Does serum anti-Müllerian hormone levels always discriminate presence of the ovaries in adult bitches? Comparison of two ELISA kits.

Anti-Müllerian hormone (AMH) is produced in the ovary, and thus, it is an excellent marker of follicle pool in females. Current interest is the clinical use of this parameter as a biomarker to assess presence or absence of an intact ovary and to diagnose ovarian remnant syndrome (ORS) following incomplete ovariohysterectomy (OHE) in bitches. The aim of this study was to evaluate serum AMH concentrations in bitches (n = 34) before and after OHE using two different commercial ELISA kits, one of which is based on detecting human AMH and the other is based on detecting human AMH and the other specified for canine AMH. Furthermore, serum AMH levels were also measured in six ORS cases to compare the diagnostic utility of the two different ELISA kits. Serum AMH concentrations measured using the human and canine kit prior to and after OHE were 0.32 ± 0.24, 0.006 ± 0.22 ng/ml (p < .001) and 12.08 ± 22.81, 9.55 ± 15.42 ng/ml (p = .868), respectively. Thus, the canine-based kit was not able to reveal the significant drop in serum AMH levels. In conclusion, the human-based ELISA kits successfully detected the drop in serum AMH concentrations. Reliable results can only be achieved from well-designed ELISA kits, and AMH levels might be a useful diagnostic tool for the evaluation of presence or absence of ovaries as well as for the detection of ORS cases in bitches.

The effects of magnesium sulphate on the contractile activity of uterus in an animal model of preeclampsia.

PURPOSE: This study was undertaken to evaluate the effects of magnesium sulfate (MgSO4) on the contractile activity of the uterus in a pregnant rat model of preeclampsia induced by N-nitro-arginine methyl ester (L-NAME). MATERIALS AND METHODS: Twenty-eight, 160-220 gram, three to four month old female Sprague-Dawley rats were used in this study. After conception was confirmed by vaginal smears on the first day of pregnancy, the animals were allocated into four groups according to the chemicals fed in their drinking water as control (nothing administered), L-NAME (50 mg/kg L-NAME), MgSO4 (600 mg/kg MgSO4), and MgSO4 + L-NAME group (600 mg/kg MgSO4 + 50 mg/kg L-NAME). The pregnant uterus strips were isolated on the 19th day and the contractile activity of uterus was examined by applying 0, 0.1, 0.2, 0.4, 0.8, and 2.5 mIU/ml oxytocin to each group and responses are recorded accordingly. RESULTS: There were no statistically significant differences regarding fetal parameters and peak amplitudes of the oxytocin stimulated pregnant rat myometrial strips among groups. In L-NAME group at 0 and 0.1 mIU/ml oxytocin, the contraction frequency in a ten-min period was statistically lower than the control group (Z = -2.850, p = 0.004; Z = -2.902, p = 0.004, respectively). In MgSO4 group only at 0 mIU/ml oxytocin, the frequencies of the contractions in ten-min period were statistically lower than the control group (Z = -2.973,p = 0.003). In L-NAME + MgSO4 group at 0, 0.1 and 0.2 mIU/ml oxytocin concentrations the frequencies of the contractions in ten-min period were statistically lower than the control group (Z = - 4.018, p = 0.000; Z = -3.237, p = 0.001; Z = -2.902, p = 0.004, respectively). In L-NAME + MgSO4 given group at each oxytocin concentrations, the frequencies of the contractions in ten-min period were lower but not statistically different than the L-NAME group. CONCLUSION: MgSO4 has no significant effect on the amplitude of spontaneous or oxytocin induced myometrial contractions, but decreased the frequency of spontaneous contractions. At each doses of oxytocin, MgSO4 has no significant effect on the frequency of contraction in a pregnant rat model of preeclampsia induced by L-NAME.

Vascular endothelial (VEGF) and epithelial growth factor (EGF) as well as platelet-activating factor (PAF) and receptors are expressed in the early pregnant canine uterus.

The aim of this study was to investigate the course of expression of platelet-activating factor (PAF), PAF-receptor (PAF-R), epidermal growth factor (EGF), EGF-R, vascular endothelial growth factor (VEGF), VEGF-R1 and VEGF-R2 in uterine tissue during canine pregnancy. For this purpose, 20 bitches were ovariohysterectomized at days 10-12 (n = 10), 18-25 (n = 5) and 28-45 (n = 5) days after mating, respectively. The pre-implantation group was proven pregnant by embryo flushing of the uterus after the operation, the others by sonography. Five embryo negative, that is, non-pregnant, bitches in diestrus (day 10-12) served as controls. Tissue samples from the uterus (placentation sites and horn width, respectively) were excised and snap-frozen in liquid nitrogen after embedding in Tissue Tec(®). Extraction of mRNA for RT-PCR was performed with Tri-Reagent. In the embryos, mRNA from all factors except VEGF was detected. In the course of pregnancy, significantly higher expression of PAF and PAFR as well as VEGF and VEGFR2 during the pre-implantation stage than in all other stages and a strong upregulation of EGF during implantation were characteristic. The course of EGF was in diametrical opposition to the course of the receptor. These results point towards an increased demand for VEGF, EGF and PAF during the earliest stages of canine pregnancy.

The effectiveness of gender determination using polymerase chain reaction and radioimmunoassay methods in cattle.

The aim of this study was to use polymerase chain reaction (PCR) by amplifying DNA from bovine (Bos taurus) fetal cells recovered through uterine puncture and subsequent amniotic fluid aspiration and to compare the effectiveness of the PCR method with amniotic dihydrotestosterone (DHT) levels in gender determination. Amniotic DHT levels between sexes were significantly higher in males than in females in all periods except the period 91 to 120 d. The differences among the amniotic DHT levels at different gestation periods (61 to 90, 91 to 120, 121 to 150, 151 to 180, 181 to 210 d) were not significant in females but were significant in males in the period 61 to 90 d compared with three other periods. Sensitivity was equal to 97.8% (95% CI=88.2% to 99.6%), and specificity was equal to 85.4% (95% CI=80.0% to 97.6%). These two values correspond with a cutoff of DHT in amniotic fluid. Distributions of the two sex groups were classified according to the 192.1 pg/mL cutoff value. A total of 93 amniotic fluid samples were examined by PCR analysis. The sex determination of 91 samples by PCR and electrophoresis was in agreement with the visual sexes of the fetuses. In two amniotic fluid samples, DNA was not isolated, and thus no sex determination was made. Fetal gender was correctly identified by PCR in 44 of 45 males and in 47 of 48 females. In PCR, one band (at the length of 102bp) and two bands (at the lengths of 102 and 226bp) were observed respectively for female and male fetuses. It may be concluded that the levels of amniotic DHT and PCR might be used for embryo sexing in pregnant cows.