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ET Training Manual


EMBRYO TRANSFER

Applications.

Increase reproduction of females
The main use of embryo transfer has been to amplify reproductive rates of valuable females. Domestic

animals may be valuable for many reasons, including scarcity, proven genetic value, or having unique characteristics such as disease resistance. Ideally, embryo transfer is used to satisfy both genetic and financial objectives simultaneously, i.e. milk or meat production, increase efficiency, and return financial benefits.

Import/Export
The desire to improve genetics, to increase variation in the gene pool, and to introduce new breeds, has motivated the import and export of breeding stock. Moreover, if live animals are imported, the genetic influence on the general population is limited until their progeny reach breeding age. While the genetic influence of imported semen can be distributed over a larger portion of the herd, offspring have only 50 percent of the new genes and will not become producing members of the herd for two to three years. With imported embryos, the resulting offspring have 100 percent of the desired genes, but as with artificial insemination, it will be several years until the resulting animals become producers. The relative advantages and disadvantages of importing animals, semen and embryos are summarized in Table 1. Conditions of importation vary widely and frequently require months to years to carry out, many of them in advance of a proposed sales agreement. Thus, logistics are quite complicated and costly.

TABLE 1

Comparison of importing germplasm as postparturient animals, as semen or as embryos
Advantages Animals productive quickly Disadvantages Postparturient animals Expensive Animals often succumb to disease Chance of introducing exotic disease Complex transportation logistics Limited immediate genetic influence if females are imported Semen Inexpensive Low risk of disease transmission Hybrid vigour, F1 and F2* Simple transportation logistics Passive immunity from native dam Embryos Very low risk of disease transmission Costs may be lower than animals Simple transportation logistics Passive immunity from native dam Need for ET technology Long wait until animals productive Need to grade up to get pure-bred animals* Need for Al technology Long wait until animals productive

* If changing from one breed to another

Donor selection
There are two broad criteria for selecting donors for most embryo transfer programmes: (1) genetic superiority and (2) producing large numbers of usable embryos. In some cases, the sole criterion for selection is scarcity, and embryo transfer is used to increase numbers of animals available. Healthy, cycling animals with a history of high fertility make the most successful donors. Donors at least two months post-partum produce more embryos than those closer to calving. Young adults seem to yield slightly more usable embryos than maidens under some conditions. Extremely fat animals make poor donors, because they do not respond well to superovulation and because their reproductive tracts are more difficult to manipulate. Sick animals usually do not produce many good embryos.

Selection of sire
Since half of the genes come from the male, it is extremely important to use genetically superior sires. Selecting the male is usually more important than selecting the donor female because males will normally be bred to many females and can be selected more accurately than females.

Management of donors
One of the most important factors to a successful embryo transfer programme is the management of the donor animals, and the most important aspect of this is nutrition. Nutrition is critical to maximise both the number of transferable embryos recovered and the conception rates following transfer.
Donors must be fed on a rising plane of nutrition beginning at least five weeks

before the flush date. Recipients must also be on a rising plane of nutrition three to four weeks before the programme. As a general rule of thumb, if no pasture feed is available the following applies: ? ? ? If silage is used instead of hay, an equivalent DRY MATTER amount must be fed (silage has a very high moisture content). Hard feed (oats, barley or commercial pellets) should be introduced gradually over a week, not introduced suddenly, so ruminal microflora can become acclimatised. Even when pasture feed is available, feeding up to 2 kg of hard feed will definitely improve results if given according to the above time-frame. Concentrates such as pelleted feeds have the advantage of having added minerals and vitamins. Mineral and vitamin levels should be monitored and maintained during the programme by giving supplements when necessary. The levels of each of these will vary from region to region, so local knowledge is important when addressing this.

