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Our latest on our author's submission to the AMB journal: “PNA to Gene Silence for silage processing and rumen digestion,” (in press). (Unsolicited and submitted and invited for acceptance: 12/03/2019.)
The ff. publications and their current significance towards the submission of the paper: "Protein Nucleic Acids Applied to Silage (tropical and temperate) Rumen Digestion," (In press).
D. A. Flores. 1986. 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.: Introduces the standpoint of using functional amino acid feeding practices using the documented profiled amino acid supplied from digestion on fed silage diets underscoring histidine as a first limiting and tantalizing functional amino acid for feeding livestock taken together with Penn State U. studies on silaged-fed dairy livestock.
D. A. Flores. 2013. A Compilation of Ligno-cellulose Feedstock and Related Research for Feed, Food and Energy. Xlibris LLC, Bloomington IN USA.: Presents for the first time with ensilage the major thrusts of namely, water-soluble carbohydrates and pre-formed amino acids enhancement with postharvest pre-treatment and rumen digestion of silages when applied techniques are used.
D. A. Flores. 1989. Applications of Rec-DNA to Rumen Microbes for the Improvement of Low Quality Feeds Utilization. J. Biotech. 10:95-112.: Amino acid operons and their regulation is yet another approach or application to improving microbial amino acid synthesis and protein in the rumen. This was the earliest report suggesting this technology. (See also: R. Onodera. 1997. In: Rumen Microbes and Digestive Physiology in Ruminants, Pp. 83 - 94).
D. A. Flores. 1988. M. Appl. Sc. Thesis. UNSW, Sydney AU. "Strategies in Supplementation of Amino Acid Flows on Low Quality Diets in Ruminants: Manipulation of Ruminal Microbial Fermentation vis-a-vis Other Approaches to Supplementation (By-product Protein and Energy and Pre-treatment).": Intro-duces the new concepts of postharvest pretreatment, especially tropical forages, for e. g. urea ensiling. What followed was enzyme technology treatment or EFEs, solid-substrate fermen-tation or SSF and bagasse or also called: "Yeast Bagasse".
Danny Flores's latest book now off the press: “A Compilation of Lignocellulose Feedstock and Related Research for Feed, Food and Energy.”, by D. A. Flores © 2013. Xlibris LLC, IN U.S.A.
Salmon Armidale Farm & Fisheries This is a projected pilot aimed at land-based fishery farming in the B.C. Interior and for the northern Philippine islands north of Luzon. The idea is to schematize a pilot plant fisheries enhancement facility for habitat establishment and/or enhancement with a river/lake/fisheries tributaries using imported wild Pacific salmon either of the chum or sockeye variety. The lake can be fed by mountain stream or by cold water aquifers and a dammed stream or river forming a fishing lake with the pilot plant at the end of the fisheries run and the land-based "wadies" at the other end or around. This is considered the "Salmon Habitat". The official name of the project will be Salmon Habitat Enhancement Pilot Project which will then be upscaled to the actual fisheries farms operations.
Hi-Performance Health Products for Preventative Health and Sports.
We purpose the product that can be used in sustaining health in heart health, cancer(s), diabetes typeII and other related chronic inflammatory conditions. We mean to propose dietetic, PUFAs in a oleo, complex CHOs, VitD-agonists, HIS supplementation and other vitamins and minerals. Drug pharma is for injection (I.V.) and parenteral (abdomen) also called Retonibionol (R)(generic drug) for the VitD-agonist(s). The principle is for VitD usage to practice sustainable food enrichment practices with the nutraceutical VitD-agonist(s) under inclement conditions of non-enrichment and lack of dairy in the diet like in Arabic countries and where weather does not permit sufficient irradiation of the skin even with darker skinned individuals like countries in the States which have been and can prevent the incidence of certain cancers.
A Sample chapter of CHAP. 5 from our second volume of the new two-book set of: "A Compilation of Ligno-cellulose and Related Research for Feed, Food and Energy," by D. A. Flores (c). 2019. Skyebluepublications.ca, Port Coquitlam, BC Canada V3B 1G3.
To be submitted to: “A Compilation of Ligno-cellulose and Related Research for Feed, Food and Energy,” D. A. Flores. © Skye Blue Publications. Port Coquitlam BC CANADA V3B 1G3. 2019.
Chap. 10 Functional Amino Acids Fed With Organic Livestock.
By D. A. Flores
The Problem and the Potential.
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.
Sustainable feeding of rumen protected histidine for beef and dairy on lower quality protein rations with supplements. The approach to feeding protected histidine, for beef and dairy, including intra-arterial implants of synthetic bovine or caprine ghrelin, is the new organic alternative to older approaches like steroidal diethylstilbestrol (DES) implants, using a more sustainable, lower % CP basal diet, with concentrate, which would present greater acceptability to consumers.
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).
Functional Amino Acids in Livestock Feeding & Production.
Our view of research with functional amino acids (FAA) demonstrates a dichotomy between basic research and by translation is made to be research in more 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.
