Choong, Siew Shean

Haron, Abd. Wahid

Mohamad, Aidi

Yap, Keng Chee

Yusoff, Rosnina

Zainuddin, Zainal Zahari

Universiti Putra Malaysia Institutional Repository

UPM Research Report 1997-2000, Section 2-Extended AbstractsCorrelation between Vaginal Cytology and Serum Progesterone in Sumatran Rhinoceros(Oicerorhinus sumatrensis)Y. Ro5nina'S. 5. Choong', Z. Zalnal-Zahari", H. Wahid', M. Aidi3, KC.YAP'Facultyof VeterinaryMedicineUniversiti Putra Malaysia43400 UPM, Serdang, SelangorMalaysiaE-mail olCorrespondingAuthor:rosnina@vet.upm.edu.myKey words: Sumatran rhinoceros, vaginal cytology, progesterone, oestrous cycle, oestrus.IntroductionThe Sumatran rhinoceros (Dicerorhi-nus sumatrensisi, with a population ofless than 400 in the world, is the mostcritically endangered of all the fiveexisting rhino species. Its distributionthat used to range from northern Indiato Borneo is now fragmentarily foundin Sumatra, Peninsula Malaysia, andSabah. In other parts of South-East-Asia, such as Sarawak., Thailand,Myanmar, and Laos, their existence hasyet to be confirmed and the viability ofany population is unlikely. Poachingfor horns, habitat degradation for tim-ber and land conversion into agricul-ture, industries and other human usesare major threats to these rhinos(WWF, 1996). Worldwide, captivebreeding for over a hundred years hasyet to produce a progeny. Aggressivecourtship behaviour may be the majorfactor for difficult breeding in Suma-tran rhinos. Such occasion usuallyoccurs due to untimely introduction ofthe male to a female that is not in oes-trus (Zainal-Zahari et al., 1995). Inorder to determine oestrus, variousmethods including plasma hormonalassay (Zainal-Zahari et al., 1995), ex-cretory metabolites assay (Heistermannet al., 1998; Wasser et al., 1996;Schwarzenberger et al., 1993; Hindle etal., 1992) and ultrasonography (Schaf-fer, et al., 1994) have been deployed.These methods may although proved tobe effective, are usually costly, tediousand require professional handling.Thus, vaginal cytology, a fast, inexpen-sive and an easy method that had beenproven effective in dogs (Wright et al.,1990; England, 1992), sheep (Zourguiet al., 1976), Yucatan pigs (Rodgers etal., 1993) silver foxes, Vulpes vulpes(Boue et al., 2(00) was employed onSumatran rhinoceros to test for its ap-plicability in detecting oestrus in thisspecies.204Materials and MethodsThe research was carried out for sixmonths at the Sumatran RhinocerosConservation Centre, Sungai Dusun,Malaysia. Four adult female rhinoceros(R i- R2, R3'~) were the subjects ofour study. Collection of exfoliativevaginal cells was carried out twice aweek in accordance with blood sam-pling for hormonal assay for a periodof six months. The animal was coaxedinto a stanchion and then restrained.Once the tail is directed to one side. thevulva is thoroughly cleansed with mildchlorhexidine and water, and subse-quently dried using a sponge. A sizel8-rubber Foley catheter, measuring 35cm with a metal stilet inside, was in-serted gently into the vagina as far asthe fornix. The stilet was immediatelyremoved and a syringe filled withphysiological saline was attached to thecatheter. The anterior end of the vaginawas flushed with 20-40 ml of thephysiological saline and re-infused 2-3times before being aspirated and trans-ferred into a calibrated capped tube.Then, the aspirated fluid was centri-fuged for 10 minutes atl,600 r.p.m. tosediment the epithelial cells.The supernatant was discarded, leavingonly 0.1-0.2 ml of the mixture. A dropof the mixture was placed on a slide tomake a smear. Two smears were madefrom each sample and air-dried. Priorto staining, the air-dried smear wasrehydrated with distilled water for 5minutes and then fixed with 70% etha-nol for 3minutes. The first 200 cellswere counted, identified and classifiedinto parabasal, small intermediate,large intermediate, superficial