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.     

Early pregnancy diagnosis in goats by determination of pregnancy-associated glycoprotein concentrations in plasma samples

Different RIA systems available for measuring the concentrations of pregnancy-associated glycoproteins (PAGs) in dairy goats were compared in order to evaluate their accuracy in early pregnancy diagnosis. Plasma concentrations of PAGs were determined by 3 heterologous RIA systems with a bovine PAG standard and tracer in combination with antisera anti-ovine PAG (RIA 1), anti-caprine PAG55 + 62 (RIA 2), anti-caprine PAG55 + 59 (RIA 3), and by 2 homologous RIA systems that employed caprine PAG55 + 62 and caprine PAG55 + 59 and their specific antisera (RIAs 4 and 5, respectively). In all of the RIAs, the mean concentrations of PAGs were significantly higher (P < 0.01) in pregnant than in nonpregnant goats from Day 21 onwards after breeding. On Day 21, the accuracy rates of early pregnancy diagnoses were 56% (RIA 1), 96% (RIA 2), 99% (RIA 3), 95% (RIA 4) and 90% (RIA 5), whereas on Day 28 these rates were > 99% for RIAs 2, 3, 4 and 5. The RIAs for PAGs depend on proteins from the placenta being present in maternal plasma and require only a single sample of blood, to distinguish pregnant goats from those that fail to return to estrus for other reasons. The homologous and semi-heterologous assays are highly accurate as early as Day 21 of pregnancy.     

Early pregnancy diagnosis in sheep using near-infrared spectroscopy on blood plasma

The objective of this study was to evaluate the ability of near-infrared reflectance spectroscopy (NIRS) to discriminate between pregnant and nonpregnant ewes in early stages of pregnancy after artificial insemination (AI) from blood plasma. Samples were collected using jugular puncture at 18 and 25 days after AI from 188 Rasa Aragonesa and Ansotana ewes. Plasma samples were analyzed for pregnancy-associated glycoprotein (PAG) and progesterone (P4) using ELISA commercial kits. The spectra of plasma samples were recorded in the visible and near-infrared ranges. The performance of these tests were compared, using as criterion standard the pregnancy status determined using transabdominal ultrasonography at 45 days after AI. Pregnancy rate was 47.9% (90/188). At Day 18, sensitivity was similar in NIRS and P4 tests (98.9% vs. 100%; not significant) and greater than PAG (32.2%; both P < 0.001). Specificity was similar in NIRS and PAG tests (both 100%) and greater than that of P4 (84.7%; P < 0.001). At Day 25, sensitivity and specificity of NIRS and PAG were both 100%. It can be concluded that NIRS was an accurate method of diagnosis of pregnancy at Days 18 and 25 after AI in ewes.     

Comparison of accuracy of ultrasonography, progesterone, and pregnancy-associated glycoprotein tests for pregnancy diagnosis in semidomesticated reindeer

The aim of the study was to compare transrectal ultrasound with progesterone (P4) and pregnancy-associated glycoproteins (PAGs) as pregnancy detection methods for semidomesticated reindeer (Rangifer tarandus tarandus) in field conditions. Female reindeer (n = 195) were scanned transrectally by a 7.5-MHz linear array transducer, and blood was sampled either in December 2005 (n = 33), December 2006 (n = 92), or January 2007 (n = 70) during early or mid gestation. Plasma levels of P4 and PAGs were assessed by radioimmunoassay (RIA). Based on calving records, the sensitivity, specificity, predictive values, and the overall accuracy of the three tests were calculated. The overall calving rate calculated from the calving records was 86.2%. The overall accuracy of transrectal ultrasound was 99.5%. The sensitivity and specificity of transrectal ultrasound were 99.4% and 100%, respectively. In the plasma P4 test, the threshold level of 5.0 nmol/L gave the highest overall accuracy (94.9%). The sensitivity of the P4 test decreased from 96.4% to 81.5%, when the threshold level increased from 5.0 nmol/L to 8.0 nmol/L, while the specificity remained at 85.2% over the range of these cutoff values. The overall accuracy of the plasma PAG test decreased from 96.4% to 64.1% when the plasma PAG threshold level increased from 0.5 ng/mL to 3.5 ng/mL, whereas sensitivity decreased from 99.4% to 58.3%. Specificity increased from 77.8% to 100% when the plasma PAG threshold level reached 3.0 ng/mL. Transrectal ultrasound showed higher diagnostic values than those of plasma P4-RIA and PAG-RIA in diagnosing pregnancy of reindeer, with the advantage that diagnoses can be made in real time in field conditions.     

