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Involution of Bursa of Fabricius in Male and Female Chickens: A Light Microscopic Histoquantitative Study
PHYSIOLOGY AND REPRODUCTION Involution of Bursa of Fabricius in Male and Female Chickens: A Light Microscopic Histoquantitative Study 2lVANA MILICEVIC,1 DRINKA VUJIC, KATARINA ISAKOVIC, MILEVA MICIC, and NOVICA M. MILICEVIC1 Institute of Histology, Medical Faculty, University of Beograd, 11000 Beograd, Yugoslavia and Immunology Research Center, Beograd, Yugoslavia (Received for publication May 6, 1985)
1986 Poultry Science 65:2318-2323
INTRODUCTION
In the past three decades, the function of the avian cloacal bursa has been studied in detail, and the central role of this organ in the development and maturation of B lymphocytes established (Chang et al, 1955; Glick et al, 1956; Jankovic and Isakovic, 1964; Cooper et al, 1965). More recent studies have demonstrated that the bursa also has immune functions similar to those of peripheral lymphoid organs (Sorvari and Sorvari, 1977; Odend'hal and Breazile, 1979; Naukkarinen and Sorvari, 1980, 1982). However, the histology of the bursa has not been studied so systematically. The embryonal development of this organ has been extensively analyzed by light and electron microscopy (Ackerman and Knouff, 1959; Ackerman, 1962; Aramaki, 1968; Metcalf and Moore, 1971; Eskola et al, 1977), but much less attention has been paid to the later stages of ontogenic development, especially to the process of bursal involution, which begins around 3.5 to 5 months of age (Glick, 1977). Only HoffmannFezer and Lade (1972) have studied this process in detail. The lack of precise quantitative studies, which could elucidate the involution of
1
Institute of Histology.
this complex organ, is still notable. The only modern stereological analysis of the chicken bursa concerns the postnatal development of this organ up to 84 days (Romppanen, 1982). It is well known that the sex steroids strongly influence bursal development and function (Meyer et al, 1959; Aspinall et al, 1961; Glick, 1970) and that the bursa begins to involute with the approach of sexual maturity (Hoffmann-Fezer and Lade, 1972; Glick, 1977). However, no data are available about the structural changes of involuting bursa with respect to sex. Therefore, the aim of our study was to analyze, using the modern stereological approach, the different tissue compartments of the involuting bursa in male and female chickens. MATERIALS AND METHODS Experimental Animals and Tissue Preparation, Newly hatched Prelux Bro chickens were obtained from a local hatchery. These chickens are a domestic, Yugoslavian selection, bred for the purpose of producing meat. They were given commercial feed and water ad libitum. At ages of 3 and 6 months, five male and five female chickens were sacrificed with an overdose of an intravenous anaesthetic into the wing vein. The chickens and their bursae were weighed. Bursae were fixed in Carnoy's fixative. After fixation, the bursa was cut in slices per-
2318
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on March 23, 2015
ABSTRACT A histoquantitative study of the bursa of Fabricius in 3- and 6-month-old male Prelux Bro chickens (domestic selection) was made. All compartments of normal bursal tissue were examined. At 3 months of age, there were no significant differences in bursal weight and structure between males and females. The only difference observed was the higher volume density of follicular cortex and the lower proportion of follicular medulla in males compared to females. In 6-month-old male and female chickens, a different degree of bursal involution was observed. In the bursa of males, an advanced stage of involution was observed, whereas only the initial signs of bursal involution were found in females. (Key words: bursa of Fabricius, involution, sex differences, stereological analysis)
BURSAL INVOLUTION IN CHICKENS
pendicular to the longitudinal axis, i.e., to the mucosal surface. Three sections taken from proximal, middle, and distal parts of the bursa were selected for morphometric measurements. The tissue was processed in paraffin, and sections were cut at 5-/im thickness and stained with hematoxylin-eosin. Morphometric Measurements. Histoquantitative study of the bursal tissue was done according to Romppanen (1982). The point-counting method was used (Weibel, 1979). Point hits on lymphoid follicles, follicular cortex, follicular medulla, stroma, and bursal epithelium were registered at a magnification of 160X using a square grid with 25 points. The number of point hits on the tissue compartments was registered, and the corresponding volume density was calculated. A total of 600 points were counted for each bursa (200 points/section). Weights (milligrams) of different bursal tissue compartments were calculated by multiplying the corresponding volume density by the bursal weight. Surface density (mm 2 /mm 3 ) of the bursal epithelium was calculated from the equation of Weibel (1979): S v = kI L (factor k = 77/2, and I I = number of intersections per unit length of the system of test lines). The bursal epithelial surface at the luminal side [interfollicular epithelium (IFE), follicle-associated epithelium (FAE)] was measured using a square grid with 9 points. The magnification was 160x. Number of FAE areas ( N V F A E ) ( m m - 3 ) was computed from the following equation (Weibel, 1979):
