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The effect of dietary molybdenum supplementation on tissue copper concentrations, mohair fibre and carcass characteristics of growing Angora goats
ANML FEE0 SCIENCE AND TECHNCXOCY
Animal Feed Science Technology 67 (1997) 83-90
Short communication
The effect of dietary molybdenum supplementation on tissue copper concentrations, mohair fibre and carcass characteristics of growing Angora goats H. Galbraith a,* , W. Chigwada ‘, J.R. Scaife a, W.R. Humphries b a University of Abe&en,
Department of Agriculture, 581 King Street, Aberdeen AB24 5UA, UK b Rowen Research Instihde, Budsbum Aberdeen AB2 9SB, UK Acce@ed 28 October 1996
The purpose of this experiment was to better chamcterize the effects of the interaction between copper (Cu), molybdenum (MO) and sulphur 6) in the diet on growth, metabolism and fibre characteristics in Angora goats. 15 Angora goats aged 9 months and weighing 21.5 kg on average were used in a ten-week study and allocated to three dietary treatments: Treatment C (IOMJ metabolisable energy, 178g crude protein, 5.5 mg Cu, 0.57 mg MO, and 3.4g S): Treatment Ml (with 7.5 mg MO) or Treatment M2 (with 15mg MO) per animal per day. Dose-dependent increases in the concentrations of MO (P < 0.01) and Cu (P < 0.05) in plasma were recorded in response to increased dietary intake of MO. Supplementation of the control diet with increased concentrations of MO did not produce effects (P > 0.05) on growth rate, feed conversion efficiency, carcass weight or mohair fibre yield and diameter. Haematological status and concentration of Cu in liver and Cu and S in f&e at the end of the study were also not affected (P > 0.05). Concentrations of trichloroacetic acid (TCA) soluble “available” copper in plasma were not significantly different although significant (P < 0.05 and P < 0.01) reductions in the ratio of “available” to total Cu concentrations were observed. This effect was stabilised and maintained after 30 days. It is suggested that the additional Cu in plasma was largely TCA insoluble and possibly in the form of thiomolybdate complexes which may be poorly excreted and not available for uptake to the metabolic sites. It is evident that adequate “available” Cu was present in plasma and that exposure to elevated MO intake was not severe or long enough to produce clinical
* Corresponding author. 0377~8401/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO377-8401(96)01140-6
84
H. Galbraith et al. /Animal
Feed Science Technology 67 (1997) 83-90
symptoms B.V.
or to affect growth, haematological
status or fibre prodwtion.
0 1997 Elsevier Science
Keywords:
Copper; Molybdenum; Sulphur; Angora goats; Haematological status; Growth
1.ldrodnction The role and importance of copper (Cu) in the metabolism of animals are well known. Cu deficiency and toxicity in ruminants occur frequently in many parts of the world. The development of Cu deficiency depends on both the dietary Cu concentrations and the concentrations of antagonists which interfere with its absorption from the gut and subsequent utilisation for metabolic processes (Goonerame et al., 1989). Simple Cu deficiency caused by low dietary Cu intakes is often responsible for the development of Cu-responsive disorders including abnormalities in pigmentation and keratinization of wool and hair, anaemia, skeletal abnormalities and enzootic ataxia. (Bremner and Davies, 1980). However, widespread Cu deficiency has been observed in ruminants grazing herbage with apparently adequate Cu concentrations (Bremner and Davies, 1980). This “conditioned” or “induced” Cu deficiency is caused by the presence of antagonists which reduce the availability of Cu. Molybdenum (MO) and sulphur (S) have been identified as the most important antagonists of Cu (Humphries et al., 1983). The Cu content of ruminant diets usually varies between 4 and 10 mg kg- ’ DM or greater (Gooneratne et al., 1989) and it is subject to many different influences. It is well recognised that the risk of copper toxicity in sheep may also be increased by consump tion of feeds which contain high Cu (Gooneratne et al., 1989). The MO concentration of herbage frequently ranges between 0.5 and 5 mg kg-’ DM (Goonerame et al., 19891, tends to be more variable than that of copper, and, unlike that of Cu, the concentration of MO increases as the soil pH increases. The S content of herbage depends largely on the amount of S in protein and rarely exceeds 3 g kg- ’ DM. Methionine and cysteine usually comprise over 90% of the organic S in herbage (Goonerame et al., 1989). The adverse effect of increased dietary MO and S on the utilisation of Cu by ruminants has been attributed to the formation of thiomolybdates (TM) in the rumen (Dick et al., 1975; Suttle, 1974; Goonerame et al., 1989). Recent findings (Gooneratne et al., 1989) show that tetrathiomolybdate 0TM) appears to dominate in the rumen at moderate to high MO and S intake. TM bind to Cu in the rumen to form copper thiomolybdates (Cu-TM) which are insoluble and therefore unavailable for absorption. TM also bind onto the rumen solids and, at high TM concentrations, also occur in the rumen liquid phase. Excess TM is absorbed into the blood and disturbs systemic Cu metabolism, This causes characteristic increases in plasma Cu concentration, much of which is protein bound, insoluble in acid and apparently not available for uptake into tissues (Price et al., 1987; Goonerame et al., 1989). Sheep are known to be sensitive to Cu poisoning and administration of ammonium tetrathiomolybdate reduces the severity of the condition (Humphries et al., 1986).
