Diagnosis of pregnancy by radioimmunoassay of a pregnancy-specific protein in the plasma of dairy cows

The accuracy and efficiency of progesterone (P4) and bovine pregnancy-specific protein B (bPSPB) radioimmunoassays (RIA) in detecting pregnant and nonpregnant dairy cows were compared at different stages of pregnancy. The study included 145 French Friesian heifers and cows from a single herd. A total of 175 artificial insemination (A.I.) and blood sampling procedures were performed. Animals were bled 24 d post AI for P4 RIA. They were bled at 24, 26, 30 to 35, and 70 +/- 9 after AI for bPSPB RIA. Females were declared nonpregnant when plasma P4 concentrations were lower than 1.5 ng/ml. With the bPSPB RIA, cows were nonpregnant when at least one of the B Bo x 100 replicates was higher than 95% in the RIA. When compared with palpations per rectum at 70 d, the accuracy of positive diagnoses (no. positive and pregnant/no. positive diagnoses) by P4 RIA at Day 24 was 67.2% (82 122 ). The accuracy of negative diagnoses was 98% (52 53 ). Accuracy of positive diagnoses by bPSPB RIA increased with gestation age (P<0.05) from 86.2% (50 58 ) on Day 24 to 98.8% (83 84 ) at time of palpation per rectum. Accuracy of negative diagnoses increased (P< 0.001) from Day 24 (71.8%; 84 117 ) to Days 30 to 35 (100%, 83 83 ). Efficiency in detecting nonpregnant females was much higher (P < 0.001) with the bPSPB RIA on Days 30 to 35 (90.2%; 83 92 ) than with the P4 RIA on Day 24 (56.5%, 52 92 ). It is concluded that 30 days after AI, the bPSPB RIA is an efficient test both for pregnancy prediction and detection of nonpregnant dairy cows.     

Detection of pregnancy in sheep by radioimmunoassay of sera for pregnancy-specific protein B

A radioimmunoassay (RIA) for bovine pregnancy-specific protein B (bPSPB) has been shown to be a reliable test for pregnancy in cows. Pregnant ewes have a blood antigen that cross-reacts in this RIA. Two studies were conducted to determine the accuracy of detection of pregnancy in sheep using the bPSPB RIA. In Study 1, 33 ewe lambs were bred over a 70-d period in late fall. At 26, 56, and 83 d after the end of the breeding period, blood samples were collected for assay in the bPSPB RIA, and the Pregmatic 3 ultrasonic device was used to detect pregnancy. Pregmatic 3 detected pregnancy in 14, 27 and 28 ewes and nonpregnancy in 19, 6 and 3 ewes at Days 26, 56 and 83 past the breeding period, respectively. The bPSPB assay detected pregnancy in 32, 31 and 30 ewes and nonpregnancy in 1, 2 and 2 ewes at Days 26, 56 and 83 past breeding, respectively, Thirty ewes lambed and three did not. In Study 2, 180 multiparous ewes were bred over a 60-d period in summer. At 35 d after the end of the breeding period, blood samples were collected for assay in the RIA, and a real-time ultrasonic scan was done to detect pregnancy. Real-time ultrasonic testing detected pregnancy in 163 ewes and nonpregnancy in 17 ewes; whereas, the RIA detected pregnancy in 161 ewes and nonpregnancy in 19 ewes. One hundred fifty-nine ewes lambed and 21 did not. The bPSPB RIA detected pregnancy earlier and more accurately than the Pregmatic 3 ultrasonic device and was equally as accurate as the real-time scanning instrument. These studies demonstrate an accurate serological test for a pregnancy-specific antigen in sheep.     

Effect of fetal mass, number and stage of gestation on pregnancy-specific protein B concentrations in the bovine