Early pregnancy diagnosis in sheep by progesterone and pregnancy-associated glycoprotein tests

The aim of this study was to compare the accuracy of the progesterone (P4) and pregnancy associated glycoprotein (PAG) tests for determination of early pregnancy in sheep. Estrus was synchronized in 182 Awassi x Merino ewes and blood samples were collected at Days 0 (day of the insemination), 18, 22, 29, 36, and 50 after artificial insemination (AI). Plasma P4 concentrations at Days 0 and 18 were determined by double antibody radioimmunoassay, while PAG concentrations at Days 22, 29, 36 and 50 were determined by a heterologous, double-antibody radioimmunoassay (RIA) using the bovine PAG 67 kDa subunit as tracer and standard and rabbit antiserum raised against a mixture of caprine 55 and 59 kDa PAG subunits as the first antibody. The discriminatory value for diagnosis of pregnancy by the P4 and the PAG-RIA tests was > or = 1 ng/ml. Based on lambing data, the accuracy for diagnosing pregnant (sensitivity) and non-pregnant ewes (specificity) and predictivity of both tests were calculated. The sensitivity, specificity, positive and negative predictive values for P4 and PAG tests were 100, 95.4, 81.6, and 100% at Day 18 (P4) and 93.5, 100, 100 and 98.7% at Day 22 (PAG), respectively. For diagnosis of non-pregnant ewes the PAG test had significantly higher specificity than the P4 test (P < 0.05). It is concluded that ovine pregnancy can be reliably diagnosed at Day 22 after AI by using a heterologous radioimmunoassay of PAG.     

Autoimmunization of ewes against pregnancy-associated glycoproteins does not interfere with the establishment and maintenance of pregnancy

Pregnancy-associated glycoproteins (PAGs) are a large grouping of placental proteins that belong to the aspartic peptidase gene family. Although useful to detect pregnancy in ruminant species, the function of these molecules is unclear. Several PAGs expressed by trophoblast binucleate cells can enter the maternal circulation, suggesting that they could have a systemic role in altering maternal physiology. The objective of this work was to examine whether these circulating placental antigens were important in pregnancy by actively immunizing ewes against them. PAGs were purified by pepstatin-affinity chromatography and conjugated to the immunogenic protein, keyhole limpet hemocyanin (KLH). Ewes were immunized with PAG-KLH conjugate (n = 22) or with KLH alone (n = 9), and bred to intact rams. Blood samples, collected on Day 0 (day of estrus), Day 10, Days 15 to 25 and weekly throughout pregnancy, were analyzed for PAG by an ELISA. On Day 30, pregnancy was confirmed by ultrasound. Ewes immunized against PAG-KLH produced a range of reactive anti-PAG titers, whereas all immunized ewes had high anti-KLH immunoreactivity. PAGs became detectable in the anti-KLH (control) ewes at Day 21.6 ± 2.2 of pregnancy. Those ewes immunized against PAGs (n = 7), that had very low immunoreactivity toward PAGs, had measurable PAG by Day 22.9 ± 1.3, and their PAG serum profiles throughout pregnancy did not differ from the controls. Those exhibiting moderate to high anti-PAG immunoreactivity (n = 15), had significantly lower PAG concentrations than controls, with antigen not becoming detectable until Day 48.1 ± 15.6. The decrease in circulating PAG in the immunized animals did not correlate with changes in pregnancy rates, lamb number or lamb birth weight. These results suggest that while PAGs may play a role in maintaining pregnancy, their major contribution is likely to be at the fetal-maternal interface. Their actions at extra-placental sites are presumably of more secondary importance.     

Uterine secretion of prostaglandin F2 alpha in response to oxytocin in ewes: changes during the estrous cycle and early pregnancy.

