Immunoprotective efficacy of 3 Klebsiella pneumoniae type I fimbriae proteins in a murine model.

Klebsiella pneumoniae is a primary cause of clinical mastitis in dairy cows, with prevention being crucial, as treatments often fail due to antimicrobial resistance. Recent studies identified type I fimbrial antigens of K. pneumoniae as promising vaccine candidates, but there are limited research data. In this study, 3 fimbriae genes (fimA, fimC and fimG) were cloned and recombinantly expressed in Escherichia coli and their protective efficacy against K. pneumoniae evaluated in a mouse model. All 3 recombinant fimbriae proteins elicited strong humoral immune responses in mice, significantly increasing IgG, IgG1 and IgG2a. Notably, using a model of mice challenged with an intraperitoneal injection of bacteria, FimG significantly reduced bacterial loads in the spleen and lung, whereas FimA and FimC had limited protection for these organs. Either active or passive immunization with FimG produced substantial protective effects in mice challenged with K. pneumoniae LD100; in contrast, the mortality rate in the FimA-immunized group was similar to that of the control group, whereas FimC had weak protection. We concluded that the FimG recombinant protein vaccine had a favorable protective effect, with potential for immunization against K. pneumoniae mastitis.

Integrated Analysis of Transcriptome Profiles and lncRNA-miRNA-mRNA Competing Endogenous RNA Regulatory Network to Identify Biological Functional Effects of Genes and Pathways Associated with Johne's Disease in Dairy Cattle.

Paratuberculosis or Johne's disease (JD), a chronic granulomatous gastroenteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), causes huge economic losses and reduces animal welfare in dairy cattle herds worldwide. At present, molecular mechanisms and biological functions involved in immune responses to MAP infection of dairy cattle are not clearly understood. Our purpose was to integrate transcriptomic profiles and competing endogenous RNA (ceRNA) network analyses to identify key messenger RNAs (mRNAs) and regulatory RNAs involved in molecular regulation of peripheral blood mononuclear cells (PBMCs) for MAP infection in dairy cattle. In total, 28 lncRNAs, 42 miRNAs, and 370 mRNAs were identified by integrating gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In this regard, we identified 21 hub genes (CCL20, CCL5, CD40, CSF2, CXCL8, EIF2AK2, FOS, IL10, IL17A, IL1A, IL1B, IRF1, MX2, NFKB1, NFKBIA, PTGS2, SOCS3, TLR4, TNF, TNFAIP3, and VCAM1) involved in MAP infection. Furthermore, eight candidate subnets with eight lncRNAs, 29 miRNAs, and 237 mRNAs were detected through clustering analyses, whereas GO enrichment analysis of identified RNAs revealed 510, 22, and 11 significantly enriched GO terms related to MAP infection in biological process, molecular function, and cellular component categories, respectively. The main metabolic-signaling pathways related to MAP infection that were enriched included the immune system process, defense response, response to cytokine, leukocyte migration, regulation of T cell activation, defense response to bacterium, NOD-like receptor, B cell receptor, TNF, NF-kappa B, IL-17, and T cell receptor signaling pathways. Contributions of transcriptome profiles from MAP-positive and MAP-negative sample groups plus a ceRNA regulatory network underlying phenotypic differences in the intensity of pathogenicity of JD provided novel insights into molecular mechanisms associated with immune system responses to MAP infection in dairy cattle.

Rapid and reliable detection of Leishmania antibodies in canine serum with double-antigen sandwich homogeneous chemical luminescence.

BACKGROUND: Leishmaniasis, caused by Leishmania spp. parasites, is an important zoonotic disease globally, posing severe threats to humans and animals. In the absence of effective vaccines, reliable serological diagnostic methods are critical for disease control. However, the enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay have limitations due to complexity, time required and/or sensitivity. Therefore, our objective was to develop an accurate, rapid and user-friendly detection method of canine leishmania antibody based on double-antigen sandwich homogeneous chemical luminescence. METHODS: Homogeneous chemiluminescent technology was employed, and expressed recombinant fusion proteins containing full-length K9, K39 and K26 repeat sequences were used as diagnostic antigens. To establish a dual-antigen sandwich serological assay capable of detecting various antibody types, a factorial design was used to optimize concentrations of diagnostic antigen-receptor microspheres and of biotinylated diagnostic antigens, as well as of reaction solution composition and reaction duration. To evaluate and validate this newly developed method, we collected 41 Leishmania-positive serum samples, 30 Leishmania-negative control serum samples and 78 clinical serum samples for which no diagnostic information was available. Comparative analyses were performed using parasitological testing and an indirect ELISA as reference methods, focusing on diagnostic sensitivity and specificity. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the purification of the diagnostic antigens, which exhibited clear bands without impurities. Based on results from the 41 Leishmania-positive samples and 30 Leishmania-negative samples, there was sufficient sensitivity to detect samples diluted up to 256-fold, with analytical specificity of 100%. Overall diagnostic sensitivity was 100% and diagnostic specificity was 93.3%. Diagnostic performance was highly consistent between the newly developed method and the indirect ELISA (Kappa = 0.82, P < 0.01). Testing could be completed within 35 min with the new method CONCLUSIONS: We have developed a novel double-antigen sandwich homogeneous chemical luminescence method to detect canine Leishmania antibodies, with high sensitively and specificity, a short incubation interval and a simple protocol. This streamlined approach not only offers a sensitive and efficient method for clinical diagnosis but also has great potential for use in automated testing.

