Veterinary Science Communications, 3 (1979) 177--186 177

Elsevier Scientific Publishing Company, Amsterdam - - P r i n t e d in The Netherlands

9 ECSE, EEC, EAEC, Luxembourg, 1979
EUROPEAN C O M M U N I T Y
PREVIEW ARTICLE
E M B R Y O T R A N S F E R A N D T W I N N I N G IN C A T T L E

I. G O R D O N and M. P. BOLAND

D e p a r t m e n t of A g r i c u l t u r e , U n i v e r s i t y College, D u b l i n (Ireland)

(Accepted 13 S e p t e m b e r 1979)

ABSTRACT

Gordon, I. and Boland, M.P., 1979. E m b r y o t r a n s f e r and t w i n n i n g in

cattle. Vet. Sci. Commun., 3: 177-186.

Recent d e v e l o p m e n t s in cattle embryo t r a n s f e r are discussed,

p a r t i c u l a r l y those that are r e l e v a n t to the c o m m e r c i a l a p p l i c a t i o n of
the technique. Cattle t w i n n i n g for b e e f p r o d u c t i o n could offer
i m p r e s s i v e e c o n o m i c a d v a n t a g e s w h e r e n u t r i t i o n is not a limiting factor
and i n t e n s i v e m a n a g e m e n t is possible. E v i d e n c e is d i s c u s s e d w h i c h
suggests that cattle twins i n d u c e d by e m b r y o t r a n s f e r m a y h o l d several
a d v a n t a g e s over n a t u r a l l y - o c c u r r i n g twins.

INTRODUCTION

The m a i n use of cattle embryo t r a n s f e r is as a means of i n c r e a s i n g

the calf output of g e n e t i c a l l y v a l u a b l e females. Back in the e a r l y
1970's, the b o o m in the 'exotic' breeds b r o u g h t an i m m e d i a t e need to
m u l t i p l y these cattle q u i c k l y to m e e t w o r l d d e m a n d and the h i g h prices
for calves made the s u r g i c a l p r o c e d u r e s used at the time a c o m m e r c i a l
proposition. P r e s e n t day i n t e r e s t in cattle e m b r y o t r a n s f e r is m u c h
more in trying to get m o r e calves from o u t s t a n d i n g d a i r y cows, In
North America, more than iO,0OO p r e g n a n c i e s a n n u a l l y are r e p o r t e d to
r e s u l t from e m b r y o t r a n s f e r at this time (Seidel & Seidel, 1978).
A major p r o b l e m in cattle b r e e d i n g has always b e e n that only one
calf per year c o u l d be p r o d u c e d from a top cow in the h e r d and p e r h a p s
no more than 3 to 6 calves in a lifetime. Now, w i t h the aid of embryo
transfer, it is p o s s i b l e for the o u t s t a n d i n g animal to p r o d u c e a life-
time's calf crop in a single year, a l t h o u g h this level of success i s
not a c h i e v e d as r e a d i l y as commercial t r a n s f e r clinics m i g h t wish. In
fact, statistics for success rates are d i f f i c u l t to obtain, b u t
 178

generally show between 2 and 4 pregnancies per superovulated donor.

JUdged in terms of pregnancy rates, a figure of 55 - 60% for single
egg surgical transfers would seem to be as high as one can expect
(Betteridge, 1977).

Non-Surgical Embryo Recovery

Cattle embryo transfer was pioneered by Rowson and colleagues at

Cambridge (Rowson, Moor & Lawson , 1969); they developed the surgical
collection and transfer techniques which were used commercially around
the world in the early 1970's. With this, however, after more than
two or three surgical interventions, donor cows are liable to become
infertile due to the build-up of scar tissue and adhesions. Recently
developed non-surgical techniques, although calling for manipulative
skills and considerable practice, do permit the collection of embryos
from donor cattle repeatedly without damage; egg recovery rates would
seem comparable to those previously obtained with surgical procedures
(Newcomb, Christie & Rowson, 1978; Seidel & Seidel, 1978). ~t is now
possible to proceed in two ways using these new techniques; using
superovulated and non-superovulated donors. In the latter instance,
donors have been used ten or more times within a year. There is also
the possibility of taking embryos from top cows in the herd in the
weeks prior to putting them in-calf in the normal way; in this way,
the farmer may increase the supply of top-quality replacements without
disturbing the established calving pattern of the herd.

