Aust J. BioI. Sci., 1976, 29, 419-28
Fatty Liver and Kidney Syndrome in Chicks I. Effect of Biotin in Diet
Judith A. Pearson, A. R. Johnson, R. L. Hood and A. C. Fogerty Division of Food Research, CSIRO, P.O. Box 52, North Ryde, N.S.W. 2113.
Abstract
Fatty liver and kidney syndrome, a disorder of young chicks, was studied under laboratory conditions. Affected chicks had enlarged livers (hepatomegaly), an increased content of lipid in the liver, and an increased level of palmitoleic acid in the liver lipids. The disorder was observed mainly in chicks from young parent flocks, and was associated either with commercial diets which were subsequently found to be low in biotin, or with specially formulated low-biotin diets. A third factor, imposition of stress, was required to initiate the disorder. There was evidence of increased lipogenesis causing an increase of triacylglycerols in the liver lipids and an increased production of saturated fatty acids, particularly palmitic acid. Increased levels of palmitoleic acid resulted from an increased desaturation of palmitic acid. Under stress, affected chicks had low blood glucose levels, suggesting that gluconeogenesis was impaired. Since biotin-dependent enzymes are involved in both gluconeogenesis and lipogenesis, it would appear that the relevant enzymes respond differently to a deficiency of biotin.
Introduction The fatty liver and kidney syndrome (FLKS) of young chickens was first reported in Europe (Marthedal and VeIling 1958) and has since been observed in other countries. Some form of stress, such as excessive noise or low temperature, is usually involved, and when outbreaks of FLKS occur in commercial broiler chicken flocks, apparently healthy birds may die within a few hours of the imposition of the stress. The disorder affects chicks between 2 and 6 weeks of age. Post-mortem examination reveals pale, enlarged liver and kidneys and sometimes excess fluid around the heart. The carcass fat is often pink. The mortality in affected flocks is usually low (up to 6 %, Hemsley 1965), although recent outbreaks in Australia have caused losses as high as 30 %. Several groups of workers (Marthedal and VeIling 1958; Blair et al. 1969; Husbands and Laursen-Jones 1969; Riddell et al. 1971; Johnson et al. 1972; Blair and Whitehead 1974; Whitehead et al. 1974; Blair et al. 1975; Whitehead et al. 1975) have shown that diet is mainly responsible for FLKS in chicks. In a recent collaborative study (Payne et al. 1974) we have established that a marginal deficiency of dietary biotin is the causal factor of FLKS, and that the disorder can be eliminated from commercial flocks by increasing the level of biotin in the diet. Since much of the work reported in the present paper was carried out prior to this finding, the results have been re-examined with the benefit of hindsight. During the course of our study, workers in Edinburgh (Blair and Whitehead 1974; Whitehead et al. 1974; Whitehead et al. 1975) also concluded that FLKS was connected with dietary biotin. We now know that the three main factors involved in FLKS are the biotin level of the diet, the age of the parent flock (Hemsley and Marshall 1973) which influences the biotin status of the chicks at the time of hatching, and stress (Johnson et al. 1972; Whitehead et al. 1975).
J. A. Pearson, A. R. Johnson, R. L. Hood and A. C. Fogerty
420
Materials and Methods Results of several experiments are reported in this paper, and similar husbandry procedures were used for each experiment. Groups of 1-day-old, sexed, broiler chicks were wing-banded and placed in commercial-type chick brooders. Towards the end of this study chicks from young parent flocks were used as it had been shown that these chicks were more susceptible to FLKS (Hemsley and Marshall 1973). Each group was fed ad libitum on the test diets. At weekly intervals the chicks were weighed, and when they were between 1 and 5 weeks old equal numbers from each group were held overnight with access to water but not to food. In some experiments non-fasted chicks were used. After measurement of its body weight each chick was killed by exsanguination, dissected, and the liver, kidneys and heart were examined for symptoms of FLKS. The liver was weighed and its condition ('fatty', 'enlarged', or 'normal') noted. The lipid content of the liver was determined by extraction (Bligh and Dyer 1959) and the lipid was analysed by conventional methods. The fatty acid composition was determined by gas-liquid chromatography of the freshly prepared methyl esters (Hood et al. 1972). In one study the content and fatty acid composition of liver phospholipids and triacylglycerols were determined by a method involving separation by thin-layer chromatography, addition of methyl pentadecanoate as an internal standard, and gas-liquid chromatography (Christie et at. 1970). Table 1. Percentage composition of commercial diets used in experiments Ingredient 1 Wbeat Sorghum Meat meal, 50 % protein Blood meal Feather meal Poultry meat meal Fish meal Soya bean meal Rapeseed meal Sunflower meal
Diet 2A 3
45·0 25·5 7·5 3·0
+
1·5 7·5 4·0
+ +
+ +
Ingredient
Diet 2A 3
4
13·5 62·7 50·0 8·0 7·0 10·5 4·0 3·2 1·5 1·5 2·0 2·8 2·0 5·0 2·5
ASupplier did not provide percentage composition.
Cottonseed meal Oats Pollard Yeast Milk powder Tallow Premix Coccidiostat Biotin level (J-lg/kg)
4
+ +
2·5 5·5 6·7 7·0 1·0
+
+ + +
0·5
3·25 149
111
278 112
1·0 0·75 1·0
+ +
0·6
+
+, Component was present.
Lipogenesis studies with [1- 14C]acetate (Radiochemical Centre, Amersham, England) were carried out using in vitro liver-slice incubation techniques (Hood et at. 1972). Incorporation of radioactivity into individual fatty acids was determined by gas radiochromatography using a Packard Model 894 Gas Proportional Counter. Measurements of the activities of liver desaturase enzymes were made on liver homogenates (Johnson et at. 1967). All incubation experiments using liver tissue were performed at least in duplicate. Biotin assays were initially undertaken by a commercial organization using microbiological techniques, but later biotin determinations were carried out using an isotope dilution assay employing avidin to bind the biotin (Hood 1975). Blood serum samples were analysed for glucose, cholesterol, bilirubin, creatinine, protein, and albumin content by conventional clinical techniques (single channel Auto Analyzer I, Technicon methods). In the initial experiments commercial diets were used; diets known to have produced FLKS in the field were compared with 'control' diets which had not produced FLKS. Biotin assays were later performed on these diets, the compositions of which are given in Table 1. In later experiments special low-biotin diets were prepared containing wheat 78· 1 %, casein 8 %, meat-meal 10 %, dicalcium phosphate 2 %, DL-methionine 0·1 %, and L-lysine 0·3 %, together with a supplement of 1· 5 % containing trace nutrients and vitamins (Payne et at. 1974). The equivalent control diets were identical except that additional biotin was added. In the tables data are expressed as means ± standard error of the means.
