Repeated recoveries of embryos from ewes by laparoscopy

THERIOGENOLOGY

REPEATED W.A.C.

RECOVERIES

OF EMBRYOS

FROM EWES BY LAPAROSCOPY

McKelvey,

J.J. Robinson, R.P. Aitken and I.S. Rowett Research Institute Bucksburn, Aberdeen AB2 9SB, Scotland

Received

for publication: Accepted:

Robertson

August 13, 1985 ApriZ 16, 1986

ABSTRACT

Eight ewes were synchronised in oestrus and induged to superovulate were recovered on on three occasions over a period of two months. Eggs Day 7 (Day 0 = oestrus) of each cycle by a laparoscopic technique which avoids the adhesion formation normally associated with the conventional Egg recovery rates were 35% (22/62), 76% (13/17) and surgical procedure. 66% (21/32) for the three consecutive recovery sessions. This technique allows ewes to be used repeatedly as embryo donors and should be valuable to research workers and those involved with commercial embryo transfer in sheep.

Key Words:

laparoscopy,

embryo

recovery,

ewes

INTRODUCTION The standard technique used for the recovery of ovine embryos involves retrograde flushing of the uterine horns and oviducts at This method was first described some thirty years ago (1). laparotomy. As was commonly found by other researchers (2,3),our experience has also been that this technique usually results in the formation of postoperative adhesions of the genital tract, the greater omentum and the body wall. These adhesions can reduce the subsequent fertility of a donor ewe. They also make the surgery involved in the repeated recovery of embryos progressively more difficult and eventually impossible. The result is a reduction in the reproductive lifespan of each donor ewe. Attempts have been made to overcome this problem by permanently implanting cannulae into the uterus and oviducts, but these were found interfere with egg transport and the ovarian response to gonadotrophins

to

Acknowledgements The authors wish to acknowledge Messrs. C. Fraser and C.A. Simpson. aIn this paper ova.

the word "egg"

JUNE 1986 VOL. 25 NO. 6

the technical

is used to include

assistance

embryos

given by

and unfertilized

855

THERIOGENOLOGY

There have been no reports of successful transcervical recoveries (4,5). of embryos from ewes using a flushing catheter inserted via the cervix. Although embryos have recently been recovered from two goats using a transcervical catheter (6), it was not possible to assess embryo recovery rates because ovulation rates were not recorded. Recent developments in the use of laparoscopy for intrauterine insemination of ewes (7) and insertion of embryos into recipients (8) have shown that conventional surgery is no longer necessary, or indeed desirable, for these procedures. The present study was carried out to determine the feasibility of recovering embryos from ewes using a laparoscopic technique. The aim was to reduce or eliminate the adhesion formation that accompanies the conventional procedure and thus enable repeated recoveries to be made from each animal. The technique has been adapted from that first described by Tervit and Havik (2) for the recovery of uterine-stage embryos following a full laparotomy and from that reported in our recent brief communication (9) describing the first successful recovery of embryos from ewes using laparoscopy. MATERIALS

AND METHODS

Ewe Management

Eight mature multiparous Finnish Landrace x Dorset Horn ewes were individually penned during the spring/early summer period at the Rowett Research Institute located at 57“N 2'W. They were maintained on a complete diet supplying 14 megajoules (MJ) of metabolisable energy and 195 g crude protein daily. Ewes were induced into oestrus and superovulated on three occasions over a period of, two months. Synchronisation of oestrus was achieve2 by simultaneous withdrawal of progestagen-impregnated vaginal pessaries 12 after they were inserted. Pessary withdrawal was preceded 28 h d dose of pregnant mare earlier by an i.m. 'njection of a superovulatory serum gonadotrophin' (1500 IU on the first occasion and 1000 IU on each of Each ewe was challenged every 4 h the two subsequent occasions). following pessary withdrawal using a vasectomised ram to detect the onset Those ewes exhibiting behavioural oestrus were of behavioural oestrus. artificially inseminated into the cervix on two occasions at 48 and 56 h pessaries using semen freshly collected from removal of after Suffolk rams. Eggs were recovered on Day 7 (Day 0 F oestrus) and ewes were given a luteolytic dose 'of prostaglandin analogue on Day 9. Progestagen sponges were then re-inserted a few days later in preparation for the next superovulatory cycle. Laparoscopic