Herd-level prevalence of bovine leukemia virus, Salmonella Dublin, and Neospora caninum in Alberta, Canada, dairy herds using ELISA on bulk tank milk samples.

Endemic infectious diseases remain a major challenge for dairy producers worldwide. For effective disease control programs, up-to-date prevalence estimates are of utmost importance. The objective of this study was to estimate the herd-level prevalence of bovine leukemia virus (BLV), Salmonella enterica ssp. enterica serovar Dublin (Salmonella Dublin), and Neospora caninum in dairy herds in Alberta, Canada, using a serial cross-sectional study design. Bulk tank milk samples from all Alberta dairy farms were collected 4 times, in December 2021 (n = 489), April 2022 (n = 487), July 2022 (n = 487), and October 2022 (n = 480), and tested for antibodies against BLV, Salmonella Dublin, and N. caninum using ELISA. Herd-level apparent prevalence was calculated as positive herds divided by total tested herds at each time point. A mixed-effect modified Poisson regression model was employed to assess the association of prevalence with region, herd size, herd type, and type of milking system. Apparent prevalence of BLV was 89.4%, 88.7%, 86.9%, and 86.9% in December, April, July, and October, respectively, whereas for Salmonella Dublin apparent prevalence was 11.2%, 6.6%, 8.6%, and 8.5%, and for N. caninum apparent prevalence was 18.2%, 7.4%, 7.8%, and 15.0%. For BLV, Salmonella Dublin, and N. caninum, a total of 91.7%, 15.6%, and 28.1% of herds, respectively, were positive at least once, whereas 82.5%, 3.6%, and 3.0% of herds were ELISA positive at all 4 times. Compared with the north region, central Alberta had a high prevalence (prevalence ratio [PR] = 1.13) of BLV antibody-positive herds, whereas south Alberta had a high prevalence (PR = 2.56) of herds positive for Salmonella Dublin antibodies. Furthermore, central (PR = 0.52) and south regions (PR = 0.46) had low prevalence of N. caninum-positive herds compared with the north. Hutterite colony herds were more frequently BLV positive (PR = 1.13) but less frequently N. caninum-positive (PR = 0.47). Large herds (>7,200 L/d milk delivered ∼>250 cows) were 1.1 times more often BLV positive, whereas small herds (≤3,600 L/d milk delivered ∼≤125 cows) were 3.2 times more often N. caninum positive. For Salmonella Dublin, Hutterite colony herds were less frequently (PR = 0.07) positive than non-colony herds only in medium and large strata but not in small stratum. Moreover, larger herds were more frequently (PR = 2.20) Salmonella Dublin-positive than smaller herds only in non-colony stratum but not in colony stratum. Moreover, N. caninum prevalence was 1.6 times higher on farms with conventional milking systems compared with farms with an automated milking system. These results provide up-to-date information of the prevalence of these infections that will inform investigations of within-herd prevalence of these infections and help in devising evidence-based disease control strategies.

Escherichia coli infection induces ferroptosis in bovine mammary epithelial cells by activating the Wnt/ß-catenin pathway-mediated mitophagy.

Iron overload causes mitochondrial damage, and then activates mitophagy, which may directly trigger and amplify ferroptosis. Our objective was to investigate whether Escherichia coli (E. coli) isolated from clinical bovine mastitis induces ferroptosis in bovine mammary epithelial cells (bMECs) and if so, the underlying regulatory mechanism. E. coli infection caused mitochondrial damage, mitophagy, and ferroptosis. Rapamycin and chloroquine increased and suppressed ferroptosis, respectively, in E. coli-treated bMECs. Moreover, E. coli infection activated the Wnt/ß-catenin pathway, but foscenvivint alleviated it. In conclusion, E. coli infection induced ferroptosis through activation of the Wnt/ß-catenin pathway-promoted mitophagy, and it also suppressed GPX4 expression.