Experiment 1 was conducted to determine when the ovine uterus develops the ability to secrete prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin and how development is affected by pregnancy. Pregnant and nonpregnant ewes received an injection of oxytocin (10 IU, i.v.) on Day 10, 13, or 16 postestrus. Jugular venous blood samples were collected for 2 h after injection for quantification of 13,14-dihydro-15-keto-PGF2 alpha (PGFM). In nonpregnant ewes, concentrations of PGFM increased following oxytocin on Day 16 but not on Day 10 or 13. Concentrations of PGFM did not increase following treatment on Day 10, 13, or 16 in pregnant ewes. Therefore, the ability of oxytocin to induce uterine secretion of PGF2 alpha develops after Day 13 in nonpregnant but not in pregnant ewes. Experiment 2 was conducted to precisely define when uterine secretory responsiveness to oxytocin develops. Pregnant and nonpregnant ewes received oxytocin on Day 12, 13, 14, or 15. In nonpregnant ewes, concentrations of PGFM increased following treatment on Days 14 and 15, but not earlier. Peripheral concentrations of progesterone showed that uterine secretory responsiveness to oxytocin developed prior to the onset of luteal regression. As in experiment 1, the increase in concentrations of PGFM following administration of oxytocin was much lower in pregnant than in nonpregnant ewes; however, some pregnant ewes did respond to oxytocin with an increase in PGFM. In experiment 3, pregnant ewes received an injection of oxytocin on Day 18, 24, or 30 postmating. Concentrations of PGFM increased following oxytocin on Days 18 and 24. The conceptus appears to delay and attenuate the development of uterine secretory responsiveness to oxytocin.     

The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers

The pregnancy-associated glycoproteins (PAGs) are a large gene family expressed in trophoblast cells of ruminant ungulates. The detection of PAGs (more specifically, PAG-1) in maternal serum has served as the basis for pregnancy detection in cattle. Unfortunately, PAG-1 and/or antigenically-related PAGs exhibit a long half-life in maternal serum (>8 d) and can be detected 80-100 d post-partum, thereby producing false positives in animals bred within 60-d of calving. The goal of the present studies was to develop a monoclonal-based assay that targeted early-pregnancy PAGs whose persistence in maternal serum post-partum might be relatively short-lived. Three anti-PAG monoclonal antibodies that recognized distinct subsets of PAGs were selected and used as trapping reagents in a 'sandwich' type of enzyme-linked immunosorbant assay (ELISA). A polyclonal antiserum with broad specificity was used for detecting bound PAGs. A total of 42 cows and heifers were bled daily on day 15, days 22 to 28, and then weekly throughout pregnancy and for 10 weeks (approximately 70 d) into the post-partum period. The ELISA was able to detect PAG in maternal serum of all animals unambiguously by day 28 post-insemination (PAG concentration: 8.75 +/- 3.04 ng/mL). In maternal serum, PAG concentrations peaked during the week of parturition at 588.9 +/- 249.9 ng/mL, and after calving, PAG was completely cleared (half-life: 4.3 d) by eight-week post-partum in 38 of 40 of the animals tested and was at very low concentrations in the remaining two (1.4 and 4.9 ng/mL, respectively). In summary, a monoclonal-based assay has been established that is sensitive enough to detect PAG in maternal serum by the forth week of pregnancy, but does not suffer from carry-over of antigen from a previous pregnancy.     

Myometrial activity and plasma progesterone and oxytocin concentrations in cycling and early-pregnant ewes

Myometrial activity and plasma progesterone (P) and oxytocin (OT) were measured in early pregnant (n = 5) and cycling (n = 5) ewes. Electromyography (EMG) leads and jugular and inferior vena cava (IVC) catheters were surgically placed in ewes about 1 wk before data collection. When ewes returned to estrus, they were bred to either an intact or vasectomized ram. Continuous EMG data were collected, and blood samples were collected twice daily from day of estrus (Day 0) until Day 18. Ewes bred with an intact ram were checked surgically for pregnancy on Day 20. Computerized, quantitative analysis of EMG events showed no difference in signal from the right to left uterine horns, and no differences between pregnant and cycling ewes (p less than 0.05) until Days 14-18 when nonpregnant ewes returned to estrus and had increased EMG activity. The mean number of EMG events 180-900 s in length decreased in pregnant ewes, but this difference was not significant (p less than 0.05). Jugular plasma progesterone (P) levels confirmed corpus luteum (CL) formation in all ewes, and no differences in P between pregnant and nonpregnant ewes were measured until Days 14-18, when cycling ewes underwent luteolysis and pregnant ewes maintained CL. IVC plasma oxytocin concentrations were increased in pregnant ewes compared to concentrations in nonpregnant ewes on Days 5-13 (p less than 0.05), and the difference was largest at Day 6 (means +/- SEM pg/ml: pregnant = 68.7 +/- 13.9, nonpregnant = 30.9 +/- 19.9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptors for prostaglandins F2 alpha and E2 in ovine corpora lutea during maternal recognition of pregnancy.

