Manganese (Mn) is an essential trace element for birds, which plays an important role in the metabolism of carbohydrates, amino acids, and as a necessary cofactor of numerous enzymes, hydrolases, and normal bone formation, in poultry (Keen et al. 1999; Moomaw et al. 2009). Inorganic Mn supplements, including manganese oxide (MnO) or sulfate (MnSO4), are routinely added to conventional poultry diets to meet the Mn requirements of birds for their optimum growth. However, the bioavailability of these inorganic Mn supplements (mineral salts) is very poor, leading to high concentrations in the excreta. It may be related to the formation of complexes with other substances in the digestive tract, such as phytate, which reduces the solubility of these elements and their absorption, thereby increasing their excretion (Bao et al. 2007). Numerous studies have been conducted to show the Mn homeostasis and excretion (Bertinchamps et al. 1966; Suzuki and Wada, 1981), tissue distribution (Underwood, 1977), and mechanism of absorption (Lonnerdal et al. 1987; Southern et al. 1987). It has been shown that ileum was the main site of Mn absorption for broilers (Khakpour et al. 2019), therefore, Mn bioavailability from Mn supplements used in poultry diets (Baker and Halpin, 1987; Fly et al. 1989). Baker and Halpin (1987) reported that the Mn bioavailability from MnSO4 is roughly similar to that of MnCl2 and it is higher than that of MnO and MnCO3. The high amount of Mn content observed in broiler feces is due to a high level of supplementation and/or indigestibility of the Mn source used. The supplement indigestibility is affected by endogenous or exogenous factors. The change in mineral balance caused by physiological status alteration and low Mn bioavailability of Mn supplement source led to endogenous and exogenous Mn indigestibility, respectively bioavailability. Therefore, additional Mn supplementation for poultry diets may be necessary to maintain the correct Mn level (Scott et al. 1976). The purpose of the present study was to evaluate the relative bioavailability of MnO from different sources in broilers diets. Followed by an investigation of the effect of Mn bioavailability on tibial characteristics, and serum inorganic phosphorous (P), calcium (Ca), and Mn concentration.
MATERIALS AND METHODS
The experiment was carried out based on a completely randomized design (CRD) using 660 male broiler chickens (Ross 308) assigned to 12 dietary treatments by 6 replicates including 10 birds each. The birds were fed the experimental diets for 21 to 42 days and water was offered ad libitum. The experimental diets consisted of one basal diet (as control), and 10 treatment groups which were supplemented with 400 or 800 mg/kg of feed of 1-5 MnO sources with 35%, 25%, 45-55%, 30%, and 40% purities, respectively; MnSO4 was also used (800 mg/kg of feed) in 1 treatment group as a reference standard with 100% bioavailability (Table 1). The guide for care and use of laboratory animals was followed, and the project was approved by the animal experimentation ethics committee (CETEA) of the Federal University of Minas Gerais, (protocol number 111/2009).
Determination of Mineral relative bioavailability value (RBV) and apparent ileal availability of minerals (AIAM)
The RBV was determined using basal diet as the standard source by slope ratio comparisons (Littell et al. 1995; Littell et al. 1997). Variable responses to each source were used to calculate RBV of Mn content of diets and tibia bone. Based on diets and tibia bone Mn, RBV were calculated as equation (1):
RBV= (Tibia Mn–Y intercept) × 100 / (slope of regression line relating diet or tibia Mn×intake Mn) equation (1)
Differences between sources were determined by differences in their respective regression coefficients. For determining a biological apparent ilea digestibility of basal diet with mineral sources, chromic oxide was used (Cr2O3; 0.3 percent in all diets) as an indigestible marker. Whole ileal digesta were individually collected, and measured for Cr2O3 and mineral concentrations. Apparent ideal mineral digestibility (AIAM) in experimental diets was calculated using equation (2) (Kadim and Moughan, 1997; Kadim et al. 2002).
