar et al., 2015) Cows.

• (3.6, 7.2 or 10.8 milligram chromium/head per day)

• Values of RR and RT for cows take Cr supplement and cows without any supplement were with no differences • According to the previous results Cr has no impact on somatic cell count and (RT, PR, RR) in lactating cows.

• (An-Qiang et al., 2009) • (Qi et al., 2018) Pigs.

• Cr propionate (200ppb).

• No effect on RR, ST and BT in heat stressed pigs.

• [START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF]

2-Growth performance

Cows.

• Cr yeast.

• Cr-methionine at 0.05 mg Cr/kg. • Significant increase (p < 0.05) as reported in the DMI.

• [START_REF] Alsaiady | Effect of chelated chromium supplementation on lactation performance and blood parameters of Holstein cows under heat stress[END_REF] • [START_REF] Arthington | Supplemental dietary chromium does not influence ACTH, cortisol, or immune responses in young calves inoculated with bovine herpesvirus-1[END_REF][START_REF] Zhi-Sheng | Effect of Chromium Picolinate Supplementation on Early Lactation Performance, Rectal Temperatures, Respiration Rates and Plasma Biochemical Response of Holstein Cows under Heat Stress[END_REF] Calves.

-

• 0.05 mg/kg BW0.75 Cr.

-

• 4 mg/head.
-

• The supplement of Cr to heat stressed dairy calves resulted in increasing total DMI (P = 0.002), ADG (P = 0.02) but has no effect on feed efficiency (P = 0.93) or final body weight (P = 0.14).

• Dietary supplementation of Chromium improved feed intake and growth rate in Holstein dairy calves exposed to high environmental temperature (THI=83.3 units), Also, the addition of Cr to a milk substitute or diets during pre and post-weaning cattle increases the feed intake, dry matter intake and average body weight gain at the end of the experiment of 16.4 %, 11.85 and 7.07 % respectively.

• Cr supplement improves growth rate and body weight through enhancing the absorption and digestion of nutrients. Under a variety of stress conditions, Cr requirement of calves is increased.

• Feed efficiency and average daily gain increased in case of growing calves fed Cr.

• This result is due to increase the total levels of

• (Mousavi et al., 2019a )
• (Kargar et al., 2018a) • [START_REF] Ghorbani | Performance and metabolic responses of Holstein calves to supplemental chromium in colostrum and milk[END_REF] • (Chang and Mowat, 1992)

• [START_REF] Burton | Effects of supplemental chromium on production of cytokines by mitogen-stimulated bovine peripheral blood mononuclear cells[END_REF] -• 0.5 mg/kg of supplemental Cr as Cr propionate.

immunoglobulins in serum.

• Cr supplementation to Murrah buffalo calves showed no significant effect on feed conversion ratio (FCR) over a period of 120 days, average daily gain (ADG) and dry matter intake.

• Under normal conditions, Cr has no influence on feed intake or total DMI.

• (Kumar et al., 2015) • [START_REF] Bunting | Effects of dietary inclusion of chromium propionate and calcium propionate on glucose disposal and gastrointestinal development in dairy calves[END_REF] Pigs • 200ppb of Chromium propionate.

• Final body weight has increased in heat stressed finishing pigs. • [START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF] 3-Milk yield and composition Dairy cows.

• (4g/head/day) of Dietary chelated chromium. • Cr at 0.05 mg Cr/kg).

• Moderate levels of Cr.

• 10 mg/day Cr from 21 days pre-partum to 35 postpartum. • Chromium picolinate.

• Adding to the diet of heat loaded dairy cows has no effect on milk fats, protein, lactose, and solids non-fat percentage.

• A significant rise (p < 0.05) in milk protein, fat and lactose levels by 1.043,1.066,1.02 %, respectively is recorded when heat stressed dairy cows are supplied with Cr. • Increase in feed intake, dry matter efficiency and a decline in the insulin levels leading to a significant increase in milk secretion as a result of increasing the mammary nutrient flow. • The activity of Insulin growth factor (IGF) that has functional and structural homology to insulin receptors are supported through the improvement of milk production which also mimic the action of bovine somatotropin (bST).

