Gene expression of pregnancy-associated glycoproteins-1 (PAG-1), interferon-tau (IFNt) and interferon stimulated genes (ISGs) as diagnostic and prognostic markers of maternal-fetal cellular interaction in buffalo cows.

The aim of this study was to determine the presence of Pregnancy-associated glycoprotein -1 (PAG-1) mRNA expression in the maternal circulation of pregnant buffaloes during the early stage of pregnancy. Contemporaneously, the mRNA expression levels of Interferon-tau (IFNt) and some Interferon stimulated genes (ISGs) (interferon stimulated gene 15 ubiquitin-like modifier interferon, ISG15; Mixoviruses resistance 1 and 2, MX1 and MX2; 2',5'-oligoadenylate synthase 1,OAS1) were evaluated in order to expand our knowledge of the molecular processes involved in the early stages of pregnancy and to identify potential biomarkers of maternal-fetal cellular interaction in buffalo. The study was conducted on 38 synchronized and artificially inseminated buffalo cows (d 0), divided ex post into 3 groups: Pregnant (n = 17), Non-pregnant (n = 15) and Embryo mortality (n = 6). Blood samples were collected on d 14, 19, 28 and 40 after artificial insemination (AI) for peripheral blood mononuclear cells (PBMCs) isolation. Expression levels of mRNA of PAG-1, IFNt, ISG15. MX1, MX2 and OAS1 were measured using RT-qPCR. No significant changes were observed in IFNt and PAG gene expressions between groups, while significant differences (p < 0.001) were found for ISG15, MX1, MX2, and OAS1. Pairwise comparisons revealed that the differences between groups occurred on days 19 and 28 post-AI. ISG15 proved to have the best diagnostic performance for distinguishing between pregnant animals and animals that experienced embryo mortality with the ROC analysis. According to the results of the univariate analyses, day 19 was identified as the most indicative to discriminate between groups while the most reliable genes for this differentiation were ISG15, MX1 and MX2. MX2 proved to be the best gene for discriminating pregnant buffaloes using the discriminant analysis, while MX1 was the gene that best predicted embryo mortality. Our results showed that among PAG-1, IFNt and ISGs expression as diagnostic and prognostic markers of maternal-fetal cellular interaction in buffalo cows, ISGs proved to be the best peripheral biomarkers for predicting pregnancy and embryonic mortality during the peri-implantation period. These insights into the mechanisms behind maternal-fetal interaction and the development of a method for the early detection of embryo distress may enable us to implement effective strategies to support embryo survival.

American Bison (Bison bison) reproductive endocrinology: serum Pregnancy Associated Glycoproteins (PAG), Progesterone, Estrone and Estrone-Sulfate in non pregnant animals and during gestation.

This study describes concentrations of Pregnancy Associated Glycoproteins (PAG), progesterone (P4), estrone (E1) and estrone-sulfate (E1S) in American Bison sera. In 2 ranches, mature American Bison were sampled once a year for 2 yr. Subsequent American Bison cows calving days were reported. PAG concentration was determined by Radio-Immuno Assay, whereas P4, E1 and E1S were assayed using Liquid Chromatography and Mass Spectrometry. Concentrations were compared between American Bison bulls (B, n = 7), Nonpregnant cows (NP, n = 32), first (1TP, n = 3), second (2TP, n = 26) and third (3TP, n = 15) trimester of pregnancy. Seven American Bison bulls and 92 cows were sampled, 51 calved during these 2 yr. Calving occurred mostly in spring (74.5%), but also in summer (13.7%) and fall (11.8%). PAG and P4 were higher in 2TP and 3TP than B and NP (P< 0.0001). P4 was non-basal in B and NP. E1 and E1S were correlated (P< 0.0001; r = 0.76) and increased in 2TP and 3TP when compared with B and NP (P< 0.01). Moreover, E1S was higher in 3TP than in 2TP (P< 0.0001) and correlated to pregnancy day (P< 0.0001; r = 0.60). Breeding American Bison in Belgium induces a calving seasonality loss. P4 slowly increases in 1TP and remains steady and high in 2 and 3TP. P4 non-basal and variable concentrations in B or NP disable its use as gestation marker. American Bison produce PAG in the 2 and 3TP, but Estrone-sulfate assay seems to be the best pregnancy marker during the 2 last trimesters as it could help to estimate the gestation period.

