Comparison between blood serum and salivary cortisol concentrations in horses using an adrenocorticotropic hormone challenge.

REASONS FOR PERFORMING STUDY: In horses, serum cortisol concentration is considered to provide an indirect measurement of stress. However, it includes both free and bound fractions. The sampling method is also invasive and often stressful. This is not the case for salivary cortisol, which is collected using a more welfare-friendly method and represents a part of the free cortisol fraction, which is the biologically active form. OBJECTIVES: To compare salivary and serum cortisol assays in horses, in a wide range of concentrations, using an adrenocorticotropic hormone (ACTH) stimulation test, in order to validate salivary cortisol for stress assessment in horse. METHODS: In 5 horses, blood samples were drawn using an i.v. catheter. Saliva samples were taken using swabs. Cortisol was assayed by radioimmunoassay. All data were treated with a regression method, which pools and analyses data from multiple subjects for linear analysis. RESULTS: Mean ± s.d. cortisol concentrations measured at rest were 188.81 ± 51.46 nmol/l in serum and 1.19 ± 0.54 nmol/l in saliva. They started increasing immediately after ACTH injection and peaks were reached after 96 ± 16.7 min in serum (356.98 ± 55.29 nmol/l) and after 124 ± 8.9 min in saliva (21.79 ± 7.74 nmol/l, P<0.05). Discharge percentages were also different (225% in serum and 2150% in saliva, P<0.05). Correlation between serum and salivary cortisol concentrations showed an adjusted r(2) = 0.80 (P<0.001). The strong link between serum and salivary cortisol concentrations was also estimated by a regression analysis. CONCLUSIONS: The reliability of both RIAs and regression found between serum and salivary cortisol concentrations permits the validation of saliva-sampling as a noninvasive technique for cortisol level assessment in horses.

Pregnancy-associated glycoprotein and progesterone concentrations during pregnancy failure in Bedouin goat from the southwest of Algeria.

Thirteen female Bedouin goats living in arid land of Algeria Sahara desert were used in this study. These goats were pregnant but they sustained an abortion because of unidentified causes. None of the goats showed any signs of general disease. Plasma concentrations of caprine pregnancy-associated glycoproteins (cPAGs) and progesterone (P4) were determined during pregnancy using radioimmunoassay. The cPAGs concentration was undetectable (<0.8 ng/ml) throughout the first 2 weeks of gestation. From week 3 after mating, cPAGs concentration was detectable with significant individual variations (p < 0.05) reaching a maximum secretion (436.1 ng/ml). Throughout gestation, cPAGs concentration remained relatively constant but decreased few days before abortion, on an average of 9.2 ± 1.2 days (n = 11), except for two females where the concentrations decreased later (1-2 days before abortion). One or two peaks of cPAGs concentrations (in 4/13 and in 9/13 females, respectively) have been measured few weeks before abortion (77-124 days after mating), when a decline of cPAGs was detected. The P4 concentration increased after mating, and was high from the first week till the end of pregnancy. The P4 concentration (9.1 ± 0.9 ng/ml) decreased rapidly (<0.5 ng/ml) after 4 ± 0.7 days (n = 6) or 9.4 ± 1.6 days (n = 7) before abortion. A positive relationship (p < 0.01) was found between P4 and cPAGs concentrations during gestation. Results indicate that cPAGs and P4 measurements can be used for monitoring gestation and for abortion prediction.

Factors affecting plasma prolactin concentrations throughout gestation in high producing dairy cows.

