Biological Potency of Selenium from Sodium Selenite, Selenomethionine, and Selenocystine in the Chick* M. OSMAN AND J. D. LATSHAW Department of Poultry Science, Ohio State University, 674 W. Lane Ave., Columbus, Ohio 43210 (Received for publication August 25, 1975)
POULTRY SCIENCE 55: 987-994, 1976
INTRODUCTION
W
HEN selenium (Se) is supplemented in poultry diets, it is usually added in an inorganic form such as selenite or selenate. The limited amount of information available indicates that the major portion of Se in plants is present in an organic form, selenomethionine (Allaway et al, 1967; Olson et al., 1970). Studies with eggs also show that the predominant form of selenium in a practical laying diet is selenomethionine (Latshaw and Osman, 1975). Both selenite and selenomethionine can serve as sources of Se to prevent deficiencies, as can selenocystine. The effectiveness of each on a unit weight basis is not yet clear. Cantor et al. (1975a) found that selenomethionine was 18 to 61% as effective as selenite, depending on the experiment, in preventing exudative diathesis in chicks. Selenocystine was from 69 to 78% as effective. Noguchi et al. (1973) found that selenomethionine was
"Ohio Agricultural Research and Development Center Journal Article Series No. 87-75.
only about 60% as effective as selenite in preventing exudative diathesis when both were added to the diet to supply .06 mg. Se per kg. diet. In this same study selenite also resulted in higher plasma glutathione peroxidase levels. Omaye and Tappel (1974) found that 0.1 mg. Se per kg. diet from either selenite or selenomethionine produced similar glutathione peroxidase values in several tissues but that this enzyme activity was slightly higher in the heart and liver of chicks fed selenite. Cantor et al. (1975b) evaluated the ability of Se from selenite, selenomethionine, and selenocystine to prevent pancreatic fibrosis in chicks. Se from selenomethionine was more effective than from the other two sources. A selenium analysis showed that the pancreas also retained more selenium from selenomethionine. This increased retention did not increase the glutathione peroxidase activity, however. The main purpose of this study was to evaluate the relative effectiveness of Se from sodium selenite, selenomethionine, and selenocystine to produce growth and prevent exudative diathesis (ED). A second objective
987
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ABSTRACT Experiments were conducted to determine the relative effectiveness of selenium (Se) from sodium selenite, selenomethionine, and selenocystine for promoting weight gain and preventing exudative diathesis. The chicks used were hatched from eggs low in Se. They were fed a basal diet made up mostly of corn (low in Se) and torula yeast or the basal diet supplemented with various levels of Se from sodium selenite, selenomethionine, or selenocystine. At 10 u,g. of added Se per kg. of diet, sodium selenite and selenocystine were about equal in promoting weight gain and preventing exudative diathesis. Selenomethionine was less effective. Tissues from chicks fed the various Se sources providing 60 u.g. Se per kg. of diet for four weeks were analyzed for Se. The content of tissues from chicks fed sodium selenite or selenocystine was similar. Chicks fed selenomethionine had a higher concentration of Se in the pancreas and breast muscle than chicks fed the other two Se sources, but a lower concentration in the kidney, liver, and heart. The level of Se in the kidney, liver, or heart which a Se source produces seems to be more important for preventing exudative diathesis than that which is found in the pancreas or muscle.
988
M . OSMAN AND J . D . LATSHAW
was to determine the distribution of Se in tissues when fed different Se sources. PROCEDURE
The chicks were given tap water and fed ad libitum throughout the four week experimental period. Body weight was determined at one day of age and each week thereafter. Chicks were scored for the incidence of ED at each weighing, and mortality was recorded daily. At the end of the fourth week chicks fed the basal diet and those fed 60 u,g. Se per kg. from each of the compounds were sacrificed and tissues were saved. The tissues collected were breast muscle, heart, kidney, liver, and pancreas. An equal weight of breast
1. Purchased from Calbiochem, La Jolla, California.
Percent of Ingredient the diet Yellow corn 64.45 Torula yeast (50% protein) 28.52 Mono and dicalcium phosphate1 0.50 Limestone 2.29 Iodized salt 0.40 0.27 Methionine hydroxy analogue Arginine 0.37 Animal fat 3.00 Vitamin premix2 0.15 Trace mineral premix3 0.05 1 16.5% Ca and 21.0% P. 2 Vitamin premix provides the following levels of micronutrients per kilogram of diet: retinyl palmitate, 1500 I.U.; cholecalciferol, 200 I.U.; menadione sodium bisulfite, 0.53 mg.; thiamine, 1.8 mg.; riboflavin, 3.6 mg.; pantothenic acid, 10.0 mg.; niacin, 27 mg.; pyridoxine, 3.0 mg.; biotin, 0.09 mg.; choline, 1300 mg.; folacin, 0.55 mg.; and vitamin B 12 , 0.009 mg. 3 Trace mineral premix provides the following levels of trace minerals per kilogram of diet: manganese (MnSo4), 50 mg.; iron (FeS0 4 , FeO, FeC0 3 ), 50 mg.; copper (CuO), 5 mg.; cobalt (CoCo3), 0.50 mg.; and zinc (ZnS04, ZnO), 50 mg.
