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Improved arterial oxygenation with feed restriction in rapidly growing broiler chickens
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Camp. Biochem. Physiol.Vol. 99A, No. 3, pp. 481+485, 1991 Printed in Great
0 1991Pergamon Press plc
Britain
IMPROVED ARTERIAL OXYGENATION WITH FEED RESTRICTION IN RAPIDLY GROWING BROILER CHICKENS JOHNT. BEE~ES,*~GARY BALLAM,~STEPHENHOFMEISTRR,* CHERYLPICKETT,* KENNETHMORRIS*and ANDREWPEACOCK*$ *Cardiovascular Pulmonary Research Laboratory, University of Colorado Health Sciences Center, Denver, CO 80262, U.S.A.; $The Lovelace Medical Foundation, Albuquerque, NM 87108, U.S.A.; &Southampton General Hospital, Southampton, SO9 4XY, U.K. (Received 22 October 1990)
Abstract-l. Rapidly growing broiler chickens fed ad lib. until 56 days, but feed restricted until 60 days of age, had higher arterial oxygen saturations, lower respiratory frequencies, total ventilations that were not different, and higher tidal volumes compared to those fully fed for 56 days, 2. Arterial oxygen saturation correlated negatively with respiratory frequency, but was not related to total ventilation or tidal volume. 3. Hypoventilation appeared not to be the cause of arterial oxygen desaturation. 4. Arterial oxygen desaturation correlated with the degree of right ventricular hypertrophy.
MATERIALSAND METHODS
INTRODUCTION A fatal ascites syndrome in rapidly growing broiler chickens (Hemandez, 1987; Huchzermeyer 1986, 1988; Julian 1986; Peacock 1989) may be due to right heart failure secondary to hypoxic pulmonary hypertension (Hernandez 1987; Peacock 1989) even at sea level. In a flock of male chickens being prepared for breeding, the standard industry practice of reducing food intake, was followed by decreased incidence of ascites and severity of hypoxemia (Peacock et al., 1990). However, it was not known whether increased maturity of the chickens or reduction in their feed caused the improvement. Possibly, reducing growth rate improved ventilation in relation to body size or metabolic rate. Because we have found that limiting food intake reduces pulmonary hypertension in chickens exposed to high altitude (Peacock et al., 1989), we considered that limitation of food intake likely improved oxygenation. Further, we considered that relative hypoventilation associated with rapid growth caused hypoxaemia in the heavily fed chickens. Our approach was to examine, in an industrial setting at sea level, ventilation, hypoxaemia, hematocrit, and right heart hypertrophy in groups of chickens varying in age. One group was selected just before feed restriction and another shortly afterward, yielding two groups of comparable age and identical treatments except for food offered. The study supports the concept that feed reduction, and not increased maturity per se, is associated with improved oxygenation, but it suggests that mechanisms other than hypoventilation are responsible for the improvement. tCorrespondence: John T. Reeves, CVP Research Laboratory B-133, University of Colorado, Health Sciences Center, Denver, CO 80262, U.S.A. 481
Strategy
Chickens were studied in groups of different ages where the age of individuals within a group varied less than 24 hr. Because the commercially observed peak incidence of the ascites syndrome is at 5-9 weeks of age in broiler chickens (Peacock et al., 1990), three of the five groups were within this age range. In order to observe the effects of feed restriction, two of the groups were chosen close in age, where both groups had unrestricted feed for 56 days, at which age one group was studied, and the other group was studied at 60 days after 4 days of feed restriction. The group number at 56 days was larger than for the other groups because our prior experience indicated that at this age there was likely to be a wide range of arterial oxygen saturations and right to left ventricular weight ratios. Noninvasive ventilatory and arterial oxygen saturation measurements were made during life and the extent of right ventricular hypertrophy was measured after death the same day. Because of the need for cardiac ventricular weights, a cross sectional rather than a longitudinal study design was required. General Chickens were a dominant white male type of a very fast growing broiler strain (N = 42) and of ages 18 and 74 days after hatch. The number and age of chickens in each group are shown in Table 1.All chickens were selected at random from within commercial breeder flocks and were studied in the facility near sea level where. they had been reared from hatch. All were fed on an identical regimen of commercial starter crumble (21% protein and 1450 calories/pound of feed) and water was always provided. At 56 days, following the standard industry practice for breeding stock, feed was withheld for 4 days, after which limited feed was provided. Ventilation was measured using a closed whole body plethysmograph similar to that reported by Vizek et al. (1987). When the chicken in the closed plethysmograph inspires, warming of the inspired gas to body temperature increases the volume of the gas and the pressure within the plethysmograph. If the body temperature and that within the
482 Table 1. Ventilatory measurements in rapidly growing chickens Hct (%)
VE (L/min)
33 1
OR.’ 0.17
f 63 4
2.53* 0.06
36 2
2.02 0.31
48 5
72. 4
55 8
0.37 0.03
20
3.40 0.07
36 1
3.10 0.40
52 3
57 5
48 5
0.44 0.04
60 SEM
6
3.02’ 0.03
35 1
3.15 0.64
33’ 6
76’ 4
0.31 0.02
74 SEM
5
2.01’ 0.09
27’
I.87 0.08
32. 2
7s 4
0.33 0.03
A@= (days) 18 SEM
N
46 SEM
8
56 SEM
4
(?A 0.44. 0.02
I
c”mT, 7:; 14, 2
IL’%* 23 61 3
RVfLV (Ye) 0.30 0.01
(3 Indicates difference (P --c0.05) from measurement at 56 days by analysis of variance.