Non-Surqical Transfer of Embryos

Cattle embryo transfer technology is still based on surgical proced-

ures. Under field conditions, it is estimated that current pregnancy
rates using non-surgical procedures are generally about half those
with surgery (Seidel & Seidel, 1978), although pregnancy rates up to
about 60% have been achieved by some using s Cassou 'gun' technique
(Sreenan, 1978; Trounson, Rowson a Willadsen, 1978). Every e f f o r t
should be taken to ensure that the embryo is placed in the ipsilateral
horn and that it remains in situ after the Cassou 'gun' is withdrawn.
With transfers performed by surgery, it is easier to be certain about
the deposition of the embryo; occasionally, embryos may be lost in non-
surgical transfer~process. On the basis of present evidence, however,
there s e e m s no reason why an acceptable non-surgical technique should
9 not become available for commercial use in the near future.
 179

Frozen Cattle Embryos

One of the important technological advances in cattle embryo

transfer in recent years has been the freezing of embryos and their
storage at the temperature of liquid nitrogen. Freezing studies were
initiated after Whittingham, Leibo & Mazur (1972) reported success
with mouse eggs. Work in Australia (Bilton & Moore, 1976), Denmark
(Lehn-Jensen & Greve, 1978), Holland (Trounson, Brand & Aarts, 1978),
North America (Shea, Ollis & Jacobson, 1977 ; Trounson et al., 1978)
and at Cambridge (Wilmut & Rowson, 1973; Willadsen, Polge & Rowson,
1978) has extended these studies to cattle embryos. Attempts to cool
pig embryos below 15~ have not been successful (Polge, Wilmut &
Rowson, 1974) and early cleavage stage cattle eggs survive poorly when
cooled to O~ (Trounson, Willadsen & Rowson, 1976; Trounson et al.,
1976). It is still unknown why there should be these differences
between farm species and between developmental stages in the sensitiv-
ity of the bovine embryo to cooling to O~ 9 On the basis of present
evidence, it is concluded that the optimal stage for freezing cattie
embryos is as the blastocyst prior to hatching (day 7/8 embryos) when
survival rates of 80% have been reported. Results obtained by the
transfer of frozen embryos may be some way below those achieved by
direct transfers (Seidel & Seidel, 1978) but this may be a matter of
distinguishing the more viable embryos after thawing out.
Although there is no reason why single embryo~ should not be frozen
in plastic straws in the same way as bull semen, it is not presently
possible to use the embryo in transfer immediately after thawing. The
cryoprotective agent used in freezing, dimethyl sulphoxide (DMSO), has
to be removed slowly from the embryo before transfer is made; it remains
to be seen whether techniques enabling embryos to be transferred
straight fDom the Cassou straw can be developed. If that were possible,
then there is ~o reason why inseminators from the A.I. Centres could
not perform non-surgical transfers in m u c h the same way as they carry
out inseminations at the moment.

Bull or Heifer Calves to Order ?

Although apparent success in separating X from Y ' c h r o m o s o m e b e a r i n g

spermatozoa has been reported in the literature from time to time, no
procedure has yet stood the test of time. One recent adVance in cattle
embryo transfer technology is the ability to 'sex' the embryo Cby sex
chromosome analysis) at the week-old s t a g e (Moustafa, Hahn: &~Roseiius,
180

1978). Previously, this had only been reported for the two-week old
cattle embryo (Hare et al., 1976), which appears to be much less viable
after freezing and storage than the week-old embryo (Trounson et al.,
1978). The possibility exists now to 'sex' embryos prior to freezing
and to build up frozen stocks of embryos of known gender. As yet,
applications for this new technique in the field have not been reported.
The need for delicate micro-manipulations in obtaining a smaple of
cells for 'sexing' places an obvious limit to the extent the technique
can be employed in routine transfer operations.

