Early Pregnancy Diagnosis and Fetometry by Real Time Ultrasonography in Teddy Goat

Faiz Muhammad Khand1, Allah Bux Kachiwal2, Zubair Ahmed Laghari2, Shakeel Ahmed Lakho1, Pershotam Khattri2, Saeed Ahmed Soomro2, Nazar Ali Korejo2 and Ambreen Leghari1* 1Shaheed Benazir Bhutto University of Veterinary and Animal Sciences Sakrand, Sindh, Pakistan 2Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University Tandojam, Sindh, Pakistan Article Information Received 15 March 2019 Revised 26 July 2019 Accepted 28 January 2020 Available online 17 July 2020


INTRODUCTION
G oats are the small ruminants playing leading role in developing the national economy and meeting the basic requirements of people. Economic losses in milk and kid production can be minimized or avoided through early diagnosis of pregnancy in goats as it aids for culling or rebreeding for food allotment, and for clinical and research purposes (Buckrell, 1988). Early pregnancy diagnosis has been increasing awareness to commercial farmers to increase their worth and minimize their extra uses of feed supplements. Accurate pregnancy diagnosis may provide essential information for effective herd management practices (Doize et al. 1997). A variety of approaches has been explored for the early detection of pregnancy in mammalian species. However recently many techniques have been used to detect the early pregnancy like radiography, estrone sulphate (Refsal et al., 1991), progesterone assay (Murray and Newstead, 1988), vaginal biopsy (Richardson, 1972), palpation of uterus via laparotomy (Ishwar, 1995), pregnancy specific antigen (Humblot et al., 1990). These all are used in late pregnancy, time consuming and are very difficult to perform for early diagnosis of pregnancy. Real time B

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Corresponding author: drambreen.leghari@gmail.com 0030-9923/2021/0001-0001 $ 9.00/0 Copyright 2021 Zoological Society of Pakistan mode ultrasonography is a non-invasive, accurate and rapid alternate for diagnosing of pregnancy and studying the development of the conceptus in livestock. B-mode real time ultrasound has been used for early pregnancy diagnosis and counting fetal numbers in goats (Dawson, 1999). Determination of fetal sexing and number of fetuses through ultrasonography can improve the reproductive management of farms. Especially the identification of gender of goat is much diffused as compared to cattle (Curran, 1992;Stroud, 1996). Measuring the embryo or fetal parts provides information on the growth status of the embryo or the fetus. Some parameters of fetus and uterus of female are used to measure the fetal age in different goats, CRL in saanen goat (Abdelghafar et al., 2010;Amer, 2008) and TD was measured by Lee et al. (2005) and (Singh et al., 2004) but these all are species specific. Information of these parameters of teddy goat is lacking. Teddy goat is the one of the famous goat breeds of Pakistan, his reproductive performance is very fast growing, low input and better efficiency (Kuthu et al., 2013).
To the best of our knowledge, this the first study to estimate the gestational age in teddy goats by using B-mode real time ultrasound. The present study aimed to determine the earliest day of pregnancy and to estimate the gestational age of teddy goats by B-mode ultrasound measurement of embryonic or fetal parts (CRL, TD and UD) of teddy goat. This study is also used to identify number and sex of fetuses.

Animals
The study was performed using 12 teddy goats (2.4 ± 0.347 years age; weighing 22 ± 3.47 kg) maintained at the research farm of Department of Surgery and Obstetrics, Sindh Agricultural University, Tandojam. The does were synchronized for estrus with injection of Lutalaze (Dinoprost tromethamine, Pfizer, Beljium) 1 ml/dose intramuscularly. The does were examined regularly for estrus sign. On the detection of estrus, a fertile buck was allowed for natural breeding and the day of breeding was considered as day 0 of gestation.

Ultrasound scanning
All does were subjected weekly to ultrasonographic scanning from day 17 onward after mating. Real time B mode scanner equipped with multi frequency (5-10 MHz) with transrectal probe was used (HS-2000, Honda Electronics Co., Ltd., Toyohashi, Japan). Animals were being kept off fed for 12 h prior to scanning. The does were tightly restrained by one person against railing in standing position. Using the transrectal approach, feces was removed from rectum (does usually defecate as soon as a finger or transducer enters the rectum) and a well lubricated 5 MHz with corboxymethyl-cellulose conducted gel was introduced into rectum.

Detection of conceptus
The first ultrasonographic signs of a possibly pregnant uterus were non-echogenic areas in the uterus which represent the fluid in the embryonic membranes with conceptus which was later confirmed as pregnancy.

Measurement of crown-rump length
The measurements were taken from the most upper part of the skull to the end of the sacrum, when the fetus was fully extended. As the pregnancy progressed, the fetus adopted curved position, and the measurements were taken in two phases firstly from the head to the heart area and secondly from the heart area to the sacrum.

