Complete-cycle nutrition-enhanced goat hybridization breeding method

Abstract

The invention relates to the technical field of livestock and poultry breeding, in particular to a goat hybridization breeding method capable of achieving complete-cycle nutrition enhancement. The core of the method is that systematic nutrition intervention runs through the whole breeding process. Before hybridization, performing differential nutrition pretreatment on the parents for 60 days: feeding the female parents with a breeding nutrition enhancer added with components such as D-chiro-inositol and N-acetylcysteine, and supplementing sperm motility enhancer containing acetylated L-carnitine and ergothioneine to the male parents. In the gamete treatment stage, the oocytes are subjected to in-vitro maturation culture in a special culture solution containing components such as D-chiro-inositol and a growth differentiation factor 9; after the sperms are subjected to gradient centrifugal screening, completing in-vitro fertilization in an optimized fertilization culture solution; after the embryos develop into blastocysts in the sequential culture system, carrying out laparoscopic transplantation, and carrying out staged precise nutrition culture on the pregnant ewes. According to the method, through multi-level nutrition enhancement, the reproductive capacity of the goats and the growth performance of offspring are effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of animal genetics, breeding and reproduction, and mainly relates to a method for hybrid breeding of goats, specifically a method for hybrid breeding of goats with enhanced nutrition throughout the entire breeding cycle. Background Technology

[0002] Goat hybridization breeding usually refers to mating parent breeds with different genetic advantages and utilizing the hybrid vigor of their offspring F1 generation in order to obtain a population with better performance than the parents in economic traits such as fertility, growth rate, and feed utilization.

[0003] However, existing hybridization techniques largely rely on natural mating or conventional artificial insemination, which limits the stability and maximization of offspring performance. To improve efficiency, embryo transfer technology has been applied to goat hybridization. However, this technology still faces bottlenecks in practice: First, its success rate is highly dependent on the natural physical condition of the donor and paternal parents, and gamete quality fluctuates, resulting in a limited number of high-quality embryos. Second, conventional in vitro embryo production processes lack targeted nutritional pretreatment for gametes, leaving room for improvement in the efficiency of the culture system. Third, existing methods mostly focus on the embryo manipulation itself, lacking a comprehensive, continuous, and systematic nutritional regulation design throughout the entire breeding chain, from parental nutritional preparation, gamete optimization, in vitro embryo culture, to gestation period cultivation. This results in the ineffective connection of potential at each stage, ultimately limiting the synergistic improvement of reproductive efficiency and offspring growth performance, and preventing the coordinated and full development of reproductive and growth performance.

[0004] Therefore, in view of the problems of unstable gamete quality, limited embryo production efficiency, and failure to maximize synergistic performance in the existing technology, the present invention aims to provide a full-cycle, systematic nutritional enhancement hybridization breeding solution. Summary of the Invention

[0005] This method centers on parental germplasm optimization and full-cycle nutritional regulation. Differentiated basal diets and specialized nutritional fortifiers are designed for the female Lüliang Black Goat and the male Jintang Black Goat, respectively, to improve the original quality of gametes through nutritional pretreatment. Building upon this, precise gamete processing technologies such as ultrasound-guided oocyte collection and Percoll density gradient centrifugation for sperm screening are integrated, along with a sequential embryo culture system, forming a complete technology chain from parental breeding and gamete optimization to embryo construction. Ultimately, through embryo transfer and targeted nutritional management during pregnancy, the reproductive and growth performance of goats is synergistically improved.

[0006] In a first aspect, the present invention provides a nutritional fortifier composition for goat hybridization, the composition comprising a reproductive nutritional fortifier and a sperm motility nutritional fortifier; wherein the reproductive nutritional fortifier contains the following ingredients per kilogram of maternal basal diet: D-chiroinositol 0.028-0.032g, acetylated L-carnitine 0.18-0.20g, melatonin 0.4-0.5mg, yeast selenium 0.18-0.20mg, N-acetylcysteine ​​0.9-1.0g, and L-arginine 1.8-2.0g; wherein the sperm motility nutritional fortifier contains the following ingredients per kilogram of paternal basal diet: acetylated L-carnitine 0.18-0.20g, L-citrulline 0.45-0.50g, ergothioneine 0.4-0.5mg, and zinc glycine 0.007-0.008g.

[0007] Secondly, the present invention provides a method for crossbreeding goats with enhanced nutrition throughout the entire life cycle. The method involves feeding selected maternal and paternal lines with a basic diet containing the reproductive nutrient fortifier and sperm motility nutrient fortifier described in the first aspect for nutritional pretreatment, followed by gamete preparation optimization, in vitro fertilization and blastocyst culture, laparoscopic embryo transfer and pregnancy nutrition cultivation to complete the crossbreeding of goats.

[0008] Preferably, the optimized gamete preparation includes oocyte collection and maturation culture, and sperm collection and screening; wherein sperm screening includes secondary screening using density gradient centrifugation.

[0009] Preferably, the present invention provides a method for crossbreeding goats with enhanced nutrition throughout the entire breeding cycle, the specific steps of which are as follows:

[0010] (a) Parental selection and nutritional pretreatment

[0011] (1) Selection of parent stock: The maternal parent should be a healthy goat aged 2-3 years with good reproductive performance; the paternal parent should be a robust goat aged 1.5-2 years with good semen quality.

[0012] (2) Nutritional pretreatment of the maternal parent: Nutritional intervention of the maternal parent began 60 days before hybridization. A compound feeding program was adopted, which combined the maternal parent basal diet with a reproductive nutrient fortifier. The maternal parent basal diet consisted of 30% corn, 18% soybean meal, 30% alfalfa hay, 10% wheat bran, 5% whole fat extruded soybean, 3% brewer's yeast, 2% rapeseed meal, and 2% seaweed powder. The reproductive nutrient fortifier formula contained 0.028-0.032g D-chiral inositol, 0.18-0.20g acetylated L-carnitine, 0.4-0.5mg melatonin, 0.18-0.20mg yeast selenium, 0.9-1.0g N-acetylcysteine ​​(NAC), and 1.8-2.0g L-arginine per kilogram of maternal parent basal diet. The reproductive nutrient fortifier was thoroughly mixed with the maternal parent basal diet in proportion and fed for 60 days. At the same time, the maternal parent was ensured to have 2 hours of exercise per day.

[0013] (3) Paternal nutritional pretreatment: Paternal nutritional intervention began 60 days before hybridization, using a compound feeding program consisting of a paternal basal diet and a sperm motility nutrient fortifier. The paternal basal diet consisted of 35% corn, 22% soybean meal, 25% oat hay, 6% wheat bran, 5% whole fat extruded soybean, 3% fish meal, 2% seaweed powder, and 2% brewer's yeast. The sperm motility nutrient fortifier formula contained 0.18-0.20g acetylated L-carnitine, 0.45-0.50g L-citrulline, 0.4-0.5mg ergothioneine, and 0.007-0.008g zinc glycine per kilogram of paternal basal diet. The sperm motility nutrient fortifier was thoroughly mixed with the paternal basal diet in the specified proportions and fed continuously until the semen collection was completed.

[0014] (II) Gamete preparation and optimization treatment

[0015] (1) Oocyte collection and maturation culture: Oocytes were collected from the pretreated maternal ovaries using ultrasound-guided oocyte collection technology, and oocytes with abnormal morphology were removed and placed in maturation culture medium for in vitro culture.

