Paper - Guinea pig development 21 to 35 days

THE DEVELOPMENT OF THE EXTERNAL FORM OF THE GUINEA—PIG (OAVIA OOBAYA) BETWEEN THE AGES OF 21 DAYS AND 35 DAYS OF GESTATION1

MARY T. HARMAN AND MARJORIE PRICKETT DOBROVOLNY’ Komsas State College of Agr-icelttzrc and Applied Science

fiVE CHARTS AND TWO PPlates (Eleven fiGURES)

AUTHORS’ ABSTRACT

The authors are preparing a table for the normal developments of the guinea-pig. This paper deals with the external form and the development of the external characteristics of guinea-pigs between 21 days and 35 days, copulation age. About one hundred embryos were used from thirty-six litters.

Length and weight are good criteria for determining age, although there is some variation. The flexurcs, the disappearance of the tail, the development of hair follicles at specific locations, the degree of development of the mouth and external ear are better criteria for the determination of age.

At 27 days there is a sudden increase in both weight and length. This increase is rather uniform from this period until the end of 35 days. At this time the embryo has the appearance of a newborn guinea-pig.

CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494

Review of literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494

Material and methods . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . 494

Breeding conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495

Removal of embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495

Data and observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496

Size of the embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496

The 21-day embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 502

The 24-day embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 506

The 25-day embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508

The 27-day embryo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508

The 29-day fetus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 508

The 31-day fetus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 509

The 32-day fetus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510

The 35-day fetus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 511

Occurrence of resorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513

1 Ti; iaiitilgls v;{;£"£io'i;;¢k11o§&1eag§i their iilaetitédnsés kc} 1115;. ri'.i't}'E”£;e};,i of is Animal Husbandry Department, for furnishing the animals for the breeding purposes, as well as valuable advice with regard to the care of the colony.

‘Contribution no. 147 from the Department of Zoology, Kansas State College, Manhattan.

INTRODUCTION

The authors are attempting to make a normal table for the developme11t of the guinea-pig embryo which is similar to the normal table for other mammals edited by Keibel.

The development of the external form of the guinea-pig up to the twentieth day of gestation has been given in a previous publication (Harman and Prickett, ’32). It is our purpose in this paper to continue the study of the factors which contribute to the development of the external form up to the thirty~fifth day of gestation and to supplement the data given by Draper (’20) and Ibsen (’28) as to the size of the embryos between the ages of 21 days and 35 days.

Review 0 f litemt/we

A rather complete study has been made of the external form at consecutive stages of a number of other animals, including man, the rat, the pig, the rabbit, and the chick. Among the contributors to the study of the development of specific tissues, organs, or systems of the guinea-pig are Adloii (’04) and Tims (’0l) concerning the teeth; Lams (’13), study of the egg and early segmentation; Huber (’18), on the chorda d.orsalis; Salzer (1895), on the head blood vessels; Lohle (’13) and Granzer (’08), concerning the mouth and related parts; Keibel (1888), Grrosz (’04), Grruber (’06), Dimpfl (’06), Anderson (’O9), and Dowd (’28), on parts of the uro~ genital system; Widalrowicli (’07), on the central nervous system; and Harman and Derbyshire (’32), concerning the suprarenal bodies.

MATERIAL AND METHODS

The material for this research was secured from young female guinea-pigs, the majority of which were discards from the genetics research. The young virgin females were supplied us through the cooperation and courtesy of the Animal Husbandry Department of the Kansas State College. EXTERNAL FORM OF GUINEA-PIG, 21 TO 35 DAYS

Breeding conditions

Breeding conditions were kept as uniform and as favorable as circumstances permitted. The animals were kept in an environment where there was little variation in the temperature. Regularly each day the animals were fed a balanced ration consisting of a rolled oats mixture, alfalfa hay, fresh green grass or sprouted oats, and water. All of the essential food elements are included in this diet, so that little or no variation in development arising from a nutritional cause would occur. Daily, each female showing an open vagina was placed in a cage with a male and closely observed. If copulation occurred, the female was examined for the presence of a vaginal plug, and the record was made accordingly, as, for instance, (I 26 9 X G 17 3) May 30, 1931; 9.30 A.M. vaginal plug.

Embryos of a known copulation age were obtained by killing the female when the desired period had elapsed since copulation.