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Environmental factors such a rain, sudden coldness, excess heat, stress etc. can also lead to a lower embryo recovery rate. It is important that the donors will not be expose to these kinds of conditions as far as possible. Certain species, such as goats and deer seem to be particularly susceptible to stress. A disease prevention and vaccination programme is also important to minimise any chances of the donors or recipients contracting any disease prior to and during the programme. At the end of a programme the donors are put on a low maintenance diet to lower their body weights prior to starting the next programme.

Selection of recipients
A common question in cattle ET is whether to use cows or heifers as recipients. The big advantage in using cows is there is less difficulty with calving. Conversely, heifers are easier to manage than cows because they are not lactating. Heifers generally have higher fertility than cows, especially dairy cows. On the other hand, it is more difficult to transfer embryos into heifers than cows. This does not apply in other species where the implants are done surgically. Generally maidens do as well as older animals in these species. Whatever the type of animal used, the main factor is that they must be normal fertile animals. Any animals that have failed to become pregnant to natural mating are unsuitable for use as recipients.

Management of recipients
On-the-farm recipient programmes must be tailored to the resources available. The following are essential:
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Cycling animals, not exposed to males. Animals in a good state of nutrition, preferably gaining weight. Herd health programmes. A simple, permanent means of easily identifying each animal. Facilities to keep many animals in close proximity to the treatment area for synchronization treatments, oestrus detection and embryo transfer. In the case of sheep, the donors and recipients should be run with vasectomised males to promote cycling behaviour. An excellent oestrus detection programme. Conscientious personnel.

Oestrus detection
Every donor and recipient should be checked visually for oestrus at least twice each day when they are expected in heat. Each animal will be in one of three categories each time oestrus is checked: (1) not in oestrus, (2) suspicious, or (3) in standing oestrus. Cows in oestrus stand when mounted by others. Suspicious signs include ruffled rump hair, restlessness, bawling, walking the fence, nudging, mounting, sniffing, tail raising, discharge of clear mucus from the vulva, and swelling and inflammation of the vulva. Not every animal showing one of these characteristics should be recorded as suspicious, but donors should be watched closely for standing oestrus and recorded as suspicious if it displays most of these characteristics.

In cattle, aids to oestrus detection, such as paint on the tailhead or “Kamar” heat detectors, are useful as long as they do not become a replacement for visual detection. Such aids are recommended for donors for the oestrus resulting from superovulation.. In sheep and goat programmes it is best to run donors and recipients with teaser males to identify those in heat.

Superovulatory treatments
Superovulation is the term used when OvagenTMis used to ensure that a donor releases a number of oocytes at oestrus, rather than the usual one oocyte.

Figure 1 : A sheep ovary following superovulation with Ovagen.

The protocol for superovulation is to use a combination of an intravaginal progesterone releasing device (CIDR or DIB) and prostaglandin to synchronise the cycle of the donors. Eight injections of OvagenTMat 12 hour intervals causes the donor to superovulate.

Figure 2 : A typical bovine superovulation programme, showing the reason for each treatment.
DAY 0 2 6 7 8 TREATMENT Insert intravaginal CIDR. Inject Oestradiol Benzoate Begin 12 hourly Ovagen injections Ovagen continues Ovagen continues Inject Prostaglandin Remove CIDR 9 10 Ovagen continues Donor comes into heat and is mated or artificially inseminated. 17 Donor is "flushed" to recover embryos Fertilises the eggs Induces heat Induces heat EFFECT Prevents the donor from coming into heat Synchronises the donors follicle development Causes the donor to superovulate

Dosage rates of OvagenTM in cattle depend on several factors. Some embryo transfer practitioners who commonly use FSH prefer to use a descending dose regime to lessen the negative effects of LH on embryos. With OvagenTM this practice is not necessary as Ovagen? has extremely low levels of LH compared with other FSH products on the market. However, given that the total dose is correct for a particular animal, a descending dose regime program will not affect the outcome of the program. The following recommended doses are guidelines only and should be adjusted for body weight, age, breed, and local experience

Type of Animal

Total recommended Ovagen?

dosage of

Total Volume per dose four day programme

for

Dairy cows (excluding Jerseys) Dairy heifers (excluding Jerseys)

16 mL – 20 mL Ovagen? 13 mL – 15 mL Ovagen?

