Quantification of stress sensitive markers in single fecal samples do not accurately predict excretion of these in the pig

All feces produced during 24 h were collected from five pigs and cortisol and immunoreactive cortisol metabolites (CICM), and IgA were quantified. Within pigs, the concentrations of CICM and IgA varied extensively between random samples obtained from a single fecal dropping, and deviated in most cases significantly from the true concentration measured in total fecal output (CV 6.7-130%). The CICM and IgA contents varied considerably (CV 8.1-114%) within and between individual fecal droppings from the same pig compared to the total fecal excretion. In conclusion, single random samples could not be used to reliably quantify the total fecal concentration or excretion of CICM or IgA in pigs. Analyses of all feces collected during shorter periods than 24 h did not provide an accurate estimate of the daily excretion of CICM. Thus, the concentration of stress sensitive molecules in random single fecal samples as an indicator of animal welfare should be interpreted with prudence.     

Efficiency of fecal steroid hormone measurement for assessing reproductive function in the Hokkaido brown bear (Ursus arctos yesoensis)

The present study aimed to establish simple systems for measuring fecal steroid hormones in order to monitor the reproductive profiles of captive Hokkaido brown bears. The efficiency of fecal sample processing at the steps of dehydration and extraction and the correlation between steroid concentrations in matched fecal and blood samples were studied. Then, monthly changes in fecal estradiol-17 beta and progesterone in female bears, and testosterone in male bears were examined. The procedure was finalized as follows. Fecal samples were dried at 100 degrees C for 3 hr and extracted with diethyl ether. The diethyl ether in the extracts was evaporated and residues were reconstituted in ethanol for the assays. Hormone concentrations were quantified using enzyme immunoassays. Concentrations of progesterone and testosterone in fecal and plasma samples were correlated in the systems. The changes in fecal progesterone and testosterone concentrations were similar to those in serum concentrations of bears as reported previously. In contrast, fecal estradiol concentrations did not correlate with plasma levels probably because of the time lag in excretion. However, the changes in estradiol-17 beta concentrations in feces in the present study were similar to those reported in serum. In conclusion, fecal progesterone and testosterone assay systems appear practical for monitoring ovarian and testicular activities without immobilization, though methodological improvements and further validation may be required. For the fecal estradiol-17 beta assay, there is a need to solve the problem of excretion time lag before the system can be used in the study of reproductive physiology.     

Fecal progestagens to detect and monitor pregnancy in captive female cheetahs (Acinonyx jubatus)

The purposes of the present study were to establish a noninvasive monitoring assay of fecal progestagen measurement to detect pregnancy and to identify the components of fecal progestagens in early, middle and late pregnancy in cheetahs. Feces were collected from 7 female cheetahs and analyzed from 30 days before the last copulation to parturition in 9 pregnancies. Blood was collected from one cheetah. Fecal progestagen and serum progesterone concentrations were determined by enzyme immunoassay (EIA). The profiles of the fecal progestagen concentrations were similar to the serum progesterone profile. Fecal progestagen and serum progesterone concentrations remained at the baseline until copulation. In the mean fecal progestagen profile during pregnancy (92.8 ± 0.4 days; from the last copulation to parturition), the concentrations increased 3-4 days after the last copulation and remained high until parturition. To investigate changes in the components of progestagen metabolites in the tripartite periods of gestation, fecal progestagens were analyzed by HPLC-EIA. Marked immunoreactive peaks consistent with 5α-pregnan-3α/β-ol-20-one and 5α-pregnan-3,20-dione and small peaks consistent with 5β-pregnan-3α/β-ol-20-one were detected. There were no distinct difference in the components of progestagens among the first, second and third trimesters of pregnancy. The hormone assay, as an indicator of fecal 5α-reduced pregnanes, is useful for detecting pregnancy and monitoring pregnant luteal activity in cheetahs.     

