Sains Malaysiana 51(6)(2022): 1625-1634

http://doi.org/10.17576/jsm-2022-5106-02

Faecal Concentrations of Progesterone and 17β-Oestradiol of Female Malayan Tapir from Different Faecal Hormone Extraction Methods

(Kepekatan Najis yang Mengandungi Progesteron dan 17β-Oestradiol Tapir Betina Malaya daripada Kaedah Pengekstrakan Hormon Najis yang Berbeza)

MUHAMMAD NAJIB RUSLAN¹, MASHITAH SHIKHMAIDIN^1,2,3,*, SALFARINA RAMLI⁴, NALISHA ITHNIN⁵, INTAN NASUHA AZHAR¹, NUR HAFIZAH MOHAMMED¹ & SYAIZWAN ZAHMIR ZULKIFLI¹

¹Department of Biology, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang,

Selangor Darul Ehsan, Malaysia

²Institute of Tropical Agriculture and Food Security (ITAFos), Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

³Division of Life Sciences (Molecular Biology Major), Department of Bioactive Material Sciences and Research Center of Bioactive Materials, Jeonbuk National University, 54896 Jeonju, Korea

⁴Department of Pharmaceutical Pharmacology and Chemistry, Faculty of Pharmacy, Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA, Cawangan Selangor, 42300 Puncak Alam, Selangor Darul Ehsan, Malaysia

⁵Sime Darby Plantation Technology Centre Sdn Bhd, 1^st Floor Block B, UPM-MTDC TIC III, Jalan Lebuh Silikon, 43000 UPM Serdang, Selangor Darul Ehsan, Malaysia

Received: 10 June 2020/Accepted: 22 November 2021

ABSTRACT

Non-invasive hormone monitoring of endangered wildlife provides an essential tool to optimize breeding strategies, mainly in captive management. However, the protocol of reproductive hormone monitoring on pregnancy status using non-invasive faecal samples in Malayan tapir is still inconclusive. Therefore, we compared the metabolites reproductive hormones; 17β-oestradiol and progesterone in the faecal samples extracted using methods of Schwarzenberger et al. (1996) (Methods A and B), Brown et al. (2001) (Method C) and Shutt et al. (2012) (Method D) from pregnant and non-pregnant captive Malayan Tapir. Faecal samples from four female of Malayan Tapir (Tapirus indicus) were collected for five months at Sungai Dusun Wildlife Reserve (n = 2), Zoo Taiping (n = 1) and Zoo Negara (n = 1), Malaysia. Analysis by liquid chromatography-mass spectrometry (LCMS) confirmed that progesterone was detected in all fecal sample extracted by extraction Method A, while 17β-oestradiol was undetectable in all methods. The measurement of reproductive hormones for pregnancy status via Enzyme-Linked Immunosorbent Assay (ELISA) analysis showed no significant difference (p > 0.05) for progesterone between the fecal extraction methods. Yet the data obtained were not able to validate the pregnancy status, due to similarity in concentration for both pregnant and non-pregnant tapirs. While for 17β-oestradiol, significant results were observed in all extraction methods and in pregnancy status (p < 0.05). Method C was found to be the most reliable extraction method (p < 0.05) to indicate pregnancy status. From the results, it showed that solvent used, boiling process and multiphase extraction plays important role in fecal extraction process in Malayan Tapir.

