Development of a surgical technique for long-term catheterisation of bovine fetuses.

The aim of this study was to further develop and extensively describe a surgical technique in order to realise long-term fetal blood sampling in the bovine species. Eleven Holstein-Friesian 6- to 8-month pregnant cows (4-10 years old) were used for this study. Gestational age on the day of surgery varied from approximately 180 days (n = 1) to 240 days (minimum: 232 days, maximum 252 days; n = 10). The fetal medial tarsal artery was catheterised in pregnant cows with a polyvinyl catheter in dorsal recumbency under general anaesthesia. Although 5 out of 11 operations (45.5%) performed between 232 and 252 days of gestation were lost due to different causes mainly associated with peritonitis and septicaemia, the mean interval between operations and calvings was 42.5 days (between 27 and 95 days). It is important to emphasise that a well-trained surgical team is needed for bovine fetal cannulation in order to be able to decrease the risk factors during the operations. Due to the fact that after 5 unsuccessful cases none of the pregnancies were lost, this skill can be reached, and our technique can enable bovine fetal blood sampling for long-term endocrinological and physiological investigations before and during parturition.

Investigation of PAG2 mRNA Expression in Water Buffalo Peripheral Blood Mononuclear Cells and Polymorphonuclear Leukocytes from Maternal Blood at the Peri-Implantation Period.

: The main objective of this study was to assess PAG2 mRNA expression in maternal blood cells at the peri-implantation period in water buffalo; moreover, we wanted to evaluate the earliest time in which PAG-2 could be detected in maternal blood. Thirty-two lactating buffaloes artificially inseminated (AI) were utilized. Blood was collected at Days 0, 14, 18, 28, 40 after AI (AI = day 0). Pregnancy was diagnosed by ultrasound at Days 28 and 40 post AI. Out of 32 buffaloes, 14 were pregnant (P group) and 18 were not pregnant (NP group). The plasma PAG-2 threshold of 1.0 ng/mL in the P group was reached at day 40 post AI. PAG2 mRNA expression differed between the P and NP groups, and was either evaluated in Peripheral Blood Mononuclear Cells (PBMC) or Polymorphonuclear Leukocytes (PMN), starting from day 14. However, both the estimated marginal means and multiple comparisons showed that PAG2 mRNA expression was higher in PMN than PBMC. In the present study, PAG-2 appeared in the blood (40 Days post AI), and an early expression of PAG2 mRNA at Day 14 post AI was also observed. Although further research is undoubtedly required, PAG2 mRNA in peripheral blood leukocytes could be using to better understand the role that PAGs play during pregnancy in buffalo.

Investigation into homocysteine [corrected], vitamin E and malondialdehyde as indicators of successful artificial insemination in synchronized buffalo cows (Bubalus bubalis).

The aim of this study was to describe modifications in plasma homocysteine (Hcy), vitamin E (VitE) and malondialdehyde (MDA) concentrations in the first 56 days after artificial insemination (AI) in buffalo. Thirty-five buffalo cows were divided, ex post, into three groups on the basis of pregnancy diagnosis: pregnant, not pregnant, with embryonic mortality. Pregnancy was diagnosed by ultrasonography and plasma concentrations of pregnancy-associated glycoproteins (PAGs). Our results showed that, in pregnant buffaloes, included those with embryonic mortality, MDA increased progressively while VitE decreased. In non-pregnant buffaloes, MDA and Vit E were unchanged. Hcy concentrations also remained unchanged within each group throughout the study period, but were lower in non-pregnant buffaloes than in the pregnant ones and in those with embryonic mortality. In conclusion, present data suggest that successful pregnancy in buffalo cows might be linked to Hcy metabolism and oxidative stress involvement.

Accuracy of diagnosing double corpora lutea and twin pregnancy by measuring serum progesterone and bovine pregnancy-associated glycoprotein 1 in the first trimester of gestation in dairy cows.

Progesterone (P4) and bovine pregnancy-associated glycoprotein 1 (bPAG-1) concentrations during gestation are dependent on the number of CL and fetuses, respectively. The objective of this present study was to measure and evaluate the usefulness of measuring the P4 and bPAG-1 concentrations in cases of single versus twin pregnancies and one versus two CL at the first 4 months of gestation. We hypothesized that both the number of the CL and the number of fetus might have an effect on P4 and pregnancy protein concentrations, and we can set up clinically useful threshold levels to predict twin gestations. Eighty-four Holstein-Friesian dairy cows were enrolled in this prospective observational clinical trial. Blood was collected at time point 1: between Days 29 and 42, time point 2: between Days 57 and 70, time point 3: between Days 85 and 98, and time point 4: between Days 113 and 126 of gestation, and bPAG-1 and P4 concentrations were measured. Binary logistic regression analyzing serum P4 concentrations differed at time point 2 compared with baseline level, but the area under the curve (AUC) had low sensitivity. The bPAG-1 concentrations were statistically different at each time point of gestation. The AUC cutoff values of serum bPAG-1 concentrations were sufficiently sensitive to differentiate between twin gestations from singleton ones. At time points 3 (cutoff value of 3.4 ng/mL) and 4 (cutoff value of 56.5 ng/mL), statistically significant differences with low sensitivity, high specificity, and a high AUC were found. On the basis of these results, the diagnosis of twin pregnancy using pregnancy protein measurements is clinically insufficient before Day 85 of gestation; however, the ability to confirm the early twin pregnancy diagnosis with bPAG-1 measurements appears to be promising. To achieve high sensitivity, further studies are required.

