Feeding di-ammonium phosphate as a phosphorous source in finishing lambs reduced excretion of phosphorus in feces without detrimental effects on animal performance

Objective Phosphorous (P) sources with greater bioavailability might increase animal production efficiency and decrease environmental pollution. The objective of current study was to determine animal performance, nutrient digestibility, blood metabolites and fecal P concentration in finishing lambs fed a diet with either di-calcium phosphate (DCP) or di-ammonium phosphate (DAP) as a P source. Methods Twelve 4-month-old male lambs (initial body weight 24.87±3.4 kg) were randomly allocated to a diet with either DCP or DAP (~261 g/kg of total diet P) fed ad libitum for 93 days. Diets were iso-nitrogenous and iso-energetic and had same calcium (Ca) and P concentrations. Results The DAP contained 19.7 g/kg of dry matter (DM) Ca, 185.4 g/kg DM P and 14,623 ppm fluorine, while DCP contained 230.3 g/kg DM Ca, 195.2 g/kg DM P and 1,039 ppm fluorine. The diet with DAP contained 60 ppm fluorine while the diet with DCP contained 13 ppm fluorine. Lambs fed the diet with DAP tended to have a greater daily DM intake compared to those fed diet with DCP (p = 0.09). Lambs fed DAP had greater plasma P concentration and alkaline phosphatase activity (p≤0.01) compared with lambs fed DCP. Dry matter and organic matter digestibility of the diets were similar between two treatments at days 60 and 90, while they were greater in lambs fed DCP (p<0.05) at day 30 of the trial. Feeding DAP increased P digestibility (58.7% vs 50.2%; p<0.05) and decreased fecal P concentration in lambs compared with feeding DCP (3.1 vs 3.8 g/kg DM; p<0.05). Conclusion Providing ~261 g/kg of total diet P as DAP in the diet of finishing lambs improved the bioavailability of P in the body and decreased excretion of P in feces without affecting lamb performance.


INTRODUCTION
Phosphorus (P) is an essential mineral in the animal body (e.g. component of bone, many metabolic active compounds, genetic material and of cell structures) and therefore essential in the diet. Phosphorus is, however, an expensive mineral and represent a risk for environ mental pollution [1,2]. Most of the P applied in agriculture as fertilizer or in feed is derived from phosphate rock reserves, a nonrenewable resource, which are estimated to be depleted in 50 to 100 years [2]. Optimizing the supply of P in the animal diet and improving the P availability in the body when using different P sources offer an opportunity to reduce these impacts. However, deficiency in dietary P concentration or availability should be avoided to prevent metabolic disorders and poor livestock performance [3].
Dicalcium phosphate (DCP) is the common source of P in premixes for lamb rations [4]. Diammonium phosphate (DAP) contains very low cal cium (Ca), unlike DCP, which could improve formulation of diets with high levels of legume forages that contain high Ca. Russell et al [3] and Parsad et al [5] reported that DAP can be used as a source of both P and nitrogen in ruminant diets (con taining ~235.0 g/kg P and ~212.0 g/kg nitrogen). Fertilizer grade DAP might be a suitable P source for wintering diets of sheep, which are generally low in both P and N [6]. DAP is also cheaper than DCP [2]. Despite these potential beneficial properties of DAP, few studies have determined the effects of DAP in the diet of lambs. However, P sources may contain im purities (e.g. fluorine and heavy metals) that can cause toxic effects to the animal [6], which should be determined before its use in animal feeding. We hypothesized that it would be possible to replace DCP as a common P source in the diet with DAP, without detri mental effects on the performance of growing lambs. The objectives of current study were to measure calcium, P and heavy metals concentration in DCP and DAP and to determine animal performance, nutrient digestibility, blood metabolites and fecal P concentration in finishing lambs fed a diet with either DCP or DAP as the P source.

Sheep management and feeding
Twelve 4monthold Mehraban male lambs (initial body weight [BW] 24±3.4 kg) were housed in individual cages (0.9×1.5 meter) during the current study. Lambs were vaccinated for antrotoxemia and foot and mouth disease, and orally drenched for internal parasites. A control diet was formulated according to NRC [1] recommendations and fed during the 2 weeks acclimatization period before the beginning of experiment. Feed dry matter intake (DMI) and BW were recorded during second week of this period, which were used as covariate for final statistical analysis. Lambs were randomly allocated to 2 dietary treatments containing either DCP or DAP as a P source. The both diet were formulated according to NRC [1] to have similar crude protein, metabolizable energy (ME), Ca and P concentrations (Table 1) and 261 and 305 g/kg of P in the diets was supplied by either DCP or DAP and the rest from basal diet. The diets were fed ad libitum (30 to 50 g/kg orts allowed) around 08:30 h every morning for 93 days.
Lambs had free access to water throughout the experiment. Lambs were weighed at day 0, 21, 30, 44, 62, 78, and 93 of the experiment before morning feeding. During experiments, vitamin supplements such as A, D 3 , and E were not used in the diets due to carrier material used but they supplied through muscle injection. Animals in current study were cared for according to the guidelines of the Iranian Council of Animal Care [7].

