Civcivlerde Bursa Fabriciusun Embriyonik Gelişiminin Morfohistolojik Değerlendirmesi

Kanatlı hayvanların primer lenfoid organı olan Bursa Fabricius (BF) humoral bağışıklıktan sorumludur. Kanatlılarda, BF’nin embriyonik gelişimini tamamlaması ve hayvanın sağlıklı bir yaşamını sürdürmesi açısından belirli bir olgunluğa ulaşması çok önemlidir. Bu çalışmada, civciv embriyolarında BF gelişimi ve morfometrik ölçümleri araştırılmıştır. BF, inkübasyonun 10, 13, 16 ve 21. günlerinde 24 Babcock beyaz Leghorn civciv embriyosundan alındı. Bu çalışmada ortalama BF ağırlığı 10. günde 0.006±0.001 g iken, bu değer 21. günde 0.037±0.007 g idi. Ortalama kraniyo-kaudal çap belirlenen inkübasyon günlerine göre istatistiksel olarak farklıydı. Bu değer 10. günde 2.39±0.10 mm iken, 21. günde 7.55±0.66 mm idi. Ortalama latero-lateral çap 1.94±0.15 mm ile 4.71±0.96 mm arasında değişirken, ortalama dorso-ventral çap 1.48±0.28 mm ve 2.29±0.42 mm arasındaydı. Histolojik olarak bursal kesitlerde inkübasyonun 10. gününde merkezi bir lümen ve plika gelişimi gözlendi. 13 günlük embriyoların BF’lerinde epitelyal tomurcuklanmalar, iki farklı epitel yapısı ve üç katmanlı duvar tabakasının olduğu bulundu. 21. günde BF’nin histolojik gelişiminin tamamlandığı gözlemlendi. Sonuçta, elde edilen bu veriler BF’nin kümes hayvanlarının sağlıklı yaşamını sürdürmesi açısından önemli bir lenfoid organ olduğu sonucuna varıldı. Ayrıca elde edilen morfometrik ölçümlerin bu organ üzerinde yapılabilecek çalışmalar için referans veriler olarak kullanılabileceği düşünülmektedir.

Bursa Fabricius (BF), the primary lymphoid organ of poultry, is responsible for humoral immunity. In poultry, it is very important for BF to complete its embryonic development and reach to a certain maturity in terms of maintaining a healthy life of the animal. In this study, BF development and its morphometric measurements were investigated in chick embryos. BF was taken from 24 Babcock white Leghorn chick embryos on the 10th, 13th, 16th and 21st days of incubation. The average BF weight was 0.006±0.001 g on the 10th day, while this value was 0.037±0.007 g on the 21st day. Average cranio-caudal diameter was different according to the determined incubation days, statistically. While this value was 2.39±0.10 mm on the 10th day, it was 7.55±0.66 mm on the 21st day. The average latero-lateral diameter was ranged from 1.94±0.15 mm to 4.71±0.96 mm, while the average dorso-ventral diameter was in the range of 1.48±0.28 mm and 2.29±0.42 mm. Histologically, a central lumen and plica development were observed on the 10th day of incubation in the bursal sections. 13-day-old embryos were found to have epithelial buddings, two different epithelial structures and three layers of wall in the BF. On the 21st day, we were observed that histologic development of BF was completed. In conclusion, obtained these data concluded that BF is an important lymphoid organ in terms of maintaining a healthy life of the poultry. It is also believed that the obtained morphometric measurements can be used as reference data for further studies on this organ.

Giriş

Lymphoid organs play an important role in the body's defense against pathogens. B-cells are responsible for humoral immunity in living organism[1]. These cells are producted in Bursa Fabricius (BF) which is the primary lymphoid organ of poultry [2-5]. B-cells develop in the BF, differentiate and immunoglobulin isotropic exchange is performed[2-4] here. BF is controlled by bursin hormone and various cytokines [6].

