Investigation of diagnostic use of serum anti-Müllerian hormone concentration in dioestrus and anoestrus bitches before and after ovariohysterectomy and the relationship with ovarian follicle numbers.

The aims of the study were to (1) compare the serum concentration of anti-Müllerian hormone (AMH) with the number of follicles in ovaries and (2) determine the serum AMH con- centration before and after ovariohysterectomy in dioestrus and anoestrus bitches. Sixteen bitches were divided into two groups: Group I (n=8) consisted of dioestrus and group II (n=8) anoestrus bitches. The blood samples for AMH assesment were taken before ovariohysterectomy (day 0) and on day 1, 5 and 10. Both in group I and II, serum AMH concentrations on day 1 and 5 were significantly different compared to day 0 (p⟨0.05). However, the concentrations at day 10 were under the minimum detectable concentration (1.0 ng/mL) and this finding revealed that ovaries are the only source of AMH synthesis. Follicle counts were not statistically different between the groups (p>0.05). Significantly positive correlation in serum AMH with secondary follicle num- bers (r=.942, p⟨0.01), as well as negative correlation with antral follicle numbers (r=-.765, p⟨0.05) were determined in the group I. In the group II, positive correlations between serum AMH concentration and secondary follicle numbers (r=.960, p⟨0.01) and early antral follicles (r=.726, p⟨0.05) were noted. Assesment of AMH concentration seems to not only provide the diagnosis of the presence of ovaries but also correlate with the number of secondary follicles in young dioestrus and anoestrus bitches.

GnRH and its receptor (GnRH-R) are expressed in the canine placenta and uterus.

In reproductive tissues, GnRH participates in the regulation of cell growth and proliferation by direct binding to the GnRH-R, which is essential for embryo implantation. However, there is no study on the expression and cellular localization of GnRH and GnRH-R in the canine uterus and placenta. Therefore, bitches were ovariohysterectomized 10 to 12 days after mating (vaginal cytology and progesterone measurement), the uteri were flushed, and if embryos were detectable, bitches were allocated to the embryo positive group (E-pos.; preimplantation, n = 5). Other bitches were operated at later stages and, dependent on the gestational age, either allotted to the post-implantation group (Day 18-25 after mating, n = 9), or the mid-gestation group (Day 30-40 after mating, n = 3). Dogs negative in embryo flushing served as controls (E-neg.; controls, n = 5). Samples of the entire uterine wall were taken from the middle of the horn in E-neg. and E-pos. groups, and from placental and interplacental uterine sites in post-implantation and mid-gestation groups. GnRH-R expression was localized at the mRNA and protein levels by immunohistochemistry and in situ hybridization. The expression of GnRH and GnRH-R mRNA was assessed by semiquantitative polymerase chain reaction. Additionally, both GnRH and GnRH-R mRNA were expressed in all tissues examined until mid-gestation. Relative expression of GnRH was higher than that of GnRH-R (P < 0.05). During the post-implantation stage, GnRH-R expression was significantly higher in uteroplacental than in interplacental tissues. In the uterus, GnRH-R stained strongly in the surface and glandular epithelial cells, and seemed to be weaker in myometrium and stroma. Placental signals were predominantly localized in fetal trophoblast cells and to a lesser extent in maternal decidual cells. These findings suggest a local regulatory function of GnRH during early canine pregnancy.

Endometrial echotexture parameters in Turkish Saanen Goats (Akkeci) during oestrus and early pregnancy.

This experiment was conducted to evaluate endometrial echotexture in oestrus and early pregnancy and its association with ovarian hormones and foetal count in goats. Akkeci goats (Saanen×Kilis crossbreed, n=40) were randomly divided into two groups. Ten does (NAT) were mated on natural oestrus and 30 does (SYN) were subjected to synchronisation-prior to mating. The uterus was scanned on the days of sponge insertion (d -14), sponge removal (d -2) and mating (d 0) as well as 17 (d 17) and 30 (d 30) days after mating. Mean gray level (MGL), homogeneity (HOM) and contrast (CON) values were calculated. Blood samples were collected on days ultrasonography was performed. Data were analyzed by Chi-square, ANOVA, regression tests. HOM value reached the highest level on the mating day and then continuously decreased (P<0.0001). Overall, HOM values were greater for SYN does than for NAT does after mating. CON values were virtually stable during the experimental period. MGL value fluctuated during the breeding period (P<0.03) at a similar fashion in NAT and SYN does. Foetal count was not correlated with plasma hormones and echotexture parameters. Plasma progesterone concentration was correlated with echotexture parameters (r=-0.28 for HOM; r=0.29 for CON; r=0.25 for MGL; P<0.05 for all) during post-mating. In conclusion, echotexture parameters changed during the breeding period, in association with plasma progesterone concentration. Future studies should test if the echotextural changes during embryonic fixation days can be used as a marker for early detection of pregnancy in does.