Removal of embryos

The female was killed by the use of illuminating gas. Previous to the removal of the embryos a diagram was made of the uterus in place, showing the comparative size and location of each embryo with its membranes. The embryos in the right horn of the uterus were designated as R1, R2, R3, etc., numbering from the union of the horns distally toward the ovary. In the left horn they were similarly identified as L1, L2, L3, etc.

Weights were taken of each embryo alone, of the fetal membranes, of the maternal placenta, and of the amnionic fluid. The embryo was measured and it and its membranes were labeled and placed in Bouin’s fixative solution. After fixation, each embryo was remeasured, for upon fixation the curvature of all embryos of any one age is approximately the

same, while the unfixed embryos may assume varying degrees of curvature.

DATA AND OBSERVATIONS

A total of approximately 100 embryos was secured from thirty-six pregnant females. These embryos were studied first with respect to the weight and length in relation to the age of the litter. The relationship was noted, each to the other, of the embryo, membranes, fluids, and uterus. After fixation, each embryo was examined for developmental changes characteristic for each of the nine age groups studied. The age groups were from 1 to 3 days apart. If the changes

TABLE 1

The weight of the smallest and the largest embryo of any given age and the average weight of all emb7'yo.9 of that age. The weights are of the fresh unpresereed material. The length-s are of the same embryos before and after fixation

WEIGHT OF EMBRYOS i LENGTH or EMBRYO IN MILLIME.TERB

IN GRAMS , AGE M , M , Before fixing 1 After fixing A ‘ mum ' mum : Average ‘ 3 Average  ! .. .___,,_.__.[_._. ..__.. _____ .._.____ __ __.. ._ _._ . ____ ...__._ .__. .__.. -__.. __ _ . .__-! ???? _. _ . 21 0.290 ! 0.165 7 0.310 10.9 , 3.9 . 12.3 3.5 2, 3 3 ! 3.7

24 ,0.473 0.235 0.700 f 16.3 4 14.9 A 17.3 . 12.9 t 11.5 14.7 25 20.432 1 0.360 , 0.660 16.5 16.0 g 17.0 1 14.3 12.3 15.4 27 10.733 0.475 0.930 ’ 13.4 , 15.7 . 20.6 ‘ 16.2 0 13.6 g 17.2

r

. 1.167 0.540 A 1.410 lg 20.0 7 17.2 2 21.3 = 20.9 I I

29 [ 17.3 24.0 31 2 1.971 . 1.125 % 2.520 25.9 21.2 29.2 g 23.7 19.3 g 27.6 32 ;2.957 2.410 13.310 31.5 . 27.6 34.4 26.3 24.6 29.9

l

l 34 l 3.443 . 2.300 4.300 36.2 34.0 ,2 37.3 , 32.3 . 27.5 g 37.3 35 j4.741 3.950 I 5.320 39.4 ‘ 37.5 ; 41.2 5 36.5 34.2 39.0

of the external characteristics took place. rapidly, the interval between the groups was less. This was done to locate as nearly as possible the exact time at which the various changes take place.

Size of the eml9’ry0s

Embryos at the age of 21 days of gestation and older are large enough to be removed readily and accurately from the uterus and membranes. Reference to table 1 shows an increase in the average weight of the embryo from 0.290 gm. at

21 days to 4.741 gm. at 35 days. EXTERNAL FORM OF GUINEA-PIG, 21 TO 35 DAYS 497

Individuals within any one age group may deviate markedly from the average in weight; for instance, at 21 days the average weight is 0.290 gm., while the maximum weight is 0.810 gm. and the minimum weight is 0.165 gm. However, as reference to table 4 shows, those individuals deviating so

U1 ""1" I I 1 I I H-.. -1 4 ,._____.__.. ..... .. Maximum _ ... __r Average /I f ,._.__ I 1

Weight of embryo in grams no o: I I l

21 2.5 50 35 Age of embryo in days

Chart 1 Showing weight of embryo at successive ages.

greatly from the average in weight may be very close to the average in length.

These weights plotted (chart 1) show that, with some fluctuations, there is a rather gradual increase in the average weight at any one day over that of the preceding day. This daily increase is small until the twenty-ninth day, when the daily gain is increasingly greater, causing the curve to rise rather abruptly from 29 days to 35 days. The curve of the maximum weights follows closely the curve of the average, although it shows more fluctuations. The curve of the minimum weights is rather irregular.

