Enhanced maintenance of pregnancy using leukaemia inhibitory factor in embryo culturing

The present invention relates to the use of leukemia inhibitory factor (LIF) in the enhancement of development and maintenance of mammalian embryos, particularly sheep embryos. It has been observed that following the introduction into foster mothers of embryos cultured in vitro in the presence of LIF, the maintenance of pregnancy is enhanced relative to that seen following introduction of embryos that had not been cultured with LIF prior to introduction into foster mothers.

The present invention relates to the use of Leukemia Inhibitory Factor 
(LIF) in the enhancement of development and maintenance of animal or 
mammalian embryos and the use of same to enhance impregnation. 
LIF is a protein that has previously been cloned, produced and purified in 
large quantities in recombinant form from both E. coli and yeast cells 
(International patent application Ser. No. PCT/AU88/00093). LIF has been 
defined as a factor, the properties of which include: 
1. the ability to suppress the proliferation of myeloid leukaemic cells 
such as M1 cells, with associated differentiation of the leukaemic cells; 
and 
2. the ability to compete with a molecule having the defined sequence of 
murine LIF or human LIF (defined in International patent application Ser. 
No. PCT/AU88/00093) for binding to specific cellular receptors on M1 cells 
or murine or human macrophages. 
A major difficulty associated with present in vitro fertilisation (IVF) and 
embryo transfer (ET) programs, particularly in humans, is the tow success 
rate "achieved" on implantation of fertilised embryos. Currently, in human 
IVF programs, the implantation rate may be as low as 10%, leading to the 
present practice of using up to four fertilised embryos in each treatment 
which, in turn, leads occasionally to multiple births. Accordingly, there 
is a need to improve the implantation rate in human IVF programs. 
Similarly, in IVF and ET treatments in domestic animals such as sheep, 
cattle, pigs and goats, it is highly desirable for economic reasons to 
have as high an implantation rate as possible so as to reduce the numbers 
of fertilised embryos lost and unsuccessful treatment procedures 
performed. Furthermore, as with human IVF procedures, the practice of 
transferring more than one embryo to the recipient animal to ensure 
pregnancy can result in unwanted multiple births. 
One major constraint with embryo transfer is the need to hold embryos in 
culture media for either relatively short periods of time, perhaps only a 
few hours prior to transfer or for longer periods of some days, after 
micromanipulation. 
In the development of a mammalian embryo, the fertilised egg passes through 
a number of stages including the morula and the blastocyst stages. In the 
blastocyst stage, the cells form an outer cell layer known as the 
trophectoderm (which is the precursor of the placenta) as well as an inner 
cell mass (from which the whole of the embryo proper is derived). The 
blastocyst is surrounded by the zona pellucida, which is subsequently lost 
when the blastocyst "hatches". The cells of the trophectoderm are then 
able to come into close contact with the wall of the uterus in the 
implantation stage. Prior to formation of the embryo proper by the inner 
cell mass by gastrulation, the whole cell mass may be referred to as 
"pre-embryo". 
Embryo mortality has been attributed to incomplete hatching of the 
blastocyst from the zona pellucida and/or unsuccessful implantation of the 
embryo to the uterine wall, possibly due to spontaneous differentiation of 
the embryonic stem cells (ES) during their period in culture prior to 
transplantation. 
In accordance with the present invention, it has been found that when LIF 
is included in an in vitro embryo culture medium, the hatching process is 
enhanced leading to an increased number of embryos completing the 
development stage by undergoing developmental changes associated with 
implantation. Thus, LIF is an embryo protective agent. As a result, the 
implantation rates for IVF and ET programs can be, and are, significantly 
improved by the use of LIF in the in vitro embryo culture medium. 
Furthermore, media containing LIF is suitable for use in early manipulative 
procedures on the oocyte/embryo such as in vitro fertilisation, embryo 
splitting and nuclear transfer where survival rates of embryos are low. 
LIF also has important applications in the growth of totipotent stem cell 
lines for cloning for inclusion into the media used for the transport of 
cooled or frozen embryos/semen. 
