Effect of ovulation rate and timing of ovulation after different hormone treatments on pregnancy rate in dairy cows.

Primiparous and multiparous lactating crossbred dairy cows with a mature corpus luteum and a follicle with >10 mm in diameter were treated with cloprostenol. Those cows that showed oestrus within 5 days after treatment were inseminated (Group P). The other cows (Group PG) were treated with GnRH 2 days after cloprostenol treatment and timed artificial insemination (AI) was performed on the consecutive day, or were inseminated (Group G) after detected oestrus and treated with GnRH immediately after AI. The control cows (Group C) after detected oestrus were only inseminated. All of the AIs using frozen semen were done between 6 and 7 a.m. while the ultrasonographic examinations after AI were performed between 4 to 6 p.m. The ovaries of each cow were scanned by means of transrectal ultrasonography from the day of AI until ovulation. Daily blood samples were collected for progesterone measurements. The ovulation and pregnancy rates among the groups changed between 84.6% and 95.5%, as well as 44.4% and 60%, respectively, however the differences were not statistically significant. All the cows were evaluated according to date of ovulation after AI and the pregnancy rate was 55.4% (Group 1: ovulation occurred between AI and 9-11 h after AI), 54.5% (Group 2: ovulation occurred between 9-11 h and 33-35 h after AI) and 35.5% (Group 3: ovulation occurred between 33-35 h and 57-59 h after AI), respectively. There was a trend (P=0.087) for 2.2 greater odds of staying open among cows inseminated between 33 to 35 h and 57 to 59 h before ovulation compared to cows inseminated within 9 to 11 h before ovulation. If ovulation occurred before AI, the pregnancy rate was only 22.2%, therefore determination of optimal time for AI is of great importance.

Using real-time PCR to identify pregnancy-associated glycoprotein 2 (PAG-2) in water buffalo (Bubalus bubalis) blood in early pregnancy.

This study investigates for the first time mRNA pregnancy-associated glycoprotein 2 (PAG-2) expression in blood cells during early pregnancy in water buffalo. The PAGs constitute a large family of glycoproteins expressed in the outer epithelial layer of the placenta in eutherian species. All PAGs are not concomitantly expressed throughout pregnancy; some of them are expressed in the earlier phases, whereas others appear later and are expressed over a shorter period. Twenty-one lactating buffaloes were analyzed-17 females were synchronized with PRID and artificially inseminated (AI), whereas four females were synchronized but not inseminated (control group). Blood was collected at Days 0, 18, 28, 40, and 75 from AI (AI = Day 0). Expression of PAG-2 mRNA in blood samples was measured with real-time polymerase chain reaction. Pregnancy diagnosis was performed on Day 28 (D28) and Day 40 (D40) after AI by ultrasonography (US) and by PAG-1 RIA method. The females diagnosed pregnant at D28 and confirmed pregnant at D40 were defined as D28(+)D40(+) group; the females diagnosed pregnant at D28 but not confirmed pregnant at D40 were defined as D28(+)D40(-) group; and the females that were diagnosed as nonpregnant on either days were defined as D28(-)D40(-) group. PAG-2 mRNA at Day 0 was not observed in any groups. The D28(+)D40(+) group showed the highest expression, starting on Day 18 and increasing progressively up to Day 75. PAG-2 mRNA was also expressed on Day 18 in both D28(+)D40(-) and D28(-)D40(-) groups, but their levels were lower than those of D28(+)D40(+) group and almost constant over time. PAG-2 mRNA was never detected in the control group. The significant difference in the expression of PAG-2 mRNA between the D28(+)D40(+) group and the D28(-)D40(-) group, starting from Day 18, suggests that these animals might have conceived, but have experienced early embryonic loss; therefore, the PAG-2 mRNA was still present in blood circulation although at lower levels, as found in the D28(+)D40(-) group. In conclusion, this study shows that PAG-2 mRNA can be detected in peripheral maternal blood cells earlier than circulating PAG-1 molecules and could be useful for studies on early pregnancy and embryonic mortality.

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.

Monitoring the circadian rhythm of serum and salivary cortisol concentrations in the horse.

Daily fluctuations of cortisol concentration in the blood or saliva have been repeatedly reported. However, several contradictions in the existing literature appear on this subject. The present study was performed to definitively establish options for testing adrenocortical function. To the best of our knowledge, this is the first study to evaluate parallel circadian rhythms in salivary and serum cortisol concentrations during a 24-h period. Twenty horses were examined under the same conditions. Blood and saliva samples were taken every 2 h for 24 h to determine the daily changes in cortisol concentrations of saliva and serum at rest and to determine the relationship between salivary and serum cortisol levels. Cosinor analysis of group mean data confirmed a significant circadian component for both serum and salivary cortisol concentrations (P < 0.001 in both cases). The serum cortisol circadian rhythm had an acrophase at 10:50 AM (95% CI, 10:00 AM-11:40 AM), a MESOR of 22.67 ng/mL, and an amplitude of 11.93 ng/mL. The salivary cortisol circadian rhythm had an acrophase at 10:00 AM (95% CI, 9:00 AM-11:00 AM), a MESOR of 0.52 ng/mL, and an amplitude of 0.12 ng/mL. We found a significant but weak association between salivary and serum cortisol concentrations; the Pearson correlation coefficient was 0.32 (P < 0.001). The use of salivary cortisol level as an indicator of hypothalamic-pituitary-adrenal axis activity may be warranted. However, the salivary cortisol levels are more likely to be correlated with free plasma cortisol than with the total plasma cortisol concentration.

