METHODS FOR MEASURING AND MONTORING A MEAT CHARACTERISTIC OF A LIVESTOCK ANIMAL FROM A SAMPLE OBTAINED FROM A NON-MEAT LOCATION OF THE LIVESTOCK
20220026451 · 2022-01-27
Inventors
- Sally Stevens LLOYD (Falcon, AU)
- Jose L. VALENZUELA (East Perth, AU)
- Roger Letts DAWKINS (Canning Vale South, AU)
Cpc classification
G02B21/367
PHYSICS
G01N33/92
PHYSICS
International classification
G01N33/92
PHYSICS
G02B21/36
PHYSICS
Abstract
The invention is directed to a method of livestock breeding, meat production and marketing, histology, and genetics using a biopsy sample from a non-meat location of the livestock to provide information about the meat quality of the livestock.
Claims
1. A method of determining meat quality of an animal during life, comprising: a. obtaining a tissue sample by biopsy from a non-meat location of the animal; and b. analyzing the biopsy tissue sample to determine at least one fat characteristic of the tissue sample; wherein the determined fat characteristic of the biopsy tissue sample is correlated to a fat characteristic of meat of the animal.
2. The method of claim 1, wherein the at least one fat characteristic of the tissue sample is selected from the group consisting of area of adipose tissue (AAT), adipose cell number/area, adipose cell size, adipose cell distribution, intramuscular fat melting temperature, and combinations thereof.
3. The method of claim 1, wherein the biopsy tissue sample is selected from the group consisting of a sample of the Sacrocaudalis dorsis medialis muscle and a tailhead subcutaneous fat sample.
4. The method of claim 1, wherein the step of analyzing the biopsy tissue sample to determine at least one fat characteristic of the tissue sample, comprises: staining the tissue sample to visualize adipose cells in the tissue sample and viewing through an optical microscope.
5. The method of claim 1, wherein the determination of the at least one fat characteristic is conducted using a computer-assisted method.
6. The method of claim 5, wherein the computer assisted method comprises analyzing a digital photograph taken through a microscope of the stained biopsy to determine the adipose tissue area and the muscle tissue area which can be distinguished by color.
7. The method of claim 6, wherein the computer assisted method comprises utilizing an artificial intelligence algorithm to calculate the adipose tissue area from the stained biopsy.
8. The method of claim 1, wherein said marbling microscore, between 0 and 10, and wherein said marbling microscore is determined based on said adipose tissue area and said muscle tissue area.
9. The method of claim 1, further comprising: determining Meat Standards Australia Marbling (MSA MB) score from a chart similar to
10. The method of claim 1, wherein said chart, comprises: an x-axis with a range of marbling microscore is from 0 to 10; a y-axis with a range of MSAMB scores from 200 to 1200; and a regression equation relating said range of marbling microscore to said range of MSAMB.
11. The method of claim 1, wherein the treatment comprises one of: slaughtering the animal; or continuing feeding regimen of the animal.
12. The method of claim 1, wherein the tissue sample is a subcutaneous fat sample of the animal; and wherein the fat characteristic is the melting temperature of the subcutaneous fat sample of the animal; and wherein the melting temperature of intra-muscular fat of the animal at a loin is correlated to the melting temperature of the subcutaneous fat sample of the animal.
13. A method of determining a treatment of an animal, comprising: determining a Meat Standard Australia marbling score (MSA MB) from a marbling microscore; and treating the animal by a method comprising one of: slaughtering the animal; or adjusting an exposure condition of the animal.
14. The method of claim 13, further comprising: receiving a customer input expressed in terms of MSA MB; comparing said customer input to said MSA MB to determine if said MSA MB is below, at, or above said customer input; and wherein the treatment of the animal based on the MSA MB and the customer input comprises one of: slaughtering if the comparison is at or above said customer input; or adjusting the exposure condition if the MSA MB is below said customer input.
15. The method of claim 13, wherein if the treating of the animal comprises the adjusting the exposure condition of the animal; repeating the determining of the MSA MB after a time period; and determining an additional treatment of the animal, which comprises: slaughtering the animal; or adjusting the exposure condition of the animal if the MSA MB is below said customer input.
16. A method of producing an animal breeding plan for a population of at least one animal, comprising: determining a MSA MB score for at least one animal in a population; ranking said MSA MB scores in descending value to create a ranking of said population; and determining the animal breeding treatment based on the ranking of said population.
17-23. (canceled)
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION
[0042] The present invention provides a more accurate, objective and reproducible measure of carcass marbling, forming the basis of a more efficient market in meat at the producer, wholesale and retail level. More efficient markets will provide economic benefits for producers and consumers alike. The present invention provides, among other things, a method to treat a livestock animal based on a biopsy sample obtained at a non-consumer portion of an animal during the life of the animal. The capacity to measure and therefore monitor an individual animal's progress in the deposition of IMF and the level of IMF Tm will permit producers to make better decisions as to continued feeding of animals, environmental conditions, and time of slaughter. Decisions may be tailored to the meat quality requirements of a particular market and live animals may be sold on the basis of their current IMF and IMF Tm status as measured by the invention, to the benefit of both producer and purchaser. Notably, commercial animal producers, animal seedstock producers, commercial feed manufacturers and semen and embryo suppliers will be able to make better and earlier judgements as to the efficacy of their products and practices using the methods of the present invention. Further, genetic improvement of livestock through breeding systems will be enhanced by a more accurate and earlier system of measuring desired marbling and IMF Tm outcomes.
[0043] An aspect of the invention is a method of determining meat quality of an animal during the life of the animal. The method includes obtaining a tissue sample by biopsy from a non-consumer meat location of the animal. The biopsy can be analyzed to determine at least one fat characteristic of the tissue sample. At least one characteristic of the meat of a meat location of the animal can be determined based on the fat characteristic of the tissue sample.
[0044] The fat characteristic of the tissue sample can be selected from the group consisting of an AAT, adipose cell number/area, adipose cell size, adipose cell distribution, the fat melting temperature, which can be the intramuscular fat melting temperature, and combinations thereof. The melting temperature of the subcutaneous fat sample of the animal can provide information about the intra-muscular fat melting temperature of the animal at a loin (i.e. a meat location of the animal) or other meat locations. Other characteristics of the meat at a meat consuming location of the animal can include marbling. More than one characteristic can be determined from a sample. Other characteristics can include chemical markers of “taint” flavors. Meat consuming locations include locations of cuts of meats for consumer consumption and include a forequarter cut or a hindquarter cut. Suitable forequarter cuts include for example, brisket, blade, and cube roll. Suitable hindquarter cuts include for example rump, round, tenderloin, and strip loin. Non-consumer meat locations are locations of the animal not intended for consumer consumption or not preferred for consumer consumption, and include muscle samples taken from the Longissimus dorsi at 10.sup.th-11.sup.th rib level, the muscle Sacrocaudalis dorsis medialis (located in the tailhead).
