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Chap.  Functional Amino Acids With Organic-Fed Livestock.

By D. A. Flores
     Ovine (sheep) as models for studies to define the most limiting amino acids in typical silage diets, is of interest here. We will later also discuss histidine (HIS) as an essential amino acid thought to be limiting on silage diets with dairy animals. Flores et al. (1986a) measured initially the duodenal amino acid flows on ensiled vs. control (fresh-frozen, to 'simulate' fresh grazed, alfalfa) as indicators of differences with threonine (THR) and lysine (LYS) to be most different (HIS was also reported to be marked in difference) with all the essential amino acid flows showing differences across the board. No infusion or feeding of rumen-protected supplements have been conducted with growing lambs or sheep to measure daily intake of animals (g DM/ d/ kg BW.75) with these type of studies. It should be noted that in dairy cows it has been established that LYS, methionine (MET) and HIS most limiting amongst amino acids, with rumen-protected lysine and methionine available commercially for ration supplementation.
     In this Chapter, examples involving lower quality protein silages with concentrate and protein supplemented fibrous agricultural residues (FAR), marine seagrasses and recent experiments with pelleted duckweed, that protecting the feed of high biological value (B. V.) and escape from rumen digestion are yet to be established, and are examples that have recently been encountered, that may apply here.
     The practical benefits from this new or proposed research agenda are the following: (1) exploring feasibility in improving sustainability of feeds for meat and dairy in livestock production, (2) producing healthier, leaner livestock as meat animals, (3) supporting a healthier, more productive lactation cycle, including supporting "designer" cows for boosted therapeutic milk food proteins (MFPs), "just around the corner," - amongst reproductive and geneticist biotechnologists wishing to undertake this evolutionary step in milk food product delivery as a nutraceutical towards filling your dairy case at your favourite grocery store.
     An experiment using post-ruminal (abomasal or duodenal) infusions of glucose and HIS on grass silage (fed ad libitum) plus concentrate did not increase dry matter intake, increased milk yield, protein yield, lactose yield and MF yield and had no influence on protein and lactose concentrations (M. Korhonen et al., 2000).  It is to be determined in a study yet to be proposed feeding protected HIS throughout the lactation cycle and how this relates to whole body measurements of lean body mass (LBM) and mobilization of protein stores and also the changes in MF output and how this relates to whole body measurements of fat stores and rate of fat tissue catabolism and with the energies of heat from whole body measurements.  Protected HIS serves to feed requirements for the first limiting amino acid on silage diets with dairy livestock.  It was found that infused HIS alone (i.e. without glucose) introduced abomasally resulted in unchanged % protein, with % lactose and % MF lowered in composition (C.-H. Kim et al., 2001; S. Hadrova et al., 2012).
     A study with the hormone, ghrelin, in sheep resulted in weight gain, increased feed intake and reducing metabolic utilization of fat (T. Sugino et al., 2004).  This finding would suggest for beef cattle, sheep or goats that synthetic bovine ghrelin intra-arterial implants could serve to further fatten cattle, used, in the finishing stages of the production cycle with protected HIS - supplemented diets at lower or more limited % CP levels - or lower planes of protein nutrition.
     Studies with ghrelin in early lactation is consistent with increased body fat stores, MF content, together with increased lipolysis (consistent with increased fat mobilization for milk production) and thus indicates that the observation of increased milk production is real, i.e. associated with increased content in the milk fat component (S. Borner et al., 2013).
     There may be a lingering concern over residual HIS in animal tissues including LBM and milk products but feeding levels are to supplement dietary deficiencies in the first place (6-8 g histidine / day).  The issue of ghrelin remaining in the meat and milk product as residue may be resolved by allowing for clearance time after the feeding period during holding stock in paddock or during transportation.
Over-producing Yeast Cultures with Histidine.

