Chapter 76 Edwin G. Robertson Harrogate Genetics International, Harrogate, Tennessee, USA Just as the Burger King slogan “Have It Your Way” applies to fast food, so too will the experienced embryo collection practitioner say it applies to the technique of collecting bovine embryos. Since Drs Robert Rowe, Peter Elsden, and Martin Drost reported the successful nonsurgical collection of bovine embryos in 1976, embryo transfer has become common practice in the cattle industry. After visiting Dr Rowe during September 1977 in Middleton, Wisconsin to observe his technique, I’ve spent 36 years collecting over 180 000 embryos from 30 000 cows and transferring over 80 000 embryos. During that time, my techniques have evolved. As I’ve taught beginners and visited other practitioners to observe their techniques, I quickly came to realize that “my way” was not “the only way.” Finding a procedure that is comfortable in your hands will lead to confidence that will translate into you becoming a successful embryo transfer practitioner. I have observed embryo transfer techniques worldwide and can honestly say that many techniques can be utilized to successfully collect and transfer embryos. However, I’m pretty much about “simple,” “easy,” and “quick,” so through my career I have always looked for techniques that limit my exposure to error as well as those that save time. So, the techniques I will describe are “my way,” the most efficient and effective techniques in my hands. I hope you will pick up a few helpful ideas that will increase your skills and productivity. Previous chapters have discussed superovulation, site of fertilization, and transport of sperm up the oviduct and embryo/ova down the oviduct into the uterus. We will start our discussion assuming that the embryos have arrived into the uterus by day 4.5 or 5.0. Since embryos are most versatile at day 6–7, most practitioners plan collections 6.5–7 days after the donor’s standing heat is first observed. When several donors are to be collected on the same day, there will usually be at least 12–24 hours variation in the onset of standing heat among them. Since embryos may hatch by day 7.5–8.0 and become less useable, it is usually wise to collect the donors first that exhibit standing heat the earliest. Also realize that collection on or before day 6 may result in embryos being too immature to endure the freezing process very well. Many commercially prepared collection media are available today. I use a ready-to-go medium from Bioniche called Vigro Complete Flush™, which contains a surfactant (polyvinyl alcohol) and antibiotics (gentamicin and kanamycin). Some practitioners use lactated Ringers which they have available in their veterinary clinics and simply add 0.1% bovine serum albumin as a surfactant. For short-term holding, this appears to work well and is much more economic. If embryos are collected for export, some countries will not allow animal byproducts such as bovine serum albumin to be added (due to the scare over bovine spongiform encephalopathy, or BSE). In these collections, use of a complete medium that uses polyvinyl alcohol as the surfactant is acceptable. In my early years, I carried a ramp with me to the farms and placed it in the chute, or I had the cattleman elevate the front of the chute 30–35 cm. Elevation shifts the viscera posteriorly and tends to lift the uterus upward for ease of accessing it for manipulation. I also learned a lesson the hard way about withholding feed from donors and recipients. Although it sounded like a good idea to reduce feed intake prior to my arrival in order to lessen feces, I quickly learned that one of an embryo transfer practitioner’s best friends is a full rumen. The full rumen not only elevates the uterus upward and caudally but it also prevents negative abdominal pressure caused by an empty rumen. Negative abdominal pressure causes air to be sucked around your arm and into the rectum and colon during the flush, making manipulation of the uterus very difficult. Ramps and feed are two great friends. Once the donor is in the chute or stanchion, a lidocaine (3–5 mL) epidural is administered using a 3.75 cm × 18-gauge needle. No preparation is needed for the epidural in my opinion. Little is accomplished by clipping or washing the hair unless one is willing to surgically prepare the tailhead. I never do any preparation for epidurals on donors or recipients. Immediately after the epidural, I palpate each ovary and record my estimate as to the number of corpora lutea that are on each. Unovulated follicles are noted as well as the size of ovaries if they are unusual. This information is helpful once the results are in whether good or bad. Tranquilization is another friend in some opinions, an enemy in others. Most often, I give 10 mg (0.1 mL) Rompun (xylazine) subcutaneously or intramuscularly just before the epidural. Although it causes a slight increase in uterine tone, I’ve never considered that to be a problem. Even though I’ve collected many cows without xylazine, I like it because it takes the edge off excitable cows and it makes life easier for me because they stand still throughout the collection process. I never use disinfectants (enemies) around the collection materials. I simply wipe the perineal region after the tail is tied off to the side. In the absence of water, I simply wipe the perineal area clean with a disposable towel. Once prepared, the cow is ready for collection. Most equipment is sterilized by the manufacturer using gamma radiation. Ethylene oxide was found to be embryotoxic years ago and is no longer used. If catheters and tubing are not autoclavable, simple washing with Alconox™ laboratory detergent and several rinses with distilled water is sufficient. Many practitioners