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Vitamin B12 in developing chick embryos from vitamin B12-deficient and control eggs
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
90,
250-253 (1960)
Vitamin B,, in Developing Vitamin Blz-Deficient LOUISE
J. DANIEL,
CAROL
Chick Embryos from and Control Eggs
A. HILARY
AKD
DAVID
W. YESAIR’
From the Department of Biochemistry and Nutrition and The Graduate Nutrition, Cornell Universit,y, Ithaca, New I’ork Received
School of
May 3, 1960
The vitamin B12 contents of the normal and vitamin Blr-deficient chick embryos and yolk sacs were determined at 12, 15, 18, and 21 days of age, by the use of Ochromonas malhamensis. From the 12th day of incubation on there was a decrease in the vitamin B~z content of the yolk sac with the greatest reduction occurring between the 15th and 18th days. The greatest increase in the vitamin BOXcontent of the control embryo occurred during the same period. In the deficient embryo the largest increase occurred between the 18th and 21st days. The vitamin BIz content of the whole egg (embryo plus yolk sac) decreased between the 15th and 21st days of incubation. The fate of the vitamin B12 not, recovered is unknown, but it, is possible that it was metabolized to a substance not utilized by the assay organism.
vitamin Bla activity during the incubation of the egg. The results to be reported here were obtained using Ochromonas malhamensis. This organism was chosen because it> is the most specific for vitamin B1z and does not, respond to vitamin I&-like factors or deoxyribosides (5).
INTRODUCTION
In the course of an extensive study of the effect of a vit#amin B,, deficiency on the developing chick embryo (1,2), determinations of t,he vitamin Bll contents of the embryos and yolk sacs2 were made at various time int,ervals aft#er t,he start, of incubation. While this study was in progress, two papers appeared in t’he literature on the vitamin B12 content,
of
t,he
developing
chick
EXPERIMENTAL
embryo.
Fischer, Benson, and Swendseid (3) using leichmannii as the assay organism found the vitamin B12 activit)y to increase in the egg during incubat’ion. On the other hand, Skarzynski et al. (4) reported no increase in the vit,amin B,, conteut of the egg during incubation when Ochromonas malhamensis or Lactobacillus leichwas used, whereas with Ezcglena mannii gracilis t,here was a progressive increase in
hODUC!TION
Lactobacillus
1 Public Health Service Research Predoctoral Fellow of the National Institute of i%eurological Diseases and Blindness. Present address: Lederle La.horatories Division, American Cyanamid Co., Pearl River, N. Y. 2 All contents of the egg except the embryo.
4ND OF
PREPAR.4TIOS
EMBE~YOS
Twenty-four Single Comb White Leghorn hens were placed in individual laying cages on raised wire floors. Twelve hens received a vitamin BIPdeficient diet (1) and 12 hens received the deficient diet plus 9 pg. BJlb. diet. After 1 weeks on the experimental diets, the hens were artificially inseminated t.wice weekly using pooled semen from Single Comb White Leghorn roosters, and the eggs secured subsequently were incubated at weekly intervals. At t,he end of the 5th week the hatchahilit,y of the deficient eggs was about 5O’A. From the 6th t,hrough the 12th Lveeks, living embryos were taken from eggs at, 12, 15, 18, and 21 days of age. The embryo was dissected from the yolk sac, a small quantity of blood was with250
VITAMIN
B~.L IS
drawn from the exposed heart, and the liver \vtls removed. Five embryos were pooled at 12 days (a total of four composite samples of both normal and deficient embryos), 3-4 embryos per composite at 15 days (seven composite samples of each), 2-3 embryos per sample at 18 and 21 days of age (seven composite samples of each at 1,ot.h ages). Composite samples of yolk sacs were made in a similar fashion. The samples of embryos were homogenized in a Waring hlendor and lyophilized, and the dry weights were recorded. They were ground three times in a Wiley Mill with Dry Ice using successively finer mesh delivery t.uhes. After a second lyophilization they were st,ored at -1i”C. lInti ready for assay. The yolk sacs were homogenized, I?-ophilized, weighed, ground in a mortar, and lyophilized again before st,oring at - 17°C. Aft,er 5 weeks on the diet, two eggs were taken from each hen on both the control and deficient dirts. After 9-10 weeks, 13 infertile eggs from each group of hens were removed from the incubator. The individual eggs were prepared for assay 1)~ the same procedure used for the yolk sacs.
