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  May 23, 2024
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Cara-Oxen Feeding Enterprises of Canada

For the Welfare of Water Buffaloes in the Philippine Islands
Port Coquitlam
Toll free: 16049419022

Phone: 16049458408
Fax: 16049419022
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1. Introduction



The genetics for purposes of elucidating DNA regulatory functions would include studying ncDNA sequences and transcriptional regulation of DNA. Organisms being proposed under applied control both plant crop and microbial regarding transcriptional regulation using classical gene silencing techniques are referred to as novel non-GMO class of genetically regulatory organisms (GRO) in this paper.


Using applied gene regulatory control regarding organismal metabolism termed the field of study of metabolomics can be at the levels of higher organismal and microbial metabolism, as with probiotics in feed processing, in the rumen stomach, and dual-purpose crops for food-feed purposes in mixed animal-crop farming.


It has been stated that knowledge at the molecular level for breeding should pave the way for improving production, environmental adaptation and disease resistance in livestock [1].  Metabolomics uses the computational bioinformatics with the goal to study the whole organismal genome for metabolism and its applied problems to bring about developments in efficiency of metabolism for productive traits, disorders for health conditions and adaptation to environmental and disease resistance, including infectivity and immunity in whole animals.


Note, this paper only covers whole body GRO genomic manipulation in passing.  Future dairy hi-producers for water buffaloes are not be discussed in this paper, viz., hi-producing non-GMO, GRO (non-cloned) cows producing “medicinal” milk using novel hi-quality feedstocks (e. g. vertical farmed marine seagrasses for the future).


The following non-GMO, GRO manipulations discussed here are:


1)      Rumen Microbial Probiotics.  Due to a predominant use of some bacteria and yeast as rumen probiotics it has been suggested that improving cellulolysis of coarse low-quality feeds for water buffaloes be discussed with non-GMO, GRO yeast probiotics.

2)      Dual-Purpose Cropping.  With dual-purpose cropping using non-GMO, GRO PNA-Auxin/Auxin-like Regulatory Factors (ARF) boosted crops for improved protein nitrogen (N) levels, for their improved nutritive value (N.V.), for e. g., straws, stovers and haulms or vines.

3)      Solid-substrate Fermentation (SSF) for Ensilage with “Dry” and “Wet” Feed Types Using Probiotics and Boosted for Functional Amino Acids (FAA).  The use of molecular-level genetic manipulations for feeds processing with cellulolytic yeast for single cell protein (SCP) and increasing the digestibility of bagasse from sugarcane, straws (e. g. rice, corn), stover (e. g. corn, sorghum) and haulms (e. g. legume vines).


2. Non-GMO, GRO Probiotic Feeding with Water Buffaloes.


There was a recent call for understanding the rumen microbiome more to enable scientists to increase fermentation efficiency and select species of greater interest [2].


Probiotics are defined here as any microbial species that confers, when administered into the digestive tract per os, a beneficial effect on digestion.  Here we will refer specifically to non-GMO probiotic strains as with yeast, viz. Saccharomyces cerevisiae strains in the rumen.


There is a moratorium, even now, on GMO probiotics use due to stringent regulatory precautions.

This includes dangers from lethal pathogens that could result from explosive generation of millions of recombinant bacteria in a matter of hours from what are described as “gene shuffling” techniques to improve probiotic bacteria [3].


Use of probiotics has been to improve animal productivity using both bacteria and yeast to stabilize rumen fermentation, reduce incidence of diarrhea, improve growth and the feed efficiency of young stock.  It has been proposed that genetic manipulation of microorganisms have enormous potential in developing countries in view of feed type resource availability.  Most basal feeds in such countries are low-quality fibrous feeds, as coarse feed byproduct or residues.  It should be noted that particular emphasis be made on increasing cellulolytic activity for more efficient utilization of these lower-grade feeds.


In view of a moratorium to halt GMO-type research, there will be a need to consider non-GMO approaches including protoplast fusion techniques together with X-ray or chemical mutagenesis, and now coming on the scene, RNA-based gene silencing referred to here GRO manipulations.


