Effects of dietary chromium supplementation on calf performance, metabolic hormones, oxidative status, and susceptibility to diarrhea and pneumonia

Accepted Manuscript Title: Effects of dietary chromium supplementation on calf performance, metabolic hormones, oxidative status, and susceptibility to diarrhea and pneumonia Authors: F. Mousavi, S. Karimi-Dehkordi, S. Kargar, M. Khosravi-Bakhtiari PII: DOI: Reference:

S0377-8401(18)30234-7 https://doi.org/10.1016/j.anifeedsci.2019.01.004 ANIFEE 14128

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Please cite this article as: Mousavi F, Karimi-Dehkordi S, Kargar S, Khosravi-Bakhtiari M, Effects of dietary chromium supplementation on calf performance, metabolic hormones, oxidative status, and susceptibility to diarrhea and pneumonia, Animal Feed Science and Technology (2019), https://doi.org/10.1016/j.anifeedsci.2019.01.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effects of dietary chromium supplementation on calf performance, metabolic hormones, oxidative status, and susceptibility to diarrhea and pneumonia

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F. Mousavi,a S. Karimi-Dehkordi,a1,2 S. Kargar,b,2 M. Khosravi-Bakhtiaric Department of Animal Science, College of Agriculture, Shahrekord University, Shahrekord

34141–88186, Iran b

Department of Animal Science, School of Agriculture, Shiraz University, Shiraz 71441–65186,

Iran

Agri-Animal Production Co., Isfahan, 13895–81799, Iran

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cFKA

Corresponding author: [email protected] (S. Karimi-Dehkordi)

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Authors contributed equally to this paper.

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Highlights:

Chromium-methionine (Cr) increased feed intake and weight gain in dairy calves.

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Respiration rate but not rectal temperature decreased by supplementing Cr.

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Cr decreased days with pneumonia and days of medication during the post-weaning.

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Antioxidant status and insulin sensitivity were not affected by supplementing Cr.

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ABSTRACT

The objective of this study was to determine the effect of chromium (Cr) supplementation on the health status, blood attributes, and insulin sensitivity in dairy calves reared under hot summer conditions. Twenty-four newborn Holstein calves were assigned randomly to a control group (Cr-) and a group (Cr+) receiving 0.05 mg Cr-methionine per kg BW0.75. The starter feed

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contained 0.95 mg Cr/kg dry matter. Dietary level of Cr was within the ranges (0.08 to 1 mg Cr/kg body mass daily or more) reported to affect metabolism). Supplemental Cr was included in colostrum and milk before weaning (d 1 to 63), and in the starter feed from d 64 to 91. Dry

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matter intake and average daily gain increased but respiration rate was lower in Cr+ calves. Cr supplementation decreased the number of days with pneumonia and medication days during the post-weaning period; however, Cr did not affect diarrhea frequency or the number of days with

diarrhea. Supplementing Cr increased blood serum concentration of total proteins. Furthermore, blood serum concentration of globulins tended to increase in Cr+ calves during the pre-weaning

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and overall periods. Cr supplementation increased catalase activity during the post-weaning

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period. Glucose disappearance rate tended to increase in Cr+ calves. Overall, supplementary

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feeding of Cr may benefit growth performance and health status without a major effect on blood

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temperature summer conditions.

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metabolites, antioxidant status, and insulin sensitivity in dairy calves under high ambient

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Keywords: Calf, Chromium-methionine, Insulin sensitivity, Summer

1. Introduction

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Heat stress decreases the motility of the gastrointestinal tract and feed intake (Kumar et al., 2015) associated with decreased energy intake, poorer growth and higher susceptibility to

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diseases in dairy calves (Kargar et al., 2018b). Blood concentration of insulin, insulin-like growth factor-I (Guo et al., 2016) and triiodothyronine (T3; Baccari et al., 1983) decreased but that of cortisol increased (Kumar et al., 2015) under heat stress conditions. Concentration of blood constituents [e.g., glucose, insulin, T3 and thyroxine (T4)] can be used to monitor the

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changes in metabolism and growth performance as a result of altered feed intake during heat stress (Yari et al., 2010; Kumar et al., 2015; Mousavi et al., 2018). Heat stress can also trigger cellular lipid peroxidation (Stott et al., 1976) and oxidative stress due to increased production of

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the reactive oxygen species (ROS) which are neutralized by the antioxidant defense system (Morgan and Liu, 2011; Buelna-Chontal and Zazueta, 2013). An imbalance between the

prooxidants and antioxidants may impair the ability of the defense system to protect the body against the harmfull effects of ROS (Puertollano et al., 2011). Little is known concerning the

effect of Cr supplementation on the antioxidant defense system in dairy calves when the ambient

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temperature is high.

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Decreased feed intake during heat stress may be associated with decreased Cr level in the

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body through enhanced urinary excretion which may increase Cr requirements (Yari et al., 2010;

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Pantelić et al., 2018; Kargar et al., 2018c); therefore, the utilization of Cr in animal feed may be a viable approach to alleviate stress-associated effects (Yari et al., 2010; Kumar et al., 2015;

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Pantelić et al., 2018) and improve calf performance and welfare under heat stress conditions

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(Kargar et al., 2018a,c). In dairy calves, dietary Cr supplementation (0.05 mg Cr-methionine/kg

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BW0.75) during the summer months improved their heat tolerance, and increased blood concentration of total proteins, globulins and insulin as well as insulin to glucose ratio (Kargar et

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al., 2018a,c) but decreased blood glucose levels; no effects were reported on blood β-hydroxy butyric acid (βHBA), cholesterol, T3, T4 or T3 to T4 ratio (Kargar et al., 2018a,c; Mousavi et al.,

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2018).

