Life Produce for Dairy and Meat Information Enterprises of Canada

Moving GMO to Non-GMO, GRO Dairying in Cows
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From GMO to Non-GMO, GRO Producing Dairy Cows.

1  Introduction

The interest is expressed here on the possibility of recombinantly modifying the DNA of dairy cows for milk food production, specifically with an interest in milk proteins: bovine beta-lactoglobulin, bovine casein (bC) and bovine lactoferrin (or bLF). The fact that dairy foods are essential for preventing: major cardiovascular disease risk factors, hypertension, type-2 diabetes and some cancers (H. Davoodi et al., 2013), underscores a need to investigate this further.

In general, increasing protein in milk dairy production by dietary means is by increasing energy concentration in the diet such as improving digestibility, and improving the VFA profile (including acetate).  Another is by making favourable conditions for microbial cell protein (MCP) synthesis and/or dietary protein escape (or bypass); we will take the opportunity here to emphasize the dietary approach of lowering of plant-mediated proteolytic activity resulting in greater escape and MCP flowing to the small intestines (SI), amongst others.

Precision gene editing has not been attempted in regards to application to the dairy cow for manipulating milk food protein yield or output as indicated by the scientific literature, thus far.

A first commercial example of a crop genetically modified organism (GMO) via precision gene editing was by corporation Cibus of San Diego, CA in the U. S. A. with a type of oil rapeseed

for herbicide resistance using their Rapid Trait Development System or RTDS (C. Ainsworth. 2015).

Consequent with advancing development in many countries around the world is the: 1) growth in population, 2) increase in the standard of living, 3) higher demand for animal-derived protein and 4) increases in lifestyle and environmental stressors that may lead to occupational and genetic-related disease incidence (M. J. Boland, 2012). This bodes well for initiatives with biotech to serve markets in developing areas with growing populations with milk possessing medicinal properties all touted to have some protective effect against those diseases of growing incidence in growing populations exposed to various stressors.

We will concern ourselves here with some aspects of protein nutrition and feeding of dairy cows and the prospect of producing the first GMO cow with boosted levels of bC, beta-lactoglobulin and bLF, (and omega-3 fatty acids, for that matter).

2 Need for More Demographic Data Justifying GMO Cow’s Milk Food Production.

Before DNA precision engineering research on milk cows can proceed, the issue is what should determine guidelines for administering the recommended daily allowances (RDAs) for con-sumption of this new functional milk or nutraceutical food product.

Data should be gathered as is possible for Centres for Disease Control (CDCs) across demographic areas with indicators as to incidenc, for e. g. biomarkers and adenocarcinomas, to recommend such guidelines for intake (e. g. fluid oz./day) of type of milk food product that significantly protects against systemic inflammation in the case of atherosclerosis or colon, breast or prostrate cancers with typical diets and lifestyle habits and including: 1) exposure, if any, to carcinogenic substances or terratogens in regards to their level of incidence or exposure in the environment, 2) what genetic links of individual responses there are to milk food products, connecting it to the emerging field of nutrigenomics, and 3) other dietary food with milk consumption could of course have interactions, to what extent, could confound demographic data further.

The following needs further elucidation using larger-scale demographics: 1) use of simulated nutraceutical milk food proteins (e. g. bC, beta-lactoglobulin and bLF) with each treatment used and 2) the use of the various dairy products: milk, cheese, youghurt, cream and butter and to identify their differing effects due to food source or as an ingredient.

There is already data in the literature indicating use or consumption of regular (or simulated) milk food products that confer anti-oxidant abilities in the body, having an associated lower incidence in systemic inflammation and in cancers, including colon cancer, and are thus justifiably to be given priority in biotech’s agenda to make it to commercial reality. This similarly as to what happened with “golden” rice aimed at curing blindness in countries where Vit A deficiency still occurs (M. L. Winston, 2002).

