Pathogenesis And Control Of Pulmonary Hypertension Syndrome In Broilers Using Intermittent Lighting Schedule, Dietary Furosemide And Vitamin C

Despite investigation of the syndrome for many years, ascites or pulmonary hypertension syndrome (PHS) is still a condition that inflicts financial loss on poultry farmers around the world. Approximately 4% of all broilers die from PHS, amounting to a loss estimated at $1 billion annually. So, prevention and cure of PHS are especially important. There are multiple aetiologies of pulmonary hypertension syndrome(PHS). The main cause of which is rapid growth. Thus every drug has its limit to cure the disease. Remanet of drug in meat also is a problem. Early feed restriction could slow broiler growth at early age, reduce ascites incidence, but could induce stress too. Lighting restriction at early age could decrease ascites incidence and stress effectively.1 light (L):3dark (D) lighting program is recognized as best schedule to prevent ascites, but is difficult to apply in China. To implore a simple and effective lighting schedule would be of benefit to prevent ascites. Since the mechanism of Lighting restriction remain unclear, The choose of lighting schedules are difficult.In recent years, most ascites research has centred on production capability and ascites incidence of broilers. The investigations on the aetiology of ascites in chickens include oxygen level, thyroxin, growth hormone and cortin. Pulmonary vascular remodeling is an important pathological feature of hypoxia pulmonary hypertension in mammal, leading to increased pulmonary vascular resistance and reduced compliance. Since vascular remodeling becomes progressively more important as the disease advances, an possibly more fruitful approach may be to target pulmonary vascular remodeling. It has been fould that Pulmonary vascular remodeling also occurred in PHS in broilers. It's mechanism remain unclear. The present study was conducted to evaluate the association of changes of protein kinase Ca (PKCa), platelet-derived growth factor BB(PDGF-BB) and its receptor (PDGF-βreceptor),proliferating cell nuclear antigen (PCNA) and oxygen free radical(OFR) with alterations in pulmonary vascular structure for understanding the pathogenesis of pulmonary vascular remodeling in vito and in vitro, to implore the effect of intermittent lighting schedule,furosemide and vitamin C on pulmonary vascular remodeling respectively. If PKC-a and PDGF actually play a critical role in pulmonary vascular remodeling. Both of them may be to target pulmonary vascular remodeling.Test 1 Effects of intermittent lighting schedules on incidence of pulmonary hypertension syndrome and pulmonary vascular remodeling in broilers.The purpose of the present study was to examine the efficacy of imposing intermittent lighting schedules on reduction of the incidence of ascites(or Pulmonary Hypertension Syndrome,PHS)induced by cool ambient temperature and to investigate its effect on the pulmonary vascular remodeling of broiler chickens. A total of three hundred and twenty broilers were randomly allocated to four rooms at 9 days of age. In one room, the continuous lighting (CL) schedule of 24 L:0D was maintained, and ambient temperature was kept normal. While in other three rooms continuous lighting (CL) schedule of 24L: 0D and intermittent lighting (IL) schedules of 21L:3 D and 19L:5D dark were imposed respectively during the night from 9 to 30 days of age. Chickens in the later three rooms were subjected to low temperature to induce PHS. PHS cases, body weights and feed intake were measured weekly for each group. The ratio of vessel wall area to total area (WA/TA) and mean medial thickness in pulmonary arterioles (mMTPA) were determined . The results indicated that the cool ambient temperature could increase morbidity of PHS in broilers, the values of WA/TA and mMTPA also were increased significantly, whereas the IL schedule could successfully decrease the morbidity of PHS and the values of WA/TA and mMTPA. Birds in IL groups were lighter than that in LC group during light restriction period, but both the final body weights and total feed conversion ratio were not significantly affected by the lighting program in all birds subjected to low temperature. It is concluded that introduction of IL schedules during the early growing period is of benefit to reduce PHS morbidity induced by cool ambient temperature in fast-growing broilers, and attenuated pulmonary vascular remodeling may be involved in the underlying mechanisms.Test 2 Effects of intermittent lighting schedules on the incidence of ascites, the lipid peroxidation and enzymatic antioxidant activity