     The approach molecularly to design overproducing HIS microbial hosts as is with      Corynebacterium glutamicum would be to transform the cell with a tac promoter involved in L-histidine biosynthesis which is a stronger promoter leading to overproduction of the essential amino acid in culture (Y. Cheng et al., 2013).

     Yellow grease also known as industrial waste cooking oil (WCO) (and waste vegetable oil, WVO) from collected deep fryers are used as soap stock (SS) via saponification of WCO to Ca2+ salts to be blended with L-histidine to form granules or pellets (spherical) in a plant manufacturing pelleting machine gun apparatus. In general the performance parameters of SS from yellow grease indicates no change in dry matter intake (DMI), fat digestibility (%), and % digestible fibre with an increase in net energy (NE) intake (D. R. Brown, 1988). The evidence pointed out that fat protected amino acids with passage through the rumen like protein was that there was a decrease in OM digestibility in the rumen without a change in total tract digestibility and that there wasn't a shift in carbohydrate tract digestibility due to yellow grease feeding (R. A. Zinn and Y. Shen, 1996).

     The pith, the essential ingredient in addition to sucrose for S. cerevisiae culture, the target culture for high-histidine (HI-HIS) production, is processed as follows according to A. Sidana and U. Farooq (2014). The sugarcane stalks are thoroughly washed and the pith is mechanically extracted in the sugar milling setting with pressing as in the sugar-making process. The pith is then air-dried thoroughly in sealed cloth bags. The pith is ground with a fine industrial grinder and filtered twice through the fineness of muslin cloth; then bleaching over 1 wk. will occur in deionized water (reverse osmosis) for 4 L per 1/4 kgs powder with spinning at 150 rpms. Before storage in a cool, dry place in bags it is spray dried in an industrial chamber. The culturing procedure involves yeast sparged into 200 mls distilled water per 25 g bagasse white powder, that has been pre-filtered, a second time, similar to fineness in muslin cloth and Whatman no. 1 paper, with added per 8 g sucrose (sugar) with pH adjusted [H2SO4 (aq)] to 5.6. The batch culture is stirred for 1 wk. at 25 deg. C; sporulation occurs in time as nutrient depletion ensues with further spray drying of wet media to recover yeast product and stored in a cool, dry place (5 deg C).
     A non-GMO (viz. organic) supplement would call for techniques like protein-nucleic acid (PNA) conjugated with a "carrier" like B12 to transbound the microbial cell wall and membrane with gene silencing elements using the palindromic binding principle between nucleic acid strands with central and outward symmetry binding in "reverse" symmetry and thus silencing effectively the expressed mRNA copy from the target DNA sequence or gene. The operator/promoter (O/P) binding elements or products will be blocked from translation that "feedback" and inhibit histidine (HIS) biosynthesis at the rate-limiting biosynthetic steps.
     When this technology is realized for yeast supplement fermentation, with added PNA-B12 reagent added to boost HIS content considerably, will a non-GMO type supplement be possible through this major breakthrough with PNA-B12 for functional amino acid feeding practices of the organic kind or variety.  It is perceived that PNA-B12 is a fine biochemical or "biologic" but may also be considered "a synthetic chemical entity of sorts" and presents the option of being treated with RNAase/protease, with the residue remaining washed from yeast organic matter (OM). 
    It has been said in passing that use, for e. g. of PNA-B12 with respect to its "growing condi-tions," is more efficient in terms of a dose/response relationship with its attendant inputs than the alternative recombinant DNA approach of maintaining and evaluating cultures for fermentation vs. fine biochemical synthesis of this type of reagent. It is less costly in terms of inputs and energy with the processes involved.  
