Introduction

Zinc is a cofactor of many catalytic enzymes in all living cells and is an important nutrient involved in cell division. It plays roles in growth and organ development especially in pregnant women and infants [1,2,3]. Zinc deficiency during pregnancy can lead to miscarriage, intrauterine growth retardation, deformity in the fetus, and even the occurrence of stillbirths. Infants and young children with congenital Zn deficiency are predisposed to anorexia, inattention, weight loss, retardation, reduced intelligence, and immune competency [4, 5].

Zn deficiency causes a variety of health problems that include growth retardation, immunodeficiency, hypogonadism, and neuronal and sensory dysfunctions [6]. Dietary supplementation of Zn can effectively raise Zn content in tissues and serum, and serum alkaline phosphatase (AKP) activity [7, 8]. The effectiveness of Zn supplementation is affected by its chemical form, and the bioavailability of organic Zn is higher than that of inorganic Zn in animals [9]. Hence, yeast-Cu2+, yeast-Zn2+, and yeast-Cd2+ products have been intensively studied in animal models [10]. Bacillus subtilis has a very strong capacity to bind metal elements (including Zn Cu, Fe, Mn) [11], so metal elements are enriched and Bacillus subtilis-metals can be a new source of metal supplements. For example, Zhao et al. showed that Bacillus subtilis can enrich divalent Zn ions, converting inorganic Zn to organic Zn [12]. However, as a relatively new product, there is few reports on the assessment of Bacillus subtilis-Zn in animal production. Thus, this study produced congenital Zn-deficient rats as the research object to evaluate the effects of administration of three dosages of Bacillus subtilis-Zn on feed intake, growth rate, organ development, nutrient utilization efficiency, Zn contents in internal organs in rats, ileum morphology, and microbial diversity in cecal contents. On the basis of those results, the optimal level Bacillus subtilis-Zn in the diet was defined, the effects of this optimum level were compared with those by the recommended dose of CuSO4, and it was concluded that it is possible to reduce Cu level in the diet by using Bacillus subtilis-Zn.

Materials and Methods

Rats and Experimental Design

Trial 1: Establishment of a congenital Zn-deficient rat model

A total of 72 female pregnant SD rats at the same age (7 weeks old) were allocated to two groups. One group was fed a basal diet deficient of Zn (13 mg Zn per kg air-dried diet), and the other group (the control group) was fed the basal diet supplemented with ZnSO4 for the total Zn content at 38 mg/kg diet, referred as Zn standard diet as recommended by AIN-93 [13]. Either group had six replicates (cages) with six rats per replicate. The feeding trial started from 10 days of pregnancy of the rats and continued until the end of lactation when the offspring rats were at 24 days of age.

The basal diet was formulated to meet the nutrient, except for Zn, requirements of rats according to the AIN-93 standard. The ingredients and nutrient compositions of the basal diet are shown in Table 1. The Zn content in the basal diet was measured using a plasma emission spectrometer, and the content was 13 mg/kg diet, referred to Zn-deficient diet.

Table 1 Ingredients and nutrient compositions of the basal diet (air-dry basis)
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During the experimental period, rats accessed freely to the feed and fresh drinking water (deionized water). The room temperature of the animal house was kept at 22 ± 2 °C, the relative humidity at 55 ± 5%, and good ventilation. The rats were exposed to natural day/night cycles.

On the last day of the feeding trial, the offspring rats were fasted overnight, and the total body weight of rats for each replicate was recorded. Then a total of 12 offspring rats from either group, two from each replicate, were randomly selected for blood sampling from the eyeball. The serum was harvested by centrifugation at 1000×g for 15 min and stored at – 80 °C for analyses of biochemical indicators later on.

Trial 2: Bacillus subtilis-Zn intervention in Zn-deficient rats

A total of 18 female offspring rats from the control group in trial 1 were randomly selected and used as a control group (group I) to feed the Zn standard diet. Ninety female offspring rats from the Zn deficiency group in trial 1 were randomly selected and divided into five groups, every group 18 rats. Group II was fed the basal diet drenched with ZnSO4 solution to provide Zn equivalent to the total Zn content at 38 mg/kg diet, group III fed the basal diet only (Zn deficiency group), and groups IV, V, and VI were fed the basal diet drenched with Bacillus subtilis-Zn to provide extra 2, 17, and 32 mg/kg Zn respectively. Each group had three replicates (cages), six rats in each replicate. Bacillus subtilis-Zn was provided by the Institute of High Quality Waterfowl, Qingdao Agricultural University, which contains Bacillus subtilis 3 × 109 CFU/g and Zn 2511.55 mg/kg. The product was suspended in deionized water and drenched daily at 2 mL/100 g body weight of the rat. The Zn level in the basal diet was actually measured, and Zn contents in the other diets were calculated.

