Serhan Serhat Ay

Laparoscopic ovariectomy in dogs: comparison between single portal and two-portal access.

OBJECTIVE To compare surgical times and perioperative complication rates of single portal access and 2-portal laparoscopic ovariectomy (LapOVE) in dogs using a bipolar vessel sealer/divider device, and to evaluate the performance of novice laparoscopists for right ovariectomy. STUDY DESIGN Controlled clinical trial. ANIMALS Female dogs (n=42). METHODS Dogs were divided into groups: 1=single portal and 2=2 portal. LapOVE was performed using a 5 mm vessel sealer/divider device and a 10 mm operating laparoscope (Group 1) or a 5 mm laparoscope (Group 2). Dog characteristics (weight, body condition score, ovarian ligament fat score), operative time, and perioperative complication rate were compared between groups. Right ovariectomy duration was evaluated for 2 novice laparoscopists. RESULTS No significant difference was found in mean total surgical time between group 1 (21.07 min/s) and group 2 (19.06 min/s). Factors significantly affecting times included body condition scores, ovarian ligament fat score, ovarian bleeding, and surgeon expertise. Minor complications (bleeding from ovaries or after splenic trauma) occurred and were similar in both groups. Bleeding was correlated to body condition score and ovarian ligament fat score. Interindividual differences were found among surgeons for right ovariectomy time. CONCLUSIONS Single portal access LapOVE using vessel sealer/divider device is feasible, safe, and does not significantly increase total surgical time in comparison with 2-portal approach. Laparoscopic skills may play a role in ability to perform single portal LapOVE. CLINICAL RELEVANCE LapOVE can be performed using single portal access.

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.

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 β (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.

Cytokines, growth factors and prostaglandin synthesis in the uterus of pregnant and non-pregnant bitches: the features of placental sites.

Uterine tissue from pregnant bitches was investigated by qualitative RT-PCR for the gene expression of local factors potentially important for the implantation of canine embryos. For this purpose, 10 bitches identified as being at the time of implantation or early placentation by means of ultrasonography before ovariohysterectomy (days 20-35, n = 10) provided tissues for comparison to tissue collected in a previous study and identified as early pregnant (n = 10) or non-pregnant (n = 4) by embryo flushing after ovariohysterectomy (days 10-12 after mating; Schäfer-Somi et al. 2008). Uterine tissue was excised from the middle of the left horn from early pregnant and non-pregnant animals, including from interplacental and placentation sites. The following genes were investigated: CD-4, -8; cyclooxygenase (COX)-1, -2; granulocyte macrophage-colony stimulating factor (GM-CSF); hepatocyte growth factor (HGF); insulin-like growth factor (IGF)-1, -2; transforming growth factor (TGF) and tumour necrosis factor (TNF)-alpha; interferon (IFN)-gamma; interleukin (IL)-1beta, -2, -4, -6, -8, -10, -12; leukaemia inhibitory factor (LIF) and leptin. Gene expression for CD-8, COX-1, TGF-beta, HGF, IGF-1, IL-2, -4,-10, IFN-gamma and LIF were detected in the pre-implantation uterus, and all except IL-2 and -10 were still detectable during the implantation and placentation stage. During implantation, mRNA for IGF-2 and GM-CSF were additionally detected. The dioestrous uterus differed from the pregnant uterus because of the absence of CD-8, IL-4 and IFN-gamma and the expression of CD-4, TNF-alpha and IL-6. The results suggest that IL-4, IFN-gamma, CD-8, GM-CSF and IGF-2 are regulated in a pregnancy-specific manner and that GM-CSF and IGF-2 probably have growth supporting and immune modulating functions during implantation of the canine embryo.

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.

Uterine progesterone receptor and leukaemia inhibitory factor mRNA expression in canine pregnancy.

