Research in Veterinary Science 1991, 50, 216-221
Effects of vitamin or mineral deficiency on the morphology of medullary bone in laying hens S. WILSON, S. R. I. DUFF*, Agricultural and Food Research Council Institute of Animal Physiology and Genetics Research, Roslin, Midlothian EH25 9PS
Adult laying hens were fed diets deficient in phosphorus, calcium, calcium and phosphorus, and vitamin D 3 to determine their effects on bone histology and parathyroid gland size. The phosphorus deficient diet caused an insignificant decrease in parathyroid size while the other diets caused significant increases. A considerable amount of individual variation in medullary bone volume and osteoid seam width was observed in all groups but, despite this, the calcium, calcium and phosphorus and vitamin D 3 deficient diets clearly resulted in increased osteoid. Birds receiving diets deficient in calcium and phosphorus, and in vitamin D 3 for longer periods were observed to have partially or completely resorbed medullary bone. Osteodystrophia fibrosa was noted in vitamin D 3 deficient birds which had no follicular activity.
demonstrated increased osteoid in the MB. However, this observation was not made when hens were fed a more severely phosphorus deficient diet for a shorter time (Antillon et al 1977). Bloom et al (1958) reported that in decalcified and undecalcified sections of bone from hens fed a diet deficient in calcium, the MB matrix was composed mainly of osteoid. This increase in the amount of osteoid has also been described by Zambon]n Zallone and Mueller (1969), Antillon et al (1977) and de Bernard et al (1980). Antillon et al (1977) also investigated the effects of a diet deficient in vitamin D 3 and reported the presence of wide osteoid seams and eventually the virtual absence of any MB. As well as adversely affecting bone, diets deficient in calcium or vitamin D 3 cause enlargement of the parathyroid glands (Taylor 1965, Antillon et a11977). The purpose of the present investigation was to determine the effects of deficiencies in calcium, phosphorus and vitamin D 3 on bone histology and parathyroid gland size. The results of this investigation may therefore assist in the evaluation of field material, such as that reported by Randall and Duff (1988).
THE presence of medullary bone (MB) composed of thick seams of unmineralised or poorly mineralised matrix was observed in an osteopenic hen examined by Randall and Duff (1988). The authors suggested that this histological appearance had arisen either as a result of diurnal variation in mineralisation related to the eggqaying cycle or a mineralisation defect due to Materials and methods vitamin or mineral deficiency. A subsequent study (Wilson and Duff 1990) concluded that although Hens of a modern commercial egg-laying strain, diurnal variation in mineralisation did occur in MB, aged between 36 and 44 weeks, were divided into detection of such changes required the use of the more groups of eight individuals, and fed diets as indicated sensitive technique of fluorochrome bone labelling, in Table 1. The duration of experimental diets was and indeed could not be detected by the routine de- determined by the length of time taken for the calcified and undecalcified histological techniques deficiency to affect egg production. The phosphorus deficient diet did not affect egg production and was employed by Randall and Duff (1988). It is well known that vitamin and mineral continued for 21 days. deficiencies induce osteopenia in laying hens. A diet Birds were killed by an overdose of barbiturate deficient in .phosphorus has been shown to cause a administered intravenously, either three hours or 13 reduction in MB mineral in experiments using, respec- hours after oviposition, so that a representative tively, femur density techniques and mineral com- sample of both the active and inactive periods of shellposition of bone ash (Bar and Hurwitz 1983, Garlich formation was achieved. This was confirmed by et al 1988). Riddell et al (1969), in a histological establishing the position of the next egg in the reproexamination of MB from phosphorus deficient hens, ductive tract. In addition to the eight birds in each group described previously, several hens fed for a longer period diets deficient in calcium and phosphorus, and vitamin D 3 which had apparently ceased *Present address: PDSA Northern Regional Centre, 131 Chester egg production, were also killed. These groups were Road, Sunderland 216
Medullary bone in laying hens
217
TABLE 1 : Composition and duration of diets Diet Control Phosphorus deficient Calcium deficient Calcium + phosphorus deficient Vitamin D3 deficient
Total calcium (%)
Total phosphorus (%)
Vitamin D3 (iu kg-1)
Duration (days)
3" 39 3- 39 O. 46
O- 63 O. 40 O. 63
2000 2000 2000
-21 10
O" 47 3.56
O" 40 O. 65
2000 0
7 12
diameters decreased in phosphorus deficient hens, but not significantly so. All other treatments caused significant increases.
