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ABSTRACT. The aim of this study was to establish radioimmunoassay (RIA) for saliva estrone sulfate (E 1 S), and to elucidate changes in saliva E 1 S during pregnancy in the sow. Saliva E 1 S was extracted using a commercially available solid phase column, and the E 1 S fraction obtained was subjected to RIA. The sensitivity of the RIA was 29.7 pg/tube. The intra-and inter-assay coefficients of variation were 5.5-8.4% and 13.1-19.5%, respectively. Mean recovery for E 1 S added to saliva samples was as high as 99.9%. A significant positive correlation (r=0.54, n=69, p<0.01) existed between saliva and plasma E 1 S concentrations. During gestation, the changing patterns of saliva and plasma E 1 S concentrations were essentially the same, and two peaks of E 1 S concentrations were observed, one around day 30 and another just before parturition, although E 1 S concentrations in saliva remained at only 2.4-38.1% (mean 11.4%) of those in plasma E 1 S. Thus, the present study has made it possible to measure saliva concentrations of E 1 S and demonstrated a high degree of positive correlation between saliva and plasma E 1 S concentrations. These results suggest that diagnosis of early pregnancy and of normal or abnormal fetal development could be made by measurements of E 1 S in saliva. -KEY WORDS: estrone sulfate, pregnancy, radioimmunoassay, saliva, sow.J. Vet. Med. Sci. 59(9): 759-763, 1997 saliva from the cotton. Saliva samples obtained were mixed with sodium azide (5 mg/ml) and kept frozen at 20°C until assayed for E 1 S. Immediately after saliva collection, blood samples were also collected from the median tail vein into heparinized test tubes. Plasma was separated by immediate centrifugation (1,700 × g, 15 min) and kept frozen at 20°C until assayed for E 1 S.Extraction of E 1 S: Saliva and plasma E 1 S was extracted by a slight modification of the method described by Nakamura [10]. Briefly, saliva and plasma samples (1-4 ml) were diluted with 2 ml borate buffer (0.2 M, pH 8.0) containing 0.05% bovine serum albumin and 0.06% bovine γ-globulin (BSA buffer), and then poured into a 5 ml disposable syringe attached to solid phase columns (SepPak ® PLUS C18 Cartridge, Millpore Co., Ltd., U.S.A.). The sample solutions were allowed to flow into the columns at a rate of 0.2-1 ml per min. The columns were then washed with 4 ml distilled water and with 3 ml diethylether, successively and the effluents were discarded. Finally, five ml acetone was poured into the columns, and the resultant effluent (E 1 S fraction) was recovered into test tubes. The effluents were evaporated to dryness at 50°C under nitrogen gas, and the residues were dissolved in 200 µl of BSA buffer.RIA for E 1 S: Saliva and plasma E 1 S were measured by a modification of RIA described by Nakamura [10], as shown in Fig.

ABSTRACT. We attempted to measure the gestagen concentration in the feces of pigs by using a commercial bovine milk progesterone quantitative test EIA kit, and investigated the possibility of applying of this method of gestagen concentration measurement to early pregnancy diagnosis in the sow. Feces were collected from the rectum of the pig, and 0.5 g of the feces was placed in 20 ml of distilled water, stirred, and centrifuged. The supernatant was used as the fecal solution for measurement of gestagen. The procedure used for measuring gestagen in feces was the same as that for the measurement of progesterone in milk, except that a standard fecal gestagen solution (0.5-30.0 ng/ml) was prepared by the authors in the laboratory. The sensitivity of measurement using this method was 0.80 ng/ ml, or 32.0 ng/g of fecal weight. The recovery was 105.2-105.6%. Intra-assay coeffecients of variation (CVs) were 2.8-8.5%. The interassay CVs were 7.4-10.2%. Gestagen concentrations in feces measured by the present method and progesterone concentrations in peripheral plasma, collected at the same time as the feces, were highly correlated (r=0.98, p<0.001). The criteria for diagnosis of pregnancy based on the fecal gestagen level was positive for a gestagen level of 200 ng/g and negative for a gestagen level of < 200 ng/ g. When fecal gestagen measurements were applied to early pregnancy diagnosis in 149 sows, the accuracy of diagnosis from day 21 to day 25 after the last mating was 96.2% for positive cases (102/106) and 95.3% for negative cases (41/43). Thus, the results of this study show the quantitative measurement