[0045] In some embodiments, the biopsy samples can be stored in a preservative. Suitable preservatives include formalin. In some embodiments, the concentration of the preservative can be between 5 vol. % and about 20 vol. %, in some embodiments about 10 vol. %. Methods for treatment and analysis of tissue samples are described in Survana, K., Layton, C. & Bancroft, J. Bancroft's theory and Practice of Histological techniques, (Churchill Livingstone, 2012), (“Survana 2012”) incorporated by reference in its entirety). In some embodiments, the samples can be stored between about −25° C. and about 30° C., in some embodiments about 5° C. before the samples are analyzed. Before the samples are analyzed, they can be warmed to between about 0° C. and about 50° C., in some embodiments about 25° C.
[0046] The livestock animal can be a cow, pig, sheep, chicken, bison, lamb, goat, or other consumable animal. The biopsy of a tissue sample can be taken at the tailhead of an animal. Other suitable biopsy locations include in the Ischiatic tuber region and at the 10.sup.th-11.sup.th level of the animal. Other suitable locations to obtain the biopsy sample can include non-consuming portions of an animal. The tissue sample can be of the Sacrocaudalis dorsis medialis muscle, a tailhead subcutaneous fat sample or combinations thereof. The animal can be subjected to a nerve blocking agent to minimize pain to the animal during the biopsy procedure. Typically, the area to be biopsied is prepared by removing or minimizing hair in the area and disinfecting the area. Tools used to obtain the biopsy include biopsy needle, which preferably has a diameter of at least 11 swg, in some embodiments between about 11 swg and about 6 swg, and a length of between about 60 mm and about 120 mm.
[0047] In some embodiments, more than one sample can be collected for a single animal. In some embodiments, between 1 and 20 samples of the animal can be taken at a single time.
[0048] The biopsy tissue sample can be analyzed to determine the fat characteristic(s) of the tissue sample, and the animal. The analysis can be performed by staining the tissue sample to visualize adipose cells in the tissue sample, which can be viewed through an optical microscope. Descriptions of techniques to stain tissue samples can be found in any histology lab manual, and would therefore be understood by one skilled in the art. However, other suitable resources include Survana 2012. Staining can be performed using any suitable stain as would be understood by one having skill in the art. Examples of suitable staining materials include Haematoxylin & Eosin (H&E) staining, Sudan black, gomori trichrome, martis scarlet, blue trichrome, desmin, or Oil Red staining.
[0049] The fat characteristic(s) of the tissue sample from the non-consuming meat location can be determined using visual methods (traditional) or can be determined using a computer-assisted method. The method can include analyzing a digital photograph taken through an optical microscope of the stained biopsy to determine the AAT and the muscle tissue area. These areas can be distinguished by color. These areas can be determined by a user or by using a computer. In some embodiments where a computer assisted method is utilized, the computer system, which includes a processor, and programs therein can be used to calculate the AAT and/or the muscle tissue area from the biopsy. In some embodiments, the computer assisted method can use an artificial intelligence algorithm to calculate the AAT and/or the muscle tissue area from the stained biopsy.
[0050] A marbling microscore of between about 0 and 10 can be assigned to a sample based on the ratio of the adipose tissue area and the muscle tissue area. When no fat is present, the score assigned is 0. A maximum score of 10 is provided at 30% or higher fat levels. A microscore of 1 corresponds to <2%, 2 corresponds to 3%, 3 corresponds to 4%, 4 corresponds to 8%, 5 corresponds to 12%, 6 corresponds to 16%, 7 corresponds to 20%, 8 corresponds to 24%, 9 corresponds to 28%, or 10 corresponds to >30%. If necessary, the marbling microscore calculated using the present invention can be correlated to a standards score, for example the Meat Standards Australian Marbling (MSA MB) score, the Australian Marbling (AUS MB) score, or standards from other jurisdictions.
[0051] One skilled in the art could utilize this chart to determine the correlation between the microscore of the present invention and other score standards for other jurisdictions without deviating from the present invention.
[0052] Once the standards score is determined from a biopsy, the treatment of the animal can be determined prior to slaughter. Suitable treatments include, slaughtering the animal, extending the feeding period of the animal, adjusting a diet of the animal, maintaining the diet of the animal, and/or modifying the amount of exercise of the animal. For example, if the microscore is not within an acceptable range for a characteristic of the animal, then the diet of the animal can be adjusted. If the animal is within the acceptable range for a characteristic of the animal, then the animal can be slaughtered. Furthermore, the microscore of the animal can be retested periodically, for example every day, every three days, every week, biweekly, bimonthly, every three weeks, every month, every two months, every quarter, three times per year, every five months, bi-yearly, every seven months, every eight months, every nine months, every ten months, every eleven months or yearly, or periodically during a range within these values. In other words, the animal can be tested at any point where a user desires additional data about the animal. When more than one sample has been obtained, the animal's progress can be tracked to determine if interventions based on previous results are altering the microscore of the animal and to determine if the intervention should be continued or if the treatment should be changed. For example, if a microscore indicated that the animal's characteristic of marbling was low, the animal's diet could be changed. If on a subsequent test for that animal, the microscore had not changed, or if the microscore became worse than a previous data point, the treatment for the animal can be changed—whether that means the animal is slaughtered or the diet is adjusted.
[0053] In some embodiments, the treatment can be determined based on a customer's input. For example, if the customer desires meat from a meat-consuming location that has significant marbling (e.g. a MSA MB score of between about 400 and about 1100), then an animal(s) can be tested before slaughtering to determine if the customer's input is met. If a customer's condition is not met, then the treatment of the animal can be modified or other animals can be tested to determine if the customer's condition has been met by the other animal.
[0054] An aspect of the invention is a method of determining a treatment of an animal. The method includes determining a standards score from a marbling score of a sample obtained from a non-meat location of an animal during the life of the animal. The treatment of the animal is determined based on the standards score. The treatment can be slaughtering the animal, adjusting an exposure condition of the animal, maintaining an exposure condition of the animal, or adjusting an exercise regimen for the animal.
[0055] The fat characteristic of the tissue sample can be selected from the group consisting of an AAT, adipose cell number/area, adipose cell size, adipose cell distribution, the fat melting temperature, which can be the intramuscular fat melting temperature, and combinations thereof. The melting temperature of the subcutaneous fat sample of the animal can provide information about the intra-muscular fat melting temperature of the animal at a loin (i.e. a meat location of the animal) or other meat locations. Other characteristics of the meat at a meat consuming location of the animal can include marbling. More than one characteristic can be determined from a sample. Other characteristics can include chemical markers of “taint” flavors. Meat consuming locations include locations of cuts of meats for consumer consumption and include a forequarter cut or a hindquarter cut. Suitable forequarter cuts include for example, brisket, blade, and cube roll. Suitable hindquarter cuts include for example rump, round, tenderloin, and strip loin. Non-consumer meat locations are locations of the animal not intended for consumer consumption or not preferred for consumer consumption, and include muscle samples taken from the Longissimus dorsi at 10.sup.th-11.sup.th rib level, the muscle Sacrocaudalis dorsis medialis (located in the tailhead).
[0056] In some embodiments, the biopsy samples can be stored in a preservative. Suitable preservatives include formalin. In some embodiments, the concentration of the preservative can be between 5 vol. % and about 20 vol. %, in some embodiments about 10 vol. %. Methods for treatment and analysis of tissue samples are described in Survana 2012. In some embodiments, the samples can be stored between about −25° C. and about 30° C., in some embodiments about 5° C. before the samples are analyzed. Before the samples are analyzed, they can be warmed to between about 0° C. and about 50° C., in some embodiments about 25° C.