fed the deficient diet for two to three weeks and three to six weeks, respectively. Birds were weighed before and after the experiment. The parathyroid glands were removed, fixed in buffered neutral formalin and processed to paraffin wax. Interrupted serial sections were cut and the largest section measured using image analysis (Seescan Solitaire Plus; Seesean Imaging). The proximal extremities of the right femur and tarsometatarsus, and the proximal halves of phalanx 1 of digitus tertius (middle toe) and the proximal phalanx of digitus maj or were collected. Samples of the mid-diaphysis of the femur and tibiotarsus were also collected, and all specimens were fixed in buffered neutral formalin before decalcification in formic acid and formalin. Paraffin embedded sections were prepared and stained with haematoxylin and eosin. The corresponding tissues from the left appendicular skeleton, plus the notarium and free thoracic vertebra, were collected, fixed in 70 per cent alcohol and embedded in plastic for preparation of undecalcified, 6 /zm sections (Mawhinney and Ellis 1983). Undecalcified sections were stained with Von Kossa with neutral red counterstain.
Histopathology Group 1 (controls). Decalcified histological preparations showed that half the birds sampled had slender metaphyseal trabeculae, and were therefore judged to be osteoporotic. The MB in all cases was uniformly basophilic (Fig 1). Its volume, however, was highly variable between individuals. The general appearance was considered normal, with the exception of one bird in which the presence of occasional areas of aeellular MB was noted. Examination of undecalcified sections from the same birds revealed a remarkable degree of variation in the volume of osteoid present in MB; some birds had no apparent osteoid (Fig 2), while others had obvious but fine seams (Fig 3). Group 2 (phosphorus deficient). All eight birds were judged to be osteoporotic from decalcified sections. The general appearance was similar to the control group, although three individuals were seen to have acellular MB, which was more obvious and widespread than that seen in the control birds (Fig 4). Undecalcified sections showed a similar degree of variation to the controls in the amount of osteoid present.
Results
All birds killed 13 hours after oviposition were found to have a calcified egg in the shell gland, while those killed three hours after oviposition had an uncalcified egg in the shell gland or magnum. Bodyweights and parathyroid diameters are shown in Table 2. Bodyweights were not significantly different from controls in any treatment group. Parathyroid
Group 3 (calcium deficient). Seven birds out of eight were judged osteoporotic from decalcified sections. The MB was eosinophilic and highly variable in volume. Bone trabeculae were composed of
TABLE 2: Mean bodyweights and parathyroid gland diameters ( ± SD) Control Mean bodyweight before experiment(g) Mean bodyweight after experiment(g) Mean parathyroid gland diameter (mm)
2148.72 (± 178-99) 2" 004 ( ± 0.280)
P
C
C+ P
Vitamin D3
2159' OO ( ± 173-83)
2153.70 (± 162.20)
2136' 12 (± 212.86)
2163- 27 (± 180.32)
2180.00 ( ± 174.98)
2132- 92 (± 158-78)
2127- 99 (± 225.18)
2162.09 (± 194.17)
1. 974 (±0.260)
2" 511 (±0.390)
Diets were deficient in: P Phosphorus, C Calcium, C +P Calcium and phosphorus, and vitamin D3
2" 540 (=~0.440)
2" 916 (±0.300)
218
S. Wilson, S. R. L Duff
FIG 1: Photomicrograph of proximal femur from control bird. Trabecula (T) and medullary bone trabeculae (arrows). Decalcified section. Haematoxylin and eosin x 95
FIG 2: Photomicrograph of proximal femur from control bird. Scarce medullary bone osteoid (arrows). Undecalcified section. Von Kossa x 95
lamellae of eosinophilic matrix with an intervening layer of fairly basophilic matrix (Fig 5). The axial core of eosinophilic bone was considered to be trabecular bone whereas the other two lamellae constituted MB. Undecalcified sections confirmed the presence of thick osteoid seams lining the trabecular bone, and in some cases showed the MB to be composed almost
entirely of osteoid (Fig 6). The amount of osteoid was, however, extremely variable, those birds with least osteoid having a similar appearance to the control birds.