Vit,amin IS12 was assayed using Ochron~onns ,t,2ulhamensis3 according to the met hot1 of Ford i(j). The samples were estrnctcd by taking I g. of lyophilized po\vder, 50 ml. of 0.01 X HCI, 1 mg. IVaCS. and adjusting the pH to .5.0 with ROH. They were then steamed for 30 min., cooled, filtered, and made to the :tppropriat~e volume to give 3 concrntration of approxim:rt.rly 0.025 mpg. I%,n/Illl.
The vit,amin HI2 contents of the whole egg, the embryo, and the yolk sac are presented in Table 1. It is evident that, eggs from hens on a vitamin Bls-deficient diet for 5 wwks are extremely low in vitamin Rlr \vhcn compared to the control eggs. An additional 5-wek depletion period resulted in still less B,, being transferred to the egg. The amount of vitamin Bla in the control c’ggs from hens receiving !) pg. &/lb. diet after 5 \\eeks on the experimental diet, was not’ diffcrcnt from the level found 5 wctks later. The level of vitamin RI2 in the diet of the hei1 had no effect on the dry weight, of thca whole egg during the rlltirc IO-week cxa 0chromonu.s tt~ulhumensis No. 11532 obtained from the American Type Culture Collection, Washington, I). C.
CHICK
EMBRYOS
2.51
perimental period. The low level of vitamin B1” in the deficient whole egg was, of course, reflected in t,he level of B,, in the deficient. embryo and yolk sac; this amounted to 510 7% of that, found in comparable normal tissues. The range in the vitamin B,r values obtained was rather large. However, it, has been the experience of others (‘7) t’hat, there are wide individual variations among heirs on the same diet in their ability to transfer vitamill HI2 to t)he egg. The vitamin B,? is locat’ed almost cntiwly in the yolk of the whole egg (4, 7) and is transferred from the yolk to the embryo as the chick dcvelopa. In both t’he control al1c-l deficient eggs, the most, marked redwtioll in the yolk sac B,, occurred betwell the 15th and the lSt,h days of incubation (Table II), although t)here was no great change in the rate of decrease in the dcfivicnt yolk sac* from 12 to 21 days. The redwt ion ill the I& content of the yolk sac>of the control egg (:I71 mpg.) corresponded to a marked inweasc ill the B,, content’ of the embryo (248 mpg.). With the deficient embryo, the greatest increase ill HI? (8.7 Meg.) owurred bet~ween t,he 18th and 21st days. This discrepancy may be explained by the fact, that the peak of embryo mortality in I&&eficient, eggs occurs between the 16th and 18th days (8). Thus, the average value at 21 days represents those embryos that may have possessed grcnt’er quanti‘ties of \-it.amin HI2 , and the average ~~1~s at 15 and 18 days contain values for embryos that \vould not have survived to 21 days. Support for this suggestion is obtained by cwmparing the illcrcase in the size of the deficient) embryos \vit h that of the controls. The most, marked diffcrcnw ilt change of weight of embryo oc~curred between the 15th and 18th day,< (+‘>.A1 g. for wntrol, + 1.90 g. for deficient). The vitamin Bls corwentration on a mpg, g. basis shovwl that, the c~ollc~etitrntioll of IS,, ill the embryo, twth control and drficiellt, did Iwt change appreciably with age, but tht: c,oric~cntrat,ioii of I& in the yolk sac dccreased markedly after I5 days of iltcsul)ntioli. The total vitamin I&? per vgg, c*aIcuIate(l l)y adding tht: values obtained for the (‘nbryo and yolk sac ,wparatcly, indicatc,s that
252
DANIEL,
HILAKY
AND
YESAIR
I Age of embryo
Sum of Bo in embryo + yolk S&C
Whole
egg Av. B,z per
Av.