AIAM= ((feed Mn/Cr2O3%) – (excreta Mn/excreta Cr2O3)) / (feed Mn/Cr2O3%) equation (2)
Collection and processing of samples
In order to determine the RBV food intake and total droppings, output was measured quantitatively per cage over 4 consecutive days during the 3rd week of the trial (21 to 42 d). The droppings were collected daily, dried overnight at 80 ˚C in a forced-draft oven and subsequently the collections from each pen were combined for analysis. At the end of the trial (d 42), all surviving chicks were killed by intracranial injection of sodium pentobarbitone and their small intestine were immediately exposed. The contents of the lower ileum were expressed into plastic containers by gentle flushing with distilled water. The ileum was defined as that portion of the small intestine extending from the vitelline diverticulum to a point 40 mm proximal to the ileocaecal junction. The ileum was divided into 2 halves and the digesta were collected from the lower half towards the ileocaecal junction. The digesta samples were frozen immediately after collection. Dried droppings and ileal digesta samples were ground to pass through a 0·5-mm sieve and stored at –4 ˚C until required for chemical analysis. The Mn concentrations from different sources were measured by flame atomic absorption spectroscopy (Spectro AA, VARIAN) as described by (AOAC, 1990).
Tibia bone characteristics
Tibia bone characteristics were measured according to the methods described by Zhang and Coon (1997) and Park et al. (2003). Briefly, all the bones were first weighed in the presence of air, then reweighed while suspended in water at room temperature. Bone volume was calculated with the assumption that the specific gravity of water is 1 g/cm3 at room temperature. For the fresh bone preparation, breaking strength was first measured, and bones were then dried at 100 ˚C for 24 h and weighed again. For measuring bone ash content, bones were oven-dried at 105 ˚C for 24 h and ashed in a muffle furnace at 600 ˚C for 6 h. The ash percentage was determined relative to the dry weight of the tibia. The Seedor index is the value obtained by dividing the bone weight by its length (Seedor et al. 1991; Seedor, 1993; Seedor, 1995).
Table 1 The calculated composition and nutrient content of experimental diets fed to broilers chickens (42 days)
CP: crude protein.
* Provided per kilogram of diet: vitamin A: 44000 IU; vitamin D3: 17000 IU; vitamin E: 440 mg; vitamin K3: 40 mg; vitamin B12: 70 mg; vitamin B1: 65 mg; vitamin B2: 32 mg; Pantothenic acid: 49 mg; Niacin: 122 mg; vitamin B6: 65 mg; Biotin: 22 mg and Choline chloride: 27 mg.
** Minerals: Mn (MnO): 99.20 mg; Zn (ZnO): 85 mg; Fe (FeSO4): 50 mg; Cu (Cu SuSO4): 10 mg; Se (Na2SeO3): 0.2 mg; I (KI) 13 mg and Co: 250 mg.
The Seedor index is a measure of bone density, in another word the higher the Seedor index value, the denser the bone.
The data were analyzed by the General Linear Models (GLM) procedure (SAS, 2004). Duncan's multiple range test was used to compare each experimental group with the control group of means (P<0.05).
RESULTS AND DISCUSSION
The purity of Mn concentrations from sources contained 25 to 55% for oxide, respectively, whereas the sulfate source had 30.5% Mn. Although, the oxide source of 3 had a higher concentration of Mn compared to other sources. Examining analysis of Mn sources using flame atomic absorption spectroscopy method indicated a lower degree of purity for all oxide and sulfate mineral sources, respectively (Table 2). Thus, presenting less concentration of Mn in each source could affect Mn bioavailability. Nevertheless, these differences in Mn concentrations may be related to low solubility, also decreased availability, and more excreted. Data presented those concentrations of manganese from different sources in diets fed to broilers chickens had significant effects on biological apparent ileal digestibility values dietary minerals (g/100 g) in different sources (Table 3), whereas, manganese oxide source 3 had relatively significantly higher Mn availability (P<0.05). However, manganese oxide sources 1 and 2 were significantly lower than other sources. The availability of Mn for utilization varies between inorganic Mn sources including MnO and MnSO4. In terms of experiment results, in contrast to the Baker and Halpin (1987) study, bioavailability of Mn from MnSO4 in the current experiment was lower than MnO sources. This can be attributed to the chemical form in which the Mn occurs. Estimates of the relative bioavailability were obtained by the slope ratios of linear regression equations (Table 4). When the regression slope for sulfate source was set equal to 100 %, relative bioavailability of 145 ± 5.21, 27.27 ± 3.98, 181.82 ± 5.03, 36.36 ± 7.72, and 181.82 ± 0.81 were obtained for dietary Mn concentrations of different Mn sources, respectively. The means of relative bioavailability values of Mn oxide sources ranged from 27.27 to 181.82 and were significantly different.
