Role of sulphur in cyanide detoxification in ruminants

Small Ruminant Research, 8 ( 1992 ) 277-284 © 1992 Elsevier Science Publishers B.V. All fights reserved. 0921-4488/92/$05.00 277 Role of sulphur in cyanide detoxification in ruminants C.F.I. O n w u k a l, A.O. A k i n s o y i n u a n d O.O. Tewe Division of Nutritional Biochemistry, Department of Animal Science, Universityof lbadan, Ibadan, Nigeria (Accepted 30 October 1991 ) ABSTRACT Onwuka, C.F.I., Akinsoyinu, A.O. and Tewe, O.O., 1992. Role of sulphur in cyanide detoxification in ruminants. Small Rumin. Res., 8: 277-284. Sixteen intact West African dwarf goats (10-23 kg) and eight ruminally fistulated West African dwarf sheep were fed dried cassava-based (Manihot utilissima Pohl ) diets. Dietary cyanide (HCN) intakes of 246-248 mg/kg diet were detoxified and metabolized into 2.80-4.01 mg thiocyanate/100 ml ruminant fluid; 0.04-0.07 nag/100 g serum fluid and 0.016-0.030 mg/100 g feed intake. Increasing dietary sulphur concentrations improved (P< 0.01 ) DM intake with a resultant increase in HCN intake which was highest for the 0.5% elemental sulphur diet where the least urinary thiocyanate concentration was recorded. No mortality occurred in the trials. DM intake per LW ranged from 2.05-2.99%. Results indicate significant (P< 0.01 ) correlations between sulphur concentrations and LW changes; urinary, serum and ruminal thiocyanate concentrations, while ME intake affected thiocyanate formation. Urinary N appeared to decrease non-significantly as N intake increased. Animals on the low sulphur diet had the greatest (P< 0.01 ) weight losses (7.88 + 0.31 g/d per W~TM) and the greatest ruminal and urinary thiocyanate. Thiocyanate passed from rumen to blood and from there was excreted in the urine. INTRODUCTION C a s s a v a ( M a n i h o t utilissima P o h l ) is c o m m o n l y u s e d as a n i m a l f e e d in t h e t r o p i c s ( C I A T , 1 9 8 8 ) . A c o n c e r n in u s i n g c a s s a v a as f e e d is t h e p r e s e n c e o f c y a n o g e n i c glucosides, l i n a m a r i n a n d l o t a u s t r a l i n , t h e b o n d s o f w h i c h are hydrolysed when tissues of this root crop are damaged or disrupted, thereby r e l e a s i n g free c y a n i d e ( H C N ) , o n e o f t h e m o s t p o w e r f u l p o i s o n s k n o w n . C y anide inactivatives xanthine oxidase by extraction of sulphur from the protein, w h i c h is e l i m i n a t e d as t h i o c y a n a t e ( M a s s e y a n d E d m o n s o n , 1970), while JPresent address and correspondence to: C.F.I. Onwuka, Department of Animal Nutrition, College of Animal Science and Livestock Production, University of Agriculture, P.M.B. 2240, Abeokuta, Ogun State, Nigeria. 278 C.F.I. ONWUKA ET AL. it reacts with hemoglobin forming cyanohemoglobin which is not an oxygen carrier. Oke (1969) indicated that when the cyanide poisoning process continues long enough, sickness and death result. However, cassava fed to cattle and sheep in various forms, including tapioca meal and chopped roots, has not produced obvious ill effects (Hill, 1973). Detoxification of cyanide into SCN causes an increased demand for sulphur-containing amino acids (Maner and Gomez, 1973 ). Dietary cyanide increases urinary and serum thiocyanate (Tewe et al., 1980; 1985 ). Although elemental sulphur is less available than methionine and other sulphur sources (Johnson et al., 1971 ) when added to a cassava diet it improved protein utilization efficiency by pigs. This investigation is a follow-up to an earlier study (Onwuka and Akinsoyinu, 1989a,b) and reveals the role of dietary elemental sulphur concentrations in cyanide detoxification by West African dwarf goats and sheep (Fouta djallon ) fed graded sulphur concentrations in urea-based diets. MATERIALS AND METHODS Animals and their management Eight West African dwarf (WAD) sheep, average weight 15-25 kg and fitted with permanent ruminal cannulae of approx. 