Pharmacological doses of Cr [0.08 to 1 mg Cr/kg body mass daily or even hihger; (Vincent,

2014)] had a modulating effect on carbohydrate, protein, and fat metabolism (Vincent, 2015). Cr increased insulin signaling and consequently, altered glucose metabolism if the level of dietary

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energy was adjusted to the needs. Cr, as a part of chromodulin (also called low-molecular-weight Cr-binding substance), plays an important role in the activation of insulin receptors and allows glucose entry to cells (Vincent, 2015) by recruiting membrane vesicles containing GLUT 4

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transporters from the interior of cells to the cell surface (Jovanović et al., 2017; Pantelić et al., 2018). Impaired insulin sensitivity could result in decreased efficiency of energy and protein

utilization and may predispose dairy calves to metabolic disorders occurring in later life (van den Borne, 2006). It has recently been shown that alterations in glucose-insulin dynamics in the preweaning period could affect growth performance later in life (Stahel et al., 2017). Therefore,

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more studies are needed to design a suitable nutritional management for calves in summer

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(Jovanović et al., 2017; Kargar et al., 2018c; Mousavi et al., 2018). Little data are available on

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the effect of Cr supplementation on occurrence and frequency of diarrhea and pneumonia in

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dairy calves. Organic sources of Cr decreased the morbidity rate in stressed feeder calves; no animals that received Cr died, relapsed, or failed to respond to antibiotic treatments (Mowat et

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al., 1993). Supplementary feeding of Cr-methionine (0.05 mg/kg BW0.75) to environmentally

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heat-loaded dairy calves did not affect the odds ratio of the occurrence of medication or diseases

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(diarrhea or pneumonia) but reduced the number of days with diarrhea or pneumonia and total medication days before weaning (Kargar et al., 2018b). The main goal of this experiment was to

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determine the effect of Cr supplementation on the occurrence of diseases, blood biochemical attributes, and insulin sensitivity in dairy calves durimg hot summer months. It was hypothesized

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that supplemental Cr would improve the feed intake (possibly through increasing blood glucose transfer to cells) and health-related variables in dairy calves when ambient temperature is high.

2. Materials and methods

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2.1. Climatic conditions, calves, treatments and management Experimental procedures were carried out in accordance with Protocol # 19356 as approved by the Iranian Council of Animal Care (1995). During the experiment (July 5 to October 12,

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2015), air temperature (ºC) and relative humidity (RH; %) in the calf house were recorded daily (Hobo Pro Series Temp probes, Onset Computer Corporation, Pocasset, MA) and temperature‒ humidity index (THI) was calculated according to Kargar et al. (2015). The average maximum

THI, relative humidity, and maximum temperature were 81.3, 11.5%, and 39.6°C, respectively, over the entire experimental period (Table 1), indicating a high degree of environmental heat-

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load. We tested the hypothesis whether Cr supplementation is beneficial to dairy calves under

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these envitonmental conditions.

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Twenty-four Holstein female calves (1 d of age; 42.1 ± 0.89 kg BW) were separated from

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their dams immediately after birth, weighed, and housed in a naturally-ventilated barn with sawdust-bedded pens (2.9 × 1.1 × 1.8 m; length × width × height). The bedding was renewed

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every 3 days, but manure was removed daily to keep the pens clean and dry. Each calf was fed

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with 6 L of colostrum within 2 to 8 h after birth. From d 2 of life onwards, calves were fed

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pasteurized (62.5 °C for 30 min) waste milk containing 3.34 ± 0.12% fat, 2.68 ± 0.09% CP, and 4.87 ± 0.07% lactose in steel buckets in two meals of equal volume d−1 (at 0800 and 1600 h).

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Milk was offered at a rate of 3 L/d from d 3 to d 15, 4 L/d from d 16 to d 20, 5 L/d from d 21 to d 25, 6 L/d from d 26 to d 60, and 3 L/d from d 61 to d 63 of age. Calves were weaned on d 63,

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and the study was terminated on d 91. Calves were assigned randomly to a control group with no supplemental Cr (Cr-) or a Cr-supplemented (Cr+) group (n = 12 per group). The starter feed contained 0.95 mg Cr/kg dry matter (DM; Table 1). The rate of supplementation was chosen according to Vincent (2014), who suggested that daily doses of 0.08

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to 1 mg Cr/kg of BW or even more need to be fed to obtain positive effects on metabolism. Accordingly, we provided an additional 2 mg Cr/d to determine its effect on physiological response of dairy calves. Supplemental Cr was added to 300 mL colostrum or milk fed in bottles

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up to wk 9 of age to supply 0.05 mg of Cr/kg BW0.75; and calves received the rest of colostrum or milk according to the feeding schedule described above. Cr supplement [MicroPlex 1000,

supplies 1,000 mg Cr/kg as Cr-methionine (90% methionine and 10% Cr, wt/wt); Zinpro Animal Nutrition Inc., Eden Prairie, MN] was provided by Sana Dam Co., Tehran, Iran. After weaning, the same dose of Cr was added in the starter feed. To ensure that each calf consumed the

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calculated amount of Cr, Cr-methionine was mixed with 50-g of daily starter feed allowance and

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top-dressed. Cr supplementation was adjusted based on BW of the calves every wk throughout

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the entire experimental period. Calves had free access to both fresh water and mashed starter

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feed, formulated according to the Cornell Net Carbohydrate and Protein System (CNCPS version 5.1), allowing at least 10% refusals. Ingredients and nutrient composition of the basal diet are

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shown in Table 2.

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2.2. Data collection, sampling and analyses Starter feed refusal was collected daily at 0800 h for calculation of starter intake. Starter and

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milk dry matter intakes were summed up to determine the total DM intake (DMI). Calves were weighed at birth and once weekly using an electronic balance which was calibrated by the

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manufacturer’s personell at the start of the experiment and every month thereafter. Average daily gain (ADG) and feed efficiency (kg BW gain/kg total DMI) were computed. Concentrations of Cr in the starter feed (n = 13; one sample per week) and Cr-methionine (n = 3; one sample per

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month) samples were measured using flame photometry (GBC Integra EXL ICP, Melbourne, Australia) as described by Kargar et al. (2018c). Calf health was monitored several times on a daily basis by a veterinarian unaware of the

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treatment to which the calf was assigned. Feces were classified (Kargar et al. 2018b) as firm and well-formed (score 1), soft and pudding-like (score 2), runny and pancake batter-like (score 3), or liquid and splatters (score 4). Respiration rate (RR) was measured daily at 1400 h by visual observation for three separate minutes (Kargar et al., 2015). Immediately after recording RR,

rectal temperature (RT) was measured using a thermometer (Qingdao Dacon Trading Co. Ltd.,

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Shandong, China) held in the rectum for 1 min. Calves with diarrhea (fecal score ≥ 3) or

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pneumonia (open-mouth breathing and coughing) were treated by the farm veterinarian. Calves

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with diarrhea under 2 weeks of age received ScourSTOP (25g per calf in milk for a day;

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Livestock Drugs Production Co., Garmsar, Iran), water-based oral rehydration salt solution (2.5 liter per calf; Sepid Dehdasht Co., Tehran, Iran), and Trisul (sodium sulfadiazine + trimethoprim;

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15 cc per calf; Zagros Pharmed Pars Co., Tehran, Iran). Oral rehydration salt solution and Trisul

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were administered for one or two days until fecal score was below 3. For older calves,

