DIETARY REQUIREMENTS FOR REPRODUCTION.

Transcription

1 DIETARY REQUIREMENTS FOR REPRODUCTION. III. THE EXISTENCE OF THE REPRODUCTIVE DIETARY COMPLEX (VITAMIN E) IN THE ETHEREAL EXTRACTS OF YELLOW CORN, WHEAT EMBRYO, AND HEMP-SEED.* BY BA.RNETT SURE. (From the Laboratory of Agn cultural Chemistry, University of Arkansas, Fayetteville.) (Received for publication, October 7, 1924.) In connection with his studies on the r81e of proline in nutrition, begun at the Department of Agricultural Chemistry, University of Wisconsin, in 1918, the author became interested, from work then in progress, in the supplementary value of lactalbumin to casein, fed in the same proportions &I these proteins are found to occur in milk. In the series of experiments initiated in the early part of 1919 (1) skimmed milk powder, fed at a 9.6 per cent level, was found to be excellent for growth, but was found, in the presence of a suitable salt mixture and an abundant supply of the known vitamins, to be inadequate for reproduction. The cause for failure was attributed to the possibility that the amino acid requirement of the mammalian organism for the physiological processes of fertility and lactation may be different from the requirement for growth, from the standpoint either of quantity or of quality, and so for 2 years experiments to elucidate the failure of reproduction on diets composed of purified food substances were conducted from the amino acid standpoint. Later on, after the quality of the protein in the diet had been improved, intensive efforts to produce fertility and to obtain success in lactation by increasing the known vitamins in the diet resulted in failure. No attempts were made to improve the nature of the inorganic ion complex of the synthetic diets. In these experiments it was found that the addition of one seed to the pure diets always resulted in fertility and, in a good many cases, fair success in rearing of young was secured. Downloaded from by guest on January 23, 2019 * Research paper No. 11, Journal Series, University of Arkansas. 371

2 372 Dietary Requirements for Reproduction. III The conclusion, which appeared in a previous publication in this Journal, was as follows (2): In addition to the antixerophthahnic, antirachitic, antiberi-beri, and antiscorbutic vitamins, there exists another hitherto unrecognized vitamin, that is essential for reproduction, which becomes evident only in breeding experiments where rations composed of purified food substances are employed. In that paper a reproductive vitamin was anticipated, although no positive data were then available. In this paper positive evidence is presented showing that ether extracts from yellow corn, wheat embryo, and hemp-seed, an organic factor (which, according to a suggestion formulated in a previous article (2), I choose tentatively to call vitamin E) essential for reproduction. Recently Anderegg (3) published an article, entitled Diet in relation to reproduction and rearing of young, in which he asserts that it is not necessary to assume the existence of a specific vitamin for reproduction. Since all Anderegg s diets contained large concentrations of butter fat, his conclusions are untenable, as has been pointed out by Evans (4). With high concentrations of butter fat in the diet Evans and Bishop (5) found that sterile females become pregnant and manifest a certain degree of success in rearing of young. Since McCollum and Davis (6) and Osborne and Mendel (7) have independently demonstrated that fat-soluble A, or the antixerophthahnic vitamin is present in butter fat and cod liver oil, but is absent in cottonseed, linseed, and olive oils, it occurred to me that the reproductive dietary complex might, by chance, be contained in vegetable oils. This whole paper deals, then, with attempts to secure fertility and to obtain successful lactation with laboratory-prepared oils, and with commercial vegetable oils. EXPERIMENTAL. Preparation of the Ethereal Extracts of Wheat Embryo, Yellow Corn, and Hemp-Seed. Wheat Germ Oil.-The wheat germ oil was extracted in a large Soxhlet apparatus. About 1,000 gm. of wheat embryo were allowed to extract for 7 hours until the solvent siphoned six to eight times. The ether was then distilled off and the residual ether in the wheat oil expelled by blowing a current of air from an electric fan for 3 hours.