BF begins to develop as a bump in the dorsal diverticulum of the cloacal proctodeum. On the 5th day of embryogenesis, a lumen is formed in the organ and connected to the proctodeum with a small handle[7]. BF was found to have a T-lymphocyte area on the dorsal side of the canal opening to the cloaca and therefore, this organ was considered a secondary lymphoid organ. However, atrophy of the organ and low antibody production does not make this feature very important[2,4,8]. It is reported that traces of cicatrix are seen in the 28^th week of the organ which starts to regress after 8 weeks [9].

There are three layers in the wall of BF which are tunica mucosa, tunica muscularis and tunica serosa respectively from inside to outside[4]. In BF, lymph follicles containing cortex, medula and corticomedular regions are located in the evagination of the tunica mucosa towards the lumen (plica)[10]. There are two types of epithelium as follicle-related epithelium (FAE) and interfollicular epithelium (IFE) in BF. FAE, which has a pseudostratified columnar epithelium without basal lamina, covers the lumen-facing surfaces of the developing follicles. IFE, a single layer of columnar epithelial layer, covers between two follicular structures[11].

It is very important for this organ to complete its embryonic development and reach a certain maturity in terms of maintaining a healthy life of the animal. The purpose of this research was to evaluate organogenesis and morphometric measurements (organ weight, cranio-caudal, latero-lateral and dorso-ventral diameters, organ volume) of BF in chick embryos. Also, this study will be able to model embryological studies in humans by using chick embryos during incubation. It is thought that the data and information obtained from the study may support the studies planned on this organ.

Gereç ve Yöntem

Ethical approval for this study was taken from The Ethical Committee of Health Sciences of Karamanoglu Mehmetbey University (protocol number: 2019/51).

The Eggs: BF used in this study were obtained from 24 Babcock white Leghorn chick embryos on the 10^th, 13^th, 16^th and 21^st days of incubation. Embryonic periods determined in the present study were selected considering organ development. 40 fertilized eggs were purchased for this study. For the disinfection of these initially visibly clean eggs and incubator, formalin fumigation was carried out with a mixture of 40 mL formalin (40%) and 20 g potassium permanganate per cubic meter for 30 minutes (12). These eggs were divided into four groups to be opened at determined embryonic periods for this study. 10 eggs were placed in each group because some of the eggs placed in the incubator were thought to be infertile. The eggs were numbered and placed in hatching machine (temperature: 37.4-37.6 ⁰C; humidity: 55%-65%). According to the determined embryonic days, the eggs were opened from each of these groups until 6 live embryos were obtained for evaluation in terms of organ development.

Macroscopic Evaluation of Eggs and Embryos: In this study, embryo weight, the pre-hatching egg weight, and first egg weight were weighed with precision scales. The relative embryo weights (%) were calculated by the following formula (Equation 1). Also, daily egg weight loss was obtained.

Collection and Anatomical Assessment of BF Samples: The dissected BF were removed from the peripheral tissues and their weights were measured with precision scales. Relative organ weights (%) were calculated with formula (Equation 2). Cranio-caudal, latero-lateral and dorso-ventral diameters were measured by digital caliper (OEM KMP150, 0-150 mm, Loyka).

Histological Processing of BF Samples: BF taken on the 10th, 13th, 16th and 21st days of incubation was fixed in 10% neutral-buffered formalin solution. These samples washed overnight in running water to remove the fixation solution. Then, these samples were dehydrated in graded alcohol series, cleared in xylene (3 times), and embedded in paraffin blocks. Serial sections of 5 μm thickness were taken at regular intervals using a microtome from these blocks. For histological examination, the sections were stained with Crossmon’s trichrome stain and examined with a light microscope (Leica DM-2500 attached to a DFC-320 digital camera)[13].

Statistical Analysis: The statistical analyses was performed using SPSS software version 21. The variables were investigated using visual (histograms, probability plots) and Kolmogorov-Smirnov/Shapiro-Wilk's test were performed whetherthe data are normally distributed or not. Data are expressed as means±standard deviation (SD). One-way ANOVA was used to compare parameters. Levene test was used to assess the homogeneity of the variances. Statistically, significance was accepted as P<0.05. When an overall significance was observed, further post-hoc tests were performed using Tukey's test[14].