2.00 mL – 2.50 mL Ovagen? 1.63 mL – 1.88 mL Ovagen?

Jersey cows Jersey heifers

10 mL – 12 mL Ovagen? 8 mL – 10 mL Ovagen?

1.25 mL – 1.50 mL Ovagen? 1.0 mL – 1.25 mL Ovagen?

Bos taurus beef cows Bos Taurus beef heifers

16 mL – 18mL Ovagen? 12 mL – 15 mL Ovagen?

2.0 mL – 2.25 mL Ovagen? 1.50 mL – 1.88 mL Ovagen?

Bos Indicus cows Bos Indicus heifers

12 mL – 14 mL Ovagen? 10 mL – 12 mL Ovagen?

1.50 mL – 1.75 mL Ovagen? 1.25 mL – 1.50 mL Ovagen?

Note. Some cow families are highly sensitive to FSH treatment; if no previous donor or family flushing history is known, begin on the low end of the given recommended range.

Optimum dosage of Ovagen also vary between breeds of sheep, goats and deer, as well as being influenced by management practices.

Insemination
Following superovulatory treatment, the donor should be observed closely for signs of oestrus. Superovulated cows sometimes do not display oestrous behaviour as clearly as untreated cows, therefore such oestrous detection aids as KaMaR indicators may be helpful. If liquid semen is available, between 10 and 50 × 106 motile spermatozoa are inseminated 12 hours after the donor is observed in oestrus, and a similar quantity 12 hours later. If frozen semen is used, one ampoule or straw each time is inseminated 12 and 24 hours after the donor was first noticed in oestrus. Some people recommend two doses per insemination, particularly at the second insemination. Frozen semen is thawed in a water bath at 35°C (95°F) and inseminated immediately. If only one insemination is to be done, it should be at 24 hours after oestrus was first detected. Cervical AI can also be carried out in elk and red deer. However, it is less successful in sheep and goats where laparoscopic AI is used.

Recovery of embryos
In most cases, embryos are recovered six to eight days after the beginning of oestrus (day 0). Embryos can be recovered non-surgically as early as four days after oestrus from some cows, but prior to day 6 recovery rates are lower than on days 6 to 8. Embryos can also be recovered on days 9 to 14 after oestrus; however, they hatch from the zona pellucida on day 9 or 10, making them more difficult to identify and isolate and more susceptible to infection and damage. Cattle are flushed non-surgically. The donor is given an epidural injection.

A catheter is inserted through the cervix into the uterus so that it is in the position indicated in Figure 3. The uterus is then flushed using Emcare Complete embryo flushing fluid. The recovered fluid is passed through a filter, so that only the embryos remain in the fluid in the filter cup.

FIGURE3 Position of Foley catheter for uterine horn flush

Figure 4. Emcare flushing fluid attached to the flush tubing, catheter and filter.

Because of their small cervices, sheep, goats and deer are flushed using a surgical procedure. This requires a general anaesthetic and a sterile technique to avoid infection and subsequent scarring of the uterus and ovaries, which may result in infertility.

Figure 5. Surgical flushing in a goat.

Embryology. As the embryos are found in the Emcare flushing medium, they are transferred into a tissue culture dish containing Emcare Holding Medium. Emcare Holding Medium is a patented medium that contains Zwitterions. Replacement of phosphate in the medium with ICPbio’s zwitterionic buffer MOPS (3-[N-morpholino] propane sulphonic acid) offers a metabolically inert buffering ion which does not cross membranes, thus providing a stable micro-environment at the zona pellucida, and avoids possible inhibition of embryo development by phosphate or other ions. Emcare Holding Medium also contains ICPbio low endotoxin bovine albumin. This acts to adsorb proteases and endotoxins, together with plasticisers and such other contaminants. Consequently it confers a significant degree of protection to embryos in culture. In addition it also works as an energy source

and surfactant. It has been shown that bovine embryos have a clear metabolic requirement for exogenous albumin.