Biological and genomic characterization of 4 novel bacteriophages isolated from sewage or the environment using non-aureus Staphylococci strains.

Non-aureus staphylococci (NAS) are an essential group of bacteria causing antimicrobial resistant intramammary infections in livestock, particularly dairy cows. Therefore, bacteriophages emerge as a potent bactericidal agent for NAS mastitis. This study aimed to obtain NAS-specific bacteriophages using bacterial strains isolated from cows with mastitis, subsequently evaluating their morphological, genomic, and lytic characteristics. Four distinct NAS bacteriophages were recovered from sewage or the environment of Chinese dairy farms; PT1-1, PT94, and PT1-9 were isolated using Staphylococcus chromogenes and PT1-4 using Staphylococcus gallinarum. Both PT1-1 (24/54, 44 %) and PT94 (28/54, 52 %) had broader lysis than PT1-4 (3/54, 6 %) and PT1-9 (10/54, 19 %), but PT1-4 and PT1-9 achieved cross-species lysis. All bacteriophages had a short latency period and good environmental tolerance, including surviving at pH=4-10 and at 30-60℃. Except for PT1-9, all bacteriophages had excellent bactericidal efficacy within 5 h of co-culture with host bacteria in vitro at various multiplicity of infection (MOIs). Based on whole genome sequencing, average nucleotide identity (ANI) analysis of PT1-1 and PT94 can be classified as the same species, consistent with whole-genome synteny analysis. Although motifs shared by the 4 bacteriophages differed little from those of other bacteriophages, a phylogenetic tree based on functional proteins indicated their novelty. Moreover, based on whole genome comparisons, we inferred that cross-species lysis of bacteriophage may be related to the presence of "phage tail fiber." In conclusion 4 novel NAS bacteriophages were isolated; they had good biological properties and unique genomes, with potential for NAS mastitis therapy.

Heme oxygenase activates calcium release from the endoplasmic reticulum of bovine mammary epithelial cells to promote TFEB entry into the nucleus to reduce the intracellular load of Mycoplasma bovis.

Heme oxygenase HO-1 (HMOX) regulates cellular inflammation and apoptosis, but its role in regulation of autophagy in Mycoplasma bovis infection is unknown. The objective was to determine how the HO-1/CO- Protein kinase RNA-like endoplasmic reticulum kinase (PERK)-Ca2+- transcription factor EB (TFEB) signaling axis induces autophagy and regulates clearance of M. bovis by bovine mammary epithelial cells (bMECs). M. bovis inhibited autophagy and lysosomal biogenesis in bMECs and suppressed HO-1 protein and expression of related proteins, namely nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (keap1). Activation of HO-1 and its production of carbon monoxide (CO) were required for induction of autophagy and clearance of intracellular M. bovis. Furthermore, when HO-1 was deficient, CO sustained cellular autophagy. HO-1 activation increased intracellular calcium (Ca2+) and cytosolic localization activity of TFEB via PERK. Knockdown of PERK or chelation of intracellular Ca2+ inhibited HO-1-induced M. bovis autophagy and clearance. M. bovis infection affected nuclear localization of lysosomal TFEB in the MiT/TFE transcription factor subfamily, whereas activation of HO-1 mediated dephosphorylation and intranuclear localization of TFEB, promoting autophagy, lysosomal biogenesis and autophagic clearance of M. bovis. Nuclear translocation of TFEB in HO-1 was critical to induce M. bovis transport and survival of infected bMECs. Furthermore, the HO-1/CO-PERK-Ca2+-TFEB signaling axis induced autophagy and M. bovis clearance, providing a viable approach to treat persistent M. bovis infections.

Acute post-orchiectomy pain does not reduce alpha rams' interest in feed resources.

