A method to improve the pregnancy and farrowing rate of clone recipient sows

Abstract

This invention relates to the field of mammalian reproductive technology, specifically disclosing a method for improving the pregnancy and farrowing rate of cloning recipient sows. The method mainly includes: recipient sow selection, estrus detection, pre-embryo transfer management, embryo transfer surgery and post-operative care, gestational feeding, recipient sow farrowing, and cloned piglet birth care. By supplementing recipient sows with hormones and vitamin ADE injections before and after surgery, the pregnancy and farrowing rates of recipient sows are improved. Simultaneously, supplementing with N-carbamoylglutamate during gestation increases the total number of piglets born and the number of healthy piglets. Furthermore, strict feeding management during gestation controls the sow's birth weight, thereby reducing dystocia and stillbirths caused by underweight or overweight, further increasing the number of healthy piglets.

Description

Technical Field

[0001] This invention relates to the field of mammalian reproductive technology, and more particularly to a method for improving the pregnancy and farrowing rate of clone recipient sows. Background Technology

[0002] Somatic cell cloning technology refers to the process of inducing dormancy in animal somatic cells through inhibited culture, followed by nuclear transfer. An enucleated oocyte serves as the nuclear recipient, while a somatic cell nucleus or a cell nucleus containing a small amount of cytoplasm acts as the nuclear donor. The latter is transferred into the former to construct a recombinant embryo. The donor nucleus is reprogrammed within the cytoplasm of the enucleated oocyte, initiating cleavage, embryonic development, and birth, resulting in cloned animals. This technology is also known as somatic cell nuclear transfer. Somatic cell cloning technology can be used to replicate superior breeding stock, produce transgenic livestock, create animal disease models, and isolate embryonic stem cells, demonstrating significant application value in agriculture and medicine. Currently, over 20 species of mammals, including sheep, cattle, mice, pigs, rabbits, and horses, have been successfully cloned. However, some unresolved issues remain, the most prominent being the low birth rate of cloned animals, with most cloned embryos dying during gestation. Currently, the overall developmental efficiency of cloned pig embryos is only about 0.1-1%, and this low cloning efficiency severely hinders the widespread application of somatic cell cloning technology in the pig industry.

[0003] The prevailing view is that abnormal development in cloned embryos is caused by abnormalities in the dedifferentiation, restoration of totipotency, and epigenetic reprogramming during the initiation of embryonic development of the somatic cell genome in the oocyte cytoplasm, leading to a low blastocyst rate. However, recent research indicates that even a high blastocyst rate does not necessarily guarantee a high pregnancy and delivery rate. Therefore, the issue of low pregnancy and delivery rates with cloned embryos requires further scientific investigation. Given the limitations of current scientific and technological capabilities, in vitro cloned embryos and in vitro fertilized embryos must be transferred into recipient sows to develop into complete individuals. Ensuring normal implantation and development is a crucial factor affecting the final efficiency of embryo transfer. Embryo implantation and development are closely related to the recipient sow's parity, body condition, physiological status, and post-transfer feeding and management.

[0004] To improve the pregnancy rate and litter size of somatic cell cloned embryo transfer, 200-300 embryos are typically transferred, while ordinary sows conceive only around 20 embryos. The pregnancy rate in recipient sows after cloned embryo transfer is generally 20-50%, with litters of 2-6 piglets, and an overall cloning efficiency of 0.1-1%. Because cloned embryos develop differently from ordinary embryos, the selection of recipient sows, hormone treatment and estrus control, pre-transfer management, embryo transfer surgery and post-operative care, feeding during gestation, farrowing, and newborn care of cloned piglets all differ to achieve better overall cloning efficiency.