Figure 6. Transferring embryos into Emcare Holding Medium.

Storage conditions
Embryos are damaged if exposed to excess light (e.g. microscope lights) for prolonged periods, but normal lighting or daylight in the work area is not harmful if embryos are not exposed for more than half an hour. Embryos will survive for up to 12 hours in Emcare Holding Medium at room temperature. For longer periods, it is recommended that the embryos be incubated at 350C, or they may be cooled to 40C over two to three hours and held at that temperature for 12 to 14 hours.

Containers
Embryos should be stored in small (<5 ml), sterile, transparent, sealable, inert containers. Small test tubes, Petri dishes or multi-well plates are convenient for routine use (Figure 6), but small test tubes are recommended if embryos must be moved any distance before loading into straws. Except while they are being manipulated, embryos should be stored in a dark, dust-free incubator or cabinet (set at room temperature or 37°C).

Figure 7.
Left: Plastic multi-well plate with 16 × 17-mm wells (lid to left); Right: 60 × 15-mm and 35 × 10-mm culture dishes, and a 12 × 75-mm test tube

Evaluation of embryos
Terminology.
A newly ovulated female gamete is an oocyte. Upon fertilization, the oocyte becomes a one-cell embryo. The embryo then divides into two-cell, fourcell, etc. At the 16-cell stage, the embryo becomes a morula. When a cavity (blastocoele) forms between the cells of the embryo, it is termed a blastocyst. The first three divisions of the embryo are called cleavage divisions; thus, one-to eight-cell embryos are defined as cleavage stages. During the morula stage, cells of embryos change from spherical to polygonal in shape. This phenomenon is termed compaction. Compaction is an excellent sign that the embryo is developing normally. As the morula

develops into a blastocyst, it forms a cavity, the blastocoele, by expending energy to pump fluid between the cells.

FIGURE 8 Diagram of normal bovine embryos

The zona pellucida is a gelatine-like capsule that surrounds the oocyte and early embryo. It has receptors for sperm that are inactivated after fertilization, it keeps the cells of the pre-compaction embryo together, and protects these young cells from the immune system and from pathogens. . When the blastocoele becomes very large, the embryo expands, which thins the zona pellucida. This is the expanded blastocyst stage. After one to one days more, the expansion is so great that the embryo hatches out of the zona pellucida.

Evaluation
For many beginners, the most intimidating aspect of the embryo transfer process is morphological evaluation of embryos. There are three elements to successful evaluation of embryos: training, experience and proper equipment.

Training includes learning the correct morphology of embryos at different times post-oestrus and the meaning of deviations from normal morphology. One must also learn how to manipulate and examine embryos.

FIGURE 9

(A) Follicular oocyte with adherent follicle cells (B) Follicular oocyte after removing follicle cells. (C) Normal appearing 1-cell ovum recovered five days after oestrus. Note spermatozoa in the zona pellucida. (D) Normal, unfertilised, ovulated oocyte recovered three days after oestrus

Embryos collected six days post-oestrus should be post-compaction or socalled tight morulae. They should have 50–80 cells. Embryos should be generally spherical or ovoid, not too light nor too dark in colour and have uniform cell size. Deviations from normal include irregular cell sizes, large vacuoles in cells, areas of degeneration in the embryos, some cells not compacted with the main cell mass (termed extruded or excluded blastomeres), and a damaged zona pellucida. Most of these abnormalities are a matter of degree. If part of the embryo appears degenerate, but the bulk of the embryo appears normal, it has an excellent chance of developing into a normal calf (e.g. Figure 22B).

FIGURE 10 (A) Unfertilized oocyte recovered five days after oestrus. (B) Same ovum as in (A) with bright-field optics. (C) Cracked, empty zona pellucida recovered five days after oestrus.(D) Unfertilised oocyte recovered six days after oestrus. Note blisters of clear cytoplasm.