discard all equipment after single use. I reuse as much as feasible depending on price. The choice of catheter is affected by the type of flush (horn or body), age of the cow, and size of the cow. For years I used a standard human Foley catheter 45 cm long and either 16 or 18 French made of latex to flush cows of all sizes. Today we have many choices available to add to our ease and comfort during the collection. I currently use a 52 cm silicone 16 or 18 French catheter with a 5-mL balloon for all flushes (Bioniche). The 5-mL balloon will inflate to 20 mL if needed and it gives a rounder, more defined shape than the more elongated effect the 30-mL balloon produces (Figure 76.1). Figure 76.1 Shapes of Foley catheter balloon when inflated. Catheter placement is the first critical step in nonsurgical embryo collection. I will discuss the placement of the catheter and collection procedures for both single horn and uterine body collection techniques (Figure 76.2). The following are important considerations. Is the donor a virgin heifer or cow? If virgin, how mature is her tract? Does the donor have a healthy reproductive tract or are there abnormalities? If abnormalities (e.g., adhesions, deformities, presence of infection, breed, age), what are they and how will they potentially affect the collection? What is the temperament of donor? What are the facilities available for the flush? Figure 76.2 Schematic of flush equipment set-up. First, let’s talk about a normal collection and later discuss special circumstances, abnormalities, and tricks of the trade. Experts train bank tellers to detect counterfeit money by learning to first identify what real money looks like, then they can spot any abnormality. That’s the way I like to think of difficult collections. Once you learn to collect normal cows efficiently and with confidence, then when trouble arises you will, hopefully, not panic but will smartly navigate through the trouble and salvage a successful but difficult collection. A metal stylet must be inserted into the catheter to make it rigid enough to be passed through the cervix. A small amount of KY jelly is applied to the tip of the stylet before entry into the catheter to aid in its withdrawal after passage. The stylet should be slightly longer than the catheter (60-cm stylet, 52-cm catheter) to allow the catheter to be stretched so the stylet will fit tightly inside it (Figure 76.3). This prevents the tip of the stylet from accidentally popping out of one of the fluid ports (holes) near the catheter tip during its passage through the cervix. Cervical damage and uterine hemorrhage are enemies. Passage of the catheter is similar to that in artificial insemination (AI) except the catheter is considerably larger. In addition, stretch must be kept on the catheter or finger pressure on the cervix may manipulate the stylet tip out of the lumen through the fluid port on the side of the catheter tip. Figure 76.3 Stylet protruding through catheter. Eventually every practitioner runs into a crooked cervix which cannot be passed. An invaluable tool is a cervical dilator. I would never leave home without one even though I may use it only once or twice per year. Stone Mfg. Co. made one I designed and sells it as the Harrogate AI/ET gun and cervical dilator. (Editor’s note: Product #10050, Stone Mfg, 1212 Kansas Ave, Kansas City, MO 64127. inquiries@stonemfg.net.) The dilator can be passed through the most difficult cervix. Then, as the dilator is withdrawn by an assistant, you can quickly pass the catheter through before the cervix has time to return to its abnormal shape. Dilators are especially useful in Brahman and Brahman crossbreeds which tend to get “S” curves and/or 90° bends in the cervix as they age (Figure 76.4). Figure 76.4 (a) Normal versus crooked cervix. (b) Cervical dilator tip. Passing catheters through difficult or crooked cervices requires tenacity. Steady forward pressure must be kept on the stylet. Squeezing each cervical ring in front of the catheter often helps by “flattening” the ring, thereby making a “wide slit” out of the ring whose opening may be offset to one side or the other. The four cervical rings are usually aligned, with the opening of each ring being straight in front of the preceding one, but it is not uncommon to have one ring’s opening in the center, the next at 9 o’clock, the next at 3 o’clock, and the last at 12 o’clock. Pressing holes into slits often aids passage. Dilators are diagnostic friends in discovering where each “hole” is located. During my first 5 years, I performed all horn flushes. For the past 30 years I have used the body flush unless I had a diagnostic reason to collect single horns or if I was performing a single embryo recovery. For the body flush, understanding the anatomy of the uterine body is necessary to prevent frustration (Figure 76.5). Figure 76.5 Normal anatomy. In veterinary school, I was taught to use the external bifurcation to manipulate the uterus. Depending on the age, breed, and size of the cow, the external bifurcation can be 5–15 cm from the cervical opening, causing us to believe there is a lot of open space in the uterine body immediately anterior to the cervical os. Not so! In reality, there is only 1.25–5 cm of open space (heifers usually 1.25–1.9 cm while larger aged cows have 2.5–5 cm of space). Because the catheter has 2.5–3.75 cm of tip anterior to the cuff balloon, when we inflate the cuff the tip of the catheter protrudes into one of the horns. The balloon will occupy 1.25 cm with 2–3 mL of inflation, but will occupy 2.5–3.75 cm if 6–10 mL inflation is required to make the balloon large enough so that it won’t retract into the cervix. Catheter placement is easiest if you direct the catheter tip into the