Growth evaluation method and system for hybrid offspring of Zhuangluo No.1 and Guiafil No.1 tilapia mossambica

Abstract

The invention belongs to the technical field of aquatic animal breeding, and discloses a growth evaluation method of a hybrid offspring of Zhuangluo No.1 and Guiafil Tilapia, which comprises the following steps: carrying out cross breeding on a 'Guiafil No.1' Tilapia strain with obvious growth advantages and a disease-resistant new variety 'Zhuangluo No.1', comparing growth character indexes of offspring, and comparing the growth character indexes of the offspring in countable characters. The spine number of the dorsal fins of the four groups of offspring is 14-17, the number of the dorsal fins is 12-14, the spine number of the dorsal fins of the Nile tilapia is 14-18, and the number of the dorsal fins of the Nile tilapia is 10-14, which indicates that the four groups of offspring are closer to the Nile tilapia. In measurable characters, the body type of the hybrid progeny is between that of two parents, and the ZG group is closer to that of the female parent GG group. After 90 days, the average weight of the ZG group and the GZ group is different from that of the GG group, but the difference is not obvious (P is greater than 0.05), but the standard deviation of the ZG group is greater than that of the GZ group and the GG group. The survival rates of the four groups of filial generations are ZZ > ZG > GG > GZ under the condition that the whole group is not infected with streptococcus, and the yield is ZG > GZ > GG > ZZ.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic animal breeding, and in particular relates to a growth evaluation method and system for hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia. Background Art

[0002] Tilapia, due to its rapid growth, strong reproductive capacity, wide adaptability, and delicious meat, holds a prominent position in global freshwater aquaculture and is a recommended aquatic species by the Food and Agriculture Organization of the United Nations. However, with the expansion of aquaculture scale, increased density, and deteriorating aquaculture environments, frequent streptococcal infections have become a serious problem facing tilapia farming. This disease, with its rapid spread and high mortality rate, causes economic losses exceeding 5 billion yuan annually to my country's tilapia aquaculture industry. Currently, the prevention and treatment of streptococcal infections primarily relies on antibiotics, but long-term antibiotic use can lead to increased pathogen resistance and pose potential risks to the environment and consumer health. Furthermore, while existing vaccines offer some protection, their cost and operational complexity hinder their large-scale deployment. Therefore, comprehensive approaches to combating streptococcal infections in tilapia are urgently needed.

[0003] In response to this problem, disease-resistant breeding based on modern genetics and molecular biology techniques has become an important direction for solving streptococcal disease. Through molecular marker-assisted selection (MAS) or whole genome selection (GS), genes related to disease resistance can be accurately screened and excellent varieties can be cultivated. For example, the World Fisheries Center has successfully bred the fast-growing and stress-resistant "GIFT tilapia" through multi-generational group selection and hybridization. Domestic and foreign studies have also shown that the use of specific families for artificial infection screening can significantly improve the disease resistance of fish. In addition, through in-depth research on the transmission mechanism and pathogenic mechanism of streptococcal disease, vaccine development and precise prevention and control strategies can be further optimized. In the future, comprehensive solutions that integrate disease-resistant gene screening, breeding technology and optimized breeding management will provide important support for the sustainable development of the tilapia farming industry. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a growth evaluation method for hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia.

[0005] The present invention is achieved in that a growth evaluation method for a hybrid progeny of Zhuangluo No. 1 and Guifei No. 1 tilapia comprises:

[0006] Step 1: Evaluation of growth traits and disease resistance of hybrid offspring of tilapia "Zhuangluo No. 1" and "Guifei No. 1";

[0007] Step 2: Physiological and biochemical performance evaluation of hybrid offspring of tilapia "Zhuangluo No. 1" and "Guifei No. 1";

[0008] Step 3: Analysis of the transcriptome differences of spleens of the self-crossed and reciprocal cross progenies of “Zhuangluo 1” and “Guifei 1” after infection with Streptococcus tilapia.

[0009] Furthermore, the growth traits and disease resistance of the hybrid offspring of the "Zhuangluo No. 1" and "Guifei No. 1" tilapia were evaluated:

[0010] The first generation offspring from self-pollination of "Zhuangluo No. 1" was named ZZ; the first generation offspring from self-pollination of "Guifei No. 1" was named GG; the offspring with "Zhuangluo No. 1" as the female parent and "Guifei No. 1" as the male parent was named ZG, and the offspring with "Guifei No. 1" as the female parent and "Zhuangluo No. 1" as the male parent was named GZ. The growth traits of the hybrid offspring were compared and analyzed in detail with those of the parents.

[0011] Furthermore, the physiological and biochemical performance of the hybrid offspring of the "Zhuangluo No. 1" and "Guifei No. 1" tilapia was evaluated:

[0012] Tilapia offspring were infected with Streptococcus agalactiae by artificial intraperitoneal injection, and the enzyme activities in the livers of infected offspring were detected.

[0013] Furthermore, the differential transcriptome analysis of spleens of the self-cross and reciprocal cross progenies of "Zhuangluo No. 1" and "Guifei No. 1" after infection with Streptococcus tilapia was performed:

[0014] Transcriptome sequencing analysis of spleen tissues in the ZG and ZZ, and GZ and ZZ groups revealed a series of differentially expressed genes and the key signaling pathways they participate in during immune regulation and infection response. Changes in gene expression in key pathways such as necroptosis, TGF-β signaling, and apoptosis may play a key role in regulating immune and inflammatory responses. Further studies of these genes and the signaling pathways they regulate will help reveal the mechanisms of immune response and provide new research ideas for the treatment and prevention of Streptococcus infection.

[0015] Another object of the present invention is to provide a growth evaluation system for hybrid progeny of Zhuangluo No. 1 and Guifei No. 1 tilapia, comprising:

[0016] Disease resistance evaluation module, used to evaluate the growth traits and disease resistance of hybrid offspring of "Zhuangluo No. 1" and "Guifei No. 1" tilapia;

[0017] Physiological and biochemical performance evaluation module, used to evaluate the physiological and biochemical performance of hybrid offspring of "Zhuangluo No. 1" and "Guifei No. 1" tilapia;

[0018] The differential analysis module is used to analyze the differential transcriptome of the spleen of the self-cross and reciprocal cross progenies of "Zhuangluo No. 1" and "Guifei No. 1" after infection with Tilapia Streptococcus.

[0019] Another object of the present invention is to provide a computer device, which includes a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the growth evaluation method of the hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia.

[0020] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to execute the steps of the growth evaluation method for the hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia.

[0021] Another object of the present invention is to provide an information data processing terminal, which is used to implement a growth evaluation system for the hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia.

[0022] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0023] This study compared the growth traits of the offspring by crossbreeding the "Guifei No. 1" tilapia strain, which exhibits significant growth advantages, with the disease-resistant "Zhuangluo No. 1" tilapia strain. The results showed that the four offspring groups had 14 to 17 dorsal fin spines and 12 to 14 dorsal fin rays, respectively, compared to 14 to 18 dorsal fin spines and 10 to 14 dorsal fin rays for the Nile tilapia, indicating that the four offspring groups were relatively similar to the Nile tilapia. Among the measurable traits, the body size of the hybrid offspring was between the two parents, with the ZG group being closer to the maternal GG group. After 90 days, the average body weight of the ZG and GZ groups was different from that of the GG group, but the difference was not significant (P>0.05). However, the standard deviation of the ZG group was greater than that of the GZ and GG groups. When the whole species was not infected with Streptococcus, the survival rate of the four groups of offspring was ZZ>ZG>GG>GZ, and in terms of yield, ZG>GZ>GG>ZZ.

[0024] Evaluation of the physiological and biochemical properties of the livers of offspring infected with Streptococcus agalactiae revealed differences in enzyme activity between the different parents, which is closely related to the offspring's disease resistance. Within 72 hours of S. agalactiae injection, changes in AKP, CAT, and GPT activities in the livers of the four offspring groups showed an initial increase followed by a decrease over time. At 16 hours, the increase in AKP activity in the ZG group was significantly lower than that in the other three groups (P < 0.05). 72 hours after S. agalactiae injection, levels in GG, ZZ, and ZG all decreased, with a slight decrease in the GZ group. The GG group had the lowest alkaline phosphatase activity at 72 hours. The GG group experienced a more significant decrease in AKP content between 16 and 72 hours, while the other three groups experienced a slower decline. Tilapia disease resistance is associated with the rate of decline in AKP activity. Before infection with S. agalactiae, the ZG group had the highest CAT activity, significantly higher than the GG and ZZ groups. After infection with S. agalactiae, no significant differences in CAT activity were found among the four groups (P>0.05). Before S. agalactiae injection, there were no significant differences in GPT activity among the four groups. Sixteen hours after S. agalactiae injection, GPT activity increased significantly, with the ZG group experiencing a significantly higher increase than the other three groups (P<0.05). Seventy-two hours after infection with S. agalactiae, GPT activity was lowest in the GG group and highest in the GZ group, with significant differences between the GZ group and the ZZ and ZG groups (P<0.05). The speed of enzyme activity decline is related to tilapia's resistance to streptococcal infection; those with slower enzyme activity decline exhibit a more sensitive and rapid response to external streptococci.

[0025] Transcriptome sequencing analysis of spleen tissue from offspring infected with Streptococcus agalactiae revealed that hamp was the differentially expressed gene in both the TGF-beta signaling pathway and the phagosome between ZG and ZZ, and between GZ and ZZ. Both the apoptosis and necroptosis signaling pathways contain the birc2 gene, suggesting that these signaling pathways and shared genes may function together following bacterial infection. Furthermore, the shared expression of txn, hamp, and fth1a genes between the ZG and GZ infection groups suggests that infection has similar effects on gene expression and pathway regulation in spleen tissue. Although GZ and ZG infection exhibit certain similarities in their effects on spleen tissue, differences exist in pathway regulation, such as in the apoptosis, necroptosis, and TGF-beta signaling pathways. Infected offspring may affect gene expression and function in spleen tissue through distinct mechanisms.