FIG 3: Photomicrograph of proximal femur from control bird. Moderate osteoid (arrows). Undecalcified section. Von Kossa x95
FIG 4: Photomicrograph of proximal femur. Phosphorus deficient bird. Acellular medullary bone with pyknotic nuclei. Decalcified section. Haematoxylin and eosin x 250
Group 4 (calcium and phosphorus deficient). Six birds out of eight were osteoporotic. The appearance
Medullary bone in laying hens
219
FIG 5: Photomicrograph of proximal femur. Calcium deficient bird. Trabecular bone (TB), medullary bone (MB). Decalcified section. Haematoxylin and eosin x 95
FIG 6: Photomicrograph of proximal femur. Calcium deficient bird. Thick seams of osteoid lining trabecular bone (TB). Medullary bone (MB) composed almost entirely of osteoid. Undecalcified section. Von Kossa x 95
of this group in decalcified sections was similar to group 3, with the same variability in the volume of MB. Areas of acellularity in the MB were noted in two individuals, but not to the extent seen in group 2. Undecalcified sections confirmed the presence of osteoid seams on the trabecular bone, and demonstrated the variability of MB composition. Fig 7 shows
MB composed almost entirely of osteoid, while Fig 8 shows MB from a different bird, with little osteoid. The majority of birds in the group, however, were similar in appearance to that seen in Fig 7, Those birds which were fed the deficient diet for longer and which appeared to have ceased laying could be split into two groups; those which were
r
#
FIG 7: Photomicrograph of proximal femur. Calcium and phosphorus deficient bird. Medullary bone (MB) composed mainly of osteoid. Undecalcified section. Von Kossa x 95
FIG 8: Photomicrograph of proximal femur. Calcium and phosphorus deficient bird. Medullary bone (MB) with little osteoid. Undecalcified section. Von Kossa x 95
220
S. Wilson, S. R. L D u f f
FIG 9: Photomicrograph of proximal femur. Vitamin D3 deficient bird. Trabecular bone (TB) with thick osteoid seams. Undecalcified section. Von Kossa x 95
FIG 10: Photomicrograph of proximal femur. Vitamin D3 deficient bird. Osteodystrophia fibrosa (arrows). Decalcified section. Martius scarlet blue x 95
found to have follicular activity (diet duration two weeks) and those which had none (diet duraffon three weeks). The former group were observed to have a much reduced volume of MB, composed mainly of osteoid, while the latter had no MB. Both groups had trabecular bone lined with thick seams of osteoid.
to have an unusual capacity to exhibit hyperplasia and hypertrophy in response to deficiency of ultraviolet light or dietary vitamin D 3 or calcium (Ringer and Meyer 1976). Parathyroid hypertrophy in response to calcium deficiency occurs more rapidly in birds than in other vertebrates (Urist 1967) and has been reported frequently (Taylor 1965, Mueller et al 1970, Antillon et al 1977). The deficient diets in the present study caused significant increases in parathyroid gland size, with the exception of the phosphorus deficient diet which resulted in an insignificant decrease in size. Shane et al (1969) reported parathyroid gland atrophy in growing pullets fed a diet similar in composition to that used in the present study. In all treatments, MB volume was observed to be extremely variable between individuals. Such variation is an observation which has been reported elsewhere (Bloom et al 1958). A similar degree of variation in osteoid seam width was observed by Taylor and Belanger (1969) and Wilson and Duff (1990) and was also noted in the present study. Since half the birds in each group were killed three hours and the remainder 13 hours after oviposition, and the variations observed did not correspond to these times, it is unlikely that the variation is a reflection of different stages of shell production. However, the group sizes were small, and a study using more birds and quantitative histomorphometry would be required to clarify the issue. Despite the individual variation in osteoid seam width, it was clear that those groups receiving calcium-, calcium and phosphorus-, and vitamin
Group 5 (vitamin D 3 deficient). Seven birds out of eight were osteoporotic. The appearance in decalcified sections was similar to that of groups 3 and 4. Undecalcified sections showed all the birds in the group to have MB composed mainly of osteoid, and trabecular bone lined with thick seams of osteoid (Fig 9). There was considerable individual variation in the amount of MB present. The birds fed the deficient diet for a longer period could be split as in group 4 (diet duration three weeks in those with follicular activity, and six weeks in those with none), and were found to have a similar appearance to the previous group in undecalcified sections. Decalcified sections revealed that all birds which had no follicular activity showed signs of osteodystrophia fibrosa; resorbed bone was replaced by fibrous tissue (Fig 10).