Expression of genes involved in the embryo-maternal interaction in the early-pregnant canine uterus.

Although there is no acute luteolytic mechanism in the absence of pregnancy in the bitch, a precise and well-timed embryo-maternal interaction seems to be required for the initiation and maintenance of gestation. As only limited information is available about these processes in dogs, in this study, the uterine expression of possible decidualization markers was investigated during the pre-implantation stage (days 10-12) of pregnancy and in the corresponding nonpregnant controls. In addition, the expression of selected genes associated with blastocyst development and/or implantation was investigated in embryos flushed from the uteri of bitches used for this study (unhatched and hatched blastocysts). There was an upregulated expression of prolactin receptor (PRLR) and IGF2 observed pre-implantation. The expression of PRL and of IGF1 was unaffected, and neither was the expression of progesterone- or estrogen receptor ß (ESR2). In contrast, (ESR1) levels were elevated during early pregnancy. Prostaglandin (PG)-system revealed upregulated expression of PGE2-synthase and its receptors, PTGER2 and PTGER4, and of the PG-transporter. Elevated levels of AKR1C3 mRNA, but not the protein itself, were noted. Expression of prostaglandin-endoperoxide synthase 2 (PTGS2) remained unaffected. Most of the transcripts were predominantly localized to the uterine epithelial cells, myometrium and, to a lesser extent, to the uterine stroma. PGES (PTGES) mRNA was abundantly expressed in both groups of embryos and appeared higher in the hatched ones. The expression level of IGF2 mRNA appeared higher than that of IGF1 mRNA in hatched embryos. In unhatched embryos IGF1, IGF2, and PTGS2 mRNA levels were below the detection limit.

Vaginal cytology after induction of ovulation in the queen: comparison of postoestrus and dioestrus.

OBJECTIVE: To compare the vaginal cytology of ovulating and non-ovulating queens. PROCEDURE: The study group comprised 15 queens showing behavioural oestrus. Ovulation was induced in 7 (dioestrus group) and 8 were left untreated (postoestrus group). Vaginal smears were collected from all animals prior to ovariohysterectomy on day 7. Epithelial cells were classified as basal-parabasal, intermediate, superficial, or anucleated superficial cells and counted using computer-assisted image analysis. From each smear, 50 representative vaginal epithelial cells were chosen. Digital images of cells were taken and cell area, cytoplasm area, nucleus area, cell diameter, cell perimeter, nucleus/cytoplasm ratio and red-green-blue (RGB) values were measured using image analysis software. Measurement data were compared between groups. RESULTS: Ovulation induction was successful in all animals. The swabbing procedure in oestrus did not induce ovulation in any postoestrus queens. Mean duration of oestrus was 6.65 ± 0.44 and 4.71 ± 0.32 days (P > 0.05) in the postoestrus and dioestrus queens, respectively. Intermediate cell count averaged 21.43% in dioestrus cats and 10.76% in postoestrus cats (P < 0.05). Epithelial cells in the postoestrus group had higher cell area, cytoplasm area, cell diameter and cell perimeter measurements (P < 0.01). Red (90.9 ± 1.6), green (76.1 ± 1.3) and blue (83.6 ± 1.4) channel values in postoestrus were higher than the values (81.3 ± 0.8, 65.8 ± 0.9 and 74.0 ± 0.7, respectively) in dioestrus (P < 0.01). CONCLUSION: Induction of ovulation in oestrus queens results in a significant increase in the number of intermediate cells and a significant decrease in both the dimensions and RGB values of vaginal epithelial cells on day 7.

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.

Is apoptosis a regulatory mechanism during early canine pregnancy?

