Abstract:
METHODS OF MEASURING PHOSPHORUS STATUS IN SHEEP ** **-t J.H. TERNOUTH*, H.M.S. DAVIES*, S. WHINERAY and B.J. THOMAS SUMMARY A series of methods for estimating the phosphorus (P) status (reserves) of sheep were compared. Inorganic P values for tarsal plasma were higher than jugular values except when P-depleted sheep were being repleted. Blood assay techniques were considered to be unsuitable for measuring P status. The most repeatable of phorus expressed as mgP/mgN the P and N can be measured sensitive index as there is be resorbed as the minerals the analytical measurements for rib or mgP/gDM. The former measurement following a single acid digestion. evidence that the organic matrix of are depleted. bones was phosmay be useful as It may also be a the bone may not A rapid neutron activation analysis (NAA) technique was investigated. The metatarsal bone was irradiated for 300 s and the gamma spectrum of induced activity collected for 300 s. Using this technique, considerable depletion of metatarsal bone P was indicated in weaners fed a P-deficient diet. The NAA technique appears to have considerable potential for use as a research tool and in the field. INTRODUCTION In Queensland, sheep and cattle commonly graze to be either marginally or grossly P deficient; such siderable loss of animal production. The deficiency as losses in body weight and reduced fertility and is reduced feed intake and quality (Cohen 1975). MATERIALS AND METHODS Adult sheep deficient in phosphorus were prepared using a parotid saliva replacement technique (Temouth, unpublished) and a low P diet. The parotid saliva was replaced by an electrolyte solution devoid of P (Tomas 1974). Weaners (5 - 13 mo old) were depleted of phosphorus by feeding the same low P diet (Milton and Ternouth 1979). 'Rib biopsy samples (Little 1972) andjugularandtarsal vein blood samples were taken and neutron activation analysis measurements (Whineray et az. 1980) made at intervals from sheep fed normal or low P diets at various stages of P depletion. For the NAA measurement the anaesthetised sheep was cradled in a canvas stretcher with the hind legs hanging through two holes. The two 10 Ci neutron sources were located 4.5 cm on each, side of the midline of the metatarsal bone for a 300 s activation period. A single 7.5 cm x 7.5 cm Nal detector was positioned as close as possible to the/cranial surface of the bone and the gamma spectrum of induced activity collected for 300 s starting 65 s after the irradiation ceased. * Department of Animal Production, University of Queensland, St. Lucia, Qld 4067. Department of Physics, Queensland Institute of Technology, George St., Brisbane + Qld 4000. On leave from Massey University, Palmerston North, New Zealand. forages that are recognised deficiencies result in conis most commonly manifested due, at least in part, to ** 213 Animal production in Australia Rib bone samples were analysed for P (Fiske and Subbarrow 1925) following wet digestionin 36N H2SO4 with a Se catalyst. Cortical rib P content was related to fresh sample volume (Avogadro technique), fresh weight, dry weight and-N content (Kjeldahl technique). Plasma inorganic P was measured as described by Little et aZ. (1971). RESULTS AND DISCUSSION Plasma inorganic phosphorus A total of 102 paired samples of blood collected from 25 sheep fed normal or deficient diets showed that tarsal plasma contained more P than jugular plasma (6.62 vs 6.29 mg/lOO ml; SE = 0.008; P < 0.001). This result agrees with those of Siemon and Moodie (1972) in sheep and the coccygeal-jugular values in cattle (Parker and Blowey 1974; Teleni et aZ.1976) all of whom concluded that the differences were due to salivary secretion of P. However, during P repletion of four P-depleted adult sheep, tarsal P was not significantly different from jugular P (7.71 vs 8.04 mg/lOO ml; SE = 0.13) indicating that bone accretion of P depleted sheep was substantial and may equal salivary gland &sorption. In a second experiment the plasma P levels of 4 P-deficient and 4 P-normal sheep were compared-when fed .on low P (0.4 gP/kgDM) andnormal P (2.9 gP/kgDM) diets. Sheep fed the P-deficient diet had plasma levels of 3.68 + 0.86 mgP/lOO ml whilst the sheep fed the P-normal diets had levels of 10.11 ki.42 mgP/lOO ml. When the plasma levels of the sheep were compared on the basis of high and low status, there was no significant difference between the groups of sheep (8.34 + 1.96 and 5.45 