Plasma membrane receptors for prostaglandins (PG) F2 alpha and E2 were quantified in ovine corpora lutea obtained from nonpregnant and pregnant ewes on Days 10, 13, and 15 post-estrus, and from additional ewes on Days 25 and 40 of pregnancy. Regardless of reproductive status or day post-estrus, concentrations of luteal receptors for PGF2 alpha were 7- to 10-fold greater than those for PGE2. In pregnant ewes the concentration of receptors for PGF2 alpha was highest on Day 10 (35.4 +/- 2.8 fmol/mg) and lowest on Day 25 (22.3 +/- 2.5 fmol/mg). A difference in the concentration of luteal receptors for PGF2 alpha between pregnant and nonpregnant ewes was apparent only on Day 15 post-estrus, at which time the concentration of receptors for PGF2 alpha was higher in pregnant ewes than in nonpregnant ewes (27.1 +/- 2.7 vs. 17.7 +/- 2.7 fmol/mg). Concentrations of receptors for PGE2 in pregnant ewes were similar (p > 0.05; 2.8 +/- 0.3 to 3.7 +/- 0.2 fmol/mg) between Days 13 and 40 but were higher (p < 0.05) than in corpora lutea obtained from nonpregnant ewes on Days 10 (5.0 +/- 0.4 vs. 4.1 +/- 0.2 fmol/mg) and 15 (3.7 +/- 0.2 vs. 2.0 +/- 0.4 fmol/mg) post-estrus. Although concentrations of receptors for both PGF2 alpha and PGE2 were lowest in corpora lutea obtained from nonpregnant ewes on Day 15, this was not due to luteal regression since the weights and concentrations of progesterone in corpora lutea on Day 15 were not lower than those for corpora lutea obtained on Days 10 and 13.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pregnancy-associated glycoproteins (PAG) detection in milk samples for pregnancy diagnosis in dairy goats

In ruminants, glycoproteins synthesized in the outer layers of the trophoblast enter the maternal bloodstream. The assay of their concentration in plasma or in serum can be used for pregnancy diagnosis and to indicate feto-placental health status. In this study, concentrations of the pregnancy-associated glycoprotein (PAG) were investigated in the milk of dairy goats. The mean concentrations of PAG in milk were significantly higher in pregnant than in non-pregnant goats throughout the sampling period. Positive milk PAG levels were found in all pregnant animals from Day 32 after breeding, but remained at negative values in nonpregnant ones. This study reports for the first time a pregnancy diagnosis based on milk concentrations of PAG, that could be of great use on the farm.     

Plasma urea nitrogen in relation to pregnancy rate in dairy sheep

The aim of this field study was to investigate the relationship of plasma urea nitrogen (PUN) with the pregnancy rate in lactating Awassi × Merino ewes. One hundred and eighty-five Awassi × Merino ewes were used in the present study. Ewes were fed a diet containing 17.4% crude protein and were milked twice a day by the milking machine. The ewes were synchronized for estrus by insertion of intravaginal sponges containing 30 mg flurogestone acetate for 14 days. At the time of sponge removal each ewe was administered eCG (600 IU). All ewes were inseminated twice with fresh semen into the external os of the cervix at 48 and 56 h after sponge removal. The day of insemination was considered as Day 0 for calculating the gestational period. Blood samples were collected from each ewe at Days 0, 18 for measurement of PUN concentrations and at Day 22 after AI for measurement of pregnancy-associated glycoprotein (PAG) by radioimmunoassay (RIA). Thirty-eight ewes (20.5%) were confirmed pregnant by PAG-RIA test at Day 22 and by ultrasonography at Day 80. The mean (±S.D.) concentration of PUN in all ewes at Day 0 was 12.7±4.6 mmol/L. There were non-significant differences in the level of PUN between pregnant and non-pregnant ewes at Days 0 (12.2±4.2 mmol/L vs. 12.8±4.7 mmol/L, respectively) and 18 (9.6±2.9 mmol/L vs. 10.4±4.0 mmol/L, respectively) after AI. Mean PUN concentrations decreased significantly from Day 0 to Day 18 after AI in both pregnant and non-pregnant ewes. By using logistic regression analysis, there was no effect of PUN concentrations on the probability of pregnancy occurrence in the studied ewes (odds ratio: 0.97; 95% confidence interval: 0.9-1.05; P=0.45). In conclusion, there was no evidence of a relationship between PUN concentration and pregnancy rate for lactating Awassi × Merino ewes in the present study because of low pregnancy rate observed.     