plethysmograph are known, the pressure swings with respiration allow measurement of frequency and total ventilation. The plethysmogaph was maintained at a temperature of 23°C. The plethysmographchambers were clear perspexcylinders of diameter 17.8cm and length 30.5 cm for smaller chickens and of diameter 29.2 and length 47 cm for larger chickens. Ports in the chamber wall allowed temperature and pressure measurement as well as sampling and changing the composition of gas within the chamber. The ports could be sealed by turning a stopcock. One end of the chamber was a plate which could be opened to insert or remove the chicken, and which could be held closed by welder’sclamps and sealed air tight by silicon grease. While in the chamber, the chickens roosted on a horizontal wire mesh. Before measurement, the chicken was weighed and had its rectal temperature measured. It was then inserted into the chamber, through which fresh air flowed. AU measurements were delayed until the chicken sat and became quiet. During this preliminary period, air flow through the chamber was su&ient to maintain CO, concentrations less than 0.2% (Beckman Instruments LB-2 infrared CO, analyser) and 0, concentrations greater than 20.8% (oxygen fuel cell, model 101, Applied Technical Products). Temperatures were measured in the chicken and in the chamber by mercury thermometer. To measure ventilation, the chamber was sealed by quickly closing the stopcocks, and pressure changes within the chamber were measured using a model CD15 Validyne transducer and recorded on a 4 channel recorder (Gould Instruments). Calibration of pressure changes was by recording the deflection caused by sudden injection of 3-5 ml of air into the chamber at the onset of expiration. The ventilation tracing was digitized and analysed using an IBM PC computer, which compared the size of the pressure and the calibration signals, and counted breaths. In 16 chickens aged 56 days respiratory frequency was also counted by eye outside the chamber. Frequency increased 16 & 5 breaths per minute when the chickens were placed in the chamber. Other than the increase in respiratory frequency, there was no evidence of stress imposed by the plethysmograph. Arterial oxygen saturation was measured using an oximeter unit which, as previously reported (Peacock, in press), yielded measurements in good agreement with simultaneous and directly measured arterial blood saturation (7 = 0.97). Arterial oxygen saturations are not reported for chickens aged 18 days because of technical difficulties related to their small size. Following the ventilatory and saturation measurements, 2 ml of blood wan withdrawn from a leg irein. Hematocrit micropipettes were f&d and centrifuged an an International Micro Capillary Centrifuge (model MB) for 3 min and the hematocrlt determined, The chickens were then killed by rapid cervical didocation, and the heart was
removed. The atria, and great vessel8were di away and the ventricles separated and weighed as previously described
(Peacock et al., 1990), Reported is the weight ratio of the freshly dissected right ventricle to left ventricle plus septum, RV/(LV + S). Statistics The differences between age groups were determined by one-way analyaes of variance, and relationship between two variables was by correction cc&i&at. DiEennces or correlations were considered significant when P < 0.05.
RESULTS Measured variables are shown in Table 1. The chickens given food ad lib. grew rapidly, from 0.44 kg at 18 days after hatch to 3.40 kg at 56 days. Chickens aged 60 days, having had restricted feed for 4 days, weighed less than those at 56 days, and the 74 day old chickens, also having restricted feed since age 56 days weighed less than the 60 day old chickens (P < 0.05). Hematocrits were lowest in the 74 day old group. Both minute ventilation and tidal volume increased with weight (P < 0.05). Arterial oxygen saturation by oximeter could not be measured in the 18 day old chickens, but was sharply higher in the chickens aged 60 and 74 days, than those aged 46 or 56 days. We noted that the chickens with the lowest oxygen saturations often breathed with their beaks open. Because minute ventilation and tidal volume were related to body size, subsequent analysis expressed these variabks per kg of body weight. The highest ventilation per kg was seen in the 18 day old chickens, Fig. 1, top. Measurements
in the groups 46-74 days of
age were not different from each other. The respiratory frequencies fell with advancing age and reached a nadir in chickens 60-74 days old, Fig. 1, middle. The frequencies were sharply lower in the chickens 60 and 74 days old compared to those aged 56 days. The tidaf volumes per kilogram of body weight were lower in the chickens at 46 and 56 days of age than in the other groups, Fig. 1, bottom. In particular, the higher tidal volumes in the 60 and 74 days old chickens vs those at 56 and 46 days reflected the lower frequencies in the older chicken. The decrease in frequency and the increase in tidal volume coincided with the program of restricted feeding. These ventilatory events in the 60 and 74 day old chickens accompanied improved arterial oxygenation and reduced hematocrit. Thus the arterial oxygen saturations were higher in the 60 and 74 day old chickens than in those 56 or 46 days, Fig. 2, top, and
SaO, in rapidly growing chickens
Fig. I. Ventilation per kg of body weight (top), respiratory frequency (middle), and tidal volume per kg body weight (bottom) as related to age of the chickens. In this and Fig. 2, the numbers for each age group are indicated in Table 1,and shown are mean and one standard error. Asterisks indicate differences (P < 0.05, by ANOVA and Student-NewmanKeuls multiple comparison test) from values at 56 days. Arrow indicates restriction of food at 56 days.
