STEROID LEVEL AND PREGNANCY RATE OF ACEH COWS IN RESPONSE TO OVULATION INDUCTION USING PRESYNCH-OVSYNCH METHOD

This study aimed to observe the steroid level and pregnancy rate in Aceh cows in response to ovulation induction using presynch-ovsynch method. Ten of two months post-partum non-pregnant and clinically healthy Aceh cows were divided into two groups of five cows each. First group (K1) was treated with presynch-ovsynch method. Second group (K2) was estrus synchronized using 5 mL of PGF2α, intramuscularly, administered twice with a 12-day interval. Both K1 and K2 were inseminated with frozen fertile semen 48-hours after the last treatment. The estrous observation was done after the last injection. Blood samples were collected immediately after the insemination for estradiol level quantification and on day-7 post-insemination for progesterone level measurement. The steroid level measurement was carried out using enzymelinked immunosorbent assay (ELISA) technique. Pregnancy test was established on day-90 post insemination by rectal palpation technique. All of the cows showed symptom of estrous after the treatment. Estradiol and progesterone level of K1 vs K2 each were 294.98±110.48 vs 392.76±11.6 pg/mL (P>0.05) and 23.85±15.14 vs 12.69±5.64 ng/mL (P>0.05). Pregnancy rate of K1 vs K2 each were 60.0% vs 0.0%. Thus, presynch-ovsynch improved pregnancy rate but conversely did not elevate the estradiol level in Aceh cows. ____________________________________________________________________________________________________________________


INTRODUCTION
Aceh cattle are one of the potential local beef cattle in Indonesia.The growth rate of local beef cattle is not as high as other cross-breed cattle, however, they show productivity and give maximum economic efficiency in limited conditions (Romjali et al., 2007).Nevertheless, their population tends to decrease (FAO, 1996in Arum et al., 2013).Low reproductive efficiency including anestrus and difficulty in estrus detection contribute to the low productivity in the development of cattle farm in Indonesia (Herdis et al., 1999).Artificial insemination (AI) was disturbed due to the nonsimultaneous estrus happening and bad intensity of the estrus.Estrus synchronization was developed to overcome the difficulty of estrus detection.
The new synchronization program which does not require estrus observation is presynch-ovsynch (presynchronization-ovulation synchronization).Presynch protocol includes twice injections of prostaglandin F2 alpha (PGF2α) before the ovsynch induction (Navanukraw et al., 2004).Ovulation synchronization method used the combination of PGF2α and gonadotropin-releasing hormone (GnRH).GnRH injection applied in day-0 to induce follicular ovulation and to start a new follicular cycle, in day-7 PGF2α was injected to regress the corpus luteum.In day-9, the cows were re-injected with GnRH to induce ovulation of dominant follicle which had been recruited after the first GnRH injection.Artificial insemination was performed 16-24 hours later without identifying the estrus symptoms (Pursley et al., 1997;Efendi, 2015).Presynch was estrus pre-synchronization protocol carried out in the beginning of estrus cycle to optimize GnRH response in order to enhance pregnancy rate compared to ovsynch method (Moreira et al., 2000;El-Zarkouny et al., 2004).
GnRH administration stimulates anterior pituitary to secrete follicle stimulating hormone (FSH) and luteinizing hormone (LH).Both of the hormones play important role in folliculogenesis and ovulation.The follicles further produce estrogen which manifest as the estrus symptoms (Hafizuddin et al., 2011).Estrogen increases the sensitivity of female genital organs which is marked by changes in the vulva and mucous production (Lammoglia et al., 1998;Andriyanto et al., 2015).Lyimo et al. (1999) stated that estrus intensity correlated with estradiol level in blood with the correlation value of 0.7.Good estrus intensity indicates a success pregnancy.Therefore, the elevation of estradiol level due to presynch-ovsynch treatment was expected to be a predictor of AI success and increment of pregnancy rate.Otherwise, high LH concentration produces ovulation or luteinization in dominant follicle resulting in progesterone elevation in line with growth and formation of accessories corpus luteum (Pursley et al., 1997).Progesterone prevents early embryonic death, thus increasing the pregnancy rates (Willard et al., 2003).
Several synchronization protocols had been reported to be used in Aceh cows (Efendi et al., 2015;Siregar et al., 2015).Efendi et al. (2015) reported ovsynch protocol whereas Siregar et al. (2015) reported estrus synchronization protocol using progesterone (CIDR-B) and PGF2α.There has been yet any report on the implementation of presynch-ovsynch protocol in Aceh cows prior to this study.

