The efficiency of intracytoplasmic sperm injection (ICSI) in cattle remains limited, particularly when sex-sorted spermatozoa are used, partly due to insufficient chromatin decondensation associated with the highly compact protamine structure of bovine sperm. This study evaluated thioglycolic acid (TGA) as an alternative reducing agent to dithiothreitol (DTT) to promote controlled chromatin decondensation while preserving sperm DNA integrity and improving ICSI outcomes. Sex-sorted and conventional bovine spermatozoa were treated with different TGA concentrations (0.5-5 mM), and sperm functional parameters and chromatin integrity were assessed. Low TGA concentrations (0.5-1 mM) increased CMA3 positivity, indicating increased protamine-DNA accessibility, without compromising sperm viability or chromatin integrity, whereas higher concentrations impaired sperm function. Based on these findings, 1 mM TGA was selected for subsequent ICSI experiments. In contrast, DTT treatment increased DNA fragmentation and chromatin instability. Following ICSI, sperm treated with 1 mM TGA exhibited a higher normal fertilization rate (two pronuclei; 70.4%) compared with the DTT-treated (36.8%) and control groups (8.3%). Moreover, embryos derived from TGA-treated spermatozoa showed high cleavage competence (79.0%) and improved blastocyst development (46.0%). Gene expression analysis of Day 8 blastocysts revealed increased IGF2 and OCT4 expression, reduced Survivin expression, and increased HSP70 expression, potentially reflecting altered cellular regulation in embryos derived from TGA-treated spermatozoa. In conclusion, optimized TGA treatment may facilitate controlled sperm chromatin decondensation while preserving DNA integrity, representing a promising strategy to improve bovine ICSI outcomes using sex-sorted spermatozoa.

ABSTRACTRam sperm are more vulnerable to freezing than those of most other farm animals. During sperm freezing, the cell membrane loses some of its cholesterol, which regulates signalling mechanisms and prevents premature capacitation. Resveratrol (RES) increases the fluidity of the cell membrane, which becomes peroxidized during freezing and reduces free radicals. In this study, the effectiveness of RES, cholesterol‐loaded cyclodextrin (CLC) and their combinations in ram sperm cryopreservation were investigated. The collected semen was divided into two equal volumes: One was diluted with tris‐citric acid‐glucose medium (TCG) containing CLC, whereas the other was diluted with a CLC‐free TCG solution. After examining motility, both groups were further divided into two equal volumes, forming the following working groups: control (no RES, no CLC); RES (20 µg/mL); CLC (2 mg CLC/120 × 106 sperm); and RES + CLC (RES 20 µg/mL + 2 mg CLC/120 × 106 sperm). These groups were diluted with media containing their respective additives. Post‐thawing, the samples were analysed for motility, acrosome and membrane integrity, membrane functionality, mitochondrial activity, capacitation status, oxidative stress and DNA integrity. CLC preserved sperm total motility, acrosome and plasma membrane integrity and decreased the rate of early capacitation (p < 0.05). RES had no significant effect on sperm quality before freezing and post‐thawing (p > 0.05). However, RES + CLC increased mitochondrial activity post‐thawing (p < 0.05). In conclusion, CLC minimized sperm membrane damage caused by cryopreservation in ram sperm. RES alone was ineffective, and the combination of RES and CLC did not yield a positive synergistic effect on ram spermatological parameters.

Many wildcat species are threatened with extinction, rare or vulnerable due to habitat destruction and poaching. In addition, Ankara and Van domestic cat species originating from Türkiye are in danger of extinction and are under protection. Thus, the requirement for assisted reproductive techniques in b oth domestic and nondomestic cat species has been increasing in recent years. Assisted Reproductive Technologies (ART) such as in vitro maturation, in vitro fertilization, embryo transfer, and cloning in domestic cats (Felis catus) provide a useful and suitable model for the conservation of endangered cat species. Domestic cats can be recipients for embryo transfer and recipient cytoplasm for nuclear transfer from various small wildcat species. Thanks to ART, it is possible to ensure the continuation of the generation by producing in vitro embryos or by making intra or inter-species clones from wild cats that have lost their reproductive functions or even died recently. Many inherited genetic disorders have been identified in cats that are similar to humans. Due to their genetic closeness, they have recently begun to be used as animal models in some therapeutic studies on humans, especially on kidney and nervous system diseases. In the early years, in vitro study results were less successful than in farm animals but in recent years ART’s such as in vitro embryo production, embryo transfer, cloning, and transgenesis have made significant progress in domestic of domestic and wild cats. This review includes the assisted reproductive technologies applied in recent years and the results obtained in domestic cat and felines.

