The document discusses an overview of livestock metabolomics. It defines metabolomics as the large-scale study of small molecules present in cells, biofluids, tissues and organs. Various techniques for metabolomic analysis are described including mass spectrometry, NMR spectroscopy, GC-MS and LC-MS. Applications of metabolomics in livestock include disease diagnosis, biomarker identification, monitoring drug and surgical impacts, and understanding gene-environment interactions. Specific examples include identifying metabolic biomarkers for mastitis resistance in dairy cows and detection of milk fever in cattle. The challenges and future prospects of metabolomics research are also outlined.

1. CREDIT SEMINAR
ON
LIVESTOCK METABOLOMICS – A
OVERVIEW
Dr. G.KALAISELVI
DPV(M)17002(ABT)
PART TIME Ph.D SCHOLAR
DEPARTMENT OF ANIMAL BIOTECHNOLOGY
MADRAS VETERINARY COLLEGE, CHENNAI-7

2. Molecular phenotyping
200000 metabolites in
plant system and 2000
endogenous metabolites
in human.
1 million Genome ~
26,000-31,000 protein
encoding genes
Metabolomics
Large scale study of
small molecules present
cells, biofluids, tissues
and organs

3. ANCIENT URINE METABOLIC
BIOMARKER
Biomarker
Biological
characteristics that
are objectively
measured and
evaluated as
indicators of
normal biological
processes,
pathological processes
or pharmacological
responses to a
therapeutic
intervention

4. Types of metabolites

5. Metabolic finger
printing
Unbiased global
screening
approach
Sample
preparation,
separation and
detection
Metabolic
profiling
Identification
and
quantification of
metabolites
Quantitative
analysis of set of
metabolites
Metabonomics
The metabolic
response of
organisms to
pathophysiological
stimuli or genetic
modification
PRINCIPLE APPROACHES TO
METABOLOMICS
Metabolic foot
printing
Analysis of
extra cellular
metabolites
Lipidomics
metabolomics
focusing
exclusively on
lipids

6. APPLICATION OF METABOLOMICS

7. •Drug discovery
•Toxicology and
pharmacokinetics study
•Agricultural and plant
development
•Clinical biomarker for
disease diagnosis
Next generation
phenotyping
Genome-wide
association studies with
metabotypes (mGWAS
Network
reconstruction
methodologies and
complexity of
metabolomics
information
Clinical metabolomics
Generation of metabolic signature of disease state and host response
Monitoring of gene –environment interaction
Effect of drug and surgery
Identification of functions of an unknown gene
APPLICATION OF METABOLOMICS
IN LIVESTOCK

10. Schiess R. et al. , 2009. Targeted proteomics strategy for clinical biomarker discovery,
Molecular Oncology, 3( 33–44)
High abundant proteins
Accessible Proteins
Plasma protein dynamic range
Mass Spectrometry
capability
MASS SPECTROSCOPY CAPACITY OF
ANALYSIS METABOLITES

11. TECHNIQUES IN METABOLOMIC
ANALYSIS

13. APPLICATION OF
METABOLOMICS IN LIVESTOCK
PRODUCTION AND
REPRODUCTION

14. IMPORTANCE OF METABOLOMICS IN LIVESTOCK
PRODUCTION AND REPRODUCTION
Breeding
Physiology
Genetics
Clinical
metabolomics
Metaboloic
biomarker
Gene –
metabolite
relationship

15. Disease diagnosis and investigation (new
diagnostic tests, identification of biomarkers
for diseases)
Understanding the biochemistry behind
disease and processes and identifying
prognostic indicators
Optimising health and production (eg,
nutrition and the environment).
A family-based QTL analysis for targeted
serum metabolomic profiles was reported in
male F2 individuals (Widmann et al., 2013)
Pharmaceutical development - identifying
new targets for drugs and toxicology testing.
Animal welfare (both direct and indirect
research to improve animal production and
reproduction .
Changes in the metabolic pathways of liver
tumours induced by aquatic cadmium toxicity
study in wild flat fish (Southam and others
2008).
FOCUS ON VETERINARY FIELD

16. The rumen is home to
billions and billions of
microbes, including
bacteria, protists, fungi,
and viruses. These many
different rumen microbes
form a complex
community of organisms
that interact with one
another, helping the
animal digest its food.
ANIMAL PRODUCTION-RUMEN
MICROBIAL METABOLOMICS

17. Interactions between gut microbial
populations among themselves and with
the host and highlights the importance of
gut microbes as modifiers of the host
metabolic phenotype.
Rumen microbial diversity had a major
influence on rumen fermentation
parameters, digestibility,and the general
metabolism of the host.
 The interaction found between
trimethylamine N-oxide and methanogens
from Methanomas siliicoccales shows
potential of the metabolomics approach
for the discovery and monitoring of
biomarkers of rumen microbial functions
( Marcovi et.al.,2015)
METABOLIC PHENOTYPING

