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Genetic Resistance to Deficiency of Riboflavin in the Chick
Genetic Resistance to Deficiency of Riboflavin in the Chick W. F. LAMOREUX* AND F. B. HUTT Cornell University, Ithaca, New York (Received for publication November 1, 1947)
* Present address: Kimber Poultry Breeding Farm, Niles, Calif.
eggs laid by different families ranged from 1.67 + 1.68 to 63.37 + 6.68 percent. Such differences, and similar ones between families or strains observed by Culbertson et al. (1932) in swine, by Gowen (1936) in rats, and by Fenton and Cowgill (1947) in mice, suggest that one could increase by selection the efficiency with which animals utilize either their complete diet or certain nutrients in it. The feasibility of doing so has been demonstrated by Morris, Palmer and Kennedy (1933) in the rat, and by Danish breeders in bacon hogs as reported by Lush (1936). This study was undertaken to test the possibility of developing a strain of White Leghorns relatively resistant to a deficiency of riboflavin in the diet. EXPERIMENTAL PROCEDURE
Experiment I Beginning in 1935 an attempt was made to develop strains of White Leghorns which had relatively high or low requirements of riboflavin for the production of eggs capable of hatching. During three generations of selection a significant difference between these strains was not attained: This apparently resulted from the unfortunate choice of hatchability as a criterion for selection, a measure commonly used at that time in studies of the nutritive requirements of fowl for riboflavin. Great variation in hatchability was present throughout the experiment, but selection on that basis was quite in-
334
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A ^ R E A T differences in the response of ^ - * individuals to different diets, or to certain deficiencies in the diet, are normally expected, and are commonly assumed to result, in part, from differences in genotype. Striking results of selection for resistance to a nutritional deficiency were obtained by Serfontein and Payne (1934) when, with only one generation of selection, they were able to segregate fowls 18.6 percent of whose chicks developed "slipped tendon" (perosis), as compared with 50 percent among chicks from parents which had been susceptible to the disorder. In view of a later study by Wilgus, Norris and Heuser (1937) we may assume that much of this difference was the result of differences in genetic resistance to a deficiency of manganese in the diet. A difference between breeds in their requirement for vitamin Bi (thiamine) was later demonstrated by Lamoreux and Hutt (1939). They found that White Leghorns consistently had a greater mean age at death than Rhode Island Reds when fed only a diet very deficient in that vitamin. Great differences in the apparent requirement for another vitamin were observed by Davis, Norris, and Heuser (1938). When White Leghorn families of three full sisters each were compared by feeding a diet deficient in riboflavin, the hatchability of the fertile
GENETIC RESISTANCE TO RIBOFLAVIN DEFICIENCY
effective, probably because other factors than the deficiency of riboflavin contributed much of the variation. This experiment was therefore abandoned in 1939. Experiment II
335
A second experiment was then undertaken to determine whether or not resistant and susceptible strains of White Leghorns could be developed by using other bases for selection. Resistance of chicks to a deficiency of riboflavin was measured by feeding them a diet deficient in that vitamin during their first three to six weeks after hatching. Those chicks which survived on the deficient diet until four or five weeks of age and made relatively large gains in body weight were selected as prospective breeders in the resistant strain. On the other hand, because the least resistant chicks were unable to survive for five weeks on the deficient diet, the breeders in the susceptible strain had to be selected from among those which gained least in body weight to three weeks of age. At that time the susceptible chicks were given an adequate diet and additional supplements of riboflavin in an effort to rear them to maturity. It was apparent that this practice did not retain the most susceptible chicks as breeders because such individuals died before they reached three weeks Pounds Corn meal, table 66.65 of age. It may therefore be assumed that Peanut meal IS. 00 breeders in the susceptible strain were inCasein (riboflavin extracted) 10.00 Salt mixture 5.00 termediate in their ability to survive, Soybean oil 3.00 because some selection for viability could Sardine or cod liver oil 0.25 Choline 0.10 not be avoided in this procedure. MicroThe foundation breeding stock comprised grams five males and twenty females from a Thiamine (vitamin Bi) 300 strain of White Leghorns maintained at Pyridoxine (vitamin Be) 500 Pantothenic acid 700 Cornell. From these breeders twenty. Extracts containing factors R and S from 6 lbs. families of chicks were obtained and of yeast. reared to four weeks of age upon the deficient diet. The surviving chicks of 12 Factor R has been identified as a folic acid
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families (3 to 14 chicks each) were then selected to start two strains. Chicks from six families showed an unweighted average gain in weight of 182 grams and were considered resistant to a deficiency of riboflavin. Chicks from the other families made gains of only 126 grams and were thereafter called susceptible. No exchange of birds between these two "strains" was made after that time, and no birds from other sources were introduced. Control chicks were obtained from two different sources. Some were from hens kepts in cages and receiving an adequate all-mash diet, just as did breeders of the resistant and susceptible strains. Others were from birds kept in pens and fed an adequate diet of mash and scratch grain. Because chicks from these two sources did not differ appreciably in their response to the experimental diet, they have been combined. The diet used was devised by Dr. L. C. Norris and his associates of the Department of Poultry Husbandry, Cornell University. They kindly furnished the feed or assisted in its preparation. It was changed once during this experiment to reduce the amount of riboflavin it contained, and to furnish nutrients not previously known to be required by the chick. The diet used for chicks in the last two generations is described below.
