Alpaca Breeding Technologies | Embryo Transfer in Alpacas and Llamas

Embryo Transfer Glossary

Jorge Reyna

BSc (hons), MScVetSc (Sydney Univ.)

Lecturer in Higher Education - Learning Designer

Faculty of Science

University of Technology Sydney

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This glossary intends to clarify some basic terminology involved in embryo transfer technology and it will be useful for understanding upcoming articles in this area. My knowledge has been acquired during my time in universities and my professional life in the reproductive area since 1993. This glossary has not been put in alphabetical order but in logical order.

Embryo: Early stage of development of future offspring after fertilization of the oocyte (egg) by the sperm.

ET: Embryo transfer is one step in the process of removing one or more embryos from the reproductive tract of donor females and transferring them to one or more recipient 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 in the early 1970s in North America .

Donor: A female with superior genetic merits that are to be disseminated into the herd using superovulatory treatments to obtain multiple embryos to be transferred into recipients, thereby obtaining several valuable offspring per year.

Recipient: An inferior female that will carry a high quality embryo removed from the donor. Recipients need to be animals of proven fertility, good size and with a normal reproductive tract. The reproductive stage needs to be synchronised with donors, as it is necessary to ensure that the embryo removed from the donor will find the same conditions in the uterus of the recipient and thus a greater chance of surviving and completing gestation.

Embryo freezing: Reproductive technique that allows conservation of embryos for unlimited periods of time in liquid nitrogen in cattle and sheep, making the use of ET technologies very flexible. In alpacas it still is an area of research and it may take many years to test an efficient protocol to freeze alpaca embryos.

MOET: Multiple ovulation and embryo transfer program. This concept was introduced for the first time in 1987 by Smith from the University of Guelph . This researcher showed how ET can be a powerful technique to achieve genetic gain.

Hormone: A substance synthesized and secreted by a ductless endocrine gland that passes into the circulatory system for transport and inhibits, stimulates or regulates the functional activity of the target organ or tissue. We will give a brief description of the following hormones, as they are important to understanding how superovulatory treatments work in alpacas: FSH, LH, GnRH, PMSG, Oestrogens, Progesterone and Prostaglandin F2α.

FSH: Follicle Stimulating Hormone is classified in the group of gonadotrophins which promote growth of the ovarian follicles, also called Graafian follicles. FSH is used in superovulatory treatments in donors to induce growth of several follicles at a time to obtain embryos which are transferable to recipients.

LH: Luteinising Hormone secreted by the pituitary gland which is responsible for the rupture of the follicular wall and ovulation in the female.

GnRH: Gonadotrophin Release Hormone secreted by the anterior hypothalamic area, responsible for the release of FSH and also LH. GnRH is commonly used to induce ovulation in donors after the application of FSH, and also in recipients to synchronise reproductive status and to ensure that the recovered embryos will find the same conditions in the recipient's uterus.

PMSG: Pregnant Mares' Serum Gonadotrophins is a hormone that has an FSH activity and was extensively used in superovulatory protocols in the past. The problem is that this hormone has a long life in the blood stream and hyper-stimulates the ovaries and thereby causes unovulatory follicles, rising oestradiol levels and problems with sperm/oocyte transport in the uterus. These days its use is very restricted, as FSH has proved to be more effective in superovulatory protocols.

Oestrogens: Hormones produced by ovarian follicles that regulate the secretion of FSH and LH. Other functions are to promote sexual behavior, stimulate development of secondary sexual characteristics and also have an anabolic effect.

Progesterone: Hormone secreted by the luteal cells from the corpus luteum , the placenta and adrenal gland. When an alpaca is receptive to the male, it means that high levels of oestrogens are present in the blood. This animal will ovulate 24-30 hrs after mating and the ruptured follicle will form the corpus luteum . If fertilisation and implantation of the embryo occurs, the corpus luteum will secrete progesterone during the whole pregnancy period. If fertilisation does not occur, the corpus luteum will secrete progesterone until day 10-12 and then regress through the action of prostaglandin F2α. Progesterone is the hormone that makes the female unreceptive to the male.

Prostaglandin F2α: Hormone secreted by the uterus that induces luteolysis or destruction of the corpus luteum . When a female become pregnant, the developing embryo sends a signal to the uterus (maternal recognition of pregnancy), preventing the release of prostaglandin F2α. If the female does not become pregnant, the release of prostaglandin F2α will cause luteolysis by day 10-12.

Follicles: Rounded structures at the ovarian surface that look dark when observed by transrectal ultrasound because of their liquid content. The follicle contains the egg or oocyte that will be released upon ovulation.

Follicular waves: A term proposed by Rajakoski in 1960 in cattle which refers to the growth and regression of ovarian follicles. Ovarian follicular dynamics in alpacas occur in waves as well. What this means is that when a new group of follicles start to grow at the ovary, there will be one which will grow faster and become dominant and will inhibit the growth of the rest. This dominant follicle will be present for a few days (10-12) and then regress. Then, a new group of follicles will start to grow again (new follicular wave) and a new dominant follicle will appear. When an alpaca female with a mature dominant follicle has been mating, the stimuli will induce the release of LH within 15 minutes and this dominant follicle will ovulate in 30 hrs.

Dominant follicle: A follicle which grows quickly and exerts dominance over the rest in terms of production of two hormones: oestradiol and inhibin. These two hormones will cause regression of the subordinated follicles. Superovulatory treatments in alpacas in the presence of a dominant follicle are less efficient than in its absence, as tested in cows and ewes.