Likewise, there is a corresponding increase in the crownrump length of the embryo. The length like the weight increases more rapidly from the twenty-ninth day to the thirtyfifth day than from the twenty-first day to the twenty-ninth day (charts 2 and 3). However, the average length (after fixation) increases during this 15-day period only from 8.6 mm. to 36.5 mm., as contrasted with the weight which is approximately 16 times greater at 35 days than at 21 days.

Accompanying the increase in the size of the embryo alone there is an increase in the weight of the fetal membranes and the associated maternal tissue (table 2). This increase is not as constant as the increase in the embryo alone, largely owing to the nature of the membranes.

Considered on the basis of the percentage of the whole, the embryo shows a greater increase in weight than do any of the membranes (chart 4). At the twenty-first day the embryo represents only 15.4 per cent of the total weight of the blastecyst and the maternal tissue, while at 35 days the weight of the embryo is 42.8 per cent of the whole. The fetal placenta shows a very slight increase from 15.6 per cent at 21 days to 18.2 per cent at 35 days. Actually the increase in the fetal placenta is from 0.295 gm. to 2.025 gm. The maternal placenta increases from 0.586 gm. to 1.330, but considered on the basis of the percentage of the whole it decreases from 31.1 per cent at 21 days to 11.9 per cent at 35 days. The fluids weigh more at 35 days than at 21 days, but the percentage is irregularly constant. As chart 4 shows, the embryo during this period is developing more rapidly than the membranes. The fetal placenta develops rapidly enough to remain almost constant as to percentage weight, while the percentage weight of the maternal placenta decreases greatly, indicating that its development is practically complete. EXTERNAL FORM OF GUINEA-PIG, 21 TO 35 DAYS 499

__ Average _.__ Minimum

30

B) (J!

N O

I-' (J!

Length of embryo in millimeters 2-’ O

2.1 25 50 35 Age of embryo in days

Chal-‘t2 Showing length of embryo (before fixation) at successive ages.

I 401 1 _ _ I I /, I I / 55 I r --r: | /’ -_-__ Minimum /* : J.

Length of embryo in millimeters

31 25 :50 :55 Age of embryo in days

Chart 3 Showing length of embryos (after fixation) at successive ages. These are the same embryos dealt with in chart 2. EXTERNAL FORM OF GUINEA-PIG, 21 T0 35 DAYS 501

TABLE 2

The wvemge weight of the embryo, of the fetal placenta, of the maternal placenta, and of the fluids, and the c.-/uemge length of the emb7°yo of each age

WEIGHT 011 E WEIGHT or I

AGE L‘Ei£i$f§§’ “Z§FB‘$.'§-8” 1,533-;;;A;m g gtggggggg W§3f5§§s°” ttttttt ..Dwyé.... Emu. __(;?..;;;z_;_-.-_ Gmmé . Grams !_.___E;1;;........21 . 10.9 0.290 : 0.295 1 0.588 0.714 24 15.8 0.515 0.589 } 0.857 1 0.809 25 18.5 0.481 0.480 1 0.719 0.844 27 18.4 0.783 0.880 1.040 1 1.180 29 8 19.9 = 1.167 0.911 0.940 1 1.258 31 25.9 1.971 1.078 1.151 1.551 32 30.4 2.787 1.504 1.314 1 1.903 34 38.2 H 3.443 1 1.430 1.035 2.511 35 39.4 1 4.741 2.025 1.330 : 3.043 flUIDS

80 .,. .-_;_:- 04121“ AL 1=LA.cENTA~;§l

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50

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g FETAL PLACENTA

(D

fla

Age of embryo in days

Cha.1-1:4 Showing ‘percentage of the whole’ growth for embryo, p1a.ce11tae, and fluids, based on average weights of litters at successive ages.

The 21-day embryo

At 21 days the embryo measures, on the average, 10.9 mm. in length (fig. 5). The nape flexure places the head in a position at a right angle to the rest of the body. The sacral and cephalic flexures are present, although the cephalic flexure is becoming obscure, due to the rapid development of the head. A row of paired, slightly raised cubical thickenings locates externally the forty—one pairs of mesodermic somites. The tail showing 7 or 8 somites is about 1.2 mm. in length.

