This study aimed to investigate the effects of fibroblast growth factor 2 (FGF2) on the in vitro maturation (IVM) of porcine oocytes. Porcine oocytes were cultured in IVM medium supplemented with different concentrations of FGF2 (0, 50, 100, and 150 ng/mL). After 44 h of IVM, cumulus expansion, first polar body (PB1) extrusion rate, cleavage rate following parthenogenetic activation, and blastocyst formation rate were evaluated. We further examined the effects of FGF2 on oocytes mitochondrial function and explored whether FGF2 promotes IVM through activation of the mitogen-activated protein kinase (MAPK) pathway. Compared with the 0 ng/mL control group, the 100 ng/mL FGF2 treatment group showed significantly higher rates of PB1 extrusion, cumulus expansion index, and cleavage (P < 0.05). Moreover, oocytes treated with 100 ng/mL FGF2 exhibited significantly elevated mitochondrial membrane potential and mitochondrial content (P < 0.01). RT‑qPCR analysis revealed that mRNA expression levels of MAPK1 and MAPK3 were significantly increased (P < 0.05), while the mRNA expression of SPRY2 remained unchanged (P > 0.05). In summary, supplementation with 100 ng/mL FGF2 during IVM significantly enhanced first polar body (PB1) extrusion, cumulus expansion, mitochondrial function, and MAPK pathway activation in porcine oocytes. These findings suggest that an optimal concentration of FGF2 improves IVM efficiency and early developmental competence, possibly through MAPK‑mediated regulation of oocyte maturation and enhanced gap junction communication between cumulus cells and the oocyte, thereby improving IVM quality.

[This retracts the article DOI: 10.1016/j.omtn.2021.03.004.].

Buffaloes are vital livestock in South-East Asia, attributed to their adaptation to hot and humid climates as well as their capacity to produce high-quality milk and meat. However, the texture of buffalo meat is suboptimal and its slow growth rate restricts the development of the buffalo farming industry. Consequently, studies exploring the key biochemical factors associated with buffalo muscle development have become a research focus. CircRNAs are a class of non-coding RNAs which can function as molecular sponges, participate in protein scaffold formation, and encode short peptides. Previous studies have shown that circRNAs are capable of regulating muscle development; however, relatively few reports have addressed their association with buffalo muscle development. In this study, data from Western blotting and RT-qPCR showed that circCOPS8 significantly enhanced the differentiation of buffalo myoblasts while inhibiting their proliferation (p < 0.05). In contrast, in a mouse model of muscular injury, circCOPS8 prevented the repair of injured muscles. Additionally, RIP-qPCR assays confirmed that circCOPS8 could bind to IGF2BP3 (p < 0.05). Furthermore RT-qPCR and transcriptome sequencing results revealed that circCOPS8 inhibited cell growth by upregulating the expression of genes such as ATR (p < 0.05). Our findings suggested that circCOPS8 promoted the differentiation and apoptosis of buffalo myoblasts while inhibiting their proliferation. The inhibition of cell proliferation was primarily mediated by the binding of circCOPS8 to IGF2BP3 and the promotion of ATR gene expression. This study investigated the role and underlying mechanism of circCOPS8 in buffalo myoblasts, which will extend our understanding of non-coding RNA-mediated regulation of buffalo muscle development, with the ultimate goal of improving the meat quality of buffaloes.

In vitro maturation (IVM) of oocytes is crucial in livestock breeding. Oocytes obtained by IVM are more susceptible to oxidative stress than in vivo, leading to low maturation rates. Betaine from red beetroot acts as an antioxidant and methyl donor, regulating epigenetic modifications in cell physiology. This study investigates Betaine's effects on porcine oocyte IVM, embryo development and underlying molecular mechanisms. Results demonstrate that 16 mmol/L Betaine significantly enhances the first polar body extrusion, cleavage and blastocyst rates compared to the control and other concentrations. Betaine elevates normal cortical granule distribution, normal spindle assembly, normal chromosome arrangement and overall m6A levels during IVM. It increases the antioxidant gene expression and mitochondrial function and decreases reactive oxygen species levels. However, Betaine's beneficial effects were diminished by AMPK inhibitor compound C. In conclusion, Betaine enhances porcine oocyte IVM and early embryo development by enhancing the antioxidant capacity and mitochondrial function pathway.

