Book - Chemical embryology 2-7 (1900)
Embryology - 26 Apr 2024    Expand to Translate
|
|---|
| Google Translate - select your language from the list shown below (this will open a new external page) |
|
العربية | català | 中文 | 中國傳統的 | français | Deutsche | עִברִית | हिंदी | bahasa Indonesia | italiano | 日本語 | 한국어 | မြန်မာ | Pilipino | Polskie | português | ਪੰਜਾਬੀ ਦੇ | Română | русский | Español | Swahili | Svensk | ไทย | Türkçe | اردو | ייִדיש | Tiếng Việt These external translations are automated and may not be accurate. (More? About Translations) |
Needham J. Chemical Embryology Vol. 2. (1900)
| Online Editor |
|---|
|
| Historic Disclaimer - information about historic embryology pages |
|---|
|
Chemical Embryology - Volume Two
Section 7 The Energetics and Energy-Sources of Embryonic Development
7-1. The Energy Lost from the Egg During Development
Many investigators, realising that, owing to the changing composition of the embryonic tissues and the raw material of development, it is difficult to compare different entities if the material exchange is alone considered, have thought it worth while to investigate the energetics of the transformations in question. Sometimes this has been done, as we have already seen, by measuring the heat produced during a given developmental period by the embryonic tissues, sometimes it has been done by combusting the embryo and the yolk in the bomb calorimeter, and obtaining in this way data for the amount of energy stored in the substance under investigation. It is with researches of the latter type that this chapter will largely be concerned.
To establish first the fact that the calorific value of an equal amount of a definite substance may change during the developmental period, it is simply necessary to refer to the figures of Murray, who in 1926 estimated the fuel value of i gram of dry substance in the chick embryo between the 5th and the 21st days of incubation. The curve he obtained is shown in Fig. 256, which clearly shows that it rises in a sigmoid form, the points agreeing well enough with the earUer values of Tangl. The increase in calorific value is no doubt due to the decrease of the inorganic and the increase of the organic quota in the embryo. From Fig. 256 it can be seen that the calorific value of i gm. of dry embryo at the end of incubation is about 6-2, having risen from 5-1 at the 5th day, and Murray pointed out that it was rising up towards the level of the calorific value of the unincubated yolk and white taken together, i.e. 6-94. The variation in fuel value here shown reveals well the drawbacks of the plastic efficiency coefficient. As a measure of efficiency it does not take account of the fact that the units on which it is based are constantly changing in calorific quality. It is interesting that Murray found a divergence between the observed and calculated calorific value in the first half of incubation.
- Throughout this book "calorie" means gram-calorie and "Calorie" kilo-calorie.
This was rightly regarded by Murray as outside the Hmits of the standard errors. He concluded that either or both of the two constants used in the calculations were too high for the protein and fat of the embryo during the early stages of incubation, for, after all, these constants have been exclusively derived from experiments on adult tissues. It may, therefore, be assumed as probable that the true calorific constants for the substances present in early embryonic life (in the case of the chick) should be regarded as lower than those for the corresponding adult substances. It follows, then, that, just as the fuel value per gram of organic matter rises with age, so also the fuel value of either or both the protein and fat fractions rises, due to the increasing proportion within each group of substances with a relatively high calorific value.
Fig. 256.
The opening up of the study of energy-relations in embryogenesis is due to Tangl and his collaborators, who in a long series of papers from 1903 onwards published the results of their extensive researches with the bomb calorimeter. Eichwald's review presents some of their data. Tangl's first paper dealt with the avian egg, and he defined his aim as the attempt to see how much energy was utiHsed during development, in what manner the process went on, and what were the sources of it. He was profoundly impressed by the difficulty and the fascination of the problem of "work of development", the problem of relating the morphological and structural coming-intobeing with physico-chemical work done. It was not possible, he felt, that living animals should acquire ontogenetically their shape and form, without having to pay a fee, perhaps a heavy one, to entropy. Obviously this question is one of some difficulty, and a good deal depends on definitions, in which respect Tangl and his school were not altogether happy. "Die Menge der wahrend der Entwicklung des Embryos umgewandten chemischen Energie, nenne ich Entwicklungsarbeit", said Tangl. Thus the first figures he obtained (on the egg of the starling)
cals. Undeveloped egg ... 3067
Finished embryo ... 2312
Entwicklungsarbeit 755
showed the method he intended to adopt. In calling the total amount of energy not used for storage in the embryo "Entwicklungsarbeit", Tangl was confusing two things which it is important to keep separate, (i) the amount of energy used by the formed cells of the embryo during development for combustion processes, and disappearing as heat, and (2) the amount of energy, if any, which passes into the embryo in the form of food from the yolk and white, and which is yet not recoverable from the dried material by the bomb calorimeter. This second fraction has more right to be called "Entwicklungsarbeit" than the first one, for as soon as any new cell is formed it begins an oxidative metabolism of its own. Undeniably this is work done during development, but not a true "Entwicklungsarbeit", the energy of which would have to be put down on the balance sheet of ingoings and outgoings as missing, i.e. in some way bound up with the structure. Exactly how this could take place has been the theme of several speculations; thus some have suggested that energy would be required to maintain certain orientations of molecules at intracellular surfaces, and doubtless the form of the animal is the outward and visible sign of such inward steady states, but these postulated processes have never been demonstrated. There is also, of course, osmotic work and secretion work to be considered. I shall return later to this point; meanwhile, it is necessary to penetrate further into the facts concerning "Entwicklungsarbeit". The terminology presents difficulties, but the method adopted will be to speak of the "Entwicklungsarbeit" in Tangl's sense as Ea. and of the true "Entwicklungsarbeit", if there is any such thing, as O.E. or organisation energy. Thus Ea. will be defined as the amount of energy not stored in the embryonic tissues and not left behind in the unused raw materials at the end of development, while O.E. will be defined as the amount of energy, if any, laid up in the embryo, which, though appearing as calorific value of combusted wet tissues, would not result from the combustion of an unorganised mixture of its constituents^. If all the constituents of the finished embryo could be mixed together mechanically and the mixture then combusted, the calories contained in it might be slightly fewer than those contained in the same substances when organised into an embryo. This difference is the O.E. These conceptions are illustrated by the diagram in Fig. 259, to which reference should be made.
Tangl's first experiments, then, showed that the Ea. of the starling's egg was 755 caL, i.e. 24-6 per cent, of the original amount of energy present, corresponding to a loss of dry weight during development of 15-7 per cent.
His next experiments were done on hen's eggs of three races, all of which had dry weights when unincubated varying between 24-3 and 24-9 per cent., and calorific values of between 6906 and 7078 cal. per gram, dry substance, and between 1692 and 1734 cal. per gram wet substance. The whole egg contained 88-9 Cal. What happened on incubation is shown in Fig. 257. The energy contained in the tissues of the embryo increased steadily with its growth ; that contained in the remainder of the egg equally steadily decreased, but in addition there was, of course, a loss from the egg as a whole due to the combustions. This loss appears on the graph as a shaded area, the extent of which at the end of development represents the Ea. and amounts to 16 Cal. This is more energy than is spent by the starling embryo during its development, but the chick embryo is larger than that of the starling. In order to have a common basis of comparison, Tangl computed the Ea. as related to i gm. of embryo (wet weight), which he called the "relative Entwicklungsarbeit ' ' (hereafter referred to as the R.Ea.), and as related to i gm. of embryo (dry weight), which he called the "specifisches Entwicklungsarbeit" (hereafter referred to as the S.Ea.). When these values were calculated out for the chick, he obtained the graph shown in Fig. 258. The Ea., of course, rose during development, for the quantity of material burned on any one day rose, but the R.Ea. and the S.Ea. fell, as they were bound to do, owing to the declining metabolic rate. Tangl introduced some corrections at this stage for the weight of the membranes in the early stages, but, even after these had been made, the R.Ea. and the S.Ea. were still much higher for the earlier than for the later stages. In other words, more energy was dissipated in forming i gm. of loth day embryo (wet or dry weight) than in forming i gm. of 21st day embryo, a conclusion quite in harmony with all that is known about the change in respiratory intensity with age.
1 Definitions of this and other terms will be found summarised together on p. 999.
Fig- 257
Tangl compared the magnitude of R.Ea. in the chick embryo with the " Erhaltungsarbeit " or basal metabolism of the adult hen; an average figure for the chick was 100 cal. and for the adult hen 71, other conditions being maintained as equivalent as possible. He concluded, also, that the Ea. was derived almost entirely from fat, by dividing the amount of dry solid disappearing from the egg by the amount of chemical energy disappearing, thus finding that the calorific value of the material disappearing was, on an average, 9000 cal. per gm. He then combusted some purified egg-fat and obtained a calorific value of 9476 cal. per gm. Tangl did not fail to point out how well this fitted in with the results of the chemical analyses of Prevost & Morin; Liebermann, and Pott.
The dotted lines sh^ the figures corrected for the membranes
Fig. 258.
Table 1 16.
Cals.
Cals.
Efficiency of storage.
Day
Stored
Combusted (Ea.)
Total
Storage x loo/storage and combustion
10
0-269
1-42
1-689
15-9
10
I -04
2-76
3-8
27-4
12
2-2
5-8i
8-01
27-5
12
3-78
8-08
11-86
31-9 65-0
18
15-58
8-35
23-93
19
23-89
18-79
42-68
56-0
21
IIU
13-51
40-85
65-0
21
20-09
56-77
65-0
21
31-63
14-30
45-93
69-0
Tangl next considered the relations between Ea. and the energy stored up in the tissues of the embryo, though he did not discuss this from the point of view of efficiency. Nevertheless, the table he drew up is interesting, and is reproduced in Table 116. As development goes on, the relation between storage and combustion changes, for while at first the efficiency of storage is low, it rises as development proceeds, and reaches 66 per cent, or so by the end of incubation. This course of events has since been repeatedly confirmed. Tangl expressed it by concentrating attention on the fact that the body of the finished chick embry^o contains roughly 32 Gal. of chemical energy, and the Ea. is roughly 16 Cal., therefore about | of the original energy was used for storage, and | for combustion. The overall energetic efficiency was therefore 66 per cent. Tangl's figures for calorific value of the embryonic tissues have already been discussed in relation to the similar ones obtained by Murray — it is worth noting, however, that Tangl did not find any notable alteration in the calorific value of the unused yolk between the loth and 21st days of incubation. The distribution of chemical energy in the finished embryo was as follows:
Table 117.
^ /•
%of
the energy
Dry
content of
calories per
weight
the whole
gram dry
Organ
in grams
calories
embryo
weight
Muscles
1-3391
8,951
28-3
6687
Central nervous system ...
0-1642
986
3-1
6007
Viscera
0-9329
5'55i
17-6
5950
Skin, etc
1-1927
6,756
21-4
5537
Bones
1-4461
7,094
22-4
4907
Remainder
0-4502 5-4801
2,288
7-2
5647
Whole embryo
31,626
loo-o
5771
Membranes
0-2818
1,220
—
4329
To a large extent these figures reflect the varying fat-content of the individual parts of the body. Comparative researches on this subject at different stages of development might reveal some interesting relationships.
The paper of Tangl & v. Mituch contained a more accurate investigation of the energy relations in the hen's egg. The individual differences between energy-content of embryos from different hens, etc., were found to be exceedingly small; and the average figure for the Ea. was 22-94 Cal. This was distinctly higher than the corresponding value given by Tangl in his first paper, and it meant, of course, that the R.Ea. and S.Ea. were also higher than he had at first thought. The following table shows the figures for six embryos :
Table
118.
calories
From hen {a) From hen {b)
Ea. 20,460 22,940 23,130 23,640 24,200 23,240
R.Ea.
727 821
993
£§
743
S.Ea. 3830
3260
3810 3400
Specifi( of the
energy-content substance burnt
9,250 10,510
9,070 10,460
so that the average result was R.Ea. 805 cal. and S.Ea. 3600 cal.
Tangl's second paper was concerned with an attempt to carry out his ideas, working with various bacilli during the development of cultures. His work in this field will be found assessed in Stephenson's monograph. The third paper of the series was by Farkas, and contained a careful study of the developing silkworm egg from the energetical point of view. He made complete analyses of the eggs before and after their development, the details of which receive consideration elsewhere in this book. For the unincubated egg he got the following values :
calories per gram
calories per gram dry weight...
calories per gram fat
for the hatched larvae:
calories per gram
calories per gram dry weight...
