Patent Application
20250161245
The present disclosure relates to the field of dietary non-protein nitrogen (NPN)-sources, more in particular a mixture of urea, biuret and N-containing by-products created during the urea condensation process, or stated differently, during biuret production, in a ruminant feed supplement.
Ruminants are defined as large hoofed herbivorous grazing or browsing mammals that are able to acquire nutrients from plant-based feed by digesting it in a specialized stomach having four compartments, one of which is the rumen. Thanks to the microbial actions in the rumen prior to digestion, ruminants have the unique ability to use (low quality) protein sources and also non-protein nitrogen (NPN) sources. NPN sources provide nitrogen for ruminants, especially in low-protein diets. This gives ruminant animals a competitive advantage over other domesticated animals. The symbiotic relationship with the rumen microflora allows ruminants to upcycle nutrients to produce highly nutritious animal proteins, by means of an efficient pre-gastric fermentation of feeds, which takes place in the reticulorumen, the largest compartment of their gastrointestinal tract. Ruminants provide an optimum environment for microorganisms to thrive, and they, in turn, supply the ruminant host with microbial crude protein, which is of high quality in terms of amino acids supplement. In beef and dairy production systems, the microbial crude protein synthesized in the rumen provides the majority of the metabolizable protein required by the animal.
Increased utilization of nitrogen (N) for bacterial synthesis and capture of dietary N into anabolic products, particularly absorbed amino acids, may be influenced by dietary manipulation, mainly by energy levels in the ration. However, there is an upper limit to the overall efficiency of the process: a maximum of 50 to 60% of dietary N, or 70 to 90% of apparent digested N will be converted into amino acids and released into the portal vein. For this reason, particularly in beef cattle diets, it is extremely important to assess the optimum kinetics of N release in NPN sources for each energy source (e.g., basal forage diet, supplemental by-products, etc.) to maximize N utilization and minimize excretion.
In most beef production systems, several grains and grain byproducts are available as supplemental protein sources. However, on a per kg of N supplied, it is nearly impossible to compete with the lower costs of supplemental crude protein in the form of NPN. Indeed, non-protein nitrogen, mainly in the form of urea, is known as a cost-effective source of supplemental nitrogen (N) in ruminant production. Commercial/feed-grade urea typically contains only traces of biuret, with a biuret content not exceeding 1.0 wt %. Urea is however usually not included in more than 1 wt. % of the diet. Any shortage of protein in a diet or supplementation program is frequently covered by the use of urea-based supplements in the form of range cubes, liquid or pelleted supplements and tubs. Apart from urea, other ammonia forming chemical compounds such as ammonium sulfate have been used in the formulation of ruminal diets to replace part of plant protein sources and providing adequate amounts of ruminal degradable protein for a better efficiency of fiber digestion and the synthesis of microbial protein. The use of NPN, however, has been traditionally limited by the fast rate of N-release in the rumen of a ruminant (animal) in the form of ammonia. If the release of ammonia in the rumen exceeds the use capacity by ruminal microbiota, this N-excess will be excreted, with a consequent loss of energy and N-efficiency. Furthermore, if the ammonia concentration overpasses the liver excretion capacity of the ruminant, ammonia toxicity or death may occur.
As a result, in the prior art, it has been aimed at improving the use of NPN to reduce the rate of N-release in the rumen. In recent years, several slow-release urea products have been developed and tested in ruminant nutrition, for instance by physical means, such as by applying a coating, with variable results. Applying a coating with the aim to obtain slow release means adding fat, wax or the like to the NPN-source. This has the disadvantage that the NPN-content is diluted. A coating furthermore increases the production cost of a dietary N-source in cattle diets that should be cost effective. Another solution is to apply chemical means such as using pure biuret. The disadvantage of pure biuret is that the production thereof is very expensive, because the heating time to obtain the pure biuret out of urea has to be extended a lot.
In addition, currently, there is growing interest in decreasing the environmental impact of ruminant production, including beef and dairy, and many strategies are being pursued to achieve this goal.
In the current ruminant feeding systems, methane (CH4) production is an inevitable consequence of fermentative digestion. The microbial consortium of the rumen must dispose hydrogen that is produced during fermentation. Otherwise, hydrogen would accumulate in the rumen and thereby inhibit fermentation of ingested feeds. Therefore, the rumen microbial system is provided with methanogens that reduce carbon dioxide (CO2) to methane.
The major hydrogen (electron) sink in the rumen is methane produced by the reduction of carbon dioxide using reduced co-enzymes such as NADH as the electron source. Methane is however an energy loss for ruminants and a potent greenhouse gas, which has a 28-times greater global warming potential than carbon dioxide.
Globally, ruminants produce some 80 million tons of methane annually, accounting for some 28% of manmade methane emission. The adoption of ruminant management practices which result in major reductions of release of methane are consequently high priority.
It is a goal of the present disclosure to provide NPN-sources for ruminants, such as for a ruminant feed supplement composition which overcomes the limitations of the known prior art, useful for improving the rumen fermentation, the productivity and the performance of the ruminant animals, and the enteric methane emission, especially in low crude protein diets, which provides N in a timely manner such that the microbial growth in the rumen is enhanced and the production of energy yielding compounds is improved.
The present disclosure generally relates to a composition or mixture comprising biuret, urea, and N-containing by-products of the biuret production out of urea, and uses thereof and methods related thereto. The terms “NPN composition”, “NPN mixture”, and “composition comprising urea, biuret and N-containing by-products of the biuret production out of urea” are generally used interchangeably herein, and refer to a composition or NPN composition according to the present disclosure, as further detailed herein.
According to a first aspect of the present disclosure, the use of a mixture or composition of biuret, urea and N-containing by-products of the biuret production out of urea as a NPN-source in a ruminant feed supplement composition to reduce the methane emission produced by a ruminant animal is disclosed. It has been observed that the supplementation of a ruminant feed with urea, biuret and N-containing by-products of the biuret production out of urea as a NPN source, reduces enteric methane production and emission of ruminant animals without compromising or even improving their performance, in particular when compared to a diet or ruminant feed supplemented with urea as NPN source.
According to a second aspect of the present disclosure, the use of a mixture of biuret, urea and minor N-containing compounds created during the urea condensation process as a NPN-source in a ruminant feed supplement composition to decrease the acetate to propionate ratio produced by rumen microbes in a ruminant has been disclosed. Stated differently, the use of a composition comprising biuret, urea and N-containing by-products of the biuret production out of urea as a NPN-source in a ruminant feed supplement composition to decrease the acetate to propionate ratio produced by rumen microbes in a ruminant is disclosed, in particular when compared to a diet or ruminant feed supplemented with urea as NPN source.
According to a third aspect of the present disclosure, the use of a mixture of biuret, urea and minor N-containing compounds created during the urea condensation process as a NPN-source in a ruminant feed supplement composition to improve the efficiency of microbial protein synthesis (EMPS) has been disclosed. Stated differently, the use of a composition comprising biuret, urea and N-containing by-product of the biuret production out of urea as a NPN-source in a ruminant feed supplement composition to improve the efficiency of microbial protein synthesis (EMPS) is disclosed, in particular when compared to a diet or ruminant feed supplemented with urea as NPN source. According to a fourth aspect of the present disclosure, the use of a mixture of biuret, urea and minor N-containing compounds created during the urea condensation process as a NPN-source in a ruminant feed supplement composition to increase the dry matter (DM) and organic matter (OM) digestibility has been disclosed. Stated differently, the use of a composition comprising biuret, urea and N-containing by-products of the biuret production out of urea as a NPN-source in a ruminant feed supplement composition to increase the dry matter (DM) and organic matter (OM) digestibility is disclosed, in particular when compared to a diet or ruminant feed supplemented with urea as NPN source.