In this study we characterized the peripheral plasma pregnancy-specific protein-B (PSPB) profile throughout gestation and examined the effect of stage of gestation, fetal mass and number on this profile in Holstein cows after non surgical embryo transfer. Cows (n = 12) were divided into 2 groups: Group 1 = single embryo recipient cows (n = 5), Group 2 = twin-embryo recipient cows (n = 7). Blood was collected approximately every third day from Day 0 (Day 0 = first day of standing estrus), then daily for the last 10 d of gestation, and sampling was stopped 1 d post partum. Two twin-embryo recipient cows had abnormal pregnancies; therefore, their data were excluded from the group. The time trend concentrations of plasma PSPB were significantly affected by the stage of gestation (P < 0.001) and fetal number (P < 0.001). In both groups PSPB increased gradually, with the mean levels being significantly higher (P < 0.01) in the twin-bearing group from Day 50 onwards (0.7 +/- 0.2 vs 9.2 +/- 4.5 ng/ml, singleton and twin-bearing cows, respectively) except for Day 10 pre-partum. By mid-gestation (Day 140), mean PSPB levels increased in the singleton (P < 0.001) cows by thirty-fold (21.2 +/- 3.2 ng/ml) as opposed to a ten-fold (98.4 +/- 13.2 ng/ml) increase in the twin-bearing (P < 0.001) group. The mean PSPB concentrations between Days 30 to 20 prepartum dramatically increased by about 700 to 200% in singleton (128.8 +/- 46.3 to 745.6 +/- 66.7 ng/ml) and twin-bearing cows (375.6 +/- 130.4 to 861.5 +/- 127.9 ng/ml), respectively. The PSPB levels between Day 10 prepartum to parturition were significantly higher (P < 0.001) in the twin-bearing group than in the singleton group (745.6 +/- 66.7 to 1627.4 +/- 238.9 ng/ml vs 861.5 +/- 127.9 to 3103.0 +/- 643.0 ng/ml in singleton and twin-bearing groups, respectively). Calf birthweight was correlated (P < 0.01) to peripheral PSPB concentration in singleton cows; however, this relationship decreased with the subsequent increase in fetal number. Cows giving birth prematurely to stillborn calves or to a schistosomus reflexus calf exhibited abnormal PSPB profiles. These results indicate that peripheral PSPB levels are correlated to the stage of gestation and fetal number. In addition, the peripheral pattern of PSPB is a valuable guage for predicting fetoplacental viability.     

Acute-phase protein profile during gestation and diestrous: proposal for an early pregnancy test in bitches

Early pregnancy diagnosis in bitches has special relevance for adequate medical assistance to assure normal gestation, diagnosis of abortion or embryonic resorption, undesirable pregnancy interruption and dog owners wishing to assure medical assistance during parturition and to program participation of females in dog shows. The aim of this study was to verify acute-phase protein profile variation as a consequence of generalized inflammatory reaction due to embryonic endometrial invasion and use alterations as a method for early pregnancy diagnosis. Also the relationship between hormonal status and acute-phase proteins concentrations was assessed. Weekly serum samples were collected from 20 non-pregnant (NP) bitches and 20 pregnant females (P) to determine levels of fibrinogen, haptoglobin, ceruloplasmin, seromucoid, glycoprotein, alpha(2) globulin, progesterone and estradiol-17beta. No correlation was found between the implantation sites formed (number of pups born) and the hepatic stimulus for the acute-phase protein production. The conclusions are that acute-phase proteins can be used as an early pregnancy test for bitches from the 3rd week of gestation (14th-21st day post LH peak) for haptoglobin assay (values above 112.42 mg/dl of HbCN binding capacity), from the 4th to the 6th week (21st-42nd day post LH peak) for ceruloplasmin (values above 12.76+/-5.29 U/l), from the 4th week (21st-28th day post LH peak) for glycoprotein (values above 13.67%) and from the 4th week of gestation (21st-28th day post LH peak) for alpha(2) globulin (values above 0.61 g/dl). Fibrinogen and seromucoid increased in the P group from the 5th to the 6th week, respectively, thus not being suitable as parameters for an early pregnancy diagnosis. Relationship between ceruloplasmin and estradiol-17beta and seromucoid and progesterone were verified. For the acute-phase protein test it is important to verify bitches' healthy condition and to assure the precise mating dates to avoid false-positive and -negative results, respectively.     

Dynamics of heat shock protein 70 concentrations in peripheral blood lymphocyte lysates during pregnancy in lactating Holstein-Friesian cows

The aim of this study was to characterize the dynamics of the concentrations of heat shock protein 70 kDa (HSP70) in peripheral blood lymphocytes of lactating Holstein-Friesian dairy cows (Bos taurus) during pregnancy. The detection of pregnancy was carried out and blood samples collected on Days 40, 90, 120, 150, 180, and 210 of gestation from 46 cows (11 primiparous and 35 pluriparous, 34 seropositive and 12 seronegative to Neospora caninum). Peripheral blood lymphocytes were isolated by density gradient centrifugation. Serologic analysis of Neospora infection and determinations of HSP70 concentrations in lymphocyte lysates were carried out using commercial enzyme-linked immunosorbent assay kits. Climate variables were monitored using on-farm data loggers. Heat shock protein 70 concentrations increased in lymphocytes as gestation progressed, particularly in primiparous cows, with no effect from Neospora infection, climate variables, milk production, semen-providing bull, or outcome of gestation (singletons or twins). Our results show that HSP70 concentrations increased in lymphocytes as gestation progressed and were not affected by stressful factors, such as milk production, heat stress, chronic infection (neosporosis), or twin pregnancies.     

Plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations during gestation in Neospora-infected dairy cows

The aim of this study was to determine whether plasma pregnancy-associated glycoprotein-1 (PAG-1) concentrations in pregnancy are affected by persistent Neospora caninum infection in dairy cows. The data analyzed were derived from 22 multiparous cows: 16 N. caninum-seropositive and 6 N. caninum-seronegative animals (used as controls). Three of the 16 seropositive cows aborted during the study period and the corresponding data were analyzed separately. Pregnancy diagnoses were performed on day 40 post-insemination by transrectal ultrasound, 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, and then at parturition or at the time of abortion detection. Plasma was tested for antibodies against N. caninum and PAG-1 concentrations were determined by radioimmunoassay. In non-aborting animals, the effects of neosporosis (seropositive versus seronegative), N. caninum antibody levels, semen providing bull, sex of the newborn, and day of gestation on PAG-1 concentrations were evaluated by GLM repeated measures analysis of variance. The effect of the gestation period (first half versus second half) on the N. caninum antibody titer was established by the Student's t-test in seropositive cows. A significant positive effect of gestation day on PAG-1 concentrations was observed (d.f.=6; F=12.6; P<0.0001). For all cows, PAG-1 concentrations increased steadily during the course of gestation, with peak concentrations recorded at parturition. Neosporosis (P=0.493), N. caninum antibody levels (P=0.921), sex of the newborn (P=0.856) and semen providing bull (P=0.087) had no effect on plasma PAG-1 concentration. There was a significant 52% increase (P<0.0001) in N. caninum antibody titers during the second half of gestation compared to the first half. The fates of the three aborting cows were abortion on gestation day 215 in one, and fetus mummification diagnosed on gestation days 180 and 210, respectively, in the remaining two cows. A luteolytic dose of prostaglandin was applied 30 days after mummification diagnosis in these last two cows, and fetus expulsion was detected on days 215 and 250, respectively. Two of the aborted fetuses were submitted to laboratory analysis and the presence of N. caninum was confirmed by specific PCR. In the cows with a mummified fetus, PAG-1 concentrations were low or undetectable when the diagnosis was made. These findings suggest that N. caninum infection has no effect on placental function in chronically infected, cows not suffering abortion, while PAG-1 measurements in aborting animals provide a useful indication of feto-placental status.     

Adipose tissue progesterone concentrations in dairy cows during late pregnancy and early lactation

The objectives of the study were to measure progesterone (P₄) concentrations in subcutaneous adipose tissue from the tailhead of dairy cows in various reproductive states, and to examine the effect of epinephrine-induced fat mobilization on P₄ release from adipose tissue in vitro. Blood samples and adipose tissue biopsies were taken from 12 cows twice during late pregnancy and three times during early lactation. Concentrations of P₄ in adipose tissue were higher (P < 0.005) during pregnancy than at any of the non-pregnant stages examined and, in general, the relative changes with reproductive state reflected changes in plasma P₄ concentration and body condition score. Plasma and adipose tissue P₄ concentrations were positively correlated (P < 0.001) at estrus and during the luteal phase of the estrous cycle. When adipose tissue samples from eight cows were incubated as explants in vitro and challenged with epinephrine, non-esterified fatty acid (NEFA) and P₄ concentrations increased (P < 0.05) with increasing concentrations of epinephrine. These results demonstrate that substantial quantities of P₄ are sequestered in adipose tissue, depending on the reproductive state, and indicate that lypolysis may result in the release of P₄ from this tissue, thereby influencing plasma P₄ concentrations.     