[0005] Iron is an essential trace element in animal metabolism, participating in physiological and biochemical processes such as electron transport and oxidative phosphorylation. Iron is also a component of ribonucleotide reductase, participating in DNA synthesis in cells. The vast majority of iron exists in the body as hemoglobin, the main component of red blood cells and the primary carrier of oxygen and carbon dioxide. Hemoglobin levels reflect iron homeostasis in the body. Insufficient iron intake or absorption leads to iron deficiency anemia, characterized by a decreased number of red blood cells, reduced hemoglobin levels, loss of appetite, lethargy, and increased respiratory rate; severe anemia can lead to death. Studies have found a negative genetic correlation between sow hemoglobin levels and piglet birth weight, and a positive genetic correlation with piglet survival rate. Research indicates a negative linear correlation between hemoglobin levels in pregnant sows and reproductive performance. Since the iron content and biological value in feed ingredients are very low, iron supplements are typically added in commercial production to prevent iron deficiency.

[0006] Chinese Patent 201510261676.0 provides a method for improving sow reproductive performance, including daily management of the gestation pen, feeding during gestation, drug health care, and routine immunization during gestation. Through the above methods, especially the refined feeding management of feeding and drug health care during gestation, the survival rate and healthy piglet rate of sows can be significantly improved, with a survival rate of over 98% and an average litter size of 12-14 piglets per sow. Sows have strong lactation capacity and high milk quality during the postpartum lactation period, and the peak lactation period can be extended, enhancing the birth weight and disease resistance of newborn piglets, which has great application prospects.

[0007] Chinese Patent 201810465160.1 discloses a peptide-chelated iron compound preparation for improving the reproductive performance of sows and piglets. The preparation is characterized by the following mass ratios of each component: 15-25 parts peptide-chelated iron, 25-35 parts probiotics, and 10-15 parts yeast cell wall. This invention, through rational formulation, exhibits a synergistic effect, not only replacing injectable iron supplementation for piglets but also significantly increasing sow milk production, improving feed utilization, and enhancing the immunity of sows and piglets. It has advantages such as increasing piglet survival rate, promoting animal growth, and significantly improving the reproductive performance of sows and piglets.

[0008] Currently, iron supplements are classified into three categories: inorganic salt iron, organic acid chelated iron, and amino acid chelated iron. In existing technologies, organic iron sources have limited effects on promoting the reproductive performance of sows. Therefore, developing an iron supplement with good digestibility and high bioavailability to improve the pregnancy and farrowing rate of clone recipient sows has a promising market prospect. Summary of the Invention

[0009] In view of the above-mentioned problems in the existing technical solutions, the technical problem to be solved by the present invention is to provide a method for improving the pregnancy and farrowing rate of clone recipient sows.

[0010] The technical solution of this invention:

[0011] A method for improving the pregnancy and farrowing rate of clone recipient sows includes the following steps:

[0012] S1 selects suitable weaned or non-pregnant sows as candidate recipient sows based on the selection criteria;

[0013] S2 When the candidate recipient sow is a weaned sow, pregnant mare serum gonadotropin (PMSG) and vitamin ADE injection are administered intramuscularly on the second day after weaning, and human chorionic gonadotropin (HCG) is administered on the fourth day. When the candidate recipient sow is a non-pregnant sow, acetaminophen is administered orally by mixing it into the feed for 18 consecutive days. After stopping feeding, pregnant mare serum gonadotropin (PMSG) and vitamin ADE injection are administered on the second day, and human chorionic gonadotropin (HCG) is administered on the fourth day.

[0014] S3 conducts backfat testing, estrus testing, and health status testing on candidate recipient sows. Sows that meet the standards undergo pre-embryo transfer management. The number of prepared sows should be higher than the planned number of surgeries to prepare for unforeseen circumstances.

[0015] After passing the S4 test, the recipient sows were injected intramuscularly with cefquinoxime suspension. At the same time, a mixture of 10% lincomycin hydrochloride injection and houttuynia cordata injection was used for uterine perfusion. Vaginal purulent discharge was checked 12 hours after uterine perfusion or 2 hours before surgery. Sows with purulent discharge could not participate in transplantation.