Pregnancy rates and corpus luteum-related factors affecting pregnancy establishment in bovine recipients synchronized for fixed-time embryo transfer

The objective was to investigate the influence of corpora lutea physical and functional characteristics on pregnancy rates in bovine recipients synchronized for fixed-time embryo transfer (FTET). Crossbred (Bos taurus taurus × Bos taurus indicus) nonlactating cows and heifers (n = 259) were treated with the following protocol: 2 mg estradiol benzoate (EB) plus an intravaginal progesterone device (CIDR 1.9 g progesterone; Day 0); 400 IU equine chorionic gonadotropin (eCG; Day 5); prostaglandin F_2α (PGF_2α) and CIDR withdrawal (Day 8); and 1 mg EB (Day 9). Ovarian ultrasonography and blood sample collections were performed on Day 17. Of the 259 cattle initially treated, 197 (76.1%) were suitable recipients; they received a single, fresh, quality grade 1 or 2 in vivo-derived (n = 90) or in vitro-produced (n = 87) embryo on Day 17. Pregnancy rates (23 d after embryo transfer) were higher for in vivo-derived embryos than for in vitro-produced embryos (58.8% vs. 31.0%, respectively; P < 0.001). Mean (±SD) plasma progesterone (P₄) concentration was higher in cattle that became pregnant than that in nonpregnant cattle (5.2 ± 5.0 vs. 3.8 ± 2.4 ng/mL; P = 0.02). Mean pixel values (71.8 ± 1.3 vs. 71.2 ± 1.1) and pixel heterogeneity (14.8 ± 0.3 vs. 14.5 ± 0.5) were similar between pregnant and nonpregnant recipients (P > 0.10). No significant relationship was detected between pregnancy outcome and plasma P₄, corpus luteum area, or corpus luteum echotexture. Embryo type, however, affected the odds of pregnancy. In conclusion, corpus luteum-related traits were poor predictors of pregnancy in recipients. The type of embryo, however, was a major factor affecting pregnancy outcome.     

Failure to maintain interspecific pregnancy after transfer of Dall's sheep embryos to domestic ewes

Over a 3-year period, 32 Dall's sheep (Ovis dalli dalli) embryos were transferred into 24 domestic sheep (O. aries) recipients and 4 were transferred into 2 Dall's sheep recipients. In the first year, none of the 10 O. aries recipients was diagnosed pregnant. In the following 2 years, 9 (37%) of the domestic sheep recipients were pregnant on Day 18, 8 (33%) on Day 40, 6 (25%) on Day 90 and 4 (16%) on Day 120; 1 aborted at Day 125 and another at Day 145. Pregnancies were established only in ewes that had previously been recipients of Dall's sheep embryos. The 2 remaining pregnant sheep were treated with progesterone from Day 125 until the fetuses were determined to be dead at Day 145. Both of the Dall's sheep recipients (Year 2) established pregnancies; 1 live Dall's sheep lamb was born 174 days after mating. No differences in serum progesterone, oestrone, prostaglandin F-2 alpha metabolites or cortisol concentrations could be detected during pregnancy between recipients carrying Dall's sheep embryos, recipients receiving progesterone treatment or domestic ewes carrying domestic sheep pregnancies. Six fetuses were necropsied (1 at Day 125 and 5 at Day 145-146): all fetuses were premature and had various degrees of hydranencephaly. No significant differences were found when cotyledon numbers were compared among domestic ewes carrying Dall's sheep lambs. Dall's sheep ewes lambing naturally and domestic ewes lambing naturally. These results demonstrate that the transfer of Dall's sheep embryos to domestic ewes results in the establishment but subsequent loss of pregnancy and that these losses occur throughout gestation.     

