Bubaline embryos are believed to descend into the uterus around day 4 (oestrus = day 0) and shed their zona pellucida (“hatch”) between days 6 and 7. Consequently, most non-surgical recoveries should be made on days 5 and 6. Buffalo embryos have been recovered non-surgically by three different techniques: Foley catheter, Rusch catheter, and IMV catheter.
A two-way roundtip Foley catheter (French size No. 16 to 24) with a 30-ml inflatable balloon is used (Figure 2B). The two-way catheter has one channel for inflation of the balloon and a single channel for alternate inflow and outflow of flushing medium. A sterile stylet (such as the plunger of an insemination gun) is inserted the full length of the device to render it sufficiently rigid to allow introduction into the uterus under guidance per rectum.
The donor is restrained in a chute or in stocks. Nervous animals are given 5–10 mg of chlorpromazine or another suitable tranquilizer. Faeces are carefully removed from the rectum to avoid aspiration of air, and a preliminary estimate is made of the number of ovulations (CL). Epidural anaesthesia is administered (4–6 ml of 2-percent lidocaine) to prevent defaecation and straining. Inadvertent air can be removed from the rectum with a small stomach tube attached to a “wet vac” vacuum cleaner. The vulva and perineal region are throughly washed with plain water and blotted dry. The tail is tied out of the way. If the cervix feels small or tortuous, a cervical dilator (Figure 2A) may be used to gently expand and straighten the cervical canal. The dilator and subsequent catheters are covered with a sanitary sleeve before they are introduced into the vagina. This protective cover is perforated just before the instrument enters the external os of the cervix. The rigid, relatively sharp-pointed dilator should be used with extreme caution as it can readily perforate the uterine wall as it is “forced” through the tight cervical canal. The lips of the vulva are again parted and the covered Foley catheter, with the stylet in place, is inserted into the vagina and on into the lumen of the cervix. It is then manipulated into the appropriate horn until the inflatable balloon is situated at the base of the uterine horn (Figure 3). The balloon is slowly inflated with 15–25 ml of air in adult buffaloes and 10–15 ml of air in heifers. The endometrium can easily be split by overdistension, resulting in haemorrhage and escape of the flushing solution into the mesometrium from which it cannot be recovered.
After the catheter is in position, the stylet is removed and the catheter is connected via a Y-junction by sterile tubing to a 1 000-ml bottle of flushing solution. The remaining arm of the Y-junction is connected to a free piece of tubing. The flow of medium in both pieces of tubing is controlled by quick-release clamps. While the outlet tubing is occluded, the flushing solution enters the uterus by gravity flow with the bottle suspended one metre above the level of the uterus. The horn of the uterus is extended by elevating the utero-tubal junction and by carrying it anteriorly. When the inflow stops, the inlet tubing is clamped off and the clamp on the outlet tubing is released. The fluid is channelled directly through an embryo filter (75-μ pore size).
Alternatively, when filters are not available, the effluent may be collected in a 1-litre graduated cylinder. After the embryos have been allowed to settle for 20–30 minutes, the supernatant is carefully siphoned off and the last 75 ml are examined directly under a stereomicroscope. Slow siphoning is most easily accomplished by gently lowering a length of small diameter (e.g. 1 mm) tubing into the cylinder until the end of the tubing reaches the 75-ml mark. Siphoning is started by aspiration with a small syringe at the other end of the tubing.
In older animals with long pendulous tracts, manipulation of the cervix and uterus is facilitated by retracting the cervix into the vagina with cervical forceps. If the returning fluid is blood-tinged, the red cells may be washed directly through the filter by opening both clamps between the filter and the bottle of flushing solution. The filter should never be allowed to run completely dry leaving the embryos on the filter disk exposed to the air. A 1-cm layer of fluid can be maintained by regulation with a clamp on the tubing attached to the bottom of the filter unit. During the final collection of the flushing solution, 50 IU oxytocin may be given to aid in the recovery of the residual portion of the medium from the uterus.
In superovulated animals, the procedure is repeated for the opposite horn using a separate sterile catheter. It is hazardous to reinsert the stylet into the Foley catheter while it is in the uterus because the sharp tip might exit through one of the side openings. Some operators prefer placement of the catheter with the balloon just anterior to the internal os of the cervix, in the body of the uterus, which enables them to flush both horns simultaneously. In older animals the balloon is frequently displaced to this body location during the filling and stretching of the uterus, even when the balloon was initially placed in one of the horns. When this happens, both horns are simply flushed at the same time.
The Rusch catheter is a 68-cm long, red rubber, 18 French gauge balloon catheter made in Germany (Figure 2C). The catheter has a self-contained locking stylet and a tip which extends 4.5 cm beyond the balloon. The added length is an advantage. Placement of the catheter is similar to that of the Foley catheter. After the stylet has been withdrawn 3–4 cm, the catheter can be directed further into the horn because the rubber is stiffer than the Foley catheter. This manoeuvre is particularly advantageous in older animals with long pendulous uteri. The uterine lumen is flushed by alternating inflow and outflow.
Equipment for nonsurgical embryo recovery. A. Rusch catheter with self-contained stylet, length 68 cm; B. Foley catheter with plunger of Cassou Al syringe as stylet, length 42 cm; C. Cervical dilator, length 36 cm, diameter 6 mm, the last 4 cm of the tip are tapered down to a 3-mm rounded tip.
Location of the balloon of the recovery catheter at the base of the uterine horn
The third approach makes use of a large, rigid, stainless steel, three-way collection device (IMV, France). One channel serves to inflate the balloon, the second, the stainless steel cannula, to introduce the flushing medium, and the third, a small flexible catheter which can be advanced into the tip of the horn, to recover the flushing medium.
Each of these embryo recovery methods works best for the individuals who have devised them or for those who have acquired experience with them. The relative cost of the catheters varies considerably.