dry ureight Gram
E66
Normal --dUYS 0 0
12 15 18 21
4 (20) 7 (21)
6
(18)
6 (17)
0.39 1.84 4.25 6.72
-
7
151 402 478
! 83.6 94.7 71.2
14.i3 11.96 9.27 ’ 4.82 ,
Vitamin
738 6X 299 104
21.5
I
6.7 11.5 20.2
3.95 3.22 3.50
w.ls.
14.06 (24)d 11.55 (13)
815” 785f
57.7 54.0
5X2
14.06 (24) ‘14.56 (13)
0
0.39 4 (20) 1.65 7 (22) I 7 (20) 1 3.55 5.86 1 6 (16)
VW.
I3,?-deficient
0
12 15 18 21
8.
13.89 ! 51.0 11.87 39.5 23.5 9.70 6.11 1 10.2
3.67 3.31 2.38 1.56
51 .o+~ 46.2 ( 35.0 30.4 j
a The liver and 0.02-0.1 ml. hlood were removed from each embryo for other analyses and are not included in these values. The 1s~~contents of the livers from 1% and 21-day-old normal embryos were 16 and30 mpg./liver, respectively; and thoseof the corresponding deficient embryonic livers were 2.0 and 2.4 mpg./liver, respect,ively. 6 Yolk-sac sample consists of the egg contents cswpt the embryo. c All values are on a dry-weight bssis. d Numbers in parent,heses represent total number of embryos or eggs. e Eggs collected at end of 5th week on experimcnt.al diets. f Infertile eggs collertcd between 9th and 10th wcrk of expriment. From the 5th to the 10th week the hatchability of the deficient eggs dropped from 50% to less than 10%. Hatchability of t,he normal eggs was 78% at the 5th Tveek and 50% at the 10th week. 0 There was insuficicnt t,isslle left for analysis in 1he cast of the 12.day-old cmhryos.
there was a dewease in the Blr content, from 15 t,o 21 days of incubat,ion in t,he order of of 25-50s. These results arc contrary to those obtaiued by Fischer, Benson, and Swendseid (X), who report,ed an increase in B1.’ content, with agca. These investigators, however, did not include cyanide or hisulfite in the extraction procedure. l>enton and Kellogg (9) have shown that, t,he vit,amiu BIZ content of the egg is higher when cyanide or hisulfit,e is used. This may amount for t,he low values obtained and t,he discrepanc4rs ohserved. Skarzynski et al. (1) report,ed no change
in the amount of vitamin B,, in tjhe egg duriilg incubation when Ochromonas malhamensis or Lactobacillus l&hmawCi was used. These investigators did employ cyanide in the extra&ion of the samples. In our \vork none of the embryo samples contained liver. However, the livers of the 1% and 21-day-old embryos were assayed for B,, , and t,he amounts found (see footfuote a, Tnhle I) were negligible in comparison to t,he amount, UP accounted for. We are rather certain of good sampling, since t,he per cent nitrogeu oht,ained on quudruplicate 100-mg. samples agreed wry closely. In addition, every effort
The fate of the “lost”’ vitamin B1:! is not, known. It seems probahlc, however, that it has txzen metabolized to a product that is no longer a groxth factor for &hromonns malhamcnsis.
The authors are indrbted to the Poultry I.)(,partmcnt for supplying the eggs usrtl in this w)rk, :tntl for the technical assistance of nIiss Const:lrrw cornw:lll,
Sormd ,,I/#.
s.
+1.45
~
+2.41
+2.47
~
+2a +76
1. YESAIR,
1). W., RICOSKER.