Usage of oocytes obtained from ovaries after long-term cold storage for in vitro embryo production is a promising tool for the protection of wildlife and endangered animal species. Mammalian oocytes are susceptible to oxidative stress with regard to the high lipid content of plasma membranes. Melatonin is known as a powerful antioxidant and anti-apoptotic agent due to its ability to eliminate toxic oxygen derivatives and reduce the formation of reactive species. This study was performed to verify the optimal environmental conditions for long-term preservation of cat ovaries (Felis domesticus) by adding different concentrations of melatonin (500, 750 and 1000 mu M) to the storage medium (0.9% NaCl) as an antioxidant to be preserved at 4 degrees C for 24 h. To determine the effect of melatonin on cat oocytes collected from stored ovaries, the anti and pro-apoptotic gene levels in cumulus oophorus, the in vitro maturation rates, the cell membrane and oocytes viability were evaluated. In all melatonin added groups regardless of whether they are stored in the cold; Pro-apoptotic gene levels (BAX) were determined to be upregulated however, anti-apoptotic gene levels (BCL-2) were downregulated in cumulus cells (P<0.05). The cell membrane stability and cell viability rates of oocytes began to deteriorate in parallel with the rate of melatonin increase. In parallel with these findings, in vitro maturation rates of oocytes were negatively affected as the amount of melatonin increased (P <= 0.001). In conclusion the results showed that adding melatonin (500,750 or 1000 mu M) to the ovarian transport and storage medium had negative effect on in vitro maturation rate, viability and cell membrane structure of cat oocytes.

Usage of oocytes obtained from ovaries aft er long-term cold storage for in vitro embryo production is a promising tool for the protection of wildlife and endangered animal species.Mammalian oocytes are susceptible to oxidative stress with regard to the high lipid content of plasma membranes.Melatonin is known as a powerful antioxidant and anti-apoptotic agent due to its ability to eliminate toxic oxygen derivatives and reduce the formation of reactive species.Th is study was performed to verify the optimal environmental conditions for long-term preservation of cat ovaries (Felis domesticus) by adding diff erent concentrations of melatonin (500, 750 and 1000 µM) to the storage medium (0.9% NaCl) as an antioxidant to be preserved at 4°C for 24 h.To determine the eff ect of melatonin on cat oocytes collected from stored ovaries, the anti and proapoptotic gene levels in cumulus oophorus, the in vitro maturation rates, the cell membrane and oocytes viability were evaluated.In all melatonin added groups regardless of whether they are stored in the cold; Pro-apoptotic gene levels (BAX) were determined to be upregulated however, antiapoptotic gene levels (BCL-2) were downregulated in cumulus cells (P<0.05).Th e cell membrane stability and cell viability rates of oocytes began to deteriorate in parallel with the rate of melatonin increase.In parallel with these findings, in vitro maturation rates of oocytes were negatively aff ected as the amount of melatonin increased (P≤0.001).In conclusion the results showed that adding melatonin (500,750 or 1000 µM) to the ovarian transport and storage medium had negative eff ect on in vitro maturation rate, viability and cell membrane structure of cat oocytes.

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 cumulus-oocyte 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.

