Haematology and morphological changes in young broiler chicks with experimentally induced hypoxia

Research in Veterinary Science 1987, 43, 331-338

Haematology and morphological changes in young broiler chicks with experimentally induced hypoxia M. H. MAXWELL, Institute/or Grassland and Animal Production, Poultry Division", Roslin, Midlothian EH259PS, S. G. TULLETT, West 0/ Scotland Agricultural College, Poultry Husbandry Department, Auchincruive, Ayr KA6 5HW, F. G. BURTON, Institute/or Grassland and Animal Production, Poultry Division", Roslin, Midlothian EH259PS

The haematology, histology and ultrastructure of day-old broiler chicks subject to experimentally induced hypoxia during incubation were examined. Some birds were allowed to reach five weeks old before examination. All the red blood cell parameters, namely, haemoglobin, packed cell volume and red cell counts were raised significantly in hypoxic birds compared with control material and the results closely resembled the haematological profile of young broilers with an ascitic syndrome. By week 5 these parameters had returned to normal. Morphological changes were seen in all the organs examined from day-old hypoxic chicks. The lungs showed much congestion and large numbers of granulocytes were present in hearts, and testes. In the livers, hepatocytes contained enlarged mitochondria together with a reduction in glycogen content. Congested lungs were seen at five weeks old but recovery to normal morphology was observed in all other organs. I THE domestic fowl is sensitive to chronic hypoxia encountered at high altitude (Atland 1961). Recently, Maxwell et al (1986a,b) examined the haematology, histopathology and ultrastructure of tissues from young broilers with an ascitic syndrome and kept at low altitude and compared the findings with those reported on birds from areas of high altitude in North America (Olander et al 1967), South America (Hall and Machicao 1968, Cueva et al 1974) and South Africa (Huchzermeyer 1984). The results suggested that there were close similarities. In electron microscope studies Maxwell et al (I 986b) showed that the organs most affected by the ascitic syndrome were the . heart, liver, kidneys and lungs. Tullett and Burton (1985) and Burton and Tullett (1985) showed that eggshell porosity affected the metabolism and growth of the developing domestic fowl embryo and the subsequent status of the bloodgas and acid-base parameters. The purpose of the present study was to examine the haematological and 'Formerly pari of the Agricultural and Research Council's Poultry Research Cent re

morphological data of chicks hatched from eggs with low porosity shells. The equators of these eggs had previously been sealed with aluminium tape to induce an internal hypoxic atmosphere. The results were compared with those obtained from birds with a previously identified ascitic syndrome.

Materials and methods

Birds Domestic fowl eggs (fresh weight, 62'5 ± 0·5 g) with low porosity shells from the same strain of broiler breeder were selected on the basis of weight at a local hatchery (D. B. Marshall Ltd, Newbridge). The equators of all eggs were sealed with a 2· 5 cm wide strip of self-adhesive aluminium tape (Tullett and Burton 1985) to induce an internal hypoxic atmosphere. To assess shell porosities, weight loss from the eggs was measured according to Tullett (1981).

Experiment 1 Thirty-two eggs were divided into two equal groups before incubation, Group I eggs were incubated in a forced-draught incubator at 37' 8°C and a wet-bulb set to 30°C (Tullett and Burton 1985). Group 2 eggs were incubated as above but with oxygen supplementation from day 14.

Experiment 2 Forty taped eggs were assigned equally to two groups and the incubation procedures were followed as in experiment I. Upon hatching the birds were removed from the incubators at three days old and placed into brooders. The birds were maintained on a commercial broiler starter and grower diet and water was available ad libitum. Five hypoxic and five oxygen-supplemented control birds were killed at two, three. four, five weeks old.