21.13 mgP/lOO ml respectively). The plasma P levels clearly reflect the level of P in the recent diet and in the gut pool of P rather than the status of the sheep. It is concluded that plasma inorganic P is not a satisfactory measure of'phosphorus status. Rib biopsy and Neutron Activation Analysis Sections of rib bone collected from the 12th and 11th rib of a normal P sheep at slaughter were used to estimate the repeatability of the rib biopsy analytical techniques. The sections (2.0 cm long) were numbered in a dorsoventral direction with Section 1 starting 2 - '3 km late ral to the costo-vertebral joint and four analytical techniques compared (Table 1). The mean values for mgP/g wet weight and mgP/g dry weight were substantially lower than those previously obtained in our laboratory (Poppi and Temouth 1978) and this difference was due to the lower P status of this sheep. In'the present experiment the variability of the means was significantly smaller (P < 0.01) for the mgP/mgN and the mgP/g dry weight techniques than with either of the other techniques. The TABLE 1 Phosphorus concentrations of six sections of rib analysed by four techniques. . 214 Animal Production in Australia most repeatable technique was the mgP/mgN ratio, the error betweenduplicatesbeing less than 3%. This can be compared with errors between duplicates of the others of 4, 8 and 16% for mgP/g dry weight, mgP/gwetweight and mgP/ml wet volume respectively. The use of mgP/ml wet volume was unsatisfactory because of the difficulty inherent in measuring bone volume while mgP/g wet weight appeared less satisfactory than mgP/mgN. The reason for this is not clear. Table 2 shows the results of an experiment (Sevilla and Ternouth 1980) in which both bone biopsy and NAA techniques were employed on four occasions with sheep aged between 5 and 13 months. The NAA results (expressed as net counts) showed that the metatarsal bones were apparently not initially subjected to significant P depletion although some depletion of rib P was occurring. Subsequently both the rib and metatarsal bones were depleted further so that at 13 months of age the bones were estimated to contain 65% of the initial P levels. TABLE 2 Rib phosphorus (P) and metatarsal neutron activation analysis (NAA) measurements of 5-13 month old lambs of normal and low P status Expressing the P content of rib bones as gP/gN again appeared to be suitable for rib biopsy samples. Benzie et al. (1955) found that resorption of minerals from bone is accompanied by the development of non-osseous organic matter indistinquishable from the organic matrix. Thus as the concentration of P in the bone falls, the N level may rise so that P/N ratio is a sensitive measure of bone status. However all rib biopsy techniques suffer from the difficulties associated with the need to complete the surgery, to dry, digest and analyse the P content of the bone and, from the difficulty of obtaining serial results from the same animal. There are known to be,considerable variations in P content along a rib and between ribs (Little and Minson 1977; Poppi and Ternouth 1978). The repeatability of the NAA technique was estimated to be 5% when used on the metatarsal bone of sheep (Whineray et al. 1980)'. The metatarsal bone was chosen because the neutron sources and radiation detector can be positioned at a small and constant distance from the bone regardless of the fatness of the sheep. As this technique can be used repeatedly on the same sheep and may be completed in under 15 mins with the results immediately available, it appears'to be of conI siderable value both as a research tool and in the field. ACKNOWLEDGEMENTS We wish to thank Mr C.C. Sevilla, Mrs D. Burling and Miss M.E. Elliott for valued assistance. 215 Animal production in Australia REFERENCES BENZIE, D., BOYNE, A-W., DALGARNO, AC., DUCKWORTH, J., HILL, R. and WALKER, D.M. (1955) J. Agric. Sci. 46:425. 13:5, COHEN, R.D.H. (1973) Aust. J.?xp. Agric. and Anim. Husb. c COHEN, R.D.H. (1975) World Rev. Anim. Prod. 11:27, FISKE, C.H. and SUBBARROW, Y. (1925) J. BiolyChem. 66: 3 75. C (1972) Aust. Vet, J. 48:668. LITTLE, D.A. LITTLE, D.A., ROBINSON, P-J., PLAYNE,%J. and HAYDOCK, K. P . (1971) Aust. Vet. J. 47:153. LITTLEyD% and MINSON, D.J. (1977) Res. Vet. Sci. 23:393. 4:150. MILTON, J.T.B. and TERNOUTH, J.H. (1979) Proc. Nutr.Soc. Aust. = (1974) Vet. Rec. 95:14. PARKER, B.N.J. and BLOWEY, R.W. 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