Embryo Transfer in Alpacas

Jorge Reyna

BSc (Hons), MScVetSc (Sydney Univ.)

Lecturer in Higher Education - Learning Design

Faculty of Science

University of Technology Sydney

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INTRODUCTION

Genetic gain in alpacas is slow, as males reach puberty at one-three years, gestation is long (11.5 months), and there is a limited number of offspring from females over their whole reproductive life. Limitations of AI include the lack of a reliable technique to collect sperm due to the length of copulation, the unique mucoid character of the sperm, low concentration and motility of the spermatozoa, and the lack of techniques to store sperm in chilled or frozen form. Even with 42% success in our AI program with frozen-thawed sperm, it is not yet commercially viable (Reyna 2006c).
 

Another powerful reproductive technique is embryo transfer, which involves removing one or more embryos from the reproductive tract of donor females (valuable females) and transferring them to one or more recipient females (low value females). But the actual transfer of an embryo is only one step in a series of processes that may include some or all of the following: superovulation and insemination of donors, collection of embryos, isolation, evaluation and short-term storage of embryos, micromanipulation and genetic testing of embryos, freezing of embryos, recipient synchronisation and embryo transfer. This powerful reproductive technique was successfully accomplished for the first time by Walter Heape in 1890, starting as a research tool and becoming a commercial enterprise in cattle by the early 1970s.
 

In the case of alpacas, the first experiment was conducted in Peru in 1968 (Novoa M and Sumar K), but it was not until 1974 that the first ET cria was born (Sumar K and Franco). Later, in 1987 and 1996, more crias were reported in Peru (Palomino). The slow progress was manly due to political and economic factors which I have described in a previous article (Reyna 2005). In Australia the first ET alpaca cria was reported in 2002 (Vaughan). In the case of llamas, the first ET cria was born in the USA (Wiepz and Chapman 1985). More were born later in the UK (Bourke et al. 1992) and Chile (Gatica et al. 1994).
 

The objective of this paper is to describe the current knowledge about ET in SAC, including herd evaluation and selection of animals, superovulatory protocols, factors which affect superovulatory response, recipient synchronisation, embryo collection and assessment, recovery rates and fertility rates in ET, and also limitations and the need for further research in the area.
 

1. HERD EVALUATION

Embryo transfer as a reproductive technique is not complicated, but it is time-consuming and also requires investment in superovulatory hormones, laboratory consumables and service fees. For this reason, Herd evaluation (HE) is essential prior to starting an ET program, to minimise the risk of choosing animals which are not suitable for the procedure.

Herd evaluation (HE) is a new procedure developed by Alpaca Reproductive Technologies (ART) to ensure that animals destined for an ET program (donors and recipients) are suitable for the procedure, thereby optimising the production of quality embryos and pregnancies. Herd evaluation does not guarantee that females will respond to superovulatory treatments in the future or that recipients will carry the pregnancy successfully and produce a cria. The idea is to discard “problem animals” which lead to less efficient results.
 

Herd evaluation takes into account 4 essential criteria that allow us to choose an animal as suitable for an ET program: (1) rectal accessibility (2) morphology of the reproductive tract (3) ovarian examination, and (4) cervical accessibility. I have discussed this topic in a previous article (Reyna 2006b).
 

A) DONOR SELECTION

Two criteria are considered for selecting donors for an embryo transfer program: (1) genetic superiority, meaning animals which contribute to the genetic objectives of the program, and (2) likelihood of producing large numbers of usable embryos. For example, it makes good sense to select donors whose offspring can be sold at a profit above embryo transfer expenses. Obviously, it is inappropriate to produce animals that will not be accepted by farmers. In many cases, objective measures of genetic superiority can be used, for example conformation, fleece fineness (density, crimp, yield and persistence), precocity and growth rates, carcass weight (in Peru), fertility, and disease resistance. Because phenotypic superiority may not indicate genetic superiority, it is usually desirable to consult someone trained in animal breeding so that the best donors are selected to meet objectives.
 

Selection of donors for embryo production is frequently overlooked; indeed, in using embryo transfer to circumvent infertility one often selects against this trait. Although embryo production should be secondary to genetic superiority, it should still be considered seriously. Healthy female alpacas with a history of high fertility make the most successful donors. When there is a choice, animals without problems such as retained placenta should be used. Donors at least two months post-partum should be used as they are likely to produce more embryos. In the case of cattle, young cows seem to yield slightly more usable embryos than heifers under some conditions (Hasler 2004). In alpacas there is no evidence of how age may affect embryo production capacity.
 

