Introduction

Chromium (Cr), an essential nutrient for mammalian organisms, functions as a cofactor for insulin when in the trivalent oxidation state (Cr+3). Therefore, Cr enhances the functions of insulin in cell metabolism [1]. Various trivalent chromate compounds have been used as nutritional supplements, weight-loss agents, and muscle-development agents in human and as feed additives in domestic animals [2–4]. However, responses of human or animals to Cr supplementation have been variable [5]. Moreover, it is unlikely for healthy individuals to develop Cr deficiency [6]. Thus, the use of Cr supplements is probably unnecessary for the general public. However, there are strong evidences indicating that dietary Cr addition is beneficial for people or animals under physiological, pathological, nutritional, or environmental stress [7–8]. The beneficial roles of Cr could be due to the fact that the storage of Cr in the body tends to be decreased or depleted under stress conditions [8–9]. Under stress conditions, dietary Cr supplementation has been found to promote immunomodulatory function for both innate and adaptive immune response [9]. For example, in stressed feeder calves, dietary Cr supplementation increased the level of total serum immunoglobulin, particularly IgM, and enhanced primary antibody response to human red blood cells in the model employing the injection of human red blood cells [10]. The enhanced immune responses following Cr supplementation under the conditions of stress occur in association with changed profiles of endocrine hormones (e.g., insulin and cortisol) [11].

Cr is absorbed primarily in the small intestine [12, 13]. Thus, the absorption and utilization of Cr should be dependent on its status in intestinal tracts. The factors affecting the intestinal absorption of particles include their size, nature of the polymer, zeta potential, and vehicle [14]. Particle, when its dimension is reduced to nanometer size, exhibits new electrical, magnetic, mechanical, and biological properties [15]. Previously, Cr nanoparticles (CrNano) with average particle size ranging from 40 to 70 nm was shown to have beneficial effects on growth performance, carcass characteristics, and pork quality in finishing pigs [16] and on body compositions in rats [6]. However, the effects of CrNano supplementation on hormone and immune responses under stress condition still remain unclear. Numerous studies have shown that nanoparticles are more inclined to be recognized by the immune system and ingested by immune cells such as macrophages, monocytes, and leukocytes [17]. Hence, the objective of the present study was to investigate the effects of supplementation of graded doses of Cr in the form of CrNano on the hormone and immune responses in heat-stressed Sprague–Dawley rats.

Materials and Methods

Experimental Materials and Design

The CrNano with average size ranging from 40 to 70 nm was obtained from the Key Laboratory of Molecular Animal Nutrition, Ministry of Education, China. Eighty male Sprague–Dawley rats with an average initial body weight (BW) of 60 ± 3 g were purchased from Shanghai Laboratorial Animal Center, Chinese Academy of Science (Shanghai, China).

All rats were randomly allotted to four dietary treatment groups of 20. The first group was offered a basal diet (composition is shown in Table 1) as a control. The second, third, and fourth groups received basal diet supplemented with 150, 300, and 450 μg/kg Cr, respectively, in the form of CrNano. The rats were raised in stainless steel cage with five each. The diets and ultrapure water were provided ad libitum. The study was conducted for 8 weeks, and during the experiment, the house’s temperature was measured four times a day (0600, 1200, 1800, and 2400). The mean value of daily temperature in the house was 30 ± 3°C. The environmental humidity is 40–60% and a 12-h light/dark cycle was maintained. BW and food intake were recorded once a week over the 8-week period. Average daily gain (ADG), daily food intake (DFI), and food efficiency (FE) were calculated from these records. The protocol employed in this experiment was approved by Zhejiang University Animal Care and Use Committee.

Table 1 Composition of the Basal Diets
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Blood and Tissue Sampling

At the end of the feeding trial, ten rats from each dietary treatment group were chosen to be killed by anesthetization (ketamine, 8.7 mg/100 g BW, and xylazine, 1.3 mg/100 g BW) following an overnight fast and weighed for final BW. The abdominal cavity was opened, and blood was drawn directly from the abdominal aorta. The blood sample from each rat was divided into two portions. One portion of blood sample was used to prepare for serum by centrifugation at 1,000 rpm for 10 min and the other was heparinized to further isolate peripheral blood lymphocytes (PBL) for lymphocyte proliferation assay. The PBL was prepared by using lymphocyte separation medium (Sigma, St. Louis, MO, USA), then counted and assessed for viability by trypan blue dye exclusion, and adjusted at 2 × 106 cells/mL in RPMI-1640 medium containing 20% fetal bovine serum (FBS; GIBCO, Grand Island, NY, USA).