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Enrofloxacin (Enrocin 5%; 3 mL per calf; Razak Laboratories Co., Karaj, Iran) and Flunixin meglumine (Flunixin 5%; 5 mL per calf; Razak Laboratories Co., Karaj, Iran) were administered

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for 3 consecutive days, and another 2 days for non-responding individuals. To treat pneumonia, calves were administered with Florfenicol (10 mL per calf; Razak Laboratories Co., Karaj, Iran)

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and Flunixin meglumine (Flunixin 5%; 5 mL per calf; Razak Laboratories Co., Karaj, Iran) two times every other day; non-responding individuals were treated for 2 more days with Oxytetralcycline (Oxivet 5%; 10 mL per calf; Razak Laboratories Co., Karaj, Iran) and Tylosin (Tyloject 20%; 10 mL per calf; Razak Laboratories Co., Karaj, Iran) or Ceftionel (Ceftiofur 5%;

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6 mL per calf; Daanapharma Co., Tabriz, Iran). No calf died during the study period. Calves exhibiting signs of clinical illness or receiving medications were excluded from sampling, to avoid any confounding effects on the recorded measurements.

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On d 7, 21, 35, 49, 63, 77 and 91 of life, blood samples were collected 4 h after the morning feeding into vacuum serum separator tubes containing clot activator, and immediately placed on ice. Blood samples were centrifuged at 3,000 × g for 20 min at 4°C, and 1.5-mL serum was

pipetted into 2-mL cryotubes and stored at −20°C. Concentrations of blood constituents were determined spectrophotometrically (UNICCO, 2100, Zistchemi, Tehran, Iran) using

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commercially available kits [Pars Azmoon Co., Tehran, Iran; Catalogue Numbers: glucose (1-

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500-017), cholesterol (1-500-010), blood urea nitrogen (BUN; 1-400-029), triglyceride (1-500-

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03), total protein (1-500-028), and albumin (1-500-001)] according to the manufacturers’

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instructions. Globulin concentration was calucated by subtracting albumin from total protein concentration. Concentration of βHBA was measured using a colorimetric kit (Randox

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Laboratories Ltd., Ardmore, UK) with a Technicon-RA 1000 Auto-analyzer (DRG Co.,

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Marburg, Germany). Malondialdehyde concentration was measured using the thiobarbituric acid

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reactive substances method as described previously by Kargar et al. (2015). Concentrations of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase were determined

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according to procedures described earlier (Kargar et al., 2018b). Serum concentrations of insulin, T3, T4 and cortisol were measured on d 91 of life (ELISA, Bio-Tek Instruments Inc., Winooski,

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VT) using a commercial kit (Diaplus Inc., North York, ON, Canada).

2.3. Intravenous glucose tolerance test (GTT)

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At the end of the experiment (THI: 57.8 to 73.8), an intravenous GTT was conducted using 14 calves (7 calves per treatment that were closest to the mean treatment BW). The GTT was performed at 16-h after feed deprivation (with unlimited access to fresh water) to ensure that

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calves were on identical energy level, and to minimize the possibility of meal pattern responses on glucose and insulin metabolism. Calves were fitted with 18-gauge jugular catheters (Yasa Teb Co., Isfahan, Iran), and allowed to rest for at least 1 h after which a glucose solution (50% wt/vol) was infused (0.3 g glucose/kg BW).

Blood samples (10 mL) were taken at 0, 20, 40, 60, 90, and 120 min post-infusion and put in

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vacutainer tubes (K2EDTA, BD Vacutainer, Franklin Lakes, NJ). Before each blood sampling, 5

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mL blood was drawn into a syringe to clear the catheter, and discarded. To prevent clot

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formation, the catheters were flushed with one mL heparin stock solution. Samples were stored

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on ice and centrifuged immediately at 3,000 × g for 20 min at 4°C. Plasma was aliquoted into 2mL tubes in duplicate and stored at −20°C for glucose and insulin analyses. Glucose and insulin

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pharmacokinetic variables in response to intravenous GTT including basal concentrations,

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relative clearance rate (%/min; 20–120 min), time to half-maximal concentration (T1/2; min), the

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area under the curve (AUC; 0–120 min), and insulin sensitivity were calculated according to

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Mousavi et al. (2018).

2.4. Statistical analyses

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Data on Cr intake, DMI, ADG, feed efficiency, body weight, RR, RT, or fecal score were

summarized as one measurement (d 1-91). Data were subjected to ANOVA (PROC MIXED, SAS 9.4, SAS Inc., Cary, NC) with time (day or week) as the repeated measures for the abovementioned variables and blood metabolites. Data on fecal score were not normally distributed.

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Therefore, the GLIMMIX procedure (SAS 9.4, SAS Inc., Cary, NC) was employed to fit the Poisson distribution using a log-link function. In both MIXED and GLIMMIX models, the effects of treatment, time, and treatment by time interaction were considered as fixed and calf as

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a random effect. Autoregressive covariance structure (Type 1) was the best fit for these data as determined by the lowest Akaike’s information criterion. Data on BW, blood hormones, and GTT variables were analyzed using the same model without the time effect. Initial BW was included in the model as a covariate for the analysis of final weight.

Models for the occurrence of diarrhea, pneumonia, and needs to medication were tested by

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logistic regression using a binomial distribution in the GLIMMIX procedure (SAS 9.4, SAS Inc.,

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Cary, NC). The odds ratios were calculated to compare the likelihood of 2 treatments to

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experience any event. Frequency and duration of diarrhea, pneumonia, and administration of

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medication were tested with the Poisson distribution using the GENMOD procedure (SAS 9.4, SAS Inc., Cary, NC). Least squares means for time and treatment × time effects were separated

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using Tukey’s adjustment when the overall F-test was P < 0.05. Trends were declared at 0.05 ≤ P

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3. Results

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≤ 0.10.

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3.1. Growth performance, physiological parameters and occurrence of diseases As anticipated, Cr supplementation increased Cr intake (P < 0.001; Table 3) and increased

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progressively as calves aged (Week effect: P < 0.001) with greater Cr intake for Cr+ calves vs. Cr- calves over the trial period (Treatment × Week effect: P = 0.005). Supplementing Cr increased total DMI (P = 0.002), ADG (P = 0.02) but not feed efficiency (P = 0.93) or final body weight (P = 0.14). Irrespective of treatment, total DMI increased progressively as calves aged

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(Week effect: P < 0.001) with higher total DMI at weaning (d 63) for Cr+ calves (Treatment × Week effect: P = 0.01). Cr supplementation tended to increase ADG (Treatment × Week effect: P = 0.07) and feed efficiency (Treatment × Week effect: P = 0.03) during the first week of the

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experiment. The Cr+ calves had lower RR compared with Cr- calves (P = 0.02; Table 3). Table 4 presents the logistic models for the occurrence of diarrhea (score ≥ 3) and pneumonia, and needs for medication during the pre-weaning (d 1 to 63), post-weaning (d 64 to 91), and overall (d 1 to 91) preiods. The occurrence of diarrhea and pneumonia or the chance of administration of

medication were not influenced by Cr supplementation. Cr supplementation decreased the

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number of days requiring medication (P = 0.01) and number of days with pneumonia (P = 0.04)

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after weaning (Table 5).