First, when there is a choice, use inherently fertile animals for donors, animals which are at least two months post-partum and otherwise in good reproductive health. Second, encourage management practices that minimise or circumvent potential problems, such as having animals gaining weight at the time of embryo transfer. Third, develop strategies to deal with problems caused by the embryo transfer program itself. For example, repeated superovulation of the same donor means that she will not be going through an annual reproductive cycle; cows tend to get fat under such circumstances.

B) RECIPIENT SELECTION

In the case of recipients, you should select healthy animals of a good size, proven fertility and good maternal ability. I prefer to use animals that have llama genes (Chilean types), as they usually are of good size and fertility (Figure 1). It is important to avoid using small and medium size animals, to obviate the problems of rectal inaccessibility and the distochia which can result from the implantation of embryos from larger animals.
 

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Figure 1: Typical Chilean alpaca types are desirable females as embryo recipients due to good size, easy rectal accessibility and maternal ability

2. SUPEROVULATORY PROTOCOLS

In SAC superovulatory treatments are carried out in two steps; the first is to induce follicular growth using FSH or eCG. For this, it is necessary to ensure that there is no dominant follicle at the initiation of the treatment. In cows/ewes it has been demonstrated that a dominant follicle has a detrimental effect on the superovulatory response and affects the yield of embryos. There is no information available on SAC, but presumably follicular dominance will suppress the superstimulatory response in llamas and alpacas. The second step is to produce ovulation of the follicles which have been thus stimulated with the use of GnRH or a synthetic analogue.
 

It is important to mention that, in recent years, FSH has surpassed eCG as the method of choice for superovulating cattle. In most studies comparing the two procedures, FSH treatment has resulted in slightly higher numbers of usable embryos. However, eCG works nearly as well. Everyone agrees that eCG results in a much larger ovary, generally double the volume of one treated with FSH. This is probably related to its very long half-life (five days) in cattle (that of FSH is several hours), which results in continued recruitment of follicles after ovulation, very high progesterone levels and, probably, abnormalities in ovum transport (Figure 2).
 

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Figure 2: An alpaca ovary stimulated with eCG, generally double the normal size, showing two unovulatory follicles, 15 and 21 mm, left and right respectively.

Superovulatory treatment schemes in SAC have been summarized by Ratto (2005) as follows:
 

a. Luteal phase induced by ovulation (Bourke et al. 1995): GnRH or hCG is given when a dominant follicle ≥ 9 mm is present (day 0). At day 7, 1000 IU of ECG is administered (i.m). At day 9, a luteolytic dose of prostaglandin is given and 750 IU of hCG is given when follicles reach 9 to 13 mm in diameter.
 

b. Luteal phase stimulated by progesterone treatment (Bourke et al. 1992; Garcia et al. 1994; Bourke et al. 1995): Using progesterone or progestogens for 7 to 12 days, pFSH injections (20mg) every 12 h for 5 days, starting 48 h before progestogen withdrawal. To induce ovulation a dose of 750 IU of hCG or 8 µg of GnRH was administered.
 

c. Sexually receptive phase (Correa et al. 1997; Ratto et al. 1997): females that present sexual receptivity for 5 consecutive days receive 20 mg of pFSH every 12 h for 5 days (200 mg). After the last injection, females are treated with 750 IU of hCG to induce ovulation.
 

The number of ovulations or CL varies widely among studies, ranging from 2 to 11 per animal. The variations can be attributed to follicular status at the start of the superovulatory protocol, different types of gonadotrophins used, different protocols and purity of the hormone preparations, and also possibly also environmental conditions. We will discuss these factors later. Table No 1 presents superovulation schemes used in SAC.
 

Table 1: Superovulation schemes used in South American Camelids.

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At present I am testing several superovulatory protocols including the use of intravaginal sponges impregnated with FGA for 7 days, injections of oestradiol and multiple doses of pFSH every 12 h for 3 to 5 days. Also, the combination of a single dose of eCG + multiple pFSH injections (3-5 days) as reported in the dromedary camel (Skidmore 2004) is currently under evaluation . On the other hand, similar superovulatory responses have been achieved with a single dose of pFSH diluted on a “long term vehicle” in our first trial, but a large group of animals is required to confirm our findings.
 

