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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
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Age and gender influence the cardiorespiratory function and metabolic rate of broiler chicks during normocapnia and hypercapnia Lívia P. Espinha a,b , Fernando A. Souza a , Aretuza C. Capalbo a , Kênia C. Bícego a,b , Marcos Macari a , Luciane H. Gargaglioni a,b,∗ a
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Department of Animal Morphology and Physiology, São Paulo State University – (UNESP FCAV), Jaboticabal, SP, Brazil National Institute of Science and Technology in Comparative Physiology (INCT-FisiologiaComparada), Brazil
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a r t i c l e
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a b s t r a c t
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Article history: Accepted 29 May 2014 Available online xxx
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Keywords: Bird CO2 chemosensitivity Ventilation Thermoregulation Metabolic rate
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Pulmonary ventilation (VE ), body temperature (Tb), mean arterial pressure (MAP), heart rate (fH ) and ·
metabolic rate (VO2 ) were measured in 10 (d10)- and 21 (d21)-day-old male and female chicks exposed ·
to 7% CO2 . Under normocapnia, VE was higher in d10 chicks than in d21 due to a higher tidal volume; in females a higher respiratory frequency (fR ) was also observed. The d10 birds presented higher fH and ·
VO2 . The d21 females showed the highest CO2 ventilatory response due to increased fR . MAP did not change during hypercapnia while a hypercapnic bradycardia occurred, except in d21 females. Hypercap·
nia induced a drop in Tb in all groups and an increase in VO2 in d21 males. Overall, no gender effect is observed in cardiorespiratory and metabolic variables in d10 and d21 chicks under normocapnia, the ·
differences in VE and fH between ages may be related to distinct metabolic demands of these phases. The d21 female chicks seem to be more sensitive to hypercapnia. © 2014 Published by Elsevier B.V.
1. Introduction
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Hypercapnia is a powerful stimulus for air breathing vertebrates and evokes several compensatory responses in birds, such as an increase in ventilation (Bouverot, 1978; Mortola, 2004). When challenged by CO2 , most avian species increase tidal volume, while the response in breathing frequency is more variable (Bouverot, 1978). To detect changes in CO2 /pH, birds possess peripheral (located in the carotid bodies), central (located in the central nervous system), and intrapulmonary chemoreceptors that are highly sensitive to CO2 (Milsom, 2002). Respiratory response to hypercapnia was demonstrated to be different in adult male and female ducks. According to Dodd et al. (2007), the acute hypercapnic ventilatory response is lower in females than in males. Cardiorespiratory and metabolic responses to hypercapnia were also demonstrated in birds in early life such as in chicken (Mortola, 2009) and in duck hatchlings (Mortola and Toro-Velasquez, 2014);
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∗ Corresponding author at: Departamento de Morfologia e Fisiologia Animal, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista Júlio de Mesquita Filho, Via de acesso Paulo Donato Castellane s/n, 14870-000 Jaboticabal, SP, Brazil. Tel.: +55 16 32092656; fax: +55 16 32024275. E-mail addresses: [email protected], [email protected] (L.H. Gargaglioni).
however no information about gender differences was provided. At least in mammals, cardiorespiratory function and metabolic rate under normocapnic and hypercapnic conditions depend on gender and age in young animals (Wenninger et al., 2009). Male rats undergo changes in ventilatory responses to CO2 during early development, while females show little or no age-dependent change in ventilatory responses to hypercapnia. From 10 to 15 days after birth male rats show a more pronounced response to CO2 than females, but this difference is no longer observed in adult animals (Holley et al., 2012). It is intriguing that such differences can be observed during early life. As far as we know, the gender influence on physiological responses to hypercapnia is still a matter of investigation in prepubertal birds. Therefore, in the present study, we used precocial young birds, 10 and 21 days old chickens, to compare cardiorespiratory and metabolic responses to CO2 in males and females.
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2. Methods
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2.1. Animals
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Experiments were performed on unanesthetized 10- and 21day-old broilers (Gallus gallus – Cobb 500® ). The animals had free access to water and food and were housed in a room with a
http://dx.doi.org/10.1016/j.resp.2014.05.013 1569-9048/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Espinha, L.P., et al., Age and gender influence the cardiorespiratory function and metabolic rate of broiler chicks during normocapnia and hypercapnia. Respir. Physiol. Neurobiol. (2014), http://dx.doi.org/10.1016/j.resp.2014.05.013
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1981) assuming that chamber was fully saturated. Tidal volume and consequently VE were corrected by body mass.
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controlled environment (Cobb, 2012) and a 12:12 h light:dark cycle (lights on at 7:00 AM). The methods were approved by the local ethical committee (CEUA – Comissão de Ética no Uso de Animais – FCAV-UNESP; Protocol: 026368/11).
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2.2. Surgery
The catheter was connected to a pressure transducer on the experiment day, and the birds were allowed free movement. The pulsatile arterial pressure (PAP) was measured with a pressure transducer (TSD 104A, Biopac systems, USA) connected to an amplifier (DA 100C, Biopac systems, USA). The heart rate (fH ) and mean arterial pressure (MAP) were quantified from the PAP recording using the same system (MP100 ACE, Biopac systems, USA). PAP and
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Two days before the experiments, a 20 cm long catheter [PE-10 connected to PE-50 (Clay Adams, Parsippany, NJ)] was inserted into the abdominal aorta through the femoral artery while the animals were under anesthesia from an intramuscular injection of 30 mg/kg of ketamine + 1.5 mg/kg of xylazine. The free end of the catheter was fixed in the interscapular area. A portion of the leg and abdomen were shaved, and the skin was sterilized with betadine solution and alcohol. For body temperature (Tb) measurements, a temperature datalogger (SubCue, Calgary, AB, Canada) was implanted in the abdominal cavity. The datalogger was programmed to take a reading every 5 min. After the surgery, the animals received two doses of enrofloxacin (10 mg/kg, intramuscular) and flunixin meglumine (2.5 mg/kg, intramuscular) to prevent infection and post-surgical discomfort, respectively. The first dose was given at the end of the surgery, and the second one was administered 6–12 h later.
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2.3. Determination of pulmonary ventilation
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Measurements of pulmonary ventilation (VE ) were performed using the whole body plethysmography method (closed system; Bartlett and Tenney, 1970). Freely moving 10- and 21-day-old chickens were kept in a 5-L and 10-L chamber, respectively; these chambers were ventilated with humidified room air or with a humidified hypercapnic gas mixture containing 7% CO2 in air ·
(White Martins, Sertãozinho, Brazil). During VE measurements, the flow was interrupted, and the chamber was sealed for short periods of time (∼2 min). The pressure oscillations caused by breathing were monitored using a differential pressure transducer (TSD 160A, Biopac Systems, Santa Barbara, USA). The signals were fed into a differential pressure signal conditioner (DA 100C, Biopac Systems, USA), passed through an analog-to-digital converter, and digitized on a microcomputer equipped with data acquisition software (MP100A-CE, Biopac Systems, USA). The sampling frequency was 200 samples s−1 . The results were analyzed using the data analysis software Acqknowledge (v3.8.1 data acquisition system, Biopac Systems, USA). The volume was calibrated during each experiment by injecting 1 mL of air into the animal chamber. The tidal volume (VT ) was calculated using the following formula of Malan (1973): PT TA PB − PC V T = VK × × × PK TR (PB − PC) − TA/Tb × (PB − PR) where PT is the pressure deflection associated with each VT , PK is the pressure deflection associated with injection of the calibration volume (VK), TA is the air temperature in the animal chamber; TR is the room temperature; PB is the barometric pressure; PC is the vapor pressure of water vapor in the animal chamber; Tb is the body core temperature; and PR is the water vapor pressure at ·
Tb. VE and VT are presented at ambient barometric pressure and Tb and expressed as volumes that are saturated with water vapor at this temperature (BTPS). The Tb was monitored by a datalogger (Sub-Cue, Calgary, AB, Canada), and the air temperature in the animal chamber was monitored using a thermoprobe (model 850210, Cole Parmer, Chicago, IL). According to Malan (1973), TR may be slightly lower than TA because of the heating of the chambers by the animals. The PC (the water vapor pressure in the animal chamber) was calculated indirectly using an appropriate table (Dejours,
2.4. Measurements of arterial blood pressure and heart rate
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fH data were collected simultaneously with VE measurements.