[0016] S5 embryo transfer surgery was performed on recipient sows with strict disinfection measures, control of ambient temperature and operation time. After the operation, human chorionic gonadotropin (HCG) was injected intramuscularly. 14 days after the operation, human chorionic gonadotropin (HCG) was injected intramuscularly again.

[0017] Following S6 embryo transfer, the recipient sow was given intramuscular injections of ceftiofur sodium and houttuynia cordata daily for three consecutive days, and the wound was routinely disinfected. N-carbamoylglutamate was supplemented throughout the gestation period post-surgery, and backfat was monitored, with feeding adjusted based on the results. The sow's condition was checked daily post-surgery, including vaginal discharge, return to estrus, and abortion. Ultrasound examinations were performed twice post-surgery, on days 30 and 60.

[0018] After embryo transfer, S7 pregnant sows are cleaned and disinfected 107-110 days later and then moved to the farrowing house to await delivery. Sows that do not show signs of delivery 115 days after the transfer can be injected with cloprostenol (PG) to induce delivery. If there are still no signs of delivery after 118 days, a cesarean section is performed. After delivery, the cloned piglets are recorded and registered.

[0019] S8 involves newborn care of the cloned piglets. After disinfecting the sow's udder, the cloned piglets are allowed to nurse independently. If the cloned piglets are unable to nurse independently, assisted nursing is provided. Cloned piglets with birth defects are artificially nursed. The cloned piglets undergo health maintenance and immunization according to normal procedures. Furthermore, in step S1, the recipient sow breed is a Landrace, Large White, or a crossbred pig.

[0020] Furthermore, the criteria for weaning sows selected in step S1 are: having given birth to more than 2 litters but less than 45 litters, with a litter size of ≥12 piglets in two consecutive litters, ≥10 weaned piglets, no ectopic delivery in the previous litter, no obvious uterine inflammation, healthy limbs and hooves, normal udder, and backfat of 14-18mm; the criteria for non-pregnant sows are the same as those for weaning sows.

[0021] Furthermore, in step S2, the injection volume of PMSG is 800-1000 iu / head, the injection volume of vitamin ADE solution is 4-5 mL / head, and the injection volume of HCG is 400-500 iu / head.

[0022] Furthermore, in step S4, the dosage of cefquinoxime suspension is -1 mL / 20 kg body weight, the dosage of lincomycin hydrochloride injection is 10-20 mL / head, and the dosage of houttuynia cordata injection is 50-60 mL / head.

[0023] Furthermore, in step S5, the amount of HCG used is 400-500 iu / head.

[0024] Furthermore, in step S6, the dosage of ceftiofur sodium is 0.5-1g / head, and the dosage of houttuynia cordata injection is 20mL / head.

[0025] Furthermore, in step S6, the amino acid supplementation involves adding N-carbamoylglutamic acid to the feed at a dosage of 800-1000 g / t, the organic iron chelating agent at a dosage of 400-1000 g / t, and the vitamin supplementation involves adding vitamin E, sodium selenite, biotin, and folic acid to the feed at dosages of 80-100 g / t, 0.1-0.3 g / t, 0.1-0.5 g / t, and 6-10 g / t, respectively. Postoperative management includes fasting on the first day, feeding 0.8-1 kg / head per day on the second day, 1.2-1.5 kg / head per day on the third day, and 1.6-2 kg / head per day from the fourth to the tenth day.

[0026] Part of the iron transfer from pregnant sows to the fetus is achieved through uterine transferrin (a yellow transferrin secreted by the uterus). Reduced uterine transferrin in late pregnancy is considered a cause of anemia in newborn piglets. Sow serum iron levels are lowest in late pregnancy, and the efficiency of iron transfer to the fetus decreases during this time. Furthermore, hematocrit iron levels are positively correlated with fetal weight and placental efficiency. Therefore, iron levels affect both farrowing rates in pregnant sows and piglet survival rates. In this invention, the inventors prepared an iron complex and then complexed it with mannose under alkaline conditions to obtain an organic iron chelating agent. This chelating agent has good water solubility, high absorption rate, and stable structure, allowing for absorption in the intestine without easy degradation, significantly improving utilization. Sufficient iron absorption in pregnant sows prevents anemia in piglets at birth and results in higher survival rates. This invention also controls the birth weight of sows by managing different feeding amounts during pregnancy, thereby reducing dystocia and stillbirth caused by underweight or overweight sows, and further improving the farrowing rate.