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3.2. Blood biochemical attributes

Serum concentrations of glucose, βHBA, BUN, cholesterol, triglyceride, albumin, and

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albumin to globulin ratio were not affected by Cr supplementation (Table 6). In Cr+ calves,

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serum concentration of total proteins increased before weaning (P = 0.02), after weaning (P =

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0.07) and over the whole experiment (P = 0.008). Serum concentration of globulin tended to be higher in Cr+ calves both before weaning (P = 0.07) and over the whole trial (P = 0.06).

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Concentrations of blood glucose, corisol, T3, T4 and T3 to T4 ratio were not affected by Cr (Table 7) but Cr+ calves recorded higher blood insulin levels and insulin to glucose ratio as compared

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with Cr- calves.

Serum concentrations of MDA and GPx were not affected by Cr supplementation (Table 8);

however, the SOD concentration tended to increase in Cr+ calves at weaning (Treatment × Week effect: P = 0.06). Serum concentration of catalase in Cr+ calves was higher after weaning (P <

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0.001). The Cr+ calves also recorded a higher concentration of catalase on d 49 as compared with Cr- calves (Treatment × Week effect: P < 0.001).

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3.3. Intravenous GTT The results of the GTT are presented in Figure 1 (A and B) and description of the curves in Table 9. The average maximum THI, relative humidity, and maximum temperature were 73.8,

11.4%, and 31.8°C, respectively, on the day of GTT (Table 2), indicating no environmental heatload. Irrespective of treatment, blood glucose concentration was highest at 20 min post-glucose

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infusion, returning to basal levels within 2 hours of the infusion. No significant differences were

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found in the basal concentrations of glucose and insulin, T1/2, AUC, and insulin sensitivity index.

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However, Cr-supplemented calves tended to have (P = 0.09) higher glucose disappearance rate

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as compared to Cr- calves.

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4. Discussion

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Temperature-humidity index can exceed 80 from May through September in central Iran. In

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this study, THI values ≥ 78 were considered to exert a high degree of environmental heat-load on dairy calves (Yari et al., 2010; Kargar et al., 2018c). According to Yari et al. (2010), decreased

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RR in response to supplemental Cr may indicate an adjustment to the environmental heat-load; however, in the present study, RT and fecal score were within their physiological ranges and

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unaffected by Cr, indicating the ability of calves to cope with the potentially arisen stressors or their good conditioning allowing them to better tolerate the summer heat stress (Kumar et al., 2015; Kargar et al., 2018c; Mousavi et al., 2018).

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In the present study, increased DMI in Cr+ calves implies that Cr may be deficient when calves are reared under high ambient temperarures (Kargar et al., 2018a,c). Improved insulin action (by decreased AUC) in calves may facilitate glucose entry into cells (Yari et al., 2010) and can in

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turn increase the feed intake (NRC, 2001). Although glucose disappearance rate tended to increase by supplementing Cr in the present study (Table 9), differences in AUC for insulin and glucose and insulin sensitivity did not reach statistical significance. Others reported no Cr effect on DMI under thermo-neutral (Bunting et al., 2000; Ghorbani et al., 2012) or moderate

environmental heat-load (Kumar et al., 2015; Mousavi et al., 2018) conditions. Therefore, it may

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Cr may not lead to improvements in calf performance.

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be assumed that Cr requirements are not increased in non-stress conditions, and supplementing

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The Cr+ calves, in part due to increased DMI, had a greater rate of weight gain

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(approximately + 10%) over the entire experimental period, indicating a more efficient tissue accretion in these calves (Ghorbani et al., 2012; Kargar et al., 2018a,c). However, others (Yari et

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al., 2010; Kumar et al., 2015; Mousavi et al., 2018) did not observe any positive response on

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weight gain in Cr-supplemented calves. Such inconsistencies might be ascribed to the chemical

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form, mode of administration (in liquid feed, starter diet, and/or both), level and duration of feeding of the supplemetal Cr, and type and duration of stressful conditions in these studies.

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The Cr+ calves experienced fewer days with pneumonia and needed fewer days for recovery after weaning. This can be related to the higher feed intake or lower RR and also higher blood

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levels of total protein and globuin in Cr+ calves compared with Cr- calves (Mowat et al., 1993; Kargar et al., 2018b). Mowat et al. (1993) observed lower susceptibility to diseases and better response to antibiotic therapy when stressed feeder calves received organic sources of Cr. Kargar et al. (2018b) reported that supplementing Cr (as Cr-methionine at 0.05 mg/kg BW0.75) to

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environmentally heat-loaded dairy calves decreased the number of days with diarrhea (-0.9 d) or pneumonia (-0.7 d) and total medication days (-1.5 d) before weaning. The non-significant effect of Cr on blood glucose concentration are in line with other studies

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(Bunting et al., 2000; Yari et al., 2010; Ghorbani et al., 2012). As expected, we observed a gradual increase in blood βHBA concentration as calves aged, indicating a normal physiological fuel shift in the source of energy from glucose to short-chain fatty acids with the initiation of

ruminal fermentation and development (Bunting et al., 2000). The lack of treatment effect on blood βHBA concentration was in agreement with data reported by Yari et al. (2010) but not

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with Ghorbani et al. (2012) who observed a decrease in blood βHBA concentration in calves

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supplemented with Cr at 0.03 mg/kg BW0.75. Inconsistent with the results reported by Ghorbani

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et al. (2012), Cr supplementation did not affect blood concentrations of cholesterol and

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triglycerides (Chang and Mowat, 1992), indicating no Cr effect on fat and cholesterol metabolism in the present study. Blood concentrations of total proteins and albumin are longer

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term indicators of amino acid meabolism and the immune states, whereas BUN may more

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accurately reflect short-term dietary effects on rumen NH3-N production and hepatic N turnover

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(NRC, 2001; Yari et al., 2001). As such, blood globulin, and albumin to globulin ratio are used to assess the systemic immune function at times of high metabolic demands. The impact of Cr

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supplementation on increasing total proteins and globulins may be a reflection of increased demand and competition for amino acids between treatment groups and thereby a challenge to

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the hepatic synthesis of globulins needed to maintain normal immune function. In the present study, increased blood concentrations of total proteins and globulins are in line with Kargar et al. (2018b) but not Mousavi et al. (2018) who observed no changes in blood concentrations of total proteins and globulins when Cr-methionine (0.05 mg/kg of BW0.75) was supplemented to weaned

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calves experiencing a moderate heat load; however, the calves in the study by Kargar et al. (2018b) and in the present study experienced higher degrees of environmental heat-load. The elevated concentration of blood insulin in Cr+ calves is in line with Bunting et al.

