Book - The Development of the Albino Rat 1
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The early developmental stages of placental mammals, embracing the stages of sex cell maturation and fertilization, of segmentation, of blastodermic vesicle and germ layer formation, though subject of numerous contributions extending over many years, have in no form been completely investigated. The literature dealing with the phenomena of maturation and fertilization as observed in placental mammals has in recent years been enriched by a number of studies to the extent that for certain of the mammals — bat, rabbit, guinea-pig, mouse, and rat — the data at hand are sufficiently complete to enable a clear and comprehensive presentation, based on observed facts, and permit of comparison with similar phenomena as observed in other vertebrate and invertebrate forms. As concerns the process of segmentation in placental mammals, there are still lacking sufficiently comprehensive observations embracing a number of forms to enable a clear and succinct presentation of the rate of blastomere formation, the cytomorphosis of the cells, and of the relative position of the several segmentation stages in the genital tract. This is no doubt owing to the difficulty of obtaining the necessary material timed so as to admit of proper staging, and the impossibility of making extended observations on living material. Our knowledge of the phenomena of blastoderm vesicle formation, though comprehended in its general phases, is lacking in detail, except for a very limited number of forms. The process of germ layer formation is of such fundamental importance to a clear comprehension of later developmental stages, both in phylogeny and in ontogeny, that a brief account of observed facts in any one form may not be regarded as wholly without value.
Opportunity presented itself, while stationed at The Wistar Institute of Anatomy and Biology, to collect and fix an extended series of embryological stages of the albino rat. This material has proven sufficiently comprehensive to enable a presentation of the several developmental stages of this mammal, beginning with the pronuclear stage and extending to the stage of the anlage of the mesoderm. For this period, which extends to about the tenth day after insemination, only very few of the essential stages are lacking, though for certain of the stages confirmatory preparations would have been desirable. The material at hand, however, seemed sufficiently complete to present a connected account of the stages it is hoped to cover. The embryology of allied forms, especially of the mouse, has received much more extended study than has that of the rat, though the development of the rat has received especial consideration by Fraser, Christiani, Selenka, Duval, Robinson, Widakowich, and as concerns maturation and ovulation, by Sobotta and Burckhard, Kirkham and Burr. The pertinent literature will be considered in connection with the presentation of my own results.
Materials and Methods
The material on which this investigation is based was obtained from albino rats (Mus norvegicus albinus, Donaldson) taken from the extensive rat colony of The Wistar Institute of Anatomy and Biology. The experience gained in the breeding, feeding, and growth experiments, extending over many years, conducted by Donaldson and his associates and resulting in numerous excellent publications, was at my disposal while collecting this material. The material used was all carefully timed, so that sequence of stages was obtained with some degree of certainty. With care and experience, it is possible to regulate and observe insemination, so that stages may be approximated quite accurately. Kirkham and Burr state that "on several different occasions we have observed actual pairing" of the albino rat. Widakowich states that he was unable to obtain accurate data as to the age of the embryos except by observing coitus. According to this observer, a female rat permits many males to copulate in the course of several hours, receiving males 30 times or oftener, when suddenly she drives them away. Sobotta and Burckhard, on the other hand, admitted males a few hours after parturition, depending on the fact that many mammals ovulate soon after parturition. Though attempts were made, they were unable to observe pairing, and they state that the 'Dieners' charged with the care and feeding of the rat colony were only seldom able to observe attempts at pairing. At The Wistar Institute no difficulty is experienced in pairing albino rats. Dr. J. M. Stotsenburg, to whose experience and careful records I am greatly indebted for the trustworthiness of the material collected, made use largely of females who had born one litter. Pairing was seldom attempted a few hours post partem, as was done by Sobotta and Burckhard, but usually about 30 days after the birth of a litter, which may have been nursed or otherwise disposed of. The great majority of females used in pairing were at the time free from 'domestic cares.' The females employed were kept in separate cages for some time before giving birth to young and until the time of mating. About 30 days after the birth of a litter, a male was placed in the cage with the female. If the female was in heat, copulation usually took place soon after. The male was left with the female for an hour to an hour and a half, during which time several pairings would occur, and at the end of which time the female would try to hide from the male, climb the side of the cage and defy him with her teeth. The male albino rat is not prostrated by the sexual act, the same male serving for several successive copulations. In case the female was not in heat, this soon became evident and the male removed, to be again placed into her cage 24 or 48 hours later. The time when the. copulation was first observed was noted on the card attached to the cage and gave the time from which the age of the embryo or respective stage was reckoned. The time given is, therefore, that of 'insemination,' a term which Long and Mark have introduced to indicate "the introduction of the male sexual elements into the genital tracts of the female by the act of coitus or otherwise." This time could be accurately noted, while 'semination' which "applies to the access of the spermatozoa to the eggs in the oviducts, the coming into contact of the male and female reproductive cells" can not be accurately timed. The success attained in pairing albino rats as above stated, obviated the necessity of depending upon chance material or resorting to 'artificial insemination' as described for the mouse by Long and Mark. I am at loss to understand why Widakowich should regard the age determinations of Sobotta and Melissinos (mouse embryos) more accurate than his own, reckoned from the time of observed coitus. The slight thougli observable variation in the rate of development in a series of ova of the same animal, more marked when supposedly similar stages of several animals are investigated, precludes the accurate timing of stages.
