The Effect of Administration of Royal Jelly on the Differentiation and Growth of Newborn Rats’

HELEN DAWSON, RICHARD RUBOTTOM,2 AND LOWELL HARRIS Department of Anatomy, College of Medicine, University of Iowa, Iowa City, Iowa

Polymorphism determined by environ- mental factors is not an uncommon occur- rence among the social insects. However, the remarkable effect that an environmen- tal factor can have on altering develop- ment and producing dimorphism is no- where more clearly seen than in the honey bees. Larvae, hatched from genetically identical eggs, develop either into imper- fect females (worker bees) or perfect fe- males (queen bees) depending on whether or not the larvae are fed and maintained on a diet of royal jelly. The degree of dimorphism which results involves both morphological and physiological variations of such a marked degree that to an inex- perienced person the worker and the queen bees might be considered as belonging to two distinct species.

However, even in bees, the mechanism of this action is not understood. Ribbands (’53) and Johannson (’55) have reviewed the literature on the theories of polymorph- ism and at the present time there seem to be two opposing theories: ( 1 ) that the mechanism of the differentiation is quali- tative; ( 2 ) that it is quantitative, and the worker bee larvae undergo a ‘hutritional castration,” while the queen larvae receive an excess of royal jelly throughout their entire life.

Interestingly enough both worker and queen larvae are fed royal jelly for the first 24 to 48 hours, then the diet is changed and only the queen larvae are maintained on royal jelly. The effect of this amazing nutritional material becomes apparent almost immediately. Melampy and Willis (’39) reported that in queen larvae two to three days old the oxygen consumption was already 50% greater than in worker larvae of the same age. The differences between the two groups of

larvae become greater as time goes on. The queen larva develops into an insect nearly twice the size and weight of the worker bee and her spermotheca and ova- ries are more highly developed. In the queen bee the number of egg tubes in the ovaries number 150 to 170, while there are only 4 to 5 egg tubes in the ovaries of the worker bees. The size of other glands differ in the two groups. The queen’s man- dibular glands are about three times larger than those of the workers, yet the pharyn- geal glands are 40 to 50 times larger in the worker bees. The queen completes her development from egg to the adult state in 15 to 17 days compared to 24 to 27 days required for the worker. Queen bees live longer than workers. Ordinarily the latter live only a few weeks, except for those workers developed late in the summer who survive the winter. These bees live for about 300 days or longer. Queens, on the other hand, normally live from 4 to 6 years. Probably the most amazing capacity asso- ciated with this dimorphism is the tre- mendous egg laying ability of the queen. She is capable of laying roughly 1,500 to 2,500 eggs per day, approximately twice her body weight, not for a single day, but for weeks and months. It is not at all sur- prising then to find she also has a much higher rate of metabolism than the worker bee.

The material credited with producing these marked changes in the two types of females is royal jelly, a substance produced by the pharyngeal glands of the worker bees. In recent years a number of investi- gators have made detailed chemical and biological analyses of royal jelly in an

1This research was made possible by a grant from TMr. Rubottom held a pahtime Reseakh Fellow-

123

the Sioux Honey Association Sioux City Iowa.

ship, sponsored by the Lederle Laboratories.

124 HELEN DAWSON, RICHARD RUBOTTOM AND LOWELL HARRIS

effort to determine not only its composition but, more specifically, to identify the sub- stances responsible for effectuating di- morphism in the bees. This complex and variable material is conceded as having approximately the following composition : moisture 66.05%, protein 12.34%, total lipid 5.4%, total reducing substance 12.49%, ash 0.82%, undetermined 2.84% .

Royal jelly is a good source of the B vitamins but has little or no demonstrable amounts of vitamins A, C, D, E or K. Sig- nificant amounts of amino-acids and their derivatives are found in royal jelly in the free state as well as in the combined form. Pearson and Burgin ('41) in determining the pantothenic acid content of royal jelly, found it to be one of the richest natural sources of pantothenic acid. They suggest that this material may be responsible, in part at least, for queen development. Kitzes, et al. ('43) found that royal jelly contained 4.1 ~1 g/g (dry weight) of biotin and concluded that both biotin and panto- thenic acid might play an important role in the metabolism and differentiation of the young larvae. Gontarski ('49) likewise worked on the biotin content of royal jelly.

