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What is the major hormone discussed in North America with regard to reproduction in alpacas? Progesterone! What does it do? Why do we monitor progesterone and what can we learn from its assay? Are all methods of monitoring progesterone equal and can we accurately diagnose pregnancy with progesterone assays? These questions are commonly heard by veterinarians who are assisting owners with the management of breeding alpacas. In conjunction with WoodsEdge Wools Farm, Stockton, N.J., the following is my response to many of these queries.

Progesterone or progestin by definition is a steroid hormone produced by the corpus luteum (CL) of the ovary and is necessary for pregnancy maintenance. Following ovulation when the ovum is released from the follicle, the granulosa cells lining the follicle are transformed under the influence of luteinizing hormone to develop into a CL. The corpus luteum then begins to produce progesterone which causes the female to reject the male's advances within approximately one to four days post breeding and also stimulates a change in the endometrial lining of the uterus. Progesterone stimulates the endometrial glands to proliferate, enlarge and secrete proteins necessary to support the nutrition of the early developing embryo and its yolk sac. Under continued progesterone as well as other hormone influences, the endometrium develops and provides a base for placental contact between the fetus and uterus for exchange of nutrients, blood gases and waste products between the mother and fetus.

What does progesterone do?
In the lama, it is reported that the corpus luteum must be maintained for nearly the entire gestation to provide progesterone support (1,2,3). Removal of the CL or ovary containing the CL in pregnant lamas between one and 11 months of gestation will abort the pregnancy in approximately 24 hours (4). Should the CL be destroyed by prostagladin release associated with inflammation or severe stress, the ovarian source of progesterone would decrease and the pregnancy would be lost. Thus, it is important that we learn to monitor the lama's endogenous or serum progesterone and to supplement if the need arises.

Why do we monitor progesterone?
To monitor the serum or plasma progesterone (P4) in lamas, the assays utilized in other species have been adapted. The assays are to determine if progesterone is present and at what concentration. Purposes for which we presently utilize P4 assays are to: 1) determine if ovulation has occurred and a CL has formed; 2) determine if the CL is functional in producing sufficient P4; and 3) monitor luteal activity during gestation in lamas with problem pregnancies. One point to emphasize, as is accepted in nearly all other species, normal elevated P4 levels is not a method for pregnancy diagnosis. A positive P4 assay simply indicates that progesterone is in the lamas' serum, whether it comes from a functional CL or a drug source and does not indicate diagnosis of pregnancy. Examples of improper use of P4 to diagnose pregnancy is when your lama is bred and forms a functional CL but resorbs or loses her pregnancy. The CL may persist and continue to provide a positive P4 assay even though she is no longer pregnant (called a retained or persistent CL). Similarly, in females with conditions such as segmental aplasia (missing segments of the uterus, cervix and/or vagina) of the genital tract, a positive P4 assay will follow breeding if ovulation occurs, even though these females are incapable of conceiving. We have diagnosed by ultrasound a 5-year-old female llama with segmental aplasia and observed the persistent CL on an ovary which lacked connection to the uterine horn. This female over the previous three years had "progesterone-diagnosed pregnancies" based on elevated P4 and was even sold as a pregnant female, but she never produced a cria.

Progesterone assays are a valuable tool to monitor ovulation and hormonal activity of pregnancy, but we recommend not using it as your only source of pregnancy diagnosis. Since the female's behavior of rejecting the male is associated with progesterone influence, it is generally a sound assumption that progesterone is elevated if the female is "spitting-off" the male after breeding. Additional tests, such as ultrasound as early as 17 to 21 days or palpation at 30 to 40 days after breeding, should be utilized to verify pregnancy diagnosis instead of a P4 assay.

How Is Progesterone Measured?
To measure serum or plasma progesterone, most university or commercial laboratories utilize a radio-immunoassay (RIA) based on progesterone determinations for humans or a specific domestic species. Depending on the derivation of the antibody utilized in the specific RIA, there may be some individual variation in the reported serum P4 concentrations between various laboratories. Each laboratory needs to establish its own norms for alpacas and to provide their information to the clientele. For the lama, most laboratories presently indicate an adequate norm to be 1.0 to 2.0 ng/ml of P4 at 3 to 4 days after mating or approximately 3 days after ovulation with an increase to 3 to 6 ng/ml by Day 1 to 12 in pregnant lamas or a rapid decrease by Day 8 to 10 in nonpregnant lamas (4,5).

Due to the different RIA systems used for P4 assays, veterinarians and clients must become aware of what is normally acceptable for lama pregnancy maintenance from each individual laboratory. Some laboratories indicate 1.0 ng/ml as sufficient while others suggest more than 2.0 ng/ml. You can compare laboratory results by sending the same sample to two or more laboratories to determine normal levels for each lab. Variations of 1 to 2 ng/ml are common between laboratories.

Large commercial laboratories or university laboratories are the main resource for lama clientele to obtain P4 assays. Unfortunately, due to geographical locations, these laboratories may be distant enough from lama patients to delay receiving results several days to even weeks. Thus their usefulness declines, especially for verifying ovulation and CL formulation. To overcome this problem in our area, we have evaluated several commercially available enzyme-linked immunosorbent assay (ELISA) systems compatible with veterinary clinical laboratories (6). The one ELISA system which has performed well in our hands for quantitative diagnostic measurement of lama P4 and is in close agreement with university laboratories is the Amplified Enzyme Immunoassay: NOVO BioLabs, Ltd. (Enzygnost Serum Progesterone Kit: Hoechst-Rousell AgriVet). This kit provides 96 determination sites and is supplied with standards. It requires an additional investment of the plate reader or specialized spectrophometer microcell. When samples are run in duplicate with standards, it provides for approximately 36 to 42 tests which can be run in 35 to 60 minutes.

In an attempt to provide an even more rapid and easily ran P4 assay, we evaluated three semi-quantitative ELISA test kits over the past two years (Cite Semi-Quent: Agritech System, Inc.; Equicheck: American Diagnostic Sales, Inc. and Target: Bio Metallics, Inc.). All were found unreliable for quantitative results in lamas. Presently we recommend that alpaca breeders work closely with a single laboratory or attempt to set up the Enzygnost System if sufficient patient samples can be generated to warrant the investment in equipment.

1. Fowler, Murray, Medicine and Surgery of South American Camelids, 1st ed, Iowa State University Press, Ames, Iowa. 50010 (1989).

2. Johnson, LaRue: Llama Medicine In: The Veterinary Clinics of North America. 5:1 (March, 1989).

3. Johnson, LaRue: Llama Reproduction, G.A.L.A. Llama Medicine Seminar Manual (May, 1991).

4. Bravo, P. Walter: Reproductive Endocrinology of Female South American Camelids, Proc. of the Annual Meeting of Society for Theriogenology (1991): 303.

5. Adams, GP, Sumar J, Ginther OJ: Form and Function of the Corpus Luteum in Llamas. In: Animal Reproduction Science, 24 (1991) 127-138.

6. Neely, DP: Progesterone/Progestin Therapy in the Broodmare. Proc. Am. Assoc. Equine Pract., (1988): 203-218.

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