2163
6104 (specific energy-content)
9343
1631 5782
and for the unused materials, membranes, etc. :
calories per gram
calories per gram dry weight...
4560 5301
Or, in round numbers, expressed differently, i.e. for the whole material :
Unincubated eggs Hatched larvae
Unused material, membranes, etc. .. Material lost, i.e. used up during de velopment ...
Calories
in the
material used
71-402
31-879 22-291
17-232
%of the value for
the unincubated eggs
44-65 31-22
24-13
The analytical figures showed that 17-32 per cent, of the original dry solid, 48-24 per cent, of the original fat-content, and 0-65 per cent, of the original nitrogen-content had disappeared, so at first sight it seemed certain that the source of the energy utilised had been fat. However, as the nitrogenous end products were not estimated, and as they would remain in the eggs and so form part of the nitrogen value at the end of development, some protein may have been burned too, and perhaps some of the fat was turned into carbohydrate, of which no determinations were made.
From the figures just given, it follows, as 42,220 eggs were used, that in the development of one silkworm egg 0-408 cal. is required for waste or combustion, i.e. the Ea. is equivalent to 0-408 cal. or o- 1 74 mkg. From the same figures the R.Ea. may easily be calculated, and Farkas found that it came to 882 cal., while the S.Ea. was 3125 cal. Farkas was naturally struck with the resemblance between these figures and those which in the previous summer had been found to hold for the hen's egg by Tangl, i.e. R.Ea. 658 cal. and S.Ea. 3426 cal. Considering that the hen's egg weighs 70,000 times as much as the silkworm's egg, and the hatched chick 50,000 times as much as the hatched silkworm, the agreement was remarkable. It meant that, in order to produce i gram of finished embryo, whether of the silkworm or the chick, and whether wet or dry weight was considered, about the same amount of energy was required for combustion purposes. It meant that in each case about the same degree of wastefulness was found in embryonic development; thus the average overall number of calories per gram of finished chick (dry weight) was 5771, and the average number of calories wasted in producing this result was 3426; therefore the work was done with
an efficiency of 62-9 per cent. I 7, x 100). Tangl and his
5771 + 3426 /
associates, however, did not emphasise this aspect of the question, for they were more interested in the problem of the relations between energy and form. They did not look on the energy of the substances combusted as energy wasted, i.e. as energy lost during development, but rather as energy associated in some way with the assumption of structure and form, i.e. as energy lost for development.
Table 119.
Used during development
D^ Weight
solids Energy
Fat
Other solids
Weight
Energy
Weight
Energy
(mgm.)
(cal.)
(mgm.)
(cal.)
(mgm.)
(cal.)
I gm. larva (R.Ea.)
103-6
882-0
59-9
559-0
43-7
323-0
I gnn. dry weight
larva (S.Ea.)
367-8
3125-0
212-1
1982-0
155-2
1 143-0
I larva (Ea.)
0-048
0-41
0-028
0-26
0-02
0-15
Farkas went on to point out that, during the development of 19-54 gm. of silkworm larvae, 2-03 gm. of dry substance had disappeared from the eggs, corresponding to 17-23 Cal. of energy. The specific energy-content of the substance lost (energy per gram dry weight) was 8-51 Cal., which differed from the results obtained by Tangl on the hen, i.e. between 9 and 10 Cal. The analytical figures permitted Farkas to conclude that the Ea. of the silkworm's egg was provided to the extent of 63-4 per cent, by fat combustion and 36-6 per cent, by the combustion of some other substance or substances, of which protein was probably the most important, though, from Tichomirov's earher work, some carbohydrate was probably also utilised. Table 119 illustrates these facts. By running one series of larvae right through after hatching during a hunger period of some days, Farkas was able to get some idea of the energy relationships during this post-hatching period, during which the remains of the yolk are used. As Table 120 shows.
Table 120.
Loss of matter and energy
Embryonic period + hunger period
Embryonic period only
Hunger period only
Gm. % of undeveloped
0-1786 30-4
0-1036 17-32
0-0750 13-08
Gm.
% of undeveloped
o-io6o 79-77
0-0599 48-24
0-0461 31-53
Cal. % of undeveloped
I-4IO 40-23
0-882 24-13
0-528 i6-io
Dry weight
Fat
Energy
the values for the post-embryonic period are all lower than those for the time before hatching. Comparison of these results with the analytical figures indicated that substances of lower energy-content than either fat or protein were combusted for energy during this period.
Tangl & Farkas next published a joint paper on the development of the trout embryo They found that r egg (presumably of Salmo fario) weighed 88-2 mgm., and contained 193 cal. energy. The specific energy-content (i.e. per i gm. dry weight) was 6453 cal. For 5 1 8 eggs the energy-content before development was 99 • 85 Cal. , and after it 96-39 Cal., showing a loss of 3-46 Cal. during the process, or for one egg 6-68 cal. Neither nitrogen nor fat diminished — ^in fact, the latter rose by 37 per cent. — a circumstance which led Tangl & Farkas, after various experiments, to the suggestion that urea and uric acid were acting as sources of energy. This remarkable assumption has since turned out to be unnecessary, and will be discussed later (see p. 1 1 18). Tangl & Farkas could not calculate the R.Ea. and the S.Ea. for they were unable to ascertain the weights of the embryos at the various stages.
Tangl & Farkas made a comparison between the three kinds of eggs they had studied, as follows :
Loss in % of the original amounts from fertilisation to hatching
Trout
Hen
Silkworm
Total weight ...
Water
Dry solid
5-6 7-1
2-7
17
21
i8
26 69 17
Loss in
% of the total loss
Water
Dry solid
. ^6
85 15
77 23
The slight loss of water from the trout's egg shown by Tangl & Farkas does not contradict the findings of Kronfeld & Scheminzki, for, as Fig. 238 shows, before hatching the water-content of the larva as a whole is almost constant, although that of the yolk alone is decreasing. The Ea. of 6-68 cal. was a remarkably small proportion of the energy originally contained in the egg, only 3-5 per cent., and contrasted with the 18 per cent, which is lost by the chick embryo by the time of hatching, and the corresponding 24 per cent, of the silkworm. But it must be remembered that hatching occurs relatively early in the trout, and that for a long time afterwards the yolk is the only source of food for the larva. The R.Ea., then, as Tangl & Farkas pointed out, would have been distinctly lower than that for the other embryos. Here we touch one of the fundamental difficulties of Tangl's conceptions, for, when we define the S.Ea. as the amount of energy used for combustion during the storage of, i.e. the formation of, i gm. dry weight of the finished embryo, we omit to define what a finished embryo is. Tangl assumed throughout his work that the time of hatching was the natural index, but in the case of organisms such as the trout, which have a prolonged yolk-sac period, this is evidently wrong. It is probable that, if one were to take the yolk-sac period into consideration, one would find an R.Ea. very like those for the chick and the silkworm, but this has not so far been done.
7-2. Energy of Growth and Energy of Differentiation
The sixth and seventh papers of the series were devoted by Tangl to a study of the energetics of metamorphosis in the fly, Ophyra cadaverina, and to a general discussion of the meaning of "Entwicklungsarbeit" in relation to embryonic growth and insect metamorphosis. Here he distinguished between "Erhaltungsarbeit" or basal cataboHsm, i.e. energy of maintenance, on the one hand, and "Arbeit fiir Bildung von lebenden Substanz", on the other hand, but he regarded the former as negHgibly small during embryonic development, probably a rather important error. The "Bildungsarbeit" he divided into " Neubildungsarbeit " and "Wachstumsarbeit". Roughly corresponding to differentiation and growth respectively, these terms stood for the production of organs and the laying down of morphological and chemical structures in an architectural plan, on the one hand, and the actual increase in size of individual cells and the body as a whole, on the other hand. Tangl hit upon an ingenious method which he hoped would solve the problem of assessing how much of the " Entwicklungsarbeit " was devoted to "Neubildungsarbeit", and how much to "Wachstumsarbeit", i.e. the study of insect metamorphosis. There, Tangl argued, the weight loss was very slight, practically negligible, no food was taken in, there would be no "Wachstumsarbeit" (Wa.), and all the changes could be regarded as the rearrangement of a pre-existent pattern, alterations of the form and spatial arrangement of the cells making up the various organs. Everything would be "Neubildungsarbeit" (Na.). This proposal, interesting as it was, was from the first open to criticism. It was well known that the pupa has a definite, if feeble, respiration, and therefore could hardly be considered as losing no weight. Moreover, Tangl's calculation depended upon the assumption not only that
Ea. = Wa. + Na.,
which is probably not true, but also upon the assumption that the " Umbildungsarbeit " (Ua., i.e. transformation work) of metamorphosis was equivalent to the Na. In other words, practically no attention was paid by Tangl to the histolysis of the old arrangements, although he was expressly studying a holometabolic insect. Why should there not be a " Histolysearbeit " (Ha.)? It is well known to builders and contractors that "Histolysearbeit" may be considerable. If there were, Ua. would be equivalent to Ha. + Na., and, as no method was devised for distinguishing between these two, the subtraction of Na. from Ea. to get Wa. was invalid. Again, the insect larva and pupa contains a "fat body" which, according to Folsom and many entomologists, can be considered as the equivalent of a yolk, and which disappears in metamorphosis, Tangl was here making the same mistake as has so often been made by other investigators of closed systems such as eggs. A given substance increases in amount, therefore, they say, it cannot be in process of being used up, forgetting that there may be a balance between catabolic and anabolic factors, the net result of which happens to be in favour of the latter. So here, the "fat body" may be responsible for a good deal of Wa. in metamorphosis.
Tangl used for his work a large supply of the larvae of Ophyra cadaverina, one of the corpse flies, and carried out on it a large number of chemical analyses and bomb calorimeter measurements. Thus, during the 6 days of pupation, looo pupae combusted 3-72 Cal. of energy, or per day 0-62 Cal., and during the 13I days of metamorphosis the pupae combusted 3-82 Gal., or 0-282 Cal. per day. Thus the energy utilisation per gm. per day during the first period was 57-2 cal., and during the second period 36-0 cal. From his chemical analyses Tangl calculated that, in the pupation period, 88-7 per cent, of the energy in the solid burned was provided by fat, and that, in the metamorphosis period, 98-6 per cent, was so provided.
From the figures given, it follows that during the metamorphosis of the completely pupated larva into the completely free imago 3-82 cal, are lost per insect. To this must be added the calorific value of the excrements in the chrysalis and the chrysalis case itself, i.e. 4-44 cal., making a total of 8-26 cal. As the imago when completed weighed 7-32 mgm. (all these values, of course, were averages from a large number of observations) the R.Ea. and the S.Ea. were readily calculable, and came out as follows :
calories
R.Ea 523
S.Ea. ... ... 1566
though these should perhaps be termed R.Ua. and S.Ua. With these figures Tangl compared some other values which he calculated from the chemical work of Weinland on the fly Calliphora vomitoria thus:
calories
Ea. 24-3
R-Ea 399
S.Ea 1184
The agreement was striking, but would have been even more so had Weinland counted the abandoned chrysalis cases as part of the waste.
instead of part of the fly; when Tangl's figures were computed in that way, they came to R.Ea. 462 cal. and S.Ea. 1 144 cal., almost in exact correspondence. Tangl next compared these results with those of Farkas on the silkworm's metamorphosis, and found that, though the Ea. of the silkworm was much higher than that of either Ophyra or Calliphora (it is a much bigger insect), its R.Ea. and S.Ea. were very similar:
calories
Ea.
379
R.Ea. ...
481
S.Ea. ...
1962
from which he concluded that the energy wasted by combustion in the production of i gm. weight of imago wet or dry from the larval stage was much the same for the two diptera and the lepidopteron.
The next step in Tangl's calculations was to find out how much energy had to be given off in order to transmute the larva into the pupa. Knowing the weights of the organism at the beginning and end of the pupation period, and having the results of bomb calorimeter measurements at hand, these values were obtained:
calories
Ophyra cadaverina
^ Bombyx mori
(Tangl)
(Farkas)
ation period (larva to pupa)
Ea.
4-34
416
R.Ea
467
317
S.Ea
... 1157
1429
amorphosis period (pupa to
imago)
Ea.
3-82
379
R.Ea
... 1566
481
S.Ea
1962
By adding the results together so that the energy-consumption for the whole period, i.e. from the beginning of pupation to the birth of the adult imago, Tangl got the following figures :
Ea
R.Ea
S.Ea
which, as he did not fail to notice, are of the same order as those for embryonic development.