Unless otherwise specified, use of "LIP" herein refers to synthetic 
recombinant or naturally occurring human, murine and/or livestock or 
ruminant LIF, such as from sheep, pigs, cows, goats, donkeys and horses 
and from other animals such as dogs, cats or birds (eg. chickens) and to 
derivatives or parts thereof. Such derivatives or parts thereof include 
any one or more contiguous series of amino acids contained within any one 
of the above LIEF molecules and includes single or multiple amino acid 
substitutions, deletions and/or additions to or in the natural or 
synthetic LIF molecule. Conditions for preparing recombinant LIF are 
disclosed in International Patent Application Nos PCT/AU88/00093 and 
PCT/AU90/00001 although variations and/or modifications to these 
conditions may vary depending on the host cell used. Any such variations 
and/or modifications are within the scope of the subject invention. The 
host cells may be eukaryotic (eg. yeast, mammalian, insect, plant etc.) or 
prokaryotic (eg. Escherichia coli, Bacillus sp, Pseudomonas sp etc.) 
cells. 
Accordingly, one aspect of the present invention contemplates a method for 
enhancing the impregnation rate in an animal with one or more embryos said 
method comprising maintaining and/or developing the embryos in a medium 
containing an effective amount of leukaemia inhibitory factor (LIF) for 
sufficient time and under appropriate conditions and then implanting the 
embryos into the animal. 
By "impregnation" means the rate of successful implantations and subsequent 
development of a fertilised embryo in 
Another aspect of the present invention contemplates a method for 
maintaining embryos or pre-embryos in in ELtX culture while retaining 
viability for use in embryo transfer, IVY: and/or genetic manipulation 
which method comprises culturing said embryos in a medium containing an 
effective amount of LIF for sufficient time and under appropriate 
conditions. 
This method of maintaining the viability of embryos in culture has 
potential for allowing genetic manipulation of the whole embryo. Such 
successful genetic manipulation is restricted at the present time due to 
the limited amount of time available to perform experiments on viable 
embryos. 
The method also may be advantageous in maintaining viability of embryos 
under transport conditions and may also be beneficial in the storage of 
embryos when compared to techniques currently employed. 
Another aspect of the present invention relates to a method for enhancing 
the in vitro development of a mammalian embryo to the implantation stage, 
which method comprises the step of culturing the embryo in vitro in a 
culture medium containing an effective amount of mammalian LIF. 
As is demonstrated below the inclusion of LIF in the culture medium prior 
to the formation of the blastocyst, or both prior to and following 
blastocyst formation, also increases the number of pre-embryos completing 
the developmental stage by undergoing development changes associated with 
implantation. The addition of LIF also reduces the number of pre-embryos 
degenerating while in culture. As a result, the implantation rate for IVF 
and ET programs can be significantly improved by use of LIF in the in 
vitro culture medium. 
"Animal embryos" is used in its broadest sense encompassing embryos from 
mammals such as humans, ruminant and other livestock animals and other 
animals such as birds and fish. It will be appreciated that while the 
subject invention is exemplified herein by the development ovine embryos 
in vitro, the present invention extends to the use of LIF in the 
development of embryos of other animal or mammalian species including 
humans, ruminants and animals such as cattle, horses, donkeys, goats and 
the like. 
The present invention, also extends to a method for in vitro fertilisation 
and subsequent implantation of a mammalian embryo which is characterised 
in that the embryo is cultured in vitro in a culture medium containing an 
effective amount of mammalian LIF prior to transfer into animal or 
mammalian host, where "host" is defined as a suitably receptive female 
animal or mammal. 
A further aspect of the present invention relates to a non-human animal and 
in particular a chimaeric non-human animal or transgenic progeny of said 
animal generated by known techniques using ES cells which have been 
maintained in vitro in LIF-containing culture medium. In accordance with 
this aspect of the present invention, ES cells are derived from animal 
embryos passaged in a culture medium containing LIF wherein said ES cells 
have additional genetic material inserted therein. The transgenic animals 
contemplated include nonhuman mammals such as livestock and ruminant 
animals and domestic animals. 
