Biological Availability of Phosphorus in Raw and Acidulated Sinda and Chilembwe Rock Phosphates in Broiler Chickens

A 14 day broiler chickens feeding trial was conducted to determine bio-availability and relative biological values (RBV) of phosphorus in raw and acidulated local rock phosphates (RPs) as potential replacements for imported and more expensive di-calcium phosphate (DCP). Treatments included Raw, Sulphuric acid and Phosphoric acid acidulated Sinda and Chilembwe RPs. Bio-availability was assessed based on feed intake, body weight gains, feed conversion ratios, phosphorus retention and mineralization of tibia bones and blood serum. RBVs were calculated on the same parameters using DCP as a reference standard. Phosphorus contents in local RPs were significantly (P ≥ 0.05) lower than in DCP, but acidulating RPs with Phosphoric acid significantly increased phosphorus, but reduced calcium contents. Other minerals were comparable with what was in DCP. Acidulating RPs significantly (P ≥ 0.05) increased body weight gains and feed conversion ratios, but not feed intake. Phosphorus retentions, weights and ash contents of tibia bones were significantly (P ≥ 0.05) lower in chickens fed diets based on raw than that of those fed diets based on acidulated RPs and DCP; an indication that acidulating the RPs improved phosphorus bio-availability in broiler chickens. There were however; no statistical differences (P ≥ 0.05) among treatments in calcium and phosphorus contents in both tibia bones and blood serum.

Keywords: Sinda; Chilembwe; Rock Phosphates; Phosphoric and Sulphuric Acids; Acidulation; Phosphorus Bio-Availability

Practical poultry diets are largely made up of cereal grains and oilseed meals that are normally deficient in dietary phosphorus. This is because up to 80% of phosphorus in feed ingredients of plant origin is bound to phytic acid (Rama Rao et al. 1999 and Kies et al., 2001) and only about 30% of this amount is assumed to be available to poultry and other non-ruminants (Nelson et al., 1968) [1-3]. Thus, rather than depending on total dietary concentrations, phosphorus requirements in poultry and other non-ruminants are based on biological availabilities that estimates portion of dietary phosphorus that is absorbed and utilized by the animal (Shastak et al., 2012; WPSA, 2013) [4,5]. A number of response criterion are employed for assessing phosphorus bio-availabilities including feed intake, body weight gains, feed conversion ratios and mineralization of bones and blood. Bone mineralization is usually based on tibia or toe bone weights, ash content and concentration of phosphorus and calcium in these tissues (Shastak et al., 2012) [4]. Phosphorus bio-availability may also be evaluated based on amounts retained in animal body after subtracting excreted amounts from what was consumed in the feed (Godoy and Chicco, 2001) [6]. To avoid underestimating retained amounts, feacal samples are usually collected from the distal end of the ileum in order to exclude urinary phosphorus excretions (Sastak et al, 2012) [4]. Phosphorus bio-availabilities may also be assessed based on relative biological values (RBVs), whereby different phosphorus sources are evaluated by comparing performance of animals fed diets based on test phosphorus sources with that of animals fed diets based on a known reference standard that is usually assigned 100% availability (Lima et al., 1997) [7].

In recent years, a number of studies have established that available phosphorus requirements in broiler chickens is far less than current National Research Council (NRC, 1994) recommendations (Applegate and Angel, 2014; Li et al., 2016; Hamdi et al., 2017) [8-11]. However, commercial broiler diets are still formulated to meet current NRC recommendations for safety margins that guarantee animals from showing deficiency symptoms (Li et al., 2016; Applegate and Angel, 2014; Angel et al., 2003) [9,10,12]. However, with such intakes, there is always the danger of over-supplementation that does not only result in higher feed costs but also increased phosphorus excretions leading to environmental pollutions (Shastak and Rodehutscord, 2015) [13]. The main sources of inorganic phosphorus in poultry and other non-ruminant diets are mono-calcium, di-calcium and tri-calcium phosphates that are derived from processed rock phosphates (Lima et al., 1997; Fernandes et al., 1999; Petersen et al., 2011) [7,14,15]. In Zambia, processed phosphates are imported and because of limited foreign exchange earnings, they have become too expensive; while use of alternatives such as animal processing by-products is limited by variability in supply and content of essential nutrients including phosphorus (Waldroup, 1999) [16]. This has necessitated the need for exploitation of local RPs to replace imported phosphates. This research was conducted to evaluate use of raw and acidulated Sinda and Chilembwe RPs as alternative sources of inorganic phosphorus in broiler starter diets. Evaluations were based on mineral composition, phosphorus retention, feed intake, body weight gains and feed conversion ratios and mineralization of tibia bones and blood serum in broiler chickens fed diets based on raw and acidulated RPs using DCP as a control.