[0026] In summary, this invention provides a new development direction and research idea for tilapia farming by breeding disease-resistant tilapia varieties and exploring their immune regulation and disease resistance mechanisms, and provides important theoretical and practical support for the healthy development of freshwater aquaculture and disease prevention and control.

[0027] This study aims to crossbreed the "Guifei No. 1" tilapia strain, which exhibits significant growth advantages, with the disease-resistant "Zhuangluo No. 1" tilapia strain. The study then evaluates the growth traits of the offspring, combines artificial infection with Streptococcus agalactiae with a comprehensive analysis of physiological and biochemical parameters to analyze their disease resistance. Transcriptome sequencing of spleen tissue from offspring infected with Streptococcus agalactiae reveals the mechanisms of inflammation and immune response in tilapia induced by Streptococcus infection. The main results are as follows:

[0028] Evaluation of growth traits and disease resistance of hybrid offspring of tilapia "Zhuangluo No. 1" and "Guifei No. 1"

[0029] The present invention named the first generation of offspring from the self-pollination of Zhuangluo No. 1 ZZ; the first generation of offspring from the self-pollination of Guifei No. 1 GG; the offspring with Zhuangluo No. 1 as the female parent and Guifei No. 1 as the male parent ZG, and the offspring with Guifei No. 1 as the female parent and Zhuangluo No. 1 as the male parent GZ. A detailed comparative analysis of the growth traits of the hybrid offspring and their parents was conducted. The results showed that there were no significant differences in the number of scales on the lateral line and dorsal fin among the four groups of offspring, but the number of scales on the lateral line and dorsal fin spines showed the order GZ>GG>ZG>ZZ, with a significant difference between GZ and ZZ. In terms of growth traits at different time points, there were significant differences in body length and weight between GG and ZZ at 0 days; at 30 days, the total length, body width, and weight of the ZZ group were significantly lower than those of the other three groups; and at 60 days, the body length, body height, and weight of the ZZ group were also significantly lower than those of the other three groups. Over time, the average absolute weight gain rate of the four groups of offspring followed the order ZG > GZ > GG > ZZ. Furthermore, at 30 and 90 days, the absolute weight gain rate of the ZG group was significantly higher than that of the ZZ group, while at 60 days, the absolute weight gain rate of the ZG group was significantly higher than that of both the ZZ and GG groups. At 90 days, the order of fatness was GG > GZ > ZG > ZZ, while at 0, 60, and 90 days, the fatness of the GZ and GG groups was significantly higher than that of the 30-day group.

[0030] Evaluation of physiological and biochemical performance of hybrid offspring of tilapia "Zhuangluo No. 1" and "Guifei No. 1"

[0031] Tilapia offspring were infected with Streptococcus agalactiae via intraperitoneal injection, and enzyme activities in their livers were measured. Results showed that with prolonged infection, the activities of AKP, CAT, and GPT in the offspring livers initially increased and then decreased. At 16 hours, the ZG group showed a significantly lower fold increase in AKP activity than the other three groups. At 72 hours, AKP activity in the GZ group decreased slightly, reaching the lowest level in the GG group. CAT activity was highest in the ZG group before infection. Within 72 hours after S. agalactiae injection, CAT activity in the offspring livers of the four groups showed a similar trend of initially increasing and then decreasing. GPT activity, however, increased significantly in the ZG group 16 hours after S. agalactiae injection, with a significantly higher fold increase than in the other three groups. After 72 hours, GPT activity was lowest in the GG group, and highest in the GZ group, with significant differences from the ZZ and ZG groups. The experiment showed that the rate of change of liver enzyme activity was related to the ability of tilapia to resist streptococcal infection. Individuals with a slower decline in enzyme activity showed a more sensitive and rapid response when responding to streptococcal infection.

[0032] Differential analysis of spleen transcriptomes of self-cross and reciprocal cross progenies of “Zhuangluo No. 1” and “Guifei No. 1” infected with Streptococcus tilapia

[0033] The present invention conducted transcriptome sequencing analysis on the spleen tissues of the ZG and ZZ, GZ and ZZ groups, and found that there were a series of differentially expressed genes and the key signaling pathways in which they participated in the immune regulation and infection response process. In key pathways such as necroptosis, TGF-β signaling pathway and apoptosis, changes in gene expression may play a key role in regulating immune and inflammatory responses. Further research on these genes and the signaling pathways they regulate will help to reveal the mechanism of immune response and provide new research ideas for the treatment and prevention of streptococcal infection. The experimental results showed that GZ and ZG infections had similarities in the regulation of differentially expressed genes and pathways in spleen tissue, but there were differences in specific signaling pathways. This suggests that the type of infection may affect the way immune regulation and pathophysiological responses are responded to, and provides a scientific reference for further exploration of the pathogenesis of streptococcal infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention provides a flow chart of a method for evaluating the growth of hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia.

[0035] Figure 2 This is a structural block diagram of a growth evaluation system for hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia provided by an embodiment of the present invention.

[0036] Figure 3 This is a graph showing the changing trend of the annual production of tilapia aquaculture in China from 2000 to 2023, provided by an embodiment of the present invention.

[0037] Figure 4 1 is a graph of four groups of offspring survival rates provided by an embodiment of the present invention.

[0038] Figure 5 This is a graph of the total yield of four tissue progeny provided by an embodiment of the present invention.

[0039] Figure 6 This is a graph showing the mortality rate of the ZG group after injection of different concentrations of Streptococcus agalactiae provided in an embodiment of the present invention.

[0040] Figure 7 This is a graph showing the cumulative mortality of the first generation of four groups of offspring after injection of Streptococcus agalactiae at a concentration of 5.8×10 8 CFU / ml, as provided in an embodiment of the present invention.

[0041] Figure 8 This is a graph showing the differences in liver alkaline phosphatase activity in four groups of first-generation offspring infected with Streptococcus agalactiae, provided by an embodiment of the present invention.

[0042] Figure 9 This is a graph showing the differences in liver catalase activity after infection with Streptococcus agalactiae in four groups of first-generation offspring provided by an embodiment of the present invention.

[0043] Figure 10 This is a graph showing differences in liver alanine aminotransferase activity in four groups of first-generation offspring infected with Streptococcus agalactiae, provided by an embodiment of the present invention.

[0044] Figure 11 It is a heat map of correlation between samples provided by an embodiment of the present invention.

[0045] Figure 12 1 and 2. The bubble diagram (a) of the enrichment pathway of the same differentially expressed genes between ZG and ZZ, and the bubble diagram (b) of the enrichment pathway of the same differentially expressed genes between GZ and ZZ provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0047] like Figure 1 As shown, the growth evaluation method of a hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia provided in an embodiment of the present invention comprises the following steps:

[0048] S101: Evaluation of growth traits and disease resistance of hybrid offspring of tilapia "Zhuangluo No. 1" and "Guifei No. 1";

[0049] S102: Evaluation of physiological and biochemical performance of hybrid offspring of tilapia “Zhuangluo No. 1” and “Guifei No. 1”;

[0050] S103. Differential analysis of spleen transcriptomes of self-cross and reciprocal cross progenies of “Zhuangluo 1” and “Guifei 1” after infection with Streptococcus tilapia.

[0051] Evaluation of growth traits and disease resistance of hybrid offspring of tilapia "Zhuangluo No. 1" and "Guifei No. 1" provided in the embodiments of the present invention:

[0052] The first generation offspring from self-pollination of "Zhuangluo No. 1" was named ZZ; the first generation offspring from self-pollination of "Guifei No. 1" was named GG; the offspring with "Zhuangluo No. 1" as the female parent and "Guifei No. 1" as the male parent was named ZG, and the offspring with "Guifei No. 1" as the female parent and "Zhuangluo No. 1" as the male parent was named GZ. The growth traits of the hybrid offspring were compared and analyzed in detail with those of the parents.

[0053] Evaluation of physiological and biochemical properties of hybrid offspring of tilapia "Zhuangluo No. 1" and "Guifei No. 1" provided in the embodiments of the present invention:

[0054] Tilapia offspring were infected with Streptococcus agalactiae by artificial intraperitoneal injection, and the enzyme activities in the livers of infected offspring were detected.

[0055] Analysis of the differential transcriptome of spleens of the self-crossed and reciprocal cross progenies of "Zhuangluo No. 1" and "Guifei No. 1" provided in the embodiments of the present invention after infection with Streptococcus tilapia:

[0056] Transcriptome sequencing analysis of spleen tissues in the ZG and ZZ, and GZ and ZZ groups revealed a series of differentially expressed genes and the key signaling pathways they participate in during immune regulation and infection response. Changes in gene expression in key pathways such as necroptosis, TGF-β signaling, and apoptosis may play a key role in regulating immune and inflammatory responses. Further studies of these genes and the signaling pathways they regulate will help reveal the mechanisms of immune response and provide new research ideas for the treatment and prevention of Streptococcus infection.

[0057] like Figure 2 As shown, the embodiment of the present invention provides a growth evaluation system for hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia, comprising:

[0058] Disease resistance evaluation module, used to evaluate the growth traits and disease resistance of hybrid offspring of "Zhuangluo No. 1" and "Guifei No. 1" tilapia;

[0059] Physiological and biochemical performance evaluation module, used to evaluate the physiological and biochemical performance of hybrid offspring of "Zhuangluo No. 1" and "Guifei No. 1" tilapia;

[0060] The differential analysis module is used to analyze the differential transcriptome of the spleen of the self-cross and reciprocal cross progenies of "Zhuangluo No. 1" and "Guifei No. 1" after infection with Tilapia Streptococcus.

[0061] Another object of the present invention is to provide a computer device, which includes a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the growth evaluation method of the hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia.

[0062] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to execute the steps of the growth evaluation method for the hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia.

[0063] Another object of the present invention is to provide an information data processing terminal, which is used to implement a growth evaluation system for the hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia.

