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Circulating miRNAs in maternal plasma as potential biomarkers of early pregnancy in sheep
(Frontiers Media Sa, 2022) Hitit, Mustafa; Kose, Mehmet; Kaya, Mehmet Salih; Kirbas, Mesut; Dursun, Sukru; Alak, Ilyas; Atli, Mehmet Osman
MicroRNA (miRNA) plays an important role in the control of gene expression and is implied in many biological functions, including embryo implantation and development. The aim was to assess plasma miRNA profiles during the peri-implantation and ascertain potential candidate miRNA markers for early pregnancy diagnosis in ovine plasma. The plasma samples were obtained from a total of 24 ewes on days 12 (pre-implantation; P12, n = 4), 16 (implantation; P16, n = 4) and 22 (post-implantation; P22, n = 4) after mating, and on their corresponding days of 12 (Pre-C; C12, n = 4), 16 (Imp-C; C16, n = 4) and 22 (Post-C; C22, n = 4) of the estrous cycle. The miRNA profiles in plasma were assessed by microarray technology. We detected the presence of 60 ovine-specific miRNAs in plasma samples. Of these miRNAs, 22 demonstrated a differential expression pattern, especially between the estrous cycle and early pregnancy, and targeted 521 genes. Two miRNAs (oar-miR-218a and oar-miR-1185-3p) were confirmed using RT-qPCR in the ovine plasma samples. Protein-protein interaction (PPI) network of target genes established six functional modules, of which modules 1 and 3 were enriched in the common GO terms, such as inflammatory response, defense response, and regulation of immune response. In contrast, module 2 was enriched in the developmental process involved in reproduction, embryo development, embryonic morphogenesis, and regulation of the developmental process. The results indicate that miRNAs profiles of plasma seemed to be modulated during the peri-implantation stage of pregnancy in ewes. Circulating miRNAs could be promising candidates for diagnosis in early ovine pregnancy.
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.
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.
Early pregnancy-related changes in toll-like receptors expression in ovine trophoblasts and peripheral blood leukocytes
(Elsevier Science Inc, 2017) Kaya, Mehmet Salih; Kose, Mehmet; Guzeloglu, Aydin; Kiyma, Zekeriya; Atli, Mehmet Osman
In the present study, we aimed to 1) demonstrate the presence of all 10 toll-like receptors (TLRs) in ovine trophoblasts, and 2) investigate the expression profiles of TLR1 - 10 mRNAs in peripheral blood leukocytes (PBLs) in ewes during early pregnancy. For those purposes, ovine trophoblasts (n = 6) were collected from pregnant ewes on day 13. PBLs were collected from non-pregnant (n = 6) and pregnant ewes (n = 17) on days of mating (d) 0 and 18. TLR mRNAs in ovine trophoblasts were visualized by free-floating in situ hybridization (ISH). To assess the expression profiles of TLR1-10 in PBLs, total RNA was isolated and transcribed to cDNA. TLR1-10 mRNA levels were determined by real-time PCR in triplicate. The Relative Expression Software Tool (REST 2009) was used for statistical analysis. We detected mRNAs for TLR2, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR10 but not for TLR1, TLR3, and TLR9 in trophoblasts. TLR2, TLR5, TLR6, TLR7, TLR8, and TLR10 mRNAs were expressed by all trophoblasts, whereas TLR4 mRNA and protein in trophoblasts were more limited. In PBLs, TLR expression did not differ between day 0 and day 18 in non-pregnant ewes; however, ewes in early pregnancy exhibited significantly upregulated expression of TLR2 (23-fold), TLR4 (3.1-fold), TLR6 (1.7-fold), and TLR8 (2.2-fold) on day 18 compared with day 0. In contrast, TLR10 was downregulated (2-fold) on day 18 by pregnancy. Similar results were also obtained for TLR2, TLR4, TLR6, TLR8 and TLR10 from the comparison between day 18 non pregnant and day 18 pregnant groups. According to these results, the presence of TLRs in early ovine trophoblasts suggests that these cells play an immunological role at the maternal fetal interface. The results also suggest that tight regulation of some components of TLRs in PBLs due to embryo- and/or pregnancy related factors is necessary for successful establishment of early pregnancy in ewes. (C) 2017 Elsevier Inc. All rights reserved.