As fixing fluids, there were used Zenker's fluid, sublimatealcohol, Flemming's fluid, Bouin's fhiid, and Carnoy's fluid. After a few trials, all were discarded in favor of Carnoy's fluid, prepared by mixing G parts of absolute alcohol, 3 parts of chloroform, and 1 part of glacial acetic acid. This somewhat illogically compounded fluid penetrates rapidly and does not cause shrinkage. Tissues are fixed in it for several hours, then washed in several changes of absolute alcohol in which it has been my custom to store the tissues. The following procedure was practiced in all stages up to about 12 days after insemination: The animals were anaesthetized and the head severed from the body, to admit of free bleeding. The rat was then fastened to a board, and thorax and abdomen opened by a mid-sagittal incision, the abdominal walls pinned back, and the intestine elevated toward the thorax. With as little manipulation as possible, the ovaries were separated from their attachment, the mesometrium cut, the uterine horns elevated and the vagina severed. The whole genital tract was then placed on a clean slide and arranged in approximately normal position. Slight tension was maintained by tying a thread to the connective tissue removed with each ovary and bringing the threads along the reverse side of the slide and tying them to the vagina. If the side is clean, the mesometrium of each uterine horn may be spread out evenly and caused to adhere to the slide. Ovaries, oviducts, and uterine horns may thus be spread out in normal position and each uterine horn fixed as a straight tube. AVhen thus arranged on the slide, the preparation was placed in a relatively large quantity of Carnoy's fluid, fixed, and then transferred through several absolute alcohols. For nearly all the material used in this study, the method of fixation was as here given. In the earlier stages of material collection, attempts were made to obtain segmentation stages in warm normal salt solution. Several were thus obtained and were used to control the observations made on sections, as will be discussed later. By cutting the oviduct at about its middle, freeing it from its mesosalpinx and cutting the uterus about 1 cm. below the insertion of the oviduct, a pipette fitted with a rubber bulb and filled with warm normal salt solution can be inserted into the uterine cavity and moderate pressure made. It is usually possible to wash into a watch crystal a certain number of the contained segmenting ova. Before reading the article by Widakowich, essentially the same method as employed bj^ him, for isolating implanted blastodermic vesicles was developed. This may be quite readily done after fixation in C'arnoy's fluid and teasing under a stereoscopic binocular. Vesicles sectioned in situ, however, gave on the whole more satisfactory results, so that teasing out implanted vesicles was not resorted to.
The fixed tissues were imbedded in paraffin, using xylol as a clearing fluid. For stages including those falling within the period ranging from the first to the fourth day after insemination, the ovary and oviduct to its insertion in the uterus, were embedded e?i masse. For stages falling within the period of fifth to sixth day after insemination, the uterine horns were divided into segments measuring about 1.5 cm., and sectioned parallel to the plane of the mesometrium. For later stages, after the enlargements in the uterine horns are distinctly evident, these were removed and cut severally in the three planes. The great majority of the sections were cut at a thickness of 10 ju ; certain ones at a thickness of 5 ju ; a few at a thickness of 7 n. The sections were fixed to the slide by the water-albumen method. The great majority of the series were stained in hemalum, counterstained in Congo red. This solution, which presents certain advantages as a counterstain for embryologic tissues, is prepared as follows: 0.5 gms. of Congo red (Griibler) is placed in 100 ccm. of distilled water and the water brought to boiling. This should give a clear solution. Before cooling, add 100 ccm. of distilled water and 10 ccm. of absolute alcohol. The Congo red solution thus prepared may be kept many weeks. After staining the series in the usual way in hemalum, thej^ are differentiated in acid alcohol, and passed through several washes of 'tap water' into distilled water. they are then stained in the Conso red solution, which may be diluted with distilled water about five times. With the diluted solution, the counterstaining refpiircs one to two hours. The sections are then rinsed in distilled water, differentiated in 80 per cent alcohol, dehydrated, cleared, and mounted in damar. Certain of the series were stained in Heidenhain's iron-hematoxylin and counterstained in Congo red. The drawings accompanying this contribution were nearly all drawn on coarse 'Ross board,' with the aid of the camera lucida at a magnification of 1000 diameters, using pencil and India ink. Such drawings admit of liberal reduction, and give a detail not readily obtained otherwise. Free use has been made of the Born method of reconstruction, especially for earlier stages. The majority of the models thus obtained are here reproduced.
I desire to express my sincere thanks and apjireciation of the very material aid given me by Mr. Wayne J. Atwell, then Assistant in the Department of Histology and Embryology of the University of Michigan, in the making of the reconstructions of the oviducts included in this account.
- Melissinos and Widakowich state having used as material the albino rat, variety Mus rattus albinus. Donaldson has conclusively shown, that by reason of physical characters — blood crystals, shape of the skull, etc. — the albino rat kept as pet or laboratory animal cannot be Mus rattus albinus, but must be Mus norvegicus albinus.
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Cite this page: Hill, M.A. (2020, July 10) Embryology Book - The Development of the Albino Rat 1. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/Book_-_The_Development_of_the_Albino_Rat_1
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