According to a further aspect of the present disclosure, the use of a ruminant feed supplement composition comprising urea, biuret and N-containing by-products of the biuret production out of urea as a NPN-source to improve the dry matter intake (DMI) of ruminant animals is disclosed, in particular when compared to a diet or ruminant feed supplemented with urea as NPN source.
It has now unexpectedly been discovered by the applicant that by using a combination of urea, biuret and minor N-containing compounds created during the urea condensation process to form the biuret (also referred to as “by-products of the biuret production based on urea” or “N-containing by-products of the biuret production out of urea”), the rumen fermentation is altered directly in a way that the enteric methane production is decreased, the acetate to propionate ratio is decreased, as well as that the efficiency of microbial protein synthesis is improved. It has furthermore been found that the DM and OM digestibility is increased, and that the dry matter intake of the ruminant animal is increased, in particular when compared to a diet or ruminant feed supplemented with urea as NPN source.
According to a possible use according to the present disclosure, the mixture or composition comprises between 30-60 wt. % of urea and between 30 and 60 wt. % biuret, with wt % based on the total weight of the mixture or composition. More in particular, the mixture or composition comprises between 35 and 55 wt. % of urea and between 35 and 50 wt. % of biuret, with wt % based on the total weight of the composition. Even more in particular, the mixture or composition comprises between 37 and 52 wt. % of urea and between 39 and 46 wt. % of biuret, with wt % based on the total weight of the composition.
In a particular use according to the present disclosure, the composition comprises between 5 and 30 wt. % of N-containing by-products of the biuret production out of urea, based on the total weight of the composition.
In a specific use according to the present disclosure, the ruminant feed supplement composition further comprises:
According to a further specific use according to the present disclosure, the mixture further comprises
Another aspect of the present disclosure provides a ruminant feed composition comprising one or more nutritional ingredients and an NPN comprising biuret, urea and N-containing by-products of the biuret production out of urea. In certain embodiments, the NPN composition comprises (a) between 30 and 60 wt. % of urea; (b) between 30 and 60 wt. % of biuret; and (c) between 5 wt. % and 30 wt. % of N-containing by-products of the biuret production out of urea, based on the total weight of the NPN composition. More in particular, the NPN composition comprises (a) between 37 and 52 wt. % of urea; (b) between 39 and 46 wt. % of biuret; (c) between 3 wt. % and 6 wt. % of triuret; (d) between 0.5 and 3 wt. % of ammelide; and (e) between 3 and 10 wt. % of cyanuric acid, based on the total weight of the NPN composition. In certain embodiments, the one or more nutritional ingredients form a nutritionally balanced ration or a basal ration for a ruminant.
According to a further related aspect of the present disclosure, a method for reducing the enteric methane emissions of a ruminant animal is disclosed, said method comprising administering to the ruminant animal a NPN composition according to the present disclosure, comprising biuret, urea and N-containing by-products of the biuret production out of urea, wherein the composition comprises between 30 and 60 wt. % of urea and between 30 and 60 wt. % biuret. It has been found that the enteric methane emissions of the ruminant animal, expressed as g methane/kg dry matter intake or g methane/kg organic matter intake, is reduced by at least 5%, particularly by at least 10%, such as by about 15%, when the NPN composition is administered to a ruminant as NPN source compared to a diet comprising urea as NPN source.
According to a further related aspect of the present disclosure, a method for decreasing the acetate-to-propionate ratio produced by rumen microbes in a ruminant animal is disclosed, said method comprising administering to the ruminant animal a NPN composition according to the present disclosure, comprising biuret, urea and N-containing by-products of the biuret production, wherein the NPN composition comprises between 30 and 60 wt. % of urea and between 30 and 60 wt. % biuret. It has been found that the acetate-to-propionate ratio produced by rumen microbes in a ruminant, particularly estimated via an in vitro fermentation, such as a continuous flow fermentation with rumen fluid to simulate rumen fermentation, is decreased by at least 5%, in particular by at least 10%, such as by about 20%, when the NPN composition is administered to a ruminant as NPN source compared to a diet comprising urea as NPN source.
According to a further related aspect of the present disclosure, a method for improving the efficiency of microbial protein synthesis (EMPS) by rumen microbes in a ruminant animal is disclosed, said method comprising administering to the ruminant animal a NPN composition according to the present disclosure comprising biuret, urea and N-containing by-products of the biuret production out of urea, wherein the NPN composition comprises between 30 and 60 wt. % of urea and between 30 and 60 wt. % biuret. It has been found that the EMPS, particularly estimated via an in vitro fermentation with rumen fluid to simulate rumen fermentation was increased by at least 5%, in particular by at least 10%, when the NPN composition is administered to a ruminant as NPN source compared to a diet comprising urea as NPN source.
According to a further related aspect of the present disclosure, a method for increasing the dry matter (DM) and organic matter (OM) digestibility is disclosed, said method comprising administering to the ruminant animal a NPN composition according to the present disclosure comprising biuret, urea and N-containing by-products of the biuret production, wherein the NPN composition comprises between 30 and 60 wt. % of urea and between 30 and 60 wt. % biuret.
According to a further related aspect of the present disclosure, a method for increasing the dry matter intake (DMI) of a ruminant animal, said method comprising administering to the ruminant animal a NPN composition according to the present disclosure comprising biuret, urea and N-containing by-products of the biuret production, wherein the NPN composition comprises between 30 and 60 wt. % of urea and between 30 and 60 wt. % biuret. It has been found that the DMI, expressed in kg DM/day, is increased by at least 5% when the (NPN) composition is administered to a ruminant as NPN source compared to a diet comprising urea as NPN source.
In particular embodiments, the NPN composition according to the present disclosure and administered in the methods according to the present disclosure comprises between 35 and 55 wt. % of urea and between 35 and 50 wt. % of biuret, based on the total weight of the composition, more in particular comprises between 37 and 52 wt. % of urea and between 39 and 46 wt. % of biuret, based on the total weight of the composition. In particular embodiments, the NPN composition according to the present disclosure and administered in the methods according to the present disclosure further comprises between 5 and 30 wt. % of N-containing by-products of the biuret production out of urea, based on the total weight of the composition.
In particular embodiments, the NPN composition according to the present disclosure and administered in the methods according to the present disclosure comprises between 2.5 and 8 wt. % of triuret, and/or between 0.1 and 5 wt. % of ammelide, and/or between 2.5 and 15 wt. % of cyanuric acid, and between 0.1 and 2.0 wt. % of moisture, based on the total weight of the composition, more in particular, wherein the composition comprises between 3 and 6 wt. % of triuret, and/or between 0.5 and 3 wt. % of ammelide, and/or between 3 and 10 wt. % of cyanuric acid, and between 0.1 and 1.5 wt. % of moisture, based on the total weight of the composition.
In certain embodiments, the methods according to the present disclosure further comprises the steps of preparing or providing a ruminant feed supplement composition comprising the NPN composition, or preparing or providing a ruminant feed composition comprising the NPN composition, and administering the ruminant feed supplement or the ruminant feed composition to the ruminant animal. More in particular, the NPN composition, comprising biuret, urea and N-containing by-products of the biuret production according to the present invention is present in the ruminant feed composition in a concentration between 0.5 and 4 wt. %, more in particular between 0.5 and 3.5 wt. % or between 0.5 and 3 wt. %, even more in particular between 1 and 2.5 wt. %, with wt % based on the total dry matter basis of the ruminant feed composition. In particular, the ruminant feed composition further comprises one or more nutritional ingredients, wherein the one or more nutritional ingredients form a nutritionally balanced ration or a basal ration for a ruminant animal.
It is understood that the different methods according to the present disclosure are non-therapeutic methods.
The present inventors have surprisingly found that a specific NPN composition (or mixture) for ruminants, comprising urea, biuret and one or more N-containing by-products of a urea condensation process or biuret production process, has many advantages when fed to a ruminant as a NPN source, particularly when compared to a ruminant fed with urea as a NPN source. Moreover, for the different embodiments and aspects envisaged herein, the NPN composition or NPN mixture according to the present disclosure significantly outperforms urea as a NPN source (when compared to a ruminant fed with a diet without NPN source).