Fatty Liver and Kidney Syndrome in Chicks. I
421
Results and Discussion
FLKS involves the sudden death of an apparently healthy chick at about 3-5 weeks of age, with post-mortem findings of pale kidneys and extensive fatty infiltration of the liver. However, these are the end results of the syndrome and there must be a sequence of events culminating in the death of the chick. When commercial diets which had been implicated in field outbreaks of FLKS were used under laboratory conditions, the percentage of chicks actually dying from the disorder was small. In order to study the development of the disorder, it was necessary to devise a method for identifying FLKS in its early stages. Although there was no significant difference in body weight between normal chicks and those with FLKS at the same age, the latter had larger livers (both on a dry weight basis and on a total protein basis), i.e. they exhibited hepatomegaly. In order to compensate for the usual bird to bird variation in body weight, the ratio ofliver weight to body weight was used to normalize such variations, and a ratio which was higher than the mean for the control chicks by at least two standard deviations was used as an index for the presence of FLKS. Using this criterion it was possible to identify chicks in the early stages of FLKS, before the more obvious abnormalities, such as excess lipid in the liver and death of the chicks, were manifested. The results shown in Table 2 were obtained using chicks selected for their possible susceptibility to FLKS, i.e. from a young parent flock (Hemsley and Marshall 1973). The specially formulated low-biotin diet and the identical diet to which extra biotin had been added (see Materials and Methods) were used. FLKS was detected in the chicks on the low-biotin diet, killed at 14 days of age, and the incidence of FLKS was similar in chicks killed and examined in subsequent weeks. About 65 % of the 100 chicks on this diet developed FLKS, whereas only 3 % of the 100 chicks on the same diet supplemented with biotin showed FLKS symptoms. Equal numbers of male and female chicks were used in the experiment. Contingency analysis showed that the females were more affected than the males (35 females compared to 29 males, with a confidence level of 95%), confirming the observations of Blair et al. (1975) and Whitehead et al. (1975). The results for the two sexes are combined in Table 2. There was no significant effect of the two diets on the growth rate of the chicks. The liver weight/body weight ratio was higher in chicks at 14 days of age than at 20, 28 or 35 days, but as the ratios, and other parameters measured, remained similar during the 20-35-day period, the results for chicks within that period were pooled. In Table 2 the content of individual fatty acids in the liver lipids is expressed as milligrams in the liver per 100 g body weight because chicks were killed at weekly intervals and this method of expression was used to allow comparisons to be made between chicks of different sizes. In chicks that did not have FLKS the levels of individual liver fatty acids expressed on this basis were very similar. The chicks which died from FLKS were characterized by hepatomegaly, a higher level of lipid in the liver, and changes in the composition of the liver fatty acids. In those chicks which had not reached the terminal stage of FLKS there was no significant increase in the lipid concentration of the liver, despite an increase in the liver weight and changes in the content of some of the liver fatty acids. The greatest proportional change in the fatty acids of the livers of chicks with FLKS was in the level of palmitoleic acid (16 : I) which showed a twofold increase in affected chicks
20-35
14
+
B
+ Died B
+
21 11 24 2
10 4 6
2-99±0-08 4-68±0-22 5-92±0-20 6-25+0-07
3-58±0-08 4-39±0-30 5-00±0-28 4-1±0-2 4-4±0-2 6-3±0-3 7-3+0-7
4-7±0-4 4-9±0-2 6-1±0-7
4-1±0-1 4-9±0-9
4-7±0-2
3-19±0-10
2-77±0-04 4-26±0-24
%
Liver lipid
Liver wt x 100 Body wt
29-1±2-1 43-2±2-5 80-0±5-0 94-5+12-5
38-9±2-8 46-8±4-8 66-3±8-4
26-2±0-7 44-7±10-9
34-6±2-7
16: 0
3-5±0-5 7-4±0-7 68-5±5-8 85-5±6-5
6-6±0-8 10-5±2-0 40-5±9-7
2-1±0-1 5-7±2-2
4-2±0-5
16: 1
23-6±1-7 30-8±2-8 8-7±0-6 8-0± 1-0
27-0±2-4 33-3±2-7 17-2±2-4
22-0±0-6 33-3±4-3
31-0±1-9
18: 0
27-6±3-0 49-3±3-4 143-0±8-3 172- 5 ± 33 -5
38-9±2-8 59-3±7-8 117-2±19-0
22-7±1-0 58-3±21-2
33-9±3-0
18: 1
18-8±1-3 29-8±2- 3 24-8±1-3 31-5±2-5
24-7±2-4 26-3±1-1 28-3 ±4-2
17-0±0-5 26-0±0-6
23-7±1-9
18: 2
Amount of each major fatty acid in the liver (mg/l00 g body weight)
12-6±1-0 17-7±1-8 4-4±0-5 5-5+1-5
18-0±3-0 20-0±3-1 8-0±2-0
12-2±0-4 19-7±0-9
21-1±1-4
20: 4
affected chicks by at least two standard deviations_ B All chicks that died had typical symptoms of FLKS_ Ten chicks that died on this diet were not examined_ c Died without overnight fast.
+ Died + Died c A -, Absence of FLKS; +, FLKS was probably present_ The liver weight/body weight ratios in affected chicks were greater than the mean value for the non-
124
69 3
20-35
+
18
14
249
No_ of chicks
Age of chicks (days)
Biotin in diet (p,g/kg)
FLKS A
Table 2. FLKS and dietary biotin in fasted chicks The basic diet was formulated on wheat, casein and meat meal and used with or without additional biotin (see text). The chicks were killed after an overnight fast, at weekly intervals, from 2 to 6 weeks of age
q
~
o 61
i"-
P P-
~
P-
o o
:I:
r
?O
?
~ o
?O Ci' ::r
i"-
.8
i
~
i"-
~
423
Fatty Liver and Kidney Syndrome in Chicks. I
and a tenfold increase in chicks which died from FLKS after fasting. A similar but smaller effect occurred in the level of palmitic acid (16 : 0) and oleic acid (18: 1). There were no consistent changes in the level of linoleic acid (18 : 2) in the livers of affected chicks. This is to be expected because the level of linoleic acid is largely determined by dietary intake, which in this experiment was the same for both diets. Table 3. Effect of FLKS on the lipid composition of livers and kidneys offasted 14- to 35-day old chicks Biotin in diet (J.lgjkg)
FLKSc
149A
6
111B
+
+ Died 149A 111B
+
+ Died A
No. of chicks
Diet 1 of Table 1.
2 13 4
Ratio of triacylglycerols to phospholipids (a) In liver lipid 0·lHO·01
0'29±0'07 0'55±0'16 4·32±0·31
2
(b) In kidney lipid 0'52±0'08
3 1 2
0·60±0·10 1·0 3·88± 1·27
B Diet 2 of Table 1.
C
Percentage of 16 : 1 in the fatty acids of Triacylglycerols
Phospholipids
5·0±0·5
1'9±0'2
3'7±0'1 9·0±0·5 17·0±0·9
1'9±0'3 3·8±0·4 9·7±0·8
6'5±0'5
l'HO'l
6·2±0·5 8·3 16·3±1·7
2·1±0·5 3·5 5·0±0·9
See footnote A, Table 2.