331

M. H. Maxwell, S. G. Tullett, F. G. Burton

332

TABLE 1: Mean haematological values of hypoxic and oxygen supplemented day-old chicks (± sol

Test

Oxygen (n = 16)

Hypoxic (n = 16)

Hb(g l00ml- 1) RBC (loG mm- 3) PCV (percentage) MCV (I'm 3)

10·31 (± 1·06) 2·42 (± 0·30) 30·8 (± 2·69) 127·8 (± 12'6) 33·6 (± 3'14) 42·9 (± 6·16) 18,019 (± 5,497) 10,512 (± 3,897) 64·3 (± 17'3) 5·0 (± 3·8) 4·8 (± 3·8) 24·9 (± 12·0) 1·1 (± 1-8)

11·20 (± 1'45) 2·77 (± 0'44) 34·8 (± 2·58) 125·7 (± 18'0) 32·7 (± 2·53) 40·9 (± 5·02) 28,621 (± 10,934) 12,233 (± 4,880) 73·4 (± 8·23) 4·7 (± 2·8) 3·9 (± 2·2) 17·8 (± 6·9) 0·3 (± 0·6)

MCHC (percentage) MCH (pg) Thr (103 mm- 3) WBC (103 mm- 3)

H

E B L M

(percentage)

Significance *(*)

NS NS NS

**(*)

NS (*)

NS NS NS

Hb Haemoglobin, RBC Red blood cells, PCV Packed cell volume, MCV Mean cell volume, MCHC Mean cell haemoglobin concentration, MCH Mean cell haemoglobin, WBC White blood cells, H Heterophils, E Eoslnophils. B Basophils, L Lymphocytes, M Monocytes ***P
Treatments Blood tests, Day-old chicks from the eggs of experiment I were killed by decapitation and blood was collected into plastic tubes containing EDTA anticoagulant. Blood from the older chicks was withdrawn from the brachial vein and dispelled into similar tubes. The haematological tests included red blood cell (RBC), white blood cell (WBC) and thrombocyte counts, haematocrit (scv) and haemoglobin measurements, white cell differential counts and the calcula-

tion of indices (mean cell haemoglobin, mean cell volume and mean cell haemoglobin concentration (Maxwell 1981»,

Histology. The older chicks were killed with an overdose of pentobarbitone sodium administered intravenously. Pieces of heart, lung, liver, kidney and testes from birds in both experiments were fixed in 10 per cent formol saline, processed to paraffin wax and 4 urn thick sections were stained with haematoxylin and eosin. Some were also stained for

333

Hypoxia in broiler chicks

peroxidase after the fixation period but before wax embedding according to Maxwell (1984).

Electron microscopy. Tissues from the same organs as above were prepared and processed for electron microscopy according to the procedures of Maxwell et al (l986b). \ Results

Haematology The results of the haematological tests on the day-

FIG 3: Lung from day-old hypoxic chick. Red blood cells within atrium. x 6696

old chicks are given in Table 1. Significant effects of treatments were observed in several parameters. In the hypoxic group of chicks the PCV showed the greatest increase (P
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FIG 4: Lung from day-old control chick. Uncongested atrium. x 3648

334

M. H. Maxwell, S. G. Tullett, F. G. Burton

significant to the level where P
Morphology Prominent histological alterations occurred in the lungs from both chicks (experiment I) and older birds (experiment 2). Considerable congestion and haemorrhage was present (Fig I) but dilation of the tertiary bronchi in the day-old chicks was minimal. The extent of this congestion was widespread throughout the lung tissue of all chicks and, compared with the oxygen-supplemented birds, was also a common feature in the lungs of most hypoxic birds up to five weeks old (Fig 2). Some birds within this age group showed enlargement of the atrial and septal walls of the air spaces. Congestion within atria was also a frequent observation (Fig 3) but none was seen