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2.5. Determination of arterial pH and gases
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Arterial blood samples were obtained during normocapnia and hypercapnia from a subset of the 10- and 21-day-old chicks via the implanted arterial catheter. 95 L of blood were sampled by a cartridge (EG7+) for immediate analysis of the arterial pH (pHa), arterial carbon dioxide partial pressure (PaCO2 ), arterial oxygen partial pressure (PaO2 ) and bicarbonate (HCO3 − ) with an i-STAT portable blood gas analyzer (i-STAT Analyzer, Abbott Laboratories, NY, USA). Body temperature values were provided to the analyzer for corrections of blood gases and pH. 2.6. Metabolic rate and Tb
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The metabolic rate was measured by indirect calorimetry (oxy·
gen consumption, VO2 ) using closed system respirometry (Almeida et al., 2004). At the end of the normocapnic and hypercapnic periods, the air flow of the chamber was interrupted for 2 min, and the air was continuously sampled by an O2 analyzer (PowerLab System, ADInstruments® /Chart Software, version 7.3, Sydney, Australia). While the chamber was sealed, the oxygen fraction did not drop to less than 19.5%. The percentage of oxygen decay inside the chamber was plotted against time, and the slope of the resulting curve multiplied by the net volume of the chamber (discounted the volume of the animal) divided by the animal mass was used to calculate the rate of oxygen consumption. The VO2 was corrected by body mass and values are presented in STPD (standard conditions of temperature, pressure and dry air). The body temperature was measured using implanted intracoelomic dataloggers (SubCue, Calgary, AT, Canada), at the end of the experiments, which were connected to the computer and the obtained temperature values were corrected according to the specifications in the manufacturer’s manual. 2.6.1. Experimental protocol Two days after surgery, each animal was individually placed in a plethysmography chamber maintained at 27 ◦ C (10 days) and 25 ◦ C (21 days), and the animals were allowed to move freely while the chamber was flushed with humidified air. These temperatures were chosen based on a previous study by Meltzer (1983) that described that 27 ◦ C and 25 ◦ C are considered to be the thermoneutral temperature for 10 and 21 days, respectively. After the animals became ·
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calm (∼30 min), the control VE , PAP and VO2 were measured. Next, a humidified hypercapnic gas mixture (7% CO2 in air, White Martins, Sertãozinho, Brazil) was flushed through the chamber for 30 min, ·
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and the VE , PAP and VO2 were measured at minutes 5, 10, 20, and 30 of the hypercapnia. Blood samples were collected 15 min before the hypercapnic challenge and during the hypercapnic exposure (30 min) for pHa, PaCO2 , PaO2 , and HCO3 − measurements. The Tb was recorded in 5-minute intervals throughout the experiment.
Please cite this article in press as: Espinha, L.P., et al., Age and gender influence the cardiorespiratory function and metabolic rate of broiler chicks during normocapnia and hypercapnia. Respir. Physiol. Neurobiol. (2014), http://dx.doi.org/10.1016/j.resp.2014.05.013
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Table 1 Body mass (g) at birth, 10 and 21 days of female and male chicks.
Birth 10 days 21 days
Male (n = 12)
Female (n = 12)
P
47.1 ± 1.5 137 ± 6.7 666 ± 26
47.4 ± 2.9 129 ± 9.3 609 ± 15.5
ns ns ns
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2.6.2. Data processing and analysis The results are reported as means ± SEM. The data analyses were performed using SAS software (SAS System, version 9.1) and were tested for normality of deviation (Cramer Von-Mises criterion) and homoscedasticity (Levene test). The normocapnic and hypercapnic conditions were analyzed separately to determine the gender and age effect. The effect of hypercapnia on VT , fR , VE , MAP, fH , blood gases, HCO3 − and pHa over the time course was evaluated by two-way analysis of variance (repeated measures). For blood gases, HCO3 − and pHa, since an interaction (time × treatment) was observed, point-by-point comparisons of mean values between vehicle and drug treatments during normocapnia and hypercapnia were performed by one-way ANOVA. Values of P ≤ 0.05 were considered significant.
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3. Results
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All data met the assumption of normality of deviation (Cramer Von-Mises criterion) and homoscedasticity. Body weight of hatchlings, 10- and 21-day-old broilers is presented in Table 1. No gender difference was observed.
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3.1. Normocapnia
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The 10-day-old broiler chicks (males and females) presented ·
higher VE under normocapnic conditions (P < 0.001) compared with that in the d21 animals due to a higher VT (Fig. 1). In females, the ·
higher VE in d10 was also caused by a higher fR compared to d21 (P < 0.01). No difference was observed between males and females at the same age. Blood gases and pHa did not differ between males and females. The pHa was higher in the d21 males than in d10 males, indicating an age effect (P < 0.05) (Table 2). Gender and age did not affect MAP (Fig. 2). The 10-day-old animals presented higher fH values than did the 21-day-old (P < 0.05). There was no difference in Tb between genders at the same age and no difference between ages at the same gender. Age·
and gender-based differences in the VO2 /kg were observed in the
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Fig. 1. Ventilation (VE ), tidal volume (VT ) and respiratory frequency (fR ) of 10 and 21 days old chickens under normocapnic conditions.a,b Means followed by different letters are statistically different (P < 0.05).Values are reported as means ± SEM.
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animals (Table 3). A higher VO2 /kg was observed in the younger animals compared with the older animals (P < 0.01).
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3.2. Hypercapnia
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Hypercapnia increased VE significantly in both the d10 and d21 male and female broiler chicks due to the enhanced VT relative to that in normocapnia (effect of time: P < 0.001, no interaction effect) (Fig. 3). The 21-day-old females presented higher ventilatory response to hypercapnia compared to all groups (effect of treatment: P < 0.001, no interaction) due to a higher fR (effect of treatment: P < 0.001, no interaction). Hypercapnia caused a decrease in pHa and an increase in PaCO2 and PaO2 in all groups (P < 0.001). No changes in HCO3 − were observed during normocapnia and hypercapnia. The pHa was higher in d21 males than in d10 males in normocapnia, indicating an age effect (P < 0.05) (Table 2).
No difference was observed in MAP among groups (Fig. 4). Hypercapnia induced bradycardia in all groups (effect of time: P < 0.001), except for 21-day-old females (effect of treatment, P < 0.01, no interaction effect). Hypercapnia caused a significant decrease in Tb in all groups (effect of time: P < 0.001, no interaction). No difference was observed in Tb among groups (Fig. 5). ·
Both males and females presented higher VO2 related to body mass in d10 than in d20 chicks (Table 3). Hypercapnia induced an ·
increase in VO2 /kg in the 21-day-old males (effect of time: P < 0.05, no interaction). The 10-day-old animals presented a higher ventilatory equiv·
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alent (VE /VO2 ) in normocapnia and hypercapnia than did the 21-day-old animals (P < 0.001). The d10 males and females and the
Please cite this article in press as: Espinha, L.P., et al., Age and gender influence the cardiorespiratory function and metabolic rate of broiler chicks during normocapnia and hypercapnia. Respir. Physiol. Neurobiol. (2014), http://dx.doi.org/10.1016/j.resp.2014.05.013
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4 Table 2
Q6 Values of arterial pH (pHa), arterial oxygen partial pressure (PaO2 ), arterial carbon dioxide partial pressure (PaCO2 ), and plasma bicarbonate (HCO3 − ) of 10 and 21 days old male and female chicks during normocapnia (0% CO2 ) and submitted to hypercapnia (7% CO2 ). 0% CO2
7% CO2
Male (n = 6) 10 days pHa PaCO2 (mmHg) PaO2 (mmHg) HCO3 −
7.56 29.20 92.92 25.91
± ± ± ±
0.01 0.9 1.9 0.9
Male (n = 5) 21 days pHa PaCO2 (mmHg) PaO2 (mmHg) HCO3 −
7.59 29.04 94.50 27.47
± ± ± ±
0.01b 1.2 1.0 1.3
Female (n = 5) 7.56 28.40 92.69 25.02
± ± ± ±
0.01 1.2 1.2 0.9
Female (n = 4) 7.58 28.18 94.50 25.90
± ± ± ±
0.01 1.0 1.4 0.9
Male (n = 6) 7.32 53.60 113.33 26.93
± ± ± ±
Female (n = 5)
0.02a 1.9a 1.7a 1.7
7.33 50.52 113.20 26.00
Male (n = 5) 7.34 55.06 113.00 28.44
± ± ± ±
± ± ± ±
0.02a 2.2a 1.3a 2.2
Female (n = 4)
0.02a 2.5a 1.6a 0.6
7.36 51.90 114.75 25.00
± ± ± ±
0.02a 3.9a 1.1a 1.9
Values are reported as means ± SEM. a Means difference between 0% and 7% CO2 in the same gender. b Means difference between ages in the same gender.