This study was conducted in the School of Agricultural Sciences at the University of Zambia in Lusaka. The RPs was obtained from Sinda and Chilembwe rock phosphate deposits in Sinda and Petauke Districts of Zambia’s Eastern Province. RPs from each source was processed by crushing with a Jaw Stone Crusher (Denver Equipment Pvt. Limited) for particles to pass through a 5 mm screen followed by grinding with Fristsch Rotary Ball Mill through a 0.2 mm sieve. Ground RPs from each source were then divided into three portions of 10 kg each, with the first portion being left intact as raw, while the second and third were each acidulated with 2 litres per kg of concentrated Sulphuric acid (98% Analytical grade) and 84% Phosphoric acid: respectively. After acidulation, the RPs were washed with distilled water and left to dry in a forced air drying oven. This resulted in six treatments consisting of raw, Sulphuric acid and Phosphoric acid acidulated Sinda and Chilembwe RPs. Commercial DCP was included as a control for the study. Samples of prepared RPs were first analysed in triplicate for Calcium (Ca), Phosphorus (P), Sodium (Na), Zinc (Zn) and Lead (Pb) using standard procedures of the Association of Official Analytical Chemists (AOAC, 1998) [17].

Determination of phosphorus bio-availability and relative biological values of phosphates based on broiler chickens feeding trial.

After mineral composition analysis, prepared RPs were incorporated into starter rations for a 14-day feeding trial to determine phosphorus bio-availability based on performance of broiler chickens using DCP as a control treatment (Table 1). The trial used 252 ten-day old unsexed Hubbard broiler chickens that were initially fed a commercial broiler starter diet (Novatek Feeds Limited).

For the trial, 6 chicks of relatively similar body weights (231.7±7.81g) were randomly allocated to experimental diets and placed in battery cages that were equipped with feeders and drinkers. Each treatment was replicated in 6 battery cages. The chicks were housed in a temperature controlled room where they were exposed to 23 hours of lighting at a temperature of 28 oC each day. The temperature was reduced to 24 oC after one week of the study. Feed and water were provided ad libitum throughout the experimental period.

Performance of chickens during the trial was assessed based on daily feed intake and weekly body weight gains that were used to calculate feed conversion ratios. Retention of phosphorus was determined by analyzing for phosphorus content in excreta to assess portions of consumed amounts that were retained for utilization by the chickens. The excreta was collected during the last 4 days of the trial and after each collection, the collected amounts were frozen at -20 oC and kept until the end of the trial. After the trial, all excreta samples from each cage were thawed, dried at 60 oC for 48hrs and weighed after cooling to room temperature. The dried samples were then pooled and analyzed for phosphorus to determine amounts retained by subtracting excreted amounts from what was consumed as described by Lima et al. (1995) and Coon et al. (2007) [18,19].

On day 20, three birds from each cage were randomly selected to collect blood from the wing web vein using plain vacutainer tubes for determination of phosphorus and calcium in blood serum as described by Lima et al. (1997) [7]. On day 24, there was another random selection of three birds from each cage that were slaughtered to harvest left legs from each chicken. The legs were then boiled for 10 minutes, after which the tibia bones were cleared of attaching muscles and ligaments and defatted before being dried at 50 oC for 48 hours (Driver et al., 2006) [20]. The dried bones were then weighed after cooling to room temperature to determine tibia bone weights before being incinerated for 24 hours at 600 oC to determine ash content that was later analysed for calcium and phosphorus contents. The analysed parameters were then used to determine phosphorus bio-availabilities and to calculate relative biological values (RBV) of phosphorus in raw and acidulated local RPs using DCP as a reference standard.