in the parabronchi of atria from age-matched control birds (Fig 4). In the hypoxic birds there appeared to be an increase in lipid droplets in the alveolar wall cells. Inflammatory cells were seen in the hearts from all age groups of hypoxic birds (Fig 5) compared with control material. These cells were predominantly heterophilic in nature although a few peroxidasepositive eosinophils were seen. In hearts from day-old hypoxic chicks, areas of cellular degeneration were common (Fig 6) and increased lipid appeared between myofibrils. Associated with many of the myofibrils there appeared to be an uneven mitochondrial distribution although this was less obvious in the older birds. Many mitochondria had an abnormal morphology, some appearing ring shaped (Fig 7). Glycogen deposits were less numerous than in control material. No significant histological lesions were seen in livers or kidneys from any age group, but by electron microscope many hepatocytes in livers from hypoxic day-old chicks showed either enlarged or abnormalshaped mitochondria. Some measured up to 2· 8 /-1m in diameter (Fig 8) and ringed forms were not infrequent (Fig 9). Other abnormal features common to these cells was a reduction in glycogen, lipid droplets within nuclei and profiles of rough endoplasmic reticulum with marked dilations (Fig 10). In the kidneys, there was an excessive number of vacuoles located apically and basally within the

Hypoxia in broiler chicks

proximal convoluted tubules, some of which measured up to 2·3).1m in diameter (Fig II). Many of these vacuoles contained peroxisomes and in some instances as many as seven were counted. There was also an increase in lipid droplets which were located basally within the proximal convoluted tubule (Fig 12), although there was no damage to the mitochondria and no thickening of the glomerular basement membranes. Infiltrations of granulocytes were also seen in the testes from the hypoxic birds (Fig 13a).

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Discussion

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The red cell parameters, namely haemoglobin, rev and RBC count, in the day-old hypoxic chicks of the present study, bear a close similarity to those described in young broilers with an ascitic syndrome (Maxwell et aI1986a). All three parameters showed significantly raised levelscompared with control material and thus the degree of haemoconcentration in the present hypoxic chicks may be used as an indicator of environmental stress and may reflect the importance hypoxia plays in the development of the above ascitic syndrome. Furthermore, in both studies the heterophil levels were significantly raised, suggesting that an additional stress factor may also be involved (Gross and Siegel 1984). However, the large increase in the thrombocyte count of the hypoxic chicks as yet cannot be explained. By week 3 many of the blood parameters in the hypoxic chicks had stabilised compared with the oxygen-supplemented birds and by week 5 recovery of the haematological pr'bfile was complete. In a recent study on rats subjected to hypobaric hypoxia for two weeks, Fried and Reid (1984) demonstrated full recovery in haematocrit levels after one month.

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FIG 6: Heart muscle from day-old hypoxic chick. organisation and cellular debris. x 5832

M. H. Maxwell, S. G. Tullett, F. G. Burton

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FIG 7: Heart muscle from day-old hypoxic chick. Abnormally shaped mitochondria (arrows). x 12,528

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"z:Q FIG 9: Liver from day-old hypoxic chick. Several ring-shaped mitochondria are present in this micrograph (arrows). x 25,056

-...., FIG 11: Kidney from day-old hypoxic chick. Proximal convoluted tubule containing many basally and apically located vacuoles, some with peroxisomes. The largest vacuole measures 2·3 I'm in diameter (arrow). x 5832

FIG 10: Liver from day-old hypoxic chick. A dilated profile of rough endoplasmic reticulum (arrow). x 38,880

Hypoxia in broiler chicks

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(bl FIG 13: a. Testis from a day-old hypoxic chick. Granulocytes can be seen in the intertubular spaces (arrows). x 448. b. Testis from day-old control chick. x 448