Table 3 Oxygen consumption (mL kg−1 min−1 STPD) of 10 (d10) and 21 (d21) days old male and female chicks during normocapnia (0% CO2 ) and exposure to hypercapnia (7% CO2 ). 0% CO2
d10 d21
7% CO2
Male (n = 6)
Female (n = 6)
Male (n = 6)
Female (n = 6)
34.16 ± 3.8 17.15 ± 2.5a
31.77 ± 3.7 22.02 ± 3.1a
41.01 ± 3.0 25.99 ± 2.3a , b
41.72 ± 3.7 23.35 ± 2.8a
Values are reported as means ± SEM. a Means difference between ages in the same gender. b Means difference between 0% and 7% CO2 in the same gender.
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d21 females presented an increased VE /VO2 response to hypercapnia (P < 0.001) (Fig. 6). 4. Discussion
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This study evaluated the cardiorespiratory and metabolic responses to hypercapnia (7% CO2 in air) in male and female broiler chicks at 10 (d10) and 21 (d21) days after hatch. 4.1. Normocapnia
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Younger broiler chicks, both male and female, presented higher ventilation under normocapnia than did the older chicks. This difference was due to an enhanced VT in males and an increased VT and fR in females. It seems that a different scenario is observed in pre-pubertal rats, since there is no age effect on ventilation in females while it is observed the lowest pulmonary ventilation at days 12–13 after birth in males (Holley et al., 2012). Despite the higher ventilation in d10 chicks, no difference in the blood parameters and HCO3 − was found (Table 2). However, pH was slightly higher only in the d21 males (7.59) than in the d10 males (7.56) in normocapnia. The higher ventilation observed in the younger animals could be related to the higher mass-specific ·
Fig. 2. Mean arterial blood pressure (MAP) and heart rate (fH ) of 10 and 21 days old chickens in normocapnia. a,b Means followed by different letters are statistically different (P < 0.05). Values are reported as means ± SEM.
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metabolic rate (Table 3); however V E /VO2 is lower in d21 chicks compared to d10. The reason for lower ventilation in the older chicks (Fig. 1) may be a consequence of fast growing selection. Further studies in these animals are necessary to clarify this issue. Our values for PaCO2 are lower (Kawashiro and Scheid, 1975; Fedde et al., 2002) and pH are higher (Kawashiro and Scheid, 1975) compared to the values found for adult white leghorn. We believe that the differences may firstly be due to the distinct ages and lineages used by Kawashiro and Scheid (1975) and Fedde et al. (2002). Secondly, we have used unanesthetized and intact broiler chicks
Please cite this article in press as: Espinha, L.P., et al., Age and gender influence the cardiorespiratory function and metabolic rate of broiler chicks during normocapnia and hypercapnia. Respir. Physiol. Neurobiol. (2014), http://dx.doi.org/10.1016/j.resp.2014.05.013
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F10 (n=6) M10 (n=6) F21 (n=6) M21 (n=6)
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Fig. 4. The relative changes in mean arterial blood pressure (MAP) and heart rate (fH ) of 10 and 21 days old chickens in 10 and 21 days old chickens. Data are expressed relative (%) to levels before CO2 administration as means ± SEM. The 21-day-old female broiler chickens presented lower bradycardic response to hypercapnia compared to all groups (two-way ANOVA; effect of treatment, P < 0.01, no interaction).
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7% CO2 -2 0
-10
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Time (min) ·
Fig. 3. The relative changes in ventilation (VE ), tidal volume (VT ) and respiratory frequency (fR ) in 10 and 21 days old chickens. Data are expressed relative (%) to levels before CO2 administration as means ± SEM. The 21-day-old female broiler chickens presented higher ventilatory response to hypercapnia compared to all groups (twoway ANOVA; effect of treatment: P < 0.001, no interaction).
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Δ Tb (oC)
-30
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-2 -15 -10 -5
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and performed our experiments two days after surgery, whereas Kawashiro and Scheid (1975) performed the experiments 2 h after surgery and Fedde et al. (2002) used decerebrated animals, therefore it is possible that the differences in arterial pH and blood gases may be caused by the effect of the anesthetics or decerebration. As mentioned above, pH of d21 male chicks was 0.03 higher than d10 males. Since PaCO2 values were very similar between groups, the higher pH must be due to a higher HCO3 − levels (25.91 ± 0.9 mmol/L in d10 compared to 27.47 ± 1.3 mmol/L in d21), despite the fact that no statistical difference was observed. To the best of our knowledge, there is no data in the literature about blood gases and pH in young broilers to compare with our present results. Similar to ventilation, fH was not different between males and females in both ages, but older animals had lower fH (Fig. 2). This decrease most likely occurs because of the lower mass-specific metabolic rate in the larger animals. Despite these differences, Tb did not change among groups, indicating higher heat loss in d10 chicks. In fact, ventilation is almost 8 times higher in these chicks, which may contribute to evaporative heat loss. Additionally, the
0
5
10
15
20
25
30
35
Time (min) Fig. 5. Change in body temperature (Tb) of male (M) and female (F) 10 and 21 days old chicks exposed to 7%CO2 . Values are reported as means ± SEM.
similarity of Tb between males and females corroborates the results in young and adult rats from Florez-Duquet et al. (2001). Age and gender did not affect MAP of our chicks during normocapnia (Fig. 2). Previous studies have shown that MAP is higher in adult leghorn males than in females (Nishimura et al., 1981; Kamimura et al., 1995; Nishimura et al., 2001); however, our chicks (10–21-day-old) were much younger than those used in those studies (35–56 weeks). In the latter case, the higher MAP in males could be related to the higher plasma catecholamine levels (Ruiz-Feria et al., 2004). Furthermore, Nishimura et al. (2001) also demonstrated that the MAP of 2–3 weeks old male chicks is ∼150 mmHg and tends to increase with age. In our study, MAP of d10 and d21 chicks was ∼110 mmHg. The difference in these results could reflect the difference in strains (egg-laying versus broilers).
Please cite this article in press as: Espinha, L.P., et al., Age and gender influence the cardiorespiratory function and metabolic rate of broiler chicks during normocapnia and hypercapnia. Respir. Physiol. Neurobiol. (2014), http://dx.doi.org/10.1016/j.resp.2014.05.013
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. .
VE /VO2
Male 10d Female 10d Male 21 d Female 21d 50 45 40 35 30 25 20 ** 15 10 5 0
**
0
7 % CO2 ·
Fig. 6. Changes in ventilatory equivalent (VE /VO2 ) from the air value (21% inspired O2 and 0% inspired CO2 ) during exposure to hypercapnia (high inspired carbon dioxide concentration, 7% CO2 ), in chickens of 10 and 21 days. ** Statistically significant difference between ages (P < 0.05, ANOVA).
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4.2. Hypercapnia Hypercapnia resulted in an increased ventilation and tidal volume in all animals with no change in breathing frequency. This ·
strategy helps reduce dead space, makes VE more efficient, and has also been reported in rats (Holley et al., 2012). A previous study by Dodd et al. (2007) in adult ducks demonstrated that hypercapnia causes an augmentation in ventilation due to an increase in tidal volume (∼2-fold), and, to a lesser extent, due to an increase in breathing frequency (∼1.4-fold). In other studies with adult birds, the breathing frequency decreased during hypercapnia (Milsom et al., 1981; Jones and Purves, 1970; Bouverot and Leitner, 1972; Osborne et al., 1977; Colby et al., 1987). Therefore, the different responses to hypercapnia observed in our study and those of others may be due to the different CO2 levels. Since we have used a high level of CO2 (7%) compared to milder exposures (5%) in the other studies; our exposure may represent a major stress, which different animals could react to in various ways. Therefore, the differences observed in our study may not only reflect differences in CO2 chemosensitivity, but species, age- or gender-differences in the response to CO2 stress. At least for hypoxic stress, this chicken lineage seems to be less resistant and very susceptible to 10% O2 exposure (unpublished data). Therefore, our chicks might be more sensitive to CO2 stress, which could explain the VO2 increase during hypercapnia in d21 males and a tendency of increase in the other groups (Table 3). As expected, CO2 exposure promoted an increase in PaCO2 , PaO2 and a decrease in pHa in all chicks (Table 2), with no difference among groups. We observed a nonsignificant tendency of an increase in HCO3 − during hypercapnia. These data are similar to those found in adult rats exposed to 7% CO2 (Biancardi et al., 2008, 2014; Patrone et al., 2014). Surprisingly, d21 females presented a higher CO2 ventilatory response than did the other groups, indicating that these chicks are more sensitive to hypercapnia. Contrasting results were provided by Dodd et al. (2007) in adult ducks and Bavis and Kilgore (2001) in adult quails. According to these authors, the CO2 response in adult females is lower than that in adult and juvenile male ducks (5% CO2 – Dodd et al., 2007) and does not differ between genders in quails (6% CO2 – Bavis and Kilgore, 2001). These contrasting responses compared to the current study could be related to age, species or/and CO2 levels because we used pre-pubertal 10- and 21-dayold broiler chicks exposed to 7% CO2 . Furthermore, Holley et al.
