Embryo transfer and related technologies in sheep reproduction

Review

Embryo

transfer

and related

_technologies

in

_sheep

_reproduction

Pasqualino

Loi

a

_Grazyna

Ptak

b

Maria Dattena

a

_Sergio

Ledda’

Salvatore Naitana

Pietro

_Cappai

_a

a_{Istituto Zootecnico}

e _Caseario, 07040 _{0lmedo, Sassari,}

_Italy

b

Department

of Animal

_{Reproduction, University}

of

_Agriculture,

30059, Krakow, Poland

!

Dipartimento

di

_Biologia

Animale, Università di Sassari,

Italy

(Received 31

_July

1998;

accepted

13 October 1998)

Abstract -

This paper reviews the status

_{of embryo}

transfer and the

_{major technologies applied}

to

preimplantation

of

_embryos

in

_{sheep. Embryo production}

from

_{superovulated}

ewes is hindered

_by

an

unpredictable

response to hormonal treatment.

_Progress

in this area should be

_{expected by}

an

appro-priated

control of follicular

_development

with

_{gonadotropin-releasing}

hormone (GnRH)

agonist

or

antagonist prior

to

_{gonadotrophin}

administration.

_{Simple protocols}

for the

_{cryopreservation}

of

_sheep

embryos by

vitrification are

_already

available and the

_development

of frozen-thawed

_blastocysts

to term is close to the fresh ones. Further research is

_required

to

_identify

factors able to _promote the

mat-uration in vitro of _oocytes,

_namely

those obtained from

_prepubertal

animals. Semen and

_embryo

sexing procedures

are available in cattle

_although

much less attention was

_paid

to their

_application

to

sheep. Among

all the

_{reproductive technologies, cloning}

with

_embryonic

and foetal cells has

_progressed

dramatically

in

_sheep

and nuclear transfer has been used to

_produce

_transgenic

animals as an alter-native to

_{pronuclear injection.}

The

_production

of the first lamb cloned from a somatic cell

_opened

new

opportunities

in animal

_breeding

as well as

_exciting

lines of basic research. The overall conclusions are _{that, apart} from

_{superovulation,}

the

_application

of in vitro

_technologies

is

_likely

to evolve

_rapidly

and once

_applied,

a _great

_impact

on traditional and new animal

_productions

should be

_expected.

However, a better

_{understanding}

of the

_changes

in gene

expression,

induced in

_{embryos by}

different

in vitro

_{manipulation procedures,}

_{is necessary} to _prevent abnormal foetal

_development.

© Inra

Elsevier, Paris.

superovulation

/ in vitro maturation /

_{embryo freezing}

/ nuclear transfer /

_transgenesis

/

_sheep

Résumé ― Transfert

_d’embryons

et

_techniques

associées en

_reproduction

ovine. Cette revue traite du transfert

_d’embryons

et des

_{principales biotechnologies appliquées}

à

_l’embryon

_{ovin. La}

pro-duction

_{d’embryons, après superovulation,}

est _{limitée par la}

_réponse

non

_{reproductible}

au

traite-ment hormonal. Un

_{progrès pourrait}

venir d’un contrôle

_approprié

du

_{développement}

folliculaire

*

Correspondence

and

_reprints

E-mail:

_{izcscrip@tin.it}

avec un

_agoniste

ou

_antagoniste

de GnRH

_appliqué

avant le traitement

_gonadotrope.

Des

_protocoles

simples pour la congélation d’embryons

ovins par vitrification sont

_disponibles

et,

après

décongélation,

le

_{développement}

à terme de

_blastocystes

_congelés

est

_proche

de celui des

_embryons

frais. De

nou-velles recherches seront _{nécessaires pour} identifier les facteurs

_capables

de stimuler la maturation in vitro des ovocytes, en

_particulier

ceux d’animaux

_{prépubères.}

_{Le sexage des}

_{spermatozoïdes}

et des

embryons

est

_possible

chez les bovins, mais peu

appliqué

chez les ovins. De toutes les

_techniques

de

reproduction,

c’est celle du

_clonage

à

_partir

de cellules

_{embryonnaires}

ou foetales

qui

a le

_plus

pro-gressé ;

le transfert nucléaire a été utilisé pour

produire

des animaux

_{transgéniques,}

comme alterna-tive à

_{l’injection}

_{dans les pronoyaux. La}

_production

du

_premier

agneau cloné à

partir

d’une cellule

somatique

a ouvert de nouvelles

_perspectives

en

_élevage

et en recherche fondamentale. En

conclu-sion,

excepté

dans le domaine de la

_{superovulation,}

les

_{biotechnologies}

vont évoluer

_{rapidement ;}

leur

application

aura certainement un

_{grand impact}

sur les méthodes traditionnelles et nouvelles de

pro-duction.