TABLE 3

The percentage of the whole of each of the parts of the blastocyst and mate-rnal decid-ua

l

Days A Per cent l Per cen.ilw ; Per cent P87‘ Cent 21 1 15.4 } 15.6 31.1 , 37.9 24 : 20.0 ; 22.9 33.3 lg 23.7 25 20.7 20.6 30.9 g 27.7 27 _ 21.4 13.0 : 23.4 32.2 29 27.3 21.3 22.0 i 29.0 31 34.2 13.7 20.0 27.0 32 36.9 20.1 17.5 l 25.4 34 40.9 17.0 12.3 . 29.3 35 1 423 j 13.2 [ 11.9 1 27.3

Development of the heart and beginning of the decided enlargement of the liver causes a marked protrusion ventrally. The intestine, up to this time comparatively undeveloped, is now elongating rapidly, so that a coiled loop of it extends into the umbilical cord a short distance.

The fore limb buds, about 1.7 mm. in length, are slightly longer than the hind limb buds, which are about 1.5 mm. in length. Each is about 1.1 mm. to 1.2 mm. in diameter. At the extreme edge the buds are becoming slightly flattened, but as yet they show no digits. The limbs are directed forward with the soles and palms toward the body.

The head, quite large in proportion to the rest of the body (chart 5), measures 5.1 mm. from the tip of the nose to the TABLE 4

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TABLE 5

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40 __ 4__ I 55 ____ ..Crown-rump length /

“"‘ -......- Nose-top length I ___--Chin—top length ; Fore limb f ____ Hind limb x _u.“ Tail 30.

N 01

_n

..n—

P‘ 01

Length of embryo in millimeters to ca

10

21 25 30 35 Age of embryo in days

Chart 5 Showing average length of component parts of embryos at successive ages.

top of the head, and 3.9 mm. from the chin to the top. The latter measurement is taken almost perpendicular to the former. The mandibular processes are fused together ventrally (fig. 1) and the maxillary processes have united with the processus globularis, separating the nasal pits from the oral fossa and leaving the mouth opening roughly rectangular in shape.

The eye shows much pigmentation of both the iris and the retinal layer. There is hardly more than an indication of the folds which will form the lids.

The external ear is manifest by an irregular ridge along either side of the first cleft which is closed at the most ventral portion, but remains open in the mid-dorsal part. This remaining slit gives rise to the external auditory meatus.

The 24-day embryo

At 24 days the embryos, on the average, measure 15.8 mm. in length (fig. 6). Proportionally, the increase in head length is slightly less than the increase in total body length (chart 5). The head size is now 7.3 mm. from the nose to the top of the head, and 5.5 mm. from the chin to the top, at right angles to the former measurement. Reference to chart 5 shows that the nose-top measurement has increased slightly more than the chin-top measurement. This is due to the beginning nasal protrusion and the comparative retardation of the chin development. The appearance of the head, especially the face, has changed decidedly (fig. 2). In spite of the increase in the size of the head, the mouth measures approximately the same as the 21-day stage. The upper jaw has begun to assume the U-shaped condition as found in the adult.

This emphasizes the proportionally much smaller appearance of the mouth. The lip ridges of the upper jaw are widening and are beginning to extend down slightly over the lower jaw, which is small and receding, but, nevertheless, greatly developed as compared with the fused fiat mandibular processes found at 21 days. The now less prominent external nares are just above the mouth at the base of the nasal elevation.

A fold of tissue above and below the eye delimits the eye from the remainder of the face. These folds, the beginning of the eyelids, do not, however, extend over the eyeball. The only change in t.he external appeara.nce of the eyeball is in the increased pigmentation of the iris and the more clearly defined pupil.

Numerous vibrissae follicles appear in definite areas on the face. In a rather triangular patch between the eye, mouth, and nose there are from 6 to 17 follicles which do not make their appearance in any regular order. Just above the eye is a group of three follicles, all of which appear simultaneously.

The anlages of the external ear which appeared at 21 days as two rows of elevations have further differentiated. Of these elevations the auricular fold and then helix are undergoing the most rapid change. The auricular fold is elongating and becoming somewhat elevated, while the helix is becoming wider and more flattened.

The maximum length of the tail is reached at this age. It is now 1.5 mm. long, and still shows indistinctly the somite markings.