[0057] The livestock animal can be a cow, pig, sheep, chicken, bison, lamb, goat, or other consumable animal. The biopsy of a tissue sample can be taken at the tailhead of an animal. Other suitable biopsy locations include in the Ischiatic tuber region and at the 10.sup.th-11.sup.th level of the animal. Other suitable locations to obtain the biopsy sample can include non-consuming portions of an animal. The tissue sample can be of the Sacrocaudalis dorsis medialis muscle, a tailhead subcutaneous fat sample or combinations thereof. The animal can be subjected to a nerve blocking agent to minimize pain to the animal during the biopsy procedure. Typically, the area to be biopsied is prepared by removing or minimizing hair in the area and disinfecting the area. Tools used to obtain the biopsy include biopsy needle, which preferably has a diameter of at least 11 swg, in some embodiments between about 11 swg and about 6 swg, and a length of between about 60 mm and about 120 mm. In some embodiments, more than one sample can be collected for a single animal. In some embodiments, between 1 and 20 samples of the animal can be taken at a single time.
[0058] The biopsy tissue sample can be analyzed to determine the fat characteristic(s) of the tissue sample, and the animal. The analysis can be performed by staining the tissue sample to visualize adipose cells in the tissue sample, which can be viewed through an optical microscope. Descriptions of techniques to stain tissue samples can be found in any histology lab manual, and would therefore be understood by one skilled in the art. However, other suitable resources include Survana 2012. Staining can be performed using any suitable stain as would be understood by one having skill in the art. Examples of suitable staining materials include H&E staining, Sudan black, gomori trichrome, martis scarlet, blue trichrome, desmin, or Oil Red staining.
[0059] The fat characteristic(s) of the tissue sample from the non-consuming meat location can be determined using visual methods (traditional) or can be determined using a computer-assisted method. The method can include analyzing a digital photograph taken through an optical microscope of the stained biopsy to determine the AAT and the muscle tissue area. These areas can be distinguished by color. These areas can be determined by a user or by using a computer. In some embodiments where a computer assisted method is utilized, the computer system, which includes a processor, and programs therein can be used to calculate the AAT and/or the muscle tissue area from the biopsy. In some embodiments, the computer assisted method can use an artificial intelligence algorithm to calculate the AAT and/or the muscle tissue area from the stained biopsy.
[0060] A marbling microscore of between about 0 and 10 can be assigned to a sample based on the ratio of the adipose tissue area and the muscle tissue area. When no fat is present, the score assigned is 0. A maximum score of 10 is provided at 30% or higher fat levels. A microscore of 1 corresponds to <2%, 2 corresponds to 3%, 3 corresponds to 4%, 4 corresponds to 8%, 5 corresponds to 12%, 6 corresponds to 16%, 7 corresponds to 20%, 8 corresponds to 24%, 9 corresponds to 28%, or 10 corresponds to >30%. If necessary, the marbling microscore calculated using the present invention can be correlated to a standards score, for example the MSA MB score, the AUS MB score, or standards from other jurisdictions.
[0061] One skilled in the art could utilize this chart to determine the correlation between the microscore of the present invention and other score standards for other jurisdictions without deviating from the present invention.
[0062] Once the standards score is determined from a biopsy, the treatment of the animal can be determined prior to slaughter. Suitable treatments include, slaughtering the animal, extending the feeding period of the animal, adjusting a diet of the animal, maintaining the diet of the animal, and/or modifying the amount of exercise of the animal. For example, if the microscore is not within an acceptable range for a characteristic of the animal, then the diet of the animal can be adjusted. If the animal is within the acceptable range for a characteristic of the animal, then the animal can be slaughtered. Furthermore, the microscore of the animal can be retested periodically, for example every day, every three days, every week, biweekly, bimonthly, every three weeks, every month, every two months, every quarter, three times per year, every five months, bi-yearly, every seven months, every eight months, every nine months, every ten months, every eleven months or yearly, or periodically during a range within these values. In other words, the animal can be tested at any point where a user desires additional data about the animal. When more than one sample has been obtained, the animal's progress can be tracked to determine if interventions based on previous results are altering the microscore of the animal and to determine if the intervention should be continued or if the treatment should be changed. For example, if a microscore indicated that the animal's characteristic of marbling was low, the animal's diet could be changed. If on a subsequent test for that animal, the microscore had not changed, or if the microscore became worse than a previous data point, the treatment for the animal can be changed—whether that means the animal is slaughtered or the diet is adjusted.
[0063] In some embodiments, the treatment can be determined based on a customer's input. For example, if the customer desires meat from a meat-consuming location that has significant marbling (e.g. a MSA MB score of between about 400 and about 1100), then an animal(s) can be tested before slaughtering to determine if the customer's input is met. If a customer's condition is not met, then the treatment of the animal can be modified or other animals can be tested to determine if the customer's condition has been met by the other animal.
[0064] An aspect of the invention is a method of producing an animal breeding plan for a population of at least one animal, The method includes determining a standards score or microscore for at least one animal in a population. The standards score or the microscore are ranked in an order to determine a rank in the population. Based on the rank, an animal breeding treatment can be determined.
[0065] The rank of the animal can be determined based on a desired fat characteristic of the animal. Using this information, a breeder can determine a plan for breeding two animals so that its progeny has the desired fat characteristic.
[0066] The fat characteristic of the tissue sample for each animal can be selected from the group consisting of an AAT, adipose cell number/area, adipose cell size, adipose cell distribution, the fat melting temperature, which can be the intramuscular fat melting temperature, and combinations thereof. The melting temperature of the subcutaneous fat sample of the animal can provide information about the intra-muscular fat melting temperature of the animal at a loin (i.e. a meat location of the animal) or other meat locations. Other characteristics of the meat at a meat consuming location of the animal can include marbling. More than one characteristic can be determined from a sample. Other characteristics can include chemical markers of “taint” flavors. Meat consuming locations include locations of cuts of meats for consumer consumption and include a forequarter cut or a hindquarter cut. Suitable forequarter cuts include for example, brisket, blade, and cube roll. Suitable hindquarter cuts include for example rump, round, tenderloin, and strip loin. Non-consumer meat locations are locations of the animal not intended for consumer consumption or not preferred for consumer consumption, and include muscle samples taken from the Longissimus dorsi at 10.sup.th-11.sup.th rib level, the muscle Sacrocaudalis dorsis medialis (located in the tailhead).
[0067] In some embodiments, the biopsy samples can be stored in a preservative. Suitable preservatives include formalin. In some embodiments, the concentration of the preservative can be between 5 vol. % and about 20 vol. %, in some embodiments about 10 vol. %. Methods for treatment and analysis of tissue samples are described in Survana 2012. In some embodiments, the samples can be stored between about −25° C. and about 30° C., in some embodiments about 5° C. before the samples are analyzed. Before the samples are analyzed, they can be warmed to between about 0° C. and about 50° C., in some embodiments about 25° C.
