Journal of Reproduction and Development, Vol. 53, No. 1, 2007 —Research Note— Excretion Patterns of Fecal Progestagens, Androgen and Estrogens During Pregnancy, Parturition and Postpartum in Okapi (Okapia johnstoni) Satoshi KUSUDA1), Koki MORIKAKU2), Ken-ichi KAWADA3), Kenji ISHIWADA4)# and Osamu DOI3) 1)Laboratory of Animal Reproduction, United Graduate School of Agricultural Science, Gifu University, Gifu 501-1193, 2)Preservation and Research Center, City of Yokohama, Kanagawa 241-0804, 3)Laboratory of Animal Reproduction, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193 and 4)Yokohama Zoological Gardens Zoorasia, Kanagawa 241- 0001, Japan #Present: Kanazawa Zoological Gardens of Yokohama, Kanagawa 236-0042, Japan Abstract. The aim of the present study was to establish a simple method to monitor ovarian activity and non-invasively diagnose pregnancy in okapi (Okapia johnstoni). The feces of a female okapi were collected daily or every 3 days for 28 months. Steroids in lyophilized feces were extracted with 80% methanol, and the fecal levels of immunoreactive progestagens (progesterone and pregnanediol- glucuronide), androgen (testosterone), and estrogens (estradiol-17β and estrone) were determined by enzyme immunoassays with commercially available antisera. Using the progesterone profiles, the durations of the luteal phase, follicular phase, and estrous cycle were determined to be 11.1 ± 0.4, 5.3 ± 0.6, and 16.5 ± 0.7 days (n=22), respectively. Fecal levels of immunoreactive progesterone, pregnanediol glucuronide, and testosterone gradually increased from early pregnancy and peaked several months before parturition. More pregnanediol glucuronide was excreted in feces than progesterone during late pregnancy, but not during the estrous cycle. Although the fecal concentrations of immunoreactive estradiol-17β and estrone change a little throughout pregnancy and non-pregnancy, they rose sharply and temporarily on the day following parturition. The present study indicates that fecal assays with commercial antisera for progesterone and pregnanediol glucuronide are useful for evaluating luteal activity and diagnosing pregnancy and indicates that estrogens might have some role as a trigger of parturition. Key words: Estrous cycle, Fecal hormone, Okapi (Okapia johnstoni), Pregnancy, Progesterone (J. Reprod. Dev. 53: 143–150, 2007) kapi (Okapia johnstoni), a member of the family rainforest. According to the 2006 IUCN Red List of Giraffidae, was first described scientifically in Threatened Species [3], okapi is classified as Low 1901 [1]. An estimated 6,200–10,650 individuals of Risk/Near Threatened (LR/nt) and is not in this species live in the northern and eastern immediate danger of extinction, but they are Democratic Republic of the Congo [2]. Possible considered vulnerable because of deforestation, habitats are extremely limited to areas of dense their delicate resistance to disease [4], and civil strife in the Democratic Republic of the Congo (DRC) [5]. Accepted for publication: September 16, 2006 Published online: October 14, 2006 In captivity, okapi were exhibited for the first Correspondence: O. Doi (e-mail: [email protected]) time at the Antwerp Zoo in Belgium in 1919 [2]. 144 KUSUDA et al. Currently, the captive population numbers a total of 144 animals at 40 zoos in America, Europe, and Japan [6, 7] and 15 animals at the Epulu station of the Okapi Wildlife Reserve in the Ituri tropical rainforest, which is in the northeastern area of the DRC. At least two hundred and forty have been born in captivity since 1970 [7, 8], and captive breeding is successful. In Japan, Yokohama Zoological Gardens Zoorasia was the first zoo to exhibit female and male okapis in 1997, and they have produced a total of 2 offspring, one in 2000 [9] and another in 2003. As of February 2006, a total of 7 okapi are being kept and exhibited at Yokohama Zoological Gardens Zoorasia (5 okapi) and Tokyo Ueno Zoological Gardens (2 okapi) [7]. Endocrinological observations on reproductive physiology in female okapi have mainly been reported by the groups of Loskutoff [10-13] and Fig. 1. A female okapi, named Layla (International Studbook Schwarzenberger [14–17]. Both groups have No. 468), and her feces (inset). attempted to monitor luteal activity and diagnose pregnancy by assays of hormone metabolites in excreta. The estrous cycle of the okapi was methods utilizing enzyme immunoassay systems determined to be approximately 15 days based on with commercially available antisera for changes in the concentrations of pregnanediol progesterone (P4), PdG, testosterone (T), E2 and glucuronide (PdG) in urine [11, 13] and feces [14]. estrone (E1). Urinary levels of estrogens have been reported to increase sharply 1–2 days after complete luteal regression [12]. A peak consistent with estradiol- Materials and Methods 17β (E2) elution patterns has also been observed by high performance liquid chromatography in okapi Animal and collection of samples urine