CN110959575B - Guangdong auricularia auricula pig feeding method capable of promoting growth of farrowing muscle in gestation period - Google Patents
Guangdong auricularia auricula pig feeding method capable of promoting growth of farrowing muscle in gestation period Download PDFInfo
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- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
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Abstract
The invention belongs to the technical field of livestock and poultry breeding, and particularly relates to a feeding method for Guangdong small eared pigs in gestation period, which can promote the development of farrowing muscles. According to the invention, according to the development characteristics of a pig fetus and uterine contents thereof, the gestation period of the Guangdong auricularia auricula sow is divided into six specific periods, the feeding amount of the Guangdong auricularia auricula sow is respectively determined, and meanwhile, two gestation materials in the early period and the later period of pregnancy are combined to replace the conventional 'step-by-step high' mode in North America and China and the 'low-height-low-height' feeding mode in Europe, so that accurate feeding is implemented, and the reproductive performance of the sow is optimized. Provides related data support and theoretical guidance for establishing reasonable feeding amount for local pig breeds in different gestation periods in China.
Description
Technical Field
The invention belongs to the technical field of livestock and poultry breeding, and particularly relates to a feeding method for Guangdong small eared pigs in gestation period, which can promote the development of farrowing muscles.
Background
In recent years, with the improvement of the living standard of consumers, the quality requirement of pork is increasing. At present, the main group for commercial pig breeding in China is high lean type pig breeds and hybrid offspring thereof introduced from abroad. Its advantages are high growth speed, high lean meat percentage and high feed conversion rate, but its meat quality and anti-stress power are obviously reduced. Therefore, the pork of the local pig breed in China occupies an important position in the high-grade brand pork of the international big pork market.
The Guangdong small eared pig belongs to a good local pig breed in south China, is one of eight good local pig breeds in China, is also one of local pig breeds with the largest feeding amount in two broad areas, and has the advantages of good maternal property, strong stress resistance, tender meat, fresh taste, compact body type, stable heritability and the like. Meanwhile, the problems of low growth speed, low daily gain, low slaughter rate, low lean meat percentage, low feed conversion rate and the like of the sows are greatly limited in the rapid development and large-scale production in the pig industry due to the fact that the pregnant backfat is too thick and the coefficient of variation between primary weight litter is large.
Sows are the core of the whole pig industry, and the nutritional requirements at different stages of the breeding cycle are greatly different. Up to now, the nutritional needs in the early and middle stages of pregnant sows given by NRC (2012) are a constant value. Fetal development and its associated tissues do not grow at the same rate throughout pregnancy. It is noted that the pregnant nutritional needs of the eurotium cristatum pig are still blank. Therefore, the method has great practical significance for dividing different gestation stages of the Guangdong small eared pig and researching different feeding amounts of the Guangdong small eared pig on the body conditions of sows, the birth weight of piglets and the muscle development of offspring of the piglets, and improving the breeding efficiency and the muscle growth and development of the offspring of the Guangdong small eared pig.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide the feeding method for the Guangdong small eared spotted pigs in the gestation period, which promotes the muscle development of the offspring, and the method takes the physiological characteristics of the sows in different gestation periods as a starting point, combines the reproductive nutrition requirements of the sows, and refines the feeding amount in each gestation period so as to improve the birth weight and the muscle development of the offspring.
The purpose of the invention is realized by the following technical scheme:
a feeding method for Guangdong small eared pigs in gestation period for promoting the development of farrowing muscles comprises the following steps:
(1) on the day of hybridization, the daily feed intake (ADFI) of each gilt of the Guangdong small earbuds is 0.30 Kg;
(2) after 1-3 days of hybridization, the daily feed intake (ADFI) of each Guangdong auricularia auricula sow is 0.63 Kg;
(3) 4-30 days after the hybridization, the daily feed intake (ADFI) of each Guangdong auricularia auricula sow is 0.85 Kg;
(4) after the breeding, the daily feed intake (ADFI) of each Guangdong auricularia auricula sow is 1.19 Kg;
(5) after the breeding, the daily feed intake (ADFI) of each Guangdong auricularia auricula sow is 1.27Kg after 61-85 days;
(6) after the breeding, the daily feed intake (ADFI) of each Guangdong auricularia auricula sow is 1.98Kg after the breeding for 86-112 days;
during pregnancy, the Average Daily Feed Intake (ADFI) of each gilt of the Pleurotus Ostreatus is 1.30 Kg;
the feed comprises the following components: corn, bean pulp, barley, bran, rice bran meal, coarse bran and premix;
the feed is preferably divided into two pregnancy feeds of 1-85 days of pregnancy and 86-112 days of pregnancy;
the pregnancy material for 1-85 days comprises the following components in percentage by mass:
the pregnancy material for 86-112 days of pregnancy comprises the following components in percentage by mass:
wherein, the premix in 1kg of complete ration comprises the following components:
copper, iron, selenium, zinc, manganese, vitamin and vitamin B2Vitamin D3Vitamin E, vitamin K3Vitamin B2Vitamin B6Calcium pantothenate, nicotinic acid, vitamin B12；
The premix in 1kg of a full ration preferably comprises the following components:
75 mg of copper, 750 mg of iron, 0.20 mg of selenium, 750 mg of zinc, 187 mg of manganese, 150000IU of vitamin A and vitamin B2140 mg, vitamin D325000IU, vitamin E1000IU, vitamin K335 mg of vitamin B2140 mg of vitamin B670 mg, 350 mg calcium pantothenate, 500 mg nicotinic acid, vitamin B120.4 mg;
at present, two feeding modes exist in the gestation stage of sows, one mode is a 'step type' feeding mode which is adopted by North America as a representative mode, and the other mode is a 'concave type' feeding mode which is adopted by Denmark and European Union as a representative mode. However, the two modes are not ideal feeding modes for local pig breeds in gestation period in China. The invention deeply explores the influence of a suitable feeding mode in the gestation period on the body condition and the generation growth of the Guangdong small eared pigs, and specifically comprises the following steps: according to the development characteristics of a fetus and uterine contents thereof, the gestation period of the Guangdong auricularia auricula-judae sows is divided into six specific periods, the feeding amount of the Guangdong auricularia auricula-judae sows is respectively determined, and meanwhile, two gestation materials in the early period and the later period are combined to replace the traditional step-by-step high mode (only divided into three stages in the early, middle and later periods) in North America and domestic and low-high-low-high feeding mode (only divided into four stages) in Europe, so that accurate feeding is implemented, and the reproductive performance.