E.,
1).
ASI) UANIEI,, 79, 168 (1959). 2. YESAIR, 1). I$'., (k1LI)STEIK, .J., .4NI) I)AKIEL, I,. J., ilrch. Iliochem. Ijiophys. 84, 316 (1959). 3. FIWHER, Y. A., BENSOK. E;. RI.. ASI) SwmrJREID, RI. 15., Arch. Hiochenc. llioph!ys. 74, 158
I,. J., dtwh. fSiochen~.Iliophys.
12-15 15 18 IS-21
~ :
+1.26 +1.90 +2.x
,
+1.S t-8.7
-11.5 i -16.0 ~ ~ -13.3
11Yolk-sac sample consists of the rgg contents csecpt the embryo. 6 hftpr the start, of incul-xttion.
was made to pool egg cont,ents from different, hens, but from these same hens at each time period. Since there is marked individual variation in B,, caont,ent’ of eggs from different, hens, hut not among eggs from a given hen (7), this proredure should eliminate irregularit,ies in values that! might, result from t.he random sampling of a flock of hens.
(1958). 4. SK,~RZYNSICI, B., %aK, Z., OSTROWSKI, W., I)A\.Ik:S, &I. K.> (iREGORY, 11. E., ASIJ l
Sy~~pos2’u (C:tmt)ridge, Engl.)’ No. 13, 3(i-51 (195.5). 6. FORD, J. E., Uril. J. .yrrtrition 7, 299 (1953). 7. YAc'OWITZ, H., MILLER, II. F., KORRIS. I,. C., 9N1) HELSER, G. F., Poultry Sci. 31, 89 (195’1). S. OLCESE, O., COUCFI, J. R., QVISENHERRY, J. H., i\Nl)
1’E.IRSOh-,
I’.
I<.,
./.
.\‘utrifion
41,
423
(1950). 0. TRENTON, C. A., AND KELLOGG, Hiochem. Hiophys. 46, 105 (1953).
W. I,., Al&l.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
90,
250-253 (1960)
Vitamin B,, in Developing Vitamin Blz-Deficient LOUISE
J. DANIEL,
CAROL
Chick Embryos from and Control Eggs
A. HILARY
AKD
DAVID
W. YESAIR’
From the Department of Biochemistry and Nutrition and The Graduate Nutrition, Cornell Universit,y, Ithaca, New I’ork Received
School of
May 3, 1960
The vitamin B12 contents of the normal and vitamin Blr-deficient chick embryos and yolk sacs were determined at 12, 15, 18, and 21 days of age, by the use of Ochromonas malhamensis. From the 12th day of incubation on there was a decrease in the vitamin B~z content of the yolk sac with the greatest reduction occurring between the 15th and 18th days. The greatest increase in the vitamin BOXcontent of the control embryo occurred during the same period. In the deficient embryo the largest increase occurred between the 18th and 21st days. The vitamin BIz content of the whole egg (embryo plus yolk sac) decreased between the 15th and 21st days of incubation. The fate of the vitamin B12 not, recovered is unknown, but it, is possible that it was metabolized to a substance not utilized by the assay organism.
vitamin Bla activity during the incubation of the egg. The results to be reported here were obtained using Ochromonas malhamensis. This organism was chosen because it> is the most specific for vitamin B1z and does not, respond to vitamin I&-like factors or deoxyribosides (5).
INTRODUCTION
In the course of an extensive study of the effect of a vit#amin B,, deficiency on the developing chick embryo (1,2), determinations of t,he vitamin Bll contents of the embryos and yolk sacs2 were made at various time int,ervals aft#er t,he start, of incubation. While this study was in progress, two papers appeared in t’he literature on the vitamin B12 content,
of
t,he
developing
chick
EXPERIMENTAL
embryo.
