CN111491520A

CN111491520A - Methods for measuring reducing equivalent production of tissue to determine metabolic rate and methods of use

Info

Publication number: CN111491520A
Application number: CN201880081453.8A
Authority: CN
Inventors: 本杰明·J·伦奎斯特
Original assignee: University of Arizona
Current assignee: University of Arizona
Priority date: 2017-11-15
Filing date: 2018-11-15
Publication date: 2020-08-04
Also published as: AU2018370021A1; EP3709818A4; EP3709818A1; WO2019099717A1; CA3082604A1; BR112020009559A2; AU2018370021B2

Abstract

Methods for identifying genetically superior animals, drugs, nutritional strategies or physiological manipulations that increase the feed efficiency or productivity of an animal, for example, selecting animals with genetically superior feed efficiency or productivity based on the metabolic rate of a particular tissue, wherein the metabolic rate of certain tissues, such as skeletal muscle, is inversely proportional to feed efficiency and the metabolic rate of other tissues, such as breast, is directly proportional to milk production. Thus, animals with low skeletal muscle metabolic rates typically eat more efficiently, e.g., gain more weight per unit of food. The methods herein can be used to improve genetics, nutrition, and operation or to more efficiently produce animal products, such as meat products, milk products, egg products, wool products, and the like. The methods herein can also be used to determine estimated reproductive values for feed efficiency, growth, or yield of an animal.

Description

Methods for measuring reducing equivalent production of tissue to determine metabolic rate and methods of use

Technical Field

The invention relates to the measurement of reducing equivalents (e.g. NADH, FADH)₂NADP (H) and/or coenzyme Q) for measuring the tissue metabolic rate in animals and humans. The method of measuring reducing equivalents provides a high throughput means of determining the metabolic rate of an animal or human. The method can be used for a variety of purposes, such as assessing feed efficiency, assessing productivity of an animal, determining the likelihood of developing obesity, sorting animals, and the like.

Government support

The invention is carried out under the government support of the USDA/NIFA awarded subsidies 2016-. The government has certain rights in the invention.

Background

Basal metabolic rates can be estimated by feeding animals in metabolic chambers, but the use of metabolic chambers is difficult due to practical and economic challenges. Alternatively, an oxygen sensor may be used or by measuring the CO of the medium₂Acidification was used to measure the ex vivo metabolic rate of the tissue. However, neither of these measures has the sensitivity associated with the accumulation of signals described in the present invention, and both suffer from gas exchange between the medium and air.

The techniques described herein were previously used to assess cell viability, not metabolic rate. Thus, it was surprisingly found that measuring reducing equivalents in animal (or human) tissue can be used as a surrogate for measuring metabolic rate in animals (or humans). When it is found that the tissue metabolic rate can be used to assess the feed efficiency or tissue of an individualAn additional leap is obtained with respect to the production potential of (2). The method of the invention is characterized by measuring the reduction equivalents (e.g., NADH, FADH)₂Nadp (h), coenzyme Q, etc.) for use in determining the metabolic rate (e.g., tissue specific metabolic rate) of an animal or human. The present invention provides high throughput methods for determining metabolic rate in animals or humans.

The invention also features uses of the methods, for example, methods of measuring metabolic rate using reduction equivalence measurement. For example, the methods herein can be used to:

identifying and/or selecting animals that are feed-efficient;

elimination of animals with poor feed efficiency;

identifying and/or selecting high producing animals (e.g., high milk production, high egg production, meat production, or any other suitable animal product);

identifying an animal or subject (e.g. a human) more likely to become overweight or obese;

identifying an animal or subject (e.g., a human) with greater resistance to obesity;

developing an estimated reproductive value for feed efficiency, growth, milk production or egg production in the animal;

selecting a farm animal with higher production efficiency or better feed efficiency;

identifying an animal or human having a particular likelihood of developing obesity;

testing metabolic rate response to drugs (e.g., antibiotics), nutrients, or other molecules. These tests can be applied to evaluate the impact of such stimuli on yield, feed efficiency and compliance driving, based on the relationship between feed efficiency and yield;

assessing the effect of stress or stimulation (e.g. exercise) on the metabolic rate of the animal. Considering the relationship to feed efficiency and yield, these tests can be used to evaluate the impact of this stimulus on yield, feed efficiency and compliance driving.

Without wishing to limit the invention to any theory or mechanism, it is believed that the inventive technique is advantageous because it provides a fast, high-throughput, scalable and easy method of assessing tissue metabolic rate (via cell viability assays, e.g., measuring reducing equivalents), while allowing the user to correlate the metabolic rate with parameters such as feed efficiency and tissue-specific production formation. For example, growth can be assessed by applying it to skeletal muscle of young animals; by applying this method in adult skeletal muscle, feed efficiency can be measured or assessed; by applying it to the lactating mammary glands one can determine the milk yield.

Disclosure of Invention

As previously mentioned, the invention features measuring reducing equivalents (e.g., NADH, FADH) in animal (or human) tissue₂Coenzyme Q, etc.) to determine the metabolic rate (or tissue-specific metabolic rate) of an animal (or human). The present invention is not limited to any particular method for measuring the reduction equivalents. For example, the present invention is not limited to the use of resazurin, MTT, or any other particular reducing equivalent indicator. In some embodiments, measuring the reduction equivalent can be characterized as measuring NADH production. In some embodiments, measuring the reduction equivalent can be characterized as measuring FADH₂And (4) generating. In some embodiments, measuring the reducing equivalent can be characterized as measuring coenzyme Q. In some embodiments, measuring the reduction equivalent can be characterized as measuring NADH, FADH₂NADP (H), or coenzyme Q.

As used herein, the term "animal" can refer to any suitable animal or human, such as, for example, cattle (e.g., cows, beef cattle), goats, sheep, pigs, mice, dogs, cats, humans, non-human primates, chickens, fish, mollusks, and the like. For example, in humans, non-human primates, dogs, cats, etc., the methods of the invention can be used to determine the likelihood of obesity. The methods and systems of the present invention are not limited to the animals disclosed herein.

As used herein, the term "tissue" may refer to any suitable tissue of an animal or human, such as skeletal muscle, breast tissue, brown adipose tissue, white adipose tissue, liver, kidney, fin, and the like. The methods and systems of the present invention are not limited to the organization disclosed herein.

Without wishing to limit the invention to any theory or mechanism, if tissue mass is greater relative to the overall body weight (e.g., skeletal muscle), a lower tissue-specific metabolic rate may indicate a lower overall animal basal metabolic rate (the energy expended to maintain proper tissue function without changing tissue mass). A lower basal metabolic rate allows less dietary energy to tend toward the body to maintain energy needs and more dietary energy to tend toward growth or product (e.g., milk, egg, meat, etc.) formation. Thus, a low skeletal muscle metabolism rate in slow/non-growing adult animals indicates the potential for good feed efficiency (product quality/feed quality), as well as a high potential for product (e.g., milk, meat) production, and the like.

The invention also features the use of a method of measuring reducing equivalents for determining the metabolic rate of an animal (or human).

For example, the invention provides methods for identifying animals with a particular feed efficiency, e.g., high feed efficiency. As used herein, the term "feed efficiency" refers to the weight obtained per unit of feed or the amount of product produced per unit of feed. The invention also features methods of classifying animals based on feed efficiency. The invention also features methods of selecting (e.g., grouping, segregating, etc. methods) animals with a particular feed efficiency (e.g., high feed efficiency). In some embodiments, the feed efficiency determined by the methods herein can be used to calculate expected offspring differences.