Technique

Ewes were prepared for general anaesthesia by not being fed for a period of 36 h. Anaesthesia was induced by the intravenous administration of thiopentane sodium and maintained, following ZChronogest; Intervet Labs., p.l.c., Cambridge, England. P.M.S.G., Intervet Labs., p.l.c., Cambridge, England. CEstrumate 100 ug; ICI p.l.c., Macclesfield, England

856

JUNE 1986 VOL. 25 NO. 6

THERIOGENOLOGY Following the clipping endotracheal intubation, using halothane/oxygen. of wool from the abdominal wall anterior to the udder, the ewes were suspended head down in a laparoscopy cradle at an angle of 45" to the Two stab incisions were made 2 cm on either side of the horizontal. midline about 10 cm anterior to the udder (Figure 1) to permii entry of a After straight endoscope and a pair of laparoscopy grasping forceps . insufflation of the abdomen using C02, both ovaries were located and the corpora lutea were counted. A third stab incision was then made on the midline about 5 cm This incision was used to insert a anterior to the first two wounds. small sterile stainless steel trocar and cannula (7 mm diameter, 7 cm The trocar was immediately removed and ,a blunted 18G bovine long). paravertebral needle was then advanced into th&.abdomen through the steel cannula. The uterine horn ipselateral to an ovulating ovary was grasped as far as possible caudal to the external bifurcation of the uterus, and the blunted needle was used to make a small incision (0.25 cm) through the uterine wall into the lumen of the uterus. The blunte needle was withdrawn and a latex Foley 2-way paediatric 4, (10 FG x 25 cm long with bulb-tip length 1.5 cm; bladder catheter balloon size 3 ml) containing a straight wire stiffener was passed via the steel cannula into the abdomen and directed through the uterine incision into the lumen of the uterus (Figure 2). The catheter was advanced so that the bulb of the Foley catheter lay about 1 cm from the point of entry into the uterus. The wire stiffener was then withdrawn and the balloon of Inflation could be viewed via the laparoscope; the catheter inflated. some 5-7 ml of air was needed to distend the balloon. Having positioned the Foley catheter, the uterus was released from the grasping forceps, and these forceps were then used to pick up the ipselateral horncat its uterotubal junction. An intravenous catheter set (16 G x 13.3 cm) was pushed through the abdominal wall on the midline at a point about 6 cm anterior to the udder and directed towards the uterotubal junction. The tip of catheter set then pierced the uterotubal junction on the uterine side of the grasping forceps as near as possible to the oviduct, and the stylet was withdrawn to leave the catheter positioned in the lumen (Figure 3). The grasping forceps were left in place to constrict the base of the oviduct and prevent the retrograde flow of flushing medium along the oviduct. Culture mediumd (60 ml) was then flushed via the intravenous catheter through the uterine horn to emerge via the Foley catheter into embryo collection dishes (Figure 4); 40 ml of fluid was injected over a period of about 15 set and then the final 20 ml was injected more rapidly to produce a greater flushing effect within the uterus. The exit catheter was clamped and the embryo dishes were changed between these two flushes. Any residual fluid (about 10 ml) was expressed by blowing 30 ml of air through the uterus. The contents of each dish were then examined under a Wild Mk 5 stereomicroscope for the presence of embryos. ESemm grasping forceps; Wolf U.K., Mitcham, Surrey, England. Euromedical Industries, p.l.c., Worthing, Sussex, England. 'Angiocath.; intravenous catheter placement unit (Teflon), Deseret dMedical Inc., Sandy, Utah 84070, U.S.A. Ovum Culture Medium, Flow Laboratories, Irvine, Scotland, Catalogue 16-347-49.

JUNE 1986 VOL. 25 NO. 6

No.

857

THERIOGENOLOGY

Intravenous Catheter Grasping Forceps Foley Catheter

Set

Umbilicus

858

Figure

1.