Effect of the interval from follicle aspiration to initiation of lengthened FSH treatment on follicular superstimulatory and superovulatory responses and embryo production in lactating Simmental cows
(Elsevier Science Inc, 2019) Cirit, Umut; Ozmen, Mehmet Ferit; Kucukaslan, Ibrahim; Kose, Mehmet; Kutsal, Huseyin Gokhan; Cinar, Elif Merve
The present study evaluated follicular superstimulatory (FSS) and superovulatory (SOV) responses and in vivo embryo production in lactating Simmental cows treated with FSH starting 1 or 2 days after follicle aspiration (FA). The performance of a lengthened superovulation program, named 6dFSH-P36-hCG60, is described. At random stages of the estrous cycle, cows (n = 52) were subjected to ultrasound-guided transvaginal aspiration of all follicles >= 5 mm. After FA, cows were randomly assigned to one of two groups in which FSH treatments started 1 or 2 days after FA (groups FA-1D and FA-2D, respectively). Cows were superstimulated with a total of 500 mu g pFSH over 6 days on a decreasing dose schedule and were pre-treated with a single dose of 400 IU of eCG 24 h before the start of FSH treatments. Follicular superstimulatory (the mean numbers of follicles >= 8 mm on the day of hCG treatment) and SOV responses (the mean numbers of CL and cows with >= 3 CL at the time of collection) were similar in FA-1D and FA-2D groups. However, when compared to FA-1D group, the number of unfertilized ova tended to decrease (0.4 vs 1.7; P = 0.065) and percentage of fertilized ova tended to increase (95.8% vs 84.6%; P = 0.066) in FA-2D group. Moreover, the mean numbers and percentages of both transferable embryos (8.0 and 77.6% vs 6.4 and 57.7%) and freezable embryos (5.3 and 51.5% vs 3.5 and 31.1%) were numerically higher in FA-2D group than FA-1D group. The results of the study suggest that starting a lengthened superovulation programs in Simmental cows 2 days after FA has potential to increase percentage of fertilized ova and the number of transferable and freezable embryos, although new studies may be needed to confirm this findings. (C) 2019 Elsevier Inc. All rights reserved.
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.
Expression pattern and cellular localization of two critical non-nuclear progesterone receptors in the ovine corpus luteum during the estrous cycle and early pregnancy
(Elsevier, 2022) Atli, Mehmet O.; Akbalik, Mehmet Erdem; Kose, Mehmet; Alak, Ilyas; Atli, Zehra; Hitit, Mustafa
The study aimed to investigate the expression and cellular localization of two critical non-nuclear progesterone receptors, including membrane-associated-progesterone-receptor-component-1 (PGRMC1) and progestin and adipoQ receptor family member 7 (PAQR7) throughout the estrous cycle and early pregnancy in ovine corpus luteum (CL). Ewes were randomly grouped into cyclic (C, n = 4 per group) or pregnant (P, n = 4 per group) groups. Following slaughtering, the CL was obtained from both cyclic and pregnant ewes on days 12 (C12 and P12), 16 (C16 and P16), and 22 (C22 and P22). Western blotting and RT-qPCR were utilized to assess the expression levels of PGRMC1 and PAQR7, whereas immunohistochemistry was performed to determine the localization of PGRMC1 and PAQR7 in CL. Data were evaluated by one-way ANOVA, and the P < 0.05 was considered a significant difference. PGRMC1 was shown to be expressed in both small and large luteal cells and endothelial cells in CL, while PAQR7 expression was only found in small and large luteal cells. Compared to cycle days, pregnancy increased the expression of PGRMC1. PAQR7 did not differ during early pregnancy but reduced during the functional luteolysis stage (C16). mRNA and protein expression patterns for PGRMC1 and PAQR7 were similar on the studied days. This is the first study that demonstrates the expression and cellular localization of PGRMC1 and PAQR7 in ovine CL. We suggest that these receptors could execute a significant role in the ovine CL life span in both cyclic changes and the establishment of pregnancy.