336
W. F. LAMOREUX AND F. B. HUTT
conjugate. Factor S is believed to be a "complex" in which strepogenin is an important ingredient. RESULTS OF SELECTION Do Sexes Differ?
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FIG. 1. Mortality among White Leghorn chicks from strains selected for resistance and susceptibility to a deficiency of riboflavin, and among unselected controls.
Mortality in the fifth generation, when mortality in both strains was relatively low, was only 6 percent among 104 females in the resistant strain, compared with 13 percent among 94 males. In the sixth generation 50 percent of 173 females died, whereas 53 percent of 171 males did so. This nearly equal mortality in the sixth and critical generation makes it reasonable to combine the data from both sexes for this study. Gain in weight is normally more rapid in male chicks than in females, so a pre-
males. Because the males did not excel the females in rate of growth during the period studied, it has not been necessary to make any correction for sex. Strain Differences in. Mortality Mortality among chicks receiving a diet very deficient in riboflavin is normally expected to be much higher than when an adequate diet is fed. After 5 generations of selection (1938-1942) chicks of the sixth generation were tested for resistance and susceptibility by raising them to five
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 26, 2015
It was recognized that males and females might not be equally resistant to the deficient diet, and might, therefore, require separate study.
liminary comparison of the body weights in the two sexes is required before the rate of growth in the two strains can be compared. Surviving females of the resistant strain in the fifth generation made average gains in body weight of 60 grams, 4 grams less than that attained by males. In the sixth generation, however, corresponding gains were 72 grams among 73 females compared to only 70 grams among 64
GENETIC RESISTANCE TO RIBOFLAVIN DEFICIENCY
weeks of age on the deficient diet. The chicks tested were of two slightly different sorts in each line, since some of them came from progeny-tested dams (hens) and others from untested dams (pullets). In both of these groups mortality was lower in the resistant strain than in the susceptible one (Figure 1).
The progeny test enables the investigator to sort families into resistant and susceptible groups. This was done in the original selection of breeders for the two strains. It may be assumed that part of the difference between such groups is genetic, but further evidence is required to prove it. This was first provided by the fact that those hens selected because of the relative resistance or susceptibility of their progeny produced, from other sires not previously tested, additional generations of chicks which differed in the same way. This is illustrated in Figure 1. Only 25 percent of .the chicks from dams that previously produced resistant progeny died while receiving the deficient diet, i.e. to five.weeks of age. Among the progeny of dams selected for susceptibility 38.5 percent died, and among control chicks from hens of equal or greater age 50 percent died. Thus the mortality was twice as great among controls as among the progeny of dams selected for resistance to the vitamin deficiency. Because the mortality in the control chicks was as high as in the susceptible ones, or higher, it is clear that selection for increasing susceptibility had been ineffective. In the families of chicks that were most susceptible, all or nearly all of the birds died. Because no measure of gain in weight was obtained and successive generations were not available, such dams were not retained, and the mortal-
ity among the progeny of progeny-tested dams in the susceptible strain was therefore lower than among controls. Untested Parents The second method used for measuring the effectiveness of selection was to compare the progeny of pullets selected from the two strains on the basis of their individual performance, and that of their siblings. If the relative resistance of the individual or of the family of sisters resulted from something other than heredity, i.e. some unknown maternal influence, their offspring should be equally resistant, whether they came from the resistant or susceptible strain. That this is not the case is apparent from Figure 1, where it is shown that among the progeny of untested parents (pullets) the mortality among chicks from the susceptible strain (64 percent) was one-third higher than among those from the resistant strain (48 percent). In this comparison, unlike that between progenytested dams, no previous selection has removed the dams of most susceptible families, and chicks of the susceptible strain suffered as much mortality as did the controls. All groups of progeny from untested dams showed higher mortality than corresponding groups from the older progenytested hens despite the fast that their chicks were mixed together in the brooders. This probably results from the same factors that contribute to greater viability among the progeny of two-year-old than of yearling hens when adequate diets are used. The natural culling of the least viable hens by death during their first year was one such cause, and the elimination of dams whose progeny failed to live (in the susceptible strain) or suffered high mortality (in the resistant strain) was another and probably more effective cause.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 26, 2015
Progeny-tested Dams
337
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W. F. LAMOREUX AND F. B. HUTT
Strain Differences in Rate of Growth
Untested Parents Pullets produced chicks in the sixth generation which gained 70.1 grams in 5 weeks, which was 57 percent greater than the gain made by chicks of the susceptible strain (44.6 grams). This difference was
Progeny-tested Dams Progeny-tested dams of the resistant strain produced chicks in the sixth generation whose average gain in weight to five 100
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CONT.