The results in Table 3 were obtained from birds fed two commercial diets (1 and 2 of Table 1), one of which (diet 2) had caused field outbreaks of FLKS. Out of a total of 200 chicks on diet 2, 19 had symptoms of FLKS. None of the 200 chicks on diet 1, which had a higher biotin content, developed FLKS. It is not possible to predict an absolute level for biotin content below which the diet is deficient and liable to produce FLKS in chicks; for example, in one experiment (Table 3) a diet containing 111 Jig of biotin/kg induced FLKS in only 9· 5 % of the chicks, whereas in another (Table 2) a diet containing more biotin (124 Jig/kg) induced FLKS in 65% of the chicks. Other factors are clearly involved; these could include the availability of the biotin in the diet, the biotin status of the chick at hatching, and stress. The chicks with FLKS in Table 3 showed the same changes as those in Table 2. The increase in the lipid content of the livers of chicks with terminal FLKS can largely be accounted for by an increase in the content of triacylglycerols. However, the characteristic increase in the level of 16 : 1 occurred both in the triacylglycerols and the phospholiphids. Similar changes were observed in the kidneys, although only a few chicks were examined in detail. There was a high content of lipid in the liver of I-day-old chicks (c. 20%) which decreased to a normal value (c. 5%) in 7 days. The possibility did exist that the high lipid content of the livers in chicks with FLKS was due to failure of the birds to mobilize this lipid. This was shown not to be the case, since the lipid class distribution and fatty acid composition of the liver lipids of I-day-old chicks were completely different to those in older birds. In I-day-old chicks sterol esters comprised 80 % of the liver lipids, and oleic acid accounted for 80 % of the fatty acids of the sterol esters. These values were quite unlike those of either the normal. chicks beyond
J. A. Pearson, A. R. Johnson, R. L. Hood and A. C. Fogerty
424
10 days of age or of chicks with FLKS, since in these birds sterol esters accounted for only a small proportion of the total liver lipids, and the fatty acid compositions of the lipid classes were quite different. That the source of the chicks used was a factor in the incidence of FLKS was verified in an experiment using two commercial diets (diets 3 and 4 of Table 1) and 200 I-day-old chicks from each of three parent flocks, A, Band C. The results are given in Table 4, which shows that the diet which was low in biotin and had produced FLKS in field outbreaks also produced FLKS under experimental conditions. Table 4. Biotin in diet
Parent flock
(,ig/kg)
278 A
A B C
112B
A B C
Effect of diet and parent flock on the incidence of FLKS in chicks
Age of chicks (days)
No. of chicks examined
Chicks affected with FLKS
14 20--42 14 20-42 14 20-42
11 27 8 29 12 33
0
14 20-42 14 20-42 14 20-42
10 37 18 75 10 45
0 4 13 44
1
2 0 1
7
100 x Liver wt/body wt
------------Affected
Not affected
3·90
2·51 ±0·05 2·06±0·05 2· 54±0·06 2'17±0'04 2·57±0·03 2'06±0'04
3·41±0·18 4·08±0·24 4· 25 ±0'17 4·05 4'12±0'17
2'65±0'05 2·21 ±0'04 2·70±0·11 2·40±0·06 2· 54±0'05 2·26±0·05
3·98 3·20±0·01 2·93
Mortality due to FLKSC(%) 0 0 0 0 5 3
B Diet 4 of Table 1. Diet 3 of Table I. CAfter overnight fast. Mortality in each case calculated for entire group (l00 chicks) for 42-day period.
A
However, the effects of the diet were dependent on the source of the chicks; for example, chicks from parent flock B had a far greater incidence of FLKS than those from parent flocks A or C. Presumably this was related to the level of biotin in the chicks at hatching, but these values were not ascertained because the causal factor was not known at that stage. However, when chicks from parent flock B were fed on the diet higher in biotin the incidence of FLKS was practically eliminated. Lipogenesis studies were undertaken on some of the chicks from the above experiment, using [1-14C]acetate as tracer (Table 5). Chicks with FLKS induced by the diet low in biotin showed many of the typical changes produced by the disorder, and these changes were exaggerated in one chick which was moribund prior to killing. FLKS had little effect on the in vitro oxygen uptake by the liver or on the conversion of acetate to CO 2 , Although there was a considerable reduction in lipogenesis in vitro due to fasting, livers from fasted chicks with FLKS showed a greater rate of lipid synthesis from acetate than livers from fasted chicks which did not have the disorder. This difference could be accounted for almost entirely by an increase in the synthesis of triacylglycerols. FLKS did not appear to affect the rate of synthesis of cholesterol or cholesterol esters. In non-fasted chicks with FLKS on the low-biotin diet, more lipid was also synthesized. Moreover, because of their enlarged livers this effect was multiplied in those chicks with FLKS. Despite the increased rate of synthesis of lipid in the
------------_. 425
Fatty Liver and Kidney Syndrome in Chicks. I
chicks with enlarged livers, there was at this stage in the development of FLKS no increase in the concentration of lipid in the liver. Presumably the chicks were still able to mobilize and utilize the lipid. However, the one moribund chick (Table 5) accumulated lipid in the liver (13, 3 %, w/w). Table 5. FLKS e
Biotin in diet (fig/kg)
Total lipid
Phospholipid
Triacylglycerol
(a) Fasted chicks 14·5±1·5
10'5±I'3
1·8±0·6
17·0±3·1 36'3± 1·0 28'9
I2·9±3·2 17·5±1·9 6·0
2·2±0·7 16·7±2·6 21·6
3
(b) Non-fasted chicks 163·2±22·7
36·7±3·3
120'5±19'0
4 5
117·6±22·6 18S·6±S·9
26'8±6'7 46'5±7'0
79·2± 17·9 132'7± S'O
8
112B
6 4
+
+ Moribund 278 A I.12B
+ 3 of Table 1.
Acetate converted to each lipid class [nmol h- 1 (g liver)-lj
No. of chicks
278 A
A Diet
Lipogenesis and FLKS in chicks
B Diet 4 of Table I.
e See footnote A, Table 2.
The effect of FLKS on the synthesis of individual fatty acids in the liver is shown in Table 6. In the fasted chicks the rate of synthesis of liver lipid was so reduced that it was possible to determine the incorporation of [1-14C]acetate into fatty acids of the livers in only two of the control chicks. Table 6. Biotin in diet (flg/kg)
Effect of FLKS on rate of synthesis of individual fatty acids of the triacylglycerols of liver
FLKS e
Acetate converted to triacylglycerol fatty acid [nmol h -1 (g liver) -1]
No. of chicks 12: 0
27SA 112B
+
2 6
27SA 112B
+
4
TrD 1·9±0·9
Tr Tr 1-4±0-7
14: 0
16 : I
IS : 0
IS : I
(a) Fasted chicks 0·6±0·3 Tr 1·2±0·4 12·0±4·5 Tr 31·0±12·4
0·1 0·S±0·4
0·1 1·3±0·9
Tr
(b) Non·fasted chicks 52·7±6·4 5·2±1·3 3·2±0·7 51-7±IS'0 10-4±3'2 6-0±2-7 3-6:1:1-0 2'9±0-S 40'4±6-1
20·S±2·7 19·3±S·9 35·6±7·9
3·7±1·5 6'S±2-9 7·1±I·S
35·5 ± 11- 5 21-7±S'5 46'2±2-0
B Diet 4 of Table l. A Diet 3 of Table l. D Trace, less than 0 - I %_
14 : I
16: 0
e See footnote A, Table 2_
The increased rate of synthesis of liver fatty acids in fasted chicks with FLKS relative to fasted control chicks can be explained by an increase in the rate of synthesis of the saturated fatty acids lauric (12: 0), myristic (14: 0) and palmitic (16: 0). The rate of desaturation of saturated fatty acids to mono-unsaturated fatty acids is reduced in fasted chicks, but there were indications from the tracer incorporation into fatty acids that in the fasted chicks there was an increased desaturation in those chicks with FLKS relative to those without FLKS. In non-fasted chicks on the
J. A. Pearson, A. R. Johnson, R. L. Hood and A. C. Fogerty
426
same level of dietary biotin there were also indications of a faster rate of production of 16: 1 and 18 : 1 by the chicks with FLKS, again suggesting increased desaturation. These indications were confirmed by measuring the desaturase activity of the livers of chicks on high-biotin and low-biotin diets (Table 7), which showed that there was an increased rate of desaturation in both fasted and non-fasted chicks with FLKS. This may be due to increased availability of saturated fatty acids in these chicks. The rate of desaturation in fasted chicks was lower than that of non-fasted chicks. The addition of extra biotin to some of the in vitro incubation mixtures during the desaturase assay had no effect on the amount of desaturation. Table 7. Biotin in diet
Effect of FLKS on the desaturation of palmitic and stearic acids by chicken liver FLKS D
No. of chicks
% 16: 1 in liver lipid
Percentage desaturation Palmitate
Stearate
+
(a) Fasted chicks 6 2·2±O·2 8 2·7±O·7 5 6·8±2·2
2·6±O·2 2·l±O·3 5·3±O·9
3·3±O·4 3·l±O·4 8'6±1'5
+
(b) Non-fasted chicks 6 7·I±O·3 2 6·2±O·3 4 12·3±O·1
42·5±3·8 33·6±6·1 57·7±4·0
n.d. E n.d. n.d.