3 Low-protease Feeds and Bypass Oilseeds.

3.1 Low-protease Feeds

To improve escape and reduce protein nitrogen (N) degradation and N loss in the rumen, improving MCP N synthesis, various approaches can be used with feeds. (We will not discuss grazed, fresh grass or legume forages or grains as the application of low-protease feeds to them either falls outside the discussion or no data applicable to them has yet been found in the literature, respectively. (However, at this time, the reader should note strongly the possibilities that low-protease grains may offer in feeding as it has with other forages.)

To outline here these approaches as to their biological or chemical mechanisms, by example, these are: 1) management practices [e. g. wilting with high plant enzyme polyphenyloxidase (PPO) which improves heat damage and ruminal escape of feed protein];  2) innoculants, including fibrolytic recombinant Lactobacilli spp. (D. J. Armstrong and H. J. Gilbert, 1991); 3) other additives, including: a) biological,  e. g. tannins (J. L. Mangan, 1988) and b) chemical, e. g. formaldehyde and formic acid (A. Taghizadeh et al., 2007; E. Charmley, 2001); 4) plant breeding (e. g. low-protease and high PPO) (D. A. Flores 2013; M. R. F. Lee, 2014); and 5) finally recombinant engineering (e. g. low-protease, high PPO, high anti-protozoal (defaunating) levels to increase MCP flow (D. A. Flores, 2013; M. R. F. Lee, 2014; A. Navas-Camacho, et al., 1993).

In silages, plant-mediated proteases are not limiting due to the highly degradable nature in silage protein N, except, it is hypothesized, where heat protection, e. g. wilting, is applied just following harvest (Flores, 2013) (see below section 3.2).

3.2 Dietary Protease and Possible Effects on the “Transactional Hypothesis” of Amino Acids of the Rumen.

The “Transactional Hypothesis” combines evidence from the following: (1) peptides are transported with different rates of efficiency and ATP (energy) of transport, (2) the energy of incorporating amino acids from peptides consumes less ATP than free amino acids to microbial biomass protein and (3) that protein that is optimally less degradable and released for utilization and uptake is more efficiently taken up into MCP. 

To outline some areas for further research on the efficiency of microbial protein synthesis (EMPS) (e. g. with sheep rumen models): (1) transport processes and rates of pre-formed amino acids (PFAAs) (i. e. amino acids and peptides, APPs) through the cell membrane, (2) mitogenesis (rate of cell division) (viz. ATP, NADPH, THF, B12, and DNA synthesis), (3) and apoptosis (in higher cells alpha-TNF is related to cell death; there is yet no evidence linking FOS directly with apoptosis in lower cells, unlike equine peripheral blood cell models where alpha-TNF can be measured and related further to mitogenesis, (4) “uncoupling” with bacterial cells is due to “uncoupled” energetic efficiency which occurs with relative differential efficiencies under energy limiting conditions (P. Sharma et al., 2012), and could be used as bases for further studies into substrate and their availability regards efficiency and yield for biomass in the rumen.

It is proposed, again, here as hypothesis that forage feed heat-treated or damaged (e. g. steam or wilted) provides a higher supply of N-containing substrates to rumen microorganisms, in relation to energy availability to carbohydrate degradation due probably to their reduced protein solubility and proteolysis increasing (see, Thomson and Beever, 1980); at the given dilution rate (%/hr).  We conjecture further to this that the rate of proteolysis is optimized which otherwise results in loss from excess deamination to NH3-N; following this, re-uptake occurs of NH3-N and pre-formed amino acid-N (PFAA-N).

Studies consistent with the previously held hypothesis is the finding by Lee Baldwin’s group of UC-Davis CA U. S. A. that replacing 25% of urea-N with N from a mixture of 18 protein AAs maximized the microbial yield (DM units/carbohydrate substrate units fermented), while adding a blend of protein AAs plus peptides doubled in vitro rumen microbial cell yield; further it was found that adding AA mixtures, that is ,mixed peptides resulted in substantial reduction in proportions of individual AAs in microbial protein derived from de novo synthesis (G. A. Broderick, 2014).

