qancy Wang *, Robert N. Shoffner, Jack S. £

Department of Genetics and Cell Biology and Animal Sciences, Unive

(Revision received 16 February 19821 (Accepted 19 February 1982)

Ehromosomal rearrangements, such as unequal trm ersions, which can be readily identified at mitotic agenetic markers for the identification of parentage, relations. Furthermore, the concurrent use of chrom~ regating traits is a powerful tool for gene localizatiol tblishment of linkage relationships. Fhe alkylating reagents triethylene melamine (TEM) dS) have been used for the induction of chromoson tttanach, 1957, 1959; Cattanach et al., 1968). The 1 uction of chromosomal breakage and rearrangement ~n suggested that a high degree of alkylation may cau accur proximate enough in the twin strand of DNA in (Freese, 1961). In male mice, the germ cell stages

C l l l O m t ) ~ . O l l l i i l r ¢ i i r r a i l ~ t f l i l ¢ i i t ~ ; i L r l U

localization in eukaryotes and for the

and ethyl methanesulfonate >real rearrangements in mice

mechanism involved in the is still uncertain, but it has

cause the sites of depurination to result in scission of the

which are most sensitive to >minant lethals and translocations are the epididymal rmatids for EMS (Cattanach et ai., 1968; Fahmy and ermatids for TEM (Cattanach, 1957, 1959; Cattanach et ny, 1954).

and TEM were used as clastogenic agents to obtain ',nts in the spermatozoa of male chickens. Experiments he: (1) the effect of various dose levels of TEM and EMS y of progeny from treated males, (2) the male germ cell e mutagens, and (3) the recovery and identification of

of Laboratory Medicine and Pathology, Box 198 Mayo Memorial ota, Minneapolis, MN 55455 (U.S.A.).

nesulfonate; TEM, triethylenemelamine.

5 © Elsevier Biomedical Press

- - v - -

establishment Th~

(EMS) (Cattanach ind been to occur chaJ the induction of both dominant spermatozoa and late spermatids Fahmy, 1957) and mid spermatid al., 1968; Fahmy and Fahm~

In present study EMS chromosomal rearrangements were conducted to determine: on the embryonic mortalit' stage most sensitive to the

* Present address: Department Building, University of Minnesota

Abbreviations: EMS, ethyl methanesulfon~

0027-5107/82/0000-0000/$02.75

)mosomal structural chin ting agents: triethylene n rl methanesulfonate

Otis and Cheng

University of Min 55108 (U.S.A.)

(Received 18 August 1981) ¢ 1982)

translocatiol mitotic metaph

parentage, cell line chromosomal r~

tids

role and

M.

I, MN

centric cellent ds and hts and

lte

4emorial

trol line (RRc) were selected for treatment. A group a semen from these males prior to the mutagenic trea ility level. I'hree males were injected intraperitoneally with 400, i i, respectively. Another set of three RRc males we1

0.5, 0.8 and 1.2 mg of TEM per bird, respectivel) ~r to injection in an effort to eliminate the untreate ected daily from each treated rooster and two virgin for 19 days. Eggs were collected daily for each of

abated for a fertility diagnosis. It should be noted th~ day following a single insemination. Embryonic deve nination of a 5-day incubation period. Embryos wer se which died before the gastrula-blastula stages; d 2, trly identifiable extra embryonic membranes; and d 3 e a definite blood circulation or which died at an e 3ryos, NE, were those which were macroscopically m d2, d 3 and NE was calculated for each day's eggs, rel: ;ruination after the original injection.

series of three separate mutagenic treatments were a m n ~ n m n l r a a r r a n a e m P n t Q l~a~ecl n n t h ~ ra~n l t~ n f t h

2, those which had developec 3, those advanced enough tc even later stage, The norma normal. The percentage of F

representing successive days ol

were made for the induction ot ats. Based on the results of the initial dosage level trial at • mg injection of EMS per male and a 1.2-mg injection o! o be satisfactory. These dosage levels were used for the t carriers in three separate experiments.

males were injected i.p. with 400 mg of EMS and 16 RR~ 'e injected i.p. with 1.2 mg of TEM. Following injectior md placed in small floor mating pens for natural matin~ line females. The Minnesota Marker lines are population.' Lt traits used in linkage studies. The RRc line is a multipk xticular traits. The same type of mutagenic treatment wa, rker males for reciprocal matings to RRc females. Egg:

hen for following the mutagenic treatment

~l~tl~l'

have embr~

d l , insemm;