The methods described above (e.g., methods of identifying an animal with a particular feed efficiency, etc.) and other methods described herein can include determining a reducing equivalent yield (e.g., amount, change, etc.) in a tissue sample (e.g., skeletal muscle tissue sample) from the animal, wherein the reducing equivalent yield (e.g., amount, change, etc.) in the tissue sample is inversely proportional to the feed efficiency. In some embodiments, if the reducing equivalent yield (e.g., amount, change, etc.) in the skeletal muscle tissue sample is below a predetermined threshold, the animal from which the skeletal muscle tissue sample is obtained has a high feed efficiency compared to an animal having a reducing equivalent yield (e.g., amount, change, etc.) above the predetermined threshold. Determining the reducing equivalent yield (e.g., amount, change, etc.) in the tissue sample can include introducing a reducing equivalent indicator to the tissue sample and measuring the amount or change of the reducing equivalent indicator (which is indicative of metabolic activity).

The predetermined threshold may be an average of reducing equivalent yields for breeds, groups or species of animals. The predetermined threshold may be determined by the user, for example, by selecting a stringency based on the desired feed efficiency. The predetermined threshold may be a percentile level (e.g., 5 th percentile, 10 th percentile, 25 th percentile, 50 th percentile, etc.). The predetermined threshold may be determined using a group of animals with known reducing equivalent yields (e.g., amounts, variations, etc.) and known feed efficiencies. The predetermined threshold may classify the animal by feed efficiency.

With respect to the methods described herein, in some embodiments, the reducing equivalent is NADH. In some embodiments, the reducing equivalent is nadp (h). In some embodiments, the reducing equivalent is FADH₂. In some embodiments, the reducing equivalent is coenzyme Q. In some embodiments, the reducing equivalent is NADH or FADH₂. In some embodiments, the reducing equivalent is NADH or coenzyme Q. In some embodiments, the reducing equivalent is FADH₂Or coenzyme Q. In some embodiments, the reducing equivalent is NADH or nadp (h). In some embodiments, the reducing equivalent is nadp (h) or coenzyme Q. In some embodiments, the reducing equivalent is NADP (H) or FADH₂. In some embodiments, the reducing equivalents are NADH, FADH₂NADP (H), and coenzyme Q. In some embodiments, the reducing equivalents are NADH, FADH₂Or coenzyme Q. In some embodiments, the reducing equivalents are NADH, FADH₂NADP (H), or coenzyme Q.

The method may further comprise breeding using animals identified as having high feed efficiency. The method can also include producing an animal product (e.g., milk, meat, etc.) using the animal identified as having high feed efficiency.

The invention also provides methods of identifying animals with a particular milk yield, e.g., a high milk yield. The invention also features a method of classifying animals based on milk production. The invention also features methods of selecting (e.g., grouping, segregating, etc. methods) animals having a particular milk yield (e.g., high milk yield).

The methods described above (e.g., methods of identifying animals with high milk production, etc.) and other methods described herein can include determining reducing equivalent production (e.g., amount, change, etc.) in a breast tissue sample from an animal, wherein reducing equivalent production (e.g., amount, change, etc.) in the breast tissue sample is directly correlated with milk production potential. In some embodiments, if the reducing equivalent production (e.g., amount, variation, etc.) in the breast tissue sample is above a predetermined threshold, the animal from which the breast tissue sample is obtained has a high milk production potential compared to an animal having a reducing equivalent production (e.g., amount, variation, etc.) below the predetermined threshold. Determining the reducing equivalent yield in the tissue sample can include introducing a reducing equivalent indicator into the tissue sample and measuring the amount or change of the reducing equivalent indicator (which is indicative of metabolic activity).

The predetermined threshold may be an average of reducing equivalent yields for breeds, groups or species of animals. The predetermined threshold may be determined by the user, for example, based on a desired strictness of selection of the milk yield potential. The predetermined threshold may be a percentile level (e.g., 5 th percentile, 10 th percentile, 25 th percentile, 50 th percentile, etc.). The predetermined threshold may be determined using a group of animals with known reducing equivalent yields (e.g., amounts, variations, etc.) and known milk yields. The predetermined threshold may classify the animal by milk production potential.

With respect to the methods described herein, in some embodiments, the reducing equivalent is NADH. In some embodiments, the reducing equivalent is nadp (h). In some embodiments, the reducing equivalent is FADH₂. In some embodiments, the reducing equivalent is coenzyme Q. In some embodiments, the reducing equivalent is NADH or FADH₂. In some embodiments, the reducing equivalent is NADH or coenzyme Q. In some embodiments, the reducing equivalent is FADH₂Or coenzyme Q. In some embodiments, the reducing equivalent is NADH or nadp (h). In some embodiments, the reducing equivalent is nadp (h) or coenzyme Q. In some embodiments, the reducing equivalent is NADP (H) or FADH₂. In some casesIn embodiments, the reducing equivalents are NADH, FADH₂NADP (H), and coenzyme Q. In some embodiments, the reducing equivalents are NADH, FADH₂Or coenzyme Q. In some embodiments, the reducing equivalents are NADH, FADH₂NADP (H), or coenzyme Q.

The method may further comprise breeding using animals identified as having high milk production. The method may further comprise producing milk using an animal identified as having high milk production.

The invention also features a method of calculating a feed efficiency reproduction value for an animal. In some embodiments, the methods comprise determining feed efficiency based on a metabolic rate of a tissue sample from the animal, e.g., as described herein, wherein the metabolic rate is determined by determining a reducing equivalent yield (e.g., amount, change, etc.); and assigning an estimated expected progeny difference from the reproduction mean based on the metabolic rate of the tissue sample. In some embodiments, the estimated reproductive value is indicative of inheritance of feed efficiency of the potential parental culture. In some embodiments, the method further comprises combining the estimated feed efficiency with one or more additional estimated reproductive values (e.g., rib area, intramuscular fat, fat depth, birth weight, weaning weight, and carcass yield).

The invention also features methods of biasing or biasing the genetic composition of an animal population (e.g., a neonatal population) toward having a high feed efficiency preference. In some embodiments, the method comprises determining the metabolic rate of a tissue sample (e.g., skeletal muscle tissue) from the animal (as described herein). In some embodiments, the method further comprises selecting an animal with an optimal feed efficiency for breeding a neonatal animal population with a particular predicted feed efficiency.

The invention also features methods of biasing or skewing the genetic composition of an animal population (e.g., a neonatal population) toward having a high milk yield preference. In some embodiments, the method comprises determining the metabolic rate of a tissue sample (e.g., breast tissue) from the animal (as described herein). In some embodiments, the method further comprises selecting an animal with an optimal milk yield for breeding a new animal population with a particular predicted milk yield.

The invention also features methods for detecting the effect of a drug, dietary supplement, diet, or other composition on the feed efficiency of an animal. In some embodiments, the method comprises determining a baseline tissue-specific metabolic rate of the animal by measuring reducing equivalent production (e.g., amount, change, etc.) in a first tissue sample (e.g., skeletal muscle) from the animal; administering the drug, dietary supplement, diet or other composition to the animal; the reducing equivalent production (second tissue-specific metabolic rate) in a second tissue sample of the animal tissue is then determined. In some embodiments, the drug, dietary supplement, diet, or other composition does not affect the feed efficiency of the animal if the second tissue-specific metabolic rate is equal to the baseline tissue-specific metabolic rate. In some embodiments, the drug, dietary supplement, diet, or other composition has a positive effect on the feed efficiency of the animal if the second tissue-specific metabolism rate is less than the baseline tissue-specific metabolism. In some embodiments, the drug, dietary supplement, diet, or other composition has a negative impact on the feed efficiency of the animal if the second tissue-specific metabolism rate is greater than the baseline tissue-specific metabolism.