(2012) found more pronounced responses of male 14–15 days old rats to 7% CO2 . At least in these animals, this age represents a critical period for the development of the ventilatory chemosensitivity. Interestingly, the authors observed a higher plasma concentration of estradiol in male 12–13 days old rats, suggesting a hormonal modulation in CO2 chemoreception (Holley et al., 2012). Regarding broiler chicks, it was demonstrated that females present much higher plasma estradiol levels than males as early as just after hatching, a pattern that is maintained up to 42 days (Gonzales et al., 2003). As we observed differences in hypercapnic ventilatory response of d21 but not d10 females compared to males, the sexual hormones modulation in CO2 response in broiler chicks warrants further investigation. Increased estradiol influence might also explain why d21 females, in contrast to the other groups, did not present hypercapnia-induced bradycardia (Fig. 5). At least in adult rats, estradiol increases the sympathetic modulation of fH (Dias et al., 2010). In agreement with studies in adult dogs (Suutarinen, 1966; Koehler et al., 1980), rats (Wendling et al., 1967), and humans (Bristow et al., 1971), we observed that hypercapnia did not alter MAP of chicks (Fig. 5). However, Tenney (1956) and Bloom et al. (1977) reported increases in MAP of adult cats and calves exposed to hypercapnia, suggesting that the species and the CO2 levels could affect MAP. As commented above, CO2 exposure promoted a decrease in fH of most of our chicks, which is consistent with the responses observed in other species (Talwar and Fahim, 2000). An increased peripheral resistance could explain the lack of MAP effect despite the decreased fH . However, this effect should be assessed in more detail in future studies. Hypercapnia promoted a decrease in Tb in all chicks. Some authors believe the hypercapnia-induced reduction in Tb results from the increased heat loss instead of from reduction in the metabolic rate (reviewed by Bícego et al., 2007). The increased ventilation (Fig. 4) may contribute to higher evaporative heat loss. Our results in d21 chicks corroborate previous studies that also demon-
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strated increased VO2 during hypercapnia in adult rats (Lai et al., 1981). In conclusion, in resting conditions no gender effect was observed in the cardiorespiratory and metabolic variables analyzed in 10 and 21 days old chicks, the differences in ventilation and heart rate between ages seem to be related to the distinct metabolic demands of these phases. Female chicks, especially at age of 21 days after hatch, seem to be more sensitive to hypercapnia than the other groups, which might be related to differences in sexual hormone secretion even in early age. Uncited references
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Butler (2004) and Szdzuy and Mortola (2008). Acknowledgments This work was supported by Fundac¸ão de Amparo à Pesquisa Q5 do Estado de São Paulo (FAPESP). This study is part of the study developed by Livia P. Espinha to obtain a master’s degree at the Graduate Program in Animal Science from FCAV-UNESP. LPE was the recipient of a FAPESP (no. 2011/02280-5) graduate scholarship. References Almeida, M.C., Steiner, A.A., Coimbra, N.C., Branco, L.G., 2004. Termoeffector neuronal pathways in fever: a study in rats showing a new of the locus coeruleus. J. Physiol. 558, 283–294. Bartlett, D.J.R., Tenney, S.M., 1970. Control of breathing in experimental anemia. Respir. Physiol. 10, 384–395.
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Bavis, R.W., Kilgore Jr., D.L., 2001. Effects of embryonic CO2 exposure on the adult ventilatory response in quail: does gender matter? Respir. Physiol. 126 (3), 183–199. Biancardi, V., Bícego, K.C., Almeida, M.C., Gargaglioni, L.H., 2008. Locus coeruleus noradrenergic neurons and CO2 drive to breathing. Pflug. Arch. 455 (6), 1119–1128. Biancardi, V., Bícego, K.C., Gargaglioni, L.H., 2014. ATP in the locus coeruleus as a modulator of cardiorespiratory control in unanaesthetized male rats. Exp. Physiol. 99 (1), 232–247. Bícego, K.C., Barros, R.C., Branco, L.G., 2007. Physiology of temperature regulation: comparative aspects. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 147 (3), 616–639. Bloom, S.R., Edwards, A.V., Hardy, R.N., 1977. Adrenal and pancreatic endocrine responses to hypoxia and hipercapnia in the calf. J. Physiol. 269 (1), 131–154. Bouverot, P., 1978. Control of breathing in birds compared with mammals. Physiol. Rev. 58, 604–655. Bouverot, P., Leitner, L.M., 1972. Arterial chemoreceptors in the domestic fowl. Respir. Physiol. 15 (3), 310–320. Bristow, J.D., Brown Jr., E.B., Cunninghan, D.J., Goode, R.C., Howson, M.G., Sleight, P., 1971. The effects of hipercapnia, hypoxia and ventilation on the baroreflex regulation of the pulse interval. J. Physiol. 216 (2), 281–302. Butler, P.J., 2004. Metabolic regulation in diving birds and mammals. Respir. Physiol. Neurobiol. 141 (3), 297–315. Cobb, 2012. Broiler Management Guide., pp. 1–68, Available in http://67.43.0.82/ docs/default-source/guides/cobb-broiler-management-guide-(english). pdf?Status=Temp&sfvrsn=4 (accessed October 2013). Colby, C., Kilgore, D.L., Howe, S., 1987. Effects of hypoxia and hipercapnia on VT , f, and VI of nestling and adult bank swallows. Am. J. Physiol. 253, 854–860. Dejours, P., 1981. Principle of Comparative Respiratory Physiology, 2nd ed. Elsevier, New York. Dias, D.P., Oliveira, M., Salgado, H.C., Fazan Jr., R., 2010. Ovariectomy does not affect the cardiac sympathovagal balance of female SHR but estradiol does. Braz. J. Med. Biol. Res. 43 (10), 969–975. Dodd, G.A.A., Scott, G.R., Milsom, W.K., 2007. Ventilatory roll off during sustained hypercapnia is gender specific in pekin ducks. Respir. Physiol. Neurobiol. 156, 47–60. Fedde, M.R., Nelson, P.I., Kuhlmann, W.D., 2002. Ventilatory sensitivity to changes in inspired and arterial carbon dioxide partial pressures in the chicken. Poult. Sci. 81 (6), 869–876. Florez-Duquet, M., Peloso, E., Satinoff, E., 2001. Fever and behavioral thermoregulation in young and old rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280, 1457–1461. Gonzales, E., Kondo, N., Saldanha, E.S., Loddy, M.M., Careghi, C., Decuypere, E., 2003. Performance and physiological parameters of broiler chickens subjected to fasting on the neonatal period. Poult. Sci. 82 (8), 1250–1256. Holley, H.S., Behan, M., Wenninger, J.M., 2012. Age and sex differences in the ventilatory response to hypoxia and hypercapnia in awake neonatal, pre-pubertal and young adult rats. Respir. Physiol. Neurobiol. 180 (1), 79–87. Jones, D.R., Purves, M.J., 1970. Effect of carotid body denervation upon the respiratory response to hypoxia and hypercapnia in the duck. J. Physiol. 211, 295–309. Kamimura, K., Nishimura, H., Bailey, J.R., 1995. Blockade of beta-adrenoceptor in control of blood pressure in fowl. Am. J. Physiol. 269, 914–922. Kawashiro, T., Scheid, P., 1975. Arterial blood gases in undisturbed resting birds: measurements in chicken and duck. Respir. Physiol. 23 (3), 337–342.
