New application of tryptophan in improving weaned piglet muscle fiber type transformation

Abstract

The invention belongs to the technical field of animal nutrition, and particularly relates to novel application of tryptophan in improving weaned piglet muscle fiber type transformation. It is found for the first time that by adding a certain amount of tryptophan into the weaned piglet basic ration, weaned piglet muscle fiber type conversion can be promoted, the slow muscle fiber content is reduced, the fast muscle fiber content is increased, and therefore the weaned piglet growth performance and meat quality are further improved. The invention finds a new application of tryptophan in improving the growth potential, muscle yield and growth performance of piglets, and provides a thought for subsequent development of new feeds and additives.

Description

Technical Field

[0001] The invention belongs to the technical field of animal nutrition, and in particular relates to a new use of tryptophan to improve the conversion of muscle fiber types in weaned piglets. Background Art

[0002] In modern pig farming, the weaning stage is a critical period in the growth and development of piglets. At this time, piglets switch from breast milk feeding to solid feed and face many physiological and environmental challenges. Studies have shown that the muscle fiber type of piglets has an important impact on their growth performance, meat quality and disease resistance. Usually, piglets will experience problems such as decreased growth rate and weakened immunity after weaning, which is closely related to the transformation of muscle fiber type, all of which affect their later growth and production performance.

[0003] For pigs, tryptophan is an important essential amino acid. In addition to its role in protein synthesis, it also plays an important role in a variety of physiological processes. Studies have found that tryptophan can be converted into 5-hydroxytryptamine (serotonin) in the body. This neurotransmitter is closely related to emotions, behavior and physiological regulation. In addition, tryptophan is also involved in regulating metabolism and immune function. In recent years, studies have shown that the reasonable addition of tryptophan can improve the growth performance and health of piglets. Muscle fiber type has a significant effect on the growth performance, meat quality and feed conversion efficiency of pigs. Generally speaking, slow fibers (type I) and fast fibers (type II) differ in energy metabolism and contraction characteristics, and fast fibers usually have higher growth potential and meat quality characteristics. Therefore, it is of great practical significance to promote the transformation of piglet muscle fiber types from slow muscle fibers to fast muscle fibers.

[0004] At present, there is no research on the relationship between tryptophan and muscle fiber type transformation in weaned piglets, and development is urgently needed. Summary of the invention

[0005] The technical problem to be solved by the present invention is that weaned piglets have problems such as slow muscle development, poor growth performance and frequent diseases just after weaning.

[0006] The technical solution of the present invention to solve the above technical problem is: to provide a new use of tryptophan to improve the conversion of muscle fiber types in weaned piglets.

[0007] Among them, in the above-mentioned new use of tryptophan to improve the conversion of muscle fiber types in weaned piglets, the weaned piglets refer to piglets with an age of 25 to 28 days and a body weight of about 5 to 11 kg.

[0008] Wherein, in the above-mentioned new use of tryptophan for improving the conversion of muscle fiber types in weaned piglets, the purity of the tryptophan is 98.5%.

[0009] Among them, in the above-mentioned new use of tryptophan to improve the transformation of muscle fiber types in weaned piglets, the tryptophan has a melting point of 289-290°C, a boiling point of 342.72°C, a density of 1.34, and a pH value of 5.5-7.0.

[0010] Wherein, in the above-mentioned new use of tryptophan for improving the conversion of muscle fiber types in weaned piglets, the tryptophan content is 0.35%.

[0011] Among them, in the above-mentioned new use of tryptophan to improve the conversion of muscle fiber types in weaned piglets, the method of using tryptophan is: adding it to the basic diet of weaned piglets.

[0012] Furthermore, the formula of the basic diet includes: 68.51 parts of corn, 8.07 parts of soybean meal, 8.67 parts of corn gluten meal, 5 parts of whey powder, 5 parts of wheat bran, 0.44 parts of monocalcium phosphate, 1 part of limestone, 0.3 parts of salt, 1 part of trace element premix, 0.18 parts of threonine, 0.82 parts of lysine, 0.1 parts of methionine, 0.21 parts of tryptophan, and 0.7 parts of alanine.