In some embodiments, the method comprises administering to the animal a drug, dietary supplement, diet, or composition; and determining the metabolic rate of the animal by determining the reduction equivalent production (e.g., amount, change, etc.) in a tissue (e.g., skeletal muscle) of the animal. In some embodiments, the drug, dietary supplement, diet, or other composition does not affect the feed efficiency of the animal if the animal's metabolic rate is equal to a control metabolic rate that is the metabolic rate of the animal or group of animals that are not administered the drug, dietary supplement, diet, or other composition. In some embodiments, the drug, dietary supplement, diet, or other composition has a positive effect on the feed efficiency of the animal if the animal's metabolic rate is less than a control metabolic rate, which is the metabolic rate of the animal or group of animals that are not administered the drug, dietary supplement, diet, or other composition. In some embodiments, the drug, dietary supplement, diet, or other composition has a negative effect on the feed efficiency of the animal if the animal's metabolic rate is greater than a control metabolic rate that is the metabolic rate of the animal or group of animals that are not administered the drug, dietary supplement, diet, or other composition.

The invention also features methods of detecting the effect of a drug, dietary supplement, diet, or other composition on milk production by an animal. In some embodiments, the method comprises determining a baseline tissue-specific metabolic rate of the animal by measuring reducing equivalent production (e.g., amount, change, etc.) in a first tissue sample of breast tissue of the animal; administering the drug, dietary supplement, diet or other composition to the animal; and determining a second tissue-specific metabolic rate of the animal by measuring a reducing equivalent yield (e.g., amount, change, etc.) in a second tissue sample of the mammary tissue of the animal. In some embodiments, the drug, dietary supplement, diet, or other composition does not affect the milk yield of the animal if the second tissue-specific metabolic rate is equal to the baseline tissue-specific metabolic rate. In some embodiments, the drug, dietary supplement, diet, or other composition has a negative impact on the milk yield of the animal if the second tissue-specific metabolism rate is less than the baseline tissue-specific metabolism. In some embodiments, the drug, dietary supplement, diet, or other composition has a positive effect on the milk yield of the animal if the second tissue-specific metabolism rate is greater than the baseline tissue-specific metabolism.

In some embodiments, the method comprises administering to the animal a drug, dietary supplement, diet, or composition; and determining the tissue-specific metabolic rate of the animal by determining the reducing equivalent production (e.g., amount, change, etc.) in the mammary tissue of the animal. In some embodiments, if the tissue-specific metabolic rate of the animal is equal to the control tissue-specific metabolic rate, the control tissue-specific metabolic rate is the metabolic rate of breast tissue of the animal or group of animals to which the drug, dietary supplement, diet or other composition has not been administered, and the drug, dietary supplement, diet or other composition does not affect milk production of the animal. In some embodiments, wherein the control tissue-specific metabolic rate is the metabolic rate of the animal or group of animals to which the drug, dietary supplement, diet or other composition is not administered if the tissue-specific metabolic rate of the animal is less than the control tissue-specific metabolic rate, the drug, dietary supplement, diet or other composition has a positive effect on milk production by the animal. In some embodiments, wherein the drug, dietary supplement, diet, or other composition has a negative effect on the milk yield of the animal if the tissue-specific metabolic rate of the animal is greater than a control tissue-specific metabolic rate that is the metabolic rate of the animal or group of animals that is not administered the drug, dietary supplement, diet, or other composition.

The invention also features methods of testing a drug, dietary supplement, diet, or composition in vitro. The method can be characterized by obtaining a sample from the animal and treating the sample with a drug, dietary supplement, diet, or composition in culture to determine whether there is an effect of the drug, dietary supplement, diet, or composition on the production of reducing equivalents (e.g., metabolic rate).

As used herein, the term "baseline" refers to the metabolic rate or other parameter, and may refer to an amount predetermined by the industry or user. For example, the baseline may be predetermined by the user by testing the metabolic rate (or milk production) of the animal prior to administration of the drug or composition. In some embodiments, the baseline is predetermined by other individuals, e.g., country average, breed average, etc.

The invention also features methods for treating animals to improve milk production. The method may include determining the amount of a drug, dietary supplement, diet, or other composition administered to achieve a particular milk yield (e.g., using a method or combination of methods described herein), and the dosage of a drug, dietary supplement, diet, or other composition administered to achieve a desired milk yield.

The invention also features methods of treating animals to increase feed efficiency. The method can include determining the amount of a drug, dietary supplement, diet, or other composition administered to achieve a particular feed efficiency (e.g., using a method or combination of methods described herein), and the dosage of the drug, dietary supplement, diet, or other composition administered to achieve a desired feed efficiency.

As used herein, the terms percentile, percentile level, threshold level, and/or baseline may refer to a predetermined amount or level determined by a user or industry. For example, in some embodiments, the threshold level or percentile level is an industry average. In some embodiments, the threshold level or percentile level is set by a user. The threshold level may be unique to a particular breed or group. The threshold level or percentile level may depend on the desired feed efficiency, milk yield, etc.

In some embodiments, the threshold or percentile is the 50 th percentile or average. In some embodiments, the threshold or percentile is the 5 th percentile. In some embodiments, the threshold or percentile is the 10 th percentile. In some embodiments, the threshold or percentile is the 15 th percentile. In some embodiments, the threshold or percentile is the 20 th digit. In some embodiments, the threshold or percentile is the 25 th percentile. In some embodiments, the threshold or percentile is the 30 th percentile. In some embodiments, the threshold or percentile is the 35 th percentile. In some embodiments, the threshold or percentile is the 40 th percentile. In some embodiments, the threshold or percentile is the 45 th percentile. In some embodiments, the threshold or percentile is the 55 th percentile. In some embodiments, the threshold or percentile is the 60 th percentile. In some embodiments, the threshold or percentile is the 65 th percentile. In some embodiments, the threshold or percentile is the 70 th percentile. In some embodiments, the threshold or percentile is the 75 th percentile. In some embodiments, the threshold or percentile is the 80 th percentile. In some embodiments, the threshold or percentile is the 85 th percentile. In some embodiments, the threshold or percentile is the 90 th percentile. In some embodiments, the threshold or percentile is the 95 th percentile. In some embodiments, the threshold or percentile is the 5 th percentile. In some embodiments, the threshold or percentile is the 99 th percentile. In some embodiments, the threshold or percentile is the 5 th percentile.

The present invention is not limited to the above threshold or percentile. The present invention is not limited to the above-described method of determining the threshold or percentile.

Without wishing to limit the invention to any theory or mechanism, features and advantages of the methods of the invention include, but are not limited to: (a) the ability to test tissue specific metabolic rates; (b) testing the ability of genetic, nutritional, endocrine, and physiological effects on tissue-specific metabolic rates; (c) ability to test in vivo or ex vivo effects; (d) the ability to test the effect of any water or DMSO soluble compound on metabolic rate; (e) ease of application and measurement (including fluorescence change or color change, which can be measured from a photograph); (f) use of an accumulated signal, which is more sensitive than simple oxygen consumption measurements and allows differentiation of small differences between animals; (g) simple, including only mixtures of several solutions; and (h) the ability to amplify it to measure 1000s of sample simultaneously.

Any feature or combination of features described herein is included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Other advantages and aspects of the invention will be apparent from the following detailed description and claims.