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(2000) who supplemented their calves with Cr-propionate at 0.5 mg/kg DMI. On the other hand, Kumar et al. (2015) showed decreased concentration of blood insulin during summer in buffalo calves when Cr (as CrCl3) was supplemented at 1 and 1.5 mg/kg DMI, but not at 0.5 mg/kg DMI. The fact that blood insulin concentration and insulin to glucose ratio increased in Cr+ calves indicated that more insulin was required to clear glucose from the blood, thus less tissue

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sensitivity to insulin (Bunting et al., 2000). The lack of treatment effects on blood concentrations

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of T3, and T4, and on T3 to T4 ratio is in line with those reported by Kumar et al. (2015).

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However, according to Ghorbani et al. (2012), Cr supplementation (0.03 mg Cr/kg BW0.75)

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increased blood T4 concentration and decreased T3 to T4 ratio in dairy calves. In another study, Yari et al. (2010) reported that increasing Cr doses (from 0 to 0.02 and 0.04 mg Cr/kg BW0.75)

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dose (Yari et al., 2010).

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resulted in quadratic reductions in serum T4, whereas blood T3 decreased only at the higher Cr

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The effect of Cr supplementation on blood cortisol concentration under stress conditions in claves are not consistent. Supplementing Cr decreased blood cortisol concentration in birth-

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stressed dairy calves (Ghorbani et al., 2012) and in buffalo calves during summer (Kumar et al., 2015). In other studies, supplemental Cr did not affect (the present study; Kargar et al., 2018b,c;

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Mousavi et al., 2018) or increased (Yari et al., 2010) blood cortisol concentrations in heatstressed calves. Part of the discrepancies in these findings may be due to differences in the calf age, species, type and duration of Cr supplementation, as well as the level, type, and duration of stress. Therefore, the effect of Cr on cortisol secretion warrants further investigation.

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Excessive generation of ROS imparts damages to the tissues and cellular components. The antioxidant defense system consists of antioxidants (e.g., tocopherol, glutathione, SOD, GPx, catalase, etc.) that neutralize the oxidative stress caused by increased generation of ROS

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(Puertollano et al., 2011). Catalase deactivates H2O2, which could otherwise penetrate through bio-membranes and may inactivate several enzymes. Pedraza-Chaverrí et al. (2005) showed

time-dependent changes in the activity of antioxidant enzymes in Cr-supplemented rats. Cr has the potential to catalyse the Fenton/Haber-Weiss reaction to form hydroxyl radical formation,

and antioxidant enzymes are able to preserve homeostasis during Cr-induced oxidative stress by

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dismutation of toxic superoxide ions to hydrogen peroxide and then by its reduction to water as

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well as protecting the membrane lipids from oxidative damage (Długosz et al. 2012). Increased

A

activities of catalase and SOD by Cr in the present study can be a result of an adaptive response

M

to oxidative stress. However, these increases were within the physiological ranges, as also supported by no changes in MDA (as an indicator of lipid peroxidation) concentration, which

D

mainly depends on the availability of polyunsaturated fatty acids and antioxidant defense (Kargar

TE

et al., 2015). Accordingly, we conclude that calf antioxidant defense system under high ambient

EP

temperarure of the present study was not affected by Cr supplementation. In the present study, a lack of treatment effect on insulin sensitivity is in line with Bunting et

CC

al. (2000), who observed increased glucose disapperance rate in Cr-propionate supplemented calves with no effect on insulin sensitivity. Unlike our results, Jovanović et al. (2017) reported

A

decreased basal glucose and insulin concentrations and increased glucose utilization (by decreasing T1/2 and glucose AUC and increasing glucose disapperance rate) when Cr-yeast was supplemented to dairy calves at 0.04 mg/kg body weight. The limited effect of Cr on the GTT may be due to other factors including a lack of actual heat stress during GTT (as indicated in

16

Table 1), limited the bio-availability of Cr from Cr-methionine, or that the degree of insulin resistance was too great for the amount of Cr to have a significant effect.

SC RI PT

5. Conclusions Supplementary feeding of Cr to dairy calves during hot summer months may benefit growth performance (through increasing feed intake and weight gain) and health status (through

decreasing RR and lowering days with pneumonia or faster recovery from disease), but have

U

only minor effects on antioxidant status and glucose-insulin kinetics.

N

Acknowledgments

A

The authors thank Shahrekord University for financial support and providing the research

M

facilities. The authors express their kind appreciation to the farm staff at FKA Agri-Animal Production Company (Isfahan, Iran), for diligent animal care, and to Sana Dam Company (Tehran,

D

Iran) for donating Cr-methionine. The authors also express their appreciation to Keith E. Inskeep

TE

(Emeritus Professor; West Virginia University, USA) and Mohammad Javad Zamiri (Emeritus

EP

Professor; Shiraz University, Iran) for editing English version of this manuscript. Conflicts of interest

CC

The authors wish to confirm that there are no recognized conflicts of interest associated with this publication and there has been no financial support for this work that could have influenced

A

its outcome.

References

17

Baccari-Jr., F., Johnson, H.D., LeRoy-Hahn, G., 1983. Environmental heat effects on growth, plasma T3, and Postheat compensatory effects on Holstein calves. Exp. Biol. Med. 173, 312– 318.

SC RI PT

Buelna-Chontal, M., Zazueta, C., 2013. Review: Redox activation of Nrf2 and NF-κB: A double end sword? Cell Signal. 25, 2548–2557.

Bunting, L.D., Tarifa, T.A., Crochet, B.T., Fernandez, J.M., Depew, C.L., Lovejoy, J.C., 2000. Effects of dietary inclusion of chromium propionate and calcium propionate on glucose disposal and gastrointestinal development in dairy calves. J. Dairy Sci. 83, 2491–2498.

U

Chang, X., Mowat, D.N., 1992. Supplemental chromium for stressed and growing feeder calves.

