INTRODUCTION
In recent years, feed cost has been a critical factor in the swine industry, accounting for 50%–85% of the total production expenses [1]. Improving feed efficiency can be a sound solution for reducing feed costs. Recently, the effects of dietary concentration and the relative ratio of calcium (Ca) to phosphorus (P) on growth performance, feed efficiency, and bone mineralization have been mainly discussed with controversial [2,3]. Ca and P are widely acknowledged as essential microminerals in pig nutrition and the deposition of lean tissue, maintenance and synthesis of skeletal structure, and many other functions [4–6]. The safety of P in diets of swine is frequently calculated using small margins. Typically, P excretion will increase when it is overestimated [7,8]. In contrast, the danger of an abundant Ca supply in swine diets occurs because of its low cost and the absence of environmental issues [9].
The digestibility and absorption of both minerals are affected by the Ca and P ratios [10]. Therefore, swine diets should supply appropriate Ca and P ratios as well as the individual requirements for both minerals. An imbalance in the Ca and P ratio leads to a reduction in growth performance and bone mineralization, particularly when insufficient P is formulated in pig diets [11–14]. Furthermore, the indigestible Ca-phytate-P structure in the small intestine is created by a high Ca or Ca and P ratio, which exacerbates the function of exogenous phytases [15,16].
Limestone (calcium carbonate [CaCO3]) is mainly used as the Ca source. However, CaCO3 frequently binds to acids in the gastrointestinal tract, reducing nitrogen and P digestibility by lowering protein and P solubility [17,18]. Therefore, dietary CaCO3 levels are negatively correlated with P consumption. Moreover, high dietary CaCO3 and P supplementation in swine diets leads to excessive pig manure excretion during intensive production, resulting in environmental issues. One of the most widely employed nutritional strategies in recent years to overcome environmental P pollution and promote P utilization in farm animals is the incorporation of phytase in animal diets. Calcium chloride (CaCl2) is an alternative to CaCO3 because of its high solubility in water. This may cause differences in bioavailability and digestibility. Consequently, the main aim of our current study was to determine how changes in dietary Ca (levels and sources) affect the growth performance, nutrient digestibility, blood profile, and bone mineralization of growing pigs.
MATERIALS AND METHODS
The protocol for this study was approved by the Institutional Animal Care and Use Committee of Kangwon National University, Chuncheon, Republic of Korea (ethical code: KW-210707-1).
A total of hundred and eighty growing pigs (Landrace × Yorkshire × Duroc [LYD]) with an average body weight (BW) of 10.37 ± 0.01 kg were randomly allotted to one of the three treatments on the basis of initial BW. The experiment was conducted in a randomized complete block design at the Research Center of Animal Life Sciences at Kangwon National University. There were 10 pigs per replicate, with 6 replicates in each treatment. The treatments included low (Ca 0.60% in Phase 1 and 0.50% in Phase 2), standard (Ca 0.72% in Phase 1 and 0.66% in Phase 2), and high (Ca 0.84% in Phase 1 and 0.72% in Phase 2). The experimental diets were supplemented for 28 d in two phases: phase 1 (days 0–14) and phase 2 (days 15–28). The pigs were group-housed in partially slatted concrete floor pens with a 2.80 m × 5.00 m pen size. All pens contained a self-feeder and nipple drinker to allow access to feed and water ad libitum. The diets were formulated to provide all nutrients to meet or exceed the nutrient requirements listed in the NRC [19], except Ca (Table 1).
Phase 1: Low, 0.60%; Standard, 0.72%; High, 0.84%; Phase 2: Low, 0.50%; Standard, 0.66%; High, 0.82%.
Supplied per kg of diet: 16,000 IU vitamin A (palmitate), 2.00 mg vitamin B1 (thiamin), 5.00 mg vitamin B2 (riboflavin), 2.00 mg vitamin B6 (pyridoxine), 0.03 mg vitamin B12 (cyanocobalamin), 25.00 mg niacin, 0.40 mg folic acid, 0.05 mg biotin, 5.00 mg ethoxyquin, 2,000 IU vitamin D3 (cholecalciferol), 75.00 mg vitamin E (dl-α-tocopheryl acetate), 2.00 mg vitamin K3 (menadione).