_Cependant,

une meilleure connaissance des effets sur

_{l’expression}

de

_{gènes embryonnaires}

induits par les

manipulations

in vitro serait nécessaire pour éviter un

_{développement}

foetal anormal.

© Inra/Elsevier, Paris.

superovulation

/ maturation in vitro /

congélation d’embryons

/ transfert nucléaire /

_homogenèse

/

mouton

1. INTRODUCTION

Nearly

all the

_technologies

related to

embryo

production

and

_manipulation

in domestic

_ruminants,

from

_artificially

con-trolled

_breeding

to the

_production

of

_Dolly,

the first cloned

mammal,

have been

devel-oped

in

_sheep,

before

_being

transferred to

its more

economically important

counter-part, the bovine.

The aim of this work is to review the

technologies

used in

_sheep

_reproduction

with a

_{particular emphasis}

on the limits and the

_perspectives

for

_{improvement. Secondly,}

the

_potential

value of these new

_technologies

in

_{sheep breeding}

and

_reproduction

is described and

_{finally, already}

known and new

_aspects

of abnormal foetal

develop-ment associated with in vitro

_manipulation

are discussed.

2. SUPEROVULATION

Small-scale studies in

_sheep

embryo

transfer

_reported

40 _{years ago}

_[7]

estab-lished the basic

_techniques

for

_surgical

recovery of

embryos (reviewed by

Moore

[68]).

The Australian

_experiments

_leading

to the artificial control of oestrus

_[90]

fol-lowed

_by

the identification and

_purification

of

_{gonadotrophins, provided}

the basis for the

_development

of suitable

_protocols

for

sheep superovulation.

An enormous amount of literature

con-cerning

superovulation

in

_sheep

has been

produced

and from its

_analysis

it is evident that an accurate control of ovulation has

never been achieved.

_{Embryo yield}

after

superovulation

is

_dependent

_upon many

fac-tors that can be

_grouped

as follows.

I)

Factors

_inherently

variable and diffi-cult to

_modify

_(breed,

season,

_management).

It is easy to _{understand that very little}

improvement

can be

_expected

from factors

like breed and

_consequently

flock manage-ment

_[21]

as well as nutrition

_[61].

_Breeding

dairy

Sarda ewes in Sardinia

_imposes

a

dif-ferent

_management

from that

_required

for

breeding

Merino ewes in New Zealand or

Scottish Blackface in

Scotland;

moreover,

the ovarian response of Sarda ewes to the same commercial

_{gonadotrophin}

prepara-tion will

_always

be different from that of Merino ewes

_{[60, 72]. However,}

a

_major

effort has been made

_by

_reproductive

superovula-tion

_protocols

into a

_large

number of domes-tic breeds under a broad environmental

range (see [45]).

2)

Factors

_susceptible

on

_improvement

(gonadotrophin,

knowledge

of ovarian

phys-iology). Pregnant

mare serum

_{gonadotrophin}

(PMSG)

[6],

pituitary

extracts of different

origin

[28, 69, 96]

and human

_menopausal

gonadotrophin

[92]

are

_commonly

used for

superovulation

in

_sheep.

The

_biological

properties

of the

_{gonadotrophins}

used for

superovulation

have been described in detail elsewhere

_[13];

for review see

_[45])

Exoge-nous

_{gonadotrophin}

_interplay

with the

somatic and

_germinal

_compartments

of the follicle

_leading

to the ovulation of a number of _oocytes

_larger

than normal.

However,

there is evidence that their action is modu-lated at the ovarian

_level,

_{thereby making}

the answer

_{unpredictable.}

Furthermore,

addi-tional

_negative

effects can occur before

ovu-lation,

as a _consequence of

_inadequate

oocyte maturation

_[67]

or

later,

_during

early

embryonic development

as a result of

unbal-anced hormonal

_profiles

[3, 51].

Several

strategies

have been

_suggested

for

optimiz-ing

the

_yield

of transferable

_embryos

from

superovulated

donors: the administration of anti-PMSG antibodies

_[12],

_pituitary

folli-cle-stimulating

hormone

_(FSH)

instead of PMSG

_[66],

association of these two

gonadotrophins

[91],

single

versus

multi-ple

injections

[30, 64],

or inclusion of GnRH

or

_growth

hormone in the treatments

_[36,

111].

However,

the well-known side-effects of the

_{superovulatory}

treatment such as unovu-lated

follicles,

low fertilization _{and recovery}

rates

_(reviewed

_by

Naitana et al.

_[72])

were

left

_largely

unsolved.

_Undoubtedly,

the

bal-ance between FSH and

luteinizing

hormone

(LH) components

or the

_purity

of the

gonadotrophin preparation play

an

impor-tant role

_[24];

the

_availability

of

recombi-nant FSH

_[59]

should in

_theory

reduce the

negative

side effects linked to the

_purity

of the commercial

_preparation.