Measures, Sample Collection, and Analyses

The experiment started when the offspring rats aged 24 days, and lasted for 5 weeks; the feeding and management protocols were the same as in trial 1. The amounts of feed offered and the residue were recorded weekly for very replicate, and the voluntary feed intake (ADFI) was calculated. The initial and final body weights for each replicate were recorded after overnight fasting, and the body weight during the experimental period was recorded at weekly intervals for calculation of average daily weight gain (ADG).

In the last week of the experiment, all rats were transferred to the metabolic cages for measuring nutrient digestibility and utilization efficiency. The trial lasted for 4 days, the first day was for acclimation, and the last three days for collections of the fecal and urine excretions, as well as recording the feed intake. For each cage of rats, the feces and urine were daily collected into a container pre-added with 10 mL of 10% hydrochloric acid. The excrete over three consecutive days were weighed, pooled, and sampled to determine nitrogen (N) and Zn concentrations.

On the last day of the experiment, two rats from each replicate cage were randomly selected (a total of 6 rats for each group), and after overnight fasting, their body weights were recorded. Then the rats were euthanatized with ether. A blood sample was taken from the eyeball. Serum was harvested by centrifugation at 1000×g for 15 min and stored at – 80 °C until analysis later on.

The internal organs, heart, liver, spleen, kidneys, and lungs of rats were taken out, and their weights were recorded after drying excess moisture on their surfaces using filter paper. The fresh weights were used for calculation of the organ index as follows:

The organ index (%) = the fresh weight of organ (g) / live body weight (g) × 100%.

Nitrogen in the feed, feces, and urine was measured with a FOSS TECATOR Kjeldahl analyzer. Either extract in the diet was determined by ether extraction using a SZC-C fat analyzer. Zinc content in feed was measured using inductively coupled plasma mass spectrometry (ICP-MS).

The internal organs were dried at 105 °C and the dry weight was recorded. The organ was digested by wet digestion for measuring the contents of Zn by ICP-MS. The procedure is the following: About 0.5 g dried organ sample was weighed into an Erlenmeyer flask. Then 10 mL of nitric acid and 2 mL of perchloric acid were added and mixed. The flask was covered with glass and heated to 180 °C until the organic material was fully digested. The residual solution was then fully transferred into a 50-mL volumetric flask, and the volume was made up with deionized water. The solution was used for determining Zn concentrations.

Alkaline phosphatase activity (AKP), metallothionine, and Cu-Zn SOD activity in serum were determined using commercial kits (Nanjing Jiancheng Institute of Biological Engineering, China) following the manufacturer’s instructions.

The ileum tissue was fixed using 10% formaldehyde for 48 h. After removing the impurities such as fat, the fixed tissue was dehydrated using ethanol, xylene transparent, paraffin-embedded, sliced (LEICA RM2235), and finally stained with hematoxylin-eosin (HE). The ileum morphology was examined under OLYMPUS microscope (DP2.0), and the villus height and crypt depth were measured with a microscopic micrometer [14].

The microbiota diversity analysis on the caecum content samples was based on the Illumina HiSeq sequencing platform, using a Paired-End sequencing method to construct a small fragment library for sequencing, and performed Alpha Diversity and genus dominant microbial analysis [15]. The specific method is as follows:

Microbial DNA in the cecal content was extracted using E.Z.N.A.® Stool DNA Kit (Tiangen Biochemical Technology Co, China), following the manufacturer’s instructions. The quality and quantity of genomic DNA were assessed with a nanodrop spectrophotometer, with the A260/A280 ratio between 1.8 and 2.0 considered the criterion for the quality control. No obvious RNA banding was shown by gel electrophoresis, and genomic bands were clear and complete. DNA was frozen at – 80 °C prior to PCR amplification.