• An increase in milk production which was associated with decrease lipolysis and increase glucose uptake that will result in DMI rise.

• The milk yield has increased (p=0.013), dietary Cr has no significant on milk components (protein, fat and lactose) in dairy cows under high temperature (THI=79.61).

• [START_REF] Alsaiady | Effect of chelated chromium supplementation on lactation performance and blood parameters of Holstein cows under heat stress[END_REF] • (Nikkhah, 2012)

• (Winkelman and Overton, 2012)

4-Blood variables

Cows.

• Chromium yeast (6mg/head /day).

• 0.05 mg of supplemental Cr/kg of BW 0.75

• Chromium picolinate (3.5 mg of Cr/cow daily).

• 3.5m g/kg of Cr/cow.

• Failed to show any effect on blood serum level of hemagglutination test (HA), cholesterol, triglycerides, urea, IGF-1, BHBA, albumen, and glucose while there is a drop in total blood and protein concentration.

• Serum, insulin and NEFA reduced significantly.

• No changes in glucose, blood urea nitrogen (BUN), creatinine, and cholesterol of lactating cows under heat stress conditions.

• Significantly increased Hsp72 (11.68 μg/L) compared to those did not receive Cr (6.21 μg/L), Cr may play an anti-inflammatory role in lactating cows by promoting the release of Hsp72, increasing the production of IL-10, and inhibiting the degradation of IκBα under summer hot situation.

• [START_REF] Soltan | Effect of dietary chromium supplementation on productive and reproductive performance of early lactating dairy cows under heat stress[END_REF].

• [START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF] • [START_REF] Jin | Effects of dietary supplementation of active dried yeast on fecal methanogenic archaea diversity in dairy cows[END_REF] • (Qi et al., 2018) Calves.

• Cr-methionine (0.05 mg /kg BW 0.75).

• Chromium at level 0.05 mg/kg BW 0.75

• Dairy farm calves suffering from heat stress had no significant influence on glucose, albumin, and A/G ratio, triglyceridecholesterol, βHBA and BUN. Nevertheless, the serum concentration of globulin tended to be higher in Cr+ calves during the experimental period. • Higher serum concentrations of glucose and a higher ratio of insulin to glucose measured at the end of the experiment.

• Chromium supplementation had small effects on blood glucose and insulin in calves under normal or heat-stress conditions.

• (Mousavi et al., 2019a) • (Kargar et al., 2018b;[START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF] • [START_REF] Ghorbani | Performance and metabolic responses of Holstein calves to supplemental chromium in colostrum and milk[END_REF] • 1 and 1.5 mg/kg of DMI.

• 0.5 mg of Cr/kg of DMI.

• Cr-methionine (0.05 mg /kg BW0.75).

• 0.04 mg Cr/kg of BW0.75.

• 0.5, 1, and 1.5 mg of Cr/kg

• (0.03 mg of Cr/kg of BW0.75).

• Raising Cr doses (from 0 to 0.02 and 0.04 mg Cr/kg of BW0.75).

• Blood insulin concentration reduced in heat-stressed buffalo calves but not at 0.5 mg/kg of DMI.

• Blood concentrations of glucose and insulin decreased and increased, respectively, when dairy calves were supplemented with cr.

-Hormones:

• No notable effect on the T3, T4, T3/T4 ratio, and cortisol hormones activities However, they also reported that calves fed with Cr, recorded higher blood insulin level and insulin to glucose ratio as compared to those in the control group.

• No influences on serum levels of T3, T4 and cortisol measured at pre and post weaning.

• Similar findings were observed in buffalo calves that received 0.5, 1, and 1.5 mg of Cr/kg of DMI.

• Increased blood T4 concentration and reduced T3 to T4 ratio in dairy calves.

• Quadratic decline in serum T4, whereas blood T3 decreased only with the higher Cr dose supplementation in dairy calves.

• (Kumar et al., 2015) • [START_REF] Bunting | Effects of dietary inclusion of chromium propionate and calcium propionate on glucose disposal and gastrointestinal development in dairy calves[END_REF] • (Mousavi et al., 2019a) • (Kargar et al., 2018b) • (Kumar et al., 2015) • [START_REF] Ghorbani | Performance and metabolic responses of Holstein calves to supplemental chromium in colostrum and milk[END_REF] • [START_REF] Yari | Physiological calf responses to increased chromium supply in summer[END_REF] Cows

• (Cr-Yeast, 8mg/d).