Brief description of the drawings

The features and advantages of the present invention will become apparent upon consideration of the following detailed description, taken in conjunction with the accompanying drawings, in which:

figure 1A shows that skeletal muscle biopsy caused a linear increase in fluorescence signal over time, indicating that skeletal muscle tissue continues to produce reducing equivalents and remains viable through the incubation period. In addition, the signal was shown to be sensitive to fasting (16 hours fasting).

Figure 1B shows that liver biopsy caused a linear increase in fluorescence signal over time, indicating that liver tissue continues to produce reducing equivalents and remains viable through the incubation period. In addition, the signal was shown to be insensitive to fasting (16 hour fasting).

FIG. 1C shows the 4 hour metabolic rate (change in fluorescence/ng DNA) from FIGS. 5A and 5B. The metabolic rate of skeletal muscle biopsy was reduced by fasting (16 hours), but fasting did not affect the metabolic rate of liver biopsy. This confirms that this assay can be used to assess the effect of nutritional status on tissue-specific metabolic rate.

FIG. 1D shows that glucose (1mM) in the media increases the skeletal muscle biopsy metabolic rate, measured as the relative change in fluorescence/mg tissue. This confirms that this assay can be used to assess the effect of nutrients on tissue specific metabolic rates.

FIG. 1E shows that isoproterenol (β -adrenoceptor agonist) increases skeletal muscle metabolic rate at high glucose concentrations (2 mM.) this confirms that this assay can be used to assess the effect of drugs on tissue-specific metabolic rate.

Fig. 1F shows that the metabolic rate (expressed as fluorescence change/mg tissue) of tissue biopsies (e.g., Brown Adipose Tissue (BAT), White Adipose Tissue (WAT), white skeletal muscle, red skeletal muscle, heart, kidney, and liver) varied between tissues and with mouse age (1 month, 3 months). This confirms that the assay can be used to assess the effect of physiological parameters (including but not limited to age, exercise, hormonal status, etc.) on tissue specific metabolic rate.

Fig. 2 shows the metabolic rate (fluorescence change over time) of many tissues (e.g., Brown Adipose Tissue (BAT), White Adipose Tissue (WAT), white skeletal muscle, red skeletal muscle, heart, kidney, and liver) in fed and fasted mice. This confirms that the invention described herein is applicable to tissues.

FIG. 3 shows that breast biopsy metabolic rate can be used to predict milk production. The ex vivo mammary gland metabolic rate measured using the assay described in this patent (upper panel) predicts ex vivo mammary lactose production (lower panel) in response to heat stress (day 13-19 of pregnancy, 35 ℃/50% humidity), maintained at room temperature (RT; 22-24 ℃/50% humidity) and the feed is taken ad libitum, or maintained at room temperature (22-24 ℃/50% humidity) and the feed is limited to the feed consumed by heat stressed animals (gavage; PF).

Figure 4A shows the fluorescence changes induced by skeletal muscle biopsies collected from young and mature Angus cattle (upper panel; percent change in fluorescence relative to baseline; lower panel). Figure 4A shows a biopsy from a mature fully grown cow with a lower metabolic rate than a biopsy collected from a young grown cow. The variability of FC/4h induced by skeletal muscle biopsies from young and mature cows was robust. This variability may be important for the inheritance of increased efficiency.

Figure 4B shows the fluorescence changes induced by skeletal muscle biopsies collected from Angus and Hereford cattle (upper panel; percent change in fluorescence relative to baseline; lower panel). There was no breed difference in skeletal muscle metabolic rate between Hereford and Angus cattle. However, intraspecies-animal variability in skeletal muscle metabolic rate (FC/4h) is extensive in Hereford and Angus cattle. This variability may be important for the inheritance of increased efficiency.

FIG. 5 shows the relative change in skeletal muscle biopsy induced fluorescence, corresponding to cumulative energy expenditure over time, which increases throughout the 2-h incubation period and differs based on feeding switched to high fiber high starch (HF-HS), high fiber low starch (HF-L S), low fiber high starch (L F-HS), and low fiber low starch (L F-L S) diets.

The relative grades of animals based on skeletal muscle metabolic rate maintained in the diet treatment are determined in fig. 6 the treatment includes a High (HS) or low (L S) rumen degradable starch source and a High (HF) or low (L F) rumen degradable fiber source.

FIG. 7 shows that the growth-diverted skeletal muscle metabolic rate (as shown on the X-axis, relative fluorescence change) is directly related to the Average Daily Gain (ADG) (upper panel), independent of the average Dry Matter Intake (DMI) (middle panel), and related to feed and weight gainThe ratio (F: G) is inversely related (lower panel). (each dot represents an individual animal.) the dashed line shows the best-fit linear regression of the performance variables versus the relative fluorescence. R is shown²The values reflect the deterministic coefficients for each regression. This data determines skeletal muscle reduction equivalents (NADH, FADH)₂Etc.) as a proxy for tissue metabolic rate, which can be used to predict growth and feed efficiency in growing animals.

Detailed Description

Determination of metabolic rate

The present invention provides methods for measuring metabolic rate in an animal (or human), wherein the methods are characterized by measuring reducing equivalents (e.g., NADH, FADH)₂NADP (H), coenzyme Q, etc.). The method can include obtaining a tissue biopsy (e.g., skeletal muscle tissue biopsy, breast tissue biopsy, etc.) from the animal. The biopsy may be obtained by suitable means, e.g. using a needle biopsy tool, by rough cutting, etc. The method can include placing at least a portion of the biopsy in an appropriate medium in a plate or tray (e.g., a 12-well plate, a 24-well plate, a 96-well plate, etc.). The biopsy in the disc is then subjected to a reducing equivalent indicator, such as a dye. The biopsy is then read, for example, color (e.g., RGB analysis), absorbance, fluorescence, and/or any other suitable parameter that can be measured to assess signal change. The measured change indicates the reduction equivalent (e.g., NADH, FADH)₂NADP (H), coenzyme Q, etc.). Notably, the concentration of reducing equivalent indicators (e.g., resazurin, MTT, AlamarBlue, PrestoBlue) can be titrated to meet specific needs; titration of concentration can alter the sensitivity of the assay. The present invention is not limited to the above-described reducing equivalent indicators.

As a non-limiting specific example, some of the studies herein use the Resazurin assay to measure NADH H⁺And (4) generating. Biopsies were collected from animals and immediately placed into wells of 96-well plates containing Dulbecco's Modified Eagle Medium (DMEM) with Pen/Strep, and placed with 95% O₂、5％CO₂At 37 ℃. After 1 hour of equilibration, the biopsies were transferred to a filling with 300. mu.l of supplementWells in DMEM with 0.1% DMSO, Pen/Strep and 0.16% 10X resazurin. Immediately after the biopsy of the removed tissue, the color (rgb assay), absorbance (570-600nM) or fluorescence (excitation 530nM, emission 590nM) was measured. The plates are then "read" at intervals (color analysis or plate reader measures absorbance and fluorescence) to assess signal changes. The measured signal change is indicative of NADH H⁺The extent of production. The signal accumulates over time.

Fig. 1A, 1B, 1C, 1D, 1E and 1F show the use of resazurin-based assays on thermostated tissue collected from mice fig. 1A shows that skeletal muscle metabolic rate increases linearly over time to 4 hours and is sensitive to fasting, fig. 1B shows that liver metabolic rate (increases linearly over time and is not sensitive to fasting), fig. 1C shows that metabolic rate in skeletal muscle (expressed as fluorescence change/ng DNA) decreases with fasting, fig. 1D shows that glucose in the culture medium increases skeletal muscle metabolic rate (sensitive to ex vivo nutrient administration), fig. 1E shows that isoproterenol (β -adrenoceptor agonist) increases skeletal muscle metabolic rate at high glucose concentration (sensitive to drug application), fig. 1F shows that the metabolic rates of different tissues differ with the age of the mice (1 month, 3 months).