[0016] Further preferred, the oocyte collection technique under ultrasound guidance specifically involves: using an ultrasound probe to locate follicles on the maternal ovary, focusing on selecting follicles with a diameter of 2-6 mm for puncture and aspiration, controlling the aspiration negative pressure to 80-90 mmHg, collecting follicular fluid and recovering cumulus-oocyte complexes (COCs), removing morphologically abnormal oocytes, and placing them in a maturation culture medium for in vitro culture;

[0017] (2) Sperm collection and screening: Paternal semen was collected using the artificial vagina method, and initial screening under a microscope was performed to select sperm with a density ≥2×10⁻⁶. 9Semen with a motility ≥0.7 and a concentration of 1 / mL was selected for initial screening. The pre-screened semen was mixed with sperm diluent at a 1:3 ratio, incubated at 37°C for 10 min, and then centrifuged at 1500-2000 rpm for 10-15 min. The supernatant was discarded, and the precipitate was washed twice with diluent to obtain a pre-purified sperm suspension. Further screening was performed using density gradient centrifugation. Two different volume fractions of Percoll separation solution were slowly added sequentially to a centrifuge tube to form a double-layer gradient. The sperm suspension was gently added to the top layer, and centrifuged at 2000-2500 rpm for 20-25 min. The precipitate at the bottom of the tube after centrifugation was the high-motility sperm clump. The supernatant was discarded, and the precipitate was resuspended with sperm diluent. Finally, the sperm suspension with a motility ≥0.85 was selected for subsequent in vitro fertilization (IVF).

[0018] (III) In vitro fertilization and embryo culture

[0019] (1) In vitro fertilization: Selected high-motility sperm are mixed with mature oocytes and placed in fertilization culture medium. They are co-cultured at 38.5℃, 5% CO2 and saturated humidity for 18-20 hours to complete the sperm-egg combination process.

[0020] (2) Embryo culture and screening: After fertilization, the fertilized eggs are washed and transferred to the embryo culture medium to culture to the blastocyst stage; the embryo culture medium adopts a sequential culture system: cleavage embryo culture medium is used from day 1 to 3, with 4% fetal bovine serum and 0.2 mmol / L amino acid mixture added; blastocyst culture medium is used from day 4 to 7, with 8% fetal bovine serum, 0.5 mmol / L glucose and 0.1 mmol / L glutamine added; during the culture process, the embryo development is observed daily, embryos with developmental arrest or abnormal morphology are removed, and high-quality blastocysts with a grade of 4AA (Gardner blastocyst scoring system) are selected for transfer;

[0021] (iv) Embryo transfer and cultivation

[0022] (1) Embryo transfer: Laparoscopic embryo transfer technology is used for embryo transfer. High-quality blastocysts are selected and transferred to the uterine horn of the recipient mother after estrus synchronization treatment. Three blastocysts are transferred to each mother. At the same time, the transfer depth is precisely controlled to keep it 2-3 cm away from the tip of the uterine horn. After the operation, in order to ensure the implantation and development of blastocysts, 15 mg of progesterone is injected into each ewe intramuscularly for 14 consecutive days. More preferably, the estrus synchronization treatment is as follows: vaginal sponge plug (containing progestin) is inserted into the vagina of the recipient ewe for 12-14 days. When the plug is removed, 0.1 mg of cloprostenol is injected intramuscularly per ewe to induce estrus synchronization. Embryo transfer is performed on the 6th-7th day of the estrus cycle.

[0023] (2) Nutritional training during pregnancy: During the first 3 months of pregnancy, the basic diet of the mother remains unchanged, and 0.01g / kg body weight of docosahexaenoic acid (DHA) and 0.008g / kg body weight of choline are added to promote the development of embryonic organs. During the last 2 months of pregnancy, the energy and protein supply of the diet is increased. Specifically, on the basis of the original pregnancy diet formula, for every 1kg increase in diet, 100g of corn and 40g of soybean meal are added and mixed together. At the same time, 0.01g of ferrous fumarate is added per kilogram of diet to prevent fetal anemia. During the entire pregnancy, the mother is guaranteed to have sufficient drinking water to avoid stress response. Regular prenatal check-ups are also conducted. One week before the end of pregnancy, the mother is transferred to a sterile farrowing room to observe lambing and assist with delivery to ensure that the ewe delivers smoothly and obtains the F1 generation of hybrid goats.

[0024] Preferably: In step (a) (1) of the present invention, the father goat is Jintang Black Goat and the mother goat is Lüliang Black Goat.

[0025] Preferred: In step (ii) (1) of the present invention, the maturation culture medium is based on TCM199, with the addition of 4.5-5.0 g / L of recombinant human serum albumin (rhSA), 0.09-0.10 g / L of reduced glutathione (GSH), 0.09-0.10 g / L of D-chiroinositol, and 13-15 ng / mL of growth differentiation factor 9 (GDF9). The culture is carried out at 38.5℃, 5% CO2, and saturated humidity for 24-28 h until the oocytes expel the first polar body, thus obtaining mature oocytes.

[0026] Preferably, the sperm diluent formula in step (ii) (2) of the present invention is as follows: 4.0g glucose, 1.0g fructose, 2.0g trehalose, 1.4g sodium citrate, 0.2g sodium bicarbonate, 0.075g potassium chloride, 0.05g sodium pyruvate, 0.002g gentamicin, and distilled water to 100mL;

[0027] Preferably, in step (ii) (2) of the present invention, the two different volume fractions of the Percoll separation solution are 80% and 40%, respectively.

[0028] Preferably, in step (iii) (1) of the present invention, the ratio of mature oocytes to screened high-motility sperm is 1:10. 5 .

[0029] Preferably, in step (iii) (1) of this invention, the fertilization culture medium is based on BO-IVF fertilization medium, with the addition of recombinant human serum albumin (rhSA) 45-50 g / L, reduced glutathione (GSH) 0.055-0.060 g / L, D-chiroinositol 0.09-0.10 g / L, and growth differentiation factor 9 (GDF9) 9-10 ng / mL to improve sperm-egg fertilization efficiency. This invention specifically uses recombinant human serum albumin in the in vitro culture system. Compared with conventionally used fetal bovine serum (FBS), recombinant human serum albumin has a clearly defined composition, not only providing essential nutrients and osmotic pressure support for gametes and embryos, but more importantly, significantly reducing the risk of pathogen transmission and batch-to-batch quality differences from animal-derived serum, thus improving the stability and safety of the in vitro embryo production system.

[0030] Secondly, the goats bred through hybridization in this invention achieve a synergistic improvement in both reproductive and growth performance.

[0031] The present invention has the following beneficial effects:

[0032] 1. Construct a full-cycle collaborative technology system to achieve deep integration of parental nutrition enhancement, in vitro culture optimization and pregnancy regulation, break through the limitations of traditional single-link improvements, form a complete technical closed loop from gamete development to offspring breeding, and enhance the continuity and efficiency of the entire reproductive process.

[0033] 2. Create a continuous in-vivo and in-vitro nutrition design, so that the parent diet and the key components in the in vitro culture medium can work synergistically to lay a high-quality natural foundation for gametes and provide continuous adaptive support for embryonic development, thus solving the problem of fragmented traditional nutritional intervention and fully stimulating reproductive potential.