The advantages of a NPN composition according to the present disclosure are in particular:
In this context, and without wishing to be bound by theory, it is believed that there has been a selective pressure on the microbes in the rumen that produce acetate and propionate in such a way that more propionate and less acetate, in view of ruminant feed supplement compositions without the mixture according to the present disclosure, are produced. Propionate is the most important glucogenic precursor in ruminants. This means that lowering the acetate to propionate ratio enhances the supply of energy for metabolic use, through which more energy is available to be used by the ruminant enhancing its production of proteins. In other words, the rumen fermentation has been made more efficient in an energetic way, supporting the production of meat, milk, etc.
Biuret is typically formed through a non-catalytic thermal decomposition of urea. This process is generally referred to herein as “biuret production”, “biuret production out of urea” or “urea condensation process”. The main reaction and the side reactions in biuret production out of urea are known to the skilled person and they are represented in
As used herein, the composition or mixture according to the present disclosure, in particular the NPN composition or mixture according to the present disclosure, generally comprises urea, biuret and one or more by-products of a biuret production process. More in particular, the NPN composition or mixture according to the present disclosure, comprises between 60 wt. % and 95 wt. % of urea and biuret, and between 5 wt. % and 40 wt. % of N-containing by-products of a biuret production process, based on the total weight of the composition or mixture. Even more in particular, the NPN composition or mixture according to the present disclosure comprises between 70 wt. % and 95 wt. % or between 75 wt. % and 95 wt. % of urea and biuret, and between 5 wt. % and 30 wt. % or between 5 wt. % and 25 wt. % of N-containing by-products of a biuret production process, based on the total weight of the composition or mixture.
In particular embodiments, the NPN composition or mixture according to the present disclosure comprises between 30 and 60 wt. % of urea and between 30 and 60 wt. % of biuret, more in particular between 35 and 55 wt. % of urea and between 35 wt. % and 50 wt. % of biuret, even more in particular between 37 and 52 wt. % or between 40 and 50 wt. % of urea and between 39 and 46 wt. % or between 40 and 45 wt. % of biuret, based on the total weight of the composition or mixture. In certain embodiments, the NPN composition or mixture according to the present disclosure, further comprises between 5 wt. % and 30 wt. % of N-containing by-products of a biuret production process out of urea, in particular between 5 wt. % and 25 wt. % of N-containing by-products of a biuret production process out of urea, based on the total weight of the composition or mixture.
In certain embodiments the N-containing by-products of a biuret production process out of urea comprises one or more of triuret, ammelide and cyanuric acid.
In certain embodiments, the NPN composition or NPN mixture according to the present disclosure comprises, as N-containing by-products of a biuret production process, (a) between 2.5 wt. % and 8 wt. % of triuret, more in particular between 3 and 6 wt. % of triuret; (b) between 0.1 and 5 wt. % of ammelide, more in particular between 0.5 and 3 wt. % of ammelide; and/or (c) between 2.5 and 15 wt. % of cyanuric acid, more in particular between 3 and 10 wt. % of cyanuric acid, based on the total weight of the composition or mixture.
In certain embodiments, the NPN composition or mixture according to the present disclosure further comprises between 0.1 and 2 wt. % moisture (water), more in particular between 0.1 and 1.5 wt. % moisture, based on the total weight of the composition or mixture.
In particular embodiments, the NPN composition or mixture according to the present disclosure is a ruminant feed supplement composition.
Particular embodiments of the NPN composition or mixture or ruminant feed supplement composition according to the present disclosure comprises
Optionally, for all embodiments of the NPN composition according to the present disclosure, one or more anti-caking agents as generally known in the art can be added to the ruminant feed supplement composition, either incorporated in the feed supplement, either as a coating, to improve handling properties and to improve the quality of the ruminant feed supplement composition.
Furthermore, it is also possible to add any pre-biotics, pro-biotics, post-biotics, anti-biotics, bio stimulants known in the art for fostering/supporting the nitrogen assimilation and rumen fermentation.
An aspect of the present disclosure relates to the use of a NPN composition or mixture according to the present disclosure, or stated differently a mixture or composition of urea, biuret and minor N-containing compounds created during the urea condensation process to obtain the biuret, as a NPN-source in a ruminant feed supplement composition to, particularly at the same time or simultaneously,
According to a related aspect of the present disclosure, a composition comprising urea, biuret and N-containing by-products of biuret production out of urea, for use as an NPN-source in a ruminant feed supplement composition to decrease the enteric methane production or emissions of a ruminant animal or to decrease the enteric methane production by rumen microbes in a ruminant is disclosed.
According to a related aspect of the present disclosure, a composition comprising urea, biuret and N-containing by-products of biuret production out of urea, for use as an NPN-source in a ruminant feed supplement composition to decrease the ratio of acetate to propionate produced by rumen microbes in a ruminant is disclosed.
According to a further related aspect of the present disclosure, a composition comprising urea, biuret and N-containing by-products of biuret production out of urea, for use as an NPN-source in a ruminant feed supplement composition to improve the EMPS in a ruminant is disclosed.
According to a further related aspect of the present disclosure, a composition comprising urea, biuret and N-containing by-products of biuret production out of urea, for use as an NPN-source in a ruminant feed supplement composition to increase the DM and OM digestibility in a ruminant.
According to a related aspect of the present disclosure, a composition comprising urea, biuret and N-containing by-products of biuret production out of urea, for use as an NPN-source in a ruminant feed supplement composition to increase the dry matter intake of a ruminant animal is disclosed.
A related aspect of the present disclosure provides a method for reducing the enteric methane production of a ruminant animal, comprising the steps of
A related aspect of the present disclosure provides a method to decrease the acetate to propionate ratio produced by rumen microbes in a ruminant, comprising the steps of:
A further related aspect of the present disclosure provides a method to improve the EMPS in a ruminant, comprising the steps of
A further related aspect of the present disclosure relates to a method to increase the DM and OM digestibility in a ruminant, comprising the steps of
In certain embodiments, the enteric methane emissions of the ruminant animal are reduced by at least 5% or by at least 10%, such as by about 15%, when expressed in g/kg DMI (dry matter intake) or g/kg OMI (organic matter intake), when the NPN composition according to the present disclosure is administered to a ruminant as NPN source compared to diet comprising urea as NPN source.
In certain embodiments the acetate to propionate ratio produced by rumen microbes in a ruminant is decreased by at least 5%, in particular by at least 10%, such as by about 20%, when the NPN composition according to the present disclosure is administered to a ruminant as NPN source compared to a diet comprising urea as NPN source. In particular, the acetate-to-propionate ratio may be determined on a sample obtained from an in vitro fermentation with ruminant fluid, to simulate rumen fermentation, particularly wherein a suitable fermenter or incubator, such as a continuous flow or batch fermenter or incubator, is inoculated with ruminal fluid and fed with a ruminant feed composition to simulate rumen fermentation.
In certain embodiments, the efficiency of microbial protein synthesis (EMPS) in a ruminant is increased by at least 5%, in particular by at least 10%, such as by about 20%, when the NPN composition according to the present disclosure is administered to a ruminant as NPN source compared to a diet comprising urea as NPN source. In particular, the EMPS may be determined on a sample obtained from an in vitro fermentation with ruminant fluid, to simulate rumen fermentation, particularly wherein a suitable fermenter or incubator, such as a continuous flow or batch fermenter or incubator, is inoculated with ruminal fluid and fed with a ruminant feed composition to simulate rumen fermentation.
