A caudal flank approach for the collection of oviductal-stage bovine embryos

THERIOGENOLOGY

A CAUDAL FLANK APPROACH FOR THE COLLECTION OF OVIDUCTAL-STAGE BOVINE EMBRYOS

D.F.Wolfe,’

M.

G. Riddel1.l R.L. Carson1

1 D.A.Stringfellow.2 Mysinger, P.D. Garrett3

P.W. and

1 2

Department of Large Department of Pathobiology

College

of

Veterinary

Recel

ved

Medicine,

for

Animal Surgery and ‘Department Auburn

pub I lcat Ion: Accepted:

and Medicine of Anatomy

University.

AL

38849

September 25, 1989 April 22, 1990

ABSTRACT Oviductal-stage embryos were surgically collected from 27 superovulated adult cows of various breeds, ages, and parity. A total of 88 surgeries was performed via a caudal flank grid approach, with the animals in lateral recumbency and the reproductive tract irrigated with sterile glycerol solution prior to surgical closure. Eight cows were operated on twice and five cows were operated on three or more times. The maximum number of surgeries for a single cow was five. Successful ova collection was accomplished in each surgical attempt, and all cows submitted to this procedure subsequently became pregnant following return to the breeding herd. This technique provided greater exposure of the ovary, uterine tube, and uterine horn, with less adhesion formation than traditional ventral midline techniques. Key

words:

ova,

embryos,

bovine,

oviduct

INTRODUCTION Traditional methods of surgical embryo collection in the bovine required the cow to be placed in dorsal recumbency with the hindquarters elevated. and a 15 to 30 cm caudal midline incision to be made which undermined or bisected the udder to acheive exposure of the reproductive tract.(l.2) The limited exposure resulted In trauma to and adheslons of reproductive tissues. The adhesions involving the ovary, oviduct, broad I igaments. uterine horns and the abdominal wall produced by these techniques limited the number of collections possible per cow and lowered the reproductive efficiency of the donor females.(2-7)

Acknowledgments Publication Auburn University, This research Grant No. 401.

No.

1995, College Auburn, AL was funded by

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of

Veterinary

Alabama

Research

Medicine, Institute

167

THERIOGENOLOGY Current research in gene insertion and pronuclear DNA injection methods have been performed most successfully with single-cell zygotes that were collected by oviductal lavage through midline surgical exposure(8.9). Present interest in the microinjection of embryos for production of transgenic livestock requires surgical ova collection, because bovine embryos descend into the uterus around Day 4.5 to 5.0 (estrus=Day 0) and are therefore unavailable by nonsurgical collection techniques prior to this time(3,8,10). The purpose of this paper is to describe our success using a caudal flank grid approach for the oviductal collection of embryos. MATERIALS

AND

METHODS

Twenty-seven cows of various breeds, ages and Parity were utilized for the collection of early-stage oviductal embryos. Each cow was subjected to a 4-d superovulation treatment, and was artificially inseminated two or three times 12-h apart, Food and water were beginning at the onset of standing estrus. withheld from each cow for 36 to 48 h prior to surgery. The cows were posltloned In right lateral recumbency prior to Lidocaine HCL 2% (1.0 mg/kg I.V.) was adminlstered induction. and anesthesia was induced with to reduce card!ac arrythmia. 10% thiamylal sodium and was maintained with halothane. The left rear limb was pulled caudally. and the left caudal flank A skin incision was prepared and draped for aseptic surgery. was made on a line parallel to and 5 cm cranial to the quadriceps femoris muscle (Figure 1).

Transverseproccrssesof lumbar vertebras Paralumbar fossa Skin Incision ,Quadriceps femoris muscle

Fold of the flank Figure

168

1.

Cow positioned uterine tube. caudal ly.

for The

embryo collection left pelvic limb

is

from the pulled

left

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THERIOGENOLOGY The incision was begun 20 cm ventral to the transverse processes of the lumbar vertebrae and extended for 20 cm toward the flank fold. The subcutaneous fascia and abdominal tunic were incised. The fibers of the external and internal abdominal oblique muscles were bluntly separated as was the underlying transverse abdominal muscle to expose the peritoneum, which was bluntly perforated to expose the abdominal cavity. The tip of the left uterine horn, tube and artery were located and exteriorized by gentle traction on the broad ligament. The uterine tube was cannulated with a sterile glass pipette (Figure 2) attached to a 60-ml syringe containing flush media consisting of phosphate buffered saline with 2% fetal bovine serum (PBS-FBS) and antibiotics(l1).

ofwary

Figure

2.