In accordance with the present invention, "homologous" or "heterologous" 
systems may be employed meaning that the animal from which LIF is derived 
is the same animal (homologous) or a different animal (heterologous) from 
which the embryos are isolated. The LIF may be naturally occurring but is 
preferably recombinant or synthetic LIF. The LIF may be of any origin such 
as human, murine or livestock animal (including ruminant animal) LIF 
provided that the LIF has the desired effect. It may be, for example, that 
a LIF from one animal may not be as active as a LIF from another animal. 
It is within the skill of the addressee to readily screen for suitable 
LIFs from appropriate animals. Where recombinant LIF is used, it may be 
produced in eukaryotic or prokaryotic cells. 
The present invention is also directed to composition comprising an 
effective amount of LIF in combination with an animal (eg. mammalian) 
embryo maintaining medium. The present invention also provides a 
composition having embryotrophic and/or embryo protective properties 
comprising LIF in combination with one or more of ovine trophoblast 
protein, Interleukin 1 and/or 2, macrophage colony stimulating factors, 
platelet activating factor, a factor in the murine fibroblast 3T 3 line 
and/or plasminogen. The above composition may also be in combination with 
an embryo maintaining medium. 
An embryo maintaining medium as contemplated herein includes but is not 
necessarily limited to SOF and/or M2. 
The amount of LIF used in accordance with the present invention is that 
required to maintain and/or develop embryos and/or enhance impregnation 
and is in the range of 100 units/ml to 10,000 units/ml, preferably 500 
units/ml to 5,000 units/ml and most preferably from 1,000 units/ml to 
5,000 units/mi. A unit of LIF activity is defined in PCT/AU88/00093. 
The present non-limiting examples further illustrate the present invention:

EXAMPLE 1 
Materials and Methods 
EMBRYO COLLECTION 
Forty mature Merino ewes were treated for 14 days with a CIDR containing 
0.3 g progesterone (Riverina Artificial Breeders, NSW) and were 
superovulated by the injection (i.m.) of 5 mg of the pituitary 
follicle-stimulating hormone FSH-P (Intervet Australia Ply, Ltd, Sydney, 
NSW) and 200 iu of PMSG (i.m.) (Pregnecol; Heriot Agvet Pty, Ltd, 
Melbourne, Vic) 48 h before CIDR withdrawal, followed by decreasing doses 
of FSH-P (4, 3, 3, 2, and 1 mg) every 12 h afterwards. Ovulation was 
ensured by a 50 .mu.g injection (i.m.) of Gonadotrophin-Releasing hormone 
(GnRH;Auspep Pry, Ltd, Melbourne, Vic) 24 h after CIDR withdrawal, which 
coincided with the time that most ewes were detected in oestrus by 
vasectomised rams fitted with marking crayons. Ewes were inseminated via a 
laparoscope with approximately 100.times.10.sup.6 fresh motile sperm into 
each uterine horn 8-12 h after oestrus. The semen was collected from 4 
fertile rams, diluted 1:1 in phosphate buffered saline (Flow Laboratories, 
North Ryde, NSW) and kept at ambient temperature prior to insemination. 
Six days after oestrus the ewes were anaesthetised with an injection 
(i.v.) of the barbiturate sodium thiopentone (Intraval: May & Baker, 
Footscray, Vic) and maintained under general anaesthesia by a mixture of 
halothane (May & Baker) and oxygen. The uterus was exteriorised by a 
midventral laparotomy and each horn was cannulated with a Foley catheter 
(Promedica, Moorabin, Vic) at approx. 1 cm below the bifurcation. A 20 g 
needle attached to a 20 ml syringe filled with flushing media was passed 
into the lumen of the uterus near the uterotubal junction. Each horn of 
the uterus was flushed and the media plus embryos were collected into 
glass vessels via a Foley catheter. Embryos were pooled in M2 holding 
media containing 4 mg/ml Miles BSA (Pentex Crystalline; Miles Diagnostics, 
Kanakee, Ill., USA) and graded according to health and stage of 
development before allocation to treatment groups. 