Further elongation of the limb buds occurs, a.nd the flat~ tened distal end has begun to show shallow grooves which indicate the establishment of the digits. The digits project but slightly, giving the extreme edge a scalloped appearance. There is little or no indication of the transverse furrows separa.ting the hind limb into its component parts. The fore limb, which is slightly the longer of the two, shows noticeably a differentiation into the lower and upper arm.

The prominent external genitalia have made their appearance, and, although the sexes cannot be distinguished with certainty, there is evidence of sex differentiation.

Extending from the fore limb to the hind limb on either side is a thickening, the milk ridge. No nipples are formed along the ridge at this stage. MARY T. HAR-MAN AND M. P. DOBROVOLNY

The .25-day embryo

The tables of data show that the 25-day embryos recorded weigh on the average slightly less than the embryos 24 days of age, although there is an increase in the length. The authors have seen no explanation for this decrease, since the embryos of all the age groups were obtained under similar conditions and were given similar treatment. Moreover, the 25-day embryos show a noticeably advanced structural development.

The 27-day embryo

At 27 days the embryos exhibit few changes in appearance. The change is largely a further development of the structures already established at 24 days (fig. 7, chart 5). The nipple of the mammary gland makes its appearance at this stage, but it is small and indistinct.

T he 2.9-day fetus

The length of the fetus continues to increase gradually but rather slowly (charts 2 and 3). The term fetus may be applied to the embryo at 29 days (fig. 8). The head and limbs are larger, but no external part of the fetus increases at an especially rapid rate (chart 5), with the exception of the chintop length of the head, which at 29 days approaches the nosetop length more closely than at any other time in this 15-day period.

The embryo is still somewhat curled, particularly in the neck region; however, the sacral and cephalic fiexures are no longer present as flexures. No somite markings are visible in the tail, which now averages about 0.1 mm. shorter than at 21 days. As will be seen later, this shortening and ultimate disappearance of the tail continues, due partly to the failure of some of the somites to develop vertebrae and partly to the more rapid development of the surrounding tissue, the buttocks.

The cornea of the eye stands out in a dome-shaped prominence, indicating an increasing complexity of the internal EXTERNAL FORM OF GrUINEA—P1G, 21 TO 35 DAYS

structure. In some cases the lid folds have grown up so that they almost. cover the iris. As the folds are growing up over the eye-ball, they are fusing together at the inner and outer corners. The more rapid fusion occurs at the outer corners.

The auricular fold of the ear is elo11ga.t.i11g and bending forward, almost covering the external meatus and completely covering the antihelix and antitragus. The Ventral elevations, destined to form the helix, are flattening and elo1'1gati11g so that they extend somewhat over and into the openi11§_>'.

T Facial features closely resemble those of the fetus at birth, except for the nose, which is more prominent than at birth. In some individuals the nose is so long that it almost resembles a beak.

Previous to 29 days, the abdominal protrusion has been anteriorly in the region of the liver and it is due to the rapid enlargement of that orga11. Because of the increased development of the intestines, at 29 days, the abdomen protrudes more noticeably toward the caudal end.

The external genitalia are assuming the characteristics of one sex or the other. By close examination the sexes can be distinguislied.

The 3.1-day fetus

After the JEWGIIT.-_V—1”]i11'[l’1 day the length of the fetus and its component parts increases rapidly, causing the curves of length to rise abruptly. The length of the fetus at 31 days is 25.9 mm.~——a marked increase over the length at 29 days.

The head has undergone an enlargement, but not as noticeable as the lengthening of the whole animal. The nose—top lengtli shows a more rapid increase than at any previous period. The chin-—top length has increa.scd, but not as rapidly as in the preceding 2-day period. In general, the shape of the head is Very similar to that of the ful1—term fetus (fig. 9). The nose, as in many newborn animals, is somewhat longer in proportion than tl1e nose of the adult. As the nose has been elo11gat:i1‘:g, the month has assumed more nearly a horse-shoe shape so that the width is practically the same as at 21 dayg_

The 11ose pad is appearing’ and the median slit in the upper lip is extending up toward the nostrils. The lids of the eye have fused together completely. The ear is Very similar in appea1'anee t.o the f1:1ll_V formed ear, except that the pa.1't.s are t,hiel{ so that the depressions and elevations are not so elear-cut.

