Calcium Homeostasis and Bone Metabolism in Goats Fed a Low Protein Diet

The objective of this study was to investigate the effects of low-protein diets on blood calcium (Ca) level, bone metabolism, and the correlation between bone metabolism and blood calcium in goats. Twenty-four female Xiangdong black goats with similar body weight (19.55 ± 3.55 kg) and age (8.0 ± 0.3 months) were selected and allocated into two groups: control group (CON, 10.77% protein content) and low-protein group (LP, 5.52% protein content). Blood samples were collected on days 1, 4, 7, 16 and 36 before morning feeding to determine the concentration of calcium (Ca), parathyroid hormone (PTH), bone gla protein (BGP), C-terminal telopeptide of type 1 collagen (CTX-1), bone alkaline phosphatase (BALP), and 1, 25-dihydroxyvitamin D3 [1,25(OH)2D3]. Liver samples were collected to determine the expression of bone metabolism-related genes. There was no difference observed between LP and CON in concentration of plasma Ca or any of bone metabolism markers (P > 0.05). In the liver, the mRNA expression of bone gamma carboxyglutamate protein (BGLAP), alkaline phosphatase (ALPL), and mothers against decapentaplegic homolog-1 (SMAD1) were increased (P < 0.05) in LP as compared with CON. The correlation analysis of Ca and bone metabolism markers showed no significant correlation between Ca and bone metabolism. These results suggest that the blood Ca concentration in mature goats may keep at a stable level through nitrogen cycling when the providing protein is not enough.


INTRODUCTION
Calcium (Ca) is an essential mineral in the physical and is closely related to various physiological and pathological processes (1). It has been reported that bone metabolism is closely linked to Ca homeostasis, including bone resorption and bone formation, of which bone resorption is necessary to maintain an appropriate level of blood Ca (2). Bone is the calcium repository, containing about 98% of the total body calcium (3). Bone is a highly dynamic organ that is constantly metabolized and remodeled throughout life because bone plays a crucial role in providing support, maintaining mineral balance, and protecting soft tissue for the body (4,5). The metabolic balance of bones depends on bone formation by osteoblasts and bone resorption by osteoclasts (6). Generally, biochemical markers of bone turnover have proved to be a valuable and simple tool for studying bone metabolism status (7), such as bone alkaline phosphatase (BALP), bone gamma-carboxyglutamate protein (BGLAP), and cross-linked Cterminal telopeptides of type I collagen (CTX-1). Moreover, bone development is profoundly influenced by signaling pathways, such as the Wingless tail (Wnt) signaling pathway (8), the OPG / RANKL / RANK signaling pathway (9), and the Smads signaling pathway (10). In addition, bone resorption dissolves the mineralized matrix of bone under the control of osteoclasts and hormones such as parathyroid hormone (PTH) and 1, 25dihydroxyvitamin D3 (1,25(OH) 2 D3), releasing ionized Ca into the blood and then altering the blood Ca concentration (11,12). As most Ca is located in bone (13), and the plasma calcium concentration is tightly controlled in a narrow range to maintain numerous fundamental physiological functions of the body (14). In goats, it has been reported that the total plasma calcium ranges from 2.0 to 3.0 mmol/L (15), and Ca deficiency can lead to delayed growth and development and pathological risks such as osteoporosis, rickets, and osteochondrosis (16).
Dietary protein is essential for animal growth and health, and it is a vital source of synthetic osteoprotein amino acids (17,18). However, resource shortage and ammonia emission are the main problems in protein feed application (19,20). Thus, low-protein diets are commonly used to reduce feed costs and nitrogen emissions and maintain intestinal health (21,22). Previous studies in young goats have demonstrated that severe mineral homeostasis and bone changes occurred in a reduced protein diet (23,24). However, the effect of reduced dietary protein on Ca homeostasis and bone metabolism in mature goats is not yet fully understood. Therefore, we hypothesized that reducing dietary protein could alter the bone metabolic balance and regulate plasma Ca levels. Using mature goats as experimental animals, the objectives of this study were to determine the effects of a low-protein diet on bone markers (in the blood), bone metabolic gene expression (in the liver), plasma Ca concentration, and to evaluate the correlation between bone metabolism and blood Ca.

MATERIALS AND METHODS
The experiment was performed at the animal and crop laboratory building of the institute of subtropical agricultural ecology, Changsha, Hunan province. All experimental procedures were executed according to the Animal Care and the Use Guidelines of the Animal Care Committee, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China (ISA-2019-0115). All goats used in this study are purchased from Mulei Black Goats Breeding Farm in Liuyang, Changsha, China.