muscle was weighed from each of three chicks, and the three samples of breast muscle were then homogenized and lyophilized for one pooled sample. Two pooled samples, representing six individual chicks, were used for each tissue for each treatment. Other tissues were similarly pooled and prepared for Se analysis. The pooled samples were then analyzed for Se content by the method of Olson (1969) as modified by Latshaw (1975). Since it was observed that the maximum response occurred at such low levels of supplemental Se in the first experiment, a second experiment was designed in the same manner as the first except for the levels of Se added. Sodium selenite or selenocystine was added to the basal diet to supply 3.3, 6.7, or 10 p,g. of Se per kg. Selenomethionine was added to supply 5, 10 or 15 u,g. of Se
Downloaded from http://ps.oxfordjournals.org/ at University of Cambridge on June 8, 2015
Experiment 1 was conducted using day-old chicks. To facilitate the development of a Se deficiency in the chicks, they were hatched from eggs laid by Single Comb White Leghorn hens which were fed a practical diet low in selenium (about 0.07 mg./kg.) and which contained no added vitamin E. These hens were artificially inseminated with semen from broiler type males. Unsexed chicks were randomly assigned to electrically heated battery pens with raised wire floors, seven chicks per pen. Chicks were fed the basal diet (Table 1) or the basal diet supplemented with Se, three pens per dietary treatment. By analysis the basal diet contained 30 |xg. Se/kg. Se was supplied by sodium selenite, DL-selenomethionine,1 or DL-selenocystine.1 Each compound was added to supply 10, 20, 40, or 60 |xg./kg. of diet. To improve the accuracy of addition each compound was dissolved in distilled water, or in the case of selenocystine, distilled water decreased to pH 2 by the addition of HC1. The appropriate number of ml. of solution were then added to the diet before mixing.
TABLE 1.—Composition of the basal diet
989
SELENIUM POTENCY
TABLE 2.—Effect
of various levels of organic and inorganic selenium compounds on four week gain and feed utilization
Treatment Basal Basal + N a 2 S e 0 3
Se added (ixg./kg.)
Basal + Se-Met
Basal + Se-Cys
Feed:gain ratio 2.73a 1 1.96d 2.05cd 2.30b 2.30b 2.19bcd 2.06cd 2.17bcd 2.19bcd 2.01cd 2.04cd 2.15bcd 2.23bc
'Means not having the same letter are significantly different (P < 0.05). per kg. Each treatment was fed to three replicates of seven chicks each, and data were collected as in the first experiment. Differences in treatment means were statistically compared on the basis of Duncan's multiple range test. (Duncan, 1955). RESULTS Chicks fed the basal diet in experiment 1 gained only 166 g., significantly less than chicks fed any source of Se at any level (Table 2). Those chicks fed either sodium selenite or selenocystine showed a maximum response in weight gain at the lowest level added, 10 (xg. Se per kg. of diet. Adding more Se from these compounds did not increase growth. More Se was required for comparable growth when it was added in the form of selenomethionine. Approximately 20 |xg. Se per kg. was needed to allow growth comparable to that found when Se came from either sodium selenite or selenocystine. Chicks fed the basal diet required significantly more feed per unit of gain than those of any other treatment. The data suggest that the most efficient use of feed occurred when the chicks were fed the least amount of Se necessary to produce maximum growth. Feeding higher levels of Se increased the feed to gain ratio. This is the pattern observed
when Se was supplied by selenite and selenocystine. When selenomethionine was fed, the best feed to gain ratio was found at 20 (xg. Se per kg. diet. The incidence of ED and mortality is found in Table 3. No signs of exudative diathesis were found at the end of the first week. By the end of the second week all of the chicks fed the basal diet and many of the other chicks had signs of ED. In the milder cases the bluish area was often only about a centimeter in diameter, generally just anterior to the leg. In some chicks the bluish areas disappeared, while in others the increasing severity resulted in death. All remaining chicks fed the basal diet had ED at the end of four weeks. Some ED was present in chicks fed 10 and 20 |xg. Se per kg. of diet from selenite or selenocystine, but none was found when chicks were fed the higher levels. Most of the chicks fed 10 (jig. Se per kg. of diet from selenomethionine still had ED at the end of four weeks. All other levels resulted in about a third of the chicks having ED. Data on mortality show that 8 of the 21 chicks fed the basal diet died of ED. None died in the selenite treatments, and the one death in the selenocystine treatments was not due to ED. Of those fed 10 \xg. Se per kg. of diet from selenomethionine, 6 died. The increase in tissue Se resulting from
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10 20 40 60 10 20 40 60 10 20 40 60
Gain (g.) 166a1 274c 277c 252bc 252bc 223b 249bc 279c 262bc 267bc 268bc 281c 280c
990
M. OSMAN AND J. D. LATSHAW
TABLE 3.-
-Effect of various dietary selenium supplements on mortality and the incidence of exudative diathesis Se 1st week added M. 2 (tig./kg.) E.D. 1
Treatment Basal Basal + N a 2 S e 0 3
Basal + Se-Met
Basal + Se-Cys
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
19 12 9 7 4 17 13 9 8 15 12 12 9
M. 2 2 0 0 0 0 1 0 0 0 0 0 0 0
4 t h ' week
E.D.'
M. 2
E.D.'
M. 2
16 10 6 2 0 12 10 8 7 11 9 9 5
5 0 0 0 0 4 0 0 0 0 0 0 0
13 4 3 0 0 11 7 6 6 5 3 0 0
8 0 0 0 0 6 0 0 0 1 0 0 0
'E.D. Number of cumulative surviving chicks showing exudative diathesis/21 started. 2 (Mortality)—Number of chicks dead at end of the week/21started.
