A comparison of the effects of estrus cow serum and fetal calf serum on in vitro nuclear maturation of bovine oocytes

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A comparison of the effects of estrus cow serum and

fetal calf serum on in vitro nuclear maturation of bovine

oocytes

J Spiropoulos, Se Long

To cite this version:

A

_comparison

of the effects of

estrus

cow

serum

and fetal calf

serum on

in

vitro

nuclear maturation of bovine

_oocytes

J

_Spiropoulos,

SE

_Long

University of Bristol,

School

_{of Veterinary Science,}

Department of

Animal

_{Husbandry, Langford House,}

Bristol BS18

_7DU,

UK

(Proceedings

of the 9th

_{European Colloquium}

on

_Cytogenetics

of Domestic

_Animals;

Toulouse-Auzeville,

10-13

_July

₁₉₉₀₎

cattle

_/

female meiosis

_/

in vitro maturation

_/

_oocyte

INTRODUCTION

The use of in vitro matured

_(IVM)

_oocytes

in in vitro fertilization

_(IVF)

_programs

requires

an exact

_knowledge

of the

_timing

of nuclear maturation events. In

_cattle,

the

_timing

of IVM

_prior

to IVF has been

_{variously reported}

to be 19-22 h

_(Goto

et

_al,

₁₉₈₈₎

_{up to} 27 h

_(Xu

et

_al,

_1987).

Correct IVM is necessary to avoid

fertilization of premature or

_aging

_oocytes.

_{In IVF programs the criteria used for}

oocyte

maturation are cumulus cell

_expansion

and extrusion of the first

_polar

_body.

Neither are direct assessments of

_{cytoplasm maturation,}

which is one of the most

important

factors for successful

_IVF,

but since fertilization in vivo occurs when the

oocyte

is at

_metaphase

II

_(MII)

it is

_usually

assumed that

_completion

of meiosis is

accompanied by

acquisition

of

_{developmental}

_competence

_by

the

_oocyte.

_Thus,

in

vitro,

it is also assumed that

_complete

_oocyte

maturation is acheived

_by

the MII nuclear

_stage.

In this

_study

the

_timing

of the meiotic _process

_during

IVM of

_oocytes

is examined under conditions similar to those used for IVF _programs.

MATERIALS AND METHODS

Ovaries were collected from adult cows and heifers

_{approximately}

20 min after

slaughter

and

_transported

to the

_laboratory

in sterile

_{phosphate-buffered}

saline

(PBS)

or normal saline

supplemented

with 100

_IU/ml

_penicillin

and 100

_A

_g/ml

transferred into the culture medium. The culture medium was TCM-199 with 2.2

g/1

sodium bicarbonate

_plus

_{50 pg/ml}

_{gentamicin supplemented}

with either

20%

FCS

or

20%

estrus cow serum

(ECS).

A total of 479

_oocytes

were

_cultured,

320 with added

_granulosa

cells and 159 without.

Oocyte

culture was either in

_droplets

of medium under sterile

_paraffin

oil in a

Petri dish or in multiwell

_plates

under

_{paraffin oil,}

and in

5%

C0

2

in air at 39°C

for between 8 and 25 h.

To

_harvest,

the cumulus cells were removed either

_by

_putting

the COC in a

0.1%

_{hyaluronidase:PBS}

solution for 2-10 min and

_{flushing through}

a fine

_pipette

or

_by

a

_quick

_spin

with a whirlimixer. Chromosome

_preparations

were made from

the denuded oocytes

using

either Tarkowski’s method

₍₁₉₆₆₎

or a modification of

the method of

_Dyban

_(1983).

The modification consisted of

_placing

the

_oocyte

in

Tyrode’s

acid

_{(pH 2.5)}

for a few seconds after the

_hypotonic

treatment. This softens the zona

_pellucida.

Next the

_oocyte

is

_placed

in fixative

_(3:1,

acetic

_{acid-methanol)}

for 1-2 min and then transferred to a _wet, ice-cold slide. The fixative is allowed to

evaporate and the slides to

_dry

in air.

_Staining

was with a

10%

solution of Giemsa

at

_pH

6.8-7.0

The ECS was obtained

_{by collecting}

blood from cows as soon as

_possible

after behavioral estrus was first noted. The serum was inactived

_{by heating}

at 56°C for

30 min before use.

RESULTS

Cumulus-oocyte complexes

with added cumulus cells achieved better and more

extensive cumulus cell

expansion

than those without added

cells,

but this was not

correlated with the

_stage

of meiotic division after a

_given

culture time or the

_quality

of the chromosome

_{preparations.}

Denuding

the

_oocytes

_by

fine

_pipetting

was much better than the use of the

whirlimixer because _many

_oocytes

were lost

_using

the latter

_technique,

_presumably

by

sticking

to the wall of the tube.