337

338

M. H. Maxwell, S. G. Tullett, F. G. Burton

Morphological changes were seen in all the organs hope of mimicking the physiological conditions and examined from the day-old hypoxic chicks. Many of parameters that develop at high altitude. these changes closely resembled those in tissues from birds with an ascitic syndrome (Maxwell et al 1986a,b). The lungs showed considerable congestion References with many of the parabronchi and atria being ATLAND, P. D. (1961) Journal of Applied Physiology 16,141-143 BURTON, F. G. & TULLETT, S. G. (1985) Comparative Bioaffected. chemistry and Physiology 81A, 377-385 The existence of large numbers of granulocytes, CUEVA, S., SILLAU, H., VALENZUELA, A. & PLOOG, H. mainly heterophils, in the hearts and testes of day-old (1974) Research in Veterinary Science 16, 370-374 chicks agrees with their presence in birds with the FRIED, R. & REID, L. M. (1984) Journal of Applied Physiology 57, 1247-1253 previously reported ascitic syndrome. This inflamR. & REID, L. M. (1985) American Journal of Pathology matory-like response may be the result of chemotactic FRIED, 121, 102-111 factors being released into the hypoxic tissues. It has GROSS, W. G. & SIEGEL, H. S. (1984) Avian Diseases 27, 972-979 also been proposed that invasions of granulocytes HALL, S. A. & MACHICAO, N. (1968) Avian Diseases 12, 75-84 may playa role as contributors to ischaemic-induced HUCHZERMEYER, F. W. (1984) Poultry Bulletin June 279 M. D., YAFFE, P. & YOFFEY, J. M. (1985) Journal of myocardial injury (Lucchesi and Mullane 1986)and in KENDALL, Anatomy 142, 85-102 the present study they may similarly lead to impair- LUCCHESI, B. R. & MULLANE, K. M. (1986) Annual Reviews of ment in cardiac function. The reduction in glycogen Pharmacology and Toxicology 26, 201-224 together with abnormally large mitochondria in day- MAXWELL, M. H. (1981) Research in Veterinary Science 30, 233-238 old chick hepatocytes corresponds with the changes MAXWELL, M. H. (1984) Research in Veterinary Science 37,7-11 seen in birds with an ascitic syndrome (Maxwell et al MAXWELL, M. H., ROBERTSON, G. W. & SPENCE, S. (1986a) 1986a,b). However, such mitochondrial changes were Avian Pathology IS, 511-524 absent in kidneys from hypoxic chicks and the MAXWELL, M. H., ROBERTSON, G. W. & SPENCE, S. (1986b) Avian Pathology IS, 525-538 thickness of glomerular basement membranes OLANDER, H. J., BURTON, R. R. & ALDER, H. E. (1967) appeared normal. Avian Diseases 11, 609-620 The lung was the only organ to show morphological RABINOVITCH, M., GAMBLE, W. J., MIETTINEN, O. S. & REID, L. (1981) American Journal of Physiology 240, H62-H72 change at five weeks old. Compared with lungs from ROSSE, C. (1976) International Review of Cytology 45, 155-290 oxygen-supplemented birds they showed much TULLETT, S. G. (1981) Turkeys 29, 24-28 congestion and haemorrhage and unlike the other TULLETT, S. G. & BURTON, F. G. (1985) Comparative Bioorgans examined, recovery to normality was far from chemistry and Physiology 81A, 137-142 complete. Regeneration of tissues after the stress TURNER, M. S., HURST, J. M. & YOFFEY, J. M. (1967) British Journal of Haematology 13, 942-948 effects of hypoxia have been fully described in YOFFEY, J. M. (1974) The Bone Marrow in Hypoxia and Rebound. rodents (Turner et al 1967, Yoffey et al 1968, Yoffey C. C. Thompson 1974,1980, Rosse 1976, Rabinovitch et a11981, Fried YOFFEY, J. M. (1980) International Review of Cytology 62, 311-359 and Reid 1984, 1985, Kendall et aI1985). The present results suggest that there may be strong YOFFEY, J. M., JEFFREYS, R. V., OSMOND, D. G., TURNER, ·.M. S., TAHSIN, S. C. & NIVEN, P. A. R. (1968) Annals of the physiological links between hypoxia and the ascitic New York Academy of Sciences 149,179.-:;192 syndrome which occurs both at high and low altitudes (Maxwell et al 1986a,b). Further experiments are in Received October 31, 1986 progress involving young broilers maintained in an Accepted December 14, 1986 experimentally induced hypoxic atmosphere in the