Obviously, so far no specific element of royal jelly has been isolated and proven to be the factor responsible for causing the dimorphism in bees so as to prove be- yond the shadow of a doubt the validity of the qualitative theory.

Haydek ('43) favors a differentiation based on the quantitative differences in the food of the worker and queen larvae. He suggests the extra nutrients available to the queen larvae results in rapid growth and maturation of their ovaries. The ova- ries then secrete a hormone which circu- lates in the blood and inhibits the differ- entiation of worker bee characteristics. Ribbands ('53) likewise seems to favor this idea.

It is not surprising that royal jelly has intrigued and interested man for some time. Here is a substance which when fed continuously to female bee larvae increases the life span 20 to 25 times and promotes sexual vigor, a veritable Fountain of Youth for the queen bee. It is unfortunate, how- ever, that without adequate scientific evi- dence and proof, some people seem to have assumed that royal jelly can perform

equally spectacular feats if administered to man or animals. Such ill-advised as- sumptions without adequate proof can be detrimental and dangerous if taken seri-

In view of the meager amount of objec- tive evidence on the effect of royal jelly on differentiation and development of higher forms of life, the following experi- ment was carried out in an effort to deter- mine what effect repeated administration of royal jelly to newborn rats would have over a prolonged feeding period.

ously.

METHOD AND MATERIAL

Royal jelly is a natural food for honey bees and, in fact, is the only food of the queen. In trying to test its effects on im- mature rats it is neither possible nor prac- tical to use it as the sole source of nutrient material for the newborn of this species. However, we did want to give repeated large doses of royal jelly, limited only by the tolerance of the experimental animals and ability to inject the substance intra- peritoneally into the newborn rats. Royal jelly is an extremely biologically potent material and since relatively little informa- tion is available on dosages of royal jelly for animals we ran a series of pilot experi- ments dealing with methods of adminis- tering royal jelly (oral, subcutaneous and intraperitoneal injections) as well as in a variety of dosages and variations in fre- quency of injections. From the results of our observations we chose to administer .1 cm3 of a 2% aqueous solution of royal jelly every third day for the duration of our experiments. Thus each dose contained the equivalent of approximately 2.60 mg of raw royal jelly. The royal jelly was furnished by beekeepers through the Sioux Honey As~ociation.~ For this experiment the royal jelly was obtained from two day cells, shipped to us in opaque jars and refrig- erated immediately on arrival.

Newborn rats of the Long-Evans strain (inbred since '28) were used as the ex- perimental animals. In order to maintain the newborn rats under conditions as near normal as possible, the young animals re- mained with their mothers except for the

3The authors wish to express their appreciation to those bee keepers who furnished us with the royal jelly to carry out this research.

EFFECT OF ROYAL JELLY O N NEWBORN RATS 125

time required to weigh and inject them. The adult females were given a diet of Purina Rat Chow and these pellets were also available to the young rats as they became old enough to eat them. At 21 days of age the young were weaned and separated according to sex, and from this time to the end of the experiment they re- ceived only Purina Rat Chow as their maintenance diet.

Approximately 24 hours after birth the newborn rats were sexed, marked for later identification and weighed. In each litter, on the basis of sex and birth weight, spe- cific animals were indicated to receive in- jections of royal jelly or to serve as con- trols, All injected animals were first weighed and then received intraperiton- eally either .1 cm3 of the 2% aqueous royal jelly solution or. 1 cm3 normal sterile saline. These injections were repeated every third day for a period of either 62 to 64 days or 84 days. The rats of the control group re- ceiving no injections were weighed and handled in a manner comparable to the injected rats for the duration of the experi- ment. At the termination of the experi- ment the animals were weighed, etherized

and autopsied. Adrenals, ovaries or testes were removed, weighed and pieces taken for the histological examination. The data were analyzed statistically by employing the t-distribution method.

In this particular experiment, a total of 239 rats were treated and survived the ex- perimental period.