Sample analysis and nutrient digestibility
Fecal grab samples were collected for 4 consecutive days (one per lamb per day) at day 30, 60, and 90 at 3 h after feeding. Fecal samples were dried at 50°C for 48 h and the 4 grab sam ples per lamb per each sampling period were pooled. At days of fecal sampling, feed samples from forage and concentrate portion (50 g from each portion) collected and mixed in the lab to make experimental diets. Diets were oven dried at 50°C for 48 h and ground through 1 mm before further analysis. Orts were collected to measure nutrient intake. Standard pro cedures described by AOAC [8] were used to determine DM, ash, calcium, and P concentration. Fecal samples were ana lyzed for acid insoluble ash according to Van Keulen and Young [9] and organic matter (OM) according to AOAC [8]. Acid insoluble ash was used as an internal marker to estimate di gestibility in combination with measured DM, OM, and P [9]. In last period of sample collection, P concentration was measured in fecal samples for 4 consecutive days. The con centrations of fluorine, cadmium, lead, mercury and arsenic of DAP, DCP, and experimental diets were measured using a ContrAA 700 (Analaytikjena, Jena, Germany) by atomic ab sorption method according to procedures described by Jorhem [10].

Blood sample collection
On day 30, 62, and 93 blood samples were collected from the jugular vein of each lamb into heparinized tubes prior to feeding. Plasma Ca [11] and P [12] and serum glucose, urea nitrogen and alkaline phosphatase activity were measured using commercial kits (Pars Azmon Inc., Tehran, Iran). Glu cose was measured using enzymatic photometric method [13]. Blood urea nitrogen was measured using UreaseGLDH method [14]. The alkaline phosphatase was measured using DGKC (Germany Biochemistry Association Standard) [15]. Also, photometric method by creolphthalein complex one for measuring calcium and photometric UV test for measuring P degree were used. Biochemistry experiments were performed by AutoAnalyzer machine, Hitachi 717.

Statistical analysis
All data were analyzed by repeated measurements using Proc mixed of SAS 9.2 [16] using the following model: Where Y ijk is the observation of dependent variable; µ is the fixed effect of population mean for the variable; T i is the fixed effect of supplemented P source (i = 2, DCP and DAP); P j is the fixed effect of sampling day (j = 3 for blood and fecal sam ples and j = 6 for DMI and BW measurements); T i ×P j is the interaction between factor T at level i and factor P at level j; lamb k is the random effect of lamb within treatment (k = 6); and e ijk is the random error associated with the related ob servation.
The DMI and BW in the week before the beginning of the experimental treatments were included in the model as co variates. The covariate was excluded from the model when it was not significant (p>0.10). The covariance model for repeat ed measurements were selected based on Akaike information criterion (AIC). The adjust Tukey test was used for multiple treatment comparisons using the LSMEAN statement of SAS (SAS Institute, Cary, NC, USA). For the different statistical tests, significance was declared at p≤0.05 and trends at p≤0.10, unless otherwise stated.

Mineral and heavy metals concentration in DAP and DCP containing diets
Fertilizer grade DAP contained 19.7 and 185.4 respectively Ca and P (g/kg) and 14,623.3 ppm fluorine, while DCP con tained 230.3 and 195.2 respectively Ca and P (g/kg) and 1,039.1 ppm fluorine ( Table 1). The DCP contained more lead, while concentration of mercury, cadmium and arsenic were similar compared with DAP. The solubility of P in acid was similar for DAP and DCP. The diet with DAP contained 60 ppm fluorine while the diet with DCP contained 13 ppm fluorine (Table 2). Both experimental diets had similar Ca, P, and other nutrients measured ( Table 2).

Animal performance
Lambs fed the diet with DAP tended to have greater daily DM intake during experiment compared to those fed the diet with DCP (p = 0.09; Table 3). Average daily gain, final BW, BW change and feed conversion ratio were similar between lambs fed the two dietary treatments. Daily DM intake and BW in creased with advancing lamb age (p<0.05). Average daily gain and feed conversion ratio remained similar over the 93 days measurement period.
There was a dietary treatment×time period interaction (p< 0.05; Table 4) for DM and organic matter digestibility. At day 30, DM and organic matter digestibility were greater in lambs fed the diet with DCP compared with DAP (p<0.05), while at days 60 and 90 of the trial, DM and organic matter digesti bility were similar for lambs fed the two dietary treatments. Fecal P concentration was lower for lambs fed the diet with DAP than for lambs fed the diet with DCP at day 90 of the experiment (p<0.05; Table 4). Apparent digestibility of P was greater for lambs fed the DAP diet compared with lambs fed DCP diet (p<0.05; Table 4).