FIGURE 11 (A) Degenerate, unfertilized ovum recovered five days after oestrus. Nomarski optics. (B) Unfertilized ovum with two fragments of cytoplasm. Note large vesicles within cytoplasm. Bright-field optics. (C) Fragmented ovum, likely unfertilized recovered five days after oestrus. Bright-field optics. (D) Disintegrated ovum, probably unfertilized. Bright-field optics

FIGURE 12 (A) Normal appearing 2-cell embryo recovered four and a half days after oestrus, (B) Degenerating 2-cell embryo recovered five days after oestrus. Note clear cytoplasm in one blastomere. (C) Normal 4-cell embryo recovered two and a half days after oestrus. (D) A 2-cell embryo recovered five days after oestrus. Note clear cytoplasm.

Day-7 embryos should be early blastocysts. Day-8 embryos should have a large blastocoele and some should be expanding, i.e. the diameter should be increasing so that the zona pellucida is thinned. A distinct, inner cell mass should be present. As with day-6 embryos, various imperfections are not uncommon in perfectly acceptable embryos. FIGURE 13 (A) Normal 8-cell embryo recovered three days after oestrus. (B) Same embryo as in (A) but Nomarski optics. (C) Normal 12- to 14-cell embryo recovered four days after oestrus. (D) Severely retarded 12- to 14-cell embryo recovered six days after oestrus.

FIGURE 14 (A) Uncompacted morula recovered three days after oestrus, probably degenerating. (B) Uncompacted morula recovered three days after oestrus; dark cytoplasm. (C) Severely retarded and degenerating embryo recovered six days after oestrus. (D) Severely degenerate embryo recovered seven days after oestrus. All are bright-field optics.

The single most difficult task for people learning to classify embryos is to distinguish between tight morulae and unfertilised oocytes that look very similar in size and texture. The unfertilised ovum has a perfectly smooth cell membrane, at least over a part of the cell, while the tight morula will have a slightly scalloped appearance. FIGURE15 Newly compacted morula recovered seven days after oestrus.(B) Compacted morula recovered seven days after oestrus.(C)compacted morula recovered seven days after oestrus, fair morphological quality. (D) Poor quality morula with many degenerate cells.

FIGURE 16

(A) Normal, early, expanded blastocyst recovered seven days after oestrus.(B) Same embryo as in (A) but Nomarski optics. (C) Normal, expanded blastocyst recovered seven and a half days after oestrus. Note the thinned zona pellucida. Bright-field optics. (D) Hatching blastocyst typically found nine days after oestrus.

FIGURE 17

(A) Good quality compacted morula with a few degenerate cells.(B) Unfertilised ovum recovered seven days after oestrus, easily mistaken for a morula with a dissecting microscope.(C) Degenerate, probably unfertilised ovum(D) Degenerate, unfertilised ovum, easily mistaken for morula at lower magnification

FIGURE 18

(A) Newly compacted morula recovered six days after oestrus (good quality) but with one large and probably abnormal cell to the upper right. (B) Unfertilised ovum easily mistaken for a morula. (C) Normal blastocyst recovered seven and a half days after oestrus. (D) Unfertilised ovum with large vesicle recovered five days after oestrus, easily mistaken for a blastocyst at lower magnification. All are bright-field optics

The proper procedure for classifying embryos is to isolate them, remove debris and then separate them into groups of transferable (or freezable or splittable) and non-transferable (unfertilised or severely degenerate) groups. In cases in which classification is uncertain, ova should be examined with a compound microscope. It is often impossible to determine if an ovum is a severely degenerate embryo or is unfertilised. Even two-or three-cell embryos may in fact be fragmented, unfertilised ova. Clearly, this classification system ranks embryos reasonably well on a statistical basis. Of course, it is far from ideal from the standpoint of sorting embryos into the group that will result in calves and the group that will not. As a rule of thumb, only good and excellent embryos are suitable for splitting, and only fair, good, and excellent ones are suitable for freezing. Results of freezing fair quality embryos are marginal.