of the fecal gestagen concentration in the sow using a bovine milk quantitative test EIA kit is a practical method for early pregnancy diagnosis. -KEY WORDS: early pregnancy diagnosis, feces, gestagen, quantitative test EIA kit, sow.J. Vet. Med. Sci. 59(8): 695-701, 1997 easier to collect than blood. Measurements were carried out using a commercial bovine milk progesterone enzyme immunoassay (EIA) kit, which is quick and does not require any special equipment. The authors reported good results from the application of this method to early pregnancy diagnosis in sows [27][28][29]. In the present study, the same commercial bovine milk progesterone EIA kit was used for quantitative measurement of the gestagen concentration in samples of feces, which are even easier to collect than saliva, and the applicability of this method to early pregnancy diagnosis in the sow was investigated. MATERIALS AND METHODS Test kit:The kit used for gestagen quantitative measurement was an Ovucheck bovine milk progesterone EIA kit (Cambridge Veterinary Sciences Ltd., U.K.). The microtitre plate had 96 wells with sheep progesterone antibody pre-coated in each well. The reagents included enzyme-labeled antigen-binding solution (conjugate), substrate tablets, substrate tablet soluble buffer solution, and stopping solution, as well as four types of standard progesterone milk solutions (1.0, 5.0, 10.0, 30.0 ng/ml; standard milk solution was...

ABSTRACT. The aim of this study was to establish radioimmunoassay (RIA) for fecal estrone sulfate (E1S) and to elucidate changes in fecal E1S during pregnancy in the sow. Fecal E1S was extracted on a commercially available solid phase column, and the E1S fraction obtained was subjected to RIA. The sensitivity of the RIA was 8.5 pg/tube. The intra-and inter-assay coefficients of variation were 8.8-8.9% and 10.7-14.2%, respectively. Mean recovery for E1S added to fecal samples was as high as 95.0%. A significant positive correlation (r=0.904, n=147 p<0.0001) existed between fecal and plasma E1S concentrations. Mean E1S concentration in feces and plasma fluctuated exhibiting two peaks. The first peak of E1S concentration was evident on days 28-32 in feces and on days 26-30 in plasma. The E1S concentration in both feces and plasma remained at baseline levels during mid-pregnancy, but began to rise gradually around days 72-82 and 70-80, in feces and plasma respectively, and reached a peak concentration on days 110-114. Following parturition, the concentration of E1S in plasma declined rapidly, but there was a two-day delay before a decline in fecal E1S. Apart from this two-day delay, changes in fecal E1S were similar to those in plasma E1S. The study indicates that the measurement of E1S in feces could be a useful tool for early pregnancy diagnosis and for monitoring fetal development in sows and gilts.-KEY WORDS: estrone sulfate, feces, pregnancy, radioimmunoassay, sow.J. Vet. Med. Sci. 61(6): 661-665, 1999 our department were used for the present study. The sows were artificially inseminated on the first day of estrus. Fecal samples were collected from each sow at intervals of two days during pregnancy, as described previously by Moriyoshi et al. [13]. Briefly, a plastic glove for rectal examination was worn on the hand which was inserted into the rectum of sows. An appropriate amount of impacted feces was taken from the rectum and immediately aliquoted in 2 g amounts. The aliquots were put into plastic test tubes each containing 8 ml of 0.01 M phosphate buffer with 0.1% of bovine serum albumin (BSA; albumin, bovine fraction V, Sigma-Chemicals Co., St. Louis, U.S.A), and then taken back to the laboratory. After thoroughly shaking on a Direct Mix TS-50 (Thermal Kagaku Sangyo Co., Ltd., Japan), the solution was centrifuged at 1,700 × g for 15 min, and the supernatant was frozen at 20°C as the fecal solution until assayed for E1S. Immediately after fecal collection, blood samples were also collected from the median tail vein into heparinized test tubes. Plasma was separated by immediate centrifugation (1,700 × g, 15 min) and kept frozen at 20°C until assayed for E1S. In order to use as E1S standard for fecal samples, E1S-free feces were collected from 5 castrated boars. The E1S-free fecal solutions prepared as described above were pooled. Preparation of standard fecal E1S solution: Fecal samples were collected from 5 castrated boars and the fecal solutions prepared were pooled as described above. These were used fo...