the hematocrit was significantly lower at 74 days than in younger chickens, Fig. 2, middle. Right ventricular hypertrophy was not statistically different between the groups, Fig. 2, bottom, but there was a tendency for the ratio to be higher in the 56 day old chickens (P = 0.06). The variation in hypertrophy could have been caused by variable hypoxaemia, in that arterial oxygen saturation and the degree of right ventricular hypertrophy were closely related, Fig. 3. The right ventricular hypertrophy tended to decrease with feed restriction in that for the 56 day old group, 17 of the 20 chickens had ratios of right to left ventricular weight that were greater than the average ratio (0.32) observed in the 11 chickens in the combined 60 and 74 day old groups. Thus, restriction of feed was associated with marked improvement in variables relating to ventilation and oxygen saturation. At issue was whether the differences in the ventilatory variables were the cause, or the result, of the improvement in saturation. For all chickens aged 46 to 74 days having saturations measured (N = 38), there was no relation of saturation to total ventilation per kg body weight (r = 0.20), or to tidal volume per kg body weight (r = 0.08). However, respiratory frequency was negatively correlated to saturation (r = -0.4), N = 38, P < O.Ol), indicating that low arterial oxygen saturations occurred in
483
Fig. 2. Arterial oxygen saturation (top}, hematocrit (middle), and the degree of right ventricular hypertrophy (the ratio RV/[LV + S], bottom) as related to age of the chickens.
chickens with high respiratory frequencies, and confirming the strong negative relationship suggested by the group data in Table 1. If low ventilation and respiratory frequency had caused the low saturations, then ventilation and frequency should have been positively correlated with saturation, but no such correlations were found. Specifically, prior to feed restriction for the 46 and 56 day old chickens (N = 27), there was a trend (P = ns) for both respiratory frequency (r = -0.31) and ventilation per kg of body weight (r = -0.35) to be negatively related to saturation. There was no significant relation of saturation to tidal volume per kg
Fig. 3. Relation of the degree of right ventricular hypertrophy to arterial oxygen saturation measured on the same day in 26 chickens 46 and 56 days of age. (Not shown is data from one chicken considered to have a technical error in the weight of the right ventricle.) The correlation between the two variables was significant (Y = -0.4X + 64, r = 0.064, P < 0.01).
484
JOHN T. REEVESet
body weight (r = -O-28), suggesting that low tidal volumes had not caused the low saturations. Also, in the I6 chickens having respiratory frequency measured outside the body plethysmograph, the negative correlation coefficient between frequency and saturation (r = -0.65, P < 0.05), was significant. The absence of positive relationships suggested that the respiratory variables were not the cause of the low saturations prior to feed restriction.
DISCUSSION
The present study showed that the standard practice of feed reduction prior to breeding in rapidly growing broiler maie chickens was a~ompanied by a marked decrease in respiratory frequency and an increase in tidal volume, an increase in arterial oxygen saturation, and trends toward decreases in hematocrit and right heart hypertrophy. The study was conducted near sea level under optimal industry conditions with daily care by trained personnel who gave careful attention to hygiene of the chickens. Throughout, the chickens remained in the environment to which they had been accustomed since hatch. Measurements relating to ventilation and saturation were noninvasive. Thus the study attempted minimal interference in the usual management of the chickens. That feed restriction begimnng at 56 days was associated with improved subsequent oxygenation, was indicated by the arterial saturations in both the older groups (ages 60 and 74 days) and by the lowered hematocrit in the 74 day old group. That the improvement in oxygenation was likely due to the feed restriction was suggested by the comparison of the 56 day old group (no restriction) with the 60 day old group (feed restriction) where there was (1) a large difference between the groups in arterial oxygen saturation, respiratory frequency, and tidal volume, (2) a small difference between the groups in their ages, and (3) an experimental design which insured that the only difference in care between the two groups was the amount of food offered. If so, it was the management practice of feed restriction and not just factors related to increasing maturity of the chickens which was responsible for their improved health. In previous reports (Peacock et al., 1989, 1990) we have suggested that the ascites syndrome is caused by hypoxia because: (1) in rapidly growing chickens (Hemandez, 1987; Peacock el al., 1989, 1990) and other species (Will, 1962), chronic hypoxia elevates pulmonary arterial pressure, a known cause of right heart failure and ascites, (2) the hypoxaemia in chickens precedes the presence of the syndrome and is correlated with the degree of right heart hypertrophy (Peacock et al., 1989, 1990), (3) the degree of Helena in young chickens predicts their subsequent mortality from the syndrome (Peacock et al. lQQO),and (4) on histologic examination, the hearts and lungs of the affected chickens show only the non specific changes expected in hypoxic pulmonary hypertension (Peacock et al., 1989; Wilson, 1988). The findings in the present study were consistent with this concept, in that right heart hypertrophy was correlated with the degree of arterial oxygen desaturation prior to the restriction of feed, and the improved
al.
