LEVEL OF ESTRADIOL 17-β SERUM AND OVARIAN FOLLICULARE DYNAMICS IN SHORT ESTROUS CYCLE OF BALI CATTLE

C.M Airin1, P. P. Putro2, P. Astuti3 and E. Baliarti4 1Faculty of Veterinary Medicine, Gadjah Mada University, Jl Fauna No. 2 Karangmalang Yogyakarta - Indonesia 2Department of Obstetric and Gynaecology, Faculty of Veterinary Medicine, Gadjah Mada University, Jl Fauna No 2 Karangmalang Yogyakarta - Indonesia 3Department of Physiology, Faculty of Veterinary Medicine, Gadjah Mada University, Jl Fauna No 2 Karangmalang Yogyakarta - Indonesia, 4Department of Animal Production, Faculty of Animal Husbandry, Gadjah Mada University,Jl Fauna No 2 Karangmalang Yogyakarta - Indonesia Corresponding E-mail : [email protected]

Received December 27, 2013; Accepted February 10, 2014

ABSTRAK

Penelitian ini bertujuan untuk mengetahui kadar estradiol 17-β dan gambaran dinamika folikel yang menyertai kejadian siklus estrus yang pendek Penelitian ini menggunakan 7 ekor sapi Bali yang ada di Kebun Pengembangan Penelitian Pertanian, dan Peternakan (KP4), betina, umur 2 tahun, sehat dan bersiklus estrus normal. Pengukuran diameter folikel menggunakan ultrasonografi (USG) dan darah diambil dari vena jugularus dimulai hari pertama setiap hari dalam waktu yang bersamaan selama 3 siklus. Kadar estradiol 17-β dianalisis menggunakan metode Enzyme Immuno Assay (EIA) Hasil penelitian menunjukkan 4 ekor sapi Bali mempunyai siklus estrus pendek (n=7) diantara siklus estrus normal. Sapi Bali tersebut mempunyai 1 gelombang perkembangan folikel dengan panjang siklus 7-10 hari, diameter folikel ovulasi maksimal dan kadar estradiol 17-β menyerupai siklus normal. Kadar tertinggi estradiol 17-β pada siklus tersebut 107,77 ± 55.94 pg/ml pada hari ke 7-10 saat ukuran folikel ovulasi mencapai 10.5 ± 0,38 mm. Kesimpulan penelitian ini adalah kejadian siklus estrus pendek dapat terjadi diantara siklus normal pada sapi Bali. Kata kunci : siklus estrus pendek, sapi Bali, estradiol 17-β, serum, folikel ovulasi

ABSTRACT

The aims of the research were to confirm the short estrous cycles and determine the blood level of estradiol 17-β and ovarian follicukar dynamics in these cases. The research was conducted using seven Bali cattle, approximately 2 years of age, kept in healthy condition with normal estrous cycles. Observation of estrus symptoms was performed daily. Ovarian follicles was examined and measured using ultrasonography started at the estrus day. Blood samples were collected from jugular vein, blood sample collection and ultrasonographical examination of the ovaries were performed daily in the same time. Serum level of estradiol 17-β was performed using EIA. The short cycle estrus were observed in 4 Bali cattle (n=7) among natural estrous cycle. They have only one wave ovarian follicular development whereas the maximal size of ovarian follicles ovulation likes the normal cycle The duration of short estrous cycle was 7-10 days with normal usual estrus behavior. The peak of blood serum level was 107.77 ± 55.94 pg/ml when the diameter dominant follicle of short estrous cycle was reached 10.5 ± 0.38 mm. It can be concluded that the short estrous cycles may occur in Bali cattle after puberty among normal cycles. Keywords : Short estrous, Bali Cattle, Estradiol 17-β, Serum, Ovarian Follicle

INTRODUCTION Indonesia outside Java island. They are true tropical, have a high tolerance under poor Bali cattle can be found spreading all over environment, and late maturing animals as well