-Antioxidants:

• Significant reduction of creatine kinase as a response to yeastchromium dietary supplementation to lactating heat-stressed cows, which may be attributed to the use of different animal models, proper dose gradients of Cr, and chemical forms of Cr.

• (Mousavi et al., 2019a) Calves.

• 1.0 mg Cr/kg DM and above. • High Cr-yeast.

• 4 mg of supplemental Cr/kg of DM and Cr nicotinate. • 1.5 mg of Cr/kg.

• Positive impacts in improving both humeral and cell-mediated immunity in heat-stressed buffalo calves.

-Increased serum total Ig and IgM levels in high Cr-yeast supplemented calves following transport stress. • No change in total IgM in both high-Cr yeast and Cr nicotinate supplemented transport stressed calves.

• To heat stressed buffalo calves, decreased significantly Hsp70.

• (Kumar et al., 2015) • (Burton et al., 1993) • [START_REF] Kegley | Immune response, glucose metabolism, and performance of stressed feeder calves fed inorganic or organic chromium[END_REF].

• (Kumar et al., 2015), (Qi et al., 2018) Pigs.

• Chromium propionate supplementation (200 ppb).

• Chromium tripicolinate nanoparticles (400 ppb).

• 200 μg/kg Cr as Cr-Nano.

• Increase in blood neutrophils by 37% corresponded to the control group.

• Plasma glucose, insulin and NEFA levels in serum were not changed in chromium tripicolinate nanoparticles-treated pigs (400 ppb) compared to those in the control group.

• 13.2 and 20.6 % higher serum concentration of IgM and IgG.

• [START_REF] Mayorga | Effects of dietary chromium propionate on growth performance, metabolism, and immune biomarkers in heat-stressed finishing pigs1[END_REF] • [START_REF] Hung | Dietary nano-chromium tripicolinate increases feed intake and decreases plasma cortisol in finisher gilts during summer[END_REF] • [START_REF] Wang | Effects of chromium nanocomposite supplementation on blood metabolites, endocrine parameters and immune traits in finishing pigs[END_REF]

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Effect of heat stress on animal performance, production and reproduction.

Figure 3 .

 3 Figure 3. Chemical formula of Cr III picolinate.

Figure 4 .

 4 Figure 4. The role of Chromium (III) against cellular glucose uptake (insulin binds to its

Figure 5 .

 5 Figure 5. The beneficial effect of Chromium on glucose and non-esterified fatty acids (NEFA)

Table 1 .

 1 The main findings related to Cr supplementation in heat stressed-animals

	Parameter	Species	Form & dosage	Findings	References
	1-Thermoregulatory responses	Dairy calves.


Mathieu Lempereur
email: mathieu.lempereur@univ-brest.fr

François Rousseau

Olivier Rémy-Néris

Christelle Pons

Laetitia Houx

Gwenolé Quellec

Sylvain Brochard

A new deep learning-based method for the detection of gait events in children with gait disorders: Proof-of-concept and concurrent validity