Diagnosis of pregnancy in moose using a bovine assay for pregnancy-specific protein B

Blood samples were collected from 26 moose (Alces alces ) and evaluated for the presence of an antigen that cross-reacted with antisera to bovine pregnancy-specific protein B (P-SPB). The objective of this study was to determine if the P-SPB radioimmunoassay (RIA) was a reliable indicator of pregnancy in these animals. In the first year of the study calf production the following summer was used as the index of previous pregnancy. In the second year all females were subjected to palpation per rectum after chemical immobilization. Seven of the 10 cows sampled in the first year were also sampled in the second year. All animals determined pregnant by rectal palpation were positive for P-SPB; however, P-SPB was not detected in males.     

Zinc and copper concentrations in serum from Spanish women during pregnancy

A cross-sectional study of serum zinc (Zn) and copper (Cu) levels in 31 healthy pregnant women and 51 healthy, nonpregnant controls living in the Mediterranean area of Granada, Spain, was performed. The subjects were divided into two groups: Group A, consisted of pregnant women in three categories according to the trimester of pregnancy, and Group B consisted of nonpregnant women acting as controls. In pregnant women, serum Zn levels were found from 0.300-1.340 mg/L and serum Cu from 0.936-2.304 mg/L, whereas in the nonpregnant women group, the mean serum levels were 0.947 ±0.265 mg/L for Zn and 1.092 ±0.365 mg/L for Cu. Serum Zn progressively decreased with gestation. Mean Zn levels were 0.829 ±0.253, 0.846 ±0.329, and 0.620 ±0.142 mg/L, corresponding to the first, second, and third trimesters of pregnancy, respectively. Serum Zn concentrations were significantly lower in pregnant women as compared to controls: 0.712 ±0.236 mg/L vs 0.947 ±0.265 mg/L, respectively (p < 0.05). In contrast, Cu levels increased with period of gestation from 1.053 ±0.498 mg/L in the first trimester to 1.616 ±0.304 mg/L in the second and 1.689 ±0.344 mg/L in the third. Serum Cu levels in the second and third trimesters of pregnancy were significantly higher (p < 0.05) than those determined during the first trimester and for nonpregnant controls. Both Zn and Cu during pregnancy did not appear to be dependent on the subject’s age (p > 0.05).     

Comparison of ultrasonography, bovine pregnancy-specific protein B, and bovine pregnancy-associated glycoprotein 1 tests for pregnancy detection in dairy cows

At Days 26 to 58 after AI, 138 Holstein-Friesian dairy cows were repeatedly examined by ultrasonography, using a 7.5 MHz linear-array rectal transducer. The total calving rate was 37.6% (52/138), and late embryonic mortality occurred 8.6% of the cows (12/138). On the days of ultrasound scanning, blood samples were drawn from the jugular vein for measuring the concentration of bovine pregnancy-specific protein B (bPSPB) and bovine pregnancy-associated glycoprotein 1 (bPAG 1). When compared with calving results, there were no significant differences in accurate diagnosis of pregnant cows were found between the 3 methods. However, when recognition of an embryo proper with a beating heart was used as the criterion for positive ultrasonographic diagnosis significantly fewer (P < 0.001) pregnant cows were correctly identified than by the other 2 tests. When compared with the noncalving cows, significantly fewer (P < 0.001) false positive diagnoses were made by the 2 ultrasonographic tests than by the PSPB and bPAG 1 tests, while significantly fewer (P < 0.001) false positive diagnoses were made by the bPSPB test than by the bPAG 1 test. The accuracy of detecting nonpregnant animals by both protein tests was limited by the relatively long half-life of these proteins after calving and by early embryonic mortality.     

Diagnosis of pregnancy in wood bison using a bovine assay for pregnancy-specific protein B

Blood samples were collected from 51 wood bison (Bison bison athabascae ) and evaluated for the presence of an antigen that cross-reacted with antisera to pregnancy-specific protein B(PSPB). The objective of this study was to determine if the PSPB radioimmunoassay (RIA) was a reliable indicator of pregnancy in these animals. Pregnancy of mature females was determined either at autopsy (20 animals) or by palpation per rectum after chemical immobilization (18 animals). The antigen was not detected in either males or juvenile females. There was minor cross-reaction in sera of one of nine nonpregnant females that had been exposed to males. The antigen was found in the sera of 25 of 27 females that were confirmed pregnant. It was concluded that the PSPB RIA was a useful tool in the determination of pregnancy in wood bison.     