The thymus was removed and weighed. The spleen was aseptically removed, weighed, and crushed in a homogenizer with 5 mL RPMI-1640 medium (GIBCO, Grand Island, NY, USA). The homogenate was centrifuged at 1,000 rpm for 5 min at 4 °C, the supernatant discarded, and the pellet suspended in 5 mL sterile distilled water under slight agitation for 25 s to lyse the erythrocytes. The spleen cells were then diluted in 10 mL RPMI-1640 medium and centrifuged again. The resulting pellet of lymphocytes was washed again in 2 mL RPMI-1640 medium and filtered through a gauze (80 μm) to eliminate impurities. The recovered cells were diluted in RPMI-1640 medium containing 20% FBS, counted, and assessed for viability by trypan blue dye exclusion. The spleen cells concentration was adjusted at 2 × 106 cells/mL for subsequent test.

Determination of Serum Hormone, Immunoglobulin, and Alexin

The concentrations of insulin (INS), growth hormone (GH), insulin-like growth factor I (IGF-I), thyrotropic-stimulating hormone (TSH), free triiodothyronine (FT3), free thyroxine (FT4), and cortisol in serum were determined using commercially available 125I RIA kits (Beijing North Institute of Biological Technology, China) by the procedure of radioimmunoassay on a scintillation counter (Packard 8500, USA).

The levels of immunoglobulin A (IgA), IgG, IgM, alexin 3 (C3), and alexin 4 (C4) in serum were determined by special commercial kits (Cicheng Biochemical Reagent, Ningbo, China) with the recommended procedures.

Lymphocyte Proliferation

The mitogen-stimulated T and B cell proliferations were performed in vitro with isolated PBL and splenocytes. The concentrations of 5 μg/mL of concanavalin A (ConA; Sigma, MO, USA) for T cell proliferation and 10 μg/mL of lipopolysaccharides (LPS; Sigma, MO, USA) for B cell proliferation were used. Triplicate samples of splenocytes or PBL with or without mitogen were pipetted into individual wells of a 96-well microtiter plate and incubated for 72 h in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Four hours before harvesting, 50 μL medium containing 1 μCi 3H-thymidine was added to the cultures. The 3H-thymidine uptake by lymphocytes was measured by a liquid scintillation counter (Packard 8500, USA), and mean count per minute was determined for the triplicate samples. Mean count per minute was used to calculate the stimulation indices (SI) as follows: count per minute of stimulated cell cultures/count per minute of unstimulated cell cultures.

Anti-SRBC PFC Assay

Four days before the end of the feeding trial, five rats from each dietary treatment group were immunized via injection of the lateral tail vein with 0.4 mL of 1.25 × 108 sheep red blood cells (SRBCs) per milliliter sterile saline. At the end of the experiment, the rats were killed and the spleens were removed aseptically. Spleen cell suspensions were prepared by methods as described above. Aliquots of spleen cells were added to glass test tubes held at 47°C in a constant temperature water bath. Earle’s balanced salt solution (GIBCO; containing 5% agar and 0.05% diethyl aminoethyl dextran), SRBCs, and guinea pig complement (GIBCO) were also added into the tube and mixed by vortexing. The content in each test tube was then poured into a 100-mm Costar Petri dish and covered with a 45 × 50-mm glass cover slip. The Petri dishes were incubated for 3 h at 5% CO2 and 37°C, after which the number of plaques were counted. Results were then expressed as plaque-forming cells (PFC) per 106 cells. Each sample was assayed in triplicate.

Phagocytosis of Peritoneal Macrophages

Five rats from each dietary treatment group were selected to perform assay of phagocytosis of peritoneal macrophages. On the last day of the feeding trial, 0.5 mL of 1 × 107 chicken red blood cells (CRBC) was intraperitoneally injected into each rat, and the rats were euthanized 1 h later. The fluid of the abdominal cavity was collected to make a smear for each rat. The smear were incubated at 37°C for 30 min in a wet box, fixed with 95% ethanol, and then stained by Wright dye. The number of macrophage ingesting CRBC out of a total of at least 100 cells was calculated by direct visual enumeration using a light microscope. The phagocytic index (PI) was calculated using the following formula: PI = number of total ingested CRBC/number of macrophage ingesting CRBC.

Statistical Analysis

Data are presented as the mean±standard deviation (SD). Statistical differences among the means of the various groups were evaluated by analysis of variance using generalized linear model procedures and SAS software (SAS Institute, 1989). A probability of P < 0.05 was considered significant.