3 Barnett Sure 373 Corn OX-The corn oil was prepared in a similar manner, only the extraction was allowed to proceed for 14 hours. Later on it was found that acetone is a more efficient solvent for corn oil from the standpoint of quantitative returns, and so another preparation was obtained by extraction for 14 hours with hot acetone. Hemp-Seed OX-The hemp-seed oil was extracted in the cold by allowing crushed seed to stand in contact with large volumes of ether for 24 hours. Most of the ether carrying the oil, which settled on top, was siphoned off, and the residual solvent squeezed out through cheese-cloth. The ether was then distilled off, and the residual solvent expelled by the aid of an electric fan running for 3 hours. Two series of experiments were carried out: (I), using casein as the basal protein; and (II), using protein derived from skimmed milk powder purchased from the Merrell-Soule Company, New York. Series I. Composition of Control Rations (No Vegetable Oil Present). Ration &I. Ration 444. Casein Casein Salts Salts _... 4.O Agar-agar Agar-agar Cod liver oil O Cod liver oil O Dextrinl O Harris yeast-vitamin powder Dextrin The publication of Osborne and Wakeman s paper (8) on the extraction from yeast of a very potent water-soluble vitamin B preparation encouraged the use of such a desiccated extract in all this work. Such extracts were purchased from the Harris Laboratories, Tuckahoe, New York, which is producing a standardized product (9). After being used for 8 weeks, this vitamin preparation, to obtain normal growth, had to be increased to 0.6 per cent, and from the time of breeding until the termination of the experiments it was further increased to 1.0 per cent of the total ration. In rations of Series I, containing vegetable oils, 5 per cent of the oil employed replaced 5 per cent dextrin. 1 Dextrin carried an 80 per cent alcoholic extract of 30 gm. of etherextracted wheat embryo.

4 374 Dietary Requirements for Reproduction. III Series II. Composition of Control Rations (No Vegetable Oil Present). Ration 466. Skimmed milk powder O Ferric citrate Agar-agar O Harris yeast-vitamin powder Cod liver oil Dextrin Ration J8689. Skimmed milk powder Ferric citrate Agar-agar... 2.O Harris yeast-vitamin powder Cod liver oil Salts Casein Gelatin Dextrin The composition of the rest of the rations containing laboratoryprepared and commercial vegetable oils is shown in Charts V to XIII inclusive. After being used for 4 weeks, the yeast vitamin extract in the Series II rations was increased from 0.4 to 0.5 per cent of the total ration. In all the work reported in this paper all the animals,2 with the exception of a few individuals, made normal growth. If an animal did not-make normal growth, it was discarded and not used for breeding. Since normal growth was secured on all rations, it was thought that considerable space can be saved by omitting charts of growth and by showing only charts of the growth of litters. One chart of growth, however, was considered of interest to be insertedthat showing skimmed milk powder as a source of the water-soluble B vitamin. (See Chart IV.) Two males and three females were used for each experiment. The males and females were placed in separate neighboring compartments and bred when they were from 90 to 100 days old (10). Pregnant females were separated in individual compartments 3 or 4 days before delivery was expected, and were given wooden boxes in which to deposit and rear their litters. If the females had milk, they almost invariably became accustomed to the box and kept their young in it. As soon as milk flow stopped, * No growth was made on the ration containing fish oil, because of lack of food consumption.

