Effects of the supplemental chromium form on performance and metabolic profile in laying hens exposed to heat stress

Effects of the supplemental chromium form on performance and metabolic profile in laying hens... Abstract This experiment was conducted to compare the effect of the supplemental chromium (Cr) form on performance, egg quality, and metabolic profile in laying hens exposed to heat stress (HS). Laying hens (n = 1800; 16-wk-old; Lohmann LSL-Lite) were kept in cages in temperature-controlled rooms at either 22 ± 2°C for 24 h/d (thermoneutral, TN) or 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. Hens reared under both environmental conditions were fed 1 of 3 diets: a basal diet and the basal diet supplemented with either 1.600 mg of chromium-picolinate (CrPic, 12.43% Cr) or 0.788 mg of chromium-histidinate (CrHis, 25.22% Cr) per kg of diet, delivering 200 μg elemental Cr per kg diet. Data were analyzed by 2-way ANOVA. Exposure to HS caused decreases in feed intake (P < 0.0001), egg production (P < 0.0001), egg weight (P < 0.0001), eggshell weight (P < 0.0009), eggshell thickness (P < 0.0001), eggshell strength (P < 0.0001), and Haugh unit (P < 0.0001), deterioration in feed conversion ratio (P < 0.0001), increases in serum glucose and cholesterol concentrations (P < 0.0001 for both), decreases in serum and egg yolk Cr concentrations (P < 0.0001 for both), and decreases in serum Na (P < 0.002) and K (P < 0.01) concentrations. Both Cr sources were equally effective in alleviating performance variables under the HS condition. However, neither Cr sources alleviated deteriorations in egg quality parameters and serum electrolytes. Both Cr sources decreased serum glucose and cholesterol concentrations and increased serum and egg yolk Cr concentrations under the HS condition. In conclusion, HS adversely affected laying performance, egg quality, and metabolic profile. Both CrPic and CrHis partially alleviated the adverse effect of HS on these parameters. Inclusion of either Cr source could be a part of nutritional management strategies to overcome the adverse effects of HS performance and metabolic profile in laying hens. INTRODUCTION High ambient temperature is one of the detrimental environmental factors in poultry farms (Freeman, 1987). Heat stress (HS) is a serious welfare issue and causes economic losses due to poor performance and compromised immune and health statuses (Howlider and Rose, 1987; Sahin and Kucuk, 2003; Mashaly et al., 2004). Increased excretion and reduced bioavailability of nutrients during HS may contribute to suppressed performance and immune potency (Belay and Teeter, 1996). Consequent development of oxidative stress (de Araujo et al., 2007; Sahin et al., 2009, 2010) may be linked to decreased tissue reserves of minerals (e.g., Se, Cu, Mn, Cr) (Khan et al., 2014) and vitamins (e.g., vitamins A and E) (Sahin and Kucuk, 2003) that are integral components of the antioxidant defense system. Chromium (Cr) is involved in metabolic pathways of nutrients (e.g., carbohydrate, lipid, protein, and nucleic acid) through potentiating the insulin action (Anderson, 1987; Cupo and Donaldson, 1987; Hayirli, 2005). It contributes to anabolic profile (Lien et al., 1996, 1999, 2004) and alleviates oxidative stress and lipid peroxidation (Samanta et al., 2008; Sahin et al., 2009, 2010) in laying hens exposed to HS. Chromium chelates with organic compounds have lower toxicity and higher bioavailability than Cr in inorganic forms (Kim et al., 1996; Piva et al., 2003). Various Cr chelates (yeast, picolinate (CrPic), nicotinate, propionate, and methionine) are commercially available. The Cr form is important, particularly in case of exposure to stressors, to compensate deficiency and achieve biological response (Sahin et al., 2002; Piva et al., 2003). Chromium-histidinate (CrHis) is a newly developed chelate and has not been tested in laying hens. This experiment was therefore conducted to compare the effects of different Cr forms (CrHis vs. CrPic) on performance, egg quality, and metabolic profile of laying hens exposed to HS. MATERIALS AND METHODS Animals, Treatments, and Management The experiment involving laying hens (n = 1800, 16 wks old, Lohmann LSL-Lite) was conducted at a commercial farm (Umut Tavukçuluk A.Ş., Elazig, Turkey). The birds were kept in cages (48 cm × 45 cm × 45 cm, width × depth × height) (4 birds/cage, at a density of 540 cm2/hen, providing 12 cm feeder space per hen) in temperature-controlled rooms at either 22 ± 2°C for 24 h/d (thermoneutral, TN) or 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. The basal diet was formulated to meet nutrient requirements (NRC, 1994; Table 1). The hens in both TN and HS conditions were either not supplemented with Cr (C) or supplemented with 1.600 mg of CrPic (12.43% elemental Cr, Nutrition 21, NY) or 0.788 mg of CrHis (25.22% elemental Cr, Nutrition 21, NY) per kilogram diet in mash form, delivering 200 μg Cr per kg diet. In order to avoid confounding effects of an organic portion of the Cr-chelates, the diets C, CrPic, and CrHis were added with 1.401 mg picolinic acid+0.589 mg histidine, 0.589 mg histidine, and 1.401 mg picolinic acid per kilogram, respectively. Each treatment was replicated in 75 cages. Chickens were managed in accordance with animal welfare regulations at the Republic of Turkey Ministry of Food, Agriculture and Livestock. Feed and fresh water were offered ad libitum during the experimental period. Birds were exposed to a light: dark cycle of 16 h:8 h per day. Table 1. Ingredients and nutrient composition of the basal diet.1 Ingredient  %  Maize  54.85  Soybean meal  28.26  Corn oil  4.66  Salt  0.33  DL-methionine  0.22  Limestone  9.30  Dicalcium phosphate  1.80  Vitamin-mineral premix2  0.35  Nutrient composition (g/kg, dry matter basis)    Metabolisable energy, kcal/kg3  2800  Crude protein  177.6  Calcium  40.4  Phosphorus  6.3  Methionine3  4.0  Lysine3  11.1  Ingredient  %  Maize  54.85  Soybean meal  28.26  Corn oil  4.66  Salt  0.33  DL-methionine  0.22  Limestone  9.30  Dicalcium phosphate  1.80  Vitamin-mineral premix2  0.35  Nutrient composition (g/kg, dry matter basis)    Metabolisable energy, kcal/kg3  2800  Crude protein  177.6  Calcium  40.4  Phosphorus  6.3  Methionine3  4.0  Lysine3  11.1  1Experimental diets were constructed through reconstituting the premix in the basal diet at the expense of CaCO3 to contain histidinate (0.236 g) plus picolinate (0.560 g), CrPic (0.640 g) plus histidinate (0.236 g), and CrHis (0.315 g) plus picolinate (0.560 g) in the diets C, CrPic, and CrHis, respectively. The basal diet contained 0.17 ± 0.08 mg Cr and those supplemented with CrPic and CrHis contained 2.09 ± 0.13 mg Cr per kg diets (mean ± SD). 