The content of unsaturated fatty acids (UFAs) is significantly associated with the flavor, taste, and nutritional value of beef. Adipose tissue (AT) develops in multiple depots, and the UFA content of AT varies among different depots. To elucidate the regulatory role of long non-coding RNAs (lncRNAs) in UFA synthesis across different AT depots in buffaloes, we conducted RNA sequencing (RNA-seq) on AT samples from six distinct depots. A total of 8926 lncRNAs were identified and 1363 of them were novel. The numbers of lncRNAs identified in different AT depots were similar. Through weighted gene co-expression network analysis (WGCNA), a module including 207 lncRNAs with a high correlation with UFA content was revealed. Functional enrichment analysis showed that these lncRNAs were significantly enriched in fat deposition and fatty acid metabolism. Notably, two lncRNAs (MSTRG.11229 and MSTRG.16994) were further identified. Both lncRNAs exhibited predominant expression in sternum subcutaneous AT (SSAT) and were upregulated during adipogenic differentiation in SSAT-derived adipocytes. What is more, the expressions of the two lncRNAs presented a high correlation to adipogenesis and UFA synthesis genes as well as UFA content. Collectively, this study provides a comprehensive atlas of lncRNA profiles across six AT depots and identifies two lncRNAs with high correlation with UFA content in buffaloes. These findings offer valuable insights and lncRNAs for the regulation of UFA synthesis in buffaloes.

Apoptosis of mammalian granulosa cells (GCs) is considered to be the main cause of follicular atresia. MiRNA-mediated post-transcriptional gene regulation plays a pivotal role in cell apoptosis. Our previous studies demonstrated that miR-424 is highly expressed in atretic follicles and may promoter of buffalo follicular atresia. In this study, we investigated the role and molecular mechanisms of miR-424 in buffalo follicular atresia. GCs apoptosis, proliferation and estrogen secretion were assessed by flow cytometry, EdU and ELISA, respectively. The expression levels of relevant genes were measured by qRT-PCR. Last, miR-424 agomir and antagomir was administered to mice via tail vein injection. The results showed that miR-424 promotes follicular atresia by inducing cell cycle arrest, inhibiting GC proliferation, suppressing the secretion of estradiol, and enhancing apoptosis. SERPINB2, as a target gene of miR-424, inhibits follicular atresia by promoting GCs proliferation, hormone secretion and inhibiting GCs apoptosis. Overexpression of miR-424 or silencing of SERPINB2 significantly reduced the number of follicles at various developmental stages in mice, whereas inhibition of miR-424 yielded opposite effects. In conclusion, our findings establish miR-424 as a key regulator of follicular atresia in buffalo, through direct targeting of SERPINB2 to modulate granulosa cell functions.

Background: While economically vital, buffalo exhibits low reproductive efficiency largely due to embryonic losses during implantation. Successful implantation requires precise embryo–maternal communication and metabolic/immune adaptations in the endometrium. We aimed to identify key serum metabolic signatures and associated peripheral immune responses that characterize the endometrial receptivity window during early pregnancy in water buffalo. Methods: Blood samples from pregnant (Preg, n = 12) and non-pregnant (Non-P, n = 10) buffaloes were collected on days 15, 18, and 21 post-artificial insemination (AI). We measured leukocyte counts and hormone levels and performed untargeted serum metabolomic profiling using LC-MS. Results: Pregnant buffaloes showed significantly reduced total white blood cell count, lymphocyte (LYM%), and neutrophil (NEU%) percentages, indicating immune remodeling at the beginning of pregnancy establishment. Metabolomic analysis identified 131 differentially expressed metabolites (DEMs) associated with pregnancy status at different stages. Enriched pathways included steroid hormone synthesis, retinol metabolism, starch/sucrose metabolism, and phenylalanine biosynthesis. Crucially, alterations in unsaturated fatty acids, retinol, and phenylalanine metabolism, along with monocyte (MON%)/LYM% ratios, were strongly linked to receptivity changes and successful implantation. Conclusions: Endometrial receptivity in buffalo during the embryonic implantation window was associated with changes in immune cells and metabolism in the blood, suggesting that immunometabolism may play an essential role in modulating endometrial receptivity during the implantation window. This study provides potential clues and a metabolic framework for understanding the underlying mechanisms of buffalo embryonic implantation.