RES.
SUSC.
• CONT.
PROGENY OF HENS
FIG. 2. The average gains in weight to five weeks of age of White Leghorn chicks from strains selected for resistance and susceptibility to a deficiency of riboflavin, and of unselected controls.
weeks of age on the deficient diet (89.9 grams) exceeded the gain among comparable chicks of the susceptible strain (49.4 grams) by more than 80 percent (Figure 2). These chicks were all the progeny of cockerels not previously used as breeders, and presumably this difference between the two strains is therefore less than could be attained by using progeny-tested sires with such dams. Control chicks were intermediate with respect to gain in weight.
less than that between chicks from progeny-tested dams, a result that would obviously be expected. However, the difference is significant (value for P =0.03) and of sufficient magnitude to demonstrate clearly that selection was effective in developing a strain capable of relatively good growth while receiving a diet deficient in riboflavin. It can be seen in Figure 2 that the difference between chicks from progenytested hens and from untested pullets is
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RES.
PROGENY OF PULLETS
339
GENETIC RESISTANCE TO RIBOFLAVIN DEFICIENCY
Specific Resistance The evidence presented thus far shows that the resistant and susceptible strains and individuals within those strains differ in their ability to survive and grow while receiving the diet deficient in riboflavin. This difference might result either from a specific difference in the requirements of the two strains for riboflavin, or the two strains might show corresponding differences irrespective of the diet they receive. This problem was studied by comparing the growth of chicks from the two strains when they were fed a diet adequate in riboflavin, the same diet used in that year for rearing chicks in the station flock. Because the progeny of different hens within either strain may differ in their rate of growth, no chicks were included in this comparison unless sibs survived in the groups receiving each diet, and unweighted rather than weighted averages were used for comparing the two strains. Chicks from both strains were mixed together in the brooders to ensure comparable treatment. The results obtained in the sixth generation are shown in Table 1. If the differences observed in earlier tests with the deficient diet (Figures 1 and 2) were not the result of specific differences in requirement for riboflavin, one would expect an even greater difference on an adequate diet that permitted normal
growth. This did not occur. It is true that chicks from the resistant strain made greater gains in weight in the groups receiving each of the diets. However, when the adequate diet was fed, their gain exceeded those of chicks from the susceptible strain by only 7 grams, an insignificant difference of less than 4 percent. In contrast, on the TABLE 1.—The gain in weight to 5 weeks of age of White Leghorn chicks from the sixth generation of resistant and susceptible strains when fed either a diet deficient in riboflavin or a diet containing adequate amounts of that vitamin. Deficient diet Strain Chicks Resistant Susceptible Difference
94 18
Adequate diet
Gain in wt. Chicks (gm.) 1 71 49 22*
34 17
Gain in wt. (gm-)1 202 195 7
1
Unweighted averages. * Values for P = < 0 . 0 5 , if calculated from the average gains made by full sibs. Value for P = < 0 . 6 l , if calculated from the gains made by individuals.
deficient diet, despite the facts that few of the susceptible chicks survived and that relatively small gains were made by both strains, there was a significant difference between strains of 22 grams, almost half as great as the total gain of the susceptible strain (value for P = <0.05 if calculated from the averages of families of full sibs, and <0.01 if calculated from the gains made by individuals). These results show that little difference in growth between the two strains is to be expected when an adequate diet is fed. The marked differences shown to exist when the chicks receive a diet deficient in riboflavin are therefore most logically attributable to specific differences in the genetic requirements for riboflavin in the two strains.
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somewhat greater in the resistant than in the susceptible strain. This probably results from two facts. First, because of high mortality in the susceptible strain, the number of birds available for the selection of breeders was always smaller than in the resistant strain, and, second, the most susceptible chicks died so that no measure of growth was obtained. Selection of females on the basis of the progeny test therefore produced more apparent results in the resistant strain.