The preceding hypothesis is reflected by P. H. Robinson (1999) and a recommendation for dairies stipulating that: (1) sufficient soluble and degradable proteins to dairy cows not exceeding rumen microbial protein requirements and (2) sufficient supplementary dietary protein with a high escape value and (3) the latter having an acceptable AA profile in order to supply absorbable protein that could limit milk protein production.

3.3 Wilting of Silages.

Silage production offers harvesting for larger quantities of forage at specific times allows the farmer more control over the composition, i. e. nutritive value, of the harvested material although processing in silage fermentation can vary considerably and the resulting

silages can markedly differ in nutrient profile according to the efficiency of ensiling techniques (D. E. Beever, 1983).  To illustrate this with a case study in point from the U.K., practice was thus: direct-cut (non-ensiled) harvested material is normally ensiled with an additive versus only partially wilted material with use of an additive which is more restricted and is often only used when poor weather conditions prevail; research into digestion and utilization which we will illustrate with data from grass silages reflects the described practices for the U.K.: digestive duodenal amino acid nitrogen (DAA-N) flows (g AA-N/ g total N intake) with fresh grass was 0.63, wilted silage was 0.47 and unwilted silage was 0.54; this is somewhat reflected in the small intestinal AA-N absorption (g AA-N/ g total N intake) of 0.41, 0.31 and 0.41 (D. E. Beever, 1983). Indeed, the data illustrates how variable poor weather conditions can prevail in the U. K. making in turn nutrient profile or digestion vary from the norm.

3.4 Chemical Treatment with Formaldehyde of Silage.

Recent advances with corn silage to supplement other forage diets (that is, grasses and legumes) demonstrated that formaldehyde effectively decreases ruminal NH3-N resulting in high escape protein and can be targeted to performance or productivity of animals (A. Taghizadeh, et al., 2007).

3.5 Chemical Treatment with Direct Formic Acidification of Silage.

Direct  acidification has been a method demonstrated to be effective in improving silage feeding value; there has been widespread adoption since more than a decade  and a half since (E. Charmley, 2001). One study demonstrated with wilted silage that successive addition and effective restriction of silage improved growth of cattle on silage and performance of dairy cows; it is important to note that rapid acidification occurs within minutes at time of addition of treatment unlike natural fermentation (E. Charmley, 2001).

3.6 Biological Treatment with Tannins  of Silage.

The use of condensed tannins (or plant polyphenols – relatives to flavanoids and lignin) will be further developed by genetic manipulation or conventional plant breeding with pasture grasses or legume species with suitable tannin content to control soluble protein rumen concentration without interfering in the small intestinal digestion (J. L. Mangan, 1988).

An e. g. of this type of work described from the Plant Genetic Manipulation Group, Nottingham, U.K. is generating mesophyll protoplasts from legume species originally from green leaves not expressed in their leaves but fused with high-producing tannin species using cells from their flower petals (J. L. Mangan, 1988).

3.7 Canola Oilseed Bypass Supplements.

Unsaturated fatty acids in Ca2+ salts (bypass fat) have a positive linear effect on the milk fat percentage of lactating cows; with supplementation of bypass fat (derived of Ca2+ salts of LCFAs) there was an increase during the total lactation (early, mid and late) period, there was an increase in the total unsaturated fatty acids (USFAs) and monounsaturated fatty acids (MUFAs) and decrease in saturated fatty acids (SFAs) as a percentage of the total fatty acids of milk (P. K. Naik, 2013).

Chemically prepared bypass fat includes formaldehyde-treated protein encapsulated fatty acids with canola as an e. g. of a source of fat and contains 40.2% in its oil seeds;  the oilseed can be crushed, then treated with formaldehyde (1.2 g/ 100g protein) in plastic bags or silos and kept for about a week (P. K. Naik, 2013).