TABLE 4.—Selenium concentrations in various organ tissues Se-contents (jxg. Se/g. dry tissue) Treatment Basal Basal + 60 |xg. Se/kg. (Na 2 Se0 3 ) Basal + 60 |xg. Se/kg. (Se-Met) Basal + 60 jig. Se/kg. (Se-Cys)
Kidney
Liver
Pancreas
Heart
Muscle
0.179a' 1.333bc 1.180b 1.450c
0.148a' 0.781c 0.679b 0.768c
0.186a' 0.430b 0.711c 0.533b
0.114a' 0.465b 0.378b 0.463b
0.053a' 0.139b 0.194c 0.129b
' Means in a vertical column not having the same letter are significantly different (P < 0.05).
dietary Se is shown in Table 4. Kidney Se increased the most in response to increased dietary Se. Significantly more Se was present in the kidney when selenocystine was fed than when selenomethionine was fed. Kidneys from selenite fed chicks had an intermediate Se concentration, not significantly different from those of the other Se treatments. Both the liver and heart contained more Se from dietary selenite and selenocystine than from selenomethionine. In the liver the difference is statistically significant but not in the heart. An opposite effect was found in the pancreas and breast muscle. These tissues retained more Se when selenomethionine was fed than when selenite or selenocystine was fed. In experiment 2 increasing levels of Se from selenomethionine resulted in an almost linear
increase in growth (Figure 1). At 10 ji.g. Se per kg. of diet selenomethionine was not as effective in promoting growth as either selenite or selenocystine. Selenite was less effective than selenocystine at the 3.3 n-g. Se level, but no difference was present at 10 (xg. per kg. diet. The incidence of ED at four weeks of age (Figure 2) again showed that selenomethionine was not as effective as the other two sources. Approximately 1.5 times as much Se from selenomethionine is needed to reduce the incidence of ED to a level comparable to that from selenite. Generally, the decrease in incidence of exudative diathesis did not show a linear relationship with increasing dietary Se levels. The decrease in incidence was fairly abrupt as the requirement of Se for maximum weight gain was approached.
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10 20 40 60 10 20 40 60 10 20 40 60
3rd week
2nd week E.D.'
SELENIUM POTENCY
991
260
220
200
180
Added Selenium (yg/kg d i e t )
FIG. 1. Four-week weight gain as affected by various levels of selenium from different compounds. DISCUSSION The results of this study show that Se from selenomethionine was not as effective as Se from selenite or selenocystine. For either the production of weight gain or reduction of the incidence of exudative diathesis, selenomethionine Se was only about two-thirds as effective. This value is in fairly good agreement with that found by Noguchi et al. (1973), but generally higher than that found by Cantor
et al. (1975a). It should be noted that with the diet used by these researchers, the selenium requirement apparently is greater than in the diet used in this study. Their basal diet contained approximately 15 |xg. Se per kg. and some degree of Se deficiency was still evident after adding 60 |o,g. Se per kg. diet in the form of selenite. The basal diet used in this study contained approximately 30 jxg. Se per kg., and signs of a Se deficiency
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240
992
M . OSMAN AND J . D . LATSHAW
100
'
#Na.Se03 Ose-Cys
"
Ose-Met
60
3
6 Added S e l e n i u m
9
]2
(pg/kg d i e t )
FIG. 2. Incidence of exudative diathesis at four weeks as affected by various levels of selenium from different compounds.
were almost totally alleviated by the addition of 10 u.g. of selenite Se per kg. It is possible that the vitamin E contributed by corn in this study decreased the need for Se. Information on the ability of Se from the three different sources to prevent pancreatic
fibrosis was not obtained. However, no atrophy or other abnormal condition of the pancreas was noted during the collection of tissue for selenium analysis. The question arises as to why Se from selenite was biologically more effective than
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•
993
SELENIUM POTENCY
Schwarz and Feeney (1964) found that 75Se as selenite attached nonenzymatically to cystine and migrated with cystine in chromatographic separation. When 75Se was injected into a rabbit as selenite, 75Se was incorporated into protein (Cummins and Martin, 1967). When liver protein was hydrolyzed with enzymes and the amino acids chromatographed, 75 Se was present in the area of methionine and cystine. However, the 75 Se could be released from protein by dialysis with an alkaline solution of pH 11-12, and this treatment was not severe enough to release Se from selenocystine or selenomethionine standards. The evidence suggested no selenide formation, just an interaction between sulfur-containing amino acids and 7 5 Se0 3 = . Jenkins (1968) also found no evidence that chicks
could synthesize selenium-containing amino acids from inorganic Se. This report postulated that the Se molecule was present between the sulfur atoms of cystine. Based on the above information, in this experiment selenite was not converted to selenomethionine or selenocystine. Since the distribution of selenium in tissues is similar for selenite and selenocystine, and since the biological potency of selenite and selenocystine is similar, selenocystine is probably converted to an inorganic form. Although chicks can effectively use selenocystine prepared in the laboratory as a source of selenium it is doubtful that practical feed ingredients would contain any significant amount. Olson et al. (1970) did not find selenocystine in wheat, and animals probably cannot synthesize it. The pattern of selenium distribution in tisses may be a factor in the greater biological potency of Se from selenite and selenocystine than from selenomethionine. Selenomethionine feeding produced lower levels of Se in the kidney, liver, and heart than the other two Se compounds, although liver was the only tissue which was significantly lower. The Se content of breast muscle and pancreas, however, was higher when selenomethionine was fed. Why these different patterns occur is not yet known, but they appear to be related to the different biological potencies observed.