5 Barnett Sure 375 however, the mothers either devoured the young or scattered them on the screens, so that the litter would be found dead in shavings on the bottom of the galvanized pan. In all cases the litters were reduced to six, so as not to overburden the mammary gland and at the same time provide an exact method of comparison. DISCUSSION. When casein was used as the basal protein in Series I experiments, no fertility was obtained on Control Rations 441 and 444, containing no vegetable oils, although normal growth was secured with all animals. On the other hand, 100 per cent fertility was secured with ethereal extracts of the wheat embryo and hempseed. Only one female out of three became pregnant on Ration 450, carrying commercial olive oil. 100 per cent fertility was also obtained with commercial cottonseed oil, but the data on this oil have been omitted in this paper, since the experiments are being continued with skimmed milk powder from the standpoint of lactation and will be published later in connection with work in progress on peanut, soy bean, peach kernel, and sunflower oils. No fertility was obtained on Rations 447 and 451 which contained commercial linseed and cocoanut oils. Charts I, II, and III, however, indicate that rearing of young on Series I rations was rather unsuccessful. Two females on Ration 445, which contained a 5 per cent ethereal extract of wheat embryo, reared their litters of six young successfully for periods of 7 and 10 days, after which periods the mammary glands of the mothers became dry, and the young, of course, began to lose weight rapidly. Recently Evans stated that (4): wheat embryo, in the fatfree condition, is as effective in its galactogogic action as the whole wheat embryo, and that, it would appear, therefore, that the food material necessary for mammary function is not soluble in fats. In this connection the author would like to state here the results of two experiments out of a series not yet completed, which strongly suggest that the organic factor necessary for reproduction is a fat-soluble vitamin. On a ration containing 20 per cent whole wheat embryo three females have given birth to three normal litters and have reared them successfully through-

6 376 Dietary Requirernents for Reproduction. III 0 x * P

7 I I tldl2t.h a y Of llf0 20 In. rrm.a q l lo Days CHART 111. I

8 378 Dietary Requirements for Reproduction. III out the lactation period. Whole wheat embryo, after being extracted with ether for 7 hours, still contains 0.4 per cent fat. A ration containing 20 per cent of such ether-extracted wheat germ carries 0.08 per cent fat. On such a low fat-containing ration normal litters were born, but the young were born dead. It seems, then, that very small amounts of wheat oil are required for fertility, but considerably more for proper nourishment of the fetus during gestation. The mammary glands of the mothers giving birth to litters on the ether-extracted wheat embryo rations were found completely dry. On a diet containing 35 per cent of ether-extracted wheat embryo two females gave birth to normal litters and nursed them for a few days, but then disposed of them.3 Such results would indicate that the oil as found in the germ of wheat is necessary not only for fertility and proper nourishment of the fetus during gestation, but also for successful lactation. The detailed data of the experiments referred to above will be published in a subsequent communication. It was anticipated that the most successful results in fertility and lactation would be obtained by employing skimmed milk powder for the following reasons: (1) It contains all the proteins of milk; (2) when fortified with small amounts of ferric citrate, it furnishes a superior salt mixture; (3) when fed at a 50 per cent plane of intake, it in itself serves as an excellent source of the water-soluble B vitamin (see Chart IV) ; and (4) it is absent in the organic factor (vitamin E) essential for fertility. During the first 5 to 7 weeks all animals on skimmed milk powder rations suffered with considerable diarrhea, possibly due to gastrointestinal disturbances produced by the large amounts of lactose in the diet (11). During the diarrhea period the animals had a rough coat and looked somewhat abnormal, but their remarkable gains in weight would seem to disprove their apparent ill state of health. After 8 weeks, intestinal troubles ceased and 8 These experiments were completed during May of this year, and the results were communicated before the Arkansas Medical Society of Fayetteville, on May 20, Series I experiments, using casein as the basal protein, were completed during July of this year. Evans article on Unique dietary needs for lactation, which appeared in Science, Vol. lx, No. 1540, is dated July 4, 1924.

9 Barnett Sure 379 the animals developed a smooth coat. They made normal growth and were completely resistant towards diarrhea. Not infrequently certain individuals overcame the diuretic effect of their skimmed milk diet 4 weeks after weaning time. When a litter is born the mother is watched very carefully with respect to her attitude towards the young. When a mother has normal mammary function her maternal instinct guides her I IAaar-drsr I I I 12.0 I I I I I I I CHART IV. to be very solicitous towards the welfare of her litter. She stays in the box provided for her, keeps the young warm, and nurses them most of the time except during intervals when she must go to drink and eat her food. When, however, there is lack of milk flow and she feels she is unable to supply nourishment, the mother becomes very irritable and makes no attempt to save her baby rats. She either devours them or scatters them on