Bulgular

Anatomical Changes:The data were given in Table 1. Statistically, the lowest pre-hatching egg weight was on 21^st day whereas the highest value was on 10th day (P<0.05). The embryo weight was different according to the determined incubation days. On the 10^th and 21^st days, it was found as 3.07±0.33 g and 41.19±3.94 g, respectively. Statistically, the organ weights were similar on 10^th-13^th days and 16^th-21^st days. The daily egg weight loss was in the range of 0.53%-0.67%. In the statistical analysis of diameter measurements, whereas the smallest values were on the 10th day, the highest values were on the 21st day (P<0.05).

Histological Evaluation: On the 10^th, 13^rd, 16^th and 21^st days of incubation, histological structure of the BF was microscopically given in Figure 1. In the tissue sections, we observed organ drafts containing a lumen on day 10^th of incubation. A lot of mucosal protrusions, called plica, were seen on the inner surface of the bursa. Large basophilic cell accumulations and hematopoietic foci were determined under the epithelial layer. On the 13^rd day of incubation, the number of plicas in BF increased, and its development was more advanced than the 10^th day. There were epithelial buddings occurring by basophilic cell accumulation and the presence of ongoing basophilic cell accumulations in the mucosa layer. Occasionally, lymphocytes were noted. The separation of the BF wall layers was clearly observed. Erythrocytes filled vessels and small hematopoietic areas were found. Furthermore, it was noted that the FAE and IFE began to form in the organ. On the 16^th day of the incubation, many small lymph follicles occurring from epithelial buddings were observed in the tunica mucosa. The lymphocyte-rich medulla of these follicles was quite prominent. FAE and IFE were considerably developed according to 13th day of incubation. Interfollicular connective tissue were developing. On the 21^st day, histological development of BF was completed. Advanced follicular development, marked separation cortex-medulla-corticomedullar zone, evident FAE and IFE development besides trabeculae were observed in the organ.

Tartışma

The BF, which varies in shape and size according to poultry species, is opened by a small canal into the proctodeum region of cloaca[3,15,16]. Mahanta et al.[3] reported that the BF was caudal 2.15±0.39 mm from the left kidney and 1.53±0.22 mm from the right kidney. In this study, BF of embryos according to certain embryonic periods were oval shaped. This finding was consistent with other studies[2,4,15,17].

Mahanta et al.[3] reported that the average weight of the BF was 0.053±0.02 g. Also, average longitudinal diameter, transverse diameter and organ thickness of Kadaknath chick were 5.78±0.28 mm, 3.59±0.18 mm and 3.09±0.29 mm, respectively. In this study, the average BF weight was 0.006±0001 g on the 10th day, while this value was 0.037±0.007 g on the 21st day. Average cranio-caudal diameter was different according to the determined incubation days, statistically. While this value was 2.39±0.10 mm on the 10^th day, it was 7.55±0.66 mm on the 21^st day. The average latero-lateral diameter was ranged from 1.94±0.15 mm to 4.71±0.96 mm, while the average dorso-ventral diameter was in the range of 1.48±0.28 mm and 2.29±0.42 mm. These values indicate that the embryo and its organs develop and grow.

Optimal incubation conditions should be met in terms of reliability of the study. The relative humidity of the incubator. It is the water that evaporates from the pores in the egg shell that determines the relative humidity of the incubator. This also leads to daily egg weight loss. This rate should be 0.55%-0.70% during incubation[18]. In this study, the daily egg weight loss was between 0.53% and 0.67% (Table 1). These values were within the range of desired values, which indicates that our incubation conditions are optimal. In this study, average embryo weights were 3.07 g, 8.33 g, 24.27 g, and 41.19 g in the incubation on days 10^th, 13^th, 16^th, and 21^st, respectively. According to Table 1, the data were similar with the data of Bellairs and Osmond[19]. Differences may be due to egg size and/or chick breed.