H. Galbraith et al. /Animal
Feed Science Technology 67 (1997) 83-W
85
Information on the sensitivity of goats to excessive dietary intakes of Cu is limited. Humphties et al. (1987) reported that animals receiving excessive amounts of Cu from a calf milk replacer containing an estimated 1Omg kg-’ DM Cu and used in their diet, developed symptoms of Cu toxicity similar to those in sheep. Moreover. injection of large doses of ammonium tetrathiomolybdate, reversed the toxic symptoms. Goats may therefore be more similar to sheep than cattle in their adverse response to high dietary intakes of Cu, but all three species are sensitive to Cu deficiency under certain conditions. However, systematic information on the relationships between dietary Cu, MO and S in goats is limited. The current study explored the effects of increased dietary intakes of MO on Cu concentrations and fibre characteristics of Angora goats on a high S diet, conditions which have previously been shown to cause deficiencies in sheep (Suttle, 1974). 2. Materials and methods 2.1. Animals and diets 15 wether Angora goats aged about 9 months and weighing 2 1.5 kg on average were allocated to three dietary treatments (five per treatment) using a randomised block design. They were offered a basal pelleted diet based on molassed sugar beet pulp (Trident Feeds, Peterborough, UK), barley straw, oat feed, soya bean meal, white fish meal and a mineral and vitamin supplement in addition to 50g per animal per day of good-quality grass hay. This diet provided (per kg DM), 5.5mg Cu and 0.57mg MO, and following supplementation with sodium sulphate, 3.4g S. The basal diet was either unsupplemented (C) or supplemented with MO at either 7.5 mg (treatment Ml) or 15 mg (treatment M2) per animal per day by treating 50g of the basal diet with 1.0 ml of solutions of sodium molybdate containing these quantities of MO. The 50g portion was allowed to dry and was then offered to the goats at 09:OOh. This part of the ration was fully consumed before *the remainder of the diet was offered to the goats. This diet provided 37g of DM per kg of live weight. The final concentrations of the MO and S used were in excess of those (MO, 4mg kg- ‘; S, 3.Og kg- ‘) considered to produce reductions in Cu availability in sheep (Suttle, 1974). The goats were housed in individual pens and extraneous exposure to metal was minimised by covering surfaces with plastic tape and offering deionised water in plastic containers. The goats were weighed weekly and feed residues were monitored daily. The goats were slaughtered after 71 days by the approved method of stunning using a captive bolt pistol followed by exanguination (Shahjalal et al., 1992). Samples of liver were collected immediately after slaughter, frozen rapidly and stored at - 20” and rapidly thawed when required for analyses. 2.2. Blood andPbre
collection
Individual blood samples were collected every two weeks by jugular venepuncture into proprietary vacutainer tubes (Becton Dickinson, VACUTAINER Systems Europe, Meylan Cedex, France) containing (a) EDTA anticoagulant for determination of packed
H. Galbraith et al. /Animul
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Feed Science Technology 67 (1997) 83-W
cell volume (PCV) and haemoglobin, and (b) heparin for the measurement of Cu and MO concentrations and of caeruloplasmin activity. Mohair fibre samples were collected from a 10 cm X 1Ocm square area on the mid-side on days 35 and 70 of the study using a conventional horse-hair clipper (Shahjalal et al., 1992). The samples were washed by soaking overnight in a solution of the detergent Decon 75 (50 ml 1-l >, rinsed thoroughly in distilled water and then dried at 80°C. Diameter was measured on a random sample of 100 fibres as described by Shahjalal et al. (1992).
2.3. Analysis of Cu, MO and S Samples of plasma were prepared by conventional centrifugation of whole blood at 1OOOg. Separated blood cells were then washed and re-suspended in physiological saline. Haemoglobin was determined on whole blood samples following haemolysis using a Contraves Analyser Model 4300. PCV was measured following centrifugation in microhaematocrit capillary tubes with a Hawksley microhaematocrit reader. Concentrations of total Cu in plasma were measured following 1:1 dilution in distilled water using a Varian AA-875 series atomic absorption spectrophotometer. Trichloroacetic acid (TCA) soluble or “available” Cu was similarly measured after adding 0.5 ml plasma to 2.Oml of a solution containing TCA (lOOgl_‘), mixing and centrifuging for 15 min at 1600 g. The supematant was analysed by atomic absorption spectrophotometry. Total plasma MO concentrations were measured following acid digestion, by graphite furnace atomic absorption spectrophotometry (Mot-rice et al., 1989). Caeruloplasmin activity was measured in plasma using the method of Mason et al. (1980). Liver and washed fibre samples were digested in mixtures of nitric, perchloric and sulphuric acids before analysis of Cu by atomic absorption spectrophotometry. Caeruloplasmin activity was measured in plasma using the method of Mason et al. (1980). Total S concentrations in fibre were analysed using X-ray fluorescence spectrometry using a Philips PW104 automatic sequential wavelength dispersive spectrometer following the grinding of samples in liquid nitrogen and pelleting. 2.4. Statistical analysis The data were examined by analysis of variance using the package Minitab 7.3 (Minitab Inc. 1989) and presented as treatment means with an estimate of the pooled standard error of the difference @ED) between any two means. Where significance (P < 0.05) was obtained for overall treatment effects, the signifkances of differences between means for pairs of treatment groups were calculated by comparison with values for least significant difference. 3. Results 3.1. Growth, pelformance,
carcass characteristics
and jibre production
Supplementation of the diet with increased concentrations of MO had no effect (P > 0.05) on dry matter intake, live-weight gain or chilled carcass weight (Table 1).