Estradiol 17-β and Ovarian Folliculary on Short Estrous Cycle in Bali Cattle (C.M. Airin et al.) 23 (Toilehere et al., 2003) The length of estrous MATERIALS AND METHODS cycle is not differ from the other cattle breeds. It consits luteal and follicular phase, the average Animals and Blood Sampling length of the oestrus cycle is 21 days, with some The research was used seven Bali cattle kept evidence that it is shorter when nutrition is poorer in Agricultural Training, Research and (Geoffry et al., 2003) Development Station (KP 4), Gadjah Mada Short estrus is an estrus cycle that occurs in University-Yogyakarta. The Bali cattle had the short duration with normal estrus behavior. The same age (average 2 years) and healthy condition. duration of short estrous cycle is 7-10 days but an Blood sample collection and ovarian examination 8-day was most frequent (Odde et al., 1980). were initiated when the animals showing estrus Short estrous cycle are connected to the symptoms, i.e. standing heat (standing still while attainment of puberty or the resumption of being mounted by other cattle), transparent cyclicity postpartum (Taponen, 2002). The results vaginal discharge, changes in vulva (it becomes of some researches showed that a short estrous warm, oedematus and reddish in colour) cycle a normal phenomenon (Edq cit in Mukasa, (Toelihere, 2003). 1991). Although the ova of short cycle can be fertilized but it so difficult to predict estrus time Ultrasound Examinations normally. The short estrsu cycle signifcance Transrectal ultrasound examinations were initially lies in difficulty to predict subsequent performed by a single operator using a real-time, estrus particulary for cow not interacting with B-mode scanner with an 8.0-MHz linear-array bulls (Mukasa, 1991). The short cycle are well transducer (Honda-Japan). Ovarian maps were documented in cattle during puberty with drawn at each examination and the relative spontaneous or induced ovulation postpartum. positions and sizes of ovarian follicles greater In the short estrous cycle, the corpus luteum than 3.0 mm in diameter and CL were recorded can demise early because prostaglandin excreted daily. The dominant follicle was defined as the from uterus quickly. In day 6 after estrus, the follicle reach 5 mm of diameter (Evan, 2004; uterus more sensitive to the luteolytic effect of Umut et al., 2008). prostaglandin. The follicle plays a fundamentally important role in reproduction which development Blood Sampling and Determination of and ovulation are important to improve and Estradiol 17-β from Serum Samples control reproductive function in farm and Blood samples were collected by vacuum companion animals (Roche, 2004). Estradiol is puncture of a jugulair vessel into silicone plain produced by granulosa cells and theca sel of tubes (venoject) by 20 G needles (vacutainer) in follicle ovaria, the increase in follicular size is all bali cattle daily, serum was seperated and associated with an increase in estradiol stored at −20°C until the time of analysis. concentration. It means that the fluctuative Analysis of blood serum estradiol 17-β was only estradiol level can determine stage of performed from short estrous cycle animals. The development ovarian follicle. first step was adding 25 µl of standard and The behaviour of short estrus as same as samples into appropriate wells and 200 µl of with the normal cycle but in this period the ova enzyme conjugate into each wells. The plate was can not be fertilized. If the short estrus occured, it incubate at room temperature for 120 minutes can disrupt a recording of estrus cycle and then remove the incubation mixture by flicking difficult to predict the true time estrus with fertile plate contents into a waste container. The each ova. Information about blood level of estradiol, wells was rinsed for 3 times with distilled or ovarian follicular dynamics and length of short deionized water than dispense 100 µl substrate estrous cycles in Bali cattle is very limited. In the solution into each well. The reaction was stopped early studies, the authors had seen short estrous by add 100 µL of stop solution to each well. The cycles in normally cycling of Bali cattle (Airin, last step, plate was read used OD at 450 nm Unpublished data). within 10 minutes. The aims of the present research were to detect blood level of estradiol 17-β and ovarian Statistical Analysis follicular dynamics in short estrous cycles of Bali The data analysis was performed cattle. descriptively with individual data. The individual data would be compared between dynamics of