Fas is a membrane-bound protein which upon activation causes programmed cell death. Fas ligand (FasL) binds Fas on target cells. Both these factors are known to regulate apoptosis at implantation in different species and thus might be involved in the regulation of implantation in dogs. The aim of the study was to assess the expression of Fas and FasL in canine uterine tissue throughout pregnancy as well as in pre-implantation embryos using RT-PCR and RT-qPCR. Uterine tissues was collected from of 21 healthy pregnant bitches (group I: days 10-12, n = 5; group II: days 18-25, n = 6; group III: days 28-45, n = 6) and from 4 non-pregnant bitches (controls: days 10-12). Pregnancy stage was determined by days after mating, that is, 2-3 days after ovulation as determined by vaginal cytology and progesterone measurement. After ovariohysterectomy, uteri from group I bitches were flushed with PBS and the embryos washed and stored frozen at -80°. Tissues from the other groups were taken from the implantation and placentation sites, respectively, covered with Tissue Tek(®) and frozen at -80°. Extraction of RNA was performed with Trizol Reagent and RT-qPCR using SYBR green probes. In pre-implantation embryos, only FasL but not Fas could be detected. In all tissues from pregnant and non-pregnant bitches, both parameters were detectable. Before implantation (group I) expression of FasL resembled that of non-pregnant bitches in early dioestrus and decreased significantly during implantation and thereafter (p < 0.05). Expression of Fas did not change significantly until day 45. The relative expression of Fas exceeded that of FasL at each stage investigated, which is comparable to observations of other species; however, high standard deviations indicate high individual differences. These preliminary results point towards a regulatory function of the Fas/FasL system during early canine pregnancy.

Expression and activity of matrix metalloproteinases in the uterus of bitches after spontaneous and induced abortion.

Aim of this study was to determine the intrauterine activity of matrix metalloproteinases (MMP)-2 and -9 after cessation of the local effect of progesterone. For this purpose, pregnancy was terminated in 10 bitches at mid-gestation with the progesterone receptor antagonist aglepristone (10 mg/kg body weight, sc, Alizine®; Virbac, France) at two subsequent days (group IRA = induced resorption/abortion). The IRA group was divided into two subgroups (Group I, n = 5, days 25-35 of pregnancy; group II, n = 5, days 36-45). Five further bitches were introduced with beginning abortion (group SRA = spontaneous resorption/abortion). Seven healthy bitches between day 25 and 45 of gestation served as controls. After ovariohysterectomy at the end of abortion and between days 25 and 45 of gestation, respectively, the distribution and activity of collagenases were investigated by immunohistochemistry and gelatin zymography. At placental sites, MMP-2 activity in the endometrium was significantly lower in IRA groups than in the SRA group (33.7 ± 11.8% and 39.3 ± 5.4% vs 52.2 ± 10.2%, p < 0.05); however, MMP-2 expression was lowest in the control group (control: 21.4 ± 6.3%; p < 0.01) and similarly in the myometrium (controls: 13.1 ± 2.5%; p < 0.05). MMP-9 activity was also lower in the endometrium and myometrium of the control group in comparison to SRA and IRA groups (11.8 ± 3.2%; p < 0.01 and 28.4 ± 32.8%; p < 0.05). At interplacental sites, the amount of active collagenases in the myometrium was significantly lower in the control group. It is concluded that the blockade of the biological progesterone effect was associated with an increase in activity of both collagenases.

Expression of MHC-I and -II in uterine tissue from early pregnant bitches.

The aim of the study was to investigate the expression of major histocompatibility complex (MHC)-I and -II in uterine tissues from pregnant and non-pregnant bitches, taken at different time periods after mating. The pregnant bitches were ovariohysterectomized during the pre-implantation (group 1, n = 4), implantation (group 2, n = 7) and placentation stage (group 3, n = 7). Non-pregnant animals in diestrus served as controls (group 4, n = 7). The expression of MHC- I and -II in salpinx, apex, middle horn, corpus uteri and at implantation sites was investigated by immunohistochemistry as well as qualitative and quantitative RT-PCR; MHC-I mRNA was detected in all tissues and with quantitative RT-PCR, and no significant changes were detected until placentation. Immunohistologically, at the apex and corpus site, the average number of MHC-II positive cells increased from the pre-implantation to the post-implantation stage (apex: 1.54 +/- 1.21 to 3.82 +/- 2.93; corpus: 1.62 +/- 1.9 to 5.04 +/- 4.95; p < 0.05). The greatest numbers of MHC-II positive cells were observed at placentation sites (6.64 +/- 5.9). In parallel, a marked increase in the relative mRNA expression of MHC-II in uterine tissues was assessed from the pre-implantation to the placentation stage (relative to Glycerinaldehyd-3-phosphate-Dehydrogenase (GAPDH): 6.9 +/- 9.5, 8.4 +/- 5.8, p > 0.05). Immunohistologically, in the salpinx, significantly greater numbers of MHC-II positive cells were found in the tissues of pregnant animals than in the control group (p < 0.05). It is proposed that the increase in MHC-II is pregnancy-related, even though the impact on maintenance of canine pregnancy is still unclear.