Keywords: Gait, children, recurrent neural network, deep learning

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The current gold standard for the automatic detection of gait events in clinical practice involves the use of ground reaction forces measured by force platforms [START_REF] Veilleux | Agreement of spatio-temporal gait parameters between a vertical ground reaction force decomposition algorithm and a motion capture system[END_REF]. However, pathological gait is highly variable and the use of gait aids, small steps or foot dragging during swing phase can produce false force thresholds, all of which may reduce the accuracy of the detection. To avoid these issues, gait events are therefore often determined manually, in particular for pediatric gait which is highly variable, however this process is time consuming and its accuracy and reliability strongly depend on operator expertise. To overcome these issues, computational methods of gait event detection, mostly based on kinematic data from opto-electronic systems, have been developed. A literature review [START_REF] References Bruening | Automated event detection algorithms in pathological gait[END_REF] identified nine kinematic-based algorithms [START_REF] De Asha | A marker based kinematic method of identifying initial contact during gait suitable for use in real-time visual feedback applications[END_REF][START_REF] Desailly | Foot contact event detection using kinematic data in cerebral palsy children and normal adults gait[END_REF][START_REF] Ghoussayni | Assessment and validation of a simple automated method for the detection of gait events and intervals[END_REF][START_REF] Hreljac | Algorithms to determine event timing during normal walking using kinematic data[END_REF][START_REF] Hsue | Gait timing event determination using kinematic data for the toe walking children with cerebral palsy[END_REF][START_REF] Jasiewicz | Gait event detection using linear accelerometers or angular velocity transducers in able-bodied and spinal-cord injured individuals[END_REF][START_REF] O'connor | Automatic detection of gait events using kinematic data[END_REF][START_REF] Salazar-Torres | Validity of an automated gait event detection algorithm in children with cerebral palsy and non-impaired children[END_REF][START_REF] Zeni | Two simple methods for determining gait events during treadmill and overground walking using kinematic data[END_REF] that appeared promising for event detection in gait patterns such as equinus, slide/drag or steppage gait. The literature review and a recent study by [START_REF] Gonçalves | Identification of gait events in children with spastic cerebral palsy: comparison between the force plate and algorithms[END_REF] both revealed that the variables that result in the most accurate determination of events are sagittal resultant velocity [START_REF] Ghoussayni | Assessment and validation of a simple automated method for the detection of gait events and intervals[END_REF], horizontal position [START_REF] Desailly | Foot contact event detection using kinematic data in cerebral palsy children and normal adults gait[END_REF][START_REF] Zeni | Two simple methods for determining gait events during treadmill and overground walking using kinematic data[END_REF], and vertical/horizontal acceleration [START_REF] Hreljac | Algorithms to determine event timing during normal walking using kinematic data[END_REF][START_REF] Hsue | Gait timing event determination using kinematic data for the toe walking children with cerebral palsy[END_REF] for FS, and horizontal position [START_REF] Desailly | Foot contact event detection using kinematic data in cerebral palsy children and normal adults gait[END_REF][START_REF] Zeni | Two simple methods for determining gait events during treadmill and overground walking using kinematic data[END_REF] and sagittal velocity [START_REF] Ghoussayni | Assessment and validation of a simple automated method for the detection of gait events and intervals[END_REF] for FO. However, all those algorithms are limited by an accuracy of four frames (33.3 ms), and the fact that none can be used accurately across all types of gait pathologies. There is therefore a need for the development of learning-based approaches that do not require a priori information relative to the input data.

Most studies that have used machine learning to study biomechanical parameters in patients with musculoskeletal and neuromuscular diseases have focused on the classification of pathological movements, prediction of the risk of development of certain diseases or the effect of an intervention, or the automatic recognition of activity for outof-clinic monitoring [START_REF] Halilaj | Machine learning in human movement biomechanics: Best practices, common pitfalls, and new opportunities[END_REF]. Recently, a method using deep neural networks to detect gait events in children with cerebral palsy determined FS and FO with respective errors of 18.3 ms and 12.5 ms [START_REF] Kidziński | Automatic real-time gait event detection in children using deep neural networks[END_REF]. However, that method requires the use of a biomechanical model to compute the kinematic input data (3D joint angles using the conventional gait model described by [START_REF] Kadaba | Measurement of lower extremity kinematics during level walking[END_REF], 3D position and velocity of the markers, and 3D position, velocity and acceleration of the pelvis).

Recurrent neural networks, and more specifically long short term memory (LSTM) networks are among the most efficient in learning dynamics from time series [START_REF] Hochreiter | Long short-term memory[END_REF]. Although LSTM networks are very popular [START_REF] Schmidhuber | Deep learning in neural networks: An overview[END_REF]), a specific type of analysis such as sensitivity analysis, Taylor decomposition or backward propagation, is necessary to understand the decision process used by such networks and thus to ensure appropriate interpretation of the output from the network, especially in the clinical context.