H. Galbraith et al. /Animal Table 1 Growth performance,
Feed Science Technology 67 (1597) 83-90
87
carcass and fibm characteristics Treatments
Total dry matter intake (kg) Live weight gain (kg) LWG/DMI Chilled carcass weight (kg) Clean fibre weight (day 69) (g) Clean fibre diameter (mm)
’
Control
Ml
M2
64.7 5.74 0.09 11.4 9.23 31.5
67.7 6.86 0.10 11.8 10.2 30.0
66.7 7.14 0.11 11.3 8.8 28.1
SED
Statistical
1.25 1.40 0.002 0.54 0.95 1.22
NS NS NS NS NS NS
significance
a n = 5 goats per treatment.
Similarly, the dietary treatments had no effect on the weight of clean fibre collected on day 69, or on its diameter. 3.2. Concentrations
of Cu, MO and S in tissues and mohair
MO supplementation had no effect (P > 0.05) on mean values for haemoglobin and packed cell volume in whole blood (Table 2). However, concentrations of MO in plasma were elevated (P < 0.01) with increasing dose levels of MO in the diet, as were concentrations (P < 0.05) of total, but not TCA soluble (P > 0.051, Cu (Table 2). In contrast, the proportion of plasma Cu that was soluble in TCA was decreased (P < 0.05) in a dose-dependent manner on day 28 with a trend maintained on day 70. The dietary treatments did not affect (P > 0.05) caeruloplasmin activity in plasma, concentrations of
Table 2 Mean values for parameters
measured
in whole blood, plasma, tibre and liver samples Treatments
Whole blood Haemoglobin fg 1- ’) Packed cell volume (1 l- ’) Pla.ma Total Cu (TCu) (pmoll-‘1 TCA-soluble Cu (SCu) (pmoll-’ ) Total MO, &moll-‘1 Caeruloplasmin activity (Eu 1-l ) SCu/TCu SCu/TCu Red blood cell Cu ( pm011 - ’> Liver Cu ( firno1 kg- ’ DM> Mohair Cu ( pm01 kg-’ DM) S (mm01 kg-’ DM)
SED
Statistical significance
Day
Control
Ml
M2
70 70
117 0.30
114 0.30
114 0.30
8.4 0.018
NS NS
70 70 70 70 28 70
14.9a 13.4 0.03a 40.6 l.Oa 0.92a 15.7 631 73.0 720
16.2b 11.8 0.87b 31.2 0.89ab 0.74ab 14.5 653 64.7 770
20.9b 13.4 3.67~ 38.7 0.66c 0.63b 13.4 581 61.4 740
2.0 1.42 0.44 7.1 0.06
’ NS ** NS
70 71 69 69
0.09 2.41 48.8 9.44 5.7
** * NS NS NS NS
SCu/TCu is TCA-soluble “available” plasma CU as a proportion of total plasma Cu Means with different letters on the same horizontal line are significantly different (P < 0.05)
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Cu in red blood cells, liver and mohair samples or sulphur concentrations in mohair fibre.
4. DiseUWion The growth rate and food conversion efficiency were typical of those previously recorded in studies with Angora goats given good-quality diets (e.g. Shahjalal et al., 1992). None of the measurements for growth and carcass characteristics were affected by MO, even at high dietary S intakes. Similarly there were no significant effects of MO on the weight or diameter of clean tibre recorded at the end of the study. Anaemia has been associated with Cu deficiency in ruminants (Gooneratne et al., 1989). However, there were no differences ( P > 0.05) in the values recorded in this study for haemoglobin (114-117 gl-‘) and packed cell volume (0.3) which were close to the means of 113gl-’ and 0.33 respectively as described by Topps and Thompson (1984) for cattle and which are broadly representative of the normal order of magnitude for ruminants. The imposed treatments were not severe or prolonged enough to affect these growth and physiological parameters. The dose-dependent increase in plasma MO concentrations suggests an efficient absorption of this mineral from the rumen. The parallel elevations in total plasma Cu concentrations may indicate the formation of thiomolybdate complexes as described in other studies (Gooneratne et al., 1989). The highest inclusion level of MO in the diet, equivalent to approximately 16 mg kg - ’ DM, was considerably lower than the range of lOO-200mg kg-’ MO diet for sheep quoted by Howell (19831, although greater than the concentration of 4 mg kg-’ in the presence of 3.Og kg-’ S which reduced Cu availability in sheep (Suttle, 1974). Concentrations of TCA-soluble or “available” Cu in plasma and associated caerulosplasmin activity were not different between treatments. The increase in the amount of TCA-insoluble Cu in the plasma of Mo-treated goats may reflect the slow clearance of Cu-MO-albumin complexes from the circulation. Lack of signs of toxicity make it apparent that the concentrations of available Cu in plasma were adequate to maintain normal physiological function. The absence of significant effects of dietary MO on liver Cu suggests that the formation of TCA-insoluble Cu-containing complexes did not result in the depletion of liver Cu. The present results may be compared with those of Humphries et al. (19871, in which goats suspected of suffering from copper poisoning were treated with ammonium tetrathiomolybdate. Ammonium tetrathiomolybdate treatment increased average total plasma Cu concentrations from 22.5 to 30.2 p,moll-‘, only 60% of which was TCA soluble. The “available” Cu concentration of 18.1 pmoll-’ was greater than the values of 11.8-13.4 p,moll-’ TCA soluble Cu recorded in the present study, and consistent with those of 9.0-18.7 pmol-’ cited by McKenzie (1993). In keeping with the study of Humphries et al. (19871, supplementation of the diet with MO in the presence of adequate S, increased concentrations of total Cu and reduced the proportion of total Cu which was TCA soluble. Since the concentration of TCA-soluble “available” Cu was similar between days 28 and 70, there was probably a stabilisation of the different
H. Galbraith et al. /Animal Feed Science Technology 67 (1997) 83-90
89
Cu-containing compartments and Cu flux within the body. The results also suggest that there was little depletion of Cu in liver in the 70-day study. The concentrations of 581-630 kmol kg-’ DM are in excess of the 300 pmol kg-’ DM cited by Humphries et al. (1987).