Keywords: Malayan tapir; non-invasive fecal; oestradiol; pregnancy; progesterone

ABSTRAK

Pemantauan hormon tak invasif terhadap hidupan liar yang terancam penting untuk mengoptimumkan strategi pembiakan terutamanya dalam pengurusan haiwan tawanan. Walau bagaimanapun, protokol pemantauan hormon pembiakan terhadap status pembiakan dengan menggunakan tak invasif daripada sampel najis tapir Malaya masih tidak muktamad. Oleh itu, dalam penyelidikan ini kami mengkaji perbezaan pengukuran hormon pembiakan metabolit; 17β-oestradiol dan progesteron dalam kaedah pengekstrakan najis Schwarzenberger et al. (1996) (Kaedah A dan Kaedah B), Brown et al. (2001) (Kaedah C) dan Shutt et al. (2012) (Kaedah D). Sampel najis daripada empat betina tapir Malaya (Tapirus indicus) dikutip selama lima bulan di Hutan Simpan Sungai Dusun (n = 2), Zoo Taiping (n = 1) dan Zoo Negara (n = 1), Malaysia. Analisis dengan spektrometri kromatografi gas cecair (LCMS) mengesahkan bahawa semua sampel najis menunjukkan progesteron hanya dari Kaedah Pengekstrakan A, sementara 17β-oestradiol tidak dapat dikesan daripada mana-mana Kaedah Pengekstrakan najis. Pengukuran hormon pembiakan untuk status kehamilan melalui analisis ujian imunosorben berkaitan enzim (ELISA) tidak menunjukkan sebarang perbezaan yang signifikan bagi progesteron antara Kaedah Pengekstrakan tinja. Namun data yang ditunjukkan dalam kajian ini tidak dapat mengesahkan status kehamilan kerana nilai progesteron untuk hamil dan tidak hamil adalah hampir sama. Sementara untuk 17β-oestradiol, ia menunjukkan hasil yang signifikan dalam semua kaedah pengekstrakan dan status kebuntingan (p < 0.05) dengan kepekatan tertinggi 17β-oestradiol dengan menggunakan Kaedah C, ini menunjukkan bahawa pelarut memainkan peranan penting dalam proses pengekstrakan najis dan nampaknya kaedah ini boleh digunakan untuk mendiagnos kehamilan tapir Malaya (p < 0.05). Kami mencadangkan untuk progesteron, pengesahan immunoasei diperlukan mungkin disebabkan oleh merendahkan kepekatan progesteron. Keputusan kajian menunjukkan jenis pelarut yang digunakan, proses pendidihan dan pengekstrakan multifasa memainkan peranan penting dalam proses pengekstrakan najis tapir Malaya.

Kata kunci: Kehamilan; najis tak invasif; oestradiol; progesteron; tapir Malaya

References

Asa, C.S., Bauman, J.E., Houston, E.W., Fischer, M.T., Read, B., Brownfield, C.M. & Roser, J.F. 2001. Patterns of excretion of fecal oestradiol and progesterone and urinary chorionic gonadotropin in Grevy’s zebras (Equus grevyi): Ovulatory cycles and pregnancy. Zoo Biology 20(3): 185-195.

Bamberg, E., Möstl, E., Patzl, M. & King, G.J. 1991. Pregnancy diagnosis by enzyme immunoassay of oestrogens in feces from nondomestic species. Journal of Zoo and Wildlife Medicine 22(1): 73-77.

Beehner, J.C. & Whitten, P.L. 2004. Modifications of a field method for fecal steroid analysis in baboons. Physiology and Behavior 82(2-3): 269-277.

Berkeley, E.V., Kirkpatrick, J.F., Schaffer, N.E., Bryant, W.M. & Threlfall, W.R. 1997. Serum and fecal steroid analysis of ovulation, pregnancy, and parturition in the black rhinoceros (Diceros bicornis). Zoo Biology 16(2): 121-132.

Brown, J.L., Citino, S.B., Shaw, J. & Miller, C. 1994. Endocrine profiles during the estrous cycle and pregnancy in the Baird's tapir (Tapirus bairdii). Zoo Biology 13(2): 107-117.

Brown, J.L., Bellem, A.C., Fouraker, M., Wildt, D.E. & Roth, T.L. 2001. Comparative analysis of gonadal and adrenal activity in the black and white rhinoceros in North America by noninvasive endocrine monitoring. Zoo Biology 20(6): 463-486.