The study was conducted as a Completely Randomized Design (CRD) consisting of six treatments and a control. The feeding trial used six birds in a cage for each treatment that served as an experimental unit that was replicated six times. All collected data were subjected to Analysis of Variance (ANOVA) according to General Linear Models (GLM) of Statistical Analysis System (SAS). An F-test was used to detect significant differences among treatment means, which were separated using least significant different (LSD) according to Tukey’s test of significance at P ≥ 0.05.

Sinda and Chilembwe raw RPs contained similar amounts of phosphorus that were significantly (P ≥ 0.05) lower than what was in DCP (Table 2). Acidulating the RPs with Sulphuric acid had no effect on phosphorus content, but phosphoric acid increased amounts closer to what was in DCP. The content of calcium in Sinda raw RPs was statistically similar to what was in DCP, while Chilembwe raw had significantly (P≥0.05) lower amounts. Acidulating RPs with either acid reduced calcium content, with the reduction being greater when phosphoric acid was used as demonstrated by significantly (P≥0.05) lower calcium levels in phosphoric acid acidulated RPs. Content of lead was significantly (P≥0.05) higher in raw RPs an indication that acidulating the RPs reduced its concentration as demonstrated by having no significant (P≥0.05) differences in lead content among acidulated RPs including DCP. The amount of zinc was significantly higher in Sinda than Chilembwe RPs, with DCP having the lowest amounts. The amounts of sodium in RPs were between 0.25 and 0.39% and did not seem to have been affected by acidulating the RPs. The sodium in DCP were statistically similar to what was in Sinda RPs that was significantly (P≥0.05) higher than what was in Chilembwe RPs.

Performance of broiler chickens fed diets based on raw and acidulated Sinda and Chilembwe RPs in comparison with that of the control group showed no significant differences (P ≥ 0.05) in feed intake among all treatments (Table 3). There were however; significant differences (P ≥ 0.05) in body weight gains (BWG) with birds fed diets based on DCP and acidulated Sinda and Chilembwe RPs having significantly (P ≥ 0.05) higher weight gains than those fed diets based on raw RPs. The increase in body weight gains of chickens fed diets based on acidulated Sinda RPs was however; not significantly (P ≥ 0.05) different from that of chickens fed diets based on raw Sinda RPs. Results on feed conversion ratios (FCR) reflected that of body weight gains and also showed that chickens fed diets based on acidulated RPs and the control had significantly (P ≥ 0.05) better feed conversion ratios than that of chickens fed diets based on raw RPs.

When the results on performance of broiler chickens were expressed as relative biological values (RBVs) of phosphorus using DCP based diet as a reference standard, feed intake figures ranged from 94.7 to 100.5% and also showed no major differences among treatments. The RBV figures on body weight gains ranged from 88.3 to 100.2% with chickens fed diets based on raw RPs having significantly inferior gains than that of chickens fed diets based on phosphoric acid acidulated RPs. This was also the case when RBVs were based on FCR where the figures ranged from 88.44 to 103.36% again demonstrating that diets based on raw RPs promoted inferior phosphorus utilizations compared with that of chickens fed diets based on acidulated RPs and DCP.

The results on retention of phosphorus in broiler chickens showed the amounts of phosphorus retained in chickens fed diets based on raw Sinda and Chilembwe RPs to be significantly lower (P ≥ 0.05) than that of chickens fed diets based on acidulated RPs and the control (Table 4). Only chickens fed diets based on Sulphuric acid acidulated Chilembwe RP had phosphorus retention figures that were statistically (P ≥ 0.05) similar to what was recorded for raw RPs. When phosphorus retention results were expressed as RBVs using DCP as reference standard, they showed Sinda and Chilembwe raw RPs to have lower figures when compared with that of chickens fed diets based on acidulated RPs. Only chickens fed diets based on Sulphuric acid acidulated Chilembwe RPs had retention figures that were significantly lower than that of the control diet.