Sites of entry

Figure

2.

A 1OFG Foley catheter

for laparoscopic

is inserted

equipment.

into one uterine

horn.

JUNE 1986 VOL. 2.5 NO. 6

THERIOGENOLOGY

Intravenous

Figure

3.

Introduction junction.

Semm forceps

of intravenous

catheter

set into the uterotubal

Intravenous

Figure

4.

Complete

instrumentation

JUNE 1986 VOL. 25 NO. 6

ready

for flushing

of uterus.

859

THERIOGENOLOGY

Having flushed one horn, the process was repeated for the other horn; and, following expression of excess CO2 and removal of instruments from the abdomen, the three stab wounds were closed using single Michel clips A single injection of a long acting broad to approximate the gkin edges. spectrum antibiotic was given to each ewe following the laparoscopy. Evidence of adhesion formation resulting from the first and second collections was recorded prior to the second and third collections in all ewes irrespective of whether they had been inseminated or not. RESULTS Flushing

Procedure

Positioning the two catheters into the lumen of the uterus was found However, due to differences between to be a relatively simple procedure. ewes in the size of their uterine horns, difficulties were encountered initially in assessing the correct degree of inflation for the bulb of the There was also some variation between catheters in the Foley catheter. The operator could only assess elasticity of the self-retaining balloons. bulb inflation visually, and this proved to be less successful than the Several flushes were technique of manual palpation as used in the cow. lost because of either inadequate bulb pressure which permitted leakage past the bulb or excessive bulb pressure which ruptured the uterine horn at that site. No attempt was made to repair ruptured uterine horns. Healing was found to be complete in all cases at the time of subsequent These problems mainly occurred during the first collection recovery. session and are reflected in the much lower recovery rates achieved on that occasion. Fluid recovery was found to start following the introduction of 12-20 The volumes of media recovered from ml medium into the uterine horn. each ewe flushed in the final recovery session are shown (Table 1). In these ewes a record was kept of whether eggs were located in the first or Of 21 eggs recovered, 19 (90%) were located second portions of the flush. in the first 30 ml of fluid recovered. Embryo

Recovery

In general, the fluid recovered by this technique contained less debris than that normally obtained following retrograde flushing through Therefore, following a short period to permit settling, the oviducts. eggs were easily located under the stereomicroscope. A small quantity of blood was found in about 5% of the flushes. The number of eggs recovered in relation to the ovulation rate is given for each ewe (Table 2). The recovery rates for each of the three collection sessions were 35% (22/62), 76% (13/17) and 66% (21/32), giving an overall recovery rate of 50% (56/111). The fertilization rates for each session were 50, 100 and 71% respectively. Fertilized eggs were at the late morula or blastocyst stage of development.

aClamoxyl

860

L.A., Beecham

Animal

Health,

Middlesex,

England.

JUNE

1986 VOL.

25 NO. 6

THERIOGENOLOGY

Table

1.

Volumes of flushing following injection

medium recovered of 60 ml

Left Horn

Right Horn

Ewes

103 114 123 129 132 178 192 194

37 ml No ovulationsa No ovulationsa 48 ml 51 ml 52 ml 38 ml No ovulationsa

15 ml No ovulationsa 38 ml 15 ml 57 ml 50 ml 58 ml 51 ml

aNot flushed Adhesion

Formation

Body Wall and Omentum No adhesions were found at these sites following the first recoveries, and only one ewe was found to have an adhesion between the greater omentum and the site of entry of the endoscope at the time of the third recovery. This did not interfere with successful recovery of her embryos. Uterine

Body

None of the ewes was found to have adhesions the uterus at the time of the third recovery. Uterine

involving

the body of

Horns

Following the first recovery in which 14 horns had been flushed, six showed evidence of mild fibrin formation at the uterotubal junction but no attachment to other areas of the genital tract. Following the second recovery in which 11 horns were flushed, five showed evidence of fibrin formation, but only one horn had developed an adherent tag to another part of the same uterine horn. This tag consisted of only a few strands of fibrin and was easily broken down using the grasping forceps. In no case did any of the fibrin formation prevent a horn from being flushed. Ovary/Oviduct No fimbrial adhesions were detected following the first recovery session. Two ovaries showed evidence of fibrin tags at the time of the third collection, but all the eggs produced were success&lly recovered from both these horns, indicating that the normal function of the fimbria had not been compromised.

