Comparison of accuracy of pregnancy-associated glycoprotein (PAG) concentration in blood and milk for early pregnancy diagnosis in cows.

The aim of this study was to compare two methods of early pregnancy diagnosis by determining pregnancy-associated glycoprotein (PAG) concentration in blood and PAG concentration in milk. Blood and milk samples were obtained on days 0 (AI day), 14, 21, 28, 35, 49, 63, 77, 91 and 105 of gestation from 60 lactating Holstein Frisian cows from one herd, carrying live fetuses. To determine PAG concentration a specific RIA system (RIA-706) was used. PAG concentration in blood and milk increased after 28 days of pregnancy, with blood concentrations being significantly higher than in milk. However, the accuracy of both tests at this time point was similar (sensitivity: 92 % in blood, 93 % in milk; specificity 53 % and 60 % respectively). None of the tests were able to detect open cows properly at this stage. On day 35 of gestation sensitivity (100 % for blood, 97 % for milk) and specificity (100 % for blood, 100 % for milk) were high enough to be used for reliable pregnancy diagnosis. The accuracy (sensitivity and specificity) for PAG concentrations in blood and milk for the rest of the study was 100 %. Our investigation shows that PAG determination in milk is a stress-free and non-invasive method for early pregnancy diagnosis in cattle.

Le but de cette étude était de comparer les concentrations de glycoprotéines associées à la gestation (PAG) dans le sang et le lait en vue d'un diagnostic de gestation précoce chez la vache. Des échantillons de sang et de lait ont été prélevés aux jours 0 (IA), 14, 21, 28, 35, 49, 63, 77, 91 et 105 de la gestation sur 60 vaches Holstein Frisonnes en lactation avec un fœtus vivant. On a utilisé pour mesurer la concentration de PAG une méthode RIA spécifique (RIA-706). Les concentrations de PAG dans le sang et le lait s'élevaient à partir du 28ème jour de gestation, les concentrations mesurées dans le sang étant nettement plus élevée que celles mesurées dans le lait. L'exactitude des deux tests était à ce moment-là similaires (sensitivité: 92 % dans le sang, 93 % dans le lait, spécificité: 53 % respectivement 60 %). Aucune des deux méthodes n'était, à ce stade, à même de distinguer une vache non portante avec justesse. Au 35ème jour de gestation, la sensitivité des deux méthodes (100 % dans le sang, 97 % dans le lait) et leur spécificité (100 % dans le sang, 100 % dans le lait) était assez élevées pour permettre un diagnostic de gestation sûr. L'exactitude (sensitivité et spécificité) des mesures de concentration de PAG dans le sang et le lait durant la suite de la gestation était de 100 %. Ces études montrent donc que la mesure de PAG dans le lait représente une méthode non-invasive et exempte de stress pour le diagnostic précoce de gestation chez la vache.

Pregnancy - associated glycoproteins as a new diagnostic tool in cattle reproduction.

Pregnancy-associated glycoproteins (PAGs) are powerful pregnancy markers in domestic cattle. These proteins are expressed in mono- and binucleate trophoblast cells from the first days of gestation until calving. Different molecules were identified as being expressed at various stages of pregnancy. However, up to date, their functions and activities during pregnancy were not yet established. Specific RIA tests were developed (classic and alternative RIA) and used to measure the concentration of these glycoproteins in blood during gestation and the postpartum period in cattle. In maternal blood, PAGs rise to detectable levels from days 24 to 28 after fertilization. A recent study indicated that PAGs can also be detected in milk samples. However, concentrations in milk are much lower when compared to those of plasma.

Concentration of bovine pregnancy associated glycoprotein in plasma and milk: its application for pregnancy diagnosis in cows.