It was found that investigation with a cannulated Mediterranean breed in Brazil using 3 commercial strains of S. cerevisiae measuring digestive efficiency in rumen parameters as with liquid outflow rate and rumen volume showed no significant differences observed among treatments compared to controls (no yeast addition) [4].  They indicate a rate of solute end product generation from digestion and rumen “holding capacity” with rate of digestion relatable perhaps to intake.  It was however of interest to note that use of yeasts showed significant effects on the degradation of the cellulose and hemicellulose components of fibre in sugarcane in relation to control [4].  Another study with water buffalo cows in Colombia resulted in no significant increase in yield and milk composition in water buffalo cows [5].


3. Biosafety and GMO, Gene-Edited (GE) Porin-Based Probiotics for the Water Buffalo.


Mentioned here specifically is an approach for biosafety and the biocontainment of GMO-type microorganisms, with molecular studies, such as that of [6] where a model porin system using ruthenium II quaterpyridinium complexes and octameric channel protein subunits blocked with naturally occurring porins with negatively charged constriction zones allows for the use of stable binding analytes like specially designed recombinant low molecular weight (LMW) cationic protein plugs.


Here over-expressed charged porin channels to a level that cannot be compensated for by re-equilibration with considerable energy expenditure, causes them to be unviable without the presence of requisite cationic plugs, administered orally to a fixed concentration based on body weight (daily to twice daily) until it equilibrates.


Genetic editing (GE), a more precise form of genetic modification (GM), is applied to chromosomal DNA in microbial cells with the use of transcription factor engineering (TFE) with directed biologics complexed in PNA-B12 units to boost the production of porins and their subunits or components to a lethal dose on the cell membrane surface.


Small to medium farm settings would be apt for this technology for administration of a controlled dosage of slow-release sporulated inoculum tablets to various teathered or stanchioned animals depending on their handability or behaviourial conditioning, for e. g., using the “carrot stick” approach training animal livestock over time.


The use of futurescapes like the colonization of Outer Space is imagined with the requisite use of agronomic crop-livestock rearing systems putting a premium on limiting feed resources through byproduct or residual feeding practices and the need through probiotic interventives for the optimization of animal-extracted nutrients from feeds.



4. Mixed Animal-Crop Farming with Dual-Purpose Crops.


Mixed animal-crop farming brings about a socio-cultural transformation and could include the future use of biotechnology.  For e. g., bringing about cropping developments to exploit more feed byproducts using dual-purpose cropping.  These could be through use of biologics as has been proposed for PNA-Auxin/ARF.  They would improve nutritive value (N. V.) both for food crops and for nitrogen (N)-enrichment of farm byproducts for feeding meat and dairy and herd expansion with breeding.  An improved breed for production could be realized in hi-producing water buffalo cows with proposed PNA-B12 regulatory biologic administered sublingually or to the udder’s mammary gland as was mentioned but not stipulated for discussion here.


Dual-purpose cultivars as non-GMO, GRO using biologics for increased N. V. result in increased feed intake, digestibility and availability of nutrients and improved nutrient profile.  And these might be further transformed to functional feeds, boosted for particular amino acids, again with the proposed biologic technology directly applied in the field through regulated, controlled dust cropping.


Examples of possible candidate food-feed crops that come to mind are the cereals and oilseeds, e. g. wheat, oats, barley, triticale, canola, rice, corn, soybean, and sorghum.


Leading research on dual-purpose cropping from Australia has already been used there which should expand further within the Australasian region in countries with high rainfall.  They provide additional forage and increase in farm profitability [7]. It could be a “game changer” with smaller subsistence farmers in developing countries.


This type of cropping should be applied in countries  like the Philippines, where grazing lands are limited calling for more alternative sources of forage like sugarcane for dairy feeding.  Draught animals can be used for field preparation between planting seasons for rice and can be used during the dry season there to increase forage availability for feeding beef and dairy animal ranching.


Livestock resource planning is to follow to transform the farm herd with breeder stock, allowing further ownership for trade or sale and of animal products from meat and dairy.  Capital or asset management will also be converted to cash flow for financial assets and for infrastructural, equipment and tool upgrades on-farm.