where N A = number of FAE profiles per unit area of sectional plane and D = the diameter of the FAE area in space determined according to the equation: ^
4
D = -77r d
where d = diameter of FAE area measured by linear measurements with a ruler at a magnification 400X. The mean height (h) (^m) of the epithelium (IFE and FAE) and the mean diameter of follicles (Df0i) (|izm) were calculated as stereologic derivatives according to the following equations: by where V v = corresponding volume density (IFE or FAE) and Sy = corresponding surface density (IFE or FAE) and D fol
¥«V
2 x
V
where Vyfoii = volume density of lymphoid follicles and N V F A E = numerical density of FAE areas. For the statistical analysis of numerical data, Student's t-test and a 2 X 2 factorial analysis of variance were used. RESULTS The most voluminous bursal tissue compartment in male and female chickens was the lymphatic tissue both in relative (Table 1) and absolute amounts (Table 2). There was no
NA
VFAE - D
TABLE 1. Volume densities (%) of bursal tissue compartments (X ± SD) Age of chickens 1 6 Months
3 Moii t h s Volume densities
Males
Follicles, c o r t e x + medulla Follicular c o r t e x Follicular m e d u l l a Stroma Epithelium, I F E + F A E 2 IFE FAE
88.2 51.2 36.9 9.7 4.9 3.6 1.2
± ± ± ± ± ± ±
7.1 1.4 1.7 1.0 1.1 1.5 .6
Females
Males
85.2 44.5 40.6 9.3 5.9 4.8 1.1
79.0 ± 42.7 ± 36.2 ± 12.8 ± 7.9 ± 6.4+ 1.4 ±
± ± ± ± ± ± ±
1.9 .7* 3.0* 1.5 1.8 1.7 .2
1
Five chickens/group.
2
IFE = Interfollicular epithelium, FAE = follicle-associated epithelium.
Females 3.2 1.1 3.2 4.3 2.4 2.0 .3
'Statistically significant difference between males and females of the same age (P<.05).
86.3 46.3 39.9 8.6 5.3 3.9 1.3
± ± ± ± ± ± ±
3.1* 3.1* 2.6 .8* 1.2 1.2 .6
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The results demonstrated that there are no significant differences in bursal weight and minor changes in structure between 3-monthold male and female Prelux chickens. The only differences observed were the higher volume density of the follicular cortex and the lower proportion of follicular medulla in males compared with females. The stereological analysis
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on March 23, 2015
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statistically significant difference in the bursal weight and weight of bursal tissue compartments between 3-month-old male and female chickens (Table 2). The value for volume density of follicular cortex was higher, and the proportion of follicular medulla was significantly lower (P<.05) in males compared to females (Table 1). There was no significant difference in the volume density of stroma and epithelium (IFE and FAE) between males and females (Table 1). However, the absolute amount of IFE was greater in females (Table 2). The difference in surface density (Table 3) of the bursal epithelium, as well as in the number of areas of follicle-associated epithelium (Table 4), was not statistically significant between males and females. The number of areas of follicle-associated epithelium in a mature bursa presumably corresponds to the follicle number (Romppanen, 1982). The epithelial height (IFE and FAE) and the diameter of FAE and follicles were similar in males and females (Table 5). The results of 2 X 2 factorial analysis of variance demonstrated that in 6-month-old chickens, there was a significant main effect of age (P<.05) for follicular cortex, numerical density of FAE, surface density of FAE, and diameter of FAE. However, 6-month-old males had significantly lower values for volume density of follicles and follicular cortex (Table 1) compared with females. The absolute amounts of bursal tissue, follicles, and follicular cortex were also significantly lower in 6-monthold males (Table 2). The volume density of bursal stroma was significantly higher in 6month-old males in comparison with females u Tj 0 crt (Table 1). There were no significant differences rt jy in the surface density of the bursal epithelium u s (Table 3), numerical density of follicle-assoO c <+* U ciated epithelium (Table 4), and diameter of V II * follicle-associated epithelium and follicles (U -• < < % (Table 5) between 6-month-old male and female chickens.
