CHAPTER 63 Assisted Reproductive Technologies in Cattle
Assisted reproductive technologies (ART) have made tremendous advances, especially during the past 15 years. Artificial insemination (AI) remains the most effective method for achieving genetic progress in populations of cattle. The global market remains strong for frozen semen and embryos. Millions of cattle are bred by AI, and more than a half million embryos are transferred annually world wide. Most of the top dairy sires used for AI were derived from embryo transfer (ET). Improvements in methods of controlling the estrous cycle and ovulation have resulted in more effective programs for AI, superovulation of donor cows, and the management of ET recipients. The recent introductions of in vitro embryo production, cloning, and sexed semen have added to the ART “toolbox.”
In this chapter we discuss current reproductive technologies in cattle, with particular emphasis on procedures used in the practice of ET. Our goal is to provide the reader with information and guidelines for applying ET and related technologies. The newer technologies of in vitro embryo production and cloning are also addressed. The chapter concludes with a discussion of the use of sexed sperm and embryos to preselect calf gender.
Artificial insemination was the first assisted reproductive technology to be applied commercially for the genetic improvement of animals in the mid-1900s. The advantages of AI in terms of disease control, the import and export of frozen semen, the availability of accurate breeding records, and the elimination of dangerous bulls on farms are well established. AI has become the foundation for expanded breeding schemes such as estrus synchronization programs (synchronized breeding, including timed AI), embryo transfer, in vitro embryo production (IVP), the use of sexed semen, cloning, and transgenics. The use of AI, especially in dairy cattle, has become so routine that most of it is practiced by the producers or herd managers themselves. The main disadvantage is that the latter do not always develop sufficient skills to maintain acceptable conception rates in their herds.
The next major commercial advancement in reproductive biotechnology was embryo transfer in the late 1970s. ET enabled the acceleration of the proliferation of genetic material from the dam as well as of the sire. The ability to freeze and transport bovine embryos around the world has made ET an extremely useful technology for disease control and biosecurity programs, genetic salvage of valuable individuals, and development of new lines or breeds of cattle. ET is a factorial process that depends on a series of carefully orchestrated sequential steps. Poor performance in any of the steps directly affects the success rate and the final outcome, the number of calves weaned.
Selection of the donor is based on two major criteria: (1) genetic merit, generally determined by the owner and based on performance, and (2) reproductive soundness, as assessed by the veterinarian. The donor must be in good body condition and preferably gaining weight. She should be free from underlying diseases, a minimum of 50 to 60 days post partum, and cycling regularly. Generally, cows with a history of reproductive problems do not make good donor animals.
Donors are further evaluated by examination of the cervix, uterus, and ovaries per rectum to determine that they are free from adhesions or other palpable lesions. It is prudent to test the patency of the cervical canal with a cervical dilator for sufficient internal diameter to permit passage of a collection catheter, especially if the prospective donor is a heifer or of Bos indicus breeding. This prevents the occasional frustration of being unable to negotiate the cervix after a series of costly hormonal injections.
Vaccinations should be current for local diseases. Blood typing of both donor and sire prior to or at the time of embryo transfer for subsequent identification of offspring is highly recommended and is generally required prior to export.
Single embryos or multiple embryos may be collected from naturally ovulating or superovulated cows, respectively. For optimal efficiency 2 to 4 donors should be treated and synchronized with their recipients for each attempt; this allows sharing of the recommended potential 8 to 10 recipients per donor.
Superovulation remains the least predictable step in the process of embryo production. In the bovine tremendous variation in response occurs with age, breed, lactational status, nutritional status, season, and stage of the cycle at which treatment is initiated. Follicle stimulating hormone (FSH), which has a short half-life, necessitates twice-daily injections over a period of 4 to 5 days. Treatment is begun during the mid-luteal phase (day 8 to 12) of the donor’s cycle and employs the use of prostaglandins (PG) to synchronize the cycles of the donors and the recipients. Alternatively, treatment may be initiated on day 16 or 17 (day 0 = estrus) of the donor’s natural estrous cycle. Currently the most commonly used source of FSH in the United States is Folltropin-V.* Twice daily intramuscular injections of FSH are recommended (Table 63-1). Prostaglandins (25–35 mg PGF2α or 500 μg PG-analogue IM) are routinely given at the time of the fifth and sixth FSH injections of a 4-day regimen, which is then followed by estrus in 2 days and ovulation in 3 days. This interval from PG to the onset of estrus is 12 to 24 hours shorter in superovulated animals than in naturally ovulating cows or heifers. Consequently, recipients should be injected with PG 24 hours before the donors if this method of synchronization is used. The response to the FSH regimen ranges from zero to 20 or more ovulations with an average of 8 to 10. There appears to be no difference in response between a 4-day and a 5-day regimen. Generally, heifers require a smaller total dose and older animals a higher total dose.