18. The seminal plasma - sperm motility,
sperm membrane protection,
protection from oxidative stress, sperm
capacitation and acrosome reaction and
oocyte penetration (Therien et al.,
1995)
The presence of citric acid, A -glutamyl
transferase and acid phosphatase in
seminal plasma and leptin in serum are
also predictors of epididymal function,
prostate function and sperm
morphology (Bhat et al., 2006).
Bovine seminal
plasma protein,
clusterin, albumin
phospholipase A2,
and osteopontin
High fertile bulls
Citrate , lactate,
glycerylphospho
rylcholine, and
glycerylphospho
rylethanolamine
High in infertile
bulls
SEMINAL FLUID
METABOLOMICS

19. Sperm membrane damage biomarker-
Oxoglutarate amino tranferase
Cryo injury biomarker – fibrononectin
Excessive production of reactive oxygen
species (ROS) leads to oxidative stress –
poor fertility
 Free radical marker -CH, -NH, -SH,
C=C and OH found in male reproductive
tracts affecting sperm quality and
function. (Agarwal A, 2005)
Citrate, tryptamine / taurine, isoleucine,
leucine, aglutamyl transferase, acid
phosphatase and oxidative stress
metabolites in seminal plasma and
isoleucine, asparagine, glycogen, citrulline
in serum - detect early fertility in bulls.
SPERM PROTEOMIC
METABOLOMICS

20. Polyunsaturated fatty acids, with
linoleic acid found in larger
quantities in small follicles which is
responsible for inhibition of meiosis
in bovine oocytes
OOCYTE FOLLICULAR FLUID METABOLOMICS

21. Progesterone, Resveratrol 4′-glucoside,
Lupinisoflavone N, and Peonidin acetyl 3,5-
diglucoside , Isoflavones, anthocyanidins,
and prenylflavonoids,, peonidin acetyl 3,5-
diglucoside, 5,7-dihydroxy-6-methyl-8-
prenylflavanone and xanthohumol –
increase female fertility
Lupin isoflavone N, which is an
isoflavone found in plants.
 Isoflavones-------antioxidant character
supplementation of animal feeding with
lupines grass leads to increased ovulation
rate in sheep and also to improved
reproductive efficiency in the case of
ruminants (Tatiane Melina et.al.,2018)
Prostaglandin M. – Specific marker of
the high oocyte number groups.
 Phytoestrogenic compounds-
renylflavonoids – anti oxidant property
(Guerreiro et.al.,2018 )
FOLLICULAR FLUID METABOLIC
BIOMARKER

22. APPLICATION OF
METABOLOMICS IN
LIVESTOCK DISEASES

23. IMMUNO METABOLOMICS

24. Species Types of biomarker Condition when increased
Swine Haptoglobin Pneumonia, Sepsis, Inflammation
Swine Serum amyloid A
Pig -MAP1
Pneumonia, Sepsis
Swine C-reactive r protein Sepsis, Inflammation
Swine pig-MAP1 Sepsis
Swine 1-acid glycoprotein Inflammation
Swine Ceruloplasmin Inflammation
Swine Acid soluble glycoprotein Inflammation
Chickens Haptoglobin Infectious bronchitis
Chickens Serum amyloid A Infectious bronchitis
Cattle Lipopolysaccharidebinding protein Mastitis, Respiratory Disease
Bovine Serum Amyloid A Mastitis, Respiratory Disease,
Bovine Haptoglobin Amyloidosis,Mastitis,
Bovine 1-acid glycoprotein Respiratory Disease,
Mastitis, Respiratory Disease
Sheep Haptoglobin Caseous lymphadenitis,
Sheep Serum amyloid A Pulmonarydamage, Caseous
lymphadenitis
Sheep Fibrinogen Fibrinogen Pulmonarydamage
Sheep Ceruloplasmin CeruloplasminPulmonary
damage
Goat Haptoglobin Inflammation
Goat Serum amyloid A Inflammation
Goat Fibrinogen Inflammation
Goat Acid soluble glycoprotein InflammationGoat
BIOMARKERS
FOR LIVESTOCK
DISEASES

25. The 13 different types of proteins are
responsible for high and low mastitis
resistance in cow.
 Most of the proteins were membrane-
bound and less suitable as a biomarker
Sample preparation and protein
quantification with a high-throughput
technique like ELISA is more difficult.
Gamma-glutamyl transpeptidase1
(gGT1) and lactoferrin (LF).
Lactoferrin is a part of the innate
immune system and has antimicrobial,
antiviral, antifungal, anti-inflammatory
and anti-oxidative properties among others
by iron sequestering (Kanwar et al., 2015)
METABOLIC BIOMARKER FOR MASTITIS
RESISTANCE IN DAIRY COW

26. Magnesium, alkaline Phosphatase,
hydroxyproline, osteocalcin,
parathormone, calcitionin, and 1,25-
dihydroxy-vitamin D which are
associated with the regulation of
calcium metabolism.
Serum amyloid A protein (SAA),
Calcitonin-gene related peptide
(CGRP), Endopin 2B, Serpin
peptidase inhibitors (SPI) – Up
regulated
 Down regulated proteins -
fibrinogen beta chain, IGg
heavchain C-region (IGg -CH), and
albumin are metabolites used for
detection of milk fever. ( Collard
et.al.,2000)
METABOLOMIC BIOMARKERS FOR
DETECTION OF SUBCLINICAL MILK FEVER