[0034] 3. By integrating specific functional components with precise operation techniques, through synergistic protection of antioxidant components, precise ratio of reproductive regulatory factors, and key operations such as secondary sperm screening and precise laparoscopic transplantation, we can achieve a simultaneous leap in reproductive performance and offspring growth performance, overcoming the core pain points of low efficiency and lack of prominent offspring advantages in traditional hybridization. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a comparison chart of the daily weight gain of goats in Examples 1-3 and Comparative Examples 1-4, Comparative Example 9 and Comparative Examples 11-13.

[0037] Figure 2 This is a comparison chart of the daily feed intake of goats in Examples 1-3 and Comparative Examples 1-4, Comparative Example 9 and Comparative Examples 11-13.

[0038] Figure 3 This is a comparison chart of the weight ratio of goat feed in Examples 1-3 and Comparative Examples 1-4, Comparative Example 9 and Comparative Examples 11-13. Detailed Implementation

[0039] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0040] TCM199 culture medium was purchased from Merck Co., Ltd. (Beijing); Percoll separation medium was purchased from Solarbio Science & Technology Co., Ltd. (Beijing); BO-IVF fertilization medium was purchased from Guangzhou Huayuexing Medical Technology Co., Ltd. (Guangzhou); D-chiral inositol was purchased from Sichuan Weikeqi Biotechnology Co., Ltd. (Sichuan); acetylated L-carnitine was purchased from Wuhan Huaxiang Kejie Biotechnology Co., Ltd. (Wuhan); amino acid mixture (item number Y0005) was purchased from Beijing Lanbolide Trading Co., Ltd. (Beijing); cleavage embryo culture medium (item number V018060) and blastocyst culture medium (item number V019060) were both purchased from Shenzhen Weituo Biotechnology Co., Ltd.

[0041] Example 1

[0042] This embodiment provides a method for crossbreeding goats with enhanced nutrition throughout the entire breeding cycle, the specific steps of which are as follows:

[0043] I. Parental selection and nutritional pretreatment

[0044] The donor female line was selected from healthy, 2-3 year old Lüliang Black Goats with good reproductive performance, and the male line was selected from robust, 1.5-2 year old Jintang Black Goats with good semen quality. Nutritional intervention for the parent lines was initiated simultaneously 60 days before crossbreeding. The female line was fed a compound diet consisting of a basic maternal diet and a reproductive nutrient fortifier. The basic maternal diet consisted of 30% corn, 18% soybean meal, 30% alfalfa hay, 10% wheat bran, 5% whole fat extruded soybeans, 3% brewer's yeast, 2% rapeseed meal, and 2% seaweed powder. The reproductive nutrient fortifier formula contained 0.028g D-chiral inositol, 0.18g acetylated L-carnitine, 0.4mg melatonin, 0.18mg yeast selenium, 0.9g N-acetylcysteine ​​(NAC), and 1.8g L-arginine per kilogram of the basic maternal diet. The basic maternal diet and reproductive nutrient fortifier were thoroughly mixed in the specified proportions and fed for 60 days, ensuring the female line had 2 hours of exercise daily.

[0045] The paternal parent was fed a compound diet consisting of a basal diet and a sperm motility fortifier. The basal diet consisted of 35% corn, 22% soybean meal, 25% oat hay, 6% wheat bran, 5% whole fat extruded soybeans, 3% fish meal, 2% seaweed powder, and 2% brewer's yeast. The sperm motility fortifier was formulated with 0.18g acetylated L-carnitine, 0.45g L-citrulline, 0.4mg ergothioneine, and 0.007g zinc glycine per kilogram of the basal diet. The basal diet and sperm motility fortifier were thoroughly mixed in the correct proportions and fed continuously until the semen collection was completed.

[0046] II. Gamete Preparation and Optimization

[0047] Oocytes were collected using ultrasound-guided oocyte retrieval technology. An ultrasound probe was used to locate follicles on the maternal ovary, with a focus on selecting follicles with a diameter of 2-6 mm for aspiration. The aspiration negative pressure was controlled at 80 mmHg. Follicular fluid was collected, and cumulus-oocyte complexes (COCs) were recovered. Abnormally morphologically damaged oocytes were removed. The oocytes were then placed in a maturation culture medium based on TCM199 for in vitro culture. The maturation culture medium was supplemented with recombinant human serum albumin (rhSA) 4.5 g / L, reduced glutathione (GSH) 0.09 g / L, D-chiroinositol 0.09 g / L, and growth differentiation factor 9 (GDF9) 13 ng / mL. The culture was maintained at 38.5℃, 5% CO2, and saturated humidity for 24 h until the oocytes extruded the first polar body to obtain mature oocytes. Paternal semen was collected using a sham vaginal technique, and sperm density ≥2×10⁻⁶ was selected after microscopic screening. 9Semen with a motility ≥0.7 and a concentration of 1 / mL was prepared for use. The pre-screened semen was mixed with sperm diluent at a ratio of 1:3, incubated in a 37℃ water bath for 10 min, and then centrifuged at 1500 rpm for 10 min. The supernatant was discarded, and the precipitate was washed twice with the diluent to obtain a preliminarily purified sperm suspension. The sperm diluent formulation was: glucose 4.0 g, fructose 1.0 g, trehalose 2.0 g, sodium citrate 1.4 g, sodium bicarbonate 0.2 g, potassium chloride 0.075 g, sodium pyruvate 0.05 g, and gentamicin 0.002 g. Add distilled water to 100 mL; further screening is performed using density gradient centrifugation. 80% and 40% Percoll separation solutions from Beijing Solarbio Science & Technology Co., Ltd. are slowly added sequentially to the centrifuge tube to form a double-layer gradient. The solvent for both separation solutions is the aforementioned sperm diluent. The sperm suspension is gently added to the top layer, centrifuged at 2000 rpm for 20 min, and the high-motility sperm clusters at the bottom of the tube are collected. The supernatant is discarded, and the precipitate is resuspended in sperm diluent. Finally, the sperm suspension with a motility ≥0.85 is selected for subsequent in vitro fertilization under a microscope.

[0048] III. In Vitro Fertilization and Embryo Culture

[0049] The selected high-motility sperm were mixed with mature oocytes at a ratio of 1:10. 5 The mixture was prepared in the specified proportions and placed in fertilization culture medium. The mixture was then co-cultured at 38.5℃, 5% CO2, and saturated humidity for 18 hours to complete the fertilization process. The fertilization culture medium was based on BO-IVF fertilization medium, supplemented with recombinant human serum albumin (rhSA) 45 g / L, reduced glutathione (GSH) 0.055 g / L, D-chiroinositol 0.09 g / L, and growth differentiation factor 9 (GDF9) 9 ng / mL to enhance fertilization efficiency. After fertilization, the fertilized eggs were washed and transferred to embryo culture medium. During the blastocyst stage, a sequential culture system was used for embryo culture. From day 1 to 3, cleavage embryo culture medium supplemented with 4% fetal bovine serum and 0.2 mmol / L amino acid mixture was used. From day 4 to 7, blastocyst culture medium supplemented with 8% fetal bovine serum, 0.5 mmol / L glucose and 0.1 mmol / L glutamine was used. During the culture process, the embryo development was observed daily, and embryos with developmental arrest or abnormal morphology were removed. High-quality blastocysts that meet the Gardner blastocyst scoring system 4AA grade standard were selected for transfer.