2Per kilogram contained: vitamin A (retinyl acetate), 5,233 IU; vitamin D (cholecalciferol), 1,000 IU; vitamin E (dl-tocopheryl acetate), 1.25 IU; menadione sodium bisulfite, 2,5 mg; thiamine-hydrochloride, 1.5 mg; riboflavin, 3 mg; niacin, 12.5 mg; d-pantothenic acid, 5 mg; pyridoxine hydrochloride, 2.5 mg; vitamin B12, 0.0075 mg; folic acid, 0.25 mg; choline (choline chloride), 125 mg; Mn (MnSO4-H2O), 50 mg; Fe (FeSO4–7H2O), 30 mg; Zn (ZnO), 30 mg; Cu (CuSO4–5H2O), 5 mg; Co (CoCl2–6H2O), 0,1 mg; I (KI), 0,4 mg; Se (Na2SeO3), 0,15 mg. 3Calculated values (Jurgens, 1996). View Large Samples and Data Collection Performance and egg quality parameters were determined by the cage. Feed intake was measured weekly. The egg count and egg weight were recorded daily and feed conversion efficiency [feed consumed, g/(egg production, % x egg weight, g)] was calculated weekly. At the end of the study, egg quality parameters (eggshell thickness, eggshell breaking strength, and Haugh unit) were measured on two eggs collected randomly from each cage. The shell thickness was a mean value of measurements at three locations on the egg (air cell, equator, and sharp end) by using a dial pipe gage. The Haugh unit was calculated using following formula: Haugh unit = 100 × log (H + 7.57 − 1.7 × W0.37), where H = albumen height (mm) and W = egg weight (g) (Eisen et al., 1962) after determining albumen height by using a micrometer (TLM-N1010, Saginomiya, Tokyo, Japan) and egg weight. At the end of the study (wk 28), blood samples were collected from axillary vein of 10 randomly chosen birds from each treatment group. Samples were put into additive-free vacutainers and then centrifuged at 5,000 × g at 4°C for 10 min. Sera were transferred to microfuge tubes, kept on the ice, and protected from light to avoid oxidation during sampling. Sera were stored at −75°C for determination of Cr, aspartate-aminotransferase (AST), alanine-aminotransferase (ALT), γ-glutamyl transferase (GGT), lactate dehydrogenase (LDH), creatine kinase (CK), glucose, cholesterol, protein, albumin, globulin, and creatine concentrations. Laboratory Analyses Feed samples were analyzed for crude protein (#988.05), ether extract (#932.06), crude fiber (#962.09), crude ash (#936.07), Ca (#968.08), and P (#965.17) in triplicates (AOAC, 1990). The metabolizable energy, lysine, methionine, and cysteine contents were calculated based on their tabular values listed for the feed ingredients (Jurgens, 1996). For determination of Cr and electrolyte mineral concentrations, 0.3 g feed and egg yolk as well as 0.5 mL serum samples were first digested with 5 mL concentrated HNO3 in a Microwave Digestion System (Berghoff, Eningen, Germany) for 30 min. The specimens were subjected to graphite furnace atomic absorption spectrophotometer (AAS, Perkin-Elmer, Analyst 800, Norwalk, CT). Serum glucose, cholesterol, protein, albumin, globulin, and creatine as well as AST, ALT, GGT, LDH, and CK concentrations were measured using Biochemistry Test 9 kits (Samsung LABGEO, IVR-PT05, Seoul, Korea) on auto-analyzer (Samsung LABGEOPT10). Statistical Analyses In a 2 × 3 factorially arranged treatment within a completely randomized design experiment, data were analyzed by 2-way ANOVA using the PROC GLM procedure (SAS, 2002). The linear model to test the effect of treatments on response variables was as follows: Yijk = μ + ETi + DSj + (ETxDS)ij + eijk, where Y = response variable, μ = population mean, ET = environmental temperature, DS = dietary supplement, and e = residual error [N (σ, μ; 0, 1)]. Statistical contrasts were constructed in the model to attain the effects of supplemental Cr (C vs. Ave = CrPic and CrHis) and Cr source (CrPic vs. CrHis) and statistical significance was considered at P ≤ 0.05. RESULTS AND DISCUSSION Performance and Egg Quality The adverse effects of HS on performance variables were evident, as reflected by decreased feed intake (−17.4%), egg production (−17.4%), and egg weight (−7.0%) and worsened feed conversion (+8.4) (P < 0.0001 for all; Table 2). Overall, supplemental Cr increased feed intake (P < 0.0006), egg production (P < 0.001), and egg weight (P < 0.0001), regardless of the Cr chelate type, but did not affect feed conversion. In agreement with previous experiments involving various poultry species (Sahin et al., 2002, 2004, 2005; Orhan et al., 2012), HS depressed performance variables (Table 2). Table 2. The effect of different chromium chelates on performance and egg quality parameters in laying hens reared under heat stress. Groups1  Response variables2  Environment  Supplement  Feed  Egg  Egg  FCR  Eggshell  Eggshell  Eggshell  Haugh  (E)  (S)  intake  production  weight    weight  thickness  strength  units      (g)  (%)  (g)    (g)  (mm)  (kg/cm2)  (%)  Thermoneutral    128.5  90.7  62.7  2.26  5.80  0.38  4.59  80.7  Heat stress    106.2  74.9  58.3  2.45  5.48  0.36  4.50  75.4      1.8  1.4  0.2  0.03  0.07  <0.001  0.01  0.04    Control  112.3b  78.7b  59.7c  2.40  5.52b  0.36  4.52b  76.1c    CrPic  117.9a  83.5a  60.7b  2.34  5.63a,b  0.37  4.56a  78.4b    CrHis  122.0a  86.4a  61.0a  2.32  5.78a  0.37  4.57a  79.8a      3.2  2.4  0.6  0.05  0.1  0.01  0.01  0.6    Control  128.6  90.4  62.8  2.27  5.68  0.38  4.58  80.1  Thermoneutral  CrPic  125.8  89.7  62.4  2.25  5.80  0.38  4.59  80.7    CrHis  131.8  92.4  62.9  2.27  5.94  0.38  4.61  81.4    Control  96.1  67.0  56.7  2.54  5.37  0.35  4.46  72.1  Heat stress  CrPic  109.1  76.6  58.7  2.44  5.45  0.36  4.52  75.9    CrHis  113.3  81.2  59.4  2.37  5.63  0.36  4.54  78.3      2.3  2.0  0.2  0.05  0.11  0.01  0.02  0.52  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.0001  0.0001  0.0009  0.0001  0.0001  0.0001  S    0.0006  0.001  0.0001  0.30  0.08  0.81  0.01  0.0001  E × S    0.003  0.01  0.0001  0.35  0.98  0.42  0.30  0.0001  Groups1  Response variables2  Environment  Supplement  Feed  Egg  Egg  FCR  Eggshell  Eggshell  Eggshell  Haugh  (E)  (S)  intake  production  weight    weight  thickness  strength  units      (g)  (%)  (g)    (g)  (mm)  (kg/cm2)  (%)  Thermoneutral    128.5  90.7  62.7  2.26  5.80  0.38  4.59  80.7  Heat stress    106.2  74.9  58.3  2.45  5.48  0.36  4.50  75.4      1.8  1.4  0.2  0.03  0.07  <0.001  0.01  0.04    Control  112.3b  78.7b  59.7c  2.40  5.52b  0.36  4.52b  76.1c    CrPic  117.9a  83.5a  60.7b  2.34  5.63a,b  0.37  4.56a  78.4b    CrHis  122.0a  86.4a  61.0a  2.32  5.78a  0.37  4.57a  79.8a      3.2  2.4  0.6  0.05  0.1  0.01  0.01  0.6    Control  128.6  90.4  62.8  2.27  5.68  0.38  4.58  80.1  Thermoneutral  CrPic  125.8  89.7  62.4  2.25  5.80  0.38  4.59  80.7    CrHis  131.8  92.4  62.9  2.27  5.94  0.38  4.61  81.4    Control  96.1  67.0  56.7  2.54  5.37  0.35  4.46  72.1  Heat stress  CrPic  109.1  76.6  58.7  2.44  5.45  0.36  4.52  75.9    CrHis  113.3  81.2  59.4  2.37  5.63  0.36  4.54  78.3      2.3  2.0  0.2  0.05  0.11  0.01  0.02  0.52  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.0001  0.0001  0.0009  0.0001  0.0001  0.0001  S    0.0006  0.001  0.0001  0.30  0.08  0.81  0.01  0.0001  E × S    0.003  0.01  0.0001  0.35  0.98  0.42  0.30  0.0001  1Thermoneutral = environmental temperature of 22 ± 2°C for 24 h/d; Heat Stress = environmental temperature of 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. Control = the basal diet not supplemented with Cr; CrPic = the basal diet supplemented with 1.600 mg of CrPic (12.43% elemental Cr) per kilogram diet; CrHis = the basal diet supplemented with 0.788 mg of CrHis (25.22% elemental Cr). 2Data are ± SEM. Data with uncommon superscripts in rows (main effect of supplements) differ (P < 0.05). FCR = feed conversion ratio [g feed consumed per g egg mass (egg number × egg weight)]. View Large Positive effects of supplemental Cr on performance variables were more notable under the HS condition than under the TN condition. The increases (about 0.5 to 2.5%) in performance variables for hens supplemented with CrHis and CrPic were similar under TN the condition. However, increases in feed intake (13.5 vs. 17.9%; P < 0.003), egg production (14.3 vs. 21.2%, P < 0.01), and egg weight (3.5 vs. 4.8%, P < 0.001) for hens supplemented with CrHis were higher than those for hens supplemented with CrPic under the HS condition. Alterations in feed conversion in response dietary treatments under both conditions were insignificant. Previous studies also showed that improvement in performance parameters was significant when Cr supplemented in organic form (Sahin et al., 2001, 2002, 2004; 2009; Rao et al., 2012). This superiority is due the fact that Cr in the organic complex is more bioavailable than Cr in inorganic forms (Piva et al., 2003; Suksombat and Kanchanatawee, 2005; Hayirli, 2005; Sahin et al., 2010). Both Cr sources alleviated performance parameters under the HS condition, being CrHis more effective than CrPic (Table 2). Chromium-histidinate is a stable and histidinate allows the