According to the invention, through animal feeding experiments and slaughtering experiments, the fact that the feeding mode of the first-born Guangdong auricle lesser pig can reduce the stillbirth rate and improve the birth weight and muscle weight of piglets in the gestation period is found; on the other hand, through molecular tests and tissue morphology researches, the feeding mode disclosed by the invention can improve the relative cross-sectional area of the muscle and the number of muscle fibers of the newborn piglets, up-regulate the expression (MyoG, MyCH-IIA and MyCH-IIX) of marker genes related to muscle development, simultaneously improve the content of Glu, Asp and total amino acid in the muscle and up-regulate the expression of amino acid transporters (ASCT2 and GLAST). Therefore, the feeding mode of the Guangdong auricularia auricula pig is adopted during the gestation period, and the expression of the muscle development related gene and the amino acid transfer carrier gene is influenced, so that the growth and development of the offspring muscle are facilitated, and the birth weight is improved.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a novel feeding mode, namely a ladder type feeding mode, aiming at the gestation period of a first-born Guangdong auricularia auricula pig, the mode divides the gestation period of the Guangdong auricularia auricula pig into six specific periods according to different nutritional requirements in each period, the feeding amount of the Guangdong auricularia auricula pig is respectively determined, the nutritional requirements in each period in the gestation period are refined, the feeding amount is more accurate, the target is more definite, and the ladder type mode is adopted to gradually improve the feed intake so as to achieve the optimal reproductive performance of the sow.
(2) Under the test condition, compared with a concave feeding mode, the ladder type feeding mode adopted by the Guangdong auricle-lesser pig in the gestation period can reduce the stillbirth rate, improve the birth weight and muscle weight of piglets, improve the expression of genes related to muscle development and amino acid transfer carrier genes, and be beneficial to promoting the growth and development of the muscles of the offspring, thereby improving the birth weight.
Drawings
Fig. 1 is a graph comparing the feed intake for two feeding modes.
FIG. 2 is a graph of the analysis of the results of the effect of two feeding patterns on the gene expression of the longissimus dorsi of piglets; wherein, A: comparing mRNA expression of myosin heavy chain and myogenesis related genes in longissimus dorsi of low-weight piglets in the ladder-shaped group and the concave-shaped group; b: comparison of mRNA expression of myosin heavy chain and myogenesis associated genes in the longissimus dorsi of piglets in the ladder and trough groups for average body weight, all data are expressed as mean ± standard error, marked by x (P <0.05), marked by x (P <0.01), and n is 4-7.
FIG. 3 is a graph of the analysis of the results of the effect of two feeding modes on piglet semitendinosus gene expression; wherein, A: comparison of mRNA expression of myosin heavy chain and myogenesis-associated genes in the semitendinosus of low-weight piglets in ladder and concave groups, B: comparison of mRNA expression of myosin heavy chain and myogenesis associated genes in the semitendinosus muscle of piglets in the ladder and trough groups for average body weight, all data are expressed as mean ± standard error, marked by x (P <0.05), marked by x (P <0.01), and n is 4-7.
FIG. 4 is a graph of the analysis of the results of the effect of two feeding patterns on piglet psoas major gene expression; wherein, A: comparing mRNA expression of myosin heavy chain and myogenesis related gene in low-weight piglet psoas major muscle of ladder type group and concave type group, B: comparison of mRNA expression of myosin heavy chain and myogenesis associated genes in the psoas major muscles of piglets in the ladder and concave groups for average body weight, all data are expressed as mean ± standard error, marked by x (P <0.05), marked by x (P <0.01), and n is 4-7.
FIG. 5 is a graph of the analysis of the results of the effect of two feeding patterns on the gene expression of the longissimus dorsi amino acid vector of newborn piglets; wherein, A: comparing the mRNA expression of the gene related to the amino acid transport vector in the longissimus dorsi of the low-weight piglets in the ladder-shaped group and the concave-shaped group, B: compared with mRNA expression of genes related to amino acid transport vectors in the longissimus dorsi of piglets in the ladder group and the concave group, all data are expressed as mean values +/-standard error, the difference is marked by x (P <0.05), the difference is marked by x (P <0.01), and n is 4-7.
