THE IMAGE OF EMBRYO AND FETAL OF ETAWA CROSSBRED GOATS USING ULTRASONOGRAPHY AT DIFFERENT GESTATIONAL AGES

This study aimed to estimate the gestational age of Etawa crossbred goats based on ultrasound images of embryo and fetal using transcutaneous ultrasonography. Etawa crossbred does (n= 5) were synchronized using double PGF2α injection with 10 days interval and mated naturally. Early pregnancy was diagnosed on 20 day after mating base on isoechogenic image of the embryo (2.65±0.75 mm) and hypoechogenic appearance of gestational sac. On 30 day of the first trimester, the embryo size was 8.31±0.42 mm. The embryo reached 10.44±0.51 mm on 35 day. On 60 day, the existence of the fetal were clearly appeared along with isoechogenic of umbilical cord, hyperechogenic os cranium (25.5±0.1 mm), and body length (51.83±1.59 mm). On 75 day, the diameter of the os cranium was 33.03±0.20 mm, body length of 110±1.73 mm, and bones structures appeared hyperechogenic for os thoracalis and os vertebrae. The placenta was isoechogenoic with diameter of 23.3±0.2 mm. On 90 day, the placenta dominated the uterus (22.67±1.70 mm) and reached its maximum diameter on 145 day (33.73±2.91 mm). In conclusion, early pregnancy detection of Etawa crossbred goats using transcutaneous ultrasonography could be performed on 20 day after mating. ____________________________________________________________________________________________________________________


INTRODUCTION
Etawa crossbred goat is one of goat types that produce meat and milk developed in Indonesia which is considered having high economic value.From the price view, the milk of Etawa crossbred goat has higher price than cow milk (Sodiq and Abidin, 2008).The milk production of Etawa crossbred goat is 0.45-2.2/liter/goat/day.The lactation period length varies greatly (92-256 days) with average of 156 days.With good management, Etawa crossbred goat doe can produce milk over 200 days in a year.The average production of goat milk in Indonesia is lower (2-3 liter/goat/day) than other subtropical regions which can reach 5-6 liter/goat/day on highly productive Etawa crossbred goat (Sodiq and Abidin, 2008).
One of the purposes of the animal husbandry development is to increase the production of livestock to fulfil the domestic consumption need of meat and milk along with affordable price for society (Marzuki and Dwijatmiko, 2004).To meet the domestic consumption need, at least 5.6 billion goats/year is needed, with the addition from foreign demands.According to Marzuki and Dwijatmiko (2004), in order to reach the animal husbandry development, there are three approach strategies in the form of technical approach, integrated approach, and agribusiness approach.Technical approach is interpreted as an effort to increase population number of livestock through number of births.Increasing production and population of livestock could be performed using quality improvement of reproduction either for male and female goats; therefore well operated management of livestock reproduction is essentially needed.
Early detection of pregnancy is one of important aspects in reproductive management and should be performed after the animal mate.Early detection of pregnancy can offer rapid information of mating success so that production efficiency due to infertility could be achieved and in turn would reduce the expense of breeding program and aid livestock management economically.Furthermore, faster evaluation and differentiation between pregnant and non-pregnant livestock could increase the efficiency of reproduction and reduce the production cost (Gonzalez et al., 2004, Karen et al., 2004).
In foreign countries, transabdominal ultrasonography technique has been utilized along with great accuracy as diagnosis tool of pregnancy and estimation of the number of embryo in lamb (Buckrell, 1988;Garcia et al., 1993;Anwar et al., 2008), goat (Martinez et al., 1998;Gonzalez et al., 2004;El-Zarkouny et al., 2008), and deer (Revol and Wilson, 1991).Amrozi and Setiawan (2011) reported that the use of ultrasonography could also be performed to observe the embryo and fetal development in order to estimate the gestational age of Garut lamb.Ultrasound image of certain organ from fetus could be utilized to estimate gestational age.The measurement of fetal body and head diameter obtain high accuracy in estimating gestational age and determining birth (Goddard, 1995).
In Indonesia, study of goat reproduction is very limited, particularly about the diagnosis of early detection and examination of gestational age using ultrasonography even though early information of pregnancy status may give plenty of benefit to the goat management and reproduction business.According to Heibel and Perkins (1989), the estimation of pregnancy diagnosis and gestational age is important to achieve maximum reproductive efficiency.
According to the previous descriptions, it is necessary to perform the study of ultrasonography usage toward the observation of embryo and fetal development in order to estimate the gestational age of Etawa crossbred goats.

MATERIALS AND METHODS
The sample used in this study was five female Etawa crossbred goats with productive age (2-4 years) with normal estrus cycle and one male Etawa crossbred goat as teaser.The estrus cycle of Etawa crossbred goats had been synchronized using double intramuscular injection of PGF2α (1 mL/goat) with interval of 10 days.All Etawa crossbred goats were mated naturally when the estrus signs appeared.The diagnosis of early pregnancy and embryo fetal growth observation were performed using transcutaneous ultrasonography.