N

J. Anim. Sci. 70, 559–565.

A

Długosz, A., Rembacz, K.P., Pruss, A., Durlak, M., Lembas-Bogaczyk, J., 2012. Influence of

M

chromium on the natural antioxidant barrier. Pol. J. Environ. Stud. 21, 331–335. Ghorbani, A., Sadri, H., Alizadeh, A.R., Bruckmaier, R.M., 2012. Performance and metabolic

TE

95, 5760–5769.

D

responses of Holstein calves to supplemental chromium in colostrum and milk J. Dairy Sci.

EP

Guo, J.-R., Monteiro, A.P.A., Weng, X.-S., Ahmed, B.M., Laporta, J., Hayen, M.J., Dahl, G.E., Bernard, J.K., Tao, S., 2016. Short communication: Effect of maternal heat stress in late

CC

gestation on blood hormones and metabolites of newborn calves. J. Dairy Sci. 99, 6804– 6807.

A

Iranian Council of Animal Care, 1995. Guide to the Care and Use of Experimental Animals, Vol. 1. Isfahan University of Technology, Isfahan, Iran.

Jovanović, L., Pantelić, M., Prodanović, R., Vujanac, I., Đurić, M., Tepavčević, S., VranješĐurić, S., Korićanac, G., Kirovski, D., 2017. Effect of peroral administration of chromium

18

on insulin signaling pathway in skeletal muscle tissue of Holstein calves. Biol. Trace Elem. Res. 180, 223–232. Kargar, S., Ghorbani, G.R., Fievez, V., Schingoethe, D.J., 2015. Performance, bioenergetic

SC RI PT

status, and indicators of oxidative stress of environmentally heat-loaded Holstein cows in response to diets inducing milk fat depression. J. Dairy Sci. 98, 4772–4784.

Kargar, S., Habibi, Z., Karimi-Dehkordi, S., 2018a. Grain source and chromium

supplementation: Effects on feed intake, meal and rumination patterns, and growth performance in Holstein dairy calves. Animal. (Accepted;

U

doi:10.1017/S1751731118002793)

N

Kargar, S., Mousavi, F., Karimi-Dehkordi, S., 2018b. Effects of chromium supplementation on

A

weight gain, feeding behaviour, health and metabolic criteria of environmentally heat-loaded

M

Holstein dairy calves from birth to weaning. Arch. Anim. Nutr. 72, 443–457. Kargar, S., Mousavi, F., Karimi-Dehkordi, S., Ghaffari, M.H., 2018c. Growth performance,

D

feeding behavior, health status, and blood metabolites of environmentally heat-loaded

TE

Holstein dairy calves fed diets supplemented with chromium. J. Dairy Sci. 101, 9876–9887.

EP

Kumar, M., Kaur, H., Sarma-Deka, R., Mani, V., Kumar-Tyagi, A., Chandra, G., 2015. Dietary inorganic chromium in summer-exposed buffalo calves (Bubalus bubalis): Effects on

CC

biomarkers of heat stress, immune status, and endocrine variables. Biol. Trace Elem. Res. 167, 18–27.

A

Morgan, M.J., Liu, Z.G., 2011. Review: Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. 21, 103–115.

Mousavi, F., Karimi-Dehkordi, S., Kargar, S., Ghaffari, M.H., 2018. Effect of chromium supplementation on growth performance, meal pattern, metabolic and antioxidant status, and

19

insulin sensitivity of summer-exposed weaned dairy calves. Animal. (Accepted; doi:10.1017/S1751731118002318) Mowat, D.N., Chang, X., Yang, W.Z., 1993. Chelated chromium for stressed feeder calves. Can.

SC RI PT

J. Anim. Sci. 73, 49–55. NRC. 2001. Nutrient Requirements of Dairy Cattle. 7th ed. Natl. Acad. Press, Washington, DC. Pantelić, M., Jovanović, L.J., Prodanović, R., Vujanac, I., Đurić, M., Ćulafić, T., Vranješ-Đurić, S., Korićanac, G., Kirovski, D., 2018. The impact of the chromium supplementation on

insulin signalling pathway in different tissues and milk yield in dairy cows. J. Anim. Physiol.

U

Anim. Nutr. 102, 41–55.

N

Pedraza-Chaverrí, J., Barrera, D., Medina-Campos, O.N., Carvajal, R.C., Hernández-Pando, R.,

A

MacíasRuvalcaba, N.A., Maldonado, P.D., Salcedo, M.I., Tapia, E., Saldívar, L., Castilla,

M

M.E., Ibarra-Rubio, M.E., 2005. Time course study of oxidative and nitrosative stress and antioxidant enzymes in K2Cr2O7-induced nephrotoxicity. BMC Nephrol. 6, 4–15.

D

Puertollano, M.A., Puertollano, E., de-Cienfuegos, G.A., de-Pablo, M.A., 2011. Dietary

TE

antioxidants: Immunity and host defense. Curr. Top. Med. Chem. 11, 1752–1766.

EP

Stahel, P., MacPherson, J.A.R., Berends, H., Steele, M.A., Cant, J.P., 2017. Short communication: Parameters of abomasal emptying and glucose-insulin dynamics in

CC

Holstein-Friesian calves at 2 ages and 2 levels of milk replacer intake. J. Dairy Sci. 100, 5068–5072.

A

Stott, G.H., Wiersma, F., Menefee, B.E., Radwanski, F.R., 1976. Influence of environment on passive immunity in calves. J. Dairy Sci. 59, 1306–1311.

20

Van den Borne, J.J.G.C., Verstegen, M.W.A., Alferink, S.J.J., Giebels, R.M.M., Gerrits, W.J.J., 2006. Effects of feeding frequency and feeding level on nutrient utilization in heavy preruminant calves. J. Dairy Sci. 89, 3578–3586.

SC RI PT

Vincent, J.B., 2014. Review: Is chromium pharmacologically relevant? J. Trace Elem. Med. Biol. 28, 397–405.

Vincent, J.B., 2015. Is the pharmacological mode of action of chromium (III) as a second messenger? Biol. Trace Elem. Res. 166, 7–12.

Yari, M., Nikkhah, A., Alikhani, M., Khorvash, M., Rahmani, H., Ghorbani, G.R., 2010.

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Physiological calf responses to increased chromium supply in summer J. Dairy Sci. 93,

A

CC

EP

TE

D

M

A

N

4111–4120.

21

Table 1 Climatic conditions during the study period and at the day of glucose tolerance test (GTT).