[0027] The preparation method of the organic iron chelating agent includes the following steps:

[0028] X1 Weigh 5-6 parts by weight of 2-aminomethylpyridine and 4-6 parts by weight of triethylamine and add them to 4-10 parts by weight of ethanol. Then add 7-9 parts by weight of 3-bromopropionic acid, heat to 70-90℃ and stir for 20-30h. After the reaction is completed, cool to room temperature, filter, wash the residue with acetonitrile and concentrate to dryness to obtain 3-pyridine-methylaminopropionic acid.

[0029] X2 Weigh 2-3 parts by weight of 3-pyridine-methylaminopropionic acid and 2-3 parts by weight of tetramethylammonium hydroxide from step X1 and add them to 10-20 parts by weight of methanol. Then add 3-4 parts by weight of ferrous chloride tetrahydrate, stir for 1-4 hours and filter. After pouring the filtrate into diethyl ether, a precipitate appears. After 7 days, filter and dry to obtain the iron complex.

[0030] X3 Weigh 5-6 parts by weight of oligomannose, add 25-30 parts by weight of water to dissolve it, add 0.5 mol / L sodium hydroxide aqueous solution to adjust the pH to 9-10, stir and mix evenly, add 5-6 parts by weight of the iron complex from step X2, and keep the pH at 9-10, then add twice the volume of anhydrous ethanol for precipitation, centrifuge at 4000-5000 rpm for 5-10 min, precipitate the lower precipitate with twice the volume of anhydrous ethanol and continue centrifugation, freeze-dry the lower precipitate to obtain the mannose-iron complex as an organic iron chelating agent.

[0031] Furthermore, in step S6, the sows after postoperative management are fed according to their backfat condition. Specifically, from 11 to 30 days postoperatively, sows with a backfat of <14mm are fed 2.0-2.2 kg / day, and those with a backfat of 14-18mm are fed 1.8-2 kg / day; from 31 to 85 days postoperatively, sows with a backfat of <14mm are fed 2.6-2.8 kg / day, those with a backfat of 14-18mm are fed 2.0-2.2 kg / day, and those with a backfat of >18mm are fed 1.8-2 kg / day; after 86 days postoperatively, sows with a backfat of 18-22mm are fed 2.8-3 kg / day.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1) Since a large number of embryos are transplanted into the recipient sow, insufficient hormones in the recipient sow can affect the development and implantation of cloned embryos. Therefore, supplementing hormones before and after transplantation can maintain the hormone level in the recipient sow, provide an intrauterine environment conducive to embryo development and implantation, and thus improve the pregnancy and delivery rate;

[0034] (2) After transplantation in recipient sows, the main factor affecting litter size and number of healthy piglets is the high embryo loss rate during gestation. Therefore, in this invention, the inventors supplemented N-acetylglutamate (NCG) during gestation, which can promote endogenous arginine synthesis and effectively increase litter size and number of healthy piglets. At the same time, vitamin supplementation promotes uterine expansion and placental formation during gestation, increases uterine horn length and placental surface area, better provides nutrition for the fetus, and promotes fetal growth and development.