The aim of the present study was to investigate possible relationships between plasma concentrations of prolactin and the following factors throughout gestation in lactating dairy cows: photoperiod, season, milk production, Neospora caninum-seropositivity, twin pregnancy, and plasma concentrations of pregnancy associated glycoproteins-1 (PAG-1), progesterone and cortisol. Pregnancy was diagnosed by trans-rectal ultrasonography on Day 40 post-insemination and by palpation per rectum on Days 90, 120, 150, 180 and 210 or until abortion in aborting cows. Blood samples were collected from each animal immediately before each pregnancy diagnosis. The study population was comprised of 73 non-aborting (54 Neospora-seropositive cows) and 20 aborting cows (all Neospora-seropositive) cows. In non-aborting cows, GLM repeated measures of analysis of variance revealed that lactation number and days in milk had no effect on plasma prolactin concentrations throughout gestation, whereas high plasma prolactin concentrations were significantly associated with high plasma levels of cortisol and PAG-1, with Neospora-seropositivity, positive photoperiod (increasing day length), twin pregnancy, and with low plasma progesterone concentrations. An interaction among plasma prolactin, plasma cortisol and milk production was observed in that plasma prolactin concentrations differed significantly throughout gestation and were highest in high-producing cows with high cortisol levels. In Neospora-seropositive non-aborting versus aborting cows, mean prolactin concentrations failed to differ on Days 40, 90, 120, 150 and 180 of pregnancy, whereas prolactin values were significantly lower (P=0.03) in aborting animals on Day 210 of pregnancy. Our results indicate that a positive photoperiod and Neospora-infection lead to increased plasma prolactin concentrations throughout gestation. Reduced prolactin concentrations detected in Neospora-seropositive aborting cows compared to non-aborting animals suggests a protective effect of prolactin in N. caninum infection.

Correlation of five radioimmunoassay systems for measurement of bovine plasma pregnancy-associated glycoprotein concentrations at early pregnancy period.

The measurement of serum or plasma PAG concentrations is currently used as a specific method for pregnancy diagnosis in cattle. In this study, the correlation between five radioimmunoassay systems (RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool) developed for measurement of PAG concentrations in ruminant species was investigated in plasma from pregnant Friesian Holstein females. Plasma PAG concentrations (ng/mL) measured by different RIA systems were significantly correlated between them ( > or = 0.81; P<0.001). PAG concentrations increased significantly from Day 21 (n=27) to 30 (n=37) after AI by use of all PAG-RIA systems. From Day 30 to 80 after AI, lower PAG concentrations were observed when using the homologous system RIA-497. The addition of several proteinase inhibitors changed neither the non specific binding nor the B(0) binding to the tracer. Our results suggest that all tested PAG-RIA (RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool) are highly correlated and can be useful to follow PAG concentrations in samples collected during the first trimester of gestation.

Factors affecting plasma pregnancy-associated glycoprotein 1 concentrations throughout gestation in high-producing dairy cows.

This study was designed to establish the factors, if any, which could affect plasma pregnancy-associated glycoprotein-1 (PAG-1) expression in a study population of 87 pregnant, high-producing dairy cows. The factors examined were: semen providing breed (Holstein-Friesian vs Limousin), outcome of gestation (male vs female newborn, and singleton vs twin pregnancies), lactation number, milk production at pregnancy diagnosis, plasma progesterone concentration, season of gestation (warm period, March-November vs cool period, December-February), and day of gestation (40, 90, 120, 150, 180 and 210). Pregnancy was diagnosed by transrectal ultrasound on day 40 post-insemination and by palpation per rectum on days 90, 120, 150, 180 and 210. Blood samples were collected from each animal immediately before each pregnancy diagnosis. The relative contributions of the different factors on PAG-1 concentrations were evaluated by GLM repeated measures analysis of variance. No significant effects of the herd, foetal sex, milk production, lactation number and plasma progesterone concentrations were observed. In contrast, twin pregnancy, the use of Limousin semen and conception during the cool period were correlated with significantly increased plasma PAG-1 concentrations throughout gestation. Our data indicate that both cow well-being during early placental development, determined in our conditions by reduced heat stress when conception occurred in the cool season, and crossbreed pregnancies lead to improved PAG-1 production throughout the gestation period.

Pregnancy-associated glycoprotein secretion in North Moroccan goats.

The plasma profile of pregnancy-associated glycoprotein (PAG) and its relationship with fetal number were studied in 14 North Moroccan goats using a semi-heterologous radioimmunoassay (RIA). Peripheral blood was collected every day during the first month of pregnancy, afterward the blood samples were collected trice a week. The PAG were first detected at day 20 of pregnancy, their levels increase to week 8 of gestation then decrease slightly until parturition. Statistical differences between goats carrying one or two foetuses are observed from week 5 of pregnancy until parturition. Factorial Discriminant Analysis provides mathematical models for the discrimination between the litter size using the PAG level and the week of pregnancy. Using only one blood sample per week, high sensitivity, specificity and overall accuracy (74%, 88% and 81%) were obtained using these models. It is concluded that the PAG RIA is an effective tool for early diagnosis of pregnancy and for discrimination between the litter sizes in North Moroccan goats.