(l1g/k g)
278 A 112B
373 c 62 c
Diet 3 of Table 1. B Diet 4 of Table 1. c A basic wheat, meat meal and casein diet used with or without additional biotin (see text). ° See footnote A, Table 2. E Not determined.
A
Analyses of samples of blood serum from chicks with or without FLKS for cholesterol, bilirubin, creatinine, protein and albumin content gave no indication of liver malfunction. However, chicks with FLKS had low blood glucose levels, confirming the observations of Bannister et al. (1975). This suggests that these chicks had impaired gluconeogenesis, leading to hypoglycemia which may have been a factor in the death of the chicks. Despite reports (Balnave 1966; Roland and Edwards 1971; Edwards et al. 1973) that biotin deficiency in chicks results in elevated levels of palmitoleic acid in liver lipids, FLKS has not previously been associated with a deficiency of biotin, since the classical symptoms of biotin deficiency (dermatitis, perosis, and poor growth) have not been observed in field outbreaks of FLKS. Provided they are not subjected to stress, broiler chicks on a diet marginally deficient in biotin will grow to maturity at the same rate as chicks on a diet adequate in biotin. The involvement of two additional factors in FLKS, namely stress and low-biotin status of the chick at hatching, has made it difficult to ascertain the primary cause of this disorder. Any form of stress puts heavy demands on the gluconeogenesis pathway after liver glycogen reserves are depleted. Chicks on the biotin-deficient diet showed evidence of impaired gluconeogenesis and, consequently, of poor ability to withstand stress. The most likely cause for this is reduced activity of the biotin-dependent enzyme pyruvate carboxylase.
Fatty Liver and Kidney Syndrome in Chicks. I
427
Impaired gluconeogenesis would explain the different rates of lipogenesis in chicks with FLKS and in those without FLKS. In chicks with FLKS, lipogenesis is increased because impaired gluconeogenesis increases the availability of substrates, e.g. pyruvate, for fatty acid synthesis. A difficulty with such a suggestion is that biotin is also required as a co-factor in lipogenesis, namely in the carboxylation of acetyl-CoA. A possible explanation is that the biotin-dependent enzyme of the gluconeogenesis pathway is more markedly affected by marginal biotin deficiency than the biotin-dependent enzyme of the lipogenesis pathway. This aspect is explored in the following paper. Acknowledgments
This work was supported in part by a grant from the Australian Chicken Meat Research Committee. Dr C. G. Payne, University of Sydney, provided special diets, and Dr S. Nobile, The Vitamin Laboratories, Sydney, performed the microbiological biotin assays. Blood assays were performed by the Department of Clinical Biochemistry, Prince Henry Hospital. G. Ford, S. Kozuharov and C. Godfrey provided technical assistance. References Balnave, D. (1966). Effect of gonadal hormones and biotin deficiency on growth and lipid composition of liver and tissue in the immature female chick. Proc. XIII World Poultry Congo pp. 213-18. Bannister, D. W., Evans, A. J., and Whitehead, C. C. (1975). Evidence for a lesion in carbohydrate metabolism in fatty liver and kidney syndrome in chicks. Res. Vet. Sci. 18, 149-56. Blair, R., Bolton, W., and Duff, R. H. (1969). Fatty liver and kidney disease in broiler chickens receiving diets with varying contents of protein. Vet. Rec. 84, 41-3. Blair, R., and Whitehead, C. C. (1974). An assessment of the factors associated with fatty liver and kidney syndrome in broilers. Proc. XV. World Poultry Congo p.380. Blair, R., Whitehead, C. C., and Teague, P. W. (1975). The effect of dietary fat and protein levels, form and cereal type on fatty liver and kidney syndrome in chicks. Res. Vet. Sci. 18, 76-81. Bligh, E. G., and Dyer, W. J. (1959). A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol. 37,911-17. Christie, W. W., Noble, R. c., and Moore, J. H. (1970). Determination of lipid classes by a gaschromatographic procedure. Analyst (London) 95, 940-4. Edwards, H. M., Denman, F., and Jackson, R. (1973). Severity of biotin deficiency and fatty acid composition of tissues. Poult. Sci. 52, 2024. Hemsley, L. A. (1965). The 'Fatty liver and kidney syndrome' of young chickens. Vet. Rec. 77, 124-6. Hemsley, L. A., and Marshall, L. G. (1973). The influence of parent stock on the susceptibility of chicks to the fatty liver and kidney syndrome. Br. Vet. J. 129, Ixii-Ixiv. Hood, R. L. (1975). A radiochemical assay for biotin in biological materials. J. Sci. Food. Agric. 26, 1847-52. Hood, R. L., Thompson, E. H., and Allen, C. E. (1972). The role of acetate, proprionate and glucose as substrates for lipogenesis in bovine tissues. Int. J. Biochem. 3, 598-606. Husbands, D. R., and Laursen-Jones, A. P. (1969). Fatty liver and kidney syndrome. Vet. Rec. 84, 232-3. Johnson, A. R., Pearson, J. A., Shenstone, F. S., and Fogerty, A. C. (1967). Inhibition of the desaturation of stearic to oleic acid by cyclopropene fatty acids. Nature (London) 214, 1244-5. Johnson, A. R. et al. (1972). Fatty liver and kidney disease in young chickens. Proc. Australas. Poult. Sci. Conv. p. 15-16. Marthedal, H. E., and Velling, G. (1958). Liver and kidney disease in chickens. Proc. VIn Nordiske Veterinormetet. p. 250. Payne, C. G., Gilchrist, P., Pearson, J. A., and Hemsley, L. A. (1974). Involvement of biotin in the fatty liver and kidney syndrome of broilers. Br. Poult. Sci. 15, 489-98.
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J. A. Pearson, A. R. Johnson, R. L. Hood and A. C. Fogerty
Riddell, C., Olsen, G. V., and Grimson, R. E. (1971). Fatty liver and kidney syndrome in a broiler flock. Avian Dis. 15, 398-405. Roland, D. A., and Edwards, H. M. (1971). Effect of essential fatty acid deficiency and type of dietary fat supplementation on biotin-deficient chicks. J. Nutr. 101, 811-8. Whitehead, C. C.; Bannister, D. W., Wight, P. A. L., and Weiser, H. (1974). Studies on biotin requirements and deficiency in chicks. Proc. XV World Poultry Congo p.70-2. Whitehead, C. C., Blair, R., Bannister, D. W., and Evans, A. J. (1975). The involvement of dietary fat and vitamins, stress, litter and starvation on the incidence of the fatty liver and kidney syndrome in chicks. Res. Vet. Sci. 18, 100-4.