Fig. 2 shows the metabolic rates of various tissues in fed and fasted mice, such as Brown Adipose Tissue (BAT), White Adipose Tissue (WAT), white skeletal muscle, red skeletal muscle, heart, kidney and liver, showing the effect of physiological changes (fasting) on metabolic rate (measured by 4 hours fluorescence change/mg tissue). White adipose tissue and liver were two tissues affected by fasting when measured as a change in fluorescence at 4 hours per mg of tissue. Skeletal muscle tissue is not affected. Without wishing to limit the invention to any theory or mechanism, DNA may be a preferred correction factor.

The present invention is not limited to the above-described methods and compositions for measuring reducing equivalents.

Feed efficiency and productivity

Without wishing to limit the invention to any theory or mechanism, it is believed that a lower tissue metabolism rate is associated with higher feed efficiency and/or higher productivity (e.g., higher milk, muscle, egg, etc. production).

The present invention provides methods for identifying (and/or selecting) animals with high feed efficiency. The invention also provides methods of identifying animals with increased productivity (e.g., egg production, milk production, etc.), methods of predicting animals with high productivity, and methods of selecting animals with high productivity. The methods herein select for a lower tissue-specific metabolic rate (e.g., a lower skeletal muscle metabolic rate), and/or a lower basal metabolic rate, among others.

Other methods provided herein include, but are not limited to, methods of determining animal reproductive values.

In some embodiments, to determine the feed efficiency of an animal, tissue from the animal (e.g., skeletal muscle) is tested to determine metabolic rate by determining reducing equivalent yield (e.g., amount, change, etc.). By way of example, animals or tissues from animals (e.g., skeletal muscle) are tested with reducing equivalent indicators and evaluated by assessing changes in fluorescence, absorbance (570- "600 nM" when Resazurin is used) or by color. In some embodiments, the obtained metabolic rate is then compared to a known range of change in fluorescence, absorbance, or color, and the percentile is determined. In some embodiments, tissue-specific changes in fluorescence, absorbance, or color are corrected for DNA, protein, or mass of the sample.

In some embodiments, reducing equivalents used in the mathematical model may be used in conjunction with reducing equivalent (metabolic rate) data to determine or predict feed efficiency or productivity, e.g., weight gain, egg production, milk production, etc. In the milk production example, prospective studies of herds can be used to predict high milk yields. Mammary tissue can be obtained and tested for metabolic rate, and milk production can be determined for each subject. Candidate scoring functions may be selected to classify future test animals into either high milk yield or low milk yield categories (or other different or additional categories). For example, those animals with high mammary gland metabolic rate may be selected as animals predicted to have high milk production.

In some embodiments, the methods of the invention are characterized by selecting animals with low basal metabolic rates (e.g., low tissue-specific metabolic rates). In some embodiments, the methods of the invention are characterized by selecting animals with high basal metabolic rates (e.g., high tissue-specific metabolic rates).

Referring to fig. 3, the mammary gland metabolic rate can be used to predict milk production. Figure 3 shows the ex vivo mammary gland metabolic rate (upper panel) predicts ex vivo mammary lactose production (lower panel) in response to heat stress (day 13-19 of pregnancy, 35 ℃/50% humidity), maintained at room temperature (RT; 22-24 ℃/50% humidity) and feed taken ad libitum, or maintained at room temperature (22-24 ℃/50% humidity) and feed limited to consumption by heat stressed animals (gavage; PF).

FIGS. 4A and 4B show the metabolic rates of young calves and fully grown cattle (Angus and Hereford). Figure 4A shows that mature fully grown cattle have a lower metabolic rate than younger grown cattle. FIG. 4B shows that there is no average difference in the metabolic rates of Hereford and Angus cattle. The variability of FC/4h is extensive in young and mature cattle as well as Hereford and Angus cattle. This variability is important for the inheritance of increased efficiency.

Thus, the effect of nutrition, nutrients, drugs, environmental stimuli, and other factors (e.g., age, etc.) on metabolic rate (e.g., tissue-specific metabolic rate) can be studied. The method of the invention can also be used to study the efficiency of specific tissues important for productivity (e.g. mammary tissue important for milk production).

Obesity

The invention also provides methods for identifying susceptibility or resistance to obesity in an animal (or human), e.g., for determining whether a subject (e.g., an animal, a human, etc.) has a likelihood of developing obesity or becoming overweight, or whether an animal or a human is resistant to obesity.

As a non-limiting example, to develop a mathematical scoring function (a clinical mathematical model) to predict the likelihood of developing obesity or becoming overweight, prospective studies of groups with known outcomes may be used to develop relationships between outcomes and metabolic rates (e.g., calculating one or more candidate scoring functions, etc.). For example, a cohort of patients may be evaluated, where each individual provides a skeletal muscle biopsy that tests for metabolic rate by measuring reducing equivalent production (e.g., amount, change, etc.). The resulting skeletal muscle metabolic rate can be plotted against body weight change in response to a given lifestyle intervention. Alternatively, long-term longitudinal studies can be performed to assess changes in body weight over time.

As a non-limiting example, the results may be grouped based on known criteria for classifying an individual as non-overweight, overweight and obese. For example, in the case of humans, a Body Mass Index (BMI) of 25.0 to 29.9 is defined as overweight, and a BMI of 30 or more is defined as obese. Thus, BMI values can be used to determine the outcome of individuals in a group.

Once the candidate scoring function is selected, one or more cutoffs may be selected to classify the patient into the above categories. A non-limiting example of a mechanism for determining truncation of a class may be a Receiver Operating Characteristic (ROC) curve. The ROC curve allows the user to balance the sensitivity of the model (e.g., priority of subjects that will capture as many "positives" or "likely to become overweight/obese") with the specificity of the model (e.g., minimize false positives "likely to become overweight/obese candidates").

The selected scoring function (and optionally the cut-off value determined by the ROC curve) provides a predetermined threshold for assessing the susceptibility of the test subject to obesity.

The invention also features methods of selecting feeds, e.g., selecting feeds with high feed efficiency, selecting feeds with high productivity, etc. Reproductive selection can be characterized by a tissue-specific metabolic rate (e.g., skeletal muscle, etc.) of the test mother or father, which can be indicative of feed efficiency and/or productivity of the offspring.

Action of drugs or other stimulants

The invention also provides methods for determining the effect of genetic, environmental stimuli, medical treatments (e.g., antibiotics), physiological treatments (e.g., exercise), dietary supplements, dietary or nutritional treatments on the metabolic rate (e.g., basal metabolic rate, tissue-specific metabolic rate) of an animal of interest and, thus, on feed efficiency, productivity, and the like.

For example, a nutritional treatment may be administered to a group of animals (e.g., cattle), and skeletal muscle metabolic rate may be determined to assess what impact the nutritional treatment has on metabolic rate and potential feed efficiency, productivity, and the like.

Classification of cattle from skeletal muscle NADH reduction Rate

The invention features a high throughput method for assessing selected energy expenditure to improve efficiency. For example, the following method describes a muscle biopsy technique that classifies cattle by skeletal muscle nicotinamide adenine dinucleotide reduction rate for assessing the metabolic rate of skeletal muscle biopsies in cattle. This technique can be used to genetically select for growth across species or feed efficiency. The present invention is not limited to the methods, assays, and compositions described herein.