Fischer, Benson, and Swendseid (3) using leichmannii as the assay organism found the vitamin B12 activit)y to increase in the egg during incubat’ion. On the other hand, Skarzynski et al. (4) reported no increase in the vit,amin B,, conteut of the egg during incubation when Ochromonas malhamensis or Lactobacillus leichwas used, whereas with Ezcglena mannii gracilis t,here was a progressive increase in
hODUC!TION
Lactobacillus
1 Public Health Service Research Predoctoral Fellow of the National Institute of i%eurological Diseases and Blindness. Present address: Lederle La.horatories Division, American Cyanamid Co., Pearl River, N. Y. 2 All contents of the egg except the embryo.
4ND OF
PREPAR.4TIOS
EMBE~YOS
Twenty-four Single Comb White Leghorn hens were placed in individual laying cages on raised wire floors. Twelve hens received a vitamin BIPdeficient diet (1) and 12 hens received the deficient diet plus 9 pg. BJlb. diet. After 1 weeks on the experimental diets, the hens were artificially inseminated t.wice weekly using pooled semen from Single Comb White Leghorn roosters, and the eggs secured subsequently were incubated at weekly intervals. At t,he end of the 5th week the hatchahilit,y of the deficient eggs was about 5O’A. From the 6th t,hrough the 12th Lveeks, living embryos were taken from eggs at, 12, 15, 18, and 21 days of age. The embryo was dissected from the yolk sac, a small quantity of blood was with250
VITAMIN
B~.L IS
drawn from the exposed heart, and the liver \vtls removed. Five embryos were pooled at 12 days (a total of four composite samples of both normal and deficient embryos), 3-4 embryos per composite at 15 days (seven composite samples of each), 2-3 embryos per sample at 18 and 21 days of age (seven composite samples of each at 1,ot.h ages). Composite samples of yolk sacs were made in a similar fashion. The samples of embryos were homogenized in a Waring hlendor and lyophilized, and the dry weights were recorded. They were ground three times in a Wiley Mill with Dry Ice using successively finer mesh delivery t.uhes. After a second lyophilization they were st,ored at -1i”C. lInti ready for assay. The yolk sacs were homogenized, I?-ophilized, weighed, ground in a mortar, and lyophilized again before st,oring at - 17°C. Aft,er 5 weeks on the diet, two eggs were taken from each hen on both the control and deficient dirts. After 9-10 weeks, 13 infertile eggs from each group of hens were removed from the incubator. The individual eggs were prepared for assay 1)~ the same procedure used for the yolk sacs.
Vit,amin IS12 was assayed using Ochron~onns ,t,2ulhamensis3 according to the met hot1 of Ford i(j). The samples were estrnctcd by taking I g. of lyophilized po\vder, 50 ml. of 0.01 X HCI, 1 mg. IVaCS. and adjusting the pH to .5.0 with ROH. They were then steamed for 30 min., cooled, filtered, and made to the :tppropriat~e volume to give 3 concrntration of approxim:rt.rly 0.025 mpg. I%,n/Illl.
The vit,amin HI2 contents of the whole egg, the embryo, and the yolk sac are presented in Table 1. It is evident that, eggs from hens on a vitamin Bls-deficient diet for 5 wwks are extremely low in vitamin Rlr \vhcn compared to the control eggs. An additional 5-wek depletion period resulted in still less B,, being transferred to the egg. The amount of vitamin Bla in the control c’ggs from hens receiving !) pg. &/lb. diet after 5 \\eeks on the experimental diet, was not’ diffcrcnt from the level found 5 wctks later. The level of vitamin RI2 in the diet of the hei1 had no effect on the dry weight, of thca whole egg during the rlltirc IO-week cxa 0chromonu.s tt~ulhumensis No. 11532 obtained from the American Type Culture Collection, Washington, I). C.