greater absorbability and bioavailability (Anderson et al., 2004). Heat stress deteriorated eggshell quality indices (eggshell weight, −5.5%, P < 0.0009; eggshell thickness, −5.3%, P < 0.0001; and eggshell strength, −2.0%, P < 0.0001) and decreased Haugh unit (−6.6%, P < 0.0001) (Table 2). Supplemental Cr, especially CrHis, increased eggshell weight, eggshell strength, and Haugh unit (Table 2). However, the positive effect of supplemental Cr form on eggshell quality parameters did not differ depending on the environmental condition. Studies investigating the effect of supplemental Cr on egg quality parameters are inconsistent. Torki et al. (2014) reported no alteration in egg quality parameters. The Cr supplementation improved eggshell stiffness and Haugh unit (Table 2). The mechanism by which Cr affect Ca metabolism in eggshell formation is not elucidated. The overall effect of supplemental Cr on eggshell quality parameters was independent of environmental temperature in the present experiment and elsewhere (Karimi et al., 2015). However, several studies reported improvement in eggshell quality in quails (Sahin et al., 2001, 2002) and hens (Torki et al., 2014) reared under the HS condition and quails (Yildiz et al., 2004; Senobar et al., 2012), hens (Lien et al., 1999; Uyanik et al., 2002; Ma et al., 2014), and turkeys (Biswas et al., 2015) reared under the TN condition. These inconsistent reports could be related to the form and level of supplemental Cr. Chromium and Electrolyte Concentrations Hens housed under the HS condition had lower Cr concentrations in serum and egg yolk (P < 0.0001 for both) as well as electrolyte minerals’ concentrations in serum (Na, P < 0.002; K, P < 0.01; and Cl, P < 0.06) than those reared under the TN condition (Table 3). Supplemental Cr increased serum and egg yolk Cr concentrations (P < 0.0001 for both), but did not alter concentrations of electrolyte minerals in serum. The effect of Cr as CrHis on tissue Cr concentrations was more notable than Cr as CrPic (P < 0.05). There was no environmental temperature by dietary supplement interaction effects on tissue Cr and serum electrolyte levels. Table 3. The effect of different chromium chelates on tissue chromium and serum electrolyte concentrations in laying hens reared under heat stress. Groups1  Response variables2  Environment  Supplement  Serum Cr  Yolk Cr  Serum Na  Serum K  Serum Cl  (E)  (S)  (mg/L)  (mg/kg)  (mmol/L)  (mmol/L)  (mmol/L)  Thermoneutral    1.98  473  148  4.70  108  Heat stress    1.13  313  145  4.39  106      0.07  6  1  0.08  1    Control  1.28c  359c  146  4.46  107    CrPic  1.56b  398b  147  4.50  107    CrHis  1.82a  421a  147  4.68  107      0.12  19  1  0.11  1    Control  1.75  443  148  4.58  108  Thermoneutral  CrPic  1.95  478  149  4.63  108    CrHis  2.23  498  148  4.89  108    Control  0.82  276  144  4.34  105  Heat stress  CrPic  1.18  317  146  4.36  107    CrHis  1.40  344  146  4.46  107      0.10  6  1  0.15  1  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.002  0.01  0.06  S    0.0001  0.0001  0.50  0.32  0.74  E × S    0.59  0.77  0.53  0.79  0.30  Groups1  Response variables2  Environment  Supplement  Serum Cr  Yolk Cr  Serum Na  Serum K  Serum Cl  (E)  (S)  (mg/L)  (mg/kg)  (mmol/L)  (mmol/L)  (mmol/L)  Thermoneutral    1.98  473  148  4.70  108  Heat stress    1.13  313  145  4.39  106      0.07  6  1  0.08  1    Control  1.28c  359c  146  4.46  107    CrPic  1.56b  398b  147  4.50  107    CrHis  1.82a  421a  147  4.68  107      0.12  19  1  0.11  1    Control  1.75  443  148  4.58  108  Thermoneutral  CrPic  1.95  478  149  4.63  108    CrHis  2.23  498  148  4.89  108    Control  0.82  276  144  4.34  105  Heat stress  CrPic  1.18  317  146  4.36  107    CrHis  1.40  344  146  4.46  107      0.10  6  1  0.15  1  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.002  0.01  0.06  S    0.0001  0.0001  0.50  0.32  0.74  E × S    0.59  0.77  0.53  0.79  0.30  1Thermoneutral = environmental temperature of 22 ± 2°C for 24 h/d; Heat Stress = environmental temperature of 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. Control = the basal diet not supplemented with Cr; CrPic = the basal diet supplemented with 1.600 mg of CrPic (12.43% elemental Cr) per kilogram diet; CrHis = the basal diet supplemented with 0.788 mg of CrHis (25.22% elemental Cr). 2Data are ± SEM. Data with uncommon superscripts in rows (main effect of supplements) differ (P < 0.05). View Large Chromium absorption takes place in the jejunum. It appears that Cr distributes to yolk easier than albumen in all poultry species (Nisianakis et al., 2009). Absorbability of Cr in inorganic form is inferior to Cr with organic complex (Amatya et al., 2005). However, Piva et al (2003) showed that Cr deposition in egg yolk (0.48 mg/kg) was not different when hens were fed CrCl3, Cr-yeast, and Cr-aminoniacinate. Resulting from decreased feed intake, HS may additionally aggravate Cr mobilization from tissues (Geraert et al., 1996; Sahin et al., 2002), which could lead to its deficiency (Doisy, 1978; Hayirli, 2005). Chromium requirement increases in case of exposure to stressors (Khan et al., 2014). Indeed, hens exposed to HS had lower serum Cr concentration than those reared under the TN condition (Table 3). Supplemental Cr compensates body Cr reserves and alleviates the adverse effects of HS in quails (Sahin et al., 2005, 2010), broilers (Lien et al., 1999), hens (Lien et al., 1996, 2004, 2008), and turkeys (Steele and Rosebrough, 1981). Restoration of Cr reserves is associated with increases in serum insulin, glucose, and cholesterol concentrations in overcrowded hens (Mirfendereski and Jahanian, 2015) and heat-distressed quails (Sahin et al., 2001, 2002). In the present study, serum and egg yolk Cr concentrations increased by supplemental Cr in laying hens reared under the HS conditions. The increases in serum and egg yolk Cr concentration were in agreement with the literature (Uyanik et al., 2002; Piva et al., 2003; Sahin et al., 2004; Ma et al., 2014). Serum Metabolites and Enzymes Exposure to HS caused increases in serum glucose (13.3%, P < 0.0001), cholesterol (34.5%, P < 0.0001), and creatine (8.8%, P < 0.007) concentrations, but did not affect concentrations of serum total protein and its fractions (Table 4). Supplemental Cr decreased serum glucose (P < 0.0001) and cholesterol (P < 0.03) concentrations. Decreases in serum glucose and cholesterol concentrations were greater for hens supplemented with CrHis than those for hens supplemented with CrPic (P < 0.05). There were no environmental temperature by dietary supplement interaction effects on serum metabolic profile, except for serum cholesterol concentration. Supplemental Cr, especially Cr as CrHis, tended to decrease serum cholesterol concentration under the HS condition to a greater extent than under the TN condition, as compared to Cr as CrPic (P < 0.06). Table 4. The effect of different chromium chelates on metabolic parameters in laying hens reared under heat stress. Groups1  Response variables2  Environment  Supplement  Glucose  Cholesterol  Protein  Albumin  Globulin  Creatine  (E)  (S)  (mg/dL)  (mg/dL)  (g/dL)  (g/dL)  (g/dL)  (mg/dL)  Thermoneutral    188  113  5.62  2.01  3.30  0.34  Heat stress    213  152  5.85  2.03  3.35  0.37      2  6  0.15  0.02  0.10  0.01    Control  209a  146a  5.63  2.02  3.33  0.35    CrPic  200b  130a,b  5.75  2.03  3.28  0.36    CrHis  193c  123b  5.84  2.01  3.36  0.35      4  8  0.18  0.02  0.13  0.01    Control  195  114  5.57  2.02  3.38  0.33  Thermoneutral  CrPic  188  114  5.67  2.01  3.23  0.35    CrHis  183  111  5.63  2.00  3.28  0.34    Control  223  178  5.68  2.02  3.28  0.37  Heat stress  CrPic  212  145  5.83  2.04  3.32  0.38    CrHis  204  134  6.04  2.02  3.44  0.37      3  9  0.30  0.03  0.18  0.02  