Manuscript received 9 February 1976
Fatty Liver and Kidney Syndrome in Chicks I. Effect of Biotin in Diet
Judith A. Pearson, A. R. Johnson, R. L. Hood and A. C. Fogerty Division of Food Research, CSIRO, P.O. Box 52, North Ryde, N.S.W. 2113.
Abstract
Fatty liver and kidney syndrome, a disorder of young chicks, was studied under laboratory conditions. Affected chicks had enlarged livers (hepatomegaly), an increased content of lipid in the liver, and an increased level of palmitoleic acid in the liver lipids. The disorder was observed mainly in chicks from young parent flocks, and was associated either with commercial diets which were subsequently found to be low in biotin, or with specially formulated low-biotin diets. A third factor, imposition of stress, was required to initiate the disorder. There was evidence of increased lipogenesis causing an increase of triacylglycerols in the liver lipids and an increased production of saturated fatty acids, particularly palmitic acid. Increased levels of palmitoleic acid resulted from an increased desaturation of palmitic acid. Under stress, affected chicks had low blood glucose levels, suggesting that gluconeogenesis was impaired. Since biotin-dependent enzymes are involved in both gluconeogenesis and lipogenesis, it would appear that the relevant enzymes respond differently to a deficiency of biotin.
Introduction The fatty liver and kidney syndrome (FLKS) of young chickens was first reported in Europe (Marthedal and VeIling 1958) and has since been observed in other countries. Some form of stress, such as excessive noise or low temperature, is usually involved, and when outbreaks of FLKS occur in commercial broiler chicken flocks, apparently healthy birds may die within a few hours of the imposition of the stress. The disorder affects chicks between 2 and 6 weeks of age. Post-mortem examination reveals pale, enlarged liver and kidneys and sometimes excess fluid around the heart. The carcass fat is often pink. The mortality in affected flocks is usually low (up to 6 %, Hemsley 1965), although recent outbreaks in Australia have caused losses as high as 30 %. Several groups of workers (Marthedal and VeIling 1958; Blair et al. 1969; Husbands and Laursen-Jones 1969; Riddell et al. 1971; Johnson et al. 1972; Blair and Whitehead 1974; Whitehead et al. 1974; Blair et al. 1975; Whitehead et al. 1975) have shown that diet is mainly responsible for FLKS in chicks. In a recent collaborative study (Payne et al. 1974) we have established that a marginal deficiency of dietary biotin is the causal factor of FLKS, and that the disorder can be eliminated from commercial flocks by increasing the level of biotin in the diet. Since much of the work reported in the present paper was carried out prior to this finding, the results have been re-examined with the benefit of hindsight. During the course of our study, workers in Edinburgh (Blair and Whitehead 1974; Whitehead et al. 1974; Whitehead et al. 1975) also concluded that FLKS was connected with dietary biotin. We now know that the three main factors involved in FLKS are the biotin level of the diet, the age of the parent flock (Hemsley and Marshall 1973) which influences the biotin status of the chicks at the time of hatching, and stress (Johnson et al. 1972; Whitehead et al. 1975).
J. A. Pearson, A. R. Johnson, R. L. Hood and A. C. Fogerty
420
Materials and Methods Results of several experiments are reported in this paper, and similar husbandry procedures were used for each experiment. Groups of 1-day-old, sexed, broiler chicks were wing-banded and placed in commercial-type chick brooders. Towards the end of this study chicks from young parent flocks were used as it had been shown that these chicks were more susceptible to FLKS (Hemsley and Marshall 1973). Each group was fed ad libitum on the test diets. At weekly intervals the chicks were weighed, and when they were between 1 and 5 weeks old equal numbers from each group were held overnight with access to water but not to food. In some experiments non-fasted chicks were used. After measurement of its body weight each chick was killed by exsanguination, dissected, and the liver, kidneys and heart were examined for symptoms of FLKS. The liver was weighed and its condition ('fatty', 'enlarged', or 'normal') noted. The lipid content of the liver was determined by extraction (Bligh and Dyer 1959) and the lipid was analysed by conventional methods. The fatty acid composition was determined by gas-liquid chromatography of the freshly prepared methyl esters (Hood et al. 1972). In one study the content and fatty acid composition of liver phospholipids and triacylglycerols were determined by a method involving separation by thin-layer chromatography, addition of methyl pentadecanoate as an internal standard, and gas-liquid chromatography (Christie et at. 1970). Table 1. Percentage composition of commercial diets used in experiments Ingredient 1 Wbeat Sorghum Meat meal, 50 % protein Blood meal Feather meal Poultry meat meal Fish meal Soya bean meal Rapeseed meal Sunflower meal
Diet 2A 3
45·0 25·5 7·5 3·0
+
1·5 7·5 4·0
+ +
+ +
Ingredient
Diet 2A 3
4
13·5 62·7 50·0 8·0 7·0 10·5 4·0 3·2 1·5 1·5 2·0 2·8 2·0 5·0 2·5
ASupplier did not provide percentage composition.
Cottonseed meal Oats Pollard Yeast Milk powder Tallow Premix Coccidiostat Biotin level (J-lg/kg)
4
+ +
2·5 5·5 6·7 7·0 1·0
+
+ + +
0·5
3·25 149
111
278 112
1·0 0·75 1·0
+ +
0·6
+
+, Component was present.
Lipogenesis studies with [1- 14C]acetate (Radiochemical Centre, Amersham, England) were carried out using in vitro liver-slice incubation techniques (Hood et at. 1972). Incorporation of radioactivity into individual fatty acids was determined by gas radiochromatography using a Packard Model 894 Gas Proportional Counter. Measurements of the activities of liver desaturase enzymes were made on liver homogenates (Johnson et at. 1967). All incubation experiments using liver tissue were performed at least in duplicate. Biotin assays were initially undertaken by a commercial organization using microbiological techniques, but later biotin determinations were carried out using an isotope dilution assay employing avidin to bind the biotin (Hood 1975). Blood serum samples were analysed for glucose, cholesterol, bilirubin, creatinine, protein, and albumin content by conventional clinical techniques (single channel Auto Analyzer I, Technicon methods). In the initial experiments commercial diets were used; diets known to have produced FLKS in the field were compared with 'control' diets which had not produced FLKS. Biotin assays were later performed on these diets, the compositions of which are given in Table 1. In later experiments special low-biotin diets were prepared containing wheat 78· 1 %, casein 8 %, meat-meal 10 %, dicalcium phosphate 2 %, DL-methionine 0·1 %, and L-lysine 0·3 %, together with a supplement of 1· 5 % containing trace nutrients and vitamins (Payne et at. 1974). The equivalent control diets were identical except that additional biotin was added. In the tables data are expressed as means ± standard error of the means.