Results

Growth Performance and Food Utilization

The effects of CrNano supplementation on the growth performance and food utilization of the heat-stressed rats were summarized in Table 2. No differences for growth performance and food efficiency were observed between the control group and the groups with supplementation of 150, 300, and 450 μg/kg Cr from CrNano.

Table 2 Growth Performance and Food Utilization of Heat-Stressed Male Sprague–Dawley Rats Fed CrNano for 8 Weeks
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Serum Hormone, Immunoglobulin, and Alexin

The effects of CrNano on the levels of hormones and immunoglobulins in the animal serum were determined. Dietary supplementation of 150, 300, and 450 μg/kg Cr from CrNano significantly decreased serum concentrations of insulin and cortisol and increased serum IGF-I level in heat-stressed rats (Table 3). However, concentrations of GH, TSH, FT3, and FT4 in serum were not significantly affected.

Table 3 Hormone Levels in Sera of Heat-Stressed Male Sprague–Dawley Rats Fed CrNano for 8 Weeks
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Sera IgG levels in heat-stressed rats were significantly increased by dietary addition of 150, 300, and 450 μg/kg Cr in the form of CrNano (Fig. 1). However, supplementation of Cr from CrNano did not produce significant effects on serum concentrations of IgA, IgM, C3, and C4 (Fig. 1).

Fig. 1
figure 1

Serum contents of immunoglobulin and alexin in heat-stressed male Sprague–Dawley rats fed CrNano for 8 weeks. Data are presented as the means±SD, n = 10 per treatment. Different superscript letters connote statistically significant differences (P < 0.05)

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Weights of Thymus and Spleen

As shown in Table 4, no significant effect of supplemental Cr from CrNano on the weights of the thymus and spleen of heat-stressed rats was observed.

Table 4 Weights of the Thymus and Spleen of Heat-Stressed Male Sprague–Dawley Rats Fed CrNano for 8 Weeks
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Lymphocyte Proliferation

The mitogen-stimulated T and B lymphocyte response to ConA and LPS, respectively, of both PBL and spleen cells, harvested from dietary-treated (150, 300, and 450 μg/kg Cr from CrNano) and control rats is shown in Fig. 2. With ConA and LPS, the mean lymphoproliferative response was significantly elevated in dietary Cr-treated rats. In addition, the ConA-stimulated response of spleen lymphocytes was elevated in a Cr dose-dependent manner.

Fig. 2
figure 2

Stimulation indices with T or B cell mitogen stimulation of peripheral blood and spleen lymphocytes in heat-stressed male Sprague–Dawley rats fed CrNano for 8 weeks. Data are presented as the means±SD, n = 10 per treatment. Different superscript letters connote statistically significant differences (P < 0.05). ConA-PBL ConA-stimulated PBL, LPS-PBL LPS-stimulated PBL, ConA-SL ConA-stimulated spleen lymphocyte, LPS-SL LPS-stimulated spleen lymphocyte

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Anti-SRBC PFC Response

The antibody response of spleen cells isolated from dietary Cr-treated rats to SRBC was significantly higher than that of those isolated from control rats (Fig. 3). In addition, treatment of heat-stressed rats with Cr from CrNano appeared to improve the PFC response in a dose-dependent manner.

Fig. 3
figure 3

Anti-SRBC response in heat-stressed male Sprague–Dawley rats fed CrNano for 8 weeks, as determined by the PFC assay. Data are presented as the means±SD, n = 5 per treatment. Different superscript letters connote statistically significant differences (P < 0.05)

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Phagocytosis Ability

The phagocytic activities of macrophages isolated from both control and dietary Cr-treated rats were assessed (Fig. 4). The uptake capacity of peripheral phagocytes was increased by CrNano treatment. Although the PI was increased significantly by treatment of 150, 300, and 450 μg/kg Cr as CrNano, PI with treatment of 450 μg/kg Cr was slightly lower than that with treatment of 300 μg/kg Cr.

Fig. 4
figure 4

PI of CRBC by peritoneal macrophages from heat-stressed male Sprague–Dawley rats fed CrNano for 8 weeks. Data are presented as means±SD, n = 5 per treatment. Different superscript letters connote statistically significant differences (P < 0.05)

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Discussion

Two major types of trivalent Cr compounds, i.e., the organic and inorganic Cr compounds, have been used as nutritional supplements in human and feed additives in animals. Although the positive effects of dietary supplementation of these Cr compounds have varied among reports, it is generally believed that inorganic Cr salts are not as biologically active as organic forms of Cr [5]. The underlying reason could be the superior absorptivity of organically complexed Cr [5, 7]. It is known that nanoparticles, for their small size effects, are shown to exhibit greater absorption rate both in vivo and in vitro in comparison to macroparticles [18–19]. The beneficial effects of CrNano on growth performance, carcass characteristics, and pork quality in finishing pigs [16] and on body compositions in rats [6] have attributed to its superior absorptivity [20]. High ambient temperature is considered to be a physiological stress which promotes the loss of body Cr store [8–9]. Therefore, the supplementation of Cr with high absorptivity might contribute to alleviate the negative effects of unfavorable conditions on animal growth.