CHICK
EMBRYOS
2.51
perimental period. The low level of vitamin B1” in the deficient whole egg was, of course, reflected in t,he level of B,, in the deficient. embryo and yolk sac; this amounted to 510 7% of that, found in comparable normal tissues. The range in the vitamin B,r values obtained was rather large. However, it, has been the experience of others (‘7) t’hat, there are wide individual variations among heirs on the same diet in their ability to transfer vitamill HI2 to t)he egg. The vitamin B,? is locat’ed almost cntiwly in the yolk of the whole egg (4, 7) and is transferred from the yolk to the embryo as the chick dcvelopa. In both t’he control al1c-l deficient eggs, the most, marked redwtioll in the yolk sac B,, occurred betwell the 15th and the lSt,h days of incubation (Table II), although t)here was no great change in the rate of decrease in the dcfivicnt yolk sac* from 12 to 21 days. The redwt ion ill the I& content of the yolk sac>of the control egg (:I71 mpg.) corresponded to a marked inweasc ill the B,, content’ of the embryo (248 mpg.). With the deficient embryo, the greatest increase ill HI? (8.7 Meg.) owurred bet~ween t,he 18th and 21st days. This discrepancy may be explained by the fact, that the peak of embryo mortality in I&&eficient, eggs occurs between the 16th and 18th days (8). Thus, the average value at 21 days represents those embryos that may have possessed grcnt’er quanti‘ties of \-it.amin HI2 , and the average ~~1~s at 15 and 18 days contain values for embryos that \vould not have survived to 21 days. Support for this suggestion is obtained by cwmparing the illcrcase in the size of the deficient) embryos \vit h that of the controls. The most, marked diffcrcnw ilt change of weight of embryo oc~curred between the 15th and 18th day,< (+‘>.A1 g. for wntrol, + 1.90 g. for deficient). The vitamin Bls corwentration on a mpg, g. basis shovwl that, the c~ollc~etitrntioll of IS,, ill the embryo, twth control and drficiellt, did Iwt change appreciably with age, but tht: c,oric~cntrat,ioii of I& in the yolk sac dccreased markedly after I5 days of iltcsul)ntioli. The total vitamin I&? per vgg, c*aIcuIate(l l)y adding tht: values obtained for the (‘nbryo and yolk sac ,wparatcly, indicatc,s that
252
DANIEL,
HILAKY
AND
YESAIR
I Age of embryo
Sum of Bo in embryo + yolk S&C
Whole
egg Av. B,z per
Av.
dry ureight Gram
E66
Normal --dUYS 0 0
12 15 18 21
4 (20) 7 (21)
6
(18)
6 (17)
0.39 1.84 4.25 6.72
-
7
151 402 478
! 83.6 94.7 71.2
14.i3 11.96 9.27 ’ 4.82 ,
Vitamin
738 6X 299 104
21.5
I
6.7 11.5 20.2
3.95 3.22 3.50
w.ls.
14.06 (24)d 11.55 (13)
815” 785f
57.7 54.0
5X2
14.06 (24) ‘14.56 (13)
0
0.39 4 (20) 1.65 7 (22) I 7 (20) 1 3.55 5.86 1 6 (16)
VW.
I3,?-deficient
0
12 15 18 21
8.
13.89 ! 51.0 11.87 39.5 23.5 9.70 6.11 1 10.2
3.67 3.31 2.38 1.56
51 .o+~ 46.2 ( 35.0 30.4 j
a The liver and 0.02-0.1 ml. hlood were removed from each embryo for other analyses and are not included in these values. The 1s~~contents of the livers from 1% and 21-day-old normal embryos were 16 and30 mpg./liver, respectively; and thoseof the corresponding deficient embryonic livers were 2.0 and 2.4 mpg./liver, respect,ively. 6 Yolk-sac sample consists of the egg contents cswpt the embryo. c All values are on a dry-weight bssis. d Numbers in parent,heses represent total number of embryos or eggs. e Eggs collected at end of 5th week on experimcnt.al diets. f Infertile eggs collertcd between 9th and 10th wcrk of expriment. From the 5th to the 10th week the hatchability of the deficient eggs dropped from 50% to less than 10%. Hatchability of t,he normal eggs was 78% at the 5th Tveek and 50% at the 10th week. 0 There was insuficicnt t,isslle left for analysis in 1he cast of the 12.day-old cmhryos.