CULTURE OF EMBRYOS 
Embryos (166 assessed as healthy and 17 as poor quality) were randomly 
allocated to one of three treatment groups. Embryos in treatment 1 (80 
healthy and 5 poor quality embryos at the morula or early blastocyst 
stage) were cultured in lots of 10 for 1-2 h in 5 ml of M2 holding media 
prior to being transferred either as pairs (Group 1; n=38) or singularly 
(Group 2; n=47) into recipient ewes at Day 6 of the oestrous cycle. Group 
3 embryos (44 healthy and 6 poor quality embryos at the morula or early 
blastocyst stage), were cultured individually in SOF media for 48 h prior 
to transfer. Three separate 100 .mu.l droplets of SOF culture media were 
placed in a 35.times.10 mm plastic Petri-dish and covered with 2.5 ml 
paraffin oil (Labchem, Ajax Chemicals, Auburn, NSW). One embryo was then 
placed in each droplet and the Petri-dishes were placed in a waterjacketed 
incubator and were maintained at 39.degree. C. in an atmosphere containing 
20%O.sub.2, 5% CO.sub.2 and 75% N.sub.2. Group 4 embryos (44 healthy and 6 
poor quality morula or early blastocysts) were cultured for 48 h in SOF 
culture media as for Group 3 except that human recombinant LIF was added 
to the media at a rate of 1000 units/ml). 
The number of healthy or poor quality (degenerating) embryos, those 
achieving morula, blastocyst or hatching blastocyst stage of development 
was recorded at the time of embryo collection, every 12 h afterwards and 
immediately prior to transfer to recipient ewes at Day 8 after oestrus. 
CULTURE MEDIA 
The flushing media used was Dulbecco's phosphate buffered saline containing 
10% (v/v) foetal calf serum, Penicillin G. potassium salt (0.060g/l) and 
Streptomycin sulphate (0.050g/l). 
M2 culture media (Quinn et al, 1982) was a modified Krebs-Ringer solution 
with some of the bicarbonate substituted with HEPES buffer. The components 
were HEPES buffer (4.969 g/l), NaCl (5.533 g/l), KCl (0.356 g/l), 
CaCl.sub.2.2H.sub.2 O (0.252 g/l), KH.sub.2 PO.sub.4 (0.162 g/l), 
MgSO.sub.4.7H.sub.2 O (0.293 g/l), NaHCO.sub.3 (0.349 g/l, Penicillin G. 
potassium salt (0.060 g/l), Streptomycin sulphate (0.050 g/l) made up to 1 
liter with Millipore H.sub.2 O. 
SOF culture media (Tervit et al., 1972) consisted of NaCl (6.95 g/l), KCl 
(0.534 g/l), KH.sub.2 PO.sub.4 (0.162 g/l), CaCl.sub.2 2H.sub.2 O (0.252 
g/l), MgCl.sub.2 6H.sub.2 O (0.10 g/l), NaHCO.sub.3, (2.106 g/l), Na 
lactate (0.616 g/l), Na pyruvate (0.0363 g/l), Glucose (0.270 g/l), BSA 
(32.0 g/l), Penicillin G. potassium salt (0.060 g/l), Streptomycin 
sulphate (0.050 g/l) made up to 1 litre with Millipore H.sub.2 O. 
All media was sterilised by filtration through a 0.2 .mu.m filter 
(Millipore Pry, Ltd, Richmond, Vic). 
EMBRYO TRANSFER 
Two hundred 3 year old malden Merino ewes had their oestrous cycles 
synchronised with a 14 day CIDR treatment. An injection of 400 UI PMSG was 
given at the time of CIDR withdrawal. Vasectomised rams, fitted with 
harnesses and crayons were placed with the ewes to detect oestrus. Ewes 
observed in oestrus were randomly allocated to one of 4 recipient groups. 
Recipient ewes were treated with local anaesthetic (lignocaine, Lyppard 
Chemicals, Brighton, Vic) and using laparoscopy, the number of corpora 
lutea on each ovary was recorded. The uterus was located and the tip of 
the uterine horn, ipsilateral to an ovulating ovary was exteriorised 
through a 2 cm midventral incision. The uterus was punctured approximately 
4 cm from the tip and the embryo, bathed in Ms holding media was deposited 
via a Tomcat catheter (Size 3.5 FR, Lyppard Chemicals, Brighton, Vic) 
attached to a 1 ml syringe. The uterine horn was returned into the 
abdominal cavity and the incision sutured. 