3. FACTORS THAT AFFECT THE SUPEROVULATORY RESPONSE

There are many factors that may affect the superovulatory response in SAC as in other domestic species. Regrettably most of these factors have not been validated in field trials in SAC and still remain unknown. I will mention and describe some of the most important:
 

A) Ovarian status at the beginning of the superovulatory treatment. It has been reported in cows (Huntinen et al. 1992) and ewes (Rubianes 2000) that the presence of a dominant follicle will affect superovulatory response. Females who present a dominant follicle at the beginning of the treatment will produce fewer embryos than females without a dominant follicle. The probable reason is that the dominant follicle produces oestradiol and inhibin, which cause the subordinate follicles to regress, and even FSH injections cannot “rescue” these follicles from becoming atresic. There is no information in this area in SAC. It would be a very simple experiment to superovulate a group of animals in the presence/absence of a dominant follicle to draw a conclusion.
 

B) Combination of hormones used and purity. As I mentioned above in the superovulatory protocols, FSH treatment has resulted in slightly higher numbers of usable embryos than eCG has in cattle. The reason is that eCG has a very long half-life which results in continued recruitment of follicles after ovulation, very high oestradiol levels and, probably, abnormalities in ovum transport.
 

A special problem with FSH products is that they are quite impure, as they are obtained from swine pituitaries. Recently, other FSH products of various purities have become available. Most of them feature low contamination with luteinising hormone (LH). Although the addition of large amounts of LH to FSH reduces its efficacy for the superovulation of cattle, there are few convincing studies which show that the small amount of LH found in commercial batches of FSH decreases efficacy greatly. In fact, some LH may be needed for optimal superovulatory responses. The other impurities in FSH seem to be of little consequence, other than making it difficult to compare dosages among products, which may range from 1 to 50 percent pure. This results in widely different weights of product per dose (Seidel and Moore 1991).
 

C) Body condition and health. This is definitely an important factor which will affect superovulatory responses. In the case of cows, extremely fat animals make poor donors, both because they do not respond well to superovulation and because their reproductive tracts are more difficult to manipulate. Sick animals and/or those in poor body condition usually do not produce many good embryos (Seidel and Moore 1991). Good quality forage is an important nutritional factor associated with good response to superovulation and viable embryo production in beef cattle. Donor females grazing on highly palatable, green pastures usually have a higher superovulatory response than those on poor-quality pasture, hay or silage (Stroud and Hasler 2006). In the case of alpacas, there are no references available on this topic. An unpublished study from Peru shows that nutrition and body condition can dramatically affect embryo yield in alpacas under Altiplano conditions in Peru, i.e. above 4,000 meters above sea level (Zambrano 2003).
 

D) Lactational status. Lactation in either beef or dairy cows does not diminish the response to superovulation, provided that cows are cycling and not losing weight (Seidel and Moore 1991). Further research needs to be conducted on alpacas to elucidate if there is a lactational effect on the production of embryos.
 

E) Seasons and environmental conditions. In the ewe, seasonality in the reproductive cycles has been correlated with different responses in embryo production throughout the year and has been related to ovarian dynamics. Alpacas are considered to be non-seasonal animals, but it has been reported in Peru that the follicular population at the ovaries varies with the season. In the rainy season (December to March) there is more ovarian activity and more follicles are visible at the ovarian surface (Bravo and Sumar 1989), in comparison with the dry season (April to November). It would be interesting to conduct a study in Australian conditions monitoring the number and size of follicles in female alpacas during the whole year in order to draw a conclusion. If we do find a seasonal difference, it will also be necessary to then perform superovulatory treatments throughout the whole year to see if that affects the yield of embryos.
 

F) Age. Donor age is an important factor that affects the number of viable embryos produced upon superovulation in cattle. Virgin heifers tend to produce fewer embryos than do mature cows. After 10 years of age, the production of embryos tends to decline (Stroud and Hasler 2006). There is no information available on this topic in SAC. It may be necessary to conduct a field trial, superovulating alpacas of different ages, to draw a conclusion.
 

G) Repeated treatments. Follicle Stimulating Hormone (FSH) is the hormone commonly used in superovulatory protocols. It is a protein, which means that several applications may produce an immunological response and the production of antibodies against the hormone. Responses to superovulatory treatments may thereby be diminished in the future. Generally, donor cows respond similarly to first, second and third treatments. The response to subsequent treatments can be diminished in some individuals (Hafez 2000). There is research available on repeated superovulatory treatments which reports a reduction in response after the first two treatments in cows (Schilling et al. 1984; Bastidas and Randel 1987; Bhattacharyya et al. 1989; Dochi et al. 1998; Triveni and Kharche 2001a; Triveni and Kharche 2001b). Nevertheless, other authors report no significant differences (Busse 1995; Ahn et al. 1997; Ansari et al. 2001). In the ewe, repeated superovulation has been reported to cause a decrease in the ovarian response (Chung et al. 1987; Sharma et al. 1996). This is an area which is in dispute.
 

