Bovine embryo transfer

Embryo transfer (ET) technologies are tools for advancing the rate of genetic improvement. Artificial insemination (AI) allows the rapid genetic improvement of a breed through the use of superior sires; embryo transfer is a means to increase the contribution made by superior females. This means that a greater percentage of the herd can be derived from valuable females than would be the case if only AI were to be used.

ET is widely used in the beef and dairy industries. ET is a cornerstone of many breeding programs and has allowed many breeders to excel. However, ET is not necessarily only for breeders of pure-bred animals. Many high producing grade dairy cows animals in the United States have been used as ET donors. ET is an involved process and attention to many details is important. In the 20 years since ET has become commercially viable, many changes have occurred in embryo collection, transfer, freezing and thawing processes

Superovulation
Ordinarily at the time of ovulation only one, or possibly two (which would result in twins) ova or eggs are released from the cow's ovary or ovaries. To increase the efficiency of ET, it is important to increase the number of ova released at the time of ovulation and maximise the number of high quality, transferable embryos collected. This is accomplished through the process of superovulation. Superovulation, or superstimulation, involves injections of follicle stimulating hormone (FSH) of which there are several brands although in most cases we use a product called Folltropin-V. Usually, injections are given twice daily for four days, starting 7-14 days after the cow was in heat. It does not matter if the heat was naturally occurring or was induced using prostaglandin. Where natural heats do not coincide with convenient times for embryo collections, CIDR-B intravaginal devices may be used. Where the CIDR-B device is used, injections usually start 5 days after device insertion.

Injections with FSH can cause a dramatic increase in the number of follicles that develop on the ovaries and, therefore, the number of eggs (ova) available to be released at the time of ovulation. Up to 40% of seemingly normal cows will have a poor (1-2 embryos produced) response to super-ovulation. As the ova are released over a period of time, breeding is recommended 12 hours after the onset of standing heat and again 12 hours later. The onset of standing heat is considered time '0' and embryos are collected 7 days later.

Only high quality semen obtained from bulls of known fertility should be used in an ET program (unless there is a research purpose behind the program). Due to the effects of the hormones used in superstimulation, the environment within the reproductive tract of a superstimulated cow is less hospitable than that of a 'normal' cow to sperm cells. If poor quality semen is used, or poor insemination technics are followed, it is likely that the proportion of unfertilised ova or degenerate embryos will be increased.

The inseminator should not attempt to estimate the response of the ovaries to super-stimulation. At that time it is possible for follicles to be ruptured and ova can be lost. Cows which are to be used as embryo donors should have experienced at least one normal heat since calving and should be gaining weight. Feeding a carbohydrate rich diet will help improve the response to super-ovulation. Potential donors with a history of calving difficulties or retained placenta should be subjected to an examination of their reproductive tract prior to commencing a program. Recipients should be at least 60 days post-calving and preferably later (90 days) for optimal results.

Embryo recovery
When embryo transfer was first developed all procedures were conducted surgically, with the donor under general anesthetic. Now all procedures are performed non-surgically. Restraint is important and a reliable squeeze, preferably sheltered from the weather, is desirable. Dairy cows can usually be restrained in a head gate. Dust and cool draughts have a negative impact on embryo viability following collection.

Embryo recovery is usually performed 7 days after the donor was in estrus. To minimise discomfort to the donor and straining during the process, the tail head is prepared with an antiseptic and an epidural anesthetic is administered. The tail is tied to the side and the vulvar area is cleansed. A gloved arm is introduced to the cows rectum to allow manipulation of the reproductive tract. The vulvar lips are parted and a long, flexible, sterile catheter, stiffened internally by a sterile stainless steel stylet is introduced. Efforts are made to minimise contact of the catheter with the walls of the vagina and the stiffened catheter is passed through the cervix of the cow. Normally at 7 days after estrus the cervix is much more tightly closed than at the time of breeding and in heifers especially can be difficult to penetrate.

The catheter is gently manipulated into one of the horns of the uterus. A balloon-like cuff close to the tip of the catheter is inflated with air to seal off the tip of the uterus where the embryos are located. The cuff prevents flush fluid leaking behind the catheter's collection ports with a resulting loss of some of the embryos. The stiffening stylet is then removed from the catheter.

A sterile, slightly warmed balanced and buffered salt solution is used to flush the uterine horns and suspend the embryos. The flushing solution can be introduced through the catheter by coupling the catheter to the container of flush fluid using a sterile plastic tubing set, with a Y arrangement that allows fluid to leave the catheter and be collected in a filter. The filter reduces the volume of fluid that must be searched for embryos by allowing the flush fluid to pass through but retains any embryos.

An alternate method uses sterile syringes to introduce and remove fluid from the uterus and place it in the sterile filter dish. Both methods are used widely by embryo transfer practitioners and each has advantages and drawbacks.

The horn of the uterus is irrigated with between 250 and 500 mL. of flush fluid, almost all of which should be recovered. In general, most of the embryos collected are present in the first 50-100 mL. of fluid recovered from each uterine horn. After the first horn is flushed, the balloon is deflated, the stylet re-introduced into the catheter and the catheter directed into the second uterine horn. Flushing of the second horn is then performed.

Often the flush fluid may contain a small amount of mucus with small blood clots. A lot of blood or very cloudy flush fluid suggests a problem (i.e. endometritis - a uterine infection) within the uterus of the donor cow.