5. Conclusions
Differences between and within species in susceptibility to deficiencies or toxicities owing to Cu and other trace minerals are well recognised (Mills, 1983; Wiener and Woolliams, 1983). It is apparent (Humphries et al., 1987) that goats may be as susceptible to copper poisoning as sheep. The occurrence of swayback owing to copper deficiency in goats has been also reported (McKenzie, 1993). However, results from the present study suggest that the maintenance of “available” Cu concentration in plasma of 11.8 pmol l- ’ or greater was adequate to maintain normal haematological status, growth, fleece production and liver Cu concentrations. Further studies could usefully investigate further the quantitative relationship between dietary Cu, S and MO and the absorption, excretion and metabolism of these mineral elements in the goat.
Acknowledgements
The authors wish to thank Mr. G.F.M. Paterson, Ms. J. Pool, Mr. J. Struthers, Mr. T.W. Begg and Mr. N. Lokke for the care of experimental animals; Mr. I. Mckay, Miss M. Nicol and Mrs. M. Warren for analysis of trace elements and blood characteristics; Dr. D. Bain of the Macaulay Land Use Research Institute for analysis of sulphur; Dr. Janet Roden for statistical advice; Professor I. Bremner for providing constructive comments and Mrs. Edna Watt and Mrs Joey Parker for typing the manuscript.
References Bremner, I. and Davies, N.T., 1980. Dietary composition and absorption of trace elements by ruminants. In: Y. Ruckebusch and P. Thivend (Editors), Digestive Physiology of Ruminants. MTP Press, Lancaster, pp. 409-427. Dick, A.T., Dewey, D.W. and Gawthome, J.M., 1975. Thiomolybdates and the copper-molybdenum-sulphur interaction in ruminant nutrition. J. Agric. Sci. (Camb.), 85: 567-568. Gooneratne, S.R., Buckley, W.T. and Christensen, D.A., 1989. Review of copper deficiency and metabolism in ruminants. Can. J. Anim. Sci., 69: 819-845. Howell, J.C., 1983. Toxicity problems associated with trace elements in domestic animals. In: N.F. Suttle, R.G. Gunnn, W.M. Allen, K.A. Linklater and G. Weiner (Editors), Trace Elements in Animal Production and Veterinary Practice. Occasional Publication No. 7. British Society of Animal Production, pp. 107-l 17. Humphries, W.R., Phillippo, M., Young, B.W. and Btemner, I., 1983. The influence of dietary iron and molybdenum on copper metabolism in calves. Br. J. Nutr., 49: 77-86. Humphries, W.R., Mills, C.F., Greig, A., Roberts, L., Inglis, D. and Halliday, G.J., 1986. Use of ammonium tetrathiomolyhdate in the treatment of copper poisoning in sheep. Vet. Rec., 119: 596-598. Humphries, W.R., Morrice, P.C. and Mitchell, A.N., 1987. Copper poisoning in Angora goats. Vet. Rec., 121: 231.
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Mason, J., Lamand, M. and Kelleher, C.A., 1980. The fate of Mo-labelled sodium tetrathiomolybdate after duodenal administration in sheep; the effect on caeruloplasmin (EC 1.16.3.1) diamine oxidase activity and plasma copper. Br. J. Nutr., 43: 515-523. Mills, C.F., 1983. The physiological and pathological basis of trace element deficiency disease. In: N.F. Suttle, R.G. Gunn, W.M. Allen, K.A. Linklater and G. Weiner (Editors), Trace Elements in Animal Production and Veterinary Practice. Occasional Publication No. 7. British Society of Animal Production, pp. l-10. Morrice, P.C., Humpbries, W.R. and Bremner, I., 1989. Determination of molybdenum in plasma using graphite furnace atomic absorption spectrophotometry. Analyst, 114: 1667-1669. McKenzie, D., 1993. R. Goodwin (Editor) Goat husbandry. 5th edition. Faber and Faber, London, pp. 101-104. Price, J., Will, A.M., Paschaleris, G. and Chesters, J.K., 1987. Identification of tbiomolybdates in digesta and plasma from sheep after administration of Mo-labelled compounds into the rumen. Br. J. Nutr., 58: 127-138. Shahjalal, Md., Galbraith, H. and Topps, J.H., 1992. The effect of changes in dietary protein and energy on growth, body composition and mohair fibre characteristics of British Angora goats. Anim. Prod., 54: 405-412. Suttle, N.F. 1974. Recent studies of the copper-molybdenum antagonism. Proc. Nutr. Sot., 33: 299-305. Topps, J.H. and Thompson, J.K., 1984. Blood characteristics and the nutrition of ruminants. Ministry of Agriculture, Fisheries and Food. Department of Agriculture and Fisheries for Scotland. Reference Book 260. HMSO, London, p. 32. Wiener, G. and Woolliams, J.A., 1983. Genetic variation in trace element metabolism. In: N.F. Suttle, R.G. Gunn, W.M. Allen, K.A. Linklater and G. Weiner (Editors), Trace Elements in Animal Production and Veterinary Practice. Occasional Publication No. 7. British Society of Animal Production, pp. 27-38.