In the case of the dromedary camel, it has been reported that it can become refractory to superovulatory treatments with eCG and FSH after several years. Observations indicated that ovarian activity was completely arrested (Skidmore 2004).
 

H) Stress conditions and animal husbandry. It is crucial to minimise stress before, during and after an ET program in order to achieve the best results. In the case of cows, reproductive efficiency under stress is less than optimal. Some females under stress do not show oestrus (heat), while other animals have a reduced chance of becoming pregnant. Certain types of management situations are stressful for animals. The bodily response most characteristic of stress involves an increase in the secretion of hormones called glucocorticoids from the adrenal gland. The balance of reproductive hormones can be altered by stress and may cause delayed ovulation in dairy cows. High environmental temperatures may have a direct adverse effect upon the survival of the oocyte and the sperm, or the development of the embryo while in the reproductive tract. The oocyte and the sperm may not form a healthy embryo, or a developing embryo may die, resulting in an early abortion. This problem may result in conception rates falling below 20% during summer months.

Many husbandry practices may cause unnecessary stress near the time of ovulation. A good example is chasing or roping in a very aggressive way, or isolating the donors in a breeding stall away from the rest of the herd. In the second case it is a good idea bring another female into the vicinity to minimise the stress caused by isolation. It is crucial to minimise stress by not chasing, beating or roping the animals, and handling them as gently as possible.
 

Facilities can play an important role in minimising stress during ET procedures. Simple things like proper pens to allow the farmer to handle animals adequately, and ET restraint tables, are necessary to reduce stress. In dairy cows, using unsecured, nervous females which are free to move during the embryo transfer can adversely affect conception rates (Stroud and Hasler 2006).
 

In this vein, Luis Bethencourt of “La Hacienda Alpacas” has designed a novel ET alpaca restraint table which allows easy placement of the animal and secures the legs to avoid movement. It also helps the ET practitioner to perform ovarian examinations, flushing procedures and embryo implantation in recipients (Figure 3).
 

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Figure 3: ET Alpaca restraint table designed by Luis Bethencourt of “La Hacienda Alpacas”, Marulan, NSW. This device allows a clear visualisation of ovarian structures due to the animal’s front legs remaining a bit higher than the back legs.

I) Donor management experience and expertise. This is the single most important variable affecting superovulation results, since it encompasses all of the other techniques discussed above. Successful donor management both understands and implements the factors listed above on a routine basis. Poorly qualified managers do not have a full understanding of these factors and therefore have difficulties using them.
 

The importance of animal husbandry and management practices is often overlooked or underestimated. It is the responsibility of the ET practitioner to educate clients on the value of basic animal husbandry.
 

Finally, an important factor is sperm quality. We can provide the female with all the right conditions to produce a good number of viable oocytes, but if the male used does not have good quality sperm (in terms of motility, concentration and fecundation capacity), the yield of embryos may be poor. Natural mating after applying superovulatory protocols makes it impossible to assess sperm quality. That is why I am proposing the use of AI with fresh sperm in donors in the near future, as it will allow us to assess the quality of the sperm we are using and thereby increase the chance of obtaining a better yield of embryos.

In summary, there are many factors that affect the superovulatory response and it is hard to control all of them. That is one of the reasons why ET may work very well on one farm but not on another. The idea is to try to use a strategic plan to include all these factors (or most of them) to ensure better results.


4. RECIPIENT SYNCHRONISATION

To ensure that the embryos removed from alpaca donors will find the same uterine conditions in the recipients, it is important to synchronise ovarian status between donors and recipients by ensuring a corpus luteum of the same stage. Embryos are recovered on day 7 from donors, and recipients need to have a corpus luteum that is 7 days old to maximise the chance of pregnancy. For this purpose, exogenous GnRH, synthetic GnRH agonist, LH, hCG or seminal plasma may be used to induce ovulation of recipients. Time of ovulation is generally between 28 and 30 h post injection.
 

In order to induce ovulations in recipients, they must have a mature follicle (dominant follicle) able to respond to LH action, to ovulate and to form a healthy corpus luteum, otherwise ovulatory failure may occur and these animals will not be able to be used as recipients at the time. This means that animals destined to be recipients need to be scanned by transrectal ultrasound, in order to find the dominant follicle, to ensure a response to ovulation induction. In practical situations, sometimes the embryo transfer practitioner is not available to scan the recipients and the farmer needs to assume that there is a dominant follicle and administer the GnRH or synthetic analogue. Before the ET procedure, the practitioner needs to scan the recipients in order to find a corpus luteum and decide whether to use the animal or not. Table 2 shows dose rates of GnRH, analogues and seminal plasma used to induce ovulation in SAC.

