Abstract:
Animal Production in Australia Vol. 15 THE EFFECTS OF PHOSPHORUS UPON IN VITRO MICROBIAL DIGESTION J.T.B. MILTON* and J.H. TERNOUTH* SUMMARY The effects of different levels of phosphorus (P) upon in vitro digestion of barley straw neutral-detergent fibre (NDF) were studied in buffered ruminal fluids and caeco-colic digesta. Supplemental P added to ruminal and caeco-colic digesta from P deficient sheep stimulated microbial digestion. The microflora of the large intestine had a minimum P requirement similar to that of rumen microflora for optimal digestion of NDF (>50-80 mg P/l). INTRODUCTION Depressions in dry matter intake and digestibility have been observed with sheep fed high calcium-low phosphorus (HCaLP) diets (Field et al. 1975; Sevilla and Ternouth 1980, 1982). Milton and Ternouth (1979) considered P supplementation of P deficient sheep fed a HCaLP diet increased food intake and digestibility by stimulating microbial digestion in the rumen. As the large intestine is caudal to the major site of P absorption, P may also limit microbial digestion in the large intestine of ruminants fed HCaLP diets. Durand et al. (1982) reported ruminal fluid soluble P levels of 20 mg P/l for sheep fed a HCaLP diet for 6 weeks. The effects of different levels of P upon in vitro digestion of barley straw NDF were investigated. Graded quantities of P, with and without nitrogen (N), sulphur (S) and sugar were added to low P buffered ruminal fluids in study 1. In addition, graded quantities of P were added to a high P buffered ruminal fluid. In study 2, barley straw was incubated in low P buffered caeco-colic digesta with various levels of P. MATERIALS AND METHODS Study 1 A level of P (0, 2, 4, 6, 8 or 16 mg in 4 ml of buffer solution) was added to six groups of 4 polycarbonate tubes in block 1. Three digestion tubes in each group contained 0.5 g air-dry ground (1 mm) barley straw, the other without barley straw was a sample tube. Tubes for block 2 were prepared the same as for block 1, but with 26 mg urea, 4.9 mg sodium sulphate and 75 mg sugar added to all tubes. Four groups of 3 digestion tubes and 1 sample tube each group with 0, 2, 4 or 6 mg of P were prepared in block 3. The buffer solution without P contained 21.68 g NaHC03, 570 mg KCl, 470 mg NaCl, 60 mg MgC12 and 40 mg CaC12/1. Buffer solutions with P were achieved by substituting Na2HP04 for NaHC03 on an equal sodium basis. Low P ruminal fluid was obtained from two P depleted sheep fed a HCaLP diet (Milton and Ternouth 1979). High P ruminal fluid for block 3 was obtained from a sheep fed the same HCaLP diet with an additional 3 g P/kg for 21 weeks. Ruminal fluid was collected 4 h after feeding, strained, the pH was measured and a subsample frozen. Strained ruminal fluid was mixed (1:3.6 V/V> with P-free artificial saliva (39'C after infusion with CO2; 14.2 g NaHC03, 570 mg XL, 470 mg NaCl, 60 mg MgC12 and 40 mg CaCl# and 46 ml of this buffered ruminal fluid dispensed into tubes with a Filamatic automatic pipetting machine. Sample tubes for each P level were held at 4'C. Digestion tubes were capped, the contents gently mixed and the tubes incubated at 39'C for 48 h. The contents of the tubes i'CDept of Animal Production, University of Queensland, St. Lucia, Qld 4067. Animal Production in Australia Vol. 15 were remixed after 12 and 24 h. The pH of the fluid in each tube was measured on removal from the incubator prior to freezing the tubes. Study 2 In this single-block study, 0, 2 or 6 mg of P was added to 3 digestion and 2 blank tubes (no barley straw) and incubated with 46 ml of buffered caeco-colic digesta at 39'C for 48 h. The digesta was obtained from a sheep fed a HCaLP diet (Sevilla and Ternouth 1980>, euthanased 6 h after feeding. The pH of the digesta was measured, the digesta mixed directly with P-free artificial saliva (1:3.6 V/V> and dispensed into the tubes using a graduated cylinder. No samples of the media were retained for analysis. The 15 tubes were incubated, the contents mixed and the fluid pH measured before freezing the tubes. Analytical and statistical methods Barley straw was analysed for P (Fiske and Subbarow 1925) after digestion in sulphuric acid and the NDF content determined (Van Soest and Wine 1967). Sample tube media was centrifuged at 18OOOg and the supernatant frozen. Ruminal fluid and media supernatant were digested in 4:l nitric/perchloric acid and analysed for P (Fiske and Subbarow 1925). The Ca content of digested ruminal fluid was determined by atomic absorption spectrophotometry (Willis, 1961). The contents of digestion tubes and blanks (study 2) were added to 100 ml of neutral-detergent solution and the NDF content determined by the method of Van Soest and Wine (1967). The P and NDF content of the barley straw for both studies were 0.47 and 737 g/kg dry matter respectively. Blank NDF residues were only taken into account to calculate NDF digestibilities in study 2. Triplicate NDF digestibility and digestion tube pH values in blocks 1 and 2 were statistically