4 Precision Gene Editing of Large Animal Dairy Genomes.

It was recently stated: “Transgenic animals are an important source of protein and nutrition for most humans and will play key roles in satisfying the increasing demand for food in an ever-increasing world population.” (W. Tan et al. 2012). This would suggest, soon to come, gene editing of large animal dairy genomes within this decade or so although the discussion and findings on genetics an genomics of large animal dairy cow’s genome has just begun before a more comprehensive picture of a molecular basis for manipulations can be arrive at (see the ff. this section). Recent applications with gene editing with plants have been proposed for: improvement with nutritional value and resilience; in livestock genome editing is just beginning; specific or potential applications are: (1) polled traits regarding dehorned cattle without addi-tional DNA being present, (2) mutations of the myostatin gene for marketable muscle mass or meat and (3) adaptation in breeds such as with heat or cold tolerance, drought tolerance and resistance to particular infectious agents (D.  Caroll and R. A. Charo, 2015).

The most preferred technique of genome editing is clustered regularly interspaced palindromic repeats (CRISPR) – associated protein (Cas9) which is based on nucleases which cleave the DNA to be differentiated from homologous recombinant (HR) – mediated conventional gene targeting; the nucleases cleave chromosomal DNA with double-stranded breaks (DSB) at the site to be modified, (2) followed by either error-prone non-homologous end-joining (NHEJ) or homologous directed repair (HDR); NHEJ frequently induces targeted gene disruption (gene – knockout) while HDR – mediated repair can introduce a sequence into the genome (gene – knockin) leading to precise genomic modifications: (a) point mutations, (b) gene correction and (c) gene insertion (J. Hou. 2016) Areas of research specialization in the field of reproductive technology, i. e. embryology and allied techniques applied with precision engineering techniques include for e. g.: cell culture, IVP, embryo culture, molecular biology, cloning, gene editing, transgenesis and animal model development and validation as it is related to farm animals.

A series of techniques that are used in genome editing – mediated gene correction as e. g. is (1) in vitro fertilization (IVF) or  intracytoplasmic sperm injection (ICSI) to make the zygote, (b) microinjection techniques for HDR manipulation, (c) pre-implantation genetic diagnosis resulting in the repaired embryos, (d) the 1-2 blastocytes of interest recovered, (e) transfer of the developing embryos, (f) non-invasive prenatal genetic testing, (g) chorionic villus sampling and finally long-term follow-up (M. Araki and T. Ishii, 2014).

To begin a discussion on large animal dairy genetics and genomics we will refer here to quantitative trait loci (QTL) mapping from breeding experiments which identifies locations in the chromosomal DNA correlates phenotypic or expressed characteristics using single nuclear polymorphisms (SNPs) (single – base mutations) along the DNA. The following QTLs for the milk qualities or parameters, for: 1) milk yield, 2) protein content or 3) % composition, can be found.  Protein content loci are at chromosome 3, 6 & 20, while protein yield loci are at chromosome 1, 3, 6, 9, 14 & 20; the genes and mutations that explain these QTLs remain uncharacterized at this time; it should be mentioned that QTLs are mapped or located statistically, based on microsatellite panels or tracts of repetitive DNA in which certain motifs of DNA repeat as much as 5-50 X using condifence limits to statistical significance (M. Amills, et al., 2012).

Recent developments using high throughput SNP genotyping (or sequencing) (G. C. Schopen et al. , 2011) found significant associations between chromosome 5, 6, 11 & 14 to milk protein %,

with one interesting preliminary find pointing to a possible relationship between regulatory genes on structural genes in an operon demonstrating a trans effect in bovine of an

SNP affecting chromosome 11 & 14 for a casein protein component (i. e. CSN1S1, alpha casein subunit one – a major milk casein component) while residing in chromosome 6 – perhaps suggesting a role for transcription factors (TFs).