Up to the twe11ty—seve11t.l1 day the fore limbs have been Slig'l]tljT longer than the hind limbs, but by the t-l1i'I.'l..-}7~fiI'St- day the hind limb is somewhat the loiiger. The major divisions of the limb are well established and the limbs are l)eg'l11]]i1'1g‘ to rotate. The toes are elongating‘ and spreading apart. On the fore limb a fourth tee is well defined, while a fifth toe, the thumb, merely a tiuberele-like growtli on the inside of the leg. On the hind limb there are three toes well defined, and a fourth toe, appearing much like the ‘thumb’ on the fore limb. The nail folds and sole plates are well established a11d the wallmig pads are thickening.

The hair follicles (fig. 3) which appear at this time are quite uniformly distributed over all parts of the l)0(lj__.«-*, with the exeept.ion of the ears, a narrow strip of the eye lids, a narrow area around the lower jaw, and the lower part of the limbs.

The tail, about 0.6 mm. long, now appears as a mere bump. A t.l1iel<ening’ of the tissues in the definitive location of the mammary gland indicates the be§._,>'i111iiug' of that organ- The nipple is somewhat more distinct than at 27 days.

."l’h.(3 3.2—(.l(z3; fvatus

There is no st.1°il<i11g change in the appearaiiee of the fetus since 31 days (fig. 10). Vibrissae begiii to project above the surface. Secondary and smaller hair follicles are new interspersed rather unifo1°mly betweeii. the primary follicles Wli'liCl1 are farther apart than at 31 days, due to the growth of the skin area between them (fig. 4). EXTERNAL FORM OF GUINEA-PIG, 21 TO 35 DAYS

The 35-day fetus

VVl1ile the term fetus may be applied to the embryo at 29 days, by 35 days the proportions of the component parts of the body approach more closely those of the newborn animal (fig. 11). The weight and length curves (charts 1, 2, and 3) continue to rise at about the same degree as from 29 days to 31 days. The average length at 35 days is 39.4 mm. and the average weight is 4.741 gm. 3

The feet are well developed and all of the definitive parts are present. The nails are soft and have not lengthened to form claws. However, close examination shows that much keratin is present. The walking pads are becoming more thickened.

Follicles for the eyelashes are present along the eyelids, which are completely fused together over the eyeball. More vibrissae are appearing on the surface. No hair has ap— peared. The external ear is similar. to the car at 31 days, except that the pinna is not as thick proportionally. The nose now looks very much like the nose of the newborn animal. The tail is completely gone, leaving no more than an indication of its earlier location. The external genitalia, although not in the definitive form, show pronounced sex differentiation.

Occruvrrence of resorption Resorption of guinea-pig fetuses at an early age seems to be of rather common occurrence. Ibsen ( ’28) cites instances of fetuses nearing the end of the gestation period, but does not record any at the earlier stages. Of the forty-four litters we have recorded, 13 show resorption of part or all of the embryos. These litters at varying stages of resorption and ranging in age from 21 to 32 days are as follows:

Age Right horn Left horn 21 days R1 normal L1 and L2 normal, L3 a small mass 24 days R1 normal, R2 abnormal L1 a mass of tissue

R1 normal, R2 a sac with 3. L1 normal shapeless mass

R1 partially re-sorbed placenta, R2 and R3 normal

25 days R1 normal L1 a mangled ma.ss in membranes

27 days R1 a greatly discolored mass L1 dark and apparently dead, weight normal, L2 and L3 normal

R1 retarded and partially re- L1 and L2 normal sorbed

29 days R1 fluid not normal in appcar- L1 resorbing a11ce, R2 with blood clot on head 31 days R1 normal L1 and L3 normal, L2 resorbing

32 days R1 small, discolored, disintegrating, R2 normal

The death and resorption of the fetus begin previous to any noticeable change in the appearance of the fluids and membranes. As noted above, early evidence of resorption is in death, discoloration, and subnormal development of the embryo. The amniotic fluid becomes clouded and discolored shortly after the resorption of the fetus begins. Resorption of the placenta occurs last and is manifest by a softening, disintegration, and loss of the elasticity of the tissue, in addition to a loss of the normal pink color.

