Evaluation of serum nitric oxide level with B-MOD and doppler ultrasonography findings in the process of luteolysis in rabbits

Objective: In the presented thesis, it was aimed to evaluate the luteal perfusion change, which is one of the stages of functional luteolysis in rabbits, through the possible change in B-mode and Doppler USG images and to measure serum nitric oxide (NO) levels. Material and Methods: In this study pseudopregnancy is induced with gonadotropin-releasing hormone (GnRH; 0.2 mL Buserelin, im) to a total of 14 female rabbits. Luteolysis was induced in the first study group (Group 1, n=7) with Prostaglandin F2α (PGF2α) injection (200 µg alfaprostol) on the 9th day of pseudopregnancy. In Group 2 (n=7), placebo was injected to form the control group. B-mode and Doppler ultrasonography was performed with 18 MHz microconvex probe before injection, 40-80-360 min and 24 hours after injection in all rabbits. The area (mm2), mean grayness value (MGV) and heterogeneity values on the total ovarian tissue were calculated from the B-mode images with the help of Image-J program. Pulsatile (PI) and resistance (RI) index values were recorded over the arteriole, which was observed closest to the luteal tissues in Doppler measurements. Each measurement value was recorded separately for the right and left ovaries. Before luteolysis (days 0-3-6-9) and at the relevant hours after USG, 4mL blood sample was taken from the ear vein for serum progesterone measurement. In addition, blood samples were taken only at the relevant hours after USG for NO measurement. Blood samples were stored at -24 0C until measurement by ELISA method. Accordingly, ultrasonographic findings and serum measurement results were determined by Student T-test and Mann Whitney U test between groups, considering the ovaries in the same directions between the experimental and control groups; Again, it was evaluated statistically using analysis of variance and Freidman's Test to calculate the time-dependent variation within the group. Results: When the individual clinical responses were examined, behavioral changes such as feathering and nesting were observed in false pregnancies, and although the corpora lutea could be observed with USG, no significant difference was found between the groups in terms of serum P4 levels. When ultrasonography parameters examined: the left ovarian area of the experimental group was smaller than the control group at 360 min (P<0.05). In the intragroup evaluations, the surface area of the right ovary at the 40th minute was larger than the left ovary in both groups (P<0.05). In terms of mean grayness values (MGV), the left ovaries of the experimental group were higher than the control group at the 0th minute measurements (P<0.05). In addition, the MGV value of the left ovaries of the experimental group increased significantly until the 80th minute and decreased at the 360th minute (P<0.05). When the heterogeneity values were evaluated between the groups, it was observed that the right ovarian heterogeneity value of the experimental group was lower at the 360th minute (P<0.05). In the experimental group, the heterogeneity value increased on the right side compared to the left side at 360 min (P<0.05). In terms of Doppler USG findings, there was no difference between the groups in terms of PI and RI values. However, the RI value of the right ovary of the control group was higher at 0 min than the subsequent measurements (P<0.05). There was no difference between the groups in serum NO levels during the study. However, in the control group, an increase was observed at the 80th minute and a significant decrease was observed at the 360th minute (P<0.05). Conclusion: In the light of the findings obtained, it has emerged that there may be difficulties in the hormonal follow-up of false pregnancy induction in rabbits, and the clinical response of the cases should be monitored as well as the steroid levels. In addition, it is thought that it would be more beneficial to develop rapid test kits and make real-time measurements in the days following the induction, instead of storing the samples before the measurement and measuring them with the ELISA method after the study. Ovaries in rabbits, which have a more variable location in the abdominal cavity due to their relatively small size and long horn structure compared to other species, can be visualized non-invasively by ultrasonography applied with the right technique. However, during the application, location asymmetry and increase in artifact due to intra-caecal gas on the right side should be considered. The functional/structural asymmetry phenomenon, which has been proven in the past in other mammals and poultry after the study, seems to be possible for rabbit ovaries as well. Accordingly, it was concluded that the right ovary might be larger and more active. Based on the MGV and Heterogeneity changes detected after luteolysis, echotexture studies can be used to confirm luteal regression. However, there is a need for more detailed procedures and follow-up examinations such as daily ecotextural examinations of the corpora lutea, which will lyse and leave its place to the corpus albicans, over a longer period of time. It has also been seen that the perfusion of the ovarian tissue can be followed by Doppler ultrasonography in rabbits, and it can easily take place in clinical practice in terms of Pulsed Doppler examinations. Due to the limited number of studies about relationship between luteal regression and NO in rabbits, and the fact that this change was not observed in this trial, it was concluded that it should be investigated in a more detailed study with a different measurement technique in the future. Keywords: Luteolysis, Nitric Oxide, Pseudopregnancy, Rabbit, Ultrasonography.