5 Approaches to Precision Gene Editing of Probiotics in Dairy Production.

The process of biomolecular manipulation to deliver via genetic engineering gene copies of interest to within the chromosomal DNA is to use suicide vectors or plasmids that deliver DNA

into the cell and after transformation proceed to commit suicide because they cannot replicate in the host but manage in rare events to integrate elements (e. g. transposons – DNA with insertional end-repeats, and within, structural genes) confirmed by expressed markers such as antibiotic resistance.  It is suggested to explore, store and draw on existing plasmid gene bank sources in microbial hosts and proceed to manipulate by conventional molecular genetic recombination techniques (D. A. Flores, 1989) and still via newer precision gene editing techniques.

Probiotics have been identified to play a role in promoting dairy production.  We will not go at length to discuss new approaches, methods or recent finds.  It should be mentioned in passing

that two species of yeast can be direct-fed and that do retain in the rumen with beneficial effects: Aspergillus oryzae and Saccharomyces cerevisiae.  Handling involves drying (viz. lyophilizing) cells to preserve viability and metabolic activity and in some cases yeast cells are mixed together with their fermentation medium (J. Chiquette. 2009).

Results in dairy production indicate that yeast  probiotics are beneficial when protein is deficient, that is, deficiencies arise in MCP and bypass protein, from the diet but not when protein supplements are given (J. Chiquette, 2009). It was also suggested earlier by D. A. Flores (1988) that probiotics could be a feed substitute for low quality feed supplements (and with feed pretreatments of the basal ration) as a hypothesis.

The following are suggested approaches to gene editing of rumen microbial probiotics to improve digestion in the rumen with: (1) Saccharomyces cerevisiae, fed continuously at 5 g/d with an Fae enzyme and an E. coli etherase that can be cloned in this host to improve fibre

digestion, attacking the PCLCCs of plant tissue residues, (2) Saccharomyces cerevisiae fed continuously at 5  g/d with a grass diet low in certain essential amino acids (EAA) by boosting total EAA flows to the intestines by adding EAA operon cassettes and/or TFs by precision editing that are limiting in MPS (see: D. A. Flores 1989), fed with ATP (energy) from the increase in fibre digesters that result from yeast addition, increasing the sugars (energy pool) that would support amino acid synthesis and microbial growth and (3) Aspergillus oryzae is known to retain like yeast in the rumen and will be fed at a given rate (g/d);  to provide Aspergillus oryzae as a “surrogate” for the synthetic protein MB1 (M. Beauregard, et al., 1995); it is unknown at this

time if it can retain in the rumen, although ‘tweaking’ the energy supply pool, viz. adding an additional cellobiase gene and/or its TFs by precision editing  (note: this assumes that the cellobiase gene predominated across genus Aspergillus, one e. g. being Aspergillus niger) would increases energy availability from fibre by deregulating its feed-back inhibition (FBI) and serve this purpose (see: D. A. Flores, 1988).

6 Milk Protein Components to Manipulate and Expected Health Benefits.

6.1 Milk Protein Components.

Milk proteins in forms that are bioactive as peptides and/or proteins exert an effect either through the immune system or on the gut itself; different milk components may act syner-gistically when interpreting data to assess milk’s effect or bioactivity.  The main bioactive proteins in milk are: immunoglobulins, bC (80% of total milk protein), bLF, beta-lactoglobulin (14% of total milk protein), alpha-lactalbumin and cytokinins.

6.2 bC in Milk and Its Health Benefits.

Casein products (e. g. tripeptides) have been shown to have hypotensive effects in in vitro stu-dies on endothelial cell function via NO and inhibiting angiotensin I converting enzyme (ACE); NO inhibits inflammation; also, they are known to moderate inflamed monocyte adhesion in inflamed endothelial cells (M. S. DaSilva and I. Rudkowska, 2015).  Hypertension leads to damage of the inner or endothelial lining of blood vessels and develop-ment of atherosclerosis including the adhesion of blood monocyte cells in the process of plaque formation and eventual occlusions.