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requirement in the feed. It has not yet been shown that any vertebrates can themselves synthesize this vitamin, although the possibility exists that it may be manufactured by the bacteria in the intestine. Tatum (1944) found that every yeast and mould investigated by him was able to synthesize riboflavin. However, tests by Lamoreux and Schumacher (1940) indicated that any such synthesis was likely to be on an insignificant scale within the body of the fowl, although it went on rather rapidly in the faeces after defecation. It seems more likely that the difference between birds with high or low requirement of riboflavin lies in ability to utilize the supply of that vitamin in the feed. This should not be surprising because such a genetic difference has been demonstrated with respect to one of the other vitamins of the B complex, namely, thiamine. White Leghorns utilize thiamine more efficiently than do Barred Rocks and Rhode Island Reds, both as chicks (Lamoreux and Hutt, 1939) and as adults (Scrimshaw et ah, 1945). No similar difference between breeds of fowls has been demonstrated with respect to riboflavin. Analyses of eggs from 12 different breeds or varieties by Jackson et al. (1946) showed that, although all birds ate the same diet, the amount of riboflavin put in their eggs varied in different breeds from 2.91 to 3.71 micrograms per gram of egg. The significance of the differences was apparently not tested. SUMMARY Selection of White Leghorns relatively resistant or susceptible to a deficiency of riboflavin in the diet was practised during six generations. When fed the experimental diet, chicks from the resistant strain suffered less mortality and survivors made significantly greater gains in body weight
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DISCUSSION The production of strains of fowl having relatively low requirements for riboflavin is possible. By the standards of commercial poultrymen, however, even the most resistant chicks obtained by five generations of selection were unable to make normal growth on the deficient diet. The long period of time required and the high cost of selection as practised in this experiment indicate that commercial development of strains resistant to this nutritional deficiency is not justified under present conditions. The striking differences between individuals in their susceptibility to a deficiency of this vitamin suggest, however, that in the case of certain expensive ingredients in the diet, it may be genetically undesirable and economically unsound to feed amounts that will enable the few highly susceptible chicks in a population to thrive. It should also be recognized that, because of the expense and difficulty of practising selection of the type used here, the genotypes of only a few foundation birds could be tested. This would be a very serious handicap if the desired genes are rare, and different experiments conducted on this basis should be expected to yield different results. This was true in an experiment conducted by Lerner and Bird (1948), in which some resistance to a deficiency of riboflavin was observed. Unlike the present experiment their results show that no specific resistance to a deficiency of riboflavin was obtained. In selecting for resistance and susceptibility to the deficient diet two strains were developed which showed similar differences in rate of growth when an adequate diet was fed. The genetic differences revealed by selection in this experiment should not be interpreted as indicating any synthesis of riboflavin by the birds having the lower
GENETIC RESISTANCE TO RIBOFLAVIN DEFICIENCY
than did chicks in the susceptible strain. Because chicks from the two strains made comparable growth when fed an adequate diet, it is believed that there were genetic differences between them which were specifically concerned with the utilization of riboflavin. REFERENCES
Lamoreux, W. F., and F. B. Hutt, 1939. Breed differences in resistance to a deficiency of vitamin Bi in the fowl. J. Agric. Res. 58:307-316. Lamoreux, W. F., and A. E. Schumacher, 1940. Is riboflavin synthesized in the feces of the fowl? Poultry Sci. 19: 418^23. Lerner, I. M., and F. H. Bird, 1948. Experiments on selection for resistance to riboflavin deficiency in S. C. White Leghorns. Poultry Sci. 27: 342-346. Lush, J. L., 1936. Genetic aspects of the Danish system of progeny testing swine. Iowa Agr. Exp. Res. Bui. 204. Morris, H. P., L. S. Palmer, and C. Kennedy, 1933. Fundamental food requirements for the growth of of the rat VII. An experimental study of inheritance as a factor influencing food utilization in the rat. Minn. Agr. Exp. Sta. Tech. Bui. 92. Scrimshaw, N. S., F. B. Hutt, and M. W. Scrimshaw, 1945. The effect of genetic variation in the fowl on the thiamine content of the egg. J. Nutrition 30: 375-383. Serfontein, P. J., and L. F. Payne, 1934. Inheritance of abnormal anatomical condition in the tibial metatarsal joints. Poultry Sci. 13: 61-63. Tatum, E. L., 1944. The biochemistry of fungi. Ann. Rev. Biochem. 13: 667-704. Wilgus, H. S., Jr., L. C. Norris, and G. F. Heuser, 1937. The role of manganese and certain other trace elements in the prevention of perosis. J. Nutr. 14: 155-167.
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Culbertson, C. C , H. H. Kildee, M. D. Helser, and W. E. Hammond, 1932. Swine performance record—litter comparisons. Series I I . Iowa Agricultural Exp. Sta. Leaflet No. 28. Davis, H. J., L. C. Norris, and G. F. Heuser, 1938. Further evidence on the amount of vitamin G required for reproduction in poultry. Poultry Sci. 17: 87-93. Fenton, P. F., and G. R. Cowgill, 1947. The nutrition of the mouse I. A difference in the riboflavin requirements of two highly inbred strains. J. Nutr. 34: 273-283. Gowen, J. W., 1936. Inheritance as it affects survival of rats fed a diet deficient in vitamin D. Genetics 21: 1-23. Jackson, S. H., T. G. H. Drake, S. J. Slinger, E. V. Evans, and R. Pocock, 1946. The influence of riboflavin consumption on its concentration in hens' eggs. J. Nutrition 32: 567-581.