[0064] The present invention is specifically implemented:

[0065] 1Main farmed species of tilapia in China

[0066] The successful tilapia culture varieties include Nile tilapia, Mozambique tilapia, Aurelian tilapia, red tilapia, Fushou tilapia, "Jifu" tilapia, Oni tilapia, "Xinjifu" tilapia, "Jili" tilapia, "Guifei No. 1" tilapia, and "Zhuangluo No. 1" tilapia (Table 1).

[0067] Table 1 Introduction and cultivation of tilapia

[0068]

[0069]

[0070] 1.1 Tilapia aquaculture production in various provinces in China in 2023. As of 2023, the total production of tilapia aquaculture in China reached 1.739 million tons. The industry has developed rapidly across the country. In 2023, the tilapia aquaculture production in Heilongjiang, Gansu and Qinghai did not exceed 10,000 tons. Figure 3 As can be seen, from 2000 to 2023, the national annual tilapia production showed an overall increasing trend, despite a slight decline in 2017. National tilapia production increased from 629,000 tons in 2000 to 1,739,000 tons in 2023, with an average annual growth rate of 4.3%. Meanwhile, tilapia production in Guangdong Province increased from 249,000 tons in 2000 to 757,000 tons, with an average annual growth rate of 13.21%.

[0071] Figure 3 This is a chart showing the changing trend of annual tilapia farming production in the country from 2000 to 2023.

[0072] 1.3 Purpose and Significance

[0073] Tilapia plays an important role in my country's freshwater aquaculture industry. Thanks to its rapid growth rate, strong reproductive capacity, wide-ranging diet, excellent adaptability, and rich nutritional value, it has become one of the world's important sources of animal protein

[98] . However, with the expansion of tilapia farming scale and the increase in farming density, as well as changes in the farming environment, the frequent occurrence of streptococcal infection has become a serious problem

[24] . The high mortality rate and rapid spread of this disease pose a major threat to the aquaculture industry and cause huge economic losses to farmers. At present, there is no method to completely cure streptococcal disease. Therefore, there is an urgent need to genetically improve tilapia germplasm and enhance its own disease resistance to effectively meet this challenge.

[0074] Selective breeding for growth and disease resistance is a complementary strategy to improve fish production performance and health

[99] . The present invention uses hybrid breeding technology to hybridize the fast-growing "Guifei No. 1" and the disease-resistant "Zhuangluo No. 1", and compares and analyzes the growth traits of the offspring. The offspring are infected with Streptococcus agalactiae by artificial injection. The liver enzyme activity and spleen transcriptome sequencing are performed at different stages, namely the uninfected period (0h), the dying period (16h) and the recovery period (72h). The disease resistance of the offspring is compared and the key genes and main signal transduction pathways related to the resistance of tilapia to Streptococcus agalactiae infection are analyzed and screened, thereby exploring the analytical response mechanism of tilapia to Streptococcus infection, laying the foundation for the implementation of molecular assisted breeding and even transgenic breeding in the future.

[0075] 2. Comparative analysis of growth traits and disease resistance of hybrid progenies of “Zhuangluo No. 1” and “Guifei No. 1”

[0076] "Zhuangluo No. 1" (variety registration number GS-01-004-2018) is a new aquatic tilapia variety that has been approved by the national disease resistance by the tilapia research team of the Guangxi Zhuang Autonomous Region Fisheries Science Research Institute based on the national-level Guangxi Zhuang Autonomous Region Nanning Tilapia Breeding Farm. Its resistance to tilapia streptococcal infection has increased by an average of 25.57%, and its aquaculture survival rate has increased by an average of 19.19%

[22] . At the same time, the latest GIFT tilapia introduced by the World Fisheries Center has been bred for 8 consecutive generations, and the "Guifei No. 1" tilapia strain with obvious growth advantages has been obtained. These two germplasm resources are very ideal hybrid parents for hybrid breeding. The purpose of this invention is to compare the growth differences of "Zhuangluo No. 1", "Guifei No. 1" and their hybrid first generation to provide data support for further breeding in the later stage.

[0077] 2.1 Materials and Methods

[0078] 2.1.1 Sources of experimental materials

[0079] Both the "Zhuangluo No. 1" and "Guifei No. 1" broodstock were sourced from the National Tilapia Breeding Farm in Nanning, Guangxi Zhuang Autonomous Region. Broodstock weighing over 500g were selected in March 2022. Initial screening was conducted based on the criteria of no external injuries, normal body shape, strong physique, bright coloration, and absence of disease. Preliminary selected tilapia were examined for genital pore development with the abdomen facing upwards, and further selection criteria were assessed. Sexing was performed based on the structural differences in the genital pores of tilapia (SC / T 1105-2007). Females were selected based on a noticeably enlarged abdomen, a full posterior abdomen near the genital pore, an open genital pore with a reddish halo. Males were selected based on bright coloration and the discharge of semen when gently squeezed from the head to the tail. 400 females and 200 males of each "Zhuangluo No. 1" and "Guifei No. 1" were selected, soaked in 10% povidone-iodine for 10 minutes, and then placed in experimental cages.

[0080] The Streptococcus tilapia strain used in the infection experiments was donated by the Fish Disease Research Laboratory of the Guangxi Academy of Fisheries Sciences. Analysis by biochemical parameters, serotype, genotype, and whole-genome sequencing confirmed its identity as Streptococcus tilapia, demonstrating good regional representativeness and immunogenicity.

[0081] 2.1.2 Fry cultivation

[0082] At the tilapia breeding farm in Nanning, Guangxi Zhuang Autonomous Region, cages (2.0 m long x 1.2 m wide) were constructed in a 30 m x 20 m rectangular pond with a depth of 2.5 m. Broodstock were placed in the cages for pairing at a 2:1 ratio of male to female, with 20 cages per group. The pond and cages were cleaned and disinfected before breeding, and povidone-iodine was used for disinfection during fish handling. The water source was aerated groundwater (river water), maintaining a dissolved oxygen concentration of 6 mg / L. Each cage was fed a "Tongwei" brand tilapia floating feed containing 32% protein. The daily feed amount was determined to be 3% of the total fish weight, and was fed three times daily. A total of four groups of first-generation offspring were obtained [“Zhuangluo No. 1”♀דZhuangluo No. 1”♂(ZZ), “Zhuangluo No. 1”♀דGuifei No. 1”♂(ZG), “Guifei No. 1”♀דZhuangluo No. 1”♂(GZ), “Guifei No. 1”♀דGuifei No. 1”♂(GG)].

[0083] 2.1.3 Determination of growth traits of hybrid offspring

[0084] In this experiment, two hybrid combinations and two self-propagated combinations were produced by parental hybridization and self-pollination. The phenotypic differences of fish in different combinations are affected by multiple factors, including genetics, environmental factors, and the interaction between the two

[100] . In order to avoid the influence of the initial size of tilapia on the results of the growth comparison test, the breeding work of the four combinations in this experiment was carried out simultaneously, and the breeding environment was unified to the greatest extent to reduce the systematic error of the experiment. The size of the fry initially selected was different, but the four groups of first-generation fry emerged within 3 days, and the size of the GG fry was larger than that of the other three groups when the fry were selected. Therefore, the influence of different hatching times of the fry on the experimental results can be ruled out. Therefore, the results of this experiment can effectively reflect the level of difference in growth traits among the four combinations.

[0085] Growth trait determination is divided into two categories, including countable traits and measurable traits. After the 90th day, ten fish were randomly selected from each group to measure countable traits. Countable traits include the number of scales below the lateral line, the number of scales below the lateral line, the number of dorsal fin spines, and the number of dorsal fin rays; measurable traits include weight, total length, body length, body width, and body height. 120 fry were randomly selected from each group for comparative breeding experiments, marked with microarrays, and mixed cultured in the workshop cement pool. Feeding was given once in the morning and evening every day, with the feed amount being 3% of body weight. On the 0th, 30th, 60th, and 90th days, 30 fish were randomly selected for measurement of body weight (W), body length (SL), total length (TL), body width (BW), and body height (BH). Feeding was stopped the day before weighing. SPSS26.0 software was used to analyze the data on body weight and body length. The relative weight gain rate, absolute weight gain rate, and fatness of each combination at different time points were calculated. The calculation formula is as follows:

[0086] Body weight relative gain rate (WGR, %) = (W2-W1) / W1

[0087] Absolute weight gain rate (AGR, g / d) = (W2-W1) / (t2-t1)

[0088] Fat content (CF, %) = W / SL3 × 100%

[0089] In the above formula, t2-t1 refers to the interval days, W2-W1 refers to the weight difference, and SD refers to the standard deviation of weight. Refers to the average weight.

[0090] 2.2 Results

[0091] 2.2.1 Comparison of countable traits of the four groups of offspring

[0092] The mean values of the countable traits of each offspring are shown in Table 2. The number of dorsal fin spines and dorsal fin rays in the four groups of offspring in this experiment ranged from 14 to 17, and from 12 to 14. There were no significant differences in the number of lateral line scales or dorsal fin rays among the four groups. The mean values of the number of lower lateral line scales and dorsal fin spines were GZ > GG > ZG > ZZ, with no significant differences between GZ, GG, and ZZ (P > 0.05), but significant differences between GZ and ZZ (P > 0.05).

[0093] Table 2 Mean values of countable traits of each offspring

[0094]

[0095] Note: Data in the same row with different letters in superscript indicate significant differences (P<0.05).

[0096] 2.2.2 Comparison of measurable traits of the four groups of offspring

[0097] After 90 days of rearing, the survival rate of each offspring was above 98% during the experimental period. The statistical results of the measurable traits of the four offspring were analyzed by analysis of variance and LSD test, and the results are shown in Table 3. In this experiment, at 0 days, there were no significant differences in total length and body width among the four groups (P>0.05), while there were significant differences in body length and weight between the GG and ZZ groups (P<0.05). At 30 days, the total length, body width, and weight of the ZZ group were significantly lower than those of the other three groups (P<0.05). The ZG group had the highest average weight, followed by the GG group. At 60 days, the ZZ group had significantly lower body length, height, and weight than the other three groups (P<0.05). There were no significant differences in total length and body width among the four groups. The ZG group had the highest average weight, followed by the GZ group. At 90 days, the ZG and GZ groups showed differences from the GG group, but the differences were not significant (P>0.05). The first generation of the four groups had a body length of less than 18 cm, with a body length ratio of 2.20-2.44 to body height; and a body length of more than 18 cm, with a body length ratio of 2.28-2.45 to body height.

