Kuluçka öncesi ve erken embriyonik dönemde döllü ve dölsüz tavuk yumurtasının pander nükleusu ve embriyonik disk hücreleri üzerinde ışık mikroskobik incelemeler
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Tarih
2020
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Selçuk Üniversitesi Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada, horoz ve tavukların perifer kan eritrosit çekirdekleriyle döllü ve dölsüz yumurtaların Pander nükleusu ve blastodisk hücrelerinin hücre çekirdekleri, floresans, histokimyasal ve mikromorfometrik yöntemlerle incelenerek, horozlarla ve tavukların, dölsüz ve döllü yumurtaların DNA içerikleri arasındaki farkların belirlenmesi amaçlanmıştır. Bu amaçla, 60 adet döllü ve 30 adet de dölsüz Babcock ırkı yumurtacı tavuk yumurtası kullanıldı. Babcock ırkı 30 tavuk ve 30 horozun kanat venasından heparinli tüplere alınan perifer kan örneklerinden frotiler hazırlandı. Tespit işlemini takiben, frotilerden bir kısmı May grünwald-giemsa ile boyanırken, diğerlerinde Feulgen reaksiyonu ve floresan mikroskobik inceleme gerçekleştirildi. Dölsüz yumurtaların Pander nükleusu pipetle tüplere alınarak santrifüje edilerek dişi yumurta hücresi çekirdeğinin izolasyonuna çalışıldı. Döllü yumurtalar kuluçkanın 84. saatine kadar kuluçka makinesinde ( Prodi HB 175 ) inkübe edildikten sonra embriyonik disk sahası poly-L-lizin kaplı lamlara aktarıldı. Hazırlanan örneklerin bir kısmı May grünwald-giemsa iale boyanırken, kalan örneklerde Feulgen reaksiyonu gerçekleştirildi. Bu çalışmada, horoz ve tavuk eritrositlerinin morfometrik verileri bir birine birbirine oldukça yakın olduğu tespit edilmiştir. Bununla birlikte, horoz eritrosit çekirdeği morfolojik indeksi tavuklarınkinden önemli derecede daha büyüktür (p<0,05). Bu durum, horoz eritrositlerinin DNA içeriğinin tavuklarınkinden daha fazla olmasından kaynaklanmış olabilir. Horoz ve tavukların perifer kan eritrosit çekirdeklerinin renk analiz sonuçlarına göre horoz eritrosit çekirdeklerinin birim alanındaki (1 µm2) total renk yoğunluğuyla kırmızı ve yeşil renk yoğunlukları tavuklarınkinden fazla olmakla birlikte, bu fark istatistiki açıdan önemli değildi. Kuluçkanın erken döneminde eritrosit (84. Saat) eritrositler üzerinde yapılan renk analizleri sonuçlarına göre total renk yoğunluğu 1,01-0,92 ve 0,62-0,15 aralıklarında, kırmızı renk yoğunluğu 1,13-0,74 ve 0,71-0,17 aralıklarında, yeşil renk yoğunluğu 0,77-0,42 ve 0,29-0,05 aralıklarında, gri ton yoğunluğu ise 1,01-0,61 ve 0,54-0,15 aralıklarında toplanan ikişer grup oluşturuldu. Erkek eritrositlerinin daha fazla DNA içeriğine sahip olduğu dikkate alındığında iki gruptaki embriyoların erkek, ikinci gruplardakilerin de dişi embriyo olabilecekleri sonucuna varıldı. Bu çalışmada uygulanan yöntemlerin ve elde edilen bulguların tavuk ve horoz genomları arasında kesin ayrım yapabilmede yeterli olmadığı açıktır. Her ne kadar, erken dönemde yani kuluçka öncesi ve/veya erken kuluçka döneminde cinsiyet tayininde hedefler, sistemin ve ekonominin mantığına da uygun biçimde otomasyona ve embriyoda en az hasarla analiz gerçekleştirmeye uygun olan spektroskopik yöntemlere doğru kaysa, bunlar için kesin ve doğru veri sağlayacak yöntemler yine de sitometrik yöntemlerdir. Bu yüzden, bu konuda daha geniş popülasyonları kapsayan, hiperspektral dijital kameraların da kullanılacağı ve detaylı istatistiksel yöntemler yardımıyla gerçekleştirilecek olan daha detaylı sitometrik çalışmalara ihtiyaç vardır.