The motility, sperm membrane and acrosome integrity during post–thawing incubation is an indication of the usability of the semen. The maintenance of higher motility of sperm during post–thawing incubation simultaneously with the maintenance of intact cell membrane and acrosome integrity reflects a greater likelihood of their survival in the female genital tract to undergo capacitation and fertilize ova or even to be used in in vitro embryo production. The aim of this study was to elucidate the protective ability of royal jelly on motility, spermatozoa plasma membrane and integrity of acrosome during 4 hours of incubation of post-thawed ram semen in thawing media contains different concentrations of RJ (0.5, 1.0 and 2.0%). The post-thaw results revealed that 0.5% RJ supplementation to incubation media significantly improved (P< 0.05) total and progressive motility after the second hour of incubation compared to 0, 1 or 2% RJ groups; however, a non-significant difference (P> 0.05) was obtained regarding the sperm acrosome integrity across all the treatment groups. In conclusion, the structural and functional parameters of sperm during -thawing can be enhanced by supplementing the RJ in post-thawing media before used especially in IVF process and a dose of 0.5% RJ provide the best post-thaw sperm quality.

Percoll gradient and swim-up are the most common methods used to obtain high quality intact sperm to be used for the in vitro fertilization procedures. In this study, we evaluated the effects of swim-up and percoll gradient methods on DNA integrity and other spermatological parameters. To determine DNA breakdown, toluidine blue staining was used. Motility, and progressive motility were analyzed via computer assisted semen analyzer (CASA) system and the others (morphology, viability, membrane and acrosome integrity) were evaluated via phase contrast and florescent microscopy. The membrane integrity was tested by hypo-osmotic swelling test. Also, the separated sperms were tested for in vitro embryos production. Our results showed no differences between the two methods of separation compared to the fresh samples in regard of sperm motility (P>0.05), however, in the separated sperms with both methods, progressive motility was higher than the fresh sperm and the highest results were obtained in Percoll method (P<0.05). In comparison to swim-up, Percoll gradient method maintained the highest normal sperm morphology rate and the lowest acrosome defects (P<0.05). In addition, the membrane integrity, intact acrosome and intact chromosome rates were also higher in the sperm samples separated by percoll gradient method (P<0.05). The blastocyst rate produced with sperm separated by Percoll gradient method was significantly higher than the swim-up group (P<0.001). Sperm samples obtained after Percoll gradient centrifugation could improve motility, morphological intactness, and DNA integrity. Accordingly, it was concluded that the Percoll gradient separation method is more useful then the swim-up for in vitro sheep embryo production. Also, Toluidine blue staining could be a suitable technique for determination of DNA integrity.

The objective of the present study was to examine the effects of two different transport temperature (37 degrees C vs 4 degrees 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 degrees C and 4 degrees C. In order to determine the effects of storing time, some ovaries transported at 4 degrees C were stored at the same temperature for 24 h. Selected cumulus oocyte complexes (COCs) were matured for 48 h at 38 degrees C in four-well petri dishes containing 500 mu L 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 degrees 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 degrees C and 4 degrees C groups (P>0.05), and importantly lower at 4 degrees 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 degrees C causes apoptosis of the cumulus cells at much higher rates and (III) storing of ovaries for 24 h at 4 degrees C affects negatively IVM rate of oocytes.

The effects of two vitrification protocols on the survival of sheep blastocysts were examined in embryos produced in vitro with sheep oviduct epithelial cells co-culture (CC) or without co-culture (C). Oocytes collected from slaughtered ewes were matured for 24 h, fertilized with fresh ram semen for 20 h and cultured in SOF medium for up to 9 days in vitro. For vitrification, blastocyst stage embryos were assigned to two equilibration groups (20% ethylen glycol (EG) or 10% glycerol (G) for the first equilibration), and as the second equilibration they were kept in 20% ethylen glycol plus 10% glycerol for 5 minutes. After 30 sec in vitrification solution (25% ethylen glycol + 25% glycerol), they were immediately immersed into liquid nitrogen. After thawing procedure, embryos were transferred into 0.25 M sucrose for 5 min, washed in Hepes buffered synthetic oviduct fluid (HSOF) and cultured in SOF medium for 24 h. Cleavage rates were 75.2% in C and 74.2% in CC groups, and blastocyst rates were 14.4% in C and 17.1% in CC groups. After in vitro culture of vitrified-thawed blastocysts, survival rates were 62.1, 38.4, 30.2, and 39.3% in C-EG, CC-EG, C-G and CC-G groups, respectively. This study shows that vitrification of sheep embryos using ethylene glycol instead of glycerol as a first equilibration cryoprotectant could give reasonable survival rates and that co-culture of embryos with sheep oviduct epithelial cell has no beneficial effect on vitrification of embryos.