Fatty Liver and Kidney Syndrome in Chicks. I
421
Results and Discussion
FLKS involves the sudden death of an apparently healthy chick at about 3-5 weeks of age, with post-mortem findings of pale kidneys and extensive fatty infiltration of the liver. However, these are the end results of the syndrome and there must be a sequence of events culminating in the death of the chick. When commercial diets which had been implicated in field outbreaks of FLKS were used under laboratory conditions, the percentage of chicks actually dying from the disorder was small. In order to study the development of the disorder, it was necessary to devise a method for identifying FLKS in its early stages. Although there was no significant difference in body weight between normal chicks and those with FLKS at the same age, the latter had larger livers (both on a dry weight basis and on a total protein basis), i.e. they exhibited hepatomegaly. In order to compensate for the usual bird to bird variation in body weight, the ratio ofliver weight to body weight was used to normalize such variations, and a ratio which was higher than the mean for the control chicks by at least two standard deviations was used as an index for the presence of FLKS. Using this criterion it was possible to identify chicks in the early stages of FLKS, before the more obvious abnormalities, such as excess lipid in the liver and death of the chicks, were manifested. The results shown in Table 2 were obtained using chicks selected for their possible susceptibility to FLKS, i.e. from a young parent flock (Hemsley and Marshall 1973). The specially formulated low-biotin diet and the identical diet to which extra biotin had been added (see Materials and Methods) were used. FLKS was detected in the chicks on the low-biotin diet, killed at 14 days of age, and the incidence of FLKS was similar in chicks killed and examined in subsequent weeks. About 65 % of the 100 chicks on this diet developed FLKS, whereas only 3 % of the 100 chicks on the same diet supplemented with biotin showed FLKS symptoms. Equal numbers of male and female chicks were used in the experiment. Contingency analysis showed that the females were more affected than the males (35 females compared to 29 males, with a confidence level of 95%), confirming the observations of Blair et al. (1975) and Whitehead et al. (1975). The results for the two sexes are combined in Table 2. There was no significant effect of the two diets on the growth rate of the chicks. The liver weight/body weight ratio was higher in chicks at 14 days of age than at 20, 28 or 35 days, but as the ratios, and other parameters measured, remained similar during the 20-35-day period, the results for chicks within that period were pooled. In Table 2 the content of individual fatty acids in the liver lipids is expressed as milligrams in the liver per 100 g body weight because chicks were killed at weekly intervals and this method of expression was used to allow comparisons to be made between chicks of different sizes. In chicks that did not have FLKS the levels of individual liver fatty acids expressed on this basis were very similar. The chicks which died from FLKS were characterized by hepatomegaly, a higher level of lipid in the liver, and changes in the composition of the liver fatty acids. In those chicks which had not reached the terminal stage of FLKS there was no significant increase in the lipid concentration of the liver, despite an increase in the liver weight and changes in the content of some of the liver fatty acids. The greatest proportional change in the fatty acids of the livers of chicks with FLKS was in the level of palmitoleic acid (16 : I) which showed a twofold increase in affected chicks
20-35
14
+
B
+ Died B
+
21 11 24 2
10 4 6
2-99±0-08 4-68±0-22 5-92±0-20 6-25+0-07
3-58±0-08 4-39±0-30 5-00±0-28 4-1±0-2 4-4±0-2 6-3±0-3 7-3+0-7
4-7±0-4 4-9±0-2 6-1±0-7
4-1±0-1 4-9±0-9
4-7±0-2
3-19±0-10
2-77±0-04 4-26±0-24
%
Liver lipid
Liver wt x 100 Body wt
29-1±2-1 43-2±2-5 80-0±5-0 94-5+12-5
38-9±2-8 46-8±4-8 66-3±8-4
26-2±0-7 44-7±10-9
34-6±2-7
16: 0
3-5±0-5 7-4±0-7 68-5±5-8 85-5±6-5
6-6±0-8 10-5±2-0 40-5±9-7
2-1±0-1 5-7±2-2
4-2±0-5
16: 1
23-6±1-7 30-8±2-8 8-7±0-6 8-0± 1-0
27-0±2-4 33-3±2-7 17-2±2-4
22-0±0-6 33-3±4-3
31-0±1-9
18: 0
27-6±3-0 49-3±3-4 143-0±8-3 172- 5 ± 33 -5
38-9±2-8 59-3±7-8 117-2±19-0
22-7±1-0 58-3±21-2
33-9±3-0
18: 1
18-8±1-3 29-8±2- 3 24-8±1-3 31-5±2-5
24-7±2-4 26-3±1-1 28-3 ±4-2
17-0±0-5 26-0±0-6
23-7±1-9
18: 2
Amount of each major fatty acid in the liver (mg/l00 g body weight)
12-6±1-0 17-7±1-8 4-4±0-5 5-5+1-5
18-0±3-0 20-0±3-1 8-0±2-0
12-2±0-4 19-7±0-9
21-1±1-4
20: 4
affected chicks by at least two standard deviations_ B All chicks that died had typical symptoms of FLKS_ Ten chicks that died on this diet were not examined_ c Died without overnight fast.
+ Died + Died c A -, Absence of FLKS; +, FLKS was probably present_ The liver weight/body weight ratios in affected chicks were greater than the mean value for the non-
124
69 3
20-35
+
18
14
249
No_ of chicks
Age of chicks (days)
Biotin in diet (p,g/kg)
FLKS A
Table 2. FLKS and dietary biotin in fasted chicks The basic diet was formulated on wheat, casein and meat meal and used with or without additional biotin (see text). The chicks were killed after an overnight fast, at weekly intervals, from 2 to 6 weeks of age
q
~
o 61
i"-
P P-
~
P-
o o
:I:
r
?O
?
~ o
?O Ci' ::r
i"-
.8
i
~
i"-
~
423
Fatty Liver and Kidney Syndrome in Chicks. I
and a tenfold increase in chicks which died from FLKS after fasting. A similar but smaller effect occurred in the level of palmitic acid (16 : 0) and oleic acid (18: 1). There were no consistent changes in the level of linoleic acid (18 : 2) in the livers of affected chicks. This is to be expected because the level of linoleic acid is largely determined by dietary intake, which in this experiment was the same for both diets. Table 3. Effect of FLKS on the lipid composition of livers and kidneys offasted 14- to 35-day old chicks Biotin in diet (J.lgjkg)
FLKSc
149A
6
111B
+
+ Died 149A 111B
+
+ Died A
No. of chicks
Diet 1 of Table 1.
2 13 4
Ratio of triacylglycerols to phospholipids (a) In liver lipid 0·lHO·01
0'29±0'07 0'55±0'16 4·32±0·31
2
(b) In kidney lipid 0'52±0'08
3 1 2
0·60±0·10 1·0 3·88± 1·27
B Diet 2 of Table 1.
C
Percentage of 16 : 1 in the fatty acids of Triacylglycerols
Phospholipids
5·0±0·5
1'9±0'2
3'7±0'1 9·0±0·5 17·0±0·9
1'9±0'3 3·8±0·4 9·7±0·8
6'5±0'5
l'HO'l
6·2±0·5 8·3 16·3±1·7
2·1±0·5 3·5 5·0±0·9
See footnote A, Table 2.