collected during estrus [10]. However, the A female okapi, named Layla (International excretion patterns of fecal estrogens have not been Studbook No. 468; born on: 22 July 1996 at the clarified. Dallas Zoo; Fig. 1), was used in this study. This Pregnancy in okapi is detectable by female was temporarily kept in an approximately transabdominal ultrasonography and 1,000 m2 outdoor area during the day and in 26.88 measurements of changes in body weight [8, 9, 18]. m2 indoor chambers during its nocturnal period in Since mating occurs during pregnancy in some the Asian Tropical Forest Zone (currently being cases [1], routine hormone monitoring is more kept and exhibited in the African Tropical Rain useful for determining the time of conception. Forest Zone) at Yokohama Zoological Gardens Urinary PdG and 20α-dihydroprogesterone [11] ZOORASIA [9]. She gave birth normally (21 and fecal PdG [14–16] have been reported to be November 2000) after a gestation period of 428 excreted in significantly higher levels in pregnant days based on the day of the final mounting versus non-fertile okapi. Thus, assays of these behavior by a male (20 September 1999). The hormones have enabled pregnancy to be diagnosed animal was fed a daily diet of alfalfa hay, pellets, non-invasively. carrot, Komatsuna (Japanese mustard spinach), The aim of this study was to establish a simple lettuce, and celery. Drinking water was freely method of assaying fecal hormone concentrations available. to monitor ovarian activity and diagnose Fresh-looking feces (Fig. 1) were collected from pregnancy non-invasively. We focused on 1) the floor of the animal’s sleeping chamber by 0900 h evaluating a simple method of extracting steroids daily or every 3 days from 8 October 1999 (18 days from feces and 2) establishing generally available after observation of the final mounting behavior) to EXCRETION OF FECAL STEROIDS IN PREGNANT OKAPI 145 4 February 2002. Feces were placed in a Ziploc® FKA234-E; Cosmo Bio) were used. The main cross- bag and stored at –20 C until assay. reactivity of the antibody was 100% for E1, 40% for E1-3-sulfate, and 15% for E1-3-glucuronide. Serial Extraction of steroids dilutions of fecal extracts from samples For the assay of progestagens (P4 and PdG), demonstrated parallelism with the standard curves androgen (T), and estrogens (E2 and E1), the in P4, PdG, T, E2 and E1 assays. steroids in the fecal samples were extracted using a methanol extraction method [19]. Briefly, one or Data analysis two grain-shaped frozen feces were lyophilized for All fecal data are expressed on a per gram dry approximately 24 h and crushed using a hammer. feces basis. Data are presented as the mean ± A portion of the powder, 0.1 g was then extracted standard error of the mean (SEM). Analyses were with 80% methanol in ultra pure water (5 ml) by conducted using Student’s t-test. One estrous cycle vortex-mixing for 30 min. After centrifugation at is the period from the first significant rise in fecal 2,500 rpm for 10 min, the supernatant (methanol iP4 level of one cycle to the rise in the following fraction) was removed into a clean tube and diluted cycle. The day of sampling before the first at a ratio of 1:20 for the P4, PdG and T assays and 1:4 significant increase in fecal iP4 and iPdG levels was for the E2 and E1 assays using assay buffer (0.04 M defined as day 0. disodium hydrogen phosphate dihydrate buffer containing 0.1% bovine serum albumin and 0.05 M sodium chloride). Results Enzyme immunoassays Excretion patterns of fecal steroids during pregnancy Fecal concentrations of immunoreactive P4 (iP4), and parturition PdG (iPdG), T (iT), E2 (iE2), and E1 (iE1) were The changes in the fecal concentrations of the determined using enzyme immunoassays, as progestagens, androgen and estrogens from early described previously [19]. For the P4 assay, a P4 pregnancy to recurrence of the estrous cycle standard (Wako Pure Chemical, Osaka, Japan), P4- postpartum are shown in Fig. 2. At 18 days after 3-CMO-HRP (dilution of 1:500,000, FKA301; observation of final mounting behavior, the fecal Cosmo Bio, Tokyo, Japan), and P4 antibody iP4 and iPdG levels were already about 10 times the (1:250,000, LC-28; Teikoku Hormone, Kanagawa, postpartum basal levels. Fecal iP4, iPdG, and iT Japan) were used. The main cross-reactivity of the levels gradually increased during the greater part antibody was 100% for P4 and 62.2% for 5α- of the pregnancy, and at 1–3 months before pregnanedione. For the PdG assay, a PdG standard parturition, they reached approximately 50, 90, and (Sigma), PdG-HRP (1:100,000, FKA333; Cosmo Bio), 10 times the basal levels, respectively. The day and PdG antibody (1:800,000, FKA334-E; Cosmo after parturition, the iP4, iPdG, and iT values Bio) were used. The main cross-reactivity of the drastically decreased. Immunoreactive P4 values antibody was 100% for PdG and pregnanediol and were slightly higher than iPdG values during the 16.0% for 20α-hydroxyprogesterone.