FIG. 6 is a graph of the analysis of the results of the effect of two feeding patterns on the gene expression of the amino acid vector of psoas major of newborn piglets; wherein A is the mRNA expression comparison of the related genes of the amino acid transport vectors in the psoas major muscles of the low-weight piglets in the ladder-shaped group and the concave-shaped group, and B: compared with mRNA expression of genes related to the amino acid transport vectors in the psoas major muscles of piglets in the ladder-shaped group and the female-shaped group, all data are expressed as mean values +/-standard error, the difference is marked by x (P <0.05), the difference is marked by x (P <0.01), and n is 4-7.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
The premix in 1kg of the full ration in the examples preferably comprises the following components:
75 mg of copper, 750 mg of iron, 0.20 mg of selenium, 750 mg of zinc, 187 mg of manganese, 150000IU of vitamin A and vitamin B2140 mg, vitamin D325000IU, vitamin E1000IU, vitamin K335 mg of vitamin B2140 mg of vitamin B670 mg, 350 mg calcium pantothenate, 500 mg nicotinic acid, vitamin B120.4 mg;
example 1 feeding mode
In practice, a limited feeding mode is usually adopted to ensure the best fecundity of the breeding sows. However, the sow is not satisfied with satiety in pregnancy due to excessive feeding limitation, stereotypy behavior is frequent, and meanwhile, the sow is low in reproductive performance due to insufficient nutrition supply and intrauterine growth retardation. Therefore, the suitable feeding mode is the premise of ensuring the high-efficiency breeding efficiency of the sows. Previous studies divided gestation into early, intermediate and late stages and determined the amount fed at each stage separately. However, recent studies suggest that it is more necessary to determine the nutritional requirements of the fetus and its contents based on the developmental window period. Therefore, according to the development characteristics of a fetus and uterine contents thereof, the invention divides the gestation period of the Guangdong auricular female pig into six specific periods and respectively determines the feeding amount of the Guangdong auricular female pig, and replaces the traditional 'step-by-step high' mode (only divided into three stages in the front, middle and later periods) in North America and China and the 'low-high-low-high' feeding mode (only divided into four stages) in Europe, and the precise feeding is implemented, so that the reproductive performance of the female pig is optimized.
In the following embodiments of the invention, a ladder type feeding mode is adopted in the gestation period of the first-born Guangdong Tremella Aurantialba pigs, the specific feed intake of each Guangdong Tremella Aurantialba sow is shown in table 1 and figure 1, the concave feeding mode in the prior art is used as a comparison, the specific feed intake and nutrient components are shown in table 1 and table 2, and the influence of the ladder type feeding mode on the farrowing performance and the farrowing muscle development of the Guangdong Tremella Aurantialba pigs is deeply researched.
TABLE 1 feed intake corresponding to two feeding modes during gestation
Note: in the table, the feed intake is the real-time average daily feed intake of the sows in the test, the feed intake of the sows in the ladder group and the feed intake of the sows in the concave group in the feeding modes of 1-3 d, 4-30 d, 31-60 d, 61-85 d and 86-112 d are compared, and the feed intake of the sows in the two groups in the feeding modes of 1-3 d, 4-30 d, 31-60 d and 61-85 d is remarkably different (P is less than 0.0001).
TABLE 2 nutritional components and nutritional levels of two pregnancy feeds at the early and late stages of pregnancy
Example 2 Effect of feeding Pattern on farrowing Performance of the Cantonese auricularia auricula pig
In order to verify the effect of the invention, the breeding performance of the Guangdong auricularia auricula sows is compared between the stepped feeding mode group and the concave feeding mode group.
1. Test animal
The test animal is a Guangdong small eared pig, and the test site is a Lemin core pig farm of No. one food Co., Zhanjiang city, Guangdong province.
2. Test processing and grouping
Selecting 80 pigs with weights and backfat of the original Guangdong small eared piglets which are consistent, randomly dividing into 2 groups, repeating each group for 40 times, repeating 1 pig for each group, adopting the step type feeding mode in the embodiment 1 for the first group, and adopting the concave type feeding mode in the embodiment 1 for the second group. The feeding amount of the two groups is different in 1-85 days of pregnancy, so that the total food intake in the whole pregnancy stage is equal to the average daily food intake.
3. Data processing and analysis
The data obtained in this study were presented as mean ± sd or mean ± sd, all data were analyzed for significance using the t-test method (P <0.05 indicates significant difference, P <0.01 and P <0.001 indicates very significant difference), and data analysis and mapping were performed by GraphPad Prism software.
4. Test results
(1) Influence of different feeding modes of sows in gestation period on backfat and body weight of sows
As can be seen from Table 3, both feeding modes had no significant effect on the sow's gestational backfat and body weight and their changes (P > 0.05).
TABLE 3 Effect of two feeding modes on sow backfat and body weight1
Note:1all data are expressed as mean ± standard deviation. The table shows the comparison of backfat and body weight and their changes in different gestation stages between the ladder and concave groups.
(2) Influence of different feeding modes of sows in gestation period on reproductive performance of sows
As can be seen from Table 4, the two feeding patterns had no significant difference in the total litter size, the number litter size in live births, the weight of newborn litter, etc. of the sows (P > 0.05). The ladder-type group has a significantly lower dead-fetus rate and invalid-young rate than the concave group (P <0.05), and the number of the dead tires of the concave group is twice that of the ladder-type group, and in addition, the initial weight of the ladder-type group tends to be higher than that of the concave group (P0.06).
