Semen Protein CRISP3 Promotes Reproductive Performance of Boars through Immunomodulation.

Semen proteins play an important role in male reproductive performance and sperm fertilization ability and can be used as potential biomarkers to evaluate male fertility. The role of cysteine-rich secretory protein 3 (CRISP3) in male reproduction remains unknown. This study aimed to investigate the role of CRISP3 in the reproductive performance of boars. Our results showed that the CRISP3 protein content was significantly and positively correlated with boar fertility, sow delivery rate, and litter size. CRISP3 is highly expressed in the bulbourethral gland of adult boars and is enriched in the seminal plasma. It is localized in the post-acrosomal region of the sperm head and migrates to the anterior end of the tail after capacitation. The CRISP3 recombinant protein did not affect sperm motility and cleavage rate, but it significantly downregulated the mRNA expression of inflammatory factors IL-α, IL-1ß, and IL-6 and the protein expression of IL-α and IL-6 in lipopolysaccharide (LPS)-induced RAW264.7 cells, indicating that CRISP3 has an immunomodulatory function. In conclusion, our study suggests that semen CRISP3 protein levels positively correlate with reproductive performance, which may be achieved by regulating immune responses in the female reproductive tract.

Glutathione S-transferase kappa 1 is positively related with sperm quality of porcine sperm.

The glutathione S-transferase (GST) superfamily members play an important role in the male reproductive tract and sperm physiology. However, the expression profiles of some members of this protein family and their effect on sperm quality remain unclear. In this study, we found that GST kappa 1 (GSTK1) encoded protein is abundant in the testes and capacitated sperm acrosome. Western blot analysis revealed that the decreased abundance of GSTK1 was observed in low motile spermatozoa; moreover, GSTK1 expression decreased in sperm stored at 17°C under a long preservation time. In vitro analyses revealed that GSTK1 had no significant effect on sperm motility, capacitation, or acrosome reaction. Notably, after capacitated sperm were incubated with 4 and 8 µg/ml anti-GSTK1 antibodies, the fertilization rate significantly decreased in vitro fertilization assay. The current study demonstrates that GSTK1 is correlated with sperm quality and is a promising marker for the assessment of sperm quality and provides a basis for understanding the potential molecular mechanism for targeting pathogenic factors in male infertility.

Involvement of Porcine ß-Defensin 129 in Sperm Capacitation and Rescue of Poor Sperm in Genital Tract Infection.

In mammals, ß-defensins have been reported to play pivotal roles in sperm protection and fertilization. However, the function and mechanism of porcine ß-defensin 129 (pBD129) in the sperm remain unclear. Here, we demonstrate that pBD129 is a glycosylated protein and broadly exists in accessory sex glands and coats the sperm surface. We inhibited the pBD129 protein on the sperm surface with an anti-pBD129 antibody and found that sperm motility was not significantly affected; however, sperm acrosome integrity and tyrosine phosphorylation levels increased significantly with time (p < 0.05) during capacitation. These changes were accompanied by an increase in sperm Ca2+ influx, resulting in a significantly reduced in vitro fertilization cleavage rate (p < 0.05). Further investigation revealed that treatment with recombinant pBD129 markedly restored the sperm motility in semen contaminated with Escherichia coli. The results suggest that pBD129 is not only associated with poor sperm motility after genital tract infection but can also protect the spermatozoa from premature capacitation, which may be beneficial for semen preservation.

Characterization of Long Non-Coding RNA Profiles in Porcine Granulosa Cells of Healthy and Atretic Antral Follicles: Implications for a Potential Role in Apoptosis.

Long non-coding RNAs (lncRNAs) play important roles in multiple biological processes including ovarian follicular development. Here we aimed to gain novel information regarding lncRNAs transcriptome profiles in porcine granulosa cells of advanced atretic antral (AA) and healthy antral (HA) follicles using RNA-seq. A total of 11,321 lncRNAs including 10,813 novel and 508 annotated lncRNAs were identified, of which 173 lncRNAs were differentially expressed (DE-lncRNAs); ten of these were confirmed by qRT-PCR. Gene Ontology indicated that DE-lncRNAs associated with developmental processes were highly enriched. Pathway analysis demonstrated predicted cis- and trans-targets of DE-lncRNAs. Potential mRNA targets of up-regulated DE-lncRNAs were mainly enriched in apoptosis related pathways, while targeted genes of downregulated DE-lncRNAs were primarily enriched in metabolism and ovarian steroidogenesis pathways. Linear regression analyses showed that expression of upregulated DE-lncRNAs was significantly associated with apoptosis related genes. NOVEL_00001850 is the most-downregulated DE-lncRNA (FDR = 0.04, FC = -6.53), of which miRNA binding sites were predicted. KEGG analysis of its downregulated target genes revealed that ovarian steroidogenesis was the second most highlighted pathway. qRT-PCR and linear regression analysis confirmed the expression and correlation of its potential targeted gene, CYP19A1, a key gene involved in estradiol synthesis. Our results indicate that lncRNAs may participate in granulosa cells apoptosis and thus antral follicular atresia.

[Heat shock protein 90 in male reproduction].

Heat shock protein 90 (Hsp90), as a member of the Hsp family and widely found in the gonads of humans and animals, plays an essential role in male reproduction and induces various changes in the process of reproduction. Hsp90 regulates the division of germ cells and participates in spermatogenesis, location of germ cells, formation of sperm microtubes, protection of sperm from oxidative stress, and inducement of acrosomal reaction. Studies showed significant changes in location and expression of Hsp90 in the sperm of oligospermia and asthenospermia patients. This paper presents an overview of Hsp90 in male reproduction and male infertility and a prospect of the treatment of male infertility.