Subordinate follicles: Follicles which stop growing and eventually will regress due to the action of oestradiol/inhibin secreted by the dominant follicle.

Atresia: Morphological and biochemical degenerative process in the subordinated follicles at the ovary that leads to follicular death, also called apoptosis.

Corpus luteum : Solid structure present at the ovary after ovulation takes place. The CL is responsible for the production of progesterone, which maintains pregnancy in alpacas during the whole gestation period. After a superovulatory treatment it is possible to predict the superovulatory response by counting the corpora lutea using transrectal ultrasound. One CL is equivalent to one oocyte.

Induced ovulators: Those animals that need the copula stimuli to release LH and to ovulate. In these animals there is no oestrous cycle and the female present long periods of receptivity (>30 days) to the male, with short periods of non-receptivity (2-3 days). Camelidae are induced ovulators, as are cats. In the case of alpacas an induced ovulatory factor in the seminal plasma in males has been identified, which also produces the release of LH to ensure ovulation will take place after mating.

Spontaneous ovulators: Animals that present an oestrous cycle regularly, like cows and ewes. Ovulation takes place every 17 days regardless of whether the female is mating or not.

Superovulation: An hormonal treatment which uses FSH to induce multiple follicular grow and thus the release of several oocytes instead of one, and which promotes the possibility of being fertilised via artificial insemination or natural mating and of obtaining multiple embryos from a valuable donor. In the case of cattle, sheep and goats, these follicles will ovulate spontaneously after FSH application. In alpacas, as they are induced ovulators, a dose of LH is required to break the follicular wall and to produce ovulation after the FSH treatment.

Superovulatory treatment in alpacas: A protocol to induce growth, maturation and ovulation of several follicles at the same time in order to obtain embryos from quality donors after natural mating. In general terms, a protocol to superovulate alpacas uses FSH in decreasing doses injected 12 hrs apart for 3 to 5 days. Twenty-four hours after the last FSH injection, a hormone to induce ovulation is applied, either LH or GnRH. At the end of the hormonal treatment, animals will be mated to fertilise those oocytes and will then be flushed 7 days later to recover the embryos.

Synchronisation of recipients: Preparing the recipient animals to accept the embryo harvested from the donor. It is necessary to have a synchrony between donor and recipients, so that the embryo removed from the donor will find exactly the same environment in the uterus of the recipient and the chance of bearing a cria will increase.

Cervix: Lower part of the uterus that connects with the vagina. In the case of alpacas, the cervix presents 2-3 regular or irregular rings. In order to do flushing/ deposit of embryos or artificial insemination it is necessary to pass the cervix and introduce the catheter/insemination gun into the uterine horn. Sometimes when the cervix is irregular it is too difficult to pass it, and these animals cannot be used in ET programs.

Flushing: Procedure of collecting of embryos from the uterus of a donor female, using a Foley catheter that is introduced into the vagina, passing the cervix and reaching the uterine horns. It consists in repeated uterine injection of flushing media to remove the embryos and then collecting them to be assessed and transferred into the recipients. Flushing needs to be performed very carefully as the embryos can be damaged and will not then survive upon transfer to the recipients. In the case of alpacas, flushing is performed 7 days after mating.

Embryo recovery rate: This is a simple calculation of the ratio between the number of corpora lutea counted by transrectal ultrasound and the yield of embryos.

Embryo assessments: Morphological evaluation of embryos in order to determine it they are transferable. For this purpose a stereomicroscope is used to determine which embryos are of sufficient quality to be transferred into a recipient.

Transferable embryo: Embryo that has passed the morphological assessment of quality and will be put into a recipient to complete pregnancy. A good embryo is evenly granulated and symmetrical.

Transfer to receptors: Procedure that consists in placing the embryo deep into the uterine horn using an insemination gun. It is a simple procedure, but it requires a lot of practice to achieve good results. It needs to be performed very gently, as the gun may hit the uterine walls and may cause bleeding that will kill the embryo and/or cause uterine infections.

Transplant: Refers to organs, not to embryos. It is a term that has been incorrectly used in the past and still is incorrectly used by many people. The proper term is ‘transfer'.

Laparoscopy: A surgical procedure which uses a telescope-like instrument to have a look at the pelvic organs. Initially, embryo transfer was conducted using this technique until the middle of the 1970's, when non-surgical recovery was developed. In the case of small ruminants like sheep and goat it is still used, as it is impossible to gain access to their reproductive tracts rectally.

Laparotomy: A surgical procedure which exposes organs to be manipulated from the exterior of the animal body. Embryo transfer used laparotomy in the past to expose the reproductive tract to perform flushing. These days this technique is used in small ruminants (sheep and goats) only, due to limited access to the reproductive tract via the rectum. In the case of cows and alpacas, a Foley catheter is used and this is a simple procedure.

Transrectal ultrasound: A reproductive technique which uses the interaction of sound waves with a living tissue/organ to produce an image. Transrectal ultrasound uses a probe or transducer that is inserted into the rectum of the female and is led onto the ovarian surface, making possible a clear visualisation in real time, without direct manipulation, of the ovarian structure. It makes it possible to measure ovarian diameter, number and size of follicles, corpora lutea and to detect pathologies such as cystic ovarian disease. It can be used for early pregnancy diagnosis in alpacas. Transrectal ultrasound is a powerful tool in ET technology for determining prior ovarian status before application of superovulatory treatments, and also for evaluating the response by counting the number of corpora lutea after superovulatory treatments.

Please cite as:

Reyna, J (2006). Embryo transfer in alpacas glossary. World Alpacas Magazine (UK). October Autumn Issue. 54-55.

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