10 380 Dietary Requirements for Reproduction. III the screen, so that the young are later found cold in the shavings of the galvanized pan below the screen. After the young have been separated from the mother any length of time, they naturally die. Sometimes the mother scatters the young on the platform of the feeding pan and completely ignores them. On August 19 and 20 seven litters were born to females partaking of skimmed milk powder rations containing ethereal extracts of wheat embryo, yellow corn, and hemp-seed. On the 2nd day after delivery two females on Ration 466, containing 5 per cent of wheat germ oil, were found ignoring their litters and scattering them all over the screens, and all attempts to induce them to take care of their young resulted in failure. On the 2nd day both litters were found dead. One female on Ration 467, containing a 3 per cent ethereal extract of wheat embryo, reduced her litter of six given her to four, and these died on the following day. Fortunately, however, one female on Ration 466, one on Ration 467, one on Ration 468, containing hemp-seed oil, and one mother on Ration 469, containing corn oil, showed evidence of at least a desire to keep their litters. When the litters were weighed, however, on the 2nd and 3rd days of lactation, they showed very little, almost insignificant gains in weight, as indicated in Charts V, VI, and X. Our experience with hundreds of litters of stock rats receiving natural foodstuffs supplemented with a liberal supply of whole milk shows that very frequently there is little milk flow during the first 24 hours of lactation, but that the young invariably gain significantly on the 2nd day of lactation, and that occasionally by the end Qf the 3rd day and very often on the end of the 4th day of lactation the litters will as much as double their initial weight. When several mothers had already disposed of their litters and some were maintaining them with little increases in weight, it occurred to me that the requirement of the water-soluble B vitamin for normal mammary gland function may be ever so much greater than the requirement for body growth and stimulation of the appetite. The r&e of the water-soluble B vitamin in lactation had already been pointed out in reproduction studies of Georgia velvet bean pod meal (2). In these experiments it

11 Barnett Sure 381 was found that when dextrin carried 10 gm. of alcoholic extracts of ether-extracted wheat embryo in a 100 gm. ration, litters of four young were reared for about, a week. Increasing the concentration of the water-soluble B vitamin to 20 per cent resulted in the rearing of litters of four young for about 13 days. When, however, the dextrin in the ration carried 30 gm. of alcoholic extracts of ether-extracted wheat embryo, introducing a 30 per cent concentration of water-soluble B vitamin, a litter of four young was weaned, although it was not reared normally throughout the lactation period. A similar situation was readily anticipated in these (Series II) experiments when failure in rearing of young seemed inevitable, because of the very small gains in weight of the litters as late as the 3rd day of lactation, and because of the small amounts of milk found in the mammary glands of the mothers. An attempt was then quickly made to improve lactation by increasing the concentration of the water-soluble B vitamin with the potent yeast extract furnished by the Harris yeastvitamin powder prepared according to the Osborne and Wakeman technique (8), which is a standardized product, from 0.5 to 1.0 per cent of the total ration. At this time the same change was made in all skimmed milk powder rations on which females were bred. While no response was obtained 24 hours after the increase in the water-soluble B vitamin concentration was made, most remarkable gains were made by the litters which previously seemed doomed to failure, 48 and 72 hours after the addition. The mothers began to secrete milk and the most surprizing success was secured with all the litters. The young made practically normal growth throughout the period of lactation and were successfully weaned on the 21st to the 25th day after birth. Evans (4) reports that he can cure sterility with 1 to 6 drops of wheat germ oil daily. Lactation, however, is seriously im- paired on pure diets. The average weaning weights of the animals resulting from such lactation is almost exactly half that which is normal, i.e., 20 grams instead of 40 grams on the twentyfirst day of life. Very seldom can a young rat, unless he attains a weight of from 33 to 35 gm., be successfully weaned, because until he becomes that large he is not able to eat food and drink water. It

