The objective of the present study was to examine the effects of two different transport temperature (37°C vs 4°C) and cold storage of ovaries for 24 h on cumulus cell apoptosis and maturation rates of cat oocytes in vitro. Ovaries were collected from 15 ovariohysterectomized domestic cats and maintained and transported to the laboratory in phosphate buffer saline at 37°C and 4°C. In order to determine the effects of storing time, some ovaries transported at 4°C were stored at the same temperature for 24 h. Selected cumulus oocyte complexes (COCs) were matured for 48 h at 38°C in four-well petri dishes containing 500 μL of modified oviduct medium (mSOF) under mineral oil in a 5% CO2 incubator with nearly 100% humidified. The morphological features of apoptosis were analysed in the cumulus cells at the beginning of in vitro maturation in both transporting temperature groups and after 24 h of cold stored group. The degree of apoptosis in cumulus cells were measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL). The IVM rates of oocytes were determined using Hoechst (33342) staining. Although the apoptotic morphological features were seen rarely and in similar rates in 37 and 4°C transporting groups (19.40 and 21.55%, P>0.001), it was seen more intensely in the 24 h cold stored group (34.80%, P<0.001). The IVM findings were similar (49.77, 44.55%) at 37°C and 4°C groups (P>0.05), and importantly lower at 4°C transporting and 24 h cold stored groups (18.90%, P<0.05). In conclusion, the results of this study suggest that (I) cumulus cells of cat oocytes are partially exposed to apoptosis during transportation at warm or cold temperature, (II) storing of ovaries for 24 h at 4°C causes apoptosis of the cumulus cells at much higher rates and (III) storing of ovaries for 24 h at 4°C affects negatively IVM rate of oocytes. Keywords: Cat, Ovary, Transport temperature, Oocyte, Cumulus, Apoptosis Kedi Oositlerinin In Vitro Olgunlaştırılması ve Kumulus Hücrelerinin Apoptoz Oranları Üzerine, Ovaryum Taşıma ve Saklama Sıcaklığının Etkisi ÖzÇalışmanın amacı, ovaryumların iki farklı sıcaklıkta (37°C ve 4°C) taşınma ve soğukta 24 saat bekletilmenin kumulus hücrelerindeki apoptoza ve kedi oositlerinin in vitro olgunlaşma oranları (IVM) üzerine etkilerini incelemektir. Kısırlaştırılmış 15 kediden ovaryumlar alındı ve yarısı 37°C, diğer yarısı da 4°C'de olmak üzere, fosfat tampon tuzlu solüsyonunda (PBS) laboratuara taşındı. Soğukta bekletmenin etkilerini belirlemek içinse, 4°C 'de taşınan ovaryumların yarısı, aynı sıcaklıkta olmak üzere 24 saat bekletildi. Seçilen kumulus oosit kompleksleri (COCs), %100'e yakın nemin sağlandığı %5 CO2'li inkübatörde mineral yağ altındaki 500 μL modifiye Sentetik Ovidukt Medyumu (mSOF) içeren dört gözlü petrilerde olmak üzere 38°C'de 48 saat süreyle olgunlaştırıldı. Apoptozun etkileri, hem iki farklı taşıma grubunda, hem de soğukta 24 saat bekletilen grupta olmak üzere in vitro olgunlaşmanın hemen öncesinde kumulus hücrelerinde test edildi. Apoptosis derecesi...
The reproductive biology of the domestic dog is unique among mammalian species; because of this, in vitro maturation (IVM) rate is still very low compared with other domestic animals in spite of attempts at improvement. The aim of this study was to consider the use of recombinant human gonadotropins as a replacement for pituitary gonadotropins for IVM of dog oocytes. A total of 845 cumulusoocyte complexes were used in this study. To determine the effects of human recombinant gonadotropins, maturation medium was supplemented with two different concentrations (0.05 or 0.1 IU/mL) of pituitary (pFSH, pLH) and human recombinant (rhFSH and rhLH) gonadotropins. After the IVM period, the maturation rate of the oocytes was investigated under an epifluorescence microscope. Our findings showed no significant difference in maturation rate using either pituitary or human recombinant gonadotropin groups (P > 0.05). Applying 1.0 IU human recombinant gonadotropin caused the lowest maturation rate (34.57%; P < 0.05). In conclusion, recombinant human gonadotropins could be applied for IVM of dog oocytes. Moreover, 0.05 IU/mL rhFSH and rhLH can be successfully used in place of biologically derived hormones.
Synchronization of donor cells is an important step for the success of somatic cell nuclear transfer application and facilitates the development of embryos. Contact inhibition, serum starvation and different chemical agents are used in synchronizing different types of somatic cells. In this study, to synchronize the primary ovine adult (POF) and foetal (POFF) fibroblast cells to G0/G1 phases, the contact inhibition, the serum starvation, roscovitine and trichostatin A (TSA) methods were used. In the first part of the study, roscovitine (10, 15, 20 and 30 μM) and TSA (25, 50, 75 and 100 nM) were applied for 24 h to determine the optimal concentration for POF and POFF cells. In the second part, optimal concentrations of roscovitine and TSA for these cells were compared with contact inhibition and serum starvation methods. Cell cycle distribution and apoptotic activity analysis were performed by flow cytometry to compare this synchronization methods. Serum starvation method resulted in higher cell synchronization rate in both cells compared to other groups. Although contact inhibition and TSA also achieved high success rates of synchronized cell value, it was observed that the difference between serum starvation and these groups was significant (p < .05). When the apoptosis rates of the two cell types were examined, it was observed that the early apoptotic cells in contact inhibition and late apoptotic cells in the serum starvation were higher than the other groups (p < .05). Although the 10 and 15 μM concentrations of roscovitine gave the lowest apoptosis rates, it was observed that it failed to synchronize both the ovine fibroblast cells to G0/G1 phase. As a result, it was concluded that while roscovitine was not successful to synchronize both the POFF and POF cell lines, TSA (50 nM for POF cells and 100 nM for POFF cells) can be used efficiently as an alternative to the contact inhibition and the serum starvation methods.
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