BURSAL INVOLUTION IN CHICKENS
2321
TABLE 3. Surface density (mm2/mm3 ) of the bursal epithelium (X ± SD) Age of chickens1 3 Months
6 Months
Surface density
Males
Females
Males
Females
Epithelium, IFE + FAE 2 IFE FAE
210.0 ± 44.2 189.6 ± 38.0 20.4 ± 6.2
254.4 ± 37.1 231.7 ± 31.2 22.7 ± 5.9
215.6 ±49.4 181.3 ±42.7 34.3 ± 6.7
254.9 ± 13.2 220.6 ± 6.9 34.3 ± 6.6
1
Five chickens/group.
3
IFE = Interfollicular epithelium, FAE = follicle-associated epithelium.
Numerical density, number of FAE' areas/1 mm 3 bursal tissue Age of chickens 2 Sex
3 Months
6 Months
Males Females
,2± .01 .2 ± .04
.4 ± .01 .3 ±.01
Although in 6-month-old chickens, there was a significant main effect of age for follicular cortex, numerical density of FAE, surface density of FAE, and diameter of FAE, a difference in rate of bursal involution was observed between sexes. All changes (the decline of bur-
' FAE = Follicle-associated epithelium. 2
Five chickens/group.
TABLE 5. Epithelial height, diameter of FAE1 and follicles (X + SD) Age of chickens2 3 Months Males
6 Months Females
Males
Females
(Mm) Height of IFE 3 Height of FAE Diameter of FAE Diameter of follicles
28.1 83.6 98.9 1943.3
± 4.4 ± 9.2 ± 8.3 ±480.3
1
FAE = Follicle-associated epithelium.
2
Five chickens/group.
3
IFE = Interfollicular epithelium.
36.4 53.6 101.0 1870.0
± 8.1 ± 3.5 ± 7.2 ± 109.8
35.6 44.0 78.9 1523.3
± 7.7 ± 1.7 ± 1.7 ± 130.2
17.8 40.8 95.1 1766.6
± 5.5 ± 6.1 ± 4.7 ± 250.3
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did not reveal any signs of involution of the bursa in Prelux chickens at this age. In White Leghorns, the involution begins earlier, i.e., between 10 and 16 weeks of age (Naukkarinen and Sorvari, 1984). The development and involution of the bursa varies greatly in different chicken strains (Wolfe et al, 1962; HoffmannFezer and Lade, 1972; Glick, 1977).
TABLE 4. Numerical density (mm' of follicle-associated epithelium (X ± SD)
2322
MILICEVIC ETAL.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on March 23, 2015
ogy 68:944-949. Chang, T. S., B. Glick, and A. R. Winter, 1955. The significance of the bursa of Fabricius of chickens in antibody production. Poultry Sci. 34:1187— 1198. Cooper, M. D., R.D.A. Peterson, and R. A. Good, 1965. Delineation of the thymic and bursal lymphoid systems in the chicken. Nature 205: 143-146. Eskola, J., O. Ruuskanen, J. E. Fraki, M. K. Viljanen, and A. Toivanen, 1977. Alkaline phosphatase in the developing bursa of Fabricius. A comparative study of the cyclophosphamide and testosteroneinduced immunodeficiencies in the chick embryo. Scand. J. Immunol. 6:185—194. Glick, B., 1970. Immunity studies in testosterone propionate injected chicks. Int. Arch. Allergy Appl. Immunol. 38:93-103. Glick, B., 1977. The bursa of Fabricius and immunoglobulin synthesis. Int. Rev. Cytol. 48:345— 402. Glick, B., T. S. Chang, and R. G. Jaap, 1956. The bursa of Fabricius and antibody production. Poultry Sci. 35:224-225. Hoffmann-Fezer, G., and R. Lade, 1972. Postembryonal Entwicklung und Involution der Bursa Fabricii beim Haushuhn (Galus domesticus). Z. Zellforsch. Mikrosk. Anat. 124:406-418. jankovic, B. D., and K. Isakovic, 1964. Role of the thymus and the bursa of Fabricius in immune reactions in chickens. Int. Arch. Allergy Appl. Immunol. 