there was a dewease in the Blr content, from 15 t,o 21 days of incubat,ion in t,he order of of 25-50s. These results arc contrary to those obtaiued by Fischer, Benson, and Swendseid (X), who report,ed an increase in B1.’ content, with agca. These investigators, however, did not include cyanide or hisulfite in the extraction procedure. l>enton and Kellogg (9) have shown that, t,he vit,amiu BIZ content of the egg is higher when cyanide or hisulfit,e is used. This may amount for t,he low values obtained and t,he discrepanc4rs ohserved. Skarzynski et al. (1) report,ed no change
in the amount of vitamin B,, in tjhe egg duriilg incubation when Ochromonas malhamensis or Lactobacillus l&hmawCi was used. These investigators did employ cyanide in the extra&ion of the samples. In our \vork none of the embryo samples contained liver. However, the livers of the 1% and 21-day-old embryos were assayed for B,, , and t,he amounts found (see footfuote a, Tnhle I) were negligible in comparison to t,he amount, UP accounted for. We are rather certain of good sampling, since t,he per cent nitrogeu oht,ained on quudruplicate 100-mg. samples agreed wry closely. In addition, every effort
The fate of the “lost”’ vitamin B1:! is not, known. It seems probahlc, however, that it has txzen metabolized to a product that is no longer a groxth factor for &hromonns malhamcnsis.
The authors are indrbted to the Poultry I.)(,partmcnt for supplying the eggs usrtl in this w)rk, :tntl for the technical assistance of nIiss Const:lrrw cornw:lll,
Sormd ,,I/#.
s.
+1.45
~
+2.41
+2.47
~
+2a +76
1. YESAIR,
1). W., RICOSKER.
E.,
1).
ASI) UANIEI,, 79, 168 (1959). 2. YESAIR, 1). I$'., (k1LI)STEIK, .J., .4NI) I)AKIEL, I,. J., ilrch. Iliochem. Ijiophys. 84, 316 (1959). 3. FIWHER, Y. A., BENSOK. E;. RI.. ASI) SwmrJREID, RI. 15., Arch. Hiochenc. llioph!ys. 74, 158
I,. J., dtwh. fSiochen~.Iliophys.
12-15 15 18 IS-21
~ :
+1.26 +1.90 +2.x
,
+1.S t-8.7
-11.5 i -16.0 ~ ~ -13.3
11Yolk-sac sample consists of the rgg contents csecpt the embryo. 6 hftpr the start, of incul-xttion.
was made to pool egg cont,ents from different, hens, but from these same hens at each time period. Since there is marked individual variation in B,, caont,ent’ of eggs from different, hens, hut not among eggs from a given hen (7), this proredure should eliminate irregularit,ies in values that! might, result from t.he random sampling of a flock of hens.
(1958). 4. SK,~RZYNSICI, B., %aK, Z., OSTROWSKI, W., I)A\.Ik:S, &I. K.> (iREGORY, 11. E., ASIJ l
Sy~~pos2’u (C:tmt)ridge, Engl.)’ No. 13, 3(i-51 (195.5). 6. FORD, J. E., Uril. J. .yrrtrition 7, 299 (1953). 7. YAc'OWITZ, H., MILLER, II. F., KORRIS. I,. C., 9N1) HELSER, G. F., Poultry Sci. 31, 89 (195’1). S. OLCESE, O., COUCFI, J. R., QVISENHERRY, J. H., i\Nl)
1’E.IRSOh-,
I’.
I<.,
./.
.\‘utrifion
41,
423
(1950). 0. TRENTON, C. A., AND KELLOGG, Hiochem. Hiophys. 46, 105 (1953).
W. I,., Al&l.