RESULTS The results of intraperitoneal injection

of royal jelly into newborn, inbred Long- Evans strain female and male rats are summarized in tables 1 and 2.

Females In both the 62 to 64 day

and 84 day old female rats, the mean final body weights are greatest in the animals receiving injections of royal jelly and they are lightest in the saline control groups (table 1). If the t-value of the difference in weights between the royal jelly and the saline control group is calculated, there is found to be a significant difference for both age groups. The mean final weights of the control rats receiving no injections, in both age groups, lie between the means

Body weight:

TABLE 1 Data concerning female rats given intraperitoneal injections of royal jelly evely third day from birth

to 62 to 64 days or 84 days and controls of the same age

Experimental No. of Age in Body Adrenal Ovary group rats days weight weight weight

gm mg mg Royal Jelly 26 62-64 183.8lf 17.22 53.2 212.8 68.72 13.4

Saline 16 62-64 160.3 219.9 55.6 f 6.7 60.42 11.8

( 133-211)3 (28.0-93.6) (52.7-103.6)

(control) (122-193) (44.8-67.4 ) (32.1-85.6) - 2.304s5 + 0.68 ns8 -2.01 s

No Injection 17 62-64 177.7 f19.6 60.7'f 10.0 68.0 .t 10.7 (control) (138-209) (42.1-84.8) (45.2-91.9)

- 1.42 ns + 1.94 ns - 0.19 ns Royal Jelly 25 84 222.3 f25.2 57.3 *14.5 83.8 f 23.9

(188-285) (24.8-79.6) (64.0-150.8)

Saline 20 84 206.5 f18.5 61.0 f12.1 70.9 11.6 (control) (182-234) (36.2r84.2) (49.~3-86.0)

No Injection 15 84 211.7 216.6 62.0 k9.0 76.5 2 11.0

- 3.96 s + 0.88 ns -2.18 s

(control) (183-246) (48.2-79.0) (51.3-95.8) - 1.05 ns + 1.09 ns - 1.10 ns

1 Mean. 2 Standard deviation. 3 Range.

4t-value of difference from mean royal jelly group. J A significant difference. 6 No significant difference. 7 Number of animals was 16.

126 HELEN DAWSON, RICHARD RUBOTTOM AND LOWELL HARRIS

TABLE 2

Data concerning male rats given intraperitoneal injections of royal jelly every third day f r o m birth to 62 to 64 days OT 84 days and controls of the same age

Experimental No. of Age in Bqdy Adrenal Testes group rats days weight weight weight

g m m g m g Royal Jelly 28 62-64 243.0l-i 26.12 33.03 f 10.9 2634.2-t- 262.3

Saline 18 62-64 243.1 ‘-32.9 38.6 C10.6 2461.4 C 379.1

( 191-288 ) 4 ( 18.8-69.4 ) (2177.9-3114.6)

(control) ( 178-301 ) (28.3-79.4) ( 1701.8-3305.4 ) +0.015ns6 + 1.66 ns -1.7911s

No Injection 15 62-64 219.5 k42.7 46.3 k16.6 2428.82361.4 (control) ( 165.1-312.2) (27.9-75.6) ( 1640.3-3273.9)

-2.17 s7 + 3.05 s -2.08 s ___ Royal Jelly 26 84 335.0 rt41.7 44.4 -c11.0 2937.9t-455.2

( 274.2-424.4 ) ( 28.7-65.2 ) ( 1973.4-3626.0)

Saline 16 84 307.4 C46.3 38.8 f7.8 2832.4 rt 342.0

- 1.95 s

No Injection 17 84 306.3 rt28.6 35.7 29.0 2909.8 f 218.9

(control) (222-390) (24.3-52.8) (2036.2-3292.0) - 1.53 ns - 1.73 ns

(control) (253-365) (22.0-53.8 ) (2573.8-3327.4 ) -2.42 s -2.63 s - 0.63 ns

1 Mean. 4 Range. 2 Standard deviation. 5 t-value of difference from mean royal jelly group. 3 No. animals was 27. 6 No significant difference.