Many embryo transfer practitioners use exogenous progesterone or intravaginal controlled drug (progesterone) release devices (controlled internal drug release, CIDR)† as part of the superovulation protocol. This approach may be used simply to synchronize a group of donors in order to start the FSH treatment approximately 10 days after their synchronized heat. Alternatively, the donors may be superovulated with the CIDR in place according to the treatment schedule in Table 63-2. The advantage of the latter approach is that the donors may be implanted at any time during their cycle. However, it is critical that the donor is a reproductively normal, cycling animal.
|0||CIDR* inserted vaginally|
|2||PM||100 μg GnRH†|
|4||PM||60 mg (3.0 cc) FSH‡|
|5||AM||60 mg (3.0 cc) FSH|
|PM||60 mg (3.0 cc) FSH|
|6||AM||50 mg (2.5 cc) FSH|
|PM||50 mg (2.5 cc) FSH|
|7||AM||40 mg (2.0 cc) FSH|
|PM||40 mg (2.0 cc) FSH, 35 mg (7.0 cc) PGF2α§|
|8||AM||40 mg (2.0 cc) FSH, 25 mg (5.0 cc) PGF2α|
|9||AM||Estrus and Al|
|PM||Estrus and Al|
|16||Embryo recovery, transfer, and freezing|
* CIDR, Controlled internal drug release; EAZI-BREED CIDR progesterone insert, InterAg Company, Hamilton, NZ, or Pfizer Animal Health, New York.
† GnRH, Gonadotropin releasing hormone; Cystorelin (Gonadorelin), Merial Limited, Iselin, NJ; IM injection.
‡ FSH, Follicle stimulating hormone; Folltropin-V, Bioniche Animal Health USA, Inc., Athens, GA; IM injections.
§ PGF2α, Prostaglandin F2α; Lutalyse, Pfizer Animal Health, New York; IM injections.
It is difficult to accurately assess the number of ovulations by palpation of ovarian structures per rectum on the day of embryo recovery when the number of corpora lutea (CL) exceeds 4 to 6 per ovary or when several anovulatory follicles are also present. An excessive number of anovulatory follicles in the presence of corpora lutea appears to adversely influence the percentage of recovered embryos because of an unfavorable estrogen-to-progesterone ratio, which affects gamete and embryo transport through the tubular reproductive tract.
Donor management can be enhanced by the use of real-time ultrasonography. In addition to accurate assessment of the normalcy of the reproductive tract, including ovarian status, the presence of a dominant follicle (DF) can be ascertained. The presence of an active DF can suppress ovulation rate by as much as 40%. Ablation of the DF prior to FSH treatment allows donors to be scheduled based primarily by the calendar rather than their follicular wave patterns.
Ultrasonography is helpful in assessing the potential superovulatory response on the day prior to ovulation or at the time of AI. When only one or a few follicles are observed a back-up sire can be selected rather than semen from an extremely expensive bull.
Ultrasonography and palpation of the ovaries per rectum have been shown to have similar accuracy for determination of the number of CL at the time of embryo recovery. However, the number of anovulatory follicles can be counted more accurately by ultrasonography, and this information may aid in explaining a poor response to the owner.
Estrus Detection and Insemination
Accurate estrus detection is of great importance not only for timely insemination of the donor, but also for the determination of the degree of synchronization of estrus and ovulation between the donor and her recipients. The age of the embryo is calculated from the time of onset of standing heat.
Donors should be artificially inseminated twice with a 10- to 12-hour interval, beginning 4 to 6 hours after the onset of estrus, to cover the range of time over which the ovulations may occur. Depending on the quality of the frozen semen, a double inseminating dose may be used at each insemination. A double inseminating dose should be used in cows with a large pendulous uterus.