27.  The level of Β-hydroxy butyrate
(BHBA), blood glucose levels (Glc), total
triglycerides (TG), non esterified fatty
acids (NEFA) including palmitic acid
(PA), heptadecanoic acid (HA), stearic
acid (SA), trans-9-octadecenoic acid (T-
9-OA), myristic acid (MA), cis-9-
hexadecenoic acid (C-9-HA), long chain
unsaturated fatty acids and saturated
acids and aspartate aminotransferase
(AST) are increased.
Leucine (3-hydroxyisovaleric acid
(3HIV)), 4-aminobutyric acid (GABA) –
L -glutamic acid catabolism, melibiose
from galactose metabolism, erythritol, a
precursor of fructose 6-phosphate and l-
serine (L-ser) are decreased. (Kreipe
et.al.,2011).
METABOLIC BIOMARKERS FOR THE DETECTION OF
SUBCLINICAL FATTY LIVER SYNDROME AND KETOSIS

28. Metabolomic analyses used to
characterize metabolic disturbances in
canine liver disease (Whitfield et al.,
2005).
Canine diabetes - 42 types of
metabolites analysed (Allison
et.al.,2017)
The metabolic responses of cattle to
low level anabolic steroid treatment
using a metabolomics strategy were
studied.
Metabolite profiles were analyzed by
NMR spectroscopy
The trimethylamine-N-oxide,
dimethylamine, hippurate, creatine, and
citrate was found to be increased
indicating a coordinated response to
anabolic steroids (Dumas et al. ,2005)
CLINICAL METABOLOMICS

29. RECENT
ADVANCEMENT IN
METABOLOMICS
APPROACH

30. STEM CELL METABOLOMICS FOR INDUCED
PLURIPOTENT STEM CELL DEVELOPMENT

31. MNP was developed (Fe3O4@SiO2-FPBA)
with 4-formlyphenylboronic acid (FPBA)
grafted on its surface displaying a high
density of boronate binding sites(
Engineered MNP) ( Zhang et.al 2018)
Ribosylated metabolites extracted by
using boronate materials but adsorption
capacity and selectivity very less
Surface enhanced Raman spectroscopy
(SERS) - sensitive detection of molecules in
the low nM range owing to the signal
enhancement induced by a localized
electromagnetic field around the plasmonic
substrate
Inorganic nanoparticles such as
plasmonic nanoshells enhance ionization of
small molecules in matrix-assisted laser
desorption/ionization mass spectrometry
(MALDI-MS) for detection of low molecular
weight metabolites
NANO –METABOLOMICS

32. PLANT METABOLOMICS
(http://www.metabolomics.bbsrc.ac.uk/MeT-RO.htm). NMR spectroscopy,
GC-MS, LC-MS, and CE-MS –55000 metabolites

39. MAS NMR to analyse
human eye tissues for the
first time and a total of 29
metabolites were identified
from the cornea, iris,
ciliary body, lens and
retina.
The biochemical
contents of the tissues were
found to correspond to the
biological Functions
(Kryczka et .al .,2017)
Corneal storage and
transplant

41. Study found that 30-40% of
subjects experienced ALT
elevations (Watkins, et al.,
JAMA, 296 (1), 87, 2006 )
Metabolites safety testing of
drug (MST)

44. Nile grass rat-
African wild
rat -resistance
to diabetes
Zuker
dramatic
fatty rat-
transgenic
rat for
obesity study

47. COMMERCIALLY AVAILABLE
METABOLOMIC KIT

48. COMMERCIALLY AVAILABLE
METABOLOMIC KIT
Rs. 40 /Test
Rs. 27 /Test

49. Successful extraction of metabolites
from cellular matrices.
Structural diverse group of
compounds - difficult to develop
methods that can separate all types of
metabolites.
Ionization/Visualization of all
metabolites is also difficult to achieve
as each compound will vary in its
affinity for a detector.
Metabolites concentration will be
affected by age, gender, physical
condition and parasitic loads
Time consuming and costly
equipments
TECHNICALLY SKILLED
PERSON
CHALLENGES OF
METABOLOMICS

50. FUTURE PROSPECTS
Metabolomics is a complex
discipline so good experimental
planning a knowledge of
bioinformatics and the
appropriate use of statistics are
required for a successful and
meaningful study.
 Fast validation of any
metabolomic study is important
and development of single
specific biomarker for production
and reproduction traits of
livestock.
Plant metabolites –
nutrigenomic and
ethnoveterinary medicine
Identification and
quantification of all
metabolites in a biological
cell or system.
The effects of the
environment, drugs and
disease on the metabolome.
 To study the metabolic
changes that underlie health
and disease.
To develop new metabolic
biomarker based diagnostic
tests
Micromrtabolomics and
target metabolomics