TABLE 4 Effect of two feeding patterns on sow reproductive Performance1
| Item | Step type group | Concave group | P value |
| Number of samples (n) | 32 | 34 | |
| Gross litter size (n) | 11.09±1.80 | 11.82±2.50 | 0.18 |
| Litter size of live birth (n) | 10.59±1.93 | 10.74±2.06 | 0.78 |
| Number of dead tires (n) | 0.47±0.71 | 0.97±1.36 | 0.15 |
| Percentage of dead fetus (%) | 4.23 | 8.21 | 0.02 |
| Rate of invalid offspring2(％) | 4.51 | 9.20 | 0.01 |
| Birth weight (g) | 650.12±123.48 | 596.64±106.92 | 0.06 |
| Birth weight (g) | 6808.69±1463.66 | 6336.56±1386.20 | 0.18 |
| Coefficient of variation between cells | 0.17±0.06 | 0.19±0.07 | 0.21 |
Note:1all data are expressed as mean ± standard deviation. The indexes related to the reproductive performance of the sows in the ladder-shaped group and the female group are compared;2the invalid rate is the ratio of dead fetus and mummy rate.
From the test results, the two feeding modes have no obvious influence on the change of the back fat and the body weight of the sow and the body weight change of the back fat. But the ladder-shaped group can obviously reduce the stillbirth rate and the ineffective farrowing rate and has the tendency of improving the birth weight of piglets.
Example 3 Effect of feeding Pattern on muscle Mass of Pleurotus Cantonensis pigs
1. Test materials
(1) Test animal
The newborn piglet is hybridized with the female parent of the Guangdong small earring pig and the duroc male pig.
(2) Sample collection
On the day of delivery of the sows, 7 sows (the specific feeding method is the same as that in example 2) in the ladder-type feeding mode group and 5 sows (the specific feeding method is the same as that in example 2) in the concave feeding mode group are selected (the selected sows are all normal-litter-size sows with good mammary gland development and health), and 24 newborn piglets with average weight and low weight are selected from each sow in each group. Weighing before eating colostrum, and then slaughtering on site to collect samples. After slaughter, centrifuge, liver, spleen, lung, kidney, small intestine, supraspinatus, infraspinatus, brachiocephalus, soleus, semitendinosus, psoas major, longissimus dorsi were rapidly dissected and weighed, and one each of frozen sections and 3 molecular samples were collected in a course along the fibers of the brachiocephalus, soleus, semitendinosus, psoas major, longissimus dorsi muscle.
2. Data processing and analysis
The data obtained in this study are presented as mean ± sd or mean ± sd, and all data were analyzed for significance using the t-test method.
3. Test results
As can be seen from table 5, there was no significant difference in muscle weight in the low body weight group for both feeding modes (P > 0.05). As can be seen from Table 6, the weight of supraspinatus, brachiocephalic triceps and psoas major of normal newborn heavy piglets in the ladder-shaped group can be significantly influenced (P <0.05), and the weight of supraspinatus, brachiocephalic triceps and psoas major of normal heavy piglets in the ladder-shaped group is significantly higher than that in the concave-shaped group (P < 0.05).
TABLE 5 Effect of two feeding modes on Low-weight newborn piglet muscle weight (units, g)1
TABLE 6 Effect of two feeding modes on average body weight of newborn piglets muscle weight (units, g)1
Note:1all data are expressed as mean ± standard deviation. Table 5 compares the weights of the supraspinatus, infraspinatus, brachiocephalus, longissimus dorsi, psoas major, soleus and semitendinosus muscles of the low body weights of the ladder and concave groups; table 6 compares the weights of supraspinatus, infraspinatus, brachiocephalus, longissimus dorsi, psoas major, soleus and semitendinosus muscles for the ladder and concave groups.
Example 4 Effect of feeding Pattern on the Gene developing muscle in the Pleurotus Ostreatus pig
1. Test materials
(1) Test sample
The young Guangdong earland piglets had low and average body weight of the longissimus dorsi, semitendinosus and psoas major (samples collected in example 3).
(2) Reagent
DEPC treated Water, agar sugar powder, solid-phase RNase scavenger, RNA sample buffer, 4sGleRed nucleic acid dye, 50 XTAE buffer, etc. available from Shanghai Biotechnology engineering Co., Ltd；PrimeScriptTMRT reagent Kit with gDNA Eraser from Takara, 2X PCR Master Mix from Shanghai Biyuntian Biotechnology Limited; a3042 × RealStar Green Fast mix (with ROX II) from GenStar; trizol was purchased from Aibaimeng Biotech, Inc., Guangzhou.
(3) Instrument for measuring the position of a moving object
Electrophoresis gel imaging analyzer (BIO RAD Life medicine products, Inc., Molecular Imager ChemiDOC XRS + with Image LabTM Software model); clean bench (AIRTECH corporation, model 1809532W); PCR instrument (BIO RAD Life medicine products Co., Ltd., model S100048/48); fluorescent quantitative PCR instrument (Thermo fisher scientific, Quantstrudio 6 Flex).
2. Method of producing a composite material
(1) HE staining of muscle tissue sections
And (3) preparing frozen sections: 1) tissue fixation and dehydration: placing the trimmed tissues in 15 wt% of sucrose solution, dehydrating and precipitating at 4 ℃ in a refrigerator, and transferring into 30 wt% of sucrose solution, dehydrating and precipitating at 4 ℃ in a refrigerator; 2) OCT embedding: taking out the dehydrated tissue, slightly absorbing surface water with filter paper, placing the tissue on an embedding platform with the section upward, dripping OCT embedding agent around the tissue, placing the embedding platform on a quick-freezing platform of a freezing microtome for quick-freezing embedding, and slicing after the OCT turns white and becomes hard. Directly freezing the fresh tissue into a slice without fixing and dehydrating, and directly flattening the tissue of a target part into the OCT embedding medium embedding slice by using a scalpel; 3) slicing: fixing the embedding table on a slicing machine, firstly roughly cutting to trim and flatten the tissue surface, then starting slicing, wherein the slicing thickness is 8-10 mu m, and flatly placing a clean glass slide above the cut tissue piece to stick the tissue on the glass slide; 4) after the label is written on the slice, the slice is stored at the temperature of minus 20 ℃ for standby.