Relationship of progesterone, bovine pregnancy-associated glycoprotein-1 and nitric oxide with late embryonic and early fetal mortalities in dairy cows.

The aim of the present study was to determine the relationship of progesterone (P4), bovine pregnancy-associated glycoprotein-1 (bPAG-1) and nitric oxide (NO) levels with late embryonic (LEM; day 28 to day 42) and early fetal mortalities (EFM; > day 42 to day 56) in dairy cows. Transrectal ultrasonography (6-8 MHz) was performed in 100 Holstein-Friesian cows at days 28, 42 and 56 after artificial insemination (AI; day 0) to diagnose pregnancy and to monitor the fate of the embryo. After ultrasound scanning of each cow, a milk sample was collected for assessment of P4 by an ELISA test and a blood sample was collected for assessment of bPAG-1, by using a double-antibody radioimmunoassay, and serum NO metabolites (nitrate + nitrite). Based on ultrasonographic examinations and bPAG-1-RIA, 41 of 100 inseminated cows were confirmed pregnant at day 28 after AI. Nine cows suffered of LEM, and 6 cows suffered of EFM and the overall pregnancy loss rate was 36.6% (15/41) between days 28 and 56 of pregnancy. By logistic regression analysis, there were no significant relationships between the level of P4 and bPAG-1 at day 28 after AI and the occurrence of LEM and EFM. Also, there were no significant relationships between the levels of P4 and bPAG-1 at day 42 and the occurrence of EFM. On the other hand, a significant relationship (P<0.05) was found between NO level at day 28 and the occurrence of LEM. In conclusion, measurement of the serum NO concentration at day 28 of pregnancy might help to predict the outcome of pregnancy by day 42 in dairy cows but further studies are needed to confirm this.

Assessment of pregnancy-associated glycoprotein (PAG) concentrations in swamp buffalo samples from fetal and maternal origins by using interspecies antisera.

Pregnancy-associated glycoproteins (PAG) constitute a large family of glycoproteins found in the outer placental epithelial cell layer of the placenta in Eutherian species. In ruminants, they are noted to be structurally closely related among the different species. This study was designed to determine PAG concentrations in maternal and fetal plasma, allantoic and amniotic fluids in buffalo species. Antisera (AS) generated in rabbits against distinct PAG molecules were used in three radioimmunoassay (RIA)-PAG systems: RIA-1 (antiserum raised against bovine PAG67kDa; AS#497), RIA-2 (antiserum raised against caprine PAG55 + 62 kDa; AS#706) or RIA-3 (antiserum raised against buffalo PAG; AS#859). Samples were collected at a slaughterhouse (n = 67). PAG concentrations determined by RIA-2 gave significantly higher results in both allantoic and amniotic fluids (12.7 ± 2.1 ng/mL and 24.0 ± 7.3 ng/mL, respectively). Regarding maternal and fetal plasma, PAG concentrations obtained by RIA-2 (21.8 ± 2.4 ng/mL and 20.2 ± 2.5 ng/mL, respectively) and RIA-3 (25.0 ± 2.2 ng/mL and 21.9 ± 3.2 ng/mL, respectively) were higher than those obtained by RIA-1 (15.5 ± 1.4 ng/mL and 16.1 ± 1.8 ng/mL, respectively). The correlation among the three systems was very high. The study clearly reveals the ability of different PAG-RIA systems to measure PAG concentration in swamp buffalo samples.

Accuracy of transrectal palpation for early pregnancy diagnosis in Egyptian buffaloes.