Factors affecting plasma progesterone in the early fetal period in high producing dairy cows.

The aim of the present study was to determine whether certain animal- or management related factors could affect plasma progesterone concentrations on Day 42 of gestation in high producing dairy cows. Factors affecting early fetal loss were also analyzed. The study population was comprised of 199 pregnant cows classified as having high (> or =9 ng/ml) or low (<9 ng/ml) plasma progesterone concentrations. Through logistic regression procedures it was determined that, based on the odds ratio, cows with two or more corpora lutea were three times more likely to have high progesterone concentrations than cows with a single corpus luteum. Low producing cows during the warm season were 2.86 times more likely to have high progesterone concentrations than the remaining cows. Primiparous cows with high concentrations (>4 ng/ml) of pregnancy associated glycoprotein-1 (PAG-1) were 2.73 times more likely to have high progesterone concentrations than the remaining cows. Of the 199 pregnancies, 25 (12.6%) suffered early fetal loss: 22/136 (16.2%) during the warm season and 3/63 (4.8%) during the cool season, all in cows without additional corpora lutea. Based upon the odds ratio, cows without an additional corpus luteum were 3.67 times more likely to suffer fetal loss during the warm season than during the cool season. Our results indicate that milk production, the presence of two or more corpora lutea and plasma PAG-1 concentrations can affect plasma progesterone concentrations at the onset of the fetal period. The presence of an additional corpus luteum strongly diminished the risk of early fetal loss during the warm period.

Radioimmunoassay of bovine placental lactogen using recombinant and native preparations: determination of fetal concentrations across gestation.

Concentrations of bovine placental lactogen (bPL) were determined in fetal plasma samples by twelve double-antibody competitive radioimmunoassay systems (RIA I-XII) based on either recombinant bPL (non-glycosylated) or native bPL (glycosylated). Both preparations were used as standard and tracer, and for primary antisera production. The minimum detection limit measured by these RIA varied from 0.02 to 0.6 ng bPL mL(-1). The coefficients of correlation of different bPL RIA systems were up to 90% (P < 0.0001) when each RIA was tested against the average values of all twelve RIA systems. All developed RIA were used to investigate the incidence of different bPL isoforms in bovine fetal serum samples (n = 71). Fetal concentrations ranged from 11.8 to 35.7 ng mL(-1) at the third month and from 1.1 to 13.5 ng mL(-1) at the ninth month of gestation. They tended to decrease with advancing gestation. In general, those RIA systems that used recombinant bPL as the standard measured higher values than those using the native bPL preparation. These differences decreased toward the end of gestation (P < 0.05), suggesting a lower rate of glycosylation. Our results provide evidence of different glycosylated isoforms of bPL in fetal serum at different gestation periods.

Accuracy of ultrasonography and pregnancy-associated glycoprotein test for pregnancy diagnosis in buffaloes.

The aims of the present study were to evaluate and compare the accuracy of transrectal ultrasonography and pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) test for diagnosis of pregnancy in buffaloes. Two hundred and seventy-five buffalo cows and heifers were examined once for pregnancy diagnosis by transrectal ultrasonography using a 5 MHz linear-array transducer between Days 19 and 55 after mating. After ultrasound scanning, a blood sample was withdrawn from jugular vein of each animal for measuring pregnancy-associated glycoprotein using a heterologous double-antibody RIA. Based on palpation of the uterus per rectum at Days 75-90, 87 animals were designated pregnant and 188 as non-pregnant. The sensitivity of transrectal ultrasonography at Days 19-24 was 44.4%, reaching 100% from Day 31 after mating. The specificity of transrectal ultrasonography ranged between 92.5 and 100% from Days 19 to 55 after mating. The sensitivity of PAG-RIA test was 11.1% at Days 19-24 and reached 100% from Day 31 after mating. The specificity of PAG-RIA test ranged from 90 to 100% from Days 19 to 55 after mating. There were no significant differences between the sensitivity and specificity of the two tests in all examined periods. In conclusion, transrectal ultrasonography and PAG-RIA test are highly accurate tests for detecting pregnant buffaloes from Day 31 after mating onwards.

Comparison of five radioimmunoassay systems for PAG measurement: ability to detect early pregnancy in cows.