and cor-nified based on Schutte's classification(Christie et al., 1972). EosinophilicIndex (El) of each smear was obtainedusing the formula by (Mestre et al.,(1990). Blood samples were collectedfrom these females at an interval ofthree to four days throughout the pe-riod of this study. The site of bloodcollection was the medial coccygealblood vessels. Blood samples werecentrifuged within one hour after col-lection for IS minutes at 2.500 r.p.m.Plasma was aspirated and stored in alabeled cryovial at -20°C pendinganalysis. Plasma progesterone concen-tration was measured using a commer-cial kit, Coat-A-Count® Progesterone(Diagnostic Products Corporation, LosAngeles, California 90045-5597,U.S.A.) by radio immunoassay.Results and DiscussionA total of 330 smears from the foursubjects were examined and five typesof epithelial cells were described(parabasal, small intermediate, largeintermediate. superficial. and corni-fied), based on Schutte's cell classifi-cation (Christie et al., 1972). Parabasal,small intermediate and large intennedi-ate cells were classified as non-keratinised cells. The superficial andcornified cells were grouped as kerati-nised cells as they contain keratin pre-cursors (characteristic of oestrus), andthus stained red with Modified Shorr'strichrome.Results of vaginal cytology indicatedthat the oestrous cycle of Sumatranrhinoceros was 22±6.36 days, com-pared to 28±7.64 days as indicated byserum progesterone radio immunoas-say. From eosinophilic curves andserum progesterone profiles shown inFigures 1,2.3 and 4. oestrous cycle ofeach subject was irregular, with theexception of R2• Statistical analysisusing SPSS programme showed thatcorrelation between vaginal cytologyand serum progesterone was weak(0.235). However. when statisticalanalysis was done on individual ani-mal. correlation coefficient (r) betweenthe two reached as high as 0.745 in R..0.408 in R2, 0.183 and 0.172 in R3 andR, respectively. The eosinophilicBiological Sciencesindex (El) averaged at 57.42±12.96. Ofthe twelve peaks recorded in the fouranimals, two animals showed three firstEl peaks followed by second peaks,13.33±2.08 days after the first highestpeak. For these subjects, the percentageof non-keratinised cells ranged be-tween 49.1 and 65.0 per cent.Although the period of study was forsix months, due to breeding pro-gramme and stress factors, samplingfrom subjects ranged from 121 to 204days. Cell types, shape, size and stain-ing properties in vaginal cytology ofthe Sumatran rhinoceros is similar tothat reported in bitches (Concannon etal., 1986) and cows (Miroud et al. andNoakes, 1990).Occurrence of El peaks were notedwhen progesterone profile was at itslowest range, which could possibly bethe oestrus stage. However, this profilewas inconsistent, El peaks had oc-curred when progesterone profile wasat a relatively high level, 0.79 ng/m!and 0.97 ng/ml respectively. This mayaccount for the weak correlation coef-ficient (0.235) between vaginal cytol-ogy and progesterone profile. Also,irregularity of oestrous cycles, varia-tions of El and progesterone concen-trations between individuals and withindifferent oestrous cycles of each animalled to the weak correlation.A higher frequency of sampling maygive us a better picture of the repro-ductive status of these animals. How-ever, due to fear of imposing greatstress on the animals, samplings werelimited to only twice per week. Fur-thermore, presence of large cysts andtumours in the uteri of most of thefemales in this study (Schaffer et al.;1994) may also influence reproductiveabilities of these animals.ConclusionsResults showed that vaginal cytologywas not a reliable method to detectoestrus in Sumatran rhinoceros. Accu-racy of oestrus detection may increasewith frequency of sampling thoughstress may also hamper their reproduc-tive abilities. Furthermore, the repro-ductive