Bovine embryos descend into the uterus around day 4.5 (estrus = day 0) and shed their zona pellucida (“hatch”) between days 8 and 10. Consequently, most nonsurgical recoveries are made between days 6 and 8.
A two-way round tip balloon catheter (French size 16 to 24) with a 30 ml inflatable balloon is used. The two-way catheter has one channel for inflation of the balloon plus 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 may be given 5 to 10 mg of acepromazine or another suitable tranquilizer. Feces are carefully removed from the rectum to avoid aspiration of air, and an estimate is made of the number of ovulations (CL). Epidural anesthesia is administered (4 to 6 ml of 2% lidocaine hydrochloride) to prevent defecation and straining. Fractious animals may be given epidural anesthesia with a combination of xylazine (30 mg) and sterile saline or sterile water (7 ml or sufficient quantity). Bos indicus breeds are more sensitive to the action of xylazine and should receive 20 mg of xylazine in a sufficient quantity, usually 7 ml, of sterile saline. Inadvertent air can be removed from the rectum with a small stomach tube attached to a wet vacuum cleaner. The vulva and perineal region are thoroughly 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 may gently be used to expand and straighten the cervical canal. The dilator and subsequent catheters may be 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 when it is forced through the tight cervical canal. The lips of the vulva are again parted and the balloon catheter, with the stylet in place, is inserted into the vagina and advanced 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. The balloon is slowly inflated with 15 to 20 ml of air or flushing medium in adult cows and 10 to 15 ml of air in heifers. The endometrium can easily be split by overdistention, resulting in hemorrhage 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 1000 ml bottle or bag of flushing medium. 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 approximately 1 meter above the level of the uterus. The horn of the uterus is extended by elevating the tubouterine 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 channeled directly through an embryo filter (75 μm pore size).
In older animals with long pendulous tracts, manipulation of the cervix and uterus can be facilitated by retracting the cervix into the vagina with cervical forceps. If the returning fluid is blood-tinged, the red blood cells may be washed directly through the filter by opening both clamps between the bottle of flushing solution and the filter. The filter should never be allowed to run completely dry, leaving the embryos on the filter disk exposed to the air.
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 balloon 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 though the balloon was initially placed in one of the horns. When this happens, both horns are simply flushed at the same time (body flush).
Flushing and Holding Media
The most commonly used medium for nonsurgical embryo recovery is Dulbecco’s phosphate buffered saline (PBS). One percent heat-treated bovine serum (10 ml) is added to each individual 1 L bottle of flushing medium, which may be used at room temperature. Serum acts as a protein source for embryo growth and membrane stabilization, and renders embryos less sticky. In lieu of serum, 0.04% bovine serum albumin (BSA) may be used for the recovery medium and 0.4% for the holding medium.
Ten to 20% serum is added to the flushing medium to make a holding medium that can also be used for short-term (less than 24 hours) culture. Holding medium should be sterilized by filtration through a 22 μm Millipore filter attached to an all-plastic syringe. The rubber plungers of some syringes have been shown to be coated by an embryotoxic lubricant; hence it is recommended that all-plastic syringes be used.
Holding dishes should be kept covered to minimize contamination and evaporation. The latter will increase the osmolarity of the medium. Changing the embryos to a fresh dish of holding medium periodically (every 2 hours) further minimizes the effects of contamination and evaporation.
By the very nature of the procedure, it is vital that all aspects of quality control of media and equipment that come in contact with the embryos are strictly adhered to. It is also advisable to use commercially prepared media and sterile disposable supplies.1
Embryo Handling and Evaluation
Identification and evaluation of embryos is one of the most challenging aspects that confronts the embryo transfer practitioner, especially the beginner. Embryo quality and poor handling techniques can directly affect pregnancy rates. A stepwise procedure for embryo searching is presented at the end of this section.
Once removed from the stable protective environment of the uterus the embryo should be handled with respect regarding its temperature, pH, osmolarity, and contaminants, factors that may affect viability. Embryos depend on the ambient fluid to maintain their physiologic integrity and as a source of nutrients.