This study was conducted to describe the minimum detection limit, reproducibility, accuracy, specificity and parallelism of different pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) systems: RIA-497, RIA-706, RIA-780, RIA-809 and RIA-Pool. Their ability to distinguish between non-pregnant and pregnant females at day 30 after artificial insemination (AI) was investigated. The antisera were raised in rabbits against different PAG preparations. All RIA systems proved to be sensitive, repeatable and accurate for measuring PAG concentrations. The dilutions of plasma samples taken at an early stage of pregnancy were found to be parallel to the standard curves. No cross-reaction was observed with different carbohydrates, either with Pregnant Mare Serum Gonadotropin (PMSG) or human Chorionic Gonadotropin (hCG). The concentrations of PAG in pregnant females at day 30 after AI were shown to be higher with the use of antisera R#706, R#780, R#809 and Pool when compared with antiserum R#497. All the RIA systems gave 100% sensitivity and negative predictive values. On the other hand, the use of antisera R#780 and R#809 resulted in lower specificity and positive predictive values. The present study clearly shows that the ability of PAG-RIA systems to diagnose pregnancy specifically at day 30 after AI can be improved by using a combination of antisera raised against different forms of PAG.

Measurement of ovine pregnancy-associated glycoprotein (PAG) during early pregnancy in Lacaune sheep.

This study describes ovine pregnancy-associated glycoprotein (ovPAG) concentrations in 20 Lacaune sheep during early pregnancy. Measurements were performed by using semi-purified ovPAG as standard, tracer and immunogens for antibody production in rabbits. Antisera R780 (against ovPAG(57+59kDa)) and R805 (against ovPAG5(58+61kDa)) were used respectively in RIA-780 and RIA-805. Blood samples were collected at days 0, 18, 20, 22 and 25 after artificial insemination. From day 18 after breeding onward, the mean ovPAG concentration was significantly higher (p < 0.001) in plasma samples from pregnant ewes (n = 17) than in non-pregnant ones (n = 3). The specific activity of the tracer was 11 760 Ci/mmol in RIA-780 and 14 900 Ci/mmol in RIA-805. The minimal detection limits for RIA-780 and RIA-805 were 0.2 ng/ml and 0.3 ng/ml, respectively. The intra-assay CV of samples with low (1.0 ng/ml), medium (2.5 ng/ml) and high (4.0 ng/ml) PAG concentrations were 3%, 6% and 9% for RIA-780 and 8%, 9% and 5% for RIA-805. The inter-assay CV in the same samples were 13%, 12% and 7% for RIA-780 and 13%, 11% and 5% for RIA-805. The recovery was higher than 95% in both assays. No cross-reaction was observed with members of aspartic proteinase family as well as with other tested proteins. In both RIA-780 and RIA-805, inhibition of the binding of the tracer by antisera was parallel between standard curve and serial dilutions of pregnant ewe samples. In conclusion, the two homologous RIA systems are suitable for early quantification of ovPAG concentrations in ewe plasma samples from day 18 after breeding.

Influence of progesterone concentrations on secretory functions of trophoblast and pituitary during the first trimester of pregnancy in dairy cattle.

The essential role played by progesterone in the maintenance of pregnancy is unequivocal; however, the effects of progesterone on the secretory patterns of placental and pituitary molecules during the gestation period are not well defined. The objective of this study was to describe pregnancy-associated glycoprotein (PAG) concentrations (measured by RIA-497 and RIA-Pool) in pregnant females with progesterone concentrations lower (low-P4 group, n=20) or higher (high-P4 group, n=17) than the mean of 8.74 ng/mL on Day 21 (AI=Day 0). Luteinizing hormone (LH) and prolactin concentrations were also measured in both groups. Throughout the study period, blood samples were collected on Days 0, 21, 45, 60, and 80 from 37 females that were confirmed to be pregnant. PAG concentrations measured by both RIA-497 and RIA-Pool tended to be higher in high-P4 group than in low-P4 group from Day 30 until Day 80. On Day 80, plasma PAG concentrations that were measured using RIA-497 were observed to be higher (P<0.05) in the high-P4 group than in the low-P4 group (10.2+/-8.7 ng/mL versus 6.9+/-3.8 ng/mL). Concentrations of LH on Day 60 and prolactin on Day 80 were observed to be significantly lower (P<0.05) in the high-P4 group. There was a tendency for the concentrations of LH (Days 45 and 80) and prolactin (Days 30, 45, and 60) to be lower in cows in the high-P4 group than in the low-P4 group. Our results suggest the existence of a relationship among the concentration levels of progesterone, PAG, LH, and prolactin during early pregnancy.