[0013] Furthermore, the basic diet also includes: 1845 IU of vitamin A, 200 IU of vitamin D, 12 IU of vitamin E, 0.48 mg of vitamin K, 9.3 mg of pantothenic acid, 3.1 mg of riboflavin, 0.29 mg of folic acid, 29.0 mg of niacin, 1.0 mg of thiamine, 4.3 mg of vitamin B6, 0.05 mg of biotin, 0.016 mg of vitamin B12 and 0.43 g of choline per 1 kg of feed.

[0014] Furthermore, the basic diet also includes: trace element premix per kg of feed containing 84 mg zinc, 97 mg iron, 3.2 mg manganese, 5.2 mg copper, 0.14 mg I and 0.259 mg selenium.

[0015] Among them, in the above-mentioned new use of tryptophan to improve the conversion of muscle fiber types in weaned piglets, the muscle fiber type conversion refers to the conversion of slow muscle fibers into fast muscle fibers.

[0016] Furthermore, the muscle fiber type conversion refers to reducing the content of slow muscle fibers and increasing the content of fast muscle fibers.

[0017] The invention also provides a feed for improving the conversion of muscle fiber types of weaned piglets, comprising a basic diet and tryptophan.

[0018] The present invention also provides a method for improving the conversion of muscle fiber types in weaned piglets, comprising the step of adding tryptophan to a basic diet.

[0019] The beneficial effects of the present invention are:

[0020] The present invention provides a new use of tryptophan to improve the conversion of muscle fiber types in weaned piglets. The present invention discovers for the first time that by adding a certain amount of tryptophan to the basic diet of weaned piglets, the conversion of muscle fiber types in weaned piglets can be promoted, the content of slow muscle fibers can be reduced, and the content of fast muscle fibers can be increased, which is further reflected in improving the growth performance and meat quality of weaned piglets. Based on the new use of the present invention, tryptophan can be used to prepare feed for improving the conversion of muscle fiber types in weaned piglets, and a simple and efficient method for promoting the conversion of muscle fibers in weaned piglets and improving the growth performance and meat quality of weaned piglets is also provided. The present invention provides a basis for the high-quality production of weaned piglets and has important practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 Effects of different treatments on weight gain of weaned piglets.

[0023] Figure 2 The effects of different treatments on the muscle fiber type composition of weaned piglets, where A is the immunofluorescence image of the longissimus dorsi muscle, and B is the statistical diagram of the ratio of fast-twitch fibers to slow-twitch fibers.

[0024] Figure 3 Effects of different treatments on muscle fiber density in weaned piglets.

[0025] Figure 4 The expression of genes related to muscle fiber type transformation. DETAILED DESCRIPTION

[0026] The present invention discovers for the first time that tryptophan can improve the transformation of muscle fiber types in weaned piglets, thereby improving their muscle development and growth performance by regulating the nutritional environment and physiological state of piglets, thereby promoting healthy breeding.

[0027] Slow-twitch muscle fibers, also known as oxidative muscle fibers, contract slowly but are resistant to fatigue, while fast-twitch muscle fibers, also known as glycolytic muscle fibers, can produce fast and powerful contractions but are prone to fatigue. Since these fibers have a large number of myofilaments, their diameter is larger than that of slow-twitch muscle fibers. More myofilaments mean greater standing force. The energy production of fast-twitch muscle fibers does not rely on oxygen, and anaerobic energy production can be applied. During anaerobic energy production, the process of converting glucose into lactate is called glycolysis. Large and powerful muscles are mainly composed of fast-twitch muscle fibers. The present invention feeds weaned piglets with formulated feed, which will cause an increase in the proportion of fast-twitch muscle fibers. It can be seen that the window period is a critical period that affects the conversion of piglet muscle fiber types.

[0028] The tryptophan of the present invention can improve the muscle fiber density of weaned piglets by improving the conversion of muscle fiber types, thereby improving the weight gain of piglets.