New Ways of Evaluating Cattle Embryos

Not all bovine embryos are suitable for use in transfers due to
morphological abnormalities (Boland, Crosby & Gordon, 1978a). In our
own laboratory, as a result of work by Sreenan & Scanlon (1968), the
rabbit is employed routinely to check bovine embryo viability and for
storage. Now, German workers (Schilling & Doepke, 1979) have reported
on a staining technique which may be used to assess more accurately
the suitability of embryos for transfer; in evaluating 7-day old
embryos after freezing and thawing, thetechnique may be of especial
value. The test permits living cells to be distinguished from dead
cells under the microscope without adversely affecting the viability
of the embryo.

Producing Identical Cattle

Cattle identical twins can be of considerable value in various

aspects of animal production research. They permit some experiments
to be done with much smaller numbers of animals than usual. It has
now been shown (Willadsen, 1979) that identical twins can be produced
in sheep by taking a 2-cell egg and separating the two blastomeres
under the microscope by micromanipulation. There would seem to be no
reason why a similar approach should not be feasible in cattle.
Transplantation of nuclei, a technique developed in studies with
the large amphibian egg, currently poses substantial technical diffic-
ulties when a t t e m p t e d w i t h the much smaller mammalian egg (Bromhall,
1975); it may, however, eventually have a part to play in producing
supplies of embryos of identical genetic constitution for use in
cattle embryo transfer. Whether it ever becomes POSSible for a
nucleus, taken from tissues of an adult animal, to give rise to the
development of a normal mammal, remains to be seen. M c K i n n e l l (1978),
 181

on the basis of a careful review of literature in this area, concludes

that somatic nuclei generally do become irreversibly differentiated
during the development of an organism and are not able to replace the
zygote nucleus when transplanted into an egg.

Cattle Twinning Techniques

The induction of twinning in cattle by various methods has been the

subject of a recent review by Anderson (1978). For Ireland, in which
beef is the single most important product of the agricultural industry,
the availability of a commercially acceptable twinning technique could
be of special importance in increasing the supply of desirable beef
calves. The approach adopted in this laboratory employs slaughtered
beef heifers as the 'donors' of embryos, mated cows as 'recipients',
non-surgical transfer of a single embryo through the cervix by t h e
Cassou 'gun' and the rabbit for storing and evaluating embryos prior
to transfer. Information o n typical yields of embryos that can be
expected from super ovulated beef heifers is given in Table I . Not all
the embryos would be 'usable', but it should prove possible to get
about six per heifer, enough to carry out six twinning transfers.

TABLE I

Response to PMSG - prostaglandin Treatment in beef heifers

Treatment PM~G-PG
(2,500 iu PMSG) 1977 1978

Donor Heifers 18 12

In Heat & Bred 16 12

Mean Ovulation Rate 15.6 16.2

Mean No. Eggs Recovered iO.i 11.6

Mean No. Eggs Fertilized 9.6 8.9

Fertilized Eggs per Donor 8.6 8.9

Pregnancy and Twinning Rates

Studies involving somewhere about 2 , 0 0 0 h e i f e r cattle h a v e ~ b ~ e n con-

182

ducted in various aspects of cattle t w i n n i n g in this l a b o r a t o r y f r o m

1966, w h e n the r e s e a r c h p r o g r a m m e commenced. W i t h the d e v e l o p m e n t of
a basic technique (Boland, Crosby & Gordon, 1975) e f f o r t has been made
to get twin calves born (Boland, Crosby & Gordon, 1976, 1978b; Boland,
& Gordon, 1978). Data in Table II are for b e e f and dairy cattle, most
of w h i c h were dealt with on the College farm. There is a r e a s o n a b l e

TABLE II

Outcome of s i n g l e - e m b r y o t w i n n i n g transfers in b e e f and dairy cattle

M a t e d R e c i p i e n t Cattle

Suckler Cows DairyCows All

N u m b e r of T w i n n i n g
Transfers 20 28 48

P r e g n a n t to First
Service 13 19 32 (67%)

W i t h Twin Calves 7 9 16 (50%)

p o s s i b i l i t y that these results can be i m p r o v e d to the p o i n t of obtain-

ing a 70% p r e g n a n c y and a 70% t w i n n i n g rate, figures w h i c h have been
a c h i e v e d w i t h transfers p e r f o r m e d by s u r g e r y (Sreenan, 1977). Apart
from our own work, a r e c e n t report by S r e e n a n (1979) for 61 m a t e d
cattle r e c e i v i n g single e m b r y o transfers shows a p r e g n a n c y rate of 60%
and a t w i n n i n g rate of 60%. In practice, this means that iO0 calves,
r a t h e r than 60 to 65, are p r o d u c e d to cows c a l v i n g to first service