[0068] The livestock animal can be a cow, pig, sheep, chicken, bison, lamb, goat, or other consumable animal. The biopsy of a tissue sample can be taken at the tailhead of an animal. Other suitable biopsy locations include in the Ischiatic tuber region and at the 10th-11th level of the animal. Other suitable locations to obtain the biopsy sample can include non-consuming portions of an animal. The tissue sample can be of the Sacrocaudalis dorsis medialis muscle, a tailhead subcutaneous fat sample or combinations thereof. The animal can be subjected to a nerve blocking agent to minimize pain to the animal during the biopsy procedure. Typically, the area to be biopsied is prepared by removing or minimizing hair in the area and disinfecting the area. Tools used to obtain the biopsy include biopsy needle, which preferably has a diameter of at least 11 swg, in some embodiments between about 11 swg and about 6 swg, and a length of between about 60 mm and about 120 mm. In some embodiments, more than one sample can be collected for a single animal. In some embodiments, between 1 and 20 samples of the animal can be taken at a single time.
[0069] The biopsy tissue sample can be analyzed to determine the fat characteristic(s) of the tissue sample, and the animal. The analysis can be performed by staining the tissue sample to visualize adipose cells in the tissue sample, which can be viewed through an optical microscope. Descriptions of techniques to stain tissue samples can be found in any histology lab manual, and would therefore be understood by one skilled in the art. However, other suitable resources include Survana 2012. Staining can be performed using any suitable stain as would be understood by one having skill in the art. Examples of suitable staining materials include H&E staining, Sudan black, gomori trichrome, martis scarlet, blue trichrome, desmin, or Oil Red staining.
[0070] The fat characteristic(s) of the tissue sample from the non-consuming meat location can be determined using visual methods (traditional) or can be determined using a computer-assisted method. The method can include analyzing a digital photograph taken through an optical microscope of the stained biopsy to determine the AAT and the muscle tissue area. These areas can be distinguished by color. These areas can be determined by a user or by using a computer. In some embodiments where a computer assisted method is utilized, the computer system, which includes a processor, and programs therein can be used to calculate the AAT and/or the muscle tissue area from the biopsy. In some embodiments, the computer assisted method can use an artificial intelligence algorithm to calculate the AAT and/or the muscle tissue area from the stained biopsy.
[0071] A marbling microscore of between about 0 and 10 can be assigned to a sample based on the ratio of the adipose tissue area and the muscle tissue area. When no fat is present, the score assigned is 0. A maximum score of 10 is provided at 30% or higher fat levels. A microscore of 1 corresponds to <2%, 2 corresponds to 3%, 3 corresponds to 4%, 4 corresponds to 8%, 5 corresponds to 12%, 6 corresponds to 16%, 7 corresponds to 20%, 8 corresponds to 24%, 9 corresponds to 28%, or 10 corresponds to >30%. If necessary, the marbling microscore calculated using the present invention can be correlated to a standards score, for example the MSA MB score, the AUS MB score, or standards from other jurisdictions.
[0072] One skilled in the art could utilize this chart to determine the correlation between the microscore of the present invention and other score standards for other jurisdictions without deviating from the present invention.
[0073] In some embodiments, the breeding treatment can be determined based on a customer's input. For example, if the customer desires meat from a meat-consuming location that has significant marbling (e.g. a MSA MB score of between about 400 and about 1100), then two animals can be breed based on the animals microscores prior to slaughter of the animals in order to meet a desired characteristic.
[0074] An aspect of the invention is a method of determining an adjustment of an exposure condition for a population. The method includes organizing a population of animals into at least two groups. A plurality of pairs are created that include an animal from a first group of the at least two groups and a second group of the groups. An exposure condition from the first group is adjusted. Periodically, the standards score or microscore of the first group is compared to another group to determine the effect of the adjustment on the exposure condition.
[0075] In some embodiments, two groups are made. Animals with fat characteristics are divided into the two groups. One of the animals is subjected to an exposure condition and is adjusted. Periodically, the microscore and/or the standards score is measured in the two animals to determine if there is an effect on the microscore and/or standards score based on the exposure.
[0076] The fat characteristic of the tissue sample for each animal can be selected from the group consisting of an AAT, adipose cell number/area, adipose cell size, adipose cell distribution, the fat melting temperature, which can be the intramuscular fat melting temperature, and combinations thereof. The melting temperature of the subcutaneous fat sample of the animal can provide information about the intra-muscular fat melting temperature of the animal at a loin (i.e. a meat location of the animal) or other meat locations. Other characteristics of the meat at a meat consuming location of the animal can include marbling. More than one characteristic can be determined from a sample. Other characteristics can include chemical markers of “taint” flavors. Meat consuming locations include locations of cuts of meats for consumer consumption and include a forequarter cut or a hindquarter cut. Suitable forequarter cuts include for example, brisket, blade, and cube roll. Suitable hindquarter cuts include for example rump, round, tenderloin, and strip loin. Non-consumer meat locations are locations of the animal not intended for consumer consumption or not preferred for consumer consumption, and include muscle samples taken from the Longissimus dorsi at 10.sup.th-11.sup.th rib level, the muscle Sacrocaudalis dorsis medialis (located in the tailhead).
[0077] In some embodiments, the biopsy samples can be stored in a preservative. Suitable preservatives include formalin. In some embodiments, the concentration of the preservative can be between 5 vol. % and about 20 vol. %, in some embodiments about 10 vol. %. Methods for treatment and analysis of tissue samples are described in Survana 2012. In some embodiments, the samples can be stored between about −25° C. and about 30° C., in some embodiments about 5° C. before the samples are analyzed. Before the samples are analyzed, they can be warmed to between about 0° C. and about 50° C., in some embodiments about 25° C.
[0078] The livestock animal can be a cow, pig, sheep, chicken, bison, lamb, goat, or other consumable animal. The biopsy of a tissue sample can be taken at the tailhead of an animal. Other suitable biopsy locations include in the Ischiatic tuber region and at the 10.sup.th-11.sup.th level of the animal. Other suitable locations to obtain the biopsy sample can include non-consuming portions of an animal. The tissue sample can be of the Sacrocaudalis dorsis medialis muscle, a tailhead subcutaneous fat sample or combinations thereof. The animal can be subjected to a nerve blocking agent to minimize pain to the animal during the biopsy procedure. Typically, the area to be biopsied is prepared by removing or minimizing hair in the area and disinfecting the area. Tools used to obtain the biopsy include biopsy needle, which preferably has a diameter of at least 11 swg, in some embodiments between about 11 swg and about 6 swg, and a length of between about 60 mm and about 120 mm. In some embodiments, more than one sample can be collected for a single animal. In some embodiments, between 1 and 20 samples of the animal can be taken at a single time.
[0079] The biopsy tissue sample can be analyzed to determine the fat characteristic(s) of the tissue sample, and the animal. The analysis can be performed by staining the tissue sample to visualize adipose cells in the tissue sample, which can be viewed through an optical microscope. Descriptions of techniques to stain tissue samples can be found in any histology lab manual, and would therefore be understood by one skilled in the art. However, other suitable resources include Survana 2012. Staining can be performed using any suitable stain as would be understood by one having skill in the art. Examples of suitable staining materials include H&E staining, Sudan black, gomori trichrome, martis scarlet, blue trichrome, desmin, or Oil Red staining.