Responses to intra-luteal administration of cloprostenol in dairy cows.

The aim of the study was to determine the luteolytic dose of cloprostenol administered directly into the corpus luteum (CL; intra-luteal treatment, ILT) in dairy cattle. Cows of two control groups were treated with 500 µg of cloprostenol (Estrumate®) intramuscularly (IM-500) or via ILT with 0.2 mL of physiological solution (ILT-0). Cows of four experimental groups were treated by ILT with cloprostenol in doses 5, 25, 50 and 100 µg (ILT-5, -25, -50 and -100 groups). Progesterone concentrations (P4) and size of CL were evaluated to assess luteolysis at 0, 0.5, 1, 2, 4, 8, 24 and 48 h or at 0, 24 and 48 h after ILT, respectively. Cows in the ILT-0 and -5 groups were unaffected by ILT. The P4 concentrations were less in cows of the IM-500, as well as ILT-25, -50 and -100 groups at 48 h subsequent to ILT. The size of the CL was less in cows of IM-500, as well as ILT-25, -50 and -100 groups at 48 h after ILT. There were P4 concentrations of about 1 ng/mL 48 h after ILT in cows of the IM-500, as well as ILT-50 and -100 groups. In conclusion, the cloprostenol dose of 50 µg administered intra-luteally is a luteolytic dose in cows.

Pregnancy-associated glycoproteins (PAGs) concentrations in water buffaloes (Bubalus bubalis) during gestation and the postpartum period.

For the first time in literature this study describes the pregnancy-associated glycoprotein (PAG) profile of buffalo cows during gestation and the post-partum period using antiserum raised against PAG-molecules purified from buffalo placenta (AS#860). Ninety-eight buffalo cows, belonging to a buffalo herd subjected to a synchronization and artificial insemination (AI) program, were enrolled in this study. Blood samples were taken on days 0 (AI), 23, 25, 28, 30 and then biweekly until the end of pregnancy. Pregnancy was confirmed by ultrasonography on days 28 and 45, and by rectal palpation from day 60 onwards. Blood samples were suspended for the non-pregnant cows on day 45, while the blood of 20 buffaloes that had calved was tested every five days from the day of calving until day 50 post-calving. A cut-off value of 1.0 ng/mL was used in order to discriminate between pregnant and non-pregnant buffaloes. We used Linear Mixed models after Log(x+1) transformation to analyse the PAG concentrations. Fifty-two buffalo cows had become pregnant out of 98 synchronized (53%) and 46 remained non-pregnant (47%) as shown by ultrasonography and the PAG analysis. Significant differences (P < 0.001) in PAG concentrations were observed between the pregnant and non-pregnant buffaloes from day 23 as the PAG of the non-pregnant cows was always close to zero. Conversely, the PAG of the pregnant cows increased progressively from day AI until day 105 post-insemination and then stabilized until the end of pregnancy. Regarding pregnancy diagnosis, the sensitivity of PAG-RIA 860 system (ability of the test to correctly identify pregnant buffalo) ranged from 23% on day 23-98% on day 28 post AI; the specificity (ability to correctly identify non-pregnant buffaloes) was 100% throughout the sampling period. PAG progressively decreased from parturition to day 25 post-partum; from day 30 post-partum, the concentrations fell below 1 ng/mL and were close to 0 on the last day of observation (50 d post-partum). In conclusion, our results showed that RIA-860 is highly accurate for diagnosing pregnancy in buffaloes starting from day 28 of gestation. Furthermore, the rapid disappearance of PAG concentration after calving means that a cut-off limit in post-partum for detecting a new pregnancy is not required.

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.

Probiotic mixture supplementation in the preventive management of trinitrobenzenesulfonic acid-induced inflammation in a murine model.