Calves and C a l v i n g

Twin calves are g e n e r a l l y b o r n several days e a r l i e r than singles

(Gordon, W i l l i a m s & Edwards, 1962). Figures in Table III are from a
small group of eight F r i e s i a n s (second calvers) in the College d a i r y
herd. They w e r e fed s i m i l a r l y during late pregnancy. The twin calves
were born w i t h o u t h a z a r d and their average w e i g h t was about S4% that
of the singles. The t w i n - b e a r i n g F r i e s i a n s c l e a n s e d and p r o d u c e d
n o r m a l q u a n t i t i e s of m i l k subsequently. Feeding experiments with
t w i n - b e a r i n g cattle c a r r i e d out e l s e w h e r e in Ireland and r e p o r t e d on
by Sreenan (1977, 1979) also p r o v i d e e v i d e n c e i n d i c a t i n g that approp-
 183

TABLE III

Gestation periods and calf birthweights in Friesians producing singles

and twin-sets

Gestation Length
Live Birth-Weights
Range Mean Births Range Mean
(Days) (Kg.)
Cows with
Singles (4) 279-283 281.8 4/4" 36-47 41.3

Cows with
Twins (4) 273-279 276.8 8/8" 28-42 34.5

riate feeding can minimise problems of calf mortality and retained

fetal membranes.

How Do Induced Twins Compare with Natural Twins ?

Twins induced by embryo transfer can be expected to possess several

advantages over naturally occurring cattle twins. These include:
(i) The fact that farmers have prior knowledge that the cow
is carrying two calves and can plan accordingly. An
oestrogen assay can be u s e d to distinguish the twin-
bearing cows (Terqui et al., 1975; Adelakoun, Matton
& Dufour, 1978; Sreenan, 1979) and farmers Can adjust
feeding and management in the final two months of preg-
nancy.
(2) Embryo transfer establishes a bicornual twin pregnancy
which is to be preferred to a unicornual pregnancy. It
seems probable that some problems occasionally exper-
ienced with natural twinning arise because both fetuses
are located in the one horn of the uterus (Testart & du
Mesnil du Buisson, i966).
(3) Cows should be prepared for twin calving by extra feed
and attention. There is evidence from work in the U.K.
(Hammond, 1959; Gordon et al., 1962) the U.S.A. (Turman
et al., 1971), France (Chupin et al., 1976) and Ireland
(Sreenan, 1977, 1979) that such preparation can minim-
ise problems which are sometimes associated with natural
twins.
(4) The farmer can select mature strong cows to carry the
184

induced twins. Under nature, there is sometimes an

association between twinning and endocrine disturbances
which make twin-bearers more difficult than usual to
get in-calf (Erb, Hinze & Gildow, 1959).