status of these animals was notestablished. A number of matings hadnot resulted in pregnancy, not to men-tion production of viable offspring.Thus, further study on the reproductivestatus of these animals should be pur-sued.Benefits from the studyThe benefits obtained form this study isthe knowledge that exfoliative vaginalcytology is not a fast and reliable tech-nique to determine the oestrous cycleof this Malaysian endangered species.Since the Sumatran rhinoceros is con-sidered near extinct, it is imperativethat we pursue a research on su-perovulation and embryo transfer ofembryos, and on the collection andcryopreservation of Sumatran rhinoc-eros semen.Literature cited in the textBoue, P., Delhornmr, A. and Chaffaux, S.2000. Reproductive management of silverfoxes (Vulpes vulpes) in captivity.Theriogenology.53-9:1717-1728.Christie, D.W., Bailey, J.B. and Bell, E.T.1972. Classification of cell types invaginal smears during the canine oestrouscycle. Br. Vet. J. 128:301-310.Concannon, P.W. 1986. Clinical andendocrine correlates of canine ovariancycles and pregnancy. Current VeterinaryTherapy IX. Small Animal Practice. W.B.Saunders Co. (Philadelphia): 1214-1225.England, G:C. 1992. Vaginal cytology andcervicovaginal mucus arborisation in thebreeding management of bitches. J. Sm.Ani. Pract. 557-582.Heistermann, M., Agil, M., Buthe, A.,Hedgers, J.K. 1998. Metabolism andexcretion of oestradiol-17b andprogesterone in the Sumatran rhinocerosiDicerorhinus sumatrensisy. Anim.Reprod. Sci. 53(1-4):157-172.Hindle. J.E .•Hodges, J.K. 1990.Metabolism of oestradiol-De andprogesterone in the white rhinoceros(Ceratotherium simum simum). J. Reprod:Fertil.571-580.Mestre. J.. Wanke. M.• Sucheyre, S. 1990.Exfoliative vaginal cytology and plasmaconcentrations of progesterone,luteinising hormone and opestradiol-Iz-during oestrus in the bitch. J. Sm. Ani.fracto 31: 568-57().Miroud, K., Noakes, D.E. 1990. Exfoliativevaginal cytology during the oestrous cycleof the cow, after ovariectomy, and afterexogenous progesterone and oestradiol-17~. Br. Vet. J. 146: 387-397.Rodgers. ].B .•Sherwood, L.C., Fink. B.P .•Sadove, RC. 1993. Estrus detection byusing vaginal cytologic examination inminiature swine. Lab. Anim: Sci. 597-602.Schaffer. N.E .• Zainal-Zahari, Z., Suri,M.S.M., Jainudeen, M.R.. Jeyendran, RS.1994. Ultrasonography of thereproductive anatomy in the Sumatranrhinoceros (Dicerorhinus sumatrensis). J.Zoo Wildl. Med. 25(3): 337-348.Schwarzenberger, F., Francke. R .•Goltenboth, R 1993. Concentration offaecal immunoreactive progestagenmetabolites during the oestrous cycle andpregnancy in the black rhinoceros(Diceros bicamis michaeli). J. Reprod.Fertil. 98(1): 28S-29l.Wasser. S.K .• S. Papageorge. Foley. C.•Brown, S and J.L. 1996. Excretory fate ofestradiol and progesterone in the Africanelephant (Loxodonta africana) andpatterns of fecal steroid concentrationsthroughout the oestrous cycle. Gen.Compo Endocrinel. 102: 255-262.Wright, PJ. 1990. Application of vaginalcytology and plasma progesteronedeterminations to the management ofreproduction in the bitch. J. Sm. Ani.Pract, 31: 335-340.Zourgui, A .•Neophytou, G .• Elize, K. 1976.Results of vaginal cytology studies andexamination of the arborisationphenomenon of the vaginal mucus for thedetermination of estrus activity in sheep.Arch. Exp. Veterinanned. 30(6): 889-896.Project Publications inRefereed JournalsNone.Project Publications inConference ProceedingsNone.Graduate Research. Choong Siew Shean. 2002. Wild lifereproduction. [M.V.Se.}. Universiti PutraMalaysia.205

Correlation between vaginal cytology and serum progesterone in Sumatran rhinoceros (Dicerorhinus sumatrensis)

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Correlation between vaginal cytology and serum progesterone in Sumatran rhinoceros (Dicerorhinus sumatrensis)

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