The primary aim of this study was to test the proof-of-concept of a new gait event detection method (DeepEvent) using an LSTM network. The second aim was to compare the accuracy of this new method with a set of different methods proposed in the literature and to determine if a resulting error of less than 10 ms (1 frame at 100 Hz) could be achieved. The third aim was to identify and interpret the parameters used in the decision process.

Method

Gait data

Data from 226 children who had undergone clinical 3D gait analysis were used. All parents provided written informed consent on a form that had been approved by the Brest CHRU. The children had the following pathologies: neurological disorders (65%, 76% of whom had cerebral palsy), neuromuscular disorders (11%), orthopedic disorders (9%) and other types of disorders (15%). All data were recorded using the same motion analysis system (Vicon MX, Oxford Metrics, UK) and four force platforms (Advanced Mechanical Technology, Inc., Watertown, MA, USA) in the same motion laboratory (Brest CHRU) between 2015 and 2019. The data collected by the 15 infrared cameras (sampling rate 100 Hz or 120 Hz) were synchronized with the ground reaction forces recorded by the force platforms (1000 Hz or 1200 Hz). The markers were placed according to the protocol by by [START_REF] Kadaba | Measurement of lower extremity kinematics during level walking[END_REF]. Marker trajectories and ground reactions forces were dual-pass filtered with a low-pass Butterworth filter at a cut-off frequency of 6 Hz. All participants walked independently, with no gait aids, at their natural speeds. An average of 5 trials was recorded for each child, yielding 1156 trials in total. All FS and FO events were manually identified by an engineer [START_REF] Crowther | A method for optimal division of data sets for use in neural networks[END_REF]). Among the test data, 815 FS (446 left FS and 369 right FS) and 815 FO (369 left FO and 446 right FO) had previously been identified using the force platform data and were used as the gold standard method for comparison purposes. FS was determined as the first frame at which the vertical ground reaction force was greater than 10 N. FO was determined as the first frame at which the vertical ground reaction force was lower than 10 N.

Network architecture

3D position and velocity of the left and right heel, toe and lateral malleolus markers for each trial were used as input data for the DeepEvent recurrent network. A zero-padding technique was used to obtain the same number of frames for each trial (with a maximum duration of 1536 frames).

The neural network consisted of M bidirectional LSTM layers with N hidden units (Supplementary data) and was followed by a dropout layer. Finally, a one-time distributed dense layer was used to detect gait events. The 5 gait events (no event, left FS, right FS, left FO and right FO) were encoded using a one-hot encoding scheme. The parameters M and N were empirically chosen to maximize the performance of the network with the validation data set by an empirical approach. A sensitivity analysis was carried out to explain the neural network decisions by measuring the contribution of each input variable to the overall prediction [START_REF] Simonyan | Deep inside convolutional networks: Visualising image classification models and saliency maps[END_REF].

DeepEvent was implemented using Keras [START_REF] Chollet | Keras[END_REF]) in Python [START_REF] Van Rossum | Python tutorial[END_REF]), with crossentropy as loss function and Adaptive Moment Estimation (Adam) for optimization on a Dell Workstation 7910 (CPU Xeon e5-2680-v4 2.4 GHz) with a Nvidia GeForce GTX 1080 Ti. DeepEvent is available on Github (github.com/LempereurMat/deepevent).

The contribution of each type of input data to the decision process is presented in figures 1 and 2. The sensitivity analysis showed that DeepEvent used the input data between 0 and 20%, between 50 and 70% and between 90 and 100% of the gait cycle. During these intervals, the contribution of each typeof input data was between 0 and 0.04. Outside of these intervals, the weights were closer to 0. These timings perfectly matched the definition of the beginning of swing phase at 60% of the gait cycle. The determination of the FS mainly involved the use of the z-axis (longitudinal) position and velocity of the toe and heel markers at the beginning and end of gait cycle (weight ¿ 0.04). To predict FO, DeepEvent mainly used the x-axis (antero-posterior) velocity and z-axis position and velocity of the toe marker around 60% of the gait cycle (weight ¿ 0.03).

FS [CI]

FO [CI] Table 1: Absolute differences in milliseconds between DeepEvent, the methods in the literature, and the gold standard (automatic determination using force platform data). CI: confidence interval. Best results are highlighted in bold.