Nevertheless,

our

_opinion

is that no obvious

_improvements

should be

_expected

even from the

_adoption

of

_highly

_purified

hormones until _systems for

_controlling

follicular recruitment and selection are available.

More

_recently,

a detailed

_{understanding}

of the processes involved in the

growth

and differentiation of the

_ovulatory

follicles

_[33]

has been achieved and many

strategies

have

been

_suggested

to control the follicular wave

emergence in cattle

[15].

Unfortunately,

analogous

work has not been

_repeated

in

sheep

because there is a more commercial

interest for

_multiple

ovulation and

_embryo

transfer

_(MOET)

in cattle and intrinsic dif-ferences between

_species

in the _{process of}

follicular selection

[33].

Progress

made

_by

_reproductive

endocri-nologists dealing

with follicular

_dynamics

provided

the _necessary tools for a radical

change

in

_{superovulation}

treatments. Chronic

treatment with GnRH

_agonist

inhibits the

gonadotrophin-dependent

follicular

_growth

by downregulating

LH secretion from the

pituitary

[17].

This observation

suggests

that a treatment with GnRH

_agonist

fol-lowed

_by

the administration _{of exogenous}

gonadotrophin,

may in

theory

produce

a uni-form cohort of follicles available for

ovu-lation. This

_theory

has _yet to _{be proven in}

sheep,

but

_encouraging

results in a small

scale

_experiment

were

_obtained,

where the number of transferable

_embryos

was

increased in ewes treated with GnRH

_agonist

[16].

The

_{downregulation}

of follicular

devel-opment

with

_long-term

treatment of GnRH

analogues

or

antagonists

_(Gong,

_pers.

comm.)

prior

to the administration of

_highly

purified

gonadotrophin

appears to be a _very

promising

line of research for the

improve-ment of

_{superovulation.}

3. INSEMINATION OF THE DONORS

Regardless

of the stimulation

_regimen

used,

reduced fertilization rates after natural

mating

are a common occurrence in super-ovulated ewes,

_especially

in those females

exceeding

ten ovulations

_(reviewed

_by

Gor-don

_[45]).

This

_problem

has been overcome

by

Trounson and Moore

_[109]

with the

sur-gical deposition

of semen

_directly

into the

uterine

horns;

the

_following

introduction of

laparoscopic

intrauterine insemination

com-pletely

resolved this inconvenience

_[89].

Usually,

a

_single

insemination

_performed

around 48-50 h after _{sponge removal does}

not interfere with ovum

_pick

from the

fim-bria and

_{gives satisfactory}

fertilization and

recovery rates

_[94].

4. EMBRYO RECOVERY

AND TRANSFER

Sheep embryos

are

_{usually surgically}

recovered

_using

the method

_{suggested by}

Hunter and co-workers

_[50];

_briefly,

the

uterus and oviducts are

_exposed

to a

mid-ventral

_laparotomy

and the

_reproductive

tract is flushed with different

_shaped

catheters with sterile medium. A much smaller incision was

_required

for the

par-tial exposure of the uterine horns in the

tech-nique suggested by

Tervit and Havik

_[101].

Laparoscopic techniques

were later

intro-duced for

_embryo

_recovery to reduce the extent of

_surgical

intrusion

_[63].

While this

approach

did allow

_repeated

recoveries from

the same

_animal,

the _average

_yield

from a

single flushing

was lower than

_surgical

col-lection

_[95].

_Although

some success has been described for cervical collection of

embryos

[10],

more work is _necessary to

make it a reliable

_option

in

_sheep.

The

embryos

are

_usually

recovered at the

morula-blastocyst

stage, 6-7

days

after the induced oestrus, evaluated under a

dissect-ing microscope

before

_being

frozen or

immediately

transferred,

either

_by

laparo-tomy or

_laparoscopy

_[62],

into suitable

syn-chronous

_recipients.

The

_development

to term of fresh

_embryos

is around 70

_%,

with differences

_depending

on breeds

_(reviewed

by

Gordon

_[45]).

5. REPEATED SUPEROVLJLATION

Once a suitable

_{superovulation protocol}

is

established,

the next

_step

is to

_verify

the

responsiveness

of the same donor to

repeated

treatments. Work from Moore and Shelton

[69]

in Australia demonstrated that

multiple superovulation

can be induced in

sheep

at a

_1-year

interval without a

signifi-cant reduction in the _{ovarian response.} Tor-res and Sevellec

_[108]

later came to the same

conclusion. Whether

_{immunological}

_responses

induced

_by

the

_{gonadotrophins}

used for

superovulation

can reduce the ovarian response still remains an _open

_question.

However,

the side-effects of

_repeated

treat-ment with

_{gonadotrophins}

_[34]

are not the

major

factor

_{limiting multiple superovulation}

in

_sheep.

Adhesions caused

_by

the

_repeated

laparotomic

collection and

_protrusion

of the

endometrium at the

_puncture

site in the

laparoscopic

recovery, may reduce the num-ber of

_flushings

obtainable from one donor

[76].