[0035] (3) In the feeding management of pregnant sows during pregnancy, the role of adjusting the amount of feed according to the back fat condition is: if the sow is too thin during pregnancy, it will affect the nutritional supply of the fetus, resulting in an increase in weak piglets, deformed fetuses, or stillbirths; being too thin will also lead to insufficient energy reserves in the sow, making the sow unable to give birth, causing dystocia, and even causing the fetus to suffocate and die; if the sow is too fat, the birth process will be prolonged and the dystocia rate will increase due to the large size of the fetus and the narrowness of the birth canal. Therefore, adjusting the amount of feed according to the back fat condition in a timely manner can greatly reduce the dystocia rate of pregnant sows. Detailed Implementation

[0036] The technical solution of the present invention will be described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0037] The parameters of some raw materials in this embodiment of the invention are as follows:

[0038]

[0039] Example 1

[0040] A method for improving the pregnancy and farrowing rate of clone recipient sows includes the following steps:

[0041] S1 selects 15 sows from weaned Large White sows that have given birth to 2 or more but less than 5 litters, with more than 12 piglets in two consecutive litters, more than 10 weaned piglets, no ectopic delivery in the previous litter, no obvious uterine inflammation, healthy limbs and hooves, normal udder, and backfat of 14-18mm as candidate recipient sows.

[0042] S2 administered 1000 iu PMSG and 5 mL of vitamin ADE injection to each recipient sow intramuscularly on day 2 after weaning, and 500 iu HCG on day 4.

[0043] S3 conducts backfat testing on recipient sows, with a backfat thickness of 15-22mm. The estrus cycle and physical condition of recipient sows are also tested. Qualified sows undergo pre-embryo transfer management.

[0044] Ten recipient sows that passed the S4 test were given an intramuscular injection of cefquinoxime suspension at a dose of 1 mL / 20 kg of body weight. At the same time, each sow was given a uterine irrigation with a mixture of 20 mL of 10% lincomycin hydrochloride injection and 60 mL of houttuynia cordata injection. Vaginal purulent discharge was checked 12 hours after uterine irrigation or 2 hours before surgery. Sows with purulent discharge could not participate in transplantation.

[0045] S5 performed embryo transfer surgery on recipient sows, with strict disinfection, environmental control, and operation time. After the operation, each sow was injected intramuscularly with 500 iu of HCG, and was injected intramuscularly again 14 days after the operation.

[0046] Following S6 embryo transfer, recipient sows were given intramuscular injections of 1g / sow of ceftiofur sodium and 20mL / sow of houttuynia cordata daily for three consecutive days, with routine wound disinfection. Throughout the post-operative period, recipient sows were fed a diet supplemented with the following: 800g / t N-carbamoylglutamate, 1000g / t organic iron chelate, 100g / t vitamin E, 0.3g / t sodium selenite, 0.5g / t biotin, and 10g / t folic acid. Specific post-operative feeding management included: fasting on the first day, feeding 1kg / sow on the second day, 1.5kg / sow on the third day, and 2kg / sow daily from the fourth to the tenth day. N-carbamoylglutamate was supplemented throughout the post-operative period, and backfat was monitored, with feeding adjusted based on the results. From 11 to 30 days post-surgery, feed 2.2 kg / day for sows with a backfat thickness <14 mm and 2 kg / day for sows with a backfat thickness of 14-18 mm. From 31 to 85 days post-surgery, feed 2.8 kg / day for sows with a backfat thickness <14 mm and 2.2 kg / day for sows with a backfat thickness of 14-18 mm and 2 kg / day for sows with a backfat thickness >18 mm. After 86 days post-surgery, feed 3 kg / day for sows with a backfat thickness of 18-22 mm. Check the sows' condition daily, including vaginal discharge and signs of estrus return. Perform two ultrasound pregnancy checks on days 30 and 60 post-surgery.

[0047] After embryo transfer, S7 pregnant sows are cleaned and disinfected and then moved to the farrowing house to await delivery. Sows without signs of delivery can be induced by intramuscular injection of 2mL of cloprostenol 115 days after the transfer. If there are still no signs of delivery after 118 days, a cesarean section will be performed. After delivery, the cloned piglets will be recorded and registered.