Prolactin-induced activation of nuclear factor kappaB in bovine mammary epithelial cells: role in chronic mastitis.

We sought to determine whether prolactin (PRL) could influence the neutrophilic inflammation that characterizes chronic mastitis. Most of the genes encoding inflammatory proteins depend on the nuclear factor kappaB (NF-kappaB) for their expression. We addressed the hypothesis that immunomodulatory activities of PRL might arise from an increase in NF-kappaB activity. MAC-T cells, a bovine mammary epithelial cell line, were stimulated with increasing concentrations of bovine PRL (1, 5, 25, 125, and 1,000 ng/mL). Level of NF-kappaB binding activity was measured and mRNA was evaluated for IL-1beta, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GMCSF), IFN-gamma, and tumor necrosis factor (TNF)-alpha, cytokines known to require NF-kappaB for their maximal transcription. Prolactin activated NF-kappaB; maximal NF-kappaB activation was weaker with PRL than with TNF-alpha at 30 or 180 min poststimulation. In addition, PRL significantly amplified, in a dose-dependent manner, mRNA expression of IL-1beta, IL-6, IL-8, GMCSF, and TNF-alpha. We measured PRL concentrations in blood and milk from healthy and chronic mastitis-infected cows, and studied the relationship between the PRL concentration and the degree of inflammation in the mammary gland as indirectly assessed by somatic cell counts (SCC). Plasma PRL did not differ significantly between healthy and chronic mastitis-affected cows (63.7 and 67.5 ng/mL, respectively). Milk PRL concentration was significantly increased in chronic mastitis-affected quarters with the highest SCC, and had a positive significant correlation between SCC, as well as between the number of neutrophils present in milk samples. The present findings show that PRL promotes an inflammatory response in bovine mammary epithelial cells via NF-kappaB activation, and suggest a role for PRL in the pathogenesis of chronic mastitis.

Effect of the type and number of prostaglandin treatments on corpus luteum, the largest follicle and progesterone concentration in dairy cows.

Lactating dairy cows (n = 72) with a mature corpus luteum (CL) (diameter of > or = 17 mm) determined by ultrasonography and having a follicle with a diameter of > or = 10 mm were randomly assigned to four groups. Cows were treated with cloprostenol i.m. once or twice, or with dinoprost i.m. once or twice with an 8-h interval. The ovaries of each cow were scanned daily by transrectal ultrasonography to measure the changes in the areas of CL and the largest follicle and to determine the occurrence of ovulation. Oestrus was verified twice daily. In addition, blood sample was withdrawn from each cow daily for measuring progesterone (P4) concentrations. Significant decreases in the percentage changes relative to areas of CL and P4 concentrations or increases in the percentage changes in the area of the largest follicle on day 0 were detected in each group during the experiment. However, the type of the drug and the number of the treatments had no significant effect on those parameters. Cows ovulated with or without showing oestrus (group A) and cows exhibiting no oestrus and ovulation (group B) were also evaluated. In contrast to the mean area of the CL and the mean concentration of P4 on day 0, the mean area of the largest follicles between the two groups on day 0 differed significantly. Significant decreases in the percentage changes relative to the area of the CL and P4 concentration or increases in the percentage changes relative to the area of the largest follicle during the experiment were detected in both groups; however, there were no group differences. Treatment of dairy cows with two injections of prostaglandins (cloprostenol or dinoprost) at an 8-h interval resulted in more cows being observed in oestrus within 5 days after treatment and having significantly higher pregnancy rate than those treated with a single prostaglandin injection.

Serum osteocalcin and CTX-MMP concentration in young exercising thoroughbred racehorses.