oxygen saturation after restriction was associated with the presence of normal right heart weights. If hypoxic pulmonary hypertension is the cause of the syndrome, the important issue then becomes the cause of the hypoxia itself. Of the three known causes of hypoxaemia, intracardiac right to left shunts, hypoventilation and ventilation/perfusion (V/Q) mismatch, the shunt seems unlikely. We have found no intracardiac defects in hypoxaemic chickens (Peacock, et al., 1990). Also, an intracardiac shunt, per se, would cause systemic hypoxaemia, but would not cause within the lung itself, the hypoxia considered necessary to elevate pulmonary arterial pressure. Although the present study cannot distinguish between the remaining two likely causes of arterial oxygen desaturation, there were clues relative to ventilation. Our observation that some of the heavily fed chickens breathed with open beaks, suggested that stresses, i.e., hypoxaemia, heat, and/or high metabolic rates, were driving ventilation. Hypoxaemia appeared to be a major stress because respiratory frequencies were high when saturations were low and the frequencies were low when the saturations were high. Chickens are known to have ventilatory responses to hypoxia (Powell, 1988). Thus we considered that the high frequencies were the result and not the cause of the low saturations. High metabolic rates which accompany high feed intake or superimposed heat stress would add to the hypoxia-related tachypnea. The improvement in tidal volume was associated with feed restriction and the concurrent increase in oxygen saturation. Thus it deserves consideration as a cause of the improved saturation. We have concluded that it was not a major cause of the improved saturation because there was not a significant positive correlation between it and saturation either for the whole population or for the more hypoxaemic chickens examined prior to feed reduction. Also, because frequency appeared to be a consequence of hypoxaemia, and because frequency is a primary determinant of tidal volume, we have emphasized changes in frequency rather than those in tidal volume. Therefore our tentative conclusion is that tidal volume increased after feed reduction because respiratory frequency fell. However, even if a low tidal volume were not the cause of the hypoxaemia before feed reduction, any improvement after feed reduction should cause some improvement in arterial oxygenation. Needed to establish more definitively the role of tidal volume in the ascites syndrome and in the respiration of chickens is an examination in awake chickens of ventilatory changes with acute hypoxia and the relation of such changes to the subsequent development of hypoxic pulmonary hypertension. Although the present study suggests that ventilatory changes follow, rather than cause, hypoxaemia in rapidly growing broiler chickens, further work is clearly needed. For example, one needs to know whether susceptible chickens have impaired ventilatory drives and whether they have relative hypoventiiation at an early age. The noninvasive methods described here provide one approach to such studies. If hypoventilation can be ruled out as a cause of the hypoxaemia, then attention should be turned to inequalities of ventilation and perfusion in the rapidly growing chicken. Whatever the mechanism, it is clear from the
SaOr in rapidly growing chickens
present study that feed restriction is associated with prompt and substantial improvement in arterial oxy genation. It seems likely that feed restriction and not simply increased maturity of the chickens may have been responsible for the serial improvement in oxy genation (Peacock er al., 1990) previously observed in chickens. Acknowledgements-This study was supported in part by USA National Heart. Luna and Blood Institute grants HL-14985 and HL-17731, and by an anonymous donor. Dr Peacock was in receipt of fellowships from the ScaddingMorrison-Davies Fund of the British Thoracic Society, The Royal Society and the Wellcome Foundation. REFERENCES
Hemandez A. (1987) Hypoxic ascites in broilers: a review of several studies done in Columbia. Avian Diseases 31, 658-661. Huchzermeyer F. W. and DeRuyck A. M. (1986) pulmonary hypertension syndrome associated with ascites in broilers. Vet. Record 119, 94. Huchzermeyer F. W., DeRuyck A. M. C., Van Ark H. (1988) Broiler pulmonary hypertension syndrome-III. Commercial broiler strains differ in their susceptibility. Ondrestepoort J. Vet Res. 55, 5-9.
485
Julian R. J., Friars G. W., French H., Quinton M. (1986) The relationship of right ventricular hypertrophy, right ventricular failure, and ascites to weight gain in broiler and rooster chickens. Avian Diseases 31, 130-135. Kawashiro T. and Scheid P. (1975) Arterial blood gases in undisturbed resting birds: measurements in chicken and duck. Resp Physiol23, 337-342. Peacock A. J., Pickett C. K., Morris K. G. and Reeves J. T. (1989) The relationship between rapid growth and pulmonary hemodynamics in the fast growing broiler chicken. Amer. Rev. Resp. Dis. 139, 1524-1530. Peacock A. J., Pickett C. K., Morris K. G. and Reeves J. T. (1990) Spontaneous hypoxaemia and right ventricular hypertrophy in fast growing broiler chickens reared at sea level. Comp. Biochem. Physioi. 97A, 537-541. Powell F. and Schield P. (1988) Physiology of gas exchange in the avian lung. In Form and Function in Bircis. (Edited by King A. F. and McLelland J.), Academic Press, London. Vizek M., Pickett C. K. and Weil J. V. (1987) Interindividual variation in hypoxic ventilatory response: potential role of carotid body. J. Appl Physiol. 63, 1884-1889. Will D. H., Alexander A. F., Reeves J. T. and Grover R. F. (1962) High altitude-induced pulmonary hypertension in normal cattle. Circ. Res. 5, 172-177. Wilson J. B., Julian R. J. and Barker I. K. (1988) Lesions of right heart failure and ascites in broiler chickens. Aviun Diseases 32, 246-261.