Transfer of embryos in cattle.
In cattle, embryos are routinely transferred into the uterine horn on the same side as the ovary with the corpus luteum. . The big problem with non-surgical transfer is the difficulty in becoming proficient in this technique. First, it is necessary to be able to palpate ovaries accurately in order to select the side of ovulation. Recipients must be rejected if no corpus luteum is present or pathology of the reproductive tract is noted. Even very experienced technicians make some errors in palpating corpora lutea. The next step is to pass the embryo transfer device through the cervix. This is more challenging during the luteal phase, which is when embryos are transferred, than during oestrus, when artificial insemination is done and the cervix is more open. Heifers present a special challenge because of the small cervix; some breeds of cattle are more difficult than others, e.g. certain Bos indicus breeds require greater skill. The best training prior to undertaking non-surgical embryo transfer is experience in artificial insemination. Ideally, the trainee will have inseminated hundreds of cattle artificially, including a large number of heifers. The third step with non-surgical transfer is to be able to insert the tip of the instrument into the desired uterine horn quickly, smoothly and atraumatically. Some people never master this technique, and others require hundreds of transfers to become proficient. This is not surprising since pregnancy rates from artificial insemination are usually markedly lower for the first 50–100 cows bred by a newly trained inseminator than after he or she has become proficient. Well-trained inseminators generally require 100–200 non-surgical transfers until their pregnancy rates plateau; others usually require more. Most technicians who are successful with nonsurgical transfer had low pregnancy rates for their first 100 non-surgical transfers.

Loading straws.
We recommend loading straws for embryo transfer as illustrated in Figure 16. The first step is to take a sterile 0.25-cc straw, label it and rinse it twice with medium to removed any toxic contaminants, taking care not to wet the cotton plug and to discard the rinses. A plastic 1-cc tuberculin syringe fits snugly over the straw for aspirating and expelling fluid. The straw is filled nearly one-third full of fluid, then with a 5-mm column of air, then another column of fluid containing the embryo, one-third the length of the straw, then another short column of air, and finally more fluid to fill the straw and wet the cotton plug. Care must be taken not to compromise the sterility of the tip of the straw or the internal surfaces.

Embryo transfer equipment.
The most commonly used instrument for non-surgical transfer is the standard Cassou-type embryo transfer gun for French straws (Figure 17). There are many other basically similar devices. These are used with specially designed sheaths with a-traumatic tips. A sterile plastic chemise is placed over the loaded instrument. This keeps the gun clean as it is placed in the vagina, and the gun is pushed through the plastic bag as it enters the cervix.

Figure 19 : Non-surgical transfer equipment illustrating a 0.25-cc plastic straw, a Cassou-type transfer gun, the plastic sheath and a chemise.

Epidural anaesthesia.
Epidural anaesthesia is recommended for routine non-surgical transfer. This relaxes rectal musculature, making it easier to manipulate the reproductive tract gently as is required for high pregnancy rates. Very experienced technicians sometimes do not use epidural anaesthesia. However, under most conditions this is probably unwise because of the occasional difficult animal. Epidural anaesthesia clearly costs some minutes in time, and occasionally effective anaesthesia is not attained. The problem of waiting several minutes until the rectal muscles relax can be circumvented by having an assistant give the epidural injection about five minutes before embryo transfer while the technician is transferring the embryo to the previous recipient. The procedure for epidural anaesthesia is the same as for non-surgical embryo recovery.

Embryo Transfer in small ruminants.
Because of the small size of the cervix in small ruminants, the most effective way to transfer embryos is by laparoscopy. The recipients (whether sheep, goats or deer) are heavily sedated and placed on a surgical trolley with the hind feet elevated. Two small incisions are made in the body wall, and a laparoscope introduced so that the ovaries can be visualised. The horn of the uterus ipsilateral to the corpus luteum is exteriorised through the second incision, and the embryo is injected directly into the uterus.

