Jorge Reyna

This article intends to answer questions about the application of embryo transfer in alpacas as a powerful tool to genetically improve herds. Its main purpose is to present the information available in an objective and simple manner to inform breeders of the advantages and limitations of the technique.
1. Embryo transfer is not a novel reproductive technology. The first successful embryo transfer was performed by Walter Heape in rabbits in 1890 (Cited by Betteridge 2003). Later on, in the 1920’s, several researchers described the technique in detail. During the 1930s and 1940s many experiments were conducted in sheep and goat (Mapletoft and Hasler 2005). In the early 1970s the bovine ET industry emerged in North America (Hasler 2004).
In the case of alpacas, the first experiment was conducted in Peru in 1968 (Novoa and Sumar), but it was not until 1974 that the first ET cria was born (Sumar and Franco). Later on, in 1987 and 1996, more crias were reported in Peru (Palomino). In Australia the first ET alpaca cria was reported in 2002 (Vaughan).

2. Embryo transfer technology is not a complicated technique per se, but requires good knowledge and skills in order to be performed satisfactorily. Knowledge of alpaca endocrinology and reproductive physiology is essential in order to design an efficient superovulatory protocol, as it is not a species extensively studied (Palomino and Gomez 1993). Skills in the area of transrectal ultrasonography are required, to be able to find the ovaries and determine ovarian status prior to stimulatory hormonal treatment (Reyna 2006). Before starting a superovulatory treatment, donor animals need to be scanned to make sure no dominant follicle is present, otherwise the response to the treatment may be compromised and affect the yield of embryos, as reported in cattle (Huntinen et al. 1992) and sheep (Rubianes 2000). In the case of alpacas, this is an assumption and needs to be tested (Adams and Ratto 2001). A necessary experiment would be to superovulate alpacas in the presence and absence of a dominant follicle, and then to evaluate the yield and quality of embryos. It is likely that follicular dominance in alpacas follows the patterns reported in other domestic species.
Prior to flushing, it is necessary to scan the donors and count corpora lutea in order to determine the response to the superovulatory treatment. In the case of recipients, animals need to be scanned in order to find a dominant follicle (mature follicle) that will ovulate at hormonal induction (GnRH or LH) and thereby ensure that the reproductive stage of donor and recipient will be synchronised. This should ensure that the removed embryo will find the same conditions in the uterus of the recipient (Bourke et al. 1992). Training in rectal palpation and artificial insemination skills is necessary to perform the flushing and/or deposit of embryos in the uterine horn. All this knowledge and skill takes time and practice in order to achieve good results.
3. ET is an “expensive” reproductive technique. Superovulatory treatments in alpacas range from AUD $125 to $200, depending on the type of hormones involved. Materials required to flush the females, and general laboratory consumables, are “expensive” as well, ranging from AUD $60 to $90 per animal. The reason is that all of these materials are embryo-tested, which means that they are safe and will not kill the embryo. But the most expensive part is the ultrasound equipment, which needs to be an accurate machine with a resolution of no less than 7.5 MHz.
In my view, ET is an expensive technique if used in animals with low genetic merit. But, if you choose the right genetics, it will be a good investment and one cria born via ET may be able to cover costs and the rest will be profits. Selection of animals is crucial.

4. Response to superovulatory treatments is highly variable and unpredictable. A major problem that the ET industry in cattle has faced over the last 30 years is that the response of an individual cow cannot be predicted and is highly variable, ranging from 0 to 20 embryos with an average of 6 usable embryos. The possible reasons for this unreliable response have been discussed before by many authors, and it could be related to breed of animals, age, nutrition and body condition, lactational status, presence/absence of a dominant follicle, superovulatory protocol and purity of the hormone used, stress, environmental conditions, some molecular mechanisms at the ovary that are particular to each animal, and also donor management experience and expertise (Stroud and Hasler 2006). All of these factors have been studied extensively in cows, but not in alpacas. There is an urgent need to study all these factors in alpacas in order to improve the response to superovulatory treatments.
5. Embryo recovery rates in alpacas have been frustratingly variable. Regardless of the embryo collection technique used, based on the corpora lutea count after superovulatory treatment by transrectal ultrasound, embryo recovery rates are generally less than 50% (Del Campo and Del Campo 1995). Flushing has been performed from day 6.5 to 12 after mating (Adams and Ratto 2001), and the reason for 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.

