1944 247 Karyotypes of Domestic Cattle, Zebu And

1944 247

Karyotypes of Domestic Cattle, Zebu and Domestic Water-Buffalo (ChromosomeStudies in Domestic Mammals,IV)1 By Sajiro Makino Zoological Institute, Faculty of Science , Hokkaido Imperial University, Sapporo (With 30 figures) Received January 9, 1944

Though the ungulate animals occupy, as fully known , a very weighty position in agricultural industry and therefore the accurate knowledge of their chromosomal features is of essential importance for their breeding , their karyological investigations have not sufficiently gone on , many im portant problems still being left unsolved at present, and the whole field thus requires reinvestigation. In this view the author has already pub lished three new studies, one of which deals with the chromosomes of the horse and the others with those of the goat, sheep and pig (MAKINO, '43a, b, '44) . The present paper includes the accounts regarding the chromosomes of the following three forms of bovine mammals, domestic cattle (Bos taurus L.), zebu (or humped cattle, Bos indicus L.) and domestic water buffalo (Bubalus buffelus L.). The chromosomes of domestic cattle have been the subject of repeated investigations by previous authors and there have appeared eight papers published by VON BARDELEBEN'92, SCHOENFELD'02, VAN HOOF '13, MASUI '19 , WODSEDALEK'20 and KRALLINGER'27, '28, '31 (of. OGUMAand MAKINO '37) . Although the data presented by the last mentioned author (KRALLIN GER '31) have generally been regarded as conclusive, being commonly accepted as a final criticism of domestic cattle, the findings of the present study point out some facts which are completely inagreeable with his results. The zebu. Bos indices, on the other hand, furnishes a new mate rial for cytology, since the literature on the chromosomes had, until now, no reference to this form. Concerning the buffalo, there exists only a sole study undertaken by PKAKADZE ('39) who worked with Bubalus bubalus L., giving a brief account on the chromosome number. These three forms of bovine animals are all, as well known, the mem bers of the subfamily Bovinae of the family Bovidae. WEBER ('28) divided the Bovinae into two distinct groups. One of them includes only the genus Bubalus, while the other contains the following four genera, Bibos, Bos,

1 Contribution No . 191 from the Zoological Institute, Faculty of Science, Hokkaido Imperial University, Sapporo, Japan.

Cytoiogia 13. 1944 17 248 S. MAKINO Cytologia 13

Poephagus and Bison. The relationship existing between the above taxonomical groupings and their karyotypes is also a matter of interest to be considered in this study. Before going further, the author acknowledges here his great indebt edness to Professor OGUMA who has directed the work and always given valuable criticism during the course of the study. Gratitude must be extended to Prof. YAMANE of the Taihoku Imp. University, Dr. A. KADONO and Mr. E. TATEZAWAof the Kosyun Experimental Farm, a branch of the Government Research Institute of Taiwan, by whose kind offices the mate rial of zebu and water-buffalo became accessible. The expense defrayed for collecting the material used was borne partly by the Scientific Research Expenditure of the Department of Education and a grant out of the Nippon Keiba-Kai.

Material and Method

All the accounts herein concerned deal with the chromosomes in male germ-cells observed through the course of spermatogenesis. The testes removed by means of castration, cut into pieces and preserved with fixa tive, provided the material for the present study. The fixatives adopted were in every case CHAMPY's mixture and FLEMMING's solution without glacial acetic acid, either of which were used with an excellent result. For staining the sections were subjected to HEIDENHAIN's iron-haematoxylin method. In the case of domestic cattle (Bos taurus L.) the investigation has been carired out with both the well-established breed and the primitive breed, from the standpoint of comparison, and for the former representa tive the Holstein breed and for the latter the native breed1 found in Tyosen (Korea) were selected as the material respectively. The latter form is one of the local breeds in our country and well known as a useful draft animal. The material of the zebu or humped cattle (Bos indicus L.) and the domestic water-buffalo (Bubalus buffelus L.) was obtained in the Kosyun Experimental Farm, located at the most southern part of Taiwan (Formosa), where they have been bred for years, during the author's sojourn taking place in March of 1940.