Discussion
The regulation of, parathyroid hormone release is mediated by circulating plasma calcium levels and one of the main targets of parathyroid hormone are bone cells responsible for mobilisation of skeletal calcium. The parathyroid glands of birds have been observed
Medullary bone in laying hens D3-deficient diets had thicker osteoid seams on both medullary and trabecular bone than control birds. Increased osteoid seam width, representing a mineralisation failure of newly formed bone matrix, is characteristic of osteomalacia and has been reported by various authors (Bloom et al 1958, Zambonin Zallone and Mueller 1969,~ Antillon et al 1977, de Bernard et al 1980). The phosphorus deficient diet did not result in a mineralisation defect, in agreement with Antillon et al (1977). The phosphorus deficient diet used by Riddell et al (1969), although of a similar content to that used in the present study, was fed for 40 weeks. This may explain the increased osteoid present in that experiment. Of the birds receiving diets deficient in calcium and phosphorus or vitamin D 3 for longer, those' which had some follicular activity were observed to have a reduced volume o f MB, while in those with no follicular activity the MB was completely resorbed. MB is formed approximately 14 days before the point of lay by the synergistic action of oestrogens and androgens and under normal conditions of dietary calcium deficiency, ovulation ceases and the MB is resorbed. However, Luck (1978) reported that in hens fed a very low calcium diet, poorly mineralised MB persisted after nine weeks, even though laying ceased after one week. Osteodystrophia fibrosa is occasionally superimposed on osteomalacia, and such exuberant growth of loose fibrous tissue in the bones is usually associated with hyperparathyroidism and can be provoked nutritionally by diets deficient in calcium (Jubb and Kennedy 1963). Only those birds on the vitamin D 3 diet for an extended time (approximately six weeks) showed signs of this disorder, and these were the birds with the largest parathyroid glands. Osteodystrophia fibrosa may therefore be a consequence only of severe, prolonged exposure to deficiency in laying hens. In conclusion, the histological appearance of decalcified and undecalcified sections of bone from laying hens subjected to a variety of nutritional deficiencies has been established, and will assist in the evaluation of field material.
221
References ANTILLON, A., SCOTT, M. L., KROOK, L. & WASSERMAN, R. (1977) Metabolic response of laying hens to different dietary levels of calcium, phosphorus, and vitamin D 3. Cornell Veterinarian 67, 413-444 BAR, A. & HURWlTZ, S. (1983) Egg shell quality, medullary bone ash, intestinal calcium and phosphorus absorption, and calciumbinding protein in phosphorus deficient hens. Poultry Science 63, 1975-1979 BLOOM, M. A., DOMMN, L. V., NALBANDOV, A. V. & BLOOM, W. (1958) Medullary bone of laying chickens. American Journal of Anatomy 102, 411-453 DE BERNARD, B., STAGNI, N., CAMEROTTO, R., VITTUR, F., ZANETTI, M., ZAMBONIN ZALLONE, A. & TETI, A. (1980) Influence of calcium depletion on medullary bone of laying hens. Calcified Tissue International 32,221-228 GARLICH, J., MORRIS, C. & BRAKE, J. (1988) External bone volume, ash and fat-free dry weight of femurs of laying hens fed diets deficient or adequate in phosphorus. Poultry Science 61, 1003- 1006 JUBB, K. V. F. & KENNEDY, P. C. (1963) Pathology of Domestic Animals. London and New York, Academic Press. p 18 LUCK, M. R. (1978) PhD thesis, University of Leeds MAWHINNEY, W. H. B. & ELLIS, H. A. (1983) A technique for plastic embedding of mineralised bone. Journal of Clinical Pathology 36, 1197- 1199 MUELLER, G. L., ANAST, C. S. & BRE1TENBACK, R. P. (1970) Dietary