The Procedure of Transcutaneous Ultrasonography Usage
Ultrasonography tools (MINDRAY DP3300 VET, Shenzhen Mindray Bio-Medical Electronic Co. Ltd, China) was placed at the left side of operator which was placed at the left position of the goat.Scanning was performed using abdominal probe 3.5 Mhz (35C50EB, Shenzhen Mindray Bio-Medical Electronic Co. Ltd, China) when the doe goat was on laying position.Some part of the hairs were shaved before scanning to visualize best images, KY jelly was polished around abdomen in front of the mammae, probe was directed towards cranial of mammae until sebum which filled the inguinal gland, and then directed to dorsal and a little caudomedial.Probe was slightly pressed on the abdomen towards urinary bladder.The interpretation of form, size changes, and detected organ were performed real time.Maximal picture of head diameter and fetal body length were searched and the measurement number appeared in the ultrasonography monitor.Sonograms were kept in digital picture form.On the ultrasonography monitor, the image of embryo, fetal, uterus, and bones showed white picture (hyperechogenic/hyperechoic), placentom gave grey color (isoechogenic/hypoechoic), whereas amnion and uterus lumen showed black color (hypoechogenic/anechoic).Fetal length was measured from forehead to tail base (Ali and Hayder, 2007) or peak of the head to heart then heart to the base of tail (Abdelghafar et al., 2011).The observation of embryo and fetal growth using ultrasonography was performed in each trimester, which were in first trimester were performed in 14 th to 20 th day, subsequently in 30 th day, and 35 th day.Then in second trimester the measurements were performed in 60 th , 75 th , 90 th day, and on third trimester were performed in day 120 th and 145 th of pregnancy.

Data Analysis
The observation data of ultrasound pictures of embryo and fetal growth of Etawa crossbred goats were analyzed descriptively.