[0050] IV. Embryo Transfer and Culture

[0051] Laparoscopic embryo transfer was used. Recipient ewes underwent estrus synchronization treatment, specifically by inserting a progesterone-containing vaginal sponge into the vagina of the recipient ewe for 12 days. Upon removal of the sponge, each recipient ewe was given an intramuscular injection of 0.1 mg cloprostenol to induce estrus synchronization. On day 6 of the estrous cycle, selected high-quality blastocysts were transferred into the uterine horn, with 3 blastocysts transferred per ewe. The transfer depth was precisely controlled to maintain a position 2 cm from the tip of the uterine horn. Post-operatively, to ensure blastocyst implantation and development, each ewe was given an intramuscular injection of 15 mg progesterone daily for 14 consecutive days. During the first three months of pregnancy, the maternal basal diet remained unchanged, supplemented with 0. Docosahexaenoic acid (DHA) is added at 0.01g / kg body weight, and choline is added at 0.008g / kg body weight to promote embryonic organ differentiation and development. During the second month of pregnancy, the energy and protein supply in the diet is increased. Specifically, for every additional 1kg of diet, 100g of corn and 40g of soybean meal are added and mixed together. At the same time, 0.01g of ferrous fumarate is added per kilogram of diet to prevent fetal anemia. Throughout the pregnancy, the ewes are given sufficient water and stress is avoided. Regular prenatal checkups are conducted. One week before the end of pregnancy, the ewes are transferred to a sterile farrowing pen for lambing observation and assistance to ensure a smooth delivery and the generation of hybrid F1 goats.

[0052] Example 2

[0053] This embodiment provides a method for crossbreeding goats with enhanced nutrition throughout the entire breeding cycle, the specific steps of which are as follows:

[0054] I. Parental selection and nutritional pretreatment

[0055] The donor female was selected from healthy, 2-3 year old Lüliang Black Goats with good reproductive performance, and the male was selected from robust, 1.5-2 year old Jintang Black Goats with good semen quality. Nutritional intervention for the parent stock was initiated simultaneously 60 days before crossbreeding. The female was fed a compound diet consisting of a basic maternal diet and a reproductive nutrient fortifier. The basic maternal diet consisted of 30% corn, 18% soybean meal, 30% alfalfa hay, 10% wheat bran, 5% whole fat extruded soybeans, 3% brewer's yeast, 2% rapeseed meal, and 2% seaweed powder. The reproductive nutrient fortifier formula contained 0.03g D-chiral inositol, 0.19g acetylated L-carnitine, 0.45mg melatonin, 0.19mg yeast selenium, 0.95g N-acetylcysteine ​​(NAC), and 1.9g L-arginine per kilogram of the basic maternal diet. The basic maternal diet and reproductive nutrient fortifier were thoroughly mixed in the specified proportions and fed for 60 days, ensuring the female had 2 hours of exercise daily.

[0056] The paternal parent was fed a compound diet consisting of a basal diet and a sperm motility fortifier. The basal diet consisted of 35% corn, 22% soybean meal, 25% oat hay, 6% wheat bran, 5% whole fat extruded soybeans, 3% fish meal, 2% seaweed powder, and 2% brewer's yeast. The sperm motility fortifier was formulated with 0.19g acetylated L-carnitine, 0.47g L-citrulline, 0.45mg ergothioneine, and 0.0075g zinc glycine per kilogram of the basal diet. The basal diet and sperm motility fortifier were thoroughly mixed in the correct proportions and fed continuously until the semen collection was completed.

[0057] II. Gamete Preparation and Optimization

[0058] Oocytes were collected using ultrasound-guided oocyte retrieval technology. An ultrasound probe was used to locate follicles on the maternal ovary, with a focus on selecting follicles with a diameter of 2-6 mm for aspiration. The aspiration negative pressure was controlled at 85 mmHg. Follicular fluid was collected, and cumulus-oocyte complexes (COCs) were recovered. Abnormally morphologically damaged oocytes were removed. The oocytes were then placed in a maturation culture medium based on TCM199 for in vitro culture. The maturation culture medium was supplemented with recombinant human serum albumin (rhSA) 4.7 g / L, reduced glutathione (GSH) 0.095 g / L, D-chiroinositol 0.095 g / L, and growth differentiation factor 9 (GDF9) 14 ng / mL. The culture was maintained at 38.5℃, 5% CO2, and saturated humidity for 26 h until the oocytes extruded the first polar body to obtain mature oocytes. Paternal semen was collected using a sham vaginal technique, and sperm density ≥2×10⁻⁶ was selected after microscopic screening. 9 Semen with a motility ≥0.7 and a concentration of 1 / mL was prepared for use. The pre-screened semen was mixed with sperm diluent at a ratio of 1:3, incubated in a 37℃ water bath for 10 min, and then centrifuged at 1700 rpm for 12 min. The supernatant was discarded, and the precipitate was washed twice with the diluent to obtain a preliminarily purified sperm suspension. The sperm diluent formulation was: glucose 4.0 g, fructose 1.0 g, trehalose 2.0 g, sodium citrate 1.4 g, sodium bicarbonate 0.2 g, potassium chloride 0.075 g, sodium pyruvate 0.05 g, and gentamicin 0.002 g. Add distilled water to 100 mL; further screening is performed using density gradient centrifugation. 80% and 40% Percoll separation solutions from Beijing Solarbio Science & Technology Co., Ltd. are slowly added sequentially to the centrifuge tube to form a double-layer gradient. The solvent for both separation solutions is the aforementioned sperm diluent. The sperm suspension is gently added to the top layer, centrifuged at 2200 rpm for 22 min, and the high-motility sperm clusters at the bottom of the tube are collected. The supernatant is discarded, and the precipitate is resuspended in sperm diluent. Finally, the sperm suspension with a motility ≥0.85 is selected for subsequent in vitro fertilization under a microscope.

[0059] III. In Vitro Fertilization and Embryo Culture

[0060] The selected high-motility sperm were mixed with mature oocytes at a ratio of 1:10. 5 The mixture was prepared in the specified proportions and placed in fertilization culture medium. The mixture was co-cultured at 38.5℃, 5% CO2, and saturated humidity for 19 hours to complete the fertilization process. The fertilization culture medium was based on BO-IVF fertilization medium, supplemented with recombinant human serum albumin (rhSA) 47 g / L, reduced glutathione (GSH) 0.057 g / L, D-chiroinositol 0.095 g / L, and growth differentiation factor 9 (GDF9) 9.5 ng / mL to enhance fertilization efficiency. After fertilization, the fertilized eggs were washed and transferred to embryo culture. Embryos were cultured in liquid medium to the blastocyst stage. A sequential culture system was used for the embryo culture medium. From day 1 to 3, cleavage embryo culture medium supplemented with 4% fetal bovine serum and 0.2 mmol / L amino acid mixture was used. From day 4 to 7, blastocyst culture medium supplemented with 8% fetal bovine serum, 0.5 mmol / L glucose and 0.1 mmol / L glutamine was used. During the culture process, the embryo development was observed daily, and embryos with developmental arrest or abnormal morphology were removed. High-quality blastocysts that met the Gardner blastocyst scoring system 4AA grade standard were selected for transfer.