8 m m diameter, were used for the ruminal and serum thiocyanate studies. Sixteen intact WAD goats (eight intact does and bucks) with BW from 10-23 kg, allotted in a randomized block design into groups of four goats each on the basis of body weight and sex, were used for the metabolic studies. Goats were housed in individual modified metabolism cages and had free daily access to fresh clean water and trace-mineralized salt blocks. Sheep and goats were dewormed routinely with Banminth Wormer (a Pfizer product ) and declared uniformly healthy before the start of the experiments. Goats were weighed on the 1st and 7th days of the collection periods. Mean BW were used in computation of the various parameters. Diets Four experimental diets were mixed, the principal ingredients being varying levels of elemental sulphur and cassava flour, urea, vitamin A and D crumbles and common salt (Table 1 ). The control diet had no added sulphur. Each goat and sheep was offered 0.8 kg concentrate and 0.3 kg Cynodon hay per day for a 21-day period, comprising 14 d of preliminary adjustment and 7 d of faeces and urine collection. Collection of urine, faeces, blood and ruminal fluid For goats, modified metabolism crates as described by Onwuka and Akinsoyinu (1989b) with removable trays were used to separate urine and faeces, which were collected daily before feeding. Collection bottles were washed with ROLE OF SULPHUR IN CYANIDE DETOXIFICATION IN RUMINANTS 279 5 ml 3% H 2 S O 4 solution to prevent N loss and bacterial growth. Aliquots of daily urinary collections were bulked and stored at - 2 oC, while faeces were dried for 2 days at 70 ° C, milled and stored in air-tight bottles. Blood samples were drawn from the jugular vein of sheep on the I st and 4th days of collection. They were centrifuged at 3000 rpm and the serum preserved for thiocyanate assay. Rumen liquor was sampled (Alexander, 1969) 2 h after feeding on days l and 4 from the fistulated sheep, when they tended to be resting. Liquor was strained immediately through two layers of fine muslin cloth, the pH measured with a dual electrode pH meter, and thereafter analysed. Urine and rumen liquor were decolorised with activated charcoal before analysis. Analytical procedures Proximate chemical, including sulphur analyses, were conducted with AOAC ( 1975 ) methods. Thiocyanate concentrations in urine, serum, and rumen liquor, and bound and free hydrocyanic acid levels were determined by modifying the methods of Wood and Cooley ( 1956 ), using the Zeiss Spectrophotometer PM 2A, read at 460 m/z wavelength. Data were subjected to analyses of variances for DM intake, BW changes, N utilization and cyanide metabolism. Means found to be significant were separated with pairwise comparison of Duncan's (1955 ) multiple range test for linear contrasts. RESULTS Percentages of ingredients constituting the four experimental diets (Table 1 ) indicate that N was supplied principally by urea, and energy by cassava TABLE 1 Ingredients in the experimental diets used (g/100 g DM ) Ingredients Diet Control Urea Sulphur Cassava flour Salt (NaC1) 2Vitamin A, D crumbles Total 2 3 4 5.70 92.30 0.50 1.50 5.70 0.25 92.05 0.50 I. 50 5.70 0.50 91.80 0.50 1.50 5.70 0.75 91.55 0.50 I. 50 100.00 100.00 100.00 100.00 tValues according to Onwuka and Akinsoyinu ( 1989a ). 