Mutagenic treatments A

chromosomal rearrangements. the embryonic level, a 400-m TEM per male appeared to induction of rearrangement

A. Trial A 16 RRc sexually mature

sexually mature males were each male was ejaculated and with 4 Minnesota Marker line segregating for 18 dominant recessive for all of these particular applied to Minnesota Marker were collected from each

ngements by breeding tests and c

he embryonic level esigned to determine the effect of dosage level, and the sensitive body weights close to 3 kg from

of female treatment to

600 and were injecte

pectively. All ma ated sperm,'

hens wer the suco

that fertility ,onic development

were classifi~

18 days

karyo-

iMS on ages. 6 mated

ainated control

MS per oneally culated en was ed each tys and out the

at the F; d 1,

aed to

normal F, of

of at of

the

RRc ection

g )ns )le

2nt was

~gs ',nt.

J

• e used in natural matings; the remaining two sets ;cted marker males were used for artificial insemim h RRc or marker male was mated with 2 marker or ,'refore, on each particular day, there as a maximurr h mutagen type. Eggs were collected and dated for 15 the mutagens. For artificial insemination, semen wa I marker male and used to inseminate 1 marker and 12 consecutive days. Only one insemination was me

alts obtained from each hen would represent successi~ ,e collected from each hen for 10 days and incubated. was determined by candling on the 7th day of incul~ Lg banded, checked for chromosomal constitution, an,

C. Trial C I'he same procedure was followed except that mating.,

Of a total of 296 chicks hatched from Trial A, 200 we maturity. Genetic tests were made initially because c ~ening test, would not detect reciprocal, equal trans] t ~ r " n r n ~ r ~ e a n t ~ r , i n v a r e i ~ n c e m ~ | l d a l ~ t ; ~ n e n r

gs were limited to the first 10

200 were available for genetic tests cytological observation, as a

transloeations, symmetrical peri- versions, small deletions or duplications. In addition, the many pairs of microchromosomes would be almost

f rearrangements were mated to the RRc multiple reces- rgous carrier was expected to exhibit high embryonic Lbility. To secure reliable estimates of early mortality, leeted from each female suspect and 40 eggs from the

by high embryonie mortality in Trial A, and all the chicks td C, were then karyotyped by the feather pulp method

determine the presence of possible rearrangements, ~eations or inversions.

The days following the injection.

Recovery of rearrangements Of a

at maturit s c r e e n l n

centnc or paracentnc rover! rearrangements involving impossible to detect cytologically.

The potential carriers of sive stock. Each heterozyg, mortality and low hatchabilio approx. 20 eggs were collec~ females mated to a male suspect.

The suspects, identified recovered from Trial B and (Shoffner et al., 1967) to specifically unequal translocations

," set weekly, candled at day 7 fo os prior to blood ring formation) y 7). The remaining live embryos ed to maturity. Embryos which dk

• ker males were injected with 400 r ted with 1.2 mg of TEM. Of the ted with EMS and a similar set i~

of 2 inj insemination. Fo

2 RRc fel maximum of 12 el

days folh was collecte

1 RRc fe made to eaq

successive days aJ The perc~

incubation. H~ and reared

tion of nbryos tted to 7 were

and an et of 3 TEM, and 2

Latings, ctively. d from jection h RRc ctively, aat the a. Eggs d 1 and :s were

10

tests a

) e r l -

almost

reces- tonic

:y,

the

nethod ;ments,

< 0 , -

Z ©

C Z <

e ~

f ' l

©

,-..l

<

l rehydrated by passage through 70% ethanol and disl ned with carbol fuschin for 3-5 min and then rinsed :e in absolute alcohol and air-dried. Slides were mol t checked under the microscope for chromosomal con

;uits and discussion

re level comparison ~¢ithin the dose ranges tested, both EMS and TEl onic mortality in the dl stage rather than d 2 or d: centrations were increased, the early embryonic death ly increased. These results indicate that d~ mortali mical mutagenic effect.

plot of d~ versus day of insemination following inje effect of the chemicals on the differentiation and m,"

rn cells. For both EMS and TEM, the most sensitive s ~tion with extension of effectiveness to day 9 (Fil gests that d~ mortality reflects a drastic chromosot d disorganization of development, and that the d 2 ax drastic effects on the chromosomes, allowing furthe~

mortality is an indication of the

ection of the mutagen shows maturation stages of the male

stages are 1-7 days after the gs. 1 and 2). This pattern

)mal disruption with almost and d 3 mortality results from

further development before termi-

experiments, a dose level was needed to allow for the Since the dosage of 600 and 800 mg of EMS produced

he first 5 days following injection, the lower dosage of 400 ~r "the less toxic TEM the 1.2-mg dosage was chosen.