    With swine feeding (monogastrics) soluble fibres sources as with galactomannans from copra meal byproduct, a plentiful feed resource in places like the Philippines, Malaysia and Indonesia where coconut tree plantations are commonplace, the feed source is markedly unbalanced for amino acids, e. g., the high ratios of arginine:lysine (ARG:LYS) including the latter's availability due to heat processing with the byproduct. This begs the question whether wholesale feeding made available through practice (including eliminating possible mycotoxins in the feed) will be facilitated by "green" biotech production of yeast-based, high amino acid supplements, a cheaper proposition compared to chemically synthesized amino acids currently used. ARG has other functional value(see: below) making leaves copra meal byproduct an attractive source when formulating least cost total mixed rations for feeding swine. 
Functional Amino Acids in Livestock Feeding & Production.

     Our view of research with functional amino acids (FAA) demonstrates a dichotomy between basic nutritional research and by translation is made to be research in more productive or applied terms, for e. g., nutritional studies with functional nutritionals or supplements apropos to health in human or to livestock production studies. Here, we will discuss examples of: (1) tissue metabolism from a basic reference point, (2) lactation in dairy milking cows, and (3) sow-piglet litter performance from feeding FAA.
     The literature in the past has mentioned certain essential amino acids as with: methionine (MET), phenylalanine (PHE), lysine (LYS) and histidine (HIS) that promote human growth hormone (hGH) release in adults; this does not presume to apply necessarily to all laboratory animals or livestock species but we will lends itself to species of interest (viz. certain farm livestock) at this time. Here, we present findings regards other amino acids that are non-essential as with: arginine (ARG) and ornithine (ORN). It was found in the case of ARG that it stimulates GH secretion by suppressing endogenous somatostatin secretion; the experiment demonstrated both ARG and human growth releasing hormone (hGRH) leads to higher serum GH in humans versus either one (J. Alba-Roth et al., 1988). In the case of ORN administration affects diurnal rhythms of plasma growth hormone (GH) in mice with higher levels in the day as opposed to night time (H. Matsuo et al., 2014).
     Amino acids (AA) initiate cellular or biochemical changes that lead to protein synthesis in this regulatory way.  Meat quality is improved through supplementation with arginine (ARG) to growing-finishing pigs before slaughter, and proline (PRO) stimulates both small intestines and whole-body growth in weanling pigs (G. Wu, 2010).  Requirements of FAA is now dependent on, in terms of nitrogen balance or status and AA profiling of whole-body tissues, additional issues of: (a) functional requirements for essential bodily functions towards and for LBM accretion itself is one factor, (b) an additional finding that dietary AA are substantially catabolized by the small intestine shows a marked difference between skeletal body ratios versus what enters from the diet (G. Wu, 2010); or in the case of ruminants the emptying result from escape (dietary) or breakdown and resynthesis in the rumen.
     An experiment using post-ruminal (abomasal or duodenal) infusions of glucose and HIS on grass silage (ad libitum) plus concentrate did not increase dry matter intake (DMI), increased milk yield, protein yield, lactose yield and milk fat (MF) yield and had no influence on protein and lactose concentrations (M. Korhonen et al., 2000). It was found that infused HIS alone (i. e. without glucose) introduced abomasally resulted in unchanged % protein, with % lactose and %MF lowered in composition (C.-H. Kim et al., 2001; S. Hadrova et al., 2012). It is to be determined in a study feeding protected HIS throughout the lactation cycle how this is related to the whole body measurements of LBM and mobilization of protein stores and also the changes in whole body measurements of fat stores and rate of fat tissue catabolism and how this relates to MF output and together with the energies of heat from whole body measurements.  Protected HIS would serve to feed requirements for the first limiting amino acid on typical silage diets as with dairy livestock.
     Supplementation with milk replacer of low birth weight (LBW) piglets is typically with whey, lactose and skim milk as the main ingredients with a composition of 25% crude protein (CP), 45-50% lactose and 1-12% lipids mixing at a rate of 150-250 g/L; benefits on growth and development of LBW piglets are: (1) boosted growth rate and heavier mean weaning weights, (2) improvement of the survival rates for the LBW piglets and (3) does not apparently reduce the final weight varia-tion at final slaughter (M. De Vos et al., 2013).
