Chromosomal analysis of embryos produced by artificially inseminated superovulated cattle

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Chromosomal analysis of embryos produced by

artificially inseminated superovulated cattle

Wa King, A Verini Supplizi, Hep Diop, D Bousquet

To cite this version:

Note

Chromosomal

_analysis

of

_embryos

produced

by

artificially

inseminated

superovulated

cattle

WA

_King

A Verini

_Supplizi

₂

_{HEP Diop}

₂

D

_Bousquet

University

of Guelph, Department of

Biomedical

_Sciences,

Ontario

_Veterinary

_College,

_{Guelph, ON,}

N1G

_{2W1, Canada;}

.

Z Universite

de

_Montreal,

centre de recherche en

reproduction animale, faculte

de medecine

_{vétérinaire, CP5000, Saint-Hyacinthe, PQ,}

J2S 7C6

Canada;

’!

Instituto di Produzioni

Animali,

Facolta de Medicina

_Veterinaria,

Via San Costanzo

_l,,

06100

_{Perugia, Italy;}

’ Ecole inter-Etats des sciences et médecine

_{v6t6rinaire, département Chirurgie}

et

_{Reproduction,}

BP 5077

Dakar, Senegal;

5

BOVITEQ,

1425, Grand

_{Rang Saint-Prançois, Saint-Hyacinthe, PQ,}

J2S 7A9

_Canada;

(Received

23 June

_{1994; accepted}

14 November

₁₉₉₅₎

Summary - To determine the chromosomal

_complement

of

pre-morula

bovine

embryos,

30 Holstein heifers were

_{superovulated}

and inseminated and their

_embryos

were

_subjected

to chromosome

_analysis

on

_days

_2, 3 or 4. Of the 298

embryos/ova

_recovered,

101 had

one or more cells in

_metaphase

and of

_these,

60 could be

_{karyotyped. Eight}

of the

karyotyped embryos

were

_{chromosomally abnormal, including}

3

_triploids,

1

_tetraploid

and 4

_mixoploids.

It was concluded that the abnormalities occurred at or

shortly

after the

time of fertilization.

bovine

_/

embryo chromosome

_/

_triploid

_/

_tetraploid

_/

_mixoploid

Résumé - _{Analyse chromosomique} chez des _embryons provenant de vaches super-ovulées et inséminées artificiellement.

_Afin

de déterminer la

_{garniture chromosomique}

d’embryons

bovins aux stades de

pré-morula,

30

_génisses

Holstein ont été

_{superovulées}

et inséminées. Les

_{embryons furent}

récoltés aux _{jours 2,} 3 _ou

_{4 et}

soumis à une

_analyse

chromosomique.

Sur un total de 298

_{embryons/ovules récoltés,}

101 montraient une ou

plusieurs

cellules en

_métaphase

et 60 de ceux-ci _{ont pu} être examinés _pour leur _caryotype. Huit

_{embryons analysés présentaient}

une _garniture

_{chromosomique}

anormale : 3

_triploïdes,

1

_{tétraploïde et}

_{4 mixoploi’a’es.}

Il est conclu que les anomalies se

produisent

au moment de la

_{fertilisation}

ou

_{juste après.}

INTRODUCTION

Under intense

_agricultural

_management

_{superovulation}

is used to induce

_multiple

ovulation in cattle to

_{provide genetically}

valuable

_embryos

for collection and

trans-fer.

_However,

_only

about

60%

of the

_embryos

that are collected from

superovulated

cattle have normal

_morphology

and are considered suitable for transfer

(Schnei-der

_Jr,

et

_al,

_1980;

Schiewe et

_{al, 1987; Lopez}

Gatius et

_al,

_1988).

In

_humans,

chromosomal abnormalities in the

_embryo

or fetus are the most

_frequent

causes of

malformations and pregnancy failure

(Jacobs

et

al,

1978).

Chromosomal

analysis

of cattle

_embryos

at the morula and

_blastocyst

_stages,

when

_embryo

transfer is

usually performed,

has revealed abnormalities that are

_thought

to

_compromise

de-velopment

(King,

1991).

These abnormalities include

_{aneuploidy, mixoploidy}

and

polyploidy

(for

review see

_King,

1990).

Prior to the morula and

_blastocyst

_stages

very few observations on the chromosomal constitution of

embryos produced by

superovulated

cattle have been documented

_(King

and

Picard, 1985; Murray

et

_al,

1985).

Hence,

little is known of the situation close to the time of fertilization. The

_objective

of this

_study

was to determine the chromosomal

_complement

of

pre-morula

stage

embryos produced by superovulated

cattle. Some of the

_embryos

reported

here were included in a

_preliminary

_report

published

in abstract form

(Verini

Supplizi

et

_al,

_1988).