To amplify the V3-V4 region of 16S rRNA for Illumina deep sequencing, universal primers, 338F:5′-ACTCCTACGGGAGGCAGCA-3′ and 806R:5′-GGACTACHVGGGTWTCTAAT-3′, were used. The PCR was performed in a total reaction volume of 20 μL: H2O 13.25 μL, 10 × PCR ExTaq buffer 2.0 μL, DNA template (100 ng/mL) 0.5 μL, prime1 (10 mmol/L) 1.0 μL, prime2 (10 mmol/L) 1.0 μL, dNTP 2.0 μL, and ExTaq (5U/mL) 0.25 μL. After an initial denaturation at 95 °C for 5 min, amplification was performed by 30 cycles of incubations for 30 s at 95 °C, 20 s at 58 °C, and 6 s at 72 °C, followed by a final extension at 72 °C for 7 min. Then the amplified products were purified and recovered using a 1.0% agarose gel electrophoresis method. Finally, the library construction and sequencing steps were performed by Beijing Biomarker Technologies Co. Ltd. (Beijing, China).

The bioinformatic analysis on the microbial in the cecal content was completed at the Biomarker Biocloud Platform. To obtain the raw tags, paired-end reads were merged by FLASH [16]. Then raw tags were filtered and clustered in the next steps. The merged tags were compared to the primers, and the tags with more than six mismatches were discarded by FASTX-Toolkit [17]. Tags with an average quality score < 20 in a 50-bp sliding window were truncated using Trimmomatic and tags shorter than 300 bp were removed. We identified possible chimeras by employing UCHIME, a tool included in mother [18]. The denoised sequences were clustered using Qiime UCLUST module and tags with similarity ≥ 97% were regarded as an OTU. Taxonomy was assigned to all OTUs by searching against the Silva databases using the RDP classifier within QIIME [19, 20].

Statistical Analysis

Statistical analysis of the data was performed using SPSS 17.0 software. In trial 1, there were Zn-deficient and control groups, and the difference between two groups was analyzed using the Student t test procedure. Trial 2 had six groups, so one-way ANOVA and multiple comparisons of the differences using the LSD method were performed to compare differences among groups. In addition, there were three dosages, i.e., low, medium, and high levels of Bacillus subtilis-Zn, plus zero addition of Bacillus subtilis-Zn (group III); hence, the data from these four groups were used to derive linear and quadratic relationships between the amounts of Bacillus subtilis-Zn (0, 2, 17, 32 mg/kg) and the various measures. Once the quadratic relationship reached the level (P < 0.05), the equation is presented in the paper and was used to estimate the amount of Bacillus subtilis-Zn (i.e., the optimal dosage) that maximized or minimized the corresponding measure. All data were expressed as mean values with pooled standard error of the means (SEM). The difference between means with P < 0.05 was declared significant.

Results

Trial 1: Establishment of Zn-deficient model in young rats

As shown in Table 2, feeding the pregnant rats the Zn-deficient diet lowered the body weight, body length, liver Zn content, and metallothionine concentration and AKP and Cu-Zn SOD activities in serum than rats fed the Zn standard diet (P < 0.05 for all). The results confirmed that the model of Zn deficiency was successfully established in the young rats.

Table 2 Trial 1: Effects of feeding pregnant rats a Zn-deficient diet on growth and blood parameters of the offspring rats
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Trial 2: Zn intervention on young rats congenitally deficient of Zn

Effects on feed intake and growth rate

As shown in Table 3, there were no significant differences in feed intake, growth rate, body weight, and the feed to weight gain ratio between group I (young rats with normal Zn status and fed the Zn standard diet), and in group II (young rats with congenital Zn deficiency and provided Zn at 38 mg/kg) (P > 0.05 for all). The feed intake, daily weight gain, and body weight in group III (young rats with congenital Zn deficiency and fed the Zn-deficient diet) were reduced (P < 0.01 for all), compared with those in group II. These results indicate that the feed intake and growth rate of young rats were dominated by Zn content of the diet, and least affected by the Zn status of the animals.

