Recent Research Advances of Applied Biochemistry in Animal Nutrition

1. Recent Research Advances of Applied Biochemistry in Animal Nutrition AnusornCherdthong, PhD 137748 Applied Biochemistry in Nutritional Science Email: anusornc@kku.ac.th • E-learning: http://ags.kku.ac.th/eLearning/137748

2. Introduction • Improve animal productivity • Reduce feed cost/increase profit • Recent advance application: • Nutrigenomics • Rumen molecular biotechnology

3. Nutrigenomics • Evaluate effect of nutrient on • Gene expression • Protein synthesis • Sequencing • Transcription • Metabolites • Nutrigenomics • Transcriptomics • Proteomics • Metabolomics

4. Transcriptomics • Gene expression: RNA content among organs • Microarray technique is major method • Hybridization of cDNA and probed • Computer evaluate: different color • More genes can be investigate • Rapid • Ex: investigate 8000 genes in rat’s liver fed with casein protein or fed with FA (Endo et al., 2002; Tachibana et al., 2005)

5. Transcriptomics • Microarray technique in cattle production • Structure muscle with gene expression • Growth gene • Fetus growth gene • Muscle synthesis gene: FSTL1 and IGFBP5 Lehnert et al. (2007)

6. Transcriptomics overview Zduñczyk and Pareek (2009)

7. Proteomics • Study protein: cell, tissue and organs • Techniques: • two dimensional (2D) gel electrophoresis • mass spectrometry (MS)proteomics • Ex: • Study methionine on tissue growth • Gene expression by tandem mass spectrometer

8. Proteomics overview Zduñczyk and Pareek (2009)

9. -omics technology • Advantages: • Development feed quality and suitable with genetic • Selective specific nutrient on gene • Nutrient management • Interaction of nutrient and gene • Study aging process • Immune system • Prevention of disease • Reproductive

10. omics technology overview Zduñczyk and Pareek (2009)

11. Biotechnology in rumen • Study of ruminal microorganism • Bacteria • Protozoa • Fungi • Dynamic change as affected by diets • Number • Quality • Unknown species • Role of microbes

12. Roughage • Rumen Ecology of Ruminants Cellulase, xylanase Pectinase, esterase Attachments VFA Degradation Fermentation Microbial protein Interaction • End-products • Lactate • Formate • Acetate • Ethanol • CO2, H2 Sources: Wanapat (2004)

13. Modern molecular techniques • Based on 16S rRNA/ 18S rDNA of rumen microorganism and first literature by Stahl et al. (1988) • Rumen microbial populations could be under- estimated, require more time by traditional techniques • Molecular techniques can be used to be more accurate, sensitively and rapidly

14. Modern molecular techniques • PCR assays were developed for several species and specific strain of rumen bacteria (Reilly and Attwood, 1998) • Real- time PCR assay to study quantitative and relationship of rumen bacteria, protozoa and fungi(Skillman et al, 2006; Ozutsumi et al, 2006) • PCR- DGGE assay to study diversity of rumen microbes (Denget al., 2007) • However, DNA extraction are important to provide genomic DNA efficiency

15. Step by step of molecular techniques Rumen fluid, digesta DNA extraction* Genomic DNA Quantity Diversity PCR- DGGE* PCR* Real- time PCR* Sequence Phylogenetic trees Gel electrophoresis

16. Summary step by step of PCR DNA template, H2O, Primer, 10x buffer, MgCl2, dNTPs, Taq polymerase DNA template PCR machine Denature Annealing Extension Detected Quantity PCR product Gel electrophoresis Band compare with marker Data

17. Advantages of PCR • Amplify specific DNA • Rapid for increase DNA • Obtain DNA product for analyses other techniques Disadvantages of PCR • Don’t know quantity of DNA product • Require time for gel electrophoresis (1- 2 hr) • Carry over contamination

18. Real- time PCRtechnique • Develop from conventional PCR  many limited • 2 path for development are… • detect PCR product in soluble by fluorescence receptors (SYBR Green I Dye or probes fluorescence) • thermolcycler real- time thermolcycler not only temp. control but as light source also. • Therefore, this technique is amplify DNA and detect PCR product at the same time.

19. Summary of step by step of real- time PCR DNA template, H2O, Primer, 10x buffer, MgCl2, dNTPs, Taq polymerase, SYBR Green DNA template Real- time PCR machine Denature Annealing Extension Detected Quantity PCR product Software Data

20. Advantages of real- time PCR • Amplify specific DNA, rapid for increase DNA • Detect DNA product at the same time • Obtain DNA product for analyses other techniques • Quickly and purity for analysis Disadvantages of real- time PCR • Setting up requires high technical skill and support • High equipment cost

21. PCR- DGGEtechnique • Denaturing gradient gel electrophoresis (DGGE) are technique for study rumen diversity • PCR Amplified fragments of 16S rDNA can separate DNA fragments of the same length but different base pair sequences • Separate double strand of DNA (<500 bp) by denature agent such as urea and formaminde • Different concentration gel from low (top) to high (ground)  many band on gel