Synchronization 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 Kivircik 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 18 th 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

This study was conducted to evaluate the protective effects of cholesterol pre-incubation on ovine oocytes exposed to cold stress and vitrification. In the first experiment, cumulus oocyte complexes harvested from ovaries of slaughtered ewes were exposed to 2 mg/ml cholesterol loaded methy14cyclodextrin (CLC) in maturation medium for 2,4 or 24 h. Oocytes were then exposed to cold stress at 4 degrees C for 30 min, and the viability and chromosome abnormality of oocytes were evaluated. Our results showed no significant differences between treatment groups in regarding to oocyte viability or ooplasm integrity. However, oocytes exposed to cold stress without preincubation with CLC had significantly higher chromatin dispersion (P<0.05) than pre-incubated oocytes with CLC. In second experiment, oocytes were exposed to 2 mg/ml CLC in maturation medium for 2 h and vitrified after maturation in conventional straws (0.25 ml; CS) or in open pulled straws (OPS). Osmotic stress of vitrification media to oocytes was also evaluated. Oocytes pre-treated with CLC had a significantly higher viability rate and ooplasm integrity than non-treated oocytes after vitrification in OPS (P < 0.05). The findings of this study also show that CLC pre-incubation of ovine oocytes reduce the chromosome degeneration after vitrification in both OPS and CS.

Animal production via SCNT provides a unique tool for protection of valuable individuals, conservation of vulnerable and endangered species and production of transgenic animals. A total of 167 MI and 219 MII stage oocytes were used as the material of the study. The oocytes were enucleated at 44 h after in vitro maturation by aspiration of the polar body and the MI or MII plates. Cycling granulosa cells were used for nuclear transfer. Cell fusion was induced with DC pulses of 2.0 kV/cm 60 mu s, 0.1s apart (2x) delivered by a BTX Electrocell Manipulator 200 (BTX, San Diego, CA, USA). After fusion, the embryos were activated by 1.0 kV/cm 20 mu s DC pulses 0.1s apart (2x) followed by 2 mM 6-DMAP (6-dimethylaminopurine) incubation in culture medium for 4 h in a humidified atmosphere of 5% CO2, 5% O-2, and 90% N-2 at 38 degrees C. The somatic cell transferred embryos were cultured for 8 days in mSOF medium supplemented with 0.4% BSA in a humidified 5% CO2, 5% O-2, and 90% N2 atmosphere at 38 degrees C. After in vitro culture period, all embryos transferred to HSOF containing Hoechst 33342 (5 mu g/mL) and the cell numbers were counted under ultraviolet light using a fluorescent microscope. The fusion (66.66 vs 21.55%) and cleavage rates (15.75 vs 11.11%) were significantly higher in MII stage oocytes than MI stage oocytes (P<0.02). While SCNT embryos were developed to morula stage in MII group (14; 9.58%), all the cleaved embryos were arrested at the 2-4 cell stage in MI group. None of the embryos was developed to blastocyst stage in both groups.