Table 3 Trial 2: Effects of Bacillus subtilis-Zn on the body development of congenital Zn deficiency rats (n = 3)
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In response to administration of low, middle, and high doses of Bacillus subtilis-Zn, ADFI, ADG, and body weight (BW) of young rats were increased, depending on the dosages, compared with these for group III. Compared with group II, group V had significantly higher DAFI, ADG, and BW (P < 0.05 for all). Group VI had similar values of ADFI with those for group II (P > 0.05), but higher ADG and BW values (P < 0.05 for both). There was no significant difference in the feed to weight gain ratio among six groups (P > 0.05).

There were quadratic relationships between the amounts of Bacillus subtilis-Zn administrated (X, mg/kg) and ADFI (Y1), ADG (Y2), and BW (Y3) in rats (Table 4), and equations are shown as follows:

$$ {\displaystyle \begin{array}{l}Y1=-0.0657{X}^2+2.4198X+134.17\left({R}^2=0.9844,P=0.007\right)\\ {}Y2=-0.0018{X}^2+0.0714X+3.0496\left({R}^2=0.9969,P=0.049\right)\\ {}Y3=--0.004{X}^2+0.1223X+8.6366\left({R}^2=0.9922,P=0.001\right)\end{array}} $$
Table 4 Trail 2: Effects of Bacillus subtilis-Zn on nutrient absorption and utilization of congenital Zn deficiency rats (n = 3)
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From these quadratic relationship equations, it can be deduced that the Bacillus subtilis-Zn dosages that could maximize the BW were 18 mg/kg, 20 mg/kg for the ADG and 15 mg/kg for the ADFI. Hence, the optimal amount of Bacillus subtilis-Zn in diets was 15–20 mg/kg for the highest feed intake and growth rate, and the corresponding total dietary Zn content was 28–33 mg/kg, which is lower than 38 mg/kg recommended by AIN-93

Effects on organ index of congenital Zn deficiency rats

The organ indexes are shown in Table 3. There were no differences in the heart, liver, spleen, and kidney indexes between groups I and II (P > 0.05 for all), whereas the lung index for group II was lower compared with that for group I (P < 0.05). Feeding the Zn-deficient diet to rats (group III) reduced the liver and lung indexes (P < 0.05 for both), but did not change the other indexes compared with those for group II (P > 0.05 for all). These results indicate that the development of rat liver was dominated by the Zn intake and was not affected by the Zn status.

Compared with group II, administration of the low, middle, and high levels of Bacillus subtilis-Zn increased the kidneys index in group IV (P < 0.01), the heart kidney and lung indexes were higher in group V (P < 0.01 for both), and the heart and kidney indexes in group VI were also higher (P < 0.05 for both). There were no differences in the spleen index among groups II, III, IV, V, and VI (P > 0.05 for all).

There were significant quadratic relationships between the dosages of Bacillus subtilis-Zn (X, mg/kg) and the heart (Y1), liver (Y2), and kidney (Y3) indexes of rats, with quadratic equations as follows:

$$ {\displaystyle \begin{array}{l}Y1=-0.0005{X}^2+0.0152X+0.4173\left({R}^2=0.9477,P=0.001\right)\\ {}Y2=-0.0013{X}^2+0.0345X+2.9513\left({R}^2=0.4687,P<0.001\right)\\ {}Y3=-0.0004{X}^2+0.010X+0.9275\left({R}^2=0.4976,P=0.001\right)\end{array}} $$

Based on these quadratic equations, it could be deduced that the optimal dosages of Bacillus subtilis-Zn for maximizing the heart index were 15 mg/kg, and 13 mg/kg for both the liver and kidney indexes, and the corresponding total dietary Zn contents were 26–28 mg/kg.

Effects on nutrient utilization efficiency of congenital Zn deficiency rats

As shown in Table 4, CP, EE, and Zn utilization efficiencies were lower in group II than group I (P < 0.05 for all). All values for group III were lower (P < 0.01 for all) than these for groups I and II, indicating that congenital Zn deficiency and dietary Zn deficiency had the effects on the CP, EE, and Zn efficiencies.

Drenching rats on the Zn-deficient diet with the low, middle, and high dosages of Bacillus subtilis-Zn recovered the utilization efficacy of EE and Zn, and the medium and high doses of Bacillus subtilis-Zn had higher utilization efficiencies of CP than those in group II (P < 0.05).

