MOLECULAR ASPECTS OF HYPOXIAL STRESS IN LIVESTOCK ANIMALS By: Dr Wani Ahad M.V.Sc Scholar Animal Biotechnology

Stress The term “stress” was borrowed from the field of physics by Hans Selye (Hans Selye, 1926) And has been widely used in biology to describe a set of physiological and behavioural changes elicited by adverse stimuli He proposed that “stress is a non-specific response of the body to any change”

Stress In 1929, Cannon described stress as the sympatho-adreno medullary (SAM) system attempt to regulate homeostasis when threatened by a variety of adverse stressors (Walter Cannon, 1929) Is the condition “where the environmental demand exceeds the natural regulatory capacity of an organism’’ (Bruce McEwen and Jaap Koolhaas, 2011)

HISTORY Bernard recognized that the stability of the milieu interieur depended on ensembles of compensating mechanisms  (Bernard, 1878) Fifty years later, Walter Cannon (1929) introduced the term “homeostasis” to describe the dynamic, interactive nature of these mechanisms in maintaining the stability of the internal environment

HISTORY Endocrine and neuroendocrine events proceed in an interdependent manner to regulate multiple, variable stress responses, each unique, but influenced by previous events Summers (2001) Thermal stress on livestock particularly cattle and buffaloes decreases oestrus expression and conception rate Upadhya et al., (2007)

STRESS FACTORS ABIOTIC Environmental Managemental Changes in temperature (Heat or Cold) and relative humidity Ventilation High altitude(Hypoxia) Managemental Fear

STRESS FACTORS Hunger Social Pathology Feeding High population density Isolation High population density Mixing Pathology Pain Feeding Hunger Thirst

STRESS FACTORS BIOTIC Diseases: Bacterial Viral Fungal Parasitic

General pathways involved in stress Stressful events can activate the Hypothalamo-Pituitary-Adrenal (HPA) axis or sympatho-adrenomedullary (SAM) system The short term responses are produced by The Fight or Flight Response via SAM pathway and long term responses via HPA pathway HPA axis increases the release of Corticotrophin Releasing Hormone (CRH) from the hypothalamic paraventricular nucleus (Campagne, 2006)

Release of Neurotransmitters (i.e. NE, cholecystokinin, serotonin) Stress pathways (HPA) Stress event Hypothalamus (PVN) CRH Release of Neurotransmitters (i.e. NE, cholecystokinin, serotonin) Ant.pituitary - + ACTH Cortisol Adrenal Gland Catecholamines Energy Metabolism

High Altitude Hypoxia

Hypoxia (Hypoxiation or Anoxemia) Is the reduction of oxygen supply to a tissue below physiological levels despite adequate blood perfusion to the tissue (Illingworth et al., 2014) Is a condition in which the body or a region of the body is deprived of adequate oxygen supply (Woorons & Xavier, 2014)

Causes Low partial pressure of oxygen in the blood Low oxygen inspired air e.g high altitude Inadequate ventilation due to lung disease or depression of breathing by drugs Defective transfer of oxygen from lung alveoli to blood

Causes Low content of oxygen in the blood due to inadequate or abnormal haemoglobin e.g anemia Failure of the heart and circulation to deliver an adequate oxygen supply to the tissues, even though the content in the blood may be normal Poisoning of cells so that they cannot use the oxygen delivered to them

Classification Generalized hypoxia affecting the whole body Local hypoxia affecting a particular region of the body Acute hypoxia a sudden or rapid depletion in the availability of oxygen at the tissue level Chronic hypoxia a usually slow, insidious reduction in tissue oxygenation

Types Anemic hypoxia is due to reduction of the oxygen-carrying capacity of the blood due to decreased total hemoglobin or altered hemoglobin constituents Histotoxic hypoxia is due to impaired use of oxygen by tissues Ischemic hypoxia is the local deficiency of arterial blood in an organ

Types Hypoxic hypoxia is due to insufficient oxygen reaching to the blood Stagnant hypoxia due to the failure to transport sufficient oxygen because of inadequate blood flow e.g Hypo-volumic shock Embolic hypoxia due to an emboli in the blood vessels e,g air,blood clot etc

Counter Mechanism Hypoxia Inducible Factors (HIFs) are important transcription factors in the cellular adaptation to hypoxia by regulating different sets of genes involved in angiogenesis, metabolism and cell homeostasis (Semenza and Wang, 2011) They are heterodimeric transcription factors consisting of two structurally related subunits, one is an oxygen sensitive HIFα subunit (HIF-1α , HIF-2α or EPAS1 and HIF3α)

Hypoxia Inducible Factor and the other is the stable subunit, HIF-1β/ARNT-subunit (Aryl hydrocarbon Receptor Nuclear Translocator) (Wang et al., 1995) HIF-1α is expressed in all cells HIF2α and HIF3α are selectively expressed in certain tissues, including vascular endothelial cells, type II pneumocytes, renal interstitial cells & liver parenchymal cells (Bertout et al., 2008)

HIF1α and HIF1β structures 774/789 aa HIF-1β /ARNT b HLH A PAS B 826 aa HIF-1α TAD C ID N NLS-N NLS-C

