Complement networks in gene-edited pig xenotransplantation: enhancing transplant success and addressing organ shortage.

The shortage of organs for transplantation emphasizes the urgent need for alternative solutions. Xenotransplantation has emerged as a promising option due to the greater availability of donor organs. However, significant hurdles such as hyperacute rejection and organ ischemia-reperfusion injury pose major challenges, largely orchestrated by the complement system, and activated immune responses. The complement system, a pivotal component of innate immunity, acts as a natural barrier for xenotransplantation. To address the challenges of immune rejection, gene-edited pigs have become a focal point, aiming to shield donor organs from human immune responses and enhance the overall success of xenotransplantation. This comprehensive review aims to illuminate strategies for regulating complement networks to optimize the efficacy of gene-edited pig xenotransplantation. We begin by exploring the impact of the complement system on the effectiveness of xenotransplantation. Subsequently, we delve into the evaluation of key complement regulators specific to gene-edited pigs. To further understand the status of xenotransplantation, we discuss preclinical studies that utilize gene-edited pigs as a viable source of organs. These investigations provide valuable insights into the feasibility and potential success of xenotransplantation, offering a bridge between scientific advancements and clinical application.

SEMA4D/VEGF surface enhances endothelialization by diminished-glycolysis-mediated M2-like macrophage polarization.

Cardiovascular disease remains the leading cause of death and morbidity worldwide. Inflammatory responses after percutaneous coronary intervention led to neoathrosclerosis and in-stent restenosis and thus increase the risk of adverse clinical outcomes. In this work, a metabolism reshaped surface is engineered, which combines the decreased glycolysis promoting, M2-like macrophage polarization, and rapid endothelialization property. Anionic heparin plays as a linker and mediates cationic SEMA4D and VEGF to graft electronically onto PLL surfaces. The system composed by anticoagulant heparin, immunoregulatory SEMA4D and angiogenic VEGF endows the scaffold with significant inhibition of platelets, fibrinogen and anti-thrombogenic properties, also noteworthy immunometabolism reprogram, anti-inflammation M2-like polarization and finally leading to rapid endothelializaiton performances. Our research indicates that the immunometabolism method can accurately reflect the immune state of modified surfaces. It is envisioned immunometabolism study will open an avenue to the surface engineering of vascular implants for better clinical outcomes.

Atovaquone inhibits colorectal cancer metastasis by regulating PDGFRß/NF-κB signaling pathway.

BACKGROUND: Colorectal cancer is a common malignant tumour. Invasive growth and distant metastasis are the main characteristics of its malignant biological behaviour, and they are also the primary factors leading to death in colon cancer patients. Atovaquone is an antimalarial drug, and its anticancer effect has recently been demonstrated in several cancer models in vitro and in vivo, but it has not been examined in the treatment of colorectal cancer. METHODS: To elucidate the effect of atovaquone on colorectal cancer. We used RNA transcriptome sequencing, RT‒PCR and Western blot experiments to examine the expression of NF-κB (p-P65), EMT-related proteins and related inflammatory factors (IL1B, IL6, CCL20, CCL2, CXCL8, CXCL6, IL6ST, FAS, IL10 and IL1A). The effect of atovaquone on colorectal cancer metastasis was validated using an animal model of lung metastases. We further used transcriptome sequencing, the GCBI bioinformatics database and the STRING database to predict relevant target proteins. Furthermore, pathological sections were collected from relevant cases for immunohistochemical verification. RESULTS: This study showed that atovaquone could inhibit colorectal cancer metastasis and invasion in vivo and in vitro, inhibit the expression of E-cadherin protein, and promote the protein expression of N-cadherin, vimentin, ZEB1, Snail and Slug. Atovaquone could inhibit EMT by inhibiting NF-κB (p-P65) and related inflammatory factors. Further bioinformatics analysis and verification showed that PDGFRß was one of the targets of atovaquone. CONCLUSION: In summary, atovaquone can inhibit the expression of NF-κB (p-P65) and related inflammatory factors by inhibiting the protein expression of p-PDGFRß, thereby inhibiting colorectal cancer metastasis. Atovaquone may be a promising drug for the treatment of colorectal cancer metastasis.

