(Received 29th July 1963)

EGG TRANSFER

IN

SHEEP

EFFECT OF DEGREE OF SYNCHRONIZATION BETWEEN DONOR

AND

_RECIPIENT,

AGE OF

_EGG,

AND SITE OF TRANSFER ON

THE SURVIVAL OF TRANSFERRED EGGS

N. W. MOORE and

J.

N. SHELTON

The

_McCaughey

Memorial

_Institute,

_Jerilderie,

Jf.S. W. and

Department of

Animal

_{Husbandry, University of Sydney,}

Sydney,

Australia

(Received

29th

_July

₁₉₆₃₎

Summary.

A factorial

_experiment

is described in which fertilized

sheep

eggswere transferred ata rate oftwo _per animal to 162

_recipient

ewes all ofwhich were allowed to _go to term.

The factors included and the results obtained were:

(1)

Degree

of

_{synchronization}

between donorand

_recipient.

Transfers were made to

_recipients

in oestrus 48 hr

_before

to 48 hr

_after

their

respective

donors.

_{Highly significant}

effects were observed in the number ofeweswhich lambed

(P<0\m=.\01)

and number of_eggs

surviving

to

_{parturition (P<0\m=.\001),}

with maximum results inewesinoestrus 12 hr before to 12 hr after their

_respective

donors.

(2) Age

of_eggs.

_Eggs

were collected 48 to

60,

60 to 72 and 72 to 84

hr after the donorswerefirst observed inoestrus. Therewas a

significant

increase in the number of ewes which lambed

(P<0\m=.\02)

and lambs born

_(P<0\m=.\01)

with increase _{in age} of the transferred _eggs.

(3)

Site oftransfer. Transfers were made to the

_Fallopian

tubes or uterine horns. Tubal transfers were

significantly

more successful than uterine transfers in the number ofewes which lambed

(P<0\m=.\05)

and number oflambs born

_{(P<0\m=.\01).}

Therewere no

significant

interactions. INTRODUCTION

The

_potential

_{value of egg transfer in farm}

_animals,

both for

_practical

_purposes and as a research

tool,

has been

amply

demonstrated. In

sheep,

Rowson & Adams

₍₁₉₅₇₎

and Moore & Shelton

_(1962a)

have shown that

_large

numbers of lambs can be obtained from selected ewes, whilst Averill & Rowson

(1958)

and Moore & Rowson

_{(1959, 1960)}

have used the

_technique

to

_study

the survival and

_development

of fertilized

_sheep

_eggs.

_However,

in this

_species

precise

information is

_lacking

on _many of thefactors which affect the survival and

_development

of transferred _eggs.

J.

This_{paperpresents} the results ofan

experiment designed

to

_study

the effects

on survival of_age of_egg

transferred,

degree

of

synchronization

between the

reproductive

state of donors and

_recipients,

and the site to which _eggs are transferred.

MATERIALS AND METHODS

In the autumn of

1962,

324 fertilized _eggs collected from mature Merino

ewes,

_multiple

ovulated with a horse anterior

pituitary

_extract

(Moore

&

Shelton,

1962b)

and mated to Merino rams, were transferred ata rate oftwo

per animal to 162 mature Merino

_recipient

ewes. The

experimental

animals were run under field conditions.

Table 1

experimental design

Comparison Degree of synchronization*

Timeofoestrusinrecipi¬

ents relative to donors (hr)

Age ofeggs

Interval between onset

of oestrus and egg re¬

covery (hr)

Site_{of transfer}

Description Early Similar Late

+48 +12 ^24 +36 0 -36 +24 -12 -48 48to60 60to72 72 to84 Fallopiantubes Uterine horns Factors Product 27 54 Number of animalspercell= 3;total animals= 162.

*

Degreeof_{synchronization:} + Recipients first observed in oestrus

beforetheir_respectivedonors.

—

Recipientsfirstobservedinoestrus_after

their_respectivedonors.

Detection

_of

oestrus

'Sire-sine' harnesses were attached to rams for the detection of oestrus

(Radford,

Watson &

_Wood,

₁₉₆₀₎

and ewes were

inspected

for oestrus twice

daily

at 6a.m. and6_p.m.Donorewes were runwith intactrams and

recipients

with vasectomized rams.