[0080] The fat characteristic(s) of the tissue sample from the non-consuming meat location can be determined using visual methods (traditional) or can be determined using a computer-assisted method. The method can include analyzing a digital photograph taken through an optical microscope of the stained biopsy to determine the AAT and the muscle tissue area. These areas can be distinguished by color. These areas can be determined by a user or by using a computer. In some embodiments where a computer assisted method is utilized, the computer system, which includes a processor, and programs therein can be used to calculate the AAT and/or the muscle tissue area from the biopsy. In some embodiments, the computer assisted method can use an artificial intelligence algorithm to calculate the AAT and/or the muscle tissue area from the stained biopsy.
[0081] A marbling microscore of between about 0 and 10 can be assigned to a sample based on the ratio of the adipose tissue area and the muscle tissue area. When no fat is present, the score assigned is 0. A maximum score of 10 is provided at 30% or higher fat levels. A microscore of 1 corresponds to <2%, 2 corresponds to 3%, 3 corresponds to 4%, 4 corresponds to 8%, 5 corresponds to 12%, 6 corresponds to 16%, 7 corresponds to 20%, 8 corresponds to 24%, 9 corresponds to 28%, or 10 corresponds to >30%. If necessary, the marbling microscore calculated using the present invention can be correlated to a standards score, for example the MSA MB score, the AUS MB score, or standards from other jurisdictions.
[0082] One skilled in the art could utilize this chart to determine the correlation between the microscore of the present invention and other score standards for other jurisdictions without deviating from the present invention.
[0083] An aspect of the invention is a method of determining changes of meat quality of an animal. The method includes obtaining a first tissue sample from the animal. The tissue sample is analyzed to determine at least one fat characteristic of the tissue sample. A microscore is assigned to the fat characteristic(s). The microscore is correlated to a characteristic of meat of an animal at the time the first sample was obtained. A second sample is obtained from the animal at a second time after the first time. The second sample is analyzed to determine at least one fat characteristic of the sample. The first and second samples are both obtained from a non-consuming meat location of the animal. The change in the fat characteristic from the first time to the second time is used to determine a treatment for the animal.
[0084] The fat characteristic of the tissue sample for each animal can be selected from the group consisting of an AAT, adipose cell number/area, adipose cell size, adipose cell distribution, the fat melting temperature, which can be the intramuscular fat melting temperature, and combinations thereof. The melting temperature of the subcutaneous fat sample of the animal can provide information about the intra-muscular fat melting temperature of the animal at a loin (i.e. a meat location of the animal) or other meat locations. Other characteristics of the meat at a meat consuming location of the animal can include marbling. More than one characteristic can be determined from a sample. Other characteristics can include chemical markers of “taint” flavors. Meat consuming locations include locations of cuts of meats for consumer consumption and include a forequarter cut or a hindquarter cut. Suitable forequarter cuts include for example, brisket, blade, and cube roll. Suitable hindquarter cuts include for example rump, round, tenderloin, and strip loin. Non-consumer meat locations are locations of the animal not intended for consumer consumption or not preferred for consumer consumption, and include muscle samples taken from the Longissimus dorsi at 10.sup.th-11.sup.th rib level, the muscle Sacrocaudalis dorsis medialis (located in the tailhead).
[0085] In some embodiments, the biopsy samples can be stored in a preservative. Suitable preservatives include formalin. In some embodiments, the concentration of the preservative can be between 5 vol. % and about 20 vol. %, in some embodiments about 10 vol. %. Methods for treatment and analysis of tissue samples are described in Survana 2012. In some embodiments, the samples can be stored between about −25° C. and about 30° C., in some embodiments about 5° C. before the samples are analyzed. Before the samples are analyzed, they can be warmed to between about 0° C. and about 50° C., in some embodiments about 25° C.
[0086] The livestock animal can be a cow, pig, sheep, chicken, bison, lamb, goat, or other consumable animal. The biopsy of a tissue sample can be taken at the tailhead of an animal. Other suitable biopsy locations include in the Ischiatic tuber region and at the 10.sup.th-11.sup.th level of the animal. Other suitable locations to obtain the biopsy sample can include non-consuming portions of an animal. The tissue sample can be of the Sacrocaudalis dorsis medialis muscle, a tailhead subcutaneous fat sample or combinations thereof. The animal can be subjected to a nerve blocking agent to minimize pain to the animal during the biopsy procedure. Typically, the area to be biopsied is prepared by removing or minimizing hair in the area and disinfecting the area. Tools used to obtain the biopsy include biopsy needle, which preferably has a diameter of at least 11 swg, in some embodiments between about 11 swg and about 6 swg, and a length of between about 60 mm and about 120 mm. In some embodiments, more than one sample can be collected for a single animal. In some embodiments, between 1 and 20 samples of the animal can be taken at a single time.
[0087] The biopsy tissue sample can be analyzed to determine the fat characteristic(s) of the tissue sample, and the animal. The analysis can be performed by staining the tissue sample to visualize adipose cells in the tissue sample, which can be viewed through an optical microscope. Descriptions of techniques to stain tissue samples can be found in any histology lab manual, and would therefore be understood by one skilled in the art. However, other suitable resources include Survana 2012. Staining can be performed using any suitable stain as would be understood by one having skill in the art. Examples of suitable staining materials include H&E staining, Sudan black, gomori trichrome, martis scarlet, blue trichrome, desmin, or Oil Red staining.
[0088] The fat characteristic(s) of the tissue sample from the non-consuming meat location can be determined using visual methods (traditional) or can be determined using a computer-assisted method. The method can include analyzing a digital photograph taken through an optical microscope of the stained biopsy to determine the AAT and the muscle tissue area. These areas can be distinguished by color. These areas can be determined by a user or by using a computer. In some embodiments where a computer assisted method is utilized, the computer system, which includes a processor, and programs therein can be used to calculate the AAT and/or the muscle tissue area from the biopsy. In some embodiments, the computer assisted method can use an artificial intelligence algorithm to calculate the AAT and/or the muscle tissue area from the stained biopsy.
[0089] A marbling microscore of between about 0 and 10 can be assigned to a sample based on the ratio of the adipose tissue area and the muscle tissue area. When no fat is present, the score assigned is 0. A maximum score of 10 is provided at 30% or higher fat levels. A microscore of 1 corresponds to <2%, 2 corresponds to 3%, 3 corresponds to 4%, 4 corresponds to 8%, 5 corresponds to 12%, 6 corresponds to 16%, 7 corresponds to 20%, 8 corresponds to 24%, 9 corresponds to 28%, or 10 corresponds to >30%. If necessary, the marbling microscore calculated using the present invention can be correlated to a standards score, for example the MSA MB score, the AUS MB score, or standards from other jurisdictions.
[0090] One skilled in the art could utilize this chart to determine the correlation between the microscore of the present invention and other score standards for other jurisdictions without deviating from the present invention.