Construction of silver-coated high translucent zirconia implanting abutment material and its property of antibacterial.

High translucent zirconia (HTZ) has excellent mechanical properties, biocompatibility, and good semi-translucency making it an ideal material for aesthetic anterior dental implant abutments without antibacterial properties. In the oral environment, the surface of the abutment material is susceptible to microbial adhesion and biofilm formation, which can lead to infection or peri-implantitis and even implant failure. This study aims to promote the formation of a biological seal at the implant-soft tissue interface by modifying the HTZ surface, using the load-bearing capacity of the aluminosilicate porous structure and the broad-spectrum antibacterial effect of silver nanoparticles to prevent peri-implant bacterial infection and inflammation and to improve the success rate and prolong the use of the implant. FE-SEM (field emission scanning electron microscopes), EDS (energy dispersive spectroscopy), and XPS (X-ray photoelectron spectroscopy) results showed that aluminosilicate non-vacuum sintering can form open micro- and nanoporous structures on HTZ surfaces, and that porous aluminosilicate coatings obtain a larger number, smaller size, and more uniformly shaped silver nanoparticles than smooth aluminosilicate coatings, and could be deposited deeper in the coating. The ICP-AES (inductively coupled plasma-atomic emission spectroscopy) results showed that the early silver ion release of both the smooth silver coating and the porous silver coating was obvious, the silver ion concentration released by the former was higher than that of the latter. However, the silver ion concentration released by the porous silver coating was higher than that of the smooth coating when the release slowed down. Both smooth and porous silver coatings both inhibited E. coli (Escherichia coli), S. aureus (Staphylococcus aureus), and L. acidophilus (L. acidophilus), and porous silver coatings had stronger antibacterial properties. The silver coating was successfully constructed on the surface of HTZ, through aluminium silicate sintering and silver nitrate solution impregnation. It was found that the high concentration environment of silver nitrate solution was more advantageous for nano-Ag deposition, and the non-vacuum sintered porous surface was able to obtain a larger number of nano-Ag particles with smaller sizes. The porous Ag coating exhibited superior antibacterial properties. It was suggested that the HTZ with silver coating had clinical application, and good antibacterial properties that can improve the survival rate and service life of implants.

Real-time semi-quantitative assessment of anastomotic blood perfusion in mini­invasive rectal resections by Sidestream Dark Field (SDF) imaging technology: a prospective in vivo pilot study.

PURPOSE: Anastomotic leakage (AL) is one of the severe complications after rectal surgery, and anastomotic ischemia is one of the main factors. This prospective in vivo pilot study aimed to evaluate the effectiveness of Sidestream Dark Field (SDF) imaging in quantitative assessment of anastomotic microcirculation and to analyze its correlation with AL. METHODS: Thirty-three patients with rectal cancer who underwent laparoscopic low anterior resection from 2019 to 2020 were enrolled. Microcirculation was measured by SDF imaging at the descending colon, the mesocolon transection line (MTL), and 1 cm and 2 cm distal to the MTL. Anastomotic microcirculation was measured at the stapler anvil edge before anastomosis. Quantitative perfusion-related parameters were as follows: microcirculation flow index (MFI), perfused vessel density (PVD), proportion of perfused vessels (PPV), and total vessel density (TVD). RESULTS: All patients obtained stable microcirculation images. Functional microcirculation parameters (MFI, PPV, PVD) decreased successively from the descending colon, the colon at MTL, and 1 cm and 2 cm distal to the MTL (all P < 0.01). Extremely poor microcirculation was found at the intestinal segment 2 cm distal to the MTL. Micro-perfusion was significantly lower at the colonic limb of the anastomosis compared with the descending colon (all P < 0.001). Anastomotic leakage occurred in 3 patients (9.1%) whose anastomotic microcirculation was significantly lower than those without AL (all P < 0.01). Blood perfusion at the colonic limb of the anastomosis was significantly higher in patients with left colic artery preservation than in controls. CONCLUSION: SDF imaging is a promising technique for evaluating anastomotic microcirculation and has potential clinical significance for risk stratification of AL.

Reprogramming efficiency and pluripotency of mule iPSCs over its parents†.