24:278-295. Metcalf, D., andM.A.S. Moore, 1971. Development of the bursa of Fabricius. Pages 242—261 in Hematopoietic cells. A. Neuberger and E. L. Tatum, ed. North Holland Research Monographs, Frontier of Biology 24. Meyer, R. K., M. A. Rao, and E. L. Aspinall, 1959. Inhibition of the development of the bursa of Fabricius in the embryos of the common fowl by 19-nortestosterone. Endocrinology 64:890—897. Milicevic, Z., 1984. Interakcija imunog i endokrinog sistema u toku starenja. D.Sc. Thesis. University ACKNOWLE DGMENT of Beograd, Beograd, Yugoslavia. Naukkarinen, A., and T. E. Sorvari, 1980. Cellular This work was supported by grants from the transport of colloidal carbon in the follicleRepublic of Serbia Research Fund, Beograd, associated epithelium of the chicken bursa of Yugoslavia. Fabricius. J. Reticuloendothel. Soc. 2 8 : 4 7 3 482. Naukkarinen, A., and T. E. Sorvari, 1982. MorphoREFERENCES logical and histochemical characterization of the medullary cells in the bursal follicles of the Ackerman, G. A., 1962. Electron microscopy of the chicken. Acta Pathol. Microbiol. Scand. Sect. C bursa of Fabricius of the embryonic chick with Immunol. 90:193-199. particular reference to the lymphoepithelial Naukkarinen, A., and T. E. Sorvari, 1984. Involution nodules. J. Cell Biol. 13:127-146. of the chicken bursa of Fabricius: a light microAckerman, G. A., and R. A. Knouff, 1959. Lymphoscopic study with special reference to transport cytopoiesis in the bursa of Fabricius. Am. J. of colloidal carbon in the involuting bursa. J. Anat. 104:163-206. Leukocyte Biol. 35:281-290. Aramaki, T., 1968. Morphologic study of lymphatic Novotny, E. A., E. S. Raveche, S. Sharrows, M. tissues (XIII). Light and electron microscope obOttinger, and A. D. Steinberg, 1983. Analysis of servations on the bursa of Fabricius of the chick thymocyte subpopulations following treatment and Coturnix embryos with special reference to with sex hormones. Clin. Immunol. Immunothe lymphocytopoiesis. Keio J. Med. 17:135— pathol. 28:205-217. 155. Aspinall, R. L., R. K. Meyer, and M. A. Rao, 1961. Odend'hal, S., and J. E. Breazile, 1979. Diffusely infiltrated area of lymphoid cells in the cloacal bursa. Effect of various steroids on the development of J. Reticuloendothel. Soc. 25:315-324. the bursa Fabricii in chick embryos. Endocrinolsal mass, the reduction of volume density of follicular cortex and numerical density of FAE, the decreased height and diameter of FAE, and increased volume density of stroma) were more pronounced in males. In 6-month-old females, the most prominent changes were registered on bursal epithelium. These quantitative data substantiated previous morphological studies that demonstrated that the damage to the bursal epithelium was the earliest sign of involution and occurred before the alteration of the follicular structure (Hoffmann-Fezer and Lade, 1972; Vujic et al, 1979; Naukkarinen and Sorvari, 1984). The more advanced stage of bursal involution in males, in comparison with the females, could be attributed to the influence of sex hormones, as in vitro studies demonstrated that testosterone strongly inhibited, whereas estrogens did not inhibit, the proliferation of bursal cells (Novotny et al, 1983). Also, our quantitative histological studies revealed no signs of bursal involution in 6-month-old neonatally orchidectomized male chickens and advanced stage of bursal involution in neonatally ovariectomized females of the same age (Milicevic, 1984). However, the 2 x 2 factorial analysis of variance, used in this study for statistical analysis of all numerical data, showed that interactions between sex and age were not significant and that further studies are necessary to elucidate the mechanisms of bursal involution and influence of factors other than sex.