Inflammatory bowel diseases (IBD) are characterized by inflammatory conditions of the intestine. Probiotic bacteria (PB) can have beneficial effects in several gastrointestinal disorders. The objectives of this study were: (i) to provide an acute experimental IBD model induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in CD-1 mice, and (ii) to assess the preventive effects of Citogenex (Lactobacillus casei and Bifidobacterum lactis) supplementation on intestinal tissues and microbiota. Mice were inoculated intrarectally with saline, ethanol or different TNBS solutions. 1%TNBS induced clinical signs of colitis (P less than 0.01) and histological damage (P less than 0.01). Based on these results, mice were pre-treated with Citogenex or saline for 1, 2 or 3 weeks before 1%TNBS treatment. Probiotic pre-treatment determined a reduction of clinical signs (P less than 0.05), histological alterations of colitis (P less than 0.05) and increased beneficial bacteria (P less than 0.05). This study confirms that TNBS-induced colitis in CD-1 mice is useful for studying the mechanisms involved in IBD pathogenesis, and pre-treatment with Citogenex prevents the intestinal damage induced by TNBS.

LH peak and ovulation after two different estrus synchronization treatments in buffalo cows in the daylight-lengthening period.

The aim of this study was to determine the timing of ovulation in relation to the LH peak after synchronization using PRID or Ovsynch protocols, to assess the effects of the period of treatment on these parameters and to provide information concerning how to use the two main protocols for fixed-time artificial insemination in buffalo. Forty-eight lactating Italian Mediterranean buffalo cows were used. The buffaloes were treated in various periods as follows: February to March (n = 12 PRID, n = 12 Ovsynch), end of the breeding season, May to June (n = 12 PRID, n = 12 Ovsynch), beginning of low-breeding season according to Italian environmental conditions. To determine the LH, blood samples were taken at 4-hour intervals, starting 24 hours from PRID removal (PRID group) or 12 hours from (PGF2α) injection (Ovsynch group) up to 108 hours. The ovaries were monitored by transrectal ultrasonography to verify ovulation. The LH-ovulation interval was similar in both groups (30.10 ± 1.05 and 32.77 ± 1.15 hours, respectively, in PRID and Ovsynch group). In the PRID group, the timing of ovulation in relation to device removal was 76.83 ± 3.65 hours with a high level of variability among the animals. In the Ovsynch group, we observed a better synchronization of LH peaks and ovulations, and the timing of ovulation in relation to the last GnRH injection was 35.67 ± 1.15 hours. The percentage of animals reaching the LH peak and ovulation was lower (P ≤ 0.05) in May to June (respectively 75.0% and 54.1%) compared to February to March (respectively 95.8% and 83.3%), indicating a reduction of hypothalamus-pituitary responsiveness to the synchronization treatments in the daylight-lengthening period.

Non-invasive assessment of animal exercise stress: real-time PCR of GLUT4, COX2, SOD1 and HSP70 in avalanche military dog saliva.

Exercise has been shown to increase mRNA expression of a growing number of genes. The aim of this study was to assess if mRNA expression of the metabolism- and oxidative stress-related genes GLUT4 (glucose transporter 4), COX2 (cyclooxygenase 2), SOD1 (superoxide dismutase 1) and HSP70 (heat shock protein 70) in saliva changes following acute exercise stress in dogs. For this purpose, 12 avalanche dogs of the Italian Military Force Guardia di Finanza were monitored during simulation of a search for a buried person in an artificial avalanche area. Rectal temperature (RT) and saliva samples were collected the day before the trial (T0), immediately after the descent from a helicopter at the onset of a simulated avalanche search and rescue operation (T1), after the discovery of the buried person (T2) and 2 h later (T3). Expressions of GLUT4, SOD1, COX2 and HSP70 were measured by real-time PCR. The simulated avalanche search and rescue operation was shown to exert a significant effect on RT, as well as on the expression of all metabolism- and oxidative stress-related genes investigated, which peaked at T2. The observed expression patterns indicate an acute exercise stress-induced upregulation, as confirmed by the reductions in expression at T3. Moreover, our findings indicate that saliva is useful for assessing metabolism- and oxidative stress-related genes without the need for restraint, which could affect working dog performance.

Validation of a new pregnancy-associated glycoprotein radioimmunoassay method for the detection of early pregnancy in ewes.