[0048] S8 provides newborn care for cloned piglets, disinfects the sow's udder, and allows the piglets to nurse independently. If the cloned piglets are unable to nurse independently, they are assisted with nursing. If the cloned piglets have birth defects, they are artificially fed. The cloned piglets are also given health care and immunization according to normal procedures.

[0049] The preparation method of the organic iron chelating agent includes the following steps:

[0050] X1 Weigh 540g of 2-aminomethylpyridine and 505g of triethylamine and add them to 5L of anhydrous ethanol. Then add 765g of 3-bromopropionic acid. Heat to 80℃ and stir for 24h. After the reaction is completed, cool to room temperature, filter, wash the residue with acetonitrile, and concentrate to dryness at 45℃ and -0.9MPa to obtain 3-pyridine-methylaminopropionic acid.

[0051] X2 Weigh 250g of 3-pyridine-methylaminopropionic acid and 250g of tetramethylammonium hydroxide from step X1 and add them to 10L of methanol. Then add 375g of ferrous chloride tetrahydrate, stir for 3h and filter. The filtrate is poured into diethyl ether and a precipitate appears. After 7 days, filter and dry at 40℃ for 8h to obtain the iron complex.

[0052] X3 Weigh 600g of oligomannose and add it to 3L of water. Mix well and then add 0.5mol / L sodium hydroxide aqueous solution to adjust the pH to 10. After stirring and mixing well, add 600g of the iron complex obtained in step X2 and keep the pH at 10. Then add twice the volume of anhydrous ethanol for precipitation, centrifuge at 4500rpm for 10min, precipitate the lower precipitate with twice the volume of anhydrous ethanol and centrifuge for another 10min. Freeze-dry the lower precipitate at -30℃ and 9Pa for 48h to obtain the mannose-iron complex as an organic iron chelating agent.

[0053] The experiment was conducted in three batches, with 10 sows in each batch, for a total of 30 sows.

[0054] Example 2

[0055] Same as Example 1, except that the organic iron chelator is replaced with glycine chelated ferrous iron in the feed during gestation.

[0056] Example 3

[0057] Same as Example 1, except that the feeding amount for each recipient sow is 3 kg / day during the postoperative management process in step S6, i.e., during the sow's gestation period.

[0058] Example 4

[0059] Same as Example 1, except that N-carbamoylglutamic acid is not supplemented in the feed during pregnancy.

[0060] Compare with Example 1

[0061] Same as Example 1, except that no additional organic iron chelating agent is added to the feed during gestation.

[0062] Test Example 1

[0063] The postpartum sows were statistically analyzed according to the methods of each control case and example, including pregnancy rate, farrowing rate, total number of piglets in litter, and number of healthy piglets. Healthy piglets were defined as piglets with a birth weight of >900g. The specific results are shown in Table 1.

[0064] Table 1. Comparative analysis of sow farrowing rates under different treatment methods.

[0065] Group Head count Pregnant Pregnancy rate childbirth Delivery rate litter size Number of healthy piglets in the litter Compare with Example 1 30 10 33.33％ 8 26.67％ 4.5±0.93 3.5±0.54 Example 1 30 22 73.33％ 18 60％ 6.5±1.5 5.4±1.1 Example 2 30 20 66.67％ 15 50％ 5.2±0.77 4.07±0.8 Example 3 30 12 40％ 10 33.33％ 5.8±0.92 5.2±0.79 Example 4 30 11 36.67％ 9 30％ 5.3±0.64 4.94±0.68