[0098] Table 3 Mean values of measurable traits of the four groups of offspring

[0099]

[0100]

[0101] Note: Data in the same row with different superscript letters indicate significant differences (P<0.05).

[0102] The absolute and relative weight gain rates of the four groups of offspring at different stages during the experimental period are shown in Table 2-3. With increasing rearing time, the average absolute weight gain rates of the other three groups, except for the GZ group, gradually decreased, but there was no significant difference (P>0.05). The average absolute weight gain rates were ZG>GZ>GG>ZZ. At 30 and 90 days, the absolute weight gain rate of the ZG group was significantly higher than that of the ZZ group (P<0.05). At 60 days, the absolute weight gain rate of the ZG group was significantly higher than that of the ZZ and GG groups (P<0.05).

[0103] Table 4 shows that the average relative weight gain rates of the four groups gradually increased from 0 to 90 days of growth. At 60 and 90 days, the relative weight gain rates of the ZZ, ZG, and GZ groups were significantly higher than those at 30 days (P < 0.05). The relative weight gain rate of the GG group was highest at 90 days (2.79), significantly higher than those at 30 days (2.01) and 60 days (1.11). At 30 days, the relative weight gain rate of the ZG group was significantly higher than that of the ZZ group (P < 0.05), and at 60 days, the relative weight gain rate of the ZG group was significantly higher than that of the GG group (P < 0.05).

[0104] Table 4 Body weight growth rate of the four groups of offspring at different periods

[0105]

[0106]

[0107] Note: Different capital letters represent significant differences among different experimental groups under the same parameter within the same culture period (P < 0.05); different lowercase letters represent significant differences among the same experimental group under the same parameter within different culture periods (P < 0.05).

[0108] Table 5 shows the fatness of the four groups of offspring at different rearing stages. Within the experimental groups with the same rearing time, there were no significant differences in fatness among the four groups. At 90 days, the fatness order was GG > GZ > ZG > ZZ. At 0, 60, and 90 days, the fatness of the GZ and GG groups was significantly higher than that of the 30-day group (P < 0.05). The ZZ group had the highest fatness at 60 days, which was significantly higher than that of the 0 and 30-day groups.

[0109] Table 5 Fatness of the four groups of offspring at different stages

[0110]

[0111] Note: Different capital letters represent significant differences between different experimental groups under the same index and the same breeding time (P < 0.05); Different lowercase letters represent significant differences between the same experimental group under the same index and the different breeding time (P < 0.05)

[0112] 2.2.3 Yield of four groups of offspring

[0113] In the case of no infection with Streptococcus, Figure 4 and Figure 5 It can be seen that the survival rate of the four groups of offspring is ZZ>ZG>GG>GZ, and in terms of output, ZG>GZ>GG>ZZ

[0114] 2.3 Discussion

[0115] 2.3.1 Four groups of offspring countable traits

[0116] It has been reported that the number of dorsal fin spines of GIFT tilapia is 14-17, and the number of dorsal fin rays is 22

[101] . In this experiment, the number of dorsal fin spines of the four groups of offspring was 14-17, and the number of dorsal fin rays was 12-14; the number of dorsal fin spines of Nile tilapia was 14-18, and the number of dorsal fin rays was 10-14, indicating that the four groups of offspring were relatively close to Nile tilapia.

[0117] 2.3.2 Four groups of measurable traits of offspring

[0118] The GIFT tilapia has a perch-like appearance, with a tall, laterally flattened body. Its lateral line is two discontinuous lines, and its tail fin is blunt and rounded at the end, unbranched. Its body is covered with ctenoid scales, and its appearance is similar to that of other Nile tilapia strains. Studies have shown that the length-to-height ratio of Mozambique tilapia is 2.38-2.83

[102] , while that of Nile tilapia is 2.20-2.43 when its body length is greater than 18 cm; 2.25-2.57 when its body length is less than 18 cm, and 2.51 for Aurelia tilapia

[103] . In this experiment, the body length-to-height ratio of the four groups of first-generation offspring was 2.20-2.44 when their body length was less than 18 cm, and 2.28-2.45 when their body length was greater than 18 cm, indicating that the body shape of the four groups of first-generation offspring is closer to that of Nile tilapia. Li Jiale

[104] compared the morphology and identification of Aeolian tilapia with its parents and found that the body shape of Aeolian tilapia was similar to that of its mother, Nile tilapia. In this experiment, the body length to body height ratios were 2.46±0.47 for the ZZ group, 2.38±0.22 for the ZG group, 2.41±0.57 for the GZ group, and 2.37±0.08 for the GG group. The body shape of the hybrid offspring was between the two parents, and the ZG group was closer to the mother, GG group. A study

[105] hybridized Aeolian tilapia and Nile tilapia and analyzed the genetic relationship between the hybrid offspring and the parents. The backcross offspring was extremely inclined to the mother, which is consistent with the results of this experiment. The body shape of the hybrid offspring is more inherited from the mother.

[0119] In recent years, a new breed of Nile tilapia, "Lu Xiong No. 1", has appeared on the market. After 5 to 6 months of culture, the fry, which are 2 to 3 cm in length, can reach a weight of more than 600 g

[106] . Luo Wei

[107] and other researchers conducted a 97-day pond culture comparison experiment on 34 families of Gift tilapia. At the beginning of the experiment, the fry of each family weighed 25 to 100 g, and at the end of the culture, their weight increased to 343 to 588 g. At 0 days, the average weight of the GG group was the highest. After 30 days, the average weight of the ZG group exceeded that of the GG group. After 60 days, the average weight of the GZ group also exceeded that of the GG group. After 90 days, the average weight of the ZG group and the GZ group was different from that of the GG group, but the difference was not significant (P>0.05). However, the standard deviation of the ZG group was larger than that of the GZ and GG groups, indicating that although the ZG group inherited the characteristics of fast growth from its parents, the overall growth rate was not uniform. Liang Zhengyuan

[108] compared the growth of GIFT tilapia in cement ponds. The absolute weight gain rate of GIFT tilapia in cement ponds was 3.84 g / d. After 90 days of feeding, the absolute weight gain rates of this experiment were: ZZ 2.76 g / d, ZG 3.85 g / d, GZ 3.63 g / d, GG 3.33 g / d. The results of the ZG group and Liang Zhengyuan were similar, while the other three groups were smaller. The degree of fatness reflects the meat yield to a certain extent. After 90 days of breeding, the degree of fatness was: GG>GZ>ZG>GG. Therefore, the ZG group inherited the advantage of fast growth rate of its parents, but its fatness was lower than that of the GG and GZ groups.

[0120] 2.4 Summary

[0121] Combining the countable traits, measurable traits and yield results of tilapia, among the two groups of hybrid offspring, the growth rate of the ZG group was faster than that of the GZ group, and the yield was also higher than that of the other three groups. However, there were large individual differences within the ZG group. The hybrid offspring inherited the fast growth rate characteristics of their parents and utilized hybrid advantages to exert the breeding potential of growth rate and disease resistance.

[0122] 3. Evaluation of physiological and biochemical performance of hybrid offspring of “Zhuangluo No. 1” and “Guifei No. 1” tilapia

[0123] The core of hybrid breeding is to utilize the separation and recombination between parental genes to select offspring that inherit the excellent characteristics of both parents. Currently, the selection and breeding of disease-resistant aquatic varieties has become a research hotspot for many scientific and technological workers at home and abroad. Significant progress has been made in varieties such as Atlantic salmon, rainbow trout

[109] , flounder

[110] , and turbot

[111] . Zhu Jiajie

[79] et al. successfully selected a highly disease-resistant GIFT tilapia strain by combining family selection with artificial infection. The liver is an important tissue metabolic organ in fish, responsible for multiple metabolic and detoxification functions. When pathogens invade the body, the liver will be damaged and an inflammatory reaction will occur, which will further lead to liver metabolic dysfunction, manifested as liver granular and fatty degeneration, and ultimately affect the immune defense ability of the entire body. The present invention obtains the first generation by hybridizing two parents, and infects the first generation with streptococcus by intraperitoneal injection. Secondly, by detecting and analyzing the changes in the activity of immune-related enzymes in the liver tissue of the first generation, its ability to resist streptococcal infection can be evaluated. The research results not only provide important experimental data for breeding new disease-resistant tilapia strains, but also provide a theoretical basis for exploring functional genes related to tilapia disease resistance traits.

[0124] 3.1 Materials and Methods

[0125] 3.1.1 Sources of experimental materials

[0126] The tilapia Streptococcus strain was taken out of a -80°C freezer, thawed, and inoculated onto a chicken blood plate for culture. After culturing in a 28°C constant temperature incubator for 24 hours, the single colonies formed were Gram-stained to verify whether the bacteria were contaminated. The Streptococcus on the blood plate was then rinsed and collected with PBS buffer. The collected bacterial solution (concentration of approximately 1.0×109 CFU / mL) was injected intraperitoneally into tilapia, with 0.2 ml injected per fish. During the subsequent breeding period, the water temperature was maintained at 33°C. The brain tissue of the diseased tilapia was inoculated with a chicken blood plate to isolate the pathogen. The isolated bacteria were then rejuvenated for 2-3 consecutive rounds until the cumulative pathogenic mortality rate of the strain infection was greater than 90%. After that, the strain was used for large-scale infection trials. After rejuvenation, the strain was aseptically inoculated into sterile Tryptone Soy Broth (TSB) medium and then incubated in a 32°C incubator with shaking (180 rpm) for 12 hours. After incubation, the density of the bacterial suspension at a wavelength of 600 nm (OD600) was measured using a spectrophotometer. The medium was prepared as follows: 10 g peptone, 10 g beef extract powder, 5 g sodium chloride, and 15 g agar.