MATERIALS AND METHODS

Embryos

were

_{produced by superovulated}

Holstein heifers inseminated once or

twice with semen from a

_highly

fertile Holstein bull

during

the 24 h

following

onset of behavioural estrus.

_{Superovulation}

was induced

_by

treatment with

follicle-stimulating

hormone

_(FSH-p;

Burns-Biotech

Laboratory, Oakland,

CA,

USA)

and

cloprostenol

(Estrumate:

ICI

Pharms,

Mississauga,

ON,

Canada)

as

previously

described

_(King

et

_al,

_1987).

Females were checked for

signs

of behavioural estrus

twice

_daily.

The first detection of behavioural estrus was

designated day

zero.

Embryos

were collected

_by

post-mortem

retrograde

flush of the oviducts on

day

2

(n

=

6),

3

(n

=

23)

and 4

(n

=

1).

Only

_one oviduct from 11 of the heifers was available for use in this

_study.

For all collections the

_flushing

medium was

Dulbecco’s

_{phosphate-buffered}

saline

_(PBS,

_pH

_7.2)

_supplemented

with

2%

fetal calf

serum

_(FCS)

and antibiotics

₍₁₀₀

iu

_penicillin,

100 _pg

streptomycin/ml).

Once the

embryos

were located in the

flushing

medium

they

were washed in PBS

_containing

10%

FCS and antibiotics and transferred to Hams F10

_containing

20%

FCS and

antibiotics and colcemide

_{(0.05 pg/ml}

_medium;

_Sigma,

Saint

_Louis,

_MO,

_USA).

The

_embryos

were incubated in this medium for 4-8 h and then fixed

individually

on slides as

_previously

described

(King

et

al,

1979).

Slides were then stained with Giemsa and examined for cell number and chromosome

_composition.

Fertilization

was evaluated after fixation and was considered to have occurred if _any of the

following

were observed: mitotic

chromosomes;

2 or more

pronuclei/nuclei;

or 2 or more blastomeres. Ova that

_presented

meiotic chromosomes or lacked nuclei were

RESULTS

Flushing

the

_reproductive

tracts of the 30 females

_yielded

a total of 298

em-bryos/ova.

The mean rate of fertilization

_(percent

of total

_recovery)

was

83.2%.

In all 101

_embryos

_(33.9%)

had one or more cells in

_metaphase.

The

_karyotype

of

60 of these

_embryos

_(59.4%)

could be determined while 41 had

_{metaphase spreads}

that were either

_incomplete

or of insufficient

_quality

for

_analysis.

Of the 60

karyo-typed embryos,

52

_(86.6%)

were found to be

_diploid

_(60XX

or

_60XY)

and 8

_(13.3%)

were other than

_diploid

(table I).

The abnormal

_complements

included 3

_triploids,

1

_tetraploid

and 4

_mixoploids.

The

triploid

and

_{tetraploid embryos}

were at the 2-cell

stage

and each

_embryo

_presented

2

_metaphase

_spreads.

Three of the 4

_mixoploid

embryos

had cleaved to 2-cell

_stage

_although

4 nuclei

₍₂

_haploid

and 2

_diploid)

were

present

in each. The fourth

_mixoploid

had not cleaved but contained a

_haploid

and

a

diploid

nucleus

(fig

1).

The

_diploid

nucleus in this

_embryo

contained 62

chromo-somes. In all cases the abnormal

_embryos

were _among the least

_{developmentally}

advanced

_embryos,

estimated on the basis of cell

_number,

within the flush of the

DISCUSSION

Chromosome abnormalities have been observed in the

embryos

of most domestic animals. In

sheep

and

_pigs,

a

frequency

of

chromosomally

abnormal

embryos

of

10.4 and

_{6.6%, respectively,}

has been

_reported

_(for

review see

_King,

1990).

In

cattle the

_frequency

varies from 0 to

36.3%

_{(table II)}

according

to the

stage

of

development

and

_{morphological}

features of the

embryo.

In the

_present

_study

of

_days

2-4

_{embryos, ranging}

from 1-8

_cells,

a

_frequency

of

13.3%

was noted while Hare et al

(1980),

reported

a

frequency

1.9%

in

elongated

blastocysts

on

days

12-18. The low

frequency

at the end of the second week of

development

in the

_{elongated blastocyst}

stage suggests

a loss of abnormal

embryos

as

_development

_progresses.

Benevedes-Filho et al

₍₁₉₉₂₎

_reported

a

frequency

of

35.8%

in

day

7

embryos

which exhibited

reduced cell

number,

abnormal

_morphology

and low

_{developmental potential.}

Similarly,

King

et al

₍₁₉₈₇₎

found a

higher

rate of

chromosomally

abnormal

embryos

on

day

7 _among

morphologically

abnormal

embryos

with low cell numbers than

among

morphologically

normal ones. In the

_present

study

the abnormal

embryos

were _among the least

developmentally

advanced within individual donors

(table I).