Expression pattern of microRNAs in ovine endometrium during the peri-implantation
(Elsevier Science Inc, 2022) Kose, Mehmet; Hitit, Mustafa; Kaya, Mehmet Salih; Kirbas, Mesut; Dursun, Sukru; Alak, Ilyas; Atli, Mehmet Osman
MicroRNA (miRNA), acting as the transcriptional regulator of gene expression, has been widely demonstrated to be involved in many biological functions, including embryo implantation and devel-opment. The objective of the current study was to illuminate the expression pattern of microRNAs (miRNAs) in the endometrium during the peri-implantation in ewes. Intercaruncular endometrial samples was obtained from a total of 24 ewes on days of 12 (pre-implantation, n = 4), 16 (implantation, n = 4) and 22 (post-implantation, n = 4) of pregnancy following mating, and on their corresponding days of 12 (n = 4), 16 (n = 4) and 22 (n = 4) of the estrous cycle. The miRNA profiles were examined in the endometrium by microarray technology. We detected 116 ovine specifics miRNAs in the endometrium. Of these, nineteen were differentially expressed in early pregnancy. Four miRNAs (oar-miR-370-3p, oar-miR-411b-5p, oar-miR-379-3p and oar-miR-411a-3p) that had the most differential fold change were confirmed by RT-qPCR in ovine endometrium. The differentially expressed miRNAs targeted a total of 315 genes, resulting in 39 GO terms in molecular function, 353 in biological process, and 17 in the cellular component. The construction of the PPI network of target genes established two functional modules mostly enriched in the innate immune system, toll receptor cascades in module 1, whereas genes in module 2 were associated with GMCSF-mediated signaling events, insulin pathway, and mTOR signaling pathway. Based on the results, we may imply that miRNAs modulate ovine endometrium during the peri-implantation.(c) 2022 Elsevier Inc. All rights reserved.
Expression patterns of genes in steroidogenic, cholesterol uptake, and liver x receptor-mediated cholesterol efflux pathway regulating cholesterol homeostasis in natural and PGF2? induced luteolysis as well as early pregnancy in ovine corpus luteum
(Elsevier, 2022) Hitit, Mustafa; Kose, Mehmet; Kocak, Nadir; Atli, Mehmet Osman
The aim was to evaluate the expression of genes of steroidogenic, cholesterol uptake, and liver X receptor (LXR) mediated cholesterol efflux pathway in ovine corpus luteum (CL) during natural and prostaglandin F2 alpha (PGF2 alpha) induced luteolysis and early pregnancy. For this study, two experiments were carried out 1); ewes were grouped into two sub-groups as cyclic 12 (C12, n = 4) and 16 (C16, n = 4) and pregnant 12 (P12, n = 4), 16 (P16, n = 4), and 22 (P22, n = 4). Additionally, 2) ewes were grouped into four groups following treatment of PGF2 alpha, the duration of PGF2 alpha challenge at 1 (PG1, n = 4), 4 (PG4, n = 4), and 16 (PG16, n = 4) hours on day 12 of the cycle was compared with 0 h. The corpus luteum tissue samples were collected on the corresponding estrus cycle and pregnancy days, and RNA was extracted using Trizol. mRNA expression levels of the steroidogenic (StAR, CYP11A1, and HSD3B1) and cholesterol uptake receptors (SCARB1 and LDLR) and LXR pathway (NR1H3, NR1H2, ABCA1, and ABCG1) were assessed using quantitative PCR (qPCR), and protein of LXR pathway was investigated using western blot. In-situ hybridization was used to detect mRNA localization. Steroidogenic and cholesterol uptake mRNAs were decreased in C16, while NR1H2 and ABCG1 were increased in C16, compared to C12. Steroidogenic and cholesterol uptake mRNA was greater in P16 than in C16. NR1H2 and ABCA1 protein expression were higher in P16 than in C16. LDLR mRNA was higher in P22 than in P12, while SCARB1 was higher in P16 than in P12. NR1H2 mRNA was greater in P22 than in P12. Steroidogenic and cholesterol uptake mRNA were decreased in PGF2 alpha-induced luteolysis groups against C12. ABCG1 mRNA was higher in PG16 than in PG4 and PG1. The reduction of lipoprotein receptors rather than LXR-mediated reverse transport may contribute to the decline in progesterone (P4) in natural and functional luteolysis.