Protein Expression as a Function of [O2] Oxygen Concentration Relative HIF-1 Expression HIF-1 expression increases exponentially when O2 concentration decreases. The curve shows a point of inflection around 4-5% O2, which is the O2 concentration in normal human tissues (Semenza GL. 1997)

Mechanism Conserved proline residue in HIF-1α are hydroxylated by prolyl hydroxylase/PHD (oxygen dependent) (Ivan et al., 2001) In normal conditions hydroxylation of proline causes the binding of von Hippel-Lindau tumor suppressor (VHL) protein by an E3 ubiquitin ligase (Kaelin and Ratcliffe, 2008) The binding leads to the ubiquitination of HIF-1α & degradation by proteasome (Giaccia A J et al., 2004)

Mechanism Under hypoxic conditions prolyl hydroxylase is not activated HIF-1α accumulates and translocates into nucleus In the nucleus, it binds to HIF-1β through their HLH and PAS domains forming HIF-1 and binds to HRE present in target genes (Mole et al., 2009; Xia et al., 2009)

HIF-1 is a heterodimer HRE Angiogenesis (VEGF) Glucose metabolism Cell hypoxia HIF-1 HIF-1 HRE HIF-1 Pol II complex CBP/p300 Angiogenesis (VEGF) Glucose metabolism Cell proliferation

Role of HIF-1 Helps normal tissues as well as tumors to survive under hypoxic conditions Stimulates lipid storage and inhibits lipid catabolism through β-oxidation (Bostrom et al., 2006) HIF1α and HIF2α can modulate the expression of cytochrome c oxidase isoforms so as to maximize efficiency of the ETC (Gordan et al., 2007)

Role of HIF-1 Regulate angiogenic genes such as vascular endothelial growth factor (VEGF) (Manalo et al., 2005) HIF activation has been observed in the tissue from patients with inflammatory conditions such as arthritis, artherosclerosis, and autoimmune diseases (Nizet and Johnson, 2009) So far, more than 40 target genes have been found to be regulated by HIF-1 These genes can be classified into 3 main groups:

HYPOXIA Proliferation & migration of endothelial cells HIF-1 HYPOXIA Vascular Permeability VEGF VEGFR-1 Vasodilation Nitric oxide synthases Endothelial Sprouting Angiopoietin-1 Proliferation & migration of endothelial cells VEGF PGF Inhibitory Factors Angiopoietin-2 Extra Cellular Matrix Matrix Metalloproteinases

HIF-1 Target Genes Group 1: O2 Delivery Erythropoeitin (EPO) Nitric oxide synthase (NOS) Transferrin Transferrin receptor Vascular endothelial growth factor (VEGF) VEGF receptor -1 (Skuli et al., 2009) Group 1: O2 Delivery

HIF-1 Target Genes Group 2: Glucose /Energy Metabolism Hexokinase Phosphofructokinase Aldolase 3-Phospho Glyceraldehyde dehydrogenase Phosphoglycerate kinase Enolase (ENO) Pyruvate kinase Glucose transporter 1 Lactate dehydrogenase (Gordan et al., 2007) Group 2: Glucose /Energy Metabolism

HIF-1 Target Genes Group 3: Cell Proliferation /Viability Insulin-like growth factor 2 (IGF-2) IGF binding protein 1 IGF binding protein 3 P21/WAF1/CIP1 p35srj Group 3: Cell Proliferation /Viability

Symptoms Signs Dyspnea Restlessness Anorexia Confusion Agitation Headache Tremor Nausea Fatigue Respiratory distress Cyanosis Tachypnea Tachycardia Cardiac arrhythmias Hypertension Hypotension Lethargy Coma

Impacts of Hypoxia Brisket disease: Subcutaneous accumulation of fluid under the abdomen,brisket,neck and jowl (John H Newman et al., 2011) Hypertrophy of myocardium Valvular incompetency Polycythemia Polypnoea

Impacts of Hypoxia Hypoxia induced oxidative stress for short or long time periods affects corpus luteum development and function, leading to the decreased sheep fertility at high altitude (Parraguez VH et al., 2013) Respiratory alkalosis

Impacts of Hypoxia Cyanosis bluish tinge to the skin, lips, and nails Acute mountain sickness accompanied by loss of appetite, nausea, vomiting, fatigue, weakness, irritability or trouble sleeping (Hall D.P et al., 2014)

Impacts of Hypoxia High-altitude pulmonary edema (HAPE) usually develops with in 24 to 96 hours  (Kleinsasser et al., 2003) High-altitude cerebral edema (HACE) is a rare but potentially fatal condition (Patir H et al., 2012)

Impacts of Hypoxia Pregnant animals are at high risk Premature labor Small birth weights The average survivability of these breed has been reported to be 60% Inverse correlation between hypoxic zone & sperm concentration Decrease sperm motility (Sokol, 2006)

Impacts of Stress Early embryonic loss in livestock (Reynolds, 2005) Cortisol alters oxytocin receptor expression (Champagne, 2006) Reduced oocyte quality in cattle (Rocha, 2003) Heat stress reduces the developmental ability of embryos (Rutledge, 2001 )

Conclusion To limit the impact of extreme climatic events Genetic selection for breeds resistant to the extreme climatic conditions Optimisation of gradual shift of flock from one altitude to another Careful management and well designed housing at the level of the farming system for different altitudes are important in achieving the optimum animal performance

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