All recipient ewes were grazed with harnessed vasectomised rams for 21 days 
to provide an estimate of embryonic loss prior to or at implantation 
(Edey, 1967). Ewes were scanned using ultrasonics on Days 70 of pregnancy 
to determine the number of healthy foetuses. 
ANALYSIS 
Differences between groups were determined by Chi-square analysis. 
EXAMPLE 2 
Effect Of LIF On In Vitro Development Of Ovine Embryos To The Post-Hatching 
Stage 
EMBRYO CULTURE 
The addition of 1,000 units/ml of human recombinant LIF to SOF culture 
media significantly improved the development (more blastocysts hatching 
and less degenerating) of healthy morula and blastocyst embryos cultured 
for 48 h in vitro (Table 1 ). 
Both treatment groups had 6 embryos initially classified as poor at the 
time of embryo recovery. Over the treatment period their health did not 
improve so they were discarded and not transferred to recipient ewes. 
IMPLANTATION RATES 
When the cultured embryos were individually transferred to recipient ewes 
the 21-day non return rate (an indicator of implantation rate) of ewes 
receiving an embryo that had been cultured in SOF+LIF (Group 4) was 
significantly higher than that of ewes receiving an embryo culture in SOF 
alone (Group 3: Table 2). Furthermore, the implantation rate of the Group 
4 ewes was similar to recipient ewes that had received a single embryo 
within 2 h of collection (group 2), but the non-return rates of both 
groups were lower than that of recipient ewes that had two embryos 
transferred soon after collection (Group 1). 
Actual pregnancy rates, as determined by real time ultrasonic scanning on 
Day 70 of pregnancy are shown in Table 3. 
The results given above demonstrate that the addition of human recombinant 
LIF to culture media increases up to 4-fold the number of ovine 
blastocysts that "hatch" from the zona pellucida and decrease the number 
of embryos degenerating during their 48 h culture in SOF at an atmosphere 
of 20% O.sub.2, 5% CO.sub.2 and 75% N.sub.2. In fact 64% of the embryos 
cultured in SOF+LIF had hatched by Day 8 after oestrus which is similar to 
that found in vivo (Rowson and Moor, 1966, Bindon, 1971). LIF may have a 
role in maintaining the health of embryos under adverse conditions. 
Furthermore, during this period of culture the pregnancy rates of the 
recipient ewes after the transfer of embryos cultured in media containing 
LIF for 48 h was at least equal to that of ewes receiving an embryo 
immediately after collection. 
This stabilising role of LIF on embryos agrees with the recent reports of 
the localisation of LIF receptors on the Day 4 murine embryo and of murine 
LIF being expressed strongly in the endometrial glands of the Day 4 
pregnant and pseudopregnant mouse. This suggests that LIF is produced as a 
passive response to pregnancy rather than to the presence of the embryo. 
LIF therefore appears to be produced at the time that the embryo is 
entering the uterus from the oviduct. The results herein suggest that some 
of the embryonic mortality reported at around this time could be averted 
by an adequate supply of LIF to the embryo. Currently, in most embryo 
transfer programs in both humans and livestock animals, more that one 
embryo is transferred to each recipient to ensure a viable pregnancy. Our 
study indicates that pregnancy rates are almost 40% higher when 2 embryos 
are transferred to each ewe even though there is still about a 50% loss of 
embryos (52% of these ewes had twins, 37% had singles and 11% were not 
pregnant). Hence, there is a possibility that LIF could prevent some of 
this embryonic loss and effectively make the transfer of a single embryo 
to each recipient practical. 
TABLE 1 
______________________________________ 
Number and percentage of healthy embryos 
hatching or degenerating during 48 h culture in media 
SOF (n = 42) or SOF + 1000 units/ml LIF (n = 44). 
Time Hatched Embryos Degenerating Embryos 
in Culture 
SOF SOF + LIF SOF SOF + LIF 
______________________________________ 
0 h 0 0% 0 0% 0 0% 0 0% 
12 h 0 0% 3 7% 2 5% 0 0% 
24 h 1 2% 10* 23% 2 5% 1 2% 
36 h 6 14% 19* 43% 5 11% 1 2% 
40-46 h 7 16% 28* 64% 13* 27% 4 9% 
______________________________________ 
*Denotes significant differences between SOF & SOF + LIF treatment groups 
P &lt; 0.05. 