Observations 1. The accounts on domestic cattle (Bos taurus L.) Referring to the literature, there are observable six authors who are concerned with the chromosomes of domestic cattle. The chromosome

1 This material became accessible through the assistance of Mr . E. SIGEMOKO to whom thanks are due. 943 Karyotypes of Domestic Cattle, Zebu and Domestic Water-Buffalo 249

_??_umber of domestic cattle was reported for the first time by VON BARDE _??_EBEN ('92) giving 16 chromosomes as the diploid number. Then SCHOEN _??_PELD('02) gave an approximate number , 20-25 for the diploid complex_??_ nd 12 for the haploid. VAN HOOF ('13) found 20-24 chromosomes in _??_iploid and 12 in haploid. MASUI ('19) reported 33 diploid chromosomes _??_ nd observed one single unpaired X element. According to WODSEDALEK ('20), the diploid numbers were 37 in the male cell and 38 in the female _??_ell, the XO-XX condition of the sex chromosomes being present. It is _??_lean by reference to the recent studies that these discrepancies and very ow counts of the chromosome number as reported by earlier authors may _??_ave arisen from observations with material of inadequate preservation. They all are very far from exactness, and therefore not appreciable at all _??_t the present time. The studies made by KRALLTNGER('27, '28, '31) were _??_uite remarkable and valuable in establishing very clearly the characteristic norphology of the cattle chromosomes. He found 60 and 30 to be the _??_iploid and haploid number of domestic cattle respectively. He pointed _??_ut a V-shaped element of medium size as the X chromosome, because of _??_he fact that it is destitute of the homologous mate in the diploid complex, _??_ut his material proved to be entirely unfavourable to the study of the naturation division, due essentially to inadequate technique. Observations. The chromosome number of domestic cattle established _??_n this study shows a fair agreement to that reported by KRALLINGER ('27, '28. '31). That is, the diploid number observed in the spermatogonial livision was determined as 60 without exception and the haploid number _??_as invariably found to be 30 in both the primary and secondary spermato _??_yte division. There have been present no differences in the number of _??_hromosomes between the two different breeds herein examined. It can _??_e said with certainty that the above obtained number is the definite one _??_haracteristic to domestic cattle. Special attention was paid towards the morphological analysis of _??_he chromosomes in the diploid complement, since KRALLINGER's finding _??_ndicated an unpaired V-shaped chromosome which was pointed out by him _??_s the X element. Close examination made upon five clear metaphase _??_lates, as given in Figs. 1-5, reveals that the elements constituting the _??_liploid complement are all distinct rod-type of orthotelomitic1 nature, _??_ aking a typical radial arrangement with their taper ends towards the _??_entre of the equatorial plate. There is no slight evidence for the existence _??_f a V-shaped element. The finding of the present investigation, therefore, _??_s, in this point, not in coincidence with that of KRALLINGER ('27, '31) _??_ho emphasized the existence of a V-shaped element which was taken as

1 Based on the fundamental structure of the chromosome, recently OGUMA ('42) _??_dovocated a new terminology in which he named the chromosome having a terminal fibre _??_ttachment as the orthotelomitic chromosome. 17* 250 S. MAKINO Cytologia 13 the X on account of its unpaired condition. Thus the present investigation resulted in that the obtained evidence agrees with that of KRALLINGER ('27, '28, '31) in respect of the chromosome number, but it entirely dis agrees in an important morphological feature. Further discussion on this point will be made later. Since the chromosomes are all simple rod-type, they bear no distinc tive characteristics beyond those of length. The chromosomes show a slight variation in length, but there is present no clear distinction of the elements into large and small groups. Thus they form a closely graded series with no element outstanding in size. The even number of chromosomes occurred in the diploid complex suggests with all probability the occurrence of the X and Y chromosomes. The identification of the X and Y elements among the diploid group, however, was practically difficult, since they show no characteristic feature distinguishable from the others. Viewed from the evidences obtained in the primary spermatocyte division, however, the possible statement may be made that the X element is represented by one of the larger elements, probably that of the second largest, and the Y seems to be the one a little longer than the smallest autosomal elements. On account of its uncertain condition, the author hesitates to directly point out the X and Y elements in the diploid complex. The above observations are fully harmonious in results between the Holstein and the local breed of Tyosen, and there was found no visible difference between them either in the number of chromosomes or in the other morphological characters. And further, the present finding is very noticeable in offering the interesting fact that the karyotype of domestic cattle bears a close resemblance to that of goat which is likewise a member of the Bovidae in having 60 chromosomes which are all orthotelomitic (cf. MAKINO '43b). The metaphase plate of the primary spermatocyte shows well-defined 30 chromosomes of bivalent nature with indisputable clearness (Figs. 6-8). They dispose themselves in the equatorial plate at about equal distances from one anotner, so that the counting of the number can be made without any conflict. Of the 30 bivalents, 29 are the autosomal tetrads of ordinary structure having various sizes, and the remaining one is an XY complex assuming a remarkable heteromorphic configuration. The autosomal tetrads of larger kind arrange in the most peripheral zone of the equatorial plate and assume the form of a horizontal ring or its modified form. Those of the smaller size are found in the shape of a dumb-bell, a horse-shoe and some like them, distributing in the inner area of the equatorial plate surrounded by the larger ones. So far as the ap parent configuration is concerned, the autosomal tetrads of domestic cattle are much like those of goat in nature which have been described in the author's previous paper (MAKINO '43b). 1944 Karyotypes of Domestic Cattle. Zebu and Domestic Water-Buffalo 251