In certain embodiments, the method for decreasing the enteric methane production of a ruminant animal, for decreasing the acetate to propionate ratio produced by rumen microbes in a ruminant, for improving the EMPS in a ruminant, for increasing the DM or OM digestibility in a ruminant, and/or for increasing the dry matter intake (DMI) of a ruminant further comprises the step of replacing a portion of vegetable proteins in the feed composition with the NPN composition according to the present disclosure. It is further understood that the different methods according to the present disclosure are non-therapeutic methods.
As considered herein a ruminant or ruminant animal is a bovine, ovine or caprine animal, particularly a domesticated animal with a functional rumen. In particular embodiments, the ruminant is a sheep or a cattle, more in particular the ruminant is a beef cattle or dairy cattle.
The present disclosure further provides a feed, feed material, premix or feed additive for feeding a ruminant comprising the NPN composition according to the present disclosure. The NPN composition according to the present disclosure may be combined with a ruminant feed or feed material, or with a premix for feeding a ruminant. In context of the present disclosure the term premix or nutrient premix is used as known to the person skilled in the art and denotes a mixture comprising one or more ingredients such as vitamins, trace minerals, medicaments, feed supplements and diluents. A ruminant feed or ruminant feed material generally refers to nutritional compositions suitable for feeding to ruminants, as known to the skilled person, and includes compound feeds (i.e. generally an industrially produced feed mixture of at least two feed materials), complete feeds (i.e. a compound feed containing all main nutrients needed for a daily ration, including also all the roughage needed by the ruminant), or a concentrate feed (i.e. a compound feed which has a high content of certain substances but which is sufficient for a daily ration only if used in combination with other feed).
In particular embodiments, a ruminant feed composition comprising one or more nutritional ingredients and a NPN composition comprising (a) between 30 and 60 wt. % or between 35 and 55 wt. % of urea; (b) between 30 and 60 wt. % or between 35 and 50 wt. % of biuret; and (c) between 5 wt. % and 30 wt. % of N-containing by-products of the biuret production out of urea, based on the total weight of the NPN composition, is disclosed. More in particular, the NPN composition comprises (a) between 37 and 52 wt. % of urea; (b) between 39 and 46 wt. % of biuret; (c) between 3 wt. % and 6 wt. % of triuret; (d) between 0.5 and 3 wt. % of ammelide; and (e) between 3 and 10 wt. % of cyanuric acid, based on the total weight of the NPN composition. In certain embodiments, the one or more nutritional ingredients form a nutritionally balanced ration or a basal ration for a ruminant, as known by the skilled person.
In certain embodiments, a ruminant feed composition or feed material comprising between 0.5 and 4 wt. %, more in particular between 0.5 and 3.5 wt. % or between 0.5 and 3 wt. %, even more in particular between 1 and 2.5 wt. % of t NPN composition according to the present disclosure, is disclosed, with wt. % based on the total dry matter of the feed composition or feed material,
The present disclosure further relates to a method for feeding or rearing a ruminant comprising administering the NPN composition according to the present disclosure to the ruminant, particularly during the feeding regime, and/or feeding the ruminant with the ruminant feed composition according to the present disclosure.
The disclosure as defined here above will now be illustrated and explained in more detail in the following experimental part, which is not intended to limit the scope of this disclosure anyway.
Each NPN-source has specific characteristics, determining the release of N after entrance into the rumen. The efficiency of N-use by the rumen microbiota will however depend on the amount and the release rate of the NPN-source. This may result in a difference in the efficiency of microbial protein production in the rumen. In this example, the N-utilization in vitro of 3 different NPN-products and the mixture according to the present disclosure compared to coated and uncoated urea as NPN-sources for rumen fermentation was assessed.
A total of 6 different NPN-products and a mixture according to the present disclosure were selected to be tested in an in vitro rumen fermentation model (see Table 1) below. The mixture according to the present disclosure, in the different examples typically referred to by the code UB, generally comprises between 37 and 52 wt. % of urea, between 39 and 46 wt. % of biuret, between 3 and 6 wt. % of triuret, between 0.5 and 3 wt. % of ammelide, between 3 and 10 wt. % of cyanuric acid, and between 0.1 and 1.5 wt. % of moisture, based on the total weight of the mixture.
The in vitro model used to simulate rumen digestion and utilization of the products is the Gas Production Test protocol, adapted for protein digestibility, as described in more detail in the following paragraph.
Approximately 1 liter of rumen fluid was collected from two different rumen-fistulated animals in the morning. Rumen fluid was strained through a cheesecloth, kept at 39° C. under CO2 and then diluted 1:19 with a nitrogen-free buffer and mineral solution containing 10.03 g NaHCO3, 1.43 g Na2HPO4, 1.55 g KH2PO4, 0.15 g MgSO4·7H2O, 0.52 g Na2S, 0.017 g CaCl2·2H2O, 0.015 g MnCl2·4H2O, 0.002 g CoCl3·6H2O, 0.012 g FeCl3·6H2O and 0.125 mg resazurin per liter.
To ensure the rumen fluid was depleted of free nitrogen and to ensure nitrogen would be the limiting factor for the microbial growth at the start of the experiment, the buffered rumen fluid was pre-incubated for 4 hours at 39° C. with a carbohydrate overload of glucose, xylose and soluble starch, each at 3.33 g/l. During this pre-incubation, all nitrogen available in the rumen fluid would be incorporated into bacterial protein and other bacterial nitrogen-containing components.
Each product was weighed into different glass bottles on an isonitrogenous base, providing 15 mg N per flask. Then, 60 ml of the pre-incubated buffered rumen fluid was added, under continuous flushing with CO2. Flasks were closed with rubber stoppers and placed in a shaking water bath with 50 movements per minute at 39° C. to start the incubation period. The gas production of each flask was recorded continuously for 72 hours using a fully automated system. All test products including a blank (i.e. no product added to the flask of rumen fluid) were tested in one run in triplicate.
The cumulative gas production was measured continuously for 72 hours. The results averaged per triplicate are shown in
As can be seen in
As can furthermore be seen in
As further can be seen in
The rapid ammonia release rate of uncoated urea limits the amount of urea that may be used in a ruminant diet formulation. High inclusion levels of uncoated urea in a ruminant diet formulation will increase the ammonia concentration in the rumen rapidly, leading to ruminal accumulation and absorption of ammonia and subsequent excretion of urea in the urine.
As expected, the pure N-compounds, i.e. ammelide, triuret and cyanuric acid, which are well-known N-containing by-products of the biuret production by thermal decomposition of urea and which are also present in minor amounts in the mixture according to the present disclosure, generated a much lower cumulated gas production than uncoated and coated urea. Ammelide and cyanuric seem not to be suitable as rumen digestible nitrogen sources in dairy cattle nutrition. Their solubility and nitrogen release rate are too slow to support microbial growth. Nitrogen from biuret and triuret can be used by rumen bacteria, but it is released more slowly than coated or uncoated urea. The slow release of nitrogen from triuret and biuret may improve nitrogen utilization. However, it can be expected to escape from the rumen approximately 12 to 24 hours after ingestion (depending on the diet characteristics such as digestibility and particle size). Part of the biuret and triuret (approximately 60%) will still be unused by the rumen microbes after 24 hours, based on the cumulative gas production profiles. This amount of unused nitrogen is likely to pass the rumen and be excreted after passage through the gastrointestinal tract.
The mixture according to the present disclosure, particularly the proportion of urea, biuret and the minor N-compounds created by the production of biuret starting from urea therein, resulted in an unexpected fermentation pattern as shown by the cumulative gas production dynamics, reaching the greatest total gas production amongst all the NPN sources assessed. As can be seen in
In this example, an in vitro assay was performed using dual-flow continuous culture fermenters, aiming to simulate rumen fermentation.
Eight 1320 ml dual-flow continuous culture fermenters were used in two replicated periods. Each period consisted out of 5 days for adaptation of the ruminal fluid to the treatments and 3 days for sampling. On the first day of each period, all fermenters were inoculated with undiluted ruminal fluid filtered through two layers of cheesecloth to remove the feed particles. The fermentation conditions were constant, i.e.