TABLE 2 
__________________________________________________________________________ 
Ewes returning to service within 21 days after oestrus. 
Group 1 
Group 2 
Group 3 
Group 4 
1 Embryo 
2 Embryos 
1 Embryo 
1 Embryo 
3 h in M2 
3 h in M2 
48 h in SOF 
48 h in SOF + LIF 
__________________________________________________________________________ 
Returned to service 
0 13 26 15 
Not returned to service 
19 28 18 27 
Total 19 41 44 42 
% Not Returned 
100.sup.a 
.sup. 68.sup.b 
.sup. 41.sup.c 
.sup. 64.sup.b 
__________________________________________________________________________ 
Different superscripts denote significant differences (P &lt; 0.05). 
TABLE 3 
__________________________________________________________________________ 
Number of recipient ewes pregnant at Day 70 after oestrus. 
Group 1 
Group 2 
Group 3 
Group 4 
1 Embryo 
2 Embryos 
1 Embryo 
1 Embryo 
3 h in M2 
3 h in M2 
48 h in SOF 
48 h in SOF + LIF 
__________________________________________________________________________ 
Non-pregnant 
2 20 37 21 
Pregnant 17 22 7 21 
Total 19 42 44 42 
% Pregnant .sup. 89.sup.a 
.sup. 52.sup.b 
.sup. 16.sup.c 
.sup. 50.sup.b 
__________________________________________________________________________ 
Different superscripts denote significant differences (P &lt; 0.05). 
EXAMPLE 3 
THE USE OF LIF IN EMBRYO CULTURE AND TRANSFER 
Ovine embryos were collected from merino ewes 6 days after oestrus as 
described above. This time, however, they were cultured individually for 
10 days in either SOF, SOF+murine LIF or SOF+human LIF and development 
assessed daily. The intention was to see if and how far the embryos 
developed post hatching in media containing LIF such as whether they reach 
the expanded blastocyst or trophoblast stages of development which occurs 
at around Days 10-12 in vivo. Secondly, the effect of murine LIF on the 
development of cultured sheep embryos was also investigated. 
Embryos were collected from merino ewes in exactly the same manner as in 
Example 1. The only difference being that the ewes were superovulated by 
either the FSH products Ovagen or RFSH-50 supplied by Horizon 
Reproduction. The treatments were: 
Group 1 (n=19); Embryos cultured individually in SOF alone. 
Group 2 (n=18); Embryos cultured individually in SOF+murine LIF. mLIF added 
at 3500 units/ml by mouse stem cell bioassay which equates to approx. 8000 
units/ml using the Ml cell bioassay. 
Group 3 (n=20); Embryos cultured individually in SOF+human LIF (hLIF). hLIF 
added at 5000 units/ml by MI cell bioassay. 
The culture conditions were identical to the above examples except that 
embryo development was assessed daily rather than twice daily. The 
atmospheric condition were as described above, i.e. 20% O.sub.2, 5% 
CO.sub.2, and 75% N.sub.2. 
The results are shown in Table 4. 
TABLE 4 
__________________________________________________________________________ 
Days in Culture 
Treatment 
0 1 2 3 4 5 6 7 8 9 10 
__________________________________________________________________________ 
SOF 10 B 
19 B 
13 B 
11 B 
1 HB 
1 HB 
19 D 
9 M 6 D 
8 D 
9 B 
5 B 
9 D 
13 D 
SOF+ 9 B 
4 M 
18 B 
15 B 
14 B 
11 B 
20 D 
mLIF 9 M 
14 B 3 D 
4 D 
7 D 
SOF+ 10 B 
4 HB 
4 FB 
12 FB 
14 FB 
14 FB 
14 FB 
14 FB 
12 FB 
11 FB 
20 D 
hLIF 10 M 
16 B 
9 HB 
7 HB 
4 HB 
2 HB 
1 B 
1 B 
8 D 
9 D 
5 B 
2 B 
1 B 
1 B 
5 D 
5 D 
8 D 
9 D 
2 D 
2 D 
3 D 
3 D 
__________________________________________________________________________ 
Legend: 
M = morula 
B = blastocyst 
HB = hatching blastocyst 
FB = blastocysts free from the zona pellucida 
D = degenerating embryos 
After 3 days in culture significantly more embryos had developed to 
hatching or had completely hatched from the zona pellucida when placed in 
SOF+hLIF (Group 3) than those embryos in SOF (Group 1) or SOF+mLIF (Group 
2) (P&lt;0.01, Chi-square). Furthermore, significantly less had degenerated 
after 6 days in culture (P&lt;0.01, Chi-square). There was no significant 
effect of mLIF on the development or survival of the embryo. None of the 
blastocysts that had hatched from the zone pellucida had commenced to 
elongate prior to degeneration. 