The tibia bone weights of chickens fed diets based on the control diet were significantly heavier (P ≥ 0.05) than that of chickens fed on diets based on raw Sinda and Chilembwe RPs (Table 5). There were however; no significant differences (P ≥ 0.05) in tibia bone weights of chickens fed diets based on acidulated Sinda and Chilembwe RPs and that of chickens fed the control diet. However, the increase in tibia bone weights as a result of acidulating RPs was not enough to be statistically different (P ≥ 0.05) from that of chickens fed diets based on raw RPs. When results on tibia bone weights were expressed as RBVs using DCP as a reference standard, the figures for chickens fed diets based on raw RPs were about 15% points lower than that of the chickens fed diets based on the control. The results on tibia bone ash contents showed significant differences (P ≥ 0.05) among chickens fed diets based on raw and acidulated Sinda and Chilembwe RPs. There were however; no significant (P ≥ 0.05) differences in tibia bone ash contents between chickens fed diets based on the control and acidulated Sinda and Chilembwe RPs. When tibia bone ash contents were expressed as RBVs, the figures ranged from 99.76 to 102.44%, which was very close to 100% that was assigned for the reference standard. There were however, no significant differences (P ≥ 0.05) in calcium and phosphorus content in tibia bones of chickens fed diets based raw and acidulated RPs including that of the control diet.

The concentration of phosphorus and calcium in blood serum was also not affected by the source of inorganic phosphorus as there were no significant (P ≥ 0.05) differences among treatments including that of the control (Table 6). The concentration of phosphorus in blood serum ranged from 2.06 to 2.55 mmol/litre while that of calcium was from 1.99 to 2.44 mmol/litre and both showed no particular trends in the concentration of the two minerals as a result of acidulating the two local RPs. The RBVs also revealed no differences (P ≥ 0.05) among treatments in the content and relative biological availability of calcium and phosphorus in blood serum.

The contents of calcium and phosphorus in commercial DCP used for the current study were as expected considering that the two minerals in DCP vary between 24.0 and 27.0% and 18.0 and 20.5% for calcium and phosphorus, respectively (Lima et al., 1995) [19]. Recently phosphorus contents in DCP were reported to vary between 18.4 and 22.8% (Bikker et al., 2016) [21]. The current results were however; lower than the 22.7% and 29.0% reported as phosphorus and calcium contents in DCP that was used as a control for the Venezuelan study (Godoy and Chicco, 2001) [6]. The reported figures for phosphorus in Sinda and Chilembwe RPs are an indication that Zambian RPs are generally low in phosphorus content and these results are consistent with earlier findings that classified Zambian RPs to be low grade phosphate deposits (Aydin et al., 2009; Simukanga et al, 1994) [22,23]. The 9.87% phosphorus content in Sinda raw RP was comparable with 9.4% reported by Cheleshe et al. (2000) and Chirwa (2014) while the 10.54% in Chielembwe RPs was closer to the 12.0% reported by Appleton (2002) [24-26]. When compared with phosphates from other sources, the amounts of calcium and phosphorus in Sinda and Chilembwe raw RPs were within ranges of 10.5 to 12.3% for phosphorus, and 20.0 to 34.4% for calcium reported for Venezuelan RPs (Godoy and Chicco, 2001) [6]. The reduced calcium content noted in acidulated Sinda and Chilembwe RPs could have been due to formation of insoluble Calcium Carbonates whereas the increase in phosphorus content noted in phosphoric acid acidulated RPs was probably from the 15.9% soluble phosphorus contained in this acid (Zapata, 2004) [27].