Collection and

_transfer

_of

_eggs

The

_procedures

used for the collection and transfer of_eggs were

essentially

those described

_by

_Hunter,

Adams & Rowson

_(1955).

Experimental

design

The

_experiment

was of factorial

design (Table 1).

Degree of synchronization. Eggs

were transferred to

_recipients

first observed in oestrus 48 hr

before

to 48 hr

_after

their

_respective

donors. Potential

_recipients

were discarded if

they

did not show at least one recent ovulation at the time

Egg

transfer

in

_sheep

147

Age

of

eggs.

Eggs

were recovered in vivo

by

flushing

with

sheep

serum the

Fallopian

tubes

_together

with about 5 cm of the most distal

portion

of the uterine horns. Their _age was classified

by

the time

elapsing

between onset of

oestrus and egg recovery. Normal

_cleavage

was taken as the criterion of fertilization and

_only

_{cleaved eggs}weretransferred. Table2 showsthe distribu¬ tion ofcell _stage relative to the timeof_recovery.

Site

_of

_{transfer. Eggs}

were transferred to the

Fallopian

tubes oruterine horns. Therewas an even distribution of cell _stagesbetween the _twosites

(Table 2).

Table 2

cell stage of eggs in relation to their_age, and distribution of

cell stages accordingto site of transfer Age of 48to 60 60to72 72 to 84 Site of transfer Tubes Uterus Tubes Uterus Tubes Uterus

No. eggsatvarious cell_stages

25 22 26 29 14 13 10 26 26 43 48 >8 Total transferred 54 54 54 54 54 54

*_{Interval betweenonsetofoestrusandegg recovery (hr).}

Lambing

All the

_recipients

were allowed to _go to term.

_{During lambing they}

were

inspected

at least once

daily

when

recently

born lambs were identified.

Analysis of

results

Standard

₂

tests and

analyses

of variance were

applied

to the raw or

appropriately

transformed data. For

_simplicity

of

_analysis

the nine

_degrees

of

synchronization

were

grouped

into three

classes,

namely 'early' (recipients

48,

36 and 24 hr before

_donors),

'similar'

₍₁₂

hr

_before,

0 and 12 hr

_after)

and 'late'

_(recipients

_24,

36 and 48 hr after

_donors).

RESULTS

Eighty-three

of the 162

_{recipients subsequently}

lambed

_producing

117 lambs

(Table 3).

Degree

of synchronization

There was a

highly

significant

effect of

degree

of

synchronization

on the number of ewes which lambed

(P<0-01)

and the number of lambs born

(P<0-001).

Maximum survival occurred in

_recipients

in oestrus 12 hr before

J.

survival of _eggs which were out of

_phase

seemed unrelated to whether

they

were 'older' or

'younger'

than therelative _stage of

development

ofthe _repro¬ ductive tract of the

recipient

(Tables

4and

5).

Age

of

egg

transferred

The older the _egg, the _greater was the chance of survival

(ewes

which

lambed, P<0-02;

lambs

_{born, P<0-01;}

Table

_3).

Table 3

results-main_effects;numberofrecipient eweswhichlambed

and number of lambsborn

Main_effect Degreeofsynchronization(hr)* +48 'Early' +36 +24 +12 'Similar' 0 -12 'Late' -24 -36 -48 —linear —quadratic —cubic Age ofeggs (hr) 48to60 60to72 72 to84 —linear —quadratic Site_{of transfer} Tubes Uterus Totals

No.ewes_{which lambed}

(n = 18) 7 7 8 14 12 11 9 8 7 n.s. <001 n.s. (n = 54) 23 25 35 <002 n.s. (n=81) 48 35 <005 (n = 162) 83

No. lambs born

(n -36) 8 9 13 19 18 16 13 12 9 n.s. <0-001 n.s. (n = 108) 31 35 51 <001 n.s. (n= 162) 72 45 <0-01 (n=324) 117 *_For

analysis, comparisonwasmade of_'early','similar' and 'late' classes. For the_{'early' class,}the_recipientswereinoestrusbefore the_donors; for the 'late'class,after.

Therewerenosignificantinteractions.