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Koehler, R.C., Mcdonald, B.W., Krasney, J.A., 1980. Influence of CO2 on cardiovascular response to hypoxia in conscious dogs. Am. J. Physiol. 239 (4), 545–558. Lai, Y.L., Lamm, J.E., Hildebrandt, J., 1981. Ventilation during prolonged hypercapnia in the rat. J. Appl. Physiol. 51, 78–83. Malan, A., 1973. Ventilation measured by body plethysmography in hibernating mammals and in poikilotherms. Respir. Physiol. 17 (1), 32–44. Meltzer, A., 1983. Thermoneutral zone and resting metabolic rate of broilers. Br. Poult. Sci. 24 (4), 471–476. Milsom, W.K., 2002. Phylogeny of CO2 /H+ chemoreception in vertebrates. Respir. Physiol. Neurobiol. 141 (1–2), 29–41. Milsom, W.K., Jones, D.R., Gabbott, G.R.J., 1981. On chemoreceptor control of ventilatory responses to CO2 in unanesthetized ducks. J. Appl. Physiol. 50, 1121–1128. Mortola, J.P., 2004. Ventilatory response to hyperoxia in the chick embryo. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 137 (4), 723–730. Mortola, J.P., 2009. Gas exchange in avian embryos and hatchlings. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 153 (4), 359–377. Mortola, J.P., Toro-Velasquez, P.A., 2014. Breathing pattern and ventilatory chemosensitivity of the 1-day old Muscovy duck (Cairina moschata) in relation to its metabolic demands. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 167, 35–39. Nishimura, H., Nakamura, Y., Taylor, A.A., Madey, M.A., 1981. Renin–angiotensin and adrenergic mechanisms in control of blood pressure in fowl. Hypertension 3, 41–49. Nishimura, H., Xi, Z., Zhang, L., Kempf, H., Wideman, R.F., Corvol, P., 2001. Maturationdependent neointima formation in fowl aorta. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 130, 39–54. Osborne, J.L., Burger, R.E., Stoll, P.J., 1977. Dynamic responses of CO2 -sensitive avian intrapulmonary chemoreceptors. Am. J. Physiol. 233 (1), 15–22. Patrone, L.G., Bícego, K.C., Hartzler, L.K., Putnam, R.W., Gargaglioni, L.H., 2014. Cardiorespiratory effects of gap junction blockade in the locus coeruleus in unanesthetized adult rats. Respir. Physiol. Neurobiol. 190, 86–95. Ruiz-Feria, C.A., Zhang, D., Nishimura, H., 2004. Age- and sex-dependent changes in pulse pressure in fowl aorta. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 137 (2), 311–320. Suutarinen, T., 1966. Cardiovascular response to changes in arterial carbon dioxide tension. An experimental study on thoractomized dogs. Acta Physiol. Scand. Suppl. 266, 1–76. Szdzuy, K., Mortola, J.P., 2008. Ventilatory chemosensitivity and thermogenesis of the chicken hatchling after embryonic hypercapnia. Respir. Physiol. Neurobiol. 162 (1), 55–62. Talwar, A., Fahim, M., 2000. Hemodynamic responses to hypoxia and hypercapnia during acute normovolemic hemodilution in anesthetized cats. Jpn. J. Physiol. 50 (2), 227–234. Tenney, S.M., 1956. Sympatho-adrenal stimulation by carbon dioxide and the inhibitory effect of carbonic acid on epinephrine. Am. J. Physiol. 187 (2), 341–346. Wendling, M.G., Eckstein, J.W., Abboud, F.M., 1967. Cardiovascular responses to carbon dioxide before and after beta-adrenergic blockade. J. Appl. Physiol. 22 (2), 223–226. Wenninger, J.M., Olson Jr., E.B., Cotter, C.J., Thomas, C.F., Behan, M., 2009. Hypoxic and hypercapnic ventilatory responses in aging male vs. aging female rats. J. Appl. Physiol. 106, 1522–1528.
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Age and gender influence the cardiorespiratory function and metabolic rate of broiler chicks during normocapnia and hypercapnia Lívia P. Espinha a,b , Fernando A. Souza a , Aretuza C. Capalbo a , Kênia C. Bícego a,b , Marcos Macari a , Luciane H. Gargaglioni a,b,∗ a
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Department of Animal Morphology and Physiology, São Paulo State University – (UNESP FCAV), Jaboticabal, SP, Brazil National Institute of Science and Technology in Comparative Physiology (INCT-FisiologiaComparada), Brazil
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Article history: Accepted 29 May 2014 Available online xxx
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Keywords: Bird CO2 chemosensitivity Ventilation Thermoregulation Metabolic rate
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Pulmonary ventilation (VE ), body temperature (Tb), mean arterial pressure (MAP), heart rate (fH ) and ·
metabolic rate (VO2 ) were measured in 10 (d10)- and 21 (d21)-day-old male and female chicks exposed ·
to 7% CO2 . Under normocapnia, VE was higher in d10 chicks than in d21 due to a higher tidal volume; in females a higher respiratory frequency (fR ) was also observed. The d10 birds presented higher fH and ·
VO2 . The d21 females showed the highest CO2 ventilatory response due to increased fR . MAP did not change during hypercapnia while a hypercapnic bradycardia occurred, except in d21 females. Hypercap·
nia induced a drop in Tb in all groups and an increase in VO2 in d21 males. Overall, no gender effect is observed in cardiorespiratory and metabolic variables in d10 and d21 chicks under normocapnia, the ·
differences in VE and fH between ages may be related to distinct metabolic demands of these phases. The d21 female chicks seem to be more sensitive to hypercapnia. © 2014 Published by Elsevier B.V.
1. Introduction
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Hypercapnia is a powerful stimulus for air breathing vertebrates and evokes several compensatory responses in birds, such as an increase in ventilation (Bouverot, 1978; Mortola, 2004). When challenged by CO2 , most avian species increase tidal volume, while the response in breathing frequency is more variable (Bouverot, 1978). To detect changes in CO2 /pH, birds possess peripheral (located in the carotid bodies), central (located in the central nervous system), and intrapulmonary chemoreceptors that are highly sensitive to CO2 (Milsom, 2002). Respiratory response to hypercapnia was demonstrated to be different in adult male and female ducks. According to Dodd et al. (2007), the acute hypercapnic ventilatory response is lower in females than in males. Cardiorespiratory and metabolic responses to hypercapnia were also demonstrated in birds in early life such as in chicken (Mortola, 2009) and in duck hatchlings (Mortola and Toro-Velasquez, 2014);
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∗ Corresponding author at: Departamento de Morfologia e Fisiologia Animal, Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista Júlio de Mesquita Filho, Via de acesso Paulo Donato Castellane s/n, 14870-000 Jaboticabal, SP, Brazil. Tel.: +55 16 32092656; fax: +55 16 32024275. E-mail addresses: [email protected], [email protected] (L.H. Gargaglioni).
however no information about gender differences was provided. At least in mammals, cardiorespiratory function and metabolic rate under normocapnic and hypercapnic conditions depend on gender and age in young animals (Wenninger et al., 2009). Male rats undergo changes in ventilatory responses to CO2 during early development, while females show little or no age-dependent change in ventilatory responses to hypercapnia. From 10 to 15 days after birth male rats show a more pronounced response to CO2 than females, but this difference is no longer observed in adult animals (Holley et al., 2012). It is intriguing that such differences can be observed during early life. As far as we know, the gender influence on physiological responses to hypercapnia is still a matter of investigation in prepubertal birds. Therefore, in the present study, we used precocial young birds, 10 and 21 days old chickens, to compare cardiorespiratory and metabolic responses to CO2 in males and females.
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2. Methods
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2.1. Animals
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Experiments were performed on unanesthetized 10- and 21day-old broilers (Gallus gallus – Cobb 500® ). The animals had free access to water and food and were housed in a room with a
http://dx.doi.org/10.1016/j.resp.2014.05.013 1569-9048/© 2014 Published by Elsevier B.V.
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1981) assuming that chamber was fully saturated. Tidal volume and consequently VE were corrected by body mass.
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controlled environment (Cobb, 2012) and a 12:12 h light:dark cycle (lights on at 7:00 AM). The methods were approved by the local ethical committee (CEUA – Comissão de Ética no Uso de Animais – FCAV-UNESP; Protocol: 026368/11).