Detection of a protein of avian leukoviruses in uninfected chick cells by radioimmunoassay

A radioimmunoassay (RIA) has been used to measure the quantity of protein P27 (mol wt 27,000; group specific-1 [gs-1]) of avian leukoviruses in different types of chicken embryos and tissues of adult chickens. The RIA used was 200- to 300-fold more sensitive than the complement fixation test and was able to detect as little as 0.3 ng of P27. Among six embryos tested, which are negative for gs antigen by the complement fixation test, P27 was undetectable in three embryos, but another three contained about 5 ng of P27 per mg of cell protein by RIA. The amount of P27 in gs antigen-positive cells ranged from 22 to 57 ng per mg of cell protein. P27 was found in liver, lung, ovary, feather pulp, and spleen from adult gs antigen-positive chickens. This protein was undetectable in various tissues from gs antigen-negative chickens.     

Induction of megakaryocyte differentiation by a novel pregnancy-specific hormone.

Maturation of megakaryocytes and subsequent platelet release are normally regulated by a network of cytokines, including thrombopoietin and various interleukins. Because abnormal platelet production and activation have been implicated in gestational pathologies, additional pregnancy-specific cytokines may play important roles in the regulation of megakaryocytopoiesis. Consistent with this hypothesis, we have found that the hormone prolactin-like protein E, a placental hormone that we have recently characterized, targets megakaryocytes through a specific cell surface receptor and induces megakaryocyte differentiation through a gp130-dependent signal transduction pathway.     

Extent and pattern of pregnancy losses and progesterone levels during gestation in Swedish Red and Swedish Holstein dairy cows

Background

Pregnancy loss is a major source of infertility in dairy cows. Despite a fertilization rate after insemination (AI) of approximately 90%, calving rates are 30%–50%, indicating the occurrence of extensive embryonic and foetal losses. The aim of this study was to establish the extent and pattern of embryonic and foetal loss in Swedish Red (SR) and Swedish Holstein (SH) dairy cows, as well as, the relationship to oestrus intensity (OI) and progesterone (P4) concentration. In total, 2130 AIs and 16,176 milk P4 samples from 359 SR and 212 SH dairy cows were included in the study. Pregnancy losses were estimated using data from P4 values combined with AI information and calving data.

Results

Total pregnancy loss from AI to the day of calving was 65%. Early embryonic loss, late embryonic loss and foetal loss were estimated to be 29, 14 and 13%, respectively. There is strong evidence in the literature that P4 concentrations at different time points are associated with pregnancy loss. In the present study, cows with pregnancy losses had significantly higher P4 levels at the day of AI and significantly lower P4 concentration at days 10, 21 and 30 after AI compared to pregnant cows. Swedish Red cows had significantly lower total pregnancy losses compared to SH cows (62% and 68% respectively, P?=?0.017). Early embryonic loss was 6.7% points lower for cows inseminated at a stronger OI (OI?=?3) compared to at a weaker OI (OI?=?2, P?=?0.006). Cows inseminated at ovulation number?≥?5 had significantly lower early pregnancy losses compared to cows inseminated at first or second ovulation (11.5 and 8% points, respectively, P?<?0.05). With an increase of one SD of milk (448 kg ECM) during the first 60 days in milk, early embryonic loss increased by 4.7% points (P?=?0.006).

Conclusions

It is important to increase the number of cows calving per insemination by reducing embryo/foetal loss. This outcome can be achieved by management and breeding for optimal P4 levels at critical time points, and by considering oestrus expression in the breeding programmes to facilitate the correct timing of insemination.

     

Changes in serum thyroid hormone autoantibody concentrations during pregnancy: a case report.

A 29-year-old female patient with Graves' disease who developed thyroid hormone autoantibodies (THAA) under treatment with methimazole is presented. THAA were identified as IgG-kappa. During a first pregnancy that ended by miscarriage in the 3rd month, the titer of anti-thyroxine autoantibodies decreased by about 30%. Relapse of Graves' disease occurred 2 months later and an increase in serum THAA concentration to the initial titer was observed. THAA titer remained unchanged during treatment with methimazole and afterwards during thyroxine supplementation for radioiodine-induced hypothyroidism. During a second pregnancy, a decrease in anti-thyroxine autoantibody titer reached 45% at the time of delivery and an increase by 20% was noted 5 months later. A similar decline in THAA concentration was shown during a third pregnancy. The changes in THAA concentrations observed during pregnancy suggest an immunological influence of pregnancy on the THAA production, as previously demonstrated in other autoimmune diseases, like Hashimoto's thyroiditis.     