6. EMBRYO FREEZING

Protocols for

_{freezing sheep embryos}

have been available since the

_1970s,

after

the first lamb was born

_following

a

trans-fer of a frozen-thawed

_embryo

_[120].

In the

original protocol,

the

_embryos

were incu-bated in

_appropriated

concentration of

per-meating

cryoprotectors,

cooled 3-4 °C below the

_{freezing point,}

then ice nucleation was induced in the medium

_{by touching}

the vial with a

_pair

of

_{pre-cooled forceps.}

The

cryoprotectant

was removed

_step-wise

after

thawing

and the

_embryo

was

_ready

for

trans-fer. Once the

_procedure

was

_established,

research continued to increase

_embryo

sur-vival

_through

the

_adoption

of low

_toxicity

cryoprotectants

such as

_{ethylene glycol}

[102]

and

_particularly

after the

incorpora-tion of a

_{non-permeating}

osmotic

buffer,

sucrose

_{(reviewed by}

Rall

_[86])

into the

freezing

medium,

which allows direct

Heyman

and co-workers

_[49].

A further innovation in the

_{cryopreservation}

of

mam-malian

_embryos

was later

_{suggested by}

Rall

and

_Fahy

_[87].

In this

_experiment,

_embryos

were cooled _very

_rapidly

at thousands of

degrees

per minute in a

_high

concentration of

_{cryoprotectants}

which formed a

_glass

structure without the formation of ice

crys-tals. This

_procedure,

called

vitrification,

has been

_adapted

for _many

_species

_including

sheep

[1]

and the results in terms of survival

rate and lambs born are

_continuously

pro-gressing

([1, 99],

reviewed

_by

Niemann

[78]). Furthermore,

the vitrification

proto-cols in

_sheep

are

_{becoming increasingly}

well

defined,

thus

_eliminating

the

_expensive

pro-grammable cryogenic

units in the field

_[75].

Studies from Naitana et al.

_[73]

indicated that the

_{synchronization}

between

_recipient

and

_embryos

is _very critical when vitrified-thawed

_embryos

are transferred.

7. ALTERNATIVE ROUTES

FOR SUPEROVULATION

7.1. In vitro maturation

In vitro maturation

_(IVM)

_usually

involves selection of non-atretic enclosed

cumulus,

fully

grown oocytes and

matura-tion in either

_droplets

or Petri dishes for

approximately

24 h at 39 °C. Meiotic

mat-uration occurs

_{spontaneously}

after removal of the

_oocyte

from the follicle

_[37],

but the

addition of

_{gonadotrophins}

in vitro increases the number of

_oocytes

_{that progress} to

metaphase

II

_(Mll),

_although

it does not increase their

_{developmental}

_potential

[38].

Maturation is

_usually

_performed

in tissue culture medium

199,

as

_{pioneered by}

Moor

and collaborators

_{[65, 98].}

Foetal bovine

serum is the commonest

_supplement,

but

recently,

the addition of

_growth

factors,

in

particular

epidermal

growth

factor

_(EGF),

insulin-like

_growth

factor I

_(IGF-I)

and

transforming

growth

factors-a

_(TGF-a),

has been shown to stimulate

_oocyte

matu-ration and to reduce the

_requirement

for

gonadotrophins during

maturation

_{[42, 46,}

54].

Recent

_{experiments presented}

the

rela-tionship

between

_glutathione

and

develop-mental

_competence

_following

in vitro

mat-uration of bovine

_{oocytes [31].}

Two years later the use of

cysteamine,

which increases the

_glutathione

level in

_oocytes,

was

_already

adopted

for in vitro

_embryo

_production

_using

sheep

[115]

and lamb _oocytes

_[83].

7.2. In vitro fertilization

In

_1985,

_{Cheng reported}

a

_high

fertiliza-tion rate of

_sheep

_oocytes

matured in vitro

[22].

In this

_system,

the semen was

resus-pended

in medium

_containing

oestrus

_sheep

serum. This serum

_{supplementation}

was also used

_by

Crozet et al.

[29]

and still remains the most effective

_capacitating

factor in

sheep.

Later,

Pugh

and co-workers

[85]

reported

the birth of the first lambs

follow-ing

IVM/in vitro fertilization

_(IVF)/in

vitro culture

_(IVC)

of _oocytes collected

_during

non-breeding

season. This

_study

_clearly

demonstrated the

_importance

of

gonado-trophin priming

prior

to collection for

sub-sequent

developmental ability

of the

_oocytes.

In

fact,

in

_agreement

with an earlier

_report

of

Cheng [22],

fewer

_oocytes

from abattoir-derived ovaries were

_successfully

fertilized after IVM/IVF than

_oocytes

from ewes

receiving

low

_dosages

of FSH

_prior

to

slaugh-ter. We had the same

experience

when

work-ing

with Sarda milk

_sheep:

_oocytes

collected

at

_slaughter

_during

the

_non-breeding

season

and

_subjected

to in vitro

_techniques

had a

very low

developmental

competence.