The fermenters were fed 95 grams/day of dry matter of a diet formulated to meet or exceed current nutrients recommendations for lactating dairy cows, i.e.
And were fed in three daily administrations, i.e. at 7, 15 and 23 o'clock with a forage: concentrate diet in a range of 53:47.
Ruminal fluid was taken from two Holstein dairy cows fitted with ruminal cannulas fed with a 60:40 forage:concentrate diet with alfalfa and cracked grain (11 kg dry matter/cow/day). The afternoon before the ruminal extraction, the ruminant animals were not fed.
The NPN-dietary treatments were as follows:
To obtain isoenergetic and isonitrogenous diets using different NPN sources, a nitrogen content determination using the Kjeldahl method was used to determine the nitrogen content of each NPN product (see Table 2 below).
Due to the N-percentage differences among the products, the inclusion level of each NPN-source was different (see Table 3 below).
On the second day of sampling, 4 ml of filtered fermenter fluid was taken 2 hours after the morning feeding to determine VFA-concentrations.
During sampling days, the collection vessels were maintained at 4° C. to prevent microbial activity. Solid and liquid effluents from 3 sampling days were composited and mixed within the fermenter and homogenized. Subsamples were taken for the analyses of the volatile fatty acids (VFA). The remainder of the sample was lyophilized (freeze drying) and the dry samples were analyzed for DM, ash, N and purines to calculate bacterial nitrogen and organic matter content (OM).
Bacterial cells were obtained from the fermenter flasks the last day of each experimental period. Solid and liquid associated bacteria were isolated using a combination several detachment procedures selected to obtain the maximum detachment without affecting the cell integrity. The bacterial cells were lyophilized and the dry samples were analyzed for dry matter (DM), ash, N and purines to calculate bacterial nitrogen.
Effluent dry matter (DM) was determined by lyophilizing 300 ml aliquots in triplicate. The DM content of the diets and bacterial samples were determined by drying samples for 24 hours in an 103° C. forced air oven (see AOAC, 1990; method 950.01).
Dry samples of diets, effluents and bacteria were ashed overnight at 550° C. in a muffle furnace (see AOAC, 1990, method 976.05) and organic matter (OM) was determined by difference. Total N of diets, effluents and bacterial samples were determined by a Kjeldhal method (AOAC, 1990; method 976.05). Sample crude protein (CP) was calculated as N×6.25. Effluent CP was determined in liquid samples. Crude protein degradation was calculated as the difference between CP in the diet minus CP in the effluent and expressed as wt. % of DM. The EMPS was determined by dividing bacterial N by fermented organic matter.
The fiber components of the diets and the effluents were analyzed sequentially using a thermostable alpha-amylase and sodium sulfite and expressed without residual ash.
The samples for the VFA-analysis were prepared as described by Jouany, J. P., and J. Senaud. “Influence des ciliés du rumen sur la digestion de différents glucides chez le mouton. I.—Utilisation des glucides pariétaux (cellulose et hémicelluloses) et de l'amidon.” Reproduction Nutrition Développement 22.5 (1982): 735-752. and analyzed by gas chromatography.
In Table 4 below, the effect of the NPN-diets on the nitrogen metabolism of ruminal microorganisms, true digestibility of DM and OM, CP degradation, the efficiency of microbial protein synthesis (EMPS) in the rumen (or in other words, how much microbial protein is produced due to the microbial activity for every unit of substrate which is fed to the ruminant), in the control diet (“Control”—comprising urea) and a diet (“Diet”—comprising UB) comprising a mixture according to the present disclosure is shown.
Out of Table 4, it can be concluded that the diet comprising a mixture of urea, biuret and the minor amount of N-containing compounds originating from the biuret production out of urea according to the present disclosure have
A total of 72 crossbred yearling steers [303 kg±29 kg initial body weight (BW) were stratified by body weight (BW) and randomly allocated to three treatments. All steers consumed a basal diet of corn silage, cotton-gin trash, and a premix of vitamins and minerals.
The steers were housed in pens (8 steers/pen, 3 pens/treatment). The steers were supplemented with two different NPN-sources on an isonitrogenous basis, considering as reference the N provided by urea when included at 1 wt. % of the diet in dry matter (DM) basis.
In Table 5, the composition of the basal diet supplemented with two different types of NPN, i.e. urea (Urea) (46% N) and a mixture of urea and biuret according to the present disclosure (UB), with composition as in example 1 (41% N) is shown.
In order to determine DM of the feed, approximately 0.5 g of sample was weighed in duplicate, dried in a forced-air oven at 100° C. for 24 hours, and ashed at 550° C. for six hours.
For the determination of the fibrous component, samples were weighed in duplicate into F56 bags (Ankom Technology Crop., Macedon, NY) and analyzed for neutral detergent fiber (NDF), using heat-stable α-amylase and sodium sulfite, and subsequently for acid detergent fiber (ADF) as described by Van Soest et al. (1991) in Ankom 200 Fiber Analyzer (Ankom Technology Corp., Macedon, NY).
The concentration of N was analyzed through the Dumas dry combustion method using a Vario Micro Cube (Elementar, Manchester, UK) after samples were ball-milled using a Mixer Mill MM 400 (Retsch) at 25 Hz for 9 minutes.
The feed samples were analyzed for concentration of starch through the enzymatic-colorimetric method of Hall (2015) with the following modifications: glucose was analyzed using a quantitative colorimetric kit (G7521-11, Pointe Scientific Inc., Canton, MI), absorbance was read on 200 μl samples at OD520 in flat-bottom 96-well plates (Corning Costar 3361, Thermo Fisher Scientific Inc., Waltham, MA) using a plate reader (Fisherbrand UV/VIS AccuSkan GO Spectrophotometer, Thermo Fisher Scientific Inc., Hampton, NH).
From day −17 to day −16 relative to the beginning of the experimental period, the steers received 20% of their total supplemental NPN.
From day −15 to day −12 relative to the beginning of the experimental period, the steers received 40% of their total supplemental NPN.
From day −11 to day −8 relative to the beginning of the experimental period, the steers received 60% of their total supplemental NPN.
From day −7 to day −3 relative to the beginning of the experimental period, the steers received 80% of their total supplemental NPN.
From day −2 to the beginning of the experimental period (day 0), the steers received 100% of their total supplemental NPN.
Blood samples were collected every 14 days during the performance evaluation via jugular venipuncture into 10 ml evacuated tubes containing Na heparin (BD Vacutainer, Franklin Lakes, NJ) and placed on ice centrifuged at 1,500×g for 15 minutes at 4° C.
Plasma was transferred to polypropylene tubes (12×75 mm, Fisherbrand, Thermo Fisher Scientific Inc, Waltham, MA) and stored at −20° C. for further analysis.
When feed is eaten by a ruminant, the nutrients are initially in the form of carbohydrates, proteins and fats (or lipids). During the fermentation of the carbohydrates, carbon dioxide and methane are produced.