341
* Present address: Kimber Poultry Breeding Farm, Niles, Calif.
eggs laid by different families ranged from 1.67 + 1.68 to 63.37 + 6.68 percent. Such differences, and similar ones between families or strains observed by Culbertson et al. (1932) in swine, by Gowen (1936) in rats, and by Fenton and Cowgill (1947) in mice, suggest that one could increase by selection the efficiency with which animals utilize either their complete diet or certain nutrients in it. The feasibility of doing so has been demonstrated by Morris, Palmer and Kennedy (1933) in the rat, and by Danish breeders in bacon hogs as reported by Lush (1936). This study was undertaken to test the possibility of developing a strain of White Leghorns relatively resistant to a deficiency of riboflavin in the diet. EXPERIMENTAL PROCEDURE
Experiment I Beginning in 1935 an attempt was made to develop strains of White Leghorns which had relatively high or low requirements of riboflavin for the production of eggs capable of hatching. During three generations of selection a significant difference between these strains was not attained: This apparently resulted from the unfortunate choice of hatchability as a criterion for selection, a measure commonly used at that time in studies of the nutritive requirements of fowl for riboflavin. Great variation in hatchability was present throughout the experiment, but selection on that basis was quite in-
334
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 26, 2015
A ^ R E A T differences in the response of ^ - * individuals to different diets, or to certain deficiencies in the diet, are normally expected, and are commonly assumed to result, in part, from differences in genotype. Striking results of selection for resistance to a nutritional deficiency were obtained by Serfontein and Payne (1934) when, with only one generation of selection, they were able to segregate fowls 18.6 percent of whose chicks developed "slipped tendon" (perosis), as compared with 50 percent among chicks from parents which had been susceptible to the disorder. In view of a later study by Wilgus, Norris and Heuser (1937) we may assume that much of this difference was the result of differences in genetic resistance to a deficiency of manganese in the diet. A difference between breeds in their requirement for vitamin Bi (thiamine) was later demonstrated by Lamoreux and Hutt (1939). They found that White Leghorns consistently had a greater mean age at death than Rhode Island Reds when fed only a diet very deficient in that vitamin. Great differences in the apparent requirement for another vitamin were observed by Davis, Norris, and Heuser (1938). When White Leghorn families of three full sisters each were compared by feeding a diet deficient in riboflavin, the hatchability of the fertile
GENETIC RESISTANCE TO RIBOFLAVIN DEFICIENCY
effective, probably because other factors than the deficiency of riboflavin contributed much of the variation. This experiment was therefore abandoned in 1939. Experiment II
335
A second experiment was then undertaken to determine whether or not resistant and susceptible strains of White Leghorns could be developed by using other bases for selection. Resistance of chicks to a deficiency of riboflavin was measured by feeding them a diet deficient in that vitamin during their first three to six weeks after hatching. Those chicks which survived on the deficient diet until four or five weeks of age and made relatively large gains in body weight were selected as prospective breeders in the resistant strain. On the other hand, because the least resistant chicks were unable to survive for five weeks on the deficient diet, the breeders in the susceptible strain had to be selected from among those which gained least in body weight to three weeks of age. At that time the susceptible chicks were given an adequate diet and additional supplements of riboflavin in an effort to rear them to maturity. It was apparent that this practice did not retain the most susceptible chicks as breeders because such individuals died before they reached three weeks Pounds Corn meal, table 66.65 of age. It may therefore be assumed that Peanut meal IS. 00 breeders in the susceptible strain were inCasein (riboflavin extracted) 10.00 Salt mixture 5.00 termediate in their ability to survive, Soybean oil 3.00 because some selection for viability could Sardine or cod liver oil 0.25 Choline 0.10 not be avoided in this procedure. MicroThe foundation breeding stock comprised grams five males and twenty females from a Thiamine (vitamin Bi) 300 strain of White Leghorns maintained at Pyridoxine (vitamin Be) 500 Pantothenic acid 700 Cornell. From these breeders twenty. Extracts containing factors R and S from 6 lbs. families of chicks were obtained and of yeast. reared to four weeks of age upon the deficient diet. The surviving chicks of 12 Factor R has been identified as a folic acid
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 26, 2015
families (3 to 14 chicks each) were then selected to start two strains. Chicks from six families showed an unweighted average gain in weight of 182 grams and were considered resistant to a deficiency of riboflavin. Chicks from the other families made gains of only 126 grams and were thereafter called susceptible. No exchange of birds between these two "strains" was made after that time, and no birds from other sources were introduced. Control chicks were obtained from two different sources. Some were from hens kepts in cages and receiving an adequate all-mash diet, just as did breeders of the resistant and susceptible strains. Others were from birds kept in pens and fed an adequate diet of mash and scratch grain. Because chicks from these two sources did not differ appreciably in their response to the experimental diet, they have been combined. The diet used was devised by Dr. L. C. Norris and his associates of the Department of Poultry Husbandry, Cornell University. They kindly furnished the feed or assisted in its preparation. It was changed once during this experiment to reduce the amount of riboflavin it contained, and to furnish nutrients not previously known to be required by the chick. The diet used for chicks in the last two generations is described below.