Discussion

The aim of this study was to test the proof-of-concept of a new gait event detection approach using an LSTM network.

The results showed that DeepEvent determined FS and FO more accurately than three heuristic methods from the literature. Moreover, it was more accurate than the recently developed deep learning approach by [START_REF] Kidziński | Automatic real-time gait event detection in children using deep neural networks[END_REF] that uses more input variables (kinematic outputs from conventional gait models). The high level of accuracy of DeepEvent means that it can be used in clinical practice for the automatic detection of the gait events with a very good inter-trial reliability. This study showed that the number of sequence frame (128 frames in [START_REF] Kidziński | Automatic real-time gait event detection in children using deep neural networks[END_REF]; 1536 in DeepEvent) is an important input parameter, since increasing the number of frames in the method by [START_REF] Kidziński | Automatic real-time gait event detection in children using deep neural networks[END_REF] to the number used by DeepEvent improved the accuracy of the former method. The detection of FO was consistently less accurate than the detection of FS. This is not surprising, DeepEvent is a supervised LSTM-based approach. The accuracy of this type of algorithm is determined by the quality of the manual event annotation. In many cases, gait transitions, in particular FO, occur gradually and the identification of a single event frame is somewhat subjective, as reported by [START_REF] Kidziński | Automatic real-time gait event detection in children using deep neural networks[END_REF]. The location of the toe marker may also affect FO detection. The use of a hallux marker as in [START_REF] Stebbins | Repeatability of a model for measuring multi-segment foot kinematics in children[END_REF] could increase FO detection accuracy but this marker placement is not commonly used in clinical gait analysis.

The sensitivity analysis demonstrated the importance of both the toe and heel markers for the accurate detection of FS and FO while the lateral malleolus marker did not appear to be essential. Equally, pelvic and other markers were unnecessary because the deep learning approach could determine gait direction due to the numerous cases in the database of gait with direction along the anterior or posterior axis. In this study, all gait events were manually identified by a single experienced engineer and the neural network was trained using this information. The results now need to be externally validated by evaluating the robustness of this supervised technique using data from other laboratories.

  with 15 years' experience in gait analysis. The data included 10526 FS (5247 left and 5279 right) and 9375 FO (4654 left and 4721 right). The data were randomly divided up such that: 60% were used for training (694 trials, 6303 FS (3139 left FS; 3164 right FS)), 5614 FO (2789 left FO; 2825 right FO)), 10% for validation (116 trials, 1039 FS (514 left FS; 525 right FS), 923 FO (457 left FO; 466 right FO)) and 30% for testing (346 trials, 3184 FS (1594 left FS; 1590 right FS), 2841 FO (1411 left FO; 1430 right FO)). These proportions are typically used in deep learning methodology

Acknowledgement

The authors would like to thank Stéphane Armand (Willy Taillard Laboratory of Kinesiology, Geneva University Hospitals and Geneva University) for reviewing the manuscript and Johanna Robertson for language assistance.

Validation: concurrent validity

The results of the DeepEvent method were compared to the position-, velocity-and acceleration-based algorithms proposed by [START_REF] Zeni | Two simple methods for determining gait events during treadmill and overground walking using kinematic data[END_REF], [START_REF] Ghoussayni | Assessment and validation of a simple automated method for the detection of gait events and intervals[END_REF] and [START_REF] Hsue | Gait timing event determination using kinematic data for the toe walking children with cerebral palsy[END_REF] and the deep learning method proposed by [START_REF] Kidziński | Automatic real-time gait event detection in children using deep neural networks[END_REF]. The position-based algorithms reported by [START_REF] Zeni | Two simple methods for determining gait events during treadmill and overground walking using kinematic data[END_REF] and [START_REF] Desailly | Foot contact event detection using kinematic data in cerebral palsy children and normal adults gait[END_REF] detected FS with the same degree accuracy, however the method by [START_REF] Zeni | Two simple methods for determining gait events during treadmill and overground walking using kinematic data[END_REF] more accurately identified FO [START_REF] References Bruening | Automated event detection algorithms in pathological gait[END_REF]). The velocity-based method reported by [START_REF] Ghoussayni | Assessment and validation of a simple automated method for the detection of gait events and intervals[END_REF] identified gait events most accurately [START_REF] Gonçalves | Identification of gait events in children with spastic cerebral palsy: comparison between the force plate and algorithms[END_REF]), and the use of horizontal acceleration resulted in a more accurate determination of FS and FO than the use of vertical acceleration [START_REF] Hsue | Gait timing event determination using kinematic data for the toe walking children with cerebral palsy[END_REF]).