al matings for 18 days following the i.p. injection of EMS tbles 3 and 4. The most drastic effect on the male germ ~reater than 60%, occurs 3-10 days after the injection. re hatched, all of which appeared phenotypically normal. ty (6-7 months) and 153 surviving females were mated to

c n e m l c a l

A the germ injection sugg~ total less nal disorganization.

For the hatching level survival of hatched chicks. almost total mortality for the mg/bi rd was indicated. For

Effect of mutagens

Tria A The results of the natural

plus TEM are given in Tables cells, as indicated by dj 's

A total of 296 chicks were They were raised to matudt,

were obtained from the feather l agements. To accumulate metapha injected intraperitoneally at a dos

mbation, several developing pinfea the feather's proximal end was

(w/v) solution of sodium citrate was fixed in a 50% solution (v/~ prior to squash preparation. Aftc

verslip, slides were then placed ii distilled wat

once in q mounted pel composition

TEM drasti~ 3 (Tables

death at the d~

pected 0.05% c~/kg lucked ad im- rain of

acetic ring to ',thanol • ,s were er and 1 DPX

se em- As the spond-

male the

almost from

terns-

the

400

S .~rm

<

I

0

Z ©

Z <

V-

Z 0

0

<

I

IO 20 Days After Treatment

I. The effect of ethyl methanesulfonate (EMS) on the d I embryonk l daily insemination following i.p. injection of the mutagen in the r mg, A A; 800 mg, O

2. The effect of triethylene melamine (TEM) on the d I embryonic i daily inseminations following i.p. injection of the mutagen into tht rig, ZX A; 1.2 mg, O

C multiple recessive males to determine incidence of e ication of chromosomal rearrangements. Only 47 m les from 1 to 10 days following injection and random -18-day survivors were mated to RRc females. "[ rtality and consequent low hatchability for 7 males • e examined cytologically, 7 individuals appeared c y have had aberrations undetectahle at mitotic me

early embryo mortality as an males could be tested, so all

random individuals taken from the There was high embryonic

and 5 females. When they chromosomally normal, but

metaphase. 4 from the TEM B20756, B24503 and B23112, and 1 from the EMS

were found to be carders of at least one chromosomal d Fig. 3). In bird B24939, a distal chromosome segement mgth of the short (p) arm of chromosome No. 1 was ttified microchromosome distal to its centromere. Bird :arrier of two unequal translocations: the first involved an

(q) arm of chromosome No. 2 and a microchromosome tromosome No. 2 proximal to the centromere, and the )me No. 3 and a microchromosome. The translocated Io. 3 is very small; therefore, the change in chromosome at late metaphase. Bird B23112 carried a translocation

,'xcept that one-third or less of the distal end of the p arm involved in the rearrangement. B24503 carried a translo- les Nos. 1 and 4 in which a segment containing two dark

RRc indical males 11-18- mortalit' were may treatment group, B24939, treatment group, B25824, rearrangement (Table 5 and equal to two-thirds the len translocated to an unidentified B20756 was found to be a carrier c exchange between the long with the breakpoint of chromoso second involved chromosome segment of chromosome No length is indistinguishable similar to that of B24939, exce of chromosome No. 1 was cation between chromosomes

10o-

°! ~s0

Lfl

#

0 Days Aft~

onic mortality i in the male parent:

O).

mortality i m into the male pareJ

©).

~r of two

osome

,A\ -O-20

20 t

resulting e;

resulting 0;

the

,,y

osomal mt

)me

d2

(% )

13

24

11

d3

(% )

4 5

4 p

ip (

%)

0 0

0 H

(O (

%)

83

37

18

50

60

54

37

68

85

63

33

7 10

7

0 0

0 5

0 7

23

52

d 3

(%)

4 3

5 pi

p (%

) 0

3 0

H(f

) (%

) 73

47

18

TA

BL

E 4

LE

VE

LS

OF

FE

RT

ILIT

Y,

EM

BR

YO

MO

RT

AL

ITY

AN

D H

~ T

EM

(H

atch

-Tri

al A

)

Day

1

2 3

Set

58

56

70

F

(%)