Expression patterns of Toll-like receptors in the ovine corpus luteum during the early pregnancy and prostaglandin F2?-induced luteolysis
(Elsevier Science Inc, 2018) Atli, Mehmet O.; Kose, Mehmet; Hitit, Mustafa; Kaya, M. Salih; Bozkaya, Faruk
The aim of this study was to elucidate the expression profiles of Toll-like receptors (TLRs) in the ovine corpus luteum (CL) during early pregnancy and prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis. For this purpose, multiparous Anatolian Merino ewes were selected and randomly allotted into cyclic (including those in the induced luteolysis group, n = 20) and pregnant (n = 12) groups. All of the ewes were scheduled to be slaughtered for predetermined days/hour during the estrous cycle, early pregnancy, and PGF2 alpha induced luteolysis. The CLs were collected from both cyclic and pregnant ewes on days 12 (C12 and P12; n = 8) and 16 (C16 and P16; n = 8) and pregnant ewes on day 22 (P22; n = 4). For the induced luteolysis model, ewes were injected with PGF2 alpha on day 12 of the estrous cycle and CLs were collected at 1 h (PG1h; n = 4), 4 h (PG4h; n = 4), and 16 h (PG16h, n = 4) after injection. Quantitative polymerase chain reaction (VCR) was used to evaluate the expression profiles of TLR2, TLR4, TLR6, TLR8, and TLR10, while free-floating in situ hybridization and immunohistochemistry were used to define the spatial localization of TLR2, TLR4, and TLR7 in the CL. Data were then analyzed by one-way ANOVA and were considered statistically significant when P values were lower than 0.05. Expression of TLR2 was upregulated in both early and late stages of luteolysis (P < .05). An upregulation of TLR4 was detected at PG16h, while TLR6 was decreased at PG4h (P < .05). Expression of TLR7 and TLR8 was significantly increased during early pregnancy, at both PG16h and regressed groups (C16, P < .05). In contrast, 711210 was downregulated during PGF2 alpha-induced luteolysis and on P16 (P < .05). TLR4 and TLR7 proteins were particularly localized in endothelial cells on C12/PGOh, but prominent signals corresponding to TLR4 and TLR7 were detected in luteal cells at PG16h. The results suggest an involvement of TLRs in the luteolytic mechanism in ovine CL, as indicated by differential expression levels of TLRs during PGF2 alpha-induced luteolysis. Moreover, the present study indicates that early pregnancy-mediated changes in TLR expression in the CL may contribute to the establishment and maintenance of ovine pregnancy. (C) 2018 Elsevier Inc. All rights reserved.
