Effects Of Supplementation With N-carbamylglutamate On Pulmonary Hypertension In Holstein Heifers In Tibet

Four experiments were conducted to investigate:1)the pathophysiology of high altitude induced pulmonary hypertension(HAPH)in Holstein heifer;2)Hematological and vasodilator characteristics for high altitude acclimatization;3)the effects of N-carbamylglutamate(NCG)attenuates high altitude-induced pulmonary hypertension in Holstein heifers;4)prevalence of iron deficiency in Holstein heifers with high altitude pulmonary hypertension,and the association of serum iron with pathological parameters.Experiment 1:Pathophysiology characteristics of HAPH in Holstein heifer.In August 2014,591 Holstein heifers was transported from Xi'an,Shanxi province,China(1,027 meters)to Lhasa,Tibet,China(3,658 meters)and the mortality from brisket disease(BD)in every mouth was recorded.Ten healthy Holstein heifers(Healthy)with mean pulmonary arterial pressure(mPAP)<41 mmHg(510 ±12 kg;body weight:BW)and 10 heifers with HAPH(HAPH:mPAP>49mmHg;480 ± 18 kg BW),with a mean age of 18 ± 2 months,were selected to measure physiological parameters.Six heifers in Healthy group and ten heifers in HAPH group were euthanized,the heart dissected for histopathological analysis.Brisket and ventral edema fluid was collected for analyzing biochemical parameters.Holstein heifers affected with HAPH developed conspicuous edema in the brisket and jaw areas.Heart failure and hypertrophy of the right ventricles were found in heifers with HAPH.The RV/T was higher in HAPH than Healthy group(0.4 vs 0.25;P<0.01).In total,217 heifers died from BD giving an annual mortality incidence risk of 36.72%.Respiratory rates,oxygen saturation and blood velocity were significantly higher in Healthy group(all P<0.001).However,heart rates were higher in heifers with HAPH(P<0.05).Peripheral arterial pressures was significantly higher in HAPH group than Healthy group(P<0.01).Adenosine deaminase,L-lactate dehydrogenase and albumin were significantly higher in brisket edema fluid than ventral fluid(P<0.01).Experiment 2:Hematological and vasodilator characteristics for high altitude acclimatization in Holstein heifers ascended to high altitude.Holstein heifers resided at low altitude(LH;Beijing,China 43 m),and Holstein heifers ascended to high altitude(Lhasa city,Tibet;3,658 m)after one year exposure including hypoxia acclimated Holstein heifers(AH:mPAP<41 mmHg)and Holstein heifers affected by high altitude pulmonary hypertension(HAPH:with mPAP>49 mmHg)and crossbred cattle(CB;Holstein × Tibet cattle)born and resided in Tibet were selected to compare parameters of blood gas,blood cells and vasodilators.Each group included 15 animals with mean age of 18 ± 2 month.The HAPH had greater mPAP than other cattle classes(P<0.01).Alveolar ventilation(as indicated by partial pressure of carbon dioxide;PaCO2)was improved in CB and AH than HAPH and LH(P<0.05).In addition,LH had the greatest and HAPH had the lowest partial pressure of oxygen(PaO2)among the four cattle classes(P<0.05).Erythropoietin and mean corpuscular hemoglobin concentration were greater in AH and HAPH than LH(P<0.05).Serum iron was greater in AH than LA and HAPH(P<0.05).Hypoxia exposure increased nitric oxide and endothelin-1 level in cattle resided in Tibet compared to LH(P<0.05).Additionally,AH had greater brain natriuretic peptides than HAPH and LH,meanwhile AH had greatest C-type natriuretic peptides than other groups.Experiment 3:N-carbamylglutamate attenuates high altitude-induced pulmonary hypertension in Holstein heifers ascended to high altitude.Holstein heifers with mPAP<41mmHg were assigned to a healthy group(Healthy,n=6;mPAP:38.13 ± 1.50 mmHg;557 ± 14 kg BW)and a HAPH affected group(HAPH,n = 6;mPAP:72.28 ± 1.91mmHg;480 ± 18 kg BW)with mPAP>49 mmHg were selected.Heifers in each group were euthanized,lung tissues were collected for histopathological analysis and to evaluate the expression of endothelial nitric oxide synthase(eNOS)and endothelin-1(ET-1).Ten healthy heifers(497 ± 10.2 kg BW)and 10 HAPH affected heifers(460 ± 36.5 kg BW)were supplemented with NCG(20 g/day per heifer)for 4 w.Physiological parameters and blood samples were determined on d-1 and d 28 of feeding trial.Seven hundred Holstein heifers were transported from low elevation(2,200 m)to Lhasa.After feeding 20g/d NCG for 100 d,12 heifers in NCG group(20g NCG/d per heifer)and 17 in None-NCG group(without NCG supplementation)were randomly selected to measure physiological parameters.Expression of eNOS in small pulmonary arteriole intima were higher in the Healthy than HAPH group(P<0.01),whereas HAPH group displayed significantly thicker media and adventitia than Healthy group(P<0.05).The mRNA and protein level of eNOS were higher in the lung of Healthy than in HAPH group(P<0.01).NCG supplementation decreased mPAP and ammonia(all P = 0.001),whereas increased eNOS,arginine,plasma NO expression(all P<0.05).Dietary supplementation with NCG(100 days)decreased mPAP,heart rate,systolic pressure and morbidity of HAPH(1/12 vs 9/17;P<0.05).Experiment 4,Prevalence of iron deficiency and associations with disease phenotypes in Holstein heifers with high altitude pulmonary hypertension.We randomly selected healthy heifers(n = 10;497 ±10.2 kg BW)with mPAP<41 mmHg and HAPH affected heifers(n = 12;460 ± 36.5 kg BW)with mPAP>49 mmHg.Physiological parameters,blood cells,blood gas,total iron binding capacity(TIBC),and transferrin saturation(TRFs)were also.measured.Six heifers in each group were slaughtered and their liver,spleen,and duodenal segments were collected to measure iron deposition.Iron deficiency was more common in heifers with HAPH(100%)than in healthy animals(30%).Consistent with this,the levels of hemoglobin(P = 0.014),mean corpuscular hemoglobin(P = 0.004),hematocrit(P =0.004),TIBC(P=0.066),TRFs(P<0.001),and coccygeal arterial oxygen saturation(PaO2:P<0.001)were higher in the healthy group than in the HAPH group.Iron deposition in the liver,spleen,and intestinal mucosa was higher in the HAPH group animals than in healthy group(all P<0.05).Healthy heifers were found to have morphologically normal RBCs,while iron-deficiency induced poikilocytosis was also observed in the heifers with HAPH.Serum iron levels were inversely correlated with mPAP(r=-0.67)and systolic blood pressure(r =-0.65).but positively correlated with an increased rate of breathing(r = 0.66)and higher TIBC(r = 0.46),TRFs(r = 0.50),MCH,mean corpuscular volume(r =0.75),hematocrit(r = 0.51),and PaO2(r = 0.63).