Breeding technology and application of pure-color red cattle

Abstract

The invention provides a breeding technology and application of pure-color red cattle, and relates to the technical field of animal breeding. The breeding technology of the pure-color red cattle comprises the following specific steps: S1, detecting an MC1R gene c.901Cgt; a T site and a TYRP1 gene c.1267Ggt; according to a homozygous dominant genotype of a site A, a Gaoligong Mountain small yellow cattle female parent and a Tibetan cattle male parent are screened to ensure that the female parent and the male parent carry homozygous red dominant alleles, and meanwhile, intramuscular fat deposition related gene dominant genotype individuals are selected by combining a whole genome association analysis technology; s2, using prostaglandin F2alpha combined with gonadotropin to release hormone treatment, performing estrus cycle synchronization regulation and control on the Gaoligong Mountain small yellow cattle female parent, implementing regular artificial insemination 24-30 hours after thrombus removal, and using the Tibetan Tibetan cattle frozen semen treated by a sperm screening technology. Pure-color red cattle are bred precisely and directionally by adopting innovative technologies such as gene screening, the meat quality is improved, ecological circulation is achieved, and sustainable development of high-mountain animal husbandry is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of animal breeding, in particular to a breeding technology and application of pure-color red cattle. Background Art

[0002] Livestock development in mountainous areas has long faced challenges such as difficult selection and breeding, and low economic returns. While Gaoligong Mountain Yellow Cattle, a native breed, are adaptable to the complex terrain and changing climate, they suffer from short stature, slow growth, loose meat, and uneven fat distribution, resulting in generally low market prices. Limu Tibetan Cattle, while possessing good stress resistance and growth potential, suffer from a high rate of variegated coat color. In the beef market, buyers often use this inconsistent coat color as an excuse to lower prices, severely impacting farmers' profits.

[0003] Traditional cattle crossbreeding techniques rely heavily on selecting parents based on phenotypic traits like body shape and reproductive performance, lacking precise genetic control over traits like coat color and meat quality. Conventional artificial insemination and embryo transfer techniques, lacking genetic screening, result in significant variation in individual traits among offspring, with high-quality individuals accounting for less than 30%, making it difficult to meet market demand for high-quality beef. Furthermore, high-altitude areas face a shortage of feed resources and harsh breeding environments. Traditional farming practices not only pollute the environment but also waste resources, failing to achieve synergistic ecological and economic benefits. The sustainable development of alpine animal husbandry faces significant challenges. Summary of the Invention

[0004] Technical problems solved

[0005] In view of the shortcomings of the existing technology, the present invention provides a breeding technology and application of pure-color red cattle, which solves the problem of difficult seed selection and breeding in alpine animal husbandry.

[0006] Technical Solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions: A pure color red cattle breeding technology specifically comprises the following steps:

[0008] S1. By detecting the homozygous dominant genotypes at the c.901C>T locus of the MC1R gene and the c.1267G>A locus of the TYRP1 gene, Gaoligong Mountain Yellow Cattle females and Limu Tibetan Cattle sires were screened to ensure they carried the homozygous dominant red allele. Genome-wide association analysis was also used to select individuals with dominant genotypes for genes associated with intramuscular fat deposition.

[0009] S2. Synchronize the estrous cycle of Gaoligong Mountain steers using prostaglandin F2α combined with gonadotropin-releasing hormone. Timed artificial insemination is performed 24-30 hours after thrombosis removal using frozen semen from Limu Tibetan cattle that has been processed using sperm selection technology.

[0010] S3. Perform a biopsy of the blastocysts on day 6-7 after IVF and use real-time quantitative PCR to detect KIT gene copy number variation and ASIP gene methylation status. Select embryos with a pure color phenotype and no white spot alleles. Also, perform sex determination using SRY gene testing, prioritizing female embryos for transfer.

[0011] S4. On gestational days 45-60, 0.5%-1.0% of a methyl donor complex (containing 15 mg / kg of folic acid, 0.03 mg / kg of vitamin B12, and 1200 mg / kg of choline) is added to the maternal feed to influence fetal myofiber development and adipocyte differentiation through epigenetic regulation. The methyl donor complex also contains nano-coated conjugated linoleic acid with a particle size of 100-200 nm, capable of crossing the placental barrier.