40

68

d I

(%)

0 34

BR

YO

MO

RT

AL

1T

Y A

ND

HA

TC

HA

BI]

4 5

6 7

8 9

67

63

75

73

81

67

37

40

37

49

56

45

84

72

79

83

96

63

4 12

14

6

2 7

0 0

0 0

0 0

0 0

4 0

0 0

12

16

4 11

2

30

AT

CH

AB

II

4 5

6 7

8 9

66

63

71

68

68

7~

50

52

57

49

57

74

91

89

97

90

9 6

6 0

5 6

0 0

0 0

0 0

0 0

0 11

3

6 3

5

)NS

EC

UT

IVE

DA

YS

AF

TE

R]

12

13

14

15

77

82

70

76

70

67

89

90

7 9

5 11

6

6 0

0 0

6 3

3 0

0 0

0 87

80

92

87

NS

EC

UT

1V

ED

AY

S A

FT

ER

I

1 12

13

14

15

5 68

75

67

41

61

65

65

70

24

18

12

23

4

5 6

4 7

4 7

6 2

0 0

0 0

67

71

76

70

I T

EM

EM

S

II

TE

M

EM

S

III

TE

M

EM

S

a N

.M.,

Nat

ural

mat

ing;

A.I

., ar

tifi

cial

ins

emin

atio

n.

~OG

ICA

L R

ES

UL

TS

F

OR

TH

E

IND

U(

Win

g ba

nd

Sex

Cyt

og(

desc

ri 1

B24

939

M

t(I;

m)

B20

756

M

t(2;

m)

t(3;

m)

B23

112

F t(

1 ;m

) B

2450

3 F

t(I ;

4X

B25

824

M

t(Z

;3)~

F102

28

F t(

3;m

) F7

945

F in

v(l)

~

F131

36

F t(

2;m

) F

3572

M

t(

4;m

) F1

0845

F

t(Z

; 1~

HR

OM

OS

OM

AL

R

EA

RR

AN

Typ

e an

d ti

me

of m

atin

g (d

ays

afte

r tr

eatm

ent)

a

10 (

N.M

.)

11 (

N,M

.)

4 (N

.M.)

16

(N

.M.)

10

(N

.M.)

8 (N

.M.)

6

(A.I

.)

5 (N

.M.)

8

(A.I

.)

10 (

N.M

.)

f g w w 11 w

I

' | [ I I , i **

" I ; i.: ," " ' : ' / ; l t= Oa &L Ii

811 * . ,

It,= . 0

; , e ~t A- . .~

| i i i l I . * * mosomal rearrangements induced by the alkylating reagents in thre ,) B20756; (C) B23112; (D) B24503; (E) B25824; (F) F10028; (G) F794- ~ ]45. See Table 5 and text for the description of each specific rearrang~

(H) A

!i Fig. 3. Karyotypes of the chromosc separate hatches: (A) B24939; (B) (H) F13136; (I) F3572; (J) F10845. ment.

3 4 5

II I I | t

I I , i es

I *

X * .

=|,

hl'ee F7945;

ge-

ds was translocated from the distal end of chromoso~ ~1o. 4q (Wang and Shoffner, 1974). The breakpoint ximal to the telomere. B25824 carried a translocation [ the Z sex chromosome; the breakpoint of chromos( tromere and the breakpoint of the Z chromosome was garm. ['he carriers were genetically retested and these data ( :hability of the translocation heterozygotes is, in gene~ Je. For example, while the expected hatchability of a ( 5%, the actual value for B20756 was 10.3%. The hatc k heterozygotes ranged from 22.4 to 44.0%. The expe4 ~enetically balanced embryos survived, however, the 'r r~ 5 to 10%. Thus some rearrangements were in the siderably less. The explanations for the lower than

other equal chromosomal rearrangements in lslocations may have been induced; (2) the unequal re I high incidence of nondisjunction during meiosis wll otes; (3) there may have been more adjacent than a

hatchability of single transloca- )ected hatch would be 50% if 'normal' abortion loss ranges

'expected' range and other expected hatchability rates addition to the identified

rearrangements may have led which then produced inviable

alternate segregations in the :hromosome rearrangements, thus producing more dupli- ~tes, resulting in inviable zygotes; and (4) the viability of erozygotes resulting from alternate chromosome segrega- t of normal diploids.