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.30  0.58  0.74  0.007  S    0.0001  0.03  0.73  0.90  0.90  0.59  E × S    0.54  0.06  0.83  0.86  0.77  0.87  Groups1  Response variables2  Environment  Supplement  Glucose  Cholesterol  Protein  Albumin  Globulin  Creatine  (E)  (S)  (mg/dL)  (mg/dL)  (g/dL)  (g/dL)  (g/dL)  (mg/dL)  Thermoneutral    188  113  5.62  2.01  3.30  0.34  Heat stress    213  152  5.85  2.03  3.35  0.37      2  6  0.15  0.02  0.10  0.01    Control  209a  146a  5.63  2.02  3.33  0.35    CrPic  200b  130a,b  5.75  2.03  3.28  0.36    CrHis  193c  123b  5.84  2.01  3.36  0.35      4  8  0.18  0.02  0.13  0.01    Control  195  114  5.57  2.02  3.38  0.33  Thermoneutral  CrPic  188  114  5.67  2.01  3.23  0.35    CrHis  183  111  5.63  2.00  3.28  0.34    Control  223  178  5.68  2.02  3.28  0.37  Heat stress  CrPic  212  145  5.83  2.04  3.32  0.38    CrHis  204  134  6.04  2.02  3.44  0.37      3  9  0.30  0.03  0.18  0.02  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.30  0.58  0.74  0.007  S    0.0001  0.03  0.73  0.90  0.90  0.59  E × S    0.54  0.06  0.83  0.86  0.77  0.87  1Thermoneutral = environmental temperature of 22 ± 2°C for 24 h/d; Heat Stress = environmental temperature of 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. Control = the basal diet not supplemented with Cr; CrPic = the basal diet supplemented with 1.600 mg of CrPic (12.43% elemental Cr) per kilogram diet; CrHis = the basal diet supplemented with 0.788 mg of CrHis (25.22% elemental Cr). 2Data are ± SEM. Data with uncommon superscripts in rows (main effect of supplements) differ (P < 0.05). View Large Many studies reveal that Cr increases glucose removal from blood, reduces egg yolk cholesterol, improves eggshell quality, and enhances immunity and disease resistance (Lindemann et al., 2009). Chromium improves insulin action, leading to improvements in protein and lipid metabolism, as reflected by decreased nonesterified fatty acids, fastened serum triglyceride removal, and uptake of glucose for lipogenesis in the liver (Lien et al., 1999). These are accompanied by increasing protein accretion (Ahmed et al., 2005). Growth promoting effects of supplemental Cr is related to upregulation of expressions of skeletal muscle protein (Zha et al., 2009; Pan et al., 2013). Catabolic profile in a stress condition is characterized by a decrease in plasma protein concentration and increases in plasma glucose and cholesterol concentrations (Donkoh, 1989). Concentrations of glucose and cholesterol concentrations (Table 4) as well as enzymes (Table 5) were higher for layers reared under the HS environment than those reared under the TN environment (Table 4). Chromium plays a role in the regulation of metabolism (Prasad and Gowda, 2005) through acting as an immunostimulating substance (Sohn et al., 2000; Wenk, 2000) and exerting antioxidant effects (Sahin et al., 2005, 2010; de Araujo et al., 2007), which is critical under stressing conditions in poultry (Kim et al., 1996; Lien et al., 1996). Table 5. The effect of different chromium chelates on serum enzymes in laying hens reared under heat stress. Groups1  Response variables2  Environment  Supplement  AST (U/L)  ALT (U/L)  GGT (U/L)  LDH (IU/L)  CK (U/L)  (E)  (S)            Thermoneutral    200  1.33  24.8  1591  2438  Heat stress    203  1.37  25.8  2090  2566      5  0.10  0.5  52  60    Control  201  1.40  24.6  2015a  2511    CrPic  201  1.30  25.7  1796b  2496    CrHis  203  1.35  25.8  1709b  2499      6  0.11  0.5  81  75    Control  201  1.40  24.1  1766  2462  Thermoneutral  CrPic  198  1.30  25.5  1540  2409    CrHis  202  1.30  24.9  1466  2442    Control  202  1.40  25.0  2264  2559  Heat stress  CrPic  204  1.30  25.8  2053  2584    CrHis  204  1.40  26.7  1952  2556      8  0.17  0.8  79  106  ANOVA  ————————————————————— P ————————————————————–  E    0.64  0.81  0.12  0.0001  0.15  S    0.98  0.84  0.22  0.002  0.99  E × S    0.95  0.94  0.62  0.99  0.93  Groups1  Response variables2  Environment  Supplement  AST (U/L)  ALT (U/L)  GGT (U/L)  LDH (IU/L)  CK (U/L)  (E)  (S)            Thermoneutral    200  1.33  24.8  1591  2438  Heat stress    203  1.37  25.8  2090  2566      5  0.10  0.5  52  60    Control  201  1.40  24.6  2015a  2511    CrPic  201  1.30  25.7  1796b  2496    CrHis  203  1.35  25.8  1709b  2499      6  0.11  0.5  81  75    Control  201  1.40  24.1  1766  2462  Thermoneutral  CrPic  198  1.30  25.5  1540  2409    CrHis  202  1.30  24.9  1466  2442    Control  202  1.40  25.0  2264  2559  Heat stress  CrPic  204  1.30  25.8  2053  2584    CrHis  204  1.40  26.7  1952  2556      8  0.17  0.8  79  106  ANOVA  ————————————————————— P ————————————————————–  E    0.64  0.81  0.12  0.0001  0.15  S    0.98  0.84  0.22  0.002  0.99  E × S    0.95  0.94  0.62  0.99  0.93  1Thermoneutral = environmental temperature of 22 ± 2°C for 24 h/d; Heat Stress = environmental temperature of 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. Control = the basal diet not supplemented with Cr; CrPic = the basal diet supplemented with 1.600 mg of CrPic (12.43% elemental Cr) per kilogram diet; CrHis = the basal diet supplemented with 0.788 mg of CrHis (25.22% elemental Cr). 2Data are ± SEM. Data with uncommon superscripts in rows (main effect of supplements) differ (P < 0.05). AST = aspartate-aminotransferase; ALT = alanine-amino transferase; GGT = γ-glutamyl transferase; LDH = lactate dehydrogenase; CK = creatine kinase. View Large Among serum enzymes, only LDH was responsive to environmental temperature (Table 5). It increased by 1.3-fold due to HS (P < 0.0001). In response to Cr supplementation serum LDH concentration decreased (P < 0.002) and other enzymes remained unchanged. The decrease in serum LDH concentrations between hens supplemented with CrHis and CrPic was similar. The effects of supplemental Cr and Cr form on serum enzymes did not differ depending on environmental temperature. To overcome oxidative stress under the HS condition, Onderci et al. (2005) and Sahin et al. (2004) showed that CrPic decreased malondialdehyde levels in serum and the liver, thigh muscle and serum cholesterol, and serum glucose concentrations in quails. Hens are responsive to supplemental Cr in the regulation of the insulin action (Viera and Davis-Mcgibony, 2008). Antistress and metabolic modifier effects of supplemental Cr through modulating insulin action and mineral utilization were shown in quails, as reflected by improvements in laying performance and eggshell quality and decreased serum corticosterone, glucose, and cholesterol concentrations, and increase in serum protein concentration (Sahin et al., 2001, 2002; Torki et al., 2014). Both Cr sources decreased serum glucose and cholesterol concentrations and liver LDH levels. Similarly, Lien et al. (1996) stated that Cr supplementation to laying hen diets decreased serum glucose and cholesterol concentrations. Previous studies also showed a cholesterol-lowering effect in the serum of broilers (Kim et al., 1996) and quails (Sahin et al., 2004; Akdemir et al., 2015) supplemented with CrHis. In hens at early laying period supplemented with Cr (400 ug/kg) and L-carnitine (100 mg/kg), there was a considerable reduction in hepatic triglyceride, egg yolk cholesterol, and abdominal fat percentage (Du et al., 2005). In other studies, despite a decrease in serum cholesterol and an increase in HDL concentrations, eggshell quality remained unchanged in hens supplemented with CrPic (Lien et al., 2004, 2008). In conclusion, supplemental Cr as CrHis and CrPic alleviated the adverse effects of HS on the performance and metabolic profile of laying hens. Chromium in either could be added into the diet in order to overcome heat stress-related depression in performance and metabolic profile. 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Effects of the supplemental chromium form on performance and metabolic profile in laying hens exposed to heat stress