12 382 Dietary Requirements for Reproduction. III would seem, then, that Evans with his pure diets was only partly successful in lactation, since he was not able to wean his litters. On the skimmed milk powder Rations 465 and 483, which contained no vegetable oils, no fertility was obtained, even after the water-soluble B vitamin concentration was increased by increasing the Harris yeast-vitamin powder, in the case of Ration 465 to 1.0 per cent; and in the case of Diet 483, by fortifying the 0.5 per cent potent yeast extract with an 80 per cent alcoholic extract of ether-extracted wheat embryo to the extent of 30 per cent in the ration. Young of mothers, however, were successfully reared to weaning age on the ethereal extracts of wheat embryo, yellow corn, and hemp-seed, and the young weighed 20 gm. each on the 11th to the 14th day of life. Food consumption records clearly show that the response obtained to the increase in concentration of the potent yeast extract is not to be attributed to the fact that the mothers ate more after the 4th and 5th days of lactation. The same is true with respect to the controls. In the four control experiments (Rations 441, 444, 465, and 483) all animals ate as much as they did in the experiments in which vegetable oils were incorporated. In the control experiments, however, the females were sterile, while in the latter experiments the presence of vegetable oils led to fertility in certain cases and to fertility and successful weaning of young to the second generation in others. From records of food consumption, it was calculated that each animal was consuming about 60 mg. of the potent yeast extract per day during breeding, gestation, and lactation periods when the rations contained 0.5 per cent of the Harris yeast-vitamin powder. Increasing the concentration to 1.0 per cent gave the mothers with young 60 mg. additional of that yeast extract. It could hardly be argued that 60 mg. of the Osborne and Wakeman yeast extract prepared by the Harris Laboratories could add enough more of a mineral element or group of inorganic ions, or enough more of a nitrogenous substance or substances than that already contained in the rations to become the limiting factor in lactation. It is, of course, possible that yeast may contain several vitamins performing different physiological functions. In that case the Harris yeast-vitamin powder may carry a dietary complex which, in larger concentrations, is specifically beneficial

13 Barnett Sure 383 in lactation. Until it is shown, however, that there are present in yeast vitamins other than water-soluble B necessary for the growth and reproduction of mammals, it is most reasonable to assume at present that the water-soluble B vitamin is the factor involved. The charts clearly indicate that litters of mothers partaking of rations containing ethereal extracts of the wheat embryo (Series II experiments) fortified with larger concentrations of the water-soluble B vitamin make significant gains from the beginning of lactation, as compared with little incipient gains of litters of mothers receiving the same rations but with lesser amounts of the yeast extract. This is further evidence of the prominent role played by the water-soluble B vitamin in lactation in the Series II experiments. E$ect of an Ethereal Extract of Hemp-Seed on Reproduction. CHART V, LOT 468. Female 2001 had nine young and was given six, weighing 33 gm., to rear. On the 2nd day after birth the litter gained only 1 gm., and on the 3rd day only 2 more gm. The increase in the potent yeast extract at point x resulted in the successful weaning of five young, but the laxative effect of the hemp-seed oil, together with the gastrointestinal disturbances young animals undergo the 1st month and a half on a skimmed milk powder diet, when fed at a 50 per cent plane of intake, was more than the young could endure. The diarrhea brought them to such a miserable physical condition that it was decided to eliminate this group of young animals. Female 1999 had six young which weighed 22 gm. On the 3rd day the mother reduced them to four which are being successfully reared. E$ect of Ethereal and Acetone Extracts of Yellow Corn on Reproduction. CHARTS VI and VII, LOT 469. Ration 469 contained 3 per cent of an ethereal extract of yellow corn. After 70 days of growth the preparation of the ethereal extract was exhausted and was replaced by a 5 per cent acetone extract of yellow corn. Female 2004 had the ethereal extract of yellow corn throughout her gestation period and was given the acetone extract on the day of the delivery of her litter. This mother had ten young, and was given six, weighing 30 gm., to rear. The effect of the increase of the water-soluble B vitamin concentration on lactation is indicated in Chart VI. The litter was successfully weaned and a healthy and vigorous second generation of this group is growing up.