Çalışmanın ilk bölümünde, mezbahadan sağlanan ovaryumlardan kazanılan oositler (n=2990) olgunlaştırma medyumu içerisinde 24 saat süreyle olgunlaştırıldı. Ardından, 20 saat süreyle İn Vitro Fertilizasyona (İVF) bırakıldılar. Yarıklanma gösteren embriyolar (n=1305), Sentetik Ovidukt Fluid (SOF) medyumu içerisine alınarak altı gün süresince İn Vitro Kültüre (İVK) bırakıldılar. İVK sonrası elde edilen morula-blastosist aşamasındaki embriyolar rastlantısal şekilde üç farklı dondurma hızı grubuna eşit olarak ayrıldılar (Grup I: 0,5 °C /dk, Grup II: 0,8 °C /dk, Grup III: 1 °C /dk). Her bir gruptaki embriyolar (n=50), 1,5 M etilen glikol bulunan dondurma medyumu içerisinde farklı soğutma hızlarında donduruldu. Sonuçta 0,5 °C/dk soğutma hızının en başarılı grup olduğu belirlendi (P<0,05). Çalışmanın ikinci bölümünde, verici koyunlardan elde edilen in vivo embriyolar (Morula-Blastosist) çalışmanın birinci bölümünde bulunan en başarılı soğutma hızı (0,5 °C/dk) ile soğutularak donduruldu. Dondurulan 19 adet embriyo hormonel olarak hazırlanmış 17 alıcı koyuna transfer edildi. Transfer sonrası 60. günde yapılan ultrason muayenesinde üç adet koyunda gebeliklere ait embriyonik keseler gözlendi ve bu koyunlardan birinde de ikiz gebelik saptandı. Gebe koyunlardan bir tanesinde doğum gerçekleşti; diğer iki koyunda ise ileriki dönemde yapılan ultrason muayenesinde gebeliklerin sonlandığı gözlendi. Çalışma sonucunda, koyun embriyolarının dondurulması sırasında 0,5°C/dk soğutma hızının en başarılı hız olduğu ve bu yöntemle dondurulan embriyoların transferinden de gebelik elde edilebildiği saptandı.

This study was conducted to determine the most successful techniques on inmature and in vitro-matured cat oocytes that were parhtenogenically activated using 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX), in combination with electrical stimulation and calcium ionophore. After 44 h of in vitro maturation, the oocytes with a polar body were separated as mature (M II) and those without a polar body were considered as immature. Four different activation treatments and two control groups were used for parthenogenetic activation with both mature and immature cat oocytes. After 48 h of activation, the oocytes were examined and the non-cleaved oocytes removed. The cleaved oocytes/embryos were cultured in vitro in mSOF medium for an additional four days. After six days of in vitro culture (IVC), embryo quality was evaluated. The results in the present study suggested that (I) both in vitro matured and immature cat oocytes have a potential to develop to morula and blastocyst stages after parthenogenetic activation, (II) electrical stimulation +6-DMAP is a more useful technique for both matured and immature cat oocytes and (III) to our knowledge, this is the first report that describes morula and blastocyst formation from parthenogenetically activated immature cat oocytes.

This study was conducted to determine the most successful techniques on inmature and in vitro-matured cat oocytes that were parhtenogenically activated using 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX), in combination with electrical stimulation and calcium ionophore. After 44 h of in vitro maturation, the oocytes with a polar body were separated as mature (M II) and those without a polar body were considered as immature. Four different activation treatments and two control groups were used for parthenogenetic activation with both mature and immature cat oocytes. After 48 h of activation, the oocytes were examined and the non-cleaved oocytes removed. The cleaved oocytes/embryos were cultured in vitro in mSOF medium for an additional four days. After six days of in vitro culture (IVC), embryo quality was evaluated. The results in the present study suggested that (I) both in vitro matured and immature cat oocytes have a potential to develop to morula and blastocyst stages after parthenogenetic activation, (II) electrical stimulation + 6-DMAP is a more useful technique for both matured and immature cat oocytes and (III) to our knowledge, this is the first report that describes morula and blastocyst formation from parthenogenetically activated immature cat oocytes.