[0127] 3.1.2 Fry cultivation

[0128] Parent selection and seedling cultivation were performed according to the procedures described in Chapter 2. Three hundred fish (50-60 g) were randomly selected from each group and temporarily raised for 7-10 days to allow the fish to acclimate to the laboratory environment before conducting the experiment. Before the infection experiment began, three fish from each of the four groups were randomly selected and streaked on blood plates to detect the presence of Streptococcus tilapia.

[0129] 3.1.3 Determination of the half-lethal concentration of Streptococcus agalactiae in tilapia

[0130] The method for determining the half-lethal concentration of Streptococcus tilapia was referred to Chen Ming

[112] . When the weight of tilapia fry reached 50-60g / tail, artificial injection infection experiment was carried out, and the feed was stopped 24 hours before the experiment. 120 fish from the ZG group were evenly distributed in 4 large barrels with four concentration gradients, with 30 fish placed in each gradient. First, the Streptococcus was cultured to 5.8×1010 CFU / ml, and then diluted with PBS solution by 1 times, 10 times, 100 times, and 1000 times to form 4 concentration gradients. At the same time, PBS solution was set as the control group. A total of 5 groups of experiments were conducted, numbered A1, A2, A3, A4 and A5 respectively. Each group of fish was injected intraperitoneally with an injection volume of 0.3ml / tail. After injection, the fish were returned to the bucket and raised normally. Half of the water was replaced every 2 days, and the water temperature was maintained at 33°C using a heating tube. The number of dead fish was recorded every 2 hours. At the same time, the brain tissue of the symptomatic fish was inoculated and separated to determine whether the death was caused by streptococcal infection.

[0131] 3.1.4 Artificial intraperitoneal injection of Streptococcus tilapia

[0132] Tilapia fish were fasted for 24 hours before intraperitoneal infection with Streptococcus tilapia. The HN016 culture was diluted with PBS to a median lethal concentration (LC50). The culture was then injected into the thoracic cavity of the fish using a syringe, with an infection dose of 0.2 ml per fish. Three replicate groups of 50 fish were set up in each group. After infection, the fish were housed in a 6 m³ vat of water and maintained as normal. The water temperature was maintained at 32°C, and half of the water was replaced every two days. The number of fish that died and the time of death were counted every two hours in each group.

[0133] 3.1.5 Determination of immune-related enzyme activity

[0134] Based on the mortality curve of this experiment, tissue sampling was performed at three representative time points: before infection (0 hours), at the peak of disease onset (16 hours), and during the recovery period (72 hours). Liver tissues were randomly collected from six fish (not showing disease symptoms) in each group and stored at -80°C for future use. For enzyme activity testing, two fish were mixed into one tube, with two replicates per tube. Changes in AKP, CAT, and GPT enzyme activities in liver tissue were measured using enzyme activity assay kits from the Nanjing Jiancheng Bioengineering Institute.

[0135] 3.1.6 Statistical analysis

[0136] SPSS 26 statistical software was used to perform one-way ANOVA on the infection survival rate and enzyme activity data, and the least significant difference method was used for multiple comparisons. Differences were considered significant when P < 0.05 and extremely significant when P < 0.01.

[0137] 3.2 Results and Analysis

[0138] 3.2.1 LC50 of Streptococcus agalactiae in tilapia

[0139] Depend on Figure 6 It can be seen that when the concentration of streptococci is 5.8×1010, the mortality rate is above 85%, when the concentration is 5.8×109, the mortality rate is about 65%, when the concentration is 5.8×107, the mortality rate is less than 10%, and when the concentration is 5.8×108, the mortality rate is about 50%. Pathogens were isolated from the brains of diseased fish, and the results confirmed that they were infected with Streptococcus agalactiae. Through preliminary experiments, 5.8x108CFU / ml was selected as the half-lethal concentration.

[0140] 3.2.2 Cumulative mortality of offspring in four groups

[0141] After the injection of Streptococcus agalactiae, the cumulative mortality rate of the four groups of offspring was Figure 7 As shown in the figure, the cumulative mortality rate of the self-sufficient generation is: GG>GZ>ZG>ZZ. The peak of infection is around 16 hours, and the recovery period is reached at 72 hours.

[0142] 3.2.3 Changes in alkaline phosphatase activity in the liver

[0143] The changes of AKP activity in the liver of the first generation of the four groups after injection of Streptococcus agalactiae were shown in Figure 8 As shown by Figure 8 As shown, within 72 hours of S. agalactiae injection, changes in AKP activity in the livers of the four offspring groups showed a trend of first increasing and then decreasing over time. There was no significant difference in alkaline phosphatase activity among the four groups at 0 hours. However, by 16 hours, all alkaline phosphatase activities increased, with the ZG alkaline phosphatase (AKP) activity increasing at a significantly lower multiple than that of the other three groups (P < 0.05). 72 hours after S. agalactiae injection, the activity of GG, ZZ, and ZG all decreased, with a slight decrease in the GZ group. The GG group had the lowest alkaline phosphatase activity at 72 hours.

[0144] 3.2.4 Changes in catalase activity in the liver

[0145] Depend on Figure 9As shown, before infection with S. agalactiae, catalase (CAT) activity was highest in the ZG group and significantly higher than in the GG and ZZ groups. Within 72 hours after S. agalactiae injection, CAT activity in the livers of the offspring in all four groups showed a trend of first increasing and then decreasing over time. At 16 and 72 hours, there were no significant differences in CAT activity among the four groups (P>0.05).

[0146] 3.2.5 Changes in ALT Activity in the Liver

[0147] Depend on Figure 10 It can be seen that before the injection of Streptococcus agalactiae, there was no significant difference in the alanine aminotransferase activity of the four groups. 16 hours after the injection of Streptococcus agalactiae, the alanine aminotransferase activity increased significantly, and the increase multiple of ZG was significantly higher than that of the other three groups (P < 0.05). 72 hours after infection with Streptococcus agalactiae, the alanine aminotransferase activity of GG group was the lowest, and the alanine aminotransferase activity of GZ group was the highest, and the difference between GZ group and ZZ and ZG groups was significant (P < 0.05).

[0148] 3.3 Discussion

[0149] Compared to mammals, fish are considered lower-order organisms in biological classification. When faced with environmental stress, fish rely mainly on their nonspecific immune system as the first line of defense against pathogens to resist the invasion of pathogens

[113] . When external pathogens infect fish, their immune organs release a large amount of reactive oxygen species (ROS) into the blood circulation. These ROS are then transported to various tissues throughout the body. However, excessive production of ROS can damage the tissue and organ functions of fish

[113] , thereby triggering a series of pathological manifestations. The liver is one of the important metabolic organs of fish, responsible for storing blood, synthesizing related proteases, and detoxifying. When the liver is damaged, its function is impaired, causing fatty degeneration and leading to a decline in the immune function of the fish.

[0150] Alkaline phosphatase (AKP) is an important non-specific phosphohydrolase in the body that can regulate the absorption and utilization of nutrients in animals and plays an important role in resisting external stimuli to the body. It can hydrolyze phosphate monoesters in an alkaline environment to play a detoxifying role and exert immune defense function

[114] . It has been used as an indicator to evaluate the disease resistance of fish and has been applied in a variety of aquatic products such as sea cucumbers

[115] , grass carp

[116] and white shrimp

[117] . The present invention compared the AKP content in the liver of four groups of offspring before and after infection with pathogens and found that the AKP content increased significantly, indicating that the body of tilapia strengthened its metabolic activity to resist the infection of pathogens after infection. In addition, the AKP content in the four groups of tilapia offspring after infection showed a trend of first increasing and then decreasing. The present invention believes that this phenomenon may be related to the proliferation of pathogens, and that the liver tissue dephosphorylates the stress response to adapt to the infection of Streptococcus agalactiae. In the study by Ji Hengtao et al.

[118] , the alkaline phosphatase activity of the internal organs of Onyx tilapia and its parents increased significantly after being invaded by Streptococcus agalactiae. This result is consistent with the results of the present invention, indicating the effectiveness of the experiment of the present invention. In addition, the present invention found that: in the ZZ group, the liver AKP content at 16h and 72h after infection was higher than that of the GG group. In addition, the AKP content in the GG group decreased more significantly from 16h to 72h, while the decline rate in the other three groups was relatively slow. Therefore, the disease resistance of tilapia may be related to the rate of decline of AKP activity.

[0151] Catalase (CAT) is a key component of an organism's antioxidant defense system. It can effectively catalyze the decomposition of H2O2, prompting it to lose the effect of reactive oxygen species, thereby protecting the body. It plays an important role in maintaining the balance of free radical metabolism in the body and maintaining normal cellular metabolism. It is also an important indicator for evaluating the antioxidant system of fish. In the present invention, CAT activity was tested in liver samples of four groups of offspring 16 hours after infection with Streptococcus agalactiae. The results showed that the CAT activity of all four groups increased significantly, with no significant differences between the four groups. Further observation to 72 hours found that the CAT activity of the four groups decreased slightly. This phenomenon suggests that tilapia may be insensitive to catalase secretion after being stimulated by Streptococcus agalactiae, resulting in little change in CAT activity.

[0152] Alanine aminotransferase (GPT) is mainly present in the mitochondria of cells, especially in liver cells, where its concentration is the highest. Its concentration in the blood is about one percent of the concentration in liver cells. GPT plays a key role in the process of protein metabolism and is therefore often used as an important indicator of liver cell damage. Liu Wenzhi et al.

[119] showed that the GPT of silver crucian carp was significantly increased after being infected with herpesvirus cyprinid. Zhou Yu et al.