The situation was reversed when

treating

the

females with low doses of FSH

_(Ptak,

unpub-lished

_{observations).}

The

_positive

FSH

prim-ing

effect _{may be exerted}

_through

a direct

stimulation of the

_{cumulus/granulosa}

cells,

indirectly

through

a stimulation of follicular

7.3. In vitro culture

There are

_essentially

two culture

_systems

now used for

_{blastocyst production}

in vitro:

co-culture

_(or

medium conditioned on

_cells)

and defined

(semi-defined).

Co-culture of

sheep embryos

is

_{generally performed}

with

TCM199,

_{usually supplemented}

with serum.

The cells of choice are

_granulosa,

oviduct

epithelial

or buffalo rat liver cells. The

co-culture

_technique

was described

_primarily

by

Rexroad and Powell

_[88]

and Gandolfi and Moor

_[41].

There is evidence that

growth-promoting

factors

_(peptide

_growth

factors and/or

_cytokines)

are

_responsible

for

supporting

the

_development

of the

_embryos

in both co-culture and conditioned media

[40].

It was also discussed that these

cul-ture methods

_simply

reduce or remove

inhibitory

components,

such as

_glucose

and

oxygen

[11,

114].

In defined culture

_systems,

the

concen-tration of each

_component

is known before

the addition of

_embryos

_(unless

serum is

used).

At

_present,

the most

_commonly

used

system for the culture of

_{sheep embryos}

is

synthetic

oviduct fluid medium

_{(SOF; [103])}

enriched with amino acids and bovine serum

albumin

_(SOFaaBSA;

_[43]).

This method

yielded

blastocysts

with

_comparable

cell numbers to in vivo-derived

_{counterparts,}

in

contrast to

_{sheep embryos}

incubated with human serum which

_develop

to

_blastocysts

with lower cell numbers

_{[43, 112].}

Ovine

embryos

cultured in the _{presence of human} serum reach the

_{expanded blastocyst}

_stage

approximately

24 h earlier than their in vivo

counterparts [112].

This

_premature

blastu-lation and other abnormalities such as

frag-mentation of

_embryos,

dark _appearance and

reduced cell numbers were

_{reported by}

the same _{Australian group}

_{[113] who}

also

con-sidered increased

_{gestation length}

and some

perinatal problems

with lambs derived from

embryos

cultured with 20 % of human

serum. Such

_problems

were not confirmed for lambs obtained after culture in the

SOFaaBSA

_{system [107].}

_Despite

_progress

in the culture of ovine

_embryos,

there are

still some differences between in vitro- and

in vivo-derived

_embryos.

_Development

through

the fourth cell

_cycle,

which is

asso-ciated with the

_major

activation of the

embryonic

genome, is retarded

during

in vitro culture and the

_degree

of

_compaction

at

the morula

_stage

differs from the in vivo-derived morula

_[106].

8. OVUM PICKUP

Snyder

and Nellor

_[97]

_reported

for the first time

_laparoscopic

_{recovery of}

_sheep

oocytes

but several years later the

technol-ogy was

_optimized

in terms of _recovery rate

and

_quality

of the

_aspirated

_{oocytes [104,}

105].

In

1996,

Tervit

_[100]

summarized the results of two groups involved in the

exploitation

of ovum

_pickup

in

_sheep.

The first group showed a

_large

variation among

females in the

_production

of

_blastocysts,

with

_only

32-56 % of donors

_producing

at

least one

_blastocyst

_[8].

In the second _group

the _average donor

_produced

between 2.3 and

3.2

_{blastocysts resulting}

in about 1.5 lambs born

_[100].

It was

concluded,

that the

tech-nology

could be useful for

_producing

off-spring

from infertile ewes but needed

improvement

before it could

_replace

MOET. In our

_laboratory,

we stimulated ewes

_using

the Tervit

_protocol

and recovered 14

_oocytes

per ewe with the use of

_laparotomy

with an

average of more than three

_blastocysts

_per

donor; however,

the _oocyte and

_blastocyst

yield

gradually

decreased per session

(5

and

0.6,

respectively,

at the fifth

_session)

_[84].

9. PREPUBERTAL EWES

The

_production

of

_embryos

from

juve-niles should

_potentially

increase the rate of

genetic gain through

a _{reduction of the}

gen-eration interval. In some studies

_reporting

the

_{developmental ability}

of

_prepubertal

ewes it is observed that

_although

the nuclear maturation occurs at a

_comparable

rate for

oocytes

from both adult and

_prepubertal

ewes, normal fertilization and

_development

do not

_follow;

this can be

_largely

attributed

to abnormal

_cytoplasmic

maturation

_[56,

80, 81].