Fecal progestagen and estrone during pregnancy in a giraffe: a case report

The present study was carried out to measure fecal progestagen and estrone concentrations during pregnancy in a giraffe and examine the possibility of utilizing this assay system for pregnancy diagnosis. Fecal samples were collected from a giraffe during her third and fourth parities and mixed with methanol to prepare a fecal solution. Diluted fecal solution was used for direct enzyme immunoassay for progestagen and estrone. The newborn calf from the third parity was viable, although that from the fourth parity died 5 days after calving. In the third parity, the giraffe's progestagen and estrone concentrations increased transiently from days 30 to 120 of pregnancy. Then, they decreased and remained low until day 330. This was followed by a drastic rise in both concentrations as parturition approached. Parturition caused a reduction in the progestagen and estrone concentrations of the feces. In the fourth parity, the progestagen concentration increased gradually after mating until day 320. This was followed by a reduction in the concentration until parturition. However, the estrone concentration fluctuated, and the duration and extent of the prepartum rise in concentration were shorter and lower than those of the third parity. The hormone dynamics of the third parity suggest the possibility of early pregnancy diagnosis by measuring progestagen or estrone between days 30 and 120 after mating.     

Correlation of periovulatory serum and fecal progestins in the domestic dog.

This study was initiated to determine the relationship between canine ovarian steroids detected in serum and feces during the periovulatory interval in domestic dogs, and to examine the feasibility of a non-invasive method to estimate the time of ovulation in canid species. When bitches (n = 14) were observed to enter proestrus (based on vulvar enlargement or serosanguineous vaginal discharge), paired daily serum and fecal samples were collected for a 15- to 20-day period and stored at -20 degrees C. After extraction, progestin concentrations in both substrates were measured using an established enzyme immunoassay procedure. All samples were aligned to Day 0, the first day in which fecal progestins reached a sustained rise above 100 ng/g feces. Mean fecal progestin concentrations increased in parallel with mean serum progesterone values (r = 0.78), rising from 44.6+/-2.6 ng/g feces to 409.6+/-90.9 ng/g feces, and 5.4+/-0.9 nmol/L to 81.2+/-18.5 nmol/L, on Day -5 and Day 5, respectively. Individual fecal progestin concentrations varied markedly, but plotted against serum progesterone concentrations demonstrated correlation coefficients ranging from 0.41 to 0.97 (P<0.05). These results demonstrate that sequential changes in domestic dog serum ovarian steroid concentrations are paralleled in the feces, and that it is feasible to non-invasively monitor individual progestin changes in the periovulatory interval using fecal hormone analysis.     

Estradiol and progesterone metabolite concentration in white-tailed deer (Odocoileus virginianus) feces.

Assays of reproductive hormone metabolites require validation in each animal species. For validation of methodology in white-tailed deer (Odocoileus virginianus), fecal samples were collected from females that had been injected by blowgun with estradiol, progesterone, or a control substance. Analysis by radioimmunoassay revealed that estradiol and pregnanediol were more abundant fecal metabolites of estrogens and progestins than were estrone or progesterone. Peak excretion rates of estradiol and pregnanediol occurred within 12 and 24 hr of injection, respectively. Ovulation time was estimated by measuring the frequency of occurrence of eight behavior patterns, including copulation. Profiles were compiled for three deer over the course of estrus and early pregnancy for estradiol, estrone, progesterone, and pregnanediol using radioimmunoassay. Pregnanediol was excreted at concentrations about 1,000 times higher than those of the other three fecal steroid metabolites, and pregnanediol differed in concentration during estrus, the luteal phase, and early pregnancy. Consequently, a simpler enzyme immunoassay was adapted and used to measure pregnanediol levels over the course of estrus and early pregnancy for seven deer. Measurement of fecal pregnanediol is useful for monitoring reproductive events in female white-tailed deer.     

Measurement of glucocorticoid metabolite concentrations in faeces of domestic livestock

After 14C-labelled cortisol infusion in ponies and pigs, faecal samples were collected. Extraction of 0.5 g faeces with 5 ml 80-90% methanol yielded the highest radioactivity in the supernatant. Most of the metabolites were ether soluble. After high performance liquid chromatography (HPLC), the presence of immunoreactive metabolites was demonstrated by measuring each HPLC fraction using enzyme immunoassays for cortisol, corticosterone and 11-oxoaetiocholanolone. Only the assay for 11-oxoaetiocholanolone revealed peaks with co-eluting radioactivity. For biological validation of the test system, adrenocorticotrophic hormone (ACTH) and dexamethasone were injected intravenously successively in both species (n = 6). Cortisol concentration in blood and the 11-oxoaetiocholanolone immunoreactive substances in faeces were determined. In horse faeces, basal values of 2.3-35.2 nmol/kg were measured. After ACTH administration, an increase (more than 200% above basal values) of these metabolites was seen about 1 day after ACTH administration. After dexamethasone injection the levels decreased, reaching minimum concentrations 2 days after administration. In pigs, an increase in these metabolites was measured in only three animals after ACTH; dexamethasone did not cause a decrease. The stability of the samples after defecation was tested by storing samples from cows, horses and pigs at room temperature. It was shown that there was a significant increase in the concentration of measured cortisol metabolites in bovine, equine and porcine faeces after storage for 1 h, 4 h and 24 h, respectively. In frozen samples this effect was diminished after thawing samples at 40 degrees C; thawing the samples at 95 degrees C prevented an increase in immunoreactive substances.     