BURSAL INVOLUTION IN CHICKENS Romppanen, T., 1982. Postembryonic development of the chicken bursa of Fabricius: a light microscopic histoquantitative study. Poultry Sci. 61: 2261-2270. Sorvari, R., and T. E. Sorvari, 1977. Bursa Fabricii as a peripheral lymphoid organ. Transport of various materials from the anal lips to the bursal lymphoid follicles with reference to its immunological importance. Immunology 32:499—505. Vujic, D., Z. Rakic, K. Isakovic, and B. D. Jankovic,
2323
1979. Involution of the chicken thymus and bursa of Fabricius during aging. Period. Biol. 8 1 : 469-470. Weibel, E. R., 1979. Practical Methods for Biological Morphometry. Vol. 1. Academic Press, New York, NY. Wolfe, H. R., S. A. Sheridan, N. M. Bilstadt, and M. A. Johnson, 1962. The growth of the lymphoid organs and testes of the chicken. Anat. Rec. 142: 485-493.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on March 23, 2015
1986 Poultry Science 65:2318-2323
INTRODUCTION
In the past three decades, the function of the avian cloacal bursa has been studied in detail, and the central role of this organ in the development and maturation of B lymphocytes established (Chang et al, 1955; Glick et al, 1956; Jankovic and Isakovic, 1964; Cooper et al, 1965). More recent studies have demonstrated that the bursa also has immune functions similar to those of peripheral lymphoid organs (Sorvari and Sorvari, 1977; Odend'hal and Breazile, 1979; Naukkarinen and Sorvari, 1980, 1982). However, the histology of the bursa has not been studied so systematically. The embryonal development of this organ has been extensively analyzed by light and electron microscopy (Ackerman and Knouff, 1959; Ackerman, 1962; Aramaki, 1968; Metcalf and Moore, 1971; Eskola et al, 1977), but much less attention has been paid to the later stages of ontogenic development, especially to the process of bursal involution, which begins around 3.5 to 5 months of age (Glick, 1977). Only HoffmannFezer and Lade (1972) have studied this process in detail. The lack of precise quantitative studies, which could elucidate the involution of
1
Institute of Histology.
this complex organ, is still notable. The only modern stereological analysis of the chicken bursa concerns the postnatal development of this organ up to 84 days (Romppanen, 1982). It is well known that the sex steroids strongly influence bursal development and function (Meyer et al, 1959; Aspinall et al, 1961; Glick, 1970) and that the bursa begins to involute with the approach of sexual maturity (Hoffmann-Fezer and Lade, 1972; Glick, 1977). However, no data are available about the structural changes of involuting bursa with respect to sex. Therefore, the aim of our study was to analyze, using the modern stereological approach, the different tissue compartments of the involuting bursa in male and female chickens. MATERIALS AND METHODS Experimental Animals and Tissue Preparation, Newly hatched Prelux Bro chickens were obtained from a local hatchery. These chickens are a domestic, Yugoslavian selection, bred for the purpose of producing meat. They were given commercial feed and water ad libitum. At ages of 3 and 6 months, five male and five female chickens were sacrificed with an overdose of an intravenous anaesthetic into the wing vein. The chickens and their bursae were weighed. Bursae were fixed in Carnoy's fixative. After fixation, the bursa was cut in slices per-
2318
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on March 23, 2015
ABSTRACT A histoquantitative study of the bursa of Fabricius in 3- and 6-month-old male Prelux Bro chickens (domestic selection) was made. All compartments of normal bursal tissue were examined. At 3 months of age, there were no significant differences in bursal weight and structure between males and females. The only difference observed was the higher volume density of follicular cortex and the lower proportion of follicular medulla in males compared to females. In 6-month-old male and female chickens, a different degree of bursal involution was observed. In the bursa of males, an advanced stage of involution was observed, whereas only the initial signs of bursal involution were found in females. (Key words: bursa of Fabricius, involution, sex differences, stereological analysis)
BURSAL INVOLUTION IN CHICKENS
pendicular to the longitudinal axis, i.e., to the mucosal surface. Three sections taken from proximal, middle, and distal parts of the bursa were selected for morphometric measurements. The tissue was processed in paraffin, and sections were cut at 5-/im thickness and stained with hematoxylin-eosin. Morphometric Measurements. Histoquantitative study of the bursal tissue was done according to Romppanen (1982). The point-counting method was used (Weibel, 1979). Point hits on lymphoid follicles, follicular cortex, follicular medulla, stroma, and bursal epithelium were registered at a magnification of 160X using a square grid with 25 points. The number of point hits on the tissue compartments was registered, and the corresponding volume density was calculated. A total of 600 points were counted for each bursa (200 points/section). Weights (milligrams) of different bursal tissue compartments were calculated by multiplying the corresponding volume density by the bursal weight. Surface density (mm 2 /mm 3 ) of the bursal epithelium was calculated from the equation of Weibel (1979): S v = kI L (factor k = 77/2, and I I = number of intersections per unit length of the system of test lines). The bursal epithelial surface at the luminal side [interfollicular epithelium (IFE), follicle-associated epithelium (FAE)] was measured using a square grid with 9 points. The magnification was 160x. Number of FAE areas ( N V F A E ) ( m m - 3 ) was computed from the following equation (Weibel, 1979):