7 Significant difference.

of the royal jelly and the saline control groups and are not significantly different from either of these groups.

From the evidence available, it seems obvious that while royal jelly administra- tration does increase body weight of the injected rats as compared to rats growing normally under the usual conditions, there is nothing to indicate that royal jelly has any so-called “hormonal” stimulatory effect in accelerating growth of these rats. The weight increase observed could, more logi- cally, be attributed to the presence of the vitamin B complex and the amino-acids in the royal jelly which may serve to sup- plement the rat chow diet on which the animals are maintained.

Weights of adrenals: The mean weights of adrenals for both age groups are lightest in the royal jelly injected rats and greatest in the control groups that received no in- jections; the mean weights of the saline control groups are intermediate in both age groups. In none of the groups are these differences significant.

Weights of ovaries: Examination of the data summarized in table 1 shows the

mean weights of ovaries, in both age groups, to be greatest in the females re- ceiving royal jelly and lightest in the saline control group. Here, as in the final weight data, the royal jelly groups have signifi- cantly heavier ovaries than those of the saline control groups in both age cate- gories. In the untreated control rats, the weights of the ovaries lie between the two extremes and were not significantly differ- ent from either the royal jelly animals or the saline controls.

If the raw weights for the organs (adre- nals and ovaries) are converted into rela- tive weights, that is, the weight of the or- gan expressed as a percentage of body weight and these relative weights analyzed statistically, we find that there are no sig- nificant differences among any of the fe- male groups in either age division.

From these data there is no evidence to suggest that royal jelly (under the condi- tions of these experiments) has a selective stimulatory effect on the rat ovary or that it affects the adrenal in any significant manner that can be registered by changes in weight of the adrenals.

127 EFFECT OF ROYAL JELLY O N NEWBORN RATS

Males A summary of the data on the male rats

is presented in table 2. Examination of these data reveals that the males respond somewhat differently to the injections of royal jelly than the female rats.

In the males in both age groups, the royal jelly animals are sig- nificantly heavier than the control rats re- ceiving no injections. In the 84 day old group they are also significantly heavier than the saline controls. In the 62 to 64 day old rats, however, the mean weights of the royal jelly and the saline control rats are nearly identical, being 243.0 gm and 243.1 gm respectively.

The lightest mean weights of adrenals are found in the 62 to 64 day old royal jelly males. These weights are significantly lighter than the unin- jected controls; they are lighter than the saline controls but in this instance the difference is not statistically significant. In the 84 day old male rats, the values of the mean weights of the adrenals are re- versed. In this age group, the heaviest adrenals were found in the royal jelly in- jected rats and the lightest adrenals were in the uninjected controls. Again the saline controls occupy an intermediate position. Statistical comparison of the mean adrenal weights of the royal jelly animals to the mean adrenal weights of the uninjected controls shows this difference to be a sig- nificant one. However, there is no signifi- cant increase in weight of adrenals of the royal jelly rats over the adrenal weights of the saline control group. In the 62 to 64 day old male rats, the response is simi- lar to the response observed in both age groups of the female rats. The reaction of the 84 day old males in regard to re- sponse of the adrenals to the injection of royal jelly cannot at the present time be explained.

The mean weight of the testes is greatest in the animals receiv- ing royal jelly. If the t-values of the differ- ence from the two control groups are cal- culated, there is a significant difference in testes weight in the 62 to 64 day old group between the royal jelly and the control receiving no injections. In the other three groups, the differences are not significant.

Body weight:

Weight of adrenals:

Weights of testes:

Here as in the ovary weights in the female groups, it appears most likely that the increased weight of the testes is associated with the general increase in size of the rats due to the nutrient constituents (amino-acids and the B vitamins) present in royal jelly and cannot be attributed to any hormonal stimulation. If the raw weights of the male adrenals and the testes are converted to relative weights in terms of body weights and treated statistically to determine any significant differences, it is found that in no instance are there significant differences in the weights of these organs in either age group.