Animal Feed Science Technology 67 (1997) 83-90
Short communication
The effect of dietary molybdenum supplementation on tissue copper concentrations, mohair fibre and carcass characteristics of growing Angora goats H. Galbraith a,* , W. Chigwada ‘, J.R. Scaife a, W.R. Humphries b a University of Abe&en,
Department of Agriculture, 581 King Street, Aberdeen AB24 5UA, UK b Rowen Research Instihde, Budsbum Aberdeen AB2 9SB, UK Acce@ed 28 October 1996
The purpose of this experiment was to better chamcterize the effects of the interaction between copper (Cu), molybdenum (MO) and sulphur 6) in the diet on growth, metabolism and fibre characteristics in Angora goats. 15 Angora goats aged 9 months and weighing 21.5 kg on average were used in a ten-week study and allocated to three dietary treatments: Treatment C (IOMJ metabolisable energy, 178g crude protein, 5.5 mg Cu, 0.57 mg MO, and 3.4g S): Treatment Ml (with 7.5 mg MO) or Treatment M2 (with 15mg MO) per animal per day. Dose-dependent increases in the concentrations of MO (P < 0.01) and Cu (P < 0.05) in plasma were recorded in response to increased dietary intake of MO. Supplementation of the control diet with increased concentrations of MO did not produce effects (P > 0.05) on growth rate, feed conversion efficiency, carcass weight or mohair fibre yield and diameter. Haematological status and concentration of Cu in liver and Cu and S in f&e at the end of the study were also not affected (P > 0.05). Concentrations of trichloroacetic acid (TCA) soluble “available” copper in plasma were not significantly different although significant (P < 0.05 and P < 0.01) reductions in the ratio of “available” to total Cu concentrations were observed. This effect was stabilised and maintained after 30 days. It is suggested that the additional Cu in plasma was largely TCA insoluble and possibly in the form of thiomolybdate complexes which may be poorly excreted and not available for uptake to the metabolic sites. It is evident that adequate “available” Cu was present in plasma and that exposure to elevated MO intake was not severe or long enough to produce clinical
* Corresponding author. 0377~8401/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO377-8401(96)01140-6
84
H. Galbraith et al. /Animal
Feed Science Technology 67 (1997) 83-90
symptoms B.V.
or to affect growth, haematological
status or fibre prodwtion.
0 1997 Elsevier Science
Keywords:
Copper; Molybdenum; Sulphur; Angora goats; Haematological status; Growth
1.ldrodnction The role and importance of copper (Cu) in the metabolism of animals are well known. Cu deficiency and toxicity in ruminants occur frequently in many parts of the world. The development of Cu deficiency depends on both the dietary Cu concentrations and the concentrations of antagonists which interfere with its absorption from the gut and subsequent utilisation for metabolic processes (Goonerame et al., 1989). Simple Cu deficiency caused by low dietary Cu intakes is often responsible for the development of Cu-responsive disorders including abnormalities in pigmentation and keratinization of wool and hair, anaemia, skeletal abnormalities and enzootic ataxia. (Bremner and Davies, 1980). However, widespread Cu deficiency has been observed in ruminants grazing herbage with apparently adequate Cu concentrations (Bremner and Davies, 1980). This “conditioned” or “induced” Cu deficiency is caused by the presence of antagonists which reduce the availability of Cu. Molybdenum (MO) and sulphur (S) have been identified as the most important antagonists of Cu (Humphries et al., 1983). The Cu content of ruminant diets usually varies between 4 and 10 mg kg- ’ DM or greater (Gooneratne et al., 1989) and it is subject to many different influences. It is well recognised that the risk of copper toxicity in sheep may also be increased by consump tion of feeds which contain high Cu (Gooneratne et al., 1989). The MO concentration of herbage frequently ranges between 0.5 and 5 mg kg-’ DM (Goonerame et al., 19891, tends to be more variable than that of copper, and, unlike that of Cu, the concentration of MO increases as the soil pH increases. The S content of herbage depends largely on the amount of S in protein and rarely exceeds 3 g kg- ’ DM. Methionine and cysteine usually comprise over 90% of the organic S in herbage (Goonerame et al., 1989). The adverse effect of increased dietary MO and S on the utilisation of Cu by ruminants has been attributed to the formation of thiomolybdates (TM) in the rumen (Dick et al., 1975; Suttle, 1974; Goonerame et al., 1989). Recent findings (Gooneratne et al., 1989) show that tetrathiomolybdate 0TM) appears to dominate in the rumen at moderate to high MO and S intake. TM bind to Cu in the rumen to form copper thiomolybdates (Cu-TM) which are insoluble and therefore unavailable for absorption. TM also bind onto the rumen solids and, at high TM concentrations, also occur in the rumen liquid phase. Excess TM is absorbed into the blood and disturbs systemic Cu metabolism, This causes characteristic increases in plasma Cu concentration, much of which is protein bound, insoluble in acid and apparently not available for uptake into tissues (Price et al., 1987; Goonerame et al., 1989). Sheep are known to be sensitive to Cu poisoning and administration of ammonium tetrathiomolybdate reduces the severity of the condition (Humphries et al., 1986).