Blood metabolites
Serum urea and plasma Ca were similar between lambs fed diets with DAP and DCP (Table 5). Feeding the diet with DAP increased plasma P and serum alkaline phosphatase enzyme (p<0.05) compared with feeding the diet with DCP. There was a dietary treatment×time period interaction (p<0.05) for se rum glucose. At day 60 of feeding DAP, serum glucose was greater compared with feeding DCP (p<0.05), while serum glucose was similar at the other sampling times for lambs fed either DAP or DCP (Table 5). Blood metabolites changed as the age of lambs increased (Table 5). Lambs had lower blood glucose at day 90 compared with days 30 and 60 (p<0.0001), lower blood urea and P at day 60 compared with days 30 and 90 (p<0.0001) and higher blood alkaline phosphatase con centration at day 90 compared with day 30 and 60 (p<0.0001).
Lambs had lower blood Ca at day 30 compared with day 90, which were both lower than on day 60 (p<0.0001).

DISCUSSION
Lambs fed the diet with DAP tended to have a greater DM intake than those fed the diet with DCP in the current study, which was opposite to findings of Oltjen et al [6] who reported that feeding DAP decreased DM intake of finishing lambs. The DAP source used in the current study was in a powder form while DAP was in a granule form in the study of Oltjen et al [6]. Adding DAP as powder likely improved mixing with the  concentrates, which might improve the diet palatability and reduce feed selection and sorting by sheep. Oltjen et al [6] re ported that including DAP in pelleted concentrate rather than in granule form would increase its intake and palatability. Fur ther, only 261 g/kg of total P of diet was supplied by DAP in current study, while Oltjen et al [6] supplied 940 g/kg of P in the diet as DAP. The amount of DAP supplemented in the diet might affect the palatability of the diet. Phosphorus is involved also in the control of appetite (in a manner not yet fully un derstood) and in the efficiency of feed utilization [17]. Average daily gain and feed conversion ratio were similar for lambs fed DAP and DCP in the current study. Venedik tove et al [18] reported that feeding a diet supplemented with DAP improved weight gain and feed efficiency in calves com pared with feeding DCP and Karadzyan et al [19] reported that feeding DAP improved weight gain in steers. Karadzyan et al [19] indicated that P from DAP had greater bioavail ability than P from DCP, which was also found in the current study with lambs fed with DAP having lower fecal P and higher plasma P, which indicated that P from DAP had a greater bio availability. Phosphorus concentration in feces is affected by endogenous losses and amount of P intake [2,20,21] and plasma P is strongly related to amount of P intake and the bioavail ability of P resources [4,15]. The DM intake of lambs fed DAP and DCP were similar and therefore the difference in fecal and plasma P concentrations were likely not affected by level of intake. Annenkove et al [22], Bogdani et al [23] and Suttle [2] reported also higher bioavailability of P from DAP com pared with P from DCP.
The diet ingredients provided 739 and 695 g/kg of P lamb requirements and 261 and 305 g/kg of P requirements were provided by DAP and DCP respectively. The lack of lamb per formance responses in the current study might be due to the low inclusion level of DAP and DCP or lamb requirements might have been met independent of bioavailability. A phos phorus deficiency may be observed by slow growth, declined appetite, unthrifty appearance, listlessness, low level of phos phorus in blood (less than 4 mg/dL of plasma), and development of rickets [4], which were all not the case with either P supple ment in the diet. Further, digestibility of DM and organic matter was lower in lambs fed DAP than in lambs fed DCP (648.0 vs 690.0 and 681.0 vs 723.0 g/kg) at the end of first period (30 d), which was likely due to the greater DM intake of lambs fed DAP that can result in decreased nutrient digestibility [4]. Energy intake and availability were therefore likely similar between the two diets, another likely reason why lamb perfor mance was similar.
Feeding DAP increased serum alkaline phosphatase in lambs. Alkaline phosphatase activity gives a good indication of the extent of new bone formation and osteoblast activity [24] and was found to be decreased under poor feeding con ditions in young sheep, which may influence growth rate [25].
One concern about using of fertilizer grade DAP is its flu orine content, which if fed at high levels can exerts a cumulative toxic effect. Oltjen et al [6] reported that feeding 88 grams DAP (fluorine concentration not provided) in the diet of sheep with a BW of 45 kg resulted in toxic effects. Phosphorus to fluorine ratio in DAP used in the current study was 12.7, which was lower than the threshold of 120 recommended for mini mizing the risk of fluorosis toxicity when using P sources [4,2]. With the inclusion of DAP to provide 261 g/kg of total P in the diet, however, the fluorine concentration of diet reached 60 ppm which has been reported as the maximum tolerable level for breeding sheep [4]. A defluorination procedure may decrease the fluorine concentration in DAP. Phosphorus re sources can also contain heavy metals at toxic levels, which can be hazardous for the animal health [2]. The concentra tion of potential toxic elements in diets with either DAP or DCP in the current study were, however, below hazardous threshold levels for ruminants [26] and will therefore be safe