[0029] The present invention has no particular limitation on the source of the tryptophan, which can be purchased conventionally by a person skilled in the art; in a specific embodiment of the present invention, the tryptophan is preferably purchased from Sangon Biotech (Shanghai) Co., Ltd., and the purity of the tryptophan is preferably 98.5-101.5%, the melting point is 289-290° C., the boiling point is 342.72° C., the density is 1.34, and the pH value is 5.5-7.0.

[0030] The present invention provides a feed for improving muscle fiber type conversion of weaned piglets, comprising a basic diet and tryptophan. In the present invention, the tryptophan content is 0.35%, that is, the mass ratio of the tryptophan addition to the basic diet is 0.35:99.65.

[0031] In the present invention, the formulated feed preferably includes the following components in parts by mass: 68.51 parts of corn, 8.07 parts of soybean meal, 8.67 parts of corn gluten meal, 5 parts of whey powder, 5 parts of wheat bran, 0.44 parts of monocalcium phosphate, 1 part of limestone, 0.3 parts of salt, 1 part of trace element premix, 0.18 parts of threonine, 0.82 parts of lysine, 0.1 parts of methionine, 0.21 parts of tryptophan, and 0.7 parts of alanine.

[0032] In the present invention, the premix preferably includes, based on 1 kg of feed: 1845 IU vitamin A, 200 IU vitamin D, 12 IU vitamin E, 0.48 mg vitamin K, 9.3 mg pantothenic acid, 3.1 mg riboflavin, 0.29 mg folic acid, 29.0 mg niacin, 1.0 mg thiamine, 4.3 mg vitamin B6, 0.05 mg biotin, 0.016 mg vitamin B 12 Each kg of feed provides the following nutrients: 84 mg zinc, 97 mg iron, 3.2 mg manganese, 5.2 mg copper, 0.14 mg iodine and 0.259 mg selenium.

[0033] The formulated feed of the present invention can significantly increase the proportion of fast muscle fibers in pig skeletal muscle and increase the muscle fiber density. Moreover, the tryptophan of the present invention has no adverse effect on the growth performance of weaned piglets. It can be seen that tryptophan has a good improvement effect on the transformation of muscle fiber types in weaned piglets.

[0034] In order to further illustrate the present invention, the method for improving the muscle fiber type of weaned piglets by formulating feed provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] The equipment and reagents described in the examples are all common commercially available products.

[0036] Example 1

[0037] Thirty 28-day-old weaned piglets of a three-way crossbreed (Durham × Longhaden × Dahurian) with similar genetic background were randomly divided into two treatments, with 15 pigs in each treatment.

[0038] Two treatments were set up: (1) the control group was fed a basic feed containing 0.14% tryptophan; (2) the experimental group was fed a formulated feed; the formulated feed contained 0.35% tryptophan per kg of the basic feed, which met the nutritional requirements of pigs weighing 5-11 kg as recommended by NRC (2012). The composition and nutritional level of the basic feed are shown in Table 1.

[0039] The experimental animals had free access to food and water, and the experimental period was 35 days, including 7 days of pre-feeding.

[0040] Table 1 Basic feed composition and nutritional level of weaned piglets (5-11 kg stage) (%, feeding basis)

[0041]

[0042]

[0043] After the experiment, 5 pigs with average weight were selected for each treatment and slaughtered. The longissimus dorsi muscle was then separated and weighed, and then quickly divided into multiple 1 cm 3 The cells were stored in 4% paraformaldehyde, OCT embedding medium and -80℃ refrigerator, and used for preparing paraffin sections, frozen sections and determining the expression of genes related to muscle fiber type transformation, respectively.

[0044] (1) About weight gain of weaned piglets

[0045] The difference between the final weight and the initial weight was used to represent the weight gain of the piglets. The piglets were weighed and calculated at the beginning and end of the experiment using a pre-calibrated electronic scale in the laboratory. Figure 1 As shown. Figure 1 It can be seen that compared with the control group, the weight gain of piglets in the experimental group was significantly greater than that in the control group (P<0.01).