Celebi, M. & Demirel, M. 2003. Pregnancy diagnosis in mares by determination of oestradiol-17-b hormone in faeces. Turkish Journal of Veterinary and Animal Sciences 27(2): 373-375.

Conforti, V.A., Bravo, N.R.S., Moreira, M.R., Villar, E.C., Paulo, O.L.O.H., Veneziani, R.C.S. & Silva, M.A. 2017. High-performance liquid chromatography as a novel tool for assessing ovarian function in jaguars (Panthera onca): Development and validation of the method and quantification of ovarian steroids. Animal Reproduction 14(1): 361-361.

Ghosal, R., Kalaivanan, N., Sukumar, R. & Seshagiri, P.B. 2012. Assessment of estrus cyclicity in the Asian elephant (Elephas maximus) by measurement of fecal progesterone metabolite 5α-P-3OH, using a non-invasive assay. General and Comparative Endocrinology 175(1): 100-108.

Graham, L.H., Schwarzenberger, F., Möstl, E., Galama, W. & Savage, A. 2001. A versatile enzyme immunoassay for the determination of progestogens in feces and serum. Zoo Biology 20(3): 227-236.

Habumuremyi, S., Robbins, M.M., Fawcett, K.A. & Deschner, T. 2014. Monitoring ovarian cycle activity via progestagens in urine and feces of female mountain gorillas: A comparison of EIA and LC–MS measurements. American Journal of Primatology 76(2): 180-191.

Hodges, J.K., Brown, J.L. & Heistermann, M. 2010. Non-invasive Monitoring of Reproductive Status and Stress. Wild Mammals in Captivity. 2nd ed. Chicago, Illinois: University of Chicago Press. pp. 447-468.

Hodges, J.K., Heistermann, M., Beard, A. & Van Aarde, R.J. 1997. Concentrations of progesterone and the 5 alpha-reduced progestins, 5 alpha-pregnane-3, 20-dione and 3 alpha-hydroxy-5 alpha-pregnan-20-one, in luteal tissue and circulating blood and their relationship to luteal function in the African elephant, Loxodonta africana. Biology of Reproduction 56(3): 640-646.

Holt, W.V., Brown, J.L. & Comizzoli, P. 2014. Reproductive science as an essential component of conservation biology. In Reproductive Sciences in Animal Conservation. New York: Springer. pp. 3-14.

Kasman, L.H., McCowan, B. & Lasley, B.L. 1985. Pregnancy detection in tapirs by direct urinary estrone sulfate analysis. Zoo Biology 4(3): 301-306.

Kuckelkom, B. 1994. Assessment of pregnancy in Kiang mares (Equus hemionus holdereri) using estrogen determination in feces. Theriogenology 42(1): 37-42.

Kumar, V. & Umapathy, G. 2019. Non-invasive monitoring of steroid hormones in wildlife for conservation and management of endangered species - A review. Indian Journal of Experimental Biology 57(May): 307-314.

Kusuda, S., Ikoma, M., Morikaku, K., Koizumi, J.I., Kawaguchi, Y., Kobayashi, K., Matsui, K., Nakamura, A., Hashikawa, H., Ueda, M., Kaneko, M., Akikawa, T., Shibagaki, S. & Doi, O. 2007. Estrous cycle based on blood progesterone profiles and changes in vulvar appearance of Malayan Tapir (Tapirus indicus). Journal of Reproduction Development 53(6): 12833-1289.

Mace, G.M. & Balmford, A. 2000. Patterns and Processes in Contemporary Mammalian Extinction. Cambridge: Conservation Biology Series. pp. 27-52.

Murtagh, R., Behringer, V. & Deschner, T. 2013. LC-MS as a method for non-invasive measurement of steroid hormones and their metabolites in urine and faeces of animals. Wien Tierärztl Monat - Veterinary Medicine Austria 100: 247-254.