Site

_of

_transfer

Tubai transfers were more successful than uterine transfers in the number ofeweswhichlambed

(P<0-05)

and number oflambsborn

(i3<0-01

;Table

3).

Interactions

Egg

transfer

in

_sheep

149

Table 4

NUMBER OF EWES WHICH LAMBED AND LAMBS BORN IN RELATION TO AGE OF REPRODUCTIVE TRACTRELATIVE TO THAT OF TRANSFERRED EGGS

Site_of

transfer eggsAge(hr)of

Cell

stage

Eweswhichlambed(n= 9)

Ageof reproductivetract

relativeto_eggs Older +48to +24hr Similar +12to -12hr Tounger -24to -48 hr

No. lambsborn (n = 18)

Age of reproductivetract

relativeto_eggs Older +48to +24 Ar Similar +12to -12Ar Tounger -24/0 -48 Ar Tubes 48 to60 60to72 72to84 2to6 4to8 6to >8 Total 13 19 16 17 8 14 30 7 7 11 25 Uterus 48 to₆₀ 60to72 72to84 2to 6 4to8 6to >8 Total 13 23 Table 5

percentage of transferred eggssurviving inewes which lambed

Site of transfer Age of egg* (hr) Eggs surviving (%) Synchronization (hr) +48to ₊₂₄ +12to -12 -24to -48 Total Tubes Uterus 48to60 60to72 72 to84 Total 48to60 60to₇₂ 72to84 Total 67 80 50 80 67 65 79 63 50 62 64 64 72 64 70 88 79 75 50 50 62 56 73 77 75 75 60 60 70 64 Table of 2 Source _d.f Synchronization Ageof_egg Site of transfer Interaction Total 0-05 0-12 0-67 2-45 3-29 2 2 1 12 17 n.s. n.s. n.s. n.s.

Analysis

of

eggsurvival

Eighty-three

ewes which lambed received 166 eggs of which 117

(70-5%)

survivedtolambs. Survival in theseewes wasunaffected

by degree

of

synchroni¬

zation,

age ofeggs or site oftransfer

(Table 5).

Table 6 _presents a _summary of _egg losses. A total of

63-9%

was

lost,

the

majority (48-8%)

as a result ofloss ofwhole

pregnancies.

Table 6

summaryoflosses of eggs

No. A. Total losses

Recipientewes(total)

Eggstransferred

Eggslost ineweswhichfailedtolamb Eggslostineweswhich lambed

162 324 158 49 100-0 48-8 151

Total eggslost Total_{eggssurviving}

207 117

63-9 36-1

B. Losses ineweswhich lambed

Recipienteweswhich lambed

Eggstransferredtoeweswhichlambed

Lambs born Eggslost 83 166 117 49 1000 70-5 29-5 DISCUSSION

The absence of _any

_significant

interactions _suggests that the three factors

studied,

degree

of

_{synchronization}

_{between egg} and _{tract, age} of_{egg, and} site of transfer act

independently

ofoneanother.

Maximum survival occurred in

_recipients

first observed in oestrus within

12 hr oftheir

_respective

donors.

_However,

_recipients

did show some tolerance

to_{eggs up}to 2

days

'older' or

'younger'

than

themselves,

withnoevidence that either the older or _younger

reproductive

tract

presented

a more suitable transfer site. In this_respectthe

_sheep

issimilar tothe

rabbit,

somewhat similar

to the _rat, but dissimilar to the mouse. In the

rabbit,

Chang

(1950)

recorded maximum survivalin

_{recipients exactly synchronized}

with their

_donors,

while

Noyes

& Dickman

_(1960,

₁₉₆₁₎

found that rat _eggs of the same _age or 1

day

older than the uterus were more

likely

to survive than those 1

day

_younger. Maximum survival in the mouse was obtained

by

transferring

3|-day

_eggs to

2|-day

recipients;

it decreased with

_{exactly synchronized}

transfers of both

2\-

and

_3J-day

_eggs, whereas

_virtually

no _{eggs of}

2\

days

survived when transferred to

3J-day

recipients (McLaren

&

Michie,

1956).

Egg

survival increased

_{significantly}

with _age of _egg transferred. Whether younger eggs are more

susceptible

to the collection and transfer

procedures,

or

totransfer

itself,

is notclear.