There were significant quadratic relationships between the dosage of Bacillus subtilis-Zn (X, mg/kg) and the utilization efficiency of CP (Y1), EE (Y2), and Zn (Y3) in rats. The quadratic equations are derived as follows:

$$ {\displaystyle \begin{array}{l}Y1=-0.0185{X}^2+0.5765X+47.306\left({R}^2=0.9979,P=0.002\right)\\ {}Y2=-0.0158{X}^2+0.6883X+44.319\left({R}^2=0.9385,P<0.001\right)\\ {}Y3=-0.0224{X}^2+0.6937X+48.662\left({R}^2=0.9014,P<0.001\right)\end{array}} $$

The optimal Bacillus subtilis-Zn dosage, estimated using these equations, to maximize the utilization efficiency of CP was 16 mg/kg, 22 mg/kg for the EE utilization efficiency and 15 mg/kg for the Zn utilization efficiency, with the corresponding total dietary Zn content ranging 28–35 mg/kg.

Effects on nitrogen utilization of congenital Zn deficiency rats

The N intake, excretions in feces and urine, and retention and utilization efficiency are shown in Table 4. Compared with group I, there were no differences in any value in group II (P > 0.05 for all). Rats in group III had lower N intake, retention and utilization efficiency (P < 0.01 for all), but the similar N excretion compared with these for group II (P > 0.05 for all). The results show the congenital deficiency of Zn did not affect N metabolism, and dietary Zn deficiency reduced N intake, but N excretion remained unchanged, so N utilization efficiency was declined.

Drenching rats on the Zn-deficient diet with Bacillus subtilis-Zn recovered the N intake and retention, and the effect depended on the Bacillus subtilis-Zn dosage. Compared with group II, the N intake and retention were higher in group V (P < 0.05) and were no difference in group VI (P > 0.05). The N excretion in feces and urine were similar among groups II, III, IV, V, and VI (P > 0.05 for all).

The relationship between the Bacillus subtilis-Zn dosage (X, mg/kg) and N intake (Y) in rats showed quadratic, and the quadratic equation is as follows:

$$ Y=-0.0013{X}^2+0.0408X+2.9707\left({R}^2=0.994,P=0.017\right) $$

It can be estimated that the optimal dosage of Bacillus subtilis-Zn to maximize N intake of rats was 17 mg/kg.

Effects on Zn, Cu, and Fe contents in organs of congenital Zn deficiency rats

The Zn content in the heart, liver, brain, and kidneys are shown in Table 4. The Zn content in heart and brain for group II was not significantly different (P > 0.05 for all), but lower in liver and kidneys compared with the values of group I (P < 0.05 for both). The Zn content in these organs in group III was significantly lower than those of group II (P < 0.05 for all). It was shown that the Zn content in the heart and brain tissue of rats was not associated with congenital Zn deficiency, but was determined by the dietary Zn intake, and dietary Zn deficiency reduced Zn content in these organs.

Drenching rats on the Zn deficiency diet with the low, medium, and high dosages of Bacillus subtilis-Zn increased the Zn content in the heart, liver, and kidneys (P < 0.05 for all), compared with group III, and the middle and high dosage increased Zn content in brain (P < 0.05 for all).

The quadratic relationships between the dosage of Bacillus subtilis-Zn (X, mg/kg) and the Zn content the in heart (Y1), liver (Y2), and brain (Y3) are as follows:

$$ {\displaystyle \begin{array}{l}Y1=-0.0147{X}^2+0.5829X+20.892\left({R}^2=0.9992,P=0.001\right)\\ {}Y2=-0.0287{X}^2+0.8556X+30.79\left({R}^2=0.9693,P<0.001\right)\\ {}Y3=-0.0085{X}^2+0.2566X+21.705\left({R}^2=0.9972,P=0.030\right)\end{array}} $$

The optimal dosages of Bacillus subtilis-Zn to maximize the Zn content in heart were 20 mg/kg, and 15 mg/kg for the Zn content in both liver and brain.

Effects on ileum morphology

The ileum morphology is shown in Table 3 and Fig. 1. Compared with group I, the villus height, intestinal wall thickness, and crypt depth were increased in group II (P < 0.05 for all), the villus morphology was irregular, and the villi were sparse. There were differences in the villus height, crypt depth, the ratio of villus height to crypt depth in group III (P < 0.05 for all), the villus morphology was relatively regular, and the villi were sparse and thick. The data of group III were lower than those of group II (P < 0.05 for all). The results showed that the ileum morphology and development were associated with congenital Zn deficiency.