uterine horn opposite whichever arm you have in the rectum. For example, I use my left arm in the rectum, so it is easiest for me to place the tip of the catheter into the right horn. Inflation of the cuff and seating the catheter’s balloon is the most important part of a successful flush. I use flush media rather than air to inflate the cuff so I can see it dripping out of the valve if it leaks. The catheter should be gently introduced into the uterine horn 5 cm or so. Then, 1–2 mL of collection fluid should be injected through the valve into the cuff until you can manually feel the cuff expand. Once you locate the slightly expanded cuff with your fingertips, retract stylet about 5 cm into catheter, and then gently retract the catheter until the cuff is in the uterine body just anterior to the cervical os. Once there, continue inflating the cuff. Gently tug the catheter caudally to see if sufficient inflation has been achieved to keep the cuff from retracting into the cervix, typically 2–3 mL being required in heifers and 4–10 mL in mature cows. Once satisfied that the inflation is correct, gently remove the stylet. (NB: Always put small amounts of KY jelly on the stylet tip when it’s introduced into the catheter to prevent difficulty in withdrawing the stylet at this point.) Overinflating and underinflating the cuff are both enemies. If underinflated, the cuff will pull back into the last cervical ring of the cervix as it relaxes. Even though the catheter tip will still protrude into the uterine body, the cervical pressure on the cuff will cause the catheter lumen to collapse and no fluid can be retrieved. If that happens, you must “pinch” behind the cuff in the cervix and squirt it back out into the uterine body where you can add more inflation. The only other option is to deflate the cuff, remove the catheter, reinsert the stylet, and start over. Usually it can be pinched forward without having to remove the catheter, which could result in loss of some embryos (Figure 76.6). Figure 76.6 Underinflation. Overinflation results in only one horn being flushed unless some inflation is removed. How? See Figure 76.7. Note that if a heifer or cow has only 1.9–2.5 cm or so in her uterine body, overinflation causes the cuff to engage both the cervical os and the leaf of the internal bifurcation, preventing fluid from entering both horns. Whenever this occurs, you will only be flushing one horn and unless you figure out what’s not normal (i.e., counterfeit) embryos will be left in the other horn. Figure 76.7 Overinflation and the occlusion of one horn. Once the catheter is seated correctly, flush medium may be infused by gravity, syringe, or pump (Figure 76.8). I prefer to use a 50-mL airtight syringe connected to a disposable three-way valve so I can measure exactly how much fluid I am using. (NB: Prime the tubing to cut down on air in the uterus. Figure 76.1 displays the set-up I use today for collections.) I use a total of 400 mL of flush medium to collect both horns at the same time. Five infusions and retrievals are performed with the following amounts. Make sure to remove each infusion as completely as possible. Prior to the first infusion, the inflow side of the Y tubing should be filled (primed) with flush fluid to prevent air from being injected into the uterus, causing bubbles in the fluid. Figure 76.8 Correct catheter–balloon placement allows flush medium to flow freely throughout both horns. Follow the flush with infusion of 25 mg Lutalyse through the catheter and into uterus. Deflate cuff with a 10-mL syringe and gently remove. To assure that I get equal amounts into each horn, I hold off the first horn just in front of the catheter tip. This forces the first half of each infusion to “dam up” and then run over into the other horn. I then allow the next half of that infusion to go straight ahead into that horn. You actually learn to “feel” the fluid running under your hand and into the opposite horn as it is injected via syringe. If the fluid continues to “dam up” between your fingers and the cuff and does not run over into the opposite horn, the cuff inflation is too large and must be reduced sufficiently to allow fluid to flow between the cuff and the leaf of the internal bifurcation and into the opposite horn. Be sure the outflow side of the tubing is clamped closed before each infusion and then opened after you’ve massaged the fluid throughout the tract (Figure 76.9). After each infusion, the fluid is squeezed forward into the tip of each uterine horn and vigorously massaged to suspend the embryos. This massage forces media all the way to the uterotubal junction (UTJ) where the embryos are located. Figure 76.9 One horn can be held off to better fill the opposite horn. Once the massage is completed, the clamp is removed from the outflow tubing and the fluid is retrieved. About half of the fluid usually runs out by gravity while the other half must be reverse milked out. To reverse milk the fluid, I usually clamp the horn near the uterine tip between my second and third fingers and use my thumb to pull my fingers caudally with a motion similar to that of an inchworm. I alternate doing this between both horns and the fluid is easily moved back into the uterine body where it drains out the catheter. Sometimes I gently tug on the catheter, which produces a pumping action in the body area that aids in fluid retrieval. Each person develops his or her own technique for fluid retrieval. Repeat removal procedure after each infusion, trying to ensure the uterus is evacuated after each.
Embryo Collection and Transfer
Introduction
Timing
Collection media
Donor preparation
Equipment (and supplier)
Sterilization
Embryo collection
Body flush
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