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

2. 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”

3. 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)

4. 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

5. 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)

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

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

8. STRESS FACTORS
BIOTIC
Diseases:
Bacterial
Viral
Fungal
Parasitic

9. 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)

10. 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

11. High Altitude Hypoxia

12. 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)

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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)

19. 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)

20. 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

21. 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)

22. 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)

23. 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)

24. 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

25. 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)

26. 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:

27. 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

28. 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

29. 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

30. 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

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

32. 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

33. 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

34. 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)

35. 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)

36. 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)

37. 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 )

38. 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

39. THANK YOU