The results in Table 3 were obtained from birds fed two commercial diets (1 and 2 of Table 1), one of which (diet 2) had caused field outbreaks of FLKS. Out of a total of 200 chicks on diet 2, 19 had symptoms of FLKS. None of the 200 chicks on diet 1, which had a higher biotin content, developed FLKS. It is not possible to predict an absolute level for biotin content below which the diet is deficient and liable to produce FLKS in chicks; for example, in one experiment (Table 3) a diet containing 111 Jig of biotin/kg induced FLKS in only 9· 5 % of the chicks, whereas in another (Table 2) a diet containing more biotin (124 Jig/kg) induced FLKS in 65% of the chicks. Other factors are clearly involved; these could include the availability of the biotin in the diet, the biotin status of the chick at hatching, and stress. The chicks with FLKS in Table 3 showed the same changes as those in Table 2. The increase in the lipid content of the livers of chicks with terminal FLKS can largely be accounted for by an increase in the content of triacylglycerols. However, the characteristic increase in the level of 16 : 1 occurred both in the triacylglycerols and the phospholiphids. Similar changes were observed in the kidneys, although only a few chicks were examined in detail. There was a high content of lipid in the liver of I-day-old chicks (c. 20%) which decreased to a normal value (c. 5%) in 7 days. The possibility did exist that the high lipid content of the livers in chicks with FLKS was due to failure of the birds to mobilize this lipid. This was shown not to be the case, since the lipid class distribution and fatty acid composition of the liver lipids of I-day-old chicks were completely different to those in older birds. In I-day-old chicks sterol esters comprised 80 % of the liver lipids, and oleic acid accounted for 80 % of the fatty acids of the sterol esters. These values were quite unlike those of either the normal. chicks beyond
J. A. Pearson, A. R. Johnson, R. L. Hood and A. C. Fogerty
424
10 days of age or of chicks with FLKS, since in these birds sterol esters accounted for only a small proportion of the total liver lipids, and the fatty acid compositions of the lipid classes were quite different. That the source of the chicks used was a factor in the incidence of FLKS was verified in an experiment using two commercial diets (diets 3 and 4 of Table 1) and 200 I-day-old chicks from each of three parent flocks, A, Band C. The results are given in Table 4, which shows that the diet which was low in biotin and had produced FLKS in field outbreaks also produced FLKS under experimental conditions. Table 4. Biotin in diet
Parent flock
(,ig/kg)
278 A
A B C
112B
A B C
Effect of diet and parent flock on the incidence of FLKS in chicks
Age of chicks (days)
No. of chicks examined
Chicks affected with FLKS
14 20--42 14 20-42 14 20-42
11 27 8 29 12 33
0
14 20-42 14 20-42 14 20-42
10 37 18 75 10 45
0 4 13 44
1
2 0 1
7
100 x Liver wt/body wt
------------Affected
Not affected
3·90
2·51 ±0·05 2·06±0·05 2· 54±0·06 2'17±0'04 2·57±0·03 2'06±0'04
3·41±0·18 4·08±0·24 4· 25 ±0'17 4·05 4'12±0'17
2'65±0'05 2·21 ±0'04 2·70±0·11 2·40±0·06 2· 54±0'05 2·26±0·05
3·98 3·20±0·01 2·93
Mortality due to FLKSC(%) 0 0 0 0 5 3
B Diet 4 of Table 1. Diet 3 of Table I. CAfter overnight fast. Mortality in each case calculated for entire group (l00 chicks) for 42-day period.
A
However, the effects of the diet were dependent on the source of the chicks; for example, chicks from parent flock B had a far greater incidence of FLKS than those from parent flocks A or C. Presumably this was related to the level of biotin in the chicks at hatching, but these values were not ascertained because the causal factor was not known at that stage. However, when chicks from parent flock B were fed on the diet higher in biotin the incidence of FLKS was practically eliminated. Lipogenesis studies were undertaken on some of the chicks from the above experiment, using [1-14C]acetate as tracer (Table 5). Chicks with FLKS induced by the diet low in biotin showed many of the typical changes produced by the disorder, and these changes were exaggerated in one chick which was moribund prior to killing. FLKS had little effect on the in vitro oxygen uptake by the liver or on the conversion of acetate to CO 2 , Although there was a considerable reduction in lipogenesis in vitro due to fasting, livers from fasted chicks with FLKS showed a greater rate of lipid synthesis from acetate than livers from fasted chicks which did not have the disorder. This difference could be accounted for almost entirely by an increase in the synthesis of triacylglycerols. FLKS did not appear to affect the rate of synthesis of cholesterol or cholesterol esters. In non-fasted chicks with FLKS on the low-biotin diet, more lipid was also synthesized. Moreover, because of their enlarged livers this effect was multiplied in those chicks with FLKS. Despite the increased rate of synthesis of lipid in the
------------_. 425
Fatty Liver and Kidney Syndrome in Chicks. I
chicks with enlarged livers, there was at this stage in the development of FLKS no increase in the concentration of lipid in the liver. Presumably the chicks were still able to mobilize and utilize the lipid. However, the one moribund chick (Table 5) accumulated lipid in the liver (13, 3 %, w/w). Table 5. FLKS e
Biotin in diet (fig/kg)
Total lipid
Phospholipid
Triacylglycerol
(a) Fasted chicks 14·5±1·5
10'5±I'3
1·8±0·6
17·0±3·1 36'3± 1·0 28'9
I2·9±3·2 17·5±1·9 6·0
2·2±0·7 16·7±2·6 21·6
3
(b) Non-fasted chicks 163·2±22·7
36·7±3·3
120'5±19'0
4 5
117·6±22·6 18S·6±S·9
26'8±6'7 46'5±7'0
79·2± 17·9 132'7± S'O
8
112B
6 4
+
+ Moribund 278 A I.12B
+ 3 of Table 1.
Acetate converted to each lipid class [nmol h- 1 (g liver)-lj
No. of chicks
278 A
A Diet
Lipogenesis and FLKS in chicks
B Diet 4 of Table I.
e See footnote A, Table 2.
The effect of FLKS on the synthesis of individual fatty acids in the liver is shown in Table 6. In the fasted chicks the rate of synthesis of liver lipid was so reduced that it was possible to determine the incorporation of [1-14C]acetate into fatty acids of the livers in only two of the control chicks. Table 6. Biotin in diet (flg/kg)
Effect of FLKS on rate of synthesis of individual fatty acids of the triacylglycerols of liver
FLKS e
Acetate converted to triacylglycerol fatty acid [nmol h -1 (g liver) -1]
No. of chicks 12: 0
27SA 112B
+
2 6
27SA 112B
+
4
TrD 1·9±0·9
Tr Tr 1-4±0-7
14: 0
16 : I
IS : 0
IS : I
(a) Fasted chicks 0·6±0·3 Tr 1·2±0·4 12·0±4·5 Tr 31·0±12·4
0·1 0·S±0·4
0·1 1·3±0·9
Tr
(b) Non·fasted chicks 52·7±6·4 5·2±1·3 3·2±0·7 51-7±IS'0 10-4±3'2 6-0±2-7 3-6:1:1-0 2'9±0-S 40'4±6-1
20·S±2·7 19·3±S·9 35·6±7·9
3·7±1·5 6'S±2-9 7·1±I·S
35·5 ± 11- 5 21-7±S'5 46'2±2-0
B Diet 4 of Table l. A Diet 3 of Table l. D Trace, less than 0 - I %_
14 : I
16: 0
e See footnote A, Table 2_
The increased rate of synthesis of liver fatty acids in fasted chicks with FLKS relative to fasted control chicks can be explained by an increase in the rate of synthesis of the saturated fatty acids lauric (12: 0), myristic (14: 0) and palmitic (16: 0). The rate of desaturation of saturated fatty acids to mono-unsaturated fatty acids is reduced in fasted chicks, but there were indications from the tracer incorporation into fatty acids that in the fasted chicks there was an increased desaturation in those chicks with FLKS relative to those without FLKS. In non-fasted chicks on the
J. A. Pearson, A. R. Johnson, R. L. Hood and A. C. Fogerty
426
same level of dietary biotin there were also indications of a faster rate of production of 16: 1 and 18 : 1 by the chicks with FLKS, again suggesting increased desaturation. These indications were confirmed by measuring the desaturase activity of the livers of chicks on high-biotin and low-biotin diets (Table 7), which showed that there was an increased rate of desaturation in both fasted and non-fasted chicks with FLKS. This may be due to increased availability of saturated fatty acids in these chicks. The rate of desaturation in fasted chicks was lower than that of non-fasted chicks. The addition of extra biotin to some of the in vitro incubation mixtures during the desaturase assay had no effect on the amount of desaturation. Table 7. Biotin in diet
Effect of FLKS on the desaturation of palmitic and stearic acids by chicken liver FLKS D
No. of chicks
% 16: 1 in liver lipid
Percentage desaturation Palmitate
Stearate
+
(a) Fasted chicks 6 2·2±O·2 8 2·7±O·7 5 6·8±2·2
2·6±O·2 2·l±O·3 5·3±O·9
3·3±O·4 3·l±O·4 8'6±1'5
+
(b) Non-fasted chicks 6 7·I±O·3 2 6·2±O·3 4 12·3±O·1
42·5±3·8 33·6±6·1 57·7±4·0
n.d. E n.d. n.d.