calcium and ultimobranchial body and parathyroid gland in the chicken. American Journal o f Physiology 218, 1718-1722 RANDALL, C. J. & DUFF, S. R. I. (1988) Avulsion of the patellar ligament in osteopenic laying fowl. Veterinary Record 123, 439-441 RIDDELL, C., HELMBOLTD, C. F. & SINGSEN, E. P. (1969) A histologic study of medullary bone of laying hens under different diet and housing conditions. Avian Diseases 13, 163-165 RINGER, R. K. & MEYER, D. C. (1976)Avian Physiology. Berlin, Springer Verlag. p 360 SHANE, S. M., YOUNG, R. J. & DROOK, L. (1969) Renal and parathyroid changes produced by high calcium intake in growing pullets. Avian Diseases 13,558-561 TAYLOR, T. G. (1965) Calcium endocrine--relationships in the laying hen. Proceedings of the Nutrition Society 24, 49-54 TAYLOR, T. G. & BELANGER, L. F. (1969) The mechanisms of bone resorption in laying hens. Calcified Tissue Research 4, 162-173 URIST, M. R. (1967) Avian parathyroid physiology: including a special comment on calcitonin. American Journal of Zoology 7, 885-889 WILSON, S. & DUFF, S. R. I. (1990) Morphology of medullary bone during the egg-formation cycle. Research in Veterinary Science 48, 216-220 ZAMBONIN ZALLONE, A. & MUELLER, W. J. (1969) Medullary bone of laying hens during calcium depletion and repletion. Calcified Tissue Research 4, 136-146
Acknowledgements Thanks are due to Irene Anderson, Laura Dick and Michael Lynch for section preparation and to Dr W. Dewar for diet formulation.
Received July 19, 1990 Accepted October 9, 1990
Effects of vitamin or mineral deficiency on the morphology of medullary bone in laying hens S. WILSON, S. R. I. DUFF*, Agricultural and Food Research Council Institute of Animal Physiology and Genetics Research, Roslin, Midlothian EH25 9PS
Adult laying hens were fed diets deficient in phosphorus, calcium, calcium and phosphorus, and vitamin D 3 to determine their effects on bone histology and parathyroid gland size. The phosphorus deficient diet caused an insignificant decrease in parathyroid size while the other diets caused significant increases. A considerable amount of individual variation in medullary bone volume and osteoid seam width was observed in all groups but, despite this, the calcium, calcium and phosphorus and vitamin D 3 deficient diets clearly resulted in increased osteoid. Birds receiving diets deficient in calcium and phosphorus, and in vitamin D 3 for longer periods were observed to have partially or completely resorbed medullary bone. Osteodystrophia fibrosa was noted in vitamin D 3 deficient birds which had no follicular activity.
demonstrated increased osteoid in the MB. However, this observation was not made when hens were fed a more severely phosphorus deficient diet for a shorter time (Antillon et al 1977). Bloom et al (1958) reported that in decalcified and undecalcified sections of bone from hens fed a diet deficient in calcium, the MB matrix was composed mainly of osteoid. This increase in the amount of osteoid has also been described by Zambon]n Zallone and Mueller (1969), Antillon et al (1977) and de Bernard et al (1980). Antillon et al (1977) also investigated the effects of a diet deficient in vitamin D 3 and reported the presence of wide osteoid seams and eventually the virtual absence of any MB. As well as adversely affecting bone, diets deficient in calcium or vitamin D 3 cause enlargement of the parathyroid glands (Taylor 1965, Antillon et a11977). The purpose of the present investigation was to determine the effects of deficiencies in calcium, phosphorus and vitamin D 3 on bone histology and parathyroid gland size. The results of this investigation may therefore assist in the evaluation of field material, such as that reported by Randall and Duff (1988).