Using Caco-2 cells in an in vitro system stimulated with TNF-alpha treated with casein hydroly-

sate for 24 hrs. and inflammatory cytokines measured via enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR), peptides in fractions 1 k DaR and 1 k DaP reduced IL-8 by 68.7% and 66.1%, respectively; while using porcine colonic explant tissue in an ex vivo system stimulated by lipopolysaccharide (LPS), the 1 k DaR reduced IL-1 alpha, IL-1-beta, IL-8, TGF-beta and IL-10 providing evidence that sodium caseinate prossesses anti-inflammatory properties (A. Mukhopadhya, et al. 2014).

The recent evidence of anti-carcinogenic properties of casein in colon cancer points to: 1) the inhibition of enzymes produced by intestinal bacteria, 2) deactivation via deconjugation of

procarcinogenic glucorunides to carcinogens, 3) stimulates phagocytic activities and increase lymphocytes, 4) and new findings point to decrease in precancer markers (aberrant crypt foci) in the proximal colon of rats exposed to chemical carcinogens perhaps related directly to the molecular structure of casein . (H. Davoodi, et al., 2013).

6.3 Bovine Beta-Lactoglobulin in Milk and Its Benefits.

Beta-lactoglobulin can also serve as an ACE inhibitor and has antioxidant activities (R. I. Ricci-Cabello, et al., 2012).

6.4 bLF in Milk and Its Benefits.

This glycoprotein downregulates production of human blood monocyte cell cytokines (e. g. TNF-alpha, IL-1 beta, Il-6 and IL-8 via interference of NP-kappaB activation (M. S. DaSilva and I. Rudkowska, 2015).  This implies that bLF can be significant in reducing systemic inflammation.

In rodent animals chemically induced carcinogenesis was reduced in different organs [e. g. breast, lung, liver, colon and bladder].  There is molecular evidence showing bLF’s ability to

interact with some receptors and genetic expression of several molecules vital to cell cycle and apoptosis machinery (H. Davoodi, et al., 2013). Whey proteins, previously mentioned, which contain predominantly bLF and their anti-cancer properties may be protective in part due to their cellular levels of glutathione, an anti-oxidant (H. Davoodi, et al., 2013).

6.5 Omega-3 and Omega-6 Fatty Acids in Milk Proteins and Health Benefits.

The medicinal qualities of polyunsaturated fatty acids (PUFAS) are immunomodulatory; e. g. 

omega-3 fatty acids and their derivatives eicosapentanenoic acid (EPA) and docosahexanaenoic acid (DHA), eicosanoids, have the following mechanisms via: (1) amount and type of eicosanoids via oxylipins which are regulatory in inflammation where coronary heart disease (CHD), aging and cancer are characterized by elevated IL-1, a pro-inflammatory cytokine, (2) intracellular signaling pathways, (3) transcription factors (TFs) and (4) gene expression (A. P. Simopoulos, 2002).

Further, omega-3 chronic inflammatory diseases decreases disease activity and lowered anti-inflammatory drug use; omega-3 correlations exists between inflammatory conditions with increases in omega-6 fatty acids and decreases in omega-3 fatty acids; omega-6 exacerbates autoimmune diseases with already elevated pro-inflammatory IL-1 cytokinin and leukotriene LTB4; finally the elevated ratios can result in elevated or sustained immuno-stimulatory responses to and diminish immune-suppressing response after injury repair (A. P. Simopoulos, 2002).

7 Cow’s Milk and It’s Qualities and Processing.

It should be studied further as to taste, feel, appearance and nutritional profile or balance of milk components, for e. g., between flavours of boosted bC, bLF and beta-lactoglobulin, with iron-containing bLF.

Non-thermal technologies are advantageous in preserving bioactivity of milk components although they cost more to purchase and operate.  There is a need to increase shelf-life of non-thermally pasteurized food product while decreasing these costs.  E. g. of processing methods are: High Pressure Processing (HPP), High Hydrostatic Pressure (HHP), Ultra High Pressure (UHP), Ultraviolet (UV) light, Ultrasound, Pulsed Electric Field (PEF), Ozone, Cold Plasma, Irradiation, Pulsed Light and Oscillating Magnetic Field (Pers. Comm. 2016. IdeaConnec-tion.com).