where N A = number of FAE profiles per unit area of sectional plane and D = the diameter of the FAE area in space determined according to the equation: ^
4
D = -77r d
where d = diameter of FAE area measured by linear measurements with a ruler at a magnification 400X. The mean height (h) (^m) of the epithelium (IFE and FAE) and the mean diameter of follicles (Df0i) (|izm) were calculated as stereologic derivatives according to the following equations: by where V v = corresponding volume density (IFE or FAE) and Sy = corresponding surface density (IFE or FAE) and D fol
¥«V
2 x
V
where Vyfoii = volume density of lymphoid follicles and N V F A E = numerical density of FAE areas. For the statistical analysis of numerical data, Student's t-test and a 2 X 2 factorial analysis of variance were used. RESULTS The most voluminous bursal tissue compartment in male and female chickens was the lymphatic tissue both in relative (Table 1) and absolute amounts (Table 2). There was no
NA
VFAE - D
TABLE 1. Volume densities (%) of bursal tissue compartments (X ± SD) Age of chickens 1 6 Months
3 Moii t h s Volume densities
Males
Follicles, c o r t e x + medulla Follicular c o r t e x Follicular m e d u l l a Stroma Epithelium, I F E + F A E 2 IFE FAE
88.2 51.2 36.9 9.7 4.9 3.6 1.2
± ± ± ± ± ± ±
7.1 1.4 1.7 1.0 1.1 1.5 .6
Females
Males
85.2 44.5 40.6 9.3 5.9 4.8 1.1
79.0 ± 42.7 ± 36.2 ± 12.8 ± 7.9 ± 6.4+ 1.4 ±
± ± ± ± ± ± ±
1.9 .7* 3.0* 1.5 1.8 1.7 .2
1
Five chickens/group.
2
IFE = Interfollicular epithelium, FAE = follicle-associated epithelium.
Females 3.2 1.1 3.2 4.3 2.4 2.0 .3
'Statistically significant difference between males and females of the same age (P<.05).
86.3 46.3 39.9 8.6 5.3 3.9 1.3
± ± ± ± ± ± ±
3.1* 3.1* 2.6 .8* 1.2 1.2 .6
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on March 23, 2015
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DISCUSSION
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The results demonstrated that there are no significant differences in bursal weight and minor changes in structure between 3-monthold male and female Prelux chickens. The only differences observed were the higher volume density of the follicular cortex and the lower proportion of follicular medulla in males compared with females. The stereological analysis
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on March 23, 2015
+1 +1 +1 +1 +1 +1 +' +1 +!
statistically significant difference in the bursal weight and weight of bursal tissue compartments between 3-month-old male and female chickens (Table 2). The value for volume density of follicular cortex was higher, and the proportion of follicular medulla was significantly lower (P<.05) in males compared to females (Table 1). There was no significant difference in the volume density of stroma and epithelium (IFE and FAE) between males and females (Table 1). However, the absolute amount of IFE was greater in females (Table 2). The difference in surface density (Table 3) of the bursal epithelium, as well as in the number of areas of follicle-associated epithelium (Table 4), was not statistically significant between males and females. The number of areas of follicle-associated epithelium in a mature bursa presumably corresponds to the follicle number (Romppanen, 1982). The epithelial height (IFE and FAE) and the diameter of FAE and follicles were similar in males and females (Table 5). The results of 2 X 2 factorial analysis of variance demonstrated that in 6-month-old chickens, there was a significant main effect of age (P<.05) for follicular cortex, numerical density of FAE, surface density of FAE, and diameter of FAE. However, 6-month-old males had significantly lower values for volume density of follicles and follicular cortex (Table 1) compared with females. The absolute amounts of bursal tissue, follicles, and follicular cortex were also significantly lower in 6-monthold males (Table 2). The volume density of bursal stroma was significantly higher in 6month-old males in comparison with females u Tj 0 crt (Table 1). There were no significant differences rt jy in the surface density of the bursal epithelium u s (Table 3), numerical density of follicle-assoO c <+* U ciated epithelium (Table 4), and diameter of V II * follicle-associated epithelium and follicles (U -• < < % (Table 5) between 6-month-old male and female chickens.