The XY bivalent is highly distinct in striking contrast to the rest on account of its incomparable heteromorphic nature. It is made up of an elongated rod-shaped element, the X, and an exceedingly small element, the Y, which conjugates end-to-end with the former. It occupies the

Figs. 1-12. Chromosomes of domestic cattie (Bos _??_au_??_us). •~4000. 1-5, Spermatogonial meta phases, 60 chromosomes in each: 1-3, from the local breed of Tyosen, and 4-5, from th-e Holstein breed. 6-8, primary spermatocyte metaphases, 30 tetrads in each. 9-11, Side views of the primary spermatocyte metaphase spindles, illustrating the XY complex. 12, secondary spermatocyte metaphase, 30 dyads. position always in the outer circle of the equatorial plate, lying among the large-sized members. The X displays very conspicuously a tripartite seg mentary configuration which is to be the common characteristic of the mammalian X chromosome on account of its wide occurrence in mammals, as& furnished by the studies of OGUMA '35, '37a, b and MAKINO '41, '43a,

b, c. The entire body of the X is divided by two constrictions into three 252 S. MAKINO Cytologia13

segments of nearly equal size, connecting in a linear series. The XY bivalent, therefore, exhibits a striking configuration which is composed of four distinct small bodies in a linearly connected series (Figs. 9-11). In the most cases the X shows a weak affinity for stain, acquiring a somewhat diffused configuration with vague contour, whereas the Y is stained as sharp as the autosomal tetrad. The XY complex of this animal is extremely characteristic and notice able in the mode of conjugation. The X always stands in a linear series of segments, nearly perpendicular to the equatorial plate, holding its long axis in an approximately parallel condition, to the spindle axis. The Y element, on the contrary, lies always horizontally in the equatorial plate keeping its long axis parallel to the latter, being in end-to-end connection with the X. The clear pictures for this point are obtained from the lateral aspect of the metaphase spindle, as reproduced in, Figs. 9-11. The condi tion expressed by the X shows a close approach to that occurred in Mus (MAKINO '41), rather than to that in the related hoofed animals, goat,, sheep and horse, since in the latter forms the X always lies horizontally in the equatorial plate, in similar way as in the most species of Rattus, as clearly established in the author's previous studies (MAKING '43a, b, c). As regards the Y, on the other hand, there has ever been known no com parable case in any forms of animals. Next the consideration may be extended to the spindle fibre attach ment of the XY complex. On the basis of the fact that the X always stands in a vertical condition in the metaphase equatorial plate and, on the other hand, in view of the established fact in Mus which has the X of the similar nature (cf. MAKINO '41), the most possible expectation is that the spindle fibres attach to the free end of the X opposite to where the Y comes in contact. This view may be supported further by the evidence that the free end of the X, at least in preparations a little destained, shows a deeply stained granule, the so-called polar granule, which is always found at the locus of the fibre attachment. For the case of the Y, any formerly reported evidence seems not applicable to the present instance. As above noted, the Y lies always in a horizontal position in the equatorial plate, and this condition sufficiently leads to the supposition that the spindle fibres possibly come to contact at the inner proximity of the Y where it is in connection with the X, in striking contrast to the reported cases in all of which the attachment locus lies at the free end of the Y. Though it is practically difficult to prove the actual attachment locus in the Y element, the above interpretation may be most probable, considered from the constant behaviour of the Y showing that it always lies in a position horizontal to the equatorial plate at metaphase. The above finding in the first division clearly furnished the fact that the X element is of orthotelomitic nature having terminal fibre attachment 1944 Karyotypes of Domestic Cattle, Zebu and Domestic Water-Buffalo 253 and there is offered no evidence which shows the X to be V-shaped. This observation sufficiently gives a strong confirmation to the finding in the spermatogonial cell as already described, and at the same time it is the final evidence for the denial of the view expressed by KRALLINGER ('27, '28 , '31) who maintains the occurrence of an unpaired, V-shaped X ele ment. If the X element be the V-shape as announced by him, the configura tion of the XY bivalent at the first metaphase should be much like that of the dog in which the V-shaped X chromosome was demonstrated to occur by MINOUCHI ('28). It is highly doubtless that there must be produced, as usual, two kinds of secondary spermatocytes as the result of the segregation of the X and Y in the first division. One of them carries the X element in addi tion to the autosomal elements, while the other contains the Y instead of the X, in both of them the number of chromosomes being similarly 30 (Fig. 12). The chromosomes of the secondary spermatocyte are all of the rod-type showing the remarkable dual nature. Remarks. Beginning with the work of VON BARDELEBEN appeared in 1892, the chromosomes of domestic cattle have been repeatedly studied by several authors (SCHOENFELD '02, VAN HOOF '13, MASUI '19, WODSEDALEK '20 and KRALLINGER '27 , '28, '31), and the number of chromosomes reported by these workers ranges from 16 to 60. As already noticed, the low counts of the chromosome number given by the earlier workers such as 16 by VAN BARDELEBEN '92, 20-25 by SCHOENFELD '02, 20-24 by VAN HOOF '13, 33 by MASUI '19 and 37-38 by WODSEDALEK '20, may be accounted for with all probability by the effect of faulty preservation due to classical technique they adopted. Only the communications given by KRALLINGER ('27, '28, '31) contain valuable data which are to be appreciated at present. The chromosome number reported by KRALLINGER ('27, '28, '31) for domestic cattle is essentially agreeable with that established by the author in this study, indicating 60 as diploid and 30 as haploid, but these two observations are not in coincidence in respect of the morphology of chromo somes. KRALLINGER ('27, '28, '31) maintained that the diploid complement contains 60 elements consisting of 59 rod-shaped ones and a single V-shaped element which was regarded as the X on account of its unpaired state. Contrary to the above view, the present investigation furnished no slight indication for the co-existence of the V-shaped element in the diploid group, all the 60 elements being shown to be simple rod-shape having orthotelomitic nature. How arises such a discrepancy as to the chromo somes of one and the same animal? There are to be expected two probable explanations for the possible causes of this controversy: 1) the difference of chromosomes according to the breed of domestic cattle, and 2) the error in observations made by the investigators. The first named question was settled to a great extent in the present study by confirming that the number 254 S. MAKINO Cytologia 13