In the present study, the highest relative embryo weight and the lowest relative organ weight were statistically 21-old-day embryos (P<0.05). Consequently, these data might be considered as an indication that the embryo developed.

As a result of the measurements, increased the BF weight and embryo weight were determined in the hatching time of chicks. The increase in BF weight and embryo weight depends on the development and growth of the embryo. It was determined that egg weights decreased as the hatching time of the chicks approached. Therefore, it was thought to be that the nutrients contained in the egg were burned with oxygen and converted into carbon dioxide and water and thrown out of the egg.

BF, a lymphoepithelial organ, is not only the primary but also a secondary lymphoid organ in terms of having a T-lymphocyte area in poultry. B-lymphocytes, which are responsible for the formation of a humoral immune response to diseases, develop and differentiate in this organ[4,20,21]. The epithelial bursa draft begins to develop in 4^th-5^th days. B-lymphocyte precursors migrate to this organ draft on the 7^th day of incubation[7,22,23]. On the 9th day of embryogenesis, evaginations into the lumen of the mucosal layer are named as plica. In 10-day-old embryos, the development of lymph follicles is reported to be initiated by the accumulation of large basophilic cells under the epithelial layer[24]. In this study, a central lumen and plica development were observed on the 10^th day of incubation in the bursal sections.

Dense basophilic cell accumulation forms epithelial buddings in the mucosa layer[24]. The wall structure of the BF consists of tunica mucosa, tunica muscularis and tunica serosa[4]. There are structurally different epithelium in the BF as FAE and IFE. Whereas FAE is pseudostratified columnar epithelium that covers the luminal surface of follicles, IFE is a single layer prismatic epithelium that covers inter-follicular regions[25]. In present study, 13-day-old embryos were found to have epithelial buddings, two different epithelial structures and three layers of wall in BF.

The development of the lymph follicle is reported to be completed in the organ by the 17^th day of incubation. Each lymph follicle has a narrow cortex and a large medulla. The corticomedullar border, the continuation of IFE, separates the cortex and medulla[10]. In our study, the development of lymph follicle was clearly visible on the 16th day of incubation. On the 21^st day, we were observed that histologic development of BF was completed. Advanced follicular development, marked separation cortex-medulla-corticomedullar zone, evident FAE and IFE development, and trabeculae were seen in the organ. According to the results of this study, embryonic development of BF was found to be consistent with the findings of some researchers[4,22,26].

As conclusion, obtained results suggest that BF is an important lymphoid organ in terms of maintaining a healthy life of the poultry. Organogenesis and morphometric measurements of BF evaluated with this research by using chick embryos during incubation. It is thought that the data and information obtained from the present study may support the studies planned on this organ.