Methane emission was measured in all steers for five consecutive days using the SF6 tracer technique (Johnson et al., 1994; Henry et al., 2020). In this technique, the steers received a calibrated permeation tube filled with SF6 (3.1 g+0.2 g) before the measurement period. The average SF6 release rate was 5.2 g+1.1 mg/d. After five days of training, a vacuum gas collector canister was attached to each animal to collect a sample of daily gas production. Each day at the same hour, a new vacuum canister was replaced throughout the five days of the measurement period. Five collection canisters were used to determine environmental CH4 and SF6 concentrations. A gas sample was taken from each canister and conserved in 120 ml bottles for posterior analysis of CH4 and SF6 using gas chromatography. For the current experiment, only steers with a least three successful days of collection and measurement were considered in the final analysis. Gas samples were analyzed by gas chromatography (Agilent 7820A GC, Agilent Technologies, Palo Alto, CA). A flame ionization and electron capture detector were used for CH4 and SF6 analysis, respectively, with a capillary column (Plot Fused Silica 25 m×0.32 mm, Coating Molsieve 5A, Varian CP7536, Varian Inc., Lake Forest, CA). The injector, column and detector temperatures for CH4 analysis were 80, 160 and 200° C., respectively. For SF6, temperatures were 50, 30 and 300° C. for the injector, the column and the detector, respectively. The carrier gas for CH4 and SF6 was N2. The emissions of CH4 were calculated according to the next equation:
The gain to feed ratio (G:F) was computed as the ratio of the average daily gain (ADG) to daily dry matter intake (DMI). The feed intake was recorded daily. The individual intake was determined via the GrowSafe® intake monitoring system. In cattle management, GrowSafe Systems is a commonly used tool to closely monitor individual animal feeding data using radio frequency identification (RFID) tag technology. The animals are equipped with a unique RFID tag that is read by the feed bunks each time the animal lowers its head into the bunk to consume feed. Average daily weight gain (ADG) and feed efficiency were calculated during the performance evaluation. The ADG was determined by the difference between the final BW and the initial BW divided by the number of days of the performance evaluation (56 days). The initial BW was calculated as the average unshrunk BW of heifers on days 16 and 17. The final BW was the average unshrunk BW measured on days 72 and 73. The unshrunk BW was obtained every 14 days.
In Table 6, the effect of NPN supplementation with Urea and UB on growth performance is shown. More in particular, the initial and the final BW and the ADG, all expressed in kg, the dry matter intake (DMI) expressed in kg/day and in percentage of BW, the gain to feed ratio (G:F) expressed in kg/kg, the net energy of gain (NEg) expressed in Mcal/kg of DM and the plasma urea nitrogen (PUN) expressed in mg/dL are shown.
Table 6 demonstrates that, compared to supplementation with urea, supplementation with the UB NPN source:
Methane is an energy loss representing between 2% and 12% of the gross energy intake in ruminants.
In Table 7, the effect of the NPN supplementation with Urea and UB on enteric methane emission of beef steers is shown. More in particular, the intake expressed in kg/day, and the methane emission expressed in g/kg DMI, g/kg OMI and g/kg ADG are shown.
Out of Table 7, it can be concluded that supplementing the diet of yearling steers with UB reduced methane emission without compromising or even improving their performance. In particular, a decrease in methane emissions, when expressed as g/kg DMI or g/kg OMI, of about 15% was observed, compared to a diet containing urea as NPN source. Simultaneously, it was observed that the DMI considered over the whole duration of the experiment was increased by about 17% when using UB as NPN source, compared to the diet containing urea as NPN source.
In this example, an in vitro assay was performed using batch culture incubations, aiming to simulate rumen fermentation.
Two ruminally cannulated Angus crossbred steers (808.8±36.3 kg body weight) were used as ruminal fluid donors for the in vitro batch culture incubations. The steers were fed a corn silage and gin trash diet (70% and 30% on a dry matter basis, respectively) ad libitum at least 35 days before collecting ruminal fluid to perform the in vitro incubations. Each steer received a NPN-mixture equivalent to 100 g of urea per day comprised of an equal amount of N from the different NPN sources to adapt the rumen microbial community. Thus, each steer was fed daily 33 g of urea (46 wt. % N) or 37 g of a urea-biuret mixture according to the present disclosure (UB) comprising 41 wt. % N. These amounts were mixed in with the feed diet every two days.
The feed was prepared by drying corn silage and gin trash for 45 hours at 55° C. and ground to pass a 2 mm screen in a Wiley mill.
The in vitro batch culture incubations were conducted as a randomized complete block design using 70 wt. % corn silage and 30 wt. % gin trash on a dry matter basis conserving the diet characteristics used during the animal adaptation period. The treatments were designed to be isonitrogenous (=having the same amount of dietary nitrogen) and equivalent to 1 wt. % of urea inclusion. The NPN treatments were consequently as follows:
The in vitro incubations were conducted on three separate days (replicates). A representative sample of digesta was collected from different places in the rumen of ruminally cannulated steers and strained through 4 layers of cheesecloth, placed in pre-warmed thermos containers, and transported to the laboratory within 30 minutes after the collection. In the laboratory, the ruminal fluid from the two steers was maintained under constant CO2-flux and was combined in equal proportions. This combined ruminal fluid was mixed with McDougall's buffer (McDougall, 1948) with and without 0.077 g/liter of 10 wt. % ammonium sulfate enriched with 15N in relation 1:4, and it was used as an inoculum. Two bottles of 500 ml fitted with a side arm and a rubber septum were incubated per treatment. The bottles contained 5.6 g of substrate dry matter and 400 ml of 15N enriched inoculum, and were incubated for 24 hours at 39° C. with an agitation of 60 rpm to monitor gas production kinetics using an Ankom Gas Monitoring System from Ankom Technologies, Fairport, New York. Also, one 500-ml bottle without 15N enriched inoculum was incubated to determine the basal 15N enrichment. At 12 hours of incubation, two samples of 10 ml of rumen fluid were collected from each bottle through the septa port using a 20-ml syringe. These samples were acidified by adding 10 μl of a 20 vol/vol % of H2SO4-solution to each 10-ml sample, and frozen at minus 20° C. until further analysis. At the end of the 24 hours incubation, the final pH was recorded, the bottles were placed on ice to prevent further microbial fermentation and two 10-ml samples were collected and acidified by adding 20 μl of a 20 vol/vol % of H2SO4-solution and were frozen at minus 20° C. until further analysis. The remaining fermentation content was thereafter agitated at 60 rpm for 30 seconds and divided into two subsamples. From the first subsample, composed of whole contents, a volume of 180 ml was frozen at minus 20° C. until further analysis and from the second subsample 180 ml were used to recover the microbial pellet. Furthermore, two tubes of 100 ml per treatment containing 0.7 g of the substrate on a dry matter basis and 50 ml of inoculum without 15N enrichment and two blanks were incubated for 24 hours at 39° C. with agitation at a speed of 60 rpm. After the 24 hours incubation with ruminal fluid, 6 ml of HCl and 2 ml of a 5% pepsin solution were added. The tubes were incubated for another 48 hours at 39° C. After the incubation period, the tubes were maintained on ice and later stored for further analysis.
VFA produced by microbial fermentation in the rumen are the main source of energy absorbed by the digestive tract of ruminants. The amount and the profile of VFA in the rumen has consequences for the efficiency of energy utilization, production of methane, risks of ruminal acidosis (characterized by a decrease in pH caused by an accumulation of organic acids in the rumen and a shift in the delicate balance of rumen microorganisms). A water-based solution using ethyl acetate extraction was used to determine VFA concentrations at 12 and 24 hours. Samples were centrifuged at 10,000×g for 15 min at 4° C., 2 ml of the supernatant were mixed with 0.4 ml (5:1 ratio) of a metaphosphoric-crotonic acid solution, and samples were frozen overnight. Samples were then thawed and centrifuged again at 10,000×g for 15 min at 4° C. The supernatant was transferred into 12×75 mm borosilicate disposable culture tubes (Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) and mixed with ethyl acetate to form a 2:1 ethyl acetate: supernatant mixture. The samples were analyzed with a gas chromatograph (Agilent 7820A GC, Agilent Technologies) using a flame ionization detector and a capillary column (CP-WAX 58 FFAP 25 m×0.53 mm, Varian CP7767; Varian Inc.).