Tissue biopsy metabolic activity assessed using the redox indicator resazurin can be used as a surrogate to assess energy expenditure associated with maintenance of non-growing animals or growth rate of growing animals. These methods can assess the reproducibility, utility and sensitivity of resazurin-based assays to rank bovine skeletal muscle biopsies based on metabolic activity. A 6 year old hestan cow (BW 330 ± 11.3kg) was fed 4 dietary treatments consisting of high or low rumen degradable starch and fiber arranged with partially replicated latin square design factors. The period was 18 days, 3 days of dietary switch, 14 days of dietary acclimation and 1 day of sample collection. Semitendinosus biopsies were collected from each cow in ice-cold Dulbecco's Modified Eagle Medium (DMEM) at each time period. The analysis started within 1 hour of sample collection. To assess tissue metabolic rate, biopsies were transferred to DMEM with resazurin and incubated at 37 ℃. The fluorescence of each sample was read at time 0 and at 15 minute intervals for 2 hours. Changes in fluorescence represent skeletal muscle reduction equivalent production (e.g., NADH). The signal intensity of each animal sample increased with increasing fluorescence time (P <0.001), but there was no significant interaction between time and treatment (P >0.05), indicating that fluorescence comparisons at individual time points were sufficient. The change in fluorescence at 120 minutes was used to analyze the fixation effect of the fiber, starch and animals when considering the random effect of the cycle. The samples collected when the animals were on a high rumen degradable starch diet had greater metabolic activity than the samples collected from animals on a low starch diet (P ═ 0.023). Significant differences in metabolic activity in individual animals were also identified (P ═ 0.003). The average relative fluorescence was paired with Dry Matter Intake (DMI), Average Daily Gain (ADG) and feed to gain ratio (F: G) for each individual. The Pearson correlation coefficients associated with ADG and F: G on fluorescence change were strong (ADG 0.749; F: G-0.783) and tended to be significant (ADG P0.0864; F: G P-0.066). The Pearson correlation coefficient for DMI versus fluorescence change was weak (0.153) and no significant difference (P ═ 0.773). Thus, the method allows for the ranking of individual animals based on metabolic activity and detecting differences in metabolic activity associated with dietary changes.

Treatment of diet and acclimation period

In a partially repeated latin formula design, 6 rumen catheterized Holstein cows (BW 330 + -11.3 kg) were randomly divided into 4 dietary treatments, for each period, two treatments were repeated, while the other two treatments were not repeated, for a length of 18 days, the first 3 days were used to acclimate the animals between diets, the animals consumed the treatment diets for 14 days, and samples were collected on the last day, on a corn silage basis (29.4-35.6% DM), and a combination of barley flour (high rumen degradable starch (HS)14.1-14.8,% DM, low rumen degradable starch (L S)0.310-0.670,% DM) or corn (HS 0.00-0.380,% DM; L S10.5-12.3,% DM) and granulated beet pulp (high rumen degradable fiber (HF)3.55-6.05,% DM; low rumen fiber (L F30.5-12.3;% DM) 3) 21, 85) 3, 10.5-12.3,% DM) and granulated beet pulp (high rumen degradable fiber (HF) 3.55-6.05; HF) 3.7.7.7.7.7.7.7-7, 9, 9.7.7.7, 9, 7, 9, 7, 9.

Table 1. ingredients and nutritional compositions for each of the treated diets expressed on a dry matter basis.

¹HS-L F is high rumen degradable starch and low fiber treatment

²HS-HF as high rumen degradable starch and high fiber treatment

³L S-L F is low rumen degradable starch, and low fiber treatment

⁴L S-HF as low rumen degradable starch, and high fiber treatment

Sample preparation and Collection

A 10cm wide area 5cm to 35cm ventral to ischium is shaved and washed three times with betadine and isopropanol. Subcutaneous administration of 10ml of lidocaine was performed at 5 to 6 positions radially aligned 2cm outside the biopsy site. The target biopsy site was 20cm from the ischial site. A20 gauge biopsy needle (20 gauge) was used by making a 1cm incision through the skin with a #20 scalpel blade

Disposable core biopsy instrument) was inserted to a depth of 4cm and the needle collection sheath was pressed to obtain a sample to collect the muscle tissue sample. To obtain approximately 30mg samples, three biopsies per animal per cycle were collected to assess how variations in sample collection (sample mass, intramuscular collection sites and other unknown factors) affect the consistency of results. The samples were not weighed after collection because there was no analytical balance on the farm. The incision site was sealed with monofilament #2 suture, cleaned with isopropyl alcohol, and sprayed with an adhesive bandage. The right semitendinosus muscle was sampled in phases 1 and 3 and the left semitendinosus muscle was sampled in phases 2 and 4.

Core samples with complete structure immediately after collectionPlaced in each well of a 96-well plate filled with a pre-test solution. The solution before the test contained 30ml of DMEM (Fisher Science 21-041-. After all samples were collected, they were transferred from the pre-test solution to individual wells of a 96-well plate filled with the resazurin test assay solution. Test solution and addition of 1.6%

(Resazurin-based reagent, Thermo Scientific Y00-100) was the same. The solution was mixed immediately prior to biopsy collection, filtered using a sterile 0.22 μ M filter, and warmed to 37 ℃ prior to use.

Sample analysis

When samples are transferred to the test solution, the test solution plates are incubated in a plate reader (Spectramax M5; Molecular Devices, LL C, San Jose, CA) at 37 ℃, fluorescence is read for 2 hours at time 0 and every 15 minutes using excitation and emission wavelengths of 530 and 590nm, respectively, the resulting emissions are quantified using Soft Max Pro 6.1(Molecular Devices, LL C, San Jose, CA), and the relative fluorescence of each sample is calculated at each time point (normalized to time 0).

Sample consistency over animal period

Triplicate samples were collected over the animal period to assess the consistency of animal grades obtained from similar samples. The relative fluorescent animal internal CV is about 20%, indicating that the sample needs to be normalized by protein or DNA content or mass. Although there was some degree of difference between samples collected from the same animal consuming the same diet, when different individual samples were used, the Wilcoxon rank-sum test found no significant difference on an animal scale (P >0.05), indicating that this difference did not hinder the opportunity to rank animals by skeletal muscle metabolic activity.

Statistical analysis

The average relative fluorescence for each animal phase was calculated at each time point. Statistical analysis of mean fluorescence data was performed in R edition 3.1.0 (R core team, 2014). The analysis is structured into 2 questions: 1) whether the incubation time changes the perceived effect of treatment on the relative rate of NAD + reduction; and 2) discernable differences between animals and treatments after 2 hours. To address the first issue, a linear mixed effect model was used to test how time, fiber and starch digestibility, animals and cycles affect the normalized fluorescence readings. Fiber, starch and time are the fixing effects, and 2-and 3-membered interactions between these fixing effects were also evaluated. The treatment period and animals were randomized. Significant time for starch or fiber interaction would indicate that the process differences were inconsistent throughout the sampling period and some ideal fluorescence read time needs to be determined to make a reliable inference from the data. To solve the second problem, the data from the last time point (2h) were analyzed using a linear mixed effect model with fixed fiber, starch and animal effects and random periodic effects. Significant starch, fiber or animal effects would indicate that the assay (using only 2 hours fluorescence time) is sensitive enough to grade samples based on animal level factors (genetic potential or diet).