This relation he elaborated at length in the seventh paper of the series. As can be seen from Table 121, where all the relevant data are collected, it was very definitely the case that the period of true
Ophyra cadaverina
Bombyx mori
8-i6
795
1115
1032
3344
4115
metamorphosis, i.e. from complete pupation to free imago, had an R.Ea. and an S.Ea. of about half the value for pupation plus metamorphosis, and, more significantly, half that for embryonic development. As, in Tangl's belief, metamorphosis consisted only of "Neubildungsarbeit" with very little "Wachstumsarbeit", the conclusion naturally followed that the partition between the two was probably 50 per cent. Inspection of Table 121 shows clearly that, for the formation of a gram of dry weight of finished embryo, an approximately equal amount of energy has to be used up, and this irrespective of the position of the animal in the taxonomic scale. Tangl's remark that "die spezifische Entwicklungsarbeit der tierischen Organismen keine Funktion ihrer phylogenetischen Stellung und Organisationstufe ist" may be said to be true, though, in spite of the remarkable correspondence between the silkworm and the chick, it would be desirable to extend the number of well-authenticated cases. Tangl also laid much emphasis on the fact that the energy utilisation was much faster in embryonic development than in metamorphosis, in the case of the silkworm, for instance, being as 2-97 : o-6i, or per day per gram 198 cal, in embryonic life and 33 in metamorphosis. "Die embryonale Entwicklung", said Tangl, "beansprucht also einen viel grosseren und intensiveren Umsatz von chemischer Energie als die Metamorphose; sie erfordert eine grossere und intensivere Arbeit." Tangl admitted that an unknown proportion of his "Entwicklungsarbeit" in embryonic development and his " Umbildungsarbeit " in metamorphosis was really "Erhaltungsarbeit", "energie d'entretien", or maintenance catabolism, but he thought it possible that this fraction was identical in the two processes. Thus the way was laid open for the subtraction of the metamorphosis S.Ea. from the embryonic S.Ea. He himself never actually made this calculation, but it was obvious from his figures that the average values for metamorphosis were R.Ea. 437 and S.Ea. 1550 cal., while those for complete development, when halved, were R.Ea. 422 and S.Ea. 1270 cal. The energy of development, then, would be regarded as being approximately equally divided between diflferentiation and growthprocesses. Tangl did not, however, by any means commit himself to this conclusion, for he also suggested that the main component of the energy burned during metamorphosis was "Erhaltungsarbeit". At this point, indeed, Tangl and his associates came up against the difficulty which so often confronts those who try to relate chemical
.aS
1^
si
1 9^9 '^°?
t^jtrun m
dry
ight of nished mbryo
1 ^1-g§
I 7' r
III I III III
III I III III
H,i|- i 5>l S I I I
I .?! ^ I I I
I I I
I I I
.yawl's?.
13 " ^ C S 6
^ o
l-S i^'l I I I I I
g c c ^ CI e(
CO CO CO
I III
s s s
be no be
2°^
I ^1 ^1 I
CO
2^ 1
COCO
'1
i 1 1
W mo I 6 f-<ri
I I
« m CO
- « CO
r- CO in
UD CI «
■* lO
ci o o
CJ ocj,
CO 'J' E2
o coo
CO 1^ CO
CO eooD
CO c~) m CO O CO t}- CO t^ CO o>co
-g^
ffi ;
•s I.
o ^
11
c^^
wffi
O O --C 3 ^ S 3 ^r2 -72 LS "H O
^^■§
s w-g tr
5 s
T3Ph
1:2
Q
•S ^ C« P_ iv)
Oc^
a c« 3 ca ^ D ?
i ?n bo 'S 5 ° bo
! o2 o2 o2
i t! c3 S rt t! ni
■ S > ■ > 5 >
i 3 rt > c« 3 ta
with morphological events, namely, the difficulty of classifying morphological events in a really satisfactory way. In spite of all that had been done on metamorphosis by histologists, zoologists and naturalists, Tangl could not with certainty decide in what proportion growth and differentiation were proceeding ; terms vague enough at best, and presenting almost equal difficulties in embryonic life. We here come face to face once more with that great impediment to research in these domains, the fact that we have no quantitative measure of differentiation (on this see Section 3-2 and the Epilegomena) .
Before discussing the general outcome of Tangl's work, the eighth paper of his series must be mentioned. In it Glaser reported his estimations of the calorific value of the egg of a teleost, the minnow Fundulus heteroclitus. The figures came out as follows :
calories
1000 eggs 3273
1000 embryos ( + membranes) ... 2550
723
As 1000 finished embryos weighed 0-535 gm. dry, the S.Ea. was 1350 cal. Glaser, however, realising that sHghtly more than half the weight of the embryo at hatching was unused yolk, doubled this figure, obtained an estimate of 3280, which was in good agreement with the rest of the figures obtained by Tangl and his school. Glaser also calculated that the specific energy-content of the substance burnt was 9-0 Cal., from which he concluded that the greater part of it was fat.
7-3. The Relation between Energy Lost and Energy Stored
Subsequent work by various authors brought forward figures which are shown in Table 121, but which do not agree with those of Tangl and his associates. This is probably due to the less accurate character of the later work. For the frog the figures of Faure-Fremiet & Dragoiu, as can be seen from the table, differ somewhat from Tangl's, especially as regards R.Ea. and S.Ea., although the efficiency as calculated from them agrees well enough with the earlier work on the silkworm and the chick. This cannot be said of Faure-Fremiet's experiments on the eggs of Sabellaria and Ascaris. Perhaps the divergence here is partly due to the difficulty in deciding just when embryonic development is complete, and the impossibility of separating the embryo from the yolk. Faure-Fremiet's high levels of efficiency are probably illusory.
We may now return to the distinction made above, namely, that while no one could have taken exception to Tangl's ideas on "Entwicklungsarbeit " if it had been defined as the amount of energy disappearing in the solids combusted during a given amount of embryonic architectural work, yet throughout Tangl's writings the impression conveyed was that the " Entwicklungsarbeit " was the amount of energy disappearing for a given amount of architectural work. It is, of course, true to say that no embryonic growth, or any other kind of vital process, can go on without a certain wastage, for living machines are far from having an efficiency of 100 per cent., but there is no reason for supposing that the energy lost by combustion in the growing embryo is in any way quantitatively related to the actual increase of differentiated structures. It would, in fact, have saved a great deal of controversy if Tangl had expressed his results in terms of efficiency, for that is their real significance. To say that it involves a loss of 3100 cal. to build i gm. dry weight of silkworm, and that it involves a loss of 3280 cal. to build i gm. dry weight of minnow is simply to say that the work of storage which the fertilised egg-cell has before it cannot be accomplished without a certain amount of waste. In the case of the hen, the efficiency of storage is 62-9 per cent., in the case of the silkworm it is 63-2 per cent., in the case of the minnow it is 52-8 per cent., but this last value is certainly too small. Roughly it can be said that the efficiency of energy storage is in the neighbourhood of 66 per cent, in most of the cases known. This is so because the average calorific value of formed living tissue (average for whole body) is much the same. The important fact about Table 121, then, is not that the absolute values for Ea. come out so much aUke, because, after all, the absolute calorific values for the tissues are alike, but that the relation between these is constant, and, in fact, that embryonic development goes on, as far as ^ve can tell, with a constant efficiency in different animals.
But because Tangl apparently did not appreciate the real significance of his figures he was misunderstood from the first. Hammarsten, in an edition of his text-book which appeared during the publication of Tangl's series, showed that he did not understand Tangl's point of view. Then Bohr & Hasselbalch, in their paper on the heat production of the hen's Qgg, took over Tangl's expression "Entwicklungsarbeit", but used it in a quite different sense, namely, that of energy retained for organisation, or O.E. Bohr & Hasselbalch felt that to speak of all the energy lost by the egg during its development as "work of development" was obviously wrong, for to do so is to assume that all the energy lost has been used for development proper apart from the maintenance of life. The only real sense, argued Bohr & Hasselbalch, in which the term " Entwicklungsarbeit " can be used, is to denote that portion of energy, if any, retained by the organism in the passage of fuel material into end-products. Krogh later supported this view. If there was any "cost of production" of embryo from yolk and albumen, then direct and indirect calorimetry would be expected to give different results. These considerations were among those which led Bohr & Hasselbalch to determine the oxygen taken in by the egg and the carbon dioxide and heat given out. As we have already seen (see p. 704 and Fig. 145), the curves for observed and calculated heat production were practically superimposable between the 8th and the 19th days of incubation. There was no retention of heat by the embryo, and therefore no true Ea., i.e. no O.E. But this statement has to be qualified by the proviso that their figures showed a discrepancy of 4 per cent., which might or might not have been heat retained.
These relationships are shown in Fig. 259. The original 87 Cal. of chemical energy present in the hen's egg at the beginning of incubation divides itself into 26 Cal. of unused yolk on the 21st day, 37 Cal. of embryonic tissues and 23 Cal. given off as heat from combustion. These form 30-4 per cent., 43-2 per cent., and 26-4 per cent, respectively of the initial provision. Side by side with the column showing the amount of energy which is contained in one hatching chick, another column is placed showing the amount of energy which would be present in a bottle containing all the compounds present in the chick, in their precisely correct concentration, but in the state of powders or liquids, i.e. entirely unorganised. It is evident that, as we have not a complete analytical balance sheet of all the substances present in the embryo, still less of their calorific values, intramolecular constitution, degree of activation, etc., we cannot at present measure the difference between these two columns, especially as there is reason to believe that it would be very small. However, a portion is marked off at the top of the right-hand column, and labelled O.E. It is to be supposed that Bohr & Hasselbalch's 4 per cent., if it is not simply due to errors of technique, would take its place as part of the O.E. It will be evident that Tangl's work tells us nothing at all about the O.E., or, as we defined it before, the energy retained in a given amount of spatial intracellular and extracellular organisation. It is difficult to form a clear picture of this fraction of the energy. It is to be distinguished from Lapicque's
90000
80000
70000
60000
50000
40000
30000
20000
10000
gm. cals.
.(iLjin)
TANGLS Ea = 22940 cala. / CREa = 805cal8. , ^ SEa = 3600 cals.)
Energy added to embryo b_y coupled reactions etc. i.e. would have gone away as heat •'"■•• othermic reacti{
Energy corresponding to simple storage (same substances but, unorganised
PCals
Energy
brought into
the embrv!
by simple
storage
Energy present in the unused raw V.'t materials at the fi. end of development '^ (spare yolk)
J
Fig. 259. This diagram represents the changes between o and 20 days' incubation. For chicks allowed to hatch naturally U' will be larger ; thus chick weight in % of eggweight is 68 (Jull & Heywang; Upp), 66 (Jull & Quinn) or 64 (Halbersleben & Mussehl) according to the breed (see p. 249).
"epictesis", which is rather the work done by a secretion process, and it more resembles Shearer's conception of the work done in keeping the parts of cells and tissues together as physical systems. If this expense may be said to be in adult life of a definite though small magnitude, then, evidently, as the structure and organisation grows in embryonic life, this quota must also grow. In other words, the more organisation you have, the more molecules you maintain oriented a little differently from the position they would otherwise adopt, the more the O.E, will be. It has often been maintained, e.g. by Johansson, that animals have no expenses of this kind to meet, on the ground that, when Meyerhof caused erythrocytes to cytolyse inside a calorimeter, he observed no increased heat production, yet the same worker's results on sea-urchin's eggs could be adduced on the contrary side (and see also p. 985). In any case, the complex processes of cytolysis would have to be eliminated in some way if a serious attempt was being made to assess the O.E. directly. The conclusion to which we come, then, is that one calorie of energy contained in yolk and white can ttransform itself into one calorie of energy contained in feathers, muscles, blood and brain, without any loss of energy except that necessitated by the living cells in their quality of living cells, i.e. more or less inefficient machines. But apart from this necessary expenditure of energy, apart from the universal income tax extorted from all living cells by virtue of their constitution, the transformations of the egg seem to go on without appreciable cost, and the organisation of the animal appears from nowhere, strangely devoid of physico-chemical antecedents. Such a conclusion is intellectually unsatisfactory, and in the future attempts will certainly be made to demonstrate the existence of a definite O.E. and to measure its magnitude^.