and other morphological characters of chromosomes are not variable ac cording to the breed of cattle. The second problem seems to be the most probable source which yielded the discrepant results. The observation of KRALLINGER ('27, '28, '31) from which his conclusion has been drawn, was based on a single metaphase plate of the spermatogonial division, derived from the material of the ALLEN-BOUIN fixation (Fig. 5, i of KRALLINGER'31). This plate is extremely clear in most respects and is very excellent in showing distinctly the individual chromosomes. The V-shaped element under question is shown in solid-black, indicated as No. 21 in his drawing, and it is a medium-sized element having slightly unequal arms. The present author examined with a great care this drawing, by comparing it with the serial photographs of that plate given in this paper (Fig, 5, a-h of KRALLINGER'31), and was finally given the impression that the supposed long arm of the V-element is not actually an arm, but the outer end-part of the chromosome No. 22 which is a considerably long rod and lies overlapped with the former. In other words, the piece of the chromosome which has been drawn by KRALLINGER ('31) as the long arm of the V-element is only the outer end of the chromosome No. 22. Thus the chromosome No. 21 may not in reality be V-shaped. It is highly proba ble that the error of observation made by KRALLINGER ('27, '28, '31) may come from the optical illusion which is caused by the effect of the Allen Bouin fixation, because in the material treated with the latter named solution the overlapped chromosomes frequently fail to show a distinct differentiation into two elements, being tightly fused. The present observa tion established clearly 60 rod-shaped elements in the diploid complement for both of the two breeds examined, without proving the co-existence of the V-shaped X, and the result has been again confirmed in the observa tions upon the primary spermatocyte by finding that the X is characterized by a terminal fibre attachment, being represented by one of the larger elements. The certainty of this observation was highly increased by the study of the closely allied animal, the zebu (Bos indices), because the latter has been found to possess an essentially similar karyotype to that of domestic cattle, consisting of 60 orthotelomitic chromosomes, as detailed in the following section.

2. The accounts on the zebu, Bos indicus L. The zebu or humped cattle (Bos indicus L.) furnishes a new material for cytology, since the study of chromosomes of this animal has never before been undertaken by any investigator. According to the experiments attempted by Dr. KADONO, the zebu can readily cross with domestic cattle and yield completely fertile offsprings', It is therefore quite natural to