2Content in g/kg: M n 16.0; Zn 12.0; Fe 6.0; Cu 4.0; Co 0.3; I2 1.2; Mg 200. Vit A 0.50, IU and Vit D 0.25 IU. 280 C.F.I. ONWUKA ET AL. TABLE 2 Composition of experimental diets ( g / 1 0 0 g D M ) Diet Total S HCN (mg/kg) DM CP ME (kcal/g) Crude fibre 1 2 3 4 hay 0.1 247.0 84.5 18.3 3.044 2.7 0.2 246.0 84.8 18.3 3.037 2.9 0.6 248.0 83.9 18.6 3.023 2.9 0.8 247.0 84.4 18.2 2.991 2.6 0.4 93.5 7.8 3.176 20.2 IValues according to Onwuka and Akinsoyinu ( 1 9 8 9 a ) . TABLE 3 Performance of goats and sheep fed sulphur in cassava-based diets Performance characteristics Diet 1 mean _+s.e. D M intake g d - 1/W~7~4 D M intake as % of BW CP intake g d - 1 / W ~ T M N balance g d - l/W~k734 BW changes g d - 1/W~k734 H C N intake ( m g / k g BW) S intake ( g / d ) Ruminal SCNI ( m g / 1 0 0 g feed per d ) Serum SCN ( m g / 1 0 0 ml serum) Urinary SCN ( m g / 1 0 0 g feed per d ) 2 m e a n + s.e. 3 mean _+s.c. 4 mean -+s.c. 52.02 _+3.24 67.35 _+6.41 70.53 +0.87 70.49 -+12.63 2.05 -+0.22 2.82 -+0.22 2.98 +0.18 2.99 -+ 0.46 6.00 -+0.26 8.71 -+0.51 10.05 -+0.29 8.82 _+ 1.34 - 0 . 2 1 _+0.00 0.23 _+0.00a 0.23 _+0.00a 0.16 _+ 0.00 b - 7 . 8 8 +0.31 a - 3 . 4 2 +1.97 a 10.57 +3.63 b'¢ 9.36 -+ 3.38 ¢ 2.46 -+0.24 4.59 -+0.43 6.06 +0.14 4.56 -+ 1.71 0.38 -+0.02 0.92 -+0.14 2.69 +0.69 2.88 -+ 1.15 4.01 _+0.01 a 3.10 +0.07 b 3.60 +0.16 c 2.80 -+ 0.00 d 0.04 +0.00a 0.07 -+0.00 b 0.06 _+0.00 c 0.07 _+ 0.00 b 0.030+_0.00 a 0.029+0.00 b 0.016+-0.00 ¢ 0.024-+ 0.00 d SCN = thiocyanate. Means in a row with same superscript are not significantly different ( P > 0.05 ). flour. Based on dietary chemical composition (Table 2), the N levels were adequate for ruminants (range of 18.21 to 18.56% CP equivalent). The HCN concentrations were about equal in the four diets. N / S ratios decreased markedly with increasing dietary S levels, particularly for diets 3 and 4 (Table 3 ). BW changes followed virtually the same pattern as N / S ratios. Performance characteristics influenced by feed S and HCN are shown in Table 3. Feed and DM intake as a percentage of BW increased with S levels, increasing concentrations of HCN notwithstanding. Urinary and ruminal thiocyanate concentration decreased with higher S and HCN concentrations, while serum thiocyanate increased (Table 3 ). Ruminal thiocyanate varied in an opposite direction to serum thiocyanate (2 h after feeding), both values being, respectively, greatest and least for the S-deficient treatment, where ROLE OF SULPHUR IN CYANIDE DETOXIFICATION IN RUMINANTS . | I l l .... + + + + ++ 281 I I : I+ I I+ I + + + + + I 0 =1 ~ll I II II II II II II II II II II 0~m 0 8 8 .. "---"~ '~_ ~ gZ~ ~m~88 .R .R "-" . £ . I=l I=l " ~ O o. 0 V II i 0 ",=, o o o 0 v II .=. i 0 " " ~NNN 0 v N N ;e~e_ _ o 0 -=, II t "0 "0 0 ,-I o ~~ , ~ ,.~ ,.~ ~ r ~ r~ ~ Z ~r'~ 0 r'~ r'~ ~ ~ ~: ~ ~ ~ °~ ' , "°~ ~ •~~ ~" ~~ i i c5 A II <4." 282 C.F.I. ONWUKA ET AL. negative N balance was also recorded. Thiocyanate concentration was greatest in rumen liquor, followed by serum and urine (Table 3). The effect of supplementing with different concentrations of elemental S in urea/cassavabased diets to sheep and goats was highly significant (P<0.01) as reflected by BW changes. Table 4 shows regression relationships between selected animal performance characteristics. S concentrations were strongly correlated with DMI (P<0.05), BW gains (P<0.001) and serum thiocyanate concentrations (P< 0.05). However, thiocyanate concentrations in urine and ruminal liquor were inversely, but significantly (P< 0.01 ), correlated with S concentrations, with the result that amounts of thiocyanate lost in urine and rumen liquor decreased with increasing dietary S concentrations. S supplementation of ureabased diets improved the performance of goats and sheep but did not result in increased thiocyanate production as was observed by Maner and Gomez (1973) for rats. DISCUSSION Dietary hydrocyanic acid concentrations in this study fell within the range for peeled cassava tubers (Oyenuga and Amazigo, 1957; Tewe et al., 1976). HCN intakes of 2.46 mg to 6.06 mg/kg BW by goats and sheep in our study did not cause any death (s) ~as was observed