of broilers. The purpose of the present study was to examine the efficacy of intermittent lighting schedules at night in reducing the incidence of ascites induced by cool environment and to test its effect on the lipid peroxidation and activity of enzymatic antioxidant in broilers. A total of three hundred and twenty broilers were randomly aUocated to four rooms at 9 days of age. In one Toom, the continuous lighting (CL) schedule of 24 L:0D was maintained, and ambient temperature was kept normal. While in other three rooms continuous lighting (CL) schedule of 24L:0D and intermittent lighting (IL) schedules of 21L:3 D and 19L:5D dark were imposed respectively during the night from 9 to 30 days of age. Chickens in the later three rooms were subjected to low temperature to induce pulmonary hypertension syndrome(PHS). The results showed that Low temperature significantly increased RV/TV and the ascites morbidity, so were the plasma MDA concentration and the percentages of TWPV. Whereas the plasma SOD activity was decreased. The intermittent lighting (IL) schedules had successfully reduced PHS in broilers. The values of RV/TV, plasma MDA concentration and the percentages of TWPV were reduced significantly, whereas the activity of the plasma SOD were significantly promoted. Body weights (BW) were reduced significantly by light restriction at 20 days of age. However, IL broilers exhibited compensatory growth during the finisher period in such a way that at Day 44, a similar BW as that of their CL counterparts was attained. The results indicated that alleviated the lipid peroxidation, enhanced the enzymatic antioxidant activity and reduced the percentages of the TWPA may be the part mechanism of intermittent lighting (IL) schedule reduced ascites of broiler.Test 3 Effects of intermittent lighting schedules on expression of PKCa in pulmonary arterioles and its association with pulmonary vascular remodeling in broilers induced by cold temperature. The purpose of the present study was to examine the effects of intermittent lighting schedules on expression of protein kinase C a (PKC a) in pulmonary arterioles in broilers exposed to cold temperature and its relationship with pulmonary vascular remodeling. A total of three hundred and twenty broilers were randomly allocated to four rooms at 9 days of age. In one room, the continuous lighting (CL) schedule of 24 L:0D was maintained, and ambient temperature was kept normal. While in other three rooms continuous lighting (CL) schedule of 24L: OD and intermittent lighting (IL) schedules of 21L:3 D and 19L:5D dark were imposed respectively during the night from 9 to 30 days of age. Chickens in the later three rooms were subjected to low temperature to induce pulmonary hypertension syndrome(PHS). PHS incidence was recorded. Malonilaldehyde (MDA) concentration,packed cell volume (PCV), Mean medial thickness (mMTPA), Ratio of vessel wall area to total area (WA/TA) and right to total ventricle (RV/TV) were determined. The expression of PKCa in the pulmonary arterioles labeled by immunohistochemistry and quantified by measuring optical density (OD). The results indicated that the cool ambient temperature could increase morbidity of PHS in broilers. The value of MDA, WA/TA and mMTPA were increased significantly, the same as the OD value of PKCa, whereas the IL schedules could successfully decrease the morbidity of PHS .The value of WA/TA ,mMTPA, MDA and PKCa were decreased too. It was concluded that cold exposure enhanced arterioles PKCa expression, which might involved in the underlying mechanisms of pulmonary vascular remodeling. The introduction of IL schedules were of benefit to attenuate pulmonary vascular remodeling ,which may be correlated with the decrease of PKC a expression.Test 4 Effects of intermittent lighting schedules on expression of PDGF-βreceptor in pulmonary arterioles and its association with pulmonary vascular remodeling in broilers induced by cold ambient temperature. The purpose of the present study was to examine the effects of intermittent lighting schedules on expression of platelet-derived growth factorβreceptor (PDGF-βreceptor) in pulmonary arterioles in broilers exposed to cold temperature and its relationship with pulmonary vascular remodeling. A total of three hundred and twenty broilers were randomly allocated to four rooms. In one room, the continuous lighting schedule (CL) of 24 L: 0D was maintained, ambient temperature kept normal (group NC),while in other three rooms ,the chickens were subjected to cool ambient temperature to induce PHS. The continuous lighting schedule of 24 L:0D (group LC) and the intermittent lighting (IL) schedule of 21 L:3 D(group