THE presence of medullary bone (MB) composed of thick seams of unmineralised or poorly mineralised matrix was observed in an osteopenic hen examined by Randall and Duff (1988). The authors suggested that this histological appearance had arisen either as a result of diurnal variation in mineralisation related to the eggqaying cycle or a mineralisation defect due to Materials and methods vitamin or mineral deficiency. A subsequent study (Wilson and Duff 1990) concluded that although Hens of a modern commercial egg-laying strain, diurnal variation in mineralisation did occur in MB, aged between 36 and 44 weeks, were divided into detection of such changes required the use of the more groups of eight individuals, and fed diets as indicated sensitive technique of fluorochrome bone labelling, in Table 1. The duration of experimental diets was and indeed could not be detected by the routine de- determined by the length of time taken for the calcified and undecalcified histological techniques deficiency to affect egg production. The phosphorus deficient diet did not affect egg production and was employed by Randall and Duff (1988). It is well known that vitamin and mineral continued for 21 days. deficiencies induce osteopenia in laying hens. A diet Birds were killed by an overdose of barbiturate deficient in .phosphorus has been shown to cause a administered intravenously, either three hours or 13 reduction in MB mineral in experiments using, respec- hours after oviposition, so that a representative tively, femur density techniques and mineral com- sample of both the active and inactive periods of shellposition of bone ash (Bar and Hurwitz 1983, Garlich formation was achieved. This was confirmed by et al 1988). Riddell et al (1969), in a histological establishing the position of the next egg in the reproexamination of MB from phosphorus deficient hens, ductive tract. In addition to the eight birds in each group described previously, several hens fed for a longer period diets deficient in calcium and phosphorus, and vitamin D 3 which had apparently ceased *Present address: PDSA Northern Regional Centre, 131 Chester egg production, were also killed. These groups were Road, Sunderland 216
Medullary bone in laying hens
217
TABLE 1 : Composition and duration of diets Diet Control Phosphorus deficient Calcium deficient Calcium + phosphorus deficient Vitamin D3 deficient
Total calcium (%)
Total phosphorus (%)
Vitamin D3 (iu kg-1)
Duration (days)
3" 39 3- 39 O. 46
O- 63 O. 40 O. 63
2000 2000 2000
-21 10
O" 47 3.56
O" 40 O. 65
2000 0
7 12
diameters decreased in phosphorus deficient hens, but not significantly so. All other treatments caused significant increases.
fed the deficient diet for two to three weeks and three to six weeks, respectively. Birds were weighed before and after the experiment. The parathyroid glands were removed, fixed in buffered neutral formalin and processed to paraffin wax. Interrupted serial sections were cut and the largest section measured using image analysis (Seescan Solitaire Plus; Seesean Imaging). The proximal extremities of the right femur and tarsometatarsus, and the proximal halves of phalanx 1 of digitus tertius (middle toe) and the proximal phalanx of digitus maj or were collected. Samples of the mid-diaphysis of the femur and tibiotarsus were also collected, and all specimens were fixed in buffered neutral formalin before decalcification in formic acid and formalin. Paraffin embedded sections were prepared and stained with haematoxylin and eosin. The corresponding tissues from the left appendicular skeleton, plus the notarium and free thoracic vertebra, were collected, fixed in 70 per cent alcohol and embedded in plastic for preparation of undecalcified, 6 /zm sections (Mawhinney and Ellis 1983). Undecalcified sections were stained with Von Kossa with neutral red counterstain.
Histopathology Group 1 (controls). Decalcified histological preparations showed that half the birds sampled had slender metaphyseal trabeculae, and were therefore judged to be osteoporotic. The MB in all cases was uniformly basophilic (Fig 1). Its volume, however, was highly variable between individuals. The general appearance was considered normal, with the exception of one bird in which the presence of occasional areas of aeellular MB was noted. Examination of undecalcified sections from the same birds revealed a remarkable degree of variation in the volume of osteoid present in MB; some birds had no apparent osteoid (Fig 2), while others had obvious but fine seams (Fig 3). Group 2 (phosphorus deficient). All eight birds were judged to be osteoporotic from decalcified sections. The general appearance was similar to the control group, although three individuals were seen to have acellular MB, which was more obvious and widespread than that seen in the control birds (Fig 4). Undecalcified sections showed a similar degree of variation to the controls in the amount of osteoid present.