The mule is the interspecific hybrid of horse and donkey and has hybrid vigor in muscular endurance, disease resistance, and longevity over its parents. Here, we examined adult fibroblasts of mule (MAFs) compared with the cells from their parents (donkey adult fibroblasts and horse adult fibroblasts) (each species has repeated three independent individuals) in proliferation, apoptosis, and glycolysis and found significant differences. We subsequently derived mule, donkey, and horse doxycycline (Dox)-independent induced pluripotent stem cells (miPSCs, diPSCs, and hiPSCs) from three independent individuals of each species and found that the reprogramming efficiency of MAFs was significantly higher than that of cells of donkey and horse. miPSCs, diPSCs, and hiPSCs all expressed the high levels of crucial endogenous pluripotency genes such as POU class 5 homeobox 1 (POU5F1, OCT4), SRY-box 2 (SOX2), and Nanog homeobox (NANOG) and propagated robustly in single-cell passaging. miPSCs exhibited faster proliferation and higher pluripotency and differentiation than diPSCs and hiPSCs, which were reflected in co-cultures and separate-cultures, teratoma formation, and chimera contribution. The establishment of miPSCs provides a unique research material for the investigation of "heterosis" and perhaps is more significant to study hybrid gamete formation.

Mettl14 sustains FOXP3 expression to promote the differentiation and functions of induced-regulatory T cells via the mTOR signaling pathway.

Induced regulatory T cell (iTregs) can be generated in vitro. Thus, iTregs-based therapeutics are receiving increased attention for their potential to treat autoimmune diseases and prevent transplant rejection. However, iTregs fail to maintain FoxP3 expression and suppressive activity, which limits their clinical application. Increasing lines of evidence suggest that methyltransferase-like 14 (METTL14), a critical component of the m6A writer complex, regulates the stability and function of the Treg cells. However, beyond meeting the epigenetic modification of Treg cells, whether Mettl14 plays a role in the fate determination of iTregs is unclear. Here, we systemically investigated the potential function of METTL14 in iTregs differentiation and regulatory activity. In our study, iTregs were generated from CD4+ naïve T cells under iTreg-polarizing conditions, we found that the expression of METTL14 was increased in iTregs compared with CD4+naïve T cells. Subsequently, the expression of METTL14 was knocked down by siRNA-METTL14 interference in CD4+ naïve T cells and cultured under iTreg-polarizing conditions. According to the results, Mettl14 deficiency resulted in the disruption of iTregs differentiation evidenced by the limited FoxP3 expression. Meanwhile, inflammatory cytokines such as IFN-γ and IL-17a were upregulated in cultured iTregs. We next determined the functional change in METTL14-deficient iTregs. The results of the colitis development in Rag1-/- mice and CFSE assays revealed that loss of METTL14 significantly compromised the suppressive function of iTregs in vivo and in vitro. We further checked the altered signaling pathway in METTL14-deficient iTregs. We found that reduced METTL14 leads to activation of the mTOR pathway with increased p-mTOR and p-p70S6K, which are known to modulate the suppressive function of iTregs. In conclusion, our study revealed that Mettl14 plays a critical role in the development and suppressive function of iTregs in vitro and could thus serve as a regulatory element for stabilizing iTregs in cell-based therapy.

Microbial and metabolic features in renal transplant recipients with post-transplantation diabetes mellitus.

OBJECTIVE: Post-transplantation diabetes mellitus (PTDM) is a common complication in renal transplant recipients (RTRs). Gut microbiome plays important roles in a variety of chronic metabolic diseases, but its association with the occurrence and development of PTDM is still unknown. The present study integrates the analysis of gut microbiome and metabolites to further identify the characteristics of PTDM. METHODS: A total of 100 RTRs fecal samples were collected in our study. Among them, 55 samples were submitted to Hiseq sequencing, and 100 samples were used for non-targeted metabolomics analysis. The gut microbiome and metabolomics of RTRs were comprehensively characterized. RESULTS: The species Dialister invisus was significantly associated with fasting plasma glucose (FPG). The functions of tryptophan and phenylalanine biosynthesis were enhanced in RTRs with PTDM, while the functions of fructose and butyric acid metabolism were reduced. Fecal metabolome analysis indicated that RTRs with PTDM had unique metabolite distribution characteristics, and two differentially expressed specific metabolites were significantly correlated with FPG. The correlation analysis of gut microbiome and metabolites showed that gut microbiome had an obvious effect on the metabolic characteristics of RTRs with PTDM. Moreover, the relative abundance of microbial function is associated with the expression of several specific gut microbiome and metabolites. CONCLUSIONS: Our study identified the characteristics of gut microbiome and fecal metabolites in RTRs with PTDM, and we also found two important metabolites and a bacterium were significantly associated with PTDM, which might be used as novel targets in the research field of PTDM.