Sheep pain is an animal welfare issue monitored based on behavioral responses, including appetite. Dominant (alpha) males have priority for accessing limited feed resources, however, the effects of pain on feed interest in members of a group with defined social hierarchy are unknown. Our objective was to investigate effects of acute post-orchiectomy pain on alpha rams' interest in accessing a limited feed resource. Eighteen rams were randomly housed in pens of 3 rams. After acclimation, the first 5-d (consecutive) battery of a behavior test was performed. In this test, 180 g of the regular diet concentrate was placed in a portable trough in the center of the pen; this feed was supplemental to the diet and represented a limited, albeit strongly preferable feed resource. Rams were filmed for 5 min after the feed introduction. Hierarchical levels (alpha, beta, and gamma) were defined based on the social hierarchical index according to higher initiator and lower receptor agonistic behaviors from the social network analyses. After 15 d, a second 5-d behavioral test battery was repeated. On the following day, alpha rams were castrated. Flunixin meglumine was given immediately before surgery and a final behavioral test was performed 8 h post-orchiectomy, concurrent with an expected peak in postoperative pain. For all recordings, the latency, frequency, and duration of time that each ram had its mouth inside the feed trough were recorded, and the Unesp-Botucatu sheep acute pain scale pain scale (USAPS) was applied. The social hierarchical index was highest in alpha rams, followed by beta and gamma. The pain scores were statistically equivalent across the 11 evaluation days for beta and gamma rams, whereas there was an increase in the final evaluation for alpha. There was no difference in latency, frequency, and duration between alpha, beta, and gamma rams across evaluations. We concluded that acute post-orchiectomy pain did not decrease alpha rams' interest in accessing limited feed. Routine feeding offers a valuable chance to detect pain-related behavior using the USAPS in rams. However, dominance may confound appetite-related behaviors in assessing acute pain, as alpha rams' interest in limited feed remained unaffected by the pain.

Antioxidative Sirt1 and the Keap1-Nrf2 Signaling Pathway Impair Inflammation and Positively Regulate Autophagy in Murine Mammary Epithelial Cells or Mammary Glands Infected with Streptococcus uberis.

Streptococcus uberis mastitis in cattle infects mammary epithelial cells. Although oxidative responses often remove intracellular microbes, S. uberis survives, but the mechanisms are not well understood. Herein, we aimed to elucidate antioxidative mechanisms during pathogenesis of S. uberis after isolation from clinical bovine mastitis milk samples. S. uberis's in vitro pathomorphology, oxidative stress biological activities, transcription of antioxidative factors, inflammatory response cytokines, autophagosome and autophagy functions were evaluated, and in vivo S. uberis was injected into the fourth mammary gland nipple of each mouse to assess the infectiousness of S. uberis potential molecular mechanisms. The results showed that infection with S. uberis induced early oxidative stress and increased reactive oxygen species (ROS). However, over time, ROS concentrations decreased due to increased antioxidative activity, including total superoxide dismutase (T-SOD) and malondialdehyde (MDA) enzymes, plus transcription of antioxidative factors (Sirt1, Keap1, Nrf2, HO-1). Treatment with a ROS scavenger (N-acetyl cysteine, NAC) before infection with S. uberis reduced antioxidative responses and the inflammatory response, including the cytokines IL-6 and TNF-α, and the formation of the Atg5-LC3II/LC3I autophagosome. Synthesis of antioxidants determined autophagy functions, with Sirt1/Nrf2 activating autophagy in the presence of S. uberis. This study demonstrated the evasive mechanisms of S. uberis in mastitis, including suppressing inflammatory and ROS defenses by stimulating antioxidative pathways.

Comparative genomic analyses of Klebsiella pneumoniae K57 capsule serotypes isolated from bovine mastitis in China.

Klebsiella pneumoniae can cause severe clinical mastitis in dairy cows, with K. pneumoniae type K57 (K57-KP) being the most common capsular serotype. To identify virulence factors and antimicrobial-resistance (AMR) genes of K57-KP with varying virulence, Galleria mellonella (greater wax moth) larvae were infected as a screening model to characterize virulence of 90 K57-KP strains, with 10 and 11 strains defined as virulent or attenuated, respectively, based on larval survival rates. Next, virulence of these 21 isolates was subsequently confirmed in adhesion and lactate dehydrogenase release assays, using bovine mammary epithelial cells cultured in vitro. Finally, genes associated with virulence and AMR were characterize with whole-genome sequencing. These 21 K57-KP strains were designated into 16 sequence types based on multi-locus sequence typing and allocated in phylogenetic analysis based on single nucleotide polymorphisms. We found great genetic diversity among isolates. In addition, adhesion-associated genes (e.g., fimA, sfaA, and focA) aminoglycoside-resistance genes (aph(6)-Id, strAB) were associated with virulence. This study provided new knowledge regarding virulence of K57-KP associated with bovine mastitis, which may inform development of novel diagnostic tools and prevention strategies for bovine mastitis.