analysed using a two-way analysis of variance. Differences between P levels in block 3 of study 1 and in study 2 were determined by a one-way analysis of variance. RESULTS Study 1 The low P ruminal fluid had an initial pH of 6.7 and contained 258 mg Ca and 84 mg P/l. Digestibility values for P levels 2, 3, 4, 5 and 6 in blocks 1 and 2 were greater than P level 1 (Table 1). In block 1, digestibilities for P levels Table 1 The effect of incubating barley straw in buffered ruminal fluid of varying phosphorus content without and with nitrogen, sulphur and sugar 473 Animal Production in Australia Vol. I.5 3, 4 and .5 were similar, but greater than P levels 2 and 6. Digestibility values with P levels 3, 4, 5 and 6 were all similar in block 2 and greater than P level 2. There was an increase in digestibility with the addition of N, S and sugar to all P levels except level 1. The pH of digestion tubes generally decreased with increasing P levels in blocks 1 and 2 (Table 1). The high P ruminal fluid for block 3 had an initial pH of 6.5 and contained 120 mg Ca and 561 mg P/l. There were no significant differences between P levels in block 3 for NDF digestibility and digestion tube pH (Table 2). Table 2 The effect of incubating barley straw in buffered ruminal fluid of varying phosphorus content Study 2 The pH of the caeco-colic digesta was 7.2. The mean weight of blank NDF residue with P level 3 was 10% greater than with P levels 1 and 2. Digestion of NDF with P level 3 was greater than P level 1, but the pH of digestion tubes did not differ between P levels (Table 3). Table 3 The effect of incubating barley straw in buffered caeco-colic digesta of varying phosphorus content NS, not significant; *, PC 0.05. T Estimates based upon data of Poppi and Ternouth (1979) and low P ruminal fluid. DISCUSSION In a preliminary study similar to study 1, a 48 h incubation period gave large differences in NDF digestibility and blank NDF residue weights were negligible. Consequently, a 48 h incubation period was used in studies 1 and 2 without correction for blank NDF residues in study 1. The initial pH of the ruminal fluids were within the pH range for high rates of in vitro digestion (McLeod and Minson 1969). As volatile fatty acids (VFA) are products of microbial digestion, the pH of digestion tubes should reflect changes in digestion as modified by other buffers. Increased VFA production could explain the observed reductions in pH when NDF 474 Animal Production in Australia Vol. 15 digestion increased. The reductions in pH in study 1 were almost linear whereas NDF digestion reached a constant level between P levels 2 and 3. The P content of the media with P level 1 in block 3 was between P levels 3 and 4 in blocks 1 and 2, but NDF digestion for all P levels in block 3 were less than with P level 3 in blocks 1 and 2. As digestion did not increase with P levels higher than P level 2 in blocks 1 and 2, it is concluded that the minimum P content for optimal digestion of NDF in study 1 was 50-80 mg P/l. Other workers cited by Durand and Kawashima (19%)) considered this range to be adequate for rumen microbial cellulose digestion, but Durand and Kawashima (1980) proposed 100 mg P/l as the minimum requirement. Rees and Minson (1982) suggested that a low Ca:P ratio in the rumen could reduce microbial digestion. The high P ruminal fluid in study 1 had a Ca:P ratio of 0.2l:l whereas the ratio of the low P ruminal fluid was 3.1:1. Although these ruminal fluids were obtained from different sheep and this may confound any effect of Ca upon microbial digestion, it is noteworthy that for similar P concentrations NDF digestion in block 3 was less than in block 1. The increased digestion with added N, S and sugar in study 1 indicate that P was not the only nutrient limiting microbial digestion. These nutrients did not increase the concentration of P necessary for optimal digestion of NDF. The reason for the lower digestibility with P level 1 in block 2 is not clear. Based upon the respective concentrations of soluble P in the ruminal fluid and caeco-colic digesta of sheep fed a low P diet (Poppi and Ternouth 1979), the P content of caeco-colic digesta in study 2 was likely to be similar to the low P ruminal fluid for study 1. As NDF digestion with P level 3 in study 2 was double that with P level 1, it is concluded that the P content of the buffered caecocolic digesta had to be raised above 50 mg P/l (possibly to 120 mg P/l) to provide the P necessary for optimal microbial digestion. This experiment demonstrates that the microflora inhabiting the large intestine of chronically P deficient sheep have a similar P requirement to their counterparts in the rumen for digestion of NDF. Addition of P to buffered ruminal or caeco-colic digesta from chronically P deficient sheep increases NDF digestion. ACKNOWLEDGEMENTS The authors wish to thank Dr. D.J. Minson (CSIRO Division of Tropical Crops and Pastures) for the use of CSIRO in vitro equipment. 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