[0066] Based on the farrowing conditions of sows and the survival rate of piglets, it is evident that additional nutritional supplementation for sows during gestation is essential. N-carbamoylglutamic acid, a structural analog of N-acetylglutamic acid, can promote the endogenous synthesis of arginine. Arginine is metabolized in the body to produce nitric oxide (NO) and polyamines. NO can relax smooth muscle, promote capillary formation and growth, increase microcirculation, and improve the reproductive performance of multiparous animals, effectively increasing litter size and birth weight. Therefore, sows supplemented with N-carbamoylglutamic acid have a higher effective number of live piglets and a higher piglet survival rate. Oligomannoses contain many chemical bonds that are not hydrolyzed by amylase, and their structural properties are not easily altered. Therefore, they not only resist hydrolysis by saliva or secretions from the stomach and pancreas, but they are also not absorbed or degraded in the small intestine. When iron ions chelate with mannose to form a stable complex, a stable chemical bond structure is formed, unaffected by other substances, allowing it to reach the intestine smoothly and improving the absorption rate of iron. Iron intake directly affects the hemoglobin content of pregnant sows and is also directly related to the birth weight of piglets. Therefore, it can be seen that sows without additional iron supplementation have poorer farrowing rates and ultimately, poorer piglet survival rates. In Example 3, the lack of differentiation in feeding levels for pregnant sows may have resulted in sows being either underweight or overweight, which is not only detrimental to pregnancy and farrowing but also leads to dystocia. Consequently, both the farrowing rate and piglet survival rates were unsatisfactory.

Claims

1. A method for improving the pregnancy and farrowing rate of clone recipient sows, characterized in that, Includes the following steps: S1 selects suitable weaned or non-pregnant sows as candidate recipient sows based on the selection criteria; S2 When the candidate recipient sow is a weaned sow, pregnant mare serum gonadotropin and vitamin ADE injection are injected intramuscularly on the second day after weaning, and human chorionic gonadotropin is injected on the fourth day; when the candidate recipient sow is a non-pregnant sow, acetaminophen is administered orally by mixing it into the feed for 18 consecutive days, and pregnant mare serum gonadotropin and vitamin ADE injection are injected on the second day after feeding is stopped, and human chorionic gonadotropin is injected on the fourth day. S3 conducts backfat testing, estrus testing, and health status testing on candidate recipient sows. Sows that meet the standards undergo pre-embryo transfer management. The number of prepared sows should be higher than the planned number of surgeries to prepare for unforeseen circumstances. After passing the S4 test, the recipient sows were injected intramuscularly with cefquinoxime suspension. At the same time, 10% lincomycin hydrochloride injection and houttuynia cordata injection were mixed and then instilled into the uterus. The vaginal purulent discharge was checked 12 hours after the uterine instillation or 2 hours before the operation. Sows with purulent discharge could not participate in the transplantation. S5 performed embryo transfer surgery on recipient sows, with strict disinfection measures, control of ambient temperature and operation time. After the operation, human chorionic gonadotropin was injected intramuscularly, and human chorionic gonadotropin was injected intramuscularly again 14 days after the operation. After S6 embryo transfer, the recipient sow was given intramuscular injections of ceftiofur sodium and houttuynia cordata daily for three consecutive days. The wound was routinely disinfected. N-carbamoylglutamate, amino acids, organic iron chelates, and vitamins were supplemented throughout the gestation period. Backfat was measured and feeding was adjusted according to the results. The sow's condition was checked daily after the surgery, including vaginal discharge, return to estrus, and abortion. Two ultrasound pregnancy checks were performed on days 30 and 60 after the surgery. After embryo transfer, S7 pregnant sows are cleaned and disinfected 107-110 days later and then moved to the farrowing house to await delivery. Sows that do not show signs of delivery 115 days after the transfer can be injected with cloprostenol to induce delivery. If there are still no signs of delivery after 118 days, a cesarean section is performed. After delivery, the cloned piglets are recorded and registered. S8 provides birth care for cloned piglets, disinfects the sow's udder, and allows the cloned piglets to nurse independently. If the cloned piglets are unable to nurse independently, they are assisted with nursing. If the cloned piglets have birth defects, they are artificially fed. The cloned piglets are given health care and immunization according to normal procedures. The preparation method of the organic iron chelating agent includes the following steps: X1 Weigh 5-6 parts by weight of 2-aminomethylpyridine and 4-6 parts by weight of triethylamine and add them to 4-10 parts by weight of ethanol. Then add 7-9 parts by weight of 3-bromopropionic acid, heat to 70-90℃ and stir for 20-30h. After the reaction is completed, cool to room temperature, filter, wash the residue with acetonitrile and concentrate to dryness to obtain 3-pyridine-methylaminopropionic acid. X2 Weigh 2-3 parts by weight of 3-pyridine-methylaminopropionic acid and 2-3 parts by weight of tetramethylammonium hydroxide from step X1 and add them to 10-20 parts by weight of methanol. Then add 3-4 parts by weight of ferrous chloride tetrahydrate, stir for 1-4 hours and filter. After pouring the filtrate into diethyl ether, a precipitate appears. After 7 days, filter and dry to obtain the iron complex. X3 Weigh 5-6 parts by weight of oligomannose, add 25-30 parts by weight of water to dissolve it, add 0.5 mol / L sodium hydroxide aqueous solution to adjust the pH to 9-10, stir and mix evenly, add 5-6 parts by weight of the iron complex from step X2, and keep the pH at 9-10, then add twice the volume of anhydrous ethanol for precipitation, centrifuge at 4000-5000 rpm for 5-10 min, precipitate the lower precipitate with twice the volume of anhydrous ethanol and continue centrifugation, freeze-dry the lower precipitate to obtain the mannose-iron complex as an organic iron chelating agent.