Induction of abortion with aglepristone significantly changed the expression of progesterone and estrogen receptors in canine endometrial stromal cells.

In the present study, resorption/abortion was induced between days 25 and 45 of gestation with aglepristone (group IRA, n=10). The aim was to observe the change in the distribution of progesterone (PR) and estrogen receptors (ER), in comparison to a group of spontaneous resorptions/abortions (group SRA, n=5), and a further group of normal healthy pregnant animals, ovariohysterectomized between days 25 and 45 of gestation (controls, n=7). The receptors were assessed by means of immunohistochemistry (IHC) and RT-PCR, at the placental and interplacental sites of the uterine horn as well as in the corpus uteri. Significant differences were observed between the controls on one side and the groups of resorption/abortion on the other side. The total scores of the progesterone receptors (TPR) in the placental and interplacental part of the uterine horn, was significantly lower in the endometrial stromal cells (ESC) of the control group than in those of the SRA- and IRA-group, respectively (placenta: 5.8 vs. 6.5 and 6.7, p<0.01; interplacental sites: 5.6 vs. 6.6 and 6.6, p<0.05). In contrary, the total scores of the estrogen receptors (TER) at interplacental sites and the corpus uteri, respectively, was significantly higher in the myometrial smooth muscle cells (MSMC) and the ESC (p<0.05) of the controls. We therefore conclude, that the here observed differences between groups point to an up-regulation of TPR- and a down-regulation of TER-scores in endometrial stromal cells at different uterine sites during resorption/abortion, which indicates a special role of these cells.

Expression of genes in the canine pre-implantation uterus and embryo: implications for an active role of the embryo before and during invasion.

The aim of the present study was to assess genes expressed in maternal uterine tissue and pre-implantation embryos which are presumably involved in maternal recognition and establishment of canine pregnancy. For this purpose, 10 pregnant bitches were ovariohysterectomized between days 10 and 12 after mating. Four non-pregnant bitches served as controls. Early pregnancy was verified by flushing the uterine horns with PBS solution. The collected embryos (n = 60) were stored deep-frozen (-80 degrees C). Uterine tissue was excised, snaps frozen in liquid nitrogen and homogenized using TRI Reagent. All embryos from one litter were thawed together and also homogenized in TRI Reagent. RT-PCR was performed to prove mRNA expression of progesterone receptor, key enzymes of the prostaglandin synthesis pathway, selected growth factors, cytokines, immune cell receptors, major histocompatibility complex (MHC) and matrix-metalloproteinases (MMP). Only pregnant uteri revealed the presence of mRNA for interferon (IFN)-gamma, IL-4 and CD-8, which resembles the milieu in humans and other mammalians. Similarly, in day 10 embryos, mRNA for transforming growth factor-beta, insulin-like growth factor-1,-2, hepatocyte growth factor, leukaemia inhibitor factor, tumour necrosis factor-alpha, interleukin-1beta,-6,-8, cyclooxygenase-2, CD4(+) cells, and MMP-2 and -9 were detected, but not MHC-I or -II. We therefore suppose that the canine embryo, like its human counterpart, actively initiates measures to prevent attacks from the maternal immune system to prepare its own adhesion, nidation, growth and further development.

Matrix metalloproteinase (MMP)-2 and MMP-9 activity in the canine uterus before and during placentation.

The aim of the present study was to demonstrate the presence and localization of MMP-2 and -9 by means of RT-PCR and immunohistochemistry (IHC) within the canine uterus from the pre-implantation stage until mid-gestation and to determine MMP-2 and -9 activities by means of zymography. For this purpose, samples of the uterus and salpinx from bitches were obtained after ovariohysterectomy. Pre-implantation stages (5-12 days after mating, n = 11) were determined by verifying embryos after flushing the uterus. Further groups were determined as implantation (15-19 days after mating, n = 9), post-implantation (20-30 days after mating, n = 9) and placental stages (30-45 days after mating, n = 3). A non-pregnant group (17-30 days after mating, n = 4) served as control. MMP-2 and -9 positive cells were detected in all specimens from pregnant and nonpregnant bitches, however, with different distributions. MMP-2 was present in endothelium and smooth muscles of blood vessels and the myometrium of pregnant and nonpregnant bitches, additionally in the surface epithelium of the oviduct. The latter also stained positive for MMP-9. During placentation, MMP-2 was detected mainly in fetal blood vessels and trophoblastic cells. Higher MMP-2 activity was observed in the endometrium and myometrium of all pregnant groups compared with the nonpregnant group (p < 0.05). The pregnant groups did not differ significantly from each other (p > 0.05). MMP-9 was present in blood vessels, smooth muscle cells and epithelia, such as maternal surface epithelial cells, uterine crypts and glands. During placentation, the deep uterine glands and the epithelium of the glandular chambers were immunoreactive to MMP-9. Highest MMP-9 activities were reached in the endometrium of the pre-implantation group (23.2% of total MMP-9) and placental parts (33.3%).