[0046] (2) Regarding the transformation of muscle fiber types

[0047] The following steps were used to determine the fiber type conversion of the longissimus dorsi muscle in piglets:

[0048] First, take the sample out of the -80℃ refrigerator in advance to balance the sample temperature. The thickness of the slice is 40um. The slice is directly placed in PBS at room temperature. OCT will automatically dissolve in PBS. Then quickly block the peroxisomes and wash with PBS 3 times, 5 minutes each time. Then perform high-temperature antigen repair: 95-degree water bath, citrate (PH=6.0) repair solution, 6 minutes. After the end, let it stand for 20-30 minutes to lower the temperature; wash with PBS 3 times, 5 minutes each time; then put it in 0.2% Triton at room temperature for 20 minutes to permeabilize the membrane. After the permeabilization, add 10% goat serum to incubate for 1 hour, then transfer to the primary antibody for incubation overnight (4℃), and wash with PBS 3 times the next day, 5 minutes each time. Then incubate the secondary antibody at room temperature for 1 hour. After the incubation, wash with PBS 3 times, 5 minutes each time. Then add DAPI and incubate in the dark. After 15 minutes, wash with PBS 3 times, 5 minutes each time. Wipe off the PBS outside the specimen with filter paper, then seal the specimen with anti-fluorescence quencher, dry at 37℃, and observe and take pictures under a microscope. Figure 2 shown.

[0049] Depend on Figure 2 A shows that compared with the control group, the longissimus dorsi muscle of the experimental group piglets is composed of fast and slow muscles. Figure 2 The statistical results of B showed that the proportion of fast muscle fibers in the longissimus dorsi muscle of the experimental group piglets was significantly increased (P<0.01), while the proportion of slow muscle fibers was significantly decreased (P<0.01).

[0050] (3) Determination of muscle fiber density

[0051] The specific determination method is as follows: Paraffin section the muscle tissue, the steps are as follows:

[0052] After fixation in 4% paraformaldehyde for 24 h, the tissues were immersed in different concentrations of ethanol in sequence for gradual dehydration.

[0053] The dehydrated tissue was immersed in xylene to remove the ethanol and make the tissue transparent.

[0054] Immerse the transparent tissue in melted paraffin to saturate the tissue with paraffin. It usually needs to be heated at 60°C and the immersion time is about 2 hours.

[0055] Place the paraffin-saturated tissue into a mold, pour in fresh paraffin, and wait for it to cool and solidify.

[0056] After the paraffin block is formed, the mold is removed to obtain the paraffin-embedded tissue sample.

[0057] Use a microtome to cut the paraffin block into thin slices, usually with a slice thickness of 4-6 μm. After the slices are completed, the slices are flattened by ultrasound or water bath.

[0058] Fix the slices with a glass slide. Generally, a layer of glue (such as polyvinyl alcohol) can be added to the glass slide to ensure the adhesion of the slices.

[0059] The sections are dewaxed (usually with xylene), then go through a precipitation process, and finally stained (such as HE staining) to observe the structure.

[0060] After staining, cover with sealing glue and wait for it to dry before observation under a microscope. Figure 3 shown.

[0061] Depend on Figure 3 It can be seen that compared with the control group, the muscle fiber density of the longissimus dorsi muscle of the experimental group piglets increased significantly (P<0.05).

[0062] (4) Determination of muscle fiber content

[0063] Real-time fluorescence quantitative PCR was used for determination. The specific steps are as follows:

[0064] Total RNA was extracted from piglet muscle samples using Trizol reagent (Invitrogen, CA, USA). cDNA from RNA was amplified using a 5× All-In-One RT MasterMix (Applied Biological Materials (ABM) Inc., Shanghai, China) kit, and qRT-PCR was performed using EvaGreen 2X qPCR MasterMix (Applied Biological Materials (ABM) Inc., Shanghai, China). The operation was repeated four times for each sample, and β-actin was used as a reference gene. The RT-qPCR test results were compared according to the comparative CT method (2 -△△CT ) calculation. Primer 5.0 software was used to design PCR primer sequences and BGI was commissioned to perform biosynthesis. The primer sequences are shown in Table 2, and the PCR reaction system and reaction conditions are shown in Tables 3 and 4.