REFERENCES Allaway, W. H., E. E. Cary and C. F. Ehlig, 1967. The cycling of low levels of selenium in soils, plants, and animals. In: Selenium in Biomedicine (Muth, O.H., ed.), Avi Publishing Co., Inc., Westport, Conn. pp. 273-296. Cantor, A. H., M. L. Scott and T. Noguchi, 1975a. Biological availability of selenium in feedstuffs and selenium compounds for prevention of exudative diathesis in chicks. J. Nutr. 105: 96-105. Cantor, A. H., M. L. Langevin, T. Noguchi and M. L. Scott, 1975b. Efficacy of selenium in selenium compounds and feedstuffs for prevention of pancreatic fibrosis in chicks. J. Nutr. 105: 106-111.
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from selenomethionine in these studies. Data from Table 4 show that feeding selenomethionine results in more Se in the pancreas and muscle than feeding selenite. Since muscle comprises a large percentage of the body, more Se is probably retained from feeding selenomethionine than from selenite. Tissue Se values of hens fed practical diets in which most of the Se is in the form of selenomethionine support this view (Latshaw, 1975). If the total amount retained is not the primary factor in determining potency then the form in which Se is present may be more important. Some studies suggested that inorganic selenium could be converted into selenides or diselenides by animals. McConnell and Wabnitz (1957) injected a dog with 75 SeCl 4 and then examined acid hydrolysates of protein for 75Se distribution. After separation by paper chromatography 75Se was found in the areas of cystine and methionine. The data were interpreted to mean that selenocystine and selenomethionine had been synthesized. Rosenfeld (1962) also found similar evidence that 75Se could be incorporated into organic compounds. Synthesis of these compounds would have occurred only if Se was reduced from +4 to —2.
994
M. OSMAN AND J. D. LATSHAW
Noguchi, T., A. H. Cantor and M. L. Scott, 1973. Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks. J. Nutr. 103: 1502-1511. Olson, O. E., 1969. Fluorometric analysis of selenium in plants. J. Assoc. Offic. Anal. Chem. 52: 627-634. Olson, O. E., E. J. Novacek, E. I. Whitehead and I. S. Palmer, 1970. Investigations on selenium in wheat. Phytochemistry, 8: 1161-1165. Omaye, S. T., and A. L. Tappel, 1974. Effect of dietary selenium on glutathione peroxidase in the chick. J. Nutr. 104: 747-753. Rosenfeld, O., 1962. Biosynthesis of selenocompounds from inorganic selenium by sheep. Proc. Soc. Exptl. Biol. Med. I l l : 670-673. Schwarz, K., and E. Feeney, 1964. Selenite binding to sulfur amino acids. Fed. Proc. 23: 421.
Composition and Functional Properties of Mottled Yolksl F . E . CUNNINGHAM
Dairy and Poultry Science Department, Kansas State University, Manhattan, Kansas 66506 (Received for publication August 25, 1975)
ABSTRACT Yolk mottling was induced by feeding 0.02% Nicarbazin to 6-month old White Leghorn pullets. A second group of pullets, fed a standard layer ration, served as the control. Eggs were collected for three months on a daily basis, but the yolks were pooled on a weekly basis for analysis. Composition and functional properties of both mottled and normal yolks were determined. The percent solids of normal yolks was significantly higher than mottled yolks. The pH of normal yolks was lower than mottled yolks but not significantly. Normal yolks contained higher percentage fat, protein, and ash than mottled yolks; however, the percent carbohydrate was lower in normal yolks. There was no difference in the cholesterol content of mottled and normal yolks. Normal yolks contained significantly higher percent calcium, phosphorus, and iron than mottled yolks. Emulsifying capacity and viscosity of normal yolks were greater than for mottled yolks. Foam volume and sponge cake volume were always larger for mottled yolks than for normal yolks, but normal yolks produced more stable foam and better cake texture. Electrophoretic separation of the components of mottled and normal yolks indicated that two egg white proteins were present in mottled yolks but not apparent in normal yolks. The two proteins are believed to be ovalbumin and conalbumin. POULTRY SCIENCE 55: 994-998, 1976
INTRODUCTION
F
ACTORS commonly associated with mottled yolks were recently reviewed by Cunningham and Sanford (1973, 1974). Mot1. Contribution No. 918, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas 66506.
tling is common in commercial flocks and continues to be a severe problem of the poultry industry. Blackshear et al. (1968) reported that 56.1% of all eggs sampled from 43 flocks were mottled to some degree. Although many causes of mottling are known, very little is known about the composition or functional properties of mot-
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Cummins, L. M., and J. L. Martin, 1967. Are selenocystin and selenomethionine synthesized in vivo from sodium selenite in mammals? Biochemistry, 6: 3162-3168. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Jenkins, K. J., 1968. Evidence for the absence of selenocystine and selenomethionine in the serum proteins of chicks administered selenite. Can. J. Biochem. 46: 1417-1425. Latshaw, J. D., 1975. Natural and selenite selenium in the hen and egg. J. Nutr. 105: 32-37. Latshaw, J. D., and M. Osman, 1975. Distribution of selenium in egg white and yolk after feeding natural and synthetic selenium compounds. Poultry Sci. 54: 1244-1252. McConnell, K. P., and C. H. Wabnitz, 1957. Studies on the fixation of radioselenium in proteins. J. Biol. Chem. 226: 765-776.