Due to the limited in vitro culture duration, oocyte maturation efficiency in vitro is lower than in vivo. Nicotinamide mononucleotide (NMN), a precursor of NAD+ and a cofactor of Sirtuin deacetylases, exhibits significant antioxidant activity. This study aims to evaluate whether NMN supplementation combined with prolonging maturation time enhances cytoplasmic maturation and improves porcine oocyte quality. The blastocyst rate increased significantly after parthenogenetic activation when maturation time was extended from 44 h to 47 h or 50 h, peaking at 47 h. Prolonged maturation improved mitochondrial function and cortical granule distribution but induced ROS accumulation and apoptosis. Notably, supplementation with 100 μM NMN reversed these negative effects, significantly increasing the total number of blastocyst cells, reducing ROS levels, and upregulating cytoplasmic quality-related gene expression. In conclusion, extending maturation to 47 h with 100 μM NMN supplementation enhances oocyte maturation and embryonic developmental potential in vitro. These findings suggest that NMN, a naturally occurring nucleotide in food, may improve oocyte quality and offer insights for optimizing in vitro culture techniques as well as treating oocyte quality-related infertility.

MicroRNAs (miRNAs) are critical regulators of mammalian follicle development. Our previous work showed that miR-23a expression was significantly higher in buffalo oocytes at metaphase II (MII) than at the germinal vesicle (GV) stage, implying a role in oocyte maturation. Here, we investigated the function and mechanism of miR-23a in buffalo granulosa cells (GCs). GCs cultured in vitro were transfected with miR-23a mimics or inhibitor. The miR-23a mimic markedly increased apoptosis, reduced estradiol (E2) release, elevated progesterone (P4) secretion, and suppressed the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, whereas the inhibitor exerted the opposite effects. Dual-luciferase and rescue assays revealed that miR-23a directly targets CUG triplet repeat-binding protein 1 (CELF1). Overexpression of CELF1 reversed the pro-apoptotic effect of miR-23a, restored proliferation, normalized E2 and P4 production, and re-activated PI3K/AKT signaling. Thus, miR-23a targeted CELF1 to inhibit the PI3K/AKT pathway, promoting apoptosis, suppressing proliferation and modulating steroidogenesis in buffalo GCs, thereby controlling follicular homeostasis.

BACKGROUND:The intramuscular fat content and the level of unsaturated fatty acids influence meat quality and nutritional value. microRNAs can participate in lipid metabolic processes. Understanding how novel miRNAs regulate lipogenesis and fatty acid metabolism is key to enhancing buffalo meat quality and nutritional value. RESULTS:In this study, miRNA sequencing was conducted on 36 adipose tissues from six anatomical sites in river buffaloes (Murrah). A total of 1,682 miRNAs were identified in 36 adipose tissues, of which 987 were novel and 695 were known miRNAs. Weighted gene co-expression networks analysis (WGCNA) revealed that bta-novel-miR-25336 was highly expressed in sternum subcutaneous adipose tissue (SSAT) and had a high correlation with unsaturated fatty acids (UFA). Functional studies showed that bta-novel-miR-25336 was positively correlated with the proliferation and adipogenic differentiation of both buffalo preadipocytes and intramuscular adipocytes. Additionally, we used bioinformatics analysis and a 3' untranslated region (3'UTR) luciferase reporter assay to validate acyl-CoA dehydrogenase short chain (ACADS) as a target gene of bta-novel-miR-25336. CONCLUSIONS:In summary, bta-novel-miR-25336 may regulate biological processes associated with cell proliferation, lipid differentiation and unsaturated fatty acid synthesis through ACADS. This study provides a theoretical basis for further elucidating the regulation of lipid metabolism by novel miRNAs in farm animals.