[0061] IV. Embryo Transfer and Culture

[0062] Laparoscopic embryo transfer was used. Recipient ewes underwent estrus synchronization treatment, specifically by inserting a progesterone-containing vaginal sponge into the vagina of the recipient ewe for 13 days. Upon removal of the sponge, each recipient ewe was given an intramuscular injection of 0.1 mg cloprostenol to induce estrus synchronization. On day 6 of the estrous cycle, selected high-quality blastocysts were transferred to the uterine horn, with 3 blastocysts transferred to each ewe. The transfer depth was precisely controlled to maintain a position 2 cm from the tip of the uterine horn. Post-operatively, to ensure blastocyst implantation and development, each ewe was given an intramuscular injection of 15 mg progesterone daily for 14 consecutive days. During the first three months of pregnancy, the maternal basal diet remained unchanged, supplemented with 0. Docosahexaenoic acid (DHA) is added at 0.01g / kg body weight, and choline is added at 0.008g / kg body weight to promote embryonic organ differentiation and development. During the second month of pregnancy, the energy and protein supply in the diet is increased. Specifically, for every additional 1kg of diet, 100g of corn and 40g of soybean meal are added and mixed together. At the same time, 0.01g of ferrous fumarate is added per kilogram of diet to prevent fetal anemia. Throughout the pregnancy, the ewes are given sufficient water and stress is avoided. Regular prenatal checkups are conducted. One week before the end of pregnancy, the ewes are transferred to a sterile farrowing pen for lambing observation and assistance to ensure a smooth delivery and the generation of hybrid F1 goats.

[0063] Example 3

[0064] This embodiment provides a method for crossbreeding goats with enhanced nutrition throughout the entire breeding cycle, the specific steps of which are as follows:

[0065] I. Parental selection and nutritional pretreatment

[0066] The donor female was selected from healthy, 2-3 year old Lüliang Black Goats with good reproductive performance, and the male was selected from robust, 1.5-2 year old Jintang Black Goats with good semen quality. Nutritional intervention for the parent stock was initiated simultaneously 60 days before crossbreeding. The female was fed a compound diet consisting of a basic maternal diet and a reproductive nutrient fortifier. The basic maternal diet consisted of 30% corn, 18% soybean meal, 30% alfalfa hay, 10% wheat bran, 5% whole fat extruded soybeans, 3% brewer's yeast, 2% rapeseed meal, and 2% seaweed powder. The reproductive nutrient fortifier formula contained 0.032g D-chiral inositol, 0.2g acetylated L-carnitine, 0.5mg melatonin, 0.2mg yeast selenium, 1.0g N-acetylcysteine ​​(NAC), and 2.0g L-arginine per kilogram of the basic maternal diet. The basic maternal diet and reproductive nutrient fortifier were thoroughly mixed in the specified proportions and fed for 60 days, ensuring the female had 2 hours of exercise daily.

[0067] The paternal parent was fed a compound diet consisting of a basal diet and a sperm motility fortifier. The basal diet consisted of 35% corn, 22% soybean meal, 25% oat hay, 6% wheat bran, 5% whole fat extruded soybeans, 3% fish meal, 2% seaweed powder, and 2% brewer's yeast. The sperm motility fortifier was formulated with 0.2g acetylated L-carnitine, 0.5g L-citrulline, 0.5mg ergothioneine, and 0.008g zinc glycine per kilogram of the basal diet. The basal diet and sperm motility fortifier were thoroughly mixed in the specified proportions and fed continuously until the semen collection was completed.

[0068] II. Gamete Preparation and Optimization

[0069] Oocytes were collected using ultrasound-guided oocyte retrieval technology. An ultrasound probe was used to locate follicles on the maternal ovary, with a focus on selecting follicles with a diameter of 2-6 mm for aspiration. The aspiration negative pressure was controlled at 90 mmHg. Follicular fluid was collected, and cumulus-oocyte complexes (COCs) were recovered. Abnormally morphologically damaged oocytes were removed. The oocytes were then placed in a maturation culture medium based on TCM199 for in vitro culture. The maturation culture medium was supplemented with recombinant human serum albumin (rhSA) 5.0 g / L, reduced glutathione (GSH) 0.10 g / L, D-chiroinositol 0.10 g / L, and growth differentiation factor 9 (GDF9) 15 ng / mL. The culture was maintained at 38.5℃, 5% CO2, and saturated humidity for 28 hours until the oocytes extruded the first polar body to obtain mature oocytes. Paternal semen was collected using a sham vaginal technique, and sperm density ≥2×10⁻⁶ was selected after microscopic screening. 9Semen with a motility ≥0.7 and a concentration of 1 / mL was prepared for use. The pre-screened semen was mixed with sperm diluent at a ratio of 1:3, incubated in a 37℃ water bath for 10 min, and then centrifuged at 2000 rpm for 15 min. The supernatant was discarded, and the precipitate was washed twice with the diluent to obtain a preliminarily purified sperm suspension. The sperm diluent formulation was: glucose 4.0 g, fructose 1.0 g, trehalose 2.0 g, sodium citrate 1.4 g, sodium bicarbonate 0.2 g, potassium chloride 0.075 g, sodium pyruvate 0.05 g, and gentamicin 0.002 g. Add distilled water to 100 mL; further screening is performed using density gradient centrifugation. 80% and 40% Percoll separation solutions from Beijing Solarbio Science & Technology Co., Ltd. are slowly added sequentially to the centrifuge tube to form a double-layer gradient. The solvent for both separation solutions is the aforementioned sperm diluent. The sperm suspension is gently added to the top layer, centrifuged at 2500 rpm for 25 min, and the high-motility sperm clusters at the bottom of the tube are collected. The supernatant is discarded, and the precipitate is resuspended in sperm diluent. Finally, the sperm suspension with a motility ≥0.85 is selected for subsequent in vitro fertilization under a microscope.

[0070] III. In Vitro Fertilization and Embryo Culture

[0071] The selected high-motility sperm were mixed with mature oocytes at a ratio of 1:10. 5 The mixture was prepared in the specified proportions and placed in fertilization culture medium. The mixture was then co-cultured at 38.5℃, 5% CO2, and saturated humidity for 20 hours to complete the fertilization process. The fertilization culture medium was based on BO-IVF fertilization medium, supplemented with 50 g / L recombinant human serum albumin (rhSA), 0.060 g / L reduced glutathione (GSH), 0.10 g / L D-chiroinositol, and 10 ng / mL growth differentiation factor 9 (GDF9) to enhance fertilization efficiency. After fertilization, the fertilized eggs were washed and transferred to embryo culture medium. During the blastocyst stage, a sequential culture system was used for embryo culture. From day 1 to 3, cleavage embryo culture medium supplemented with 4% fetal bovine serum and 0.2 mmol / L amino acid mixture was used. From day 4 to 7, blastocyst culture medium supplemented with 8% fetal bovine serum, 0.5 mmol / L glucose and 0.1 mmol / L glutamine was used. During the culture process, the embryo development was observed daily, and embryos with developmental arrest or abnormal morphology were removed. High-quality blastocysts that meet the Gardner blastocyst scoring system 4AA grade standard were selected for transfer.

[0072] IV. Embryo Transfer and Culture

[0073] Laparoscopic embryo transfer was used. Recipient ewes underwent estrus synchronization treatment, specifically by inserting a progesterone-containing vaginal sponge into the vagina of the recipient ewe for 14 days. Upon removal of the sponge, each recipient ewe was given an intramuscular injection of 0.1 mg cloprostenol to induce estrus synchronization. On day 7 of the estrous cycle, selected high-quality blastocysts were transferred to the uterine horn, with 3 blastocysts transferred to each ewe. The transfer depth was precisely controlled to maintain a position 3 cm from the tip of the uterine horn. Post-operatively, to ensure blastocyst implantation and development, each ewe was given an intramuscular injection of 15 mg progesterone daily for 14 consecutive days. During the first three months of pregnancy, the maternal basal diet remained unchanged, supplemented with 0. Docosahexaenoic acid (DHA) is added at 0.01g / kg body weight, and choline is added at 0.008g / kg body weight to promote embryonic organ differentiation and development. During the second month of pregnancy, the energy and protein supply in the diet is increased. Specifically, for every additional 1kg of diet, 100g of corn and 40g of soybean meal are added and mixed together. At the same time, 0.01g of ferrous fumarate is added per kilogram of diet to prevent fetal anemia. Throughout the pregnancy, the ewes are given sufficient water and stress is avoided. Regular prenatal checkups are conducted. One week before the end of pregnancy, the ewes are transferred to a sterile farrowing pen for lambing observation and assistance to ensure a smooth delivery and the generation of hybrid F1 goats.

