Approaches to Identify Pregnancy Failure in Buffalo Cows

Simple SummaryEmbryonic mortality and pregnancy failures still represent a major issue in domestic livestock production, particularly in dairy cattle. Despite the presence of extensive work in this research area, there is still no effective, accurate and practical method able to determine timing and viability of embryo specifically during early gestation. Indeed, technologies and techniques for predicting pregnancy success must continue to be developed. The aim of this work was to find the best strategy to diagnose pregnancy failures in buffalo cows in order to improve farm reproductive management. Among the methods compared in this study (ultrasonography, progesterone, PAGs), pregnancy-associated glycoproteins (PAGs) seem to be the best marker for predicting embryonic mortality between 25 and 40 days of gestation to be utilized as a diagnostic tool to improve reproductive management in buffalo farms.The aim of this work was to find the best strategy to diagnose pregnancy failures in buffalo. A total of 109 animals belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program were enrolled in this study. Blood samples were collected at days 0, 14, 25, 28 and 40 after AI for the determination of progesterone (P4) and pregnancy-associated glycoproteins (PAGs) by the radioimmunoassay (RIA) method. Transrectal ultrasonography was performed on day 25, 28 and 40 after AI to monitor pregnancy. The animals included in the data analysis were assigned ex post in pregnant (n = 50) and mortality (n = 12) groups. By ultrasonography, the predictive sign of mortality was the heartbeat. At day 25, the PAGs concentration was significant in predicting embryonic mortality with respect to ultrasonography and P4, at the cut-off of 1.1 ng/mL. At day 28, either PAGs, at a cut-off of 2.2 ng/mL, or ultrasonography, with no detection of heartbeat, were highly predictive of embryonic mortality. PAGs were the best marker (p < 0.05) for predicting embryonic mortality between 25 and 40 days of gestation in buffalo. Its utilization as a diagnostic tool can influence management decisions in order to improve farm reproductive management

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

Purification, Characterization and Radioimmunoassay of Pregnancy-Associated Glycoproteins Isolated from Zebu Cattle

The pregnancy-associated glycoproteins (PAGs) constitute a large family of aspartic proteinases expressed in the outer epithelial cell layer of the Artyodactyla placenta. In the first part of the present work, two biochemical approaches were used to characterize PAGs isolated from zebu (Bos indicus) fetal cotyledons. The first procedure, used to isolate PAG from zebu placenta removed late in pregnancy, included extraction of proteins at neutral pH, acidic and ammonium sulfate precipitations, anion and cation exchange chromatographies. The second procedure, used to investigate PAG in placentas removed at early and mid gestational periods, included protein extractions at acid or alkaline pH followed by pepstatin-A agarose affinity chromatography. A bovine PAG radioimmunoassay was used to monitor the immunoreactivity throughout the isolation procedures. The most immunoreactive fractions issued from cation exchange and affinity chromatographies were analyzed by SDS-PAGE and Western Blot, before transfer to polyvinylidene difluoride (PVDF) membrane for NH2-microsequence determination. By use of SDS-PAGE and Western Blot, different isoforms of PAG with apparent molecular masses from 51 to 69 kDa and isoeletric points varying from 3.1 to 6.7 were identified in placentas from different gestational ages. After CM ceramic chromatography of all except 0.32 M NaCl DEAE fraction, the most immunoreactive proteins revealed N-terminal amino acid sequences (10 to 25 aa long) which were 100% identical to bovine PAG-1. The same sequence (14 aa long) was found after pepstatin-agarose affinity chromatography of proteins extracted from placentas removed earlier in pregnancy. These results converged towards the expression of one major N-terminal PAG amino acid sequence in zebu placentas at different gestational ages. In the second part of this study, two specific RIA systems were developed then used to measure plasmatic PAG concentrations during gestation and postpartum period in Azawak zebu cows. Twelve females palpated per rectum and diagnosed as pregnant were bled at 5-10 days interval approximately from Week 8 of gestation till Week 10 postpartum (pp). One zebu cow initially diagnosed as pregnant showed PAG concentrations lower than the assay sensitivity (<0.20 ng/ml) and did not calve. Another cow showed abnormally high PAG concentrations during gestation, being excluded from the general PAG profile. The 10 other zebu cows gave a very homogeneous PAG profile. In these animals, concentrations increased progressively from Week 8 to Week 35 of gestation (from 6.04.2 to 196.034.8 ng/ml), remaining relatively constant until Week 39 (210.874.8 ng/ml), when they increased sharply to reach their highest level (1,095.6607.2 ng/ml) at parturition. After delivery, PAG concentrations declined significantly (P<0.05) till the Week 2 postpartum (348.4 85.6 ng/ml) and slowly till the Week 10 postpartum. These results revealed that PAG concentrations in zebu cattle were lower than those previously described in taurine breeds between week 35 and 39 of gestation