[0091] Once the standards score is determined from a biopsy for the first sample, the treatment of the animal can be determined prior to slaughter. Suitable treatments include, slaughtering the animal, extending the feeding period of the animal, adjusting a diet of the animal, maintaining the diet of the animal, and/or modifying the amount of exercise of the animal. For example, if the microscore is not within an acceptable range for a characteristic of the animal, then the diet of the animal can be adjusted. If the animal is within the acceptable range for a characteristic of the animal, then the animal can be slaughtered. Furthermore, the microscore of the animal can be retested periodically, for example every day, every three days, every week, biweekly, bimonthly, every three weeks, every month, every two months, every quarter, three times per year, every five months, bi-yearly, every seven months, every eight months, every nine months, every ten months, every eleven months or yearly, or periodically during a range within these values. In other words, the animal can be tested at any point where a user desires additional data about the animal. When more than one sample has been obtained, the animal's progress can be tracked to determine if interventions based on previous results are altering the microscore of the animal and to determine if the intervention should be continued or if the treatment should be changed. For example, if a microscore indicated that the animal's characteristic of marbling was low, the animal's diet could be changed. If on a subsequent test for that animal, the microscore had not changed, or if the microscore became worse than a previous data point, the treatment for the animal can be changed—whether that means the animal is slaughtered or the diet is adjusted.
[0092] In some embodiments, the treatment can be determined based on a customer's input. For example, if the customer desires meat from a meat-consuming location that has significant marbling (e.g. a MSA MB score of between about 400 and about 1100), then an animal(s) can be tested before slaughtering to determine if the customer's input is met. If a customer's condition is not met, then the treatment of the animal can be modified or other animals can be tested to determine if the customer's condition has been met by the other animal.
[0093] An aspect of the invention is a method of harvesting an animal for meat production having meat with a pre-determined fat characteristic. The method includes obtaining a tissue sample from a non-meat location of an animal. The tissue sample is analyzed to determine at least one fat characteristic of the tissue sample. If the fat characteristic(s) is less than the pre-determined fat characteristic, feeding the animal with a high energy feed ration. After feeding the animal with a high energy feed ration, obtain a second sample of the tissue sample and analyzing the fat characteristic of the tissue sample until the determined fat characteristic of the tissue sample is at or greater than the pre-determined fat characteristic. When the determined fat characteristic of the tissue sample is at or greater than the pre-determined fat characteristic, slaughtering the animal to harvest meat with a pre-determined fat characteristic.
[0094] The fat characteristic of the tissue sample for each animal can be selected from the group consisting of an AAT, adipose cell number/area, adipose cell size, adipose cell distribution, the fat melting temperature, which can be the intramuscular fat melting temperature, and combinations thereof. The melting temperature of the subcutaneous fat sample of the animal can provide information about the intra-muscular fat melting temperature of the animal at a loin (i.e. a meat location of the animal) or other meat locations. Other characteristics of the meat at a meat consuming location of the animal can include marbling. More than one characteristic can be determined from a sample. Other characteristics can include chemical markers of “taint” flavors. Meat consuming locations include locations of cuts of meats for consumer consumption and include a forequarter cut or a hindquarter cut. Suitable forequarter cuts include for example, brisket, blade, and cube roll. Suitable hindquarter cuts include for example rump, round, tenderloin, and strip loin. Non-consumer meat locations are locations of the animal not intended for consumer consumption or not preferred for consumer consumption, and include muscle samples taken from the Longissimus dorsi at 10.sup.th-11.sup.th rib level, the muscle Sacrocaudalis dorsis medialis (located in the tailhead).
[0095] In some embodiments, the biopsy samples can be stored in a preservative. Suitable preservatives include formalin. In some embodiments, the concentration of the preservative can be between 5 vol. % and about 20 vol. %, in some embodiments about 10 vol. %. Methods for treatment and analysis of tissue samples are described in Survana 2012. In some embodiments, the samples can be stored between about −25° C. and about 30° C., in some embodiments about 5° C. before the samples are analyzed. Before the samples are analyzed, they can be warmed to between about 0° C. and about 50° C., in some embodiments about 25° C.
[0096] The livestock animal can be a cow, pig, sheep, chicken, bison, lamb, goat, or other consumable animal. The biopsy of a tissue sample can be taken at the tailhead of an animal. Other suitable biopsy locations include in the Ischiatic tuber region and at the 10.sup.th-11.sup.th level of the animal. Other suitable locations to obtain the biopsy sample can include non-consuming portions of an animal. The tissue sample can be of the Sacrocaudalis dorsis medialis muscle, a tailhead subcutaneous fat sample or combinations thereof. The animal can be subjected to a nerve blocking agent to minimize pain to the animal during the biopsy procedure. Typically, the area to be biopsied is prepared by removing or minimizing hair in the area and disinfecting the area. Tools used to obtain the biopsy include biopsy needle, which preferably has a diameter of at least 11 swg, in some embodiments between about 11 swg and about 6 swg, and a length of between about 60 mm and about 120 mm. In some embodiments, more than one sample can be collected for a single animal. In some embodiments, between 1 and 20 samples of the animal can be taken at a single time.
[0097] The biopsy tissue sample can be analyzed to determine the fat characteristic(s) of the tissue sample, and the animal. The analysis can be performed by staining the tissue sample to visualize adipose cells in the tissue sample, which can be viewed through an optical microscope. Descriptions of techniques to stain tissue samples can be found in any histology lab manual, and would therefore be understood by one skilled in the art. However, other suitable resources include Survana 2012. Staining can be performed using any suitable stain as would be understood by one having skill in the art. Examples of suitable staining materials include H&E staining, Sudan black, gomori trichrome, martis scarlet, blue trichrome, desmin, or Oil Red staining.
[0098] The fat characteristic(s) of the tissue sample from the non-consuming meat location can be determined using visual methods (traditional) or can be determined using a computer-assisted method. The method can include analyzing a digital photograph taken through an optical microscope of the stained biopsy to determine the AAT and the muscle tissue area. These areas can be distinguished by color. These areas can be determined by a user or by using a computer. In some embodiments where a computer assisted method is utilized, the computer system, which includes a processor, and programs therein can be used to calculate the AAT and/or the muscle tissue area from the biopsy. In some embodiments, the computer assisted method can use an artificial intelligence algorithm to calculate the AAT and/or the muscle tissue area from the stained biopsy.
[0099] A marbling microscore of between about 0 and 10 can be assigned to a sample based on the ratio of the adipose tissue area and the muscle tissue area. When no fat is present, the score assigned is 0. A maximum score of 10 is provided at 30% or higher fat levels. A microscore of 1 corresponds to <2%, 2 corresponds to 3%, 3 corresponds to 4%, 4 corresponds to 8%, 5 corresponds to 12%, 6 corresponds to 16%, 7 corresponds to 20%, 8 corresponds to 24%, 9 corresponds to 28%, or 10 corresponds to >30%. If necessary, the marbling microscore calculated using the present invention can be correlated to a standards score, for example the MSA MB score, the AUS MB score, or standards from other jurisdictions.
[0100] One skilled in the art could utilize this chart to determine the correlation between the microscore of the present invention and other score standards for other jurisdictions without deviating from the present invention.
[0101] Once the standards score is determined from a biopsy for the first sample, the treatment of the animal can be determined prior to slaughter. Suitable treatments include, slaughtering the animal, extending the feeding period of the animal, adjusting a diet of the animal, maintaining the diet of the animal, and/or modifying the amount of exercise of the animal. For example, if the microscore is not within an acceptable range for a characteristic of the animal, then the diet of the animal can be adjusted. If the animal is within the acceptable range for a characteristic of the animal, then the animal can be slaughtered.