The use of dual-purpose farm byproducts as with straws, stovers and legume haulms will allow further ensilage as haylage it is suggested, for year-round feeding.  This is perhaps the greatest development from dual-purpose cropping outside of postharvest grazing, given the possibilities of  drought conditions and global warming in tropical areas where water buffalo production predominates.  The expansion factor from additionally utilized dry matter (DM) for feeding as processed and stored will result in increases in livestock numbers.


A basic scheme for using PNA-Auxin/ARF technology involves covalently attaching the PNA-Auxin/ARF complex to their TFs, which may or may not be engineered via TFE, as either “silencers” for decreasing or as “enhancers” for boosting activity through binding of the promoter (P) on their DNA regulated operons. At this time, theoretically it is believed that the applied PNA-Auxin/ARF complex penetrates the plant’s tissues at the shoot tips to deliver the carriered conjugated TF-engineered mRNA systemically to plant cells and stimulate “storage” protein development.


5. Making Yeast Solid-Substrate Fermented (SSF) Residual Feeds.


The paper will now outline the major steps for milled bagasse pith (viz. as derinded) as substrate to be processed to what is referred here to as “Yeast Bagasse”, below. 


1) Making the semi-refined consistent particulate base.  First, is threshing of the byproduct feedstock from the field’s harvest on-farm and then fine-chopping as in making ensilage. Second, is washing thoroughly substrate followed by air-drying over 48 hrs. Third, follows with fine grinding to a semi-consistent powder (this can be adapted for each feedstock type). 


2) Mixing reaction components prior to live yeast culture (LYC) incubation.  The dried, particulate substrate is bleached with added dilute aqeous acid (H2SO4) over a 48-hr period and then mass centrifuged at industrial-scale and washed of residuals and then the pH adjusted for enzymatic conditions involving aerobic lacasse treatment over a few days in an enzyme bioreactor per batch.


3) Addition of the LYC and their incubations. The live yeast culture (LYC) is applied as follows with the remixed or reconstituted LYC sprayed onto the lofted particulate feedstock and further augered into a mixer together with the applied biologic added solution to the LYC just before it is stored for incubation with feedstock at the temperature, pH and time specified.  The biologic’s action begins the process of boosting higher levels of histidine (HIS), arginine (ARG), leucine (LEU), methionine (MET) and lysine (LYS) production in the incubating biomass with the GRO-manipulated LYC. (Note, S. cerevisiae is facultatively anaerobic.) 


4) And finally air-spray drying and storage.  The biomass is to be kept in the final holding tank and is effectively stored in a compacted form, in a cool and dry place after thorough drying by spraying onto an industrial walled chamber, collecting at the base.  It is mechanically packaged in rectangular bags for later dispensing for feeding at the dairy or beef farm feedlot.  The matter of an on-surface film on the LYC is to be flash-dried with forced N2(g) which would render it a DYC on the particulate surface with N2(g)-sealed vacuum packaging.


It is open to question at this time at what levels of amino acids can be attained and what are optimal for functional feeding regimens such as for LBM accretion, milk solids and volume output and, in general, the body condition of finishing steers and end-period production in lactating dairy cattle.  There is a need to test various basal rations plus addition of our energy-protein supplement derived from “Yeast Bagasse” pith processed which has undergone delignification, fibrolysis and protein and amino acid supplementation with SCP yeast.  What remains as the most limiting amino acids for optimum nutrition is to be demonstrated by what should be supplemented at a biologic rate of addition to the DYC and the given DM portion of pre-treated biomass.



6. Supplying Functional Amino Acids (FAA) to Silage-Fed Water Buffalo.


The beginnings of this type of technology with bagasse feedstock processed was by a Japanese concern in the Philippine Islands to feed dairy cows (viz. Holstein bred type cattle) for milk production [8].  Yeast processing however modified to be enhanced, refined and made cost-effective and tested in animal trials could be extended to cover also other byproduct residuals such as corn and sorghum stovers, haulms or legume vines, rice and wheat straws, corn and rice brans and their hulls and copra meal.