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2.2. Surgery
The catheter was connected to a pressure transducer on the experiment day, and the birds were allowed free movement. The pulsatile arterial pressure (PAP) was measured with a pressure transducer (TSD 104A, Biopac systems, USA) connected to an amplifier (DA 100C, Biopac systems, USA). The heart rate (fH ) and mean arterial pressure (MAP) were quantified from the PAP recording using the same system (MP100 ACE, Biopac systems, USA). PAP and
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Two days before the experiments, a 20 cm long catheter [PE-10 connected to PE-50 (Clay Adams, Parsippany, NJ)] was inserted into the abdominal aorta through the femoral artery while the animals were under anesthesia from an intramuscular injection of 30 mg/kg of ketamine + 1.5 mg/kg of xylazine. The free end of the catheter was fixed in the interscapular area. A portion of the leg and abdomen were shaved, and the skin was sterilized with betadine solution and alcohol. For body temperature (Tb) measurements, a temperature datalogger (SubCue, Calgary, AB, Canada) was implanted in the abdominal cavity. The datalogger was programmed to take a reading every 5 min. After the surgery, the animals received two doses of enrofloxacin (10 mg/kg, intramuscular) and flunixin meglumine (2.5 mg/kg, intramuscular) to prevent infection and post-surgical discomfort, respectively. The first dose was given at the end of the surgery, and the second one was administered 6–12 h later.
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2.3. Determination of pulmonary ventilation
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Measurements of pulmonary ventilation (VE ) were performed using the whole body plethysmography method (closed system; Bartlett and Tenney, 1970). Freely moving 10- and 21-day-old chickens were kept in a 5-L and 10-L chamber, respectively; these chambers were ventilated with humidified room air or with a humidified hypercapnic gas mixture containing 7% CO2 in air ·
(White Martins, Sertãozinho, Brazil). During VE measurements, the flow was interrupted, and the chamber was sealed for short periods of time (∼2 min). The pressure oscillations caused by breathing were monitored using a differential pressure transducer (TSD 160A, Biopac Systems, Santa Barbara, USA). The signals were fed into a differential pressure signal conditioner (DA 100C, Biopac Systems, USA), passed through an analog-to-digital converter, and digitized on a microcomputer equipped with data acquisition software (MP100A-CE, Biopac Systems, USA). The sampling frequency was 200 samples s−1 . The results were analyzed using the data analysis software Acqknowledge (v3.8.1 data acquisition system, Biopac Systems, USA). The volume was calibrated during each experiment by injecting 1 mL of air into the animal chamber. The tidal volume (VT ) was calculated using the following formula of Malan (1973): PT TA PB − PC V T = VK × × × PK TR (PB − PC) − TA/Tb × (PB − PR) where PT is the pressure deflection associated with each VT , PK is the pressure deflection associated with injection of the calibration volume (VK), TA is the air temperature in the animal chamber; TR is the room temperature; PB is the barometric pressure; PC is the vapor pressure of water vapor in the animal chamber; Tb is the body core temperature; and PR is the water vapor pressure at ·
Tb. VE and VT are presented at ambient barometric pressure and Tb and expressed as volumes that are saturated with water vapor at this temperature (BTPS). The Tb was monitored by a datalogger (Sub-Cue, Calgary, AB, Canada), and the air temperature in the animal chamber was monitored using a thermoprobe (model 850210, Cole Parmer, Chicago, IL). According to Malan (1973), TR may be slightly lower than TA because of the heating of the chambers by the animals. The PC (the water vapor pressure in the animal chamber) was calculated indirectly using an appropriate table (Dejours,
2.4. Measurements of arterial blood pressure and heart rate
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fH data were collected simultaneously with VE measurements.
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2.5. Determination of arterial pH and gases
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Arterial blood samples were obtained during normocapnia and hypercapnia from a subset of the 10- and 21-day-old chicks via the implanted arterial catheter. 95 L of blood were sampled by a cartridge (EG7+) for immediate analysis of the arterial pH (pHa), arterial carbon dioxide partial pressure (PaCO2 ), arterial oxygen partial pressure (PaO2 ) and bicarbonate (HCO3 − ) with an i-STAT portable blood gas analyzer (i-STAT Analyzer, Abbott Laboratories, NY, USA). Body temperature values were provided to the analyzer for corrections of blood gases and pH. 2.6. Metabolic rate and Tb
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The metabolic rate was measured by indirect calorimetry (oxy·
gen consumption, VO2 ) using closed system respirometry (Almeida et al., 2004). At the end of the normocapnic and hypercapnic periods, the air flow of the chamber was interrupted for 2 min, and the air was continuously sampled by an O2 analyzer (PowerLab System, ADInstruments® /Chart Software, version 7.3, Sydney, Australia). While the chamber was sealed, the oxygen fraction did not drop to less than 19.5%. The percentage of oxygen decay inside the chamber was plotted against time, and the slope of the resulting curve multiplied by the net volume of the chamber (discounted the volume of the animal) divided by the animal mass was used to calculate the rate of oxygen consumption. The VO2 was corrected by body mass and values are presented in STPD (standard conditions of temperature, pressure and dry air). The body temperature was measured using implanted intracoelomic dataloggers (SubCue, Calgary, AT, Canada), at the end of the experiments, which were connected to the computer and the obtained temperature values were corrected according to the specifications in the manufacturer’s manual. 2.6.1. Experimental protocol Two days after surgery, each animal was individually placed in a plethysmography chamber maintained at 27 ◦ C (10 days) and 25 ◦ C (21 days), and the animals were allowed to move freely while the chamber was flushed with humidified air. These temperatures were chosen based on a previous study by Meltzer (1983) that described that 27 ◦ C and 25 ◦ C are considered to be the thermoneutral temperature for 10 and 21 days, respectively. After the animals became ·
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calm (∼30 min), the control VE , PAP and VO2 were measured. Next, a humidified hypercapnic gas mixture (7% CO2 in air, White Martins, Sertãozinho, Brazil) was flushed through the chamber for 30 min, ·
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and the VE , PAP and VO2 were measured at minutes 5, 10, 20, and 30 of the hypercapnia. Blood samples were collected 15 min before the hypercapnic challenge and during the hypercapnic exposure (30 min) for pHa, PaCO2 , PaO2 , and HCO3 − measurements. The Tb was recorded in 5-minute intervals throughout the experiment.
Please cite this article in press as: Espinha, L.P., et al., Age and gender influence the cardiorespiratory function and metabolic rate of broiler chicks during normocapnia and hypercapnia. Respir. Physiol. Neurobiol. (2014), http://dx.doi.org/10.1016/j.resp.2014.05.013
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Table 1 Body mass (g) at birth, 10 and 21 days of female and male chicks.
Birth 10 days 21 days
Male (n = 12)
Female (n = 12)
P
47.1 ± 1.5 137 ± 6.7 666 ± 26
47.4 ± 2.9 129 ± 9.3 609 ± 15.5
ns ns ns
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2.6.2. Data processing and analysis The results are reported as means ± SEM. The data analyses were performed using SAS software (SAS System, version 9.1) and were tested for normality of deviation (Cramer Von-Mises criterion) and homoscedasticity (Levene test). The normocapnic and hypercapnic conditions were analyzed separately to determine the gender and age effect. The effect of hypercapnia on VT , fR , VE , MAP, fH , blood gases, HCO3 − and pHa over the time course was evaluated by two-way analysis of variance (repeated measures). For blood gases, HCO3 − and pHa, since an interaction (time × treatment) was observed, point-by-point comparisons of mean values between vehicle and drug treatments during normocapnia and hypercapnia were performed by one-way ANOVA. Values of P ≤ 0.05 were considered significant.
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3. Results
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All data met the assumption of normality of deviation (Cramer Von-Mises criterion) and homoscedasticity. Body weight of hatchlings, 10- and 21-day-old broilers is presented in Table 1. No gender difference was observed.