JUNE 1986 VOL. 25 NO. 6

861

0 0

2 0

2

2 11

5 3

6 5

4 3.

3 5

3 0

L R

L R

L R

L R

123

129

L R

194

Totals

L R

192

178

62

0 3

0 3

L R

114

132

1 4

4 5

L R

103

22

6 0

0 0

1 3

RECOVERIES OF EGGS

OVULATION RATE

(April 19, 1985)

NO. I

and egg recovery

COLLECTION

Ovulation

HORN

Table 2.

NO. II

1 1

1 1

13

0 0

2 0

17

1 1

3 2

1 2

0 0

1 2

3 2

1 2

0 0

0 0

1 0

1 1

0 0

RECOVERIES OF EGGS

OVULATION RATE

(May 16, 1965)

COLLECTION

rates for each ewe

NO. III

0 2

0 2

21

3 2

3 2

32

0 1

4 1

4 1

1 3

4 0

0 1

0

1 2

RECOVERIES OF EGGS

7 4

0 2

0 0

1 2

OVULATION RATE

(June 19, 1985)

COLLECTION

50

86

47

21

76

32

33

100

64

Recovery (%I

Overall

?

z

8

!!!

THERIOGENOLOGY

Healing

of the Uterus

Protrusion of the endometrium following uterine flushing has been recorded in the absence of sutures being placed in the uterine wall (2). Following laparoscopy, no attempt was made to reappose the serosal However, after the first session of recoveries, surfaces of the uterus. and all the sites no evidence could be found of protruding endometrium, At the of insertion of the lo-FG Foley catheter had healed completely. time of the final collection, two uterine horns showed an endometrial rosette resulting from the second collection, but these protrusions did not prevent successful collection of flushing medium from both of these ewes, nor of the embryos from one of them. DISCUSSION As an effective non-invasive technique is unlikely to be developed for the collection of embryos from ewes (lo), the only improvements which can be made over conventional surgical techniques lie in the elimination andin of the need for exteriorisation and manipulation of the genital tract These goals must be the elimination of suturing of the body wall. accompanied by egg recovery rates comparable to those achieved using conventional techniques. The procedure described in this paper meets the former criteria in full, and the egg recovery rates achieved in the second and third sessions (76 and 66%) closely approximate those recorded following a first-time use of the conventional surgical procedure (11). Although no reports have been published to indicate the recovery rates which might be expected at the second or third recovery attemp-t using conventional surgery, it seems unlikely that repeated recovery rates of this order could be achieved. The egg recovery rate achieved at the first collection was considerably lower than from the later recoveries because of failure to recover eggs from several ewes with high ovulation counts. This was due mainly to either leakage around the bal'loon or rupture of the uterine wall as a consequence of our initial lack of experience in achieving the correct balloon pressure in the Foley catheter. It is interesting, however, that healing of the uterine wall was complete in all cases at the time of the subsequent collection. Nevertheless, improvements still need to be made in assessment of balloon pressure, and more consistent results might be obtained if fluid, instead of air,were used to distend the balloon or if a pressure gauge were attached to the syringe. Although 60 ml of culture medium were flushed through each horn, the majority of eggs were recovered from the first 30 ml of fluid collected. It may therefore be unnecessary to use more than 30 ml for most horns. However, with this relatively large volume of fluid, a few minutes are required for eggs to settle completely in the collection dishes, and unless the operator is prepared to wait for confirmation of a successful recovery, it is preferable that the full 60 ml are used. In a few instances during the trial, a second aliquot of 60 ml was flushed through a uterine horn from which no eggs had been recovered. In none of these cases did this additional flush result in eggs being recovered. We have previously recovered embryos following the introduction of a 3-way flushing catheter into the uterus under laparoscopic visualisation resulted in (9). However, the larger diameter of that catheter (14 PG)