Two hundred and forty growing pigs (LYD) with an average BW of 10.32 ± 0.01 kg were randomly assigned to one of the four treatments based on initial BW. A randomized complete block design was conducted at the Research Center of Animal Life Sciences of Kangwon National University. There were 10 pigs per replicate, with 6 replicates in each treatment. Four diets differing in Ca levels (high and low) and sources (CaCl2 and CaCO3) were formulated and fed to the pigs from day 14 post-weaning (Table 2) in a 2 × 2 factorial arrangement. The pigs were fed a basal diet of mash feed ad libitum. The experimental diets were fed for 28 days in two phases: phase 1 (d 0–14), and phase 2 (d 15–28). The pigs were housed in partially slatted concrete floor pens with a pen size of 2.80 m × 5.00 m. All the pens contain a self-feeder and nipple drinker to allow ad libitum access to feed and water. The diets were formulated to provide all nutrients to meet or exceed the nutrient requirements listed in the NRC [19], except Ca.
Phase 1: Low, 0.60%; Standard, 0.72%; High, 0.84%; Phase 2: Low, 0.50%; Standard, 0.66%; High, 0.82%.
Supplied per kg of diet: 16,000 IU vitamin A (palmitate), 2.00 mg vitamin B1 (thiamin), 5.00 mg vitamin B2 (riboflavin), 2.00 mg vitamin B6 (pyridoxine), 0.03 mg vitamin B12 (cyanocobalamin), 25.00 mg niacin, 0.40 mg folic acid, 0.05 mg biotin, 5.00 mg ethoxyquin, 2,000 IU vitamin D3 (cholecalciferol), 75.00 mg vitamin E (dl-α-tocopheryl acetate), 2.00 mg vitamin K3 (menadione).
All experimental pigs were weighed individually on day one of the experiment and the last day of the experiment, and feed consumption was recorded during the entire duration of the experiment. This was used to calculate the average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G/F) at the end of each experiment.
To determine the effects of each treatment on nutrient digestibility, Cr2O3 was formulated at 0.25% in the treatments. The pigs were fed for five days with diets before collection. Fecal samples were collected four days before the end of each phase to evaluate the digestibility of dry matter (DM), crude protein (CP), crude fat (CF), ash, Ca, and P. To start sample collection, previous fecal samples were eliminated, and the fecal samples were pooled within the pen. Samples were collected and then placed in a freezer at -20°C until analysis. Fecal samples were thawed, dried at a temperature of 60°C for 72 h in a forced-air oven, ground in a 1-mm screen Wiley mill (Thomas Model 4 Wiley Mill, Thomas Scientific, Swedesboro, NJ, USA), and analyzed for calculating digestibility. Each sample was analyzed in triplicate for measuring the levels of DM (Method 930.15), CP (Method 990.03), CF (Method 960.39), ash (Method 942.05), Ca, and P (method 985.01; 16) according to the methods described by the AOAC [20].
On the last day of the experiment, one pig per replicate with a BW that was the closest to the average of the replicate was slaughtered via captive bolt stunning. The left front leg was removed, stored at −20°C, and later autoclaved for 55 min at 125°C. The tibia was then extracted from the leg. The bone marrow was removed, and the tibia was dried and soaked in petroleum ether under a chemical hood for 72 h to remove the remaining marrow and fat. The bones were dried overnight at 130°C and ashed at 600°C for 16 h.
The acid-base status of the animals was evaluated. Venous blood samples from three pigs per treatment were collected via jugular venipuncture into 3-mL non-heparinized vacuum tubes and were then analyzed within approximately 10 min after sampling for pH: sodium (Na+), potassium (K+), ionized Ca (iCa++), chloride (Cl−), bicarbonate (HCO3−), base excess (BE), hematocrit (HTC), and electrolyte balance (EB) using an i-STAT Portable Clinical Analyzer with EC8+ cartridges (i-STAT, Princeton, NJ, USA).
Data from this trial were analyzed by ANOVA using the GLM procedure in SAS (9.2, SAS Institute, Cary, NC, USA). Initial BW was used as a covariate for growth performance but was eliminated from the model when it was not significant. Each pig was an experimental unit for growth performance, feed intake, nutrient digestibility, blood EB, and bone measurement. The Tukey means comparison test was applied for treatment mean separation at p < 0.05. A probability level of less than 0.1 was considered a tendency.
RESULTS
Mortality was not observed during the experiment. The growth performance results are listed in Table 3. BW increased as the Ca level decreased (p < 0.05). ADG was also greater in the low-Ca diet group. Similarly, G/F was improved in the low-Ca diet compared to the high-Ca diet. However, there was no difference in ADFI across the treatments.
Nutrient digestibility is shown in Table 4. Although there was no difference in the DM, CP, CF, ash, and Ca digestibility as Ca level changed, P digestibility was higher in the low-Ca diet group than in the high-Ca diet group (p < 0.05).
Bone mineralization is shown in Table 5. No difference was noted in the levels of bone ash, Ca, P, and Mg as the Ca levels changed.