HE staining: 1) paraffin section dewaxing to water: sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-75% V/V alcohol 5min, and washing with tap water; 2) hematoxylin staining: staining the slices in hematoxylin staining solution for 3-5min, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue to the blue solution, and washing with running water; 3) eosin staining: the slices are dehydrated by gradient alcohol of 85 percent V/V and 95 percent V/V for 5min respectively, and are dyed in eosin dye solution for 5 min; 4) dewatering and sealing: placing the slices in anhydrous ethanol I5 min-anhydrous ethanol II 5 min-anhydrous ethanol III 5 min-xylene I5 min-xylene II 5min for transparency, and sealing with neutral gum; 5) microscopic examination and image acquisition and analysis. (the specific experimental operation is carried out by Wuhan Severe Biotech Co., Ltd.).
(2) Total RNA extraction, reverse transcription, common PCR detection and qPCR detection
Total RNA extraction: about 20mg of muscle sample was taken in a 2mL sterile enzyme-free centrifuge tube containing 800. mu.L Trizol, homogenizing for 2min, centrifuging at 12000rpm for 15min at 4 deg.C, adding 200 μ L chloroform, mixing for 15s, standing for 15min, centrifuging at 12000rpm for 15min at 4 deg.C, collecting supernatant 400 μ L, centrifuging at 1.5mL sterile enzyme-free centrifuge tube, adding 400 μ L isopropanol, vibrating gently from top to bottom, standing at-20 deg.C for 10min, centrifuging at 12000rpm for 10min at 4 deg.C, removing supernatant, washing with 500 μ L75% V/V alcohol, centrifuging at 12000rpm for 3min at 4 deg.C, removing supernatant, naturally drying RNA to translucency, adding 20 μ L DEPC water to dissolve RNA (all above operations are performed on sterile operation table, colloid system is 6 μ L DEPC water, 2 μ L dissolved RNA, 2 μ L5 RNA buffer). After the extracted RNA was confirmed to have not been degraded by electrophoresis, the RNA concentration was determined, and the RNA of all samples was diluted with DEPC water to a uniform concentration of 1000 ng/. mu.L.
Synthesis of cDNA: 1.0 mu g of total RNA of the sample is accurately absorbed by referring to the instruction of a TaKaRa reverse transcription kit for reverse transcription synthesis of cDNA.
1) The amount of DNA in the sample was digested and the reaction system is shown in Table 7, 42 ℃ for 2 min.
2) The reaction solution was prepared on ice, Master Mix was prepared first and then dispensed into each tube, and the reaction system was shown in Table 837 ℃ for 15min, 85 ℃ for 5sec, 4 ℃ for storage. The reverse transcription product is stored at-20 ℃ for later use.
TABLE 7 digestion System of DNA in RNA samples
TABLE 8 Synthesis System of cDNA
And (3) common PCR detection: primers were designed using Primer 3 software (see Table 12 for specific Primer sequences), and the designed primers were sent to Biotechnology Ltd to synthesize primers, and the synthesized primers were diluted for general PCR detection (reaction system: 1. mu.LcDNA, 10. mu.L Mix, 1.6. mu.L mixed Primer, 7.4. mu.L DEPC water; PCR program: 94 ℃ 3min, 94 ℃ 30s, 55 ℃ 30s, 72 ℃ 1min, go to step 2for 30cycles 72 ℃ 10min, 4 ℃ FOREVER). After marker by marker, the size and feasibility of the primer are verified by electrophoresis.
Real-time fluorescent quantitative PCR detection: preparing a computer system (the specific primer sequence is shown in table 12), adding the reaction system shown in table 9 into a 384-well quantitative PCR plate, carrying out PCR, observing a dissolution curve, determining that only one peak exists, and finally calculating the gene expression amount through a Ct value.
The reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 s; denaturation at 95 ℃ for 15s, annealing at 60 ℃ (according to the specific primer setting) for 30s, for 40 cycles. And (3) calculating the relative expression quantity of mRNA of each gene in the sample by adopting a 2-delta Ct method according to threshold value cycles (Cts) of the target gene and the internal reference, wherein the delta Ct (Ct target gene-Ct internal reference gene) experimental group- (Ct target gene-Ct internal reference gene) control group.
TABLE 9 real-time fluorescent quantitative PCR reaction System
(3) Data processing and analysis
The data obtained in this study were presented as mean ± sd or mean ± sd, all data were analyzed for significance using the t-test method (P <0.05 indicates significant difference, P <0.01 and P <0.001 indicates very significant difference), and data analysis and mapping were performed by GraphPad Prism software.
3. Test results
(1) Influence of different feeding modes of sows in gestation period on number and cross-sectional area of muscle fibers of newborn piglets
As can be seen from Table 10, the number of muscle fibers in the psoas major of the low-weight newborn piglets in the ladder-shaped group was significantly higher than that in the concave-shaped group (P <0.05), and there was no significant difference in the other groups (P > 0.05). As can be seen from table 11, the relative cross-sectional area of the longissimus dorsi in the primary piglets of average body weight in the ladder group was significantly higher than that in the concave group (P <0.05), with no other significant difference (P > 0.05).