mbryonic mortality and hatchability for Trials B and C, Lcial inseminations, are similar to that of Trial A. The 'ere higher in natural matings than in artificial insemina- insemination was used, the d I increased immediately after Fig. 4) but when natural matings were employed, the d 1 the third day after injection Fig. 5).

of the mutagenic effect may have been caused by the use ;xperiment. When non-virgin hens are artificially insemi-

lion all genetically from considerabl are: (1) translocations ma' to a zygotes; heterozygotes of unequal chromos cation and deficiency gametes, balanced translocation heter tion might be less than that

Trials B and C The levels of fertility, embr)

natural matings and artificial fertility and hatchability were tions. Also, when artificial insemin the mutagenic treatment (Fi increase was not seen until

The delayed expression of non-virgin hens in the ex

OF THE MALE AND FEMALE CA: ',ECOVERED FROM HATCH I

F (%) d 1 (~) d 2 +d 3 (9[

63.6 59.5 14.3 68.4 82.3 2.6 81.7 59.2 16.3 70.6 62.5 0 86.2 48.0 8.0

chromosome No. 1, of chron

anslocation between, chromosome No. i

close to 1

(Table 6) eneral, lower

double tr~

aosome

nination wa~

CHRO-

h/F (%)

:al end 4 was

,~No. 3 to the of the

aat the pected carrier

3 0 1 . . . . . .

2 0 t m m m m m m m m m m m m m m m

1-2 3 - 4

DAYS A F T E R

4. Effect of TEM and EMS on the d I embryonic mortality in mination following the injections of the mutagens in the male par,

ed, the inseminated spermatozoa will fertilize 75-1 ardless of the time of the previous mating. However, x ted with EMS- or TEM-treated males there may be a r 2 days following injection because of a toxic effec e, spermatozoa already present in the hen will be us~

when such hens are naturalb a delayed mating activity fo

effect on the drugs. During thi used for fertilization.

I • 1 3 - 1 5

AFTER TRtATM~NT.TEM&~MS

EMS on the d 1 embryonic mortality in the progeny from the natur~ )ns of the mutagens inthe male parents (Trial B).

mated 1 or t ime

1 0 0 ,

90 , >- .~ 80,

70.

Z 5 0 .

~ 40u

30= a m l

o~ 2 0 .

I 0 .

0 !1_2 3.l_4

DAYS

Fig. 5. The effect of TEM and matings following the i.p. injections of the

TEM -'~*.,,,, ........ ~ F.MS ,-"

~ CONTROL mini

i l m m mm mm m mm mm mm mm i

5 -6 7-8 940 T R E A T M E N T • T E M & E M S

the proge] parents (Trial

75-100% of 1

TEM EMS " ' " ' " " ¢ O N T R O L m l e a '

s'6 7"8 ,~o 11-12

the mut~

om daily

ve eggs

Y for h i s

natural

ction, involved chromosome No. 4 and a microchror I F10845, from a natural mating 10 days post-EMS tr~ ',hromosome No. 1 and the p arm of the Z sex chrom~ :our separate experiments have described the muta S at both the embryonic (Tables 1 and 2) and ha ging from the dominant lethals or early embryonic rn cluded that the male germ cell stages which are dating agents applied occur 1-10 days before the mat ~ever, judging from the chromosomal rearrangement ,1, it can be concluded that the male germ cell stages the clastogenic effect of either TEM or EMS oc~ nination of the maturation process. The high embryo tagens 1-4 days following the injection is due to tt aced by the chemicals. )atrick et al. (1965), Donovan et al. (1969) and Wang t the male germ cell stages occurring 1-10 days be otic DNA replication period. The specific sensitivit tale germ cells to TEM and EMS has been repor ttanach, 1959; Cattanach et al., 1968; Generoso, 1! 9). Luippold et al. (1978) and Generoso et al. (19" ;togenic effect of TEM is highly dependent upon an I ; , ' . a t ; n n T r , ,~V.l~l~;.in t h , ~ r d , ~ e t , ' ~ , ~ , ~ n ; , - . , ~ e ¢ , ~ o t A i r "I'~17kA ; ~

- - r

the severe clastogenic effect

Lg et al. (1980) have reported before maturation follow the

sensitivity of postmeiotic male and ;ported by other investigators

1969; Generoso and russell, (1977) have reported that the

intervening round of DNA clastogenic effect of TEM in the postmeiotic germ cell

that the clastogenic effect observed at the zygotic level is • oduced in the chromatin of the germ cells at the time of lted through pronuclear chromosomal replication in the he postfertilized zygote.