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Abstract

Abstract This experiment was conducted to compare the effect of the supplemental chromium (Cr) form on performance, egg quality, and metabolic profile in laying hens exposed to heat stress (HS). Laying hens (n = 1800; 16-wk-old; Lohmann LSL-Lite) were kept in cages in temperature-controlled rooms at either 22 ± 2°C for 24 h/d (thermoneutral, TN) or 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. Hens reared under both environmental conditions were fed 1 of 3 diets: a basal diet and the basal diet supplemented with either 1.600 mg of chromium-picolinate (CrPic, 12.43% Cr) or 0.788 mg of chromium-histidinate (CrHis, 25.22% Cr) per kg of diet, delivering 200 μg elemental Cr per kg diet. Data were analyzed by 2-way ANOVA. Exposure to HS caused decreases in feed intake (P < 0.0001), egg production (P < 0.0001), egg weight (P < 0.0001), eggshell weight (P < 0.0009), eggshell thickness (P < 0.0001), eggshell strength (P < 0.0001), and Haugh unit (P < 0.0001), deterioration in feed conversion ratio (P < 0.0001), increases in serum glucose and cholesterol concentrations (P < 0.0001 for both), decreases in serum and egg yolk Cr concentrations (P < 0.0001 for both), and decreases in serum Na (P < 0.002) and K (P < 0.01) concentrations. Both Cr sources were equally effective in alleviating performance variables under the HS condition. However, neither Cr sources alleviated deteriorations in egg quality parameters and serum electrolytes. Both Cr sources decreased serum glucose and cholesterol concentrations and increased serum and egg yolk Cr concentrations under the HS condition. In conclusion, HS adversely affected laying performance, egg quality, and metabolic profile. Both CrPic and CrHis partially alleviated the adverse effect of HS on these parameters. Inclusion of either Cr source could be a part of nutritional management strategies to overcome the adverse effects of HS performance and metabolic profile in laying hens. INTRODUCTION High ambient temperature is one of the detrimental environmental factors in poultry farms (Freeman, 1987). Heat stress (HS) is a serious welfare issue and causes economic losses due to poor performance and compromised immune and health statuses (Howlider and Rose, 1987; Sahin and Kucuk, 2003; Mashaly et al., 2004). Increased excretion and reduced bioavailability of nutrients during HS may contribute to suppressed performance and immune potency (Belay and Teeter, 1996). Consequent development of oxidative stress (de Araujo et al., 2007; Sahin et al., 2009, 2010) may be linked to decreased tissue reserves of minerals (e.g., Se, Cu, Mn, Cr) (Khan et al., 2014) and vitamins (e.g., vitamins A and E) (Sahin and Kucuk, 2003) that are integral components of the antioxidant defense system. Chromium (Cr) is involved in metabolic pathways of nutrients (e.g., carbohydrate, lipid, protein, and nucleic acid) through potentiating the insulin action (Anderson, 1987; Cupo and Donaldson, 1987; Hayirli, 2005). It contributes to anabolic profile (Lien et al., 1996, 1999, 2004) and alleviates oxidative stress and lipid peroxidation (Samanta et al., 2008; Sahin et al., 2009, 2010) in laying hens exposed to HS. Chromium chelates with organic compounds have lower toxicity and higher bioavailability than Cr in inorganic forms (Kim et al., 1996; Piva et al., 2003). Various Cr chelates (yeast, picolinate (CrPic), nicotinate, propionate, and methionine) are commercially available. The Cr form is important, particularly in case of exposure to stressors, to compensate deficiency and achieve biological response (Sahin et al., 2002; Piva et al., 2003). Chromium-histidinate (CrHis) is a newly developed chelate and has not been tested in laying hens. This experiment was therefore conducted to compare the effects of different Cr forms (CrHis vs. CrPic) on performance, egg quality, and metabolic profile of laying hens exposed to HS. MATERIALS AND METHODS Animals, Treatments, and Management The experiment involving laying hens (n = 1800, 16 wks old, Lohmann LSL-Lite) was conducted at a commercial farm (Umut Tavukçuluk A.Ş., Elazig, Turkey). The birds were kept in cages (48 cm × 45 cm × 45 cm, width × depth × height) (4 birds/cage, at a density of 540 cm2/hen, providing 12 cm feeder space per hen) in temperature-controlled rooms at either 22 ± 2°C for 24 h/d (thermoneutral, TN) or 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. The basal diet was formulated to meet nutrient requirements (NRC, 1994; Table 1). The hens in both TN and HS conditions were either not supplemented with Cr (C) or supplemented with 1.600 mg of CrPic (12.43% elemental Cr, Nutrition 21, NY) or 0.788 mg of CrHis (25.22% elemental Cr, Nutrition 21, NY) per kilogram diet in mash form, delivering 200 μg Cr per kg diet. In order to avoid confounding effects of an organic portion of the Cr-chelates, the diets C, CrPic, and CrHis were added with 1.401 mg picolinic acid+0.589 mg histidine, 0.589 mg histidine, and 1.401 mg picolinic acid per kilogram, respectively. Each treatment was replicated in 75 cages. Chickens were managed in accordance with animal welfare regulations at the Republic of Turkey Ministry of Food, Agriculture and Livestock. Feed and fresh water were offered ad libitum during the experimental period. Birds were exposed to a light: dark cycle of 16 h:8 h per day. Table 1. Ingredients and nutrient composition of the basal diet.1 Ingredient  %  Maize  54.85  Soybean meal  28.26  Corn oil  4.66  Salt  0.33  DL-methionine  0.22  Limestone  9.30  Dicalcium phosphate  1.80  Vitamin-mineral premix2  0.35  Nutrient composition (g/kg, dry matter basis)    Metabolisable energy, kcal/kg3  2800  Crude protein  177.6  Calcium  40.4  Phosphorus  6.3  Methionine3  4.0  Lysine3  11.1  Ingredient  %  Maize  54.85  Soybean meal  28.26  Corn oil  4.66  Salt  0.33  DL-methionine  0.22  Limestone  9.30  Dicalcium phosphate  1.80  Vitamin-mineral premix2  0.35  Nutrient composition (g/kg, dry matter basis)    Metabolisable energy, kcal/kg3  2800  Crude protein  177.6  Calcium  40.4  Phosphorus  6.3  Methionine3  4.0  Lysine3  11.1  1Experimental diets were constructed through reconstituting the premix in the basal diet at the expense of CaCO3 to contain histidinate (0.236 g) plus picolinate (0.560 g), CrPic (0.640 g) plus histidinate (0.236 g), and CrHis (0.315 g) plus picolinate (0.560 g) in the diets C, CrPic, and CrHis, respectively. The basal diet contained 0.17 ± 0.08 mg Cr and those supplemented with CrPic and CrHis contained 2.09 ± 0.13 mg Cr per kg diets (mean ± SD). 