Histological findings of ovaries, testes and adrenals: From careful examination of the histological sections of the ovaries and the testes it was impossible to distin- guish between organs from rats receiving injections of royal jelly and those of the control animals. Even in the youngest fe- males (62 to 64 days of age) the ovaries showed well-developed Graafian follicles as well as many corpora lutea in the controls as well as for those rats receiving royal jelly. The differences that were noted in the number and size of corpora lutea and maturing follicles were attributed to indi- vidual differences among the animals.

In the males the testes appeared normal. The size of the tubules and the number of tubules containing sperm seemed com- parable in the rats receiving royal jelly and the animals in the control group. Any differences observed were again attributed to be on the basis of individual variations rather than due to any effect resulting from our experimental procedures.

The examination of the adrenals of both female and male animals in both age cate- gories showed no evidence of any stress reaction in the groups receiving royal jelly injections as compared to the rats of the control groups.

DISCUSSION What effect, if any, does the administra-

tion of royal jelly have on the growth and maturation of higher animals? Based on the effect which this material produces in the queen bee, it has been suggested that equally spectacular results would occur in higher animals and man if it were admin- istered to them.

128 HELEN DAWSON, RICHARD RUBOTTOM AND LOWELL HARRIS

Royal jelly, even for the queen bee, is basically a food product. From chemical analysis of royal jelly we know it is a high protein material, containing many amino- acids and many of the B vitamins. One small fraction of royal jelly has as yet not been identified. The suggestion (Contura, '58; Chauvin, '56) that royal jelly contains hormones seems unjustifiable. It has been accepted for some time that royal jelly is produced by the pharyngeal glands of the young worker bees. The pharnygeal glands are exocrine glands, how then could royal jelly contain hormones? Pollen, which may exert estrogenic effects, is not normally present in royal jelly although it appears occasionally as a contaminant.

The overall weight increases we ob- served in our rats, both male and female, given repeated intraperitoneal injections of royal jelly from birth to 62 to 64 days and 84 days of age, as compared to the control rats, suggests that royal jelly con- tain constituents (undoubtedly nutritional) which may serve to supplement the basic diet on which all of the animals were main- tained.

The variations (Hinglais et al., '56) or similarities (Heyl, '39) in findings reported on the effect of royal jelly on growth (as evidenced by weight increments) may well be explained by variations in the adequacy of the basic diet and the inherent need and ability of individual rats (or strains of rats) to utilize the nutrient materials available.

The weights of the ovaries or testes of the royal jelly injected rats were heavier than these organs in the control groups and roughly parallel the overall weight in- crements mentioned above. In several in- stances weights of ovaries or testes in the experimental rats were significantly heav- ier than the comparable organ of the con- trols. However, the pattern, in the two age categories for the two sexes, was not consistent as is evident from the data in tables 1 and 2. If the raw weights of the ovaries or testes were reduced to the per- centage of final body weight for each in- dividual animal and these figures subjected to statistical analysis, we found that none of the mean percentage weight evaluations of the organs of the experimental rats were significantly different from the control

groups. In other words, royal jelly does not specifically stimulate or accelerate the growth of either the ovaries or the testes beyond that produced by the accelerated growth of the whole animal. Chauvin ('57) likewise reported that royal jelly adminis- tered to mice had no effect on the develop- ment of the gonads (testes and ovaries). Our findings raise a question concerning Heyl's report ('59) where after consecutive subcutaneous injections of royal jelly for only 5 days to 21 day old females, he found the ovaries nearly twice as heavy in the experimental rats as in the controls. Efforts to repeat Heyl's experiment will be attempted later.

In 1943 Stanley, Mickey, Melampy and Oertel reported that using high dosages of royal jelly produced hypertrophy of the adrenals in some of their rats. We also removed and weighed the adrenals in our animals at the end of the experiment pe- riods. With the exception of the 84 day old male rats, the royal jelly injected ani- mals had the lightest adrenals in each sex and age category. The 84 day old royal jelly rats had adrenals significantly heavier than the uninjected controls. How- ever, here again, if the adrenal weights are converted into percentages of final body weights for each individual animal and these percentage figures analyzed sta- tistically, no significant differences were found to exist.