In this study, nuclei of peripheral blood erythrocytes and nuclei of both female eggcell nuclei located at Pander nucleus, and nuclei of the blastodics cells were investigated by means of the fluorescence, histochemical ve micromorphometric methods, and it was aimed that whether there was any differences between DNA contents of male and female hen's, fertilized and unfetilized chicken eggs. For this purpose, 60 fertilized and 30 unfetilized eggs of Babcock layers. Blood smear were prepared from heparinized blood samples taken from wing veins of 30 male and 30 female chickens, via venipuncture. Some of the blood smear were stained with May grünwald-giemsa and in the remaining smears, Feulgen reaction was performed. From unfertilized eggs Pander nuclei area were taken into plastic tubes and diluted to 5 ml with prewarmed (37°C) physiological saline in order to isolate the female eggcell nuclei. Fertilized eggs were incubated until 84. hour of the incubation period (Prodi HB 175) and embryonic discc cells were isolated and taken onto poly-L-lysine coated glass slides. Some of the samples were stained with May grünwald –giemsa and in the remaining samples, Feulgen reaction was performed. In this study, morphological parameters of both of the male and females were found to be very close. Nevertheles, morphometric index of the male erythrocyte nucleus was significantly (p<0,05) larger than that of the females. This result possibly arisen from the higher DNA content of male erythrocyte nucleus. Although, the results of the color density analyses of both male and female chicken erythrocyte nuclei showed that total color density, red color density and green color density values in a unit area ((1 µm2) of the males were higher than those of the females, the differences between mean values were not statiscally significant. Results of the color analyses of the erythrocytes at early incubation period (84. hour) revealed that the total coor density accumulated between 1,01-0,92 and 0,62-0,15, the red color density ranged between 1,13-0,74 and 0,71-0,17, the green color density ranged between 0,77-0,42 ve 0,29-0,05 and the gray scale ranged between 1,01-0,61 and 0,54-0,15 ranges. Based on these results, the data were groupped into two groups wihth high and low means, for each color parameter. When regarding the male's nuclei having higher DNA content, it might be concluded that the high color intensity group for each color band has been taken from male embryos. It is clear that the investigation methods and the obtained results of hte present study are not sufficient to diccriminate male and female genome. Although, current and future trends in gender determination at pre incubation and early incubation perods inclinated to non invasive and light spectroscopic analyses those are mostly non invasive techniques, in accordance with automation and the spirit of the system and the economy, cell cytometric methods are the only methods could supply the correct and precise data for light spectroscopic analyses. Fort his reason, there is need of the further srtudies including large animal population and large number of the eggs, hyperspectral digitalcameras and more detailed statistical analyses.
In this study, nuclei of peripheral blood erythrocytes and nuclei of both female eggcell nuclei located at Pander nucleus, and nuclei of the blastodics cells were investigated by means of the fluorescence, histochemical ve micromorphometric methods, and it was aimed that whether there was any differences between DNA contents of male and female hen's, fertilized and unfetilized chicken eggs. For this purpose, 60 fertilized and 30 unfetilized eggs of Babcock layers. Blood smear were prepared from heparinized blood samples taken from wing veins of 30 male and 30 female chickens, via venipuncture. Some of the blood smear were stained with May grünwald-giemsa and in the remaining smears, Feulgen reaction was performed. From unfertilized eggs Pander nuclei area were taken into plastic tubes and diluted to 5 ml with prewarmed (37°C) physiological saline in order to isolate the female eggcell nuclei. Fertilized eggs were incubated until 84. hour of the incubation period (Prodi HB 175) and embryonic discc cells were isolated and taken onto poly-L-lysine coated glass slides. Some of the samples were stained with May grünwald –giemsa and in the remaining samples, Feulgen reaction was performed. In this study, morphological parameters of both of the male and females were found to be very close. Nevertheles, morphometric index of the male erythrocyte nucleus was significantly (p<0,05) larger than that of the females. This result possibly arisen from the higher DNA content of male erythrocyte nucleus. Although, the results of the color density analyses of both male and female chicken erythrocyte nuclei showed that total color density, red color density and green color density values in a unit area ((1 µm2) of the males were higher than those of the females, the differences between mean values were not statiscally significant. Results of the color analyses of the erythrocytes at early incubation period (84. hour) revealed that the total coor density accumulated between 1,01-0,92 and 0,62-0,15, the red color density ranged between 1,13-0,74 and 0,71-0,17, the green color density ranged between 0,77-0,42 ve 0,29-0,05 and the gray scale ranged between 1,01-0,61 and 0,54-0,15 ranges. Based on these results, the data were groupped into two groups wihth high and low means, for each color parameter. When regarding the male's nuclei having higher DNA content, it might be concluded that the high color intensity group for each color band has been taken from male embryos. It is clear that the investigation methods and the obtained results of hte present study are not sufficient to diccriminate male and female genome. Although, current and future trends in gender determination at pre incubation and early incubation perods inclinated to non invasive and light spectroscopic analyses those are mostly non invasive techniques, in accordance with automation and the spirit of the system and the economy, cell cytometric methods are the only methods could supply the correct and precise data for light spectroscopic analyses. Fort his reason, there is need of the further srtudies including large animal population and large number of the eggs, hyperspectral digitalcameras and more detailed statistical analyses.
Açıklama
Anahtar Kelimeler
Tavuk eritrositleri, Feulgen reaksiyonu, Chicken erythrocytes, Feulgen reaction
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Küçükkömürcü, M. (2020). Kuluçka Öncesi ve Erken Embriyonik Dönemde Döllü ve Dölsüz Tavuk Yumurtasının Pander Nükleusu ve Embriyonik Disk Hücreleri Üzerinde Işık Mikroskobik İncelemeler. (Yüksek Lisans Tezi). Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, Konya.
