Evaluation of the performance of three tests for the early pregnancy diagnosis in ewes and does

Background: The aim of this study was to evaluate the performance of three tests for the early pregnancy diagnosis in ewes and does. Methods: A total of 50 ewes and 50 does urine samples were collected in which 25 ewes and 25 does were pregnant, and 25 ewes and 25 does were non-pregnant from different farmers in Babugonj upazila of Barishal district and Sadar upazila of Bhola district. The pregnant and non-pregnant ewes and does were primarily selected from farmer’s record of management history. The pregnancy of ewes and does were confirmed through scanning by B-mode ultrasound machine. Three pregnancy diagnostic tests like barium chloride, copper sulfate and ballottement were evaluated considering B-mode ultrasound technique as gold standard. Results: The sensitivity of manual ballottement technique (MBT) was highest among the three tests: 96% in ewes and 92% in does. On the other hand, the specificity (96%) of the MBT was higher in ewes and that of the copper sulfate test (CST) (91.7%) was higher in does. The highest positive predictive value (PPV) of MBT (95.8%) was in ewes but the PPV of CST was highest (91.7%) in does. The negative predictive value of the MBT was highest both in ewes (92.3%) and does (95.7%). Conclusions: MBT can be considered as a screening test for the early diagnosis of pregnancy in ewes and does. MBT positive cases can be confirmed by ultrasonograpy whenever needed


Introduction
The people of Bangladesh are loving animals from very old years for domestic as well as companion animals.The productivity of farm animals is essential for profitable farming.The early pregnancy diagnosis is one of the keys for animals' management.In human, there is a lot of early pregnancy diagnostic kits, available in the market.Peoples are also habituated to use that kit as well.It is also well known that the laboratory test is needed for confirmative diagnosis.However, the scenario in case of animals is quite reverse from human.People used to practice rectal palpation in case of large animals i.e. cows, buffaloes, mares etc.In case of sheep and goat, the assumptive absence of next cyclicity is only tool for pregnancy diagnosis.Most of the time, it proofs false pregnancy diagnosis.The ewe may not show cyclicity due to nutritional deficiency, hormonal imbalance, and season etc.The rectal palpation in cows is the method for pregnancy diagnosis on 60-90 days of pregnancy (Cowie, 1948).The ultra-sonogram may use in animals at early days of pregnancy but the ultrasound machine is very unavailable and expensive.Though per-rectal palpation is the cheapest pregnancy diagnosis method, it is suggested that examining pregnant cows early in gestation by trans-rectal palpation increases the risk of iatrogenic embryonic mortality (Franco et al., 1987) Therefore a newer technology may replace the trans-rectal method of pregnancy diagnosis in the dairy industry as well as for others domestic animals.For diagnosing pregnancy, intra-rectal Doppler technique is superior to the external technique during early second trimester (Ott et al., 1981).Purohit (2010)

Study location and animals
The urine samples were collected from different farmers in Babugonj upazila of Barishal district and sadar upazila of Bhola district.The laboratory test was conducted in the Theriogenology and Animal Reproductive Biotechnology laboratory, Department of Medicine, Surgery and Obstetrics, Faculty of Animal science and Veterinary Medicine, Patuakhali Science and Technology University, Barishal Campus, Barishal.A total of 50 ewes and 50 does samples were collected in which 25 ewes and 25 does were pregnant, and 25 ewes and 25 does were non-pregnant.The pregnant and non-pregnant ewes and does were primarily selected from farmer's record of management history.The pregnancy of ewes and does were confirmed through scanning by B-mode ultrasound machine.

Pregnancy diagnosis by manual ballottement
The pregnancy was diagnosed by manual ballottement as described earlier (Cowie, 1948;Pratt and Hopkins, 1975).It is easier in thin ewes and does than does in fat animals.It was conducted as described by.Briefly by placing a hand on either side of the abdomen and squeezing or lifting upwards, the gravid uterus or fetus can sometimes be palpated through the relaxed abdominal wall.A fetus can sometimes be balloted low in the right flank during the last month of gestation.Examination becomes easy when feed and water are withheld for at least 12h before.

Collection of samples
Urine was collected from pregnant and pregnant ewes and does and brought to laboratory within icebox and stored in the refrigerator (4 to 8°C).

Copper Sulfate test
It was performed, in briefly, 0.25 ml cervical mucus was placed in CuSO4 solution.Mucus sinks indicate pregnancy positive case and mucus float pregnancy negative case.

Barium chloride test
It was conducted as described by Maslov and Smirnov (1965).Briefly, 5ml of urine will be added into 1% Barium chloride solution.Turbidity due to precipitation was indicated not pregnant animal whereas clear solution will be indicated pregnant animal.

Data analysis
The collected data was recorded and coded in Microsoft excel sheet.The sensitivity and specificity of the tests were calculated by dividing the true positive and true negative test results with the total number of pregnant and nonpregnant animals, respectively.The positive and negative predicative values of different tests were estimated by dividing the true positive and true negative test results with the total number of positive and negative results, respectively.The percent difference among sensitivities and specificities were assessed by the chi-square test using SPSS statistical Software.Differences were considered significant at a level of P < 0.05.

Evaluation of different pregnancy diagnostic technique in ewes and does
The sensitivity of manual ballottement technique (MBT) was among the three tests: 96% in ewes and 92% in does.On the other hand specificity (96%) the MBT was higher in ewes and that of the copper sulfate test (CST) (91.7%) was higher in does.The highest positive predictive value (PPV) of MBT was highest (95.8%) in ewes but the PPV of CST was highest (91.7%) in does.
Our findings have the similarity with the studies of Rao and Veena (2009) who found that the percentages of true positive reaction for pregnancy and non-pregnancy status in cows by urine barium chloride test were 80 and 10.
Though the efficacy for true positive reaction for pregnancy is comparable with the reports of Kavani (1983), Vohra and Kaikini (1992), it is higher than those reported by Verma and Mishra (1981) and lower than those reported by Moslov and Smirnov (1965) and Temblador and Landa (1971).The efficacy of this test for detecting nonpregnancy is less than those reported by Elpakov and Cyganok (1966), Akhmadeev and Vasilev (1967), Temblador and Landa (1971), Kavani (1983), and Vhora and Kaikini (1992).While in this study false negative reaction for pregnancy was only 24%, false negative for non-pregnancy was 84%.Rao and Veena (2009) found that the false negative reaction for pregnancy was only 20%, false negative for non-pregnancy was 90%.

Conclusion
The MBT can be considered as a screening test for the early diagnosis of pregnancy in ewes and does.MBT positive cases can be confirmed by ultrasonography whenever needed.
(Ott et al., 1981) also been used in the doe(Ott et al., 1981).However there are very few studies for early diagnosis of pregnancy in ewes and does (Lone et al., 2016; Thakchos, 2011; Mayura et al., 2009).In Bangladesh, there is no specific study for small ruminant (sheep and goat) pregnancy diagnosis method.Only rectal palpation is practicing in case of cows but the pregnancy diagnosis of small ruminants is very difficult.Therefore, the aim of present study is to evaluate the performance three tests for the early pregnancy diagnosis in pregnant ewes and does.

Table 1 :
Evaluation of different pregnancy diagnostic techniques in ewes considering ultrasonography as a gold standard

Table 2 :
Evaluation of different pregnancy diagnostic techniques in does considering ultrasonography as a gold standard a, b,c within the column indicates the significance difference (p<0.05)