The aim of the current study was to describe the use of a pool of different antisera raised against pregnancy-associated glycoproteins (PAGs; purified from both ovine and caprine placentas) for early pregnancy diagnosis in ovine species. Sixty-three pluriparous Sarda ewes (Ovis aries) were synchronized. Blood samples were withdrawn on Days 18, 24, 26, 28, 30, and 50 after mating. These samples were assayed for progesterone (radioimmunoassay [RIA] including an extraction step) and for pregnancy-associated glycoproteins (RIA-706 and RIA-srPool). Progesterone concentrations were under 1.0 ng/mL in all nonpregnant Sarda ewes. In pregnant ewes, mean progesterone concentrations ranged from 2.4 ng/mL (Day 24, single pregnancies) to 4.4 ng/mL (Day 28, multiple pregnancies). During all periods of examination, PAGs remained lower than 0.8 ng/mL in nonpregnant ewes. On Day 18 of pregnancy, PAG concentrations could be detected in 26 of 43 (60.5%) and in 41 of 43 (95.3%) pregnant ewes using the RIA-706 and RIA-srPool methods, respectively. From Day 24 to Day 50, using both RIA methods, PAGs could be detected in all pregnant ewes. On Day 24, the best threshold for pregnancy diagnosis was obtained by use of RIA-srPool, maximal concentration in nonpregnant ewes being 0.3 ng/mL and minimal concentration in pregnant ewes being 4.8 ng/mL. In general, progesterone and PAG concentrations were higher in multiple pregnancies than in single pregnancies. However, because of large individual variations, single pregnancies could not be differentiated from multiple pregnancies.

Effects of field bean (Vicia faba L. var. minor) dietary supplementation on plasma thyroid hormones, insulin, insulin-like growth factor-1 concentrations and mohair characteristics in growing Angora goat kids.

Twenty-two Angora kids were used to study the effect of a dietary supplementation with field bean (Vicia faba L. var. minor) on hair follicle activity, mohair characteristics and plasma total thyroxine (T4), triiodothyronine (T3), insulin (INS) and insulin-like growth factor-1 (IGF-1) concentrations. At birth, their mothers were divided in two groups (S: supplemented, C: control), the S group being supplemented with 300 g/head/day of whole field bean. At weaning, diet of kids from S group was supplemented with 80 g/head/day of whole field bean. At weaning, secondary hair follicle activity (S: 0.91 ± 0.01, C: 0.84 ± 0.02, p < 0.01) and staple length (S: 5.82 ± 0.13 cm, C: 5.16 ± 0.14 cm, p < 0.001) were greater in the S group. At 155 days of age, secondary fibre diameter (S: 16.54 ± 0.35 µm, C: 18.09 ± 0.31 µm, p < 0.01) was higher in the C kids. Concentrations of total plasma T4 and T3 were higher (p < 0.001) in S (120 ± 12 and 4.87 ± 0.92 ng/ml respectively) than in C kids (92 ± 8 and 2.97 ± 0.77 ng/ml respectively). At weaning, T4 values were negatively correlated (p < 0.05) with the secondary fibre diameter and the T3/T4 ratio was positively correlated (p < 0.05) with the secondary to primary hair follicle ratio. At 155 days of age, both T3 and T3/T4 values were positively correlated (p < 0.01) with staple length. Plasma INS (0.11 ± 0.03 ng/ml) and total IGF-1 concentrations (232 ± 13 ng/ml) were not affected by diet. Improvements of diet quality in kids positively affected fibre diameter and length and follicle activity rate. We suggest that increased circulating thyroid hormones concentrations, but not those of INS and IGF-1, may contribute to the beneficial actions of nutritional supplementation on anatomical growth and mohair production of kids.

Isolation of new pregnancy-associated glycoproteins from water buffalo (Bubalus bubalis) placenta by Vicia villosa affinity chromatography.