The results of the growth performance on Ca level and source are shown in Table 6. Final BW increased in the diets with CaCl2 compared with the case for the CaCO3diets (p < 0.05). In the CaCl2 diet group, the ADG was higher and G/F was improved, compared with the case for the CaCO3 diet group. However, there was no difference in the ADFI of the pigs.
The effects of Ca level and source on nutrient digestibility are shown in Table 7. There were no significant differences in DM, CF, and ash digestibility due to Ca level and source variation. CP, Ca, and P digestibility was higher in the CaCl2 diet group than in the CaCO3 diet group (p < 0.05).
The effects of Ca levels and sources on blood electrolytes are shown in Table 8. Cl− was upregulated in the CaCl2 diet group compared to the CaCO3diet group (p < 0.05). The HCO3−, BE, and EB levels were lower in the CaCl2 diet group than in the CaCO3diet group (p < 0.05). The HTC increased as Ca levels decreased (p < 0.05). HCO3− interacted with the Ca sources, and thus, influenced the Ca levels (p < 0.05).
The effects of Ca levels and sources on bone mineralization are presented in Table 9. Bone ash, Ca, and P were downregulated in the low-Ca diet group compared with the high-Ca diet group. There was no difference in bone ash, Ca, P, and Mg as the Ca sources changed.
DISCUSSION
Ca and P are interdependent minerals that should be used in moderate quantities. Inadequate inclusion of these two diets may have a deleterious effect on the digestibility of other minerals or nutrients [21]. For instance, excess Ca has been shown to be detrimental to obtaining optimal pig growth performance, and more marginal or lower than P requirements in swine diets exacerbate the situation [11,12,14]. Reduction in P digestibility is manifested by this adverse effect when the dietary Ca level is higher than the requirements [10]. In the gastrointestinal tract, Ca-P-insoluble compounds reduce P digestion and absorption [22,23]. Dietary P addition may help to mitigate the negative effects of excessive dietary Ca on the growth performance of pigs. To improve pig growth performance and mineral digestibility, an appropriate Ca:P ratio should be established during the feed formulation.
In Experiment 1, high concentrations of Ca lowered ADG, G/F, and P digestibility, corresponding to an estimated Ca:P ratio greater than 1.22:1. The decrease in P digestibility could be attributable to the deleterious effect of higher dietary Ca levels on ADG and G:F. [24] reported that an increase in Ca levels reduced G:F. Similarly, Wu et al. [14] observed that the ADG, ADFI, and G: F of nursery piglets fed P-deficient diets decreased with increasing Ca levels. Additional supplementation with P in the diet alleviated these negative effects. Furthermore, González-Vega et al. [11,12] and Merriman et al. [13] similarly reported the negative effects of high Ca or Ca:P ratios in 11- to 25-kg pigs, 25- to 50-kg pigs, and 100- 130-kg pigs that were supplemented without additional phytase in their diets.
In our study, we did not find any variation in bone mineralization, including bone ash, Ca, P, and Mg per tibia. Numerous studies have demonstrated that the Ca requirement for maximizing growth performance is different from maximizing bone mineralization, as numerous studies have demonstrated [16,25]. To increase bone mineralization, a higher Ca and P ratio should be supplied more than pig`s requirement [11,13,25]. Bone ash concentration did not change when the dietary Ca and P ratios were increased. This result suggests that bone mineralization changes dramatically when a low-Ca diet is formulated to maximize growth performance.
Two Ca sources and levels were used for the diets. Therefore, CaCO3 (provided as limestone) was replaced in the diets of growing pigs at varying Ca levels. In our study, different Ca sources affected the growth performance of pigs. Although dietary Ca levels and sources had no effects on ADFI, CaCl2 in pig diets showed a higher growth rate among pigs than CaCO3in pig diets throughout the trial. This may be because CaCl2 alters the dietary electrolyte balance (dEB) values. In a previous study, Austic et al. [26] observed that dEB values for optimum growth performance of pigs should be in the range of 100–300 mEq/kg.
Similarly, Patience et al. [27] found that diets with dEB values of approximately 175 mEq/kg might provide ideal optimal growth performance. Dersjant-Li et al. [28] suggested that pigs fed diets containing 200 and 500 mEq/kg showed higher growth rates than those fed diets containing 100 mEq/kg. Similarly, Haydon [29] indicated that increasing the dEB value from 25 to 400 mEq/kg can linearly increase daily feed intake. According to Budde and Crenshaw [30], however, dietary dEB values (ranging from –35 to 212 mEq/kg) did not affect piglet growth or feed intake, and Patience and Chaplin [31] suggested that a low dEB value (–20 mEq/kg) in pig diet is a better choice than a high dEB value (104 or 163 mEq/kg) with a tendency for growth rate improvement. Guzmán-Pino et al. [32] observed that pigs fed 16 and 133 mEq/kg diets had greater ADG than pigs fed 269 mEq/kg diet.