Female fertility largely depends on ovarian function. Follicles serve as the basic unit of the ovary and provide the metabolic microenvironment for oocyte development. The metabolism of follicular fluid changes dynamically with follicular development and is affected by maternal metabolism, age, and breeding season. This paper aims to summarize the metabolic characteristics of follicular fluid in growing cohort follicles, ovulatory follicles, and cystic follicles and discuss changes in follicular fluid metabolism associated with maternal metabolic stress, maternal age, and breeding season. Results provide a systematic and clear understanding of the metabolic characteristics of bovine follicular development.

Super-enhancers (SEs) are critical epigenetic regulators of tissue regeneration, yet their interplay with cellular biomechanics during myogenic differentiation remains unexplored. Here, the TPM1 locus, encoding a core actin-stabilizing protein essential for skeletal muscle regeneration, harbors an evolutionarily conserved SE (TPM1_SE) that may bridge epigenetic control and mechanotransduction. In vitro, TPM1_SE deletion impaired myogenic differentiation and diminished expression of both TPM1 and its circular RNA (circRNA) isoform, CircTPM1. Conditional deletion of TPM1_SE significantly reduce muscle mass and delayed regenerative progression. Mechanistically, TPM1_SE drives expression of linear TPM1 mRNA (mice) and CircTPM1 (bovine) via TEAD4-mediated chromatin looping, coordinating cytoskeletal reorganization during myotube formation. These effects are mediated via activation of the canonical PI3K/AKT signaling pathway through interaction with NKX2.2-a pathway mechanosensitive to cellular tension. Loss of TPM1_SE disrupted NKX2.2-PI3K/AKT signaling. Crucially, CircTPM1 directly bound MYH10, enhancing MYL3-dependent actomyosin assembly, which potentiates cytoskeletal reorganization during myotube formation. Collectively, this findings establish TPM1_SE as an evolutionarily conserved hub integrating epigenetic regulation and biomechanical output. While the murine model underscores its therapeutic potential in muscle regenerative medicine, the bovine CircTPM1-mediated mechanism highlights TPM1_SE as a promising target for genetic improvement of meat quality in livestock.

The specific expression profile and function of circular RNA (circRNA) in follicular atresia remain largely unknown. Here, the circRNA expression profiles of granulosa cells derived from healthy follicles (HFs) and antral follicles (AFs) in buffalo were analyzed by RNA-seq, and the mechanism of a differentially expressed circRNA (DEcircRNA) circTEC regulating the granulosa cell function that affects follicular atresia was further explored. RNA-seq results showed that a total of 112 DEcircRNAs were identified. Among them, circTEC was highly expressed in HF, and its circular structure was confirmed by RNase R digestion assay, reversed PCR and Sanger sequencing. Functional experiments demonstrated that circTEC promotes the proliferation and steroid hormone synthesis of buffalo granulosa cells (bGCs), and it also inhibits their apoptosis. In-depth mechanism analysis showed that the expression level of circTEC in bGCs from AFs was adversely related to miR-144-5p and consistent with FZD3. CircTEC acts as an endogenous sponge of miR-144-5p to regulate the expression of the target gene FZD3 in AFs, which promotes the proliferation of bGCs and inhibits bGCs apoptosis, thereby inhibiting follicular atresia in buffalo. In summary, our study revealed the regulatory role of the circTEC/miR-144-5p/FZD3 axis during follicular atresia in buffalo. These results provided new insights into the biological mechanism underlying follicular atresia.