2. The method as described in claim 1, characterized in that: In step S1, the recipient sows must be of Landrace, Large White, or two-way crossbred pigs; three-way crossbred sows are prohibited.

3. The method as described in claim 1, characterized in that: The criteria for weaning sows selected in step S1 are: having given birth to more than 2 but less than 5 litters, with a litter size of ≥12 piglets in two consecutive litters, ≥10 weaned piglets, no ectopic delivery in the previous litter, no obvious uterine inflammation, healthy limbs and hooves, normal udder, and backfat of 14-18mm; the criteria for non-pregnant sows are the same as those for weaning sows.

4. The method as described in claim 1, characterized in that: In step S2, the injection dose of pregnant mare serum gonadotropin is 800-1000 iu / head, vitamin ADE injection is 4-5 mL / head, and human chorionic gonadotropin is 400-500 iu / head.

5. The method as described in claim 1, characterized in that: In step S4, the dosage of cefquinoxime suspension is 1 mL / 20 kg body weight, the dosage of lincomycin hydrochloride injection is 10-20 mL / head, and the dosage of houttuynia cordata injection is 50-60 mL / head.

6. The method as described in claim 1, characterized in that: In step S5, the dosage of human chorionic gonadotropin is 400-500 iu / head.

7. The method as described in claim 1, characterized in that: In step S6, the dosage of ceftiofur sodium is 0.5-1g / head, and the dosage of houttuynia cordata injection is 20mL / head.

8. The method as described in claim 1, characterized in that: In step S6, the amino acid supplementation is achieved by adding N-carbamoylglutamic acid to the feed at a rate of 8000g / t. Postoperative management includes fasting on the first day, feeding 0.8-1kg / head per day on the second day, 1.2-1.5kg / head per day on the third day, and 1.6-2kg / head per day from the fourth to the tenth day.

9. The method as described in claim 1, characterized in that: In step S6, the sows after postoperative management are fed according to their backfat condition. Specifically, from 11 to 30 days postoperatively, sows with a backfat of <14mm are fed 2.0-2.2 kg / day, and those with a backfat of 14-18mm are fed 1.8-2 kg / day; from 31 to 85 days postoperatively, sows with a backfat of <14mm are fed 2.6-2.8 kg / day, those with a backfat of 14-18mm are fed 2.0-2.2 kg / day, and those with a backfat of >18mm are fed 1.8-2 kg / day; after 86 days postoperatively, sows with a backfat of 18-22mm are fed 2.8-3 kg / day.

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