Pregnancy diagnosis is an important part in reproduction management of ruminants. The aim of the study was to use a new method for evaluating the bPAG and cPAG in milk and blood bPAG and compare this results with the other method for pregnancy diagnosis in the cows. The study was carried out in 220 Holstein Frisian cows. Heparinised blood samples were taken from the jugular vein and stored at -20 degrees C until PAG assay by RIA. For bPAG and cPAG, RIA test, milk samples were homogenized. Pure bPAG was used as a standard tracer described by Zoli et al. (1992). The cows were diagnosed as pregnant by means of USG (Aloka SSD 210) and by rectal palpation. bPAG and cPAG concentration in milk increased after 28 day of pregnancy and showed the rapid increase near the parturition. The same results of bPAG concentration we obtained in the blood samples. The decline of bPAG concentration was faster in the milk than in the blood. The data showed that the RIA method is precise enough to measure PAG concentrations in the maternal blood and milk of cows. The data indicate that milk samples can be used for pregnancy diagnosis in cows. The sensitivity and specificity of RIA measurement of PAG are very high.

Adrenergic stimulation and blocking of hormonal secretion activity of cultured cow granulosa cells.

The aim of this study was to determine the influence of alpha- and beta-stimulators (alpha-stimulator: detomidinum HCl) as well as blockers (alpha1-blocker: doxazosin, alpha2-blocker: yohimbinum HCL, beta-blocker: carazolol) on bovine granulosa cells culture from preovulatory follicles. The cell culture was passed in TCM 199 medium with 10% FCS and antibiotics. Tested substances were added to the culture medium in different concentrations. The experiment began when at least 80% of the wells were covered (in four well culture dish of NUNCK-DK). The culture medium was collected every 24 h for hormone analysis. Hormone levels of T, E2, and P4 were determined. The culture was used up to 120 hours. Our results showed a decrease in P-4 secretion after detomidinum addition for all tested concentrations. A slight testosterone level increase was seen in the first 24 hours and then its concentration remained at a constant low level. A slight increase in 17-beta estradiol secretion was also observed. After yohimbinum addition, a statistically significant decrease of progesterone was observed for all concentrations tested. No significant changes were observed at other hormones levels when compared with the control. Doxazosin, when added into the culture medium, did not cause any statistically significant changes in hormone secretions. The addition of carazolol caused a significant decrease in progesterone secretion after culturing for 48 hours. Changes observed in other hormones levels did not differ statistically from the control. These results seem to support the hypothesis that drugs stimulating and blocking adrenergic receptors may play some role in ovarian steroidogenesis in cows.

The influence of alpha-adrenergic receptors stimulator and blockers and beta-blocker on the ovary and endocrinological activity in heifers during superovulation.

Twenty five Holstein-Friesian heifers, clinically normal and with regular oestrous cycles, were used for induction of superovulation (PMSG-PGF(2)alpha-Neutra-PMSG). Animals were divided into 5 groups receiving: I - detomidine (40 microg/kg b.w.), II - doxazosin (0.2 mg/kg b.w.), III - yohimbine HCL 1% (1 ml/50 kg b.w.), IV - carazolol (0.01 mg/ kg b.w., i.v.), and V - physiological saline (1 ml/50 kg b.w.). The heifers with PGF2 alpha-induced cycles were treated with the substances 88 hrs after being given a single i.m. injection of 2500 IU PMSG. All animals were examined by ultrasonography, and by the number and size of ovarian follicles > 3 mm in diameter. The follicles were divided into 3 groups according to the diameter. Blood plasma was stored at -20 degrees C until LH, P4, E2 and PGFM analyses. In the control (V) group, two waves of follicle growth were observed. Yohimbine produced a significant blockage of ovulation. The mean number of corpora lutea in the group III was significantly lower than that in the control group (p< 0.02). No significant differences in the number of corpora lutea were observed between the groups I, II and III. The increase in E2 concentrations could be the response to the PMSG treatment with two waves of growth of large follicles before and after ovulation. Pulsatile LH release was altered by yohimbinum injection, however, the greater amplitude of pulses immediately following yohimbinum administration are suggestive of a positive influence of the alpha-2 adrenergic receptors antagonist. Yohimbinum administration did not affect plasma concentration of examined hormones. There was a difference between the plasma levels of LH after the doxazosin injection. Single injection of the stimulators and blockers of adrenergic receptors did not affect superovulatory response in terms of the numbers of CL, unruptured follicles and embryos recovered. The affectivity of artificial insemination was not significantly different between the control group and the detomidinum groups, while in the yohimbinum group it was significantly lower.