336
W. F. LAMOREUX AND F. B. HUTT
conjugate. Factor S is believed to be a "complex" in which strepogenin is an important ingredient. RESULTS OF SELECTION Do Sexes Differ?
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FIG. 1. Mortality among White Leghorn chicks from strains selected for resistance and susceptibility to a deficiency of riboflavin, and among unselected controls.
Mortality in the fifth generation, when mortality in both strains was relatively low, was only 6 percent among 104 females in the resistant strain, compared with 13 percent among 94 males. In the sixth generation 50 percent of 173 females died, whereas 53 percent of 171 males did so. This nearly equal mortality in the sixth and critical generation makes it reasonable to combine the data from both sexes for this study. Gain in weight is normally more rapid in male chicks than in females, so a pre-
males. Because the males did not excel the females in rate of growth during the period studied, it has not been necessary to make any correction for sex. Strain Differences in. Mortality Mortality among chicks receiving a diet very deficient in riboflavin is normally expected to be much higher than when an adequate diet is fed. After 5 generations of selection (1938-1942) chicks of the sixth generation were tested for resistance and susceptibility by raising them to five
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 26, 2015
It was recognized that males and females might not be equally resistant to the deficient diet, and might, therefore, require separate study.
liminary comparison of the body weights in the two sexes is required before the rate of growth in the two strains can be compared. Surviving females of the resistant strain in the fifth generation made average gains in body weight of 60 grams, 4 grams less than that attained by males. In the sixth generation, however, corresponding gains were 72 grams among 73 females compared to only 70 grams among 64
GENETIC RESISTANCE TO RIBOFLAVIN DEFICIENCY
weeks of age on the deficient diet. The chicks tested were of two slightly different sorts in each line, since some of them came from progeny-tested dams (hens) and others from untested dams (pullets). In both of these groups mortality was lower in the resistant strain than in the susceptible one (Figure 1).
The progeny test enables the investigator to sort families into resistant and susceptible groups. This was done in the original selection of breeders for the two strains. It may be assumed that part of the difference between such groups is genetic, but further evidence is required to prove it. This was first provided by the fact that those hens selected because of the relative resistance or susceptibility of their progeny produced, from other sires not previously tested, additional generations of chicks which differed in the same way. This is illustrated in Figure 1. Only 25 percent of .the chicks from dams that previously produced resistant progeny died while receiving the deficient diet, i.e. to five.weeks of age. Among the progeny of dams selected for susceptibility 38.5 percent died, and among control chicks from hens of equal or greater age 50 percent died. Thus the mortality was twice as great among controls as among the progeny of dams selected for resistance to the vitamin deficiency. Because the mortality in the control chicks was as high as in the susceptible ones, or higher, it is clear that selection for increasing susceptibility had been ineffective. In the families of chicks that were most susceptible, all or nearly all of the birds died. Because no measure of gain in weight was obtained and successive generations were not available, such dams were not retained, and the mortal-
ity among the progeny of progeny-tested dams in the susceptible strain was therefore lower than among controls. Untested Parents The second method used for measuring the effectiveness of selection was to compare the progeny of pullets selected from the two strains on the basis of their individual performance, and that of their siblings. If the relative resistance of the individual or of the family of sisters resulted from something other than heredity, i.e. some unknown maternal influence, their offspring should be equally resistant, whether they came from the resistant or susceptible strain. That this is not the case is apparent from Figure 1, where it is shown that among the progeny of untested parents (pullets) the mortality among chicks from the susceptible strain (64 percent) was one-third higher than among those from the resistant strain (48 percent). In this comparison, unlike that between progenytested dams, no previous selection has removed the dams of most susceptible families, and chicks of the susceptible strain suffered as much mortality as did the controls. All groups of progeny from untested dams showed higher mortality than corresponding groups from the older progenytested hens despite the fast that their chicks were mixed together in the brooders. This probably results from the same factors that contribute to greater viability among the progeny of two-year-old than of yearling hens when adequate diets are used. The natural culling of the least viable hens by death during their first year was one such cause, and the elimination of dams whose progeny failed to live (in the susceptible strain) or suffered high mortality (in the resistant strain) was another and probably more effective cause.