The study investigated the expression of genes for progesterone receptor (PR) and for the cytokine leukaemia inhibitory factor (LIF) in the uterine tube and uterine horn tissues from pregnant and non-pregnant bitches. The aim was to study whether a relation existed between the likely biological effectiveness of progesterone (P(4)) and the change in the uterine expression of LIF mRNA during pregnancy, as has been described in primates. For this purpose, 20 pregnant bitches were ovariohysterectomized after being allotted to three groups according to gestational age (pre-implantation: days 10 to 12, n = 7; peri-implantation: days 18 to 25, n = 7; post-placentation: days 28 to 45, n = 7). Tissue samples were obtained from the uterine tubes, one uterine horn (including placentation sites and interplacental sites in bitches that had already implanted) and the corpus uteri, stored at -80 degrees C, and then analysed by qualitative and quantitative PCR for PR and LIF mRNA expression. From the pre-implantation to the placentation stage, a decrease in the relative expression of PR mRNA in uterine tissue was obvious and significant when expressed relative to beta-actin (11.2 +/- 6.8 vs 2.7 +/- 1.9; p < 0.05). However, over the same period, the relative expression of LIF mRNA increased (10.1 +/- 16.1 vs 50.0 +/- 32.3; p < 0.05). In addition, PR mRNA went from being detectable to no longer detectable in the uterine tube, and no longer detectable in interplacental-site uterine tissue. We conclude that LIF is important for the establishment of canine pregnancy; that decreased uterine PR mRNA expression may contribute to the increase in uterine LIF mRNA; and, that the ability of the embryo to preserve PR mRNA expression at implantation and placentation sites while expression is lost in the remainder of the uterus represent an effect important to the establishment and maintenance of pregnancy. We additionally propose that canine embryo secretory proteins have a regulatory effect on both PR and LIF before as well as at and after implantation.

Effect of deslorelin on testicular function, serum dihydrotestosterone and oestradiol concentrations during and after suppression of sexual activity in tom cats

Objectives The aim of the study was to evaluate the efficacy of a 4.7 mg deslorelin implant in tom cats. Methods Nine mature male cats were included in the deslorelin group and five cats in the control group. Before the study started, all cats were confirmed to have distinct sexually dimorphic behaviour. Blood samples were taken on the implantation day, at day 7 and at day 15, then monthly, in order to measure serum dihydrotestosterone (DHT) and 17beta(β)-oestradiol concentrations. The deslorelin group (n = 9) was divided into two subgroups: five cats (cats 1–5) were neutered in the postimplantation period during suppression of sexually dimorphic behaviour, and four cats (cats 6–9) were neutered after re-expression of sexually dimorphic behaviour. The control group cats (n = 5) were castrated without administration of the implant. Results Sexually dimorphic behaviours ceased within a mean ± SD of 13–58 days (23.30 ± 14.17) after implantation. DHT concentration decreased within 30 days. The mean duration of suppression was 26.5 ± 7.42 months and reactivation coincided with increased DHT values reaching preimplantation concentrations within 1 month. 17β-oestradiol concentrations significantly correlated with DHT concentrations (P <0.01). For cats castrated during suppression of sexual behaviour, the length of the long axes of the nuclei of Leydig cells, the diameter of seminiferous tubules and the height of the epithelium of the seminiferous tubules did not change until 3–6 months after implantation, whereas at 12 and 32 months the measured values were even lower than in the control group. For cats castrasted after reactivation, the length of long axes of the nuclei of Leydig cells and the diameter of seminiferous tubules approached the values of the control group between 4 and 6 months after reactivation. Conclusions and relevance A deslorelin implant (4.7 mg) suppresses sexually dimorphic behaviour in tom cats without any side effects and with full reversibility; however, duration of suppression is highly individual.

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.