If indeed

_{chromosomally}

abnormal

_embryos

have a slower rate of

_development

and hence a

_lower

cell

_number,

the

_present

observations

suggest

that

development

may

begin

to slow down as

_early

as

_day

2.

Chromosome

_analysis

of in

_{vitro-produced}

bovine

_embryos

has shown abnormal-ities in

12.1%

of

_embryos

at the 2-cell

_{stage, 20.0-36.4%}

at 4- to 16-cell

_stage

and

44.2%

at

_blastocyst

_(Iwasaki

et

al,

1989).

Kawarsky

(1994)

has noted a

_frequency

of abnormalities of

27.4%

on

_day

2

_{(1-8 cells)}

and

32.1%

on

day

5

(8-cell

_stage

to

morula)

in vitro. As with the in vivo

_studies,

both of these in vitro studies

sug-gest

an accumulation of

chromosomally

abnormal

embryos

over the first week of

development. Unfortunately

the limitation of in vitro culture

_prevents

_monitoring

determine if there is an elimination of abnormal

embryos

as the

_{embryo begins}

to

elongate.

All 8 abnormal

_embryos

were either 1 or 2 cells

suggesting

that the

abnormality

occurred at or close to the time of fertilization before

_completion

of the first cell

cycle.

The 3

_triploids

and 1

tetraploid

were 2-cell

embryos. Unfortunately,

the exact

origin

of the extra

_haploid

_set(s)

of chromosomes could not be determined. In

humans,

triploid

fetuses

_originate

from

_dispermic

fertilization

_(66%),

_diploid

sperm

(24%)

or

_diploid

_oocytes

(10%;

Jacobs et

al,

1978).

Tetraploid embryos

are less common and mechanisms

leading

to their

_production

are not well studied.

_They

could, however,

arise

_by

combinations of the mechanisms

_proposed

for

_triploids

as well as

_by

failure of

_cytokinesis

at first

_cleavage

or

by

endoreduplication

of the

pronuclei.

In cattle all of these

_pathways

are

_possible

since

_{polyspermic fertilization,}

diploid

sperm,

diploid

oocytes

and

endoreduplication

have been

reported

(King

et

al,

1988;

Iwasaki et

_{al, 1989;}

Yadav et

_{al, 1991;}

_Kawarsky,

_1994).

In

_pigs

it has been

_reported

that the incidence of

_polyploidy

arises due to

_ageing

of the

_oocyte

when insemination is

_delayed

_{(Bomsel-Helmreich, 1961).}

However,

this has not been confirmed in cattle.

All 4 of the

_{mixoploid embryos}

exhibited

_haploid

nuclei

₍₂

in the 3 two-cell

embryos

and 1 in the 1-cell

_embryo).

The _presence of a Y chromosome in the

haploid

cells in 2 of the

embryos

suggests

that the

_oocytes

_leading

to these

_embryos

were fertilized

by

2

_spermatozoa.

The X-chromosome

_bearing

haploid

cells _may have

originated

from a

_{spermatozoon,}

a binucleated

_oocyte

or a

_{polar body.}

The fate of

the

_haploid

cells is not known.

_King

and Picard

₍₁₉₈₅₎

and Iwasaki and Hamano

(1991)

described morula and

_pre-morula

with

_haploid

cells.

_However,

older

_embryos

with such cells have not been

reported.

It is

_possible

that these nuclei

_die,

become

quiescent

or are somehow eliminated from the

embryo.

It is also

possible

that

they

eventually

fuse with their

diploid

cohorts as

_{diploid-triploid mixoploids}

have

been

reported

in

_blastocysts

and

_elongated

_blastocysts

_(Hare

et

al, 1980; King

et

_al,

1987).

One

_aneuploid

metaphase

was observed in the 1-cell

mixoploid

(30X/62XX)

embryo.

This low incidence of

_aneuploidy

_(1/60;

_1.7%)

is consistent with the low incidence of

_aneuploid

spermatozoa

(2.8%:

Longue

and

_Harvey,

₁₉₇₈₎

and ova

(5.8%:

Yadav et

_al,

_1991).

It was concluded that

_roughly

13%

of

_day

2-4

_embryos

from

superovulated

cattle that could be

_{cytogenetically analyzed}

were

_{chromosomally}

abnormal. The

abnormalities most

_likely

arose at or soon after fertilization due to fertilization

_by

a second

_spermatozoon

or failure of

polar body

extrusion.

ACKNOWLEDGMENTS

The financial _support of Natural Sciences and

_Engineering

Research Council of Canada and the Canadian Association of Animal Breeders is

_{appreciated. Scholarships}

from the

government of

_Italy

_(AVS)

and the Canadian International

_{Development Agency}

_(HEPD)

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