24 J.Indonesian Trop.Anim.Agric. 39(1):23-29, March 2014 follicles and level estradiol 17-β in short and wave occurs on day 9 or 10 in two-wave cycles. normal estrous cycle. In the three waves, emergence second waves on Day 8 or 9, and the third wave emerges on day 15 RESULTS AND DISCUSSION or 16 (Mitesh and Govin, 2007; Adam et al., 2008). Ovarian Follicular Dynamics in Short Estrous The short estrous cycle can occur after Cyle parturition but in some studies reported that the Folliculogenesis is the process in which a short estrus cycle a normal phenomenon (Edqvist recruited primordial follicle grow and develops et al., 1984). In the short estrous cycle ova can be into a specialized graffian follicle with the fertilized and corpora lutea have short life-spans, potential to either ovulate its egg into the oviduct they have only one development follicle wave. at mid-cycle to be fertilized or to die by atresia Production of progesterone to be sub-optimal, it (Gregory, 2008). In a number spesies, follicle means that rate of pregnancy tend in the short growth is characterized by follicle wave which cycle (Galina et al.,1982). Pregnancy rates in follicular wave occurring during the normal cattle with two- versus three-wave patterns were estrous cycle (Evan, 2004). The development of compared based on the notion that the dominant anovulatory follicles comprised three preovulatory follicle in the two-wave pattern phases: growing, static and regressing phase grows for a relatively longer period and may (Noseir, 2003). contain a relatively aged oocyte. In contradictory Cattle are polyestrous animals and displays results reprorted by Blech et al. (2004), the wave estrous behaviour every 21 days but in the short of development follicle cannot influence rate of estrous cycle this phase can occurred in 7-10 pregnancy. days. In the present study indicated that short The most frequent length of short cycle was estrous cycle found in 4 Bali cattle number 1,2,3 11 days which all the short cycle was either first and 4 (n=7), its mean that 60% popullation Bali or second postpartum estrus in buffalo (Chohan, cattle of KP4 have short estrus cycle. Based on 1992). Yavas and Walton (2000) reported that follicular dynamics, the length of short estrous short estrous cycles occur in approximately 80% cycle was 7-9 days with only one follicular of all cows following the first ovulation after development wave (Figure 1a, Figure 1b and calving. The time of estrus onset depend on stage Table 1). The follicular dynamics of Bali cattle of follicular wave when corpus luteum regression no. 5, 6 and 7 were normal, they did not have is induced (Taponen, 2002). The interval can be short estrous cycle. It means that they have a shortened with administration of PGF2a when a normal cycle. The majority of bovine estrus cycle dominant follicle mature is present, this are composed of two or three folliculare waves, phenomenon can prolong if the follicle wave is whereas emergence of the first folliculare wave emerging or undergoing selection process (Roche occurs on the day of ovulation (day 0), the second et al., 1996).

Table 1. The Concentration of Blood Estradiol 17-β (pg/ml) and Diameter of Follicle Ovulatory of Bali Cattle in the Short and Normal estrus Cycle.

Diameter of Estradiol 17-β Lenght of Cycle Cattle Follicle Ovulatory (pg/ml) (mm) (mm) Short cycle 1 92.74 10.55 7 2 190.62 10.60 7 3 74.36 10.90 10 4 73.37 9.98 8 Normal cycle 5 70.20 10.40 18 6 72.40 10.02 21 7 71.40 10.05 18

Estradiol 17-β and Ovarian Folliculary on Short Estrous Cycle in Bali Cattle (C.M. Airin et al.) 25 Figure 1a. Diameter of ovarian follicular dynamics Bali cattle (no 1-2) during estrus cycle. In the grafik shown that short estrous cycle can emerge among normal estrus cycle.  = the short estrus cycle

In the present research show that the short increase in follicular size was associated with an estrous cycles occurred in Bali cattle heifers. It increase in estradiol 17-β concentration, it means meant that phenomena short estrous cycle not that the level of blood estradiol can use to usually occurring following the first ovulation determine stage of follicle development. Putro et after calving. The short estrous cycle can occur in al (2014, in press), the level of hormone in the cattle during puberty (Taponen et al., 2002). folliculare fluid as same as with blood such as tiroid hormone. Follicular fluid (FF) is an Level of Estradiol 17-β during Short Cycle avascular compartment separated from the The estrous cycle is regulated by hormones perifollicular stroma by the follicular wall within of the hypothalamus, the pituitary, the ovaries and the mammalian ovary (Abd Ellah et al., 2010; the uterus (Forde et al., 2011). Estradiol 17-β Albomohsen et al., 2011; Nasroallah et al., 2012). concentration increased during folicular phase of A progresive increase of estradiol 17-β estrous cycle. Noseir (2003) reported that the concentration was observed during growth phase