[0074] Comparative Example 1: No maternal reproductive nutrient fortifier was added, and the remaining steps were the same as in Example 2.

[0075] Comparative Example 2: The maternal parent reproductive nutrient fortifier did not contain D-chiroinositol, and the remaining steps were the same as in Example 2.

[0076] Comparative Example 3: No melatonin was added to the maternal reproductive nutrient fortifier, and the remaining steps were the same as in Example 2.

[0077] Comparative Example 4: No yeast selenium was added to the maternal parent reproductive nutrient fortifier, and the remaining steps were the same as in Example 2.

[0078] Comparative Example 5: No recombinant human serum albumin was added to the in vitro culture medium (including oocyte maturation culture medium and fertilization culture medium), and the remaining steps were the same as in Example 2.

[0079] Comparative Example 6: No reduced glutathione was added to the in vitro culture medium (including oocyte maturation culture medium and fertilization culture medium), and the remaining steps were the same as in Example 2.

[0080] Comparative Example 7: No D-chiroinositol was added to the in vitro culture medium (including oocyte maturation culture medium and fertilization culture medium), and the remaining steps were the same as in Example 2.

[0081] Comparative Example 8: No growth differentiation factor 9 was added to the in vitro culture medium (including oocyte maturation culture medium and fertilization culture medium), and the remaining steps were the same as in Example 2.

[0082] Comparative Example 9: The Percoll density gradient centrifugation secondary screening was cancelled, and the remaining steps were the same as in Example 2.

[0083] Comparative Example 10: Embryo transfer was performed using conventional surgical methods instead of laparoscopy, with the remaining steps being the same as in Example 2.

[0084] Comparative Example 11: (In vivo nutrition + in vitro routine): The parents used the enhancer of the present invention, but the oocyte maturation culture medium and fertilization culture medium used in the gamete preparation and optimization treatment / in vitro fertilization and culture stages were only TCM199 / BO-IVF fertilization medium, and no special components of the present invention (such as recombinant human serum albumin, reduced glutathione, D-chiroinositol, growth differentiation factor 9, etc.) were added. The remaining steps were the same as in Example 2.

[0085] Comparative Example 12: (In vivo routine + in vitro optimization): The parents were fed the same basal diet as in Example 2 (i.e., without the addition of reproductive nutrient fortifiers and sperm motility nutrient fortifiers), and the nutrient pretreatment time remained at 60 days. The in vitro procedures (including oocyte collection, maturation culture, sperm screening, in vitro fertilization, embryo culture, and embryo transfer) were the same as in Example 2.

[0086] Comparative Example 13: The traditional crossbreeding method was adopted. The steps were as follows: the female (Lvliang Black Goat) and the male (Jintang Black Goat) were fed the same basic diet formula as in Example 2 (i.e., without adding reproductive nutrient fortifiers and sperm vitality nutrient fortifiers), mated naturally, the embryos developed naturally, and the offspring were raised according to conventional methods. All other conditions were the same as in Example 2.

[0087] I. Reproductive performance testing

[0088] Subjects: 304 recipient female goats (2-3 year old, healthy, and with good reproductive performance, Lüliang Black Goats) were selected and randomly divided into Example 1-3 groups and Comparative Example 1-13 groups, with 19 goats in each group.

[0089] Measurement indicators: Count the total number of lambs born, the number of ewes with multiple lambs, and the number of surviving lambs; calculate:

[0090] Lambing rate = Total number of lambs / Number of ewes mated × 100%

[0091] Multi-lamb birth rate = (Number of ewes with multiple lambs / Number of ewes mated) × 100%

[0092] Survival rate = (Number of surviving lambs / Total number of lambs born) × 100%

[0093] Measurement period: Record one breeding cycle and take the average value.

[0094] Note: The survival rate of lambs is determined using the 72-hour survival method, which means that lambs that have stable breathing and heartbeat within 72 hours after birth, can suckle their mother's milk independently, have no congenital malformations, and have a birth weight of ≥2.0kg are considered to be alive.

[0095] Table 1

[0096]

[0097] Table 1 compares the reproductive performance of goats in Examples 1-3 and Comparative Examples 1-13. The results show that the lambing rate, multiple birth rate, and survival rate of Examples 1-3 were significantly better than those of Comparative Examples 1-13. Furthermore, the differences in these three indicators among Examples 1-3 were minimal, all remaining at excellent levels, fully demonstrating that the hybridization breeding method of this invention can effectively improve the reproductive performance of goats.

[0098] The example group achieved optimal goat reproductive performance through systematic nutritional intervention: parental nutritional fortification provided sufficient functional substances for gametes, precise optimization of in vitro culture improved the quality of gametes and embryos, and laparoscopic transplantation and pregnancy management ensured embryo implantation and survival, ultimately increasing lambing rate, multiple birth rate and survival rate. The performance decline in each comparative example stemmed from defects in different stages: Comparative example 1, lacking maternal reproductive nutrient fortifiers, suffered from insufficient key nutritional support for maternal oocyte development, resulting in decreased gamete quality and a significant reduction in lambing rate and multiple birth rate; Comparative example 2, lacking D-chiroinositol, affected the maturation process of maternal oocytes, reducing the proportion of high-quality oocytes and causing a decline in lambing-related indicators; Comparative example 3, lacking melatonin, disrupted the maternal reproductive endocrine balance, worsening ovulation and embryo implantation conditions, and reducing the multiple birth rate; Comparative example 4, lacking yeast selenium, resulted in insufficient maternal antioxidant capacity, increased risk of oocyte damage, and decreased survival rate; Comparative example 5, lacking recombinant human serum albumin, suffered from insufficient nutrient supply during oocyte maturation culture, reduced maturation rate, and affected lambing rate; Comparative example 6, lacking reduced glutathione, exacerbated oxidative damage to oocytes, weakened embryonic developmental potential, and reduced survival rate. Comparative Example 7 lacked D-chiroinositol, which hindered oocyte maturation, reduced fertilization success rate, and decreased lambing rate. Comparative Example 8 lacked growth differentiation factor 9, resulting in insufficient oocyte development signals, a reduced proportion of high-quality embryos, and a decline in both multiparity rate and survival rate. Comparative Example 9 eliminated secondary sperm screening, leading to the contamination of low-motility sperm and an increased rate of embryonic abnormalities after fertilization, thus reducing survival rate. Comparative Example 10 used conventional surgical transfer, which was highly invasive and lacked precise positioning, resulting in a lower embryo implantation rate and a slightly lower lambing rate. Comparative Example 11 lacked specific components in the in vitro process, reducing the efficiency of sperm-egg fusion and embryo development, and affecting both lambing and survival indicators. Comparative Example 12 did not provide nutritional fortification for the parents, resulting in insufficient gamete quality, which negated the effects of in vitro optimization and led to a decline in all indicators. Comparative Example 13 used traditional methods without targeted nutritional intervention or in vitro optimization, resulting in low gamete quality, low fertilization efficiency, and the worst reproductive performance.