DISCUSSION

As the data show, there may be variations in the size of the embryos and their membranes and fluids for any given age group. These variations may be due to a number of causes, among which are differences between copulation ages and fertilization ages, slight mechanical difierences in measurement of Weight and length, Variation in amounts of fluids adhering to embryo and membranes, and inherent tendencies for some animals to attain a greater or less size.

With reference to the relation of the time of copulation to the time of fertilization Lams ( ’13) found that fertilization EXTERNAL FORM OF GUINEA-PIG, 21 TO 35 DAYS

may occur as early as 10 hours after copulation, or the spermatozoon may not enter the egg until after 15 hours. Two pronuclei may be observed from 12 to 31 hours after coitus. He found the first segmentation spindle forming from 27 hours to 34 hours. Consequently, litters of the same copulation age may not have exactly the same fertilization age or developmental age.

The fetal and maternal placentae are so richly supplied with blood that varying amounts of it may remain in the profuse network of blood vessels when the membranes are separated from the uterus and from the embryo. Moreover, when the membranes are separated one from the other or from the embryo or from the uterus, there is no sharp line of separation. These factors in part account for the variability in weight of the membranes and fluids. There are, no doubt, inherent individual differences, also.

Even with these variations, any individual embryo can be placed in approximately the age group to which it belongs if weight and length alone are considered, for, as the curves and tables of size show, size and age have a significant relationship. On the other hand, if degree of development as well as size is considered, there will be more accuracy in placing the individual in an age group, for, as the data indicate, the degree of development is the best criterion of age.

SUMMARY

1. All of the embryos for each age show approximately the same degree of development, and degree of development is the best criterion of age.

2. The average weight increases from 0.290 gm. at 21 days to 4.741 gm. at 35 days.

3. The average length increases from 8.6 mm. (after fixation) to 36.5 mm. in the same period of time.

4. All of the flexures are present at 21 days; they become less evident at 24 days, and by 29 days they are no longer present as flexures.

5. The tail, 1.2 mm. in length at 21 days, reaches its greatest length, 1.5 mm., at 24 days, and has disappeared at the thirty-fifth day.

6. The limbs buds, somewhat elongate at 21 days, begin

to flatten at the distal end at the twenty—fourth day, shows the digital markings at 27 days, and have well-defined digits

and joints at 29 days. The sole plates and nail folds are formed by the thirty-first day.

7. The mouth is wide and open at the tWenty—first day; it is proportionally smaller and horse-shoe-shaped at 24 days, due to the development of the jaws. At 27 days it has assumed its definitive form.

8. At 21 days, the first branchial cleft has closed, except for the opening which gives rise to the auditory rneatus. Tubercles arise along either side of this Opening, andlby the thirty—second day they have developed into the definitive form of the pinna of the ear. 9

I 9. The folds of the eyelids arise at the twenty-fourth day and they fuse to cover the eyeball on the twenty-ninth day.

10. Vibrissae follicles appear at either side of the nose and above the eye at the twenty-fourth day. The vibrissae appear on the surface about the thirty-second day. A A

11. The first follicles of the hair appear at 31 days. No hair is visible on the surface at 35 days. A

12. Sex differentiation, as manifest by the external genitalia, is apparent at the twenty-fourth day, although the sexes cannot be distinguished with certainty. A

13. By the thirty-fifth day the general external appearance approaches that of the newborn guinea-pig.

LITERATURE CITED

ADLOFF, P. 1904 fiber den Zahnwechsel V011 Cavia cobaya. Anat. Anz., Bd. 25, S. 141-147. ' - —-—————— 1896 Zur Entwicklungsgeschichte des Nagethiergebisses. Z001. A11z., Ed. 20, S. 324-329.

ANDERSON, L. G. 1909 Uutzersuchungen iiber die Entstehen der Eiusseren Genitalorgane und des Afters bei den Nags-t.iere11. Ark. Z001. Stockholm, vol. 5, pp. 1~—230.

DIMPfl, H. 1906 Die Teiluug der Kloake bei Cavia cobaya. Morph. Jahrb., Bd. 35, S. 17. EXTERNAL FORM OF GUINEA-PIG, 21 TO 35 DAYS

Down, DOROTHEA R. 1928 The development of the ovary of the guinea-pig, Cavia cobaya, in embryos of eighteen to thirty days, inclusive; with some observations concerning its subsequent development. K. S. C. thesis.

