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Öğe Comparison of two different media for In vitro production of dog embryos(2010) Pabuccuoğlu, Serhat; Evecen, Mithat; Birler, Sema; Öztürk, Gül Bakırer; Hamzaoğlu, Asiye İzem; Cirit, Ümüt; Karaman, ElifKöpeklerde in vitro fertilizasyon yöntemi ile embriyo üretilebilmiş ve nükleer transfer yöntemiyle klonlanmış embriyolardan canlı bir doğum elde edilebilmiş olmasına karşın, bu alandaki teknolojilerin başarısı oldukça sınırlı düzeydedir. Bu güne kadar yapılan in vitro çalışmalarda, sadece iki adet morula ve bir adet blastosist elde edilebilmiştir. Sunulan çalışmanın amacı, iki farklı medyumun (mSOF ve TCM 199) köpek oositlerinin in vitro maturasyon (İVM), in vitro fertilizasyon (İVF) ve in vitro kültürü (İVK) üzerine etkilerini araştırmaktır. Çalışma iki aşamada gerçekleştirildi. Birinci aşamada, iki farklı medyumun köpek oositlerinin İVM’u üzerine etkisi araştırıldı. İVM sürecinin sonunda fikze edilen oositlerin nükleer olgunlaşma değerlendirmesi, aseto-orsein ile boyanarak yapıldı. Çalışmanın ikinci aşamasında ise, İVM sonrasında oositler taze sperma ile 24 saat İVF’a tabii tutuldular ve ardından yedi gün boyunca in vitro kültüre edildiler. Embriyoların gelişimsel kontrolleri her 24 saatte bir mikroskop bakısıyla kontrol edildikten sonra, yedinci günün sonunda aynı metotla fikze edilip boyanarak değerlendirildiler. Sonuç olarak, İVM oranları açısından mSOF medyum grubunda bulunan oositlerin TCM 199 grubundakilere nazaran daha başarılı olduğu belirlendi (P<0.001). İVK sürecinin sonunda TCM 199 medyum grubunda sadece bir adet oosit yarıklanma gösterirken, mSOF grubunda sekiz adet oositin yarıklandığı ve bunlardan birisinin de morulaya ulaştığı saptandı (P=0.037).Öğe Comparison of Two Different Media for In vitro Production of Dog Embryos(Kafkas Univ, Veteriner Fakultesi Dergisi, 2010) Evecen, Mithat; Cirit, Umut; Demir, Kamber; Karaman, Elif; Bakirer, Gul; Hamzaoglu, Asiye Izem; Birler, SemaEmbryo production via in vitro fertilization and nuclear transfer has been accomplished in the dog, and the transfer of the cloned embryos has recently resulted in the birth of puppies. However, the efficiency of these technologies is still very limited. Until now, only two morulas and single blactocyst have been achieved in vitro. Therefore, the aim of the present study was to examine the effects of two different media (mSOF and TCM 199) on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of immature dog oocytes. The study was performed in two steps. At the first step, the effects of two different media, on IVM of dog oocytes were investigated. At the end of the IVM period, the nuclear maturation rates were evaluated by aceto-orcein staining method. At the second step, after the IVM period the oocytes inseminated with fresh spermatozoa for 24 h and left for IVC for 7 d. At the end of the IVC period, embryonic development was assessed by microscopic observation at 24 h intervals and then fixed and stained by the same method. Consequently, maturation rates of oocytes in mSOF medium were significantly higher than those of the TCM 199 (P<0.001). After 7 d of the IVC period, only one oocyte was cleaved in the TCM 199. while eight oocytes cleaved and one of them developed to morula stage in mSOF medium group (P=0.037).Öğe Effect of Oocyte Diameter on in vitro Embryo Production in Dogs(Kafkas Univ, Veteriner Fakultesi Dergisi, 2010) Evecen, Mithat; Cirit, Umut; Demir, Kamber; Karaman, Elif; Bakirer, Gul; Hamzaoglu, Asiye Izem; Birler, SemaIn vitro embryo production has not been confidently applied to the dog successfully. Up to date, only one blactocyst have been achieved by in vitro culture. Therefore, the aim of the present study was to examine the effects of different oocyte diameters (<= 100 mu m and >100 mu m) on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of immature dog oocytes. The study was performed in two steps. At