Embryos may be maintained at room temperature for several hours without decreasing pregnancy rates significantly, provided the embryos are transferred to fresh holding medium every 2 hours. Storing embryos in a temperature-controlled portable incubator is recommended for long distance transportation. There is no obvious decrease in pregnancy rates after storage at these temperatures for 12 to 24 hours. On the other hand, embryos do not tolerate temperatures above body temperature (39° C) very well.
The physiologic range of proper pH for embryos is from 7.1 to 7.5. Thus, the pH of flushing and holding media needs to be within this range. Even a slight change in the salt concentration (osmolarity) of the medium can effectively reduce the viability of embryos. If the salt concentration of the flushing or holding media is below that of the uterine environment, embryos will absorb water and swell to reach osmotic equilibrium, which sometimes results in rupture of the cell membrane. Conversely, if the salt concentration is above that of the uterus, the embryo will shrink in size (dehydration), causing a reduction in metabolic activity. In comparing the two situations, although both are detrimental to embryos, shrinkage would be less detrimental. If PBS is prepared from a powdered mixture, care should be taken that the correct amount and quality of water is added. The normal osmolarity of uterine fluid is 270 to 300 milliosmoles.
Exposing the embryos to ultraviolet rays for a prolonged period may cause cellular death. The use of insecticide sprays in the embryology room should be avoided. Insufficient time of aeration after using ethylene oxide gas for sterilization of equipment is detrimental to live cells. Storage period, different suppliers, and batches (lot number) of sera all affect embryo growth differently.
Identification of Embryos
Evaluation of the embryo in the uterine effluent is based on identification of several morphologic features of the embryo using light microscopy. This is the only practical method to determine suitability of the embryo for transfer and freezing. These methods are subjective and depend on experience.
The embryo is spherical and is composed of cells (blastomeres) surrounded by a gelatin-like shell and acellular matrix known as the zona pellucida. The zona pellucida performs a variety of functions until the embryo hatches, and is a nice landmark for embryo identification. The zona is spherical and translucent; thus, it is clearly distinguishable from cellular debris. Because of its shape the embryo tends to roll on the bottom of the searching dish.
The overall diameter of the bovine embryo is 150 to 180 μm including a zona pellucida thickness of 12 to 15 μm. The diameter remains constant until expansion of the blastocele begins. The color of the morula and (early) blastocyst also facilitates identification because the embryo is usually darker than other uterine debris. Knowing the age of the embryo (days after onset of estrus) is also important in locating the embryo in the searching dish. The fully expanded blastocyst possesses a thinner zona pellucida and is pale (translucent) in color. A spontaneously hatched embryo is very hard to identify because the embryonic mass, without the zona pellucida, is morphologically similar to uterine debris. If the hatched embryo has collapsed, it may still be identifiable but with considerable difficulty. In summary, the important criteria in identifying embryos are (1) shape of the embryo, (2) presence of a zona pellucida, (3) size, (4) color, and (5) knowledge of the age of the embryo.
During embryonic development, cell numbers increase by geometric progression. Synchronous cell division is generally maintained up to the 16-cell stage in embryos. After that, cell division becomes asynchronous and finally individual cells possess their own cell cycle. These cells composing the embryos are termed blastomeres and are easily identified up to the 16-cell stage as spherical cells. After the 32-cell stage (morula stage), embryos undergo compaction. As a result, individual cells in the embryo are difficult to identify beyond this stage. The most obvious morphologic manifestation during compaction is the loss of a concise cellular outline. The embryo proper develops from the inner cell mass, whereas the surrounding trophectoderm primarily gives rise to the chorionic ectoderm of the placenta.
Handling the Embryos
Once an embryo is identified in the searching dish, it is immediately transferred to a small Petri dish (35 × 10 mm) containing fresh, filtered (0.22 μm pore size), sterile holding medium. Embryos are tentatively classified simply as good or bad, and may be recorded on the cover of the holding dish. This allows for a quick account of the total number of embryos found. Embryos are then serially rinsed through at least three different dishes containing fresh sterile medium using a new sterile pipet for each step. Finally, they are placed into a dish awaiting transfer or cryopreservation. Under some circumstances (e.g. for export of embryos) they must be rinsed through 10 different dishes containing sterile media and exposed to trypsin.1 All dishes must be kept covered between searches to avoid contamination, and particularly evaporation, when placed in the incubator. Evaporation of the small volume of medium in a flat dish rapidly leads to hypertonic solutions.