14 I I f :: I

15 I/I I I I I VI I I I I

16 lb D)sJV CHART VII.

17 CHAHT VIII. Lot 456. Ferr1 oitr t* 0.2 ( 1 Ham1 yeas -rit+n pvfder 0.4 I I I I I I I I I

18 Qm UO L? DPY6 CEABT Ix.

19 Barnett Sure Female 2006 had during the first half of her gestation period a 3 per cent ethereal extract and during the second half a 5 per cent acetone extract of yellow corn. This animal had nine young, was given six, weighing 30 gm., and reared her whole litter successfully for the first 18 days, but after the 18th day began to fail very badly in lactation. The only tentative explanation is that acetone may not extract the reproductive dietary complex from yellow corn as efficiently as ether. Female 2004 may have had more reserve of the ether-extracted complex stored up and this together with the acetone extractive had sufficient vitamin E to enable her successfully to nurse her young up to weaning age. The acetone extract of yellow corn had considerable solids, was thick in consistency, dark in color, and undoubtedly had extracted from the corn a different series of fatty acids from that found in the ethereal extracts, and possibly also some other lipoids. The ethereal extract was wholly in the liquid state and was lighter in color. More work, however, is necessary to settle completely the efficiency of acetone as compared with ether as a solvent of the reproductive vitamin from yellow corn. On the 30th day the three remaining young of Female 2006 weighed 39 gm. each, but were not in the very best state of health. The third female, No. 2005, of this experiment had the acetone extract during the whole gestation period. She had seven young, was given six, reduced them to three on the 2nd day, to two on the 4th day, and disposed of the remaining two in the course of the next few days. Eflect of an Ethereal Extract of Wheat Embryo on Reproduction. The greatest success in lactation was obtained with the ethereal extract of the wheat embryo. CHARTS VIII and IX, LOT 466. This ration contained 5 per cent of an ethereal extract of wheat embryo and 0.5 per cent of the Harris yeastvitamin powder. Females 1989 and 1990 had litters of nine and eleven young, respectively, and were given six young to rear, but the mothers disposed of their litters on the 2nd day. The third female, No. 1991, had ten young and was given six, weighing 27 gm., to rear. It will be noted by inspection of Chart VIII that almost as much growth was made by the litter between the 4th and 6th day as in the entire first 4 days of lactation. The response to the increase in the yeast extract is very suggestive in this case, but not conclusive. The outstanding fact, however, remains that this animal (Female 1991) reared her litter of six normally throughout the lactation period and a second generation of this group of animals is growing up successfully. The interesting fact is that the second generation is encountering considerably less diarrhea than their parents during the early part of their growth period. Females 1989 and 1990 were rebred on the higher (1.0 per cent) concentration of the Harris yeast-vitamin extract. Female 1989, which failed with her first litter on the 0.5 per cent yeast extract-containing diet, had