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3.1. Normocapnia
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The 10-day-old broiler chicks (males and females) presented ·
higher VE under normocapnic conditions (P < 0.001) compared with that in the d21 animals due to a higher VT (Fig. 1). In females, the ·
higher VE in d10 was also caused by a higher fR compared to d21 (P < 0.01). No difference was observed between males and females at the same age. Blood gases and pHa did not differ between males and females. The pHa was higher in the d21 males than in d10 males, indicating an age effect (P < 0.05) (Table 2). Gender and age did not affect MAP (Fig. 2). The 10-day-old animals presented higher fH values than did the 21-day-old (P < 0.05). There was no difference in Tb between genders at the same age and no difference between ages at the same gender. Age·
and gender-based differences in the VO2 /kg were observed in the
·
Fig. 1. Ventilation (VE ), tidal volume (VT ) and respiratory frequency (fR ) of 10 and 21 days old chickens under normocapnic conditions.a,b Means followed by different letters are statistically different (P < 0.05).Values are reported as means ± SEM.
·
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animals (Table 3). A higher VO2 /kg was observed in the younger animals compared with the older animals (P < 0.01).
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3.2. Hypercapnia
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·
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Hypercapnia increased VE significantly in both the d10 and d21 male and female broiler chicks due to the enhanced VT relative to that in normocapnia (effect of time: P < 0.001, no interaction effect) (Fig. 3). The 21-day-old females presented higher ventilatory response to hypercapnia compared to all groups (effect of treatment: P < 0.001, no interaction) due to a higher fR (effect of treatment: P < 0.001, no interaction). Hypercapnia caused a decrease in pHa and an increase in PaCO2 and PaO2 in all groups (P < 0.001). No changes in HCO3 − were observed during normocapnia and hypercapnia. The pHa was higher in d21 males than in d10 males in normocapnia, indicating an age effect (P < 0.05) (Table 2).
No difference was observed in MAP among groups (Fig. 4). Hypercapnia induced bradycardia in all groups (effect of time: P < 0.001), except for 21-day-old females (effect of treatment, P < 0.01, no interaction effect). Hypercapnia caused a significant decrease in Tb in all groups (effect of time: P < 0.001, no interaction). No difference was observed in Tb among groups (Fig. 5). ·
Both males and females presented higher VO2 related to body mass in d10 than in d20 chicks (Table 3). Hypercapnia induced an ·
increase in VO2 /kg in the 21-day-old males (effect of time: P < 0.05, no interaction). The 10-day-old animals presented a higher ventilatory equiv·
·
alent (VE /VO2 ) in normocapnia and hypercapnia than did the 21-day-old animals (P < 0.001). The d10 males and females and the
Please cite this article in press as: Espinha, L.P., et al., Age and gender influence the cardiorespiratory function and metabolic rate of broiler chicks during normocapnia and hypercapnia. Respir. Physiol. Neurobiol. (2014), http://dx.doi.org/10.1016/j.resp.2014.05.013
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4 Table 2
Q6 Values of arterial pH (pHa), arterial oxygen partial pressure (PaO2 ), arterial carbon dioxide partial pressure (PaCO2 ), and plasma bicarbonate (HCO3 − ) of 10 and 21 days old male and female chicks during normocapnia (0% CO2 ) and submitted to hypercapnia (7% CO2 ). 0% CO2
7% CO2
Male (n = 6) 10 days pHa PaCO2 (mmHg) PaO2 (mmHg) HCO3 −
7.56 29.20 92.92 25.91
± ± ± ±
0.01 0.9 1.9 0.9
Male (n = 5) 21 days pHa PaCO2 (mmHg) PaO2 (mmHg) HCO3 −
7.59 29.04 94.50 27.47
± ± ± ±
0.01b 1.2 1.0 1.3
Female (n = 5) 7.56 28.40 92.69 25.02
± ± ± ±
0.01 1.2 1.2 0.9
Female (n = 4) 7.58 28.18 94.50 25.90
± ± ± ±
0.01 1.0 1.4 0.9
Male (n = 6) 7.32 53.60 113.33 26.93
± ± ± ±
Female (n = 5)
0.02a 1.9a 1.7a 1.7
7.33 50.52 113.20 26.00
Male (n = 5) 7.34 55.06 113.00 28.44
± ± ± ±
± ± ± ±
0.02a 2.2a 1.3a 2.2
Female (n = 4)
0.02a 2.5a 1.6a 0.6
7.36 51.90 114.75 25.00
± ± ± ±
0.02a 3.9a 1.1a 1.9
Values are reported as means ± SEM. a Means difference between 0% and 7% CO2 in the same gender. b Means difference between ages in the same gender.
Table 3 Oxygen consumption (mL kg−1 min−1 STPD) of 10 (d10) and 21 (d21) days old male and female chicks during normocapnia (0% CO2 ) and exposure to hypercapnia (7% CO2 ). 0% CO2
d10 d21
7% CO2
Male (n = 6)
Female (n = 6)
Male (n = 6)
Female (n = 6)
34.16 ± 3.8 17.15 ± 2.5a
31.77 ± 3.7 22.02 ± 3.1a
41.01 ± 3.0 25.99 ± 2.3a , b
41.72 ± 3.7 23.35 ± 2.8a
Values are reported as means ± SEM. a Means difference between ages in the same gender. b Means difference between 0% and 7% CO2 in the same gender.
·
·
d21 females presented an increased VE /VO2 response to hypercapnia (P < 0.001) (Fig. 6). 4. Discussion
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This study evaluated the cardiorespiratory and metabolic responses to hypercapnia (7% CO2 in air) in male and female broiler chicks at 10 (d10) and 21 (d21) days after hatch. 4.1. Normocapnia
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Younger broiler chicks, both male and female, presented higher ventilation under normocapnia than did the older chicks. This difference was due to an enhanced VT in males and an increased VT and fR in females. It seems that a different scenario is observed in pre-pubertal rats, since there is no age effect on ventilation in females while it is observed the lowest pulmonary ventilation at days 12–13 after birth in males (Holley et al., 2012). Despite the higher ventilation in d10 chicks, no difference in the blood parameters and HCO3 − was found (Table 2). However, pH was slightly higher only in the d21 males (7.59) than in the d10 males (7.56) in normocapnia. The higher ventilation observed in the younger animals could be related to the higher mass-specific ·
Fig. 2. Mean arterial blood pressure (MAP) and heart rate (fH ) of 10 and 21 days old chickens in normocapnia. a,b Means followed by different letters are statistically different (P < 0.05). Values are reported as means ± SEM.
236
metabolic rate (Table 3); however V E /VO2 is lower in d21 chicks compared to d10. The reason for lower ventilation in the older chicks (Fig. 1) may be a consequence of fast growing selection. Further studies in these animals are necessary to clarify this issue. Our values for PaCO2 are lower (Kawashiro and Scheid, 1975; Fedde et al., 2002) and pH are higher (Kawashiro and Scheid, 1975) compared to the values found for adult white leghorn. We believe that the differences may firstly be due to the distinct ages and lineages used by Kawashiro and Scheid (1975) and Fedde et al. (2002). Secondly, we have used unanesthetized and intact broiler chicks
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F10 (n=6) M10 (n=6) F21 (n=6) M21 (n=6)
500 400 300 200 100 0
7% CO 2 -30
-20
-10
0
10
20
110 90 70
30
50
Time (min) 600
-15
-5
5
25
35
-15
-5
5 15 25 Time (min)
35
130
400 300 200 100 0
7% CO2 -30
-2 0
-10
0
10
20
110 90 70
30
50
Time (min)
200
fR (% Baseline)
15
500
fH (% baseline)
VT (% Baseline)
M10 (n=6) F10 (n=5) M21 (n=5) F21 (n=4 )
130 MAP (% baseline)
.
VE (% Baseline)
600
5
Fig. 4. The relative changes in mean arterial blood pressure (MAP) and heart rate (fH ) of 10 and 21 days old chickens in 10 and 21 days old chickens. Data are expressed relative (%) to levels before CO2 administration as means ± SEM. The 21-day-old female broiler chickens presented lower bradycardic response to hypercapnia compared to all groups (two-way ANOVA; effect of treatment, P < 0.01, no interaction).
150 100 50
M10 (n=6 ) F10 (n=6 ) M21 (n=6) F21 (n=6 )
2
0
7% CO2 -2 0
-10
0
10
20
30
Time (min) ·
Fig. 3. The relative changes in ventilation (VE ), tidal volume (VT ) and respiratory frequency (fR ) in 10 and 21 days old chickens. Data are expressed relative (%) to levels before CO2 administration as means ± SEM. The 21-day-old female broiler chickens presented higher ventilatory response to hypercapnia compared to all groups (twoway ANOVA; effect of treatment: P < 0.001, no interaction).