Fig. 1
figure 1

Effects of Bacillus subtilis-Zn on the ileum morphology of congenital Zn deficiency rats. The letters A~F in the figure respectively represent I~VI group, the same figure below.

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Drenching rats on the Zn-deficient diet with Bacillus subtilis-Zn recovered the villus height, intestinal wall thickness, and the ratio of villus higher to crypt depth, and the effect depended on the Bacillus subtilis-Zn dosage. Compared with group II, there was a significant difference in the ratio between groups (P < 0.05). The villus height of group IV was decreased (P < 0.01); the crypt depth and the intestinal wall thickness of group V were different from those of group II (P < 0.05 for all), and the crypt depth of group VI was lower (P < 0.01). The villi of each group were arranged more regularly, the intestinal glands were more developed, and the intestinal wall was thicker.

There were significant quadratic relationships between the dosages of Bacillus subtilis-Zn (X, mg/kg) and intestinal wall thickness (Y) in rats, and the quadratic equation is as follows:

$$ Y=-0.6525{X}^2+18.913X+121.83\ \left({R}^2=0.7395,P<0.001\right) $$

It could be estimated that the optimal dosage of Bacillus subtilis-Zn to maximize intestinal wall thickness of rats was 14 mg/kg.

Microbial diversity indices in cecal content

We used the diversity indexes as follows to reflect information on the abundance, coverage, and diversity of microbial community species in the cecal content. The indices for community abundance included Adaptive Coherence Estimator (ACE) and Chao1 estimator; the indices for community diversity included Shannon and Simpson. The coverage of each sample was greater than 0.99, which proved that the microbial species in the sample were almost fully identified and determined. As shown in Table 5, the OTU, ACE, and Chao1 indices were the highest in group V. There were no differences in Shannon and Simpson indices. The results showed that Bacillus subtilis-Zn could regulate the abundance and diversity of cecal microbes in the Zn-deficient rats, and the medium dose of Bacillus subtilis-Zn could improve the microbial richness in the cecum.

Table 5 Trial 2: Effects of Bacillus subtilis-Zn on cecal alpha diversity index of congenital Zn deficiency rats (n = 3)
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Microbial difference in the cecal content

A total of 107 different genera were detected in all cecal samples, and 87, 95, 96, 95, 100, and 98 genera were detected respectively in groups I~VI. Figure 2 shows the four genera with the highest relative abundance, including Bacteroides, Desulfovibrio, Allopreotella, and Lachnospiraceae for all rats. Bacteroides and Allopreotella in group II were lower than those in group I (P < 0.05 for both). Compared with group II, Bacteroides was reduced in group III (P < 0.05), and drenching rats on the Zn deficiency diet with the low, medium, and high dosages of Bacillus subtilis-Zn increased the Bacteroides enrichment (P < 0.05 for all). There were no differences in Desulfovibrio and Allopreotella in all Bacillus subtilis-Zn groups (P > 0.05 for all). The Lachnospiraceae enrichment in groups III and IV was higher than other groups (P < 0.01 for all). These results indicate that Bacillus subtilis-Zn regulates the Bacteroides and Lachnospiraceae enrichment in the caecum.