Investigation of Accelerated Lambing Possibility of Anatolian Merino Sheep
(Kafkas Univ, Veteriner Fakultesi Dergisi, 2014) Bulbul, Bulent; Kirbas, Mesut; Aktas, Ahmet Hamdi; Kose, Mehmet; Ataman, Mehmet Bozkurt; Coyan, Kenan; Kan, Mustafa
Investigation of the possibilities to increase the number of lamb gained in a year via using the accelerated lambing method and, the profitability of a farm related to this is aimed with this study. As material, 525 Anatolian Merino ewes and 40 rams, aged at 2-4, in field conditions, and 199 ewes and 15 rams at Bahri Dagdas International Agricultural Research Institute were used. Ewes in the field condition and at the Institute were divided in to two groups as accelerated lambing and control and, 200 ewes in the field condition and 75 ewes at the Institute were remained as control to get one lamb per year while 325 in the field condition and 124 at the Institute were formed treatment group. The control ewes were bred in August and September, the traditional breeding season, in a 12 month interval while 3 lambings in 2 years were applied to the ewes in the treatment group and they were bred for one month again following the period of 5 months of pregnancy, 40 days of lactation and 20 days of weaning. Ram effect, ram effect + flushing and some different protocols were used for induction and synchronization of estrus for accelerated lambing in the treatment group. As a result, more fecundity and lamb productivity achieved by accelerated lambing than once a year lambing. Synchronization methods were found to be effective on fecundity and lamb productivity in accelerated lambing applications. However, lamb yield obtained by synchronization methods used in this study were not profitable.
Investigation of interferon-tau stimulated genes (ISGs) simultaneously in the endometrium, corpus luteum (CL) and peripheral blood leukocytes (PBLs) in the preluteolytic stage of early pregnancy in ewes
(Elsevier, 2016) Kiyma, Zekeriya; Kose, Mehmet; Atli, Mehmet Osman; Ozel, Caglayan; Hitit, Mustafa; Sen, Gonca; Kaya, Muhammet
The aim of the present study was to investigate the expression profiles of Interferon-tau (IFN-T) stimulated genes (ISGs) at the mid-luteal stage of the cycle (on day 13) in the uterine endometrium and extra-uterine tissues, such as the corpus luteum (CL), and in the peripheral blood leukocytes (PBLs) of pregnant and non -pregnant ewes, The objective was to evaluate the possibility of using the regulation of ISG expression in PBLs as a possible early pregnancy indicator in ruminants. For this purpose, multiparous ewes were synchronized and either allowed to mate (pregnancy group) or detected in estrus (cyclic group; day 0). The ewes were slaughtered on day 13, and the PBLs, endometrium and luteal tissues were collected. Total RNA was isolated from eight cyclic and eight pregnant ewes, and qPCR was employed to detect the steady state levels of Interferon -stimulated gene 15 (ISG15), Myxovirus (influenza virus) resistance 1 (Mxl) and Receptor transporter protein 4 (RTP4) mRNAs. The expressions of ISG15, Mxl and RTP4 were detected in the endometrium, CL and PBLs on day 13 of the estrous cycle and pregnancy. The expressions of these ISGs were upregulated only in the endometrium of pregnant ewes compared to non-pregnant ewes, but this stimulation was not observed in the CL and PBLs. The results suggest that the embryo stimulates ISGs only in the endometrium, and the effects are not evident in the extra-uterine tissues on day 13 of pregnancy. This study suggests that the measurement of the ISG expression in the PBLs is not a reliable detection method of early pregnancy in ewes, which are in the preluteolytic stage of early pregnancy. (C) 2016 Elsevier B.V. All rights reserved.
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.
