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  October 28, 2020
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A Sample CHAP from our second volume of the new 2-vol set of: "A Compilation of Ligno-cellulose Feedstock and Related Research for Feed, Food and Energy," by D. A. Flores (c). 2020. Skyebluepublications.ca, Port Coquitlam, BC Canada V3B 1G3. 

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 focus on histidine 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 alfalfa) as indicators of differences with threonine and lysine to be most different (histidine was also reported to be marked in difference) with all the essential amino flow 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). It should be noted that in dairy cows it has been established that lysine, methionine and histidine are the most limiting amino acids with rumen-protected lysine and methionine available commercially for ration supplementation.
     In this Chapter, examples involving lower quality protein silages with concentrate, protein supplemented fibrous agricultural residues (FARs), marine seagrasses and new experiments with pelleted duckweed, protecting the feed of high biological value (B. V.) and escape from rumen digestion are yet to be established, are examples that have recently been encountered and that may apply here.
     The practical benefits from this new or proposed research agenda or goal 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 the favourite grocery store.
     An experiment using post-ruminal (abomasal or duodenal) infusions of glucose and histidine 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 whether feeding protected histidine throughout the lactation cycle and how this is related to the whole body measurements of 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 histidine serves to feed requirements for the first limiting amino acid on silage diets with dairy livestock.  It was found that infused histidine 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 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 in protected histidine - supplemented diets at lower or more limited % CP levels - or lower planes of nutrition for protein.
     Studies with ghrelin in early lactation is consistent with increased body fat stores, milk fat 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 a milk component like fat (S. Borner et al., 2013).
     There may be a lingering concern over residual histidine in animal tissues including LBM and milk products but feeding levels are to supplement deficiencies in the diet, 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 histidine 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 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; in addition, yellow grease was found to improve % digestible fibre with SS addition (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 production, is processed as such: the sugarcane stalks are thoroughly washed and the pith is mechanically extracted in a sugar mill setting and pressed in the sugar-making process; the pith is then air-dried thoroughly in sealed cloth bags. The pith is then ground with a fine industrial grinder and filtered twice through the fineness of muslin cloth; 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 is yeast sparged into 200 mls distilled water per 25 g bagasse white powder that has been pre-filtered 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. Sidana and U. Farooq, 2014).
     In actual fact 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 the gene silencing element as with the mechanism using a palindromic binding nucleic acid-based reagent with central and outward symmetry binding in the "reverse" symmetry and thus silencing the expressed mRNAs from the target DNA sequence or gene. Most likely as was mentioned earlier, 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 juncture points or steps.
     When this technology is realized for yeast supplement synthesis via its fermentation with added PNA-B12 reagent added to boost HIS content considerably will a non-GMO type supplement be possible through this major breakthrough for functional amino acid feeding practices in livestock production that qualify as the organic kind or variety. 
     It is perceived that PNA-B12 is a fine biochemical or biological but may be considered a chemical entity of sorts and has the option of being treated with RNAase and protease and the residue remaining washed from yeast culture 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 relationships with its attendant inputs than the alternative genetic recombinant approach of maintaining and evaluating cultures for fermentation vs. fine biochemical synthesis of this type of reagent. It is also less costly in terms of inputs and energy in the processes involved.  
Functional Amino Acids in Metabolism with Livestock Production.
     Following the question of how histidine (together with its amine, histamine) and other collective non-essential amino acids (NEAA) in the first of two papers by D. A. Flores et al. (1986a) on duodenal amino acid flow measurements in sheep showed consistently significantly higher supplies both in essential and non-essential amino acids, viz. the treatment differences due to supplementation, in this case, due to ensiled degradation versus the original fresh-frozen fed material, leads us to speculate whether functional amino acids (FAA) as they are known to be are via: (1) operon regulation or gene expression and e. g. of which is the tac promoter which can be recombinantly manipulated to bring about stronger regulatory promoter function to boost histidine (HIS) biosynthesis in Corynebacterium glutamicum for production (Y. Cheng 2013), cell surface signaling with the essential amino acid leucine (LEU) acting on the mTOR receptor which will be mentioned again further below, and (3) neurotransmitter functional with ovine hormonal growth releasing factor (GRF) /  growth hormone (GH) effectors, yet to be established in plasma and nitrogen balance as an indicator of lean boody mass accretion at the expense of fat deposition and whole body weight gain or loss.  
     The previous agrees with results obtained by Flores et al. (1986b) in terms of plasma amino acid levels with treatments of ensiled versus fresh-frozen feed material. The author notes here the erratum that was placed on the study reported previously with D. A. Flores et al. (1986b) showing no reported dataset for nitrogen (N) balance measurements in the experimental trials conducted with the two studies.
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 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. In particular, one essential amino acid (EAA), leucine (LEU) activates an mTOR receptor that stimulate protein synthesis and inhibits proteolysis (G. Wu, 2010).  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 or 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 histidine on grass silage (ad libitum) plus concentrate did not increase dry matter intake, 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 histidine 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 histidine 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 histidine 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 variation at final slaughter (M. De Vos et al., 2013).
     Sow's supplementation of the diet and piglet growth and development includes glutamine (GLN), arginine (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 profile 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, elevated intestinal brush border enzyme activities.  It is possible to speculate that IGF-1 (and L-CAR) systemically administered at day 3 to day 10 post-natally via osmotic mini-pump to intra-uterine growth restricted (IUGR; LBW piglets results from a condition referred to also as IUGR piglets due to increased litter size) and increased number of LBW piglets from the uterus, an everyday occurrence in the swine industry (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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