Progesterone Profile of Female Arabian Tahr (Hemitragus Jayakari) Before, During and After Pregnancy Using ELISA

INTERNATIONAL JOURNAL FOR RESEARCH IN EMERGING SCIENCE AND TECHNOLOGY, VOLUME-3, ISSUE-1, JAN-2016 E-ISSN: 2349-7610

Adnan Naim

Assistant Cell Biologist, Management of Nature Conservation, United Arab Emirates Email: [email protected]

ABSTRACT Middle Eastern subcontinent has a variety of valuable indigenous breeds of animals and birds, which possess great diversity in germplasm. Arabian Tahr (Hemitragus jayakari) has been classified as Endangered according to 2007 IUCN Red List. Various conservation programs for Arabian Tahr population is underway in different Middle Eastern countries, including United Arab Emirates. Although captive breeding is one of the way to conserve the endangered animals, but there are many novel technologies also available and in use to conserve the endangered animal species. Invasive and non-invasive methodologies are used to study the hormonal profile of animals to estimate the time of ovulation, which helps in scheduling the time for artificial insemination, and also to forecast the time of parturition. This article presents the data of the progesterone concentration in fecal samples of the two, female Arabian Tahr, before natural mating and during the gestation period. This article also presents the calculated days of gestation period, of the Arabian Tahr.

Keywords – Arabian Tahr, Endangered, Progesterone.

1. INTRODUCTION hormone and to track its level before, during and after

Arabian Tahr belongs to Mammalia Class, Artiodactyla Order pregnancy of the Arabian Tahr female. and Bovidae Family. It is a medium size, goat-like ungulate. Non-invasive techniques like estimating the concentration of Basically, they exist in the south eastern area of the Arabian steroids in milk, serum, urine and fecal sample of animals has Peninsula. However, Arabian tahr, population has also been been used extensively to study oestrus [13, 14]. Data generated observed in Oman and United Arab Emirates [2-3]. Arabian from such experiments contribute in the management of Tahr (Hemitragus jayakari) has been classified as Endangered conservation of animals, especially endangered [23-25]. (EN C2a) according to 2007 IUCN Red List [1]. Many

programs are underway for the conservation of Arabian Tahr. Measurement of concentrations of metabolized steroids in Different Assisted Reproductive Techniques (ART) are also fecal samples, as a non-invasive technique is widely used to used to conserve the germplasm of Arabian tahr. Hormonal monitor reproductive profile in captive and in wild animals. profiles of Arabian tahr are also studied during different Fecal sample collection is less stressful to the animal like weather conditions in a year and also during breeding season. blood collection [4, 5]. Steroid hormones are metabolized in Arabian Tahr, being a very aggressive animal in behavior, liver, and later excreted in feces and urine in some amount. collection of blood sample to study the hormonal profile could The extraction of steroid metabolites can be achieved by using be difficult. Therefore, collection of fecal samples and variety of methods such as ethanol, methanol etc. Fecal extraction of steroid metabolites from feces was used as a estrogen and progesterone metabolites evaluations are wel1 valuable tool to study the concentration of progesterone established methods used to study the reproductive status in a