Results
All birds killed 13 hours after oviposition were found to have a calcified egg in the shell gland, while those killed three hours after oviposition had an uncalcified egg in the shell gland or magnum. Bodyweights and parathyroid diameters are shown in Table 2. Bodyweights were not significantly different from controls in any treatment group. Parathyroid
Group 3 (calcium deficient). Seven birds out of eight were judged osteoporotic from decalcified sections. The MB was eosinophilic and highly variable in volume. Bone trabeculae were composed of
TABLE 2: Mean bodyweights and parathyroid gland diameters ( ± SD) Control Mean bodyweight before experiment(g) Mean bodyweight after experiment(g) Mean parathyroid gland diameter (mm)
2148.72 (± 178-99) 2" 004 ( ± 0.280)
P
C
C+ P
Vitamin D3
2159' OO ( ± 173-83)
2153.70 (± 162.20)
2136' 12 (± 212.86)
2163- 27 (± 180.32)
2180.00 ( ± 174.98)
2132- 92 (± 158-78)
2127- 99 (± 225.18)
2162.09 (± 194.17)
1. 974 (±0.260)
2" 511 (±0.390)
Diets were deficient in: P Phosphorus, C Calcium, C +P Calcium and phosphorus, and vitamin D3
2" 540 (=~0.440)
2" 916 (±0.300)
218
S. Wilson, S. R. L Duff
FIG 1: Photomicrograph of proximal femur from control bird. Trabecula (T) and medullary bone trabeculae (arrows). Decalcified section. Haematoxylin and eosin x 95
FIG 2: Photomicrograph of proximal femur from control bird. Scarce medullary bone osteoid (arrows). Undecalcified section. Von Kossa x 95
lamellae of eosinophilic matrix with an intervening layer of fairly basophilic matrix (Fig 5). The axial core of eosinophilic bone was considered to be trabecular bone whereas the other two lamellae constituted MB. Undecalcified sections confirmed the presence of thick osteoid seams lining the trabecular bone, and in some cases showed the MB to be composed almost
entirely of osteoid (Fig 6). The amount of osteoid was, however, extremely variable, those birds with least osteoid having a similar appearance to the control birds.
FIG 3: Photomicrograph of proximal femur from control bird. Moderate osteoid (arrows). Undecalcified section. Von Kossa x95
FIG 4: Photomicrograph of proximal femur. Phosphorus deficient bird. Acellular medullary bone with pyknotic nuclei. Decalcified section. Haematoxylin and eosin x 250
Group 4 (calcium and phosphorus deficient). Six birds out of eight were osteoporotic. The appearance
Medullary bone in laying hens
219
FIG 5: Photomicrograph of proximal femur. Calcium deficient bird. Trabecular bone (TB), medullary bone (MB). Decalcified section. Haematoxylin and eosin x 95
FIG 6: Photomicrograph of proximal femur. Calcium deficient bird. Thick seams of osteoid lining trabecular bone (TB). Medullary bone (MB) composed almost entirely of osteoid. Undecalcified section. Von Kossa x 95
of this group in decalcified sections was similar to group 3, with the same variability in the volume of MB. Areas of acellularity in the MB were noted in two individuals, but not to the extent seen in group 2. Undecalcified sections confirmed the presence of osteoid seams on the trabecular bone, and demonstrated the variability of MB composition. Fig 7 shows
MB composed almost entirely of osteoid, while Fig 8 shows MB from a different bird, with little osteoid. The majority of birds in the group, however, were similar in appearance to that seen in Fig 7, Those birds which were fed the deficient diet for longer and which appeared to have ceased laying could be split into two groups; those which were
r
#
FIG 7: Photomicrograph of proximal femur. Calcium and phosphorus deficient bird. Medullary bone (MB) composed mainly of osteoid. Undecalcified section. Von Kossa x 95
FIG 8: Photomicrograph of proximal femur. Calcium and phosphorus deficient bird. Medullary bone (MB) with little osteoid. Undecalcified section. Von Kossa x 95
220
S. Wilson, S. R. L D u f f
FIG 9: Photomicrograph of proximal femur. Vitamin D3 deficient bird. Trabecular bone (TB) with thick osteoid seams. Undecalcified section. Von Kossa x 95
FIG 10: Photomicrograph of proximal femur. Vitamin D3 deficient bird. Osteodystrophia fibrosa (arrows). Decalcified section. Martius scarlet blue x 95
found to have follicular activity (diet duration two weeks) and those which had none (diet duraffon three weeks). The former group were observed to have a much reduced volume of MB, composed mainly of osteoid, while the latter had no MB. Both groups had trabecular bone lined with thick seams of osteoid.