The development of a simple fecal immunoreactive progestin assay to monitor reproductive function in swine.

The objectives of the study were to (a) develop a simple fecal progestin extraction and radioimmunoassay method to measure immunoreactive progestin in porcine feces and (b) to characterize fecal progestin profiles during the estrous cycle and postpartum. A simple extraction method was developed in trial 1 and the mean (+/- SD) progestin recovery of the method was 84.3 +/- 3.5%. Progesterone levels measured at five different spiked concentrations (50, 100, 200, 400, and 500 ng/0.5 g feces) showed no systematic error. The sensitivity of the assay was 0.16 nmol/L of the extract. Trial 2 involved collecting fecal samples from six cycling sows every second or third day, beginning on the day of estrus (day 0) and continuing until day 22. The mean (+/- SD) fecal progestin concentrations of these sows determined by the above assay during days 0-5, days 6-10, days 11-15, and days 16-21 were 87.1 +/- 17.5, 262.6 +/- 102.1, 1188.2 +/- 454.1, and 897.3 +/- 274.1 x 10(-3) nmol/g feces, respectively. In trial 3, fecal samples from six postpartum sows were collected at weekly intervals beginning from day 7 after farrowing until day 50. The mean (+/- SD) fecal progestin concentrations were 111.0 +/- 61.1, 74.1 +/- 21.3, 66.5 +/- 26.1, 122.7 +/- 58.8 and 533.5 +/- 244.2 x 10(-3) nmol/g feces, during days 7-10, days 11-20, days 21-30, days 31-40, and days 41-50 postpartum, respectively. The results indicate that simple fecal progestin extraction and assay are feasible alternatives to the standard blood progesterone assays for monitoring reproductive function in swine.     

Noninvasive reproductive monitoring in the okapi (Okapia johnstoni).

Fecal progestagen analysis in okapis (Okapia johnstoni) was used for diagnosis of pregnancy and reproductive disorders, including a comparison of urinary and fecal progestagen analysis and endocrine data on the postpartum period. Data were generated on reliability of fecal progestagen analysis in early pregnancy diagnosis, and case reports were compiled involving single animals with missing luteal activity, abortion after twin pregnancy, and abortions due to deficient placental progestagen production. There was approximately 100-200-fold higher progestagen concentration in feces than in urine, thus explaining the high reliability of fecal progestagen evaluations in diagnosing luteal function and pregnancy. The postpartum period was characterized by lactational anestrus of several months duration, and a postpartum estrous cycle about 2-3 wk after parturition was observed in two of eight animals. An animal with five abortions due to deficient placental progestagen production was treated with altrenogest in a subsequent pregnancy and carried the fetus to term.     

Comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection

An indirect, double-antibody sandwich-type ELISA for detection of transmissible gastroenteritis virus (TGEV) was developed, using a solid phase of rabbit hyperimmune serum and a pool of 3 antipeplomer monoclonal antibodies to trap and to detect the virus, respectively. The technique was used to detect viral antigen in feces of pigs that had been infected with the virulent Miller strain, the attenuated Purdue strain, or the Erica strain (a Dutch field isolate) of TGEV. The results were compared with those of a solid-phase immunosorbent electron microscopy (SPIEM) technique for virus detection. Both techniques detected shedding of virulent virus in feces obtained from pigs on the first or second day after infection, and virus excretion continued for 6 to 8 consecutive days. Virus shedding started later in pigs infected with the attenuated Purdue strain of TGEV and lasted only 2 to 4 days. In comparison with the 2 virulent strains, infection with the attenuated strain appeared to be limited to a smaller portion of the small intestine. Of 242 fecal specimens that were tested by use of ELISA and SPIEM, 119 had positive results in both tests. Additionally, virus could be detected by ELISA in 21 and by SPIEM in 16 specimens. Fecal specimens obtained from pigs before infection always reacted negatively by ELISA for TGEV antigen; there was no cross-reactivity with fecal specimens containing porcine rotavirus or porcine epidemic diarrhea virus. The ELISA and SPIEM were found to be specific and sensitive for the detection of TGEV in feces.     