26 J.Indonesian Trop.Anim.Agric. 39(1):23-29, March 2014 Figure 1b. Diameter of ovarian follicular dynamics Bali cattle (no 3-4) during estrus cycle. In the grafik shown that short estrous cycle can emerge among normal estrus cycle.  = the short estrus cycle but progresive decrease in estradiol 17-β dynamics which the size of ovarian dominant concentration during static phase with more and follicle was 10.5 ± 0.38 mm in 7-10 days before less constant size of follicles. In the regresing ovulation. phase, there was a constant decrease in follicular In the normal estrous cycle, blood estradiol size and increase in estradiol production (Noesir, 17-β from preovulatory follicle may induce 2003). Vascularization is the important in uterine progesterone receptor which are required determining the fate of follicle and necessery for to establish progesterone dominance of folliculare health (Young and Mc Nelly, 2010). In subsequent ovulation. The dominance of the present research, the progresive increase of progesterone result inadeguate uterine blood estradiol 17-β concentration was following progesterone receptor synthesis and the estradiol ovulation without static phase. In the short estrous 17-β concentration will decrease. The uterus may cycle, the peak of blood estradiol 17-β was lose progesterone dominance earlier which this 107.77±55.94 ng/ml in 7-10 days and in the would be initiated the positive feedback loop normal cycle was reached in 18-21 days (Table 1). between oxytocin and PGF2α earlier in the estrous This fact was supported by the follicular cycle (Ottobre cit in Zoller 1993). Short et al.

Estradiol 17-β and Ovarian Folliculary on Short Estrous Cycle in Bali Cattle (C.M. Airin et al.) 27 (1990) reported that the corpus luteum formed is Variations of Follicular Fluid and Blood smaller in the short estrous, secretes less Serum in Iranian Dromedary Camels progesterone and is less responsive to stimulation. During the Peak Breeding Season. J Anim In the normal luteolysis, the timing of luteolysis Vet. Adv. 10:327-331 was influenced by the concentration of severe m- Bleach, E.C.L., R.G. Glencross, and P.G. Knight. RNA such 3β-HSD and sTAR (Gordon et al., 2004. Association between ovarian follicle 2000). Branden et al. (1988) reported that during development and pregnancy rates in dairy luteal regression, initial decreased in cows undergoing spontaneous oestrous concentration of progesterone do not appear to be cycles. Reproduction 127:621–629. due to loss of steroidogenic luteal cells. Braden, T.D., F. Gamboni and G.D. Niswender. Vascularition can influence development of 1998. Effects of prostaglandin F2α-induced follicle and life span of corpus luteum ovarium. luteolysis on the populations of cells in the Hamish (2006) reported that the grow of follicle ovine corpus luteum. Biol. Reprod. 39:245- ovarium assosiate with development individual 253 cappilary network, while it can receive nutrients Chohan, K. R., R.A Chaundhry, M.A, Awan and and oxygen by passive diffusion from stroma N.A. Naz. 1992. Incidence of short estrous blood vessel. The decreased secretion of in nili ravi buffaloes. Asian-Australasian progesterone caused decreased luteal blood flow Journal of Animal Sciences 5:583-585 by PGF2α and, thus may reduce deliver of Forde, N., M.E, Beltman, P. Lonergan, M. nutrients and substrate of steriodogenesis (Gordon Diskin., J.F. Roche and M.A. Crowe. 2011. et al., 2000). oestrous cycles in bos taurus cattle. Anim Reprod Sci. 124:163-169 CONCLUSION Gordon, D.N., J.L Juengel, J.S. Patrick, M.K Rollyson and E.W McIntush. 2000. It can be concluded that the short estrous Mechanisms controlling the function and cycles may occur in Bali cattle after puberty life span of corpus luteum. Physiol. Rev. 80 among normal cycles with length of cycle was 7- :1-29 10 days Geoffry T., T. Panjaitan, Muzani and D. Poppi. 2003. Management to Facilitate Genetic ACKNOWLEDGEMENTS Improvement of Bali Cattle in Eastern Indonesia. Proceeding Australian Centre for The authors would like to thank very much International Agricultural Research, Bali, to the chairman of the Agricultural Training, Indonesia, February, 4-7, 2002. No. 110, Research and Development Station (KP4) UGM page 23-29 for giving permission to use animals and barn Hamish, M.F. 2006. Regulation of the ovarian facilities, Sunaryanto (vet), Didik Yulianto (vet), follicular vasculatare. Reproductive Oki and Atma for technical assistance. The Biology and Endocrinology 4:18 present research is granted and funded by Hibah Mites, G. and N. Govin. 2007. Follicular Sains Veteriner from Faculty of Veterinary dynamics in Rathi (Bos indicus) cattle. Medicine, Gadjah Mada University. Veterinarski Arhiv. 77:177-186 Mukasa- Murgewa, A. Tegegne and Y. Tenklu. REFERENCES 1991, Characterization of service intervals and frequency of short oestrus cycles in Abd Ellah M.R., H.A Hussein and R. Derar. 2010. Zebu (Bos indicus) cows in Ethiophia. Ovarian follicular fluid constituents in Reprod. Nutr. Dev. 31:361-367 relation to stage of estrus cycle and size of Noseir, W. MB. 2003. Ovarian follicular activity the follicle in buffalo. Vet. World. 3:263- and hormonal profile during estrous cycle 267 in cows: the development of 2 versus 3 Adams, G.P., R. Jaiswal, J. Singh and P. Malhi. waves. Reproductive Biology and 2008. Progress in understanding ovarian Endocrinology. 1(50): 6 pages (Open Acces follicular dynamics in cattle. Journal, http:www.RBEj.com/content/1/1/ Theriogenology 69:72–80 50, page number not for citation purpose). Albomohsen, H., M. Mamouei, S. Tabatabaei and Odde, K.G., H.S Ward, G.H Kiracofe, R.M J. Fayazi. 2011. Metabolite Composition McKee and R.J Kittok.1980. Short estrous