2Per kilogram contained: vitamin A (retinyl acetate), 5,233 IU; vitamin D (cholecalciferol), 1,000 IU; vitamin E (dl-tocopheryl acetate), 1.25 IU; menadione sodium bisulfite, 2,5 mg; thiamine-hydrochloride, 1.5 mg; riboflavin, 3 mg; niacin, 12.5 mg; d-pantothenic acid, 5 mg; pyridoxine hydrochloride, 2.5 mg; vitamin B12, 0.0075 mg; folic acid, 0.25 mg; choline (choline chloride), 125 mg; Mn (MnSO4-H2O), 50 mg; Fe (FeSO4–7H2O), 30 mg; Zn (ZnO), 30 mg; Cu (CuSO4–5H2O), 5 mg; Co (CoCl2–6H2O), 0,1 mg; I (KI), 0,4 mg; Se (Na2SeO3), 0,15 mg. 3Calculated values (Jurgens, 1996). View Large Samples and Data Collection Performance and egg quality parameters were determined by the cage. Feed intake was measured weekly. The egg count and egg weight were recorded daily and feed conversion efficiency [feed consumed, g/(egg production, % x egg weight, g)] was calculated weekly. At the end of the study, egg quality parameters (eggshell thickness, eggshell breaking strength, and Haugh unit) were measured on two eggs collected randomly from each cage. The shell thickness was a mean value of measurements at three locations on the egg (air cell, equator, and sharp end) by using a dial pipe gage. The Haugh unit was calculated using following formula: Haugh unit = 100 × log (H + 7.57 − 1.7 × W0.37), where H = albumen height (mm) and W = egg weight (g) (Eisen et al., 1962) after determining albumen height by using a micrometer (TLM-N1010, Saginomiya, Tokyo, Japan) and egg weight. At the end of the study (wk 28), blood samples were collected from axillary vein of 10 randomly chosen birds from each treatment group. Samples were put into additive-free vacutainers and then centrifuged at 5,000 × g at 4°C for 10 min. Sera were transferred to microfuge tubes, kept on the ice, and protected from light to avoid oxidation during sampling. Sera were stored at −75°C for determination of Cr, aspartate-aminotransferase (AST), alanine-aminotransferase (ALT), γ-glutamyl transferase (GGT), lactate dehydrogenase (LDH), creatine kinase (CK), glucose, cholesterol, protein, albumin, globulin, and creatine concentrations. Laboratory Analyses Feed samples were analyzed for crude protein (#988.05), ether extract (#932.06), crude fiber (#962.09), crude ash (#936.07), Ca (#968.08), and P (#965.17) in triplicates (AOAC, 1990). The metabolizable energy, lysine, methionine, and cysteine contents were calculated based on their tabular values listed for the feed ingredients (Jurgens, 1996). For determination of Cr and electrolyte mineral concentrations, 0.3 g feed and egg yolk as well as 0.5 mL serum samples were first digested with 5 mL concentrated HNO3 in a Microwave Digestion System (Berghoff, Eningen, Germany) for 30 min. The specimens were subjected to graphite furnace atomic absorption spectrophotometer (AAS, Perkin-Elmer, Analyst 800, Norwalk, CT). Serum glucose, cholesterol, protein, albumin, globulin, and creatine as well as AST, ALT, GGT, LDH, and CK concentrations were measured using Biochemistry Test 9 kits (Samsung LABGEO, IVR-PT05, Seoul, Korea) on auto-analyzer (Samsung LABGEOPT10). Statistical Analyses In a 2 × 3 factorially arranged treatment within a completely randomized design experiment, data were analyzed by 2-way ANOVA using the PROC GLM procedure (SAS, 2002). The linear model to test the effect of treatments on response variables was as follows: Yijk = μ + ETi + DSj + (ETxDS)ij + eijk, where Y = response variable, μ = population mean, ET = environmental temperature, DS = dietary supplement, and e = residual error [N (σ, μ; 0, 1)]. Statistical contrasts were constructed in the model to attain the effects of supplemental Cr (C vs. Ave = CrPic and CrHis) and Cr source (CrPic vs. CrHis) and statistical significance was considered at P ≤ 0.05. RESULTS AND DISCUSSION Performance and Egg Quality The adverse effects of HS on performance variables were evident, as reflected by decreased feed intake (−17.4%), egg production (−17.4%), and egg weight (−7.0%) and worsened feed conversion (+8.4) (P < 0.0001 for all; Table 2). Overall, supplemental Cr increased feed intake (P < 0.0006), egg production (P < 0.001), and egg weight (P < 0.0001), regardless of the Cr chelate type, but did not affect feed conversion. In agreement with previous experiments involving various poultry species (Sahin et al., 2002, 2004, 2005; Orhan et al., 2012), HS depressed performance variables (Table 2). Table 2. The effect of different chromium chelates on performance and egg quality parameters in laying hens reared under heat stress. Groups1  Response variables2  Environment  Supplement  Feed  Egg  Egg  FCR  Eggshell  Eggshell  Eggshell  Haugh  (E)  (S)  intake  production  weight    weight  thickness  strength  units      (g)  (%)  (g)    (g)  (mm)  (kg/cm2)  (%)  Thermoneutral    128.5  90.7  62.7  2.26  5.80  0.38  4.59  80.7  Heat stress    106.2  74.9  58.3  2.45  5.48  0.36  4.50  75.4      1.8  1.4  0.2  0.03  0.07  <0.001  0.01  0.04    Control  112.3b  78.7b  59.7c  2.40  5.52b  0.36  4.52b  76.1c    CrPic  117.9a  83.5a  60.7b  2.34  5.63a,b  0.37  4.56a  78.4b    CrHis  122.0a  86.4a  61.0a  2.32  5.78a  0.37  4.57a  79.8a      3.2  2.4  0.6  0.05  0.1  0.01  0.01  0.6    Control  128.6  90.4  62.8  2.27  5.68  0.38  4.58  80.1  Thermoneutral  CrPic  125.8  89.7  62.4  2.25  5.80  0.38  4.59  80.7    CrHis  131.8  92.4  62.9  2.27  5.94  0.38  4.61  81.4    Control  96.1  67.0  56.7  2.54  5.37  0.35  4.46  72.1  Heat stress  CrPic  109.1  76.6  58.7  2.44  5.45  0.36  4.52  75.9    CrHis  113.3  81.2  59.4  2.37  5.63  0.36  4.54  78.3      2.3  2.0  0.2  0.05  0.11  0.01  0.02  0.52  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.0001  0.0001  0.0009  0.0001  0.0001  0.0001  S    0.0006  0.001  0.0001  0.30  0.08  0.81  0.01  0.0001  E × S    0.003  0.01  0.0001  0.35  0.98  0.42  0.30  0.0001  Groups1  Response variables2  Environment  Supplement  Feed  Egg  Egg  FCR  Eggshell  Eggshell  Eggshell  Haugh  (E)  (S)  intake  production  weight    weight  thickness  strength  units      (g)  (%)  (g)    (g)  (mm)  (kg/cm2)  (%)  Thermoneutral    128.5  90.7  62.7  2.26  5.80  0.38  4.59  80.7  Heat stress    106.2  74.9  58.3  2.45  5.48  0.36  4.50  75.4      1.8  1.4  0.2  0.03  0.07  <0.001  0.01  0.04    Control  112.3b  78.7b  59.7c  2.40  5.52b  0.36  4.52b  76.1c    CrPic  117.9a  83.5a  60.7b  2.34  5.63a,b  0.37  4.56a  78.4b    CrHis  122.0a  86.4a  61.0a  2.32  5.78a  0.37  4.57a  79.8a      3.2  2.4  0.6  0.05  0.1  0.01  0.01  0.6    Control  128.6  90.4  62.8  2.27  5.68  0.38  4.58  80.1  Thermoneutral  CrPic  125.8  89.7  62.4  2.25  5.80  0.38  4.59  80.7    CrHis  131.8  92.4  62.9  2.27  5.94  0.38  4.61  81.4    Control  96.1  67.0  56.7  2.54  5.37  0.35  4.46  72.1  Heat stress  CrPic  109.1  76.6  58.7  2.44  5.45  0.36  4.52  75.9    CrHis  113.3  81.2  59.4  2.37  5.63  0.36  4.54  78.3      2.3  2.0  0.2  0.05  0.11  0.01  0.02  0.52  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.0001  0.0001  0.0009  0.0001  0.0001  0.0001  S    0.0006  0.001  0.0001  0.30  0.08  0.81  0.01  0.0001  E × S    0.003  0.01  0.0001  0.35  0.98  0.42  0.30  0.0001  1Thermoneutral = environmental temperature of 22 ± 2°C for 24 h/d; Heat Stress = environmental temperature of 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. Control = the basal diet not supplemented with Cr; CrPic = the basal diet supplemented with 1.600 mg of CrPic (12.43% elemental Cr) per kilogram diet; CrHis = the basal diet supplemented with 0.788 mg of CrHis (25.22% elemental Cr). 2Data are ± SEM. Data with uncommon superscripts in rows (main effect of supplements) differ (P < 0.05). FCR = feed conversion ratio [g feed consumed per g egg mass (egg number × egg weight)]. View Large Positive effects of supplemental Cr on performance variables were more notable under the HS condition than under the TN condition. The increases (about 0.5 to 2.5%) in performance variables for hens supplemented with CrHis and CrPic were similar under TN the condition. However, increases in feed intake (13.5 vs. 17.9%; P < 0.003), egg production (14.3 vs. 21.2%, P < 0.01), and egg weight (3.5 vs. 4.8%, P < 0.001) for hens supplemented with CrHis were higher than those for hens supplemented with CrPic under the HS condition. Alterations in feed conversion in response dietary treatments under both conditions were insignificant. Previous studies also showed that improvement in performance parameters was significant when Cr supplemented in organic form (Sahin et al., 2001, 2002, 2004; 2009; Rao et al., 2012). This superiority is due the fact that Cr in the organic complex is more bioavailable than Cr in inorganic forms (Piva et al., 2003; Suksombat and Kanchanatawee, 2005; Hayirli, 2005; Sahin et al., 2010). Both Cr sources alleviated performance parameters under the HS condition, being CrHis more effective than CrPic (Table 2). Chromium-histidinate is a stable and histidinate allows the greater absorbability and bioavailability (Anderson et al., 2004). Heat stress deteriorated eggshell quality indices (eggshell weight, −5.5%, P < 0.0009; eggshell thickness, −5.3%, P < 0.0001; and eggshell strength, −2.0%, P < 0.0001) and decreased Haugh unit (−6.6%, P < 0.0001) (Table 2). Supplemental Cr, especially CrHis, increased eggshell weight, eggshell strength, and Haugh unit (Table 2). However, the positive effect of supplemental Cr form on eggshell quality parameters did not differ depending on the environmental condition. Studies investigating the effect of supplemental Cr on egg quality parameters are inconsistent. Torki et al. (2014) reported no alteration in egg quality parameters. The Cr supplementation improved eggshell stiffness and Haugh unit (Table 2). The mechanism by which Cr affect Ca metabolism in eggshell formation is not elucidated. The overall effect of supplemental Cr on eggshell quality parameters was independent of environmental temperature in the present experiment and elsewhere (Karimi et al., 2015). However, several studies reported improvement in eggshell quality in quails (Sahin et al., 2001, 2002) and hens (Torki et al., 2014) reared under the HS condition and quails (Yildiz et al., 2004; Senobar et al., 2012), hens (Lien et al., 1999; Uyanik et al., 2002; Ma et al., 2014), and turkeys (Biswas et al., 2015) reared under the TN condition. These inconsistent reports could be related to the form and level of supplemental Cr. Chromium and Electrolyte Concentrations Hens housed under the HS condition had lower Cr concentrations in serum and egg yolk (P < 0.0001 for both) as well as electrolyte minerals’ concentrations in serum (Na, P < 0.002; K, P < 0.01; and Cl, P < 0.06) than those reared under the TN condition (Table 3). Supplemental Cr increased serum and egg yolk Cr concentrations (P < 0.0001 for both), but did not alter concentrations of electrolyte minerals in serum. The effect of Cr as CrHis on tissue Cr concentrations was more notable than Cr as CrPic (P < 0.05). There was no environmental temperature by dietary supplement interaction effects on tissue Cr and serum electrolyte levels. Table 3. The effect of different chromium chelates on tissue chromium and serum electrolyte concentrations in laying hens reared under heat stress. Groups1  Response variables2  Environment  Supplement  Serum Cr  Yolk Cr  Serum Na  Serum K  Serum Cl  (E)  (S)  (mg/L)  (mg/kg)  (mmol/L)  (mmol/L)  (mmol/L)  Thermoneutral    1.98  473  148  4.70  108  Heat stress    1.13  313  145  4.39  106      0.07  6  1  0.08  1    Control  1.28c  359c  146  4.46  107    CrPic  1.56b  398b  147  4.50  107    CrHis  1.82a  421a  147  4.68  107      0.12  19  1  0.11  1    Control  1.75  443  148  4.58  108  Thermoneutral  CrPic  1.95  478  149  4.63  108    CrHis  2.23  498  148  4.89  108    Control  0.82  276  144  4.34  105  Heat stress  CrPic  1.18  317  146  4.36  107    CrHis  1.40  344  146  4.46  107      0.10  6  1  0.15  1  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.002  0.01  0.06  S    0.0001  0.0001  0.50  0.32  0.74  E × S    0.59  0.77  0.53  0.79  0.30  Groups1  Response variables2  Environment  Supplement  Serum Cr  Yolk Cr  Serum Na  Serum K  Serum Cl  (E)  (S)  (mg/L)  (mg/kg)  (mmol/L)  (mmol/L)  (mmol/L)  Thermoneutral    1.98  473  148  4.70  108  Heat stress    1.13  313  145  4.39  106      0.07  6  1  0.08  1    Control  1.28c  359c  146  4.46  107    CrPic  1.56b  398b  147  4.50  107    CrHis  1.82a  421a  147  4.68  107      0.12  19  1  0.11  1    Control  1.75  443  148  4.58  108  Thermoneutral  CrPic  1.95  478  149  4.63  108    CrHis  2.23  498  148  4.89  108    Control  0.82  276  144  4.34  105  Heat stress  CrPic  1.18  317  146  4.36  107    CrHis  1.40  344  146  4.46  107      0.10  6  1  0.15  1  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.002  0.01  0.06  S    0.0001  0.0001  0.50  0.32  0.74  E × S    0.59  0.77  0.53  0.79  0.30  1Thermoneutral = environmental temperature of 22 ± 2°C for 24 h/d; Heat Stress = environmental temperature of 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. Control = the basal diet not supplemented with Cr; CrPic = the basal diet supplemented with 1.600 mg of CrPic (12.43% elemental Cr) per kilogram diet; CrHis = the basal diet supplemented with 0.788 mg of CrHis (25.22% elemental Cr). 2Data are ± SEM. Data with uncommon superscripts in rows (main effect of supplements) differ (P < 0.05). View Large Chromium absorption takes place in the jejunum. It appears that Cr distributes to yolk easier than albumen in all poultry species (Nisianakis et al., 2009). Absorbability of Cr in inorganic form is inferior to Cr with organic complex (Amatya et al., 2005). However, Piva et al (2003) showed that Cr deposition in egg yolk (0.48 mg/kg) was not different when hens were fed CrCl3, Cr-yeast, and Cr-aminoniacinate. Resulting from decreased feed intake, HS may additionally aggravate Cr mobilization from tissues (Geraert et al., 1996; Sahin et al., 2002), which could lead to its deficiency (Doisy, 1978; Hayirli, 2005). Chromium requirement increases in case of exposure to stressors (Khan et al., 2014). Indeed, hens exposed to HS had lower serum Cr concentration than those reared under the TN condition (Table 3). Supplemental Cr compensates body Cr reserves and alleviates the adverse effects of HS in quails (Sahin et al., 2005, 2010), broilers (Lien et al., 1999), hens (Lien et al., 1996, 2004, 2008), and turkeys (Steele and Rosebrough, 1981). Restoration of Cr reserves is associated with increases in serum insulin, glucose, and cholesterol concentrations in overcrowded hens (Mirfendereski and Jahanian, 2015) and heat-distressed quails (Sahin et al., 2001, 2002). In the present study, serum and egg yolk Cr concentrations increased by supplemental Cr in laying hens reared under the HS conditions. The increases in serum and egg yolk Cr concentration were in agreement with the literature (Uyanik et al., 2002; Piva et al., 2003; Sahin et al., 2004; Ma et al., 2014). Serum Metabolites and Enzymes Exposure to HS caused increases in serum glucose (13.3%, P < 0.0001), cholesterol (34.5%, P < 0.0001), and creatine (8.8%, P < 0.007) concentrations, but did not affect concentrations of serum total protein and its fractions (Table 4). Supplemental Cr decreased serum glucose (P < 0.0001) and cholesterol (P < 0.03) concentrations. Decreases in serum glucose and cholesterol concentrations were greater for hens supplemented with CrHis than those for hens supplemented with CrPic (P < 0.05). There were no environmental temperature by dietary supplement interaction effects on serum metabolic profile, except for serum cholesterol concentration. Supplemental Cr, especially Cr as CrHis, tended to decrease serum cholesterol concentration under the HS condition to a greater extent than under the TN condition, as compared to Cr as CrPic (P < 0.06). Table 4. The effect of different chromium chelates on metabolic parameters in laying hens reared under heat stress. Groups1  Response variables2  Environment  Supplement  Glucose  Cholesterol  Protein  Albumin  Globulin  Creatine  (E)  (S)  (mg/dL)  (mg/dL)  (g/dL)  (g/dL)  (g/dL)  (mg/dL)  Thermoneutral    188  113  5.62  2.01  3.30  0.34  Heat stress    213  152  5.85  2.03  3.35  0.37      2  6  0.15  0.02  0.10  0.01    Control  209a  146a  5.63  2.02  3.33  0.35    CrPic  200b  130a,b  5.75  2.03  3.28  0.36    CrHis  193c  123b  5.84  2.01  3.36  0.35      4  8  0.18  0.02  0.13  0.01    Control  195  114  5.57  2.02  3.38  0.33  Thermoneutral  CrPic  188  114  5.67  2.01  3.23  0.35    CrHis  183  111  5.63  2.00  3.28  0.34    Control  223  178  5.68  2.02  3.28  0.37  Heat stress  CrPic  212  145  5.83  2.04  3.32  0.38    CrHis  204  134  6.04  2.02  3.44  0.37      3  9  0.30  0.03  0.18  0.02  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.30  