20 390 Dietary Requirements for Reproduction. III fifteen young with the second litter. She was given six, weighing 29 gm., to rear and successfully reared five young throughout the lactation period. A healthy second generation of this group is growing up. Female 1999 gave birth to twelve young (second litter), was given six, weighing 29 gm., to rear and successfully reared five young throughout the lactation period. This mother had failed with her fist litter (see Chart IX). The above evidence seems to corroborate the theory that the water-soluble B vitamin plays a hitherto unsuspected tremendous r61e in lactation, which in the Series II skimmed milk powder experiments resolves itself into a determining limiting factor in rearing of young. CHARTS X and XI are self-explanatory. Two healthy, vigorous second generations are growing up on Ration 467 which contained 3 per cent of an ethereal extract of wheat embryo. CHARTS XII and XIII, LOT 484. This ration contained 35 per cent of skimmed milk powder fortified with casein and gelatin, thus varying the quality of the amino acid content of the diet. No fertility was obtained on the control ration, No. 483, from which the wheat germ oil was absent. During the middle of the gestation periods of Females 2077 and 2079 (Lot 484) when it became apparent that several mothers were failing on skimmed milk powder rations containing ethereal extracts of wheat embryo, a strenuous attempt was made to improve the quality of Ration 434, as indicated in Charts XII and XIII. When this radical change was made in Ration 484 a similar change was made in the control ration, No. 483, with the exception that the wheat germ oil was left out. Females 2077 and 2079 reared their litters of five and six, respectively, throughout their lactation period very successfully, and healthy second generations of both of these groups of young animals are growing up. The interesting fact about this experiment is that an 80 per cent extract of ether-extracted wheat embryo in the concentration employed is able to replace 0.5 per cent of the potent yeast extract for lactation, even when the plane of skimmed milk powder intake is reduced from 50 to 35 per cent as a source of the water-soluble vitamin. Female 2078 gave birth to only three young which were disposed of by the mother in a few days, for reasons not quite apparent at this time. The mammary gland of the mother was found dry. Possibly this individual female needed a larger concentration of the water-soluble B vitamin for lactation. LOT 471. This lot received 5 per cent of commercial olive oil in the ration. Two females out of three gave birth to litters that were very pale and emaciated looking and were disposed of by the mothers in a few days. The mammary glands of the mothers were dry. It seems that commercial olive oil contains enough of the reproductive dietary complex

21 cm a , / / I I I I I I I...I. J._.I lo II! u D23Y8 CHABT X. /

22 300 oma la, DayI CHABT XI.

23 !.lzlgs CHART XII.,/,,,,, mm,,,,,,,,,,

24 394 Dietary Requirements for Reproduction. III u i i Pi D i i i Ii i i t-l

25 Barnett Sure 395 for fertility, but not enough for proper nourishment of the fetus during gestation, since the young were born in abnormal condition. No fertility was obtained with commercial linseed, cocoanut, or sesame oils when introduced to the extent of 5 per cent in the ration. CONCLUSION. The original hypothesis of the author formulated in the early part of 1919 that the total milk proteins, as found in skimmed milk powder, although excellent for growth, are inadequate for reproduction, must be abandoned because of the nature of the experimental findings reported in this paper. Skimmed milk powder, when incorporated to the extent of 50 per cent in a ration, thus introducing 17.5 per cent protein, furnishes an excellent quality and sufficient quantity of amino acids for the whole cycle of reproduction, which includes ovulation rhythm, placental function, fertility of healthy normal litters born after the complete gestation period, and successful lactation. When fortified with 0.2 per cent ferric citrate, skimmed milk powder, fed at a 50 per cent plane of intake, furnishes suthcient mineral elements of excellent quality for reproduction. After the antixerophthalmic and antirachitic vitamins in the form of 2 per cent cod liver oil, and roughage by the addition of 2 per cent agar-agar, have been supplied, two dietary factors, however, for successful reproduction are still deficient in skimmed milk powder: (1) A hitherto unrecognized organic factor, vitamin E, found in ethereal extracts of wheat embryo, yellow corn, and hemp-seed; and (2) a high concentration of the water-soluble B vitamin, a concentration much greater than that required for growth. Five young were successfully weaned on a ration which contained a 3 per cent ethereal extract of yellow corn, which was later changed to a 5 per cent acetone extract of yellow corn. A healthy second generation of this group is growing up. Five young were successfully weaned on a ration containing an ethereal extract of hemp-seed, but owing to the laxative effect of the hemp-seed oil and to the gastrointestinal disturbances young undergo the first 5 to 7 weeks on skimmed milk powder diets, it was not possible even to attempt to rear a second generation of this group of animals. The most successful results were secured with an ethereal extract of wheat embryo as a source of vitamin E. Healthy