Table 2: Ovulation induction in SAC with GnRH, analogues and seminal plasma.

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I have tested buserelin, and also seminal plasma, and obtained 80% and 70% ovulation respectively, without previous scanning of ovaries, in New South Wales conditions during summer, autumn and winter.

5. EMBRYO COLLECTION

Embryo collection or flushing is the procedure to recover the embryos from the reproductive tract by day 7 after the last mating of the donor. Embryo collection in SAC has been performed by laparotomy, laparoscopy and non-surgical collection. We will not cover laparotomy/ laparoscopy collection as it is not a common technique these days, but we need to mention that it is invasive, requires expensive equipment, can be more stressful for the animal and may cause reproductive tract adhesions that may compromise reproductive life in the future. Laparotomy/laparoscopy is important as a unique technique to collect embryos in vicuñas, due to rectal inaccessibility.
 

Non-surgical embryo collection is a simple procedure, but it requires experience in rectal palpation, good training in transrectal ultrasound, and of course a slender hand to gain access to the reproductive organs via the rectum. Twenty four hours prior to flushing, depriving animals of water and food is recommended, to avoid content in the intestinal tract and facilitate the manipulation of the uterus via the rectum. The flushing procedure has been summarized in 4 steps as follows:
 

Step 1: Place the animal in an ET restraint table with the front legs slightly higher than the back legs. The design shown in the picture was developed by Luis Bethencourt as a part of the process of improving ET and so far we have had better results that with the conventional ET “tea table”, as I call it. With the new design, the animal is more comfortable, reducing the stress of the procedure, improving visualisation of ovarian structures by ultrasound, and finally allowing us to work without the need for anesthesia to tranquilise the animal (Figure 4).
 

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Figure 4: An alpaca placed on the ET restraint table ready to be scanned and to have flushing performed. Note that the animal looks very comfortable.

Step 2: Clean the rectum and introduce 30-50 ml of ultrasonic gel to improve visibility, and then proceed to scan the ovaries using transrectal ultrasound (Figure 5).

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Figure 5: Cleaning the rectum is important before transrectal ultrasound and the addition of ultrasonic gel, to improve image quality.

Step 3: Upon finding the corpora lutea, the ET practitioner needs to be able to palpate the uterine body and horns, in order to place the two-way Foley catheter (containing a stylet which gives it rigidity) and facilitate the pass through the cervix (uterine neck)(Figure 6). When the Foley catheter has passed the cervix, sometimes the cervical rings give a cartilaginous sensation, and 10-15 ml of air is injected in order to inflate the balloon contained at the end of the Foley catheter to avoid reflux of the flushing solution during the flushing procedure (Figure 7). Some alpacas have cervices which are very easy to penetrate, and this makes flushing a very simple and quick procedure. Other animals have cervices which are hard to pass and require some experience to negotiate. In the case of irregular cervices, sometimes it is impossible to pass the Foley catheter through, and regrettably these animals cannot be used for non-surgical collections. Recently the use of a speculum in difficult animals has been shown to be effective.
 

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Figure 6: Stylet inside the Foley catheter to give it rigidity and to enable it to be passed through the cervix.

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Figure 7: Foley catheter with an inflated balloon. Upon passage through the cervix and inflation, it will seal the uterine neck and will avoid reflux of flushing solution to avoid loss of embryos through the vagina. 

Step 4: Upon placing the Foley catheter into the uterus and inflating the balloon, the two-way connector is placed so that the flushing solution enters via gravitational flow and recovers the contents from the uterus (Figure 8 and 9). At the other end, an ET filter is attached to recover the flushing solution which has passed the uterus and contains the embryos (Figure 10). When the circuit is open, letting the flushing solution enter the uterus via gravity, it is important not to overload the system as the possibility of loss of flushing solution increases with pressure. When the circuit of entrance is closed and the circuit of collection is open, the practitioner very gently massages the reproductive tract (horns and uterus) to allow the fluid to be recovered. Flushing should be performed several times (4-7), until a volume of 500 ml has passed through the system. The volume used in each single flushing depends on uterine size, and it is the decision of the ET practitioner whether 30 or 50 ml will be used.
 

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Figure 8: Passing the Foley catheter through the cervix to perform flushing.

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Figure 9: Two-way connectors attached to the Foley catheter, ready to perform embryo flushing.

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Figure 10: ET filter where the embryos are received during the flushing procedure. Before flushing, the filter must contain ¾ of the volume of the flushing solution to make the system work efficiently.