Mettl14-mediated m6A modification enhances the function of Foxp3+ regulatory T cells and promotes allograft acceptance.

N6-methyladenosine (m6A), the most prevalent form of internal mRNA modification, is extensively involved in Treg cells differentiation and function. However, the involvement of m6A in functional Treg cells for transplantation tolerance remains to be elucidated. By using an experimental transplantation mouse model, we found that m6A levels in Treg cells were altered during the induction of transplant tolerance by performing a dot blotting assay. Subsequently, we used the heterogenic Treg-specific Mettl14 knockout mice (Foxp3-Mettl14f/+ cKO) to reduce METTL14 expression and performed islets allograft transplantation. Our result revealed that reduced expression of METTL14 prevented Treg cells expansion and promoted the infiltration of CD4+ and CD8+ T cells around the allograft, which led to rapid allograft rejection in Foxp3-Mettl14 f/+ cKO mice. The expression of regulatory cytokines including IL-10 and TGF-ß was significantly decreased in Foxp3-Mettl14 f/+ cKO mice, and the suppressive function of Treg cells was also abrogated. In addition, an analysis of RNA-seq data revealed that the SOCS family (SOCS1, SOCS2 and SOCS3) is the subsequent signaling pathway affected by the METTL14 mediated m6A modification in Treg cells to modulate the suppressive function after transplantation. Taken together, our study showed for the first time that the METTL14-mediated m6A modification is essential for the suppressive function of Treg cells in transplantation and may serve as a regulatory element of Treg cell-based therapy in transplant medicine.

In vivo induction of regulatory T cells by anti-CD45RB antibody causes transferable tolerance to discordant human xenogenic islets.

BACKGROUND: The treatment of diabetes by islet cell transplantation has become an accepted therapy, with transplantation of xenogeneic islet cells an attractive alternative to the problem. Previous studies in mice have demonstrated that anti-CD45RB induce immune tolerance in human pancreatic islet cells. The current study was to define the mechanism of action of anti-CD45RB induced nonspecific immune tolerance to heteroantigens. METHODS: A total of 1500 IEQ human islets were transplanted to diabetic B6µMT-/- mice, B6 mice, and µMT-/- diabetic mice undergoing thymectomy. These mice were treated short-term with doses of anti-CD45RB. CD4+Foxp3+Tregs were detected in the blood, peripheral lymphatic organs by flow cytometry, and immunohistochemistry. In addition, anti-CD25 mAb was administered to tolerant human islet cells B6µMT-/-mice. Mice then were transplanted with other human islet cells and received CD4+CD25+Tregs isolated from tolerant human islets mice to observe islet destruction. RESULTS: Anti-CD45RB treatment-induced tolerance to islets in both immunocompetent and B-cell-deficient mice (µMT-/- mice) by processes that were dependent on CD25+ Tregs, but not B cells. Anti-CD45RB treatment increased the number of CD4+Foxp3+Tregs cells. Anti-CD45RB treatment-induced immune tolerance that was antigen nonspecific, with Tregs playing an important role. Anti-CD45RB treatment-induced tolerance generated Tregs that could be transferred to another individual to manifest nonspecific immune tolerance. CONCLUSION: The results of the experiment suggest that anti-CD45RB induced tolerance to human islet xenografts is mediated by the proliferation of Tregs. These tolerogenic Tregs can be transferred to other mice and induce nonspecific immune tolerance.

Reprogramming barriers in bovine cells nuclear transfer revealed by single-cell RNA-seq analysis.