REFERENCES

Adelakoun, T., Matton, P. & Dufour, J.J., 1978. Steroid hormone levels
in beef cows during pregnancy terminating in normal calving or
abortion and with single or multiple ovulation. Can. J. Anim.
Sci., 58:345-354.
Anderson, G., 1978. Methods for producing twins in cattle. T h e r i o g e n .
9(1):3-16.
Betteridge, K.J., 1977. Summary of factors affecting success rates in
surgical embryo transfer. In: Embryo transfer in farm animals (ed.
K. Betteridge), Dept. Agric., Canada Monograph, No. 16:29-31.
Bilton, R.J. & Moore, N.W., 1976. Storage of cattle embryos. J. Reprod.
Fert., 46:537-538.
Boland, M.P. & Gordon, I., 1978. Twinning in lactating Friesian cows by
non-surgical egg transfer. Vet. Rec., 103: 214.
Boland, M.P., Crosby, T.F. & Gordon, I., 1975. Twin pregnancy in cattle
established by non-s~rgical egg transfer. Brit. vet. J., 131:738-
740.
Boland, M.P., Crosby, T.F. & Gordon, I., 1976. Birth of twin calves
following a simple transcervical non-surgical egg transfer technique.
Vet. Rec., 99:274-275.
Boland, M.P., Crosby, T.F. & Gordon, I., 1978a. Morphological normality
of cattle embryos following superovulation using PMSG. Theriogen.
10:175-180.
Boland, M.P., Crosby, T.F. & Gordon, I., 1978b. Fetal and placental
development following surgical and non-surgical twinning in beef
heifers. Vet. Rec., 102:39-40.
Bromhall, J., 1975. Nuclear transplantation in the rabbit egg. Nature,
258:719-722.
Chupin, D, Nguyen, Huy., Azan, M., Mauleon, P. & Ortavant, R., 1976.
Hormonal induction of multiple-births; the main consequences for
breeding performance. Ann. Zootech., 25:79-94.
Erb, R.E., Hinze, P.M. & Gildow, E.M., 1959. Factors influencing pro-
lificacy of cattle. II. Some evidencethat certain reproductive
traits are additively inherited. Tech. Bull. Wash. agric. Exp. Sta.
No. 30.
Gordon, I., Williams, G. & Edwards, J., 1962. The use of serum gonad-
otrophin (PMS) in the induction of twin-pregnancy in the cow. J.
agric. Sci. Camb., 59:143-198.
Hammond, J., 1959. Twinning in cattle for beef production. Vet. Rec.
71:168-170.
Hare, W.C.D., Mitchell, D., Betteridge, K.J., Eaglesome, M.D. & Randall,
G.C.B., 1976. Sexing 2-week old bovine embryos by chromosomal
analysis prior to surgical transfer: preliminary methods and results.
Theriogen. 5: 243:253.
Lehn-Jensen, H., & Greve, T., 1978. Low temperature preservation of
cattle blastocysts. Theriogen. 9:313-322.
McKinnell, R.G., 1978. Cloning. Nuclear transplantation in amphibia.
Univ. Minnesot~ Press, Minneapolis, U.S.A.
Moustafa, L.A., Hahn, J. & Roselius, R., 1978. Experiments on sexing
day-6 and 7 bovine embryos. Berl. Munch. Tierarztl. Wscht., 91:
236-238.
 185

Newcomb, R., Christie, W.B. & Rowson, L.E.A., 1978. Non-surgical

recovery of bovine embryos. Vet. Rec., 102:414-417.
Polge, C., Wilmut, I. & Rowson, L.E.A., 1974. Low temperature preser-
vation of cow, sheep and pig embryos. Cryobiology, 11:539-540.
Rowson, L.E.A., Moor, R.M., & Lawson, R.A.S., 1969. Fertility follow-
ing egg transfer in the cow: effect of method, medium and synchron-
ization of oestrus. J. Reprod. Fert., 18:517-523.
Schilling, E. & Doepke, H., 1979. A rapid diagnostic test for the
viability of early cattle and rabbit embryos using diacetyl-
fluorescin. Naturwissenschaften (in press).
Seidel, G.E. & Seidel, S.M., 1978. Bovine embryo transfer: costs and
success rates. Advt. Anim. Breeder, 24(8):6-10.
Shea, B.F., Ollis, G.W., Jacobsen, M.E., 1977. Pregnancies following
long distance transport and transfer of frozen bovine embryos. Can.
J. Anim. Sci., 57:801-802.
Sreenan, J.M., 1977. Embryo transfer for the induction of twinning in
cattle. In: Embryo Transfer in Farm Animals (ed. K. Betteridge),
Dept. Agric. Canada Monograph No. 16:62-66.
Sreenan, J.M., 1978. Non-surgical egg recovery and transfer in the cow.
Vet. Rec., 102:58-60.
Sreenan, J., 1979. Increasing the calf crop: synchronization and twin
calving. Ir. Frm. J. 31(7):46-47.
Sreenan, J.M. & Scanlon, P., 1968. Continued cleavage of fertilized
bovine ova in the rabbit. Nature(U.K.), 217:867-868.
Terqui, M., Delouis, C., Thimonier, J. a Ortavant, R., 1975. Relation-
ship between blood oestrogen levels and the birth of single and
multiples in Charolais cattle. C.R. Acad. Sci. (Paris), 280:2789-
2792.
Testart, J. & Du Mesnil du Buisson, F., 1966. A biochemical study of
placentomas during single and twin bovine pregnancy. Ann. Biol.
anim. Bioch. Biophys., 6:483-493.
Trounson, A.0., Rowson, L.E.A. & Willadsen, S.M., 1978. Non-surgical
transfer of bovine embryos. Vet. Rec., 102:74-75.
Trounson, A.O., Willadsen, S.M., Towson, L.E.A. & Newcomb, R., 1976.
The storage of cow eggs at room temperature an~ at low temperatures.
J. Reprod. Fert., 46:173-178.
Trounson, A.O., Willadsen, S.M. & Rowson, L.E.A., 1976. The ~nfluence
of in vitro culture and cooling on the survival and development of
cowembryos. J. Reprod. Fert., 47:367-370.
Trounson, A.O., Brand, A., & Aarts, M.H., 1978. Non-surgical transfer
of deep-frozen bovine embryos. Theriogen. 10:111-115.
Truman, E.J., Laster, D.B., Renbarger, & Stephens, D.F., 1971.
Multiple births in beef cows treated with PMS and BCG. J. Anim. Sci.,
32: 962-968.
Whittingham, D.G., Leibo, S.P. & Mazur, P., 1972. Survival of meuse
embryos frozen to -196~ Science, 178:411-414.
Willadsen, S.M., 1979. A method for culture of micromanipulated sheep
embryos and its use to produce monozygotic twins. Nature (Lend.),
277:298-300.
Willadsen, S., Polge, C. & Rowson, L.E.A., 1978. The viability of deep-
frozen cow embryos. J. Reprod. Fert., 52: 391-393.
Wilmut, I. & Rowson, L.E.A., 1973. Experiments on the low temperature
preservation of cow embryos. Vet. Rec., 92:686-690.
186