Identification of multiple pregnancy-associated glycoproteins (PAGs) purified from the European bison (Eb; Bison bonasus L.) placentas.

This paper describes the first identified chorionic PAGs in the European bison (Eb), named EbPAGs, predominantly expressed during early and mid-pregnancy (45-120 day post-coitum; dpc). Many EbPAGs were extracted from various cotyledonary tissues, precipitated, chromatographed (DEAE and VVA: Vicia villosa agglutinin), electrophoresed (1D- and 2D-PAGE), analysed by heterologous (cross-species) Western blotting and then micro-sequenced by Edman degradation. Finally, twelve selected VVA-purified isoforms (Ip 3.7-7.4) were entirely characterised. Nine identified NH(2)-terminal micro-sequences were found to be PAGs. On 45 dpc, three identified forms were named: EbPAG(67AkDa) (RGSNLTHPLRNIGDLFYVGN), EbPAG(55BkDa) (RGSNLTHPL) and EbPAG(50CkDa) (SQISLRGSNLTI). On 60 dpc, the next three forms were named: EbPAG(71DkDa) (RGSNLTIHPLRNIIDLFYVG), EbPAG(55EkDa) (RGSNLTHPLRNI) and EbPAG(50FkDa) (SQISLRGS). On 120 dpc, three other forms were named: EbPAG(71GkDa) (RGSNLTHPLRNIRDLFYVG), EbPAG(60HkDa) (RGSNLTTHPLRNIKDLVVYM) and EbPAG(50IkDa) (SGSNLTTV). These EbPAG ((A-I)) sequences are unique, as they are not identical to any other PAGs purified previously in related species of the Bovidae family. However, the EbPAGs (A-I forms) have some sequence resemblance to internal sequences of various full-length polypeptide PAG precursors (in silico translated from cloned cDNAs) identified in domestic cattle. Three other novel native isoforms (J1, J2 and K): EbUPG(45kDa) J1 (SKDNYKNYIPLIVPFAT), EbUPG(45kDa) J2 (SKDNQKNYIPLIVPFAT) and EbUPG(76kDa) K (SPEFTV), were temporarily named 'unknown placental glycoproteins' (UPGs), due to their efficient VVA-purification (specific for glycoproteins only) and a lack of considerable consensus to previously sequenced placental glycoproteins in the Bovidae family. This is the first study identifying NH(2)-terminals of multiple/diverse EbPAGs and some EbUPGs purified from the synepitheliochorial cotyledonary placenta of the endangered Bison bonasus (Red List).

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.

Measurement of ovine pregnancy-associated glycoprotein (PAG) during early pregnancy in Lacaune sheep.

This study describes ovine pregnancy-associated glycoprotein (ovPAG) concentrations in 20 Lacaune sheep during early pregnancy. Measurements were performed by using semi-purified ovPAG as standard, tracer and immunogens for antibody production in rabbits. Antisera R780 (against ovPAG(57+59kDa)) and R805 (against ovPAG5(58+61kDa)) were used respectively in RIA-780 and RIA-805. Blood samples were collected at days 0, 18, 20, 22 and 25 after artificial insemination. From day 18 after breeding onward, the mean ovPAG concentration was significantly higher (p < 0.001) in plasma samples from pregnant ewes (n = 17) than in non-pregnant ones (n = 3). The specific activity of the tracer was 11 760 Ci/mmol in RIA-780 and 14 900 Ci/mmol in RIA-805. The minimal detection limits for RIA-780 and RIA-805 were 0.2 ng/ml and 0.3 ng/ml, respectively. The intra-assay CV of samples with low (1.0 ng/ml), medium (2.5 ng/ml) and high (4.0 ng/ml) PAG concentrations were 3%, 6% and 9% for RIA-780 and 8%, 9% and 5% for RIA-805. The inter-assay CV in the same samples were 13%, 12% and 7% for RIA-780 and 13%, 11% and 5% for RIA-805. The recovery was higher than 95% in both assays. No cross-reaction was observed with members of aspartic proteinase family as well as with other tested proteins. In both RIA-780 and RIA-805, inhibition of the binding of the tracer by antisera was parallel between standard curve and serial dilutions of pregnant ewe samples. In conclusion, the two homologous RIA systems are suitable for early quantification of ovPAG concentrations in ewe plasma samples from day 18 after breeding.