Downloaded from http://ps.oxfordjournals.org/ at Purdue University Libraries ADMN on May 26, 2015
Progeny-tested Dams
337
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W. F. LAMOREUX AND F. B. HUTT
Strain Differences in Rate of Growth
Untested Parents Pullets produced chicks in the sixth generation which gained 70.1 grams in 5 weeks, which was 57 percent greater than the gain made by chicks of the susceptible strain (44.6 grams). This difference was
Progeny-tested Dams Progeny-tested dams of the resistant strain produced chicks in the sixth generation whose average gain in weight to five 100
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RES.
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PROGENY OF HENS
FIG. 2. The average gains in weight to five weeks of age of White Leghorn chicks from strains selected for resistance and susceptibility to a deficiency of riboflavin, and of unselected controls.
weeks of age on the deficient diet (89.9 grams) exceeded the gain among comparable chicks of the susceptible strain (49.4 grams) by more than 80 percent (Figure 2). These chicks were all the progeny of cockerels not previously used as breeders, and presumably this difference between the two strains is therefore less than could be attained by using progeny-tested sires with such dams. Control chicks were intermediate with respect to gain in weight.
less than that between chicks from progeny-tested dams, a result that would obviously be expected. However, the difference is significant (value for P =0.03) and of sufficient magnitude to demonstrate clearly that selection was effective in developing a strain capable of relatively good growth while receiving a diet deficient in riboflavin. It can be seen in Figure 2 that the difference between chicks from progenytested hens and from untested pullets is
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RES.
PROGENY OF PULLETS
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GENETIC RESISTANCE TO RIBOFLAVIN DEFICIENCY
Specific Resistance The evidence presented thus far shows that the resistant and susceptible strains and individuals within those strains differ in their ability to survive and grow while receiving the diet deficient in riboflavin. This difference might result either from a specific difference in the requirements of the two strains for riboflavin, or the two strains might show corresponding differences irrespective of the diet they receive. This problem was studied by comparing the growth of chicks from the two strains when they were fed a diet adequate in riboflavin, the same diet used in that year for rearing chicks in the station flock. Because the progeny of different hens within either strain may differ in their rate of growth, no chicks were included in this comparison unless sibs survived in the groups receiving each diet, and unweighted rather than weighted averages were used for comparing the two strains. Chicks from both strains were mixed together in the brooders to ensure comparable treatment. The results obtained in the sixth generation are shown in Table 1. If the differences observed in earlier tests with the deficient diet (Figures 1 and 2) were not the result of specific differences in requirement for riboflavin, one would expect an even greater difference on an adequate diet that permitted normal
growth. This did not occur. It is true that chicks from the resistant strain made greater gains in weight in the groups receiving each of the diets. However, when the adequate diet was fed, their gain exceeded those of chicks from the susceptible strain by only 7 grams, an insignificant difference of less than 4 percent. In contrast, on the TABLE 1.—The gain in weight to 5 weeks of age of White Leghorn chicks from the sixth generation of resistant and susceptible strains when fed either a diet deficient in riboflavin or a diet containing adequate amounts of that vitamin. Deficient diet Strain Chicks Resistant Susceptible Difference
94 18
Adequate diet
Gain in wt. Chicks (gm.) 1 71 49 22*
34 17
Gain in wt. (gm-)1 202 195 7
1
Unweighted averages. * Values for P = < 0 . 0 5 , if calculated from the average gains made by full sibs. Value for P = < 0 . 6 l , if calculated from the gains made by individuals.
deficient diet, despite the facts that few of the susceptible chicks survived and that relatively small gains were made by both strains, there was a significant difference between strains of 22 grams, almost half as great as the total gain of the susceptible strain (value for P = <0.05 if calculated from the averages of families of full sibs, and <0.01 if calculated from the gains made by individuals). These results show that little difference in growth between the two strains is to be expected when an adequate diet is fed. The marked differences shown to exist when the chicks receive a diet deficient in riboflavin are therefore most logically attributable to specific differences in the genetic requirements for riboflavin in the two strains.
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somewhat greater in the resistant than in the susceptible strain. This probably results from two facts. First, because of high mortality in the susceptible strain, the number of birds available for the selection of breeders was always smaller than in the resistant strain, and, second, the most susceptible chicks died so that no measure of growth was obtained. Selection of females on the basis of the progeny test therefore produced more apparent results in the resistant strain.