1 For this unpublished account, the author wishes to express his hearty thanks to Dr. A. KADONO of the Kosyun Exp. Farm, Taiwan. 1944 Karyotypes of Domestic Cattle, Zebu and Domestic Water-Buffalo 255

expect the same karyotype in zebu as in domestic cattle. Observations. The diploid number counted in the spermatogonial metaphase was decidedly 60, and this number is essentially the same as that established for domestic cattle (Figs. 13-14). The other general morphological characteristics of the chromosomes of this animal are also closely identical in essential points with those of domestic cattle. All the elements forming the diploid complement are likewise simple rod-type of orthotelomitic nature, and there is found no element assuming a V-shape. Especially this evidence becomes exceedingly clear and conclusive from the observation of the equatorial plate reproduced in Fig. 13, in which were established without any slight doubt 60 sharply defined elements

Figs. 13-18. Chromosomes of zebu (Bos indicus)•~4900. 13-14, spermatogonial meta phases, 60 chromosomes in each. 15-16, primary spermatocyte metaphases, 30 tetrads in each. 17-18, side views of the primary spermatocyte metaphase spindles, showing the XY complex.

provided with an orthotelomitic attachment. As occurred in domestic cattle, the chromosomes form a graded seriation in the variation of length. The X and Y chromosomes, the occurrence of which is very likely from the even number of chromosomes, were very difficult to be pointed out with certainty in this material too, because they exhibit no noticeable characters distinguishable from the autosomal elements. From this evidence obtained in the primary spermatocyte, however, it is highly probable that the X is represented by one of the larger elements, though not by the largest, and the Y seems to be slightly larger than the members of the smallest autosomal pair. Thus, the situation in this study is nearly identical with that of domestic cattle.

The haploid number was determined as 30 in the primary spermatocyte metaphase with sufficient exactness (Figs. 15-16). The morphological 256 S. MAKINO Cytologia 13

feature of the bivalents shows nothing specially noteworthy and different from that of domestic cattle. Of 30 bivalents, 29 are the autosomal tetrads taking the form of a horizontal ring, a cross, a horse-shoe, a dumb-bell and related forms, and the remaining one is the XY complex which is very remarkable and distinctive among the rest on account of its unusual form of a heteromorphic structure. The X element is characterized by a striking segmentary structure being divided into three connecting segments by the co-existence of two constrictions. It stands nearly vertical to the metaphase equatorial plate in the form of a linear series of segments, carry ing at its inner proximity the Y element which always lies horizontally holding its long axis parallel to the equatorial plate (Figs. 17-18). Thus the structural condition displayed by the XY bivalent of this form seems not to show any noticeable difference from that recorded for domestic cattle in the former section. Though not actually observed in this study, it is very likely that the X and Y segregate to opposite poles in the first division, so that half of the secondary spermatocytes carry the X and half the Y. Remarks. The result of the present observations is evident in showing that there is present no significant difference in the chromosomes between domestic cattle and zebu, not only in the number but also in their form and other general morphological features. Likewise with the case of domestic cattle, the diploid group of zebu consists of 60 elements which are all simple rod-shape and there is no slight indication for the presence of the V-shaped element. The obtained fact may serve to a great extent as a disproof against the view of KRALLINGER ('31) who maintained the existence of an unpaired V-shaped element in the complement of domestic cattle. The result of KRALLINGER may possibly be explicable as an error of observation, as fully discussed in the previous section. As mentioned above, the karyological phenomena established in this study offer no visible difference between domestic cattle and zebu. On the other hand, attempted crosses carried out between these two animals made by Dr. KADONO in the Kosyun Exp. Farm have proved to be fully successful with fertile offsprings in every test. These evidences estab lished from two sides of investigations are highly suggestive of the close kinship of domestic cattle and zebu, not only in their systematic relation but also in their genie constitution. On the other hand, however, YAMANE and KATO ('36) stated that Holstein cattle can be clearly separated from the zebu by the morphological difference of thoracic vertebrae.

3. The accounts on domestic water-buffalo, Bubalus buffelus L. The study of chromosomes in water-buffalo has been undertaken by PKAKADZE ('39). He worked with Bubalus bubalus L.1 and reported the