POULTRY SCIENCE 55: 987-994, 1976
INTRODUCTION
W
HEN selenium (Se) is supplemented in poultry diets, it is usually added in an inorganic form such as selenite or selenate. The limited amount of information available indicates that the major portion of Se in plants is present in an organic form, selenomethionine (Allaway et al, 1967; Olson et al., 1970). Studies with eggs also show that the predominant form of selenium in a practical laying diet is selenomethionine (Latshaw and Osman, 1975). Both selenite and selenomethionine can serve as sources of Se to prevent deficiencies, as can selenocystine. The effectiveness of each on a unit weight basis is not yet clear. Cantor et al. (1975a) found that selenomethionine was 18 to 61% as effective as selenite, depending on the experiment, in preventing exudative diathesis in chicks. Selenocystine was from 69 to 78% as effective. Noguchi et al. (1973) found that selenomethionine was
"Ohio Agricultural Research and Development Center Journal Article Series No. 87-75.
only about 60% as effective as selenite in preventing exudative diathesis when both were added to the diet to supply .06 mg. Se per kg. diet. In this same study selenite also resulted in higher plasma glutathione peroxidase levels. Omaye and Tappel (1974) found that 0.1 mg. Se per kg. diet from either selenite or selenomethionine produced similar glutathione peroxidase values in several tissues but that this enzyme activity was slightly higher in the heart and liver of chicks fed selenite. Cantor et al. (1975b) evaluated the ability of Se from selenite, selenomethionine, and selenocystine to prevent pancreatic fibrosis in chicks. Se from selenomethionine was more effective than from the other two sources. A selenium analysis showed that the pancreas also retained more selenium from selenomethionine. This increased retention did not increase the glutathione peroxidase activity, however. The main purpose of this study was to evaluate the relative effectiveness of Se from sodium selenite, selenomethionine, and selenocystine to produce growth and prevent exudative diathesis (ED). A second objective
987
Downloaded from http://ps.oxfordjournals.org/ at University of Cambridge on June 8, 2015
ABSTRACT Experiments were conducted to determine the relative effectiveness of selenium (Se) from sodium selenite, selenomethionine, and selenocystine for promoting weight gain and preventing exudative diathesis. The chicks used were hatched from eggs low in Se. They were fed a basal diet made up mostly of corn (low in Se) and torula yeast or the basal diet supplemented with various levels of Se from sodium selenite, selenomethionine, or selenocystine. At 10 u,g. of added Se per kg. of diet, sodium selenite and selenocystine were about equal in promoting weight gain and preventing exudative diathesis. Selenomethionine was less effective. Tissues from chicks fed the various Se sources providing 60 u.g. Se per kg. of diet for four weeks were analyzed for Se. The content of tissues from chicks fed sodium selenite or selenocystine was similar. Chicks fed selenomethionine had a higher concentration of Se in the pancreas and breast muscle than chicks fed the other two Se sources, but a lower concentration in the kidney, liver, and heart. The level of Se in the kidney, liver, or heart which a Se source produces seems to be more important for preventing exudative diathesis than that which is found in the pancreas or muscle.
988
M . OSMAN AND J . D . LATSHAW
was to determine the distribution of Se in tissues when fed different Se sources. PROCEDURE
The chicks were given tap water and fed ad libitum throughout the four week experimental period. Body weight was determined at one day of age and each week thereafter. Chicks were scored for the incidence of ED at each weighing, and mortality was recorded daily. At the end of the fourth week chicks fed the basal diet and those fed 60 u,g. Se per kg. from each of the compounds were sacrificed and tissues were saved. The tissues collected were breast muscle, heart, kidney, liver, and pancreas. An equal weight of breast
1. Purchased from Calbiochem, La Jolla, California.
Percent of Ingredient the diet Yellow corn 64.45 Torula yeast (50% protein) 28.52 Mono and dicalcium phosphate1 0.50 Limestone 2.29 Iodized salt 0.40 0.27 Methionine hydroxy analogue Arginine 0.37 Animal fat 3.00 Vitamin premix2 0.15 Trace mineral premix3 0.05 1 16.5% Ca and 21.0% P. 2 Vitamin premix provides the following levels of micronutrients per kilogram of diet: retinyl palmitate, 1500 I.U.; cholecalciferol, 200 I.U.; menadione sodium bisulfite, 0.53 mg.; thiamine, 1.8 mg.; riboflavin, 3.6 mg.; pantothenic acid, 10.0 mg.; niacin, 27 mg.; pyridoxine, 3.0 mg.; biotin, 0.09 mg.; choline, 1300 mg.; folacin, 0.55 mg.; and vitamin B 12 , 0.009 mg. 3 Trace mineral premix provides the following levels of trace minerals per kilogram of diet: manganese (MnSo4), 50 mg.; iron (FeS0 4 , FeO, FeC0 3 ), 50 mg.; copper (CuO), 5 mg.; cobalt (CoCo3), 0.50 mg.; and zinc (ZnS04, ZnO), 50 mg.