However,

ithas been

postulated (Robinson,

1951)

and limited evidence has been

_{presented (Averill}

&

_Rowson,

₁₉₅₈₎

that the uterus does not

_provide

an environment suitable for the survival and

develop¬

Egg transfer

in

_sheep

151 Tubai transfers were more successful than were uterine transfers. This appears to

disagree

with the observations of Moore & Rowson

(1960)

and

Moore & Shelton

_{(1962a). They reported}

the survival of _eggs of less than

eight

cells transferred to the tubes to be

_{significantly}

less than that of eggs of

eight

cellsor moretransferredtothe uterus.

_{They suggested}

that the difference

was due to site of transfer.

However,

site of transfer and age of _eggs were confounded. It isnow clear that both factors are

important.

There are two

possible explanations

for the

superiority

of tubai transfers.

First,

further time

_spent

in the tubes would result in an advance in the

_stage

of

_development

_{of eggs}

_by

the time

_they

reached the uterus.

Second,

the tubai

environment _may condition the_eggs for

_{reception by}

theuterus. It is

_apparent

that

_precise

_{relationships}

between the _age of _{eggs and their}

_position

within the

_reproductive

tract must be satisfied if maximum survival is to occur. A

general

decrease in

_fertility

isacharacteristic ofalmostallworkonthehormonal control of

_breeding

in the ewe

(Robinson,

1959)

and in some instances is due to the

_premature

_appearance of_eggsin the uterus. Both Robinson

₍₁₉₆₂₎

and Shelton

_{(unpublished data)}

have evidence

_{suggesting rapid}

tubai

_transport

of eggs

following

progesterone

treatment of the

_cyclic

ewe.

Egg

losses had a

_strong

tendency

to fallonlitters as awhole. That

is,

success

or failure tended to be an all-or-none

phenomenon. By analogy

with the detailed

_{analysis by}

Brambell

₍₁₉₄₈₎

on

pre-natal

losses in the wild

rabbit,

it canbeconcludedthatwhen either the_stageof the

cycles

of donorand

recipient

are _out of

phase,

or the _eggs are in the

early

_stagesof

cleavage,

or are

placed

directly

into the uterus, the maternal environment in a

high proportion

of casesisso

unsatisfactory

asto resultintotal loss ofthe_pregnancy. On the other

hand,

when the maternal environment is

_suitable,

as in the

eighty-three

ewes which

_lambed,

_egg losses are at random andare not excessive.

In orderto obtain maximum survivalof transferred eggs in the

_sheep,

three

conditions should be satisfied.

_First,

_eggs should be collected 72 hr or more

after the onset of oestrus,

second,

transfers should be made to the

_Fallopian

tubes,

and

_third,

_recipients

should bein oestrus no more than 12 hr before or after the donors. When these conditions are satisfied some

75%

of

recipients

should

_subsequently

lamb.

ACKNOWLEDGMENTS

This

_project

wasfinanced in

_part

by

the

Sheep

and Wool Research Committe« of Australia to whom

grateful acknowledgment

is made.

Mr B.

_J.

Carlin

_provided

valuable technical assistance.

REFERENCES

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Brambell,F. W.R. ₍₁₉₄₈₎ Prenatal_mortalityin mammals. Biol.Rev.23, 370.

Chang,M.C. _{(1950) Development}andfateof transferred rabbitova or_{blastocystsin relation} tothe ovulation timeof_{recipients. J.exp. £ool.}114, 197.

Hunter,G._L.,Adams,C.E. &Rowson,L. E. A. ₍₁₉₅₅₎Inter-breedovumtransfer in_{sheep. J. agrie.} Sci._46, 143.

J.

McLaren,A.&_Michie,D. ₍₁₉₅₆₎ Studiesonthe transfer of fertilizedmouse _eggs to uterine foster-mothers. I. Factors _affecting the _implantation and survival of native and transferred eggs.

J.exp.Biol.33, 394.

Moore, N. W. & Rowson, L. E. A. _{(1959) Maintenance of pregnancy in ovariectomized} ewes by meansof_{progesterone.Nature,}Lond. 184,1410.

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