1 Synonym of B . buffelus L. 1944 Karyotypes of Domestic Cattle, Zebu and Domestic Water-Buffalo 257

diploid number of 56. This result is markedly disharmonious with that obtained in this study carried out with Bubalus buffelus L .1 The cause of this difference in result cannot be fully cleared up in this study, but the situation may find the most probable explanation under the assumption that the numerical difference reported in these two studies is the result of differences in the interpretation of V-shaped chromosomes, as detailed below. Observations. A glance at the spermatogonial metaphase of this animal gives the impression that the diploid garniture is more motley in morphology of the chromosomes than those encountered in the previous two forms, and the number of elements is rather low. Careful examination was successful in establishing that the diploid number of chromosomes found in the spermatogonial metaphase was invariably 48 (Figs. 19-20). This number is rather low as compared with those found in domestic cattle and zebu. The diploid complex of this animal is highly characterized and shows a striking contrast to those of domestic cattle and zebu, by the presence of syntelomitic2 V-shaped chromosomes. Therefore, the diploid elements can be morphologically classified into two groups with regard to their form, the syntelomitic group of V-shaped chromosomes and the orthotelomitic group of rod-shaped elements. Morphological analysis of the chromosomes revealed that the diploid complement consists of eight syntelomitic elements and 40 rod-shaped ones of orthotelomitic nature. The syntelomitic chromosomes are clearly distinguishable from one another by their apparent shape and comparative magnitude, and they are found to form four pairs of homologous elements. The largest pair (a-pair in Figs. 19-20) of this group is provided with the chromosomes of submesomitic attachment, the lengths of arms forming V's being dis similar. The elements of the second pair (b-pair) are to be considered as the subtelomitic chromosome, since their arms are exceedingly unequal in length. The members of the third pair (c-pair) are of apparently submesomitic nature. The fourth pair (d-pair) consists of chromosomes a little smaller than the others and have a submesomitic fibre attachment. The orthotelomitic chromosomes, 40 in number, are sorted, on the other hand, by comparison of their shape and size, into 19 homologous pairs, gradually decreasing in size, and an unequal pair consisting of an elongated heavy rod and a small rod-element, both being of an orthotelomitic nature. It is evidently the latter unequal elements that constitute the XY pair, the is evidently the latter unequal elements that constitute the XY pair, the out is very prominent in being the largest among the orthotelomitic ele-

1 This name was used in the study of YAMANE ('40). In the new terminology presented by OGUMA ('42), the term "atelomitic " was altered into" syntelomitic," and at the same time "mesomitic", "submesomitic" and "subtelomitic" were applied for "median", "submedian" and "subterminal" respectively. 258 S. MAKINO Cytologia 13

ments and is characterized by its heavy and rather straight appearance,

quite distinguishable from the rest (ƒÔ in Figs. 19-20), while the Y is of small rod-type being a little larger in size than the members forming

the smallest pair. It is therefore rather difficult to point out the Y with

certainty in the diploid garniture, because of the absence of distinguishable

Figs. 19-30. Chromosomes of domestic water-buffalo (Bubalus buffelus) . •~4000. 19-20 spermatogonial metaphases, 48 chromosomes in each (Y chromosome not identified). 21 - 25, primary spermatocyte metaphases, 24 tetrads in each. 26-29, side views of the primary spermatocyte metaphase_??_spindles, demonstrating the XY complex. 30, anaphase of the firs division, indicating the segregation of the X and Y elements.

features. Then the above descriptions are to be summarized as that the

male diploid complement of domestic water-buffalo contains 48 chromo

somes which consist of four homologous pairs of syntelomitic elements,

19 homologous pairs of orthotelomitic ones ranging serially from long rods

to short ones, and an unequal pair made up of the X and Y elements, both

of them being orthotelomitic. 1944 Karyotypes of Domestic Cattle, Zebu and Domestic Water-Buffalo 259

The diploid number obtained in the spermatogonial division was ascertained beyond question by the observation of the primary spermato

cyte in which the haploid number of 24 was established with indisputable clearness (Figs. 21-25). The haploid complement consists of 23 autosomal

tetrads varying in size and shape, and an XY complex of a heteromorphic

structure, made up of unequal components. Of the autosomal tetrads, four are characterized by their syntelomitic nature showing submesomitic and

subtelomitic fibre attachment, and constantly appear in the form of a

compound structure, lying always in the periphery of the equatorial plate. The largest one of them is specially prominent in the complex on account

of its remarkable shape and size. It is self-evident that these four are

the descendants derived from four homologous pairs of syntelomitic chromosomes existing in the diploid complex. The other 19 tetrads take

the form characteristic of the orthotelomitic bivalent and appear in the

shape of a horizontal ring, a thick V, a cross and their derivatives. The XY complex assumes a striking heteromorphic form quite dis

tinctive from the rest. It is composed of an elongated heavy rod, the X element, and the small rod, the Y, which is connected end-to-end with the

former. As is obvious from the spindle in side view (Figs. 26-29), the

X element constantly stands vertical to the equatorial plate with its long

axis holding in the position parallel to the spindle axis, whereas the Y

element lies horizontally in the equatorial plate coming in contact with

the X as to make a right angle with the latter. The X displays a remark able segmentary structure being divided into three parts in a linearly

connected series (ƒÔy in Figs. 27-29). The XY complex of this stage,

therefore, takes the configuration composed of four small bodies in a

connecting series. So far as the XY complex is concerned, the situation as thus mentioned is much like those occurring in domestic cattle as well

as in zebu.