The left ovary and uterine and a bent glass pipette tube.

is

tube are threaded

exteriorized, into the

uterine

Approximately 6 cm distal to the uterotubal junction, a scalpel handle was bluntly introduced into the uterine lumen (Figure 3) and withdrawn to permit insertion of a lo-mm glass cannula for fluid collection (Figure 4). Thirty milliters of the PBS-FBS media were gently flushed from the syringe into the oviductal pipette, through the uterine horn, out the cannula and into a sterile glass dish (Figure 5;12-14). The uterine incision was closed with a continuous Cushing pattern of 3-O chromic gut.

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169

Before the ovary, uterine tube and uterus were returned to the abdominal cavity, they were irrigated with 3% (v/v) glycerol in sterile saline to reduce adhesion formation(l6). The peritoneum and transverse abdominal muscle were apposed with a simple continuous pattern of No.2 chromic gut, followed by identical closure of the fascia of the internal and external abdominal oblique muscles. The subcutls was not sutured, while the skin was sutured with a continuous interlocking pattern of 0.64~~1 nonabsorbable suture.

Figure

3.

The Into

handle the

of a scalpel lumen of the

The cow was rolled right rear limb pulled flush the right uterine 27 cows for a total of

is used to uterine horn.

an

opening

into left lateral recumbency, with caudally; the procedure was repeated This procedure was performed tube. 88 collections.

Postoperatively, the cows were allowed ad for 6 h and then were given both hay and water. confined to stalls for 3 to 5 d postoperatively they were returned to their normal environment Sutures were removed on the tenth postoperative

170

make

the to on

Iibitum grass hay The cows were at which tlme at pasture. day.

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THERlOGENOLOGY

Figure

4.

Bent glass media.

tube

for

collection

of

uterine

flush

loss tube in uterine horn

glass pipette entering uterine tube Figure

5.

A bent and Its dish.

glass tube exterior

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Is threaded Is directed

into into

a

the uterine horn sterile glass

171

THERIOGENOLOGY RESULTS as judged by our ability to Good surgical exposure. exteriorize the ovary and tip of the ipsilateral uterine Average total surgical was accomplished In all animals. to flush and collect both uterine tubes through bilateral Mild swelling, which incisions was approximately 1 h. days was the only incisional persisted for 1 to 3. complication.

horn, time flank

Successful ova collection was accomplished in all cows. Five cows were bilaterally operated on three or more times. and The maximum number of eight cows were operated on twice. surgeries (bilateral approaches) for a single cow was five. From a total of 818 corpora hemmorhagica counted during 854 ova were collected. Of these, 453 were fertilized surgery, yielding an average 4.9 fertilized ova per bilateral surgery. Only minor adhesion formation was noted at subsequent and these did not restrict surgical exposure, ova laparotomies. All cows in this study collection or fertilization rate. following return to the breeding subsequently became Pregnant, herd. DISCUSSION Numerous authors have reported on the collection of ova surgically via the caudal ventral midllne(2,3,7,10.14,17). The disadvantages of this approach were the difficulty in achieving adequate exposure to the reproductive tract and postoperative adhesions resulting from surgical trauma and hemorrhage The embryo production potential of valuable donor (2,3,10,14). and some animals were rendered infertile cows was thus reduced, due to adheslons induced by these traumatic procedures (3,141 The caudal flank approach with the animal in lateral recumbency facilitates the exposure necessary to reduce surgical trauma The use of sterile glycerol and resultant adhesion formation. for irrigating the reproductive tract appears to be superior to heparin or high molecular weight dextrans that have been used for the reduction of adhesion formation (14). Even though repositioning and a second incision are we have found that the caudal flank grid approach requ i red, provides ease and speed of collection performance, and in combination with sterile glycerol irrigation, reduces fertility-limiting adhesions associated with reproductive surgery In the cow.

tract

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