28 J.Indonesian Trop.Anim.Agric. 39(1):23-29, March 2014 cycle and assosiated reum progesterone cloprostenol and gonadotropin-releasiong levels in beef cows. Theriogenology. 14 : hormone. Theryogenology. 58 : 1291-1302 105-12 Toelihere, M.R. 2003. Increasing the Success Rate Putro, P. P., Pudji Astuti, C. M. Airin, Aladria and and Adoption of Artificial Insemination for Supriyanto. 2014. Comparasion of thyroid Genetic Improvement of Bali Cattle. hormone level in the folliculare fluid and Proceeding Australian Centre for serum cattle. Proceeding The 16th Congress International Agricultural Research, Bali, Asian Australian Animal Production, Indonesia, February, 4-7, 2002. No.110, Yogyakarta, Indonesia, November 14-16, page 48-52 2014 (in press) Cirit, Ü., S. Bacinoglu, M Taş, K. Demir, A. Baş, . Roche J.F. 2004. Follicular waves in cattle. K. Ak and I.K. Ileri. 2003. Evaluation of Veterinary Research Communication short estrus synchronization methods in 28:107-110 dairy cows. Anim Reprod Sci. 109:65-76 Short R.E., R.A. Bellows, R.B. Staigmiller, J.G. Young J.M, and A.S. McNeilly. 2010. Theca: the Berandelli and E.E. Custer. 1990. forgotten cell of the ovarian follicle. Physiological mechanisms controlling an Reproduction 140:489–504 estrus and infertility in postpartum beff Zoller W.G, H.A Garverick, M.F. Smith, R.J. cattle. J. Anim Sci. 68:799-816 Moffatt, B.E. Salfen and R.S. Youngquist, Taponen J., M. Kulcsar, T. Katila, L. Katai, G. 1993. Concentrations of progesterone and Huszenicza and H. Rodriquez-Martinez. oxytocin receptors in endometrium of 2002. Short estrous and estrous signs after postpartum cow expected to have a short or premature ovulations induced with normal oestrus cycle. J. Reprod. Fertil. 97:329-337

Estradiol 17-β and Ovarian Folliculary on Short Estrous Cycle in Bali Cattle (C.M. Airin et al.) 29