The present study was undertaken to evaluate the accuracy of transrectal palpation (TRP) for diagnosing early pregnancy in buffaloes and the false diagnoses of the TRP test by using the pregnancy-associated glycoprotein radioimmunoassay (PAG-RIA) test. Pregnancy was diagnosed in 168 buffalo-cows once by TRP and PAG-RIA test between days 31 and 55 after breeding. The sensitivity of TRP for detecting pregnant buffalo-cows was 37.5% at days 31-35, increased to 93.8% at days 46-50 and reached 100% at days 51-55 (P < 0.01). All cases of false negative diagnoses (n = 10) had PAG concentration higher than the threshold (≥1.8 ng/mL) for diagnosing pregnancy. The specificity of TRP for detecting non-pregnant buffalo cows ranged between 90.9%, and 100% between days 31 and 55. All cases of false positive diagnoses (n = 5) made by TRP had PAG concentrations lower than the threshold for diagnosing pregnancy. It could be concluded that TRP is an accurate method for diagnosing pregnant and non-pregnant buffalo cows from day 46 after breeding.

Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples.

BACKGROUND: The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions. METHODS: Six bovine foetuses were chronic cannulated on the aorta via the medial tarsal artery. Infusion of rabbit anti-bPL IgG was performed during late gestation. Pooled rabbit anti-bPL antisera had a maximal neutralization capacity of 25 microg bPL/mL of immunoglobulin. Interference of rabbit anti-bPL immunoglobulin with radioimmunoassay measurement using guinea pig anti-bPL as primary antibody was first evaluated in vitro. Polyclonal anti-bPL antibodies raised in rabbit were added in foetal sera to produce 100 samples with known antibodies titers (dilutions ranging from 1:2,500 till 1:1,280,000). RESULT(S): Assessment of the interference of rabbit anti-bPL antibody showed that bPL concentrations were significantly lower (P < 0.05) in samples added with dilutions of rabbit antiserum lower than 1:80,000 (one foetus) or 1:10,000 (four foetuses). It was also shown that the recovery of added bPL (12 ng/mL) was markedly reduced in those samples in which exogenous rabbit anti-bPL were added at dilutions lower than 1:20,000. Concentrations of foetal bPL were determined in samples from cannulated foetuses. In foetuses 1 and 6, bPL concentrations remained almost unchanged (<5 ng/mL) during the whole experimental period. In Foetus 3, bPL concentrations decreased immediately after IgG infusion and thereafter, they increased until parturition. CONCLUSION(S): The use of a bPL RIA using a guinea pig anti-bPL as primary antiserum allowed for the measurement of bPL concentrations in foetal plasma in presence of rabbit anti-bPL IgG into the foetal circulation. Long-term foetal catheterization allowed for the study of the influence of direct infusion of anti-bPL IgG on peripheral bPL concentrations in bovine foetuses.

Evaluation of GnRH treatment 12 days after AI in the reproductive performance of dairy cows.

The objective of this study was to evaluate the effects of GnRH administered at Day 12 post-AI on the reproductive performance of dairy cows. Holstein-Friesian dairy cows (n=103) on a large Hungarian dairy farm were allocated randomly to treated (n=54) or control (n=49) groups. Twelve days after AI, treated cows received a GnRH agonist i.m., while the control group received a placebo (physiological saline). Progesterone radioimmunoassay was used to determine the correct timing of artificial insemination (Day 0) and the incidence of luteal insufficiency on Day 12. Ultrasonography and radioimmunoassay for pregnancy-associated glycoprotein were used to detect pregnancy and late embryonic/fetal mortality between Days 32 and 55 after AI. Three cows from each group were inseminated when progesterone concentrations were >1.0 ng/mL, and six cows (four from the treated and two from the control group) had luteal insufficiency (progesterone<1.0 ng/mL) on Day 12. Late embryonic/fetal mortality occurred in three treated cows and in two control cows. When these cows were removed from the model, calving rates after first service were 59.6% (28/47) and 59.1% (26/44) for treated and control cows, respectively (P>0.05). There was no significant difference between treated and control cows when they were inseminated before or after Day 100 from calving. In summary, administration of a GnRH agonist on Day 12 after AI did not improve reproductive performance in dairy cows. However, our approach may be used for the field evaluation of different treatment protocols.

Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy.

Pregnancy associated glycoproteins (PAGs) are extensively glycosylated secretory proteins of ruminant trophoblast cells. In cattle placenta several PAG cDNAs are expressed, but the variety of correspondent proteins and their degree of glycosylation are not well characterized. Thus, we purified PAGs by using a protocol which included a lectin (Vicia villosa agglutinin) affinity chromatography. Due to their specific glycosylation pattern, PAGs derived from binucleate trophoblast giant cells were highly enriched by this protocol. PAGs were purified from cotyledons of 2 day 100 placentas and from a single placenta at day 155 and 180. In all samples three major bands (75; 66; 56 kDa) were detected by one-dimensional SDS-PAGE. Mass-spectrometric analysis identified the 75 kDa band as a mixture of PAG-7 and PAG-6, the 66 kDa band as PAG-1 and the 56 kDa band as PAG-17. N-terminal sequencing of the day 100 sample confirmed the mass spectrometric identifications. Enzymatic release of N-glycans with peptide-N-glycanase-F from PAGs reduced the molecular weight to approximately 37 kDa which corresponds to the theoretical molecular mass of PAGs. Limited peptide-N-glycanase-F treatment revealed that all four N-glycosylation sites are quantitatively occupied in PAG-1. Compared to PAG-1 the number of potential N-glycosylation sites is lower in PAG-17 (three sites) and higher in PAG-6 and -7 (five and six sites, respectively). This suggests that the number of attached N-glycans is the main determinant of molecular mass of bovine PAGs. The degree of glycosylation may be a major factor regulating the plasma half life of PAGs.