Development of a fecal sample collection strategy for extraction and quantification of fecal immunoglobulin A in dogs

OBJECTIVE: To develop a fecal sample collection strategy and quantification method for measurement of fecal IgA concentrations in dogs. SAMPLE POPULATION: Fecal samples from 23 healthy pet dogs of various breeds. PROCEDURES: Immunoglobulin A was extracted from fecal samples. An ELISA for the measurement of fecal IgA concentrations was established and analytically validated. Intraindividual variation of fecal IgA was determined by calculation of coefficients of variation. A sample collection strategy was developed on the basis of results of intraindividual variation of fecal IgA concentrations. A reference range for fecal IgA concentrations was determined. RESULTS: The method for extraction and quantification of fecal IgA was determined to be sufficiently sensitive, reproducible, accurate, and precise. On the basis of the intraindividual variability of our results, the determined fecal sample collection strategy required analysis of a total of 4 fecal samples/dog, with each fecal sample collected on 2 consecutive days with 28 days between sample collection periods (ie, days 1 and 2 followed by days 28 and 29). Reference range values for fecal IgA concentration were 0.22 to 3.24 mg/g of feces. CONCLUSIONS AND CLINICAL RELEVANCE: Methods of fecal IgA extraction and quantification used in our study allow for identification of dogs with consistently low fecal IgA concentrations. Use of these techniques will enable future investigations into possible associations between low fecal IgA concentrations and signs of gastrointestinal disease in dogs.     

应用ELISA(双抗体夹心法)检测猪流行性腹泻病猪粪便中的病毒抗原

应用猪流行性腹泻(PED)—ELISA直接法(双抗体夹心法)对120头健康猪和5头猪传染性胃肠炎(TGE)病猪粪便标本进行检测,均不出现交叉反应;对15头PED病毒实验感染仔猪粪便标本检测,全部呈阳性;将对3(?)份ELISA阳性粪便标本和3份阴性标本的检测结果与电镜观察结果比较,其阳性符合率为97.37％,阴性符合率为100％;对在PED发病季节从不同地区采集的腹泻病猪粪便标本112份检测结果,阳性率为60.71％,而阴性反应的标本,绝大多数是在病愈后15～20d采集的。     

Effect of fatty acids added to the milk replacer on white scour and excretion of fatty acids in Holstein calves

In order to examine the relationship between white scour and fatty acids, we added fatty acids to the milk replacer. Twenty healthy Holstein calves were divided into 4 groups, five calves per group; a control group with no fortified fatty acid, and 3 groups fed either with oleic, stearic, or palmitic acid, respectively. The calves were fed milk replacer (5% of the calf's body weight) twice a day but the fatty acids (2 wt % of milk replacer) were added only once. The fecal and blood samples were obtained at 0, 12, 24, 36, and 48 h after feeding of the acids. All five calves in the palmitic acid group, and 3 out of 5 calves each in the stealic and the oleic acid groups had whitish feces after feeding fatty acid. The stearic acid group had a significantly elevated stearic acid concentration in the feces during 24–36 h compared to the pre-feeding level. The fecal concentration of palmitic acid increased significantly at 24–36 h in the palmitic acid group. We concluded that the long-chain saturated fatty acids are one of the causes of white scour in calves.     