8 Allergenicity of Milk Products.

Allergenicity to cow’s milks is variable but a significant issue. 1 in 50 or 2% of infants are allergic to milk or other dairy products; by 4 yrs or older most outgrow milk allergies but it has to be

determined as to the nature of lingering allergic symptoms although rare;  e. g.  of symptoms are hives, eczema, swelling, vomiting, diarrhea, noisy breathing or wheeze, asthma and anaphylaxis;  it has been found that beta-lactoglobulin, alpha-lactalbumin and caseins are the major allergens of milk (G. Bu, et al., 2013).

It is possible that boosted levels could have hypersensitized allergenicities of which a genetic basis if it exists should be further studied or established. Also, it should be added that processing, e. g. heat pasteurization versus non-thermal pasteurization and its efficacy against vis-à-vis for allergenicity should be further investigated with this GMO milk product of the future.

9  The Milk Food Product Delivery System.

There is at this time no comparison available for taste or product marketability tests comparing GMO cows milk “as is” compared to the commonly known enriched, purified nutraceutical formulations already out there in the market.  Today we can ask, will consumer preferences primarily be due to: 1) product quality (viz. the new taste, feel, odor, appearance, etc.) and/or 2) labelling (i. e. GMO versus non-GMO). What would be the influence of socio-economic factors of the consuming public, the government regulatory bodies and manufacturers of larger agri-food multinationals or government-run consortiums of less-developed country settings. Examples of scenarios are: 1) determining the convenience and cost of production (e. g. milk product processing with bioactive non-thermal pasteurization), 2) acceptability to farmers and their practices including cost of acquiring and maintaining livestock in their herds and 3) systems of intellectual property (IP) rights with government regulation.  It will be likely that these developing countries with very relatively young, large populations in the workforce who are health-conscious can provide the momentum to launch and sustain marketing and sales drive with their demand. Culling cows after their prime amongst high producers could be minimized by using only elite breeding stock for precision gene editing and avoid animal rights activists and their stand on cruelty against animals.

Non-GMO, GRO Probiotics in the Rumen: "new" biotech has arrived. 

To study the energy and protein (MCP & feed escape) output of biomass from the specialized rumen stomach of dairy cows we consider plant material substrates, protein/amino acid supplements and microbial cell growth in addition to escape biomass.  

There may be now theoretical bases for studying factors that could help determine the efficiency and action of microbial cell protein (MCP) synthesis.  These have been pointed out at SkyeBlue to be aspects of microbial physiology & biochemistry: (1) transport processes & rates; (2) mitogenesis or rate of cell division as possible functions of ATP, NADPH, THF, B12, and DNA synthesis; (3) apoptosis or cell death such as fructan oligosaccharide (FOS) related to alpha-TNF and cell death at least in equine peripheral blood cell models but not lower cells, and, finally, the most reasonable of our possible mechanisms or functions, (4) uncoupling of energetic efficiency.

To discuss the plant material as substrate for microbial digestion and growth, the following factors figure in: (1) ligneous components in feedstock and digestibility of fibre, (b) boosting fructan content or water-soluble sugars, (c) plant protease activity in crops and resulting pre-formed amino acid and peptide availability to rumen microbes and their synthesis and identifying further the matter of synthesis of limiting essential amino acids in microbial metabolism.

These are crucial to understanding how to increase milk output in dairy cows to start studying the underlying molecular mechanisms in microbial and plant material, we believe, is nearing a breakthrough now after a number of decades study. 

We are currently investigating possible uses of yeast (fungi) which are subsistent as rumen probiotics for essential amino acids metabolism in microbes and their growth (and GEdited "surrogate" proteins high in limiting essential amino acids) to rebalance amino acids profiles reaching the small intestines (S.I.) of cows. We are also investigating use of fungal species and their increased capacity for fibre digestion via various lignases ( esterases, etherases, and possibly, lyases).Biocontainment both via the porin plugged model are being further investigated for this purpose and immuno-based quarantining of axenic animal handlers/staffers. 