References

1. Akter SH, Khan MZI, Jahan MR, Karim MR, Islam MR. Histomorphological study of the lymphoid tissues of broiler chickens. Bangladesh J Vet Med 2006; 4: 87-92.
2. Türkmenoğlu İ, Demirkan İ, Demirkan AÇ, Akosman MS, Akalan MA. Morphology and volume measurement of bursa fabricius by stereology in Merlin (Falco Columbarius). Kocatepe Vet Derg 2019; 12: 27-32.
3. Mahanta D, Mrigesh M, Sathapathy S, et al. Gross and morphometrical studies on the thymus, spleen and bursa of fabricius of day old Kadaknath chick. JEZS 2018; 6: 555-558.
4. Sari EK, Altunay H, Kurtdede N, Bakir B. The structure of bursa of fabricius in the long-legged buzzard (Buteo Rufinus): histological and histochemical study. Acta Vet-Beograd 2015; 65: 510-517.
5. Song H, Peng KM, Li SH, et al. Morphological characterization of the immune organs in ostrich chicks. Turk J Vet Anim Sci 2012; 36: 89-100.
6. Otsubo Y, Chen N, Kajiwara E, et al. Role of bursin in the development of B lymphocytes in chicken embryonic bursa of fabricius. Dev Comp Immunol 2001; 25: 485-493.
7. Funk PE, Palmer JL. Dynamic control of B lymphocyte development in the bursa of fabricius. Arch Immunol Ther Exp 2003; 51: 389-398.
8. Çolakoğlu F, Dönmez HH. Kanatlıların sindirim kanalı lenfoid dokusu. Atatürk Üniv Vet Bil Derg 2018; 13: 106-111.
9. Bickford AA, Kuney DR, Zander DV, McMartin DA. Histologic characterization of the involuting bursa of fabricius in single-comb white Leghorn chickens. Avian Dis 1985; 29: 778-797.
10. Olah I, Glick B. Follicle‐associated epithelium and medullary epithelial tissue of the bursa of fabricius are two different compartments. Anat Rec 1992; 233: 577-587.
11. Davenport WD, Allen ER. Dome epithelium and follicle‐associated basal lamina pores in the avian bursa of Fabricius. Anat Rec 1995; 241: 155-162.
12. Olsen R, Kudirkiene E, Thofner I, et al. Impact of egg disinfection of hatching eggs on the egg shell microbiome and bacterial load. Poult Sci 2017; 96: 3901-3911.
13. Selçuk ML, Tıpırdamaz S. A morphological and stereological study on brain, cerebral hemispheres and cerebellum of New Zealand rabbits. Anat Histol Embryol 2020; 49: 90-96.
14. Karagöz Y. SPSS 22 Uygulamalı Biyoistatik. 2nd Edition, Ankara: Nobel Tıp Kitabevi, 2015.
15. Hassan SA, Tememy A, Hussein JS, Rasool BS. Histological study on bursa of fabricius of quail birds (Coturnix coturnix japonica). Egyptian Poult Sci 2011; 31: 613- 620.
16. Khan MZI, Masum M, Khan MZI, et al. Histomorphology of the lymphoid tissue of broiler chicken in Kelantan. Sains Malaysiana 2014; 43: 1175-1179.
17. Kumar P, Das P, Ranjan R, Minj AP. Postnatal development of bursa of fabricius of Khaki Campbell Duck. Indian J Vet Anat 2014; 26: 30-32.
18. Özparlak H, Çelik B, Balta D. Yumurtaya verilen siklofosfamid ve C vitamininin tavuk embriyoları üzerindeki bazı etkileri. Selçuk Üniv Fen Fak Fen Derg 2018; 44: 157-173.
19. Bellairs R, Osmond M. The Atlas of Chick Development. 2nd Edition, London: Elsevier, 2005.
20. Sandıkçı M, Karagenç L. Tavuk ve bıldırcın embriyolarında bursa Fabricius ve timusta bazı kök hücre belirteçlerinin incelenmesi. Ankara Üniv Vet Fak Derg 2013; 60: 157-163.
21. Chrząstek K, Madej JP, Mytnik E, Wieliczko A. The influence of antibiotics on B-cell number, percentage, and distribution in the bursa of fabricius of newly hatched chicks. Poultry Sci 2011; 90: 2723-2729.
22. Berktay EA, Çelik İ. Sunset Yellow FCF'nin (E110), Tavukların Timus ve Bursa Fabricii'sinin Embriyonik Gelişimi Üzerindeki Etkisinin Histolojik ve Enzim Histokimyasal Yöntemlerle Belirlenmesi. Yüksek Lisans Tezi, Konya: Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, 2014.
23. Casteleyn C, Doom M, Lambrechts E, et al. Locations of gut-associated lymphoid tissue in the 3-month-old chicken: A review. Avian Pathol 2010; 39: 143-150.
24. Ratcliffe MJH. The ontogeny and cloning of B cells in the bursa of Fabricius. Immunol Today 1985; 6: 223-227.
25. Kanasiya S, Karmore S, Barhaiya RK, et al. Histoarchitectural studies on bursa of fabricius of Kadaknath birds. J Anim Res 2018; 8: 107-110.
26. Kocaöz N, Çelik İ, Ünsal S. Kuluçkadan çıkıştan sonra tavuk bursa Fabricii'sinde oluşan histolojik değişiklikler. Vet Bil Derg 1997; 13: 43-51.