EXAMPLE 4 
This example details a repeat of Example 2 but placing the embryos in fresh 
media every second day in an attempt to induce further development of the 
hatched blastocyst. 
The treatments were as follows and the results are shown in Table 5. 
Treatments: Control - SOF 
Group 1 - SOF+mouse LIF (5000 units/ml by Ml bioassay) 
Group 2 - SOF+human LIF (5000 units/ml by Ml bioassay) 
TABLE 5 
______________________________________ 
Treat- Days in Culture 
ment 0 1 2 3 4 5 6 7 8 
______________________________________ 
SOF 2 B 2 B 2 B 8 D 
n = 8 6 M 6 M 5 M 
1 D 
SOF+ 3 B 8 B 1 HB 8 D 
mLIF 5 M 5 D 
n = 8 3 D 
SOF+ 3 B 1 HB 2 FB 5 FB 6 FB 6 FB 6 FB 8 D 
hLIF 5 M 5 B 3 HB 1 HB 2 D 2 D 2 D 
N = 8 2 M 1 B 2 D 
1 M 
1 D 
______________________________________ 
This experiment again demonstrates that hLIF improved the health of morula 
and blastocysts held in culture. While the hatched blastocysts grew from 
about 1 mm to 2 mm they did not commence to elongate. 
EXAMPLE 5 
The aim of this example was to investigate if the addition of LIF to SOF 
culture media improves the health of very early stage embryos. 
In this experiment, embryos were taken from donor ewes at various times 
after oestrus and cultured for specific periods of time in SOF or SOF+hLIF 
(1000 units/ml) before their development was assessed by counting their 
number of cells. All embryos (excepting those of group 5) were cultured in 
atmospheric conditions of low O.sub.2 tension (7%) to allow the earliest 
stage embryos to pass through the 8/16 cell block. Embryo health was 
assessed by spreading the cells on a slide and ciunying cell numbers. 
Treatments: 
Group 1. 2-4 cell embryos collected 2 days after oestrus and cultured for 6 
days in either SOF (n=10) or SOF+hLIF (n=14). 
Group 2. 2-4 cell embryos collected 2 days after oestrus and cultured for 
2.5 days in either SOF (n=15) or SOF+hLIF (n=18). 
Group 3. 8/16 cell embryos collected 4 days after oestrus and cultured for 
2 days in either SOF (n=11 ) or SOF+hLIF (n=9). 
Group 4. Morula/blastocysts collected 6 days after oestrus and cultured for 
2 days in either SOF (n=19) or SOF+hLIF (n=20). 
All these embryos were cultured in the specific atmosphere of 7% O.sub.2, 
5% CO.sub.2, 88% N.sub.2. 
Group 5. Morula/blastocysts collected 6 days after oestrus and cultured for 
2 days in either SOF (n=19) or SOF+hLIF (n=18). 
These embryos were cultured in an atmosphere of 20% O.sub.2, 5%CO.sub.2, 
75% N.sub.2. In other words this group is a repeat of the embryos cultured 
in Examples 2, 3 and 4. 
The results are shown in Table 6. 
TABLE 6 
______________________________________ 
Mean cell number (.+-. sem) 
Treatment SOF SOF + hLIF 
______________________________________ 
Group 1 41.2 .+-. 3.94 69.1* .+-. 8.42 
Group 2 7.4 .+-. 0.52 8.5 .+-. 0.60 
Group 3 17.7 .+-. 1.94 24.9 .+-. 5.20 
Group 4 75.7 .+-. 10.40 82.7 .+-. 11.49 
Group 5 56.2 .+-. 5.90 62.6* .+-. 8.42 
______________________________________ 
*Denotes significant differences between groups within rows (P &lt; 0.05) 
Students ttest. 