We can illustrate metabolism in its functionality in terms of: (1) levels: (a) regulatory (e. g. hormonal) and (b) tissue metabolism (e. g. lactation, lean body mass (LBM) accretion and litter quality), and: (2) types of functionality with: (a) neurotransmitters, (b) cell signaling, (c) operon regulation and (4) reproductive physiology. In the case of gene expression involving, regulatory elements of the operon or gene cassette in regards to transcription factors (TF) and others, and the O/P (operator/promoter) regions; in the case of cell signaling is hormonal action on cell receptor function with a resulting effect on tissue metabolic activity including synthesis; in the case of neurotransmitter/neuropeptide function in the central nervous system (CNS): hypothalamic growth releasing factor (hGRF) and human growth hormone (hGH) from the pituitary, chronic inflammation and cancers; and finally in the case of reproductive physiology, an e. g. would be avian productive function in egg laying in chickens.
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, 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, 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).
The Future of Probiotic Use in Cattle.
The use of probiotics in cattle was first introduced many years ago but problems have arose with recent findings that the genetically modified microbes do not "take" to rumen conditions and thus will not subsist for any length of time.
There are various theories as to why this is so and that the changes in DNA in some way make them unviable in a highly competitive microbial rumen environment due to primarily greater energy expenditure (viz. ATP) because of added DNA for their maintenance and function.
It is thus proposed that new technologies with CRISPR/cas9 genetic editing (GE) which elegantly replace or introduce new genes such as with transcriptional factor (TF) genetic engineering will serve to introduce improved (viz. single base mutated) gene copies or introduce new gene copies altogether.
Other e.g.s. of probiotic technologies to use are:
(1) The introduction of replacement genetic cassettes and/or use of TF genetic engineering for limiting amino acid production in amino acid rumen microbial metabolism to boost microbial cell protein (MCP) production.
(2) The introduction of replacement genetic cassettes and/or use of TF genetic engineering to synthesize water-soluble carbohydrates (WSCHOs) and peptide sequences to produce more MCP known to limit their efficiency (g MCP / kg DOMI - digestible organic matter intake).
(3) The porin model to contain the microbial populations within the rumen is used in effect, after they are drenched per os and as proteins "plug" the open pore model that is made and coded for by genes that produce transbounding membrane subunit proteins (e. g. in a hexa- or pentapeptide complex) designed using nanotechnology with a specified diameter (viz. in Angstroms) that closely fits the plugged hole model. A pharma approach is used by raising the microbes in medium with the porin from sporulation. But the spores are fed as feed pellets or tablets with chewing followed by drinking the suspension medium with the porin biopharmaceutical.
(4) Immunocontrol for biosafety of porin microbials via vaccination of both associated animals against quarantine and by their animal handlers in direct contact with the quarantined facility will result in further biosafety controls of probiotic-fed animals.
Probiotics have a role in lower quality wet silage feeds stored over the Winter months in colder climes and also in the tropics of warmer climes where silage-making is becoming more popular due to its ease of mechanization during harvesting, storage and feeding by targeting it to rumen microbial cell protein (MCP).
To expand further on the tri-innoculant approach espoused or recommended by the author [see: D. A. Flores (1989)], the following is proposed using plasmids as vectors:
 The Fibrolytic Approach: With the porin model using a soluble pore blocker to be used with fibre digesting or energy-generating hosts and to be gene edited (GE) in modified subunit form on plasmid DNA. For e. g. that can be used in practice is already developed with L. delbruckeii probiotic host with donor DNA, e. g. Fibrobacter succinogenes for cellulose, xylanase, mucillage binding at the biofilm level, and the property to survive bile salts and acid in digestion.
 The "Storage" Protein Approach: (E. g. casein, immunoglobulin) with fibre-digesting, energy-generating hosts with the porin model, using our practical, e. g., L. delbruckeii.
 The Ligninolytics Approach: On the back-burner at this time as there is no conclusive proof using esterases and etherases (and lyases - if they are better documented) and bring about mineralization what we call "end-zymes" to the endpoint of CO2 + CH4. We decline to suggest use of direct boosting of ligninolytics that habituate as the rumen is a closely regulated, highly competitive and commensal environment. We instead propose use of a plasmid construct in the same host L. delbruckeii with ligno-cellulolytic capabilities although lack of enzyme technology [(see also: D. A. Flores (2013)] hinders use at this time.
Marine Grasses as Forage Feedstock.
Marine Grasses as with common pasture and prairie high-sugar grass and legume crops have the potential to support high-producing cows, beef and goats, for both dairy and meat, and what they in particular produce, i.e. medicinal dairy milk products and high-quality beef.
It will involve incubator-driven multi-trayed 'E-Z' growers to cachet crops and provide harvest "by the roll" for hod dispensation to feeder cattle including what are called "Hi-Producers" in dairy cattle boosted for protein utilization (e. g. digestive, metabolic and intake - DMI) with these designer, non-pollinating hi-sugar crops.
The sugars have been shown to further boost rumen microbial amino acid production on forages via stimulation of efficiency of microbial cell protein (g) / digestive organic matter intake (kg) (MCP g/ DOMI kg) rate of digestion, although we do not go into proposed hypothesis as to its mechanisms.
There are other applications of high-sugar grass feeding to which this may apply to boost overall N utilization in large and small animals using the nascent technology of hydrobiology of land-based marine grass raising for fodderstock and other feedstock for energy use such as kerosene, biodiesel and medium-chain alcohols of high density or energy content.