Figure 20 : Laparoscopic implantation of an embryo into a sheep.

Figure 21 : Laparoscopic transfer into a red deer.

Synchronisation of reproductive cycles.
The stage of the reproductive cycle of the recipient must correspond to that of the donor or physiological stage of development of the embryo. Two questions arise: to what extent is asynchrony of reproductive cycles tolerated, and what methods can be used successfully for synchronizing reproductive cycles pharmacologically? Many studies indicate that pregnancy rates decline with asynchrony of donor and recipient In most studies with morphologically normal, unfrozen embryos, pregnancy rates were similar with perfect synchrony and asynchrony of 1.5 days. If there is more asynchrony than this, the pregnancy rates fall off. There are many methods of synchronizing reproductive cycles of recipients to match those of donors. In some circumstances, natural synchrony is feasible, but in most cases some recipients will need to be synchronized to augment those whose oestrous cycles match the donor's naturally. The most widely accepted procedure for synchronizing recipients is administration of Progesterone releasing intravaginal devices (CIDRs or DIBs). These are usually inserted for 8 to 10 days, and an injection of prostaglandin given 24 hours before the removal of the device. The recipients will come into heat 36 to 48 hours after removal.

Cryopreservation of embryos
Embryos can be cryopreserved in liquid nitrogen. If procedures are carried out correctly, pregnancy rates are 75–85 percent of those for fresh embryos transferred under similar circumstances. The following protocol has worked well in a variety of settings, but attention to detail is required. 1. Start with good to excellent quality embryos recovered six to eight days after the donor's oestrus. Embryos should be frozen within three to four hours of recovery. 2. Wash embryos through at least three changes of medium (ten washes if they are to be exported. 3. Embryos should be evaluated morphologically and only Grade 1 embryos frozen. 4. They are then put into Emcare Ethylene Glycol freeze medium for a maximum of 10 minutes. All of these steps are done at room temperature. 5. Rinse pre-labelled 0.25- French straws in the Emcare freezing medium to remove any toxic residues. Next, fill the straw half-way with freezing medium, then an air bubble of 4 mm, then another column of Emcare freezing medium containing the embryo so that the straw is 90 percent full when the cotton end is wetted. The end of the straw is then sealed with polyvinyl chloride powder (PVC). The straw is placed into the freezing machine vertically, with PVCsealed end up.

6. Cool straws to -6°C. The rate of cooling during this step can be slow or rapid. 7. Seed straws after they have been at -6°C for five minutes, and keep them at -6°C for an additional 10 minutes. Be sure that they remain seeded. Seeding is accomplished by touching the side of the embryo container with forceps or a cotton bud dipped into liquid nitrogen (Figure 29A). 8. Cool straws from -6°C to -30°C at 0.5°C/minute. When straws reach -30°C, put onto hold for an additional 10 min and then plunge them into liquid nitrogen (Figure 29B).

Figure 22. Inducing formation of ice crystals (seeding) by touching the walls of the straw with a cotton bud cooled in liquid nitrogen.

Figure 23. Transferring a straw with a frozen embryo in an insulated container of liquid nitrogen from the freezing machine to the liquid nitrogen tank (B)

Thawing embryos.
The straws are thawed by removing them from the liquid Nitrogen and holding them in air for five seconds. They are then held in a waterbath at 300C for 20 seconds. The straw is then dried and loaded into the transfer gun, in the case of cattle, or into the transfer pipette in small ruminants. Thawed embryos must be implanted into recipients within 10 minutes.

Splitting embryos
Embryos can be bisected from the two-cell stage through the hatched blastocyst stage. Identical twins are obtained, which are very useful for research as well as for certain commercial goals. Under above average commercial conditions, about 50 percent more calves result per two demiembryos than per whole embryo. Most of these procedures consist of two stages: immobilizing the embryo and bisecting it.
FIGURE 24 Immobilizing a blastocyst by means of suction using a micropipette and bisecting the embryo with a fragment of razor blade (A), and removing one demiembryo from the zona pellucida.