The rumen microbial ecosystem is made up mainly by species of bacteria, fungi, and protozoa. The microbes ferment ingested feeds by the ruminant, and, by fermentation, produce end products that are utilized by the ruminant as well as by the microbes themselves for their own proliferation and growth. The species diversity and their relative proportions within the microbial community depend primarily on the diet consumed by the animal. It is thus important that a healthy mixture of different feedstuffs is fed to the animal to keep the rumen and its microflora functioning efficiently. The speed of digestion of the feed depends on the quality and the supplement of the feed and is affected by the number and type of microbes, the pH in the rumen, the nutrients limiting the growth of the microbes and the removal of microbes from the rumen. Energy and protein are the major nutrients that limit the microbial growth, and therefore, the rumen fermentation. The microbial population needs energy and protein for their growth and multiplication. Protein, when digested, is broken down into peptides, which are short chains of amino acids. Further digestion of peptides results in individual amino acids and eventually ammonia. Rumen microbes are the major source of protein for the ruminant animal. If there is enough energy available, the rumen microbes break down the rumen degradable protein to amino acids and then to ammonia which is a major N-source for microbial growth. The microbes also convert NPN to ammonia, which contributes to the ammonia pool available for microbial growth. Microbial protein is continuously flushed from the rumen into the intestine where undergoes enzymatic digestion. The amino acids produced from the digested microbial protein are absorbed through the small intestine. The amount of microbial protein flowing into the intestines therefore largely depends on the availability of fermentable energy and N in the diet consumed by ruminants.
The total nitrogen (N) and the isotopic supplement were measured in the whole content and the bacterial pellet by an isotopic ratio mass spectrometry using a Vario Micro cube from Elementar Analyzer System GmbH. The bacteria pellet subsample was slowly centrifuged at 1,000×g for 10 minutes at 4° C. The resulting supernatant was fast centrifuged at 20,000×g for 20 minutes at 4° C. to obtain the pellet of bacteria. This bacteria pellet was then washed with a 0.9% saline solution and centrifuged at 20,000×g for 20 minutes at 4° C., discarding the supernatant. The saline wash procedure was repeated twice. Finally, the bacterial pellet was freeze-dried. Separately, the 180-ml whole content subsample was completely freeze-dried. The dried samples of the whole content and of the bacterial pellet were manually grinded and weighted into an 8×5-mm pressed standard-weight tin capsule using a Mettler-Toledo Excellence Plus XP Micro Balance from Mettler-Toledo GmbH, Laboratory and Weighing Technologies. Then, 35 μl of a 10 g/l solution of K2CO3 were added to each sample and dried overnight in a forced-air oven at 60° C. for complete NH3—N evaporation. The percentage of atom 15N in the dried samples was analyzed in an isotope ratio mass spectrometer (IsoPrime 100 from IsoPrime) and expressed as the fractional abundance of isotopic fractions (15N/14N) multiplied by 10.
Ruminal ammonia production largely reflects the amount of degradable protein in the rumen. Ruminal ammonia nitrogen (ammonia-N) is a critical nutrient for supporting microbial growth and fermentation. Ensuring the availability of ammonia-N in the rumen both on a quantitative and timely manner is fundamental for achieving maximum rate of fermentation and feed digestibility.
The concentration of ammonia-N at 12 and 24 hours in the incubation fluid was measured after centrifuging at 10,000×g for 15 minutes at 4° C. using an Avanti J-E centrifuge from Beckman Coulter® Incorporated. One ml of phenol reagent was pipetted into 12×75 mm borosilicate disposable culture tubes (Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA). A 20-μl aliquot of the supernatant from the centrifuged sample was transferred to the phenol-containing culture tubes. After vortexing, 0.8 ml of hypochlorite solution was added to the mixture and was vortexed again. The culture tubes were covered with glass marbles and placed in a water bath at 95° C. for 5 minutes. The absorbance was read in 96-well, flat-bottom plates at a wavelength of 665 nm using a DU-500 plate reader from Beckman Coulter® Inc. The fermentation residue reserved into the tubes of 100 ml were filtered, dried at 105° C. in a forced air oven for 24 hours and ashed at 550° C. for 6 hours to determine the undigested organic matter.
Digestibility represents the proportion of ingested nutrients consumed that are absorbed by the animal. Dry matter is defined as the loss of weight of samples when dried in an oven above 100° C. during 12-24 hours, whereas organic matter defines the weight loss of the dry matter when combusted (the dry matter minus the ash content). Dry matter consists of all nutrients, whereas organic matter consists of all nutrients with the exception of ash. Organic matter digestibility is defined as the proportion of organic matter in the feed that is digested in the rumen or total ruminant digestive tract. Ruminal organic matter digestibility can be used to estimate energy available for fueling microbial growth and the resulting synthesis of microbial protein in the rumen. The in vitro dry organic matter digestibility (IVDMD) and the in vitro organic matter digestibility (IVOMD) was calculated with the following formulae:
In Table 8 below, the concentration of the total VFA and the concentration of ammonia-N in: a control diet without NPN supplementation (“control”), a corn silage-based diet either with urea supplementation (“urea”) or with supplementation of a urea-biuret mixture according to the present disclosure (“UB”) after 12 hours of incubation is shown. In Table 9 below, the same is shown after 24 hours of incubation. In Table 9, also the pH of the fluid, the in vitro dry matter degradability (IVDMD) and the in vitro organic matter degradability (IVOMD) after 24 hours of incubation are shown. The pH of the rumen has profound effects on the growth of the ruminal microbes and the digestion that takes place in this forestomach. Rumen pH can affect the extent of rumen fermentation by altering microbial populations and tissue absorptive capacity. Low ruminal pH for prolonged periods each day for instance can affect feed intake, microbial metabolism and feed digestion, and has also been related to inflammation, diarrhea and milk fat depression.
Out of Tables 8 and 9, it can be concluded that
In Table 10, the concentration of bacterial N, bacteria efficiency (BE) or, stated differently, the efficiency of microbial protein synthesis (EMPS), i.e. g of bacteria N/kg of OM truly digested for (i) a control diet without NPN supplementation (“control”), (ii) a corn silage-based diet with urea supplementation (“urea”) or (iii) a corn silage-based diet with supplementation with a urea-biuret mixture according to the present application (“UB”), after 24 hours of incubation is shown. These parameters define the capacity of rumen microbes to use N.
Out of Table 10, it can be concluded that the supplementation of the control diet with NPN increases the amount of N assimilated by bacteria. Supplementation with the urea/biuret mixture according to the present application particularly resulted in an increased bacteria efficiency/increased efficiency of microbial protein synthesis. These results are in line with the results of example 2.
When feed is eaten by a ruminant, the nutrients are initially in the form of carbohydrates, proteins and fats (or lipids). During the fermentation of the carbohydrates, carbon dioxide and methane are produced.
The amount of methane produced was determined by measuring total gas production and the contribution of methane to it. The production of gas was recorded using an Ankom Gas Monitoring System from Ankom Technologies. This system was adapted to 500 ml bottles during the entire fermentation process. The kinetics of the production was fitted to a Gompertz model to determine the rate of the total maximal gas production (M), fractional rate of gas produced (kf) and lag phase (L). A gas subsample was analyzed to measure methane concentration and calculate the total methane production during the fermentation. The concentration of methane was determined using a gas chromatograph Agilent 7820 GC from Agilent Technologies with flame ionization and a capillary column (Plot Fused Silica 25 m×0.32 mm, coating Molsieve 5A, Varian CP 7536 from Varian Inc.).
In Table 11, the effect of NPN supplementation on the production of methane in a corn silage-based diet is shown. More in particular, methane gas production expressed in mM/g organic matter incubated (OMi) and expressed in mM/g organic matter digested (OMd), in a control diet without NPN supplementation (“control”), a corn silage-based diet either urea supplementation (“urea”) or with a urea/biuret mixture according to the present application (“UB”), are shown.
Table 11 shows that supplementation with a UB mixture according to the present disclosure resulted in a decreased production of methane compared to the control diet or the diet with urea as NPN. Methane production decreased by about 4-5% when expressed per g of OMi or per g of OMd, compared to urea as NPN.