The modified

Dyban’s

method for chromosome

_preparations

_{(Dyban, 1983)}

was

preferred

to that of Tarkowski

₍₁₉₆₆₎

because the

_spreading

was more controllable

and there was less chance of

_{losing chromosomes,}

thus

_making

assessment of chromosomal abnormalities more reliable.

There was no statistical difference between the

_percentage

of

_oocytes

at diakinesis

(fig

1)

after 8-12 h of culture

₍

_X

_z

=

_1.76;

_P =

0.1)

_{or at}

_metaphase

II

(fig 2)

after

DISCUSSION

An

_interesting

_finding

in the present

study

was that the extent of the

_expansion

of the cumulus cells was not found to correlate with the

stage

of nuclear maturation.

Therefore,

it would seem unwise to use this

phenomenon

as a criterion of IVM for

IVF.

No difference was found in nuclear maturation time

_using

the two different sources of serum

_{supplementation.}

Lu et al

₍₁₉₈₇₎

_reported

_higher

IVF rates when

ECS was used as the

_supplement.

_Therefore,

it _may be that the ECS contributes

to

_cytoplasmic

maturation.

The results from the

_present

_study

of

_timing

of nuclear maturation are in broad

agreement

with those

_previously

_reported.

Sus et al

₍₁₉₈₈₎

found that

62%

of bovine

COCs in serum-free TCM-199

_progressed

to diakinesis after 8.25-12.5 h of culture

and

72%

reached MII after 19.5-25.75 h of culture. Similar

_percentages

reached ?VIII after 24-27 h of culture in TCM-199

_supplemented

with

15%

FCS

_(61.8%)

(Leibfried

and

_First,

₁₉₇₉₎

or when

_supplemented

with 20

_mg/ml

of bovine serum

growth

protein

(71.4%) (Liehman

et

_al,

_1986).

A somewhat

_longer

maturation time of 30 h was

_{reported by}

Xu et al

₍₁₉₈₆₎

when

_they

cultured COCs in Ham’s F12 with 20% FCS.

In

_vivo,

the

_oocyte

resumes meiosis after the

luteinizing

hormone

(LH)

peak

and,

in

_cattle,

reaches MI after 8-19 h and MII after 20.5-25 h

_(Kruip

et

_al,

_1983).

Therefore,

in vitro maturation times are _very similar to those

_occurring

in vivo. It

seems

_likely,

_{therefore, that,}

once the

oocyte

resumes

_meiosis,

_progress to MII is

spontaneous and at a fixed rate. In

_vivo,

ovulation takes

place

24-30 h after the LH

_peak

_{(Hopkins, 1989),}

that

_is,

some time after the

_oocyte

will have achieved

nuclear maturation. It is

_possible

that this extra time allows for the

_completion

of

cytoplasmic

maturation.

REFERENCES

Dyban

AP

₍₁₉₈₃₎

An

improved

method for chromosome

_preparations

from

preimplanta-tion mammalian

_embryos,

_oocytes or isolated blastomeres. Stain Technol

_58,

69-72 Goto K,

Kajihara

Y, Kosaka

_S,

Koda

_M,

Nakanishi Y,

Ogawa

K

₍₁₉₈₈₎

_Pregnancies

after

co-culture of cumulus cells with bovine

_embryos

derived from in vitro matured follicular oocytes. J

_Reprod

Fertil

_83,

753-758

Hopkins

SM

₍₁₉₈₉₎

Reproductive

patterns of cattle. In:

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and

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Lea and

Febiger

Kruip

TAM,

Cran

_DG,

van Beneden

TH,

Dieleman SJ

₍₁₉₈₃₎

Structural

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in bovine oocytes

during

final maturation in vivo. Gamete Res 8, 29-47

Leibfried

_L,

First NL

₍₁₉₇₉₎

Characterisation of bovine follicular _oocytes and their

_ability

to mature in vitro. J Anim Sci 48, 76-87

Liehman

_P,

Greve

_T,

Xu KP

₍₁₉₈₆₎

Nuclear and

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Lu

_I<H,

Gordon

I,

Gallagher

NI,

McGovern H

₍₁₉₈₇₎

_Pregnancy

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embryos

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Sus

_U,

Wuthrich

_{K, Stranzinger}

G

₍₁₉₈₈₎

Chromosome

_{configurations}

and time sequence

Tarkowski AK

₍₁₉₆₆₎

An

_air-drying

method for chromosome

_preparations

from mouse eggs.

Cytogenetics

5, 394-400

Xu

_KP,

Greve

_T,

Smith

_{S, Hyttel}

P

_{(1986) Chronological}

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505-519