6. Superovulatory hormones may produce an immunological response that could lead to the production of antibodies. Follicle Stimulating Hormone (FSH) is the hormone used commonly 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 reduced in some individuals. Repeated superovulation at 15-20 day interval leads to a poor response in cows. The recommendation is 45-60 day intervals, although there are studies which shows that shorter intervals work well (Seidel and Moore 1991). In the case of alpacas there is no information available in this regard, which means it is necessary to conduct some experiments with the frequency of superovulation and to see if the response is affected after a few treatments. I am inclined to say that maybe superovulation 4 times per year (every 80-90 days) would be reasonable in alpacas, but studies will help to draw a conclusion.
If there is a concern about the effect of repeated superovulation treatments an alternative is to do Single Embryo Flushing (SEF), which means that no hormone is used, the female is mated and the embryos collected and transferred into a recipient. Single Embryo Flushing requires less investment than hormonal treatments. My experience with SEF has shown that 10-20% of females can release 2 oocytes at a time, even if twin carriers are unusual. If you decide to use SEF, then your valuable females can remain not pregnant and effectively function as embryo factories. This procedure can be performed maybe 8 times per year, which means, on conservative estimates, 4 valuable crias on the ground per year.
Another approach is the combination of two regimens, using hormones 4 times per year and SEF as well. This will maximise the number of valuable crias per year.

7. Superovulatory treatments in alpacas are not a secret. The superovulatory treatment uses pretty much the same hormones and injections given to cows. The difference is that in cows ovulation occurs spontaneously. In the case of alpacas a second hormone needs to be injected after the last FSH injection to produce release of the oocytes from the follicles (LH or GnRH). At the moment there is information available in scientific journals/theses on how to induce follicular growth and ovulation in alpacas. About 90% of the protocols use FSH and 10% use PMSG to induce growth of follicles at the ovaries. In the case of FSH, multiple doses (from 6 to 10) are given every 12 hrs (3-5 days treatment) with a total dose ranging from 50 to 200 mg. With PMSG, a single dose is given which is expressed in international units (IU) ranging from 750 to 1,500 IU. It is certainly true that the efficiency of protocols varies and this can be attributed to purity of the hormone preparations, nutritional status of the donors, age, lactational status, possible presence of a dominant follicle, season, stress and many other conditions which require further study.

The real limitation in this area in alpacas is that, even with the few references available, there is no reliable superovulatory protocol as in cows and ewes. A possible reason for this is that research in this area has not been extensive. The limitation of the current publications is basically the small samples of animals used. In the case of Peru, the main factor is the cost of hormones and, in the case of overseas laboratories it is the inconvenience of limited access to experimental animals.

8. The yield of embryos after superovulatory treatment is highly variable. In cows it is possible to obtain 4.8 embryos per animal, with 20% of these embryos not usable (degenerated) (Hasler 2004). In alpacas, there are no statistics available. This will probably remain the case for the next decade, as the use of ET on a commercial scale is restricted. Research papers on both llamas and alpacas have reported ranges of viable embryo yields from 1.3 to 3.9 (Bourke et al. 1992; Correa et al. 1997; Palomino 1997; Bravo et al. 2004). The limitation of these papers is related to the small size of the samples (from 4 to 24 animals). It is possible that Peru will provide this information in the future, as many well-established alpaca companies are getting ready to start ET programs and the larger number of animals will provide statistically significant data to rely on.

9. Fertility upon embryo transfer in alpacas is still not well established. Fertility rates upon ET in cattle go from 70% to 80% (Hasler 2004). In alpacas there are 5 reports only, with fertility and conception rates ranging from 0% to 100% (Novoa and Sumar 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 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 donors.

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 signal is a chemical signal that, if not released, will cause the uterus to not recognize the embryo and the corpus luteum to regress by day 12 by 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.