Method
Ten non-pregnant cows aged 1.5-3.0 years old, with a minimum of two months post-partum, and clinically healthy Aceh cows were divided into two groups of five cows each.First group (K1) was treated with presynch-ovsynch method.Presynch method started in day-0 with first injection of 5 mL PGF2α (Lutalyse TM , Pharmacia & Upjohn Company, Pfizer Inc.) intramuscularly, after 14 days was followed by second injection of PGF2α, and was further followed by ovsynch protocol 11 days later.Ovsynch protocol began with the injection of 1 ml GnRH followed by the injection of 5 ml PGF2α 7 days later, and followed by the injection of 1 mL GnRH (Fertagyl TM Merck, Animal Healthy) 48 hours later.After 12-24 hours, cows were inseminated with frozen semen and repeated 6 hours later.The motility of frozen semen used was more than 40%.The second group (K2) was estrus synchronized using 5 mL of PGF2α, injected intramuscularly, administered twice with a 12-day interval followed by artificial insemination 48 hours later.Pregnancy diagnostic was established on day-90 post insemination by rectal palpation technique.

Blood Collection and Preparation
Blood collection for estradiol examination was done immediately after the artificial insemination while blood collection for progesterone measurement was carried out in day-7 post-insemination.Blood serum was collected from cow's jugular vein using 5 ml disposable syringe and then inserted into a reaction tube and further stored in an ice box.Blood samples were then carried to the laboratory for serum collection.Tube was tilted 45 degrees and kept in 4° C and was subsequently centrifuged (1200 g) for 10 minutes.Serum was collected using micropipette and stored inside a micro tube in freezer with temperature -20 C.

Estradiol and Progesterone Concentration Measurements
Steroid hormone measurement was carried out using the enzyme linked immunosorbent assay (ELISA) method using DRG progesterone/estradiol ELISA kit (DRG Instrumenst GmbH, Germany).Each ELISA well (well micro titer plate) was filled with 25 μL of standard solution, sample, and control solution, then incubated for 5 minutes at room temperature.Subsequently, each well was added and mixed with 200 μl conjugate estradiol/progesterone-HRP reagent and then incubated for 6 minutes at room temperature and then shaken rapidly to remove the contents of the well.The next procedure was flushing the plates with ELISA washer three times by adding 400 μL washing solution in each well.After that, each well was added with 200 μl substrate solution and incubated for 15 minutes in room temperature.The enzymatic reaction was stopped by adding 100 μL stop solution into each well.Absorbance values were read by ELISA reader in 10 minutes with absorbance of 450 nm.

Data Analysis
Steroid levels were analyzed using t-test, whereas estrus and pregnancy percentage were reported descriptively.