The coordinate-based algorithm by [START_REF] Zeni | Two simple methods for determining gait events during treadmill and overground walking using kinematic data[END_REF] uses the anterior/posterior position of the heel and toe marker relative to the sacrum marker. The equation for the definition of FS is: t F S = max(X Heel -X Sacrum ) and for FO is: [START_REF] Ghoussayni | Assessment and validation of a simple automated method for the detection of gait events and intervals[END_REF] uses the sagittal plane resultant velocity and applies a gait speed-dependent threshold to detect FS: F S T hreshold = 0.78 × W alkSpeed and FO: F O T hreshold = 0.66 × W alkSpeed. [START_REF] Hsue | Gait timing event determination using kinematic data for the toe walking children with cerebral palsy[END_REF] uses the peak horizontal deceleration of the heel marker to identify FS:

) and the peak horizontal acceleration of toe marker to detect FO:

Kidziński et al. ( 2019) uses the 3D joint angles using the conventional gait model described by [START_REF] Kadaba | Measurement of lower extremity kinematics during level walking[END_REF], 3D position and velocity of the markers, and 3D position, velocity and acceleration of the pelvis. Their network is built with 3 LSTM layers using a sequence of 128 frames. The results from DeepEvent were also compared to a modified version of the method by [START_REF] Kidziński | Automatic real-time gait event detection in children using deep neural networks[END_REF], in which the number of frames was increased in order to obtain the same number of frames as in DeepEvent (1536).

Absolute differences in time were compared between the four algorithms in the literature, the modified Kidziński et al. ( 2019) algorithm, DeepEvent, and the force platform data.

Results

Proof-of-concept

Three bidirectional LSTM layers with 800 hidden units were chosen to maximize the performance of the network.

With these parameters, the trained model had a validation cross-entropy of 0.0286 and an accuracy of 100%. The receiver operating characteristic analysis showed that the fully trained model achieved an area under the curve of 0.9955 for the left FS, 0.9971 for the right FS, 0.9958 for the left FO and 0.9955 for the right FO indicating that the model accurately determined FS and FO.

Computational times for training DeepEvent, the original and the modified [START_REF] Kidziński | Automatic real-time gait event detection in children using deep neural networks[END_REF] methods were on average about 20, 8 and 42 minutes respectively.

Concurrent validity and parameters used in the decision process

DeepEvent predicted FS within 5.5 ms and within 10.7 ms of the gold standard, and was more accurate than the three methods from the literature, as well as the modified [START_REF] Kidziński | Automatic real-time gait event detection in children using deep neural networks[END_REF] method (Table 1). These errors are acceptable since they are well below or just superior to the threshold of 10 ms.

Conflict of interest statement

None of the authors had any financial or personal conflict of interest with regard to this work.

Supplementary data

The Long Short Term Memory (LSTM) is composed of four different gates that can change the information to the cell state in order to build a long-term memory (figure supplementary data). The first gate is called the "forget gate" (f t ) that decides for each element of the previous cell state vector C t-1 how much information to forget:

with σ the sigmoid function 1 1+e -t and x t the input data (3D position of the left and right lateral malleolus, 3D position of the left and right toe, 3D position of the left and right heel, 3D velocity of the left and right lateral malleolus, 3D velocity of the left and right toe and 3D velocity of the left and right heel). The second step concerns the "input gate" (i t ) and the "candidate gate" (g t ) . The first one is used to update value in C t-1 while the second one is used to create a "candidate" that will be added to C t-1 . Then, the cell state is updated:

C t = f t C t-1 + g t i t , : pointwise operator with tanh a hyperbolic tangent function. Finally, the "output gate" (O t ) gives the output value (h t ):

In the four gates, the values of the weight matrix W and the bias b were obtained during the training part of the recurrent neural network.