H. Galbraith et al. /Animal
Feed Science Technology 67 (1997) 83-W
85
Information on the sensitivity of goats to excessive dietary intakes of Cu is limited. Humphties et al. (1987) reported that animals receiving excessive amounts of Cu from a calf milk replacer containing an estimated 1Omg kg-’ DM Cu and used in their diet, developed symptoms of Cu toxicity similar to those in sheep. Moreover. injection of large doses of ammonium tetrathiomolybdate, reversed the toxic symptoms. Goats may therefore be more similar to sheep than cattle in their adverse response to high dietary intakes of Cu, but all three species are sensitive to Cu deficiency under certain conditions. However, systematic information on the relationships between dietary Cu, MO and S in goats is limited. The current study explored the effects of increased dietary intakes of MO on Cu concentrations and fibre characteristics of Angora goats on a high S diet, conditions which have previously been shown to cause deficiencies in sheep (Suttle, 1974). 2. Materials and methods 2.1. Animals and diets 15 wether Angora goats aged about 9 months and weighing 2 1.5 kg on average were allocated to three dietary treatments (five per treatment) using a randomised block design. They were offered a basal pelleted diet based on molassed sugar beet pulp (Trident Feeds, Peterborough, UK), barley straw, oat feed, soya bean meal, white fish meal and a mineral and vitamin supplement in addition to 50g per animal per day of good-quality grass hay. This diet provided (per kg DM), 5.5mg Cu and 0.57mg MO, and following supplementation with sodium sulphate, 3.4g S. The basal diet was either unsupplemented (C) or supplemented with MO at either 7.5 mg (treatment Ml) or 15 mg (treatment M2) per animal per day by treating 50g of the basal diet with 1.0 ml of solutions of sodium molybdate containing these quantities of MO. The 50g portion was allowed to dry and was then offered to the goats at 09:OOh. This part of the ration was fully consumed before *the remainder of the diet was offered to the goats. This diet provided 37g of DM per kg of live weight. The final concentrations of the MO and S used were in excess of those (MO, 4mg kg- ‘; S, 3.Og kg- ‘) considered to produce reductions in Cu availability in sheep (Suttle, 1974). The goats were housed in individual pens and extraneous exposure to metal was minimised by covering surfaces with plastic tape and offering deionised water in plastic containers. The goats were weighed weekly and feed residues were monitored daily. The goats were slaughtered after 71 days by the approved method of stunning using a captive bolt pistol followed by exanguination (Shahjalal et al., 1992). Samples of liver were collected immediately after slaughter, frozen rapidly and stored at - 20” and rapidly thawed when required for analyses. 2.2. Blood andPbre
collection
Individual blood samples were collected every two weeks by jugular venepuncture into proprietary vacutainer tubes (Becton Dickinson, VACUTAINER Systems Europe, Meylan Cedex, France) containing (a) EDTA anticoagulant for determination of packed
H. Galbraith et al. /Animul
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Feed Science Technology 67 (1997) 83-W
cell volume (PCV) and haemoglobin, and (b) heparin for the measurement of Cu and MO concentrations and of caeruloplasmin activity. Mohair fibre samples were collected from a 10 cm X 1Ocm square area on the mid-side on days 35 and 70 of the study using a conventional horse-hair clipper (Shahjalal et al., 1992). The samples were washed by soaking overnight in a solution of the detergent Decon 75 (50 ml 1-l >, rinsed thoroughly in distilled water and then dried at 80°C. Diameter was measured on a random sample of 100 fibres as described by Shahjalal et al. (1992).
2.3. Analysis of Cu, MO and S Samples of plasma were prepared by conventional centrifugation of whole blood at 1OOOg. Separated blood cells were then washed and re-suspended in physiological saline. Haemoglobin was determined on whole blood samples following haemolysis using a Contraves Analyser Model 4300. PCV was measured following centrifugation in microhaematocrit capillary tubes with a Hawksley microhaematocrit reader. Concentrations of total Cu in plasma were measured following 1:1 dilution in distilled water using a Varian AA-875 series atomic absorption spectrophotometer. Trichloroacetic acid (TCA) soluble or “available” Cu was similarly measured after adding 0.5 ml plasma to 2.Oml of a solution containing TCA (lOOgl_‘), mixing and centrifuging for 15 min at 1600 g. The supematant was analysed by atomic absorption spectrophotometry. Total plasma MO concentrations were measured following acid digestion, by graphite furnace atomic absorption spectrophotometry (Mot-rice et al., 1989). Caeruloplasmin activity was measured in plasma using the method of Mason et al. (1980). Liver and washed fibre samples were digested in mixtures of nitric, perchloric and sulphuric acids before analysis of Cu by atomic absorption spectrophotometry. Caeruloplasmin activity was measured in plasma using the method of Mason et al. (1980). Total S concentrations in fibre were analysed using X-ray fluorescence spectrometry using a Philips PW104 automatic sequential wavelength dispersive spectrometer following the grinding of samples in liquid nitrogen and pelleting. 2.4. Statistical analysis The data were examined by analysis of variance using the package Minitab 7.3 (Minitab Inc. 1989) and presented as treatment means with an estimate of the pooled standard error of the difference @ED) between any two means. Where significance (P < 0.05) was obtained for overall treatment effects, the signifkances of differences between means for pairs of treatment groups were calculated by comparison with values for least significant difference. 3. Results 3.1. Growth, pelformance,
carcass characteristics
and jibre production
Supplementation of the diet with increased concentrations of MO had no effect (P > 0.05) on dry matter intake, live-weight gain or chilled carcass weight (Table 1).