the first step (experiment I), effects of two different oocyte diameters on IVM of dog oocytes were investigated. The nuclear maturation rates were evaluated by aceto-orcein staining method at the end of the IVM. At the second step (experiment II), in vitro matured oocytes were fertilized with fresh spermatozoa for 24 h and in vitro cultured for 7 d. At the end of the IVC period, embryonic development was assessed by microscopic observation at 24 h intervals and fixed for staining by aceto-orcein staining method after 7 days. In comparison relating in IVM and IVF rates, larger oocytes have higher maturation (P<0.05) and cleavage (P<0.01) rates than the smaller ones. Unfortunately, none of the oocyte was reached to morula or blactocyst stage in both groups. In conclusion, it is demonstrated that the oocyte diameter may be a helpful selection criteria for dog in vitro embryo production.Öğe Effect of oocyte diameter on in vitro embryo production in dogs(2010) Hamzaoğlu, Asiye İzem; Evecen, Mithat; Pabuccuoğlu, Serhat; Birler, Sema; Cirit, Ümüt; Öztürk, Gül Bakırer; Karaman, ElifKöpeklerde, in vitro embriyo üretimi amacıyla henüz başarılı bir yöntem bulunmamaktadır. Günümüze kadar yapılan in vitro çalışmalarda, sadece bir adet blastosist elde edildiği bildirilmiştir. Sunulan çalışmada, köpeklerde farklı oosit çaplarının (?100 ?m ve >100 ?m) in vitro maturasyon (İVM), in vitro fertilizasyon (İVF) ve in vitro kültür (İVK) sonrası embriyonik gelişim üzerine etkisinin araştırılmasını amaçlandı. Çalışma iki aşamada gerçekleşti. İlk aşamada (Deney I), farklı oosit çaplarının İVM üzerine etkisi araştırıldı. Bu aşamanın sonunda oositlerin olgunlaşma durumları, aseto-orsein boyama metoduyla belirlendi. Çalışmanın ikinci aşamasında ise (Deney II), İVM sonrasında oositler taze sperma ile 24 saat İVF’a tabii tutuldular ve ardından yedi gün boyunca in vitro kültüre edildiler. Embriyoların gelişimsel kontrolleri her 24 saatte bir mikroskop bakısıyla kontrol edildikten sonra, yedinci günün sonunda aynı metotla fikze edilip boyanarak değerlendirildiler. İVM-İVF sonuçları karşılaştırmasında, büyük çapa sahip olan oositlerin, küçük olanlara göre daha yüksek oranda olgunlaşabildiği ve daha fazla oranda bölünmeler gösterebildiği saptandı (P<0.01). Her iki grupta hiçbir oosit morula ya da blastosist aşamasına kadar ulaşmadı. Sonuç olarak, in vitro köpek embriyosu üretmek amacıyla seçilecek oositler için çapın yardımcı bir ölçüt olabileceği söylenebilir.Öğe Effect of transport and storage temperature of ovaries on in vitro maturation of bitch oocytes(Elsevier Science Bv, 2006) Tas, Muzaffer; Evecen, Mithat; Ozdas, Ozen Banu; Cirit, Umut; Demir, Kamber; Birler, Sema; Pabuccuoglu, SerhatIn this study, the effects of ovary transport and storage temperature on in vitro maturation of bitch oocytes were investigated. Ovaries were collected from 23 mature bitches and one randomly selected ovary of each pair (n = 23 pairs) was transported in physiologic saline at 4 degrees C, while the other one at 35-38 degrees C for 2-4 h. A total of 316 cumulus oocyte complexes (COCs) were obtained from the 4 degrees C group and 301 COCs from the 35-38 degrees C group. All COCs were matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), essential and non-essential amino acids at 38 degrees C in ahumidified 5% CO2, 5% O-2, and 90% N-2 atmosphere for 72 h. At the end of the in vitro maturation period, nuclear maturation of oocytes was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), undetermined nuclear maturation (UDNM), and MI + MII. The nuclear maturation rates to MI, MII, and MI + MII stages were 60.44%, 10.75%, and 71.20% in the 4 degrees C group and 37.20%, 7.64%, and 45.85% in the 35-38 degrees C group, respectively. The data demonstrated that oocytes obtained from ovaries transported at 4 degrees C had higher maturation rates than from the ones transported at 35-38 degrees C (p < 0.001). (c) 2005 Published by Elsevier B.V.