A

CC

EP

TE

D

M

A

N

U

SC RI PT

Study period Day of GTT Variables Minimum Average Maximum Minimum Average Maximum Temperature (T), °C 18.3 29.0 39.6 14.3 23.0 31.8 Relative humidity (RH), % 11.5 23.7 35.8 11.4 23.4 35.3 1 THI 61.5 71.3 81.3 57.8 65.8 73.8 1 Temperature-humidity index = 0.8 × maximum T + (minimum RH/100) × (maximum T − 14.4) + 46.4 (Kargar et al., 2015).

22

Table 2 Ingredients and chemical composition of the experimental starter diet on a DM basis

1Palmac®

D

M

A

N

Diet 8.0 43.9 9.2 29.4 4.7 0.5 1.7 1.1 0.5 0.5 0.5

SC RI PT

U

. Ingredient composition, % of DM Alfalfa hay Corn grain, ground Barley grain, ground Soybean meal Extruded soybean Fat supplement1 Calcium carbonate Sodium bicarbonate Mono-calcium phosphate Vitamin and mineral mixture2 Salt Chemical composition, % of DM Dry matter, % Crude protein (CP) Non-fibrous carbohydrate (NFC)3 Neutral detergent fiber (NDF) Acid detergent fiber Ether-extract (EE) Ash Calcium4 Phosphorous4 Chromium, mg/kg DM Metabolizable energy,4 Mcal/kg of DM Net energy for maintenance,4 Mcal/kg of DM Net energy for growth,4 Mcal/kg of DM

89.2 21.6 53.1 14.7 6.3 4.3 4.3 0.98 0.52 0.95 3.10 2.32 1.76

A

CC

EP

TE

80-16 (IOI Oleochemical Industries Sdn Bhd, Prai, Malaysia). Product contained: 2% C12:0, 5% C14:0, 80% C16:0, 2% C18:0, 9% C18:1, and 3% C18:2. 2Contained per kilogram of supplement: 975,000 IU of vitamin A, 750,000 IU of vitamin D, 1,800 IU of vitamin E, 143 g of Zn, 76 g of Mn, 48.6 g of Cu, 19.5 g of Se, 18.4 g of Fe, 8 g of Ca, and 1.3 g of Co. 3Non-fibrous carbohydrate = 100 - (CP + NDF + EE + ash) (NRC, 2001). 4Calculated from NRC (2001).

23

Table 3 Growth performance and physiological parameters of summer-exposed dairy calves supplemented with (Cr+) or without (Cr-) chromium.

Treatment (T) CrCr+ 0.87b 1.12a 1.35b 1.51a b 0.72 0.79a 0.53 0.52 41.25 43.00 106.83 114.39 41.4a 39.4b 39.08 39.06 1.92 1.95

SEM 0.03 0.04 0.02 0.02 0.89 2.45 0.60 0.02 0.04

1Excluding

T <0.001 0.002 0.02 0.93 0.17 0.14 0.02 0.43 0.62

P-value Week (W) <0.001 <0.001 <0.001 <0.001 — — 0.01 0.13 <0.001

T×W 0.005 0.01 0.07 0.03 — — 0.51 0.16 0.27

SC RI PT

Item Chromium intake,1 mg/d Total dry matter intake,2 kg/d Average daily gain, kg/d Feed efficiency3 Initial body weight Final body weight Respiration rate, breath/min Rectal temperature, °C Fecal score4

chromium coming from drinking water and milk. dry matter intake = milk DM + starter feed DM. 3Feed efficiency was calculated by dividing average daily gain by average total daily DM intake (milk DM + starter feed DM). 4Fecal scores were recorded as 1 = firm and well-formed; 2 = soft and pudding-like; 3 = runny and pancake batter; 4 = liquid and splatters (Kargar et al., 2018b). a,bMeans within a row with different superscripts are significantly different (P < 0.05).

A

CC

EP

TE

D

M

A

N

U

2Total

24

Table 4 Logistic model for diarrhea1, pneumonia, and medication occurrence during the pre-weaning (d 1 to 63), postweaning (d 64 to 91), and overall (d 1 to 91) periods in summer-exposed dairy calves supplemented with (Cr+) or without (Cr-) chromium.

SEM

Odds ratio2

0.0602 0.7376 0.1505

0.44 1.87 0.45

1.06 2.09 1.16

0.47, 2.42 0.36, 2.07 0.54, 2.48

0.88 0.41 0.69

0.0528 −0.8327 −0.1742

0.42 0.32 0.35

1.05 0.43 0.84

0.48, 2.33 0.09, 1.89 0.36, 1.93

0.89 0.26 0.68

−0.0296 −0.9820 −0.1923

0.25 0.26 0.23

0.97 0.37 0.83

0.57, 1.63 0.09, 1.52 0.47, 1.44

0.91 0.16 0.49

95% CI

P-value

SC RI PT

Diarrhea occurrence Pre-weaning (Cr- vs. Cr+) Post-weaning (Cr- vs. Cr+) Overall (Cr- vs. Cr+) Pneumonia occurrence Pre-weaning (Cr- vs. Cr+) Post-weaning (Cr- vs. Cr+) Overall (Cr- vs. Cr+) Medication occurrence3 Pre-weaning (Cr- vs. Cr+) Post-weaning (Cr- vs. Cr+) Overall (Cr- vs. Cr+)

Coefficient

U

Variable and comparison

score ≥ 3 (1 to 4 scale). odds ratio (OR) indicates the probability of either having diarrhea or needing medication for the Cr- diet than for the Cr+ diet. If the OR is > 1, the Cr- diet in the comparison is more likely to have diarrhea or to be medicated than the Cr+ diet by a factor of the difference above 1. If the OR is < 1, the Cr- diet has a lower probability of occurrence than the Cr+ diet. 3Medication occurrence for both diarrhea and pneumonia.

N

1Fecal

A

CC

EP

TE

D

M

A

2The

25

Table 5 Poisson regression for frequency and duration of diarrhea, pneumonia, and days medicated in summer-exposed dairy calves supplemented with (Cr+) or without (Cr-) chromium.

Treatment (T) CrCr+

P-value T

— — —

— — —

1.08 0.08 1.17

1.00 0.00 1.00

0.28 0.04 0.27

0.84 1.00 0.69

3.08 0.25 3.33

2.75 0.00 2.75

0.17 0.12 0.16

0.63 1.00 0.41

8/12 4/12 9/12

7/12 3/12 7/12

— — —

— — —

0.75 0.42 1.17

0.83 0.25 1.08

0.32 0.51 0.27

0.81 0.48 0.84

3.25 1.67a 4.92

3.42 0.75b 4.17

0.16 0.27 0.14

0.82 0.04 0.38

6.33 1.92a 8.25

6.17 0.75b 6.92

0.11 0.17 0.10

0.87 0.01 0.23

SC RI PT 8/12 0/12 8/12

N

A

M

D

TE

EP

1

SEM

10/12 1/12 10/12

U

Item Number of calves diagnosed at least once for diarrhea Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Diarrhea frequency Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Days with diarrhea Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Number of calves diagnosed at least once for pneumonia Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Pneumonia frequency Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Days with pneumonia Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Total days medicated1 Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91)

Medicated days for both diarrhea and pneumonia. within a row with different superscripts are significantly different (P < 0.05).