[Plasma concentrations of folic acid, vitamin B12 and progesterone of cyclic bitches, bitches during pregnancy and induced abortion and bitches with pyometra]. / Plasmakonzentrationen von Folsäure, Vitamin B12 und Progesteron während des Sexualzyklus, Gravidität, Abortinduktion und Pyometra bei der Hündin.

The aim of this study was to investigate the plasma concentrations of folic acid, vitamin B12 and progesterone at different stages of the sexual cycle and pregnancy, during induced abortion and in bitches with pyometra. Bitches (n = 97) were assigned to groups as follows: a) oestrous cycle (n = 42) b) pregnancy (n = 25) c) induction of abortion (n = 10 and d) pyometra (n = 20). Oestrous cycle stages were determined by vaginal inspection and cytology. Pregnancies were estimated by ultrasound (5.0 Mhz; linear transducer; Schimadzu) at days 15-25, 35-45 and 46-63 of pregnancy. Treatments for the induction of abortion were started between days 25 and 35 after mating (5 microg/kg cabergoline daily, Galastop; 5-10 microg/kg Alfaprostol every other day, Gabbrostim). Diagnosis of pyometra was confirmed by ultrasound and vaginoscopy. Folic acid and vitamin B12 concentrations did not differ among different stages of the oestrous cycle. The mean concentration of folic acid during early pregnancy (days 15-25) exceeded levels of later stages (days 46-63): 9.4 +/- 3.7 microg/ml and 4.7 +/- 1.8 microg/ml, respectively (p < 0.01). A positive correlation between folic acid and vitamin B12 was determined in pregnant dogs ( r = 0.925; p < 0.02). Before the induction of abortion, the concentration of folic acid was 9.6 +/- 5.2 microg/ml; during abortion it decreased to 5.0 +/- 3.2 microg/ml (p < 0.01). A significant correlation (r = 0.925; p < 0.02) between progesterone and folic acid was obtained in bitches with abortion. The mean concentration of folic acid in bitches with pyometra significantly differed from that of bitches at different stages of the oestrous cycle (p < 0.05). The mean concentration of folic acid was significantly lower in metoestrous bitches when compared to bitches with pyometra (p < 0.05). The decrease of serum concentrations of folic acid during pregnancy and induced abortion show that fetal growth and abortion caused higher consumption of folic acid. Concerning bitches did not show any deficiency symptoms, which is why it can be concluded that this decrease is physiological.

A screening for the occurrence of autoreactive antibodies in sera of pregnant and non-pregnant bitches.

Sera of healthy pregnant (group I, n = 11) and non-pregnant (group II, n = 11) bitches were screened for autoantibodies (AAb). In both groups, blood samples were drawn every fifth day between days 5 and 55 after mating. Serum was analysed via indirect immunofluorescence (IIF) with the Canine ANA HEp-2 Screening Kit. In all animals, anticytoplasmic AAb were detected. Utilizing primate-heart substrate slides AAb against contractile proteins of the cytoplasm could be observed. The predominating fluorescence pattern in pregnant animals resembled above all desmin, which was proven via Western blot. The sera were then pre-incubated with tropomyosin, actin, vimentin, vinculin and keratin solutions, and assessed on HEp-2 slides and on human and canine fibroblasts as well. The latter substrate was used to verify whether the detected Ab were in fact AAb. Utilizing tropomyosin, revealed elimination of the cytoplasmic fluorescences on all three substrates. It is therefore assumed, that in sera of healthy dogs, AAb against contractile structure proteins of the cytoplasm are present regularly. The majority of pregnant bitches presented with higher end-point titres (EPT), than to be found in non-pregnant dogs. AAb against desmin played the key role in those patterns. In addition, sera were screened for thyroid specific AAb, namely thyroglobulin, thyroid peroxidase (TPO), T3 and T4, and for AAb against insulin by ELISA or Western blot (TPO). Only in two of the pregnant bitches a weak positive reaction (1:100) for T3-AAb was detected.