Fig. 2
figure 2

Structure and distribution of horizontal dominant microorganisms in genus level

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Discussion

Bacillus subtilis-Zn promotes the growth rate of congenital Zn deficiency rats

This study found that feeding rats with the Zn-deficient diet reduced feed intake, slowed the growth rate, and increased the F/G ratio (but not significant). It had a significant impact on liver development, but had little effect on the development of heart, spleen, kidneys, and lungs of rats. The administration of Bacillus subtilis-Zn to rats could increase the feed intake and growth rate of congenital Zn-deficient rats, which was beneficial to the development of liver, kidneys, and heart; the unchanged feed to weight gain ratio indicates that the main improvement was to increase feed intake. We noted obviously quadratic relationships between the Bacillus subtilis-Zn and feed intake, growth index, and Zn content in organs in the present study, and on the basis of these relationships, we estimated that the optimal dosage of Bacillus subtilis-Zn for increasing feed intake and weight gain of rats was 15 to 20 mg/kg, and the total Zn content in the diet was 28–33 mg/kg. Misztak et al. [21] and Shah et al. [1] found that Zn deficiency in pregnant rats reduced feed intake, impaired immune competency of rats, and affected fetal development. Studies by Misztak et al. [21] and Fukada et al. [22] also showed that Zn deficiency could cause a reduction of feed intake in growing rats, and a decline of nutrition utilization, hence, the body weight, body length, and length of the patella were all reduced. These results are in agreement with our findings in the present study. Thus, dietary supplementation of Zn to meet the Zn recommendation is necessary. Wang [8] compared the effects of Zn sources on the performance of Zn-deficient rats and found that yeast Zn could increase animal’s feed intake and daily weight gain, and reduce the feed to weight gain ratio. Wu [23] found that the body weight and feed efficiency value in zinc-deficient and zinc-excessive rats were lower, the zinc content in serum and liver, thymus, and spleen index also were lower. These results confirm the findings of the present study: The addition of appropriate levels of Bacillus subtilis-Zn in the Zn-deficient diet can increase feed intake and growth rate in rats and promote the development of the organs.

Bacillus subtilis-Zn improves nutrient utilization efficiency of congenital Zn deficiency rats

This study found that feeding rats with the Zn-deficient diet not only reduced feed intake, but also led to an increase in fecal N excretion; the utilization efficiency of N, fat, and Zn were all decreased; supplementing Bacillus subtilis-Zn can reverse, more effectively, the detrimental effects of Zn deficiency on nutrient utilization than ZnSO4 did. Murugesan et al. [24] proposed that good growth performance of animals depends on the adequate digestion, absorption, and utilization of dietary nutrients. Dietary Zn deficiency may affect the activities of related enzymes and hormones, resulting in decreases in appetite and nutrient utilization of animals. Supplementation of Bacillus subtilis-Zn could counteract these effects by enhancing the activity of related enzymes and hormones, increasing feed intake and nutrient utilization. Li [25] proposed that Zn is an indispensable part of many enzymes and hormones, such as alkaline phosphatase for bone growth development, aminopeptidase that regulates protein digestion, and numerous enzymes involved in nutrient metabolisms, and growth hormone, insulin, and reproductive hormones that maintain the secondary sexual characteristics. As the results of increased utilization efficiency of N and the other nutrients by supplementing Bacillus subtilis-Zn, N excretion decreased, so less pollution to the environment. Our results prove that Bacillus subtilis-Zn is an ecological feed additive in favor for animal’s performances and environment.

Bacillus subtilis-Zn increases Zn content in organs of congenital Zn deficiency rats

The present study found that feeding rats with the Zn-deficient diet reduced Zn content in the tissue and organs, while supplementing an appropriate dose of Bacillus subtilis-Zn increased the contents of Zn, despite the varying responses among heart, liver, kidneys, and brain tissue. We estimated that the optimal dose of Bacillus subtilis-Zn to increase the Zn content was 15 to 20 mg/kg for the Zn-deficient diet, and the total dietary Zn level should be 28–33 mg/kg. A study showed that Zn deficiency not only leads to reduced Zn content in animals, but also affects the transport and bioavailability of other elements [26]. For example, Zhao et al. [27] showed that Zn content and other metal elements content in the tissues of pregnant rats on a Zn-deficient diet were lower than those in Zn supplementation and control groups. Kong [28] reported no differences in growth performance and tissue Zn content of rats between yeast Zn and ZnSO4 when both Zn supplements were provided adequately to meet the Zn requirement. Bacillus subtilis has a capacity to enrich various metal elements (including Zn, Cu, Fe, Mn) [11], and Bacillus subtilis-Zn is an organic Zn product, so that the utilization of Zn could also be improved, and the amount of deposition in tissues could be increased. In addition, our results clearly show that the effects of the highest dose of Bacillus subtilis-Zn (Zn level 32 mg/kg) were lower than those on the middle dose (Zn level 17 mg/kg), indicating that the body Zn requirement has a certain threshold range, and an overdose is not conducive to nutrient digestion and absorption.