Camp. Biochem. Physiol.Vol. 99A, No. 3, pp. 481+485, 1991 Printed in Great
0 1991Pergamon Press plc
Britain
IMPROVED ARTERIAL OXYGENATION WITH FEED RESTRICTION IN RAPIDLY GROWING BROILER CHICKENS JOHNT. BEE~ES,*~GARY BALLAM,~STEPHENHOFMEISTRR,* CHERYLPICKETT,* KENNETHMORRIS*and ANDREWPEACOCK*$ *Cardiovascular Pulmonary Research Laboratory, University of Colorado Health Sciences Center, Denver, CO 80262, U.S.A.; $The Lovelace Medical Foundation, Albuquerque, NM 87108, U.S.A.; &Southampton General Hospital, Southampton, SO9 4XY, U.K. (Received 22 October 1990)
Abstract-l. Rapidly growing broiler chickens fed ad lib. until 56 days, but feed restricted until 60 days of age, had higher arterial oxygen saturations, lower respiratory frequencies, total ventilations that were not different, and higher tidal volumes compared to those fully fed for 56 days, 2. Arterial oxygen saturation correlated negatively with respiratory frequency, but was not related to total ventilation or tidal volume. 3. Hypoventilation appeared not to be the cause of arterial oxygen desaturation. 4. Arterial oxygen desaturation correlated with the degree of right ventricular hypertrophy.
MATERIALSAND METHODS
INTRODUCTION A fatal ascites syndrome in rapidly growing broiler chickens (Hemandez, 1987; Huchzermeyer 1986, 1988; Julian 1986; Peacock 1989) may be due to right heart failure secondary to hypoxic pulmonary hypertension (Hernandez 1987; Peacock 1989) even at sea level. In a flock of male chickens being prepared for breeding, the standard industry practice of reducing food intake, was followed by decreased incidence of ascites and severity of hypoxemia (Peacock et al., 1990). However, it was not known whether increased maturity of the chickens or reduction in their feed caused the improvement. Possibly, reducing growth rate improved ventilation in relation to body size or metabolic rate. Because we have found that limiting food intake reduces pulmonary hypertension in chickens exposed to high altitude (Peacock et al., 1989), we considered that limitation of food intake likely improved oxygenation. Further, we considered that relative hypoventilation associated with rapid growth caused hypoxaemia in the heavily fed chickens. Our approach was to examine, in an industrial setting at sea level, ventilation, hypoxaemia, hematocrit, and right heart hypertrophy in groups of chickens varying in age. One group was selected just before feed restriction and another shortly afterward, yielding two groups of comparable age and identical treatments except for food offered. The study supports the concept that feed reduction, and not increased maturity per se, is associated with improved oxygenation, but it suggests that mechanisms other than hypoventilation are responsible for the improvement. tCorrespondence: John T. Reeves, CVP Research Laboratory B-133, University of Colorado, Health Sciences Center, Denver, CO 80262, U.S.A. 481
Strategy
Chickens were studied in groups of different ages where the age of individuals within a group varied less than 24 hr. Because the commercially observed peak incidence of the ascites syndrome is at 5-9 weeks of age in broiler chickens (Peacock et al., 1990), three of the five groups were within this age range. In order to observe the effects of feed restriction, two of the groups were chosen close in age, where both groups had unrestricted feed for 56 days, at which age one group was studied, and the other group was studied at 60 days after 4 days of feed restriction. The group number at 56 days was larger than for the other groups because our prior experience indicated that at this age there was likely to be a wide range of arterial oxygen saturations and right to left ventricular weight ratios. Noninvasive ventilatory and arterial oxygen saturation measurements were made during life and the extent of right ventricular hypertrophy was measured after death the same day. Because of the need for cardiac ventricular weights, a cross sectional rather than a longitudinal study design was required. General Chickens were a dominant white male type of a very fast growing broiler strain (N = 42) and of ages 18 and 74 days after hatch. The number and age of chickens in each group are shown in Table 1.All chickens were selected at random from within commercial breeder flocks and were studied in the facility near sea level where. they had been reared from hatch. All were fed on an identical regimen of commercial starter crumble (21% protein and 1450 calories/pound of feed) and water was always provided. At 56 days, following the standard industry practice for breeding stock, feed was withheld for 4 days, after which limited feed was provided. Ventilation was measured using a closed whole body plethysmograph similar to that reported by Vizek et al. (1987). When the chicken in the closed plethysmograph inspires, warming of the inspired gas to body temperature increases the volume of the gas and the pressure within the plethysmograph. If the body temperature and that within the
482 Table 1. Ventilatory measurements in rapidly growing chickens Hct (%)
VE (L/min)
33 1
OR.’ 0.17
f 63 4
2.53* 0.06
36 2
2.02 0.31
48 5
72. 4
55 8
0.37 0.03
20
3.40 0.07
36 1
3.10 0.40
52 3
57 5
48 5
0.44 0.04
60 SEM
6
3.02’ 0.03
35 1
3.15 0.64
33’ 6
76’ 4
0.31 0.02
74 SEM
5
2.01’ 0.09
27’
I.87 0.08
32. 2
7s 4
0.33 0.03
A@= (days) 18 SEM
N
46 SEM
8
56 SEM
4
(?A 0.44. 0.02
I
c”mT, 7:; 14, 2
IL’%* 23 61 3
RVfLV (Ye) 0.30 0.01
(3 Indicates difference (P --c0.05) from measurement at 56 days by analysis of variance.