Because the demi-embryos will be the same sex, one or two may be transferred per recipient. It is probably best to place one demi-embryo in

each uterine horn rather than placing both ipsilateral to the corpus luteum. Twinning results in more calves per recipient. The main disadvantage is that there is increased morbidity and mortality when cows have two calves, and the calves will be slightly smaller than singles.

Washing procedures for work areas, glassware and equipment
Glassware and equipment made only of metal (e.g. cervical expanders or stylets for Foley catheters) should be scrubbed thoroughly on all surfaces in a basic detergent, soaked in an acidic detergent, and subsequently rinsed 12 times in tap water and 12 times in distilled, deionized water. Adequate rinsing is essential because all detergents are embryocidal. Glass- and metalware should be wrapped with aluminium foil to protect from contamination any surface that may come in contact with embryos or the reproductive tract of the cows, and then sterilized by dry heat at 180°C for two hours, or autoclaved at a temperature of 121°C at a pressure of 104 kilopascals for at least 30 minutes. All reusable heat-labile items, such as catheters, tubing, or searching dishes, should be disassembled and washed in the same manner as glassware. After they have dried completely, they should be wrapped and sealed in a gas-permeable paper. The recommended sterilization procedure is exposure to at least 500 mg of ethylene oxide per 1 000 cm3 for 24 hours. Ethylene oxide is highly embryocidal and residues can take 24 hours to months to dissipate, depending on the concentration of ethylene oxide, duration of sterilization, material sterilized, the type of packaging, and aeration conditions. Aeration for one week at room temperature should be adequate for most materials. A heated aerator or an evacuation hood are useful pieces of equipment, but are not required. Staff must take precautions to avoid contact with ethylene oxide because it is highly mutagenic. Solutions should be prepared with pure water. This can be obtained from sophisticated deionizing and filtration systems or from water distilled in glass stills. At least two distillations and sometimes more are required to obtain suitable water. Basic salt solutions (e.g. sodium chloride) can be decanted into 500-ml (or smaller) screw-cap bottles and sterilized by moist heat as outlined above. Bottle caps should be loosened before sterilization and tightened afterwards. More complex solutions should be sterilized by membrane filtration with 0.22-?m pore size, taking care to use positive pressure to avoid frothing

and unacceptable changes in pH. It may be helpful to filter solutions preliminarily using a 0.45-?m pore size to reduce the likelihood of clogging the finer membrane during sterilization.

Rinsing prior to use.
As an added precaution, all equipment, whether taken directly from the manufacturer's package or sterilized at the embryo transfer unit, should be rinsed just prior to use with sterile medium. Under no circumstances should equipment be used for more than one cow or more than one group of embryos without first being washed and sterilized. Equipment to be sterilized and used again should be disassembled and put to soak in soapy water as soon as possible after use.

KEY INGREDIENTS FOR SUCCESSFUL EMBRYO TRANSFER PROGRAMMES
Sometimes embryo transfer programmes are failures, usually because pregnancy rates are very low. Probably the main reason for failure is insufficient investment in training personnel. The second most common problem is insufficient animal resources. Facilities and equipment are also important, but are frequently over-emphasized.

Examples of reasonable goals of embryo transfer programmes
? To meet commercial objectives, i.e. to make a profit by providing services where commercial demand exists ? To train personnel who are in demand to meet other goals ? For research purposes, where embryo transfer is deemed the best approach to testing a hypothesis ? For preserving genetic material of indigenous breeds in danger of extinction. ? For importing embryos to provide new genetic resources and then increasing the numbers of animals of the new breed quickly ? For national livestock improvement programmes, such as MOET schemes, in which embryo transfer fits into a well-thought-out overall programme ? To test otherwise outstanding males and females suspected of being carriers of undesirable recessive genetic traits


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