[0012] S5. Within 72 hours after birth, calves were inoculated with a core bacterial preparation derived from Limu Tibetan cattle by gavage, containing dominant strains of Ruminococcus, Bacteroides, and Prevotella, at a dose of 1 × 10 9 CFU / mL, inoculated for 3 consecutive days, to promote the digestion ability of calves on alpine roughage;

[0013] S6. Dynamically adjust the feed formula based on the cattle's real-time weight, backfat thickness, and muscle development. Add 0.02% β-hydroxy-β-methylbutyric acid and 0.01% chromium picolinate to the feed 60 days before slaughter to promote intramuscular fat deposition and improve meat color.

[0014] Preferably, the female parent of the Gaoligong Mountain Yellow Cattle screened in S1 must also satisfy the requirement of having an 82 bp insertion mutation in the 3'UTR region of the Myostatin gene, and the male parent of the Limu Tibetan Cattle must carry the dominant allele of the K232A site of the CAST gene.

[0015] Preferably, the dosage of prostaglandin F2α in S2 is 25 mg / head, the dosage of gonadotropin-releasing hormone is 100 μg / head, and the time of withdrawal of the vaginal suppository is 8-9 days after placement of the vaginal suppository.

[0016] Preferably, the methyl donor complex in S3 is encapsulated in liposomes to improve bioavailability. The liposomes are composed of lecithin and cholesterol in a mass ratio of 3:1, and have a particle size of 150-200 nm.

[0017] Preferably, in the directed regulation of the calf's intestinal flora, the preparation method of the core flora preparation includes: collecting rumen fluid from healthy Limu Tibetan cattle, enriching it through anaerobic culture, separating flora components of different densities by density gradient centrifugation, and screening out flora components with a relative abundance of fiber-decomposing bacteria ≥40% through 16SrRNA gene sequencing analysis.

[0018] An application scenario of pure-color Red Bull, including the following innovative applications:

[0019] Development of stress-resistant gene resources: Utilizing high-altitude adaptability genes such as EPAS1 and HIF1A carried by pure-color red cattle, through somatic cell cloning technology, we can provide genetic resources for stress-resistant breeding of other cattle breeds;

[0020] Functional beef product development: Utilizing the conjugated linoleic acid (CLA) and omega-3 fatty acids found in pure red cattle muscle, beef products with health benefits such as lowering cholesterol and preventing cardiovascular disease should be developed. The beef products should contain CLA ≥ 0.8g / 100g and omega-3 fatty acid ≥ 0.5g / 100g.

[0021] Biofertilizer recycling system: The manure of pure red cattle is fermented through anaerobic fermentation technology to produce biogas. The remaining biogas residue and liquid biogas are fermented with microbial agents to make organic biofertilizer for alpine pasture cultivation. The microbial agents contain Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis, with an effective viable count of ≥ 2×10 8 CFU / g.

[0022] Preferably, in the development of the stress-resistant gene resource, the somatic cell cloning technology adopts a manual cloning method, and the reconstructed embryos are activated by treating with 5 μM ionomycin for 5 minutes, followed by treating with 2 mM 6-DMAP for 4 hours.

[0023] Preferably, in the development of the functional beef product, supercritical CO2 extraction technology is used to enrich the functional components in the beef, and the extraction conditions are pressure 30 MPa, temperature 45°C, and CO2 flow rate 20 L / h.

[0024] Preferably, in the biofertilizer recycling system, the conditions for anaerobic fermentation are a temperature of 35±2° C., a pH value of 7.0-7.5, a C / N ratio of 25-30:1, and a fermentation period of 25-30 days.

[0025] Beneficial effects

[0026] The present invention provides a breeding technology and application of pure-color red cattle, which has the following beneficial effects:

[0027] 1. The present invention provides a breeding technology and application for pure-color red cattle. This technology uses Gaoligong Mountain yellow cattle as the maternal parent and Limu Tibetan cattle as the paternal parent. By detecting the homozygous dominant genotypes of the c.901C>T locus of the MC1R gene and the c.1267G>A locus of the TYRP1 gene, parents are accurately screened to ensure that the offspring's coat color is pure red, completely resolving the industry's pain point of being pressured by buyers due to coat color issues. Combined with genome-wide association analysis technology to screen genes related to intramuscular fat deposition, as well as epigenetic regulation during pregnancy and precise nutritional supplementation during fattening, the pure-color red cattle bred have rich marbling, conjugated linoleic acid (CLA) content ≥0.8g / 100g and ω-3 fatty acid content ≥0.5g / 100g in meat, reaching the standard of high-end beef. The market price is 40%-60% higher than that of ordinary cattle, significantly improving the product's market competitiveness and farmers' income.