Many progresses have recently been achieved in animal somatic cell nuclear transfer (SCNT). However, embryos derived from SCNT rarely result in live births. Single-cell RNA sequencing (scRNA-seq) can be used to investigate the development details of SCNT embryos. Here, bovine fibroblasts and three factors bovine iPSCs (3F biPSCs) were used as donors for bovine nuclear transfer, and the single blastomere transcriptome was analysed by scRNA-seq. Compared to in vitro fertilization (IVF) embryos, SCNT embryos exhibited many defects. Abnormally expressed genes were found at each stage of embryos, which enriched in metabolism, and epigenetic modification. The DEGs of the adjacent stage in SCNT embryos did not follow the temporal expression pattern similar to that of IVF embryos. Particularly, SCNT 8-cell stage embryos showed failures in some gene activation, including ZSCAN4, and defects in protein association networks which cored as POLR2K, GRO1, and ANKRD1. Some important signalling pathways also showed incomplete activation at SCNT zygote to morula stage. Interestingly, 3F biPSCNT embryos exhibited more dysregulated genes than SCNT embryos at zygote and 2-cell stage, including genes in KDM family. Pseudotime analysis of 3F biPSCNT embryos showed the different developmental fate from SCNT and IVF embryos. These findings suggested partial reprogrammed 3F biPS cells as donors for bovine nuclear transfer hindered the reprogramming of nuclear transfer embryos. Our studies revealed the abnormal gene expression and pathway activation of SCNT embryos, which could increase our understanding of the development of SCNT embryos and give hints to improve the efficiency of nuclear transfer.

Corrigendum: Mettl14-mediated m6A modification enhances the function of Foxp3+ regulatory T cells and promotes allograft acceptance.

[This corrects the article DOI: 10.3389/fimmu.2022.1022015.].

Electrical Impedance Tomography Predicts Weaning Success in Adult Patients With Delayed Upper Abdominal Surgery: A Single-Center Retrospective Study.

Objective: To evaluate the predictive value of electrical impedance tomography (EIT) in patients with delayed ventilator withdrawal after upper abdominal surgery. Methods: We retrospectively analyzed data of patients who were ventilated >24 h after upper abdominal surgery between January 2018 and August 2019. The patients were divided into successful (group S) and failed (group F) weaning groups. EIT recordings were obtained at 0, 5, 15, and 30 min of spontaneous breathing trials (SBTs) with SBT at 0 min set as baseline. We assessed the change in delta end-expiratory lung impedance and tidal volume ratio (ΔEELI/VT) from baseline, the change in compliance change percentage variation (|Δ(CW-CL)|) from baseline, the standard deviation of regional ventilation delay index (RVDSD), and global inhomogeneity (GI) using generalized estimation equation analyses. Receiver operating characteristic curve analyses were performed to evaluate the predictive value of parameters indicating weaning success. Results: Among the 32 included patients, ventilation weaning was successful in 23 patients but failed in nine. Generalized estimation equation analysis showed that compared with group F, the ΔEELI/VT was lower, and the GI, RVDSD, and (|Δ(CW-CL)|) were higher in group S. For predicting withdrawal failure, the areas under the curve of the ΔEELI/VT, (|Δ(CW-CL)|), and the RVDSD were 0.819, 0.918, and 0.918, and 0.816, 0.884, and 0.918 at 15 and 30 min during the SBTs, respectively. Conclusion: The electrical impedance tomography may predict the success rate of ventilator weaning in patients with delayed ventilator withdrawal after upper abdominal surgery.

Genome collinearity analysis illuminates the evolution of donkey chromosome 1 and horse chromosome 5 in perissodactyls: A comparative study.