Eight spp. of fungi, namely, Aspergillus niger, Candida albicans, Saccharomyces cerevisiae and Fusarium spp. and four other unidentified spp. called F1, F2, F3 and F5 produced remarkable results according to the authors in the study comparing with standard media like potato dextrose agar, Sabouraud dextrose agar and corn meal agar [9].


There can be the possibility of adding a hydrolytic enzyme system from Actinomycetes spp. which are effective in breaking down with treatment the lignocellulosic polymers of cellulose, hemicellulose and lignin from this microbial spp. [10].


Functional amino acids (FAA) as supplements can be supplied at a minimal cost from waste or byproduct versus more expensive media used currently and may replace animal-based protein meal supplements (e.g.s. meat meal, feather meal, chicken litter meal, blood meal and bone meal) given that the modern phenomenon of zoonoses of diseases from their pathogens to higher animals and eventually to humans, could be catastrophic if left unchecked in the future including keeping check of farm protocols used under more intensive conditions due to the greater pressure to produce more with today’s populations.



7. Methods for the Improvement of Silage-Making.


Taking the “wet” nature of green feed material for ensilage, interventives are perscribed to ensure better quality with the onset of fermentation towards a stabler biomass.   It is suggested that improving the approach of indirectly creating acidity and lowering pH using both enzyme preparations and probiotic fibrolytic spp., with non-GMO, GRO biologics for microbial spp. to enhance fibrolysis would constitute a breakthrough.  It has been found that pretreatment with addition of exogenous fibrolytic enzymes or EFEs (cellulases/hemicellulases) at ensiling improve digestibility, fermentation and aerobic stability of ensilage [11]. SCT silage in another study were shown to have greatest improvement in quality with treatment using hemicellulase as the EFE together with acidifying inoculant spp. of L. fermentum and P. acidilacti; improvement using both cellulase and hemicellulose as EFEs with the previous two inocula also showed improvement [12]. 


Further investigation is needed with SCT and high moisture corn (HMC), which have a lower total nitrogen (TN) content and requiring supplementation as a dual-purpose crop.  Their protein-N should be preserved with added organic acids, together with field drying or wilting, in order to limit the feed protein-N in its breakdown during ensilage.


Although not yet performed, nutrification with single cell protein (SCP) (e. g. yeast, fungi) via residual substrate fermentation is recommended further using biologics to boost cellulolysis of bagasse pith, rice straw, rice bran, corn stover and copra meal together with urea-N added which can:  1) acidify and preserve the biomass, 2) increase digestibility and 3) improve intake.



8. Proposals to Develop Sugarcane as Feed for Water Buffaloes in the Philippines.


Now discussed are various proposed sugarcane developments as initiatives for feeding in support of furthering water buffalo production in the case of the Philippine Islands.


More strategies have been pointed out for use with biotechnological and with chemical/physical methods to upgrade the byproducts of sugarcane, for both bagasse pith and SCT, according to the following initiatives:


1)      Ligninolytic/cellulolytic/hemicellulolytic Lactobacillus spp. in cultures (e. g. with cellulases, hemicellulases and Faes).

2)      Possible protoplasmic fusion of mutant strains with the wild-type with back-crossing regarding reduction of O-methyltransferase activity with a considerable reduction in CCoAOMT activity and in lignin in the whole plant and refuged together to ensure standability and crop protection.

3)      Mutants high in fructan crossed with the wild-type resulting in higher sugar due to changes in fructan metabolism which benefits both ensilage and rumen fermentation.

4)      Mutants low in plant protease activity crossed with the wild-type resulting in greater availability in peptides and amino acids together with the practice of wilting with postharvest and ensilage of the SCT.

5)      Use of exogenous fibrolytic enzymes (EFE) including xylanases, cellulases and other secondary activities from preparations with yeasts and, in addition, the lignase referred to as lacasses with cofactor low mass mediators.

6)      SSF of “dry” residuals using aerobic fungal cultures such as Coprinus, Cyathus stercoreus and Dichomitus squalens.