[120] also confirmed that the transaminase content of European eels would also increase sharply after suffering from "mad swimming disease". In the present invention, the liver GPT content of the four groups of offspring increased significantly after being infected with Streptococcus agalactiae, which indicates that after being infected with Streptococcus agalactiae, the secretion amount of GPT increased and the secretion speed accelerated. The GPT content began to decline between 16 and 72 hours, with the GG group declining the fastest. Ji Huantao

[118] believed that after infection with streptococci, the transaminase level was low and the damage to the liver was small, and small liver damage indicated stronger disease resistance. In this experiment, the GG transaminase activity was the lowest at 72 hours. It is believed that after infection with streptococci, the speed of enzyme activity decline is related to the resistance of tilapia to streptococcal infection. The slower the enzyme activity declines, the more sensitive and rapid the body's response to external streptococci.

[0153] 3.4 Summary

[0154] The activities of AKP and GPT enzymes in tilapia will increase significantly after infection with Streptococcus agalactiae, and will gradually decrease during the recovery period. The activity of AKP and GPT enzymes in the ZZ group decreases faster than that in the GG group. Therefore, the strength of the group's disease resistance can be judged by detecting changes in the activity of AKP and GPT liver enzymes in the early stage of infection.

[0155] 4. Analysis of differential transcriptomes in the spleen of self-crossed and reciprocal crosses between Zhuangluo No. 1 and Guifei No. 1 after infection with Streptococcus tilapia

[0156] In recent years, tilapia has become a popular freshwater aquaculture species in my country, highly regarded for its high yield, adaptability, and ease of harvest. However, over the past decade or so, with the expansion of large-scale aquaculture and changes in the aquaculture environment, tilapia aquaculture diseases have become increasingly common. Tilapia streptococcosis is particularly prominent, with high mortality rates and rapid spread, resulting in significant economic losses for aquaculture operators. This disease has become a major threat to tilapia aquaculture, severely undermining the enthusiasm of many farmers. Currently, the prevention and treatment of tilapia streptococcosis relies primarily on antibiotics. However, overuse of antibiotics can lead to drug residues in food, impacting food safety and exportability, while also polluting the aquatic environment. Therefore, the breeding of disease-resistant tilapia varieties is urgently needed to improve disease resistance and address this issue, which may be one of the keys to alleviating the current predicament.

[0157] Transcriptomics is a comprehensive science that studies all transcribed RNAs in cells or tissues of a specific organism under specific physiological conditions. As one of the core technologies in this field, RNA sequencing (RNA-Seq) has become an important tool for revealing the dynamics and regulatory mechanisms of gene expression. RNA-Seq technology can identify differentially expressed genes under different biological states or external stimuli. With the continuous advancement of molecular biology technology, RNA-Seq technology has become a cost-effective and data-rich method for gene expression analysis. Transcriptome sequencing has been widely used in studies of species such as grass carp, semi-smooth tongue sole, channel catfish, and vannamei shrimp, enabling the screening of differentially expressed genes (DEGs) before and after infection with different pathogens and the analysis of related immune regulatory pathways.

[0158] The spleen is a crucial immune organ in tilapia, responsible for the immune response to invading pathogens. To further investigate the immune response of tilapia to infection with Streptococcus tilapia, this study proposes infecting the first-generation offspring obtained by hybridizing Zhuangluo No. 1 and Guifei No. 1 with Streptococcus tilapia, then performing transcriptome sequencing on spleens collected at different stages. By comparing the sequencing results between the first-generation offspring, the authors aim to reveal the analytical response mechanism of tilapia infected with Streptococcus tilapia and identify key genes regulating disease resistance in tilapia, laying the foundation for future molecular-assisted breeding and even transgenic breeding.

[0159] 4.1 Experimental Materials

[0160] 4.1.1 Sources of experimental materials

[0161] In March 2022, "Zhuangluo No. 1" and "Guifei No. 1" were self-crossed and reciprocally crossed, and when the fry were bred and grown to a size of 50-60g, an artificial injection of streptococcus infection experiment was carried out.

[0162] 4.1.2 Rejuvenation and cultivation of Tilapia Streptococcus

[0163] The rejuvenation and culture process of Tilapia Streptococcus is the same as that in Chapter 3

[0164] 4.1.3 Tilapia Streptococcus infection experiment and sample collection

[0165] After three rejuvenation treatments, the tilapia Streptococcus strain was inoculated into TSB medium for expansion. The culture was incubated in a 32°C shaker for 12-14 hours. The concentration of the culture was determined using a microplate reader, measuring OD600. Based on the previously determined LC50, the culture was diluted with PBS solution to the previously determined LC50. In the experimental group, each fish was injected with 0.2 ml of the diluted culture, while the control group received the same volume of PBS solution. The inoculated fish were then cultured and observed in 6-m³ plastic vats containing 32°C water.

[0166] Each of the four first-generation groups was divided into three replicate groups, each containing 80 fish. Samples were collected at 0, 16, and 72 hours post-infection. Spleen tissue from three fish in each replicate group was pooled and placed in a single sampling tube for subsequent biological analysis.

[0167] 4.1.4 RNA extraction, library construction and sequencing

[0168] For the present invention, RNA extraction is a key step in gene expression analysis. In order to obtain high-quality RNA samples, spleen tissue samples were first ground in a mortar filled with liquid nitrogen. RNA was then extracted using a kit, and the concentration and integrity of the RNA were tested and evaluated using an Agilent 2100 analyzer. The A260:A280 of each RNA sample was ≥1.8, and the A260:A230 was ≥2.2. When constructing the RNA sequencing library, equal amounts of RNA samples were selected from three replicates at each time point and mixed to reduce possible deviations from individual samples and improve the accuracy of the experimental results.

[0169] For DEG analysis, the DEGSeq2 software package was used to compare the four groups of spleen samples. DEGSeq2 is a statistical method based on the negative binomial distribution model, which is used to identify significant differences in gene expression. By setting the threshold of the P-adjust value to less than 0.05, statistically significant differentially expressed genes were screened. The transcriptome data were analyzed using the Novo Bioinformatics Platform. The KEGG enrichment analysis of differentially expressed genes was performed using the KEGG database. When P-adjust < 0.05, the pathway was considered to be significantly enriched in the differentially expressed genes, thereby better understanding the biological significance and regulatory mechanism of gene expression in the spleen.

[0170] 4.1.5 Transcriptome Sample Quality Control

[0171] After the samples passed the quality inspection, the mRNA with polyA tail was first enriched by Oligo (dT) magnetic beads for sequencing library preparation. After the library construction was qualified, 2×150bp paired-end sequencing was performed using the Ⅰllumina high-throughput sequencing platform NovaSeq 000 to obtain raw data (Raw Reads). To ensure the quality and reliability of the information analysis data, the raw data were further filtered using fastp software to remove the raw data (Raw Reads) with adapters, reads containing uncertain base information (N), and low-quality reads (the number of bases with Qphred ≤ 20 accounted for more than 50% of the total read length). The bases with a quality value of less than 10 were also deleted to obtain high-quality clean reads.

[0172] 4.1.6 Data Processing

[0173] 4.1.6.1 Analysis of Differentially Expressed Genes

[0174] Based on existing gene and genome annotation files, HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts) version 2.0.5 was used to compare GZ, ZG, and ZZ. Subsequently, String Tie (1.3.3.b) was used for novel gene prediction. Transcripts were assembled and quantified using the Network Flow algorithm and an optional de novo assembly strategy to ensure transcript integrity and accuracy. Feature Counts were then used to calculate the number of reads mapped to each gene, and FPKM (fragments per bilobase of exon model per million mapped reads) was calculated based on gene length to reflect gene expression levels. Finally, statistical analysis was performed using DESeq2 software (1.16.1) to identify differentially expressed genes. This software uses a negative binomial distribution model to compare gene expression data between the two sample combinations. P values were corrected using the Benjamini & Hochberg method, and differentially expressed genes with adjusted P values < 0.05 and |log2(fold change)| > 1 were selected.

[0175] 4.1.6.2 Analysis of Differentially Expressed Genes

[0176] Diamond (0.9.13) was used to align the target gene sequence with the selected reference protein sequence to complete functional annotation. First, the upregulated differentially expressed genes in the ZG, GZ, and ZZ groups within the time range of 0h-16h were identified, as well as the downregulated differentially expressed genes in the three groups within the time range of 16h-72h. Secondly, the common differentially expressed genes in the three groups of upregulated and downregulated differentially expressed genes were identified. Taking the ZG group as an example, the upregulated and downregulated differentially expressed genes in the ZG group identified in the first step were compared to identify the common differentially expressed genes in the two groups. The same procedure was followed for the other two groups. Finally, the common differentially expressed genes in the ZG and GZ groups were compared with those in the ZZ group, and the common differentially expressed genes in the ZG and ZZ groups, and in the GZ and ZZ groups, were selected. The results obtained from the above operations were subjected to KEGG enrichment analysis based on the statistical significance threshold of P < 0.05 to screen out KEGG enriched pathways, and the pathways related to immune disease resistance and the differentially expressed genes in these pathways were analyzed.

[0177] 4.2 Test results

[0178] 4.2.1 Quality control analysis of spleen tissue transcriptome

[0179] This study used sequencing by synthesis (SBS) technology to sequence the transcriptomes of 36 samples using the Illumina high-throughput sequencing platform. As shown in Table 6, the sequencing data volume exceeded 5.65 Gb, the proportion of filtered data to the original data was less than 0.03%, the Q20 and Q30 base ratios were both greater than 90%, and the GC content ranged from 41.63% to 47.84%.

[0180] Table 6 RNA-seq data statistical results

[0181]

[0182]

[0183] From the correlation heat map between samples, we can see that the correlation between samples is high, ranging from 0.88 to 0.98, indicating that the sequencing quality is high. The correlation between GZ and ZG at 0h and other groups is slightly lower, ranging from 0.64 to 0.75. The correlation coefficients between samples in other groups are relatively high, ranging from 0.88 to 0.98. Based on the comparison results, subsequent analysis and research can be carried out. The comparison results are as follows Figure 11 shown.