INTERNATIONAL JOURNAL FOR RESEARCH IN EMERGING SCIENCE AND TECHNOLOGY, VOLUME-3, ISSUE-1, JAN-2016 E-ISSN: 2349-7610

variety of mammalian species [6-17]. Enzyme-Linked 100 µl of a substrate solution of citrate buffer, H2O2 and 2,2’- immunosorbent assay (ELISA) is a well-established technique azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and is commonly used method to study the hormone were added to each well and the plates were covered and concentrations from animal excretions [19-21, 26]. incubated while shaking at room temperature for 30 min. The plates were then read with a single filter at 405 nm on the 2. MATERIALS AND METHODS microplate reader (Bio-Tek). Blank absorbance was subtracted 2.1 Fecal sample collection: Fecal samples were collected from each absorbance value of the sample to disregard the on 3 days a week by the animal keepers and stored in plastic non-specific binding. In all assays, absorbance values were zip bags. The bags were labeled with an animal ID, animal obtained, standard curves were generated, and samples were name and date. The bags were stored and maintained at 4 extrapolated into the equation to get a value in µg per well degree Celsius, till it was used for extraction. using MS Excel. The data was analyzed as per the instructions in the manual from Clinical Endocrinology Lab, UC, Davis 2.2 Methanol extraction of the hormone from the and Conservation and Research Center, Smithsonian National fecal sample: Before extracting the hormone from the fecal Zoological Park [18]. Samples were analyzed in triplicate sample, the bags containing the feces were kept outside at (same sample in three wells), average value was calculated and room temperature. The whole sample was smashed using a standard deviation was calculated. The line graph was plotted wooden hammer and 0.5 gram was weighed and transferred to against the standard and blank. the 15 ml conical centrifuge tubes. 5 ml of 80% methanol was added to the conical tube holding the fecal sample and the tube 3. RESULTS was put on a rotor for 30 minutes. After 30 minutes the tubes 3.1 The calculated days of gestation for female #1 were centrifuged at 1400 rpm for 20 minutes. 1-2 ml of the (chip # 5282165578) was 157 days. supernatant was collected in labeled 2 ml tube and stored at 4 Figure 1 shows the hormonal profile generated from the data degree Celsius before it is used for ELISA. obtained by running ELISA from the fecal sample of female #1 (chip # 52821-65578) on daily basis from 1st October 2008 2.3 Antibody preparation and ELISA: Anti- to 12th March 2009. The profile suggests that the female was progesterone antibodies (R4859, polyclonal) and HRP conceived on 30th September 2014 and delivered a healthy conjugates were purchased from Clinical Endocrinology Lab, baby on 07th March 2015 with a gestation period of total 157 UC, Davis and used as per their manual instructions days. The figure-1 also shows that the concentration of steroid metabolite in the fecal sample maintained a higher level than the average value during the gestation period. ELISA was performed using Nunc Maxisorb plates. The plates

were pre-coated with 50µl of diluted stock of antibody

(1:10000) in coating buffer (0.05 M, pH9.6 Sodium Female-1 #52821-65578 50 Conc ug/g bicarbonate buffer). The plate was incubated for 12-14 hours 40 at 4-degree Celsius. The unbound antibody was washed away 30 by using wash solution (0.15 M NaCl, 0.05% Tween 20). The

plate was incubated at room temperature (24-30-degree 20 of wet feces wet of

Celsius) for 1 hour post-wash and later standards, samples and 10

metabolites (progestins) ug/g ug/g (progestins) metabolites

control were added to each well. Each sample was diluted 1:5 0 3-Oct 17-Oct31-Oct14-Nov28-Nov12-Dec26-Dec 9-Jan 23-Jan 6-Feb 20-Feb 6-Mar

in phosphate buffer and 50 µl diluted sample was added per steroid Days well. 50 µl progesterone-horseradish-peroxidase (HRP) was Figure -1 added per well and the plate was further incubated for 2 h at

room temperature. Subsequently, the plates were washed and

INTERNATIONAL JOURNAL FOR RESEARCH IN EMERGING SCIENCE AND TECHNOLOGY, VOLUME-3, ISSUE-1, JAN-2016 E-ISSN: 2349-7610

3.2 The calculated days of gestation for female #2 (chip # site when the female delivers the baby. The data obtained in 056098100289796) was 156 days. this study suggests that steroid metabolites excreted in the Figure 2 shows that hormonal profile generated from the data feces can be used as non-invasive method of studying and obtained by running ELISA from the fecal sample of female monitoring the reproductive stage in Arabian Tahr. #2 (chip # 056098100289796) on daily basis from 1st October 2008 to 12th March 2009. The generated profile suggests that ACKNOWLEDGEMENT the female was conceived on 12th October 2014 and delivered I would like to thank Mr. Muslim Ali and Mr. Noufal a healthy baby on 16th March 2015 with a gestation period of Nasiruddin for performing the extraction of hormones from the total 156 days. The figure also shows that the concentration of fecal samples. steroid metabolite in the fecal sample maintained a higher REFERENCES level than the average value during the gestation period. [1] Caprine Specialist Group 1996. Hemitragus jayakari. In: IUCN 2007. 2007 IUCN Red List of Threatened Species. Female -2 #056098100289796 20 www.iuchredlist.org. Conc. ug/g

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