[0099] II. Growth Performance Measurement

[0100] Subjects for testing: F1 generation lambs obtained through the above reproductive performance test were selected as the base population. Nineteen healthy F1 generation goats aged 3 months were randomly selected from each of the following groups: Examples 1-3, Comparative Examples 1-4, Comparative Example 9, and Comparative Examples 11-13.

[0101] Weigh and record the changes in weight and feed intake of goats from 3 months to 6 months of age, and then calculate the daily weight gain and daily feed intake. The experimental results are shown in Table 2.

[0102] Average daily weight gain = (final weight - initial weight) / number of days in the trial;

[0103] Average daily feed intake = total feed intake / number of days in the experiment.

[0104] Feed conversion ratio = Average daily feed intake / Average daily weight gain

[0105] Table 2

[0106]

[0107] Table 2 compares the growth performance of goats in Examples 1-3 and Comparative Examples 1-4, 9, and 11-13. The results show that compared to Example 1, the average daily weight gain and average daily feed intake of each comparative example were significantly reduced. Simultaneously, the feed conversion ratio of each comparative example was significantly higher than that of Example 1, while the Example groups, with their superior growth performance, exhibited a lower feed conversion ratio. This result fully demonstrates that the hybridization breeding method of this invention can not only significantly enhance the growth performance of offspring goats, increasing growth rate and feed intake, but also effectively improve feed utilization and reduce breeding costs, demonstrating significant application advantages.

[0108] Through numerous experiments, we have discovered that the growth performance of F1 generation goats is not determined by a single factor, but rather by the synergistic cooperation of parental nutritional fortification, in vitro manipulation optimization, and the entire process of gestation and rearing. The absence or imperfection of any one of these factors will directly lead to a decline in growth performance.

[0109] In this embodiment, our core design is to ensure that each stage supports the others, forming a closed loop. During the parental stage, multiple components in the maternal reproductive nutrient fortifier work synergistically to create a high-quality developmental foundation for the oocytes. D-chiroinositol improves metabolic status, melatonin regulates reproductive rhythms, and yeast selenium enhances antioxidant capacity. These components collectively guarantee the inherent quality of the gametes. The paternal nutrient fortification also enhances sperm motility, providing a prerequisite for the formation of high-quality embryos. In subsequent in vitro procedures, Percoll density gradient centrifugation removes low-motility sperm, preventing abnormal fertilization from affecting embryonic development. Simultaneously, the special components in the in vitro culture medium continue the parental nutrition, further improving embryo quality. Combined with scientific nurturing during gestation, the embryo's growth potential is fully released, ultimately resulting in ideal daily weight gain and feed intake for the F1 generation goats.

[0110] The performance of each comparative example precisely confirms the indispensability of this synergistic system. Comparative example 1, lacking the maternal reproductive nutrient fortifier, lacked the synergistic support of multiple components, resulting in congenital nutritional deficiencies in oocytes and decreased gamete quality. Even with normal subsequent in vitro manipulation and culture, the growth potential of offspring was limited from the source, with daily weight gain significantly lower than in the previous example. Comparative example 2, lacking D-chiroinositol in the maternal fortifier, disrupted the synergistic metabolic regulation between components, hindering oocyte maturation and weakening the crucial foundation for embryonic development, leading to a decline in offspring growth performance. Comparative example 3, lacking melatonin, experienced an imbalance in maternal reproductive endocrine rhythms, deteriorating the early embryonic development environment and indirectly affecting offspring growth capacity. Comparative example 4, lacking yeast selenium, weakened the maternal antioxidant synergistic defense, increasing the risk of oocyte and embryo damage and inhibiting offspring growth potential. Comparative example 9 eliminated secondary sperm screening. The introduction of low-motility sperm led to an increased rate of abnormal embryonic development, and congenital developmental defects significantly reduced the daily weight gain and food intake of offspring. Although Comparative Example 11 underwent parental nutritional fortification, no special components were added during the in vitro stage, abandoning the synergistic continuation of in vitro optimization and in vivo nutrition. As a result, gametes could not fully realize their potential in the in vitro environment, leading to a reduction in embryo quality and consequently, lower offspring growth performance compared to the previous example. Comparative Example 12 only optimized in vitro operations but neglected parental nutritional fortification, resulting in congenitally low gamete quality. Even with further improvements in subsequent in vitro procedures, it was difficult to compensate for congenital defects, and growth indicators were significantly lower. Comparative Example 13 used a traditional hybridization method without targeted parental nutritional fortification or in vitro optimization. Gamete quality, fertilization efficiency, and embryonic development conditions were all at a naturally low level, lacking synergistic support throughout the entire process, resulting in the worst offspring growth performance.

[0111] These comparative results fully demonstrate that only by working together in all aspects of parental nutrition, in vitro manipulation, and gestational care, and by cooperating with each other among components and processes, can the growth potential of offspring be maximized. The improvement in growth performance brought about by this synergy cannot be achieved by improving a single aspect or by missing any key part.

[0112] The above-described embodiments are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that for those skilled in the art, any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the scope of protection of the present invention.

Claims

1. A nutritional fortifier composition for goat crossbreeding, characterized in that: The composition comprises a reproductive nutrient fortifier and a sperm motility nutrient fortifier; the reproductive nutrient fortifier contains the following ingredients per kilogram of maternal basal diet: D-chiroinositol 0.028-0.032g, acetylated L-carnitine 0.18-0.20g, melatonin 0.4-0.5mg, yeast selenium 0.18-0.20mg, N-acetylcysteine ​​0.9-1.0g, and L-arginine 1.8-2.0g; the sperm motility nutrient fortifier contains the following ingredients per kilogram of paternal basal diet: acetylated L-carnitine 0.18-0.20g, L-citrulline 0.45-0.50g, ergothioneine 0.4-0.5mg, and zinc glycine 0.007-0.008g.

2. A method for crossbreeding goats with enhanced nutrition throughout the entire breeding cycle, characterized in that: The method described herein involves feeding selected maternal and paternal parents with a basal diet containing the reproductive nutrient fortifier and sperm motility nutrient fortifier as described in claim 1 for nutritional pretreatment, followed by gamete preparation optimization, in vitro fertilization and blastocyst culture, laparoscopic embryo transfer and pregnancy nutrition cultivation to complete goat hybridization breeding.

3. The method for full-cycle nutritional enhancement in goat crossbreeding according to claim 1, characterized in that: The optimized gamete preparation includes oocyte collection and maturation culture, and sperm collection and screening; Sperm screening includes a secondary screening process using density gradient centrifugation.