In

fact,

a lower metabolism of

glu-tamine for lamb

_oocytes,

whether matured in

vivo or in

vitro,

and a

_higher

incidence of

polyspermic

fertilization were

_reported

_[79].

However,

due to the observations of

differ-ent _groups

_[4],

the rate of

_development

of lamb _oocytes in vitro from

_blastocysts

was

similar to those observed for

_embryos

derived from adult donors. Based on

appro-priate

follicle stimulation

_{protocols using}

6-8-week-old

_lambs,

_up to ten

_pregnancies

per donor

oocyte

collection session after transfer of IVF

_embryos

is

_speculated

_[5,

35].

10. SPLITTING

The

_procedure

for the

_{multiplication}

of

the

_cleavage

_stage of

_{sheep embryos}

has

been described in detail

_by

Willadsen

_[116].

However,

the loss of nuclear

_totipotency,

as a result of cell differentiation in

devel-opmentally regulated

embryos,

is the main limit of blastomere

_separation.

In

_fact,

embryos

are scheduled to _compact and blas-tulate after five to six cell

_cycles

and

con-sequently,

the cell number is

_{proportionally}

reduced in

_manipulated

_embryos.

In

fact,

the survival to term of

_blastocysts

derived from

_embryos

_containing

_1/2,

1/4 and 1/8

of the blastomeres was

_80,

50 and 6

%,

respectively

[117].

A

_{simpler procedure,}

known as

_{embryo splitting,}

was later

intro-duced to divide

_{morula-blastocyst}

_stage

sheep embryos

[44, 119]

with no deleterious

effect on

_{embryo viability}

_provided

that the

blastocyst

is

_{symmetrically}

divided

_[23].

Embryo

splitting

has been

_simplified

dra-matically

and has been used in the

com-mercial

_embryo

transfer

_industry

in

_sheep

[110]. However,

the insurmountable limit of

_{embryo splitting}

is the limited number of obtainable

_offspring

_per

_manipulated

embryo.

11. PRESELECTION OF GEN DER BY SPERM SEPARATION

AND EMBRYO SEXING

The

_yield

of sorted X and Y

_bearing

sperm in cattle with fluorescence-activated

cell sorter

_(FACS)

_technology

has

_improved

dramatically

[52]

and small scale trials of artificial insemination with selected semen

have also been carried out in

_sheep

_[27].

Once

_{again, although}

in

_theory

ram semen may be FACS

processed

with

_comparable

efficiency,

its use in artificial insemination

in

_sheep

is limited

_by

the

_high

cost of the

technology.

The situation does not

_change

for

_embryo

sexing by polymerase

chain reaction

(PCR).

Commercial kits for field

_sexing

cattle

embryos

by

PCR are available at

reason-able

_prices

and

_{probably, given}

the

_high

analogy

between

_species,

most _{of them may}

be

_successfully

used for

_{sexing sheep}

embryos;

however,

only

very limited or

pre-liminary

reports have been

_published

_[74].

12. CLONING

Since the first

_report

of lambs

_produced

by

nuclear transfer

_[118]

the

_sheep

has

always

represented

a central

_experimental

model for basic and

_applied

research. No

tentative will be made in this review to

describe the

_technique

of nuclear transfer because this

_topic

has been

_exhaustively

treated

_by

_Heyman

_{[47, 48].}

The

_present

discussion will be limited to

_sheep

nuclear transfer.

The

_key

to the

_{right interpretation}

of the

poor

development

of nuclear transfer

embryos

obtainable a few _{years ago} was

provided by

classical

_hybrid

cell fusion

experiments

[53]

where a dominant effect

of mitosis on the other cell

_{cycle phases}

was

demonstrated. The Mll _oocyte follows this basic

_rule,

as was

_clearly

demonstrated

_by

the

_{disorganization}

of the nuclear

_envelope

and the condensation of the chromatin

imposed

to the transferred nucleus

(prema-ture chromosome

_{condensation, PCC),}

regardless

of its cell

_cycle

_{stage. PCC,}

medi-ated

_by

the

_MPF,

results in extensive dam-age of the chromatin when the

replication

machinery

is on, as in the

_majority

of blas-tomeres in

_cleavage

_stage

_embryos

[18].

The nuclear

_{envelope plays}

a central role in

regulating

the

_duplication

of DNA

[14],

and

its

_{disorganization}

in MIT

_cytoplast

is

fol-lowed

_by

the

_replication

of

_previously

repli-cated DNA with the

_exception

of the _very few nuclei that are in G

(reviewed by

Campbell

et al.

_[19]).

From these

_important

observations,

it was concluded that the

nor-mal

_development

of

_embryos

reconstructed

by

nuclear transfer could

_only

be obtained

by

fusing

synchronized

G blastomeres into

MIT

_cytoplasts

or

_{alternatively, by fusing}

unsynchronized

blastomeres with

pre-acti-vated S

_{phase cytoplasts}

[18].