plethysmograph are known, the pressure swings with respiration allow measurement of frequency and total ventilation. The plethysmogaph was maintained at a temperature of 23°C. The plethysmographchambers were clear perspexcylinders of diameter 17.8cm and length 30.5 cm for smaller chickens and of diameter 29.2 and length 47 cm for larger chickens. Ports in the chamber wall allowed temperature and pressure measurement as well as sampling and changing the composition of gas within the chamber. The ports could be sealed by turning a stopcock. One end of the chamber was a plate which could be opened to insert or remove the chicken, and which could be held closed by welder’sclamps and sealed air tight by silicon grease. While in the chamber, the chickens roosted on a horizontal wire mesh. Before measurement, the chicken was weighed and had its rectal temperature measured. It was then inserted into the chamber, through which fresh air flowed. AU measurements were delayed until the chicken sat and became quiet. During this preliminary period, air flow through the chamber was su&ient to maintain CO, concentrations less than 0.2% (Beckman Instruments LB-2 infrared CO, analyser) and 0, concentrations greater than 20.8% (oxygen fuel cell, model 101, Applied Technical Products). Temperatures were measured in the chicken and in the chamber by mercury thermometer. To measure ventilation, the chamber was sealed by quickly closing the stopcocks, and pressure changes within the chamber were measured using a model CD15 Validyne transducer and recorded on a 4 channel recorder (Gould Instruments). Calibration of pressure changes was by recording the deflection caused by sudden injection of 3-5 ml of air into the chamber at the onset of expiration. The ventilation tracing was digitized and analysed using an IBM PC computer, which compared the size of the pressure and the calibration signals, and counted breaths. In 16 chickens aged 56 days respiratory frequency was also counted by eye outside the chamber. Frequency increased 16 & 5 breaths per minute when the chickens were placed in the chamber. Other than the increase in respiratory frequency, there was no evidence of stress imposed by the plethysmograph. Arterial oxygen saturation was measured using an oximeter unit which, as previously reported (Peacock, in press), yielded measurements in good agreement with simultaneous and directly measured arterial blood saturation (7 = 0.97). Arterial oxygen saturations are not reported for chickens aged 18 days because of technical difficulties related to their small size. Following the ventilatory and saturation measurements, 2 ml of blood wan withdrawn from a leg irein. Hematocrit micropipettes were f&d and centrifuged an an International Micro Capillary Centrifuge (model MB) for 3 min and the hematocrlt determined, The chickens were then killed by rapid cervical didocation, and the heart was
removed. The atria, and great vessel8were di away and the ventricles separated and weighed as previously described
(Peacock et al., 1990), Reported is the weight ratio of the freshly dissected right ventricle to left ventricle plus septum, RV/(LV + S). Statistics The differences between age groups were determined by one-way analyaes of variance, and relationship between two variables was by correction cc&i&at. DiEennces or correlations were considered significant when P < 0.05.
RESULTS Measured variables are shown in Table 1. The chickens given food ad lib. grew rapidly, from 0.44 kg at 18 days after hatch to 3.40 kg at 56 days. Chickens aged 60 days, having had restricted feed for 4 days, weighed less than those at 56 days, and the 74 day old chickens, also having restricted feed since age 56 days weighed less than the 60 day old chickens (P < 0.05). Hematocrits were lowest in the 74 day old group. Both minute ventilation and tidal volume increased with weight (P < 0.05). Arterial oxygen saturation by oximeter could not be measured in the 18 day old chickens, but was sharply higher in the chickens aged 60 and 74 days, than those aged 46 or 56 days. We noted that the chickens with the lowest oxygen saturations often breathed with their beaks open. Because minute ventilation and tidal volume were related to body size, subsequent analysis expressed these variabks per kg of body weight. The highest ventilation per kg was seen in the 18 day old chickens, Fig. 1, top. Measurements
in the groups 46-74 days of
age were not different from each other. The respiratory frequencies fell with advancing age and reached a nadir in chickens 60-74 days old, Fig. 1, middle. The frequencies were sharply lower in the chickens 60 and 74 days old compared to those aged 56 days. The tidaf volumes per kilogram of body weight were lower in the chickens at 46 and 56 days of age than in the other groups, Fig. 1, bottom. In particular, the higher tidal volumes in the 60 and 74 days old chickens vs those at 56 and 46 days reflected the lower frequencies in the older chicken. The decrease in frequency and the increase in tidal volume coincided with the program of restricted feeding. These ventilatory events in the 60 and 74 day old chickens accompanied improved arterial oxygenation and reduced hematocrit. Thus the arterial oxygen saturations were higher in the 60 and 74 day old chickens than in those 56 or 46 days, Fig. 2, top, and
SaO, in rapidly growing chickens
Fig. I. Ventilation per kg of body weight (top), respiratory frequency (middle), and tidal volume per kg body weight (bottom) as related to age of the chickens. In this and Fig. 2, the numbers for each age group are indicated in Table 1,and shown are mean and one standard error. Asterisks indicate differences (P < 0.05, by ANOVA and Student-NewmanKeuls multiple comparison test) from values at 56 days. Arrow indicates restriction of food at 56 days.