Isolation and characterization of eight pregnancy-associated glycoproteins present at high levels in the ovine placenta between day 60 and day 100 of gestation.

Pregnancy-associated glycoproteins (PAG), structurally related to aspartic proteinases, are expressed in the outer epithelial cell layer (chorion/trophectoderm) of the ungulate placenta. The aim of the present study was to isolate as many PAG molecules as possible from placentae collected between day 60 and day 100 of gestation and to characterize their amino-terminal amino-acid sequences. Three heterologous radioimmunoassays were used to monitor PAG immunoreactivity throughout the isolation procedures. Sequential use of DEAE-cellulose, gel filtration, and CM ceramic chromatographies led to the isolation of several fractions rich in PAG immunoreactivity. The fractions with a large amount of proteins were also purified by chromatofocusing. The analysis of immunoreactive fractions by SDS-PAGE, Western blotting and two-dimensional electrophoresis followed by amino-terminal microsequencing on PVDF membranes allowed to identify eight different ovPAG with apparent molecular masses ranging from 55 to 66 kDa and isoelectric points from 4.0 to 6.8. The N-terminal sequences were determined and their comparison to those previously identified revealed that four of them are identical to those encoded by previously known cDNA, while the additional four sequences appear to be novel since they have not yet been described.

Effects of an experimental Trypanosoma congolense infection on the reproductive performance of West African Dwarf goats.

Thirty-six West African Dwarf (WAD) goats were used to assess the effects of an experimental Trypanosoma congolense infection on their reproductive system. Estrous cycles were synchronised and when confirmed pregnant (n = 31), the does were randomly allocated into control and trypanosome-infected groups. After infection, the animals were carefully observed till parturition. Trypanosome infection caused an increase of rectal temperature, a significant drop in PCV (infected: 23.3 +/- 0.3%; control: 28.5 +/- 0.4%; P < 0.0001) and abortions in 27.8% of the infected does. Kids born from infected does had a lower birth weight than kids born from control goats (0.9 +/- 0.1 kg versus 1.6 +/- 0.1 kg; P < 0.0001). Eight out of 13 kids (61.5%) that were born alive from infected does died during their first week of life. Plasma pregnancy-associated glycoprotein (PAG) and progesterone concentrations were lower in the infected animals than in the controls. In general, PAG concentration in does which aborted dropped before abortion. Our results revealed that artificial T. congolense infection affected reproductive performance of WAD goats with abortions, premature births and perinatal losses being observed. Neither transplacental transmission of T. congolense nor histopathological lesions of the placenta could be demonstrated.

Pregnancy-associated glycoprotein concentrations during pregnancy and the postpartum period in Azawak Zebu cattle.

Specific RIA systems were developed and used to measure pregnancy-associated glycoprotein (PAG) concentrations during gestation and the postpartum period in Azawak Zebu cows. Twelve females were palpated per rectum and diagnosed as pregnant. Blood samples were taken at 5-10-day intervals from approximately Week 8 of gestation until Week 10 postpartum (pp). One Zebu cow (Z15) initially diagnosed as pregnant showed PAG concentrations lower than the assay sensitivity (<0.20 ng/ml) and did not calve. Another cow (ZSand) showed abnormally high PAG concentrations during gestation and was excluded from the general PAG profile. The 10 other Zebu cows exhibited a very similar PAG profile. In these animals, concentrations increased progressively from Week 8 to 35 of gestation (from 6.0+/-4.2 to 196.0+/-34.8 ng/ml), remaining relatively constant until Week 39 (210.8+/-74.8 ng/ml), when they increased sharply to reach their highest level (1095.6+/-607.2 ng/ml) at around parturition. After delivery, PAG concentrations declined significantly (P<0.05) until Week 2 postpartum (348.4+/-85.6 ng/ml) and slowly until Week 10 postpartum. Our results revealed that the PAG pattern in Zebu cattle was similar to those of taurine breeds during the first two trimesters of pregnancy, but differed in the peripartum period.