Fecal steroid analysis for evaluating ovarian function in the greater kudu (Tragelaphus strepsiceros) and lesser kudu (Tragelaphus imberbis)

Seasonal reproductive-endocrine norms have not been described for the genus Tragelaphus, which consists of seven species of African antelope. Longitudinal patterns of progesterone metabolite excretion were assessed by radioimmunoassays in fecal samples collected noninvasively (three to seven samples per week) from greater kudu (Tragelaphus strepsiceros, n = 4) and lesser kudu (Tragelaphus imberbis, n = 4). Progesterone metabolite excretion patterns revealed seasonal estrous cycles in both species, and discrimination of pregnant versus nonpregnant females was achieved in lesser kudu. These data reveal the value of fecal progesterone metabolites for establishing reproductive-endocrine norms in both zoo-maintained and free-living antelopes of the genus Tragelaphus.     

Identification of infant and adult swine susceptible to enterotoxigenic Escherichia coli by detection of receptors for F4(K88)ac fimbriae in brush borders or feces.

We attempted to determine F4(K88)-adhesive and non-adhesive phenotypes of infant (neonatal <3 day old and weaned <4 week old pigs) and adult (>6 month old) swine by ELISA using immobilized F4(K88)ac fimbrial antigen or whole F4(K88) + E. coli cells (strains M1823 and 1476) and isolated small intestinal brush borders or easily-obtainable fecal samples from the same animals. Nineteen of 22 neonates (86%), 17 of 20 weaners (85%), and 26 of 39 adults (67%) were classified identically as F4(K88) receptor-positive or negative by the ELISA. The ELISA with feces from adult swine was found to be almost equally specific (87%) as that with feces from neonatal (90%) and weaned (91%) pigs. However, the sensitivity of the assay was low (38%), indicating that fecal samples from adults contained less receptor-material than necessary for comparable phenotyping. The receptor-positive brush borders from neonates and weaners reacted significantly better (P < 0.02, < 0.001 respectively) with purified F4(K88) antigen than did those from adults. There was good agreement between the average ELISA values for feces from infant and adult swine regardless the source of coating antigen applied. With this assay we can determine F4(K88) phenotypes of infant swine using easily-collected fecal samples rather than isolated brush borders. It was also concluded that tested feces is not an acceptable alternate source of the receptor-material to brush borders from F4(K88)-susceptible adult swine.     

Effect of micronized pea and enzyme supplementation on nutrient utilization and manure output in growing pigs

An experiment was done to determine manure output, N and P excretion, and apparent digestibilities of AA, CP, P, and DM in growing pigs fed barley-based diets containing micronized or raw peas with or without supplementation with enzyme containing primarily beta-glucanase and phytase (Biogal S+). Eight barrows (21.5 +/- 1.2 kg of initial BW) fitted with T-cannulas at the distal ileum were used in a 40-d trial and housed in metabolism cages. Pigs were assigned in a replicated 4 x 4 Latin square design to 4 experimental diets: 1) barley-raw peas control (BRP), 2) barley-micronized peas (BMP), 3) BRP plus enzyme, and 4) BMP plus enzyme (BMP+E). Pigs received 2.6 times maintenance energy requirements based on BW at the beginning of each experimental period. During each experimental period, pigs were acclimatized to their respective diets for 5 d followed by a 3-d period of total fecal and urine collection and another 2-d period of ileal digesta collection. Samples were analyzed for DM, AA (diets and digesta only), N, and P. Wet fecal output of BRP plus enzyme-fed pigs tended to be lower (P = 0.07) than the amount produced by BMP-fed pigs. The amounts of dry feces and urine produced were not different among treatments (P > 0.10). Supplementing the BRP and BMP diet with enzyme increased (P = 0.002) the daily P retained per pig. Pigs fed the enzyme-supplemented diets tended to have lower (P = 0.06) fecal P excretion and greater urinary P excretion (P = 0.001) compared with pigs fed the nonsupplemented diets, but total P excretion was not influenced by diet (P > 0.10). Pigs fed the BMP+E diet retained more (P = 0.006) N per day than pigs fed the BMP diet. However, N excretion was not influenced by dietary treatment (P > 0.10), although BMP+E-fed pigs excreted 13.2% less N in the feces compared with those fed the nonenzyme supplemented controls. Inclusion of micronized peas with or without enzyme supplementation did not affect urinary or fecal N excretion (P > 0.10) compared with the BRP. Dietary treatment had no effect (P > 0.10) on ileal or fecal DM or CP digestibilities. Apparent ileal digestibilities of AA were usually lower (P < 0.05) in the BRP diet compared with the other diets. Enzyme supplementation improved P digestibility at the ileal and fecal level. The current results indicate that utilizing micronized peas in barley-based pig grower diets enhances P retention.     