by Coop and Blackley ( 1949 ) with sheep fed 2.40 mg HCN/kg BW. Presumably, the effect of S supplementation resulted from N balance and BW changes of the goats and sheep, because S is used in the fgrmation of S amino acids as well as cyanide detoxification. Dietary elemental S levels of up to 0.75% were used in this study. Akinsoyinu et al. ( 1975 ) and Shirley (1976) used 0.3% methionine and 0.2% S levels, respectively, in ruminant feeding and lower levels of urea than the 5.7% used in this study. HCN contents of the feeds also called for added S to facilitate detoxification into thiocyanate. NRC ( 1981 ) recommended 0.16 to 0.32% S for normal diets with 10-20% protein values fed to goats. This precludes the added requirement for urea and HCN metabolism by goats. The insufficiency of S in treatment 1 may have reduced the amount of protein used for growth. This, added to the reduced feed intake, may also account for why serum thiocyanate was lowest in treatment 1 which had no added elemental S and, thus, indicative of reduced detoxification of HCN into SCN. BW losses in treatments 1 and 2 were likely to have resulted from low feed intake and the inability of dietary S levels to cope with the added requirements imposed by urea and HCN in the feed. Thiocyanate, a metabolic product of cyanide detoxification (Maner and Gomez, 1973 ), followed a sequence in ruminal liquor, serum and urine which seemed to indicate that the detoxification reaction, i.e., cyanide-thiosulphate S transferase (rhodanese) enzyme activity (Wood and Cooley, 1956) oc- ROLEOFSULPHURINCYANIDEDETOXIFICATIONIN RUMINANTS 283 curred in the rumen and that thiocyanate was passed from there to serum and urine as for rats (Tewe and Maner, 1985 ), and ruminants (Cheeke and Shull, 1985 ). Passage of thiocyanate into urine is apparently slow. Renal absorption of thiocyanate from serum is a very slow process in ruminants. The increase in thiocyanate content observed may in part be due to the increased HCN and S intakes. The low mean urinary thiocyanate concentrations observed in treatment 3 imply that the diet contained optimum N and S for growth and detoxification of HCN, because a protein-deficient diet results in lower feed intake and, thus, lower cyanide ingestion, just as for treatment 1 of this study (Table 3 ). CONCLUSIONS Sulphur appears to be vital for cyanide detoxification into thiocyanate in small ruminants. Thiocyanate is formed in the rumen, carried into serum and lost slowly in urine. A dietary level of 0.5% elemental S ensured adequate cassava cyanide detoxification in sheep and goats in these trials. The addition of S to urea-based diets also kept the ruminants in positive nitrogen balance. ACKNOWLEDGEMENTS The technical assistance of Mr. Romanus N. Nwokorie of the Nutritional Biochemistry Laboratory is appreciated. Thanks are given to Mrs. Linda Onwuka and Miss O. Odedairo for preparation of the manuscript. REFERENCES Akinsoyinu, A.O., Mba, A.U. and Olubajo, F.O., 1975. DL-methionine supplementation effects of urea nitrogen utilization by growing West African dwarf goats. Nig. J. Anim. Prod., 2: 64. Alexander, R.H., 1969. Establishment of a laboratory procedure for the "in vitro" determination of digestibility. West Scotland Agric. Coll. Bull. No. 42. AOAC, 1975. Official Methods of Analysis. 12th Edition. Association of Offic. Agric. Chem., Washington D.C., USA. Cheeke, P.R. and Shull, L.R., 1985. Natural toxicants in feeds and poisonous plants. The AVI Publishing Co., Inc., pp. 173-180. CIAT, 1988. Cassava Research in Africa. A bibliography. Centro International de Agriculture Tropical. Cali, Colombia, 474 pp. Coop, I.E. and Blakley, R.L., 1949. Metabolism and toxicity of cyanide and cyanogenic glucosides in sheep. 1. 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