It is necessary at this point to consider again the theoretical work of Rubner on the energy relations in embryonic life. His experiments on the storage of food-material in early post-natal life led him, as we have seen, to the conclusion that the formation of I kilo of mammahan tissue required 4808 Cal. (i.e. total absorption, combustion plus storage), and that in pre-natal life it required about 4000 cal. (2500 for combustion and 1500 for storage). It is evident that the efficiency here is very low, but attention may for the time being be concentrated on the absolute magnitude of the combustion quota. Tangl noticed that Rubner's "law of intra-uterine developmental energy" seemed different from what his own results on the eggs of the lower animals would have led him to expect. Thus to build i kilo of silkworm during its embryonic life
Calories
t/ (Rubner) or Ea. (Tangl) 875
W (Rubner) or specific energy-content 1352
2227
^ There may also conceivably be a quota of energy used in establishing the O.E.; this quota will form part of the Ea.
are required. This contrasts very markedly with the parallel calculation of Rubner for several mammals (horse, cow, sheep, pig, and
dog) : Calories
f/ or Ea. ... ... ... ... 2480
M^ or specific energy content ... 1504
3984
One of the main differences between the two results is that the
figures of Tangl were the result of a large amount of experimental
work, whereas those of Rubner were approximations calculated on
the basis of various more or less doubtful assumptions (see p. 494) .
It followed that the efficiencies were divergent. The storage
expressed in per cent, of the absorption of nourishment, Rubner's
W " energetischer Nutzungsquotient" or jj 7-7^ ~ 100, was, for Rubner's
mammalian embryos, on an average 34-3, and for man was as low as 5-2, while, as will have already been noted from Table 121, for the silkworm it was 63-2 and for the chick 67-0. Tangl concluded that in the two latter cases quite other governing processes operate than in the case of mammals, but it is probable that future work will not support Rubner's "law". It is extremely difficult to see why intra-uterine development should be so much less efficient than development within an egg; the statement, indeed, has almost the status of a biological paradox. At the same time, it would be interesting to know what takes place as regards energy exchange in, for instance, an ovo-viviparous selachian.
It may be noted, however, that Rubner's law was found by Tangl to hold quite well for the early post-embryonic life of the silkworm.
Experimentally found Calories
Calculated on Rubner's basis Calories
S.Ea.orL^,
Wi
.■;: '4085
IIOIO
5010
15728 16020
[t would, therefore, appear that Rubner's law holds in extra-uterine or post-embryonic development only. If this is so, it is possible that the efficiency of the mammalian embryo may be very like that of the non-mammalian embryo, and in any case higher before than after birth.
A number of other workers have occupied themselves with the energy relations of various embryos. Their experiments permit of the following table, which summarises the average efficiencies at present known.
Table 122.
A.E.E. (apparent energetic efficiency)
[Synonyms: Rubner's " energetische
Nutzungsquotient"'; Terroine
& Wurmser's "rendement
energetique brut"']
Calculated
0/ /o
Investigators
Horse embryo
33"3
Rubner
Cow embryo
33'i
^,
Sheep embryo
,,
Pig embryo
40-0
Dog embryo
34-9
,,
Cat embryo
33-0
,j
Rabbit embryo
27-7
„
Average value for mammals ... 34-3
(N.B. These values were not proved by Rubner to hold for pre-natal life, and he thought the average might there be slightly higher, say, between 38 and 41 %) Human embryo ... ... ... ... 5-2
Experimentally Determined Lecithic
Chick embryo ...
Chick embryo ...
Silkworm embryo
Minnow embryo
Frog embryo (to hatching only)
Frog embryo (to disappearance of external gills only) Frog embryo (to end of yolk-sac)
Alecithic Sabellaria embryo Ascaris embryo
Experimentally Determined
Cow adult
Pig adult
Mould, Aspergilltis niger
62-9
Tangl
67-0
Tangl and Murray
63-2
Farkas
52-8
Glaser
82-0
Faure-Fremiet & V. du Streel
75-5
Barthelemy & Bonnet
5I-I
Faure-Fremiet & Dragoiu
97-9
Faure-Fremiet
95-1
,,
^.E.E.
l^-t
Kellner & Kohler
Fingerling, Kohler & Reinhardt
70-0
Terroine
& Wurmser
Several points of interest are to be noted about this table. In the first place, Terroine & Wurmser drew attention to the figure found by Faure-Fremiet and Vivier du Streel for the development of the frog, namely, 82 per cent., but this figure, though shown in the above table, cannot be compared with the rest, for it only applies to the development that takes place before hatching. It therefore resembles the figure obtained by Barthelemy & Bonnet for the frog, i.e. 75-5 per cent, for development up to the time of disappearance of the external gills. Now it is evident that, in order to get a true efficiency value for any embryo that has a prolonged post-natal yolk-sac or "autophagic" period, the term "finished embryo" can only be applied to the young organism at the end of this time. Since the energy contained in envelopes, excreta, etc., may for the present purpose be regarded as neghgible, and since at no time can embryo be separated from yolk, the efficiency is given at any moment by the amount of energy in the larva (i.e. the whole system) expressed in per cent, of the amount of energy present in the whole egg at the beginning. Absorption may be regarded as having taken place instantaneously at fertilisation. Therefore an efficiency of 82 per cent, by hatching simply means that 1 8 per cent, of the original energy has been lost by combustion, and an efficiency of 75-5 per cent, by the time of disappearance of the external gills means that 24-5 per cent, has been lost by that time. As in each case the " embryo" is partly embryo and partly yolk, these figures do not mean that the earlier periods of development in the frog have higher efficiencies than the later ones; on the contrary, they mean nothing. Happily, FaureFremiet & Dragoiu followed the development of Rana temporaria right through to the end of the autophagic period with the bomb calorimeter, and their figure corresponds well enough with that found by Tangl for the chick and with other work.
7-4. Real Energetic Efficiency
Terroine & Wurmser, in an important paper on the energy relations of growth, introduced certain new conceptions into the subject. They defined the "rendement energetique" analogously to the "plastic efficiency coefficient" as:
Energy laid up in the organism Energy in the raw _ Energy in the raw materials
materials at zero hour at the end of development which is only another way of writing
Energy stored Energy stored
Energy absorbed Energy stored + Energy in soHd burnt which, multiplied by loo, is the percentage efficiency. This they termed the "rendement energetique brut", or "apparent energetic efficiency" (A.E.E.). They proceeded to point out, however, that this A.E.E, involves a fallacy, for it does not take into account the basal metabolism — and only if this is done can the "rendement energetique reel" be computed. Tangl's Ea., as has been pointed out, is only a measure of the total embryonic catabolism. In just the same way the "rendement energetique brut" fails to allow for the fact that some of the energy absorbed by the embryo is expended in basal metabolism, maintenance energy, "energie d'entretien", etc., to which the embryo is committed by the mere circumstance of being aHve at all. Thus of the energy in the material combusted only a certain fraction ought really to be included in the calculation of the efficiency, for the rest is earmarked for the upkeep of that part of the building already constructed. The "rendement energetique brut" does not take into account the fact that every cell embarks upon a basal metabohsm as soon as it is completed. A calculation of the true growth energy must therefore allow for this, according to the following formula :
Energy laid up in the organism /Energy in the raw ) - ( Energy in the raw materials ^ Energy of V ^materials at zero hour/ \at the end of development maintenance/
U'
U-{Ur+Ue)
The denominator is now the energy absorbed for growth and nonbasal metabolism only. Armsby also advocated taking the basal metabolism into account. Some doubt may naturally be raised as to whether the usual notions of basal metabohsm can be applied to a system so rapidly changing as the embryo. Basal metabolism is that amount of energy given off in the maintenance of a steady state, but can the embryo be considered to be in a steady state even over a short period? However, from another point of view, the embryo has a certain amount of surface, and the minimum production of heat by its cells would be expected to be sufficient to fit in with this; or conversely, it possesses a certain surface corresponding to the minimum heat production of its cells. In either case some approximation to the basal metabohsm might perhaps be obtained by calculating what the surface would require. Terroine & Wurmser, in the case of the mould Aspergillus niger were enabled to alter its growth-rate by cultivating it on a medium of abnormal pH, but in the case of the chick embryo no such interference with the normal course of events is possible. In 1927 I made some exploratory calculations, using Meeh's formula and Rubner's constant. The calculation could evidently not be exact, because we do not know how these quantities vary during the embryogeny of the chick. The relevant figures are shown in Tables 123 and 124.
In Table 123 the calculated surface of the embryo is obtained according to the formula :
s - K\^m,
where S is the surface, K Rubner's constant for the chicken, 10-4, and W the weight taken from Murray's data, and Voit's figure
Table 123.
calories
evolved Total calories evolved
Surface of the embryo in basal (Bohr & Hasselbalch)
^ '■ ^ metabolism ,• '^ ^
Total Daily in- Daily in- Output Daily in sq. mm. crements elements per day crements
123456
Day o — __ t
1 — _ _ Heat _
2 — absorbed
3 100-5
198 380 586
4 '9» 182^ i?2 24 24.
^ 3BO 1% ^ 4 It
' ^^ llo If, - fe
5?o tst 11^ llo
" 2'5io =70 =ci7 396 1^6
3,080 570 537 156
^3 3,750 ^ g^g 780
H 4,490 74 y ,001
'5 5,290 860 111 ^240 2^y
ID 0,150 —
- I'Z 980 924 \f,: 250
^^"^ '.040 981 710 ,^„
19 9,280 -3 ,^-; i960
20 10,700
o 1050 -g 200
420 1350 —
Total 10,599
of 0*943 cal. per sq. mm. surface for the chick's basal metabolism is accepted. Col. 4 represents the quantity of inevitable loss on the weight of embryo formed each day. Now if the number of cal. evolved as measured by Bohr & Hasselbalch in each period of 24 hours is compared with the heat production calculated from the oxygen consumption (Col. 9, Table 124), it will be seen that the agreement is fair, though the experimental is always rather lower than the calculated value. The fact that the calculated value assumes fat only to be burnt would not entirely account for this. Returning to Table 123, however, it can at once be seen that the basal metabolism invariably exceeds the total amount of heat evolved. Thus there is not enough heat eliminated to account for the quantity that ought to be produced in maintenance energy alone. The basal metabolism as here calculated must be far too high, for if all the increments are added up the result is 61,000 calories, or about four times as much as the total energy known to be lost by combustion. We must therefore suppose either that the surface formula does not hold in embryonic life or that the high temperature (37°) in which development proceeds leads to a lower basal metabolism than would be expected. Lusk says that the minimum requirement for energy is seen to be present when the fasting organism is surrounded by an atmosphere having a temperature of 30 to 35°. Most important of all, however, is the probability that Rubner's constant for the chick does not hold for the embryonic chick. It is quiescent, its muscles have no tonus or very little, its respiratory muscles are inactive, and its heart alone is constantly requiring a supply of energy. Since the metabolism is proportional to the superficial area of the animal, it may well be asked what is happening in an embryo at the minute stage when its percentage growth-rate is 1400 (Schmalhausen).
7-5. Apparent Energetic Efficiency
Evidently it is not possible to calculate the "rendement energetique reel" (R.E.E.) for the chick. But Terroine & Wurmser pointed out that some of the discrepancy existing between Tangl and Rubner might be removed if it were known. For the growing cow the R.E.E. was calculated by Kellner & Kohler, and for the growing pig by Fingerling, Kohler & Reinhardt. They estimated the magnitudes of [a) the energy stored in the organism following the addition to a fundamental ration of a given foodstuff in known quantity, calculated on the basis of carbon and nitrogen balance and formation of tissue; (b) the total energy catabolised measured by indirect calorimetry, and {c) the "energie d'entretien"
computed from the surface law. The result was that the efficiencyrose, giving
5
R.E.E.
Cow Starch Gluten
Oil
Cellulose ...
%
58-9 45-2
63-1
Investigators Kellner & Kohler
Starch Cellulose ...
84-0 52-0
Fingerling, Kohler & Reinhardt
"Ces donnees", said Terroine & Wurmser, "rentrent parmi les plus interessantes actuellement possedes. On ne manquera pas de constater qu'une fois prise la precaution d'ecarter la depense d'entretien, si elevee chez un homeotherme, on aboutit a des chiffres tres voisins de ceux de Tangl, Farkas, et Glaser, pour le developpement de I'oeuf." At first sight this correspondence is fallacious, for we are comparing the R.E.E. of the two mammals with the A.E.E. of the various eggs studied. Nevertheless, the calculation given above shows that the basal metabolism of the chick embryo is probably many times lower than might be supposed, so that the R.E.E. would differ Httle from the A.E.E. Furthermore, as Terroine & Wurmser point out, the eggs studied are mostly those of poikilothermic animals, whose basal metabolism is known to be very low, and, as for the chick, we know that, for the greater part of its pre-natal life, it behaves as a cold-blooded animal. Rubner's low mammalian values, then, when corrected for basal metabolism, would approach the values of Tangl and his associates, and we shall probably not be far wrong if we assess the R.E.E. of all embryos, mammalian as well as non-mammalian, at about 66 per cent.