Optimized total RNA isolation from bovine sperm with enhanced sperm head lysis.

Increasing evidence of sperm RNA's role in fertilization and embryonic development has provided impetus for its isolation and thorough characterization. Sperm are considered tough-to-lyse cells due to the compact condensed DNA in sperm heads. Lack of consensus among bovine sperm RNA isolation protocols introduces experimental variability in transcriptome studies. Here, we describe an optimized method for total RNA isolation from bovine sperm using the TRIzol reagent. This study critically investigated the effects of various lysis conditions on sperm RNA isolation. Sperm suspended in TRIzol were subjected to a combination of mechanical treatments (sonication and passage through a 30G needle and syringe) and chemical treatments (supplementation with reducing agents 1,4-dithiothreitol and tris(2-carboxyethyl) phosphine hydrochloride (TCEP)). Microscopic evaluation of sperm lysis confirmed preferential sperm tail versus sperm head lysis. Interestingly, only TCEP-supplemented TRIzol (both mechanical treatments) had progressive sperm head lysis and consistently yielded total sperm RNA. Furthermore, RNA integrity was confirmed based on the electrophoresis profile and an absence of genomic DNA and somatic cells (e.g., epithelial cells, spermatids, etc.) with RT-qPCR. Our findings highlighted the importance of sperm lysis, specifically of the sperm head using TCEP with mechanical treatment, in total RNA isolation and presented a bovine-specific sperm RNA isolation method to reduce experimental variabilities.

A soy lecithin nanoparticles-based extender effectively cryopreserves Holstein bull sperm.

Plant-based semen extenders, typically derived from soybean lecithin, are easier to modulate more and consistent in their composition than animal-based extenders. As large lecithin particles can, however, reduce effectiveness and solubility in bull semen extenders, sonication was used to create nano-lecithin (NL) particles of soybean lecithin. The objective was to determine the effects of lecithin type and concentration on the quality of frozen-thawed bovine sperm. We hypothesized that reducing the size of lecithin improves its interactions with the sperm and enhances the parameters that favor its motility, viability and fertility. Semen was collected from six mature Holstein bulls and ejaculates meeting minimum standards were pooled. Eight Tris-based extenders that contained 1, 2, 3, or 4 % of either conventional lecithin (L1-L4) or NL (NL1-NL4), plus two control extenders (one animal-based extender containing 20 % egg yolk [EY] and a commercial lecithin-based extender [BioXcell®]) were compared. Among soybean lecithin-based extenders, NL3 had the highest total and progressive sperm motility, and average path, straight-line and curvilinear sperm velocity, and was comparable to EY. Additionally, sperm mitochondrial activity was the highest in NL3, whereas sperm viability was highest in EY, NL3, and L4. Following in vitro fertilization of in vitro-matured bovine oocyes, NL3 had cleavage and hatching rates comparable to BioXcell®, but a lower blastocyst rate than EY. Overall, NL3 performed better than the other extenders for most end points, with efficiency comparable to EY. We, therefore, concluded that reducing lecithin particle size to a nano level improves sperm cryopreservation with optimal performance with 3 % NL.

MicroRNA miR-223 modulates NLRP3 and Keap1, mitigating lipopolysaccharide-induced inflammation and oxidative stress in bovine mammary epithelial cells and murine mammary glands.

Bovine mastitis, the most prevalent and costly disease in dairy cows worldwide, decreases milk quality and quantity, and increases cow culling. However, involvement of microRNAs (miRNAs) in mastitis is not well characterized. The objective was to determine the role of microRNA-223 (miR-223) in regulation of the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and kelch like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) oxidative stress pathway in mastitis models induced by lipopolysaccharide (LPS) treatment of immortalized bovine mammary epithelial cells (bMECs) and murine mammary glands. In bMECs cultured in vitro, LPS-induced inflammation downregulated bta-miR-223; the latter interacted directly with the 3' untranslated region (3' UTR) of NLRP3 and Keap1. Overexpression of bta-miR-223 in bMECs decreased LPS and Adenosine 5'-triphosphate (ATP)-induced NLRP3 and its mediation of caspase 1 and IL-1ß, and inhibited LPS-induced Keap1 and Nrf2 mediated oxidative stress, whereas inhibition of bta-miR-223 had opposite effects. In an in vivo murine model of LPS-induced mastitis, increased miR-223 mitigated pathology in the murine mammary gland, whereas decreased miR-223 increased inflammatory changes and oxidative stress. In conclusion, bta-miR-223 mitigated inflammation and oxidative injury by downregulating the NLRP3 inflammasome and Keap1/Nrf2 signaling pathway. This study implicated bta-miR-223 in regulation of inflammatory responses, with potential as a novel target for treating bovine mastitis and other diseases.