muscle was weighed from each of three chicks, and the three samples of breast muscle were then homogenized and lyophilized for one pooled sample. Two pooled samples, representing six individual chicks, were used for each tissue for each treatment. Other tissues were similarly pooled and prepared for Se analysis. The pooled samples were then analyzed for Se content by the method of Olson (1969) as modified by Latshaw (1975). Since it was observed that the maximum response occurred at such low levels of supplemental Se in the first experiment, a second experiment was designed in the same manner as the first except for the levels of Se added. Sodium selenite or selenocystine was added to the basal diet to supply 3.3, 6.7, or 10 p,g. of Se per kg. Selenomethionine was added to supply 5, 10 or 15 u,g. of Se
Downloaded from http://ps.oxfordjournals.org/ at University of Cambridge on June 8, 2015
Experiment 1 was conducted using day-old chicks. To facilitate the development of a Se deficiency in the chicks, they were hatched from eggs laid by Single Comb White Leghorn hens which were fed a practical diet low in selenium (about 0.07 mg./kg.) and which contained no added vitamin E. These hens were artificially inseminated with semen from broiler type males. Unsexed chicks were randomly assigned to electrically heated battery pens with raised wire floors, seven chicks per pen. Chicks were fed the basal diet (Table 1) or the basal diet supplemented with Se, three pens per dietary treatment. By analysis the basal diet contained 30 |xg. Se/kg. Se was supplied by sodium selenite, DL-selenomethionine,1 or DL-selenocystine.1 Each compound was added to supply 10, 20, 40, or 60 |xg./kg. of diet. To improve the accuracy of addition each compound was dissolved in distilled water, or in the case of selenocystine, distilled water decreased to pH 2 by the addition of HC1. The appropriate number of ml. of solution were then added to the diet before mixing.
TABLE 1.—Composition of the basal diet
989
SELENIUM POTENCY
TABLE 2.—Effect
of various levels of organic and inorganic selenium compounds on four week gain and feed utilization
Treatment Basal Basal + N a 2 S e 0 3
Se added (ixg./kg.)
Basal + Se-Met
Basal + Se-Cys
Feed:gain ratio 2.73a 1 1.96d 2.05cd 2.30b 2.30b 2.19bcd 2.06cd 2.17bcd 2.19bcd 2.01cd 2.04cd 2.15bcd 2.23bc
'Means not having the same letter are significantly different (P < 0.05). per kg. Each treatment was fed to three replicates of seven chicks each, and data were collected as in the first experiment. Differences in treatment means were statistically compared on the basis of Duncan's multiple range test. (Duncan, 1955). RESULTS Chicks fed the basal diet in experiment 1 gained only 166 g., significantly less than chicks fed any source of Se at any level (Table 2). Those chicks fed either sodium selenite or selenocystine showed a maximum response in weight gain at the lowest level added, 10 (xg. Se per kg. of diet. Adding more Se from these compounds did not increase growth. More Se was required for comparable growth when it was added in the form of selenomethionine. Approximately 20 |xg. Se per kg. was needed to allow growth comparable to that found when Se came from either sodium selenite or selenocystine. Chicks fed the basal diet required significantly more feed per unit of gain than those of any other treatment. The data suggest that the most efficient use of feed occurred when the chicks were fed the least amount of Se necessary to produce maximum growth. Feeding higher levels of Se increased the feed to gain ratio. This is the pattern observed
when Se was supplied by selenite and selenocystine. When selenomethionine was fed, the best feed to gain ratio was found at 20 (xg. Se per kg. diet. The incidence of ED and mortality is found in Table 3. No signs of exudative diathesis were found at the end of the first week. By the end of the second week all of the chicks fed the basal diet and many of the other chicks had signs of ED. In the milder cases the bluish area was often only about a centimeter in diameter, generally just anterior to the leg. In some chicks the bluish areas disappeared, while in others the increasing severity resulted in death. All remaining chicks fed the basal diet had ED at the end of four weeks. Some ED was present in chicks fed 10 and 20 |xg. Se per kg. of diet from selenite or selenocystine, but none was found when chicks were fed the higher levels. Most of the chicks fed 10 (jig. Se per kg. of diet from selenomethionine still had ED at the end of four weeks. All other levels resulted in about a third of the chicks having ED. Data on mortality show that 8 of the 21 chicks fed the basal diet died of ED. None died in the selenite treatments, and the one death in the selenocystine treatments was not due to ED. Of those fed 10 \xg. Se per kg. of diet from selenomethionine, 6 died. The increase in tissue Se resulting from
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10 20 40 60 10 20 40 60 10 20 40 60
Gain (g.) 166a1 274c 277c 252bc 252bc 223b 249bc 279c 262bc 267bc 268bc 281c 280c
990
M. OSMAN AND J. D. LATSHAW
TABLE 3.-
-Effect of various dietary selenium supplements on mortality and the incidence of exudative diathesis Se 1st week added M. 2 (tig./kg.) E.D. 1
Treatment Basal Basal + N a 2 S e 0 3
Basal + Se-Met
Basal + Se-Cys
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
19 12 9 7 4 17 13 9 8 15 12 12 9
M. 2 2 0 0 0 0 1 0 0 0 0 0 0 0
4 t h ' week
E.D.'
M. 2
E.D.'
M. 2
16 10 6 2 0 12 10 8 7 11 9 9 5
5 0 0 0 0 4 0 0 0 0 0 0 0
13 4 3 0 0 11 7 6 6 5 3 0 0
8 0 0 0 0 6 0 0 0 1 0 0 0
'E.D. Number of cumulative surviving chicks showing exudative diathesis/21 started. 2 (Mortality)—Number of chicks dead at end of the week/21started.