Semen cryopreservation is associated with sperm vulnerability to oxidative stress and ice crystal-induced damage, adversely affecting in vitro fertilization (IVF) success. This study aimed to investigate the effects of freezing diluent supplemented with antioxidant limonin (Lim), myo-inositol (MYO), and the ice crystal formation inhibitor L-proline (LP) through sperm motility, morphological integrity, and antioxidant capacity. The Lim (150 mM), MYO (90 mM), and LP (100 mM) significantly ameliorated the quality of post-thaw sperm in Debao boar, and combined treatment of these agents significantly enhanced sperm motility, structural integrity, and antioxidant capacity compared with individual agents (p < 0.05). Notably, the combined use of these agents reduced glycerol concentration in the freezing diluent from 3% to 2%. Meanwhile, the integrity of the sperm plasma membrane, acrosome membrane, and mitochondrial membrane potential was significantly improved (p < 0.05), and the result of IVF revealed the total cell count of the blastocysts was also greater in the 2% glycerol group (p < 0.05). In conclusion, the newly developed freezing diluent for semen, by adding Lim (150 mM), MYO (90 mM), and LP (100 mM), can enhance the quality of frozen–thawed Debao boar sperm and reduce the concentration of glycerol from 3% to 2% as high concentrations of glycerol can impair the quality of thawed sperm and affect in vitro fertilization outcomes. In conclusion, the improved dilution solution formulated demonstrated efficacy in enhancing the quality of porcine spermatozoa following cryopreservation and subsequent thawing.

Background: While economically vital, buffalo exhibits low reproductive efficiency largely due to embryonic losses during implantation. Successful implantation requires precise embryo-maternal communication and metabolic/immune adaptations in the endometrium. We aimed to identify key serum metabolic signatures and associated peripheral immune responses that characterize the endometrial receptivity window during early pregnancy in water buffalo. Methods: Blood samples from pregnant (Preg, n = 12) and non-pregnant (Non-P, n = 10) buffaloes were collected on days 15, 18, and 21 post-artificial insemination (AI). We measured leukocyte counts and hormone levels and performed untargeted serum metabolomic profiling using LC-MS. Results: Pregnant buffaloes showed significantly reduced total white blood cell count, lymphocyte (LYM%), and neutrophil (NEU%) percentages, indicating immune remodeling at the beginning of pregnancy establishment. Metabolomic analysis identified 131 differentially expressed metabolites (DEMs) associated with pregnancy status at different stages. Enriched pathways included steroid hormone synthesis, retinol metabolism, starch/sucrose metabolism, and phenylalanine biosynthesis. Crucially, alterations in unsaturated fatty acids, retinol, and phenylalanine metabolism, along with monocyte (MON%)/LYM% ratios, were strongly linked to receptivity changes and successful implantation. Conclusions: Endometrial receptivity in buffalo during the embryonic implantation window was associated with changes in immune cells and metabolism in the blood, suggesting that immunometabolism may play an essential role in modulating endometrial receptivity during the implantation window. This study provides potential clues and a metabolic framework for understanding the underlying mechanisms of buffalo embryonic implantation.

Background/Objectives: As a well-known source of energy from feed, the significance of fatty acids in regulating the reproductive potential of livestock has received attention in recent years, especially follicular development. Moreover, successful ovulation is a process that is crucial for reproduction and fertility in domestic animals. Therefore, it is important to reveal the signatures of fatty acids in follicular fluid during mammalian ovulation, and this provides a possible method to prevent the occurrence of ovarian cysts in domestic animals. Methods: Pre-ovulatory follicles (n = 6) and peri-ovulatory follicles (n = 6) during normal ovulation, as well as cystic follicles (n = 6) in ovulation-deficient ovarian cyst were isolated and characterized, while follicular fluid was collected for targeted fatty acid metabolomics detection and analysis. Results: We have illustrated the anatomical and biochemical characterization of pre-ovulatory, peri-ovulatory, and cystic follicles. Subsequently, we identified changes in 51 fatty acids profiles in the follicular fluid. The highest proportion of fatty acids in the follicular fluid at three different ovulation stages is polyunsaturated fatty acids, among which the abnormality of the linoleic acid metabolism pathway was involved in ovulation defects in cystic follicles. Remarkably, we found that linoleic acid was significantly increased while arachidonic acid was significantly decreased in cystic follicles. Conclusions: Polyunsaturated fatty acids play a significant role in the follicular ovulation stage of sows. Among them, linoleic acid and arachidonic acid are closely related to the ovulation defects of cystic follicles, which suggests that identifying changes in important metabolic signatures may give us a better understanding of the pathogenesis of ovarian cyst.