DRAPER, R. L. 1920 The prenatal growth of the guinea-pig. Anat. Rec., vol. 18, pp. 369-392.

GANZER, H. 1908 Anatoniie nnd Entwicklung des Gebisses vom Meerschweinchen (Czwia cobaya), 8. 1-31. Berlin.

GROSZ, S. 1904 ljber den Perinealsack V011 Cavia cobaya und seine Driisen. Zeitschr. f. Wissenseh. Zool., Bd. 78, S. 261.

G-RUBER, O. 1906 Ban und Entwicklung der §i,usse1'en Genitalien bei Cavia cobaya. Morph. Jahrb., Ed. 36, S. 3-26.

HAM.-IAN, MARY T., AND RUSSELL CLAY DERBYSHIRE 1932 The development of the suprarenal glands in the guinea.-pig (Cavia cobaya). Am. J. Ana.t., vol. 49, pp. 335-349.

HARMAN, IVIARY T., AND MARJORIE Pnronerrrr 1932 The development of the external form of the guinea.-pig (Gavin cobeya) between the ages of eleven days and twenty days of gest-ation. Am. J. Anat., vol. 49, pp. 351-378.

I-IUBER, G. CARL 1918 On the. anlage and morphogenesis of the chords. dorsalis in Mamrnalia, in particular the guinea—pig (Cavia. cobaya). Anat. Rec., vol. 141, pp. 21T—2-64.

IBSEN, H. L. 1928 Prenatal growth in guinea,—pigs, with special reference to environmental factors affecting Weight at birth. J. Exp. Zoi)'l., vol. 51, pp. 51-91.

KEIBEL, FR. 1888 Die Entwicklurigsvorgirhige am hintoren Ende des MeerschWei11chenembryos. Archiv f. Aunt. u. Physiol.., Leipzig, 1896, S. 55.

Lr‘-'u’.\1§'3, HONORE 1913 lfltude de l’0euf de coloaya aux premieres stadee de Pembryogenese. Arch. Biol., Pa.ris—]3ruXelles, T. 28, pp. 229—323.

LOHLE, B. 1913 Die Bildung dee Ga.urne11s bei Ca.»-'ia. cobaya. (A. fleischmann-' Die liopfregion der Arnniotcn, Forts. 11). Morph. Ja.hrb., Bd. 46, S. 595-65-1.

SALZER, H. 1895 Ueber die F.ntwick1u11g dor Kopfvenen dee Meerschweinchens.

_ Morph. Ja.I1I'b., Bd. 23, S. 232-255.

Tms, H. w. M. 1901 Tooth genesis in the Cavirlne. J. Linn. Soc. Zool., vol. 28, pp. 261-290.

WIDAKOWIUII, V. ].907' Ueber Entwicklungsdifferenzen (lee Ze11trnl11orvensyg.

toms drier gleichalteriger Embryonen von Cavia cobaya. Arb. Nenrol. Inst. W':ie11, Bd. 16, S. 452-468. EXPLANATION OF PLATES

The drawings of plate 1 (figs. 1 to 4) were made with the aid of a camera lucida. figures 5 to 11 of plate 2 are photomicrographs.

PLATE 1 EXPLANATION or sreuass

1 Face View of a 21-day embryo. X 10. EN, external nares; PG, processus globularis; E, eye; MP, maxillary process; 0, oral fossa; M, mandibular process.

2 Face View of a 24-day embryos" X 8. L, beginning of eyelid; N, nasal protrusion; V, vibrisae follicles; 0, oral opening; I, iris of eye; EN, external nares.

3 Skin surface of a 31-day fetus, showing the primary hair follicles, PF. )( 13.

4 Skin surface of a 35-day fetus, showing the size and distribution of the primary hair follicles, PF, and the secondary hair follicles, SF. X 13.

517 U3-Q3301

PLATE 2

EX PLA NATION

Twe11ty-0119-(lay e.n1b-rye. X 2.

Twenty-f0ur—day embryo. )< 2. Twe11ty~seve11—da.y embryo. >1 2. Twe11ty-11ine—day fetus. X 2.

OF fiGURES

9 Tl'1irty-0110-day fetus. 10 Thirty-two-day fetus. 11 Thirty-five-da,y fetus.