Bone responds to exercise with changes in bone (re-)modelling, which might be monitored non-invasively with biochemical bone markers. The aim of this study was to evaluate the influence of exercise on serum osteocalcin and serum carboxy-terminal cross-linked telopeptide of type I collagen generated by matrix metalloproteinases (CTX-MMP) concentration in young racehorses. Seventy-one 2 to 4-year-old Thoroughbreds were included in this prospective infield study. Blood sampling was performed six times (i.e. six sampling cycles) during a 9-month period. Serum samples were analysed with commercial osteocalcin and CTX-MMP radioimmunoassays. Two-year-old racehorses had higher serum osteocalcin and CTX-MMP values than 3-year-old horses. Gender and training amplitude did not significantly influence serum osteocalcin and CTX-MMP values. Two-year-old horses showed an increase in osteocalcin values between cycles 2 and 3 and an increase in serum CTX-MMP values between cycles 1 and 2. Serum osteocalcin and CTX-MMP concentrations decreased between cycles 4 and 5, and 5 and 6. Three-year-old horses showed an increase in serum osteocalcin levels between cycles 3 and 4 and an increase in serum CTX-MMP concentrations between cycles 1 and 2, and 3 and 4. Serum osteocalcin levels decreased between cycles 5 and 6, whereas serum CTX-MMP levels decreased between cycles 4 and 5, and 5 and 6. Two- and three-year-old horses showed a decreased osteocalcin/CTX-MMP ratio between cycles 1 and 2. Moreover, 2-year-old horses showed an increase in the osteocalcin/CTX-MMP ratio between cycles 2 and 3. Sore shin formation did not significantly influence serum osteocalcin and CTX-MMP values. Serum osteocalcin and CTX-MMP are promising bone markers for monitoring exercise induced changes in equine bone metabolism.

Effect of different doses of prostaglandin on the area of corpus luteum, the largest follicle and progesterone concentration in the dairy cow.

Lactating dairy cows with a mature corpus luteum (CL) (diameter of >/=17 mm determined by ultrasonography) and having a follicle with a diameter of 10 mm (n = 49) were randomly assigned to three groups. The first group was treated with a single dose of exogenous prostaglandin (25 mg), while the second group was treated with 35 mg on day 0, and the third group served as control in order to evaluate the effect of rectal manipulation on the CL during ultrasonographic examination. Blood samples were collected daily for analysing progesterone (P4) concentrations. In group 1 the incidence of oestrus and artificial insemination (AI) in 10 days after treatment was 95% (19 of 20). The conception rate was 31.6%, and the average time to oestrus after treatment was 3.7 day. In group 2 the incidence of oestrus and A.I. was 84.2% (16/19). The conception rate was 31.2%, and the average time to oestrus after treatment was 2.8 day. In the untreated group only two cows (2/10) exhibited oestrus during the examined period and none of them became pregnant. There were no significant differences between the two treated groups in terms of reduction in the area of CL and P4 concentrations and of an increase in the area of the dominant follicles. At the same time, the decrease in the percentage changes relative to the area of CL and to the concentrations of P4 were statistically significant in both treated groups.

An improved radioimmunoassay for measurement of pepsinogen in porcine blood samples.

The study was conducted to develop a sensitive and specific radioimmunoassay (RIA) for the measurement of pepsinogen in porcine serum, and to use this test for the determination of pepsinogen concentrations in serum samples from fetuses and pigs of different ages. Compared to a previously described RIA, major improvements were made concerning the use of specific polyclonal antibodies and the use of an appropriate buffer. The assay was able to detect pepsinogen concentrations of >/=0.2 ng/mL. The recovery of pepsinogen was close to 95%. The intra-assay coefficients of variations ranged between 3.9 and 7.5% whereas the interassay ranged between 8.8 and 11.9%. These percentages correspond to a satisfactory accuracy and reproducibility of the assay. No cross-reactions were observed with the main commercially available products of the aspartic proteases family except porcine pepsin cross-reacted over 62.5 microg/mL. Pepsinogen concentrations increased steadily with increasing age of the fetuses and the pigs (P<0.05). Pepsinogen concentrations (+/-SE) in fetuses of 90-100 (n=24) and 100-110 days of pregnancy (n=36) were 0.5+/-0.1 and 5.3+/-1.3 ng/mL, respectively. In pigs of 21, 98, and 213 days of age, the pepsinogen concentrations were 290.6+/-10.8, 343.1+/-17.9 and 383.5+/-15.3 ng/mL, respectively. The results demonstrate that RIA is accurate and can be used easily to assess pepsinogen concentrations in pig sera. The test may constitute a valuable tool in epidemiological surveys and in studies related to gastric diseases in pigs.

Effect of induction of late embryonic mortality on plasma profiles of pregnancy associated glycoprotein 1 in heifers.