In contrast to the findings of Experiment 1, we found that lower levels of dietary Ca or lower Ca and P ratios were harmful to growth performance. This is because lower levels of dietary Ca are insufficient to satisfy the Ca requirement for growth. In both experiments, dietary Ca levels that were either low or too high showed an aberrant growth rate, which was consistent with the narrow-calculated Ca and P ratios. Excessive dietary P and low dietary Ca compared with greater dietary Ca levels may bind with abundant dietary P and deficient dietary Ca, which reduce ADG [25]. Contrary to the findings of the present study, González-Vega et al. [12] and Merriman et al. [13] reported that low levels of dietary Ca had no effect on pig growth performance. Previous studies used heavier experimental animals, whereas our study examined different calculated Ca and P ratios. A well-balanced dietary nutrient composition is required for younger pigs than for older pigs. As a result, younger pigs in our study may require more dietary Ca for their growth and maintenance in comparison to those in the previous study.
The diet supplemented with CaCO3 reduced the apparent digestibility of CP, Ca, and P compared to diets supplemented with CaCl2. For optimal growth of pigs, both amino acids and zinc should be included in the diet as key nutrients [33,34]. Piglets have low acid activity in their stomach at weaning, which, along with other factors such as low lactic acid concentration and/or irregular large meal intake, can lead to an increased gastric pH, even more than 5.0 [35].
In this regard, the existence of several carbonate sources in the pig diet, such as sodium bicarbonate or CaCO3 has a high acid-binding capacity, which may increase the pH of the stomach of weaned pigs [35]. After weaning, increased gastric pH decreases protein digestibility [36]. Pepsinogen protein digestive enzyme is transformed into pepsin at pH 5.0 in the stomach. However, sources of carbonate increase gastric pH. Moreover, the solubility of other minerals, such as Ca, P, and Zn, could also be affected by elevated gastric pH by encouraging the formation of Zn–Ca–phytate precipitates [37]. The negative effects on performance observed in these growing pigs may be due to the decreased CP, Ca, and P digestibility of the CaCO3 diet. The discrepancy in the results observed in the literature regarding the effects of using carbonate sources on pig performance was explained by the digestibility of intrinsic effects in minerals. However, to evaluate the relationship in this argument, further research with additional CP, Ca, and P supplements is required.
In contrast to pigs fed with CaCO3 diets, blood chloride, HCO3−, BE, and EB concentrations were shown to be lower in pigs fed with CaCl2 diets for 14 and 28 days. The acidogenic behavior of the CaCl2 diet and the influence of the acid-base balance in pigs were demonstrated in this experiment. For instance, Patience et al. [27] reported that supplementation of pig diets with chloride sources can decrease BE and HCO3− concentrations. Similarly, Patience and Chaplin [31] and Dersjant-Li et al. [38] found that blood BE, HCO3−, and pH were decreased when pigs fed on CaCl2 sources in their diet were compared with pigs fed on CaCO3 sources in their diet, respectively.
Different from Exp. 1, we observed less deposition of bone ash, Ca, and P in Exps. 2. Greater bone mineralization in Exp. 2 compared to Exp. 1, caused by the different dietary Ca levels. The existence of Ca and P can influence the deposition of bone minerals, and they accumulate at a constant ratio (2.2:1) during the formation of hydroxyapatite [4]. The fact that pigs can accumulate considerably more bone Ca and P than is required for optimal pig growth performance has been suggested in several studies [11–13,25]. This means that the Ca and P concentrations required to achieve great muscle growth are lower than those required to achieve maximum skeletal tissue synthesis. As a result, our observations indicate that additional Ca supplementation in the diet is not required for bone mineralization if P meets the requirements.
CONCLUSION
If dietary P is at or above this requirement, dietary Ca levels could have detrimental effects on growth performance. Although the lowest dietary Ca level decreased bone Ca and P levels, bone parameters were less sensitive to dietary Ca levels than growth performance. A Ca and P ratio of 1.04–1.22: 1 is required for maximizing growth performance in growing pigs. Dietary Ca sources also changed the acid–base balance and apparent digestibility of pigs, as changes in blood chloride, HCO3−, BE, and EB concentrations reduced apparent CP, Ca, and P digestibility. This was observed when the piglets were fed a Ca-carbonated diet. In this study, CaCl2 was a better choice for Ca source in the diet rather than CaCO3. However, further studies are required to elucidate the mechanisms underlying these responses.