Bacillus subtilis-Zn can regulate ileum morphology of congenital Zn deficiency rats

The present study found that zinc deficiency in rats had detrimental effects on the ileum morphology, while supplementing an appropriate dose of Bacillus subtilis-Zn could promote villus development, reduce crypt depth, and increase intestinal wall thickness, thereby increasing the VH/CD ratio, regulating intestinal morphology, promoting intestinal gland development and digestive absorption. The data showed that the optimal dosage of Bacillus subtilis-Zn to maximize intestinal wall thickness of rats was 14 mg/kg, and the total content of Zn in the diet was 27 mg/kg. Weaning-induced problems in intestinal function appear from the present results to be caused more by the changes in intestinal structure and specific loss of digestive enzymes [29]. Li et al. [30] found that an addition of high-dose zinc oxide in the diet can effectively increase the height of the anterior and middle villi of the small intestine in 21-day-old weaned piglets, and reduce the crypt depth in the posterior segment of the small intestine, thereby reduce the villus height/crypt depth ratio in the small intestine. It indicates that high zinc can effectively improve the intestinal morphology of piglets. In addition, our results in the present study clearly showed that the effects of the highest dose of Bacillus subtilis-Zn (Zn level 32 mg/kg) were weaker than these on the middle dose (Zn level 17 mg/kg), indicating that the overdose disfavor the intestinal development.

Bacillus subtilis-Zn can regulate microbial diversity in cecal contents of congenital Zn deficiency rats

The nutritional inadequacy, body injury, and disease status can change the microbial diversity in the intestines and even affect the animal’s health. Recently, it has been reported that the intestinal microbiota depends not only on genetic factors but also on environmental factors such as age, diet, and life [31, 32]. The present study showed that Bacillus subtilis-Zn at dose 30 mg/kg increased the OTU, ACE, and Chao1 indices in Zn-deficient rats, thereby increased microbial diversity and reduced of the abundance of potentially pathogenic bacteria (for example, Escherichia coli and Salmonella). We also found in the present study that Bacteroides, Desulfovibrio, Allopreotella, and Lachnospiraceae were dominant species in the caecum. Bacillus subtilis-Zn can increase the enrichment of Bacteroides in caecum contents and reduce the enrichment of Lachnospiraceae. Bacteroides plays an important role in the degradation of cellulose or hemicellulose in the rumen [33], which could be associated with improvement of animal’s growth and development, and nutrient digestion and absorption [34]. Previous studies showed that Bacillus subtilis can accelerate the process of anaerobic fermentation, change the diversity of microorganisms, lead to the enrichment of beneficial bacteria, reduce the abundance of potentially pathogenic bacteria, and change intestinal morphology and body growth [35, 36].

Bacillus subtilis-Zn is superior to ZnSO4 in repairing congenital Zn deficiency rats

This study concludes that administration of 15–20 mg/kg Bacillus subtilis-Zn to a Zn deficiency diet can significantly promote the feed intake and growth rate of congenital Zn-deficient rats. This dosage was sufficient to maximize the Zn content in the liver. Together with 13 mg/kg Zn in the basal diet, the total content of Zn in the diet was 28 to 33 mg/kg. We noticed that when Bacillus subtilis-Zn was provided at the appropriate dose, the rats’ intake and growth rate, heart, liver, kidney, lung indexes, and Zn utilization were all higher than those of ZnSO4 group (Zn content at 38 mg/kg), and the Zn contents in the organs were similar. These results demonstrate that Bacillus subtilis-Zn is a superior source of Zn to ZnSO4. Bacillus subtilis-Zn is a new source of Zn with organic Zn content of over 95%. Other research reports support the propos of using organic Zn products. For example, Wang et al. [37] showed that the bioavailability of yeast Zn was significantly higher than that of Zn sulfate. In addition, using the optimal dose of Bacillus subtilis-Zn, the total content of Zn in the diet was 28 to 33 mg/kg, which is 14 to 27% lower than 38 mg/kg Zn recommended by the AIN standard (1993). This can reduce dietary Zn content and help to reduce the amount of Zn emitted from the farming industry to the environment. In addition, the present study found that Bacillus subtilis-Zn (30 mg/kg) was superior to ZnSO4 in promoting intestinal morphology and microbial diversity.

Conclusions

Bacillus subtilis-Zn has the dual functions of probiotics and organic zinc, which can promote animal growth and development, improve nutrient utilization, and optimize intestinal flora structure and microbial diversity, and is a superior Zn source to ZnSO4.