10. Anatomical peculiarities of the alpaca reproductive tract could limit the use of some animals for ET programs. There are female alpacas that present an irregular uterine neck (cervix). In this case it is very difficult to go across the cervix to the uterus to perform the flushing procedure and/or inseminate the recipients. Regrettably these animals cannot be used in ET programs. It is thus very important, if you are planning to buy a good female to start an ET program that you ensure she has a cervix that can be penetrated easily. The only way to test this is by introducing a Foley catheter. Transrectal ultrasound will not help in this case. Also, animals of a good size are desirable, even in the case of recipients, as small animals give less easy access to the reproductive organs via the rectum.

11. Nutrition is a key factor in a MOET program and donors should be on a rising plane of nutrition. Four weeks of special nutrition leading up to flushing is recommended. An unpublished study in Peru showed how nutrition and body condition could dramatically affect embryo yield in alpacas (Zambrano 2003). In the Andes there are several resources to use to improve fertility and superovulatory response, such as “La Maca” (Lepidium meyenii). In Australian conditions, lupins can be an important nutritional ingredient which that may lead to a better superovulatory response when used prior to superovulation. When I was doing my Master’s research in transrectal ultrasound in sheep at Sydney University, I recall that on one occasion the ewes were fed with lupins over the week-end and when I scanned them their ovaries looked distinctly different, with many “extra” follicles. This anecdote suggests that the effect of lupins on ovarian dynamics may be real. In ewes, feeding with lupins for 1 to 3 weeks immediately before mating may increase the oocyte yield and thus, the number of lambs conceived and born (Rutherglen 1995). This is why I would feed animals destined to be donors with lupins for 3 weeks before hormonal treatment, especially in drought periods which restrict the green feed intake.
Summary
Embryo transfer is not a novel technique and has been an extensive area of research since 1890. It is not a complicated technique but requires a good knowledge of reproductive physiology and also skills in transrectal ultrasound and rectal palpation. It is also important to say that it is a good investment as long as the right genetics are selected. Problems that the ET industry faces are the unpredictable response of donors to superovulatory treatments and also the possibility of reducing the superovulatory response if it is used too frequently. At the moment, even with few references (mostly coming from South America), there is no reliable protocol for superovulating alpacas. Response to superovulation is highly variable and unpredictable, as in cattle and sheep. The recovery rate of embryos, regardless of the technique used, is < 50%, but fertility rates for ET have not been extensively described and are highly variable. Anatomical peculiarities of the alpaca cervix could limit the use of some animals in ET programs. Finally, nutrition is a key factor in improving the superovulatory response.

The future
Embryo transfer in alpacas could lead to the development of a new breed of extra fine Australian alpaca, as has been done with Merino sheep. The only way to achieve this is to extensively apply MOET in our herds. This could make us leaders in the alpaca industry worldwide, even if we do not achieve a population of animals as large as in Peru. This would provide a good income for breeders and would promote the alpaca industry all around the country.

I have talked with many researchers in the area of ET, and few of them share the point of view that we should wait until the ET technique is refined and knowledge of reproductive physiology in alpacas is advanced. My question is, how many decades more?. With a background in both research and production, I personally believe embryo transfer in alpacas is a necessary technology to use. We know genetic improvement is slow, due to the long gestation period. The ability to improve the herd is limited from the female side to 8-10 offspring in the animal’s entire reproductive life. With an efficient MOET program we could have several crias per year from a valuable female. A scientific background is important, but practical knowledge is needed to solve problems on a day-to-day basis. I have learnt many things that are not published anywhere since I started my work on ET in alpacas.

In conclusion, I would say that there is a lot to do in the area of embryo transfer in alpacas and it is an exciting area of work. Many questions are unanswered at the moment. If you decide to enter the fascinating world of embryo transfer on your farm, make sure you understand all these limitations and do not expect miracles. If you select the right animals and you follow the recommendations on how to prepare and manage them during and after ET, it will translate into profits for your farm and the large costs will become a wise investment.

Acknowledgements
The author thanks Peter Krockenberger for his editorial assistance.