Inoculation with Actinomyces pyogenes and administration of prostaglandin (PG)F(2alpha) were used to induce late embryonic mortality (LEM) in heifers (n=8) on Days 30-38 of pregnancy in order to compare the profile for bovine pregnancy associated glycoprotein 1 (PAG1), progesterone and 15-keto-13,14-dihydro-PGF(2alpha) (PGFM). Two pregnant heifers were used as controls. Inoculation into the uterine body caused LEM, as established by ultrasonography in each heifer within 24h of treatment. When the inoculum was injected into the first part of the cervix, LEM occurred in one of two heifers (Heifer A) between 48 and 72 h after treatment. Similarly, PGF(2alpha) treatment caused LEM in three of four heifers. In six of eight heifers, PAG1 started to decrease steadily when it was accompanied by the subsequent death of the embryo. Inoculation through the cervix caused luteolysis in three of four heifers within 6-10 days after induction. After induction of LEM, PGFM concentrations showed a two to 3.8 fold increase in three of four heifers during the following six days, and from that time changed within normal ranges. The results of this study indicate that a PAG1 assay may provide an alternative method to ultrasonography for determining LEM in the cow.

Comparison of the ability of three radioimmunoassay to detect pregnancy-associated glycoproteins in bovine plasma.

CONTENTS: Pregnancy-associated glycoproteins (PAGs) constitute a large family of glycoproteins that are synthesized in the superficial layer of the ruminant placenta according to a spatial and temporal expression pattern. When PAGs are released in the maternal blood they can be used for pregnancy diagnosis, pregnancy follow-up and for the monitoring of the trophoblastic function. Three different radioimmunoassay systems (RIA 1, RIA 2 and RIA 3) using antisera produced against PAG I67 (RIA 1), PAG55+62 (RIA 2) and PAG55+59 (RIA 3) were used in this investigation in order to measure the PAG concentration in plasma samples withdrawn from pregnant cows and heifers during different periods following artificial insemination (AI). These systems were able to detect PAG molecules in the maternal blood as early as 21 days after AI in different concentrations (RIA 1: 0.43 +/- 0.24 ng/ml, mean +/- SD; RIA 2: 0.48 +/- 0.24 ng/ml; RIA 3: 0.64 +/- 0.37 ng/ml). On days 32 and 42 RIA 2 (4.30 +/- 1.32 ng/ml and 5.56 +/- 1.95 ng/ml) and RIA 3 (4.17 +/- 1.15 ng/ml and 5.60 +/- 1.89 ng/ml) presented significantly (p < 0.0001) higher PAG concentrations than those of RIA 1 (2.43 +/- 0.81 ng/ml and 4.01 +/- 1.48 ng/ml), respectively. After day 21, significant correlations (p < 0.0001; r >/= 0.929) were determined between the three systems. Additionally the three individual PAG profiles presented in this study showed that PAG molecules secreted in the maternal blood between 21 and 50 days after AI were better recognized by the RIA 2 and RIA 3 systems. This study clearly indicated that the ability of a RIA test to recognize PAG molecules in the maternal blood can be improved by carefully selecting the antiserum.

Increase of plasma eCG binding rate after administration of repeated high dose of eCG to cows.

Equine chorionic gonadotrophin (eCG) is still used to promote follicular growth in cattle and, more recently with an increased frequency of administration, in ovum pick-up protocols. The aim of this experiment was to verify the possible effect of high frequency of administration on the immune response to eCG. The profiles of eCG binding rate, in the blood of two groups (A, B) of 4 primiparous cross breed beef cows (3-3.5 years old) submitted weekly for 5 to 10 weeks to repeated high doses (1000-2000 IU) of equine chorionic gonadotrophin, are presented in this paper. A sensitive radiometric method was used to detect antibodies in plasma. The profiles clearly indicated a marked increase of eCG binding rate after 3 to 5 injections of the exogenous hormone to the females. The statistical analysis of the results established that treatments induced a significant increase (P < 0.01) in binding rates after 6 and 3 injections in group A and B respectively. These binding rates remained elevated for at least 1 week following the last injection and decreased afterwards. The values of plasma binding rates following repeated eCG administration differed significantly between groups (0.90+/-1.04 and 1.04+/-0.11 for groups A and B before treatment versus 11.77+/-0.92, 6.70+/-0.85 for groups A and B after treatment, P < 0.01) and from one cow to another (P < 0.01) with some cows presenting no significant immune response while others were more reactive against the hormone (at least 3 injections).