Öğe Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes(Elsevier Science Bv, 2010) Evecen, Mithat; Cirit, Uemuet; Demir, Kamber; Ozdas, Ozen Banu; Tas, Muzaffer; Birler, Sema; Pabuccuoglu, SerhatUnlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The present study investigated the effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The donor bitches were categorized into three groups based on stage of estrus cycle: follicular (proestrus or estrous), luteal (diestrus) and anestrus. One ovary of each pair collected from 39 mature bitches was transported in Phosphate Buffer Saline (PBS) at 4 degrees C while the other was transported at 37 degrees C. A total of 1138 Grade I COCs obtained from all ovaries were grouped and matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH), essential and non-essential amino acids at 38.5 degrees C in a humidified 5% CO2, 5% O-2, and 90% N-2 atmosphere for 72 h. The nuclear maturation rates were evaluated by aceto-orcein staining. Oocytes harvested from follicular and luteal ovaries have a significantly higher maturation rates (MI+MII) than the oocytes from anestrual ovaries in the 37 degrees C group (p<0.05). However, oocytes harvested from anestrual ovaries transported at 4 degrees C had the highest maturation (MI + MII) rate, and the difference between anestrual and luteal ovary groups was significant (p < 0.05). The oocytes from anestrual ovaries transported at 4 degrees C have significantly higher maturation rates than those transported at 37 degrees C (p < 0.0001). However, the transport temperature (37 or 4 degrees C) did not significantly affect the maturation (MI + MII) rates of oocytes harvested from the luteal (p = 0.61) and follicular (p = 0.48) stage ovaries. It can be concluded from this study that (1) both transport temperature and transport temperature x estrus cycle stage interaction effected the maturation rates, while estrus cycle stage alone did not, and (2) transporting canine ovaries at 4 degrees C can improve in vitro maturation rates in oocytes harvested from anestrous ovaries. (C) 2009 Elsevier B.V. All rights reserved.Öğe Effects of serum starvation and ionomycin activation on the development of somatic cell nuclear transfer embryos in sheep(Chartered Inst. of Building Services Engineers, 2019) Demir, Kamber; Pabuccuoǧlu, Serhat; Cirit, Ümüt; Evecen, Mithat; Karaman, Elif; Özdaş, Özen Banu; Alkan, S.; Atalla, Hatem; Birler, SemaSynchronization of donor cells and activation of the reconstructed oocytes are important factors affecting the success rate in somatic cell cloning. In this study, it was aimed to investigate the effects of serum starvation in donor cell synchronization and ionomycin treatment in the activation of reconstructed oocytes after somatic cell nuclear transfer in Kıvırcık sheep. Cumulus cells were obtained from a slaughtered sheep ovaries and used as donor cells after serum starvation for 4 days (0.5% FCS; SS) or without serum starvation (10% FCS; S). After reconstruction, oocytes were activated by ionomycin for 5 min plus 6-dimethylaminopurine for 3 h (I+) or only with 6-dimethylaminopurine for 3 h (I-). All cleaved embryos (n= 44) at the second day of in vitro culture were transferred into synchronized recipient ewes (n= 10). Cleavage rates of the embryos were 37.3, 44.1, 34.6 and 44.7% in SS/I+, S/I+, SS/I- and S/Igroups, respectively. Recipient ewes had serum progesterone levels >1 ng/ml at 18th day were 33.3, 50.0, 50.0 and 100.0%, respectively. Only one pregnancy in the S/I- group continued after 40 days however the cloned lamb (7.1%, regarding to embryos transferred) died 10 days before term due to a maternal problem (uterine torsion). The results of this study reveal that somatic cell synchronization by serum starvation and ionomycin treatment for the activation of oocytes can be omitted for the success of somatic cell nuclear transfer in sheep.