JUNE 1986 VOL. 25 NO. 6

863

THERIOGENOLOGY

a greater degree of damage to the uterine wall, and we experienced some difficulty in advancing the tip far enough along the uterine horn to achieve efficient flushing of the uterotubal junction area. The present method was developed in an attempt to eliminate these problems. An alternative site for collecting fluid which has been introduced into the uterine horns at laparoscopy is the OS cervix (121, but the normal vaginal microflora render sterility difficult or impossible to maintain with this method of collection, and we therefore favour direct recovery from the uterus using a catheter. The problem of adhesion formation following conventional surgical embryo recovery procedures in the ewe has been an impediment to research workers involved in reproductive physiology. This problem also affects the more widespread uptake of embryo transfer technology in the sheep-breeding industry. The perfection of non-surgical recovery of bovine embryos heralded rapid commercialisation of the technique in that species. The results of the present experiment demonstrate that embryos can be collected repeatedly from ewes by a laparoscopic technique which virtually eliminates the formation of adhesions. This means that valuable ewes can now be presented for repeated embryo donations with little risk of reducing their subsequent fertility. REFERENCES 1.

Hunter, G.L., Adams, C.E. and Rowson, L.E.A. in sheep. J. Agric. Sci., Camb. %:143-149

2.

Tervit, H.R. and Havik, from the sheep uterus.

3.

In: Elsden, R.P. Embryo collection by surgical means. (ed). Embryo Transfer in Farm Animals, Monograph No.16, Canada, Ottawa, 1977, pp. 10-12.

4.

Ball, P.J.H., Simkin, M.E. and Foote, R.H. Effect of cannulae in the ampulla of the oviduct and in the tubo-uterine junction on reproductive phenomena in sheep. Theriogenology %:389-398 (1979).

5.

Limitations of intrauterine Mutiga, E.R., Baker, A.A. and Jillella, D. balloon catheters for ova collection in sheep. Theriogenology 0:213-220 (1983).

6.

BonDurant, R.H., Skirrow, S., Anderson, G.B., Hanson, F. and Rogers, W.H. Non-surgical collection of blastocysts from dairy goats. Theriogenology -22:423-431 (1984).

7.

Current problems and future potential of artificial Maxwell, W.M.C. In: Lindsay, D.R. and Pearce, insemination programmes. D.T. (eds). Reproduction in Sheep, Australian Academy of Science, Canberra, 1984, pp. 291-298.

8.

McKelvey, W.A.C., Robinson, J,J. and Aitken, R.P. A simplified technique for the transfer of ovine embryos by laparoscopy. Vet. Rec. (1985). -117:492-494

864

Interbreed (1955).

ovum

P.G. A modified technique for flushing N.Z. Vet. J. 4:138-140 (1976).

transfer

ova

Betteridge, Agriculture

JUNE 1986 VOL. 25 NO. 6

K.

THERIOGENOLOGY

9.

McKelvey, W.A.C. by laparoscopy.

Collection of embryos and Robinson, J.J. Vet. Rec. -115:158 (Abstr.) (1984).

In: Controlled 10. Gordon, I. Embryo Transfer in Sheep. Animals, Pergamon Press, Oxford, 1983, pp. 269-270.

from

Breeding

ewes

in

Farm

In: The Management 11. Willadsen, S.M. Embryo Transplantation in Sheep. and Diseases of Sheep, Commonwealth Agricultural Bureaux, UK, 1979, pp. 76-77. 12, Capehart, J.S., Bowen, M.J., Bassett, J.W., Shelton, J.M. and Kraemer, A modified technique for the collection of uterine stage ovine D.C. Theriogenology embryos. -21:227 (Abstr.) (1984).

ADDENDUM The authors wish to acknowledge that, subsequent to the completion of this manuscript and its acceptance by the journal's reviewer, the following "Successful non-surgical collection of ovine abstract was published: embryos 'I by S.A. Coonrod, B.R. Coren, B.L. McBride, M.J. Bowen, and D.C. Kraemer (Theriogenology a:149 abstr. (1986)).

JUNE 1986 VOL. 25 NO. 6

865