Introduction

Weaning of dairy calves is bound with a concurrent subjection of calves to  a  range of environmental stressors. Behavioral and physiological responses to weaning play a significant role in the well-being of calves. The development of weaning protocols aimed at precluding such stressors must be supported based on the scientific knowl- edge of the morphological,  physiological and psychological mechanisms. Despite in- creasing the reproductive and productive performance of the herd, sudden weaning is a source of stress for the calves (Ungerfeld et al., 2011). Usually, weaning involves chang- es in the social and physical environment, which in turn causes stress for calves (En- ríquez et al., 2011). Different weaning meth- ods have been proposed to avoid the impact of weaning on behavior, performance and well-being. Around weaning time animals are susceptible to different pathogens. An- tibiotics have been used to overcome such issues. However, the use of antibiotics in an- imal husbandry has certain limitations due to the antibiotic resistance to microorganisms. To replace calf feeds with antibiotics, many additives have been proposed, one of which is probiotics (Economou and Gousia, 2015). Probiotics are microbial food supplements which improve host interstitial microbial balance (Musa et al., 2009) and are alterna- tives to antibiotics in reducing diarrhea and improving immunity in calves (Heinrichs et al., 2009). Markowiak and Śliżewska (2018) reported that probiotics can enhance the weight gain of calves and piglets. Potential probiotic strains  are  normal  inhabitants  of a gut and have the ability to adhere to and colonize the epithelial cells of the gut (Musa et al., 2009). Probiotics dietary supplementa- tion can significantly ameliorate feed intake

and conversion rate, and daily weight gain in sheep, goat, cattle, pig, and horses (Casey et al., 2007; Chiofalo et al., 2004; Gobesso et al., 2018; Adriani et al., 2016; Torres-Rodri- guez et al., 2007). Probiotics can affect ani- mal health by competing against pathogens for colonization sites and nutritional sources and the production of toxics or stimulation of the immune system (Rai et al., 2013). These supplements have been associated with im- provement in the immune system, and the stimulation of non-specific immune respons- es (Rai et al., 2013). The probiotics that in- crease immunoglobulin levels have a more positive influence on growth performance and the ability to resist disease (Roselli et al., 2017). A major convenience of such supple- ments is a proliferative effect on beneficial intestinal bacteria. Some gut microflora have been described to have positive effects  on the whole body, including improved weight gain and immune function and decreased presence of pathogens (Liao and Nyachoti 2017). There are numerous growth promoter substances supplemented with animal feed improve animal production  and  potential-  ly reduce the cost of animal management. These substances contain antibiotic growth promoters, such as flavomycin, probiotics, acidifiers, enzymes, herbal products, be- ta-agonists, microflora enhancers and im- mune-modulators. Probiotic preparations have shown encouraging results in different animal production areas. Generally, probiot- ics can be added to feed or water as mono  or mixed cultures of live microorganisms (Todorov et al., 2008). Chromium (Cr) is an essential trace mineral participating in the metabolism of carbohydrates, lipids, proteins and nucleic acids (Li et al., 2013). The role of Cr has been researched for potential use as

feed additives in animal production. Chro- mium deficiency in farm animals grows during physiological and environmental stress (Ohh and Lee 2005). Bailey (2014) indicated that chromium enrolls an import- ant act in glucose metabolism and immuno- modulation, which can, conclusively, affect animal health and performance. Previous studies revealed that proliferative activi- ties in peripheral blood cells from chelated chromium supplemented calves were higher than those from calves fed inorganic chro- mium (CrCl3); also, they noticed the effect of chelated chromium on  the  non-specif- ic immune response regarding neutrophil phagocytosis in dairy cows and chromium was able to enhance the humoral immune response. IL-2, TNF-α and INF-γ concen- trations in the blood of periparturient cows with chelated chromium supplemented diet were significantly decreased (Al-Saiady et al., 2004). The objective of this study was to determine the beneficial effects of dietary supplementation of probiotic (Protexin) and chromium-methionine chelate (Cr-Met) on triiodothyronine (T3), thyroxine (T4), total protein, albumin, zinc, and growth body- weight gain in dairy Holstein calves after and before weaning.

Materials and Methods

Animal ethics

All animal experiments were  approved  by the State Committee on Animal Ethics, Shiraz University, Shiraz, Iran (IACUC no: 4687/63). We further followed the recom- mendations of the European Council Direc- tive (86/609/EC) of November 24, 1986, re- garding the standards of protecting animals used for experimental purposes.