Cytokine profile in cases with premature elevation of progesterone serum concentrations during ovarian stimulation

The aim of this study was to investigate the concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), leptin, tumor necrosis factor-alpha, interleukin (IL)-1beta and IL-6, in cycles with a premature rise of serum progesterone. 25 intracytoplasmic sperm injection (ICSI) cycles with (Group 1) and 25 ICSI cycles without a premature progesterone elevation (Group 2) were included. The cut-off value of serum progesterone on the day of human chorionic gonadotropin (hCG) administration was 0.9 ng/ml. The indication for ICSI was male factor infertility exclusively. On the day of hCG injection, serum IL-6, VEGF and bFGF were significantly higher in Group 1 (7.7+/-24.5 pg/ml, 290.2+/-161.4 pg/ml and 15.7+/-8.2 ng/ml respectively) than in Group 2 (1.7+/-0.7 pg/ml, 175.2+/-92.1 pg/ml, and 9+/-1.6 ng/ml respectively). On the day of follicular puncture, serum cytokine concentrations were similar in the two groups. IL-6 intrafollicular concentrations were higher in Group 1 (14.7+/-20.7 pg/ml) than in Group 2 (9+/-9.3 pg/ml, p=0.031). There were no differences regarding the ICSI outcome. Patients with serum progesterone above 0.9 ng/ml, have elevated serum concentrations of IL-6, VEGF, and bFGF, as well as elevated intrafollicular concentrations of IL-6. The outcome of ICSI cycles is not associated with premature elevation of progesterone when the cut-off value is set at 0.9 ng/ml.     

Free and phospholipid-bound choline concentrations in serum during pregnancy,after delivery and in newborns

The aims of this study were to determine whether serum free choline and phospholipid-bound choline concentrations change during the pregnancy or after childbirth and to determine if the serum choline concentrations of the mother and newborn are correlated. Serum free and bound choline concentrations were 10.7 +/- 0.5 microM and 2780 +/- 95 microM in control, non-pregnant women, and rose significantly (p < 0.001) to 14.5 +/- 0.6 microM and 3370 +/- 50 microM or to 16.5 +/- 0.7 microM and 3520 +/- 150 microM after 16-20 weeks or 36-40 weeks of pregnancy, respectively. Serum free and phospholipid-bound choline fell by 14-22% (p < 0.05-01) after either vaginal delivery or caesarian section, and remained low (by 15-42%; p < 0.05-0.001) for 12 h and then rose toward the baseline within 24 h. In amniotic fluid, free choline and phospholipid-bound choline concentrations were 22.8 +/- 1.0 and 19.6 +/- 0.8 microM or 24.0 +/- 1.5 and 516 +/- 43 microM at 16-20 weeks of gestational age or at term, respectively. In newborns, serum free choline concentrations were higher (p < 0.001) and phospholipid-bound choline concentrations were lower (p < 0.001) than in their mothers. These results show that serum free choline and phospholipid-bound choline concentrations are elevated during the pregnancy, which may be required for an adequate maternal supply of choline to the fetus. These observations are clinically important to determine the ideal dietary intake of choline during the pregnancy.     

Impacts of protein supplementation during late gestation of beef cows on maternal skeletal muscle and liver tissues metabolism

Since nutritional requirements are increased at the end of gestation to meet the demands of the pregnant uterus, pregnant beef cows are susceptible to mobilization of body reserves (mainly fat and amino acids (AAs)) and to alter the metabolism of nutrients in the liver and muscle to support such demands. The objective of this study was to evaluate the effect of CP supplementation on maternal nutrient metabolism in the late gestation of beef cows grazing a low-quality pasture. Forty-three pregnant Nellore cows gestating male fetuses (average age = 6 years; average weight = 544 kg) at 193 ± 30 (mean ± SD) days (d) of gestation were divided into eight groups (experimental units, with four to five cows each). Treatments were (1) control (CON, n = 4): pasture-based (PB) diet without CP supplementation and (2) supplemented (SUP, n = 4): PB diet daily supplemented with 2 g/kg of BW of a 43.5% CP supplement. Liver and skeletal muscle biopsies were performed at 265 days of gestation and samples were collected for mRNA expression. On day 280 of gestation, blood samples were collected to assess plasma levels of AA. The CON-fed cows tended to have greater (P = 0.057) total circulating AA than SUP-fed cows. The circulating glycogenic AA was greater (P = 0.035) in CON than in SUP cows. CON cows was greater for histidine (P = 0.015), methionine (P = 0.007) and alanine (P = 0.036) than SUP cows. The CON- and SUP-fed showed no differences for gluconeogenesis, fatty acid transport and signaling axis markers in the liver. The mRNA expression of markers for skeletal muscle synthesis, p7056k (P = 0.060) and GSK3B (P = 0.096), tended to be greater in cows from CON than SUP group. No differences were found for mRNA expression of markers for skeletal muscle degradation. We conclude that CP supplementation to CP-restricted late-pregnant beef cows reduces the maternal tissue mobilization and changes the profile of plasma circulating AA and the mRNA expression of markers for the synthesis of skeletal muscle tissue.     