[0102] The second sample can be obtained after the first sample, which can be one hour, 12 hours, 24 hours, 3 days, 5 days, 6 days, one week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 52 weeks, 1.5 years, or 2 years after the first sample is obtained, or during a time period ranging between two of these time periods. If on a subsequent test for that animal, the microscore had not changed, or if the microscore became worse than a previous data point, the treatment for the animal can be changed—whether that means the animal is slaughtered or the diet is adjusted.
[0103] In some embodiments, the treatment can be determined based on a customer's input. For example, if the customer desires meat from a meat-consuming location that has significant marbling (e.g. a MSA MB score of between about 400 and about 1100), then an animal(s) can be tested before slaughtering to determine if the customer's input is met. If a customer's condition is not met, then the treatment of the animal can be modified or other animals can be tested to determine if the customer's condition has been met by the other animal.
EXAMPLES
Example 1
[0104] An official MSA marble score was obtained for carcasses from the abattoir determined following the procedures described in the Handbook of Australian Meat (Meat and Livestock Australia, 2005) and reported as MSA MB (100 to 1200). A microscore for microscopic assessment of the level of marbling in histological samples stained with H&E was created. The score ranges from 0 to 10, taking into consideration the proportion of adipose tissue to muscle tissue. The size of adipocytes was also noted. The scoring was done by observation of the entire histological slide with an optical microscope under a total magnification of 100×. The exercise was done between two people, one evaluator and one person providing the samples and recording the scores. Control samples were chosen for microscore level 1, 3, 5, 7 and 9 and used as blind duplicates during the scoring process. Using Leica Application Suit version 4.12 software, an objective measure was obtained. The area of adipose tissue was selected by color. Obvious connective tissue was eliminated subjectively. The percentage fat was recorded as AAT %.
Relationship Between MSA MB Score, “Microscore” and AAT %
[0105] Examples of degrees of marbling of the Longissimus dorsi are illustrated in
[0106] For MSA MB between 270 and 450 the AAT % was in the range 0.15% to 6.5%. As the marble score increases the area of adipose tissue increases, with more and larger collections of adipocytes.
[0107] Histological examination of less marbled animals reveals connective tissue as the possible confounding factor. In some less marbled animals, there are significant areas of connective tissue that would be mistaken for fat under macroscopic examination.
Relationship Between Subcutaneous Fat Tm and IMF Tm
[0108] Thirteen sample sirloins (Wagyu breed) were tested to determine the IMF Tm and the Tm of the overlying subcutaneous fat.
Relationship Between “Microscore”, Subcutaneous Fat Tm and “Days on Feed”
[0109] Most livestock involved in meat production are placed on a high energy feeding regime prior to slaughter. The duration of this regime is referred to as “days on feed” (“DOF”). The carcasses from two groups of cattle (one group with DOF of less than 200, and one with a DOF more than 350) were tested.
[0110] Variation of marbling with DOF is illustrated in
[0111] There were also qualitative differences. Group 1 had small adipocytes located in the perimysium (
[0112] Despite a clear relationship between DOF and marbling, there is substantial variation between animals at similar DOF. Animals fed between 430 and 470 days have microscores varying between 3 and 10. An example is seen by comparing
[0113] The effects of DOF on Tm of subcutaneous fat is illustrated in
Relationship Between Marbling in the Loin and Tailhead
[0114]
[0115] In
[0116]
Relationship Between the Tm of Subcutaneous Fat Overlying the Loin and the Ischiatic Tuber
[0117] The subcutaneous fat overlying the Ischiatic tuber (IT) can also be used for in vivo sampling to monitor changes in Tm during feeding.
Marbling
[0118] The progression of marbling, its characteristics and pattern are illustrated in
[0119] At some boundaries between adipose and muscle bundles, the perifascicular myocytes are atrophic and show changes of shape and an increased affinity to eosin. This is most often observed near the ends of branches of adipocytes (
Sample Use
[0120] An operator of a feedlot purchases cattle at approximately 10 months of age and provides the newly purchased cattle with a high energy feed ration. Prior to the present invention, the operator can monitor live weight gain, but otherwise has very limited means of judging the meat quality changes in the live animals so bases decisions as to whether continue feeding an animal or slaughtering it, on live weight gain. If the operator adopts the invention, on the animals entering the feedlot a biopsy is taken from the tailhead of each animal. The muscle sample from the Sacrocaudalis dorsis medalis is histologically assessed as described above and a marbling “microscore” is determined. Either the IMF Tm from the muscle sample or the subcutaneous fat Tm taken from a biopsy from fat overlying the Ischiatic Tuber is also determined as described above.
[0121] The feedlot operator repeats the biopsy procedure every three months. He is then able to more accurately assess the carcass value of each animal at each three month point, than if he used only live weight gain data. The feedlot operator can therefore determine whether further feeding of each animal is likely to be worthwhile.
[0122] The feedlot operator also has customers for beef carcasses who require product within specific meat quality parameters described in terms of marbling score and/or IMF Tm. For instance a customer may require carcasses with a MSA MB of 600 to 750 and an IMF Tm less than 38° C. The feedlot operator can select animals for slaughter on the basis of the biopsy determinations confident that they would meet those criteria, whereas if he selected animals without the information provided by the invention, a proportion of animals slaughtered would not meet the customer's requirements.
Selection for Rapid Marbling
[0123] A farmer is producing for a market segment which requires beef with medium level of marbling (e.g. MSA MB of around 600). The less DOF required to reach that level of marbling, the lower his cost of production.
[0124] The farmer wishes to test which of his bulls is likely to produce progeny which meet the marbling objective in the lease DOF. Using the invention he is able to monitor the level of marbling in the live progeny and determine which bulls produce better outcomes. Without the invention the farmer would be required to undertake an experiment involving more animals, animals being slaughtered at less than optimum condition, and delay receiving a definitive answer to his question.
Relationship
[0125] Postmortem samples of muscle and fat were taken from carcasses of animals harvested for routine food production. Within 2 days after birth, calves are DNA tested, confirming dam and sire and allowing later traceability of the carcasses. The calves remain with their mothers until four to six months old. At weaning the males are castrated and continue grazing kikuyu, ryegrass pasture and hay until they reach a weight of 300 kg, when they are fed pellets and ryegrass hay ad libitum. The pellets are 9 mm EasyBeef (Milne Feeds, Perth, Australia), containing lupins, barley, oats, wheat and triticale, with a nutritional composition based on dry matter of crude protein (min.) 14.5%, metabolizable energy (est.) 11.0 MJ/kg, crude fiber (max.) 20.0%, urea (max.) 1.5%, and monensin 26.6 ppm.
[0126] The calves were then divided into three groups. Group 1: Long DOF, N=17, 10 Heifers and 7 steers with Wagyu content from 50% to 96% were fed for 350 to 500 days (avg 427). Group 2: Low DOF, fed for less than 300 days (AVG 116) (N=44. 5 heifers and 39 steers, with an average MSA MB of 379). Black wagyu content was generally less than 50%. Most were 50% or more Akaushi. Group 3: Full blood and crossbreed Wagyu sirloins submitted to the 2015 Australian Wagyu Association's Branded Beef Competition from various producers around Australia (N=13. IMF 11% 79 to 54%).
[0127] After slaughter, postmortem samples of about 2-gram muscle sample was taken from the tail of each carcass. C 19 haplotypes were determined using PCR, following the method described by Williamson et al. 2017. The information resulting from this analysis was used to confirm the carcass identity and validate the data for each individual.