22. DNA template, H2O, Primer*, 10x buffer, MgCl2, dNTPs, Taq polymerase DNA template PCR machine * Universal primer Denature Annealing Extension Separate DNA double strand by denature agent 16/18s rDNA DGGE technique Gel bands Program analysed Gens blank data based • Bioedit • Phylip • Clusterx • RDP II • BLAST • DDBJ • Etc. Sequencing Phelogenetic tree Summary of step by step of PCR- DGGE

23. Source: Watanabeet al (2004)

24. Step by step of DNA sequencing DNA band Reamplification by PCR DNA sequence analysis BLAST RDP II DDBJ Etc. 5’-ACACTGAGATCCC-3’ Base sequences Online search from data based Microbes species

25. Phelogenetic tree Program analysed • Bioedit • Phylip • Clusterx Source: Modified from Mackie et al. (2003)

26. Advantages of PCR- DGGE • To know diversity of rumen microbes • Separate the same length but base sequence DNA are different • New species of rumen microbes Disadvantages of PCR- DGGE • Setting up requires high technical skill and support • High equipment cost or analysis such as sequencing • Don’t know quantity

27. Rumen bacterial studies • Most of bacteria studies are cellulolytic such as F. succinogenes, R. albus, R. favefacience • Use 16s rDNA as indicate for molecular analysis • First time, Koike (2003) using PCR to detect bacterial quantity on various diets and time • Found that, F. succinogenesare predominant celluloltytic

28. Rumen bacterial studies • Real- time PCR using by Tajima et al. (2001) to detect rumen bacteria of cowsforwhichthedietwasswitchedfromhaytograinwerequantitativelymeasured • Quantity DNA bacteria from rumen fluid collection on d 0 (hay), 3 (fed grain 3 d) and 28 (fed grain 28 d) • They found that… • Cellulollytic bacteria tended to be decreased • Amylolytic bacteria tended to be increased

30. Wanapat and Cherdthong (2009)

31. Wanapat and Cherdthong (2009)

33. Cherdthong et al. (2011a)

34. Cherdthong et al. (2011a)

35. Beef cattle: • Increase cellulolytic bacteria • Increase microbial synthesis • Increase digestibility

36. Dairy cows: • Propionate • Increase microbial synthesis • Increase digestibility • Increase milk production (4 kg)

37. Cherdthong et al. (2011b)

38. 9 7 6 Cherdthong et al. (2011b)

39. Rumen bacterial studies • PCR- DGGEusing by Mackie et al. (2003) to study diversity of Oscillospira ssp. in cattle, sheep, andreindeer • PresenceofOscillospiraspeciesinvariousrumenecosystemsdependentondiet. • Oscillospiraspecies separate to three groups show in the rumen bacterial phylogenetic tree(show as Fig.) • Mao et al. (2007) investigated the effects of disodium fumarateon microbial communities by PCR- DGGE technique

40. Phylogenetic tree of 16S rDNA Oscillospira sequences Sporobactertermitidis Oscillospiraspp. F Group I Oscillospiraspp. A Oscillospiraspp. H Oscillospiraspp. G Group II Oscillospiraspp. E Oscillospiraspp. D Oscillospiraspp. C Oscillospiraspp. OSC3 Oscillospiraspp. OSC4 Group III Oscillospiraspp. OSC1 Oscillospiraspp. OSC5 Oscillospiraspp. B Oscillospiraspp. OSC2 Clostridium orbiscindens AlterdSchaeder flora strain ASF500 RuminococcusAlbus Ruminococcusflavefaciencs Aquifexpyrofuilas Source: Modified from Mackie et al. (2003)

43. Rumen protozoa studies • PCR- cloning- sequencing to study protozoal communities the first publication by Karnati et al. (2003) • Design specific primer for amplify 18s rDNA only from DNA microbe in rumen fluid • Sylvester et al. (2005) using real- time PCR to quantify bypass protozoa to duodenum • Found that protozoa about 5.9 and 11.9 % (NDF 16 and 21%) were of N bypass

44. Rumen protozoa studies • Morgavi et al. (2006) using PCR- DGGE technique to classify rumen protozoa such as Epidimiumcaudatum, Entodiniumcaudatumand Isotricaprostoma • Real- time PCR use to study relationship between protozoa and bacteriaby Ozutsumi et al. (2006) • To compare bacteria population on faunated and unfaunated • They found R.Albus, R.flavefaciens, P. ruminicola and CUR-E high in unfaunated

45. Rumen fungi studies • 18s rRNA for comparing rumen fungi limited use due to highly conserved nature • Internal transcribed spacer region 1 (ITS- 1) of rRNA gene used for studying genetic diversity • Initially used by Li and Heath (1992) could separate fungi to 2 cluster • Cluster 1Orpinomyces, Neocallimastrix andPiromyces • Cluster 2 AnaeromycesandCaecomyces

46. Rumen fungi studies • DenmanandMcSweeney (2006)usedreal-timePCRSYBRGreenassaytotargettotalrumenfungipopulationswithincattlerumen. • Detected of DNAandcrudeproteincontentswithrespecttothefungalbiomassofbothpolycentricandmonocentricfungal • They found… • Theanaerobicfungaltargetwasobservedtoincrease 3.6- foldfrom 0 to 12 h afterfeeding

50. 1 2 3 Conclusion Nutrigenomic: nutrient vs gene Molecular rumen microbiology improved feed efficiency in ruminant Application of modern techniques in small holder farmer and commercial level