In the present study, no change in growth rate and food efficiency was observed in the heat-stressed rats with dietary treatment of CrNano. The effect of CrNano on growth performance in rats reported in this study is inconsistent with our previous 6-week study under no heat stress condition in which both average daily gain and food efficiency were improved by the supplementation of 300 μg/kg Cr from CrNano [6]. In fact, supplementation of bioavailable source of Cr is not uniformly efficacious on growth performance. Hasten et al. [21] observed no change in growth rate in Sprague–Dawley rats fed basal diets supplemented with Cr chloride, Cr picolinate, and Cr nicotinate. Results from Van de Ligt et al. [22] and Lien et al. [23] showed that Cr (in the form of Cr tripicolinate and propionate, respectively) supplementation produced no effect on growth rate and feed efficiency in pigs during the postweaning period. However, Barajas et al. [24] reported that supplementation of Cr methionine improved average daily gain and feed/gain ratio of transport-stressed bull calves. Therefore, no definite conclusion on the effect of Cr supplementation on growth performance can be drawn from these existing studies. In addition, the effect of CrNano supplementation on growth performance in physiological and heat-stressed conditions remains to be further investigated.

Cr is usually claimed to be associated with the function of insulin [1]. In the present study, dietary addition of 150, 300, and 450 μg/kg Cr from CrNano significantly decreased the serum insulin levels in heat-stressed rats. This is in agreement with our previous results of CrNano in finishing pigs [25] and rats [6] experiencing no external stresses. Under no externally applied stress conditions, Cr picolinate supplementation was found to decrease sera insulin levels in pigs [2, 26], but not in rats [21, 27]. The decrease of peripheral insulin levels may be due to the enhanced binding of insulin to its receptors on targeted tissues such as muscle and fat since Cr is thought to facilitate the interactions between insulin and insulin receptors and promote insulin internalization into cells [1]. Insulin causes cellular uptake and use of glucose after binding to its receptor. Once acquired by cells, glucose is used as an energy source which, along with the anabolic actions of GH and IGF-I, drives protein synthesis and proper maintenance and functioning of all the body’s organs [9]. The elevated serum IGF-I level measured in heat-stressed rats offered the diets with supplemental CrNano may have played a role in the protein and energy metabolism. The lack of any significant effect of CrNano on serum GH in heat-stressed rats, however, suggests that the effect on IGF-I may have been independent of GH though a single blood sample is not a sensitive enough technique to assess treatment effects on GH given the pulsatile nature of GH release in most mammals [25, 28].

Cortisol is generally regarded as a calorigenic agent in many mammalian species and acts antagonistically to insulin because it prevents entry of glucose into peripheral tissues (e.g., muscle and fat) to spare it for tissues of higher demand (e.g., brain and liver) [9, 11]. Cold and heat exposure, isolation, strenuous exercise, disease, and numerous other forms of emotional, physical, metabolic, and environmental stresses increase plasma cortisol. Supplemental Cr has been demonstrated to reduce serum cortisol in pigs [25], guinea pigs [29], lambs [30], horses [31], calves [32], and humans [33]. In vitro, high concentration of Cr from Cr picolinate can directly inhibit cortisol secretion in agonist-stimulated adrenocortical cell line [34]. The present result is in line with these reports, indicating that Cr from CrNano may also have an “antistress” effect by lowering plasma cortisol. However, there are also a number of reports indicating no effect of Cr supplementation on serum cortisol levels [35–37]. The disparity of response may be related to the level and type of stress experienced by the animals. Furthermore, the ultimate target of dietary Cr supplementation may be the dynamic balance of cortisol, insulin, and probably, other endocrine modulatory effects [11].

Less data is available about the effects of Cr on thyroid hormone which increases metabolic rate causing increased oxygen consumption, heat production, and metabolism of fats, proteins, and carbohydrates in the liver, kidney, heart, and muscle [9]. In this study, no significant effect of supplemental Cr from CrNano on TSH, T3, and T4 were observed in heat-stressed rats, indicating a lack of role of CrNano in the metabolisms of thyroid hormones.