Novarino, W. & Grant, R.S. 2005. Report to Rufford Small Grant (for Nature Conservation) in Association with the Whitley Laing Foundation. Population Monitoring and Study of Daily Activities of Malayan Tapir (Tapirus indicus). Indonesia: Biology Department, Faculty Mathematics and Science, Andalas University.

Palme, R., Möstl, E., Brem, G., Schellander, K. & Bamberg, E. 1997. Faecal metabolites of infused 14C‐Progesterone in domestic livestock. Reproduction in Domestic Animals 32(4): 199-206.

Palme, R., Rettenbacher, S., Touma, C., El‐Bahr, S.M.  & Möstl, E. 2005. Stress hormones in mammals and birds: Comparative aspects regarding metabolism, excretion, and non-invasive measurement in fecal samples. Annals of the New York Academy of Sciences 1040(1): 162-171.

Palme, R., Touma, C., Arias, N., Dominchin, M.F. & Lepschy, M. 2013. Steroid extraction: Get the best out of faecal samples. Wien Tierarztl Monatsschr 100(9-10): 238-246.

Peter, I.D., Abd Wahid Haron, F.F.A., Jesse, M.A., Han, M.H.W., Fitri, W.N., Yahaya, M.S. & Alamaary, M.S.M. 2018. Opportunities and challenges associated with fecal progesterone metabolite analysis. Veterinary World 11(10): 1466.

Pukazhenthi, B., Quse, V., Hoyer, M., van Engeldorp Gastelaars, H., Sanjur, O. & Brown, J.L. 2013. A review of the reproductive biology and breeding management of tapirs. Integrative Zoology 8(1): 18-34.

Polegato, B.F., Zanetti, E.D.S. & Duarte, J.M.B. 2018. Monitoring ovarian cycles, pregnancy and post-partum in captive marsh deer (Blastocerus dichotomus) by measuring fecal steroids. Conservation Physiology 6(1): cox073.

Schaftenaar, W., de Boer, A.M. & Glatston, A.R. 2006. Monitoring of the oestrous cycle in a captive Malayan tapir (Tapirus indicus). Veterinary Record 159(13): 421.

Schwarzenberger, F. 2007. The many uses of non‐invasive faecal steroid monitoring in zoo and wildlife species. International Zoo Yearbook 41(1): 52-74.

Schwarzenberger, F., Möstl, E., Palme, R. & Bamberg, E. 1996. Faecal steroid analysis for non-invasive monitoring of reproductive status in farm, wild and zoo animals. Animal Reproduction Science 42(1): 515-526.

Shutt, K., Setchell, J.M. & Heistermann, M. 2012. Non-invasive monitoring of physiological stress in the Western lowland gorilla (Gorilla gorilla gorilla): Validation of a fecal glucocorticoid assay and methods for practical application in the field. General and Comparative Endocrinology 179(2): 167-177.

Thitaram, C. & Brown, J.L. 2017. Monitoring and controlling ovarian activity in elephants. Theriogenology 109(2): 42-47.

Torres-Pelayo, V.D.R., Rovirosa-Hernández, M.J., García-Orduña, F., Chavira-Ramírez, R.D., Boeck, L., Canales-Espinosa, D. & Rodríguez-Landa, J.F. 2011. Variation in the extraction efficiency of estradiol and progesterone in moist and lyophilized feces of the black howler monkey (Alouatta pigra): Alternative methods. Frontiers in Physiology 2: 97.

Touma, C., Sachser, N., Möstl, E. & Palme, R. 2003. Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice. General and Comparative Endocrinology 130(3): 267-278.

Traeholt, C., Novarino, W., Saaban, S., Shwe, N.M., Lynam, A., Zainuddin, Z., Simpson, B. & Mohd, S. 2016. Tapirus indicus. The IUCN Red List of Threatened Species 2016. https://www.iucnredlist.org/.

Zemanova, M.A. 2019. Poor implementation of non-invasive sampling in wildlife genetics studies. Rethinking Ecology 4: 119-132.

^*Corresponding author; email: mashitah@upm.edu.my