The average value of the A.E.E. will presumably vary with varying conditions. Is it affected by temperature ? The only answer to the question is contained in the papers of Barthelemy & Bonnet, whose experiments were conclusive. These investigators, as we have already seen, studied calorimetrically the development of the frog's egg{Ranafusca)up to the disappearance of the external gills, and caused the development to take place at different temperatures. Their results were as follows:
Efficiency (A.E.E.)
Time of • Energy in n embryos
Temperature development ' ' Energy in n eggs
° C. days ^ a ^
9 30 74-78 Average 75
II 22 71-76 „ 73
14 20 71-84 ,, 75
21 8 70-82 „ 75
Evidently the temperature exercises no effect on the proportion of energy used for storage and that used for combustion. More or less analogous results had previously been found for the development of Proteus vulgaris by Rubner, for muscular contraction by Hill, for the growth of Sterigmatocystis nigra by Terroine & Wurmser, and for the germination of seeds by Terroine, Bonnet & Joessel. The invariability of the A.E.E. of embryonic growth would seem, then, to be a special case of a general biological law.
The next question which arises is whether the efficiency varies from time to time during the development of the embryo ; we have already seen that the P.E.C. shows such a variation. Table 124 gives the calculations for determining this (Needham). The increments of calories, i.e. the amounts of potential energy stored in the embryonic body each day, are checked against the energy present in the extraembryonic part of the t§^g as determined with the bomb calorimeter by Tangl & von Mituch. It will be seen that the rest of the egg loses, in addition to combustion, 250 cal. between the 8th and the 9th days, while the embryo gains 232; a sufficient agreement. The figure of 34,000 cal. seen at the bottom of Col. 4 representing the number of calories contained in the finished embryo agrees sufficiently well with the value given by Tangl of 32,000; the latter was measured directly, the former was obtained by the addition of all the increments.
Cols. 6 to 9 give the figures relating to the energy lost in combustion. This is obtained, assuming that 100 per cent, instead of the true 92 per cent, of the total solid burnt is fat, and that i gm. of fat produces on its combustion 9300 cal. The total of this column amounts to 17,000 cal., not very far from the 16,500 cal., the Ea. of Tangl. In Col. 10 Tangl's values for Col. 9 are given, and it may be noticed that they are close to the newer ones. An error exists here owing to the fact that no account has been taken of the energy left behind in incompletely combusted materials, but as the chief of these is uric acid, and — using the data of Stohmann & Langbein for the calorific value of uric acid, 2750 cal. per gm. mol. — the calories locked up in this way only amount to 16 on the loth day, or much less than i per cent, of the total combusted; this error is neghgible. Finally Col. 1 1 shows the A.E.E., which is diagrammatically represented in Fig. 260. Starting at a low level, it slowly rises, gaining in speed till at the 14th day it is rising rapidly, but soon afterwards it falls off. The value for the whole of development works out at 66-5, which is exactly what Tangl found. Since the basal metabolism is included in this estimate, and since that might naturally be expected to be high in the early stages when the embryo is very minute, and has a large surface in proportion to its size, one can understand that the efficiency, the A.E.E., would then be very low.
Another way of interpreting ^ig. 260 would be by the recapitulation theory. Perhaps the most striking chemical attribute of the bacteria
and yeasts is their high energy ^^^ Apparent Energetic
turnover : Horace Brown, for example, showed that a yeast cell 65|would ferment its own weight of maltose at 30° C. in 2-2 hours, and at 40° C. in 1-3 hours. This 55 metabolic level would be about 100 times as high as that of an adult man. And Haacke has cal- 45 culated that certain lactose fermenting bacilli destroy from 1 78 to 14,980 times their own weight ^^s- 2^° of lactose per hour. Parallel with this furious onslaught on the
nutrient material of their environment goes a very low efficiency^,
figures for which are available in a number of papers :
Efficiency
Investigator
Organism
(%)
Stephenson & Whetham
Timothy Grass Bacillus
27-0
Becking & Parks
Nitrobacter
7-9
,,
Mtrosomonas
5-9
,,
B. niethanicus
15-1
Ruhland
B. pycnoticus
20-5
Waksman & Starkey
Thiobacillus thiooxidans
6-2
Beijerinck
Thiobacillus denitrificans
8-7
In discussing these facts Stephenson suggests that the breakdown of a substance such as sugar by the yeast-cell or a bacillus is conditioned mainly by the concentrations of cells (enzymes) and substrate, irrespective of whether the cells can benefit by the energy liberated. Thus the energy liberated by micro-organisms would be no measure of their metabolic needs but simply the result of unprotected enzymes acting upon the appropriate pabulum. "If such a view be correct"
^ But it must be understood that micro-organisms have really no definite efficiency; it varies according to their environment, and they have no means of adjusting it. Bacteria "killed" by ultra-violet light continue to oxidise at almost the normal rate, though incapable of growing, and in yeast cultures fermentation and growth are quite dissociable. When growth ceases the efficiency is nil, but in certain conditions it may be as high as 59 % (Terroine & Wurmser on moulds) says Stephenson "one may regard the evolution of the metazoal organism as involving a process whereby the energy liberated in chemical activity, which in a microbe runs to waste, is so organised and disciplined that it is liberated when and where it can subserve function, such as muscular work or maintenance of temperature; apart from such organised expenditure the liberation of energy in the higher animal is cut down to a minimum represented by its basal metabolism or energy of maintenance." She left the question open as to whether this latter quota might be, even in mammals, an expenditure which the animal was unable to prevent or, conversely, of some deeper significance. Her picture of the gradual increase of organisation in evolution, is one of much interest and may apply also to the ontogenetic passage from low to high efficiency seen in Fig. 260. Possibly the chick embryo in its earliest stages may resemble the micro-organisms in being unable to keep its enzymes apart from its substrates, although it may be noted that its efficiency is not lower than 40 per cent, at the worst^.
A third way of considering the phenomenon of rising efficiency is that of Terroine and his collaborators. In their studies of the germination of seeds (see Appendix iii) they found that — roughly speaking — the A.E.E. was highest when the reserves were mainly in the form of carbohydrate, mediocre when the reserves were in the form of fat, and lowest when they were made up of protein. Now the seedling itself, which corresponds to the developing embryo, may be considered as made up almost entirely of cellulose, i.e. carbohydrate, and Terroine and his colleagues therefore concluded that the A.E.E. varied with the nature of the food-materials, i.e. was a measure of the degree of chemical difference between the reserve materials and the finished structure. The least wastage occurred when carbohydrate was used, more when fat was used, and most when protein was used. In their work on germination it was assumed (for the sake of argument) that the composition of the seedling and the reserves was throughout the same, and this was justifiable enough as the A.E.E., estimated at various moments in germination, was always found to be the same. But in the bird's egg, the composition of the embryo does not remain the same; profound changes are taking place all the time in its constituent substances and their balance, nor do tlie yolk and white remain chemically constant. Perhaps the embryo at the 5th day of development has much more to do to whatever it is absorbing to turn it into itself than has the embryo of the 15th day, and consequently the wastage is greater.
1 Cf. the condition seen in the unfertilised echinoderm egg (p. 626) . But the embryo in the early stages seems not only to combust an excess of nutritive material, but also to fail to retain properly those building-stones which it does not combust, judging from the high proportion of amino-acid nitrogen in the allantoic liquid at that time (see p. 1096). Could this also be of ancestral significance? (see Table 163).
This seems at first sight to be in contradiction with the facts known about the "white yolk" (see p. 286) which, as Spohn & Riddle's work showed, resembles the embryonic tissue much more than it does the yellow yolk. But it is reasonable to suppose that this phase would be passed through by the 5th day, at which time the A.E.E. curve begins, and we might predict that when the efficiency of the earlier stages is known, it will turn out to be higher, perhaps as much as 60 per cent. The A.E.E. curve would then become trough-shaped, like the P.E.C. curve (see Fig. 254).
The argument due to Terroine would thus be that in the earlier stages the raw materials are more unlike the embryonic body in composition than they are later. An interesting calculation which shows that this is to some extent true is shown in Table 125, where first of all the absolute amounts of the three main cell-constituents, carbohydrate, protein and fat, are set down, both for the embryo and for the raw materials, i.e. for the remainder of the egg. Then these figures are expressed as percentages of the sum of the three in each case and given in Cols. 8 to 13, so that we have side by side the variations in balance of the three classes of substance throughout development.
Difference between the two figures {embryo and remainder) in Table 125.
At the beginning At the end
(4th day) (20th day)
Carbohydrate (Cols. 8, 1 1 ) ... ... 2-3 0-3
Protein (Cols. 9, 12) ... ... ... 29-5 9-5
Fat (Cols. 10, 13) 31-8 9-8
It can hardly be a coincidence that all three should work out Hke this, but too much emphasis must not be laid on the calculation in view of the fact that the sets of figures used for it are derived from the results of several workers using not very homogeneous material. A more serious shortcoming of such a calculation is that it assumes that the embryo must be absorbing throughout an aHquot part of the raw materials, although we may be reasonably certain that this never happens at all. And again the efficiency curve involves qualitative differences between the raw materials and the embryo itself, so that not only is the balance between carbohydrate and protein changing through development, but also the balance of integral portions of the carbohydrate fraction. Nevertheless it is interesting that the rough calculation of Table 125 does confirm the conception of an increasing similarity between the embryo and its raw materials with age, and it would be extremely valuable to collect data for a profounder examination of the problem not only in the chick but in many different animals.
It may be noted also that, if the embryo continued to behave as wastefully all through incubation as it does in the beginning, there would not be nearly enough energy in the egg to provide for it, unless the egg were increased to about twice its present size. Even then there would be no reserve yolk at hatching.
Since the A.E.E, rises with age, it resembles the percentage of total solids, the percentage of fat, the latent period of growth in tissue culture fragments, the total metabolism and the rate of the heart beat; a miscellaneous collection of factors. But, having Murray's rule in mind, and remembering that embryonic development is symmetrically diphasic in character, we may enquire whether it moves rapidly at first, then slowly, like the growth-rate, or slowly at first, then rapidly, like the metabolic rate. Evidently the rise in A.E.E. resembles the fall in metabolic rate. Thus it would seem as if the furious intensity of combustion with which the embryo begins its life was associated with great wastefulness, while later on greater economy would accompany greater frugality. The calorific value of the embryonic tissue also rises during development, and Murray's graph (Fig. 256) shows that it goes up in a curve shaped rather like that for the A.E.E. Thus the richer in potential energy the embryonic body becomes per unit weight, the more efficient is the transfer of energy from the yolk and white.
Though the curves for P.E.C. and A.E.E. are different, it is interesting to find that the average P.E.C. for all development is o-68, while the average A.E.E. is 66 per cent. Out of 100 gm. of solid presented to it, the embryo can store 68; out of 100 cal. presented to it, the embryo can store 66.
The conclusion that the A.E.E. of the chick embryo increases with age, and that, in the early stages of development, storage of energy is very inefficient, is in agreement with the views of Armsby. That it rises, as we have seen, to approximately 70 per cent, at hatching, is interesting in view of the efficiencies found by other workers on the early post-natal life of mammals, thus :
Armsby stated that in later post-natal life the efficiencies were higher still, but though he calculated them from Kern & Wattenberg's data on the sheep, Tschirwinski's data on the pig, and Armsby & Fries' data on the cow, he did not give any actual values. From a general point of view, therefore, it is probable that as more data come to light it will be found that the efficiency of the organism considered as a machine for storing energy rises from fertilisation to death. Nothing is known about the rapidity with which the adult level of efficiency is reached, but Armsby & Fries considered that this would probably occur not long after weaning (see also Brody).