0.58  0.74  0.007  S    0.0001  0.03  0.73  0.90  0.90  0.59  E × S    0.54  0.06  0.83  0.86  0.77  0.87  Groups1  Response variables2  Environment  Supplement  Glucose  Cholesterol  Protein  Albumin  Globulin  Creatine  (E)  (S)  (mg/dL)  (mg/dL)  (g/dL)  (g/dL)  (g/dL)  (mg/dL)  Thermoneutral    188  113  5.62  2.01  3.30  0.34  Heat stress    213  152  5.85  2.03  3.35  0.37      2  6  0.15  0.02  0.10  0.01    Control  209a  146a  5.63  2.02  3.33  0.35    CrPic  200b  130a,b  5.75  2.03  3.28  0.36    CrHis  193c  123b  5.84  2.01  3.36  0.35      4  8  0.18  0.02  0.13  0.01    Control  195  114  5.57  2.02  3.38  0.33  Thermoneutral  CrPic  188  114  5.67  2.01  3.23  0.35    CrHis  183  111  5.63  2.00  3.28  0.34    Control  223  178  5.68  2.02  3.28  0.37  Heat stress  CrPic  212  145  5.83  2.04  3.32  0.38    CrHis  204  134  6.04  2.02  3.44  0.37      3  9  0.30  0.03  0.18  0.02  ANOVA  ————————————————————— P ————————————————————–  E    0.0001  0.0001  0.30  0.58  0.74  0.007  S    0.0001  0.03  0.73  0.90  0.90  0.59  E × S    0.54  0.06  0.83  0.86  0.77  0.87  1Thermoneutral = environmental temperature of 22 ± 2°C for 24 h/d; Heat Stress = environmental temperature of 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. Control = the basal diet not supplemented with Cr; CrPic = the basal diet supplemented with 1.600 mg of CrPic (12.43% elemental Cr) per kilogram diet; CrHis = the basal diet supplemented with 0.788 mg of CrHis (25.22% elemental Cr). 2Data are ± SEM. Data with uncommon superscripts in rows (main effect of supplements) differ (P < 0.05). View Large Many studies reveal that Cr increases glucose removal from blood, reduces egg yolk cholesterol, improves eggshell quality, and enhances immunity and disease resistance (Lindemann et al., 2009). Chromium improves insulin action, leading to improvements in protein and lipid metabolism, as reflected by decreased nonesterified fatty acids, fastened serum triglyceride removal, and uptake of glucose for lipogenesis in the liver (Lien et al., 1999). These are accompanied by increasing protein accretion (Ahmed et al., 2005). Growth promoting effects of supplemental Cr is related to upregulation of expressions of skeletal muscle protein (Zha et al., 2009; Pan et al., 2013). Catabolic profile in a stress condition is characterized by a decrease in plasma protein concentration and increases in plasma glucose and cholesterol concentrations (Donkoh, 1989). Concentrations of glucose and cholesterol concentrations (Table 4) as well as enzymes (Table 5) were higher for layers reared under the HS environment than those reared under the TN environment (Table 4). Chromium plays a role in the regulation of metabolism (Prasad and Gowda, 2005) through acting as an immunostimulating substance (Sohn et al., 2000; Wenk, 2000) and exerting antioxidant effects (Sahin et al., 2005, 2010; de Araujo et al., 2007), which is critical under stressing conditions in poultry (Kim et al., 1996; Lien et al., 1996). Table 5. The effect of different chromium chelates on serum enzymes in laying hens reared under heat stress. Groups1  Response variables2  Environment  Supplement  AST (U/L)  ALT (U/L)  GGT (U/L)  LDH (IU/L)  CK (U/L)  (E)  (S)            Thermoneutral    200  1.33  24.8  1591  2438  Heat stress    203  1.37  25.8  2090  2566      5  0.10  0.5  52  60    Control  201  1.40  24.6  2015a  2511    CrPic  201  1.30  25.7  1796b  2496    CrHis  203  1.35  25.8  1709b  2499      6  0.11  0.5  81  75    Control  201  1.40  24.1  1766  2462  Thermoneutral  CrPic  198  1.30  25.5  1540  2409    CrHis  202  1.30  24.9  1466  2442    Control  202  1.40  25.0  2264  2559  Heat stress  CrPic  204  1.30  25.8  2053  2584    CrHis  204  1.40  26.7  1952  2556      8  0.17  0.8  79  106  ANOVA  ————————————————————— P ————————————————————–  E    0.64  0.81  0.12  0.0001  0.15  S    0.98  0.84  0.22  0.002  0.99  E × S    0.95  0.94  0.62  0.99  0.93  Groups1  Response variables2  Environment  Supplement  AST (U/L)  ALT (U/L)  GGT (U/L)  LDH (IU/L)  CK (U/L)  (E)  (S)            Thermoneutral    200  1.33  24.8  1591  2438  Heat stress    203  1.37  25.8  2090  2566      5  0.10  0.5  52  60    Control  201  1.40  24.6  2015a  2511    CrPic  201  1.30  25.7  1796b  2496    CrHis  203  1.35  25.8  1709b  2499      6  0.11  0.5  81  75    Control  201  1.40  24.1  1766  2462  Thermoneutral  CrPic  198  1.30  25.5  1540  2409    CrHis  202  1.30  24.9  1466  2442    Control  202  1.40  25.0  2264  2559  Heat stress  CrPic  204  1.30  25.8  2053  2584    CrHis  204  1.40  26.7  1952  2556      8  0.17  0.8  79  106  ANOVA  ————————————————————— P ————————————————————–  E    0.64  0.81  0.12  0.0001  0.15  S    0.98  0.84  0.22  0.002  0.99  E × S    0.95  0.94  0.62  0.99  0.93  1Thermoneutral = environmental temperature of 22 ± 2°C for 24 h/d; Heat Stress = environmental temperature of 34 ± 2°C for 8 h/d, from 08:00 to 17:00 h, followed by 22°C for 16 h (HS) for 12 wks. Control = the basal diet not supplemented with Cr; CrPic = the basal diet supplemented with 1.600 mg of CrPic (12.43% elemental Cr) per kilogram diet; CrHis = the basal diet supplemented with 0.788 mg of CrHis (25.22% elemental Cr). 2Data are ± SEM. Data with uncommon superscripts in rows (main effect of supplements) differ (P < 0.05). AST = aspartate-aminotransferase; ALT = alanine-amino transferase; GGT = γ-glutamyl transferase; LDH = lactate dehydrogenase; CK = creatine kinase. View Large Among serum enzymes, only LDH was responsive to environmental temperature (Table 5). It increased by 1.3-fold due to HS (P < 0.0001). In response to Cr supplementation serum LDH concentration decreased (P < 0.002) and other enzymes remained unchanged. The decrease in serum LDH concentrations between hens supplemented with CrHis and CrPic was similar. The effects of supplemental Cr and Cr form on serum enzymes did not differ depending on environmental temperature. To overcome oxidative stress under the HS condition, Onderci et al. (2005) and Sahin et al. (2004) showed that CrPic decreased malondialdehyde levels in serum and the liver, thigh muscle and serum cholesterol, and serum glucose concentrations in quails. Hens are responsive to supplemental Cr in the regulation of the insulin action (Viera and Davis-Mcgibony, 2008). Antistress and metabolic modifier effects of supplemental Cr through modulating insulin action and mineral utilization were shown in quails, as reflected by improvements in laying performance and eggshell quality and decreased serum corticosterone, glucose, and cholesterol concentrations, and increase in serum protein concentration (Sahin et al., 2001, 2002; Torki et al., 2014). Both Cr sources decreased serum glucose and cholesterol concentrations and liver LDH levels. Similarly, Lien et al. (1996) stated that Cr supplementation to laying hen diets decreased serum glucose and cholesterol concentrations. Previous studies also showed a cholesterol-lowering effect in the serum of broilers (Kim et al., 1996) and quails (Sahin et al., 2004; Akdemir et al., 2015) supplemented with CrHis. In hens at early laying period supplemented with Cr (400 ug/kg) and L-carnitine (100 mg/kg), there was a considerable reduction in hepatic triglyceride, egg yolk cholesterol, and abdominal fat percentage (Du et al., 2005). In other studies, despite a decrease in serum cholesterol and an increase in HDL concentrations, eggshell quality remained unchanged in hens supplemented with CrPic (Lien et al., 2004, 2008). In conclusion, supplemental Cr as CrHis and CrPic alleviated the adverse effects of HS on the performance and metabolic profile of laying hens. Chromium in either could be added into the diet in order to overcome heat stress-related depression in performance and metabolic profile. 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