Alpaca embryos are visible to the eye and appear as milky white spherical structures swimming in the embryo filter in the flushing solution. As soon as flushing is finished, the solution recovered from the filter is placed on a square Petri dish in order to locate the embryos under the stereomicroscope (Figure 11), and then placed into four-well dishes containing a holding solution for quick evaluation (Figure 12).
 

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Figure 11: Locating embryos in the square Petri dish containing the flushing solution recovered from the filter.

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Figure 12: Four-well embryo dish containing the embryo-holding solution, where the embryos will be placed for quick evaluation before transfer.

6. EMBRYO RECOVERY RATES

Regrettably, embryo recovery rates in SAC are very low and in the best scenario we expect 50% efficiency, independent of the method used. In other words, if we count 12 corpora lutea by transrectal ultrasound and/or rectal palpation in both ovaries, then we may be able to recover 6 embryos. Flushing has been performed from day 6.5 to 12 after mating (Adams and Ratto 2001), and the cause of these low recovery rates is unknown. It could perhaps be related to oestradiol increase after superovulatory treatment, as it is well established that this affects oocyte/sperm transport in other domestic species. The use of PMSG especially causes large unovulatory follicles that secrete oestradiol.


7. EMBRYO ASSESSMENTS

Embryo assessment is made using a stereomicroscope at 15X magnification. At day 7, in the case of SAC, embryos recovered are in hatched blastocyst stage and their size may range from 250 to 700 µ m (Correa et al. 1997). The classification of embryos is done according to the International Embryo Transfer Society Protocol, as: excellent or good, fair, poor, and dead or degenerating (IETS 1983). Under the stereomicroscope, a good alpaca embryo will look compact, amber and regular in shape (Figure 13). At flushing, embryos can appear a little bit shrunken (Figure 14) due to the change in osmotic pressure, but after a few minutes in the holding solution they will appear round and uniform. South American Camelid embryos are very resistant and can live up to 24 hours in a holding medium at 37 ˚C in an incubator. In this case it is important to protect against light (Palomino 1997). Personally, I prefer to transfer to recipients immediately upon evaluation to avoid further stress to the embryo.
 

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Figure 13: Good quality alpaca embryos appear compact, amber and regular in shape.

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Figure 14: At flushing, embryos appear a little shrunken, but after a few minutes become spherical and regular.

8. PREPARATION OF EMBRYOS FOR TRANSFER

Upon evaluation, embryos need to be placed on a 0.25 ml straw in order to be placed at the uterine horn using a French artificial insemination gun. I manipulate embryos with a digital pipette. The correct way to pack the embryo for transfer is as follows:

• Cut the plug from the straw

• Introduce a small volume of holding solution

• Introduce a small volume of air

• Place the embryo

• Introduce another small volume of air

• Introduce another small volume of holding solution
 

Figure 15 shows the correct way to pack the embryo into the 0.25 cc straw.

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9. EMBRYO FREEZING


Embryo freezing in SAC has not been successfully accomplished. One of the reasons it is hard to freeze SAC embryos is their quick development to hatched blastocyst stage. In the case of cattle, early embryos pass into the uterus about 4 days after fertilisation and they hatch until about 8 days. Hatching is the process in which the embryo escapes the zona pellucida. There is a long interval of time in which flushing can be performed in this species. Alpaca and llama embryos mature faster than cattle and hatching occurs between 5 and 6.5 days after conception. This is when they are released into the uterus and it is possible to collect them as hatched blastocysts by flushing.
 

In the case of cattle, the procedure to freeze the embryos that are contained on a zona pellucida is relatively easy and efficient. The freezing of hatched embryos in cattle has not been possible. The reason is the greater amount of water contained in a hatched blastocyst and its size. This means that the hatched embryos are more vulnerable to the toxicity of cryoprotectants, as it will take longer to expose the cells to the solution in order to replace the large amount of water with cryoprotectant. A cryoprotectact is a small molecule that easily penetrates inside the cells and depresses the freezing point of water. Glycerol and ethylene glycerol are examples used in cryobiology. A cryoprotectact makes it possible to freeze the embryos, avoiding the formation of crystals which damage the cells.
 

A new approach by Paul Taylor (2005), an expert on ET in llamas in the USA, was to try to use a microinjection of cryoprotectant directly into the embryo, but he found that the microinjection equipment used to date was unsuitable. Then he decided to find a device that combined sucking and puncturing functions. He called it the co-axial microinjection system (CMIS). This system allows him to obtain a pregnancy from the third embryo processed. Although there is a need to find the best solution and timing of freezing for commercial uses, there is great potential in the future for further research to develop a SAC freezing protocol which will change the whole alpaca industry worldwide, as we will then be able to import and export good genetics in a liquid nitrogen tank. The only drawback of this technique is that it requires experience in micromanipulation of embryos to perform it successfully.
 