BACKGROUND: It is important to resolve the evolutionary history of species genomes as it has affected both genome organization and chromosomal architecture. The rapid innovation in sequencing technologies and the improvement in assembly algorithms have enabled the creation of highly contiguous genomes. DNA Zoo, a global organization dedicated to animal conservation, offers more than 150 chromosome-length genome assemblies. This database has great potential in the comparative genomics field. RESULTS: Using the donkey (Equus asinus asinus, EAS) genome provided by DNA Zoo as an example, the scaffold N50 length and Benchmarking Universal Single-Copy Ortholog score reached 95.5 Mb and 91.6%, respectively. We identified the cytogenetic nomenclature, corrected the direction of the chromosome-length sequence of the donkey genome, analyzed the genome-wide chromosomal rearrangements between the donkey and horse, and illustrated the evolution of the donkey chromosome 1 and horse chromosome 5 in perissodactyls. CONCLUSIONS: The donkey genome provided by DNA Zoo has relatively good continuity and integrity. Sequence-based comparative genomic analyses are useful for chromosome evolution research. Several previously published chromosome painting results can be used to identify the cytogenetic nomenclature and correct the direction of the chromosome-length sequence of new assemblies. Compared with the horse genome, the donkey chromosomes 1, 4, 20, and X have several obvious inversions, consistent with the results of previous studies. A 4.8 Mb inverted structure was first discovered in the donkey chromosome 25 and plains zebra chromosome 11. We speculate that the inverted structure and the tandem fusion of horse chromosome 31 and 4 are common features of non-caballine equids, which supports the correctness of the existing Equus phylogeny to an extent.

The effects of cryopreservation on the acrosome structure, enzyme activity, motility, and fertility of bovine, ovine, and goat sperm.

The study was designed to investigate the effects of cryopreservation on bovine, ovine, and goat sperm motility, acrosome structure, enzyme activity, and fertilization ability. Percentage of sperm with hyaluronidase enzyme (HYD) activity was detected by a modified sodium hyaluronate-gelatin membrane. The N-α-benzoyl-DL-arginine-p-nitroanilide (BNPNA) method was used to assess the sperm acrosome enzyme (ACE). The mean percentage of sperm acrosome integrity dropped significantly (P < 0.01) after cryopreservation. The ACE activity of bovine sperm (100.48) was higher (P < 0.01) than that of ovine (57.88) or goat sperm (50.30), while the percentage of sperm with HYD activity of bovine (71.10%) and ovine (67.60%) sperm was higher than that of goat sperm (58.52%) after cryopreservation (P < 0.01). Sperm motility was positively correlated with the activity of the two acrosome enzymes before and after cryopreservation (P < 0.01). Cryopreservation had a negative effect on acrosomal morphology, motility, and acrosomal enzyme activity in their sperm. The fertilization ability of ovine and goat sperm decreased significantly after cryopreservation, but that of frozen bovine sperm did not differ significantly when compared with fresh sperm. There was no significant difference between ovine and goat sperm indices, except for percentage of sperm with HYD activity.

Establishment of bovine expanded potential stem cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.

Correlation between in vitro fertilization and artificial insemination in Holstein bulls.

OBJECTIVE: Owing to the lack of a breeding index for efficient and quick fertility evaluations of Holstein bulls when using traditional or genome-wide detection methods, this study aimed to determine whether in vitro fertilization (IVF) could be used as an indicator of conception rate of artificial insemination (AI). METHODS: Conventional and sexed frozen semen from nine bulls were used for IVF and AI. RESULTS: The IVF and AI conception rates of each bull were confirmed to be positively correlated between the conventional frozen and sexed frozen semen. The correlation coefficient R values of nine bulls between IVF and AI methods were 0.73 and 0.97 for the conventional frozen and sexed frozen semen, respectively. The average conception rate of three bulls undergoing AI was 69.5% and 64.2%, 61.8% and 58.8%, and 48.2% and 46.2% in first-, second-, and third-born cows when conventional frozen and sexed frozen semen were used, respectively, which showed a positive correlation with the fertilization rate in the same parity. We propose an evaluation standard to assess the fertilization ability of bulls based on their IVF test results, which is categorized into three grades: grade one, normal fertility bull with an AI conception rate of 40%±5% and IVF rate of 45% to 60%; grade two, higher fertility bull with an AI conception rate of 50%±5% and IVF rate of 61% to 80%; and grade three, highest fertility bull with an AI conception rate of 60%±5% and IVF rate of >80%. CONCLUSION: These findings reveal that IVF results can be used as a breeding index for bulls to evaluate their AI conception ability, which may shorten the time required to select bulls for breeding.