High motility spermatozoa are good for cryopreservation and artificial insemination (AI) of mammalian semen. In this study, normal motility (NM) and low motility (LM) Mediterranean buffalo spermatozoa were compared using quantitative proteomics and phosphoproteomics techniques to screen for important proteins and phosphorylated proteins related to the motility of spermatozoa and to identify candidate protein molecular markers related to the quality of Mediterranean buffalo semen. Proteomics results identified 2550 proteins, with 119 proteins upregulated and 146 proteins downregulated in the LM spermatozoa versus the NM spermatozoa. The differentially abundant proteins were mainly involved in carbohydrate metabolism, glycolysis/gluconeogenesis, and tricarboxylic acid cycles. The phosphoproteomics analysis revealed 412 proteins, 1228 phosphorylated peptides, and 1465 phosphorylation modification sites. Compared to the NM group, 119 peptides were downregulated in the LM group, corresponding to 98 proteins, and 84 phosphorylated peptides were upregulated in the white matter, corresponding to 61 proteins. Differentially phosphorylated proteins were primarily involved in spermatogenesis, flagellate sperm motility, and glycolysis/gluconeogenesis. The combined proteomics and phosphoproteomics results identified the common proteins HMGB4, POC1B, PKM, LDHA, TBC1D21, and CBY2, whose main roles were related to spermatogenesis, sperm flagellar structure, and energy metabolism, which can be used as potential markers of Mediterranean buffalo sperm quality.

An efficient promoter with specific transcriptional activity plays significant roles in the regulation of expression of exogenous genes. The efficient promoter specific to skeletal muscles can achieve high expression of exogenous genes in skeletal muscles. This is of great significance for the targeted improvement of livestock meat quality by combining gene editing and traditional breeding techniques. To identify efficient promoters specific to the skeletal muscles of buffalo, in the present study, a total of 14 genes, CACNG1, LRRC30, CACNG6, MYOG, VGLL2, MYOD1, KCNA7, DUPD1, PRR32, LBX1, IGFN1, ACTN3, PITX3, and MURC, were firstly screened as skeletal-muscle-specific expressed genes based on high-throughput sequencing data. Among them, only two genes - namely, VGLL2 and CACNG1 - were identified to be specifically and efficiently expressed in the skeletal muscles of buffalo by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Then, the transcriptional activity of different truncated fragments of the upstream putative promoter region of VGLL2 and CACNG1 were evaluated by the dual luciferase reporter gene detection system in mouse C2C12 cells and buffalo skeletal muscle cells. As a result, both core promoters of VGLL2 and CACNG1 were identified to have specifically and efficiently transcriptional activity in skeletal muscle tissue while the transcriptional activity of the core promoters of VGLL2 was more efficient. These results provide significant information for the targeted improvement of meat quality in buffaloes and other livestock animals.

The intramuscular fat content and the unsaturated fatty acid (UFA) composition are both critical indicators of buffalo meat quality. While microRNAs regulate fatty acid metabolism, their specific roles in buffaloes remain unclear. Our previous WGCNA identified bta-miR-30f as a hub miRNA positively correlated with UFA levels. In the present study, bta-miR-30f was found to be highly expressed in sternum subcutaneous adipose tissue and mature adipocytes. Functional studies indicated that bta-miR-30f increased lipid accumulation via enhanced adipogenesis and UFA levels, upregulating key genes including PPARG, C/EBPα, SCD, and FADS1/2. It also promoted cell proliferation. Mechanistically, the dual luciferase assay confirmed that bta-miR-30f interacted with RAD23B, whose knockdown similarly increased lipid deposition and UFA content in buffalo intramuscular preadipocytes. Thus, this study demonstrated that bta-miR-30f enhances adipogenesis differentiation and UFA accumulation by targeting RAD23B in buffalo intramuscular preadipocytes, which provides significant information for the genetic improvement of meat quality in buffaloes.