The present study describes the isolation and characterization of new pregnancy-associated glycoprotein molecules (PAG) from midpregnancy and late-pregnancy placentas in the water buffalo (Bubalus bubalis). After extraction, the homogenates are subjected to acid and ammonium sulfate precipitations followed by DEAE chromatography. Subsequently, the water buffalo PAG (wbPAG) from these solutions are enriched by Vicia villosa agarose (VVA) affinity chromatography. As determined by western blotting with anti-PAG sera, the apparent molecular masses of the immunoreactive bands from the VVA peaks range from 59.5 to 75.8kDa and from 57.8 to 73.3kDa in the midpregnancy and late-pregnancy placentas, respectively. Amino-terminal microsequencing of the immunoreactive proteins has allowed the identification of three distinct wbPAG sequences, which have been deposited in the SwissProt database: RGSXLTIHPLRNIRDFFYVG (acc. no. P85048), RGSXLTILPLRNIID (acc. no. P85049), and RGSXLTHLPLRNI (acc. no. P85050). Their comparison to previously identified proteins has shown that two of them are new because they have not been described before. Our results confirm the suitability of VVA chromatography for the enrichment of the multiple PAG molecules expressed in buffalo placenta.

Blood testosterone levels in Italian Mediterranean buffalo bulls managed in two different breeding conditions.

In Italian buffalo cows the spontaneous cyclic ovarian activity is mainly high in autumn, while during spring and early summer it is very low. However many farmers separate males from females in the October-February period to obtain births in winter-spring. In order to verify if blood testosterone concentration in adult buffalo bulls is affected by season and by different management of the contact with females, 20 adult buffalo males, bred in central Italy were submitted to monthly blood sampling for 1 year, from September to August. The bulls were kept together with females all the time (group A; n=9) or were held separated from cows from October to February (group B; n=11). The mean (+/-S.E.M.) serum testosterone concentrations were higher in spring and summer than in autumn and winter in group B (2.07+/-0.1 ng/mL versus 0.99+/-0.08 ng/mL, P<0.01) but in group A the seasonal difference was not significant (2.09+/-0.13 versus 1.48+/-0.28). The management of the contact with females affected testosterone values (P<0.01): in the separation period (October-February) the mean serum concentration in group B was lower than in March-September, when the cows were together with the bulls (0.94+/-0.09 ng/mL versus 1.95+/-0.1 ng/mL, P<0.05). This is not true for group A (1.49+/-0.20 ng/mL versus 2.00+/-0.13 ng/mL, NS). It is concluded that contact with females exerted a major stimulus for the testicular androgen secretion in buffalo bulls, even if other seasonal factors (climate, food intake) may affect control of gonadal activity.

Unnecessary arterial Doppler examination of the legs. Clinical decision rules may help?

Health resources consumption depends mainly on physicians prescription habits. We identified reduction of unnecessary prescribed lower limbs arterial Doppler examinations as an area of potential cost control and quality improvement. We designed therefore a screening method based on clinical decision rules derived from epidemiological considerations: study of our records showed that patients with normal clinical examination and low risk factors score could be considered free from arterial disease by clinical grounds only, and that patients with normal clinical examination and very high risk score needed an extensive noninvasive evaluation. By offering a screening clinical examination (needing a working time shorter than a Doppler examination) with short waiting lists, we were able to safely exclude many normal patients from extensive Doppler examination, improving effectiveness by reducing total examination time by 22% and service quality by a Doppler examinations scheduling based on clinical severity judgement.

Changes in mandible and femur proteoglycans. Physico-chemical properties after hydrocortisone treatment.

Male Wistar rats were injected with 5 mg/kg/day of hydrocortisone during 21 days. Femur and mandible proteoglycans (PG) were isolated and their physico-chemical properties were compared with bone PG from a control and a starved group. The PG molecular weight decreased as a result of the hydrocortisone treatment but to a lower degree than in starvation, whereas the PG density seemed to be more affected by corticosteroid injection. These results allow us to conclude that bone alterations found after glucosteroid treatment may be due--at least partially--to changes in the PG physico-chemical properties of that tissue.

Changes in mandible and femur proteoglycans. Physico-chemical properties after hydrocortisone treatment

Male Wistar rats were injected with 5 mg/kg/day of hydrocortisone during 21 days. Femur and mandible proteoglycans (PG) were isolated and their physico-chemical properties were compared with bone PG from a control and a starved group. The PG molecular weight decreased as a result of the hydrocortisone treatment but to a lower degree than in starvation, whereas the PG density seemed to be more affected by corticosteroid injection. These results allow us to conclude that bone alterations found after glucosteroid treatment may be due--at least partially--to changes in the PG physico-chemical properties of that tissue.