In summary, this experiment shows that the addition of hLIF to the culture 
media improves the health when they are held under sub-optimal conditions 
i.e. less than ideal atmospheric conditions (Group 5) or for long periods 
of time under improved atmospheric conditions (Group 1). 
EXAMPLE 6 
The aim of this example was to determine if the addition of hLIF to the 
transfer media improves the subsequence implantation rates of recipient 
ewes. 
In this experiment embryos were collected from superovulated Merino ewes 6 
days after oestrus. They were placed in either SOF or SOF+hLIF (5000 
units/ml) and individually intransferred to recipient ewes either within 1 
h of collection or after being held for between 6-8 h in the media at 
39.degree. C. The embryos were transferred in the culture media. The 
pregnancy rates were confirmed by ultrasonic scanning 65 days after 
oestrus. 
The results are shown in Table 7. 
TABLE 7 
______________________________________ 
Time 
Group Media in culture Pregnant 
Not Pregnant 
______________________________________ 
1 SOF &lt;1 h 21 (58%.sup.a) 
15 
2 SOF + LIF &lt;1 h 21 (51%.sup.a) 
20 
3 SOF 6-8 h 8 (21%.sup.b) 
30 
4 SOF + LIF 6-8 h 21 (53%.sup.a) 
19 
______________________________________ 
Different superscripts denote significant differences (P &lt; 0.01; 
Chisquare) 
This experiment demonstrates that hLIF has little effect upon implantation 
rates if the embryos are transferred immediately after collection. 
However, if the embryos are left in culture for between 6-8 h prior to 
transfer, which quite often is the situation in the field, the addition of 
hLIF to the media maintains the viability embryos which would otherwise 
have degenerated. 
EXAMPLE 7 
In a commercial ET program, 248 recipient Merino or Crossbred ewes each 
received 2 embryos that had been held in either M2 or M2+hLIF (5000 
units/ml). The culture time varied from about 1 to about 6-8 hours. The 
donor ewes were Merinos. 
The results are shown in Table 8. 
TABLE 8 
__________________________________________________________________________ 
N 2 Embryos 
1 Embryo Not Pregnant 
__________________________________________________________________________ 
M2 
Merinos 
148 44 40 64 
Xbred 20 10 4 6 
Total 168 .sup. 54 (32%) 
.sup. 44 (26%) 
.sup. 70 (42%) 
M2 + LIF 
Merinos 
56 24 12 20 
Xbred 44 24 7 13 
Total 100 .sup. 48 (48%) 
.sup. 19 (19%) 
.sup. 33 (33%) 
__________________________________________________________________________ 
Embryo Mortality: 
M2 = 55% 
M2 + LIF = 43% 
The addition of LIF to the M2 culture media significantly increased the 
implantation rates (P&lt;0.05: Chi-square). This indicates that LIF has 
potential as an embryo protective agent when added to media used for ET 
programs. 
Those skilled in the art will appreciate that the invention described 
herein is susceptible to variations and modifications other than those 
specifically described. It is to be understood that the invention includes 
all such variations and modifications. The invention also includes all of 
the steps, features, compositions and compounds referred to or indicated 
in this specification, individually or collectively, and any and all 
combinations of any two or more of said steps or features. 
REFERENCES 
Bindon, B. M. (1971) Systematic study of preimplantation stages of 
pregnancy in the sheep. Aust. J. biol. Sci. 24: 131-147. 
Edey, (1967) J. Reprod. Fertil. 13: 437-443. 
Quinn, P., Barros, C. and Whittington, D. G. (1982) Preservation of hamster 
oocytes to assay the fertilising capacity of human spermatozoa J. Reprod. 
Fert. 66: 161-168. 
Rowson, L. E. A. and Moor, R. M. (1966) Development of the sheep conceptus 
during the first fourteen days. J. Anat. 100: 777-785. 
Tervit, H. R., Whittington, D. G. and Rowson, L. E. A. (1972) Successful 
culture in vitro of sheep and cattle ova. J. Reprod. Fert. 30: 493-496.