The segregation of the X and Y takes place in the first division as

shown in Fig. 30, and this observation is sufficient to allow the supposition

that half of the secondary spermatocytes carry the X and half of them

the Y.

Remarks. The fact that domestic water-buffalo (Bubalus buffelus) is a member of the Bovinae together with domestic cattle (Bos taurus)

and zebu (Bos indices), naturally leads to the expectation that similar

karyotypes would be found among them. Contrary to expectation, how ever, the present observations showed that domestic water-buffalo mani

fests marked differences in its chromosome morphology as compared with

domestic cattle and zebu, both of which were found to have an identical

karyotype. not only in their number but also in their structural configura 1 Similar relationship was also reported by Sasaki ('34) by means of the serological study. 260 S. MAKINO Cytologia 13

tion. As detailed in the former descriptions, both the domestic cattle and zebu possess the diploid number of 60, all being of orthotelomitic rod-type, whereas domestic water-buffalo shows 48 chromosomes in diploid, which consist of eight syntelomitic V-shaped elements and 40 rod-shaped ones of orthotelomitic nature. The morphological comparison of chromo somes undertaken among these animals reveals that the numerical and morphological differences found between them are too great to be accounted for on the basis of the fragmentation of a chromosome into two, or the formation of V-shaped multiples due to the union of rod-shaped elements two by two. In connection with the above fact, mention made by WEBER ('28) concerning the systematic arrangement of the bovine genera is of great interest in showing that the Bovinae are divided into two definitely separated groups, one of which is represented by only a single genus, Bubalus, whereas the other group embraces the following four genera, Bibos, Bos Poephagus and Bison. The study of ZUITIN ('35) regarding the chromosomes of the yak (Poephagus grunniens L.), seems to show, though not fruitfully worked out, the occurrence of similar karyotypes in the yak and cattle. The cytological evidences above presented seem to be in favour of the systematic relation of the Bovinae as shown by WEBER ('28). The knowledge of the chromosomes of the Bovinae is still too scanty at present, to admit further consideration relative to phylo genetical relationship of chromosomes. Prior to the present investigation, PKAKADZE ('39) briefly reported the chromosomes of buffalo. According to his descriptions, the diploid chromosome number established for this animal was 56. As compared with the result obtained in this study which shows the diploid number of 48, the numerical difference of chromosomes found between these two studies is very large. The present investigation has succeeded in conclu sively establishing that the 48 diploid chromosomes consist of eight V-shaped elements and 40 rod-shaped ones. All the probable cases being considered, the most possible interpretation for this numerical difference is that the number reported by PKAKADZE ('39) is the result of misconcep tion of the V-shaped chromosomes for rods. When the eight V-shaped chromosomes are considered to be 16 rod-shaped ones, the chromosome number, 48, is found to be 56, the number reported by PKAKADZE ('39). A quite similar error has been made in the chromosome counting of sheep by several previous workers, such as KRALLINGER'31, BUTARIN '35, NIVIKOV '35 , BRUCE '35, including PKAKADZE himself ('36); they reported 60 chromosomes of rod-shape as the diploid number of sheep, but the recent studies by BERRY ('41) and the author (MAKING '43b) evinced that the above result is entirely erroneous and that this animal possesses in reality 54 chromosomes which comprise six V-shaped elements and 48 rod-shaped ones. The number 60 reported by earlier authors is the result of the 1944 Karyotypes of Domestic Cattle, Zebu and Domestic Water-Buffalo 261

interpretation of the six V-elements as 12 rods (for details, see BERRY '41 and MAKINO '43b).

Notes on the chromosomes of bovine animals

The chromosome study in the Bovidae has been extended to five genera including some eight species (cf. OGUMA and MAKINO '37, MAKINO '43b). The established facts in the studied species are briefly summarized in the following table.

Those marked with asterisks cannot be considered as valid and reinvestigation is required. For details, see Makino ('43b). r denotes the rod-shaped (orthotelomitic) element, and V the V-shaded (syntelomitic) element. The author's previous study (MAKING '43b) has succeeded in estab lishing that the numerical difference of chromosomes existing between goat (Capra hircus) and sheep (Ovis cries) is sufficiently explicable on the basis of chromosome-linkage due to the union of rod-elements two by two into V-shaped multiples, and on this basis it has become evident that there is a closely related karyological relationship between goat and sheep (for details, refer to MAKINO '43b). The karyotype of goat is, in its morphologi cal constitution, much like that of domestic cattle (Bos taurus), because all the formative elements are of simple rod-type in either of them. A remarkable and most noticeable difference between them, however, was found to lie in the morphology of the sex chromosomes. In goat the X is represented by the largest element of all and the Y is very minute in size being the smallest in the complex. The X of domestic cattle, on the contrary, is not the largest in size but probably the second largest or a little shorter, and the Y is slightly larger than the smallest elements. In addition to this, the mode of conjugation between the X and Y is very characteristic and peculiar in domestic cattle (for details, see the former descriptions). The present investigation pointed out the similarity of the karyotype between domestic cattle and zebu. In other words, the chromosomes of domestic cattle show no significant difference from those of zebu, not only 262 S. MAKINO Cytologia 13

in their number but also in their form and other morphological characters.