10. EMBRYO TRANSFER PROCEDURE

The embryo transfer procedure is the last step and consists in placing an embryo removed from the donor at the uterine horn of the previously synchronised recipient, using French insemination equipment. It is very important prior to ET to find the corpus luteum at the ovary of the recipient (Figure 16). No corpus luteum means that the recipient will not be able to keep the pregnancy, and thus not be useful for the procedure.

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Figure 16: Corpus luteum in a recipient ready to receive an embryo. The CL appears as a round grey solid structure. On the left side of the picture, an unovulatory follicle may be observed.

The embryo transfer step is as simple and quick as inseminating a cow. The right hand needs to be inside the rectum to lead the insemination gun, which contains the straw with the embryo, to pass the cervix, which is manipulated with the left hand (Figure 17). The embryo is deposited deep at the ipsilateral horn from the ovary containing the corpus luteum. It needs to be done very gently to avoid any damage (bleeding) of the uterine mucosa which may kill the embryo and/or produce an infection resulting in no pregnancy. This step requires a lot of training to be carried out successfully. There are three factors which affect the success of ET: quality of embryos, synchronisation of reproductive stage between the donor and recipients, and also the technique used to place the embryo into the uterus (Palomino 1997). Other factors, like contamination of the instruments and trauma to the uterus (bleeding), may cause low fertility upon embryo transfer.

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Figure 17: Embryo transfer is an easy and quick procedure when you have the necessary skills. The right hand helps in passing the French insemination gun through the cervix and ensures that the embryo is deposited deep into the uterine horn which contains the CL.

11. FERTILITY UPON ET IN SAC

Fertility upon embryo transfer in alpacas is still not well established. Fertility rates upon ET in cattle vary from 70% to 80% (Hasler 2004). In alpacas there are 5 reports only, with fertility and conception rates ranging from 0% to 100% (Novoa M and Sumar K 1968; Sumar and Franco 1974; Palomino et al. 1987; Palomino 1997; Huanca 2005). The limitation of this data is that the samples are too small, and incomparable regarding conditions and superovulatory protocols. Fertility upon ET in alpacas is considered to be > 70%, but embryo loss is a problem that can be as high as 30% within the first 60 days of gestation (Huanca 1993). The embryo can be produced properly and the technique of transfer properly performed, but some unknown factors may interfere with the pregnancy. In other words, an efficient protocol to superovulate and produce high quality embryos from valuable alpaca donors will not necessarily lead to good pregnancy rates in recipients. Recipients need to be animals of proven fertility and to have a corpus luteum that is in synchrony with the donor’s.
 

It has been proposed that high early embryo mortality in alpacas could be related to maternal recognition of pregnancy. When fertilization occurs, the conceptus (embryo) must signal its presence to the uterus and block the release of PGF2α, avoiding the destruction of the corpus luteum (luteolysis) to ensure progesterone production during the whole period of pregnancy (Hafez 2000). This is a chemical signal which, if not released, will cause the uterus to not recognize the embryo and the corpus luteum to regress by day 12 via action of the PGF2α. The embryo will then not survive. A recent study in Peru has suggested that oestradiol has an important role in maternal recognition of pregnancy in alpacas. The application of a single injection at day 9-11 post ovulation significantly reduced embryo mortality (Chipayo et al. 2003). It might be interesting to test if pregnancy rates are increased in recipients upon the application of oestradiol.
 

12. STRUCTURE OF COST FOR ET IN AUSTRALIA:


The structure of cost for an embryo transfer program can be split into 4 areas: hormones, flushing materials, ET materials and practitioner fees. Table 3

Table 3: Structure of unitary costs of an embryo transfer program in Australia

alpaca et

* Flushing materials are included in the flushing fees.

Now that we have a unitary structure of costs for ET, let’s propose a simple hypothetical scenario.
 