Table 2 Analyzed Mn concentrations from different sources
* By producer company and ** In our laboratory.
Table 3 Bioavailability and concentrations of manganese from different sources in diets fed to broilers chickens (as-fed basis)
1Apparent ileal availability of minerals (AIAM)= ((feed Mn/Cr2O3 %) - (excreta Mn/excreta Cr2O3)) / (feed Mn/Cr2O3 %).
SEM: standard error of the means.
Table 4 Regression coefficient and relative bioavailability value (RBV) of manganese sources
1 RBV= (Mn sources–Y intercept) ×100 / (slope of regression line relating diet or Mn sources×Mn intake).
These results agreed with the study of Luo (1989) claiming that among several inorganic manganese sources used in broiler diets, manganese oxide would be lower in bioavailability in comparison to the other sources. Another way, these differences between relative bioavailability values of sources would be depended on the chemical or physical form Mn sources. Besides certain components of the sources can interfere with their absorption or relative bioavailability. Otherwise, Mn contents in bird’s manure are far more than nitrogen. Hence, Mn in poultry manure is far more than crop requirements, causing accumulation in the soil, especially in regions of intensive poultry production (Meijer and Kroger, 1973; Hahn, and Baker, 1993; Mohanna and Nys, 1998). The less soluble compounds, such as carbonates and oxides, have lower relative biological availabilities (Fly et al. 1989; Wedekind et al. 1992; Ammerman et al. 1998). Also, Mn element have specific physiological and biochemical functions in the body and, as a result, their deficiency leads to reduced production and reproductive performance and maybe impaired fat and carbohydrate metabolism and immune response (Underwood and Suttle, 1999). There were significant (P<0.05) effects of manganese sources on the apparent retention of Mn (Table 5). The apparent Mn retention and retained Mn of oxide form of sources 4 and 3, were significantly higher than Mn oxide sources 5, 1, 2, also Mn sulfate, and control diet. Our experiment showed that Mn retention and retained Mn of sulfate sources were significantly lower than oxide sources (P<0.05). This may be mostly reflected in the trends observed for apparent Mn retention. Mn content of each source can form complexes with multivalent anions to produce insoluble salts, thereby reducing the availability of some Mn sources and showing significant differences.
Table 5 Mean apparent Mn retention (%) and apparent retained Mn content (mg/kg) of manganese sources
* Apparent mineral retention %= (diet mineral×feed intake) - (excreta mineral×total excreta) × 100 / mineral diet × feed intake.
** Apparent retained mineral content (mg/kg)= diet mineral (mg/kg) × apparent mineral retention %.
SEM: standard error of the means.
Table 6 Effect of dietary manganese source and concentration on blood minerals (mg/dL)
SEM: standard error of the means.