Dietary supplementation of 150, 300, and 450 μg/kg Cr from CrNano has not affected the relative weights of the thymus and spleen. In addition, no macroscopic physical damage was observed during the experiment. This indicates that the dosage of CrNano used in this study may have no negative toxic effect on rats.

The documented effects of dietary Cr supplementation on immune response in mammals have been inconsistent. The increased serum IgG level in heat-stressed rats in the present study is in line with our previous result in finishing pigs experiencing no external stress [25], and with Chang and Mowat [10] and Moonsie-Shageer and Mowat [37] who demonstrated that total IgG was increased in calves supplemented with high-Cr yeast under transport stress. These studies also indicated that total IgM was elevated by supplemental Cr. However, in this study, serum total IgM was unaffected by supplementation of any dosage of Cr from CrNano. Kegley et al. [38] observed no change in total IgM but a decline in total IgG in transport-stressed calves fed diets supplemented with Cr nicotinate. The effect of supplemental Cr on serum total immunoglobulin may be associated with the experimental period. Van de Ligt et al. [22] have studied the effect of Cr tripicolinate supplementation of diet on porcine immune response during the postweaning period and found that total IgG was not affected by Cr on day 0 or on day 7, but on day 28, total IgG had risen and was affected by Cr, and total IgM followed a similar pattern.

The effect of Cr on lymphocytes has been investigated in several studies. Lymphocyte blastogenesis in response to phytohemagglutinin (PHA) in stressed calves receiving supplemental Cr nicotinate [35] and to ConA in morbid calves receiving supplemental chelated Cr [39] was found to be increased. Burton et al. [40] reported that supplementation of chelated Cr in diets of dairy cows resulted in an increase in lymphocyte blastogenesis of 57% during late gestation and parturition and of 31% at the time of peak milk yield. In this study, the lymphoproliferative response of PBL and splenocytes to ConA and LPS were significantly elevated in heat-stressed rats offered diets supplemented with Cr from CrNano, indicating that CrNano may have an immunostimulatory effect. However, Van de Ligt et al. [22] reported that no difference was detected in ear-swelling in response to the PHA mitogen in weanling pigs. Van Heugten and Spears [41] indicated that Cr chloride, Cr nicotinate, and Cr picolinate supplementation in weanling pigs enhanced lymphocyte blastogenesis in response to pokeweed mitogen, but not in response to PHA. Thus, the varied effects of Cr on lymphocyte may be associated with the differences of animals, mitogens, and stress levels used among reports.

The PFC assay allows the evaluation of immunomodulating compounds and presents the appropriate conditions to emphasize changes in immune functions [42]. In this study, dietary supplementation of Cr from CrNano significantly increased antibody production in response to SRBCs. This is in accordance with previous reports of increased antibody production in response to human red blood cells and ovalbumin in stressed calves with supplementation of high-Cr yeast [37, 43]. In contrast to these results, antibody production in response to ovalbumin when measured 14 days following immunization was decreased by dietary supplementation of Cr nicotinate to weanling pigs [41]. However, dietary addition with Cr chloride or Cr picolinate did not affect antibody response to either ovalbumin or SRBCs in weanling pigs [41]. These results indicated that different forms of Cr may have varied in their immunomodulatory roles. In addition, the immunomodulatory role of Cr seems to be antigen-dependent.

Phagocytes acts as regulator and effector cells in the immune system and phagocytosis represents the indispensable step of the immunological defense system [44]. Lee et al. [45] have shown a dose-dependent effect of Cr picolinate on phagocytosis of Escherichia coli by incubation of pulmonary alveolar macrophages in medium in the presence or absence of insulin. Our results showed that dietary supplementation of 150, 300, and 450 μg/kg Cr from CrNano enhanced the phagocytosis of peritoneal macrophages, indicating immunostimulatory effects. However, 450 μg/kg Cr from CrNano showed a slightly lower PI than 300 μg/kg Cr from CrNano. It suggested that high dosage of CrNano may have immunosuppressive effect on macrophages, which should await further verification.

The apparent effect of CrNano on immune status in the heat-stressed rats in this study may be explained by the notion that nanoparticles are inclined to be recognized by the immune system in animals and humans [17]. Evidence has shown that nanoparticles can be absorbed via gastrointestinal lymphatics and enhance the duration of immune response and concurrently stimulate humoral, cellular, and mucosal immune responses [46].

Conclusion

The present endocrine and immune parameter results suggest that dietary chromium supplementation in the form of chromium nanoparticles affects hormone and immune status in heat-stressed rats. Although the underlying molecular and cellular mechanism is not clear, our study indicates that the use of chromium nanoparticles might promote the healthiness of animals under stress conditions.