CC

a,bMeans

Table 6

A

Blood metabolites of summer-exposed dairy calves supplemented with (Cr+) or without (Cr-) chromium.

26

Treatment (T) CrCr+

EP

CC

T

P-value Week (W)

T×W

4.73 2.76 3.44

0.70 0.40 0.56

0.77 0.27 0.36

0.49 0.29 0.21

0.10 0.40 0.17

0.11 0.41 0.21

0.01 0.03 0.02

0.51 0.85 0.46

<0.001 <0.001 <0.001

0.46 0.55 0.69

25.72 33.23 27.95

26.67 33.46 28.70

1.70 1.98 1.37

0.72 0.93 0.72

0.02 0.61 0.006

0.81 0.50 0.91

85.50 68.89 80.45

87.65 69.70 82.28

5.15 5.75 4.16

0.78 0.92 0.77

<0.001 0.84 <0.001

0.44 0.15 0.55

31.66 30.99 30.77

30.32 27.90 29.77

3.01 2.55 2.10

0.68 0.42 0.74

0.05 0.06 0.06

0.17 0.48 0.77

6.20a 6.47 6.28a

0.12 0.13 0.10

0.02 0.07 0.008

0.008 0.49 0.01

0.59 0.60 0.76

3.23 3.37 3.27

3.29 3.45 3.34

0.05 0.05 0.04

0.51 0.33 0.32

0.15 0.16 0.11

0.37 0.10 0.41

2.47 2.72 2.54

2.91 3.01 2.94

2.69 0.13 0.13

0.07 0.18 0.06

<0.001 0.81 0.002

0.35 0.48 0.61

1.31 1.24 1.29

1.18 1.16 1.17

0.08 0.05 0.06

0.29 0.34 0.25

0.001 0.30 0.002

0.42 0.08 0.38

D

U

N

A

M

5.71b 6.08 5.81b

SC RI PT

93.52 90.60 92.59

within a row with different superscripts are significantly different (P < 0.05).

A

a,bMeans

SEM

96.23 94.39 95.66

TE

Item Glucose, mg/dL Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) β-hydroxy butyric acid, mmol/L Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Urea nitrogen, mg/dL Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Cholesterol, mg/dL Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Triglyceride, mg/dL Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Total protein, g/dL Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Albumin, g/dL Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Globulin, g/dL Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Albumin:globulin Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91)

27

Table 7 Blood hormones on d 91 in summer-exposed dairy calves supplemented with (Cr+) or without (Cr-) chromium . SEM 4.07 0.04 16.27 0.67 2.23 0.20

P-value T 0.03 0.04 0.43 0.74 0.38 0.46

within a row with different superscripts are significantly different (P < 0.05).

A

CC

EP

TE

D

M

A

N

U

a,bMeans

Treatment (T) CrCr+ 11.93b 26.07a 0.13b 0.28a 189.0 208.0 7.13 6.80 27.60 30.50 1.47 1.25

SC RI PT

Item Insulin, µIU/mL Insulin:glucose Triiodothyronine (T3), ng/dL Thyroxine (T4), µg/dL T3:T4 Cortisol, µg/dL

28

Table 8 Blood antioxidant variables of summer-exposed dairy calves supplemented with (Cr+) or without (Cr-) chromium . T

P-value Week (W)

T×W

0.52 0.19 0.45

0.57 0.27 0.49

0.098 0.032 0.071

0.79 0.11 0.68

0.40 0.06 0.12

0.17 0.34 0.10

21.21 26.64 22.63

21.08 24.28 22.53

0.743 1.206 0.625

0.90 0.28 0.90

0.004 0.88 0.004

0.01 0.70 0.06

27.34 27.02 27.03

36.03 29.31 34.00

3.929 4.394 3.274

0.17 0.74 0.18

0.32 0.31 0.48

0.15 0.91 0.30

7.14 5.72b 6.65

6.18 7.79a 6.75

0.19 <0.001 0.84

<0.001 0.002 <0.001

0.002 0.16 <0.001

U

SC RI PT

SEM

0.425 0.187 0.322

A

within a row with different superscripts are significantly different (P < 0.05).

A

CC

EP

TE

D

M

a,bMeans

Treatment (T) CrCr+

N

Item Malondialdehyde, µmol/mL Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Superoxide dismutase, U/mL Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Glutathione peroxidase, U/mL Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91) Catalase, KU/L Pre-weaning (d 1 to 63) Post-weaning (d 64 to 91) Overall (d 1 to 91)

29

A

CrCr+

240 200

SC RI PT

Glucose concentration, mg/dL

280

160 120 80

U

40

0

20

N

0 40

60

80

100

120

A

Time post-glucose infusion, min

M

B

D TE

25

EP

20

15

CrCr+

CC

Insulin concentration, µIU/mL

30

10

A

5

0 0

20

40

60

80

100

120

Time post-glucose infusion, min Fig. 1. Blood glucose (A) and insulin (B) concentrations relative to intravenous glucose tolerance test in calves supplemented with (Cr+) or without (Cr-) chromium. Data are LSM ± SEM, n = 7 per group.

30

Table 9

SC RI PT

Glucose and insulin responses to an intravenous glucose tolerance test (on d 92) in summer-exposed dairy calves supplemented with (Cr+) or without (Cr-) chromium.

A

CC

EP

TE

D

M

A

N

U

Treatment (T) P-value Item1 CrCr+ SEM T Glucose Basal level, mg/dL 82.90 82.50 2.58 0.91 Disappearance rate, %/min 0.77 1.03 0.10 0.09 T1/2, min 103.16 74.44 13.35 0.14 AUC, mg/dL × 120 min 15071 14242 915.4 0.53 Insulin Basal level, µIU/mL 15.00 15.20 4.90 0.97 Disappearance rate, %/min 3.01 3.02 0.40 0.98 T1/2, min 27.4 27.1 3.83 0.94 AUC, µIU/mL × 120 min 1072 1232 237.4 0.63 Insulin sensitivity, mg/min × µIU/mL 0.13 0.15 0.03 0.68 1 T1/2 = time to half-maximal concentration; AUC = area under the concentration-time curve.

31