Integrated Comparative Transcriptome and circRNA-lncRNA-miRNA-mRNA ceRNA Regulatory Network Analyses Identify Molecular Mechanisms Associated with Intramuscular Fat Content in Beef Cattle.

Intramuscular fat content (IMF), one of the most important carcass traits in beef cattle, is controlled by complex regulatory factors. At present, molecular mechanisms involved in regulating IMF and fat metabolism in beef cattle are not well understood. Our objective was to integrate comparative transcriptomic and competing endogenous RNA (ceRNA) network analyses to identify candidate messenger RNAs (mRNAs) and regulatory RNAs involved in molecular regulation of longissimus dorsi muscle (LDM) tissue for IMF and fat metabolism of 5 beef cattle breeds (Angus, Chinese Simmental, Luxi, Nanyang, and Shandong Black). In total, 34 circRNAs, 57 lncRNAs, 15 miRNAs, and 374 mRNAs were identified by integrating gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Furthermore, 7 key subnets with 16 circRNAs, 43 lncRNAs, 7 miRNAs, and 237 mRNAs were detected through clustering analyses, whereas GO enrichment analysis of identified RNAs revealed 48, 13, and 28 significantly enriched GO terms related to IMF in biological process, molecular function, and cellular component categories, respectively. The main metabolic-signaling pathways associated with IMF and fat metabolism that were enriched included metabolic, calcium, cGMP-PKG, thyroid hormone, and oxytocin signaling pathways. Moreover, MCU, CYB5R1, and BAG3 genes were common among the 10 comparative groups defined as important candidate marker genes for fat metabolism in beef cattle. Contributions of transcriptome profiles from various beef breeds and a competing endogenous RNA (ceRNA) regulatory network underlying phenotypic differences in IMF provided novel insights into molecular mechanisms associated with meat quality.

Construction of a circRNA- lincRNA-lncRNA-miRNA-mRNA ceRNA regulatory network identifies genes and pathways linked to goat fertility.

Background: There is growing interest in the genetic improvement of fertility traits in female goats. With high-throughput genotyping, single-cell RNA sequencing (scRNA-seq) is a powerful tool for measuring gene expression profiles. The primary objective was to investigate comparative transcriptome profiling of granulosa cells (GCs) of high- and low-fertility goats, using scRNA-seq. Methods: Thirty samples from Ji'ning Gray goats (n = 15 for high fertility and n = 15 for low fertility) were retrieved from publicly available scRNA-seq data. Functional enrichment analysis and a literature mining approach were applied to explore modules and hub genes related to fertility. Then, interactions between types of RNAs identified were predicted, and the ceRNA regulatory network was constructed by integrating these interactions with other gene regulatory networks (GRNs). Results and discussion: Comparative transcriptomics-related analyses identified 150 differentially expressed genes (DEGs) between high- and low-fertility groups, based on the fold change (≥5 and ≤-5) and false discovery rate (FDR <0.05). Among these genes, 80 were upregulated and 70 were downregulated. In addition, 81 mRNAs, 58 circRNAs, 8 lincRNAs, 19 lncRNAs, and 55 miRNAs were identified by literature mining. Furthermore, we identified 18 hub genes (SMAD1, SMAD2, SMAD3, SMAD4, TIMP1, ERBB2, BMP15, TGFB1, MAPK3, CTNNB1, BMPR2, AMHR2, TGFBR2, BMP4, ESR1, BMPR1B, AR, and TGFB2) involved in goat fertility. Identified biological networks and modules were mainly associated with ovary signature pathways. In addition, KEGG enrichment analysis identified regulating pluripotency of stem cells, cytokine-cytokine receptor interactions, ovarian steroidogenesis, oocyte meiosis, progesterone-mediated oocyte maturation, parathyroid and growth hormone synthesis, cortisol synthesis and secretion, and signaling pathways for prolactin, TGF-beta, Hippo, MAPK, PI3K-Akt, and FoxO. Functional annotation of identified DEGs implicated important biological pathways. These findings provided insights into the genetic basis of fertility in female goats and are an impetus to elucidate molecular ceRNA regulatory networks and functions of DEGs underlying ovarian follicular development.