L1) and 19 L:5 D (group L2) were imposed respectively from 9 to 30 days of age. PHS incidence was recorded. Mean medial thickness (mMTPA), ratio of vessel wall area to total area (WA/TA) in pulmonary arterioles ,ratio of right to total ventricle (RV/TV), malonilaldehyde (MDA) concentration and packed cell volume (PCV) were determined respectively. The expression of PDGF-βreceptor in the pulmonary arterioles labeled by immunohistochemistry and quantified by measuring optical density (OD). The results indicated that the cool ambient temperature could increase the value of WA/TA and mMTPA in pulmonary arterioles, the same as the OD value of PDGF-βreceptor, whereas the intermittent lighting (IL) schedule could successfully decrease the value of WA/TA,mMTPA and PDGF-βreceptor. It was concluded that cold exposure enhanced PDGF-β receptor expression in pulmonary arterioles, which might involved in the underlying mechanisms of pulmonary vascular remodeling. The introduction of intermittent lighting schedules were of benefit to attenuate pulmonary vascular remodeling , which may be correlated with the decrease of PDGF-βreceptor expression in pulmonary arterioles.Test 5 Effects of intermittent lighting schedules on expression of PCNA in pulmonary arterioles and its association with pulmonary vascular remodeling in broilers induced by cold temperature. The objection of the present study was to examine the effects of intermittent lighting schedules on expression of proliferating cell nuclear antigen (PCNA) in pulmonary arterioles in broilers exposed to cold temperature and its relationship with pulmonary vascular remodeling. A total of three hundred and twenty broilers were randomly allocated to four rooms at 9 days of age. In one room, the continuous lighting (CL) schedule of 24 L:0D was maintained, and ambient temperature was kept normal. While in other three rooms continuous lighting (CL) schedule of 24L: 0D and intermittent lighting (IL) schedules of 21L:3 D and 19L:5D dark were imposed respectively during the night from 9 to 30 days of age. Chickens in the later three rooms were subjected to low temperature to induce pulmonary hypertension syndrome(PHS). PHS incidence was recorded. The mean medial thickness (mMTPA), ratio of vessel wall area to total area (WA/TA) in pulmonary arterioles ,Ratio of right to total ventricle (RV/TV), packed cell volume (PCV) were determined. The expression of PCNA in the pulmonary arterioles labeled by immunohistochemistry and quantified by counting the number of the positive PCNA cells and total vascular smooth ceils using computer-image analytic software and then calculating the PCNA index (PI). The results indicated that the cool ambient temperature could increase the value of WA/TA and mMTPA significantly, the same as the PI, whereas the IL schedules could successfully attenuate pulmonary vascular remodeling and PI. It was concluded that introduction of intermittent lighting schedule during the early growing period was of benefit to attenuate pulmonary vascular remodeling induced by cool ambient temperature in fast-growing broilers, and the inhibition of vascular smooth cell proliferation might be involved in the underlying mechanisms.Test 6 Effects of dietary furosemide on incidence of pulmonary hypertension syndrome and pulmonary vascular remodeling in broilers induced by cool environmental temperatures. The objective of the present study was to evaluate the influence of dietary furosemide (FURO) on the incidence of Pulmonary Hypertension Syndrome(PHS) induced by cool ambient temperature and to investigate its effect on the pulmonary vascular remodeling of broiler chickens. A total of two hundred and forty broilers were randomly allocated to three groups. The birds in group A were fed a grower diet and kept in normal ambient temperature from 14 to 44 day of age. Group C and group B were subjected to cool ambient temperature to induce PHS and were fed a grower diet with (group C) or without 0.015 percent furosemide (group B) from 30 to 44 day of age. PHS incidence, body weights were measured weekly for each group. Heart, Lung and blood samples were taken from 10 birds per group randomly selected at 14, 23, 30, 37 and 44 days of age, for the determination of the ratio of vessel wall area to total area (WA/TA), mean medial thickness in pulmonary arterioles (mMTPA), etc. It was found that birds exposed to cold had higher PHS morbidity. However, it was reduced by dietary furosemide supplementation. The value of right to total ventricular weight ratios (RV/TV) and packed red blood cell volume (PCV) in group B were significantly higher than that in group A, also were the value of WA/TA