H. Galbraith et al. /Animal Table 1 Growth performance,
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carcass and fibm characteristics Treatments
Total dry matter intake (kg) Live weight gain (kg) LWG/DMI Chilled carcass weight (kg) Clean fibre weight (day 69) (g) Clean fibre diameter (mm)
’
Control
Ml
M2
64.7 5.74 0.09 11.4 9.23 31.5
67.7 6.86 0.10 11.8 10.2 30.0
66.7 7.14 0.11 11.3 8.8 28.1
SED
Statistical
1.25 1.40 0.002 0.54 0.95 1.22
NS NS NS NS NS NS
significance
a n = 5 goats per treatment.
Similarly, the dietary treatments had no effect on the weight of clean fibre collected on day 69, or on its diameter. 3.2. Concentrations
of Cu, MO and S in tissues and mohair
MO supplementation had no effect (P > 0.05) on mean values for haemoglobin and packed cell volume in whole blood (Table 2). However, concentrations of MO in plasma were elevated (P < 0.01) with increasing dose levels of MO in the diet, as were concentrations (P < 0.05) of total, but not TCA soluble (P > 0.051, Cu (Table 2). In contrast, the proportion of plasma Cu that was soluble in TCA was decreased (P < 0.05) in a dose-dependent manner on day 28 with a trend maintained on day 70. The dietary treatments did not affect (P > 0.05) caeruloplasmin activity in plasma, concentrations of
Table 2 Mean values for parameters
measured
in whole blood, plasma, tibre and liver samples Treatments
Whole blood Haemoglobin fg 1- ’) Packed cell volume (1 l- ’) Pla.ma Total Cu (TCu) (pmoll-‘1 TCA-soluble Cu (SCu) (pmoll-’ ) Total MO, &moll-‘1 Caeruloplasmin activity (Eu 1-l ) SCu/TCu SCu/TCu Red blood cell Cu ( pm011 - ’> Liver Cu ( firno1 kg- ’ DM> Mohair Cu ( pm01 kg-’ DM) S (mm01 kg-’ DM)
SED
Statistical significance
Day
Control
Ml
M2
70 70
117 0.30
114 0.30
114 0.30
8.4 0.018
NS NS
70 70 70 70 28 70
14.9a 13.4 0.03a 40.6 l.Oa 0.92a 15.7 631 73.0 720
16.2b 11.8 0.87b 31.2 0.89ab 0.74ab 14.5 653 64.7 770
20.9b 13.4 3.67~ 38.7 0.66c 0.63b 13.4 581 61.4 740
2.0 1.42 0.44 7.1 0.06
’ NS ** NS
70 71 69 69
0.09 2.41 48.8 9.44 5.7
** * NS NS NS NS
SCu/TCu is TCA-soluble “available” plasma CU as a proportion of total plasma Cu Means with different letters on the same horizontal line are significantly different (P < 0.05)
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Cu in red blood cells, liver and mohair samples or sulphur concentrations in mohair fibre.
4. DiseUWion The growth rate and food conversion efficiency were typical of those previously recorded in studies with Angora goats given good-quality diets (e.g. Shahjalal et al., 1992). None of the measurements for growth and carcass characteristics were affected by MO, even at high dietary S intakes. Similarly there were no significant effects of MO on the weight or diameter of clean tibre recorded at the end of the study. Anaemia has been associated with Cu deficiency in ruminants (Gooneratne et al., 1989). However, there were no differences ( P > 0.05) in the values recorded in this study for haemoglobin (114-117 gl-‘) and packed cell volume (0.3) which were close to the means of 113gl-’ and 0.33 respectively as described by Topps and Thompson (1984) for cattle and which are broadly representative of the normal order of magnitude for ruminants. The imposed treatments were not severe or prolonged enough to affect these growth and physiological parameters. The dose-dependent increase in plasma MO concentrations suggests an efficient absorption of this mineral from the rumen. The parallel elevations in total plasma Cu concentrations may indicate the formation of thiomolybdate complexes as described in other studies (Gooneratne et al., 1989). The highest inclusion level of MO in the diet, equivalent to approximately 16 mg kg - ’ DM, was considerably lower than the range of lOO-200mg kg-’ MO diet for sheep quoted by Howell (19831, although greater than the concentration of 4 mg kg-’ in the presence of 3.Og kg-’ S which reduced Cu availability in sheep (Suttle, 1974). Concentrations of TCA-soluble or “available” Cu in plasma and associated caerulosplasmin activity were not different between treatments. The increase in the amount of TCA-insoluble Cu in the plasma of Mo-treated goats may reflect the slow clearance of Cu-MO-albumin complexes from the circulation. Lack of signs of toxicity make it apparent that the concentrations of available Cu in plasma were adequate to maintain normal physiological function. The absence of significant effects of dietary MO on liver Cu suggests that the formation of TCA-insoluble Cu-containing complexes did not result in the depletion of liver Cu. The present results may be compared with those of Humphries et al. (19871, in which goats suspected of suffering from copper poisoning were treated with ammonium tetrathiomolybdate. Ammonium tetrathiomolybdate treatment increased average total plasma Cu concentrations from 22.5 to 30.2 p,moll-‘, only 60% of which was TCA soluble. The “available” Cu concentration of 18.1 pmoll-’ was greater than the values of 11.8-13.4 p,moll-’ TCA soluble Cu recorded in the present study, and consistent with those of 9.0-18.7 pmol-’ cited by McKenzie (1993). In keeping with the study of Humphries et al. (19871, supplementation of the diet with MO in the presence of adequate S, increased concentrations of total Cu and reduced the proportion of total Cu which was TCA soluble. Since the concentration of TCA-soluble “available” Cu was similar between days 28 and 70, there was probably a stabilisation of the different
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Cu-containing compartments and Cu flux within the body. The results also suggest that there was little depletion of Cu in liver in the 70-day study. The concentrations of 581-630 kmol kg-’ DM are in excess of the 300 pmol kg-’ DM cited by Humphries et al. (1987).