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requirement in the feed. It has not yet been shown that any vertebrates can themselves synthesize this vitamin, although the possibility exists that it may be manufactured by the bacteria in the intestine. Tatum (1944) found that every yeast and mould investigated by him was able to synthesize riboflavin. However, tests by Lamoreux and Schumacher (1940) indicated that any such synthesis was likely to be on an insignificant scale within the body of the fowl, although it went on rather rapidly in the faeces after defecation. It seems more likely that the difference between birds with high or low requirement of riboflavin lies in ability to utilize the supply of that vitamin in the feed. This should not be surprising because such a genetic difference has been demonstrated with respect to one of the other vitamins of the B complex, namely, thiamine. White Leghorns utilize thiamine more efficiently than do Barred Rocks and Rhode Island Reds, both as chicks (Lamoreux and Hutt, 1939) and as adults (Scrimshaw et ah, 1945). No similar difference between breeds of fowls has been demonstrated with respect to riboflavin. Analyses of eggs from 12 different breeds or varieties by Jackson et al. (1946) showed that, although all birds ate the same diet, the amount of riboflavin put in their eggs varied in different breeds from 2.91 to 3.71 micrograms per gram of egg. The significance of the differences was apparently not tested. SUMMARY Selection of White Leghorns relatively resistant or susceptible to a deficiency of riboflavin in the diet was practised during six generations. When fed the experimental diet, chicks from the resistant strain suffered less mortality and survivors made significantly greater gains in body weight
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DISCUSSION The production of strains of fowl having relatively low requirements for riboflavin is possible. By the standards of commercial poultrymen, however, even the most resistant chicks obtained by five generations of selection were unable to make normal growth on the deficient diet. The long period of time required and the high cost of selection as practised in this experiment indicate that commercial development of strains resistant to this nutritional deficiency is not justified under present conditions. The striking differences between individuals in their susceptibility to a deficiency of this vitamin suggest, however, that in the case of certain expensive ingredients in the diet, it may be genetically undesirable and economically unsound to feed amounts that will enable the few highly susceptible chicks in a population to thrive. It should also be recognized that, because of the expense and difficulty of practising selection of the type used here, the genotypes of only a few foundation birds could be tested. This would be a very serious handicap if the desired genes are rare, and different experiments conducted on this basis should be expected to yield different results. This was true in an experiment conducted by Lerner and Bird (1948), in which some resistance to a deficiency of riboflavin was observed. Unlike the present experiment their results show that no specific resistance to a deficiency of riboflavin was obtained. In selecting for resistance and susceptibility to the deficient diet two strains were developed which showed similar differences in rate of growth when an adequate diet was fed. The genetic differences revealed by selection in this experiment should not be interpreted as indicating any synthesis of riboflavin by the birds having the lower
GENETIC RESISTANCE TO RIBOFLAVIN DEFICIENCY
than did chicks in the susceptible strain. Because chicks from the two strains made comparable growth when fed an adequate diet, it is believed that there were genetic differences between them which were specifically concerned with the utilization of riboflavin. REFERENCES
Lamoreux, W. F., and F. B. Hutt, 1939. Breed differences in resistance to a deficiency of vitamin Bi in the fowl. J. Agric. Res. 58:307-316. Lamoreux, W. F., and A. E. Schumacher, 1940. Is riboflavin synthesized in the feces of the fowl? Poultry Sci. 19: 418^23. Lerner, I. M., and F. H. Bird, 1948. Experiments on selection for resistance to riboflavin deficiency in S. C. White Leghorns. Poultry Sci. 27: 342-346. Lush, J. L., 1936. Genetic aspects of the Danish system of progeny testing swine. Iowa Agr. Exp. Res. Bui. 204. Morris, H. P., L. S. Palmer, and C. Kennedy, 1933. Fundamental food requirements for the growth of of the rat VII. An experimental study of inheritance as a factor influencing food utilization in the rat. Minn. Agr. Exp. Sta. Tech. Bui. 92. Scrimshaw, N. S., F. B. Hutt, and M. W. Scrimshaw, 1945. The effect of genetic variation in the fowl on the thiamine content of the egg. J. Nutrition 30: 375-383. Serfontein, P. J., and L. F. Payne, 1934. Inheritance of abnormal anatomical condition in the tibial metatarsal joints. Poultry Sci. 13: 61-63. Tatum, E. L., 1944. The biochemistry of fungi. Ann. Rev. Biochem. 13: 667-704. Wilgus, H. S., Jr., L. C. Norris, and G. F. Heuser, 1937. The role of manganese and certain other trace elements in the prevention of perosis. J. Nutr. 14: 155-167.
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Culbertson, C. C , H. H. Kildee, M. D. Helser, and W. E. Hammond, 1932. Swine performance record—litter comparisons. Series I I . Iowa Agricultural Exp. Sta. Leaflet No. 28. Davis, H. J., L. C. Norris, and G. F. Heuser, 1938. Further evidence on the amount of vitamin G required for reproduction in poultry. Poultry Sci. 17: 87-93. Fenton, P. F., and G. R. Cowgill, 1947. The nutrition of the mouse I. A difference in the riboflavin requirements of two highly inbred strains. J. Nutr. 34: 273-283. Gowen, J. W., 1936. Inheritance as it affects survival of rats fed a diet deficient in vitamin D. Genetics 21: 1-23. Jackson, S. H., T. G. H. Drake, S. J. Slinger, E. V. Evans, and R. Pocock, 1946. The influence of riboflavin consumption on its concentration in hens' eggs. J. Nutrition 32: 567-581.
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