A comparison may be made between the embryo and other engines. Its business is to store as much energy as is given it with as little loss as possible. The object of the steam engine is to produce as much mechanical work from the energy given it with as little loss as possible. The efficiency of this process is not great; in the locomotive engine, which is notoriously wasteful, it may not exceed 1 5 per cent. Wimperis and Bird give 25 per cent, for the gas engine with suction producer, and the best recorded efficiency for a Diesel engine with high maximum pressure is 40 per cent. But a much better comparison is between the embryo and the electric accumulator, for this does not alter the form of the energy passing through it. Cooper's average estimate is that an electric accumulator will give back 74 per cent, of the energy put into it, and another figure (Davidge & Hutchinson) is 70 per cent. It is interesting that the average A.E.E. of developing embryos should be of the same order.
7*6. Synthetic Energetic Efficiency
Now Terroine & Wurmser's formula for calculating the R.E.E. was
Energy stored in the embryo /Energy in raw materials \ _ /Energy in unused , Energy in solid burned \' \at zero hour / \raw materials for basal metabolism )
or, in their notation : ^ r,
This is perfectly satisfactory so long as we only consider complete combustions to carbon dioxide and water. Rapldne, however, pointed out that the developing organism may have at its disposal other sources of energy, for endothermic reactions are known to occur in vivo, which raise the chemical potential of their products. Confusion arose here owing to the fact that some workers used the term "energy sources" to apply to the solids burned to give the heat lost from the egg, while others used it to apply only to those reactions, whatever they may be, which gave the energy of organisation, or O.E. Bohr & Hasselbalch showed finally that of the total solid lost not more than 4 per cent, can participate in the O.E., but when we take into account the energy not furnished by complete combustions it is legitimate to suppose that a larger proportion of the total energy turnover may be used for O.E. We cannot expect to find this, of course, by bomb calorimetry, for the organisation is destroyed by drying. Rapkine focused attention upon coupled and spontaneous endothermic reactions, and considered that their existence in the embryo explained inter alia ( i) the atypical respiratory quotients which he had himself observed in echinoderm eggs (see p. 648), (2) the low calorific quotients of Meyerhof (see p. 651), (3) the initial heat absorption in Bohr & Hasselbalch's measurements (see p. 704), and (4) the synthesis of fatty substances which proceeds in many eggs. Applying these ideas to the efficiency formula, Rapkine suggested that the numerator U' (calories in unit weight of finished embryo) should be replaced by U' minus the energy contained in an exactly equivalent weight of original raw material. This would represent the elevation of calorific value which has gone on during development : ^ r, _
U-iUn+Uj,)'
It can be seen at once that this will give an efficiency of a very low order, but not altogether comparable with those which have already been discussed, such as the A.E.E. and the R.E.E. For what they measure is the relation between the energy in the substance stored in the embryo or transformed into its tissues, on the one hand, and the energy absorbed by the embryo from the raw materials, on the other hand, either allowing for the basal metabolism or not allowing for it. Rapkine's efficiency coefficient, on the contrary, measures the relation between the energy furnished to the embryo from coupled reactions, etc. (energy which would have been dissipated as heat if the endothermic processes had not caught and held it), on the one hand, and the energy absorbed by the embryo from the raw materials, on the other hand, allowing for the basal metabolic requirements. It thus has to do, not with energy storage as a whole, but with the storage of energy from a particular source, i.e. coupled reactions with one endothermic component^. During the absorption of 100 cal. of energy, 66 will be stored and 33 lost, but only 9, say, out of that 66 will be saved from the loss by the endothermic processes. Rapkine's coefficient may, therefore, be called the S.E.E. (synthetic energetic efficiency). As a concrete example, in the case of the chick, the values as averaged from many observations can be read off from Fig. 259, and the usual fraction for R.E.E. is
(86.85) - (^6^+ .7 (say)) = ^"^ P" =^"' For the S.E.E. the numerator would at first sight seem to be a minus quantity, for in Murray's work, for instance, the calorific value of I gm. of dry unincubated mixed yolk and white was 6-94 Cal. and that of I gm. of dry finished embryo was 6-2 Cal., so that no increase in specific energy-content would appear to have taken place. However, the finished embryo is not comparable with the unincubated yolk and white, for a notable proportion of the fat in the latter disappears by combustion, and some of it is left behind as spare yolk at hatching. The following calculation is therefore required to give the energy value of an amount of yolk and white roughly comparable with the finished embryo :
The finished embryo of the chick weighs 6*oo gm. dry weight and has in it 37-5 Cal.
i.e. 6-2 Cal. per gram dry weight. The egg at the beginning of development has inside it 12-45 g™- ^^Y weight and 86-85 Cal., i.e. 6-94 Cal. per gram dry weight ... 86-85 Cal. = 100 %
For combustion 2-5 gm. fat are used, which at
9-3 Cal. per gram is 23-25 Cal 2325 Cal. = 26-4%
For yolk unused at the end of development, i.e. about 4-75 gm. dry weight of which 40-5 % is fat and 50 % protein, i.e. :
For fat ... 17-8 Cal.
For protein ... 11-4 Cal.
29-2 Cal. ... ... 29-20 Cal. = 30-4 %
52-45 Cal.
86-85 -52-45=34-40 Cal.
1 It should be noted that this is the only kind of energy-storage which will raise the chemical potential of the embryonic body.
An amount of yolk and white, therefore, equivalent to the finished embryo has 34-4 Cal. as against its 37-5 Cal. But from this 34-4 Gal. must be subtracted a correction for the skeletal system of the finished chick, which contains very little energy.
The 37-5 Cal. is the heat contained in, not 6-oo gm. dry weight but 4-5 gm. dry weight, for the bones weigh 1-5 gm. dry weight (Tangl), i.e. 75 % of 34-4 Cal. will give the energy of an amount of yolk and white equivalent to the finished embryo =25-7 Cal.
Then 37-5 -25-7 = 1 1-8 =energy stored in embryo by the endothermic components of coupled reactions, i.e. increase of chemical potential.
This gives for the S.E.E. :
U' — X 37-5 — 25-7
jT- — — - or -— — — ^^^^T^ ^^-^-7 TT '^ 100 = 27- 1 5 per cent.
U-{Un + Ue) 86-85 - (26-4 + 17 (say)) ' ^ ^
But this calculation is only of illustrative significance, for the data are taken from various different sets of material. It is evident, nevertheless, that the S.E.E. will always work out at a very low level, certainly under 30 per cent.
Wurmser, independently following a like train of thought, found 26 per cent., instead of Terroine & Wurmser's 70 per cent., for the growth oi Aspergillus nigra. Rapkine suggests that it might be possible to discover what these coupled reactions are which store extra energy in the embryo and provide most of the O.E., by placing various hydrogen donators in the presence of embryonic tissues at different stages of development and following electrometrically their dehydrogenation. Cahn gives the following argument to show that they do not interfere much in the formation of the protein part of the embryo :
I gm. of the total proteins of the whole egg on the gth day has a calorific value of 4-91 Cal. and therefore for 5-846 gm. ... 28-700 Cal.
I gm. of the proteins of the remainder of the egg on the 21st day has a calorific value of 4-72 Cal. and therefore for 2-36 gm. 1 1-150 Cal.
1 gm. of the proteins of the embryo on the 2 ist day has a calorific value of 5-28 Cal. and therefore for 3-22 gm. ... ... 17-000 Cal.
28-150 Cal.
(The figures are Cahn's.) So there is a difference of 550 cal. and as, judging from Fridericia's uric acid figures (which are probably too high), about 135 mgm. of protein are combusted and therefore 500 cal. liberated, the balance was regarded by Cahn as exact enough to allow of the conclusion that there was no measurable O.E. in this case.
Attention may be drawn to the similarity shown in Fig. 259 between the fraction of energy contributed to the embryo by synthetic processes and the O.E. Both of these are, of course, quite hypothetical as regards magnitude. At present there does not seem to be any way of measuring the O.E. directly, but a word may be said about certain attempts at doing so which have been made in the past.
In the first place, it has been argued that if any energy is bound up with structure or organisation, this energy ought to be liberated when the structure or organisation is destroyed. Accordingly from time to time attempts have been made to discover the thermal effect of death, and of these the most recent and successful is that of Lepeschkin who killed yeast-cells in various ways as instantaneously as possible and measured the extra heat eliminated. It worked out at 2 cal. per gram of dry weight. If this energy may be in any way regarded as O.E. or the equivalent of O.E. then it might be expected to be rather greater in a metazoal, i.e. more heterogeneous, population of cells, such as the avian embryo, but even so could not exceed 20 gm. cal. in the finished chick. This amount would appear as a line rather than a solid block in Fig. 259. Rychlevska has also some relevant information obtained by combusting tissues without preliminary drying.
Secondly, there is the notion that the O.E. could be found by estimating the work required to stop its formation. Under the head of osmotic pressure, we have already had occasion to examine the work of Spaulding and of Vies & Dragoiu on the "travail osmotique d' arret" of cell-cleavage. Arguing in exactly the same way, FaureFremiet, Henri & Wurmser determined the amount of radiant energy required to stop the segmentation of Ascaris eggs. An exposure to ultra-violet light of wave-length 2800 A. sufficed. Thus 12-10^ ergs per square centimetre were required to stop development, and the receiving surface of the egg was 2-3 . lO"^ sq. cm., so that the quantity of energy received by an egg and sufficient to block its development was equal to:
12 . 10^ X 2-3 . 10-^ = 12 X 2-3 = 27 ergs.
Faure-Fremiet contrasted this value with the value for work done during the whole of development calculated from his Ea. results. Thus 280 cal. X 4-18 . 10^ ergs = 1 1-6 . 10^ ergs per gram dry weight, and, as the specific gravity of^ Ascaris eggs is i-o8 and their diameter 82/Lt, the average weight of one egg must be 3-11 . 10-^ gm. During the development of one Ascaris embryo, then, 503 . lO"^ cal. or 2100 ergs are used up. But the segmentation period is only a comparatively short part of the whole period, so that, allowing for this, a value of 39 ergs was obtained, i.e. of the same order as that found by blocking the first cleavage with ultra-violet fight. It is evident, however, that these calculations can tell us nothing at all about the O.E., for the processes of embryonic development are not reversible, and as there is no equilibrium point there can be no sense in determining the amount of energy required to stop the processes from going on.
7-7. The Sources of the Energy Lost from the Egg
Although practically nothing is known about the origin of the energy which forms the O.E., a good deal can be said about the origin of the energy which furnishes the Ea., or total catabolism of embryonic life. As we have already seen in the section on the general metabolism of the embryo, there is some reason for supposing that, during pre-natal life, the principal constituents of the embryonic body reach their maxima in a definite succession, i.e. inorganic substances, carbohydrate, protein, fat. The possible significance of this from a general point of view was there discussed, and comparison was made between these data and the varying intensities of absorption of these important components. But an ontogenetic succession of carbohydrate, protein, and fat makes its appearance not only when these three substances are considered as elements in the architecture of the embryo but also when they are considered as reserves of energy for the Ea,, fuelsources for purposes of combustion. Considered in this way, it is certain that in the case of the chick, for instance, carbohydrate attains its highest point in the first week, protein in the second and fat in the third.
For a long time it was generally considered that fatty substances were the sole sources of energy for the developing chick embryo. This view, which was satisfactory only as the roughest of approximations, grew naturally out of the early researches of Pott; Liebermann; and Tangl & von Mituch. Estimations of the fat in the embryo and that in the rest of the egg showed that a good deal had been lost in transit, actually from 2-1 to 2-76 gm. per egg on an average. Calculation demonstrated, as we have already seen, that this figure corresponded satisfactorily with the carbon dioxide evolved throughout incubation, and the heat produced during the same time. For example, Liebermann had shown that the fat of the egg contained 71-67 per cent, of carbon, and the amount used per egg Tangl & von Mituch found to be on an average 2-11 gm. According to Hasselbalch, the total amount of carbon dioxide produced during incubation amounted to 5-939 gm. From this they calculated the amount of fat which ought to have been used, and the result was 2-26 gm., which agreed moderately well with Liebermann's figure, and with one which they themselves found, 2-76 gm. Another line of investigation which gave support to this view was the work of Bohr & Hasselbalch on the respiratory quotient of the hen's egg, which gave from the 7th day onwards a figure of 0-73. The following curious argument occurs in Liebig's Animal Chemistry of 1842, and shows once more the same point of view. "The egg of a fowl can be shewn to contain no other nitrogenised compound except albumen. The albumen of the yolk is identical with that of the white, the yolk contains besides only a yellow fat in which cholesterine and iron may be detected. Yet we see in the process of incubation, during which no food and no foreign matter except the oxygen of the air is introduced or can take part in the development of the animal, that out of the albumen feathers, claws, globules of the blood, fibrine, membrane and cellular tissue, arteries and veins are produced. The fat of the yolk may have contributed to a certain extent to the formation of the nerves and the brain, but the carbon of this fat cannot have been employed to produce the organised tissues in which vitality resides because the albumen of the white and yolk already contains for the quantity of N present, exactly the proportion of carbon required for the formation of these tissues."