Animal Management and Dietary Treatments
Twenty-four female Xiongdong black goats with a similar body weight of 19.55 ± 3.55 kg, about 8 ± 0.3 months old were used in this trial All goats were randomly assigned to either a control diet group (CON, 10.77% protein content, n = 12) or  Table 1.
The total acclimatization period lasted for 14 days until all goats reached the stable dry matter intake. The experimental period lasted for 36 days. Each goat was housed in a single cage and fed twice daily (8:30 am and 5:30 pm) with the same amount of diet. All goats had free access to fresh water. A registered veterinarian slaughtered all goats with an intravenous injection of sodium pentobarbital (50 mg/kg BW) before feeding in the morning. All goats were weighed before being slaughtered at the end of the experimental period.

Sample Collection and Analysis
Blood samples were collected from the jugular vein using vacuum blood collection vessels (containing anticoagulant) on days 1, 4, 7, 16, and 36 before morning feeding of the actual experimental period to investigate the periodic changes of bone metabolism markers. Plasma was obtained after centrifugation (2,500 × g, 10 min, 4 • C) and then stored at −80 • C until analyses. Plasma concentrations of 1,25(OH) 2 D3, BGP, PTH, CTX-1, and BALP were measured using colorimetric methods with a spectro-photometer. The assay kits of PTH and BALP were purchased from MEIMIAN (Jiangsu, China). According to the manufacturer's specification, the assay kits of BGP and CTX-1 were purchased from MyBioSource Inc. (San Diego, SC, USA). The assay kits of 1,25(OH) 2 D3 were purchased from Immundiagnostik AG (Bensheim, Germany). Plasma concentration of Ca was analyzed using a blood Ca concentration detection kit (Solarbio Life Sciences Co. Ltd., Beijing, China). Liver samples were collected after slaughter to determine the gene expression that related to bone metabolism. Total RNA was extracted from liver tissue with the commercial kit (AG21017, Accurate Biology, Changsha, China), and then RNA was converted to cDNA using the RNA reverse transcription kit (AG11705, Accurate Biology, Changsha, China). The obtained cDNA template was stored at−80 • C until real-time quantitative PCR analysis was performed.

Real-Time Quantitative RT-PCR
Primers are shown in Table 2 Primers are shown in Table 2. The primer of GAPDH was designed online (https://www.sangon.com/newPrimerDesign), and others were referred to Li et al. (25). Relative quantification of the target gene expression was calculated from the melting curve and normalized against the abundance of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, using the 2 − Ct method (26), PCR and agarose gel electrophoresis verified all primers, and GAPDH has been tested across all tissue samples as a housekeeper gene.

Statistical Analysis
All data were checked for normality and variance homogeneity before further statistical analysis. The data of blood Ca and bone markers required for ANOVA, the MIXED procedure of SAS 9.4 (SAS Inst. Inc., Cary, NC, USA) were used for analyzing, the model included the fixed effects of treatments, sampling time and their interaction, and the goat as a random effect. The bone, metabolism-related genes data was analyzed by an independent samples t-test using SAS 9.4 (SAS Inst. Inc., Cary, NC, USA). In addition, the Pearson correlation analyses were performed to calculate the correlation between blood Ca and bone metabolism markers. The significant difference was declared at P < 0.05, and the tendency was considered at 0.05 ≤ P < 0.10 for all statistical analyses.

Ca and Bone Metabolism Markers in Plasma
There was no difference in concentration of plasma Ca, bone markers, or any of hormones tested between LP and CON at any of the time sampled (P > 0.05, Figure 1).

