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    Early Maturation of Corpus Luteum in Rabbits - Effect of Sildenafil Citrate on Luteolytic Capacity in the Early Luteal Period
    (Univ Fed Rio Grande Do Sul, 2023) Ucar, Eyyup Hakan; Peker, Cevdet; Erdogan, Gunes; Kose, Mehmet; Atli, Mehmet Osman
    Background: Prostaglandin F-2 alpha (PGF(2)alpha) binds to the specific receptor (PTGFR) on the corpus luteum (CL) in mammals, inducing regression of the CL structure (luteolysis) and initiating a new cycle. While PGF(2)alpha is effective only on mature CL, the immature CL structure (early luteal phase) resists PGF(2)alpha. In this study, sildenafil citrate, which is used to increase blood flow in the genital organs for treating specific pregnancy issues in women, was administered during the early luteal phase in a rabbit model to test the hypothesis of enhancing blood flow to the CL, thereby promoting earlier maturation and enabling a response to PGF(2)alpha. Materials, Methods & Results: The study was conducted in 2 sub-studies: clinical and molecular. A large number of rabbits were initially included in the sub-studies to ensure a sufficient number of pseudo-pregnant rabbits. Ovulation in rabbits was induced with buserelin acetate and was considered as day 0 of the study. The sub-studies were continued with rabbits whose pseudo-pregnancies were confirmed according to progesterone (P-4) results. As a result, the studies were continued with a total of 41 pseudo-pregnant New Zealand female rabbits, 21 of which were included in the clinical sub-study and 20 in the molecular sub-study. In both sub-studies, on day 3 of the luteal period, rabbits in the treatment group received 5 mg/kg sildenafil citrate and all rabbits received a single dose of exogenous PGF(2)alpha on day 4 to induce luteolysis. In the clinical sub-study, echotexture and intraovarian blood flow changes in the ovaries were determined by ultrasonography (USG) examination. In the molecular sub-study, the expression changes of Hypoxia Inducible Factor 1 Alpha (HIF1A) and Vascular Endothelial Growth Factor (VEGF) related to angiogenesis, Steroidogenic Acute Regulatory Protein (StAR) related to P-4 metabolism, Prostaglandin-Endoperoxide Synthase 2 (PTGS2) related to prostaglandin (PG) mechanism and 15-Hydroxyprostaglandin Dehydrogenase (HPGD) genes at mRNA level were determined using Real Time Polymerase Chain Reaction (RT-PCR) in CL tissues obtained with ovariohysterectomy (OVH) at 1 and 12 h after PGF(2)alpha injection. In addition, blood samples were collected for determine P-4 levels from all rabbits. In the clinical sub-study; there was no difference between the groups in mean gray values (MGV), whereas there was a significant decrease in both pulsatile index (PI) and resistance index (RI) values at 40 min after PGF(2)alpha injection (P < 0.05). In the molecular sub-study, it was determined that sildenafil citrate had no significant effect (P > 0.05) on the expression levels 1 and 12 h after PGF(2)alpha injection. According to the results of the molecular sub-study, no significant effect of sildenafil citrate on the mRNA expression levels in the investigated genes was detected (P > 0.05). However, within each group, differences were found according to OVH time after PGF(2)alpha injection. It was observed that PTGS2 and HPGD mRNA expressions decreased at the 12(th) h compared to the 1(st) h, while HIF1A expression increased (P < 0.05). Discussion: According to the results obtained from clinical and molecular sub-studies, it was determined that a single dose of sildenafil citrate (5 mg/kg) applied on the 3(rd) day of the luteal period did not contribute to the maturation process of the CL, did not increase blood flow, and was insufficient to break the resistance of the CL against PGF(2)alpha applied on the 4(th) day of the luteal period. However, a significant decrease in the PI value at the 40(th) min after PGF(2)alpha injection suggests that sildenafil citrate has a supportive effect, and that this decrease is also seen in the RI value, suggesting that its effect is insufficient against the vasoconstrictive effect of PGF(2)alpha.
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    Early pregnancy diagnosis using a commercial ELISA test based on pregnancy-associated glycoproteins in Holstein-Friesian heifers and lactating cows
    (Tubitak Scientific & Technological Research Council Turkey, 2016) Kaya, Mehmet Salih; Kose, Mehmet; Bozkaya, Faruk; Mutlu, Hasan; Ucar, Eyyup Hakan; Atli, Mehmet Osman
    In this study, we aimed to investigate the efficacy of a commercial ELISA test kit for detecting pregnancy-associated glycoproteins (PAGs) in peripheral blood for early pregnancy diagnosis and compare plasma PAG levels during early pregnancy in both Holstein-Friesian heifers and lactating cows. A total of 231 plasma samples were collected from heifers and lactating cows on days 25, 28, and 32 after insemination. Pregnancies were confirmed 30 days after the collection of plasma samples. Plasma PAG levels were measured using a commercial ELISA test kit for diagnosing bovine pregnancy. The effects of examination date and animal status (heifers vs. lactating cows) on PAG levels in heifers and lactating cows were analyzed using two-way ANOVA. Plasma PAG levels were significantly higher in heifers than in lactating cows; levels also increased significantly with the date of examination, i.e. days 25, 28, and 32 for heifers (P < 0.001), but not for lactating cows (P > 0.05). Although the sensitivity, specificity, and accuracy of pregnancy diagnosis using the ELISA test were acceptable in both groups, the performance of the test was superior in pregnant heifers compared to lactating cows.