[0028] 2. The present invention provides a breeding technology and application for pure-color red cattle. By utilizing high-altitude adaptability genes such as EPAS1 and HIF1A carried by pure-color red cattle, somatic cell cloning technology is used to provide key gene resources for stress-resistant breeding of other alpine cattle breeds, thus breaking through the technical bottleneck of seed selection and breeding in alpine areas. The feces of pure-color red cattle are converted into biogas and organic biofertilizer through anaerobic fermentation, establishing an "aquaculture-energy-planting" ecological cycle system. This not only solves the problem of aquaculture waste pollution, but also reduces the dependence of feed cultivation in alpine areas on external fertilizers, achieving efficient resource utilization, and transforming alpine animal husbandry from a traditional extensive type to a green, sustainable modern industry, effectively promoting the coordinated development of the economy and ecology in alpine areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The Gaoligongshan yellow cattle female parent of the present invention;

[0030] Figure 2 The male parent of the Limu Tibetan cattle of the present invention;

[0031] Figure 3 The present invention is a hybrid calf. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] Embodiment: The present invention provides a breeding technology for pure-color red cattle:

[0034] S1. By detecting homozygous dominant genotypes at the c.901C>T locus of the MC1R gene and the c.1267G>A locus of the TYRP1 gene, Gaoligong Mountain Yellow Cattle females and Limu Tibetan Cattle males were screened to ensure they carried the homozygous dominant red allele. Genome-wide association analysis was also used to select individuals with dominant genotypes for genes associated with intramuscular fat deposition. The selected Gaoligong Mountain Yellow Cattle females were also required to have an 82-bp insertion mutation in the 3'UTR region of the Myostatin gene, and the Limu Tibetan Cattle males were required to carry the dominant allele at the K232A locus of the CAST gene.

[0035] S2. Synchronize the estrous cycle of Gaoligong Mountain steers using prostaglandin F2α combined with gonadotropin-releasing hormone (GnRH). Timed artificial insemination is performed 24-30 hours after vaginal plug removal using frozen semen from Limu Tibetan cattle that has undergone sperm selection. The dosage of prostaglandin F2α is 25 mg / head, and the dosage of GnRH is 100 μg / head. The vaginal plug is removed 8-9 days after placement.

[0036] S3. Blastocysts were biopsied on day 6-7 after in vitro fertilization (IVF). Real-time quantitative PCR was used to detect KIT gene copy number variation and ASIP gene methylation status. Embryos with a pure color phenotype and no white spot alleles were selected. SRY gene testing was performed for sex identification, with female embryos preferentially transferred. The methyl donor complex was encapsulated in liposomes to enhance bioavailability. The liposomes were composed of phosphatidylcholine and cholesterol in a 3:1 mass ratio and had a particle size of 150-200 nm.

[0037] S4. On gestational days 45-60, add 0.5%-1.0% of a methyl donor complex (containing 15 mg / kg of folic acid, 0.03 mg / kg of vitamin B12, and 1200 mg / kg of choline) to the maternal feed to influence fetal myofiber development and adipocyte differentiation through epigenetic regulation. The methyl donor complex also contains nanoparticles of conjugated linoleic acid (CLA) with a particle size of 100-200 nm, capable of crossing the placental barrier.

[0038] S5. Within 72 hours after birth, calves were inoculated with a core bacterial preparation derived from Limu Tibetan cattle by gavage. The bacterial preparation contained dominant strains of Ruminococcus, Bacteroides, and Prevotella. The inoculation dose was 1 × 10 9 CFU / mL, inoculated continuously for 3 days to promote the digestion ability of calves on alpine roughage. In the targeted regulation of calf intestinal flora, the preparation method of the core flora preparation includes: collecting rumen fluid of healthy Limu Tibetan cattle, enriching it through anaerobic culture, separating flora components of different densities by density gradient centrifugation, and screening flora components with a relative abundance of fiber-decomposing bacteria ≥40% through 16SrRNA gene sequencing analysis;

[0039] S6. Dynamically adjust the feed formula based on the cattle's real-time weight, backfat thickness, and muscle development. Add 0.02% β-hydroxy-β-methylbutyric acid and 0.01% chromium picolinate to the feed 60 days before slaughter to promote intramuscular fat deposition and improve meat color.