[0184] 4.2.2 Differentially expressed genes in the spleen transcriptome

[0185] Sequencing results within and between the ZG, GZ, and GG groups were used to identify differentially expressed genes, laying the foundation for subsequent analysis of differentially expressed gene functions. Within-group analysis included the following: Splenic tissue sequencing results from the ZG, GZ, and ZZ groups at 16 and 72 hours post-infection with S. agalactiae were compared with those from 0 hours, respectively, to identify differentially expressed genes. Detailed results are shown in Table 7. The results revealed a decrease in the number of differentially expressed genes between 16 and 72 hours in the ZG, GZ, and ZZ groups.

[0186] The number of up-regulated and down-regulated genes in the ZG and ZZ groups decreased during the 0-16h and 16-72h timeframes. Comparison of the differentially regulated genes upregulated in ZG16 / ZG0 with the differentially regulated genes downregulated in ZG72 / ZG16 revealed 65 differentially regulated genes, as well as comparison of the differentially regulated genes upregulated in ZZ16 / ZZ0 with the differentially regulated genes downregulated in ZZ72 / ZZ16. Comparison of the differentially regulated genes upregulated in GZ16 / GZ0 with the differentially regulated genes downregulated in GZ72 / GZ16 revealed 282 differentially regulated genes.

[0187] Table 7 Statistics of differentially expressed genes at different time points in the ZG, GZ and ZZ groups after infection with Streptococcus agalactiae

[0188] DEG set AllDEG Up-regulated Down-regulated ZG16 / ZG0 3959 1890 2069 ZG72 / ZG16 2773 1287 1486 GZ16 / GZ0 3892 1652 2240 GZ72 / GZ16 3507 1770 1737 ZZ16 / ZZ0 5529 2806 2723 ZZ72 / ZZ16 2647 1394 1253

[0189] 4.2.3 Gene enrichment and pathway analysis

[0190] Figure 12 The chart shows the biological pathways in which differentially expressed genes in the spleen are enriched under two different conditions. Each bubble represents a specific pathway, its size represents the number of differentially expressed genes in the pathway, and its color represents the significance of the enrichment (p-value). The darker the color, the higher the significance, i.e., the stronger the correlation. Figure 12 (a) In the bubble diagram of the enriched pathways of the same differentially expressed genes between ZG and ZZ, we can see the proportion and significance of genes in each pathway. For example, the proportion of genes in the pathways "MAPK signaling pathway" and "Cytokine-cytokine receptor interaction" is higher and the significance is also stronger. Figure 12 (b) The bubble plot of the enriched pathways for the same differentially expressed genes between GZ and ZZ also shows the gene proportions and significance of the pathways. In this plot, the significance and gene proportions of the "TNF signaling pathway" and "Apoptosis pathways" are both high.

[0191] The enriched pathways for differentially expressed genes between ZG and GZ, as well as differentially expressed genes in ZZ, that were upregulated between 0 and 16 hours and downregulated between 16 and 72 hours are shown in Tables 8 and 9, respectively. KEGG enrichment results for the same differentially expressed genes in the ZG, GZ, and ZZ groups were all enriched in the necroptosis, TGF-beta signaling, and apoptosis pathways. The differentially expressed genes in the TGF-beta signaling and phagosome pathways between ZG and ZZ were both hamp. The differentially expressed genes in the apoptosis, necroptosis, salmonella infection, TGF-beta signaling, and MAPK signaling pathways between GZ and ZZ all included tnfb; the birc2 gene was also present in both the apoptosis and necroptosis pathways. The genes txn, hamp, and fth1a were also shared between the ZG and GZ groups.

[0192] Table 8 Enriched pathways of differentially expressed genes in ZG and ZZ that were up-regulated between 0h and 16h and down-regulated between 16h and 72h

[0193]

[0194] Table 9 Enriched pathways of differentially expressed genes in GZ and ZZ that were up-regulated between 0h and 16h and down-regulated between 16h and 72h

[0195]

[0196] 4.3 Discussion

[0197] 4.3.1 Spleen Tissue Transcriptome Quality Control

[0198] Sun Xiaomei et al.

[121] used local yellow cattle and hybrid cattle as experimental subjects, revealed differences in immune responses through spleen transcriptome sequencing, and determined the quality of sequencing data by evaluating the proportion of pure data, Q30 quality value, and alignment with the cattle reference genome. The results showed that the pure data ratio was as high as 97.49% to 98.02%, and the average quality value of Q30 was between 90.417% and 91.433%, indicating that high sequencing quality was generated during the sequencing process. Peng Lina et al.

[121] used high-throughput sequencing technology to obtain raw data, performed data quality control by removing low-quality reads, used HISAT2 software to align the filtered data with the rat reference genome, and then used StringTie to assemble and reconstruct the transcriptome for subsequent analysis. Through transcriptome sequencing analysis, differentially expressed genes that affect rats under aerobic exercise in different temperature environments were screened out. Wang Jiandong et al.[121,122] obtained a total of 1,049,301,886 raw sequencing reads in their study. After quality control and filtering, 1,032,097,278 high-quality sequencing reads were obtained. The unique alignment rate for all samples was above 83.76%, the maximum FDR (false discovery rate) for the sequencing data was 0.0263% (less than 0.1%), the minimum Q20 and Q30 contents of each sample were 97.39% and 93.05%, respectively, and the GC content ranged from 50.16% to 52.40%. These indicators reflect the high quality of the sequencing data and ensure the accuracy and reliability of subsequent analysis.

[0199] The sequencing data generated by the present invention exceeded 5.65 Gb, and the remaining data after quality control accounted for less than 0.03% of the original data. In terms of sequencing quality, the Q20 and Q30 base ratios were both greater than 90%, indicating high-quality bases generated during the sequencing process. Furthermore, the GC content ranged from 41.63% to 47.84%, demonstrating a reasonable base composition range. These indicators demonstrate the high quality of the data generated by the present invention, providing a solid data foundation for subsequent differential expression analysis, gene function annotation, and enrichment analysis. The inter-sample correlation heatmap reveals high inter-sample correlations, ranging from 0.88 to 0.98. However, the correlations between the GZ and ZG groups at 0 h and the other groups were slightly lower, ranging from 0.64 to 0.75. This may be due to differences in certain biological characteristics of the 0 h group compared to the other groups. The above analysis demonstrates that the data quality of the present invention meets or exceeds the standards of most similar studies, demonstrating the reliability of the sequencing technology and data processing procedures used. Furthermore, previous studies have often analyzed inter-sample correlations to assess experimental reproducibility and data consistency.

[0200] 4.3.2 Differentially expressed genes in the spleen transcriptome

[0201] Teng Wei

[123] found that there were 701 hibernation-related differentially expressed genes in the spleen of Rhinolophus ferrugineus, including 177 genes highly expressed during hibernation and 524 genes low expressed during hibernation. Among these genes, differentially expressed genes related to immune function were particularly selected, including 22 highly expressed immune genes and 51 low expressed immune genes. Typical examples include genes such as H1.2, Ccl2 and Cd44. Peng Lina et al.

[124] screened differentially expressed genes that were affected by aerobic exercise in rats under different temperature environments through transcriptome sequencing analysis. Compared with aerobic exercise at room temperature, a total of 248 differentially expressed genes were identified in low-temperature environments, of which 140 were upregulated and 108 were downregulated. The differentially expressed genes were mainly concentrated in biological processes such as immune response, immunoglobulin production, innate immune response and bacterial response. Lu Liyue et al.

[125] analyzed differentially expressed genes in the spleen of septic mice and found that in the lipopolysaccharide (LPS)-induced sepsis mouse model, the mRNA expression of the spleen changed significantly. A total of 1616 differentially expressed genes (DEGs) were identified, of which 919 genes were downregulated and 697 genes were upregulated. These changed genes involved multiple biological pathways related to inflammatory response, genetic information processing, signal transduction and cellular processes, indicating that LPS can significantly affect the immune system and activate multiple signaling pathways related to inflammation. Combined with the above literature, these findings provide important basic information for understanding the effects of spleen gene expression and provide a theoretical basis for in-depth research on the immune response mechanism and its molecular evolution pattern.

[0202] In the comparative analysis of differentially expressed genes in the spleen transcriptome, we first matched the differentially expressed genes upregulated in ZG16 / ZG0 with those downregulated in ZG72 / ZG16 to identify genes shared between the two groups. This result was then compared with the differentially expressed genes upregulated in ZZ16 / ZZ0 and downregulated in ZZ72 / ZZ16, ultimately screening out 65 differentially expressed genes. Using the same approach, we matched the differentially expressed genes upregulated in GZ16 compared with GZ0 and downregulated in GZ72 compared with GZ16, and compared these with the matching results of the differentially expressed genes upregulated in ZZ16 compared with ZZ0 and downregulated in ZZ72 compared with ZZ16, ultimately identifying 282 genes that were differentially expressed in common between the two groups.

[0203] 4.3.3 Gene enrichment and pathways

[0204] Studies have shown that necroptosis is a new type of programmed cell death pathway. Unlike traditional apoptosis and necrosis, necroptosis is a controlled cell death process that may play an important role in inflammation and immune response

[126] . The TGF-β signaling pathway has been shown to play an important role in the regulation of inflammation, regulating cell proliferation, differentiation, and the production of inflammatory factors

[127] . Apoptosis is a type of programmed cell death that plays a key role in regulating the timing and extent of immune response, helping the body to eliminate infected or damaged cells and maintain immune balance

[128] . Therefore, by analyzing key signaling pathways such as necroptosis, TGF-β signaling pathway, and apoptosis, we can gain a more specific understanding of the mechanisms of inflammation and immune response caused by bacterial infection.

[0205] Comparing ZG and ZZ groups, both differentially expressed genes in the TGF-beta signaling pathway and the Phagosome pathway included hamp. Comparing GZ and ZZ groups, differentially expressed genes in the apoptosis, necroptosis, Salmonella infection, TGF-beta signaling pathway, and MAPK signaling pathway all included tnfb. Both the apoptosis and necroptosis signaling pathways contained the birc2 gene. Genes txn, hamp, and fth1a were also shared between the ZG and GZ groups. These results suggest that ZG and GZ infection have similar effects on gene expression and pathway regulation in spleen tissue, providing a foundation for further research.

[0206] Guo Wentao et al.