Presynchronization in Sheep Ensures Synchronization of Next Estrus and Improves Fertility
(Chulalongkorn Univ, 2023) Bulbul, Bulent; Kose, Mehmet; Kirbas, Mesut; Coyan, Kenan; Ataman, Mehmet Bozkurt; Bekci, Emine Aysun; Umutlu, Seyit
In this study, fertility was evaluated either in the first hormonally synchronized or in the subsequent natural estrus among 202 sheep during the breeding season. Intramuscular (im) PGF(2 alpha )was administered 11 days apart in both the PG (n=50) and Pre-PG (n=50) groups, while im PGF(2 alpha )was given at the removal of an intravaginal progesterone sponge (after 11 days) in the Sponge (n=51) and Pre-Sponge (n=51) groups. Estrus was monitored following the last hormonal application (day 0) in all groups and starting from day 16 in the Pre-PG and Pre-Sponge groups for 5 days. Ewes were mated during the first hormonally synchronized estrus (PG and Sponge groups) or in the next (natural) estrus (Pre-PG and Pre-Sponge groups). The estrus rate and litter size in the Pre-Sponge group were higher than those in the other groups (P<0.05). In the Pre-Sponge group, pregnancy and lambing rates were higher than in the PG and Sponge groups, and fecundity was higher than in the PG group (P<0.05). It was concluded that progesterone and PGF(2 alpha )-based estrus synchronization may negatively affect fertility. Additionally, the next estrus is also synchronized after using progesterone and PGF(2 alpha )-based synchronization protocols. Fertility loss caused by the application of exogenous hormones can be mitigated by mating at the next natural estrus. Furthermore, the next natural estrus can be utilized in situations where hormone use is inappropriate but synchronization is desired.
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.
Superovulation in cows synchronized with two different progesterone plus oestradiol protocols
(Archiv Fur Tierzucht, 2013) Bulbul, Bulent; Kirbas, Mesut; Dursun, Sukru; Kose, Mehmet
A total of 26 Brown Swiss cows were used to compare the synchronization and superovulatory response of follicle stimulating hormone treated cows that were synchronized with progesterone+oestradiol valerate or benzoate. Control cows (n=8) were superstimulated with follicle stimulating hormone using twice daily injections with decreasing doses from day 10-13 after determined reference oestrus. Cows in treatment groups were received either ear implant (n=9) containing norgestomet+oestradiol valerate or progesterone releasing intravaginal device (n=9) containing progesterone+oestradiol benzoate, at random stage of the oestrus cycle, for 9 days. Seven days after the implant and progesterone releasing intravaginal device insertion, follicle stimulating hormone was injected as described in the control group. There was no significant difference between the groups for superovulation responses. In conclusion, both protocols synchronized the oestrus cycle in follicle stimulating hormone treated cows and, any of the protocols evaluated in this study can be used as a pretreatment for superstimulation started on the seventh day of the implant or progesterone releasing intravaginal device insertion in Brown Swiss cows.
Toll-like receptor 2 and 4 expression in the bovine corpus luteum during the different stages of the estrous cycle
(Brazilian Coll Animal Reproduction, 2017) Atli, Mehmet Osman; Kose, Mehmet; Kaya, Mehmet Salih; Aydilek, Nurettin; Guzeloglu, Aydin; Wiltbank, Milo C.
The aim of this study was to elucidate the presence of components of the innate immune system in the bovine corpus luteum (CL) by detecting the expression and cell-specific localization of TLR2 and TLR4 during different stages of the estrous cycle in a control study design. Bovine CL samples were collected from a local slaughterhouse and assigned to three groups as follows: developing CL (dCL; n = 6, approx. days 3-6), mature CL (mCL; n = 5, approx. days 8-12), and regressing CL (rCL; n = 5, approx. days 17-19). An upregulation of TLR2 mRNA was detected only in rCL (P < 0.05). Localization of the TLR2 protein was particularly apparent in luteal cells and a prominent immunofluorescent signal corresponding to TLR2 was detected only in rCL. TLR4 mRNA were higher in mCL and rCL compared to dCL (P < 0.05). The presence of the TLR4 protein in bovine CL was clearly detected in the luteal cells of both mCL and rCL. The results of this study suggest a role for TLRs in the development, maintenance, and regression of bovine CL. TLR signaling mediated pathway in luteal cells may involve in the regression of CL via regulation of TLR2 and TLR4.

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