[0040] The application scenarios are as follows:

[0041] Development of stress-resistant gene resources: Utilizing high-altitude adaptability genes such as EPAS1 and HIF1A carried by pure-color red cattle, somatic cell cloning technology is used to provide gene resources for stress-resistant breeding of other cattle breeds. Somatic cell cloning technology uses manual cloning. Reconstructed embryos are activated by treating with 5μM ionomycin for 5 minutes, followed by 2mM 6-DMAP for 4 hours.

[0042] Functional beef product development: Utilizing the conjugated linoleic acid (CLA) and ω-3 fatty acids found in pure red cattle muscle, beef products with health benefits such as lowering cholesterol and preventing cardiovascular disease are developed. The CLA content in the beef products is ≥0.8g / 100g, and the ω-3 fatty acid content is ≥0.5g / 100g. Supercritical CO2 extraction technology is used to enrich the functional ingredients in the beef. The extraction conditions are a pressure of 30MPa, a temperature of 45°C, and a CO2 flow rate of 20L / h.

[0043] Biofertilizer recycling system: The manure of pure red cattle is fermented through anaerobic fermentation technology to produce biogas. The remaining biogas residue and liquid biogas are fermented with microbial agents to make organic biofertilizer for alpine pasture cultivation. The microbial agents contain Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis, with an effective viable count of ≥2×10 8 CFU / g, the specific conditions for anaerobic fermentation are temperature 35±2℃, pH value 7.0-7.5, C / N ratio 25-30:1, and fermentation period is 25-30 days.

[0044] Comparative Example: Comparative Test and Result Analysis

[0045] like Figure 1-3 In order to verify the effect of the hybridization improvement method, a comparative experiment was set up. The comparison group used the traditional hybridization method, as shown in the following table:

[0046] Table 1: Growth performance comparison

[0047] index Numerical Average birth weight of treatment groups (kg) 35.2 Average birth weight of the control group (kg) 32.1 Average final weight of treatment groups (kg) 278.5 Average final weight of the control group (kg) 240.3 Daily weight gain of treatment group (g) 450.6 Daily weight gain of control group (g) 385.4

[0048] Table 2: Comparison of meat quality indicators

[0049] index Numerical Marbling score of treatment groups (1-9) 7.2 Marbling score of the control group (1-9) 4.1 CLA content in treatment groups (g / 100g) 0.95 CLA content in the control group (g / 100g) 0.42 ω-3 fatty acid content in treatment groups (g / 100g) 0.61 ω-3 fatty acid content in the control group (g / 100g) 0.25

[0050] Table 3: Comparison of coat color purity and economic benefits

[0051] index Numerical Pure color rate of treatment group (%) 100 Pure color rate of control group (%) 30 Average output value of a single cow in the treatment group (yuan) 23,800 Average output value of a single cow in the control group (yuan) 14,418 Output value increase rate (%) 65.1

[0052] Table 4: Statistical significance analysis

[0053] index p-value Final weight difference (p value) <0.001 Difference in marbling score (p value) <0.001 Difference in CLA content (p value) <0.001 Difference in ω-3 fatty acid content (p value) <0.001

[0054] As can be seen from the table data, the pure-color red cattle bred by this technology are significantly better than those bred by traditional methods in terms of growth performance, meat quality and economic benefits, and all differences are statistically significant (p<0.001). In particular, the coat color purity reaches 100%, effectively solving the industry pain point of price reduction due to coat color issues. The output value of a single cow has increased by 65.1%, fully demonstrating the innovativeness of this technology.