ds. Beth L. Trend and Dr. Tingchung Wang are highly !064 in the Scientific Journal Series of the Minnesota ;tation. This research is part of the NC-89, Regional he triethylene melamine was kindly supplied by Dr. Leon anesulfonate was purchased from the Kodak Company,

lnaucea Patrick

that meiotic female (Cattanach 1969 clast~ replication. To explain the stage, they h.ave suggested derived from the lesions produced the treatment but is mediated zygote and is finalized in the

Acknowledgements

The editorial work of M appreciated. Paper No. 12 Agricultural Experiment Station. Poultry Breeding Project. The trietl A. Snyder, and ethyl methanesulfl New York.

Trial B. All were checked cytolog ;hromosome rearrangements. Birc the injection of TEM, had one une nd a microchromosome, and bir~ [lowing an EMS injection, had an t (Table 5, Fig. 3). from Trial C, 3 were found to be c m a natural mating 5 days after T arm of chromosome No. 2 and a r 2, from an artificial insemination

microchromosome, treatment, i

chromosome (T genic eff

hatching le' mortality c

most sen maturation o

ements recover which ar

occur 4-10 ,ohic mor!

ieth'

chicks rom a )cation om an centric

nequal n, had )some. t-TEM and in p arm 3). d and

3-6). can be te two ltozoa. ttching ~ptible re the zed by

Patrick in cock sperm, Poultry Sci., 44, 587•

Shoffner, methodolo

Wan identification

Wan

Sl

tion of chromosome aberrations it 317-326. 9oration of phosphorus and seleniun

Otis (1967) Avian chromosom

for interchange analysis and se

induction and detection of diploi<

IlI.U Ut;UXylIUUIlUt. , ;I~;Ig; ill,.;lU ~ V I ~ / ' $ ) , bl) t ; l l l t i l tU&Lldt d l i U b~ l l l l l l d . l ~Jltl~llJ

my, O.G., and M.J. Fahmy (1957) Mutagenic response to tl~ permatogenesis in Drosophila melanogaster, Nature (London), 31, :se, E.B. 91961) Transitions and transversions induced by depurin~ U.S.A.), 47, 540. eroso, W.M. (1969) Chemical induction of dominant lethals in fe eroso, W.M., and W.L. Russell (1969) Strain and sex variation in ethal induction with ethylmethane sulfonate, Mutation Res., 8, 58 eroso, W.M., S.W. Huff and S.K. Scott (1971) Chemical induced :illing in mouse oocytes in advanced stages of follicular developm eroso, W.M., M. Krishna, R.E. Scotomayor and N.L.A. Cach~ ~hromos0me aberrations in mouse pachytene' spermatocytes treated 3enetics, 85, 65-72. han, A., and R.N. Shorrner (1966) Sex chromosomes in the domes Meleagris gallopavo ), and the Chinese pheasant ( Phasianus colchi :~pold, H.E., P.C. Coock and J.G. Brewen (1978) The producti, rarious mammalian cells by triethylene melamine, Genetics, 88, 31 ick, H., R.A. voitle, H.M. Hyre and W.G. Martin (1965) Incorpo

R.N., A. Krishan, G.J. Haiden, R.K. Bammi and J.S. aaethodology, Poultry Sci., 46, 333-334. ag, N. and R.N. Shoffner (1974) Trypsin G- and C-banding dentification in the chicken, Chromosoma, 47, 61-69. ag, N., J.R. Sheppard, T. Wang and R.N. Shoffner (1980) The i ~permatozoa in Gallus domesticus, Mutation Res., 73, 279-290.

ranslocations in mice by triethylene melan

ty of the mouse testis to the mutageni~

58) IV. The effects of triethylene melamin~ 67, 54.

7. Isaacson (1968) Ethyl-methane-sulfonal es., 6, 297. turk (1969) In vivo and in vitro incorpor

spermatozoa and seminal plasma of the fox the alkyl-m¢

180. by depurinating agent,

female mice, in the sensitivJ 589-598.

dominant 1~ )ment, Mutati

Cacheiro (1977) ated with trieth

the domestic fowl (G+ colchicus), Cyto 8

London),

iethylene

¢ of male

9mosome

bhorus-32 ,48, 159. :s during

~cad. Sci.

461 470. iominant

+ and cell 1-420.

lation of e (TEM),

), turkey

in

;elenium

omosome

s e x

~loid