7)      Use of ensilage postharvest treatment using anhydrous NH3(g) or in situ from urea.  This ensures development of lactic acid producing bacteria rather than yeasts, which convert sugars to alcohols and results in significant in dry matter (DM) losses.

8)      Use of potent alkali such as ammonium hydroxide.

9)      Use of physical explosive steam treatment.

10)   Addition of acetic/propionic acid to lower acidity of the fermenting ensiling biomass.


As expected, in the Philippines harvesting of sugarcane every 10-12 months can be ensiled for feeding as a major method for dairy cows as regulated by their regional national dairy authorities as well as for meat animals.


It has been mentioned in the Philippines, that the cost of chemicals can be prohibitive, with use of enzymes or EFEs and biological yeast and fungal SSF with sugarcane SCT and bagasse pithas promising alternatives.



9. Concluding Remarks.


Although this paper attempts to update possible new, advanced technologies proposed in the way of direct-applied non-GMO, GRO biologics for microbial cultures for postharvest treatment, also referred to as pretreatment, and the direct-applied manipulation of ruminal microbial digestion.  Use of bioinformatics in genomics research will continue to be required with yeast and other predominant microbial spp. in rumen digestion for eventual TFE for use in direct-applied PNA-carriered technology. And there are boosted dual-purpose cultivars to supplement lower or very low levels of protein content in feedstuffs. But there have been no attempts, thus far, to test or prove these proposed applications using further research and developments using various feeds.

These will help launch new metabolomics research in the field for boosting productive traits for feeds processing, plant crops, and rumen microbial digestion as they impact animal production traits. 

The rumen stomach is a closed environment and with its high growth rate and continuous feed input and output delivery, spells such an extremely competitive environment.  Attempt to manipulate, in order to perturb this type of environment, in the past have led to apparent ‘wash-out’ and failure to carry out any process manipulation in the rumen.  Further ecosystem genomics could serve to shed more light on a framework for constraints in manipulating rumen microbial digestion.


The hypothesis is proposed here of the possible perturbation of microbial metabolism either by gene “silencing” and/or “enhancement” to boost overall metabolic activity via “priming” key enzymatic metabolic activity or activities, then providing the requisite energy from enzyme-generated substrates for energy (ATP), further supporting the microbial cell’s fermentation catabolic and anabolic activities for synthesis, and with turnover, of rumen contents.


It is suggested that use of “primed” microbial metabolic activity, for e. g., major rumen fibrolytic spp., boosted for overall metabolic activity, with “catalytic” use of starch-urea (as a commercial preparation) for its readily available energy and ammonia-N, to be balanced with other requirements for PFAA (peptides and amino acids), for e. g., from supplemental soybean and corn meals (both presumably untreated) over the basal part of the feed ration.


More research is needed to demonstrate, if additional “catalytic” dosings are needed to sustain the effect of subsequent “priming” events.  Further testing to prove this is required to verify these additional feeding requirements with the biologic per os together with the rest of the basal ration and supplementation.  More involved studies could further combine added concentrates as supplements to demonstrate the relative effect on each other.





[1] Zhang,L, Colli, L. and Barker, J. S. F. 2020.  Asian Water Buffaloe : domestication, history and genetics.  Animal Genetics 51 : 177-191.


[2] Tedeschi, L. O., de Almeida, A. K., Atzori, A. S., Muir, J. P., Fonseca, M. A., and Cannas, A.. 2017. A Glimpse of the Future in Animal Nutrition Science. 1. Past and Future Challenges. Revista  Brasiliera de Zootecnia 46: 438-451,


[3] Cummins, J. and Ho., M.-W. 2005. Genetically Modified Probiotics Should Be Banned.  Microbial Ecology in Health and Disease 17: 66-68.


[4] Boas Soares, W. V., Franzolin, R. and Sousa, N. H.. 2007.  Effect of Addition of Different Strains of Yeast (Saccharomyces cerevisiae) on Liquid Phase Overflow Rate, Ruminal Volume and In situ Degradability in Buffaloes Fed on the Sugarcane Basis. Ital. J. Anim. Science 6 (Suppl 2): 536-539.