Pregnancy-associated changes in plasma concentration of the endocrine disruptor di(2-ethylhexyl) phthalate in a sheep model

The plasticizer di(2-ethylhexyl)phthalate (DEHP), used for producing polyvinyl chloride (PVC), acts as an endocrine disruptor with toxic effects on reproductive and developmental processes. Exposure to DEHP in humans is mainly by environment and food. Thus, our aim was to determine plasma levels in livestock animals using the ewe (Ovis aries) as a model. In a first trial, 150 samples from ewes of different ages (2 to 7 yr) and reproductive status (pregnant and nonpregnant) were analyzed by high-performance liquid chromatography (HPLC). DEHP was detected in 34.7% of the samples, with a mean level of 0.45 ± 0.01 μg/mL (range, 0.05 to 2.81 μg/mL). The percentage of nonpregnant animals with DEHP traces was higher in animals older than 4 yr (n = 66, 37.9%) than in younger animals (n = 69, 17.4%; P < 0.05), although the mean levels in ewes with residues were similar (0.16 ± 0.01 vs. 0.16 ± 0.02 μg/mL). All the pregnant ewes (n = 15) showed presence of DEHP, with higher plasma levels than that in nonpregnant females (1.42 ± 0.18 vs. 0.16 ± 0.01 μg/mL; P < 0.0001). For confirming the effect of pregnancy on mobilization of DEHP from body fat, 101 ewes of the same age were sampled in a second trial at a different farm. The percentage of animals with DEHP traces was higher in pregnant ewes (n = 32, 71.9%; P < 0.005) than in nonpregnant ewes (n = 37, 35.1%) or in ewes that recently gave birth (n = 32, 21.9%), although mean levels were similar (0.42 ± 0.02, 0.33 ± 0.02, and 0.34 ± 0.05 μg/mL, respectively). In conclusion, current results indicate a high incidence of ewes reared in the field showing accumulation of phthalates; percentage of animals with presence of DEHP increases with age, due to an extended period of exposure, but mainly during pregnancy, due to the mobilization of body reserves.     

Altered secretion of pregnancy-associated glycoproteins during gestation in bovine somatic clones

Somatic nuclear transfer (NT) in cattle is often accompanied by severe placental anomalies, hypertrophy, and hydrallantois, which induce a high rate of pregnancy losses throughout gestation. These placental deficits are associated with an abnormal increase of the maternal plasma levels of pregnancy-associated glycoprotein (PAG), produced by the trophoblastic binucleate cells (BNC) of the placenta. The objective of this study was to analyze the origin of the abnormally elevated PAG concentrations in the peripheral circulation of NT recipients during pathological pregnancies. Concentrations of PAG were measured both in maternal blood, in chorionic and cotyledonary tissular extracts from control recipients (after artificial insemination, AI, or in vitro fertilization, IVF) and clone recipients on Day 32, Day 62, and during the third trimester of gestation. Three different radioimmunoassay (RIA) systems were used. One homologous RIA for PSP60, similar to bovine PAG-1 (PAG_67kDa), and two heterologous RIA with PAG_67kDa as standard and tracer, and antisera anti-caprine PAG (AS#706 and AS#708). Circulating and tissular concentrations of bovine placental lactogen (bPL), a glycoprotein also produced by BNC, were determined by RIA at the same stages. The number of BNC in the placental tissues was determined by cell counting after immunostaining with anti PSP60 antibody on tissue sections from control and NT pregnancies. Maternal plasma PAG concentrations were not different among groups on Day 32, but they were significantly higher in NT than in control pregnancies on Day 62 with all three RIA and during the third trimester with two RIA (RIA-PSP60 and RIA with AS#708). Circulating bPL concentrations were undetectable on Days 32 and 62 and were not different in the third trimester between NT and control pregnancies. Tissular amounts of PAG on total proteins were not different between the two groups at all stages studied. No difference was determined in the percentage of PSP60-positive BNC in placental tissues between controls and NT on Day 62 and during the third trimester of pregnancy. Western blots of tissular extracts from placenta showed no major molecular weight changes of PAG in NT pregnancies compared to controls. No differences in maternal circulation concentrations or tissular content of bPL were observed between control and NT pregnancies. In conclusion, the specific increase of PAG in maternal plasma concentrations during abnormal NT pregnancies do not result from a higher proportion of BNC, or an increased protein expression of PAG and could be due to changes in the composition of terminal glycosylation which result into a clearance decrease of PAG from the circulation.     