While proto-cols for the

_{synchronization}

of blastomeres

are available in the mouse

_[82],

this and

other

_procedures

are unreliable in

_sheep

embryos (Loi, unpublished

observations).

Pre-activated

_cytoplasts

increased the fre-quency of

development

to the

blastocyst

stage and to the term of cloned

_sheep

embryos

[57].

This

_procedure,

however,

is

quite time-consuming

because a

_two-step

manipulation

is

_required

for

_embryo

recon-struction. In

_subsequent

work carried out in

our

_laboratory,

a

_{high frequency}

of

devel-opment to the

_blastocyst

stage was obtained

by transferring

unsynchronized

blastomeres from 16-32 cell

_embryos

into MIT enucle-ated _oocytes activated with

_ionomycin

and a

_protein

kinase

inhibitor,

6-dimethy-laminopurine

(6-DMAP) [58].

In

_previous

work,

activated mouse _eggs treated with

6-DMAP showed an accelerated

_pronuclear

organization

[71],

thereby indicating

the

inhibition of

_kinase(s)

involved in the nuclear membrane

_assembly

and chromatin

structure.

_Moreover,

these effects occurred without _any interference to DNA

replica-tion

_[55].

Our

_hypothesis

was found to be

correct and no structural and functional

alter-ations were detected in the nuclei of

recon-structed

_embryos

treated with 6-DMAP. With this

_{protocol, unsynchronized}

blas-tomeres can be

_safely

transferred into MII

without _any nuclear

_damage.

As in the uni-versal

_recipient

and in the G1

_protocol,

nuclei are

_exposed

to

_{reprogramming}

fac-tors

_throughout

the first cell

_cycle.

How-ever, the number of blastomeres

compos-ing

an

_embryo

limits the size of the

obtainable clones. The establishment and

maintenance in culture of

_permanent

cell lines derived from ovine

_embryos

could pro-vide an unlimited number of

genetically

identical nuclei for nuclear transfer. Such

an

_approach

was first undertaken in

_{sheep by}

Galli and co-workers

_[39],

who transferred

embryo-derived

cells into enucleated

oocytes. Four normal

embryos

were

trans-ferred and one _pregnancy was established

until

_day

45.

_Subsequent

nuclear transfer

experiments

carried out at the Roslin

Insti-tute with cultured cells derived from

embryos,

foetuses and adult

_sheep

overcame

the established limits of

_reproductive

biol-ogy

[20]

and the first lamb cloned from

somatic cell was

_produced

_[122].

These

insights opened

new

_{opportunities}

in

ani-mal

_breeding

and also confirmed nuclear transfer as the proper tool to unravel the

complex

mechanism of

_{developmental}

biol-ogy. This formidable achievement

persuaded

embryologists

to focus their attention on the nuclear transfer of foetal cells

[115]

and therefore

_{technological improvements}

should be

_expected

in the near future.

13. GENETIC MANIPULATION

Direct

_injection

of DNA into

_pronuclear

stage

embryos

was used to obtain the first

transgenic sheep

which

_produced

human

anti-emophylic

factor IX in milk

_[25].

Direct

injection

of DNA was later used for a

vari-ety

of

_{applications.}

The

_major

limit of this

technique

is the small

_proportion

of animals

that

_integrate

the _transgene into their genome.

Exogenous

DNA is

_probably

sites of the chromosomes induced

_by

the

injected

fluid. This random

_integration

would

_certainly

account for the wide _range of situations

_resulting

after

_pronuclear

injec-tion: extensive

_damage

_incompatible

with further

_development

of the

_embryo,

lethal mutation caused

_by

the

_interruption

of a

_key

gene,

silencing

of the transgene and

_finally,

in less than 1 % of the

_animals,

_{unpredictable}

expression

of the transgene

(reviewed by

Wilmut and Clark

_[121]).

In

_spite

of its

empirical

basis,

direct

_injection

of

_foreign

DNA has been used to create

_sheep

express-ing

pharmaceutical

proteins

in their milk

[123].

More accurate

_genetic

modification

can be introduced

_by

_gene

_targeting

of cul-tured cells. This

_approach

_{is based upon} recombination of the introduced gene with

complementary

sequences of the

targeted

chromosome in order to insert the

_foreign

DNA

_exactly

in the

_{predicted position.}

Homologous

recombination is now

_routinely

used for the

_genetic

modification of mouse

embryonic

stem cells.

_Successfully

modi-fied cells are then introduced into or

aggre-gated

with normal

_embryos

to

_produce

chimeras;

when

_genetically

modified cells contribute to _{the germ line of the}

_chimera,

the new character is transmitted to the

off-spring. Unfortunately,

stem cells have not

been isolated from

_embryos

of domestic

ani-mals until now. The

_production

of live lambs from nuclear transfer of a cultured cell line

[20]

has

_opened

the

_possibility

to induce

precise genetic changes

in

_species

other than mice. In

fact,

a landmark paper

_published

later

_by

the same _group

_clearly

demonstrated the

_possibility

to

_produce

_transgenic

lambs

by

transferring

genetically

modified nuclei

into enucleated

_{oocytes [93].}

Nuclear

trans-fer mediated

_transgenesis

offers several

advantages

comparing

to

_pronuclear

injec-tion.