It might always have been doubted, however, that fat was the only important source of energy: there were hints to the contrary in the literature. William Harvey had said, "and therefore the yolke seems to be a remoter and more deferred entertainment than the white, for all the white is quite and clean spent before any notable invasion is made upon the yolke". Another important observation was that of Prevost & le Royer in 1825, '^^o obtained from the allantoic fluid of a 1 7-day old chick a nitrogenous substance which gave an insoluble nitrate and resembled urea in all particulars. There had clearly been some catabolism of proteins. And since at about the same time Jacobson; Sacc; and Stas found uric acid in considerable amounts in the allantoic fluid of developing chicks during the third week of incubation, there was no doubt about it. In 1925 I pointed out that, although fat was the predominating energy-source in the chick embryo, it could certainly not be the only one, for many arguments indicated carbohydrate and protein as the energy sources of the initial stages.
1 . In the first 8 days of incubation there is a striking fall in the amount of free glucose. The curves of Sato; Idzumi; By waters; and Tomita, all show a rapid fall from about 0-4 gm. per cent, to o- 1 or less.
2. Simultaneously with this disappearance of free glucose, the lactic acid in the egg, which has previously been low, reaches a peak and immediately afterwards descends to its previous level (Tomita). From an initial level of 0-02 mgm. per cent, it attains on the 5th day a maximum of 0-13 mgm. per cent., and regains its original value about the 14th day. That it was intimately connected with glucose metabolism he proved by injecting glucose, and observing an increase of lactic acid.
3. When the figures of Bohr & Hasselbalch were examined (Fig. 144, p. 703), it was seen that, although the respiratory quotient during the last two weeks of incubation is 0-73, in the earlier stages it is nearly as high as unity^.
4. The disappearance of glycogen in very early stages of development has been investigated by Lewis and Konopacki. The former grew tissue cultures of cells from very young chick embryos, and could never find glycogen present in them after the first 50 hours of development. Konopacki, working on the frog, obtained exactly similar results. He found that after fertilisation and the formation of the perivitelline space the glycogen greatly diminished in quantity and remained very low until the neurula stage was reached, after which the glycogen rose again. Similar results have recently been reported by Vastarini-Cresi. All these workers made use of histochemical methods.
5. We saw evidence pointing in the same direction in the work of Warburg, Posener & Negelein. They found a very marked preferential consumption of carbohydrate on the part of 3-5 day chicks' tissue. The production of ammonia when the tissue was suspended in bicarbonate Ringer in vitro was at once suppressed, if sugar was provided for it. Negelein also found that the power of glycolysis is very high in early development, and lower in the adult condition,
- And Dickens & Simer found quotients of unity for 5th day chick embryos in vitro.
6. Perhaps it is no coincidence that the respiratory quotient which is found in the early cleavage stages of small marine eggs such as those of the sea-urchin is in the near neighbourhood of unity. Warburg's respiratory quotient of 0-9 for the first 24 hours of the development of Arbacia pustulosa eggs and Shearer's respiratory quotient of 0-95 for Echinus microtuberculatus are relevant here. \ higher figure still was found by Faure-Fremiet, whose results with the eggs of Sabellaria alveolata worked out at a respiratoiy quotient of i-o. As indices of the nature of the combustions going on these figures have to be accepted with caution, but it is natural to suppose that a stage which lasts 5 days in the chick may last only a few hours in lower animals which develop faster, so that the time to look for utilisation of carbohydrate might be from fertilisation to the gastrula stage.
7. Then Simon and Susanna Gage fed laying hens on Sudan III and found that, although red yolks were laid, the embryos showed no trace even of a pink colour until the i oth day of incubation. After that time they became rapidly more coloured, until at hatching they were quite red. One would suppose that, in order to be combusted, fat would have to be absorbed into the embryo, and would take the dye with it, as indeed did happen after the loth day. Murray's estimations of fat storage in the embryonic body confirm this (see Fig. 362), and point to an awakening of fat metabolism after the mid-point of incubation. Then Tallarico and Remotti have brought forward evidence showing that the lipase of the yolk increases markedly in activity towards the end of incubation, while the latter worker finds a precise correspondence between proteolytic activity of yolk and intensity of protein combustion at the 8th day of development.
8. The work of Riddle on the yolk and the yolk-sac is interesting in this connection. From the 15th to the 20th day the yolk is practically the only supply of the chick, and his chemical study of it during this period showed that there is a preferential absorption of fat. This agrees exactly with the absorption curve. The neutral fat decreases markedly in the yolk and the protein substances increase.
9. The earliest statement that protein in the hen's e^gg must be a source of energy is due to Sznerovna. She found a constant relationship between the nitrogen in the embryo and the nitrogen in the allantoic fluid, as 1 7 to i .
10. Bialascewicz & Mincovna, working on the frog's egg, found that up to hatching there had been practically no loss of fat, but that a loss of protein had certainly taken place. Parnas & Krasinska found no loss of fat during this time. Faure-Fremiet & Dragoiu also studied the frog's egg; they agreed with Bialascewicz & Mincovna in finding a loss of protein before hatching, but they also observed a loss of glycogen and of fat, indicating that all three substances had acted as sources of energy.
11. The same conclusion was arrived at by Farkas for the egg of the silkworm, Bombyx mori.
12. Dakin & Dakin found a utilisation of proteins during the development of the eggs of the plaice, and Greene observed the same thing in the king-salmon.
13. If urea and uric acid are indicators of protein metabolism, so also is the phenomenon of specific dynamic action. We have already seen evidence that there is a period in the development of the chick when this phenomenon appears (p. 935). Gayda's work on the heat given out by toad embryos throughout their development, showed that the heat eliminated during periods in which the weight was doubled, plotted against the time, gave a curve with a peak in the centre, to which the values rose and from which they descended. Thus development was more economical at the beginning and end of development than at the middle, just as in the chick.
14. Pigorini's work on the embryos of Bombyx mori, in which the glycogen was estimated throughout their development, fell into line with the other researches on carbohydrate utilisation mentioned above, and confirmed the older work of Vaney & Conte. The lastnamed investigators found that not only glycogen but also fat fell during the development of the silkworm.
15. The only work which has been done on the chemical constitution of the echinoderm egg during its development fits in well with the high respiratory quotients found for the beginning. Ephrussi & Rapkine found a fall in protein, fat and carbohydrate during its development.
16. In the chapter on respiration and heat production, attention was drawn to the fact that Meyerhof's calorific quotients, obtained during the early development of the echinoderm egg, did not correspond with the theoretical values, either for carbohydrate, protein, or fat combustion. Shearer's later values, which were based on a rather greater heat production, came nearer, and finally Rogers & Cole, finding still more heat, gave data from which calorific quotients very near the carbohydrate level can be calculated. This was first pointed out by Needham in 1927, who suggested that these researches could be interpreted as a progressive ehmination of leaks.
17. Indirect evidence about the utilisation of protein by the embryo can be gained from the work of Scheminzki, who determined the resistance of the trout egg to damage by electric currents during its development. The whole period was 55 days, and for the first 30 days there was practically no change in the resistance, but after that time it rose tremendously, the strength of current required to produce precipitation of the ichthulin in one minute increasing six times in the last 25 days of development. The effect of the current was to render the egg-membrane permeable to cations, which diffuse out and cause the ichthulin to be precipitated. Jarisch showed that lipoids and fats in systems poor in salt favour the precipitation of globulin, so if the current dismisses the cations from the egg, the precipitation of ichthulin will be more favoured the more fatty substances there are present. Scheminzki's curve becomes, then, in some measure an index of the amount of fat absorbed by the embryo, and the fact that it is of so gradual a slope during the first two-thirds of development may be interpreted as showing a greater intensity of fat absorption (and combustion?) towards the end of development than towards the beginning. These findings may be compared with those of Gage & Gage on the chick embryo.
18. Besides Tomita and Grafe, a few other investigators drew attention in the past to evidence showing that fat was not the only energy source of the chick embryo. Droge considered that protein must take a share in the work, and Sakuragi specifically went into the question of the other energy sources of the embryo. In the German summary to his Japanese paper, he says, "Obwohl bisherige Autoren, welche sich mit Stoff- und Energiewechsel von bebriitenden Hiihnereiern beschaftigen, die Bedeutung des Kohlehydrates fur Energiewechsel ganz vernachlassigten, glaubt der Verfasser, dass der schon vorhandene Traubenzucker in den ersten Bebrutungstadien besondere Wichtigkeit und grosse Bedeutung dafur hat, und dass der erste chemische Vorgang in den bebriitenden Eiern in der Zersetzung von Traubenzucker besteht". Sakuragi estimated the free and combined sugar, the fat, the various fractions of nitrogen, and the glycogen at the different stages of development, and interpreted his figures as showing that throughout development carbohydrate was combusted, the fat at the late stages being turned into carbohydrate before being burnt. His arguments for this process, however, were not convincing.
19. A very striking support for the view that a succession of energy sources takes place in the development of the chick is to be found in the analyses of the white yolk which were carried out by Riddle and by Spohn & Riddle. As has already been mentioned (see p. 286), the white yolk, the earliest pabulum of the embryo, is much richer in salts and in protein than the yellow yolk, which forms its later nutriment. These facts fit in exactly with what has already been said about the constitution of the embryo and its sources of energy. It is almost safe to predict that the white yolk will be found to have a higher percentage of total carbohydrate, or perhaps of free sugar, than the yellow yolk.
20. When the ammonia, urea and uric acid in the hen's egg are estimated throughout incubation, the absolute amounts rise in regular curves corresponding to the growth of the embryo. When further analysis is made, however, it is found that in all cases the amount of these nitrogenous end-products, when related to wet weight, rise up to a certain point, and then remain at a steady level, while, if they are related to dry weight, they rise to a peak from which they fall again in the later stages of development. Thus in each case it could be said that I gm. dry weight of embryo excretes a maximum of nitrogenous waste at a definite point in development. The obvious conclusion is that a peak of maximum protein catabolism exists, coming significantly at 8'5 days of development, i.e. exactly between the periods which, on the evidence given above, we believe to be associated with the predominant catabolism of carbohydrate and fat respectively. Fig. 261 shows these relationships, plotting the protein combusted in milligrams per cent, of the dry or wet weight of the embryo at the time against the age. In Fig. 262 the curve of Fiske & Boyden is also given. The investigations of Bialascewicz & Mincovna have also made it likely that a similar peak exists in the frog embryo. Fig. 368, constructed from their data, illustrates this strikingly, for in it the quantity of nitrogen in milligrams excreted by one embryo in each 24 hours is plotted against the age in hours from fertilisation. An unmistakable peaked curve is seen, and confirmation of the views already expressed is furnished by the curve for combustion of fatty acids, which rises steadily but later than the nitrogen excretion curves.
O wet weight
©dry ,.
B mgmsyocoagulable protein dissappearing perday: wet weight: calc. from Sakurao'
The work of Bialascewicz and Mincovna will be considered in more detail in Sections 9-9 and 11-3.
The conception of an ontogenetic succcession of energy sources has to reckon, however, with a few facts which do not easily fit it. Perhaps the most difficult phenomenon to explain from this point of view is the apparent combustion of carbohydrate exclusively by mammalian embryos (Bohr) . It must be admitted that the evidence for this is slender, but even if it were true, it would not be surprising, as most Living cells combust carbohydrate if they can get it, and the continuous perfusion system of viviparity may provide such a supply. Perhaps mammalian embryos might be regarded as having prolonged their carbohydrate period to cover the whole of their pre-natal life, and, if this were so, the peaks in basal metabolic rate found by Wood; DuBois, and others on mammals shortly after birth might be associated with maximum intensities of protein combustion.
Probably other substances besides protein, fat and carbohydrates may be utilised to supply energy in some forms of life. For example, the recent discovery by Heilbron of great amounts of spinacene, a cholesterol-like substance, in selachian eggs, may lead to the solution of the problem of the energy-source of these eggs. What they combust has so far been quite unknown.