(l1g/k g)
278 A 112B
373 c 62 c
Diet 3 of Table 1. B Diet 4 of Table 1. c A basic wheat, meat meal and casein diet used with or without additional biotin (see text). ° See footnote A, Table 2. E Not determined.
A
Analyses of samples of blood serum from chicks with or without FLKS for cholesterol, bilirubin, creatinine, protein and albumin content gave no indication of liver malfunction. However, chicks with FLKS had low blood glucose levels, confirming the observations of Bannister et al. (1975). This suggests that these chicks had impaired gluconeogenesis, leading to hypoglycemia which may have been a factor in the death of the chicks. Despite reports (Balnave 1966; Roland and Edwards 1971; Edwards et al. 1973) that biotin deficiency in chicks results in elevated levels of palmitoleic acid in liver lipids, FLKS has not previously been associated with a deficiency of biotin, since the classical symptoms of biotin deficiency (dermatitis, perosis, and poor growth) have not been observed in field outbreaks of FLKS. Provided they are not subjected to stress, broiler chicks on a diet marginally deficient in biotin will grow to maturity at the same rate as chicks on a diet adequate in biotin. The involvement of two additional factors in FLKS, namely stress and low-biotin status of the chick at hatching, has made it difficult to ascertain the primary cause of this disorder. Any form of stress puts heavy demands on the gluconeogenesis pathway after liver glycogen reserves are depleted. Chicks on the biotin-deficient diet showed evidence of impaired gluconeogenesis and, consequently, of poor ability to withstand stress. The most likely cause for this is reduced activity of the biotin-dependent enzyme pyruvate carboxylase.
Fatty Liver and Kidney Syndrome in Chicks. I
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Impaired gluconeogenesis would explain the different rates of lipogenesis in chicks with FLKS and in those without FLKS. In chicks with FLKS, lipogenesis is increased because impaired gluconeogenesis increases the availability of substrates, e.g. pyruvate, for fatty acid synthesis. A difficulty with such a suggestion is that biotin is also required as a co-factor in lipogenesis, namely in the carboxylation of acetyl-CoA. A possible explanation is that the biotin-dependent enzyme of the gluconeogenesis pathway is more markedly affected by marginal biotin deficiency than the biotin-dependent enzyme of the lipogenesis pathway. This aspect is explored in the following paper. Acknowledgments
This work was supported in part by a grant from the Australian Chicken Meat Research Committee. Dr C. G. Payne, University of Sydney, provided special diets, and Dr S. Nobile, The Vitamin Laboratories, Sydney, performed the microbiological biotin assays. Blood assays were performed by the Department of Clinical Biochemistry, Prince Henry Hospital. G. Ford, S. Kozuharov and C. Godfrey provided technical assistance. References Balnave, D. (1966). Effect of gonadal hormones and biotin deficiency on growth and lipid composition of liver and tissue in the immature female chick. Proc. XIII World Poultry Congo pp. 213-18. Bannister, D. W., Evans, A. J., and Whitehead, C. C. (1975). Evidence for a lesion in carbohydrate metabolism in fatty liver and kidney syndrome in chicks. Res. Vet. Sci. 18, 149-56. Blair, R., Bolton, W., and Duff, R. H. (1969). Fatty liver and kidney disease in broiler chickens receiving diets with varying contents of protein. Vet. Rec. 84, 41-3. Blair, R., and Whitehead, C. C. (1974). An assessment of the factors associated with fatty liver and kidney syndrome in broilers. Proc. XV. World Poultry Congo p.380. Blair, R., Whitehead, C. C., and Teague, P. W. (1975). The effect of dietary fat and protein levels, form and cereal type on fatty liver and kidney syndrome in chicks. Res. Vet. Sci. 18, 76-81. Bligh, E. G., and Dyer, W. J. (1959). A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol. 37,911-17. Christie, W. W., Noble, R. c., and Moore, J. H. (1970). Determination of lipid classes by a gaschromatographic procedure. Analyst (London) 95, 940-4. Edwards, H. M., Denman, F., and Jackson, R. (1973). Severity of biotin deficiency and fatty acid composition of tissues. Poult. Sci. 52, 2024. Hemsley, L. A. (1965). The 'Fatty liver and kidney syndrome' of young chickens. Vet. Rec. 77, 124-6. Hemsley, L. A., and Marshall, L. G. (1973). The influence of parent stock on the susceptibility of chicks to the fatty liver and kidney syndrome. Br. Vet. J. 129, Ixii-Ixiv. Hood, R. L. (1975). A radiochemical assay for biotin in biological materials. J. Sci. Food. Agric. 26, 1847-52. Hood, R. L., Thompson, E. H., and Allen, C. E. (1972). The role of acetate, proprionate and glucose as substrates for lipogenesis in bovine tissues. Int. J. Biochem. 3, 598-606. Husbands, D. R., and Laursen-Jones, A. P. (1969). Fatty liver and kidney syndrome. Vet. Rec. 84, 232-3. Johnson, A. R., Pearson, J. A., Shenstone, F. S., and Fogerty, A. C. (1967). Inhibition of the desaturation of stearic to oleic acid by cyclopropene fatty acids. Nature (London) 214, 1244-5. Johnson, A. R. et al. (1972). Fatty liver and kidney disease in young chickens. Proc. Australas. Poult. Sci. Conv. p. 15-16. Marthedal, H. E., and Velling, G. (1958). Liver and kidney disease in chickens. Proc. VIn Nordiske Veterinormetet. p. 250. Payne, C. G., Gilchrist, P., Pearson, J. A., and Hemsley, L. A. (1974). Involvement of biotin in the fatty liver and kidney syndrome of broilers. Br. Poult. Sci. 15, 489-98.
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Riddell, C., Olsen, G. V., and Grimson, R. E. (1971). Fatty liver and kidney syndrome in a broiler flock. Avian Dis. 15, 398-405. Roland, D. A., and Edwards, H. M. (1971). Effect of essential fatty acid deficiency and type of dietary fat supplementation on biotin-deficient chicks. J. Nutr. 101, 811-8. Whitehead, C. C.; Bannister, D. W., Wight, P. A. L., and Weiser, H. (1974). Studies on biotin requirements and deficiency in chicks. Proc. XV World Poultry Congo p.70-2. Whitehead, C. C., Blair, R., Bannister, D. W., and Evans, A. J. (1975). The involvement of dietary fat and vitamins, stress, litter and starvation on the incidence of the fatty liver and kidney syndrome in chicks. Res. Vet. Sci. 18, 100-4.
Manuscript received 9 February 1976