_First,

half of the animals are _necessary to

_generate

one

_transgenic

lamb.

Further-more, no

_recipients

are wasted for

_gestation

of nontransgenic offspring.

A common

prob-lem after

_{pronuclear injection}

in the

_delayed

integration

of

_{microinjected}

DNA results in

mosaic

animals;

in contrast,

transgenic

ani-mals

_{produced by}

nuclear transfer are

entirely

transgenic.

Finally,

nuclear

trans-fer allows the sex of the animal to _be

pre-determined,

thus

_increasing

the

_efficiency

two-fold,

when the sex of the

_transgenic

ani-mal is

_critical,

for

_instance,

in milk

produc-tion.

14. ABNORMAL FOETAL

DEVELOPMENT

The birth of

_{unusually large}

lambs,

asso-ciated with abnormal deliveries and

_high

incidence of

_perinatal

death,

has been

reported

to occur after nuclear

transplanta-tion,

asynchronous

transfer and transfer of in vitro

_produced

_embryos,

_particularly

in the

presence of serum

_(reviewed

_by

Walker et

al.

_[113]).

_Apparently,

an

_{inappropriate}

envi-ronment where the

_embryo

is stored for a

relatively long

time or an extensive

manip-ulation,

such as _genome

_{reprogramming}

after nuclear

transfer,

disturbs genes

con-cerned with the

_regulation

of foetal

_growth.

Like all the laboratories

_dealing

with the

majority

of

_embryo

_{manipulation,}

we

expe-rienced all the

_aspects

of the so-called

_large

calf-lamb

_syndrome.

_Apparently,

there is

no or

_only

_{very little maternal contribution}

to

labour,

_probably

as a _consequence of the

absence of hormonal

_signals

_{produced by}

the foetal adrenal

_gland

at the end of the

pregnancy.

Moreover,

we have observed the occurrence of undershot lower

_jaws

in lambs derived from in vitro

_produced

and cloned

_embryos;

the same

_phenotype

was

noted from Schnieke and co-workers

_[93]

in all the lambs derived from a cell line.

Interestingly,

the same

_phenotype

was

described in

_entirely

derived stem cell mouse

embryos

as a result of

_deregulation

of

imprinted

genes

during

in vitro culture

[32].

15. CONCLUSION

The domestication of

_{sheep played}

a

major

role in the

_development

of the

Nowa-days,

sheep

breeding

has an

_important

impact

on the _economy of

_large

regions

such as

_Italy,

France,

Spain

and Greece. The last two decades have

_dramatically

_changed

the

management

of

_sheep

_breeding,

_mainly

dairy

sheep, through improved

pasture,

nutrition and

_veterinary

assistance.

How-ever,

despite

these

improvements,

artificial

insemination is the

_{only reproductive}

tech-nology applied

in _{selection programmes.} A

large

number of reviews on the

impact

of

reproductive technologies

on

_genetic

improvement

has been written so far but it is

difficult to draw

_precise

indications from them. MOET can increase

genetic

gain

from 15 to 40 % in small and _up to 100 % in

_large

flocks

_according

to some authors

_[77]

or

have

_negligible

effects

_according

to others

[9].

All these considerations are _{based upon}

theoretical models because to our

knowl-edge,

no field trial has been undertaken to

evaluate the

_impact

of these

_reproductive

technologies

on

_{sheep breeding strategies.}

However,

several

_sheep

breeds,

namely

in

the Mediterranean

basin,

have evolved into

dairy

models closer to the cattle with

mod-em

_husbandry

methods

_{including milking}

machines. This situation lets us

_perceive

that

_{reproductive technologies}

other than

AI are

_going

to be considered

_by

geneti-cists.

_Among

the

_technologies

described

here,

some have a limited

_perspective

for

improvement, particularly

the

_surgical

or

laparoscopic

procedures.

The

_principal

ones,

however,

like

_{superovulation}

and all in vitro

technologies

including

cloning,

still have a

large

margin

for

_improvement.

Technolo-gies

like

_cloning

or

transgenesis

which are

expensive

and

_{technically complex,}

are

already employed

by biotechnological

com-panies

and the

_production

of

_{pharmaceutical}

proteins

from

_transgenic

animals _{is very} close to

_reaching

commercial

_{applications.}

Nevertheless,

it is essential that factors

responsible

for abnormal foetal

develop-ment,

resulting

from in vitro

_{manipulation,}

are identified and removed before a

large-scale

_application

of these

_technologies

can

be made.

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