DISCUSSION
This study investigated the changes of bone metabolism, and blood Ca concentrations in goats fed a low-protein diet. Numerous studies have supported the dietary protein involvement in regulating Ca metabolism by regulating kidney calcium emissions and intestinal calcium absorption, which ultimately manifests as changes in blood calcium concentration Frontiers in Veterinary Science | www.frontiersin.org   (27,28). The current study showed no change in plasm Ca in goats fed a low protein diet. Similarly, it was reported that providing a low-protein diet did not affect Ca retention in healthy postmenopausal women (29). In a study using Sprague Dawley rats as the experimental animals, plasma concentrations of Ca were not altered when fed a low-protein diet (5% protein) as compared with the control group (20% protein) (30). Previous studies have shown that urine Ca production was positively related to Ca absorption, and Ca absorption and urine Ca excretion were in balance when dietary minerals met the demands (31). Therefore, the absolute amount of Ca retention was unaffected by reduced protein in our study, which resulted in a stable concentration in blood Ca. Moreover, it has been reported that blood Ca concentration in rats fed a low-protein diet was significantly decreased after 6 weeks but gradually returned to normal levels after 8 weeks (32), which was partly contradictory with our results. This difference would be ascribed to inconsistencies in the source of protein and experimental methodology (33). Meanwhile, different animal species may have various adaptations of the change of dietary protein levels. In goat studies, it has been reported that young male goats fed a reduced-protein for 6-8 weeks significantly reduced plasma Ca concentrations due to reduced intestinal Ca uptake and microbial metabolism (34)(35)(36), which was inconsistent with our results. The difference may be related with the different physiological state of the experimental goats gone through. In our study, we used the goats with an age of 8 months, which is considered an age of rumen-function well (37). In addition, we conducted the animal trial for 36 days, but no changes were observed between initial and final body weight in both CON and LP (final body weight: 19.97 vs. 20.23 kg when fed low-protein diet and control diet, respectively, unpublished data.), which may also indicate the goats we used are relatively mature. Because of the existing functional rumen, the nitrogen cycling mechanism of mature goats could provide N through microbial metabolism when the dietary protein is not enough. By possessing such efficient recycling mechanisms, mature goats could maintain rumen microbe's N supply and synthesize microbial protein as the source for host protein (34,38,39). The current study showed that feeding a low protein diet had no change in bone metabolism markers in mature goats, including the markers reflecting the bone absorption and formation. These results were inconsistent with previous studies that a protein-restricted diet increased CTX-1 but decreased plasma 1,25(OH) 2 D3 concentrations in monogastric species and young small ruminants (23,40). To better understand the bone metabolism mechanism of goats fed a low-protein diet, we measured the mRNA expression level of bone metabolismrelated genes in liver, which is closely related to bone metabolism (25). It has been reported that BGLAP and ALPL, promote osteoblast activity and bone mineralization (41,42). The increased gene expression of BGLAP and ALPL in LP indicated that the increased level of liver osteoblastic genes may compensate for the side effect from a low protein diet and keep the balance of bone turnover. Moreover, BMP2 and SMAD1 are the essential components of the BMP-Smad-Runx2 axis, which plays a vital role in bone development and formation (43). LRP6 plays a pivotal role in mediating bone cells and bone formation activity in the typical Wnt signaling pathway (44,45). In our study, the low protein diet increased expression of BMP2, SMAD1 and LRP6. Therefore, it is possible that the goats with insufficient dietary protein may maintain the balance of bone metabolism through the BMP-Smad Runx2 axis and the Wnt signaling pathway.
The negative association between BGP and BALP was expected. In general, BALP is increased during osteoblast proliferation and extracellular matrix (ECM) maturation, and it is decreased during advanced mineralization (46, 47). In contrast, the BGP synthesis only occurs during mineralization (46,47). The results showed that PTH was positively correlated with BGP and negatively correlated with CTX-1, which were expected. These results can be explained by the fact that PTH can stimulate bone resorption to regulate Ca homeostasis (11). Duque et al. (48) showed that the osteocalcin concentration was significantly increased in SAM-P/6 mice under the treatment of 1,25(OH) 2 D3. This result was consistent with the positive correlation between BGP and 1,25(OH) 2 D3 observed in this experiment. There was no association between blood Ca and bone metabolism markers, which was inconsistent with the previous results obtained in monogastric species, such as using human, mice and chicken (49,50). It has been shown that the blood Ca was regulated by exchange from bone, absorption in the intestine, and reabsorption in the kidney, which are all tightly correlated with PTH and 1,25(OH) 2 D3 (51). The lack of association between blood Ca and bone metabolism markers observed in our study may be due to the exist of rumen, which is engaged in calcium transport and absorptions. It has been reported that the Ca transport in the rumen are probably not regulated by 1,25(OH) 2 D3, nor mediated by the 1,25 (OH) 2 D3 related binding proteins and Ca channels (52). In terms of reabsorption in the kidney, ruminant renal seems insensitive to a dietary Ca restriction induced challenge of Ca homeostasis than monogastric animals (53,54).

CONCLUSION
In summary, no change was observed in concentrations of plasma Ca or bone metabolism markers in goats fed a low protein diet. Correlation analysis showed that no significant relationship between Ca and any bone metabolism markers. These results suggest that there may be compensatory homeostasis mechanisms in mature non-pregnant goats that maintain relatively stable bone metabolism markers and Ca concentration when feeding a low protein diet for a certain period of 36 days. Moreover, a low protein diet up-regulated the gene expression of BGLAP, ALPL and SMAD1, suggesting that the goats with insufficient dietary protein may maintain the balance of bone metabolism through the BMP-Smad Runx2 axis and the Wnt signaling pathway.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Care Committee, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China.