Farm A has 3 females valued at $ 5,000 each and they decide to start an ET program using a quality male stud. Female 1 produces no embryos, female 2 produces 4 embryos, and female 3 only 2 embryos. In total we have 6 embryos to transfer. The cost of hormonal treatment will be 3 x 118.50 = AUD $ 355.50. Female 1 will not be flushed as we previously determined by ultrasound that she did not respond to the superovulatory treatment. The costs of flushing materials are included in the flushing fees. The flushing fees will be 2 x 375 = AUD $ 750. A total of 15 recipients were synchronised, 15 x 19.05 = AUD $ 285.75. The cost of transferring 6 embryos will be 6 x 275 = AUD $ 1,650. The total cost of the ET program will be: 355.50 + 750 + 285.75 + 1650 = AUD $ 3,041.25. If we assume that 50% of embryos generate crias on the ground, we are expecting 3 animals in a year’s time which could be worth at least AUD $ 2,000 each, which gives us AUD $ 6,000 in revenue and AUD $ 2,958.75 in profit. This is a conservative scenario, but realistic. The profits can be maximised if we use better genetics. The worst scenario would be that if the animals did not respond there would be a cost for hormones and a fee for the practitioner to cover their expenses and laboratory materials like flushing solution etc. This is one of several examples we can discuss. Of course, if we do not use the right genetics it will be hard to make profits. It is important to use herd evaluation to try to narrow the possibility of using “problem animals”.
 

In summary, ET requires an investment in time and resources that will lead to good profits if we carefully select our animals to be good donors/ recipients and good sires. Herd evaluation is an important process which will lead to efficient embryo production if the other variables described above are managed properly.
 

13. LIMITATIONS OF ET IN SAC

 I have covered the limitations of ET in alpacas in a previous article (Reyna 2006d):
 

• Embryo transfer technology is not complicated per se, but requires knowledge and skill in order to be performed satisfactorily.

• Embryo transfer is a time-consuming procedure and requires an investment in hormones, laboratory materials and practitioner fees.

• Response to superovulatory treatments is highly variable and unpredictable.

• Embryo recovery rates in alpacas have been frustratingly variable.

• Superovulatory hormones may produce an immunological response which can lead to the production of antibodies and diminished or no response to future superovulatory treatments.

• A reliable superovulatory protocol as described in cows and ewes has not been developed in SAC.

• The yield of embryos after superovulatory treatment is highly variable.

• Fertility upon embryo transfer in SAC is still not well defined.

• Anatomical peculiarities of the SAC reproductive tract could limit the use of some animals for ET programs.

• Only a low percentage of female SAC are able to respond to superovulatory treatments by producing multiple embryos, and it is not possible to differentiate between good and bad donors.

• Recipient females are not always able to carry pregnancies from ET. These animals can waste time and money.

• Stress during ET procedures in SAC, as in other domestic species (cows and ewes), has a detrimental effect and may cause poor response to superovulatory treatments and poor pregnancy rates. Managing stress before, during and after ET is crucial.

• Nutrition is a key factor in a MOET program and donors/recipients should be on a rising plane of nutrition. The optimal body condition is between 2.5 and 3.5.
 

Further research is a priority in ET in SAC, including new superovulatory protocols which should be based on the peculiar reproductive physiology of the species. It will be important to determine the effect of the dominant follicle on the yield of embryos, and how the repeated use of superovulatory treatments may affect the future reproductive life of donors. Study of the effect of the seasons on the production of embryos is necessary. Also, more research on embryo freezing and in vitro production of embryos using ovum-pick up in live animals would be desirable.
 

14. CONCLUSIONS

Notwithstanding all the limitations of ET in SAC, it is still a powerful tool to accelerate the genetic gain of our herds. It will lead to the development of an extra fine Australian alpaca which can give local breeders a good income if animals are exported overseas, and which will create prestige for our industry. This will make alpaca breeding more attractive to investors and will promote it around the country.
 

The future of the ET industry in SAC will depend on the possibility of conducting research in the area of superovulatory treatments and trying to elucidate the unanswered questions in order to improve the efficiency of MOET. Industry and government organisations are the key to supporting more research by offering scholarships, grants, animals and incentives to new professionals in the area. The more professionals in the area there are, the healthier the ET market will be due to competition. The greater the number of animals which are superovulated, flushed and transferred, the more likely that prices will become more affordable for small and medium breeders interesting in improving their genetics.
 

I encourage young professionals in the area of veterinary science and agricultural science, and technicians with experience in ET, to enter this exciting world. As always, I am available to answer any enquiry you may have.
 

15. ACKNOWLEDGMENTS

The author wishes to thank Luis and Suzy Bethencourt of “La Hacienda Alpacas” for providing animals, equipments and laboratory consumables to test superovulatory protocols. I would also like to thank Peter Krockenberger for his editorial assistance.

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Please cite as:

Reyna, J (2006). Embryo Transfer in South American Camelids. The Camelid Quarterly (Canada), Vol 5, No 4. 79-93.


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