5. Conclusions
Differences between and within species in susceptibility to deficiencies or toxicities owing to Cu and other trace minerals are well recognised (Mills, 1983; Wiener and Woolliams, 1983). It is apparent (Humphries et al., 1987) that goats may be as susceptible to copper poisoning as sheep. The occurrence of swayback owing to copper deficiency in goats has been also reported (McKenzie, 1993). However, results from the present study suggest that the maintenance of “available” Cu concentration in plasma of 11.8 pmol l- ’ or greater was adequate to maintain normal haematological status, growth, fleece production and liver Cu concentrations. Further studies could usefully investigate further the quantitative relationship between dietary Cu, S and MO and the absorption, excretion and metabolism of these mineral elements in the goat.
Acknowledgements
The authors wish to thank Mr. G.F.M. Paterson, Ms. J. Pool, Mr. J. Struthers, Mr. T.W. Begg and Mr. N. Lokke for the care of experimental animals; Mr. I. Mckay, Miss M. Nicol and Mrs. M. Warren for analysis of trace elements and blood characteristics; Dr. D. Bain of the Macaulay Land Use Research Institute for analysis of sulphur; Dr. Janet Roden for statistical advice; Professor I. Bremner for providing constructive comments and Mrs. Edna Watt and Mrs Joey Parker for typing the manuscript.
References Bremner, I. and Davies, N.T., 1980. Dietary composition and absorption of trace elements by ruminants. In: Y. Ruckebusch and P. Thivend (Editors), Digestive Physiology of Ruminants. MTP Press, Lancaster, pp. 409-427. Dick, A.T., Dewey, D.W. and Gawthome, J.M., 1975. Thiomolybdates and the copper-molybdenum-sulphur interaction in ruminant nutrition. J. Agric. Sci. (Camb.), 85: 567-568. Gooneratne, S.R., Buckley, W.T. and Christensen, D.A., 1989. Review of copper deficiency and metabolism in ruminants. Can. J. Anim. Sci., 69: 819-845. Howell, J.C., 1983. Toxicity problems associated with trace elements in domestic animals. In: N.F. Suttle, R.G. Gunnn, W.M. Allen, K.A. Linklater and G. Weiner (Editors), Trace Elements in Animal Production and Veterinary Practice. Occasional Publication No. 7. British Society of Animal Production, pp. 107-l 17. Humphries, W.R., Phillippo, M., Young, B.W. and Btemner, I., 1983. The influence of dietary iron and molybdenum on copper metabolism in calves. Br. J. Nutr., 49: 77-86. Humphries, W.R., Mills, C.F., Greig, A., Roberts, L., Inglis, D. and Halliday, G.J., 1986. Use of ammonium tetrathiomolyhdate in the treatment of copper poisoning in sheep. Vet. Rec., 119: 596-598. Humphries, W.R., Morrice, P.C. and Mitchell, A.N., 1987. Copper poisoning in Angora goats. Vet. Rec., 121: 231.
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Mason, J., Lamand, M. and Kelleher, C.A., 1980. The fate of Mo-labelled sodium tetrathiomolybdate after duodenal administration in sheep; the effect on caeruloplasmin (EC 1.16.3.1) diamine oxidase activity and plasma copper. Br. J. Nutr., 43: 515-523. Mills, C.F., 1983. The physiological and pathological basis of trace element deficiency disease. In: N.F. Suttle, R.G. Gunn, W.M. Allen, K.A. Linklater and G. Weiner (Editors), Trace Elements in Animal Production and Veterinary Practice. Occasional Publication No. 7. British Society of Animal Production, pp. l-10. Morrice, P.C., Humpbries, W.R. and Bremner, I., 1989. Determination of molybdenum in plasma using graphite furnace atomic absorption spectrophotometry. Analyst, 114: 1667-1669. McKenzie, D., 1993. R. Goodwin (Editor) Goat husbandry. 5th edition. Faber and Faber, London, pp. 101-104. Price, J., Will, A.M., Paschaleris, G. and Chesters, J.K., 1987. Identification of tbiomolybdates in digesta and plasma from sheep after administration of Mo-labelled compounds into the rumen. Br. J. Nutr., 58: 127-138. Shahjalal, Md., Galbraith, H. and Topps, J.H., 1992. The effect of changes in dietary protein and energy on growth, body composition and mohair fibre characteristics of British Angora goats. Anim. Prod., 54: 405-412. Suttle, N.F. 1974. Recent studies of the copper-molybdenum antagonism. Proc. Nutr. Sot., 33: 299-305. Topps, J.H. and Thompson, J.K., 1984. Blood characteristics and the nutrition of ruminants. Ministry of Agriculture, Fisheries and Food. Department of Agriculture and Fisheries for Scotland. Reference Book 260. HMSO, London, p. 32. Wiener, G. and Woolliams, J.A., 1983. Genetic variation in trace element metabolism. In: N.F. Suttle, R.G. Gunn, W.M. Allen, K.A. Linklater and G. Weiner (Editors), Trace Elements in Animal Production and Veterinary Practice. Occasional Publication No. 7. British Society of Animal Production, pp. 27-38.