KURZFASSUNG

Gordon/ I. und Boland, M.P.,1979. Embryo transfer und Zwillings-

geburten beim Rind. Vet. Sci. Commun., 3 : 1 7 7 - 1 8 6 (in Englisch).

Neuere Entwicklungen auf dem Gebiet des Embryotransfer beim

Rind werden diskutiert. Besonderer Wert wird dabei auf Techniken
gelegt, die sich f~r eine kommerzielle Anwendung eignen.
Zwillingsgeburten beim Rind f~r die Fleischproduktion bietet
beeindruckende ~konomische Vorteile in Best~nden, wo Ern~hrung
kein limitierender Faktor und i n t e n s i v e s M a n a g e m e n t m~glich ist.
Es werden Daten diskutiert, die darauf hinweisen, dab Zwillings-
k~iber yon Embryotransferstudien eine Reihe yon Vorteilen gegen~ber
nat~rlich geborenen Zwillingen aufweisen.

RESUME

Gordon, I. et Boland, M.P., 1979. Transplantation Ovulaire et

Gemellarite chez les Bovins.Vet. Sci. Commun., 3: 177-186 (en anglai

Les d~veloppements r~cents en mati~re de transplantation

embryonnaire chez les bovins sont discut~s en particulier ceux
ayant une appliavantages ~conomiques i~ o0 la nutrition n'est pas
un facteur limitant et l'~levage intensif possible. Ii est
clairement montr~ que les jumeaux obtenus par transplantation
ovulaire offrent plus d'int~r~t que les jumeaux spontan~s.

RIASSUNTO

Gordon, I. e Boland, M.P., 1979. Transferimento embrionale e

produzione di gemelli nel bovino. Vet. Sci. Commun., 3: 177-186
(in Inglese).

Vengono discussi i recenti sviluppi delle tecniche di transfe-

rimento degli embrioni nel bovino, soprattutto di quelli che
sono imp~rtanti ai fini dell'applicazione commerciale. La produ-
zione di gemelli per una pi~ alta produzione carnea potrebbe of-
frire rilevanti vantaggi economici dove l'alimentazione non rap-
presenta un fattore limitante e dove ~ possibile l'allevamento
intensivo. Vengono anche discusse le prove secondo cui i parti
geme!lari indotti nel bovino mediante trasferimento embrionale
potrebbero presentare molti vantaggi rispetto ai parti gemellari
naturali.