Furthermore, few studies have investigated the impact of inorganic Mn sources supplemented to broiler diets on the ileal digestibility or retention of Mn, or tended to be contradictory (Khakpour et al. 2019). Although the proportion of most trace elements absorbed in the intestine is rather low, homoeostasis of elements such as Mn is regulated via endogenous secretions (Sebastian et al. 1996; Sandstrom, 1997; Saima et al. 2009; Rutherfurd et al. 2012). Significant differences (P<0.05) were observed between treatments in Mn contents of blood sera on d 35 to 42 of the experiment (Table 6). Therefore, mean Ca concentrations of birds fed Mn oxide 4 source were significantly higher than those fed Mn sulfate or basal diet. Also, mean Mn concentrations of the group fed Mn sulfate source is significantly higher than those fed Mn oxide or the basal diet (P<0.05). The effect of different sources of dietary Mn, Ca, P, on ash concentrations, diameter, and length of tibia bone are presented in Table 7. The Mn concentrations in tibia bone obtained from Mn sulfate source were significantly higher than all oxide sources (P<0.05). These results agreed with Yan and Waldroup (2006) that reported the significant effects of Mn source on Mn content of tibia bone, in contrast to the other study (Wong-Valle et al. 1988; Henry et al. 1989; Smith et al. 1995). Moreover, Mn sources did not affect the ash, diameter, and length of tibia bone. Tibia bone Mn concentrations responded linearly to Mn intake in the 35 to 42 days of birds age (Table 8). The relative bioavailability values from Mn oxide sources were 22.28 to 142.85% and for Ca within the range 50.18 to 76.75% (P>0.05). The birds fed source 2 and 5 Mn oxide forms had 43 and 23% higher levels of tibia Mn than those fed the sulfate form, respectively. However, these differences for bioavailability values for the reason that inorganic minerals are present in chyme as ions that interact with other dietary constituents to form differences available or less unavailable complexes for absorption. Because the inorganic complexes formed by various sources have different characteristics or functions that might affect their absorption in the bird gut.
Table 7 Effect of dietary manganese source and concentration on tibia manganese, Ca, P and ash concentrations of 35-42 d old broiler chickens
SEM: standard error of the means.
Table 8 Relative bioavailability values of manganese sources based on slop ratio of tibia bone manganese concentrations
1 RBV= (tibia Mn–Y intercept) × 100 / (slope of regression line relating diet or tibia Mn×Mn intake).
Table 9 Relative bioavailability value (RBV) of tibia bone Mn on broiler chickens
SEM: standard error of the means.
The relative bioavailability value of Mn oxide relative to Mn sulfate obtained in our study, apart from oxide sources of 2 and 5, agrees with the other reports. In this regard, we can refer to the study of Black et al. (1984) and Wong-Valle et al. (1988) in which they reported bioavailability of 65.6, 81.9, 91.3, 75.0, and 70.3% for different Mn oxide sources compared to Mn sulfate based on multiple linear regressions on bone Mn concentration for broiler chicks (Ammerman et al. 1998). The reason for this difference in bioavailability can be attributed to higher Mn bioavailability of 2 and 5 oxide sources compared to sulfate form that may be associated with lower levels of interacting minerals such as Ca and Fe in these sources. Though bioavailability responses to these sources did not show the same trend for all indices measured. According to results, retention of Mn in diet and tibia bone was significantly different between Mn sources (P>0.05). In the case of, retention of Mn element, in tibia, there was more sensitivity to Mn sources. Tibia Mn has come to be recognized as the variable of choice in the calculation of relative bioavailability value (Cook, 1973; Ranhotra et al. 1976). These variable responses on each source were used to calculate RBV of Mn content of diets and tibia bones. Based on diets and tibia bone Mn, RBV were calculated as follows:
RBV= (tibia Mn–Y intercept) × 100 / (slope of regression line relating diet or tibia Mn×Mn intake)
Utilizing this method, the RBV of Mn sources were significantly different (Table 9) (based on reference diet=100%). These differences may be depended on physico-chemical properties of Mn sources such as the cation exchange capacity and compound absorptive properties (Van Der Klis et al. 1993; Bach Knudsen, 2001).
In conclusion, the results of the present study clearly demonstrated that Mn concentrations from Mn oxide sources were that were examining analysis by using flame atomic absorption spectroscopy method in our laboratory lower degree of purity. Thus, this could be contributed to reduced relative bioavailability values, absorption, and also, Mn retention. Furthermore, there were indigestible that could be from altered mineral balance caused by physiological changes such as decreased Mn bioavailability and will increase dietary Mn requirements, thence, there must be Mn supplemented addition to poultry diets. Therefore, our data indicated that different inorganic Mn oxide sources have differences regarding the relative bioavailability values, retention, or absorption in the ileum of birds. Nevertheless, these aforementioned differences of Mn concentrations and relative bioavailability values may be related to low solubility, physiological and biochemical functions, or chemical form in which the Mn occurs.
The authors wish to acknowledge Sepahan Daneh Parsian Co. for funds in support of this research.