But the expectation is possible that closer examinations would succeed to

find out the difference of specific characteristics of the chromosomes be

tween them by an intimate study of the individual elements by way of comparison, as succeeded by the author in the cases of Mus and Rattus

(MAKINO '41, '43c). Regrettably, the present study has not been supplied with sufficient material to admit such a precise comparative investigation. The study of YAMANE and KATO ('36) indicated that there is a clear

demarkation between domestic cattle and zebu in regard to the morpholo gical feature of the thoracic vertebrae. On the other hand, crossing experi ments attempted between domestic cattle and zebu proved to yield fertile offsprings in a quite normal manner. This evidence, being coupled with the fact of the karyological harmony, is highly suggestive of that these two animals under consideration are nearly allied in their genic constitu tion. A parallel condition has been known to occur in the following crosses, Ovis pollii karelini•~Ov. steato pyga (BUTARIN '35), Camelus bactrianus•~C, dromedarius (PKAKADZE '32) and Mus musculus•~M. molos sinus (MAKING '41).

The karyotype of domestic water-buffalo (Bubalus buffelus) is proved in this study to be greatly dissimilar from that of domestic cattle, as well as from that of zebu. It is remarkable that the chromosome number of domestice water-buffalo is the lowest in the Bovidae, so far as the observa tions go. The numerical and morphological difference found between them are too great to be interpreted on the basis of the fragmentation of a chromosome into two, or the production of V-shaped multiples due to the union of rod-elements two by two. In connection with this cytological evidence the taxonomical arrangement of the genus in the subfamily

Bovinae is very interesting. According to WEBER ('28), the genus Bubalus, being well separated from the other genera, occupies an eccentric position in the taxonomical arrangement of the Bovinae. That is, the Bovinae are divided into two definite groups, one of which is represented by a single genus, Bubalus, and the other includes the remaining four genera, namely Bibos, Bos, Poephagus and Bison.

The karyological study of the yak (Poephagus grunniens) has not been fruitfully worked out by ZUITIN ('35), but from his study the expecta tion is possible that there occurs a closely related karyotype in the yak and domestic cattle. It has been known that the cross between the yak and domestic cattle gives rise to hybrids consisting of fertile females and sterile males in the first generation (ZUITIN '35).

Summary

The karyotypes of domestic cattle (Bos taurus), zebu (Bos indices) and domestic water-buffalo (Bubalus buffelus) were studied in male germ 1944 Karyotypes of Domestic Cattle, Zebu and Domestic Water-Buffalo 263

cells during the course of spermatogenesis. The number of chromosomes in domestic cattle was found to be 60 in diploid and 30 in haploid. All the chromosomes are characterized by a simple rod-type of orthotelomitic nature and there is no evidence for the presence of the V-shaped element. The chromosomes of zebu show no visible difference from those of domestic cattle either in the number of chromosomes or in their form and other general morphological features. Likewise to domestic cattle the diploid complex consists of 60 rod-shaped elements and the haploid number is 30. The karyological feature of domestic cattle is thus identical with that of zebu. The karyotype of domestic water-buffalo was found to be markedly dissimilar to that of either domestic cattle or zebu, not only in the number of chromosomes but also in their structural configuration. In striking contrast to the former two forms, this animal exhibits the diploid number of 48 and the haploid number of 24. The complement is characterized in having 8 syntelomitic V-shaped elements and 40 rod-shaped ones of orthotelomitic nature. The comparison of the karyotype revealed that the numerical and morphological differences of chromosomes existing between them are too great to be accounted for on the basis of chromosome-fragmentation or of chromo some-linkage. Throughout the species under study there are constantly present the sex chromosomes of an usual XY-type. All the species are identical as regards the morphological nature of the X and Y elements. Morphologic ally the X as well as the Y is evidently of orthotelomitic nature. They conjugate in meiosis as usual and segregate to opposite poles in the first division. The X is remarkable in assuming the characteristic tripartite structure being divided into three connecting segments with nearly equal size showing two distinct constrictions. The mode of conjugation between the X and Y is very peculiar and characteristic in the three bovine animals herein concerned.

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