4. A method for full-cycle nutritional enhancement in goat crossbreeding according to any one of claims 1-3, characterized in that: The specific steps of this method are as follows: (a) Parental selection and nutritional pretreatment (1) Selection of parent stock: The maternal parent should be a healthy goat aged 2-3 years with good reproductive performance; the paternal parent should be a robust goat aged 1.5-2 years with good semen quality. (2) Nutritional pretreatment of the maternal parent: Nutritional intervention of the maternal parent begins 60 days before hybridization. A compound feeding program is adopted, which combines the maternal parent basal diet with a reproductive nutrient fortifier. The maternal parent basal diet consists of 30% corn, 18% soybean meal, 30% alfalfa hay, 10% wheat bran, 5% whole fat extruded soybean, 3% brewer's yeast, 2% rapeseed meal, and 2% seaweed powder. The reproductive nutrient fortifier is added to each kilogram of maternal parent basal diet with the following ingredients: D-chiral inositol 0.028-0.032g, acetylated L-carnitine 0.18-0.20g, melatonin 0.4-0.5mg, yeast selenium 0.18-0.20mg, N-acetylcysteine ​​0.9-1.0g, and L-arginine 1.8-2.0g. The reproductive nutrient fortifier is thoroughly mixed with the maternal parent basal diet in the specified proportion and fed for 60 days. At the same time, the maternal parent is guaranteed 2 hours of exercise per day. (3) Paternal nutritional pretreatment: Paternal nutritional intervention began 60 days before hybridization, using a compound feeding program consisting of a paternal basal diet and a sperm motility nutrient fortifier. The paternal basal diet consisted of 35% corn, 22% soybean meal, 25% oat hay, 6% wheat bran, 5% whole fat extruded soybean, 3% fish meal, 2% seaweed powder, and 2% brewer's yeast. The sperm motility nutrient fortifier was added per kilogram of paternal basal diet with the following ingredients: 0.18-0.20g acetylated L-carnitine, 0.45-0.50g L-citrulline, 0.4-0.5mg ergothioneine, and 0.007-0.008g zinc glycine. The sperm motility nutrient fortifier was thoroughly mixed with the paternal basal diet in the specified proportions and fed continuously until the semen collection was completed. (II) Gamete preparation and optimization treatment (1) Oocyte collection and maturation culture: Oocytes were collected using ultrasound-guided oocyte collection technology. Specifically, the ultrasound probe was used to locate the follicles on the maternal ovary. Follicles with a diameter of 2-6 mm were selected for puncture and aspiration. The aspiration negative pressure was controlled at 80-90 mmHg. Follicular fluid was collected and the cumulus-oocyte complex was recovered. Oocytes with abnormal morphology were removed and placed in maturation culture medium for in vitro culture. (2) Sperm collection and screening: Paternal semen was collected using the artificial vagina method, and initial screening under a microscope was performed to select sperm with a density ≥2×10⁻⁶. 9 Semen with a motility ≥0.7 and a concentration of 1 / mL was selected for initial screening. The pre-screened semen was mixed with sperm diluent at a 1:3 ratio, incubated at 37°C for 10 min, and then centrifuged at 1500-2000 rpm for 10-15 min. The supernatant was discarded, and the precipitate was washed twice with diluent to obtain a pre-purified sperm suspension. Further screening was performed using density gradient centrifugation. Two different volume fractions of Percoll separation solution were slowly added sequentially to a centrifuge tube to form a double-layer gradient. The sperm suspension was gently added to the top layer, and centrifuged at 2000-2500 rpm for 20-25 min. The precipitate at the bottom of the tube after centrifugation was the high-motility sperm clump. The supernatant was discarded, and the precipitate was resuspended with sperm diluent. Finally, the sperm suspension with a motility ≥0.85 was selected for subsequent in vitro fertilization (IVF). (III) In vitro fertilization and embryo culture (1) In vitro fertilization: Selected high-motility sperm are mixed with mature oocytes and placed in fertilization culture medium. They are co-cultured at 38.5℃, 5% CO2 and saturated humidity for 18-20 hours to complete the sperm-egg combination process. (2) Embryo culture and screening: After fertilization, the fertilized eggs are washed and transferred to the embryo culture medium to culture to the blastocyst stage; the embryo culture medium adopts a sequential culture system: cleavage embryo culture medium is used from day 1 to 3, with 4% fetal bovine serum and 0.2 mmol / L amino acid mixture added; blastocyst culture medium is used from day 4 to 7, with 8% fetal bovine serum, 0.5 mmol / L glucose and 0.1 mmol / L glutamine added; during the culture process, the embryo development is observed daily, embryos with developmental arrest or abnormal morphology are removed, and high-quality blastocysts with a grade of 4AA are selected for transplantation; (iv) Embryo transfer and cultivation (3) (1) Embryo transfer: Laparoscopic embryo transfer technology is used. The recipient mother needs to be synchronized with estrus first. Specifically, a vaginal sponge plug containing progesterone is inserted into the vagina of the recipient ewe for 12-14 days. When the plug is removed, 0.1 mg of cloprostenol is injected intramuscularly into each recipient ewe to induce synchronized estrus. When the recipient ewe enters the estrus cycle on the 6th-7th day, the selected high-quality blastocysts are transferred into the uterine horn. Three blastocysts are transferred into each mother. At the same time, the transfer depth is precisely controlled to keep it 2-3 cm away from the tip of the uterine horn. After the operation, in order to ensure the implantation and development of blastocysts, 15 mg of progesterone is injected intramuscularly into each ewe for 14 consecutive days. (2) Nutritional training during pregnancy: During the first 3 months of pregnancy, the basic diet of the mother remains unchanged, and 0.01g / kg body weight of docosahexaenoic acid and 0.008g / kg body weight of choline are added to promote the development of embryonic organs. During the last 2 months of pregnancy, the energy and protein supply of the diet is increased. Specifically, on the basis of the original pregnancy diet formula, for every 1kg increase in diet, 100g of corn and 40g of soybean meal are added and mixed together. At the same time, 0.01g of ferrous fumarate is added per kilogram of diet to prevent fetal anemia. During the entire pregnancy, the mother is guaranteed to have sufficient drinking water to avoid stress response. Regular prenatal check-ups are also conducted. One week before the end of pregnancy, the mother is transferred to a sterile farrowing room to observe lambing and assist with delivery to ensure that the ewe delivers smoothly and obtains the F1 generation of hybrid goats.

5. The method for full-cycle nutritional enhancement in goat crossbreeding according to claim 4, characterized in that: In step (ii) (1), the maturation culture medium uses tissue culture medium No. 199 as the base medium, with the addition of recombinant human serum albumin 4.5-5 g / L, reduced glutathione 0.09-0.10 g / L, D-chiroinositol 0.09-0.10 g / L, and growth differentiation factor 9 13-15 ng / mL. The medium is cultured at 38.5℃, 5% CO2, and saturated humidity for 24-28 h until the oocytes expel the first polar body, thus obtaining mature oocytes.

6. The method for full-cycle nutritional enhancement in goat crossbreeding according to claim 4, characterized in that: The sperm diluent formula in step (ii) (2) is as follows: 4.0g glucose, 1.0g fructose, 2.0g trehalose, 1.4g sodium citrate, 0.2g sodium bicarbonate, 0.075g potassium chloride, 0.05g sodium pyruvate, 0.002g gentamicin, and distilled water to 100mL.

7. The method for full-cycle nutritional enhancement in goat crossbreeding according to claim 4, characterized in that: In step (ii) (2), the two different volume fractions of the Percoll separation solution are 80% and 40%, respectively.

8. The method for full-cycle nutritional enhancement in goat crossbreeding according to claim 4, characterized in that: In step (iii) (1), the ratio of mature oocytes to selected high-motility sperm is 1:

105.

9. The method for full-cycle nutritional enhancement in goat crossbreeding according to claim 4, characterized in that: In step (iii) (1), the fertilization culture medium is based on BO-IVF fertilization medium, with the addition of recombinant human serum albumin 45-50g / L, reduced glutathione 0.055-0.060g / L, D-chiroinositol 0.09-0.10g / L, and growth differentiation factor 99-10ng / mL to improve the sperm-egg binding efficiency.

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