TABLE 10 Effect of two feeding modes on the number of muscle fibers and the cross-sectional area of low-weight piglets1
Note:1all data are expressed as mean ± standard deviation. The table compares the number of fibers and relative cross-sectional areas of the longissimus dorsi, semitendinosus and psoas major muscles in the low-weight piglets of the ladder and concave groups.
TABLE 11 Effect of two feeding modes on average weight piglet muscle fiber number and Cross-sectional area1
| Item | Step type group | Concave group | P value |
| Number of samples (n) | 7 | 5 | |
| Number of muscle fibers (n) | |||
| Longissimus dorsi | 404.00±62.05 | 376.86±156.06 | 0.51 |
| Semitendinosus muscle | 584.94±89.43 | 521.63±203.20 | 0.85 |
| Psoas major | 567.90±98.37 | 628.37±94.82 | 0.35 |
| Relative cross sectional area | |||
| Longissimus dorsi | 1.00±0.12 | 0.70±0.31 | 0.02 |
| Semitendinosus muscle | 1.00±0.44 | 0.77±0.28 | 0.38 |
| Psoas major | 1.00±0.31 | 0.91±0.19 | 0.57 |
Note:1all data are expressed as mean ± standard deviation. The table compares the number of fibers and relative cross-sectional areas of the longissimus dorsi, semitendinosus and psoas major muscles in the ladder and concave groups of average weight piglets.
(2) Influence of different feeding modes on expression of genes (MyHC I, MyHC IIA, MyHC IIX, MyHC IIB, MEF2, MyoG and MSTN) related to growth of longissimus dorsi muscle of piglets
As can be seen from fig. 2A, the relative gene expression level of MyCH-IIX in the ladder group tends to be higher than that in the notch group (P ═ 0.12), while the relative gene expression level of MyCH-I in the notch group tends to be higher than that in the ladder group (P ═ 0.10). As can be seen from fig. 2B, the relative expression of MEF2 and MyoG in the longissimus dorsi of the primary piglets in average body weight of the ladder group was significantly higher than that of the concave group (P < 0.05).
(3) Influence of different feeding modes on expression of genes (MyHC IIA, MyHC IIX, MyHC IIB, MEF2, MyoG, MSTN, MyoD and Myf5) related to growth of semitendinosus muscle of newborn piglets
As can be seen from fig. 3A, the relative expression level of the gene of the low-weight newborn piglet semitendinosus concave group MyCH-IIA is significantly higher than that of the ladder group (P <0.05), but the relative expression level of the gene of the ladder group MyCH-IIB tends to be higher than that of the concave group (P ═ 0.06), and there is no significant difference (P > 0.05). As can be seen from fig. 3B, the relative expression level of the genes of the medium-weight primary piglet semitendinosus concave type group MyCH-IIB was significantly higher than that of the ladder type group (P <0.05), and the relative expression level of Myf5 tended to be higher than that of the ladder type group (P ═ 0.11), while there was no significant difference (P >0.05), but the relative expression levels of the MEF2 and the MyoG genes in the ladder type group tended to be higher than that of the concave type group (P ═ 0.14, P ═ 0.18).
(4) Influence of different feeding modes on expression of genes (MyHC I, MyHC IIA, MyHC IIX, MyHC IIB, MEF2, MyoG, MSTN and Myf5) related to the development of lumbar muscle of newborn piglets
As can be seen from FIG. 4A, the relative expression levels of the genes of MyCH-IIA and MyCH-IIX in the low-weight newborn piglet psoas major muscle ladder type group are obviously higher than those in the concave type group (P <0.05), and no other significant difference exists (P > 0.05). As can be seen from fig. 4B, the relative gene expression level of MSTN in the ladder-shaped group of psoas major muscles of newborn piglets was significantly higher than that in the concave group (P <0.05), the relative gene expression level of MyCH-I in the concave group tended to be higher than that in the ladder-shaped group (P ═ 0.14), and there was no significant difference (P > 0.05).
Example 5 Effect of feeding mode on hydrolyzed amino acids in the muscle of Pleurotus ostreatus pigs in Guangdong and their transport Carriers
1. Test materials
(1) Test sample
The young Guangdong earland piglets had low and average body weight of the longissimus dorsi and psoas major (samples collected in example 3).
(2) Reagent
DEPC treated water, agar sugar powder, a solid phase RNase scavenger, an RNA loading buffer solution, a 4sGleRed nucleic acid dye, a 50 XTAE buffer solution and the like are purchased from Shanghai biological engineering Co., Ltd; PrimeScriptTMRT reagent Kit with gDNA Eraser from Takara, 2X PCR Master Mix from Shanghai Biyuntian Biotechnology Limited; a3042 × RealStar Green Fast mix (with ROX II) from GenStar; methanol (mass spectrum grade), acetonitrile (mass spectrum grade), trizol were purchased from ai bai meng biotechnology limited, guangzhou.
(3) Instrument for measuring the position of a moving object
Electrophoresis gel imaging analyzer (BIO RAD Life medicine products, Inc., Molecular Imager ChemiDOC XRS + with Image LabTM Software model); clean bench (AIRTECH corporation, model 1809532W); PCR instrument (BIO RAD Life medicine products Co., Ltd., model S100048/48); fluorescent quantitative PCR instrument (Thermo fisher scientific, Quantstrudio 6 Flex).
2. Method of producing a composite material
(1) Total RNA extraction, reverse transcription, common PCR detection and qPCR detection
The specific method is shown in example 4, and the specific primer sequences are shown in Table 12.