Tarantula cubensis extract alters the degree of apoptosis and mitosis in canine mammary adenocarcinomas

In the present study, 13 clinical cases of canine mammary adenocarcinoma were evaluated in order to understand the effect of Tarantula cubensis extract (TCE) on tumor tissue. Punch biopsies were taken from the tumors before treatment with TCE. Subcutaneous injections of TCE were administered three times at weekly intervals (3 mL per dog). Between days 7 and 10 after the third injection, the tumor masses were extirpated by complete unilateral mastectomy. Pre- and post-treatment tumor tissues were immunohistochemically assessed. The expression of B-cell lymphoma 2 (Bcl-2) was found to be higher in pre-treatment compared to post-treatment tissues (p < 0.01) whereas Ki-67 expression was lower in post-treatment tissues (p < 0.01). No significant differences in fibroblast growth factor or vascular endothelial growth factor expression were observed between pre- and post-treatment tissues (p > 0.05). The apoptotic index was determined to be low before treatment and increased during treatment. These results suggest that TCE may be effective for controlling the local growth of canine mammary adenocarcinoma by regulating apoptosis.

The Effects of Separate and Combined Use of PGF2α and GnRH Hormones and the Addition of Βeta-Carotene on Fertility Parameters in Dairy Cows with Ovarian Cysts

Background: Ovarian cysts are commonly observed pathologies, which interfere with normal cyclic activity and adversely affect fertility in cows. Beta-carotene is effective in the reduction of reproductive problems by inducing the natural defence mechanisms of the body. There are several methods that can be used for the treatment of ovarian cysts. The separate and combined use of GnRH and PGF2α commonly uses in the treatment of ovarian cysts. Therefore, in the presented study the effects of Beta-carotene (βC) addition for the treatment of ovarian cysts either with GnRH solely or GnRH and PGF2α in combination on the fertility parameters of dairy cows were investigated. Materials, Methods & Results: Seventy-six Holstein Friesian cows having ovarian cysts diagnosed by ultrasonography (USG) were divided into three groups. Cows in Group I (GI, n = 27), were injected with GnRH (Buserelin acetate, 5 mL, im), PGF2α (Tiaprost-trometamol, 5 mL, im) and βC (20 mL/cow, into 4 regions by im route). In Group II (GII, n = 25) GnRH (Buserelin acetate, 5 mL, im) and PGF2α (Tiaprost-trometamol, 5mL, im) were administrated while GnRH (Buserelin acetate, 5 mL, im) solely in Group III (GIII, n = 24). Cysts were monitored via USG, and blood samples were collected on the on day of treatment (day 0) and on the 7th and 14th days following the administrations. Cows shoving oestrous were inseminated and pregnancy diagnoses were performed on the 40th day following insemination. Treatment results showed that there were statistically no significant differences between GI and GII (P > 0.05). Only numerical difference obtained in time from therapy to pregnancy and overall pregnancy index (P > 0.05). Overall pregnancy rate (85 %), first service pregnancy rates (40 %) and overall pregnancy index (2.11) in GI were found significantly higher than GIII (53.3 %; 20 %; 4.12) [P 0.05). It was found that βC administrations significantly increased βC levels in GI than GII and GIII on the 7th and 14th days (P < 0.05). Discussion: One of the most common problems encountered in modern dairy production is the development of ovarian cysts. Treatments for ovarian cyst are numerous and variable, and have changed considerably over the years. In the present study, GnRH and PGF2α were administered together as a combination, and as a result of this combined use, higher percentages were obtained for both pregnancy rate and fertility parameters in GI and GII, in comparison to the group administered with GnRH alone (GIII). Better outcome from combination therapy (GnRH and PGF2α) may be due to the fact that luteal thickening in the walls of cysts was determined by ultrasonography, but P4 values were not identified immediately and the treatments were not categorized in accord with these values. No statistically significant differences were determined between GI and GII concerning the fertility parameters investigated, however numerical and proportional differences were observed. βC levels were significantly higher on day 7 and 14 after treatment in GI which were administered βC additionally to the treatment protocol for ovarian cysts. This statistical difference suggests that administration of βC in combination therapy is also effective in the treatment of ovarian cysts. In conclusion, it was determined that high pregnancy rates were obtained by the combined treatment of ovarian cysts (GnRH + PGF2α) and number of inseminations per conception were at desired limits. Better percentile and numerical fertility parameters were achieved in the group, which additionally received βC, high numbers of infertility cases, βC supplementation could be a viable option for treatment.