Animal management

In this work, 28 dairy Holstein calves with

an initial body weight of 93.46 kg ±11.82 (mean±SD) were selected in a completely random design (n=7) and placed in one of the four experimental groups (from 21 days before weaning to 21 days after). The exper- imental groups (G) were comprised of (G1) control (G2) Protexin (G3) Cr-Met (G4), and Protexin + Cr-Met. All experimental animals were weaned on an average 70 days of life according to the farm policy. This experi- ment was carried out in Fars province of Iran from May to September 2016. The animals were kept in an individual pen and fed starter ration formulated according to NRC (2001) requirements and water ad libitum. The type of probiotic used in our study is a commer- cial multistrain symbiotic called Protexin (Probiotics International Ltd., South Peth- erton, UK). It contains the following strains of probiotics and prebiotics: Lactobacillus plantarum, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, Lac- tobacillus rhamnosus, Bifidobacterium bifi- dum, Streptococcus salivarius ssp. thermo- philus, Enterococcus  faecium, Aspergillus oryzae, and Candida pintolopesii. We used a recommended dose of the product, which is 2 gr/calf daily. Chromium-methionine che- late (Cr-Met) was obtained from ZINPRO company and we used a recommended dose of the product (2 gr/calf daily). Fresh water was readily available at all times. The study started 21 days before weaning (average 70 days). All the calves received supplements in milk, before weaning, and 15 ml was add-  ed to water and drainage by syringe in their mouth to ensure that they have received sup- plements daily for 3 weeks after weaning.

Diet

Fed starter chemical analysis and feedstuff composition are presented in Table 1.

Table 1. Chemical analysis for treatment diets and feedstuff composition for farm mixed starter

* Each kg of vitamin premix provided (mg/kg), iron 30.0; manganese, 150.0; cobalt 3; iodine, 0.0; selenium, 0.5;vitamin A, 25,000 IU/kg; vitamin D, 5,000 IU/kg; vitamin E, 200 IU /kg.

Blood sampling

Blood samples were taken from the jugu- lar vein from all calves at the same time in  the day into plain vacutainer tubes, 21 days before weaning and 3, 7 and 21 days after weaning. The collected blood samples were quickly kept in an ice pack and sent to the laboratory. Samples were rapidly centrifuged at 3,000 rpm for 15 min. Sera were harvested and aliquots were stored at -20 ℃ until me- tabolites analysis.

Measurement of the biochemical parameters Serum T3 was measured using a competitive enzyme immunoassay kit (Padtan Elm Co., Tehran, Iran). The intra-  and inter-assay CVs of the assays were 12.6% and 13.2%, respec- tively. The sensitivity of the test was 0.3 nmo- l/L. Serum T4 was specified using a competi- tive enzyme immunoassay kit (Monobind Inc., Lake Forest, CA, USA). The intra- and inter- assay CVs of the assays were 3.0% and 3.7%, respectively. The sensitivity of the test was 5 nmol/L. Serum was analyzed for total protein by Biuret method (Commercial kit; Pars Az- moon, Tehran, Iran) and for albumin by the bromocresol green method (Commercial kit; Pars Azmoon, Tehran, Iran). Zinc serum was analyzed by atomic absorption spectrophotom-

etry (Shimadzo AA-670, Kyoto, Japan).

Statistical analyses

Data were expressed as the mean ± devia- tion (SD). A repeated measures linear mixed model was used to compare the mean con- centrations of different serological factors within similar weeks among the four differ- ent experimental groups. Statistical analy- sis was achieved using SPSS 16 (SPSS Inc., Chicago, Illinois). The level of statistical significance was set at P-value<0.05.

Results

Weight gain

The effect of treatment group  and  time on the weight gain is shown in Table 2. The effect of time was observed on the mean of weights (P<0.05), yet no effect of different diets and no interaction between different di- ets and time was found (P>0.05).

Average weight gain was at its lowest seven days before weaning (93.4± [88.7- 98.1(CL)] kg) and at its highest seven days after weaning (132.8± [126-139.7(CL)] kg).

Total protein and albumin

The effect of different   diets  and  time  on the concentration of Total protein and albumin are shown in Figures 1 and 2, re- spectively. No effect of different diets and

time was found (P>0.05) for total protein and albumin and no interaction between dif-

ferent diets and time was observed (P>0.05).

Table 2. Mean (± SEM) weight (kg) 7 days before weaning, at wean- ing and 7 days after weaning n=7 each group, * P-value<0.05

Groups

day                                         -7                                                         0                                      7

           
Control                                                         97.57±0.6                                          109.28±0.8                      137.14±0.5

Probiotic                                                        94.14±0.5                                          106.42±0.6                      133.57±0.6

           
Probiotic +Cr-Met                                                93.42±0.6                                          102.85±0.6                       98.57±0.8

Cr-Met                                                         88.71±0.7                                           98.57±0.4                       129.71±0.8

Figure 1. Mean (± SEM) serum concentrations of total protein (gr/dL) 21 days before weaning and 3, 7 and 21 days after weaning n=7 each group, * P-value

Thyroid hormones

The effect of treatment groups and time on the concentration of thyroid hormones is shown in Figures 3 and 4, respectively. An effect of different diets and time on the mean T3 con- centration was observed (P<0.05) but no inter- action between group and time on the mean T3 concentration was observed (P>0.05).

The mean T3 concentration was at its low- est in Protexin + (Cr-Met) (2.7± [2.5-3(CL)]

µg/L) group, the group where the mean T3 concentration was lower than the control and Protexin groups (P<0.05).