Comparison of accuracy of ultrasonography,progesterone, and pregnancy-associated glycoprotein tests for pregnancy diagnosis in semidomesticated reindeer

The aim of the study was to compare transrectal ultrasound with progesterone (P4) and pregnancy-associated glycoproteins (PAGs) as pregnancy detection methods for semidomesticated reindeer (Rangifer tarandus tarandus) in field conditions. Female reindeer (n = 195) were scanned transrectally by a 7.5-MHz linear array transducer, and blood was sampled either in December 2005 (n = 33), December 2006 (n = 92), or January 2007 (n = 70) during early or mid gestation. Plasma levels of P4 and PAGs were assessed by radioimmunoassay (RIA). Based on calving records, the sensitivity, specificity, predictive values, and the overall accuracy of the three tests were calculated. The overall calving rate calculated from the calving records was 86.2%. The overall accuracy of transrectal ultrasound was 99.5%. The sensitivity and specificity of transrectal ultrasound were 99.4% and 100%, respectively. In the plasma P4 test, the threshold level of 5.0 nmol/L gave the highest overall accuracy (94.9%). The sensitivity of the P4 test decreased from 96.4% to 81.5%, when the threshold level increased from 5.0 nmol/L to 8.0 nmol/L, while the specificity remained at 85.2% over the range of these cutoff values. The overall accuracy of the plasma PAG test decreased from 96.4% to 64.1% when the plasma PAG threshold level increased from 0.5 ng/mL to 3.5 ng/mL, whereas sensitivity decreased from 99.4% to 58.3%. Specificity increased from 77.8% to 100% when the plasma PAG threshold level reached 3.0 ng/mL. Transrectal ultrasound showed higher diagnostic values than those of plasma P4-RIA and PAG-RIA in diagnosing pregnancy of reindeer, with the advantage that diagnoses can be made in real time in field conditions.     

Ultrasound fetal measurements and pregnancy associated glycoprotein secretion in early pregnancy in cattle recipients carrying somatic clones

Somatic cloning in the bovine species leads to high levels of fetal losses which occur throughout pregnancy. These losses are most often associated with fetal overgrowth, a syndrome known as large offspring syndrome (LOS), and excessive maternal plasma pregnancy serum protein 60 (PSP60), a protein similar to a pregnancy-associated glycoprotein of 67 kDa (PAG I67) produced by the bovine placenta. Predicting the outcome of pregnancies initiated from cloned embryos has become an important issue both to prevent potential harm to the mother because of excessive fetal size at birth and also to get a better understanding of the relationships between growth, differentiation and placental functions in developing cloned fetuses. Here, we report on a systematic analysis of fetal and placental development in the first trimester of pregnancy performed by ultrasonographic imaging and by measurement of the maternal concentrations of pregnancy associated glycoproteins (PAGS), using four different radioimmunoassays (RIA) (two homologous RIA systems with PSP60 and PAG I67; two heterologous RIA systems with PAG I67 as standard and tracer, and antisera anti-caprine PAGs). We showed that crown-rump length (CRL) in clones appeared smaller than controls at 35, 50 and 62 days (P<0.05). At 62 days of pregnancy, CRL in cloned fetuses that died before 90 days was smaller compared to the other cloned fetuses (P<0.05) whereas the width of the fetal sack and the biparietal diameter (BPD) was larger in fetuses that developed LOS in late gestation (P<0.05). Maternal PAGs concentrations were statistically different between controls and all clone recipients as early as Day 34, suggesting early abnormal placental glycoprotein synthesis for clone pregnancies regardless of pregnancy outcome. This work provides a practical, non-invasive tool to follow up clone pregnancies and suggests that primary growth retardation and abnormal placental function precedes excessive fetal and placental growth at later stages of pregnancy.     