[0055] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A breeding technology for pure red cattle, characterized in that: The specific steps include: S1. By detecting the homozygous dominant genotypes at the c.901C>T locus of the MC1R gene and the c.1267G>A locus of the TYRP1 gene, Gaoligong Mountain Yellow Cattle females and Limu Tibetan Cattle sires were screened to ensure they carried the homozygous dominant red allele. Genome-wide association analysis was also used to select individuals with dominant genotypes for genes associated with intramuscular fat deposition. S2. Synchronize the estrous cycle of Gaoligong Mountain steers using prostaglandin F2α combined with gonadotropin-releasing hormone. Timed artificial insemination is performed 24-30 hours after thrombosis removal using frozen semen from Limu Tibetan cattle that has been processed using sperm selection technology. S3. Perform a biopsy of the blastocysts on day 6-7 after IVF and use real-time quantitative PCR to detect KIT gene copy number variation and ASIP gene methylation status. Select embryos with a pure color phenotype and no white spot alleles. Also, perform sex determination using SRY gene testing, prioritizing female embryos for transfer. S4. On gestational days 45-60, 0.5%-1.0% of a methyl donor complex (containing 15 mg / kg of folic acid, 0.03 mg / kg of vitamin B12, and 1200 mg / kg of choline) is added to the maternal feed to influence fetal myofiber development and adipocyte differentiation through epigenetic regulation. The methyl donor complex also contains nano-coated conjugated linoleic acid with a particle size of 100-200 nm, capable of crossing the placental barrier. S5. Within 72 hours after birth, calves were inoculated with a core bacterial preparation derived from Limu Tibetan cattle by gavage, containing dominant strains of Ruminococcus, Bacteroides, and Prevotella, at a dose of 1 × 10 9 CFU / mL, inoculated for 3 consecutive days, to promote the digestion ability of calves on alpine roughage; S6. Dynamically adjust the feed formula based on the cattle's real-time weight, backfat thickness, and muscle development. Add 0.02% β-hydroxy-β-methylbutyric acid and 0.01% chromium picolinate to the feed 60 days before slaughter to promote intramuscular fat deposition and improve meat color.

2. The breeding technology of pure red cattle according to claim 1, characterized in that: The female parent of the Gaoligong Mountain Yellow Cattle screened in S1 must also satisfy the requirement of having an 82 bp insertion mutation in the 3'UTR region of the Myostatin gene, and the male parent of the Limu Tibetan Cattle must carry the dominant allele of the K232A site of the CAST gene.

3. The breeding technology of pure red cattle according to claim 1, characterized in that: The dosage of prostaglandin F2α in the S2 is 25 mg / head, the dosage of gonadotropin-releasing hormone is 100 μg / head, and the time of withdrawal is 8-9 days after the vaginal suppository is placed.

4. The breeding technology of pure red cattle according to claim 1, characterized in that: The bioavailability of the methyl donor complex in S3 is improved by liposome encapsulation technology. The liposome is composed of lecithin and cholesterol in a mass ratio of 3:1, and the particle size is 150-200 nm.

5. The breeding technology of pure color red cattle according to claim 1, characterized in that: In the directed regulation of the calf's intestinal flora, the preparation method of the core flora preparation includes: collecting rumen fluid from healthy Limu Tibetan cattle, enriching it through anaerobic culture, separating flora components of different densities using density gradient centrifugation, and screening flora components with a relative abundance of fiber-decomposing bacteria ≥40% through 16SrRNA gene sequencing analysis.

6. Application of the pure-color red cattle bred according to any one of claims 1 to 5, characterized in that Including the following innovative applications: Development of stress-resistant gene resources: Utilizing high-altitude adaptability genes such as EPAS1 and HIF1A carried by pure-color red cattle, through somatic cell cloning technology, we can provide genetic resources for stress-resistant breeding of other cattle breeds; Functional beef product development: Utilizing the conjugated linoleic acid (CLA) and omega-3 fatty acids found in pure red cattle muscle, beef products with health benefits such as lowering cholesterol and preventing cardiovascular disease should be developed. The beef products should contain CLA ≥ 0.8g / 100g and omega-3 fatty acid ≥ 0.5g / 100g. Biofertilizer recycling system: The manure of pure red cattle is fermented through anaerobic fermentation technology to produce biogas. The remaining biogas residue and liquid biogas are fermented with microbial agents to make organic biofertilizer for alpine pasture cultivation. The microbial agents contain Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus licheniformis, with an effective viable count of ≥ 2×10 8 CFU / g.

7. The application scenario of the pure color Red Bull according to claim 6, characterized in that: In the development of the stress-resistant gene resource, the somatic cell cloning technology adopts the manual cloning method, and the reconstructed embryos are activated by treating with 5 μM ionomycin for 5 minutes, followed by treating with 2 mM 6-DMAP for 4 hours.

8. The application scenario of the pure color Red Bull according to claim 6, characterized in that: In the development of the functional beef product, supercritical CO2 extraction technology is used to enrich the functional components in the beef. The extraction conditions are pressure 30MPa, temperature 45°C, and CO2 flow rate 20L / h.

9. The application scenario of the pure color Red Bull according to claim 6, characterized in that: In the biofertilizer recycling system, the conditions for anaerobic fermentation are a temperature of 35±2° C., a pH value of 7.0-7.5, a C / N ratio of 25-30:1, and a fermentation period of 25-30 days.

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