1
Δ Tb (oC)
-30
0 -1 7% CO2
-2 -15 -10 -5
265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283
and performed our experiments two days after surgery, whereas Kawashiro and Scheid (1975) performed the experiments 2 h after surgery and Fedde et al. (2002) used decerebrated animals, therefore it is possible that the differences in arterial pH and blood gases may be caused by the effect of the anesthetics or decerebration. As mentioned above, pH of d21 male chicks was 0.03 higher than d10 males. Since PaCO2 values were very similar between groups, the higher pH must be due to a higher HCO3 − levels (25.91 ± 0.9 mmol/L in d10 compared to 27.47 ± 1.3 mmol/L in d21), despite the fact that no statistical difference was observed. To the best of our knowledge, there is no data in the literature about blood gases and pH in young broilers to compare with our present results. Similar to ventilation, fH was not different between males and females in both ages, but older animals had lower fH (Fig. 2). This decrease most likely occurs because of the lower mass-specific metabolic rate in the larger animals. Despite these differences, Tb did not change among groups, indicating higher heat loss in d10 chicks. In fact, ventilation is almost 8 times higher in these chicks, which may contribute to evaporative heat loss. Additionally, the
0
5
10
15
20
25
30
35
Time (min) Fig. 5. Change in body temperature (Tb) of male (M) and female (F) 10 and 21 days old chicks exposed to 7%CO2 . Values are reported as means ± SEM.
similarity of Tb between males and females corroborates the results in young and adult rats from Florez-Duquet et al. (2001). Age and gender did not affect MAP of our chicks during normocapnia (Fig. 2). Previous studies have shown that MAP is higher in adult leghorn males than in females (Nishimura et al., 1981; Kamimura et al., 1995; Nishimura et al., 2001); however, our chicks (10–21-day-old) were much younger than those used in those studies (35–56 weeks). In the latter case, the higher MAP in males could be related to the higher plasma catecholamine levels (Ruiz-Feria et al., 2004). Furthermore, Nishimura et al. (2001) also demonstrated that the MAP of 2–3 weeks old male chicks is ∼150 mmHg and tends to increase with age. In our study, MAP of d10 and d21 chicks was ∼110 mmHg. The difference in these results could reflect the difference in strains (egg-laying versus broilers).
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6
. .
VE /VO2
Male 10d Female 10d Male 21 d Female 21d 50 45 40 35 30 25 20 ** 15 10 5 0
**
0
7 % CO2 ·
Fig. 6. Changes in ventilatory equivalent (VE /VO2 ) from the air value (21% inspired O2 and 0% inspired CO2 ) during exposure to hypercapnia (high inspired carbon dioxide concentration, 7% CO2 ), in chickens of 10 and 21 days. ** Statistically significant difference between ages (P < 0.05, ANOVA).
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4.2. Hypercapnia Hypercapnia resulted in an increased ventilation and tidal volume in all animals with no change in breathing frequency. This ·
strategy helps reduce dead space, makes VE more efficient, and has also been reported in rats (Holley et al., 2012). A previous study by Dodd et al. (2007) in adult ducks demonstrated that hypercapnia causes an augmentation in ventilation due to an increase in tidal volume (∼2-fold), and, to a lesser extent, due to an increase in breathing frequency (∼1.4-fold). In other studies with adult birds, the breathing frequency decreased during hypercapnia (Milsom et al., 1981; Jones and Purves, 1970; Bouverot and Leitner, 1972; Osborne et al., 1977; Colby et al., 1987). Therefore, the different responses to hypercapnia observed in our study and those of others may be due to the different CO2 levels. Since we have used a high level of CO2 (7%) compared to milder exposures (5%) in the other studies; our exposure may represent a major stress, which different animals could react to in various ways. Therefore, the differences observed in our study may not only reflect differences in CO2 chemosensitivity, but species, age- or gender-differences in the response to CO2 stress. At least for hypoxic stress, this chicken lineage seems to be less resistant and very susceptible to 10% O2 exposure (unpublished data). Therefore, our chicks might be more sensitive to CO2 stress, which could explain the VO2 increase during hypercapnia in d21 males and a tendency of increase in the other groups (Table 3). As expected, CO2 exposure promoted an increase in PaCO2 , PaO2 and a decrease in pHa in all chicks (Table 2), with no difference among groups. We observed a nonsignificant tendency of an increase in HCO3 − during hypercapnia. These data are similar to those found in adult rats exposed to 7% CO2 (Biancardi et al., 2008, 2014; Patrone et al., 2014). Surprisingly, d21 females presented a higher CO2 ventilatory response than did the other groups, indicating that these chicks are more sensitive to hypercapnia. Contrasting results were provided by Dodd et al. (2007) in adult ducks and Bavis and Kilgore (2001) in adult quails. According to these authors, the CO2 response in adult females is lower than that in adult and juvenile male ducks (5% CO2 – Dodd et al., 2007) and does not differ between genders in quails (6% CO2 – Bavis and Kilgore, 2001). These contrasting responses compared to the current study could be related to age, species or/and CO2 levels because we used pre-pubertal 10- and 21-dayold broiler chicks exposed to 7% CO2 . Furthermore, Holley et al.
(2012) found more pronounced responses of male 14–15 days old rats to 7% CO2 . At least in these animals, this age represents a critical period for the development of the ventilatory chemosensitivity. Interestingly, the authors observed a higher plasma concentration of estradiol in male 12–13 days old rats, suggesting a hormonal modulation in CO2 chemoreception (Holley et al., 2012). Regarding broiler chicks, it was demonstrated that females present much higher plasma estradiol levels than males as early as just after hatching, a pattern that is maintained up to 42 days (Gonzales et al., 2003). As we observed differences in hypercapnic ventilatory response of d21 but not d10 females compared to males, the sexual hormones modulation in CO2 response in broiler chicks warrants further investigation. Increased estradiol influence might also explain why d21 females, in contrast to the other groups, did not present hypercapnia-induced bradycardia (Fig. 5). At least in adult rats, estradiol increases the sympathetic modulation of fH (Dias et al., 2010). In agreement with studies in adult dogs (Suutarinen, 1966; Koehler et al., 1980), rats (Wendling et al., 1967), and humans (Bristow et al., 1971), we observed that hypercapnia did not alter MAP of chicks (Fig. 5). However, Tenney (1956) and Bloom et al. (1977) reported increases in MAP of adult cats and calves exposed to hypercapnia, suggesting that the species and the CO2 levels could affect MAP. As commented above, CO2 exposure promoted a decrease in fH of most of our chicks, which is consistent with the responses observed in other species (Talwar and Fahim, 2000). An increased peripheral resistance could explain the lack of MAP effect despite the decreased fH . However, this effect should be assessed in more detail in future studies. Hypercapnia promoted a decrease in Tb in all chicks. Some authors believe the hypercapnia-induced reduction in Tb results from the increased heat loss instead of from reduction in the metabolic rate (reviewed by Bícego et al., 2007). The increased ventilation (Fig. 4) may contribute to higher evaporative heat loss. Our results in d21 chicks corroborate previous studies that also demon-
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·
strated increased VO2 during hypercapnia in adult rats (Lai et al., 1981). In conclusion, in resting conditions no gender effect was observed in the cardiorespiratory and metabolic variables analyzed in 10 and 21 days old chicks, the differences in ventilation and heart rate between ages seem to be related to the distinct metabolic demands of these phases. Female chicks, especially at age of 21 days after hatch, seem to be more sensitive to hypercapnia than the other groups, which might be related to differences in sexual hormone secretion even in early age. Uncited references
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Q4
Butler (2004) and Szdzuy and Mortola (2008). Acknowledgments This work was supported by Fundac¸ão de Amparo à Pesquisa Q5 do Estado de São Paulo (FAPESP). This study is part of the study developed by Livia P. Espinha to obtain a master’s degree at the Graduate Program in Animal Science from FCAV-UNESP. LPE was the recipient of a FAPESP (no. 2011/02280-5) graduate scholarship. References Almeida, M.C., Steiner, A.A., Coimbra, N.C., Branco, L.G., 2004. Termoeffector neuronal pathways in fever: a study in rats showing a new of the locus coeruleus. J. Physiol. 558, 283–294. Bartlett, D.J.R., Tenney, S.M., 1970. Control of breathing in experimental anemia. Respir. Physiol. 10, 384–395.
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