26 396 Dietary Requirements for Reproduction. III litters were born to females partaking of rations containing 5 and 3 per cent of wheat germ oil. Some of the litters would have died early during lactation, but were saved by increasing the water-soluble B vitamin concentration of the diets of their mothers at the critical period. With the higher concentration of the water-soluble B vitamin, young start out with excellent initial growth. Six healthy, vigorous second generations are growing up on the wheat germ oil experiments, while twelve females on conirol experiments, with no vegetable oild present, did not even become pregnant. SUMMARY. 1. Positive evidence is presented shobying that the ethereal extracts of yellow corn, wheat embryo, and hemp-seed contain an organic factor, designated as vitamin E, which is essential for reproduction. 2. Fertility was also secured with commercial cottonseed oil and commercial olive oil, <hen introduced to the extent of 5 per cent of the ration, but not with commercial cocoanut, linseed, or sesame oils. 3. The requirement of the water-soluble B vitamin for normal mammary gland function is much greater than that for growth. 4. Skimmed milk powder, when introduced to the extent of 50 per cent in the ration, furnishes amino acids sufficient in quantity and of excellent quality for reproduction. 5. Skimmed milk powder, fortified with 0.2 per cent ferric citrate, fed a,t a 50 per cent plane of intake, furnishes sufficient mineral elements of excellent quality for reproduction. BIBLIOGRAPHY. 1. Sure, B., J. Biol. Chem., 1920, xliii, Sure, B., J. Biol. Chem., , lviii, Anderegg, L. T., J. Bid. Chem., 1924, lix, Evans, H. M., Science, 1924, lx, Evans, H. M., and Bishop, K. S., J. Metabol. Research, 1923, iii, McCollum, E. V., and Davis, M., J. Biol. Chem., 1913, xv, 167; 1914, xix, Osborne, T. B., and Mendel, L. B., J. Biol. Chem., , xvi, Osborne, T. B., and Wakeman, A. J., J. Biol. Chem., 1919, xl, Bailey, E. M., Connecticut Agric. Exp. Station., Bull. 840, Dutcher, R. A., and Francis, E., Proc. Sot. Exp. Biol. and Med., , xxi, 189. Steenbock, H., Sell, M. T., and Nelson, E. M., J. Biol. Chem., 1923, Iv, Hamilton, T. S., and Card, L. E., J. Agric. Research., 1924, xxvii, 597.

27 DIETARY REQUIREMENTS FOR REPRODUCTION: III. THE EXISTENCE OF THE REPRODUCTIVE DIETARY COMPLEX (VITAMIN E) IN THE ETHEREAL EXTRACTS OF YELLOW CORN, WHEAT EMBRYO, AND HEMP-SEED Barnett Sure J. Biol. Chem. 1924, 62: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1

28 CORRECTIONS. On page 454, Vol. LIII, No. 2, August, 1922, 14 lines from the bottom, for r4: = Ia!l, = x loo 1X1 P -60 reti [a]: = - yi 7 O lines from the bottom, for NH, 4.94 read Amino N Last line, for x 100 1x1 =88 read [a]: = On page 455, 13th line, for 1.98 read th line, for C IBH,JTO~ read ClaH,2NOs X MO= _ d8 1X1 9 lines from the botton, for read lines from the bottom, for read On page 457, 4 lines from the bottom, for CaHTOrKa+H20 read CsH101h +H20. On page th line, for (99.0 9m.) read (3.0 On page 460, 8th line, for sm.). [a1'" = " x 100 ', D = + 66 read Ia]; = %;F = - 66O. IX8

29 CORRECTIONS, On page 371, Vol. lxii, No. 2, December, 1924, 7th line, for skimmed milk powder, fed at a 9.6 per cent level read skimmed milk p owder, fed at a 2Y.4 per cent level, introducing 9.6 per cent milk proteins. On page 378, 25th line, for absent read de$cient. On page 379, 3rd line, for diuretic read diarrhea-producing or laxative. On page 663, Vol. lxiii, No. 3, April, 1925, the structural formula on the left-hand side at the bottom of the page should have a single bond where the double bond now appears.