Primer sequences for genes of interest in Table 12
(2) LC-MS detection of muscle tissue hydrolysis amino acid
Sample extraction: taking 40mg (mass deviation is less than 10%) of a tissue sample from liquid nitrogen into a 2mL centrifuge tube, adding 400 mu L of ultrapure water, homogenizing the tissue (frequency is 30 times/second, 2min, and is repeated three times), taking 200 mu L of homogenate and 800 mu L of MeOH methanol/ACN acetonitrile (V: V, 1:1) into a 1.5mL centrifuge tube (simultaneously taking 50 mu L of homogenate and another 2mL centrifuge tube to be used as a QC sample), whirling for 30s, carrying out water bath ultrasound at 4 ℃ for 10min, then incubating at-20 ℃ for 1H, 14500rpm, centrifuging at 4 ℃ for 15min, taking supernatant, carrying out nitrogen drying, and finally adding 200 mu L of ACN acetonitrile/H2O water (V: V, 1:1), vortex for 30s, water bath ultrasound at 4 ℃ for 10min, 14500rpm, centrifuge for 15min at 4 ℃, filter the sample through 0.22 μm organic filter membrane in the sample bottle, and analyze by UHPLC-Orbitrap-MS/MS.
Liquid chromatography and mass spectrometry conditions: the chromatographic separation column was C18 Hypersil Gold (100 mm. times.2.1 mm, 1.9 μm), mobile phase A: 0.1% V/V Formic Acid (FA) + ultrapure water; b: acetonitrile (AC), flow rate set at 0.2mL/min, elution gradient: 0-7min, 5% -50% B; 7-8min, 50% -75% B; 8-9min, 75-80% B; 9-11min, 80-90% B; 11-15min, 90% -95% B; 15-20min 95% -5% of B. The sample size was 2. mu.L. Mass spectrum conditions: ESI ion source, using positive ion scanning mode; spraying voltage: 4000(+), 3000 (-); the atomizing gas is sheath gas (N2> 95%), and the pressure is 30 bar; the auxiliary gas pressure is 10 bar; the transfer capillary temperature was 320 ℃. A Full scan (Full scan) mode is adopted, the scanning mass-to-charge ratio range is 100-1000m/z, and the resolution is 35000; data dependent secondary scanning (ddms2), resolution 17500, high energy collision induction voltage set to NCE mode (10eV, 30eV, 50 eV).
Establishing an amino acid standard curve: 1.000mg of each of the 20 kinds of amino acid standards was precisely weighed, dissolved in ultrapure water and made up to 10mL to obtain an amino acid standard solution having an initial concentration of 100. mu.g/mL. Then diluted with ultrapure water to 6 concentration gradients of 20, 10, 1, 0.1, 0.01, 0.001. mu.g/mL, respectively. Table 13 lists the calibration equations for the 20 amino acids.
TABLE 13 amino acid labeling equations
Y represents the peak area of the sample; x represents the amino acid concentration (. mu.g/mL).
(3) Data processing and analysis
The data obtained in this study were presented as mean ± sd or mean ± sd, all data were analyzed for significance using the t-test method (P <0.05 indicates significant difference, P <0.01 and P <0.001 indicates very significant difference), and data analysis and mapping were performed by GraphPad Prism software.
3. Test results
(1) Effect of different feeding patterns on free amino acids in the longissimus dorsi of newborn piglets
As can be seen from table 14, the content of Glu and TAA in the longissimus dorsi of the low-weight piglet ladder group was significantly higher than that of the concave group (P <0.05), and the content of Asp tended to be higher than that of the concave group (P ═ 0.08); as can be seen from Table 15, the content of Lys, Glu, Asp and TAA in the longissimus dorsi of the piglet with average body weight in the two feeding modes is obviously higher than that in the notch group (P < 0.05).
TABLE 14 Effect of two feeding patterns on the hydrolysis of amino acids in the longissimus dorsi of low-weight piglets1(mu g/g fresh sample)
Note:1all data are expressed as mean ± sd, n is 4-7.
Table 15 effect of two feeding patterns on average weight piglets longissimus dorsi hydrolyzed amino acids1(mu g/g fresh sample)
Note:1all data are expressed as mean ± sd, n is 4-7.
(2) Influence of different feeding modes on free amino acid in lumbar muscle of newborn piglet
As can be seen from Table 16, the content of Glu in the psoas major of the low-weight piglet stepped group in the two feeding modes is remarkably higher than that of the concave group (P < 0.01); as can be seen from Table 17, the content of Glu in the psoas major of the ladder-shaped group of piglets with average body weight was significantly higher than that of the concave-shaped group (P <0.01), and the content of Thr, His and Asp was significantly higher than that of the concave-shaped ladder-shaped group (P <0.05) in the two feeding modes.
TABLE 16 Effect of two feeding modes on hydrolyzed amino acids in psoas major of low-weight piglets1(mu g/g fresh sample)
Note:1all data are expressed as mean ± sd, n is 4-7.
TABLE 17 Effect of two feeding patterns on average weight piglet psoas major hydrolyzed amino acids1(mu g/g fresh sample)
Note: 1 all data are expressed as mean ± sd, n is 4 to 7.