the hematocrit was significantly lower at 74 days than in younger chickens, Fig. 2, middle. Right ventricular hypertrophy was not statistically different between the groups, Fig. 2, bottom, but there was a tendency for the ratio to be higher in the 56 day old chickens (P = 0.06). The variation in hypertrophy could have been caused by variable hypoxaemia, in that arterial oxygen saturation and the degree of right ventricular hypertrophy were closely related, Fig. 3. The right ventricular hypertrophy tended to decrease with feed restriction in that for the 56 day old group, 17 of the 20 chickens had ratios of right to left ventricular weight that were greater than the average ratio (0.32) observed in the 11 chickens in the combined 60 and 74 day old groups. Thus, restriction of feed was associated with marked improvement in variables relating to ventilation and oxygen saturation. At issue was whether the differences in the ventilatory variables were the cause, or the result, of the improvement in saturation. For all chickens aged 46 to 74 days having saturations measured (N = 38), there was no relation of saturation to total ventilation per kg body weight (r = 0.20), or to tidal volume per kg body weight (r = 0.08). However, respiratory frequency was negatively correlated to saturation (r = -0.4), N = 38, P < O.Ol), indicating that low arterial oxygen saturations occurred in
483
Fig. 2. Arterial oxygen saturation (top}, hematocrit (middle), and the degree of right ventricular hypertrophy (the ratio RV/[LV + S], bottom) as related to age of the chickens.
chickens with high respiratory frequencies, and confirming the strong negative relationship suggested by the group data in Table 1. If low ventilation and respiratory frequency had caused the low saturations, then ventilation and frequency should have been positively correlated with saturation, but no such correlations were found. Specifically, prior to feed restriction for the 46 and 56 day old chickens (N = 27), there was a trend (P = ns) for both respiratory frequency (r = -0.31) and ventilation per kg of body weight (r = -0.35) to be negatively related to saturation. There was no significant relation of saturation to tidal volume per kg
Fig. 3. Relation of the degree of right ventricular hypertrophy to arterial oxygen saturation measured on the same day in 26 chickens 46 and 56 days of age. (Not shown is data from one chicken considered to have a technical error in the weight of the right ventricle.) The correlation between the two variables was significant (Y = -0.4X + 64, r = 0.064, P < 0.01).
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JOHN T. REEVESet
body weight (r = -O-28), suggesting that low tidal volumes had not caused the low saturations. Also, in the I6 chickens having respiratory frequency measured outside the body plethysmograph, the negative correlation coefficient between frequency and saturation (r = -0.65, P < 0.05), was significant. The absence of positive relationships suggested that the respiratory variables were not the cause of the low saturations prior to feed restriction.
DISCUSSION
The present study showed that the standard practice of feed reduction prior to breeding in rapidly growing broiler maie chickens was a~ompanied by a marked decrease in respiratory frequency and an increase in tidal volume, an increase in arterial oxygen saturation, and trends toward decreases in hematocrit and right heart hypertrophy. The study was conducted near sea level under optimal industry conditions with daily care by trained personnel who gave careful attention to hygiene of the chickens. Throughout, the chickens remained in the environment to which they had been accustomed since hatch. Measurements relating to ventilation and saturation were noninvasive. Thus the study attempted minimal interference in the usual management of the chickens. That feed restriction begimnng at 56 days was associated with improved subsequent oxygenation, was indicated by the arterial saturations in both the older groups (ages 60 and 74 days) and by the lowered hematocrit in the 74 day old group. That the improvement in oxygenation was likely due to the feed restriction was suggested by the comparison of the 56 day old group (no restriction) with the 60 day old group (feed restriction) where there was (1) a large difference between the groups in arterial oxygen saturation, respiratory frequency, and tidal volume, (2) a small difference between the groups in their ages, and (3) an experimental design which insured that the only difference in care between the two groups was the amount of food offered. If so, it was the management practice of feed restriction and not just factors related to increasing maturity of the chickens which was responsible for their improved health. In previous reports (Peacock et al., 1989, 1990) we have suggested that the ascites syndrome is caused by hypoxia because: (1) in rapidly growing chickens (Hemandez, 1987; Peacock el al., 1989, 1990) and other species (Will, 1962), chronic hypoxia elevates pulmonary arterial pressure, a known cause of right heart failure and ascites, (2) the hypoxaemia in chickens precedes the presence of the syndrome and is correlated with the degree of right heart hypertrophy (Peacock et al., 1989, 1990), (3) the degree of Helena in young chickens predicts their subsequent mortality from the syndrome (Peacock et al. lQQO),and (4) on histologic examination, the hearts and lungs of the affected chickens show only the non specific changes expected in hypoxic pulmonary hypertension (Peacock et al., 1989; Wilson, 1988). The findings in the present study were consistent with this concept, in that right heart hypertrophy was correlated with the degree of arterial oxygen desaturation prior to the restriction of feed, and the improved
al.