The amounts of sodium in Sinda and Chilembwe RPs were higher than 0.03% recorded for DCP; an amount that was comparable with 0.028% reported for DCP by Hyghebeart et al. (1980) [28]. Other studies have reported sodium contents in RPs to vary between 0.18 and 0.44% (Luiz et al., 2009); although Waldroup (1999) reported a narrower range of 0.10 to 0.17% [16,29]. Higher amounts of sodium are reported in sodium phosphates where they were found to average 5.28% in de-fluorinated sodium phosphates, 28.74% in disodium phosphates and 14.70% in monosodium phosphates (Hyghebeart et al., 1980) [28]. This may be an indication that sodium contents in RPs tend to vary with the source, type and processing conditions to which the rock phosphates are exposed (Lima et al., 1995; Abouzeid, 2008) [19,30]. The same could be said for the content of lead and zinc and it is unlikely that their contents in Sinda and Chilembwe RPs could have been affected by acidulation of RPs. It must however, be noted that the amounts of lead and zinc in Sinda and Chilembwe RPs were all below allowed maximum toxic thresholds for use in broiler chicken diets (NRC, 1980; AAFCO, 1999) [31,32].

The lack of significant differences in feed intake among chickens fed different treatment diets was in concert with findings of Rama Rao and Reddy (2003) and Mello et al. (2012) who also reported no significant differences in feed intake among broiler chickens fed starter diets based on different sources of inorganic phosphorus [33,34]. In more recent years, a similar study also reported feed intake not to be affected by levels of available phosphorus in the diets, despite influencing body weight gains and feed conversion ratios (Zieai et al., 2011) [35]. The lack of significant differences in feed intake among chickens fed different treatment diets in the current study could have been due to the fact that all treatments had adequate supply of available phosphorus that was above minimum requirements (Cardoso et al., 2010) [36]. This is supported by findings of Bikker et al. (2016) who only noted variations in feed intake, body weight gains and feed conversion ratios among broiler chickens fed different treatment diets when retainable phosphorus levels were less than 2.7g/kg diet [21]. No significant differences were recorded in the performance of chickens when retainable phosphorus levels ranged from 0.27 to 6.0 g/kg diet. Similar observations were reported by Askari et al. (2015) when different dietary calcium and phosphorus levels could not affect feed intake, body weight gains and feed conversion ratios [37]. Hamdi et al. (2017) also observed no differences in the performance of broiler chickens on phosphorus availability among different mineral phosphorus sources [11]. A feeding trial with pigs also reported neither the source nor the amount of phosphorus in the diet to have any influence on feed intake, daily gain and the feed to gain ratios (Petersen et al. 2011) [15]. However, other studies have reported reduced feed intake, body weight gains and feed efficiency under higher calcium and low phosphorus intakes (Shastak et al., 2012) [4]. In the current study, the reported inferior body weight gains and feed conversion ratios among chickens fed diets based on raw RPs could either have been due to reduced phosphorus bio-availability or intake of some toxic elements such as mercury, lead or fluorine in the RPs (Godoy and Chicco, 2011; Odongo et al., 2002; Tahir et al., 2011) [38-40]. Presence of toxic elements in Sinda and Chilembwe RPs may be ruled out on the basis that there was no mortality among chickens fed different treatment diets and the fact that all measured potentially toxic elements in the diets were below allowed maximum thresholds for toxicity (NRC, 1980) [31]. The calcium to Phosphorus ratios could also have affected the response of chickens to different treatment diets in the study (Leske and Coon, 2002; Yan and Waldroup, 2006, Mello et al., 2012) [33,41,42].