Interspecies pregnancy of Spanish ibex (Capra pyrenaica) fetus in domestic goat (Capra hircus) recipients induces abnormally high plasmatic levels of pregnancy-associated glycoprotein

Interspecies embryo transfer could be a valuable tool in preservation programs of endangered species. In this work the results of both interspecific-monospecific (ibex-in-goat) and interspecific-bispecific (mixed-species; ibex+goat-in-goat) embryo transfers in the capra genus are reported. The aim of this work was to compare the PAG plasmatic profiles occurring in these interspecific gestations to those encountered in normal (i.e. intraspecies) pregnancies of Spanish ibex and domestic goat. Spanish Ibex females were superovulated with 9 mg NIADDK-oFSH-17 and embryos were surgically collected 5.5 d after estrus. Two embryos were transferred per recipient. Domestic goat recipients were previously mated either to vasectomized domestic bucks (n=17 females; interspecific-monospecific gestations) or to fertile ones (n=9 females; interspecific-bispecific gestations). Intraspecific pregnancies were obtained by natural mating between males and females of the same species (Spanish ibex: n=6; domestic goat: n=1). Pregnancy rate diagnosed by progesterone was low in both interspecific-monospecific (7/17) and interspecific-bispecific (3/9) transfers. None of the monospecific (0/7) and 2 (2/3) of the bispecific established pregnancies developed to term. Ibex-in-ibex PAG profile showed 2 similar peaks of 60 to 70 ng/mL on Days 34 and 153 of pregnancy, while goat-in-goat had the maximum value (60 to 70 ng/mL) at Day 50, decreasing slightly afterwards until parturition. Mixed-species gestations (ibex+goat in goat) showed a first peak of 500 to 1000 ng/mL on Day 70 and a second one (200 to 500 ng/mL) on Day 140 of pregnancy. Four ibex-in-goat gestations that terminated with the expulsion of dead fetuses at Days 110 to 170 had their maximum PAG values (100 to 700 ng/mL) on Days 60 to 90. We conclude that it is possible to achieve pregnancies after transfer of ibex embryos into domestic goats, but this requires a great change of the PAG profiles, which increase significantly. Live ibex kids can be produced when embryos from both species share the uterus. This is the first report of successful interspecific pregnancies in the capra genus.     

Concentrations of prostaglandins E2, F2 alpha and 6-keto-prostaglandin F1 alpha in the utero-ovarian venous plasma of nonpregnant and early pregnant ewes

The effect of pregnancy on concentrations of prostaglandins E2, F2 alpha and 6-keto-prostaglandin F1 alpha (PGE2, PGF2 alpha and 6-keto-PGF1 alpha) in utero-ovarian venous plasma was examined in ewes on Days 10 through 14 after estrus, an interval which includes the critical period for maternal recognition of pregnancy. The utero-ovarian vein ipsilateral to a corpus luteum was catheterized on Day 9 or 10 in 6 pregnant and 8 nonpregnant ewes. Five blood samples were collected at 30-min intervals for 2 h beginning at 0500 and 1700 h daily. Sampling began at 0500 h on the day after catheterization. The mean and variance within each 2-h collection period were calculated for each ewe. The natural logarithm of the variance in each collection period (ln variance) was used as an estimate of the fluctuations in secretory activity by the endometrial-conceptus complex. Patterns of the mean concentrations of PGE2 were different between pregnant and nonpregnant ewes (P less than 0.01); PGE2 being higher in the pregnant ewes beginning on Day 13. There was a trend for the patterns of ln variance in PGE2 to differ (P less than 0.1) with pregnancy status over the entire period; ln variance was greater in pregnant ewes beginning on Day 13. The patterns of the mean concentrations and ln variances for PGF2 alpha and 6-keto-PGF1 alpha did not differ between pregnant and nonpregnant ewes. There were significant increases in both of these prostaglandins over time, independent of pregnancy status (P less than 0.01). The association of higher concentrations of PGE2 in utero-ovarian venous plasma with early pregnancy is consistent with the hypothesis that PGE2, originating from the uterus and/or conceptus, is one factor involved in maintenance of the corpus luteum of pregnancy.