[0065] Table 2 shows the primer sequences for fluorescence quantitative PCR

[0066]

[0067]

[0068] Note: MYHCⅠ is myosin heavy chain Ⅰ; MYHCⅡa is myosin heavy chain Ⅱa; MYHCⅡx is myosin heavy chain Ⅱx; MYHCⅡb is myosin heavy chain Ⅱb; β-actin is the internal reference gene.

[0069] Table 3 Real-time fluorescence quantitative PCR reaction system

[0070] Components volume EvaGreen 2X qPCR MasterMix 10uL Upstream primer (10 μM) 0.6uL Downstream primer (10 μM) 0.6uL Template DNA 1-2uL Enzyme-free water Make up 20uL

[0071] Table 4 Real-time fluorescence quantitative PCR reaction conditions

[0072]

[0073] Real-time fluorescence quantitative PCR results Figure 4 shown.

[0074] Depend on Figure 4 It can be seen that compared with the control group, the expression level of MYHCⅠ (myosin heavy chain Ⅰ) in the longissimus dorsi muscle of the experimental group was significantly decreased (P<0.01), the expression level of MYHCⅡa (myosin heavy chain Ⅱa) was significantly increased (P<0.01), the expression level of MYHCⅡx (myosin heavy chain Ⅱx) did not change significantly, and the expression level of MYHCⅡb (myosin heavy chain Ⅱb) was significantly increased (P<0.05).

[0075] It can be seen from the above examples that the formulated feed of the present invention can increase muscle fiber density of weaned piglets, improve muscle fiber type conversion, and achieve the purpose of improving the growth performance of weaned piglets. At the same time, it is highly safe for pigs and has no adverse effects.

[0076] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A new use of tryptophan to improve the conversion of muscle fiber types in weaned piglets.

2. The new use of tryptophan for improving muscle fiber type conversion in weaned piglets according to claim 1, characterized in that: The weaned piglets are piglets with an age of 25 to 28 days and a body weight of about 5 to 11 kg.

3. The new use of tryptophan for improving muscle fiber type conversion in weaned piglets according to claim 1, characterized in that: The purity of the tryptophan is 98.5%.

4. The new use of tryptophan for improving muscle fiber type conversion in weaned piglets according to claim 1, characterized in that: The tryptophan has a melting point of 289-290° C., a boiling point of 342.72° C., a density of 1.34, and a pH value of 5.5-7.

0.

5. The new use of tryptophan for improving muscle fiber type conversion in weaned piglets according to claim 1, characterized in that: The tryptophan content is 0.35%.

6. The new use of tryptophan for improving muscle fiber type conversion in weaned piglets according to claim 1, characterized in that: The method for using tryptophan is to add it into the basic diet of weaned piglets.

7. The new use of tryptophan for improving muscle fiber type conversion in weaned piglets according to claim 6, characterized in that: The formula of the basic diet includes: 68.51 parts of corn, 8.07 parts of soybean meal, 8.67 parts of corn gluten meal, 5 parts of whey powder, 5 parts of wheat bran, 0.44 parts of monocalcium phosphate, 1 part of limestone, 0.3 parts of salt, 1 part of trace element premix, 0.18 parts of threonine, 0.82 parts of lysine, 0.1 parts of methionine, 0.21 parts of tryptophan, and 0.7 parts of alanine.

8. The new use of tryptophan for improving muscle fiber type conversion in weaned piglets according to claim 1, characterized in that: The muscle fiber type conversion refers to the conversion of slow muscle fibers into fast muscle fibers.

9. The present invention also provides a feed for improving the conversion of muscle fiber types in weaned piglets, characterized in that: Includes basal diet and tryptophan.

10. The present invention also provides a method for improving muscle fiber type conversion in weaned piglets, characterized in that: Includes steps to add tryptophan to the basal diet.

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