RESULTS AND DISCUSSION
The results of five Etawa crossbred does which had been synchronized their estrus cycle using prostaglandin and mated naturally showed 100% positive pregnancy.Post mating observation using ultrasonography showed that early detection can be firstly detected in day 20 th after mating, as reported in Figure 1.On the 14 th to the 19 th day the embryonic vesicle continued to develop, indicated by the appearance of hypoechogenic of embryonic vesicle fluid.According to Amrozi and Setiawan (2011), early indication of pregnancy is embryonic fluids inside the uterus.Study by Sayuti et al. (2016) showed that early pregnancy diagnosis in kacang goat could be firstly detected using transcutaneous ultrasonography on the 14 th day after mating marked by hypoechogenic fluid from embryonic vesicle.Buckrell (1988) reported that hypoechogenic visualization starts to appear in embryonic vesicle on the 14 th -19 th days of pregnancy.Pregnancy could be marked by the development of embryo vesicle and the existence of fetal in uterus (Amrozi and Tumbelaka, 2014).
An animal is expected to be pregnant when it contains gestational sac, conceptus, cotyledon, and other parts of the fetal inside the uterus (Abdelghafar et al., 2007;Amer, 2008;Anwar et al., 2008).The image was obviously different between the uterus of pregnant Etawa crossbred goat and non-pregnant Etawa crossbred goat after mating (Figure 2).The uterus of non-pregnant Etawa crossbred goat showed isoechogenic uterus lumen, no hypoechogenic appearance from embryonic vesicle and no image of esoechogenic embryo as early indication of diagnosing pregnancy.Kahn (2004) reported that the early indication of pregnancy in goat could be observed in 12 th day with the appearance of anechoic (black) embryonic fluid from embryonic vesicle and 24 th day when the embryo can be firstly detected using transrectal ultrasonography method.This study showed that the embryo of Etawa crossbred goat could be detected using transcutaneous ultrasonography at 20 th day.The embryo size was 2.65±0.75mm with isoechogenic appearance and gestational sac with hypoechogenic appearance.This result was obtained more quickly than when performed on Kacang goats using similar ultrasonography technique.The isoechogenic embryo images in Kacang goat could be detected on the 24 th day during pregnancy with embryo size of 6.77 mm (Sayuti et al., 2016), and 26 days after mating on Boer goat (Rivas et al., 2005).Martinez et al. (1998) reported that goat embryo could be detected on day 19 th after mating using 5 MHz transducer.At 20 th day after mating, early detection of pregnancy in lamb could be detected by the appearance of the embryo; moreover it will be more accurate when being detected at 25 th day (Schrick and Inskeep, 1993), or 26 th day (Buckrell et al., 1986).Ishwar (1995) reported that early detection on lamb and According to Ali and Hayder (2007), length measurement of embryo and amnion sac on Ossimi lamb could only be detected at first trimester of pregnancy, in which embryo could be firstly detected between 24 th -27 th days of pregnancy.
Embryo continued to grow and started to increase in size.On 30 th day of Etawa crossbred goat pregnancy, embryo with isoechogenic appearance had 8.31±0.42mm (Figure 1).The embryo size reached 10.44±0.51mm on 35 th day of pregnancy (Figure 3).In the first trimester, embryo underwent development.According to Christensen (2015) goat embryo period starts in approximately 12 th day when blastocyst attaches to the uterus layer and approximately 42 th day when the embryo grows very fast so that it marks the end of the embryonic period.Then, the embryonic period is replaced by the fetal period.
In the second trimester of pregnancy, on 60 th day after mating, the existence of fetus was obviously appeared from the isoechogenic umbilical cord and hyperechogenic os cranium with diameter of 25.5±0.1 mm and body length on its longest axis of 51.83±1.59mm which was surrounded by hypoechogenic appearance from the pregnancy fluid.On 75 th days, the fetal growth was more obvious with the marked growth of s cranium which was 33.03±0.20 mm and body length reached 110±1.73mm (Figure 4).
Furthermore, the bone structure was observed by the hyperechogenic appearance from os thoracalis and os vertebrae.Placentom with isoechogenic appearance had diameter of 23.3±0.2mm and was obviously observed on the 75 th days of pregnancy, and got more dominating on the 90 th days of pregnancy with size of 22.67±1.70mm.On the 145 th days of pregnancy the placentom diameter reached its maximal size which was 33.73±2.91mm, as seen on Figure 5.
According to the observation of Abdelghafar et al. (2011), fetus length size of Saanen goat which was performed on 57 th days after mating reached 8.73 cm.On the 42 th -48 th days of pregnancy, the head bone image had been observed, while on the 46 th -55 th days of pregnancy the vertebrae and costa bones were obviously appeared in Ossimi lamb (Ali and Hayder, 2007).The ossification and changing process of echoic images from turbid appearance of fetal fluid could be utilized to determine the age of pregnancy (Hafez, 2000).Anwar et al. (2008), stated that placentom and leg bud were observed 100% between 45 th and 50 th days of pregnancy.Amrozi and Setiawan (2011) reported that the placentom of Garut lamb appeared on 34 th days.Placentom showed development pattern which continuously increased in significant pattern until eventually the placentom growth became insignificant (relatively constant) after the 56 th days.According to Buckrell et al. (1986) placentom could be detected using transrectal ultrasonography (5 MHz) on 30 th and 32 th days of pregnancy (Doize et al., 1997).On the 40 th days, the placentom could be identified on lamb marked by echoic circular structure (Russel, 1985).Doize et al. (1997) reported that lamb placentom reached its maximal size at 74 th days and described as resembling a cup.
Ultrasonography observation at third trimester of pregnancy were performed at 120 th and 145 th days when at those days the fetal images were unclear due to increased size of the fetus and the placentom was dominating the lumen of the uterus (Figure 5).Beside to estimate the gestational age, ultrasonography could also be used to observe the fetal position.In this study, the normal/abnormal position of the goat could be identified at 120 th days just before the birth through hyperechogenic os radius ulna appearances which getting more pointed to the birth canal (Figure 6).

CONCLUSION
Early detection of pregnancy in Etawa crossbred goat using transcutaneous ultrasonography can be performed at 20 th days after mating.The gestational age estimation of the Etawa crossbred goat using transcutaneous ultrasonography can be determined by measuring the embryo length, body length of the fetus, diameter of the os cranium, and placentom.

Figure 1 .Figure 2 .
Figure 1.The transcutaneous ultrasonography image of the Etawa crossbred goat uterus after mating at the first trimester (H= Day after mating, V= Embryonic vesicle, E= Embryo, GS= Gestational sac)

Figure 3 .Figure 4 .
Figure 3.The transcutaneous ultrasonography image of Etawa crossbred goat pregnancy at 35 th day (H= Days of pregnancy, E= Embryo, GS= Gestational sac) This study is part of Competitive Research Grant (Penelitian Hibah Bersaing) Year 2015 Number: 035/SP2H/PL/Dit.Litabmas/2015 dated 05 February 2015.Therefore, the authors would like to express sincere appreciation to Syiah Kuala University for funding this study and to the administrator of Experimental Animal Unit (UPT Hewan Coba) Faculty of Veterinary, Syiah Kuala University and UD.Puna

Figure 5 .Figure 6 .
Figure 5.The hyperechogenic appearance of placentom dominating lumen uterus at the last trimester of pregnancy (H= Days of pregnancy, PC= Placentom)