RESULTS AND DISCUSSION
The research results are presented in Table 1.There was no difference of estrus percentage between K1 and K2 at 100.0%.Estrus symptoms were in accordance with symptoms reported by Sonmez et al. (2005).Estradiol level at the moment of artificial insemination in K1 and K2 were 294.98±110.48 and 392.76±11.6 ng/mL respectively (P>0.05), whereas progesterone level in K1 and K2 on day-7 post-insemination were 23.85±15.14and 12.69±5.64ng/mL respectively (P>0.05).The pregnancy percentage of K1 and K2 were 60.0% and 0.0% respectively.The estrus response of Aceh cows after being induced by synchronization method was relatively effective.Siregar et al. (2015) also reported the high estrus response in cows induced  20.24 pg/mL in Punganur cows).When compared with the concentration of estradiol in the same breed, this result was relatively higher than that reported by Siregar et al. (2016) and Thasmi et al. (2017).Siregar et al. (2016) reported estradiol concentration of Aceh cows in estrus period was 223.13±9.50pg/ml whereas Thasmi et al. (2017) reported estradiol concentration between fertile cows and cows with repeated breeding at the estrus period were 110.4±20.1 and 101.6±17.4pg/mL respectively.Indonesia local cows tend to have higher estradiol level than other breed.Airin et al. (2014) informed that estradiol concentration of Bali cows at the moment of estrus period was as high as 107.77±55.94pg/mL.
No significance difference (P>0.05) of estradiol concentration between K1 and K2, which was in accordance with Pulley et al. (2013) which reported that there was no difference of estradiol concentration between control and cows induced by equine chorionic gonadotropin (eCG) hormone.This hormone functionally has similar activity with GnRH.Akbari et al. (2011) also reported that the difference of estradiol concentration between Holstein cows induced either by presynchovsynch or presynch-heatsynch was not significant.Ribeiro et al. (2012) revealed that presynch protocol did not influence estradiol concentration at the time of artificial insemination with concentration in double ovsynch and presynch protocol were 6.4 and 5.8 pg/ml, respectively.
From the data above, even though there were no data comparing estradiol concentration between cows induced by PGF2α and presynch, it can be expected that GnRH treatment was not able to elevate estradiol concentration.Follicular phase occurred in a very short time that time difference of blood collection allows different measurements results.Blood collection performed during artificial insemination allowed errors in determining the estradiol peak level.
Progesterone concentration of Aceh cows in this study was higher compared to the progesterone concentration reported by Siregar et al. (2016) and Arimbawa et al. (2012).
Progesterone concentration of Aceh cows throughout one cycle that was revealed by Siregar et al. (2016) in day-13 until day-16 of estrus cycle was 0.10±1.67ng/mL.Progesterone levels of Bali cows in day-7 and day-8 of estrus cycle were 3.95±0.04and 5.48±0.06ng/mL, respectively (Arimbawa et al., 2012).The mean progesterone concentration of coastal cows in the luteal phase undergoing ovulation induction with ovsynch method was 6.684 ng/mL (Zumarni, 2012).Additionally, progesterone concentration in this study was also higher compared to the report by Amiruddin et al. (2013) in similar breed cows.The mean progesterone concentration of Aceh cows in day-7 of estrus cycle undergoing superovulation induction by FSH was 17.40±5.84ng/mL.The elevation of progesterone level in this research was possibly due to the administration of GnRH which caused accessories corpus luteum formation which resulted in the drastic increase of progesterone concentration than usual (Pursley et al., 1997).Stevenson et al. (2004) conveyed that GnRH administration induced estrus, LH surge, ovulation, and normal corpus luteum formation in dairy cows.
Even though there was no statistically significant difference of progesterone concentration between K1 and K2 (P>0.05), the progesterone concentration of K1 was still higher than K2.This was caused by the double administration of PGF2α with 14-days interval in K1 which in turn able to give optimal response to GnRH from ovsynch induction (Moreira et al., 2000;El-Zarkouny et al., 2004).Vasconcelos et al. (1999) revealed that the success of ovsynch depends on the protocol usage at the beginning of the cycle.The use of protocol between day-5 to day-12 of the cycle (initial luteal phase) increased fertilization, therefore increasing pregnancy rates.Progesterone concentration in both groups were above average >5 ng/mL which means they were above the basal level (Appavu and Holtz, 1992).The concentration indicated that corpus luteum was in functional phase.It was because from day-4 the progesterone concentration increased gradually and reached its peak in the middle of the estrus cycle (Akusu et al., 2006).
Pregnancy percentage of Aceh cows in K1 was increased significantly than K2 (60.0%vs 0.0%).Pregnancy percentage of Aceh cows with different synchronization method had been reported by Siregar et al. (2015) and Efendi et al. (2015).Siregar et al. (2015) reported that pregnancy percentage of Aceh cows undergoing synchronization protocol using PGF2α and progesterone were 80.0% and 80.0% respectively, whereas Efendi et al. (2015) revealed that pregnancy percentage of Aceh cows undergoing synchronization protocol using ovsynch and PGF2α were 80.0% and 60.0%, respectively.The differences of study results were possibly due to the different treatment time.This study was done in May until June when temperature and humidity in Aceh Province reached its highest peak that causing environmental heat stress and affecting the number of conception.The increased pregnancy percentage in K1 compared to K2 (60.0%vs 0.0%) was probably because the GnRH administration prevented early embryonic death and increased the pregnancy rate.Increased pregnancy rate correlated with GnRH ability to elevate progesterone concentration through accessories corpus luteum formation (Pursley et al., 1997).Yildiz (2010) reported that GnRH administration in day-9 postinsemination could increase pregnancy number compared to control group (77.7% and 50.0%respectively).Moreira et al. (2000) revealed that the pregnancy rate of cows induced with presynch-ovsynch method was 12% higher that cows undergoing only ovsynch induction.

CONCLUSION
Presynch-ovsynch method improved pregnancy percentage but did not elevate the steroid level in Aceh cows.

Table 1 .
Estrous response, steroid level, and pregnancy percentage of Aceh cows undergoing either presynch-ovsynch or PGF2α