Cytobiology with DNA precision engineering and IVF can be used to introduce O/P regions in cassettes coding for the synthesis and secretion of milk food proteins (MFP), casein and lactoferrin, two proteins that have been studied to have an effect in protecting against certain cancers and which should be introduced in cow's milk in semi-industrialized country settings (e. g. India, Russia and China) where life-style would indicate these medicinal properties as a strong selling point similar to "golden" rice with beta-carotene by an agro-industrial giant like Monsanto (USA). GMO cows and their sires will eventually be backcrossed (various schema that can be used here) to build a sufficient 'gene pool' to bring about heterosis along with the gene edited genomic backgrounds as with growth rate, carcass quality, feed conversion efficiency and vigour, as examples.  It is suggested that facilities at the Macdonald Campus Farms of McGill U., UNE-Armidale regional and in North, Central and Coastal Queensland (AU) will take up these research goals in future with a go-ahead in GMO cow production.

There is evidence now (see: Genus Farms Milling and Foods Canada, this website) that non-GMO production of seagrass farmed fodders is possible with advancing techniques to overcome rate of growth or C-sinking or optimizing the composition (viz. energy content and availability, protein nutritive value or what we have dubbed at "SkyeBlue" as "proteinogenicity" of the diet).  

The ff. will be used to overcome or supplement the low-protein problem from the digestion of low-quality feeding regimes.  The following are to be employed when feeding the GMO cow: (1) supplementation  with HIS over-producers in yeast functional feeding; (2) supplementation with MET over-producers in yeast functional feeding; (3) interventive low-protease feeds to conserve amino acid availability in forages; (4) low-buffering capacity (Bc) from prebiotic interventives using enzyme technology active in plant forage; (5) using ensilage inoculants for fibrolysis with co-cultured feedback disinhibition to improve silage quality from acidity with fermentation of sugars to lactic acid; (6) yeast for rumen probiotics boosted for fibrolysis and increased CP yield in the microbial rumen stomach using mutagenic protoplast fusion (viz. genetically manipulated (or GM) but considered non-GMO and with surrogate "storage" proteins that have limited solubility and attack by microbial and plant proteases, and finally, (7) use of hi-amino acid forages from GM of their metabolic capacity to produce and store selected amino acids, e. g. MET and HIS in alfalfa. 

The GMO cow will be supported by these "green" technologies with various of these low-quality feeds designed to deliver total feed efficiencies that are admirable, compared to more normal feeding situations proposed here.   

The Water Buffalo for Non-GMO, GRO Manipulation for Hi-Producing Milk and Meat. 

There is now the new but exciting prospect presenting itself for the water buffalo species for the Carabao (Philippine) and Murrah, Surti and Nili-ravi (of India) for both traditional breeding for dairy hi-production and slaughter and both GMO through gene editing and non-GMO with the new PNA-B12 biologic or with our own theoretical PNA-K2(x5) biologic and also a new discovery with new proposed proteosomal-level regulatory manipulation to treat disease and address and also more production-oriented examples in higher animals (viz. humans and livestock) using mechanisms in the cell to degrade proteins such as with our "labelled" ubiquito-proteosomal - generated regulatory organisms or as UP-GROs, i. e. up-regulation of organismal metabolism for improved health or production standards.     

The need for pushing ahead with the science of bioinformatics with the water buffalo to decoding and studying the regulatory structural genes for milk synthesis and hormonal production for building carcass muscle in animals, and their ncDNA for the sequences in regulatory "cassettes" apart from the structural genes that produce even useful products in livestock production. This represents the genomics field of interest.  Also, the other is proteomics which involves studying areas as the ubiquitine proteosomal pathway in biochemistry. The scope is interventive in terms of care and management over dairy and as proposed now for meat or as it is know in the Philippines, carabeef.

(c) D. A. FLORES. SKYE BLUE INTERNET. Port Coquitlam. BC. Canada V3B 1G3.

Last update of this entry: January 22, 2024