     Sow's supplementation of the diet and piglet growth and development includes glutamine (GLN), ARG and carnitine (CAR) as a precursor to IGF-1.  The feeding of L-GLN and L-CAR positively affects piglet birth weights and insures when feeding balanced amino acid profiles to requirements during gestation leads to a more uniform litter size (M. De Vos et al., 2013).  L-CAR or IGF-1 orally administered leads to small intestinal (S. I.) growth and maturation, e. g. increased crypt cell proliferation, villus heights, and elevated intestinal brush border enzyme activities (M. De Vos et al., 2013).
     Both L-GLN and L-ARG seem to be crucial factors contributing to IUGR piglets; supplementation of L-ARG in IUGR piglets and lambs both accelerated growth.  Supplementation of precursor L-GLN in piglets between 0-21 days accelerated piglet growth and reduced pre-weaning mortality (M. De Vos et al., 2013).
1.  J. Alba-Roth et al., 1988.  Arginine stimulates growth hormone secretion by suppressing endogenous somatostatin secretion. J. Clin. Endocrin. Metabol. 67: 1186-1189. 
2.  S. Borner et al., 2013. Plasma Ghrelin is Positively Associated with Body Fat, Liver Fat and Milk Fat Concentration But Not with Feed Intake of Dairy Cows After Parturition. J. Endocrinology 216: 217-229. 
3.  D. R. Brown. 1988. Soapstock in Ruminant Diets. Mix30. The High Energy Liquid Feed. Tech. Bulletin. Pp. 1-3.
4.  Y. Cheng et al., 2013. Modification of Histidine Biosynthesis Pathway Genes and the Impact on Production of L-histidine in Croynebacterium glutamicum. Biotechno. Lett. 35: 735-741. 
5.  M. De Vos et al., 2013. Nutritional Interventions to Prevent and Rear Low-Birthweight Piglets. J. Anim. Physl. Anim. Nutrt. 98. http://online library.wiley.com/doi/full/10.1111/jpn.12133.  
6.  D. A. Flores et al., 1986a. Digestion in the Rumen and Amino Acid Supply to the Duodenum of Sheep Fed Ensiled and Fresh Alfalfa. Can. J. Anim. Sci. 66: 1019-1027. 
7.  D. A. Flores et al., 1986b. The significance of silage protein degradation and plasma amino acid ratios in the control of food intake by lambs fed ensiled and fresh alfalfa. Can. J. Anim. Sci. 66: 1029-1038. 
8.  S. Hadrova et al., 2012. The Effect of Duodenal Infusion of Histidine on Milk Yield, Milk Composition and Plasma Amino Acids in Dairy Cows. J. Anim. Feed Sci. 21: 555-565.
9.  C.-H. Kim et al.,  2001. Effects of Intravenous Infusion of Amino Acids and Glucose on the Yield and Concentration of Milk Protein in Dairy Cows. J. Dairy Res. 68: 27-34.
10.  M. Korhonen et al., 2000. Response to Graded Postruminal Doses of Histidine in Dairy Cows Fed Grass Silage Diets. J. Dairy Sci. 83: 2596-2608.
11.  H. Matsuo et al., 2014. Effects of time of L-ornithine administration on the diurnal rhythms of plasma growth hormone, melatonin and corticosterone levels in mice. The J. of Biol. and Med. Rhythm Res. 32: 225-234. 
12.  A. Sidana and U. Farooq. 2014. Sugarcane Bagasse: A Potential Medium for Fungal Cultures. Chinese Journal of Biology. Article ID 840505. 5pp. 
13.  G. Wu, 2010. Functional amino acids in growth, reproduction and health. Adv. Nutr.1:31–37
14.  R. A. Zinn and Y. Shen. 1996. Interaction of Dietary Calcium and Supplemental Fat on Digestive Function and Growth Performance in Feedlot Steers. J. Ani. Sci. 74: 2303-2309. 

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