to have an unusual capacity to exhibit hyperplasia and hypertrophy in response to deficiency of ultraviolet light or dietary vitamin D 3 or calcium (Ringer and Meyer 1976). Parathyroid hypertrophy in response to calcium deficiency occurs more rapidly in birds than in other vertebrates (Urist 1967) and has been reported frequently (Taylor 1965, Mueller et al 1970, Antillon et al 1977). The deficient diets in the present study caused significant increases in parathyroid gland size, with the exception of the phosphorus deficient diet which resulted in an insignificant decrease in size. Shane et al (1969) reported parathyroid gland atrophy in growing pullets fed a diet similar in composition to that used in the present study. In all treatments, MB volume was observed to be extremely variable between individuals. Such variation is an observation which has been reported elsewhere (Bloom et al 1958). A similar degree of variation in osteoid seam width was observed by Taylor and Belanger (1969) and Wilson and Duff (1990) and was also noted in the present study. Since half the birds in each group were killed three hours and the remainder 13 hours after oviposition, and the variations observed did not correspond to these times, it is unlikely that the variation is a reflection of different stages of shell production. However, the group sizes were small, and a study using more birds and quantitative histomorphometry would be required to clarify the issue. Despite the individual variation in osteoid seam width, it was clear that those groups receiving calcium-, calcium and phosphorus-, and vitamin
Group 5 (vitamin D 3 deficient). Seven birds out of eight were osteoporotic. The appearance in decalcified sections was similar to that of groups 3 and 4. Undecalcified sections showed all the birds in the group to have MB composed mainly of osteoid, and trabecular bone lined with thick seams of osteoid (Fig 9). There was considerable individual variation in the amount of MB present. The birds fed the deficient diet for a longer period could be split as in group 4 (diet duration three weeks in those with follicular activity, and six weeks in those with none), and were found to have a similar appearance to the previous group in undecalcified sections. Decalcified sections revealed that all birds which had no follicular activity showed signs of osteodystrophia fibrosa; resorbed bone was replaced by fibrous tissue (Fig 10).
Discussion
The regulation of, parathyroid hormone release is mediated by circulating plasma calcium levels and one of the main targets of parathyroid hormone are bone cells responsible for mobilisation of skeletal calcium. The parathyroid glands of birds have been observed
Medullary bone in laying hens D3-deficient diets had thicker osteoid seams on both medullary and trabecular bone than control birds. Increased osteoid seam width, representing a mineralisation failure of newly formed bone matrix, is characteristic of osteomalacia and has been reported by various authors (Bloom et al 1958, Zambonin Zallone and Mueller 1969,~ Antillon et al 1977, de Bernard et al 1980). The phosphorus deficient diet did not result in a mineralisation defect, in agreement with Antillon et al (1977). The phosphorus deficient diet used by Riddell et al (1969), although of a similar content to that used in the present study, was fed for 40 weeks. This may explain the increased osteoid present in that experiment. Of the birds receiving diets deficient in calcium and phosphorus or vitamin D 3 for longer, those' which had some follicular activity were observed to have a reduced volume o f MB, while in those with no follicular activity the MB was completely resorbed. MB is formed approximately 14 days before the point of lay by the synergistic action of oestrogens and androgens and under normal conditions of dietary calcium deficiency, ovulation ceases and the MB is resorbed. However, Luck (1978) reported that in hens fed a very low calcium diet, poorly mineralised MB persisted after nine weeks, even though laying ceased after one week. Osteodystrophia fibrosa is occasionally superimposed on osteomalacia, and such exuberant growth of loose fibrous tissue in the bones is usually associated with hyperparathyroidism and can be provoked nutritionally by diets deficient in calcium (Jubb and Kennedy 1963). Only those birds on the vitamin D 3 diet for an extended time (approximately six weeks) showed signs of this disorder, and these were the birds with the largest parathyroid glands. Osteodystrophia fibrosa may therefore be a consequence only of severe, prolonged exposure to deficiency in laying hens. In conclusion, the histological appearance of decalcified and undecalcified sections of bone from laying hens subjected to a variety of nutritional deficiencies has been established, and will assist in the evaluation of field material.
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Acknowledgements Thanks are due to Irene Anderson, Laura Dick and Michael Lynch for section preparation and to Dr W. Dewar for diet formulation.
Received July 19, 1990 Accepted October 9, 1990