Grafe in 1910 thought that there might be some connection between the period of carbohydrate utilisation in the chick's development and the fact that at that time the most profound morphogenetic changes were going on. And it has been suggested that the fat period at the end might be associated with preponderance of change of size over change of shape. But perhaps the time has not yet arrived for correlations of this kind. Again, some connection may appear between the succession of energy-sources in ontogenesis and the numerous observations of susceptible stages in development, such as those of Stockard and of Parnas & Krasinska. This work has brought out with exceptional clearness the fact that development may be discontinuous, and in all cases passes through critical stages when disastrous effects will follow an interference innocuous at other times. The beginning of gastrulation is such a critical stage. Stockard says, "The present extremely crude state of our knowledge of the chemistry of development will permit of no . . . satisiactory statement of the principles underlying differences in developmental rate". Critical stages in development may turn out to be associated with changes from one type of substance to another type as a source of energy. An intermediate link in the chain of events would be the rapid growth-rate of one or more organs, leading to a teratological result if development was at that moment interfered with.
Fig. 261
Fig. 262.
The ultimate nature of the succession of energy sources presents a problem of some interest. It is possible that carbohydrate is first combusted because it requires no preparation. Proteins must be deaminated, fats must be desaturated, and probably the embryo in its earlier stages cannot do either of these things, but, on the other hand, glucose lies ready for use, and it is significant that what is then combusted is free, not combined, carbohydrate^. There is already evidence that the power of desaturation of fats only arises at a comparatively late stage of development, e.g. the loth to the 15th day in the chick (see p. 1 1 7 1 ) . And we may look on the unsaturated fatty acids which are notably present in the yolk (see p. 295) as a preparation for these conditions.
Or it may be that some conception of "ease of combustion " will prove helpful. Quastel & Whetham, studying the action of B. coli on various organic substances, found that carbohydrates were much better hydrogen donators than substances of the protein or fatty type. The following figures, taken from their paper, are striking.
Reduction coefficient (The reciprocal of the molar concentration required to reduce i c.c. of 1/5000 methylene blue in presence of a standard Substance amount of organism in half an hour)
"Carbohydrate". Glucose ... 5000
Fructose . . . 5000
Lactic acid ... 583
"Protein". Alanine ... ... i
Glycine ... ... o-8
Glutaminic acid ... 25
"Fat". Nonylic acid ... ... 0*4
Heptylic acid ... ... 0-4
The succession of energy sources might, then, be related in some way to the changing rH of the embryonic cells or to other important factors in the oxidation-reduction processes going on in them. A study of the oxidation-reduction properties of embryonic cells throughout development would be interesting. Aubel & Wurmser have made some progress in this direction.
The ontogenetic succession could be either "ovogenic" or "embryogenic". On the former view, the energy sources would succeed one another simply because the dynamic equilibria and the relative concentrations of substances in the yolk and white necessitated it. The embryo would play a passive part, combusting protein and fat only since it could not get carbohydrate. On the latter view, the succession of energy sources would be intimately connected with the changing potentialities of the growing embryo. Energy must be the same from whatever source it comes but the embryo — on this view
1 And yet even here a preliminary combination with phosphoric acid may be necessary.
Table 126. Energy sources
Animal
ck (Callus domesticus) g {Rana temporaria)
ok trout {Savelinus fontinalis)
nt Salamander {Cryptobranchus allegiieniensis)
ke {Tropidonotus natrix) ... ... ... Losses not known but combusted material considered to be
(of at non ... ... . ... — 26-8 lo-i — 13-0 4-2
.worm {Bombyx mori) ... ... ... 1-98 ii'Si 8-08 0-74 9-2 4-37
[ct [Pleuronectes platessa) ... ... ... — 0-213 0-0057 — 0-174 0-0204
Amount of substance present at beginning (mgm.)
Amount of substance present at end (mgm.)
Carbo- Prohydrate tein
Fat
Carbo- Prohydrate tein
Fat
335 6375 0-040 1-14
5600 2-55
170 4890 0-037 0'84
3450 1-59
- 13-7
6-4
- IO-6
6-7
— 40-25
ii-i8
— 38-28
12-74
0-033 calculated
playing an active part — would combust such and such substances at such and such periods of its development because it would not have at those times the capacity for combusting others. The molecular orientations on its intracellular surfaces would differ at different stages of its development, and its enzyme systems would vary profoundly in activity.
At present there is not enough evidence to allow us to make a final choice between these views. The ovogenic hypothesis would commit us to the belief that, if sufficient carbohydrate were present during the protein and fat combustion periods, the utilisation of these latter by the embryo would greatly diminish or disappear. On the embryogenie hypothesis we should have to believe that, howev^er much carbohydrate were present during the protein period, the embryo would continue to combust protein, for a close relationship would exist between its source of energy and its stage of development. In favour of the ovogenic hypothesis might be cited the case of the viviparous embryo, which may possibly combust carbohydrate throughout its development. But dangers beset any direct comparison between embryos in ovo and in utero. Mammalian embryos have a continuous perfusion system, non-mammalian embryos have not ; so that in one case the proportion of embryo to nutriment does not alter, and in the other case it does. Mammalian embryos can have all their combustible material supplied to them in solution ; if the avian embryo lived in the same style it would need an ^gg vastly larger than its present size to contain its physiological sugar solution. The fat of the avian egg is tabloid food.
of various developing embryos.
Substance lost, i.e. combusted (mgm.)
Substance combusted in °/o of total material burnt
Substance combusted in % of substance originally present
Carbo- Prohydrate tein
Fat
Total
Carbohydrate
Protein
Fat
Carbohydrate
Protein Fat
20 69 0-029 0-300
2150 0-095
2250 0-424
3-02 6-84
5-57 70-70
91-4
22-4
lo-o
7-2
7-5 38-8 26-0 4-0
- 3-1
p
4-8
63
37
21-96 ?
- 1-97
p
0-97
—
100?
?
—
4-9 ?
Investigator
Murray; Needhz
4-0 Barthelemy & Br
net; Needham
Gortner; Tangl
Farkas Gortner; McCk don
principally carbohydrate by Bohr, who obtained a regularly high respiratory quotient Sommer & Wets least 0-90) Bohr
— i3'8 5'9 ? — 60-0 30-0 — 55-0 60-0 Greene; Miesche 1-24 2-1 1 3-71 5-31 ? <io-o 64-0 — i8-7 46-0 Tichomirov; Kel
— 0-039* ? 0-031 — loo-o — — 15-0 — Dakin & Dakin
from oxygen consumption.
The active autohegemonic powers of growth which the embryo has been shown to possess by the experimental embryologists might seem to favour the embryogenic hypothesis. In its support could also be adduced the fact that, during the protein period in the avian egg, there is plenty of carbohydrate being absorbed in the bound form of ovomucoid. It will be seen in Section 8-2 that the free sugar — probably the only carbohydrate fraction burnt by the embryo — does not entirely disappear until the 12th day of development. Yet, as Fig. 261 shows clearly, it is between the 8th and the 9th day that the peak of utilisation of protein occurs.^ The embryo then by no means awaits the exhaustion of its carbohydrate supplies before beginning to combust protein. This fact is strong evidence in favour of the view that the embryo and not the supply of food at its disposal is in command of the situation. In order, however, to make more certain of this, I carried out some injection experiments. Eggs were injected with a solution of glucose containing 500 mgm. per c.c. By injecting into the air-space the mortahty of embryos is much reduced. As will be seen from Fig. 263, no significant effect was produced upon
^ Moreover, at that moment the egg also contains about 140 mgm. per cent, of glucose in the bound form of ovomucoid.
the uric acid curve. If the embryo had been burning protein because carbohydrate was absent or not easily obtained, then the uric acid curve should have been depressed after the injection of glucose, but this was never the case.
So far the order in which the three great types of biologically important molecule are combusted for the Ea. during the development of the embryo has alone been taken into consideration, and not the relative proportions in which they are used for this purpose. In the case of the chick, the following figures were obtained by Needham in 1927:
Total material Correction for
disappearing during material disapthe whole of in- pearing but not cubation (mgm.) combusted (mgm.) Result
Carbohydrate... 166
Protein ... 68
Fat
Same in per
cent, of the
total material
catabolised
2171
40
o
105
126
68
2066
2260
5-57 3-02 91-4
SIGNIFICANT LIMITS
This is simply a more accurate statement of a fact which has frequently been mentioned before, namely, that the main source of Ea. in the hen's e^g: is fat. Other embryos, however, do not utilise fat to the same extent, and Table 126 summarises the data which show this. It largely explains itself. The first two items are the only ones in which our knowledge is complete, though doubtless not final. Attention may be directed to the fact that the amount of carbohydrate combusted in per cent, of the total amount of food-stuflT combusted is of the same order in the frog as in the chick. But this is not the case for protein and for fat, for in the former case 7 1 per cent, is protein and 22 per cent, fat, while in the latter case 6 per cent, is protein and 91 per cent. fat. Here then is a distinct difference between the metabolism of the embryonic chick and the embryonic irog. The problem is to decide upon what biological difference to fix as the correlate of this biochemical difference. Most probably the difference is associated with the difference between terrestrial and aquatic forms. Generally speaking, aquatic embryos use a great deal of protein during their ontogeny, but terrestrial ones are sparing of it.
This generalisation may, at any rate, be regarded as a legitimate working hypothesis, and fits in with Gray's distinction between aquatic and terrestrial embryos, in that the former can obtain water for their tissues from their environment, while the latter cannot, so that an apparatus has to be provided for storing the necessary water in the egg, and supplying it at a steady rate to the embryo. The egg of the trout, for instance, contains, like that of the chick, enough solid to make one embryo, but, unlike that of the chick, not nearly enough water. In the same way, the embryo of the trout, on the view here propounded, need exercise no economy in the combustion of protein, since it has an unlimited space into which to excrete the resulting waste products, but the embryo of the chick, on the other hand, has only a very limited means of disposing of such compounds, and accordingly obtains its Ea. from substances that burn away completely to carbon dioxide and water. This subject will be more fully discussed in Section 9-15.
SUMMARY OF DEFINITIONS
Ea (Tangl) = U (Rubner) = the " Entwicklungsarbeit," i.e. the chemical energy in that fraction of the raw
materials of the egg, which is combusted by the embryo during its development. REa (Tangl) = the Relative Ea, i.e. the Ea calculated for i gm. wet weight of embryo. SEa (Tangl)=U( (Rubner) = the Specific Ea, i.e. the Ea calculated for i gm. dry weight of embryo (Tangl) or I kilo dry weight (Rubner). OE = " Organisation-Energy," i.e. the energy stored in the embryo which, though appearing as calorific value of combusted wet tissues, would not result from the combustion of an unorganised mixture of its constituent chemical substances. Wa (Tangl) = '* Wachstumsarbeit," i.e. that fraction of the Ea which is due to the function of Growth. Na (Tangl)=" Neubildungsarbeit," i.e. that fraction of the Ea which is due to the function of Differentiation. Ua (Tangl)=" Umbildungsarbeit, i.e. that fraction of the Ea of insect metamorphosis which is due to the functions of Transformation and Rearrangement. Ha=" Histolysearbeit," i.e. that fraction of the Ea of insect metamorphosis which is due to the function of Histolysis. U (Terroine) = the amount of chemical energy stored in the raw materials of the egg. Ui! (Terroine') = the amount of chemical energy in the unused raw materials at the end of development. U' (Terroine) = the amount of chemical energy stored in the finished embryo. W (Rubner) = C/' calculated for i kilo wet weight of embryo. Wi (Rubner) = U' calculated for i kilo dry weight of embryo. Uk (Terroine) = that fraction of the Ea u hich is due to basal metabolism. AEE= Apparent Energetic Efficiency, i.e. the relation between the chemical energy in the material stored, and that in the material combusted during development, (U'/Ea). REE = Real Energetic Efficiency, i.e. the relation between the chemical energy in the material stored and that in the material combusted during development for non-basal metabolism only (U'/Ea- Ui). SEE= Synthetic Energetic Efficiency, i.e. the relation between the chemical energy furnished to the embryo by coupled reactions with one endothermic component, and that in the material combusted during development for non-basal metabolism only. PEC = Plastic Efficiency Coefficient, i.e. the relation between the material {not the energy) stored in the embryo, and the material combusted by the embryo.
Cite this page: Hill, M.A. (2024, April 26) Embryology Book - Chemical embryology 2-7 (1900). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_Chemical_embryology_2-7_(1900)
- © Dr Mark Hill 2024, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G