Measurement of trunk diameter
Fetal trunk diameter was measured from first thoracic vertebrae to lumber vertebrae. The trunk measurements comprised the transversal diameter at the level of the last rib, the width of three vertebrae with their intervertebral spaces and the width of three ribs with its intercostal spaces were taken, since a single vertebra or rib was too short to be measured accurately.

Detection of fetal number
The single and twins were detected by number of fetal heads and independent movements of fetuses which was further confirmed after parturition for checking the accuracy of ultrasonographic machine.

Fetal sex determination
The sex of fetus was determined by the external genetalia (penis, prepuce scrotal bag, nipples and genital swelling) and localizing the genital tubercle (GT). The accuracy of the fetal sexing was determined by differentiation of GT from day 40 to 109 of gestation and then followed up to birth.

Statistical analysis
The ultrasonic measurements of CRL, TD, and UD were expressed as the mean ± S.E.M. The paired t-test was used to compare the data within the group. Linear regressions analysis was used to determine relationship between gestational age and ultrasonic measurements (CRL, TD, and UD). The results were analyzed using the SPSS software (SPSS Inc., version16.0, Chicago, Il, USA).

RESULTS
All the does were estrus synchronized and breed were detected positive for pregnancy which will final confirmed at delivery. The first sign of pregnancy was an elongated or somewhat circular anechoic area in uterus which was detected on day 19 of gestation, while proper embryo with echoic structure within uterine fluid was detected on day 21 and the CRL, TD and UD were measured afterwards.
The CRL, TD and UD of fetus by transrectal ultrasonography were measured from 3 rd week to 15 th week of gestation of teddy goat and results are presented in Figure 1. It was observed that the average crown rump length in pregnant goat was 21.3 ± 0.24, 8.64 ± 0.14 and 21.55 ± 0.32 mm on 3 rd week of gestation, respectively. However, this trend smoothly increased up to 14 th week of gestation.
Further analyses showed a wide variation in the weeks of gestation for CRL, TD and UD with a highest coefficient of correlation (r 2 =0.98; p<0.01, r 2 =0.98; p<0.01, r 2 =0.97;p<0.01 respectively) was observed. The relationship between gestation age and the size of CRL from 3 rd week up to 15 th week was described by the following regression equation (r 2 =0.98; p<0.01): y=8.39+0.56x. while, the relationship between gestation age and the TD from 3 rd week up to 15 th week was described by the following regression equation (r 2 =0.98; p<0.01) y=2.94+0.276x. However, the relationship between gestation age and the UD from 3 rd week upto 15 th week was described by the following regression equation (r 2 =0.97; p<0.01,): y=11.47+0.457x. Figure 1 shows a linear relationship between CRL, TD, UD and gestation age.
Furthermore, the result of analysis of variance showed that there was highly significant difference in CRL, TD, and UD, among different stages of pregnancy at 5% confidence interval. The images reveal that measurements of CRL of fetus were recorded during the gestation at weekly interval. The measurements were taken from the crown (most upper part of the skull) to the buttocks (end of the sacrum) when fetus was fully extended.
The fetal sex and numbers were determined by transrectal ultrasonography in pregnant goats. The fetal sex from first to twelve pregnant goats were recognized as 09 female and 10 males. Whereas, the fetal numbers from twelve goats were observed as single in five goats and twins in seven goats. No deference was detected in the accuracy of fetal sexing among different type of pregnancies.
The identification of fetal sex by location of the genital tubercle (GT) varied from 6 th to 7 th week of the gestation, for most of the fetuses (10/19), the GT migration was completed in the 7 th week of gestation. For some fetuses (9/19) the completion of GT migration accrued in the 6 th week of the pregnancies.