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    Expression of circulating oar-miR-485-5p and oar-miR-493-5p during the estrous cycle and early pregnancy in ovine plasma
    (Brazilian Coll Animal Reproduction, 2024) Ucar, Eyyup Hakan; Hitit, Mustafa; Kose, Mehmet; Atli, Mehmet Osman
    In the current study, we aimed to assess the expression levels of two circulating microRNAs (miRNA) (oar-miR-485-5p and oar-miR-493-5p) in the ovine plasma during the peri-implantation. After mating, we collected the plasma samples from a total of 8 ewes on day 22 of pregnancy (P22; n = 4) and day 22 of the estrous cycle (C22; n=4). We used mature miRNA sequences for oar-miR-485-5p and oar-miR-493-5p out of one hundred fifty, which were retrieved from our microarray results of previous study. We showed that the miRNA expression of oar-miR-485-5p and oar-miR-493-5p were upregulated in P22 (P<0.05) when compared to C22. Those two miRNAs targeted 311 target genes in the peri-implantation period of pregnancy. Furthermore, we revealed 151 GO/pathway terms in biological process (BP) and 25 GO/pathway terms in molecular function (MF), while we demonstrated 13 GO/pathway terms in cellular component (CC). We revealed three hub genes as interleukin 2 (IL2), interleukin 18 (IL18), and C -X-C Motif Chemokine Ligand 10 (CXCL10). In conclusion, both miR-485-5p and oar-miR-493-5p have the potential to be a biomarker to understand peri-implantation of the ovine pregnancy in the aspect of pregnancy -reflected changes in maternal plasma.
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    Plasma profile of Pregnancy Associated Glycoproteins during Postpartum period in Konya Merino ewes lambing single and twin
    (Univ Zulia, Facultad Ciencias Veterinarias, 2023) Ucar, Eyyup Hakan; Kose, Mehmet; Kirbas, Mesut; Bozkaya, Faruk; Atli, Mehmet Osman
    This study investigated the plasma profile of Pregnancy Associated Glycoproteins (PAGs) after lambing in Konya Merino ewes using specific cattle pregnancy test kit. A total of 16 Konya Merino ewes were used as a material. Four groups were set up, ewes birthing a male lamb (SM group, n=4), a female lamb (SF group, n=4), twin male lambs (TM group, n=4) or twin female lambs (TF group, n=4). Blood plasma samples were taken on days 0 (lambing day), 3, 7, 10, 14 and then weekly until day 35. All samples were analysed using a bovine pregnancy test kit to detect of PAGs level. It was determined that there was a strong negative correlation between PAGs levels in peripheral blood and days after lambing (r2=0.969; P<0.01). However, no relationship was found between PAGs level and lamb gender or birth type. In conclusion, plasma PAGs level decreases rapidly in Konya Merino ewes regardless of lamb gender and birth type. Moreover, the results showed that the plasma profile of PAGs after lambing in Konya Merino ewes can be monitored with a bovine commercial ELISA-based pregnancy test kit, and the test results can be used in decisions and assessments based on the levels of PAG molecules.
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    Relative abundance and localization of interferon-stimulated gene 15 mRNA transcript in intra- and extra-uterine tissues during the early stages of pregnancy in sheep
    (Elsevier, 2020) Alak, Ilyas; Hitit, Mustafa; Kose, Mehmet; Kaya, M. Salih; Ucar, Eyyup Hakan; Atli, Zehra; Atli, Mehmet O.
    The aim of this study was to investigate relative abundance and localization of ISG15 mRNA transcript in intra-uterine (trophoblast, endometrium) and extra-uterine (hypothalamus, anterior pituitary, corpus luteum) tissues before and during the period of conceptus implantation. Multiparous ewes (n = 16) were randomly allotted into four groups: pregnant or estrous cyclic on days of 12 and 16 (n = 4 per group) following estrus. Relative abundances of ISG15 mRNA transcript were determined in the endometrium, corpus luteum, hypothalamus, and anteriorpituitary using real time quantitative PCR. Localization of ISG15 mRNA transcript was evaluated using in situ hybridization. The presence of ISG15 mRNA transcript was only visualized in intrauterine tissues including the endometrium and trophoblast on day 12 of pregnancy. The ISG15 mRNA transcript was detected in all tissues evaluated on day 16 of pregnancy. The abundance of ISG15 mRNA transcript was greater in the endometrium on day 12 of pregnancy than at other days when evaluations occurred while in all other tissues except the hypothalamus there were large abundances of ISG15 mRNA on day 16 of pregnancy. It is concluded that the ISG15 mRNA transcript is only present in intra-uterine tissues before conceptus implantation. The ISG15 mRNA transcript, however, is present in extra-uterine tissues of ewes during implantation probably due to an increased amount of interferon-tau in blood circulation that is produced by the developing embryo. Results also indicate, for the first time, that pregnancy is associated with an intra-hypothalamus and anterior pituitary increased abundance of ISG15 mRNA transcript in ewes.