Ozet Koyun embriyolarinin in vitro uretilmesi uzerine olgunlasma ve fertilizasyon medyumlarina yapilan katkilar ve ko-kulturiin etkilerinin incelendigi bu calismada mezbahada kesilen koyunlarin ovaryumlanndan elde edilen primer oositler (n=454) kullanildi. Oositler 4 gruba ayrilarak farkli katkilarin bulundugu TCM-199 medyumunda 26 saat olgunlastirildi. Grup 1 (SH): %20 oraninda koyun serumu, 10 ug/ml FSH, 10 ug/ml LH ve 1 ug/ml estradiol 17(3 ilaveli TCM-199, Grup 2 (S): %20 oraninda koyun serumu ilaveli TCM-199, Grup 3 (H): 10 pg/ml FSH, 10 pg/ml LH ve 1 ug/ml estradiol 17(3 ilaveli TCM-199, ve Grup 4 (M): hormon ve serum ilavesi olmayan TCM-199. Olgunlasmayi takiben oositler tekrar 3'er alt gruba ayrilarak %2 (D), %5 (O)ve %10 (Y) koyun ostrus serumu ilaveli SOF medyumu icerisine alindi ve uzerlerine elektro^jakulator yardimiyla alinmis ve yikanmis taze koc spermasi ilave edilerek 18 saat in vitro fertilize edildi, Fenilizasyonu takiben oositler kendi iclerinde tekrar 2'ser alt gruba [Grup 1 (KK): Koyun ovidukt epitel hucreleri ile ko-kultur; Grup 2 (K): Koyun ovidukt epitel hucreleri kullanilmayan kultur grubu] ayrildi ve 7 gun kulture edildi. Bu surenin sonunda embriyolarin gelismeleri degerlendirildi. Calismada en yuksek cleavage (yanklanma) ve morula oranlan SH+Y+KK grubunda {sirasiyla %62.5 ve %43.8), en dusuk oranlar ise (sirasiyla %8.7 ve %4.3) M+D+K grubunda elde edildi. Bu sonuclara gore, koyun oositlerinin in vitro olgunlasunlmasinda %20 oraninda koyun serumu ve hormonlarin, fertilIzasyon medyumunda %10 oraninda ostrustaki koyun serumunun kullanilmasi ve fertilIzasyon sonrasi koyun ovidukt epitel hucreleri ile ko-kultur uygulanmasinin yanklanma ve morula oranlarini iyilestirdigi soylenebilir. Anahtar kelimeler: Koyun, in vitro uretim, medyum kalkilan, ko-kultur EFFECTS OF SUPPLEMENTS TO MATURATION AND FERTILIZATION MEDIA AND CO-CULTURE WITH SHEEP OVIDUCTAL EPITHELIAL CELLS ON IN VITRO PRODUCTION OF SHEEP EMBRYOS Summary In this study in which the effects of maturation and fertilization media supplements and co-culturing on in vitro production of sheep embryos were investigated, primary oocytes (n=454) collected from ovaries of slaughtered ewes were used. Oocytes divided randomly into 4 groups were matured for 26 h in TCM 199 medium with different supplements: Group I (SH): TCM 199 medium supplemented by 20% sheep estrous serum, 10 ug/ml FSH, 10 ug/ml LH and 1 ug/ml estradiol 17fi, Group 2 (S): TCM 199 medium supplemented by 20% sheep estrous serum. Group 3 (H): TCM 199 medium supplemented by 10 ug/ml FSH, 10 ug/ml LH and 1 pg/ml estradiol 170, and Group 4 (M): TCM 199 medium without serum and hormones. After maturation, oocytes which were again divided into 3 subgroups were transferred in SOF medium supplemented by 2% (D), 5% (O) and 10% (Y) sheep estrous serum and fertilized in vitro for 18 h with fresh ram semen collected by electro-ejaculation and washed. After fertilization, oocytes were divided into 2 subgroups [Group 1 (KK): Co-culture with sheep oviductal epithelial cells; Group 2 (K): Culture without sheep oviductal epithelial cells] and cultured for 7 days. At the end of this period embryos were evaluated for their developments. The highest cleavage and morula rates were obtained in SH+Y+KK group (62.5% and 43.8%, respectively) and the lowest rates (8.7% and 4.3%, respectively) in M+D+K group in this study. According to the results, we concluded that supplementation of in vitro maturation media with 20% sheep estrous serum and hormones, in vitro fertilization media with 10% sheep estrous serum and co-culturing with sheep oviductal epithelial cells after fertilization could improve the cleavage and morula rates. Key words : Sheep, in vitro production, media supplements, co-culture