Absorption, distribution, metabolism, and excretion of dirlotapide in the dog

Three once-daily oral doses of 0.2 mg/kg [¹⁴C]dirlotapide were administered to beagle dogs to study the absorption, distribution, metabolism, and excretion of dirlotapide. Mean ¹⁴C recovered at 2.5 and 4.5 h after the last dose was 90%. Mean ¹⁴C in urine, bile, and feces was <1%, 1.7%, and 56% of the dose, respectively. In tissues, 26% of the ¹⁴C dose was present in the gastrointestinal tract, 6.0% in liver, and <1% each in kidney, gall bladder, heart, and brain. To further characterize drug disposition, a single 2.5-mg/kg oral dose of [¹⁴C]dirlotapide was administered to beagle dogs. More than 84% of the dose had been eliminated by 72 h in feces, with 21% of the dose present in feces as parent dirlotapide. Less than 1% of the dose was excreted in urine. In bile collected during the first 24-h postdose from three dogs, 32% and 11% of the ¹⁴C dose was present in samples from male and female dogs, respectively. Based upon metabolite profiling of plasma, excreta, and bile samples, dirlotapide was extensively metabolized to more than 20 metabolites. Biliary/fecal excretion and the potential for enterohepatic recycling of metabolites are suggested.     

Disappearance and appearance of an indigestible marker in feces from growing pigs as affected by previous-and currentdiet composition

Background: Indigestible markers are commonly utilized in digestion studies, but the complete disappearance or maximum appearance of a marker in feces can be affected by diet composition, feed intake, or an animal's BW.The objectives of this study were to determine the impact of previous(Phase 1, P1) and current-(Phase 2, P2)diet composition on marker disappearance(Cr) and appearance(Ti) in pigs fed 3 diets differing in NDF content.Results: When pigs were maintained on the 25.1, 72.5, and 125.0 g/kg NDF diets, it took 5.1, 4.1, and 2.5 d, respectively,for Cr levels to decrease below the limit of quantitation; or 4.6, 3.7, or 2.8 d, respectively, for Ti to be maximized. These effects were not, however, independent of the previous diet as indicated by the interaction between P1 and P2 diets on fecal marker concentrations(P 0.01). When dietary NDF increased from P1 to P2, it took less time for fecal Cr to decrease or fecal Ti to be maximized(an average of 2.5 d), than if NDF decreased from P1 to P2 where it took longer for fecal Cr to decrease or fecal Ti to be maximized(an average of 3.4 d).Conclusions: Because of the wide range in excretion times reported in the literature and improved laboratory methods for elemental detection, the data suggests that caution must be taken in considering dietary fiber concentrations of the past and currently fed diets so that no previous dietary marker addition remains in the digestive tract or feces such that a smal amount of maker is present to confound subsequent experimental results, and that marker concentration have stabilized when these samples are col ected.     

The shedding of group A rotavirus antigen in a newly established closed specific pathogen-free swine herd.

A longitudinal study was undertaken in a newly established specific pathogen-free (SPF) swine herd to determine the dynamics of rotavirus antigen shedding in a closed swine facility. Pregnant SPF gilts which populated the herd, and their offspring, were monitored weekly for three consecutive lactations. Fecal samples were assayed for the presence of group-specific viral antigen by a solid phase immunoassay (ELISA). Results indicate that in the week prior to farrow, 35% of samples from gilts/sows contained rotavirus antigen. During nursing, 37% of the gilts'/sows' fecal samples also contained virus antigen. Over the course of three farrowings, every gilt/sow in the herd excreted virus antigen. Virus antigen was present in 25% of the samples tested from nursing pigs and in 70% of the samples tested from pigs in the postnursing period; 95% of the litters excreted virus antigen either while nursing or postweaning. Seasonal incidence in virus antigen excretion was noted with proportionally more suckling pigs virus antigen-positive in summer and proportionally more sows/gilts positive during winter. Diarrhea occurred only rarely in the sampled population. Although piglets shed rotavirus subclinically, ELISA positive feces from piglets of each lactation caused severe disease when fed to neonatal gnotobiotic pigs. Electropherotyping of these passaged viruses indicated minor variation in RNA banding patterns over time.