Phosphorus bio-availability in non-ruminants may also be assessed as a percentage of the amounts consumed in feed that is retained in animal tissues or relative biological values based on comparison of performance of animals fed a test phosphorus source and that of others given diets based on a known reference standard that is assumed to have 100% availability (Lima et al., 1997) [7]. Both methods were used in this study to evaluate bio-availability of phosphorus in raw and acidulated Sinda and Chilembwe RPs. The results on retainable phosphorus in this study were generally within range of several studies in literature (Bikker et al., 2016; Li et al., 2016; Shastak and Rodehutscord, 2015) [10,13,21]. The reported figures were generally low may be because of the method used in estimating retainable phosphorus that was based on measuring excretion from the entire gut, which sometimes is said to underestimate amounts as it does not account for urinary excretions (Shastak et al., 2012; WPSA, 2013) [4,5]. The current results were however consistent with findings of De Groote and Huyghebeart (1997), who also reported apparent availabilities of reported phosphorus retention figures to vary between 55 and 92% when commercial RPs and animal processing by-products were used as sources of inorganic phosphorus (Van Der Kliss and Versteegh, 1996) [44]. Even pre-caecal digestibilities were found to range from 45 to 72% (Bikker et al., 2016) and from 44.57 to 59.56% (Hamdi et al., 2017) when different types of mono calcium phosphates used as sources of inorganic phosphorus in broiler chickens between 19 and 21 days of age [11,21]. It must be noted that there are considerable variations in literature on retained phosphorus among various studies, mostly as a result of variations in the nature of bio-assays, type and age of animals and assay conditions (Li et al., 2016) [13]. The low retained phosphorus levels in chickens fed diets based on raw Sinda and Chilembwe RPs may be an indication of reduced phosphorus bio-availability in unprocessed RPs (Lima et al., 1997) [7]. It could also have been influenced by many other factors including calcium levels and the calcium to phosphorus ratios in the diet, experimental techniques, chemical form of P, concentration of non phytate phosphorus, availability and interactions of various nutrients to name a few among many others (Li et al., 2016; Ziaei et al., 2011) [13,35]. High calcium and low phosphorus levels in the diet have been reported to reduce absorption and retention of phosphorus especially if it goes beyond 1.5:1 (Liu et al., 1998) [45]. Thus; optimum calcium to phosphorus ratio is crucial for increased uptake and retention of phosphorus in broiler chickens.

Increased dietary phosphorus bio-availability is documented to result in increased bone tissue synthesis and thickness (Petersen et al., 2011) [15]. Thus, the increase in tibia bone weights and ash contents in chickens fed diets based on acidulated RPs and the control could be an indication of increased phosphorus deposition in bone tissue. This is supported by earlier findings that showed tibia bone weights, ash content and the concentration of Ca and P in bones to increase with increasing levels of available phosphorus in the diet (Petersen et al., 2011; Shastak et al., 2012) [4,15]. However, the current study failed to demonstrate a corresponding increase in calcium and phosphorus deposition in both tibia bones and blood serum of chickens fed diets based on raw and acidulated RPs. Similarly, Rama Rao and Reddy (2001) also failed to show any relationship in tibia bone ash, Ca and P contents with variations in dietary phosphorus from different inorganic phosphorus sources. This led to the conclusion that Ca and P levels in diets does not seem to affect performance of chickens including their blood and bone parameters [36,46]. However, when pigs were fed diets with marginal concentrations of inorganic phosphorus (0.07 to 0.14%), they showed a correspondingly increase in bone ash, calcium and phosphorus concentrations [15]. These findings are supported by Summers et al., (1959) who concluded that body weight gains and feed conversion ratios are more sensitive measures of phosphorus bio-availability than bone and blood mineralization parameters [47]. This conclusion is consistent with more recent findings that have demonstrated that phosphorus concentrations in fat free bones may not be affected by dietary sources [4,34,37,46] . They however, contradict findings of Huyghebeart et al. (1980) and Lima et al. (1997) who reported tibia bone phosphorus contents to be a good indicator of its retention in animal tissues [19,28].

The content of phosphorus in Sinda and Chilembwe RPs was significantly lower than in DCP and acidulating the two local RPs with phosphoric acid increased phosphorus contents to levels that were statistically similar to what was in DCP. However, acidulating the RPs with either acid reduced calcium contents. The content of other minerals was not affected by acidulation and were comparable with what was in DCP. Except for Sulphuric acid acidulated Chilembwe RP, acidulating both Sinda and Chilembwe RPs improved retention and bio-availability of phosphorus as demonstrated by improved body weight gains, feed conversion ratios and retention of phosphorus in broiler chickens. This was further supported by increased weights and ash contents in tibia bones of chickens fed diets based on acidulated RPs and DCP.

The authors would like to acknowledge the Schools of Agricultural Sciences and Mines of the University of Zambia for the support rendered during implementation of this research. Funding through the United Nations University Institute for Natural Resources in Africa (UNU-INRA) is greatly appreciated. The technical personnel in the two laboratories and workers at the field station are acknowledged for the assistance rendered to the research team.