Correlation between serum and fecal concentrations of reproductive steroids throughout gestation in goats

Non-invasive techniques such as the measurement of fecal steroids are now widely used to monitor reproductive hormones in captive and free-ranging wild-life. These methods offer great advantages and deserve to be used in domestic animals. The aim of the present study was to determine the endocrine profile of dairy goats throughout pregnancy by the quantification of fecal progestins and estrogens and assess its correlation with serum concentrations. Blood and fecal samples were collected weekly from 11 adult, multiparous goats, from mating through pregnancy and 2 weeks post-partum. The extraction of estradiol and progesterone fecal metabolites was performed by dilution in ethanol. The radioimmunoassay (RIA) in solid phase was used to quantify serum 17beta-estradiol (estradiol) and progesterone, as well as their fecal metabolites. The mean concentrations of both fecal and serum estradiol started to increase between weeks 7 and 11, reached peak values near parturition and then decreased sharply (range: 19.8+/-5.8 ng/g of feces to 608.6+/-472.4 ng/g of feces and 0.007+/-0.005 ng/ml to 0.066+/-0.024 ng/ml). An increase in both fecal and blood progestagens occurred in the second week, mean concentrations remained greater until week 20, and then decreased in the last week of gestation and 2 weeks post-partum (range: 108.8+/-43.6 ng/g of feces to 3119.5+/-2076.9 ng/g of feces and 0.12+/-0.04 ng/ml to 13.10+/-4.29 ng/ml). The changes in blood and fecal hormone concentrations were analyzed and compared throughout gestation for each single goat, for each breed and for the whole group. Results indicated that matched values of serum and fecal hormone concentrations were correlated (r=0.79; p<0.001 for progesterone and r=0.84; p<0.001 for estradiol mean concentrations in the whole group). Regression analysis showed that logarithmic model allows significant prediction of serum from fecal concentrations with an R(2)=0.729 (y=0.013ln x-0.021) for estradiol and R(2)=0.788 (y=3.835ln x-18.543) for progesterone. Neither fecal nor serum concentrations were affected by the breed but a significant effect of the number of fetuses on progestin concentrations was found. Therefore, the profiles of progesterone and estradiol fecal metabolites reflect the serum concentrations of the same hormones in pregnant goats.     

An immunochemical demonstration of a pregnancy-specific protein in the horse and its use in the serological detection of early pregnancy

Two-dimensional crossed immunoelectrophoresis of sera from pregnant and non-pregnant horses, using antisera developed against early pregnant mare serum, revealed the presence of two immunologically related proteins one of which appeared to be specific to the pregnant state. This pregnancy-specific protein had beta 2-electrophoretic mobility and was first detectable at Day 6 after successful mating with a stallion. The second protein had gamma 2-electrophoretic mobility and was present in sera from pregnant and non-pregnant horses. The proteins were termed beta 2-horse pregnancy protein and gamma 2-horse protein respectively. The latter appeared to be immunologically related to the former in that the precipitin lines of the 2 proteins showed continuity. Samples from 16 mares mated with a stallion were investigated for the beta 2-protein during the first 3 weeks after mating. Of the 11 successful matings, confirmed by ultrasonic scanning at 90 days or by a successful outcome, 10 mares showed the presence of the protein. In all of 14 non-pregnant sera taken from mares not recently mated, the protein was not detectable. The validity of detection of beta 2-protein as an indication of pregnancy was clinically significant at the 10% level. The presence of the protein in 2 out of the 5 recently mated mares that did not become pregnant may be indicative of a biochemical pregnancy that failed at a later stage of gestation.