Time of day

Since the plate can be read at multiple time points, the effect of time on fluorescence within the sample was tested (see fig. 5). The reduction reaction that causes the fluorescence of resazurin is irreversible and as a result, the signal accumulates over time, allowing small short-term differences to be amplified over incubation time accumulation. The total signal increased linearly with time (P <0.0001), indicating that tissue biopsy continued to be metabolized at a constant rate throughout the incubation period. Time had no different effect on the fluorescence in the diet based on starch (P-0.5106) or fiber (P-0.8072), which supports the conclusion that the rating of the samples was similar if evaluated at any time point within 2 hours.

Diet

Skeletal muscle biopsies taken when the cows were on a high-rumen degradable starch diet had higher relative fluorescence (P ═ 0.023) than biopsies taken when the cows were fed a low-rumen degradable starch diet. The link between starch source and skeletal muscle metabolic activity may be caused by changes in energy utilization. However, using rumen degradable fiber alters the calculated ME in the diet more than rumen degradable starch, making this interpretation less likely. Alternatively, an increase in tissue metabolic activity with an increase in rumen degradable starch may be associated with a different distribution of absorbed volatile fatty acids. In post-absorption systems, different VFAs are used for energy with different efficiencies. The approximate VFA contribution to the muscle depends on the individual VFA metabolism. Less than 30% acetate, 40-55% propionate, and a minimum amount of butyrate may be used peripherally. The contribution of glucose to skeletal muscle in ruminants is due to gluconeogenesis. Hepatic uptake of propionate, valerate and isobutyrate allows for increased gluconeogenic substrates. If different starch sources contribute to different distributions of absorbed VFA, the absorbed VFA distribution may contribute to the influence of rumen degradable starch on skeletal muscle metabolic activity. Regardless of the mechanism driving this eating effect, the results suggest that this assay can be used to understand the metabolic effects of quantitative changes.

Animal(s) production

Comparison between animals showed that skeletal muscle metabolic activity was different between individuals (P ═ 0.003). The effect of the diet was consistent among the animals and the animals ranked similarly in the diet (see figure 6). This consistency indicates that the inherent differences in skeletal muscle metabolic activity may be distinguishable under various conditions, making it possible to inexpensively and durably screen animals for growth-related energy expenditure or retention in non-growing animals.

The metabolic activity of ruminants is a function of body weight and metabolic flux in an inactive, hot central environment. These differences in metabolic flows contribute to variability in feed efficiency. The individual metabolic flux can be influenced by genetic potential, activity and behavior, environment and cow feeding practices. Referring to fig. 7, the average skeletal muscle metabolic activity (expressed as relative fluorescence) for each individual was paired with Dry Matter Intake (DMI), Average Daily Gain (ADG), and calculated feed to grain ratio (F: G). The Pearson correlation coefficients associated with fluorescence changes for ADG and F: G are strong (ADG 0.749; F: G-0.783) and tend to be significant (ADG P0.0864; F: G P-0.066). The Pearson correlation coefficient for the relationship between DMI and fluorescence change was weak (0.153) and no significant difference (P ═ 0.773).

Skeletal muscle biopsy metabolic activity is an indicator of the energy expenditure for growth and maintenance of these young, fast-growing cows. In these studies, a positive correlation with growth and feed to gain rate indicates that the measure of metabolic activity is more affected by growth energy expenditure than maintenance energy expenditure. Samples taken from mature animals can more effectively address the genetic potential of feed efficiency. Given the fact that this assay is sufficiently sensitive to detect differences between animals, it can be a useful screening tool to allow genetic selection based on feed efficiency of mature cattle while maintaining.

Example 1

The following example describes a method of identifying animals with high feed efficiency. The present invention is not limited to the methods and compositions described herein.

Farmers own a herd of cattle and are interested in selecting the animal with the highest feed efficiency for breeding purposes. For each adult animal, she obtained skeletal muscle tissue samples and measured metabolic rate by measuring reducing equivalents.

The metabolic rate of skeletal muscle tissue is inversely proportional to feed efficiency, so farmers choose to feed animals with the lowest metabolic rate of 25%, which will have the highest feed efficiency. For example, for a group of 100 animals, the farmer selects 25 animals with the lowest skeletal muscle metabolic rate. Those animals in the 25 th percentile were selected for breeding purposes. The remaining 75% of the animals were not used for breeding.

Example 2

The following example describes a method of identifying animals with high milk production. The present invention is not limited to the methods and compositions described herein.

Farmers own a herd of cattle and are interested in selecting the animal with the highest milk yield for breeding purposes. For each animal, he obtained a mammary tissue sample and measured the metabolic rate by measuring the reduction equivalents.

The metabolic rate of mammary tissue is directly related to the potential milk yield. Farmers have previously conducted studies to determine the amount of milk produced per day (gallons per day) based on the specific metabolic rate of mammary tissue. For example, a metabolic rate of 1000 (change in fluorescence per mg of tissue) or more, then the animal is predicted to produce at least 6 gallons of milk per day. A metabolic rate of 2000 (change in fluorescence per mg of tissue) or more, then it is predicted that the animal will produce at least 9 gallons of milk per day. 3000 metabolic rate (change in fluorescence per mg of tissue), then the animal is predicted to produce at least 12 gallons per day.

Half of the cattle have a metabolic rate of 1000 (change in fluorescence/mg tissue) or less. Only 10% of the cattle have a metabolic rate of 2000 (change in fluorescence/mg of tissue) or higher and farmers select only those animals that are expected to produce at least 9 gallons of milk per day.

Example 3

The following example describes a method for detecting the effect of a drug on the efficiency of animal feed. The present invention is not limited to the methods and compositions described herein.

Farmers have 40 cattle and are interested in determining how drug a affects the feed efficiency of cattle. For each animal, she obtained skeletal muscle tissue samples and measured metabolic rate by measuring reduction equivalents. She calculated the average metabolic rate for this group of animals.

She then administered drug a to 20 animals once a day for two weeks and placebo to the remaining 20 animals. At the end of two weeks, she obtained skeletal muscle tissue samples from each animal and measured metabolic rate by measuring reduction equivalents. She calculated the average metabolic rate of the group of animals treated with drug a and the average metabolic rate of the group of animals given placebo.

Farmer calculated that treatment with drug a reduced the average metabolic rate of the animals by 20%. This is also associated with a 20% change in the average feed efficiency of the animals.

Example 4

The following example describes a method for detecting the effect of a drug on milk production by an animal. The present invention is not limited to the methods and compositions described herein.

Farmers own 100 cows and are interested in determining how drug B will affect the milk yield of the animals. For each animal, he obtained a mammary tissue sample and measured the metabolic rate by measuring the reduction equivalents. He calculated the average metabolic rate of the group of animals.

He then administered drug B twice daily to 50 animals for 4 weeks and placebo to the remaining 50 animals. At the end of four weeks, he obtained a mammary tissue sample from each animal and measured the metabolic rate by measuring the reducing equivalents. She calculated the average metabolic rate of the group of animals treated with drug B and the average metabolic rate of the group of animals given placebo.

Farmers calculated that treatment with drug B increased the average metabolic rate of the animals by 10%. This correlates to a 10% increase in milk production.

The disclosures of the following U.S. patents are incorporated herein by reference in their entirety: PCT/US 16/48006.

Various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also within the scope of the appended claims. Each reference cited in this application is incorporated herein by reference in its entirety.