BURSAL INVOLUTION IN CHICKENS
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TABLE 3. Surface density (mm2/mm3 ) of the bursal epithelium (X ± SD) Age of chickens1 3 Months
6 Months
Surface density
Males
Females
Males
Females
Epithelium, IFE + FAE 2 IFE FAE
210.0 ± 44.2 189.6 ± 38.0 20.4 ± 6.2
254.4 ± 37.1 231.7 ± 31.2 22.7 ± 5.9
215.6 ±49.4 181.3 ±42.7 34.3 ± 6.7
254.9 ± 13.2 220.6 ± 6.9 34.3 ± 6.6
1
Five chickens/group.
3
IFE = Interfollicular epithelium, FAE = follicle-associated epithelium.
Numerical density, number of FAE' areas/1 mm 3 bursal tissue Age of chickens 2 Sex
3 Months
6 Months
Males Females
,2± .01 .2 ± .04
.4 ± .01 .3 ±.01
Although in 6-month-old chickens, there was a significant main effect of age for follicular cortex, numerical density of FAE, surface density of FAE, and diameter of FAE, a difference in rate of bursal involution was observed between sexes. All changes (the decline of bur-
' FAE = Follicle-associated epithelium. 2
Five chickens/group.
TABLE 5. Epithelial height, diameter of FAE1 and follicles (X + SD) Age of chickens2 3 Months Males
6 Months Females
Males
Females
(Mm) Height of IFE 3 Height of FAE Diameter of FAE Diameter of follicles
28.1 83.6 98.9 1943.3
± 4.4 ± 9.2 ± 8.3 ±480.3
1
FAE = Follicle-associated epithelium.
2
Five chickens/group.
3
IFE = Interfollicular epithelium.
36.4 53.6 101.0 1870.0
± 8.1 ± 3.5 ± 7.2 ± 109.8
35.6 44.0 78.9 1523.3
± 7.7 ± 1.7 ± 1.7 ± 130.2
17.8 40.8 95.1 1766.6
± 5.5 ± 6.1 ± 4.7 ± 250.3
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did not reveal any signs of involution of the bursa in Prelux chickens at this age. In White Leghorns, the involution begins earlier, i.e., between 10 and 16 weeks of age (Naukkarinen and Sorvari, 1984). The development and involution of the bursa varies greatly in different chicken strains (Wolfe et al, 1962; HoffmannFezer and Lade, 1972; Glick, 1977).
TABLE 4. Numerical density (mm' of follicle-associated epithelium (X ± SD)
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MILICEVIC ETAL.
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ogy 68:944-949. Chang, T. S., B. Glick, and A. R. Winter, 1955. The significance of the bursa of Fabricius of chickens in antibody production. Poultry Sci. 34:1187— 1198. Cooper, M. D., R.D.A. Peterson, and R. A. Good, 1965. Delineation of the thymic and bursal lymphoid systems in the chicken. Nature 205: 143-146. Eskola, J., O. Ruuskanen, J. E. Fraki, M. K. Viljanen, and A. Toivanen, 1977. Alkaline phosphatase in the developing bursa of Fabricius. A comparative study of the cyclophosphamide and testosteroneinduced immunodeficiencies in the chick embryo. Scand. J. Immunol. 6:185—194. Glick, B., 1970. Immunity studies in testosterone propionate injected chicks. Int. Arch. Allergy Appl. Immunol. 38:93-103. Glick, B., 1977. The bursa of Fabricius and immunoglobulin synthesis. Int. Rev. Cytol. 48:345— 402. Glick, B., T. S. Chang, and R. G. Jaap, 1956. The bursa of Fabricius and antibody production. Poultry Sci. 35:224-225. Hoffmann-Fezer, G., and R. Lade, 1972. Postembryonal Entwicklung und Involution der Bursa Fabricii beim Haushuhn (Galus domesticus). Z. Zellforsch. Mikrosk. Anat. 124:406-418. jankovic, B. D., and K. Isakovic, 1964. Role of the thymus and the bursa of Fabricius in immune reactions in chickens. Int. Arch. Allergy Appl. Immunol. 24:278-295. Metcalf, D., andM.A.S. Moore, 1971. Development of the bursa of Fabricius. Pages 242—261 in Hematopoietic cells. A. Neuberger and E. L. Tatum, ed. North Holland Research Monographs, Frontier of Biology 24. Meyer, R. K., M. A. Rao, and E. L. Aspinall, 1959. Inhibition of the development of the bursa of Fabricius in the embryos of the common fowl by 19-nortestosterone. Endocrinology 64:890—897. Milicevic, Z., 1984. Interakcija imunog i endokrinog sistema u toku starenja. D.Sc. Thesis. University ACKNOWLE DGMENT of Beograd, Beograd, Yugoslavia. Naukkarinen, A., and T. E. Sorvari, 1980. Cellular This work was supported by grants from the transport of colloidal carbon in the follicleRepublic of Serbia Research Fund, Beograd, associated epithelium of the chicken bursa of Yugoslavia. Fabricius. J. Reticuloendothel. Soc. 2 8 : 4 7 3 482. Naukkarinen, A., and T. E. Sorvari, 1982. MorphoREFERENCES logical and histochemical characterization of the medullary cells in the bursal follicles of the Ackerman, G. A., 1962. Electron microscopy of the chicken. Acta Pathol. Microbiol. Scand. Sect. C bursa of Fabricius of the embryonic chick with Immunol. 