The very recent report by Garn and Haskel ('59) which was concerned with the relationship of fat thickness and de- velopmental status in children, pointed out that increased nutrition in the form of excess calories in growing children does accelerate growth of the individual and results in earlier maturation. In our ex- periments we also found that the royal jelly injected rats, both female and male, were heavier and had larger gonads than rats of the comparable control group. The fact that we found no evidence of accele- ration of maturation by microscopical ex- amination is probably due to the age range (62 to 64 days) arbitrarily selected. If we had taken gonadal tissue specimens at weekly intervals from younger animals i t is possible that microscopic evidence of acceleration of maturation might have been obtained.

EFFECT OF ROYAL JELLY O N NEWBORN RATS 129

CONCLUSION

Administration of 0.1 cm3 of a 2% aque- ous solution of royal jelly intraperitoneally to newborn rats of the Long-Evans strain every third day until the animals reached the age of 62 to 64 days and 84 days gave the following results.

Overall growth, as evidenced by body weight and weight of gonads, was accele- rated. There was no indication, however, that the growth curves of the experimental animals were distorted from those of the control groups or that royal jelly specifi- cally stimulated gonadal development or maturation. Adrenal weights in the experi- mental animals were less than in the con- trol groups, except in the 84 day old males where they were heavier than in the con- trols. If the organ weights (gonads and adrenals) of the individual animals are converted to percentage of body weights and these figures statistically analyzed, no significant differences are found between any of the experimental animals and their comparable controls.

From histological examination of the ovaries and testes it was impossible to dis- tinguish between rats receiving royal jelly and rats of the control groups. Histological examination of the adrenal glands showed no evidence of any stress reaction in any of the rats which would be attributed to the effect of royal jelly in the dosage we used.

After careful analysis of our data we conclude that royal jelly will accelerate growth as evidenced by weight gains of the magnitude observed in our experimental animals under the conditions of our experi- ment. We believe that this increment in growth can be attributed to the readily

available nutrient materials, in the form of amino-acids and the B vitamins present in the royal jelly which serve to supple- ment the basic diet on which the animals were maintained.

LITERATURE CITED

Chauvin, R. 1956 11. Composition biochemique de la gelbe royale. L’Apiculteur, 100: 45.

1957 La gelke royale. 111. Action sur les mammiferes et sur l’homme. Ibid., 101: 89.

Contura, J. M. 1958 La gelbe royale, le Mexique et l’homme. Ibid., 102: 10.

Garn, S. M., and J. A. Haskell 1959 Fat changes during adolescence. Science, 129: 1615.

Gontarski, H. 1949 Mikrochemische Futtersaf- tuntersuchungen und die Frage der Koniginnen- entstehung. Hess. Biene, 85: 89.

Haydak, M. H. 1943 Larval food and develop- ment of castes in the honeybee. J. Econ. Ent., 36: 778.

Heyl, H. L. 1939 An observation suggesting the presence of a gonadotropic hormone in royal jelly. Science, 89: 540.

Hinglais, H., M. Hinglais and J. Gautherie 1956 Recherches sur les propribtes anaboliques de la gelbe royale. Experiences chez le rat. Compte Rendu des Seances. SOC. Biol., 150: 2130.

Johannson, T. S. K. 1955 Royal jelly. Bee

Kitzes. G., H. A. Schuette and C. A. Elvehjem World, 36: 1; 21.

1943 The B vitamins in honey. J. Nutrition, 26: 241.

Melampy, R. M., and E. R. Willis 1939 Res- piratory metabolism during larval and pupal development of the female honey bee (Apis mellifica L.). Phys. Zool., 12: 302.

Pearson, P. B., and C. J. Burgin 1941 The pan- tothenic acid content of royal jelly. Proc. SOC. Exp. Biol., 48: 415.

Ribbands, C. R. 1953 The behavior and social life of honeybees. Bee Research Association Limited. 530 Salisbury House, London Wall, London, E. C. 2, p. 350,

Stanley, A. J., R. M. Mickey, R. M. Melampy and E. Oertel 1943 Biological effects of the lipid fraction of royal jelly. Proc. La Acad. Sci., 7: 75.