While the preferred embodiments of the invention have been illustrated and described, it will be readily apparent to those skilled in the art that modifications may be made thereto without departing from the scope of the appended claims. Accordingly, the scope of the invention is to be limited only by the following claims. The reference numbers recited in the claims are exemplary and are merely for convenience of examination by the patent office and are not to be limiting in any way. In some embodiments, the drawings presented in this patent application are drawn to scale, including angles, dimensional ratios, and the like. In some embodiments, the drawings are merely representative, and the claims are not limited by the dimensions of the drawings. In some embodiments, a description of the invention described using the phrase "comprising" includes embodiments that may be described as "consisting of, and thus meets the written description requirements for using the phrase" consisting of to claim one or more embodiments of the invention.

Reference signs in the claims are provided merely as a clarifying example and are intended to be exemplary, and do not limit the scope of the claims in any way to the specific features in the drawings that have the corresponding reference signs.

Claims

1. A method of identifying an animal with high feed efficiency, the method comprising determining reducing equivalent yield in a skeletal muscle tissue sample from the animal, wherein reducing equivalent yield in the skeletal muscle tissue sample is inversely proportional to feed efficiency,

wherein if the reducing equivalent production in the skeletal muscle tissue sample is below a predetermined threshold, the animal from which the skeletal muscle tissue sample is obtained has a high feed efficiency compared to an animal having a reducing equivalent production above the predetermined threshold.

2. The method of claim 1, wherein the predetermined threshold is an average of reducing equivalent yields for a breed, herd, or species of the animal.

3. The method of any one of claims 1-2, wherein the predetermined threshold classifies the animal by feed efficiency.

4. The method of any one of claims 1-3, wherein the predetermined threshold is determined using a group of animals with known reducing equivalent production and known feed efficiency.

5. The method of any one of claims 1-4, wherein the predetermined threshold is a percentile level.

6. The method of any one of claims 1-5, wherein the predetermined threshold or percentile level is selected by a user based on a desired feed efficiency selection stringency.

7. The method of any one of claims 1-3, wherein determining reducing equivalent production in the skeletal muscle tissue sample comprises introducing a reducing equivalent indicator into the tissue sample or a culture medium in which the tissue sample is incubated, and measuring the amount or change of the reducing equivalent indicator that is indicative of metabolic activity.

8. The method of any one of claims 1-7, wherein the reducing equivalent is NADH.

9. The method of any one of claims 1-7, wherein the reducing equivalent is FADH₂。

10. The method of any one of claims 1-7, wherein the reducing equivalent is coenzyme Q.

11. The process of any one of claims 1-7, wherein the reducing equivalent is NADP (H).

12. The method of any one of claims 1-7, wherein the reducing equivalents are NADH, FADH₂NADP (H), and coenzyme Q.

13. The method of any one of claims 1-12, further comprising using animals identified as having high feed efficiency for breeding.

14. The method of any one of claims 1-12, further comprising using the animal identified as having high feed efficiency for producing an animal product.

15. The method of claim 14, wherein the animal product is milk or meat.

16. A method of identifying an animal with high milk production, the method comprising determining reducing equivalent production in a breast tissue sample from the animal, wherein reducing equivalent production in the breast tissue sample is directly correlated with milk production potential,

wherein if the reducing equivalent production in the breast tissue sample is above a predetermined threshold, the animal from which the breast tissue sample is obtained has a high milk production potential compared to an animal having a reducing equivalent production below the predetermined threshold.

17. The method of claim 16, wherein the predetermined threshold is an average of reducing equivalent yields for a breed, herd, or species of the animal.

18. The method of any one of claims 16-17, wherein the predetermined threshold classifies animals by milk production potential.

19. The method of any one of claims 16-18, wherein the predetermined threshold is determined using a group of animals with known reducing equivalent production and known milk production.

20. A method as claimed in any one of claims 16 to 19, wherein the predetermined threshold is a percentile level.

21. The method according to any of claims 16-20, wherein the predetermined threshold or percentile level is selected by a user based on a desired milk yield potential selection stringency.

22. The method of any one of claims 16-21, wherein determining reducing equivalent production in the breast tissue sample comprises introducing a reducing equivalent indicator into the tissue sample and measuring the amount or change of the reducing equivalent indicator indicative of metabolic activity.

23. The method of any one of claims 16-22, wherein the reducing equivalent is NADH.

24. The method of any one of claims 16-22, wherein the reducing equivalent is FADH₂。

25. The method of any one of claims 16-22, wherein the reducing equivalent is coenzyme Q.

26. The method of any one of claims 16-22, wherein the reducing equivalent is nadp (h).

27. The method of any one of claims 16-22, wherein the reducing equivalents are NADH, FADH₂And coenzyme Q.

28. The method of any one of claims 16-27, further comprising using animals identified as having high milk production potential for breeding.

29. The method of any one of claims 16-27, further comprising using the animal identified as having high milk production potential for producing milk.

30. A method of calculating a reproductive value for a feed efficiency estimate for an animal, the method comprising:

a. determining feed efficiency based on a metabolic rate of a tissue sample from the animal, wherein metabolic rate is determined by determining reducing equivalent production; and

b. assigning estimated expected progeny differences from the breed mean based on the metabolic rate of the tissue sample; wherein the estimated reproductive value indicates inheritance of feed efficiency of the potential parental culture.

31. The method of claim 30, further comprising combining the estimated feed efficiency with one or more additional estimated reproductive values.

32. The method of claim 31, wherein the additional estimated reproduction value is selected from the group consisting of: rib area, intramuscular fat, fat depth, birth weight, weaning weight, and carcass yield.

33. A method of detecting the effect of a drug, dietary supplement, diet, or other composition on the feed efficiency of an animal, the method comprising:

a. determining a baseline tissue-specific metabolic rate for the animal by measuring reducing equivalent production in a first tissue sample from the animal;

b. administering the drug, dietary supplement, diet or other composition to the animal;

c. determining a second tissue-specific metabolic rate of the animal by measuring reducing equivalents in a second tissue sample from the animal;

wherein the drug, dietary supplement, diet, or other composition does not affect the animal's feed efficiency if the second tissue-specific metabolic rate is equal to the baseline tissue-specific metabolic rate;

wherein the drug, dietary supplement, diet or other composition has a positive effect on the animal's feed efficiency if the second tissue-specific metabolic rate is less than the baseline tissue-specific metabolic rate;

wherein the drug, dietary supplement, diet, or other composition has a negative impact on the feed efficiency of the animal if the second tissue-specific metabolic rate is greater than the baseline tissue-specific metabolic rate.

34. The method of claim 33, wherein the tissue sample is skeletal muscle.

35. A method of detecting the effect of a drug, dietary supplement, diet, or other composition on the feed efficiency of an animal, the method comprising:

a. administering the drug, dietary supplement, diet or composition to the animal;

b. determining the metabolic rate of the animal by determining the reducing equivalent production in the animal tissue;

wherein the drug, dietary supplement, diet, or other composition does not affect the feed efficiency of the animal if the animal's metabolic rate is equal to a control metabolic rate that is the metabolic rate of the animal or group of animals that is not administered the drug, dietary supplement, diet, or other composition;

wherein the drug, dietary supplement, diet or other composition has a positive effect on the feed efficiency of the animal if the animal's metabolic rate is less than a control metabolic rate, which is the metabolic rate of the animal or group of animals that is not administered the drug, dietary supplement, diet or other composition;

wherein the drug, dietary supplement, diet, or other composition has a negative impact on the feed efficiency of the animal if the animal's metabolic rate is greater than a control metabolic rate that is the metabolic rate of the animal or group of animals that is not administered the drug, dietary supplement, diet, or other composition.

36. The method of claim 35, wherein the tissue sample is skeletal muscle.