After flushing is complete, the balloon is deflated and the catheter removed from the reproductive tract of the donor. Occasionally, antibiotics may be introduced through the catheter prior to its' removal. Prostaglandin is given to the donor cow to help evacuate any debris (including embryos that could not be flushed free from the uterus) and to bring her into heat. The time between embryo collection and the next heat is quite variable and can be as little as 14 days to as long as 45 days (25 days is more common). Assuming normal reproductive health, donors can be flushed every 35- 60 days or so.

Searching for embryos in the flush fluid must be performed with the assistance of a microscope. Day 7 embryos are approximately 160 uM. in diameter (6 such embryos in a row would be one millimeter). The contents of the filter dish may be emptied into a gridded dish and the filter is then rinsed thoroughly with flush fluid to remove any embryos trapped under
blood clots or mucus.

Recovered embryos are removed from the flush fluid to holding medium, which is similar to flush fluid with some modifications which allow holding of embryos at room temperature for 8-12 hours without an adverse effect on their future viability.

Transfer to recipients
Good recipient animals are essential if an ET program is to succeed.Generally heifers and young cows with normal reproductive histories are used. Most embryos are transferred into recipients 7 days after the recipients were in standing heat. The cervix of some heifers can be difficult or impossible to penetrate at 7 days following estrus and for that reason young cows are often more desirable. In any case it is important that the recipients are in good body condition, in a positive energy balance (i.e. gaining slightly) and have adequate trace mineral supplementation. Supplementation with vitamin E and selenium is useful in marginal or deficient areas. Any factors which decrease natural conception rates will have a very adverse effect on the results of an ET program.

The estrous cycle of the recipients must be in synchrony with that of the donor. Estrous cycles can be synchronised using intra-vaginal devices (CIDR-B) and prostaglandin (Estrumate). The number of recipients to be programmed depends upon their availability and the previous ET history (if any) of the donor. As one can often anticipate 5 transferable embryos per collection attempt, it is worth setting up 8 potential recipients (if they are avialble). This helps cover the eventuality that not all will respond to favourably to synchronisation. Surplus embryos can be frozen for transfer at a later date, possibly into recipients that return to heat after an intial embryo transfer.

The recipients should be watched three times per day for 20 minutes per time to identify those animals in heat. A prepared teaser animal may help in heat detection. Each recipient should be uniquely identified with an eartag or tattoo and the time they are observed in heat should be recorded. Ideally, only recipients observed to be in heat more than a day before or a day after the donor should be used.

At the time of embryo transfer, potential recipients are palpated to ensure
the presence of a corpus luteum (CL) on one of the ovaries. The CL produces progesterone and is responsible for the initial maintenance of pregnancy. Embryos about to be transferred are loaded into 0.25mL straws (if recently collected) or removed from liquid nitrogen and thawed, if they had previously been frozen. The straw containing the embryo is then loaded into a transfer instrument, similar to an AI gun, fitted with a special sheath with a metal tip and side delivery openings. A second protective sheath is fitted over the transfer device to avoid carrying vaginal bacteria and debris into the uterus. The vulva of the recipient is cleansed, and the transfer device introduced into the vagina. Once at the cervix, the instrument is pushed through the protective sheath and carefully introduced through the cervix and threaded partially down the uterine horn corresponding to the ovary containing the CL. The embryo is then discharged gently from the gun.

Depending on the preference of the person transferring the embryos, an epidural anesthetic may be given to decrease discomfort and straining by the recipient.

Embryo freezing
If there is an over-abundance of embryos relative to donors, extra embryos
can be frozen for transfer at a later date. In general if a collection yields grade 1 (good and excellent grade) and grade 2 (fair) embryos, in excess of what can be used fresh, it is better to transfer the grade 2 embryos. Grade 2 embryos often give comparable pregnancy rates to grade 1 embryos when transferred fresh but they tend to not survive freezing and thawing as well as grade 1 embryos.

Non-pregnant recipients will usually return to heat 14 days following ET however the occasional animal will show signs of heat yet will sustain a pregnancy. Pregnancy diagnosis by ultrasound or rectal palpation may be conducted at 35-42 days following ET. By that time, non-pregnant recipients should have shown at least one heat.

Embryo sexing
Creekside Animal Clinic is now introducing embryo sexing. The sex of bovine embryos can be determined at the time of embryo collection. The procedure involves obtaining a small biopsy from the embryo. The biopsy is tested using the polymerase chain reaction (PCR) for the presence of a specific segment of DNA present on the Y (male) chromosome. The procedure is highly accurate though in rare cases the sex of the embryo is not identified. Consequently, only embryos of the desired sex are transferred or frozen for transfer at a later date. Embryo sexing is especially beneficial to dairy producers who want only heifer calves. By transferring only female embryos, the number of recipients required is reduced by 50%. The cost of determining an embryo's sex is far out weighed by the savings associated with the transfer of fewer embryos and the maintenance of fewer recipients. Dairy producers are often frustrated when they pay for embryo transfers, recipient synchronisation and feed costs, and after 9 months their ET program yields bull calves.

Prior to undertaking a program, all management aspects - especially nutrition and donor-recipient programming - should be discussed so that the client is aware of how to optimise his chances for a successful outcome.