TABLE 4.—Selenium concentrations in various organ tissues Se-contents (jxg. Se/g. dry tissue) Treatment Basal Basal + 60 |xg. Se/kg. (Na 2 Se0 3 ) Basal + 60 |xg. Se/kg. (Se-Met) Basal + 60 jig. Se/kg. (Se-Cys)
Kidney
Liver
Pancreas
Heart
Muscle
0.179a' 1.333bc 1.180b 1.450c
0.148a' 0.781c 0.679b 0.768c
0.186a' 0.430b 0.711c 0.533b
0.114a' 0.465b 0.378b 0.463b
0.053a' 0.139b 0.194c 0.129b
' Means in a vertical column not having the same letter are significantly different (P < 0.05).
dietary Se is shown in Table 4. Kidney Se increased the most in response to increased dietary Se. Significantly more Se was present in the kidney when selenocystine was fed than when selenomethionine was fed. Kidneys from selenite fed chicks had an intermediate Se concentration, not significantly different from those of the other Se treatments. Both the liver and heart contained more Se from dietary selenite and selenocystine than from selenomethionine. In the liver the difference is statistically significant but not in the heart. An opposite effect was found in the pancreas and breast muscle. These tissues retained more Se when selenomethionine was fed than when selenite or selenocystine was fed. In experiment 2 increasing levels of Se from selenomethionine resulted in an almost linear
increase in growth (Figure 1). At 10 ji.g. Se per kg. of diet selenomethionine was not as effective in promoting growth as either selenite or selenocystine. Selenite was less effective than selenocystine at the 3.3 n-g. Se level, but no difference was present at 10 (xg. per kg. diet. The incidence of ED at four weeks of age (Figure 2) again showed that selenomethionine was not as effective as the other two sources. Approximately 1.5 times as much Se from selenomethionine is needed to reduce the incidence of ED to a level comparable to that from selenite. Generally, the decrease in incidence of exudative diathesis did not show a linear relationship with increasing dietary Se levels. The decrease in incidence was fairly abrupt as the requirement of Se for maximum weight gain was approached.
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10 20 40 60 10 20 40 60 10 20 40 60
3rd week
2nd week E.D.'
SELENIUM POTENCY
991
260
220
200
180
Added Selenium (yg/kg d i e t )
FIG. 1. Four-week weight gain as affected by various levels of selenium from different compounds. DISCUSSION The results of this study show that Se from selenomethionine was not as effective as Se from selenite or selenocystine. For either the production of weight gain or reduction of the incidence of exudative diathesis, selenomethionine Se was only about two-thirds as effective. This value is in fairly good agreement with that found by Noguchi et al. (1973), but generally higher than that found by Cantor
et al. (1975a). It should be noted that with the diet used by these researchers, the selenium requirement apparently is greater than in the diet used in this study. Their basal diet contained approximately 15 |xg. Se per kg. and some degree of Se deficiency was still evident after adding 60 |o,g. Se per kg. diet in the form of selenite. The basal diet used in this study contained approximately 30 jxg. Se per kg., and signs of a Se deficiency
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240
992
M . OSMAN AND J . D . LATSHAW
100
'
#Na.Se03 Ose-Cys
"
Ose-Met
60
3
6 Added S e l e n i u m
9
]2
(pg/kg d i e t )
FIG. 2. Incidence of exudative diathesis at four weeks as affected by various levels of selenium from different compounds.
were almost totally alleviated by the addition of 10 u.g. of selenite Se per kg. It is possible that the vitamin E contributed by corn in this study decreased the need for Se. Information on the ability of Se from the three different sources to prevent pancreatic
fibrosis was not obtained. However, no atrophy or other abnormal condition of the pancreas was noted during the collection of tissue for selenium analysis. The question arises as to why Se from selenite was biologically more effective than
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•
993
SELENIUM POTENCY
Schwarz and Feeney (1964) found that 75Se as selenite attached nonenzymatically to cystine and migrated with cystine in chromatographic separation. When 75Se was injected into a rabbit as selenite, 75Se was incorporated into protein (Cummins and Martin, 1967). When liver protein was hydrolyzed with enzymes and the amino acids chromatographed, 75 Se was present in the area of methionine and cystine. However, the 75 Se could be released from protein by dialysis with an alkaline solution of pH 11-12, and this treatment was not severe enough to release Se from selenocystine or selenomethionine standards. The evidence suggested no selenide formation, just an interaction between sulfur-containing amino acids and 7 5 Se0 3 = . Jenkins (1968) also found no evidence that chicks
could synthesize selenium-containing amino acids from inorganic Se. This report postulated that the Se molecule was present between the sulfur atoms of cystine. Based on the above information, in this experiment selenite was not converted to selenomethionine or selenocystine. Since the distribution of selenium in tissues is similar for selenite and selenocystine, and since the biological potency of selenite and selenocystine is similar, selenocystine is probably converted to an inorganic form. Although chicks can effectively use selenocystine prepared in the laboratory as a source of selenium it is doubtful that practical feed ingredients would contain any significant amount. Olson et al. (1970) did not find selenocystine in wheat, and animals probably cannot synthesize it. The pattern of selenium distribution in tisses may be a factor in the greater biological potency of Se from selenite and selenocystine than from selenomethionine. Selenomethionine feeding produced lower levels of Se in the kidney, liver, and heart than the other two Se compounds, although liver was the only tissue which was significantly lower. The Se content of breast muscle and pancreas, however, was higher when selenomethionine was fed. Why these different patterns occur is not yet known, but they appear to be related to the different biological potencies observed.