The main objective of example 5 was to assess the impact of increasing doses of a mixture of urea and biuret according to the present disclosure, hereinafter UB, with composition as in example 1 (41% N, YARA International) on in vitro fermentation, nutrient digestibility, and methane production in a corn silage-based diet.
Two ruminally cannulated steers were used as ruminal fluid donors for the in vitro batch culture incubations. The steers were fed corn silage, cotton gin trash, and a premix of vitamins and minerals (70, 28, and 2% on a dry matter basis, respectively) ad libitum at least 35 d before collecting ruminal fluid to perform the in vitro incubations. To adapt steers to the presence of NPN, each steer received the N equivalent to 100 g of urea per day (46 g of N daily/steer) but divided into two NPN sources. Thus, each steer was fed daily with 33 g of urea or 37 g of a urea/biuret mixture according to the present application. These amounts were mixed in with the diet daily. In vitro incubations were conducted on three separate days (replicates) using the same silage fed to the steers as the incubation substrate. The sources of NPN to be tested were analyzed for total N content and incubated on an isonitrogenous basis.
The incubation substrates (treatments) to test were control (CON, without NPN supplementation) and four increasing inclusions (from 1× to 4×) of the urea-biuret mixture according to the present application (UB). Treatments at 1× were designed to be iso-nitrogenous and equivalent to 1% of the inclusion in dry matter basis of urea (Yara International), in the diet, except for the CON treatment.
In vitro incubations were conducted on three separate days (replicates) using the same silage fed to the steers as the incubation substrate. A representative sample of rumen digesta was collected from different places in the rumen from the ruminally-cannulated steers and strained through 4 layers of cheesecloth, placed in pre-warmed thermos containers, and transported to the laboratory within 30 min of collection. In the laboratory, ruminal fluid from the two steers was maintained under constant CO2 flux and was combined in equal proportions. Combined ruminal fluid was mixed with McDougall buffer in relation 1:4 (i.e., inoculum).
Two bottles of 120 mL per treatment containing 0.7 g and 50 mL of inoculum were incubated for 24 h at 39° C. with gentle agitation (60 rpm). Further, two bottles without substrate were incubated as blank bottles. Gas pressure was recorded at 24 h, and a sample of gas was stored for further analysis. At the end of the 24 h of incubation, the final pH of fermentation fluid was recorded, and two 10-mL samples were collected and acidified by adding 20 μL of a 20% (vol/vol) of H2SO4 solution and were frozen at −20° C. until further analysis.
Measurement of in vitro organic matter digestibility (IVOMD) was performed by incubating a separate set of duplicate 100-mL polypropylene tubes for 24 h at 39° C. on each of the replicate days. These flasks contained 0.7 g of the substrate and were inoculated with 50 mL of a 4:1 McDougall's buffer:ruminal fluid mixture. After 24-h incubation with ruminal fluid, a pepsin-HCl solution was added, and tubes were incubated for another 48 h before filtering, drying at 105° C. in a forced air oven for 24 h, and ashing at 550° C. for 6 h to determine the undigested organic matter.
Concentrations of NH3-N in the incubation fluid were measured after centrifuging at 10,000×g for 15 min at 4° C. (Avanti J-E, Beckman Coulter Inc., Palo Alto, CA) following the methodology described by Broderick and Kang (1980). Briefly, 1 mL of a phenol reagent was pipetted into 12× 75 mm borosilicate disposable culture tubes (Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA). A 20-μL aliquot of the supernatant from the centrifuged sample was transferred to the phenol-containing culture tubes. After vortexing, 0.8 mL of a hypochlorite solution was added to the mixture and vortexed again. The culture tubes were covered with glass marbles and placed in a water bath at 95° C. for 5 min. Absorbance was read on 200 μL samples at OD620 in flat-bottom 96-well plates (Corning Costar 3361, Thermo Fisher Scientific Inc., Waltham, MA) using a plate reader (Fisherbrand UV/VIS AccuSkan GO Spectrophotometer, Thermo Fisher Scientific Inc., Hampton, NH). All assays were conducted in duplicate determinations with subsequent analyses performed when CVs were above 5%.
Total gas production was measured using a manual transducer (Digital Test Gauge, Ashcroft Inc., Stratford, CT, USA), and a subsample was analyzed to measure CH4 concentration by gas chromatography to calculate total CH4 production during the course of the fermentation. To determine concentrations of CH4, a gas chromatograph (Agilent 7820A GC, Agilent Technologies, Palo Alto, CA) using flame ionization and a capillary column (Plot Fused Silica 25 m×0.32 mm, Coating Molsieve 5A, Varian CP7536; Varian Inc., Lake Forest, CA) was used. Temperatures of the injector, column, and detector were 80, 160, and 200° C., respectively, and N2 was the carrier gas flowing at 3.3 mL/min. The split ratio for the injected CH4 sample was 100:1.
Additionally, total volatile fatty acids (VFA) concentrations were measured by gas chromatography in the incubation fluid at the end of the fermentation to assess potential changes in the profile of VFA being produced, following the methodology described by Ruiz-Moreno et al. (2015). Briefly, samples were centrifuged at 10,000×g for 15 min at 4° C. Two milliliters of the supernatant were mixed with 0.4 mL (5:1 ratio) of a metaphosphoric: crotonic acid (internal standard) solution, and samples were frozen overnight. Samples were thawed and centrifuged again at 10,000×g for 15 min at 4° C. The supernatant was transferred into 12 mm×75 mm borosilicate disposable culture tubes (Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) and mixed with ethyl acetate to form a 2:1 ethyl acetate: supernatant mixture. Culture tubes were vigorously shaken and followed by a 5 min rest time to allow the separation of the ethyl acetate. A subsample of the ethyl acetate was transferred into small vials prior to analysis. Samples were analyzed with a gas chromatograph (Agilent 7820A GC, Agilent Technologies) using a flame ionization detector and a capillary column (CP-WAX 58 FFAP 25 m×0.53 mm, Varian CP7767; Varian Inc.). The column temperature was maintained at 110° C., and the injector and detector temperatures were 200 and 220° C., respectively.
The results are represented in Table 12.
1IVOMD: In vitro organic matter digestibility; OMd: Organic matter degraded.
Increasing doses of UB linearly increased the concentration of NH3—N, the rumen fluid pH, and the IVOMD. Also, the concentration of total VFA, acetate, propionate, and butyrate quadratically increased when increasing UB inclusion. Additionally, increasing the inclusion of UB linearly reduced the acetate: propionate ratrio and gas and CH4 production (Table 12).
In the present experiment, the inclusion of UB increased the NH3—N concentration resulting in greater IVOMD and concentration of total and individual VFA, indicative of a greater fermentation. Rumen microbes can utilize NH3—N to synthezise microbial protein promoting greater fermentation. Additionally, the greater concentration of NH3-N is in line with the linear increase in the rumen fluid pH.
Increasing the UB inclusion promoted other metabolic routes different than acetate formation, with a linear decrease in acetate proportion upon increasing the UB supplementation.
Furthermore, increasing the UB supplementation linearly decreased gas and CH4 production but increased VFA and IVOMD, suggesting changes in rumen microbial communities, possibly Archaeas and fibrolytic bacteria, explaining the lower production of CH4 and the lower proportion of acetate. Without wishing to be bound by theory, the greater concentration of NH3—N during the incubation results in a greater fermentation rate, thereby increasing the concentration of VFA and IVOMD. However, the faster accumulation of NH3—N may result in a toxic effect on some rumen microbial communities affecting the gas and CH4 production.
Further particular statements (features) and embodiments and uses of a NPN composition according to the present disclosure are set herein below.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22157277.9 | Feb 2022 | EP | regional |
| 22382539.9 | Jun 2022 | EP | regional |
| 22382836.9 | Sep 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/054080 | 2/17/2023 | WO |