Figure 2. Mean (± SEM) serum concentrations of albumin (gr/dL) 21 days before weaning and 3, 7 and 21 days af- ter weaning (n=7) in each group, * P-value

The mean T3 concentration was at its maximum 21 days before weaning (3.6± [3.4-3.7(CL)] µg/L). The effect of time was observed on the mean T4 concentration (P<0.05) and an interaction between differ- ent diets and time on the mean T4 concentra- tion was observed (P<0.05).

The mean T4 concentration was at its highest 21 days before weaning (11.1± [102.12(CL)] µg/L).

Zinc

The effect of treatment groups and time on the concentration of Zinc is shown in Fig-

ure 6. The effect of time was observed on the mean zinc concentration (P<0.05) (see Fig- ure 5) and an interaction was seen between different  diets  and time (P<0.05). The mean

Figure 3. Mean (± SEM) serum concentrations of triiodo- thyronine (T3) (µg/L) 21 days before weaning and 3, 7 and 21 days after weaning (n=7) in each group, * P-value<0.05.

zinc concentration was highest, 21 days after weaning (0.25± [0.22-0.28(CL)] mg/L), at which point, it was higher compared with 3 and 7 days after weaning (P<0.05).

Figure 4. Mean (± SEM) serum concentrations of thyrox- ine (T4) (µg/L) 21 days before weaning and 3, 7 and 21 days after weaning (n=7) in each group, * P-value<0.05.

Figure 5. Mean (± SEM) serum concentrations of zinc (mg/L) 21 days before weaning and 3, 7 and 21 days after weaning (n=7) in each group, * P-value

Discussion

The results of the current study showed that there was no effect of different diets on average weight gain in calves fed chromium methionine (Cr-Met) and probiotics or both. Kegley et al. (2000) showed that beef steers

fed chromium methionine chelate did not show any additional improvement on growth performances and feed efficiency compared with steers that were not supplemented with dietary chromium; on the other hand, chro- mium supplementation enhanced a glucose

clearance rate and increased a serum insulin concentration of growing steers following intravenous insulin and glucose injection. In another study, Korean native steers supple- mented with chromium methionine showed improvement in feed efficiency during growth periods; however, no difference was discovered in daily gain and dry matter in- take although serum insulin concentration was the highest in chromium  supplement- ed steers (Lee et al., 2016). This means that chromium methionine chelate has the poten- tial to induce a positive effect on nutrient uti- lization in a relatively well-nourished animal without any adverse effect on growth per- formances. Lashkari et al. (2018) described that the daily gain in stressed feeder calves fed chelated chromium supplemented diet, was increased in the feedlot due to a notably reduced morbidity compared to calves with no chromium supplementation. But, anoth- er chelated chromium supplementation for stressed steers was not able to improve the growth performance and feed competency during growth, finishing and overall periods (De Oliveira et al., 2016). Therefore, it is still uncertain how a chelated chromium supple- mentation for growing cattle and calves does react under stressful states like transportation and weaning. The study by Roodposhti and Dabiri (2012) revealed that the average daily weight gain was greater in calves fed probi- otics, prebiotics, and synbiotics at weeks 6,  7 and 8. Lesmeister et al. (2004) reported improvements in average daily weight gain (ADG) when a 2% supplemental probiotic was added to a calf diet. Probiotic bacteria also increased weight gain and feed efficien- cy in calves (Angelakis, 2017). The results of our study showed that experimental an- imals did not have zinc deficiency, T3 and T4 were in the range of the reference values.

López et al. (2018) reported that T3 and T4 normal values in calves under 3 months are 1.50 ± 0.48 (ng.ml-1) and 8.21 ± 2.10 (μg.

dl-1), respectively. Paulazo et al. (2019) in- dicated that experimentally induced zinc de- ficiency may decrease circulating TSH and T4 levels and Zinc may alter the binding of thyroid hormones. Concentrations of thyroid hormones are also influenced by such factors as fat- or starch-enriched diet (Greco et al., 2015). Pattanaik et al. (2001) observed re- duced levels of circulatory thyroid hormones in sheep and goats when fed with protein-re- stricted diets. Accordingly, because the ani- mals of the present study did not have a pro- tein deficiency, their thyroid hormones were in a normal range. Another study by Barman et al. (2019) showed a trend towards reduced serum thyroid hormones in calves fed lower dietary protein levels, further pointing to a positive relationship between protein nutri- tion and thyroid status. Moreover, with the advancement of age, the T3 level in serum also increased. Since the animals were in the active growth phase, the thyroid gland must have been activated to secrete more T3 and T4, boosting their levels in the blood (Ha- jimohammadi et al. 2015; Constable et al. 2017). We observed that the total protein and albumin were in reference values and there was no effect of different diets and time on mean TP and albumin concentration. Total protein and albumin follow the availability of protein and their concentration reduced in the face of protein deficiency (Sangpuii et al., 2019). Albumin has a practically short half- life and can reflect protein deficiency prob- lems over a duration of one or two months. We suggest that experimental animals do not have protein deficiency issues.

In conclusion, separate and mix feeding of dairy Holstein calves with chromium methi-