oxygen saturation after restriction was associated with the presence of normal right heart weights. If hypoxic pulmonary hypertension is the cause of the syndrome, the important issue then becomes the cause of the hypoxia itself. Of the three known causes of hypoxaemia, intracardiac right to left shunts, hypoventilation and ventilation/perfusion (V/Q) mismatch, the shunt seems unlikely. We have found no intracardiac defects in hypoxaemic chickens (Peacock, et al., 1990). Also, an intracardiac shunt, per se, would cause systemic hypoxaemia, but would not cause within the lung itself, the hypoxia considered necessary to elevate pulmonary arterial pressure. Although the present study cannot distinguish between the remaining two likely causes of arterial oxygen desaturation, there were clues relative to ventilation. Our observation that some of the heavily fed chickens breathed with open beaks, suggested that stresses, i.e., hypoxaemia, heat, and/or high metabolic rates, were driving ventilation. Hypoxaemia appeared to be a major stress because respiratory frequencies were high when saturations were low and the frequencies were low when the saturations were high. Chickens are known to have ventilatory responses to hypoxia (Powell, 1988). Thus we considered that the high frequencies were the result and not the cause of the low saturations. High metabolic rates which accompany high feed intake or superimposed heat stress would add to the hypoxia-related tachypnea. The improvement in tidal volume was associated with feed restriction and the concurrent increase in oxygen saturation. Thus it deserves consideration as a cause of the improved saturation. We have concluded that it was not a major cause of the improved saturation because there was not a significant positive correlation between it and saturation either for the whole population or for the more hypoxaemic chickens examined prior to feed reduction. Also, because frequency appeared to be a consequence of hypoxaemia, and because frequency is a primary determinant of tidal volume, we have emphasized changes in frequency rather than those in tidal volume. Therefore our tentative conclusion is that tidal volume increased after feed reduction because respiratory frequency fell. However, even if a low tidal volume were not the cause of the hypoxaemia before feed reduction, any improvement after feed reduction should cause some improvement in arterial oxygenation. Needed to establish more definitively the role of tidal volume in the ascites syndrome and in the respiration of chickens is an examination in awake chickens of ventilatory changes with acute hypoxia and the relation of such changes to the subsequent development of hypoxic pulmonary hypertension. Although the present study suggests that ventilatory changes follow, rather than cause, hypoxaemia in rapidly growing broiler chickens, further work is clearly needed. For example, one needs to know whether susceptible chickens have impaired ventilatory drives and whether they have relative hypoventiiation at an early age. The noninvasive methods described here provide one approach to such studies. If hypoventilation can be ruled out as a cause of the hypoxaemia, then attention should be turned to inequalities of ventilation and perfusion in the rapidly growing chicken. Whatever the mechanism, it is clear from the
SaOr in rapidly growing chickens
present study that feed restriction is associated with prompt and substantial improvement in arterial oxy genation. It seems likely that feed restriction and not simply increased maturity of the chickens may have been responsible for the serial improvement in oxy genation (Peacock er al., 1990) previously observed in chickens. Acknowledgements-This study was supported in part by USA National Heart. Luna and Blood Institute grants HL-14985 and HL-17731, and by an anonymous donor. Dr Peacock was in receipt of fellowships from the ScaddingMorrison-Davies Fund of the British Thoracic Society, The Royal Society and the Wellcome Foundation. REFERENCES
Hemandez A. (1987) Hypoxic ascites in broilers: a review of several studies done in Columbia. Avian Diseases 31, 658-661. Huchzermeyer F. W. and DeRuyck A. M. (1986) pulmonary hypertension syndrome associated with ascites in broilers. Vet. Record 119, 94. Huchzermeyer F. W., DeRuyck A. M. C., Van Ark H. (1988) Broiler pulmonary hypertension syndrome-III. Commercial broiler strains differ in their susceptibility. Ondrestepoort J. Vet Res. 55, 5-9.
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Julian R. J., Friars G. W., French H., Quinton M. (1986) The relationship of right ventricular hypertrophy, right ventricular failure, and ascites to weight gain in broiler and rooster chickens. Avian Diseases 31, 130-135. Kawashiro T. and Scheid P. (1975) Arterial blood gases in undisturbed resting birds: measurements in chicken and duck. Resp Physiol23, 337-342. Peacock A. J., Pickett C. K., Morris K. G. and Reeves J. T. (1989) The relationship between rapid growth and pulmonary hemodynamics in the fast growing broiler chicken. Amer. Rev. Resp. Dis. 139, 1524-1530. Peacock A. J., Pickett C. K., Morris K. G. and Reeves J. T. (1990) Spontaneous hypoxaemia and right ventricular hypertrophy in fast growing broiler chickens reared at sea level. Comp. Biochem. Physioi. 97A, 537-541. Powell F. and Schield P. (1988) Physiology of gas exchange in the avian lung. In Form and Function in Bircis. (Edited by King A. F. and McLelland J.), Academic Press, London. Vizek M., Pickett C. K. and Weil J. V. (1987) Interindividual variation in hypoxic ventilatory response: potential role of carotid body. J. Appl Physiol. 63, 1884-1889. Will D. H., Alexander A. F., Reeves J. T. and Grover R. F. (1962) High altitude-induced pulmonary hypertension in normal cattle. Circ. Res. 5, 172-177. Wilson J. B., Julian R. J. and Barker I. K. (1988) Lesions of right heart failure and ascites in broiler chickens. Aviun Diseases 32, 246-261.