Ozet Calismada in vitro olgunlastirilmis kedi oositlerinin in vitro fertilize olabilme ve akabinde in vitro kultur ortaminda morula ve blastosist donemine ulasabilme kabiliyetlerinin arastirilmasi amaclandi. Arastirmanin materyalini, kisirlastirilmis sokak kedilerinin ovaryumlarindan kazanilan oositler olusturdu. Ovaryumlar iki saatlik sure icerisinde 38°C' deki PBS solusyonu icerisinde laboratuara tasindi. SOF medyumu icerisinde (%0.4 BSA+10µg/ml FSH + 10µg/ml LH), gaz karisimi (%5 0 2 ,%5 C0 2 ,%90 N 2 ) ve %100'e yakin nemin saglandigi 38.5°C'lik inkubator ortaminda 48 saat sure ile olgunlasmaya birakildi (IVM). Ardindan. Swim-Up yontemiyle kapasite edilen donmus kedi spermasiyla 20 saat in vitro fertilizasyona birakildi (IVF). In vitro olarak yedi gun sureyle kulture edilen oosit/embriyolar fikse edildi ve boyandi. Faz-kontrast mikroskopta x400 buyutmede gelisim durumlari saptandi. Calismada toplam 106 adet primer oosit kullanildi. In vitro olgunlastirilan bu oositlerde, in vitro ferlilizasyonu takip eden 24 saatlik dilimde 21 adet oositin yariklandigi gozlendi. Boylece cleavage orani, %19.81 olarak belirlendi. Kultur periyodunun sonunda, embriyolarin 13 tanesinin (%12.26) morulaya ve sekizinin (%7.54) blastosiste ulastigi saptandi. Yariklanma sonuclarinin dusuk kalmis olmasina ragmen, calismada yariklanma gozlenen oositlerin tamaminin (21/21) morula-blastosiste gelistigi belirlendi. Sonuc olarak, kedi oositlerinin SOF medyumu icerisinde in vitro olgunlastirilmasi, fertilizasyonii vc kulturunu takiben transfer edilebilir duzeyde embriyo elde edilebildigi goruldu. Sunulan bu calisma ile ulkemizde ilk defa kedi oositlerinin IVM ve IVF'u ile morula ve blastosist duzeyinde embriyolar elde edilmis oldu. Anahtar Kelimeler: Kedi, Oosit, In Vitro Fertilizasyon, Embriyo IN VITRO FERTILIZATION OF CAT OOCYTES Summary The aim of the study was to investigate the ability of in vitro matured cat oocytes to be fertilized in vitro and reach to morula and blastocyst stages in vitro. The oocytes collected from spayed stray queens served as the material of the study. The ovaries were brought to the laboratory within two hours in PBS solution at 38°C. Recovered oocytes were left for maturation in SOF medium (%0,4 BSA + l0µg/ml FSH + 10 µg/ml LH added) In an incubator at 38.5°C for 48 hours under gas mixture (5% 0 2 . 5% C0 2 . 90% N 2 ) and almost 100% humidity. After iVM for 48 h oocytes were co-incubated with frozen-thawed spermatozoa for 20 h. which were capacitated by Swim-Up procedure under the same conditions. After in vitro culture for seven days, oocytes/embryos were then fixed and stained. Developmental status of the oocytes/embryos were evaluated under a phase-contrast microscope at x400 magnification. Totally 106 primer oocytes were used for in vitro maturation. After 24 h in vitro fertilization period. 21 oocytes were cleavaged (19.81%). At the end of culture period. 13 (12.16%) and eight (7.54%) of embryos reached to morula and blastocyst stages respectively. In spite of the low cleavage rate, all of the cleaved embryos have reached the morula - blasocyst stage (21/21). As a result of this study, it was observed that transferable embryos could be produced by in vitro maturation, fertilization and culture of cat oocytes in SOF medium. Key Words: Cat. Oocyte. In Vitro Fertilization. Embryo