[0128] The fat melting temperature samples were taken at the Ischiatic tuber region and at 10.sup.th-11.sup.th rib level and stored at 5° C. for fat melting point analysis following the method described in U.S. Pat. No. 10,359,380 (incorporated by reference in its entirety). Muscle histology samples were taken from the muscle Longissimus dorsi, at 10.sup.th and 11.sup.th rib level and from Sacrocaudalis dorsalis medialis muscle for H&E and oil red O staining, and were processed as follows: [0129] H&E: 10 mm×10 mm×3 mm samples stored in formalin 10% and then processed as 94 described in Survana 2012. The information recorded was the location of adipose 95 tissue (Perimysium, endomysium, epimysium), Adipocyte size and patterns of 96 adipocyte accumulation were noted. [0130] Oil Red O: 3 mm×3 mm×3 mm samples, frozen in liquid nitrogen and OCT and 99 processed following the method described in Kiernan et al. 9. The aim was to visualize 100 and describe the presence of intramyocellular lipid droplets.
[0131] The official MSA grading score, provided by the abattoir and determined by naked eye following the procedures described in the Handbook of Australian Meat (Meat & Livestock Australia, Handbook of Australian Meat, 7.sup.th edition (International Red Meat Manual) (2005), incorporated by reference in its entirety), was determined for each carcass. The H&E samples were read using a binocular microscope. The degree of marbling was scored from 0 to 10, taking into consideration the proportion of adipose tissue to muscle tissue. The scoring was done by two observers in turn: one blind evaluator and the other providing the samples and recording the scores. Reference samples were chosen for microscore 1, 3, 5, 7 and 9 and used as blind duplicates during the scoring process. Results were confirmed using Leica Application Suite version 4.12 software. The AAT was selected by color. Connective tissue was not included.
[0132] To establish their complete independence from the perimysium, serial sections of Longissimus dorsi were examined every 16 microns (
[0133] Histology has been widely used in human medicine to visualize, describe and understand morphological changes at a cellular level in muscle, for example in muscular dystrophies, which often involve excessive intramuscular fat deposition. The same techniques have been applied to bovine muscle to achieve similar goals, specifically to understand marbling.
[0134]
[0135] Table 1 below provides the Tm for multiple samples from nine sheep.
TABLE-US-00001 TABLE 1 Sample no. Tm 1 37.7 2 35.8 3 38.5 4 43.6 5 39.8 6 38.3 7 40.7 8 43.4 9 43.2
Other Results
[0136] Invasive marbling is associated with concomitant decreases in Tm of fat reflecting increasing Mono Unsaturated Fatty Acids and particularly oleic acid. Thus, it is believed that the two processes are directly related. At the simplest level, lower Tm's in oils, permits greater fluidity and therefore active invasion resulting in fine marbling. This pragmatic explanation is consistent with historical practices such as the massaging of “soft” fat during the later stages of feeding of Wagyu. It also explains the fact that non-Wagyu, like Angus, retain higher Tms and are known for seam or coarse marbling. There is additional complexity. For example, there will be time dependent induction of allelic gene products encoded within Wagyu specific haplotypes such as 60.1 and 30.4. Foremost, is SREBFI which regulates the desaturase but also many other genes including many known to be involved in muscle differentiation and other potentially relevant processes. The challenge is to unravel the multitude of interacting haplotypes, genes, products and regulators especially because marbling is age and feed dependent. Experience with human muscular dystrophy teaches that current explanations for marbling are too simplistic. However, a strategy is suggested by (1) the fact that these interactions must be represented within these two haplotypes (in contrast to non-Wagyu haplotypes), (2) the opportunity to monitor activities quantitatively over time by sampling muscle and fat from the tailhead.
[0137] The microscore and MSA MB were well correlated for highly marbled beef, especially when coarse rather than fine. However, at the extremes we prefer the microscore. In carcasses with MSA MB 400 and below, the correlation is not as strong. This can be explained by the macroscopic nature of MSA MB, restricted to the naked eye visualization of white areas at least as large as 10 to 15 adipocytes. This limitation also applies to marbling assessments using a digital camera. Other types of connective tissue also appear white while the carcass is cold and are impossible to distinguish during marbling assessment. In less marbled carcasses, where the proportion of connective tissue over fat is higher, marbling can be overestimated. In contrast, a microscopic assessment of intramuscular fat is able to distinguish these details, allowing differentiation of structures and levels of marbling more completely. Therefore, the microscore is more accurate than MSA MB for the vast majority of standard, non-Wagyu carcasses.
[0138] Quantification by the naked eye can also be misleading with extreme degrees of marbling, in that it underestimates the more desired fine marbling. Some producers and some countries use an extended scale such as AUS MB 10-13. By whatever measure however, the finer the marbling the harder to quantify by the naked eye. Histological assessment will be helpful in developing measures of fineness by including the aggressiveness of the invasion and therefore the formation of residual islands of muscle and a snowflake appearance.
[0139] Marbling and Tm change with DOF but are also significantly affected by genetics. It would be useful to have methods of monitoring amounts and composition of intramuscular fat in relation to age, DOF, breed composition, suitability for breeding and animal welfare.
[0140] Worldwide, marbling is assessed in the Longissimus dorsi muscle after quartering the carcasses, but marbling occurs at different rates depending upon the muscle sampled. Obviously, biopsies of the Longissimus dorsi would not be practical. Ultrasound scanning the LD has been found unreliable for assessing marbling in long fed Wagyu. The tailhead is more accessible than the LD, more practical for biopsies under epidural anesthesia and therefore a potential site for in vivo monitoring.
[0141] The patterns of intramuscular fat deposition within LD or SDM appear to be very similar. The microscore of the SDM and the MSA MB are well correlated (r=0.89).
[0142] Therefore, the SDM is a suitable site for monitoring progression of marbling. The healthiness of beef fat, as measured by Tm, improves with feeding due to the desaturation of stearic acid into oleic acid. This process is driven primarily by the enzyme SCD (Bota 26) and regulated by SREBF1 (Bota 19). The same enzyme, regulated by is involved in desaturation of both subcutaneous and intramuscular adipose tissue. Therefore, a user can monitor changes in Tm of intramuscular fat by sampling subcutaneous fat with a simple punch biopsy, as used in humans. A substantial deposit of subcutaneous fat over the Ischiatic tuber (IT) develops in “finished” cattle and provides a practical location for sampling.
[0143] Ranges have been discussed and used within the forgoing description. One skilled in the art would understand that any sub-range within the stated range would be suitable, as would any number within the broad range, without deviating from the invention.
[0144] The foregoing description of the present invention, related to a method of treating a livestock animal by sampling a non-consuming portion of the animal, has been presented for purposes of illustration and description. Furthermore, the description is not intended to limit the invention to the form disclosed herein. Consequently, variations and modifications commensurate with the above teachings, and the skill or knowledge of the relevant art, are within the scope of the present invention. The embodiment described hereinabove is further intended to explain the best mode known for practicing the invention and to enable others skilled in the art to utilize the invention in such, or other, embodiments and with various modifications required by the particular applications or uses of the present invention. It is intended that the appended claims be construed to include alternative embodiments to the extent permitted by the prior art.