[5] Ramirez, J. F., Medina, S., Garcia, N. Cifuentas. T. 2007. Effects of the Supplementation with Yeast (Saccharomyces cerevisiae) on Milk Yield and Milk Components of Water Buffalo Cows from Northeast of Colombia.  Ital. J. Anin. Science 6 (Suppl 2) : 502-503.


[6] Perera, A. S., Wang, H., Basel, M., Pokhiel, M. R., Gamage, P. S., Kalita, M., Wendel, S., Sears, B., Welideniya, D., Liu, Y., Aakeroy,, C., Turro, C., Troyer, D . L. and Bossmann., S. H. Chanel Blocking of MspA Revisited. Langmiur 29: 308-315.


[7] Anon. 2009. Bolstering Feed Supply to Improve Profitability and Sustainability.  Dual-Purpose Crops Fact Sheet. July.  Grains Research and Development Corporation (GRDC), Australia.


[8] Flores, D. A. 2022. Sugarcane as Feed.  In : Lignocellulosic Biotechnology and Related Research. Pp. 49-64. Mill City Press Inc.  Maitland FL USA.


[9] Sidana, A. and Farooq, U. 2014. Sugarcane Bagasse : a potential medium for fungal cultures.  Chinese Journal of Biology 5 pp.


[10] Saini, A., Aggarwal, N. K., Sharma, A., and Yadav, A.. 2015. Actinomycetes : a source of ligninolytic enzymes. Enzyme Research 15pp.


[11] Dean, D. B., Adesogan, A. T., Krueger, N. and Littell, R. C. 2005. Effect of Fibrolytic Enzymes on the Fermentation Characteristics, Aerobic Stability and Digestibility of Bermudagrass Silage. J. Dairy Sci. 88 : 994-1003.


[12] Singh, D., Johnson, T. A., Tyagi, N., Malhotra, R., Behare, P. V., Kumar, S. and Tyagi, A.K. 2023. Synergistic Effect of LAB Strains (Ll. Fermentum and P. Acidilacti) with Exogenous Fibrolytic Enzymes on Quality and Fermentation Characteristics of Sugarcane Tops Silage. Sugar Tech 25 : 141-153.


SKYEVIEW: The aim is to attain hi-bred carabeef either with crossbred cattle with both advantages in production traits with the Indian varities or their cross-breeds and the Philippine native carabao cattle of leanness, low cholesterol and tenderness/juiciness in addition to taste tests and their natural avidity for sustainable feeding using byproduct crop-based feedstuffs and their hardiness in case of environmental stressors in disease-resistance and drought hardiness.  In part reading the previous body of information on feeding carabeef and caramilk producing cattle starting with the smaller farmer who ranches with nearby sugarcane and even rice paddied water hyacinth for SCT and water hyacinth ensilage processing for year-round feeding has instigated use of both: 1) advanced (proposing) probiotic biologic-regulated innoculants (see: D. A. Flores. 2022. In: Lignocellulosic Biotechnology and Related Research.  Related research for feed, food and energy. Chap. 12., pp. 131-152), and 2) dual-purpose practice for more productive grass-fed cattle on their grasslands where available for hi-quality beef and carabeef. 

SKYEVIEW: At SkyeBlue we recognized readily the ominous problem if ever water hyacinth is allowed to clog rivers, canals or waterways, lakes and even ponds due to its unstymied capacity to replicate and spread like a common aqua weed on the water surface killing of suffocating ecology in such areas in the Philippines; thus their general policy against its propagation.  But still we ask for a moratorium on bio-controlled and bordered access control of paddy farming of this otherwise valuable fodder for silage-making with pigs and cattle production for the smaller farmer away from other bodies of water like streams and rivers. Indeed, our story continues praising this variety perhaps where land allows to grow for fodders (silage), fertilizer with composting of land amendments and even for production of biogas (viz. methane) for home cooling/heating and cooking and other modern attainments in household appliances in the tropics.

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