and mMTPA which served as describing pulmonary vessel remodeling. Birds fed 0.015 percent furosemide had significantly lower RV/TV, WA/TA and mMTPA than that in group B, also were body weights, whereas PCV were increased. It was concluded that dietary furosemide reduced morbidity of PHS induced by cool ambient temperature, presumably by reducing pulmonary arterial pressure. Attenuated pulmonary vascular remodeling might be involved in the underlying mechanisms too.Test 7 Effects of dietary vitamin C on mortality of pulmonary hypertension syndrome and pulmonary vascular remodeling in broilers under normal environmental temperatures. The objective of the present study was to evaluate the influence of dietary vitamin C on the mortality of pulmonary hypertension syndrome(PHS) under normal ambient temperature and to investigate its effect on the pulmonary vascular remodeling of broiler chickens. A total of one hundred and fifty broilers were randomly allocated to three groups in one room, in which temperature kept normal. From 21 to 37d, the birds in control group (C) were fed a grower diet, treat 1 (T₁) and treat 2 group(T₂) were fed a grower diet with 200mg/kg and 500mg/kg respectively. PHS mortality and body weights were measured weekly for each group. Heart, Lung and blood samples were taken for the determination of SOD and MDA concentration in blood plasma, packed cell volume (PCV), mean medial thickness in pulmonary arterioles (mMTPA), and the ratio of vessel wall area to total area (WA/TA), right ventricle to total ventricular weight. It was found that birds fed 200mg/kg vitamin C had lower PHS mortality than that in group C. The value of WA/TA,mMTPA of pulmonary artery with outer diameter from 80 to 200μm and PCV vaule were significantly reduced, also were the MDA level, whereas SOD concentration were significantly increased. The birds fed 500mg/kg vitamin C had same PHS mortality as control group. The percentages of WA/TA and mMTPA of pulmonary artery with outer diameter from 50 to 80μm and the concentration of MDA were increased significantly, whereas PCV and SOD concentration were reduced. It was concluded that decreasing PCV value, weeding up extra oxygen free radical (OFR) and attenuating pulmonary artery remodeling might be involved in the underlying mechanisms of prevention PHS by low dose vitamin C, Whereas promoting OFR level and inducing pulmonary artery remodeling might be the cause of high dose vitamin C failing to prevent PHS.Test 8 Effects of protein kinase C inhibitor-Calphostin C on proliferation induced by PDGF-BB in pulmonary artery smooth muscle cells of broilers. To xplore the effects of protein kinase C inhibitor-Calphostin C on proliferation in pulmonary artery smooth muscle cells (PASMC) of broilers induced by PDGF-BB. Pulmonary vascular smooth muscle cells were cultured in vitro and stimulated by PDGF-BB. The effects of Calphostin C on PASMC proliferation were analyzed by using flowcytomerty and cell counting. The PASMC proliferation induced by PDGF-BB was significantly inhibited by Calphostin C and the cells were stagnated on G₀/G₁ phase. The result of Calphostin C inhibition on the PASMC proliferation suggested that proliferation induced by PDGF-BB may be correlated with PKC pathway. Calphostin C may be an agent for prevention of vascular remodeling.Test 9 Effects of melatonin on proliferation of pulmonary artery smooth muscle cells in broilers induced by xanthine-xanthine oxidase conditioned medium. To investigate the effects of X/XO conditioned medium on proliferation of pulmonary artery smooth muscle cells (PASMC) in broilers, and melatonin could or not attenuate PASMC proliferation induced by X/XO conditioned medium. Pulmonary artery smooth muscle cells were cultured in vitro with X/XO endothelial cells conditioned medium to induce proliferation of PASMC. The effect of melatonin on PASMC proliferation were analyzed using flowcytomerty. The MDA level in medium were detected. Compared to normal PAEC medium, X/XO conditioned medium lead to PASMC proliferation, stimulate PASMC from G₀/G₁ phase to S and G₂/M phase. The MDA level in PASMC cultured with X/XO conditioned medium were significantly higher than that in PASMC cultured with normal PAEC medium. The PASMC proliferation induced by X/XO conditioned medium was successfully inhibited by melatonin, which added in medium ahead, and the number of cells on G₀G₁ phase were higher, whereas on S and G₂/M phase lower. The MDA level in melatonin group was less than X/XO group. Eliminating of OFR may be involved in the underlying mechanisms of the melatonin on inhibiting proliferation of PASMC in broilers induced by X/XO conditioned medium.