[129] found some differentially expressed genes by comparing transcriptome data at different infection periods. These genes are involved in pathways such as immune response, apoptosis and oxidative stress, which are similar to the findings in the present invention. Xie Zhenglu et al.

[130] studied the effects of streptococcal infection on the transcriptome and pathways of mouse spleen tissue. The results showed that the differential genes and pathways in spleen tissue after infection mainly involved pathways such as immune response, inflammation and cell death. This suggests that GZ and ZG infections have certain similarities in regulating pathways in spleen tissue. Zhu Jiajie [9] studied the pathophysiological effects of Streptococcus agalactiae infection on the spleen and liver of Nile tilapia. The results showed that apoptosis and inflammatory responses occurred in spleen tissue after infection, which is related to the differentially expressed genes and pathway regulation found in the present invention. Combining the results of the present invention with the analysis of previous studies, it can be concluded that after bacterial infection, changes in gene expression of signaling pathways such as necroptosis, TGF-β signaling pathway and apoptosis in spleen tissue may play a key role in regulating immune and inflammatory responses. Further investigation of these differentially expressed genes and the signaling pathways they regulate may help reveal the mechanisms of immune response and provide new research ideas for the treatment and prevention of infectious diseases. In summary, GZ and ZG infection show certain similarities in the expression of differentially expressed genes and pathway regulation in spleen tissue, particularly in pathways such as immune response, inflammation, and cell death. This suggests that GZ and ZG infection share certain common pathophysiological characteristics in the spleen and may affect gene expression in the spleen through similar mechanisms. Furthermore, there are some differences in the expression of differentially expressed genes and pathways in spleen tissue after GZ and ZG infection, such as those in the apoptosis, necroptosis, and TGF-beta signaling pathways. This suggests that GZ and ZG infection may differentially regulate pathways in the spleen.

[0207] This study compared the growth traits of the offspring by crossbreeding the "Guifei No. 1" tilapia strain, which exhibits significant growth advantages, with the disease-resistant "Zhuangluo No. 1" tilapia strain. The results showed that the four offspring groups had 14 to 17 dorsal fin spines and 12 to 14 dorsal fin rays, respectively, compared to 14 to 18 dorsal fin spines and 10 to 14 dorsal fin rays for the Nile tilapia, indicating that the four offspring groups were relatively similar to the Nile tilapia. Among the measurable traits, the body size of the hybrid offspring was between the two parents, with the ZG group being closer to the maternal GG group. After 90 days, the average body weight of the ZG and GZ groups was different from that of the GG group, but the difference was not significant (P>0.05). However, the standard deviation of the ZG group was greater than that of the GZ and GG groups. When the whole species was not infected with Streptococcus, the survival rate of the four groups of offspring was ZZ>ZG>GG>GZ, and in terms of yield, ZG>GZ>GG>ZZ.

[0208] Evaluation of the physiological and biochemical properties of the livers of offspring infected with Streptococcus agalactiae revealed differences in enzyme activity between the different parents, which is closely related to the offspring's disease resistance. Within 72 hours of S. agalactiae injection, changes in AKP, CAT, and GPT activities in the livers of the four offspring groups showed an initial increase followed by a decrease over time. At 16 hours, the increase in AKP activity in the ZG group was significantly lower than that in the other three groups (P < 0.05). 72 hours after S. agalactiae injection, levels in GG, ZZ, and ZG all decreased, with a slight decrease in the GZ group. The GG group had the lowest alkaline phosphatase activity at 72 hours. The GG group experienced a more significant decrease in AKP content between 16 and 72 hours, while the other three groups experienced a slower decline. Tilapia disease resistance is associated with the rate of decline in AKP activity. Before infection with S. agalactiae, the ZG group had the highest CAT activity, significantly higher than the GG and ZZ groups. After infection with S. agalactiae, no significant differences in CAT activity were found among the four groups (P>0.05). Before S. agalactiae injection, there were no significant differences in GPT activity among the four groups. Sixteen hours after S. agalactiae injection, GPT activity increased significantly, with the ZG group experiencing a significantly higher increase than the other three groups (P<0.05). Seventy-two hours after infection with S. agalactiae, GPT activity was lowest in the GG group and highest in the GZ group, with significant differences between the GZ group and the ZZ and ZG groups (P<0.05). The speed of enzyme activity decline is related to tilapia's resistance to streptococcal infection; those with slower enzyme activity decline exhibit a more sensitive and rapid response to external streptococci.

[0209] Transcriptome sequencing analysis of spleen tissue from offspring infected with Streptococcus agalactiae revealed that hamp was the differentially expressed gene in both the TGF-beta signaling pathway and the phagosome between ZG and ZZ, and between GZ and ZZ. Both the apoptosis and necroptosis signaling pathways contain the birc2 gene, suggesting that these signaling pathways and shared genes may function together following bacterial infection. Furthermore, the shared expression of txn, hamp, and fth1a genes between the ZG and GZ infection groups suggests that infection has similar effects on gene expression and pathway regulation in spleen tissue. Although GZ and ZG infection exhibit certain similarities in their effects on spleen tissue, differences exist in pathway regulation, such as in the apoptosis, necroptosis, and TGF-beta signaling pathways. Infected offspring may affect gene expression and function in spleen tissue through distinct mechanisms.

[0210] In summary, this invention provides a new development direction and research idea for tilapia farming by breeding disease-resistant tilapia varieties and exploring their immune regulation and disease resistance mechanisms, and provides important theoretical and practical support for the healthy development of freshwater aquaculture and disease prevention and control.

[0211] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A growth evaluation method for hybrid progeny of Zhuangluo No. 1 and Guifei No. 1 tilapia, characterized in that: The following steps are involved: Step 1: Evaluate the growth traits and disease resistance of the hybrid offspring of "Zhuangluo No. 1" and "Guifei No. 1" tilapia; Step 2: Evaluate the physiological and biochemical performance of the hybrid offspring of "Zhuangluo No. 1" and "Guifei No. 1" tilapia; Step 3: Analyze the differences in spleen transcriptomes of self-cross and reciprocal cross progenies of "Zhuangluo No. 1" and "Guifei No. 1" after infection with Tilapia Streptococcus.

2. the growth evaluation method of the hybridization progeny of Zhuangluo No. 1 and Guifei No. 1 tilapia as claimed in claim 1, is characterized in that, The step 1 comprises: The offspring of the self-pollination of "Zhuangluo No. 1" was named ZZ; the offspring of the self-pollination of "Guifei No. 1" was named GG; the offspring of the hybridization of "Zhuangluo No. 1" as the female parent and "Guifei No. 1" as the male parent was named ZG; the offspring of the hybridization of "Guifei No. 1" as the female parent and "Zhuangluo No. 1" as the male parent was named GZ; The growth traits of ZZ, GG, ZG and GZ, including body length, weight, feed conversion rate (FCR) and survival rate, were analyzed in detail compared with those of their parents; Under the same breeding conditions, record and compare their disease resistance.

3. the growth evaluation method of the hybridization progeny of Zhuangluo No. 1 and Guifei No. 1 tilapia as claimed in claim 1, is characterized in that, The step 2 includes: Infect with Streptococcus agalactiae by artificial intraperitoneal injection to simulate streptococcal infection conditions; The changes in the activities of key enzymes, such as superoxide dismutase (SOD), catalase (CAT), and lactate dehydrogenase (LDH), in the livers of offspring tilapia after infection were detected; Combined with the levels of serum inflammatory factors (such as IL-1β and TNF-α), the physiological and biochemical responses of hybrid offspring to streptococcal infection were evaluated.

4. the growth evaluation method of the hybridization progeny of Zhuangluo No. 1 and Guifei No. 1 tilapia as claimed in claim 1, is characterized in that, The step 3 comprises: Transcriptome sequencing was performed on spleen tissues of the ZG and ZZ groups, and the GZ and ZZ groups; Through bioinformatics analysis, we screened immune-related differentially expressed genes and annotated the functions of these genes; Analysis of key signaling pathways involved in differentially expressed genes, including necroptosis, TGF-β signaling pathway, and apoptosis; Correlate changes in gene expression with immune regulation and disease resistance, and propose mechanistic hypotheses.

5. the growth evaluation method of the hybridization progeny of Zhuangluo No. 1 and Guifei No. 1 tilapia as claimed in claim 4, is characterized in that: Transcriptome sequencing uses Illumina high-throughput sequencing technology, with a sequencing depth of 20Gb per sample; The screening of differentially expressed genes used the criteria of Fold Change ≥ 2 and p -value < 0.05; The functional annotation of differentially expressed genes was performed using the GO database and KEGG pathway.

6. the growth evaluation method of the hybridization progeny of Zhuangluo No. 1 and Guifei No. 1 tilapia as claimed in claim 1, is characterized in that: In the disease resistance evaluation, the transcription level of differentially expressed genes and the corresponding protein expression level were combined to verify the function of key genes using ELISA and qPCR; Through comprehensive analysis of hybrid offspring and parents, potential disease resistance mechanisms were proposed, providing a theoretical basis for the breeding of new disease-resistant tilapia varieties.

7. A growth evaluation system for hybrid progeny of Zhuangluo No. 1 and Guifei No. 1 tilapia implementing the growth evaluation method for hybrid progeny of Zhuangluo No. 1 and Guifei No. 1 tilapia as described in any one of claims 1-6, characterized in that: The growth evaluation system of the hybrid offspring of Zhuangluo No. 1 and Guifei No. 1 tilapia comprises: Disease resistance evaluation module, used to evaluate the growth traits and disease resistance of hybrid offspring of "Zhuangluo No. 1" and "Guifei No. 1" tilapia; Physiological and biochemical performance evaluation module, used to evaluate the physiological and biochemical performance of hybrid offspring of "Zhuangluo No. 1" and "Guifei No. 1" tilapia; The differential analysis module is used to analyze the differential transcriptome of the spleen of the self-cross and reciprocal cross progenies of "Zhuangluo No. 1" and "Guifei No. 1" after infection with Streptococcus tilapia.

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