90:193-199. particular reference to the lymphoepithelial Naukkarinen, A., and T. E. Sorvari, 1984. Involution nodules. J. Cell Biol. 13:127-146. of the chicken bursa of Fabricius: a light microAckerman, G. A., and R. A. Knouff, 1959. Lymphoscopic study with special reference to transport cytopoiesis in the bursa of Fabricius. Am. J. of colloidal carbon in the involuting bursa. J. Anat. 104:163-206. Leukocyte Biol. 35:281-290. Aramaki, T., 1968. Morphologic study of lymphatic Novotny, E. A., E. S. Raveche, S. Sharrows, M. tissues (XIII). Light and electron microscope obOttinger, and A. D. Steinberg, 1983. Analysis of servations on the bursa of Fabricius of the chick thymocyte subpopulations following treatment and Coturnix embryos with special reference to with sex hormones. Clin. Immunol. Immunothe lymphocytopoiesis. Keio J. Med. 17:135— pathol. 28:205-217. 155. Aspinall, R. L., R. K. Meyer, and M. A. Rao, 1961. Odend'hal, S., and J. E. Breazile, 1979. Diffusely infiltrated area of lymphoid cells in the cloacal bursa. Effect of various steroids on the development of J. Reticuloendothel. Soc. 25:315-324. the bursa Fabricii in chick embryos. Endocrinolsal mass, the reduction of volume density of follicular cortex and numerical density of FAE, the decreased height and diameter of FAE, and increased volume density of stroma) were more pronounced in males. In 6-month-old females, the most prominent changes were registered on bursal epithelium. These quantitative data substantiated previous morphological studies that demonstrated that the damage to the bursal epithelium was the earliest sign of involution and occurred before the alteration of the follicular structure (Hoffmann-Fezer and Lade, 1972; Vujic et al, 1979; Naukkarinen and Sorvari, 1984). The more advanced stage of bursal involution in males, in comparison with the females, could be attributed to the influence of sex hormones, as in vitro studies demonstrated that testosterone strongly inhibited, whereas estrogens did not inhibit, the proliferation of bursal cells (Novotny et al, 1983). Also, our quantitative histological studies revealed no signs of bursal involution in 6-month-old neonatally orchidectomized male chickens and advanced stage of bursal involution in neonatally ovariectomized females of the same age (Milicevic, 1984). However, the 2 x 2 factorial analysis of variance, used in this study for statistical analysis of all numerical data, showed that interactions between sex and age were not significant and that further studies are necessary to elucidate the mechanisms of bursal involution and influence of factors other than sex.
BURSAL INVOLUTION IN CHICKENS Romppanen, T., 1982. Postembryonic development of the chicken bursa of Fabricius: a light microscopic histoquantitative study. Poultry Sci. 61: 2261-2270. Sorvari, R., and T. E. Sorvari, 1977. Bursa Fabricii as a peripheral lymphoid organ. Transport of various materials from the anal lips to the bursal lymphoid follicles with reference to its immunological importance. Immunology 32:499—505. Vujic, D., Z. Rakic, K. Isakovic, and B. D. Jankovic,
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1979. Involution of the chicken thymus and bursa of Fabricius during aging. Period. Biol. 8 1 : 469-470. Weibel, E. R., 1979. Practical Methods for Biological Morphometry. Vol. 1. Academic Press, New York, NY. Wolfe, H. R., S. A. Sheridan, N. M. Bilstadt, and M. A. Johnson, 1962. The growth of the lymphoid organs and testes of the chicken. Anat. Rec. 142: 485-493.
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