DISCUSSION
Early pregnancy diagnosis in goats is needed for culling or rebreeding of barren does with minimum necessary delay and it provides a valuable tool in selection of animals for increased fecundity (Singh et al., 2004). The present study was carried out to determine the accuracy of early pregnancy diagnoses, number of fetuses, gender of fetuses and the gestational age of fetus utilizing measurement of CRL, TD and UD through ultrasonography. The result of present investigations showed that the early pregnancy was detected at the 3 rd week (day 19) of gestation of goat through B-mode real time ultrasonography using a 5-10 MHz transducer probe in teddy goats. While in many other studies diagnosed the pregnancy between 4 and 5 weeks after mating of goat through real time ultrasonography by the use of 5.5 MHz trans abdominal transducer (Taverne, 1984;Baronet and Vaillancourt, 1989;Lavoir and Taverne, 1989;Haibel 1990;Kähn et al., 1992;Hesselink and Taverne, 1994). In present investigation the average CRL of fetus from 21.3 to 69.34 mm was measured in between 3 rd to 15 th week of gestation by trans-rectal ultrasonography. The results are in agreement with findings reported by Martinez et al. (1998) who found CRL on day 40 as 34.2±0.6 mm. While Schrick and Inskeep (1993) reported the CRL as 17.0±2.0, 25.0±2.0 and 36.0±4.0 mm on day 30, 35 and 40 day, respectively. The difference in ultrasonographic measurements of fetal CRL might be due to different goat breeds, nutritional status and management because previous studies were conducted on large Egyptive native dairy goat (Amer, 2008) and medium size goat breeds.
In the present study strong positive correlation (r 2 =0.98; p<0.01) between CRL and the gestational age was observed from 3 rd (21 days) week to 15 th week (105 days) week of gestation. Similar observations (r 2 =0.98) were also reported by Kahn et al. (1992). However, in another study, Karen et al. (2009) reported that CRL was highly significant correlated (P≤ 0.0000; r 2 0.94) with the gestational age from 25 to 70 days.
The results of present findings showed that the TD of fetus was in the range of 8.64 to 31.65 from 3 rd week (21 days) to 15 th week (105 days) of gestation of teddy goat. Further, it was observed that 14.4 mm TD at 41 days of gestation, 18.65 mm at 55 day and 20.54 mm at 61 day of gestation of teddy goats. Singh et al. (2004) reported the TD as 15.2 mm, 21.9 mm and 19.8 mm on day 50, 61 and 64 day, respectively. The difference in TD of fetus might be due to different goat breed. In the present study

O n l i n e F i r s t A r t i c l e
strong positive correlation (r 2 =0.98; p<0.01) between TD and the gestational age was observed from 3 rd (21 days) week to 15 th week (105 days) week of gestation. Similar observations (r 2 =0.98) were also reported in Merino ewes by Sergeev et al. (1990). However, in another study Gonzalez et al. (1998) also reported that TD was highly significant correlated (P≤ 0.0000; r 2 0.94) with the gestational age from 6 weeks to 20 weeks of gestation. Only the study by Lee et al. (2005) has evaluated the correlation coefficient (r 2 = 0.89) between TD and the gestational age in goats between Days 60 to 135 was lower than that obtained in the present study. The difference in intervals of examination (15 vs. 3-5 days, respectively) in the two studies might be the reason.
The results of present findings showed that the UD of fetus was in the range 21.55 ± 0.32 to 60.76 ± 1.41 from 3 rd week (21 days) to 15 th week (105 days) of gestation of teddy goat. Further, it was observed that 36.65 mm UD at 56 days of gestation, 39.94 mm at 63 day and 53.02 mm at 82 day of gestation of teddy goats. The results are in followed by Singh et al. (2004) who found that UD on day 56 as 36.65 mm. While, Singh et al. (2004) reported the UD as 39.94, 43.02 and 53.02 mm on day 56, 63 and 82 day, respectively in Jamuna pari goats which are little bit different as compared to present findings which may be due to breed difference, health status or nutrition. In the present study strong positive correlation (r 2 =0.98) between UD and the gestational age was observed from 3 rd (21 days) week to 15 th week (105 days) week of gestation.
In this study the period of fetal sexing based on final position of GT (genital tubercle) and the migration of genital tubercle stated that maximum migration was done in 6 th and 7 th week of gestation and the result of this study was found similar to findings of . The sexing of goat fetuses begins only after 7 th week of gestation due to wide range of time to completion of genital tubercle migration these observations are supported by . In this study the first embryo was detected on day 20 and all embryos were counted accurately until day 23, whereas at least one embryo was identified in day 19 of post breeding but not all embryos counted accurately until day 26 (Kaulfuss et al., 1996;Schrick and Inskeep, 1993). While another study (Medan et al., 2004) stated that at 60 days of gestation the fetal number were detected by real-time B-mode ultrasonography which was 91.7% accurate in diagnosis. Overall accuracy of ultrasonography for pregnancy diagnosis of does in the whole experiment, total of 12 does was scanned through ultrasonography for pregnancy diagnosis. All 12 does diagnosed as pregnant and kidded. The sensitivity of the whole experiment reached 100% and the specificity also reached to 100%. More than 95% accuracy of diagnosing pregnant were reported in ewe (Grace et al., 1989;Logue et al., 1987) and accuracy of 100% in diagnosing pregnant status was also reported by White et al. (1985) in ewe between 50 to 100 days of gestation by using real time ultrasonography.

CONCLUSION
Early diagnosis of pregnancy was detected on 19 day of gestation through transrectal ultrasonography after breeding and all embryos were detected on 23 day, among all the parameters recorded, the trunk was best parameter for determination of gestational age throughout gestational period and fetal sexing was 7 th week of gestation period in teddy goats.

O n l i n e F i r s t A r t i c l e
Early Pregnancy Diagnosis and Fetometry