(3) Effect of different feeding patterns on the expression of the amino acid transporter genes (EAAT3, GLAST, ASCT2, CAT1, CAT2, LAT1) of the longissimus dorsi of newborn piglets
As can be seen from fig. 5A, the relative expression level of the gene EAAT3 in the longissimus dorsi of the concave group of the low-weight piglets in the two feeding modes is significantly higher than that in the ladder group (P <0.01), while the relative expression level of the gene ASCT2 in the ladder group is significantly higher than that in the concave group (P <0.05), and the relative expression level of the gene GLAST tends to be higher than that in the concave group (P ═ 0.07); as can be seen from fig. 5B, the relative expression level of ASCT2 gene in longissimus dorsi of the ladder-shaped group of piglets with average body weight in the two feeding modes tended to be higher than that in the concave group (P ═ 0.08).
(4) Influence of different feeding modes on expression of neonatal piglet psoas major amino acid transporter genes EAAT3, GLAST, ASCT2, CAT1, CAT2 and LAT1)
As can be seen from fig. 6A, the relative expression amounts of the genes GLAST and ASCT2 in the psoas major of the low-weight piglet notch group in the two feeding modes are significantly higher than those in the concave group (P <0.05), and the relative expression amount of the gene CAT1 tends to be higher than that in the concave group (P ═ 0.06); as can be seen from FIG. 6B, the relative expression level of CAT1 gene in the lumbar major of the ladder-shaped group of piglets with average body weight in the two feeding modes is significantly higher than that in the concave group (P < 0.05).
The research result shows that the ladder type feeding mode is adopted in the gestation period of the Guangdong small eared piglet to obviously improve the muscle weight of supraspinatus, brachiocephalus and psoas major of the newborn piglet, improve the newborn weight of the newborn piglet, obviously reduce the stillbirth rate and the invalid farrowing rate of the newborn piglet and improve the reproductive performance of the sow.
The test results show that compared with the feeding mode of a concave group, the ladder type feeding mode remarkably increases the muscle weight, the muscle fiber quantity and the cross sectional area of the newborn piglets and correspondingly increases the expression of related genes of muscle development; meanwhile, the ladder type feeding mode can obviously increase the content of hydrolyzed amino acid in muscle, particularly the content of glutamic acid and aspartic acid, and correspondingly can also increase the expression of related genes of the amino acid transport carrier. According to the results, the step-type feeding mode can improve the birth weight of the newborn piglets by improving the number of muscle fibers in muscles, the cross sectional area, the amino acid content, the muscle development correlation and the amino acid transport carrier correlation gene expression, and can play a positive role in the intestinal development and the subsequent growth development of the piglets, thereby improving the economic benefit.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> southern China university of agriculture; guangdong Yi local pig research institute Co Ltd
<120> feeding method for Guangdong small eared piglet in gestation period for promoting growth of farrowing muscle
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Claims (3)
1. A feeding method for Guangdong small eared pigs in gestation period for promoting the development of farrowing muscles is characterized by comprising the following steps:
(1) on the day of hybridization, the daily feed intake of each gilt of the Guangdong small eared pig is 0.30 Kg;
(2) 1-3 days after the hybridization, the daily feed intake of each gilt of the Guangdong small earrings is 0.63 Kg;
(3) 4-30 days after the hybridization, the daily feed intake of each gilt of the Guangdong small eared pig is 0.85 Kg;
(4) after the breeding, the daily feed intake of each gilt of the Guangdong small eared pig is 1.19Kg after 31-60 days;
(5) after the breeding, the daily feed intake of each gilt of the Guangdong small eared pig is 1.27Kg after 61-85 days;
(6) after the breeding, the daily feed intake of each gilt of the Guangdong small eared pig is 1.98Kg after the breeding for 86-112 days;
during pregnancy, the average daily feed intake of each gilt of the Pleurotus Ostreatus is 1.30 Kg;
the feed is divided into two pregnancy feeds for 1-85 days of pregnancy and 86-112 days of pregnancy;
the pregnancy material for 1-85 days comprises the following components in percentage by mass:
40.00 percent of corn, 8.90 percent of bean pulp, 8.00 percent of barley, 20.00 percent of bran, 3.98 percent of rice bran meal, 15.00 percent of coarse bran, 4.12 percent of premix and 100.00 percent of total;
the pregnancy material for 86-112 days of pregnancy comprises the following components in percentage by mass:
44.00 percent of corn, 17.00 percent of bean pulp, 5.00 percent of barley, 12.00 percent of bran, 10.68 percent of rice bran meal, 6.00 percent of coarse bran, 5.32 percent of premix and 100.00 percent of total.
2. The feeding method for Guangdong auricle-lesser pig in gestation period for promoting the development of the offspring muscle according to claim 1, characterized in that:
wherein, the premix in 1kg of complete ration comprises the following components:
copper, iron, selenium, zinc, manganese, vitamin A and vitamin B2Vitamin D3Vitamin E, vitamin K3Vitamin B6Calcium pantothenate, nicotinic acid, vitamin B12。
3. The feeding method for Guangdong auricle-lesser pig in gestation period for promoting the development of the offspring muscle according to claim 2, characterized in that:
wherein, the premix in 1kg of complete ration comprises the following components:
75 mg of copper, 750 mg of iron, 0.20 mg of selenium, 750 mg of zinc, 187 mg of manganese, 150000IU of vitamin A and vitamin B2140 mg, vitamin D325000IU, vitamin E1000IU, vitamin K335 mg of vitamin B670 mg, 350 mg calcium pantothenate, 500 mg nicotinic acid, vitamin B120.4 mg.
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| 妊娠母猪1-112天如何饲喂以及每天用料标准https://www.pig66.com/2019/303_0506/17990229.html;WJ1990;《猪友之家》;20190506;第1-4页 * |
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