REFERENCES Allaway, W. H., E. E. Cary and C. F. Ehlig, 1967. The cycling of low levels of selenium in soils, plants, and animals. In: Selenium in Biomedicine (Muth, O.H., ed.), Avi Publishing Co., Inc., Westport, Conn. pp. 273-296. Cantor, A. H., M. L. Scott and T. Noguchi, 1975a. Biological availability of selenium in feedstuffs and selenium compounds for prevention of exudative diathesis in chicks. J. Nutr. 105: 96-105. Cantor, A. H., M. L. Langevin, T. Noguchi and M. L. Scott, 1975b. Efficacy of selenium in selenium compounds and feedstuffs for prevention of pancreatic fibrosis in chicks. J. Nutr. 105: 106-111.
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from selenomethionine in these studies. Data from Table 4 show that feeding selenomethionine results in more Se in the pancreas and muscle than feeding selenite. Since muscle comprises a large percentage of the body, more Se is probably retained from feeding selenomethionine than from selenite. Tissue Se values of hens fed practical diets in which most of the Se is in the form of selenomethionine support this view (Latshaw, 1975). If the total amount retained is not the primary factor in determining potency then the form in which Se is present may be more important. Some studies suggested that inorganic selenium could be converted into selenides or diselenides by animals. McConnell and Wabnitz (1957) injected a dog with 75 SeCl 4 and then examined acid hydrolysates of protein for 75Se distribution. After separation by paper chromatography 75Se was found in the areas of cystine and methionine. The data were interpreted to mean that selenocystine and selenomethionine had been synthesized. Rosenfeld (1962) also found similar evidence that 75Se could be incorporated into organic compounds. Synthesis of these compounds would have occurred only if Se was reduced from +4 to —2.
994
M. OSMAN AND J. D. LATSHAW
Noguchi, T., A. H. Cantor and M. L. Scott, 1973. Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks. J. Nutr. 103: 1502-1511. Olson, O. E., 1969. Fluorometric analysis of selenium in plants. J. Assoc. Offic. Anal. Chem. 52: 627-634. Olson, O. E., E. J. Novacek, E. I. Whitehead and I. S. Palmer, 1970. Investigations on selenium in wheat. Phytochemistry, 8: 1161-1165. Omaye, S. T., and A. L. Tappel, 1974. Effect of dietary selenium on glutathione peroxidase in the chick. J. Nutr. 104: 747-753. Rosenfeld, O., 1962. Biosynthesis of selenocompounds from inorganic selenium by sheep. Proc. Soc. Exptl. Biol. Med. I l l : 670-673. Schwarz, K., and E. Feeney, 1964. Selenite binding to sulfur amino acids. Fed. Proc. 23: 421.
Composition and Functional Properties of Mottled Yolksl F . E . CUNNINGHAM
Dairy and Poultry Science Department, Kansas State University, Manhattan, Kansas 66506 (Received for publication August 25, 1975)
ABSTRACT Yolk mottling was induced by feeding 0.02% Nicarbazin to 6-month old White Leghorn pullets. A second group of pullets, fed a standard layer ration, served as the control. Eggs were collected for three months on a daily basis, but the yolks were pooled on a weekly basis for analysis. Composition and functional properties of both mottled and normal yolks were determined. The percent solids of normal yolks was significantly higher than mottled yolks. The pH of normal yolks was lower than mottled yolks but not significantly. Normal yolks contained higher percentage fat, protein, and ash than mottled yolks; however, the percent carbohydrate was lower in normal yolks. There was no difference in the cholesterol content of mottled and normal yolks. Normal yolks contained significantly higher percent calcium, phosphorus, and iron than mottled yolks. Emulsifying capacity and viscosity of normal yolks were greater than for mottled yolks. Foam volume and sponge cake volume were always larger for mottled yolks than for normal yolks, but normal yolks produced more stable foam and better cake texture. Electrophoretic separation of the components of mottled and normal yolks indicated that two egg white proteins were present in mottled yolks but not apparent in normal yolks. The two proteins are believed to be ovalbumin and conalbumin. POULTRY SCIENCE 55: 994-998, 1976
INTRODUCTION
F
ACTORS commonly associated with mottled yolks were recently reviewed by Cunningham and Sanford (1973, 1974). Mot1. Contribution No. 918, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas 66506.
tling is common in commercial flocks and continues to be a severe problem of the poultry industry. Blackshear et al. (1968) reported that 56.1% of all eggs sampled from 43 flocks were mottled to some degree. Although many causes of mottling are known, very little is known about the composition or functional properties of mot-
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Cummins, L. M., and J. L. Martin, 1967. Are selenocystin and selenomethionine synthesized in vivo from sodium selenite in mammals? Biochemistry, 6: 3162-3168. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42. Jenkins, K. J., 1968. Evidence for the absence of selenocystine and selenomethionine in the serum proteins of chicks administered selenite. Can. J. Biochem. 46: 1417-1425. Latshaw, J. D., 1975. Natural and selenite selenium in the hen and egg. J. Nutr. 105: 32-37. Latshaw, J. D., and M. Osman, 1975. Distribution of selenium in egg white and yolk after feeding natural and synthetic selenium compounds. Poultry Sci. 54: 1244-1252. McConnell, K. P., and C. H. Wabnitz, 1957. Studies on the fixation of radioselenium in proteins. J. Biol. Chem. 226: 765-776.
