Use of Luteinizing Hormone ELISAs in Breeding Elephants
American Association of Zoo Veterinarians Conference 2008
Dennis Schmitt1,2, DVM, PhD, DACT; Scotti Charmason1, MS; Ellen Wiedner2, VMD, DACVIM; Danielle Graham2, DVM
1Agriculture Department, Missouri State University, Springfield, MO, USA; 2Ringling Bros. Center for Elephant Conservation, Polk City, FL, USA


Successful artificial insemination (AI) of elephants depends heavily on determining the unique luteinizing hormone (LH) surges that occur during the follicular phase of the elephant’s estrous cycle. Natural breeding of elephants also can benefit from a rapid and accurate determination of the two LH surges found in elephants. There are three ELISAs available for determining the LH surge; two are commercially available assays and one is a laboratory in-house assay. They vary in their cost, time to complete the assay, and ease of performing the procedures. Detection of the initial non-ovulatory peak in luteinizing hormone (LH1) is best accomplished by use of an in-house LH assay, or use of the LH assay available from Dr. Nancy Dahl (UC-Davis, Davis, CA, USA), both of which are quantitative assays for detection of LH. For cow-side use during estrus, the qualitative ELISA Witness® LH Ovulation Timing Test Kit (Symbiotics Corporation, Kansas City, MO, USA) detects LH in elephants within 20 min. This assay requires a minimum of laboratory precision to detect the ovulatory LH peak (LH2).


Elephants are the only species known to exhibit a double LH peak during a single estrous cycle.2,4 Increased success of artificial insemination in elephants occurred partly in response to the ability to detect the LH1 surge about 21 days prior to the ovulatory LH2 surge that occurs at the end of a two- to three-day estrus.1 The first reports regarding detection of the double LH surges were performed in laboratories using custom ELISA technology that requires exacting procedures and two days to complete the quantitative assays.2,4 A semi-quantitative elephant LH ELISA that can be performed in the field in about 2.5 h was developed at UC-Davis.3 A qualitative LH assay was developed for use in dogs and cats that uses a latex strip ELISA. The time for development of the test is 20 min and detects a LH surge greater than 1 ng/ml using serum.

Elephants have LH1 and LH2 surges in the 4–16 ng/ml range,2,4 well within the detectable range for all of the assays described. The detection of the LH1 peak usually is from daily samples submitted weekly; this allows some efficiency of assay resources and provides at least a two-wk notice of LH2. However, accurate and timely detection of LH2 is needed at least daily and at times twice daily during estrus. The use of an LH assay which can be performed ‘cow-side’ and accurately detect LH2 is essential for successful AI and can be helpful in determining estrus status for natural breeding. The Witness® LH Ovulation Timing Test Kit from Symbiotics was developed for use in dogs and cats, but is effective in other species, including elephants, and meets these requirements.


Detection of LH1 provides information for predicting the LH2 surge and performance of assays that require more laboratory time and precision are useful since detection of LH1 is not as time-sensitive as LH2 detection. Both of the quantitative assays have unique advantages. An in-house assay can be set up, but requires greater preparation time, precision of laboratory procedures is more demanding, often takes two days to perform, and is more susceptible to environmental variables. The assay developed by UC-Davis costs about $5.00 per well, takes about 2.5 h to perform and is more stable. However, for quantitative results, the overhead costs of the standard curve requires about 16 wells ($90), plus two wells for each unknown sample. The UC-Davis assay can be set up as a qualitative test with high and low controls and no standard curve. This requires from three to six wells for a single sample. The Witness® LH Ovulation Timing Test Kit has a control built into each test strip and costs about $25.00 per sample. Because ‘cow-side’ testing is possible using the Witness® LH Ovulation Timing Test Kit, I recommend its use for detection of LH2, although the UC-Davis Elephant ELISA is competitively priced and can be performed in a nearby temporary laboratory. Because timing is critical in detecting LH2 and performing subsequent AI, I recommend using the Witness® LH Ovulation Timing Test Kit at the time of estrus, preceded by either one of the other assays for detecting LH1, depending on availability of laboratory labor and equipment.

Literature Cited

1.  Brown, J.L., F. Goritz, N. Pratt-Hawkes, R. Hermes, M. Galloway, L.H. Graham, C. Gray, S.L. Walker, A. Gomez, R. Moreland, S. Murray, D.L. Schmitt, J.G. Howard, J. Lehnhardt, B. Beck, A. Bellem, R. Montali, and T.B. Hildebrandt. 2004. Successful artificial insemination of an Asian elephant at the National Zoological Park. Zoo Biol. 23:45–63.

2.  Brown, J. L., D.L. Schmitt, A. Bellem, L.H. Graham, and J. Lehnhardt. 1999. Hormone secretion in the Asian elephant (Elephas maximus): characterization of ovulatory and anovulatory luteinizing hormone surges. Biol. Reprod. 61:1294–1299.

3.  Dahl, N.J., D. Olson, D.L. Schmitt, D.R. Blasko, R.S. Kristipati, and J.F. Roser. 2004. Development of an enzyme-linked immunosorbent assay (ELISA) in the elephant (Loxodonta africana and Elephas maximus). Zoo Biol. 23:65–78.

4.  Kapustin, N., J.K. Critser, D. Olson, and P.V. Malven. 1996. Nonluteal estrous cycles of 3-week duration are initiated by anovulatory luteinizing hormone peaks in African elephants. Biol. Reprod. 55:1147–1154.


Speaker Information
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Dennis Schmitt, DVM, PhD, DACT
Agriculture Department
Missouri State University
Springfield, MO, USA

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