Development of a real-time reverse transcription recombinase polymerase amplification assay for rapid detection of spring viremia of carp virus.

Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.

Application of recombinase polymerase amplification method for rapid detection of infectious laryngotracheitis virus.

Infectious laryngotracheitis is a significant respiratory disease of chickens that causes huge economic losses due to high morbidity and mortality and reduced egg production. A real-time recombinase polymerase amplification (RPA) assay was developed to accurately detect ILTV. The specific probe and primer sets were carefully designed and screened. The real-time RPA assay was carried out at 39 °C for 30 min, and results were obtained within 15 min. The results of the specificity assay showed no fluorescence signals with other avian-related viruses. The sensitivity of the assay was 1 × 102 copies/µL. The low CV value showed that the assay was reproducible. A total of 115 clinical samples were tested using the real-time RPA assay and the real-time PCR assay in parallel; the coincidence rates of the two detection methods were 100%. The results indicated that the real-time RPA assay is a specific, sensitive, rapid, and useful tool for epidemiological studies and clinical diagnosis, especially in the field and in resource-poor areas.

Characteristics of Circular RNA Expression Profiles of Porcine Granulosa Cells in Healthy and Atretic Antral Follicles.

Circular RNAs (circRNAs) are thought to play essential roles in multiple biological processes, including apoptosis, an important process in antral follicle atresia. We aimed to investigate the potential involvement of circRNAs in granulosa cell apoptosis and thus antral follicle atresia. CircRNA expression profiles were generated from porcine granulosa cells isolated from healthy antral (HA) and atretic antral (AA) follicles. Over 9632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). Back-splicing, RNase R resistance, and stability of DE-circRNAs were validated, and miRNA binding sites and related target genes were predicted. Two exonic circRNAs with low false discovery rate (FDR) high fold change, miRNA binding sites, and relevant biological functions-circ_CBFA2T2 and circ_KIF16B-were selected for further characterization. qRT-PCR and linear regression analysis confirmed expression and correlation of the targeted genes-the antioxidant gene GCLC (potential target of circ_CBFA2T2) and the apoptotic gene TP53 (potential target of circ_KIF16B). Increased mRNA content of TP53 in granulosa cells of AA follicles was further confirmed by strong immunostaining of both p53 and its downstream target pleckstrin homology like domain family a member 3 (PHLDA3) in AA follicles compared to negligible staining in granulosa cells of HA follicles. Therefore, we concluded that aberrantly expressed circRNAs presumably play a potential role in antral follicular atresia.

[Role of CYCS in the cytogenesis and apoptosis of male germ cells and its clinical application].

In order to maintain the stability of intracellular environment, various cells die autonomously in vivo, which is called apoptosis. As a key factor of cell apoptosis in the mitochondrial pathway, cytochrome c, somatic (CYCS) plays an important role in the process of male spermatogenesis. Based on more than 40 recent studies on CYCS by Chinese and foreign scholars, this review focuses on the involvement of CYCS in the mechanisms of the apoptosis and anti-apoptosis of germ cells and in the regulation of their quality and activity and also outlines the role of CYCS in marking male reproductive performance as well as in male infertility and its underlying mechanisms.

Rapid and sensitive real-time recombinase polymerase amplification for detection of Marek's disease virus.

Marek's disease (MD) is one of the most devastating diseases of poultry. It's caused by the highly infectious alphaherpesvirus MD virus serotype 1 (MDV-1). In this study, a rapid and easy-to-use assay based on recombinase polymerase amplification (RPA) was developed for MDV detection. Primer-probe sets targeting the highly conserved region of Meq gene were designed and applied to the RPA assay. The assay was carried out on a real-time thermostatic fluorescence detector at 39 °C for 20 min. As revealed by the results, no cross-reactions were found with the Newcastle disease virus (NDV), chicken infectious anemia virus (CAV), infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avain influenza virus (AIV), avian leucosis virus (ALV), avian reovirus (ARV), Marek's disease virus serotype 2 (MDV-2) and turkey herpes virus (HVT), indicating appropriate specificity of the assay. Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 102copies/µL. To further evaluate the clinical performance, 94 clinical samples were subjected to the RPA assay and 28 samples were tested MDV positive, suggesting that the real-time RPA assay was sufficient enough for clinical sample detection. Thus, a highly specific and sensitive real-time RPA assay was established and validated as a candidate for MDV diagnosis. Additionally, the portability of real-time RPA assay makes it suitable to be potentially applied in clinical diagnosis in the field, especially in resource-limited settings.

Synthesis, Photothermal Effect and Cytotoxicity of Fe3O4@Au Nanocomposites.

It is currently a very active research area to develop multifunctional nanocomposites (NCs) which integrate the novel properties from various nanomaterials for multimodal imaging and simultaneous therapy. These theranostic nanoplatforms can provide complementary information from each imaging modality for accurate diagnosis and, at the same time, afford an imaging-guided focused tumor therapy. Among them, core/shell Fe3O4@Au NCs have attracted wide attention due to their unique advantages in magnetic targeting, multimodal imaging and photothermal therapy. This study developed a layer-by-layer assembling approach to synthesize Fe3O4@Au NCs with high photothermal conversion efficiency. The as-synthesized NCs showed significant photothermal ablation capability to HeLa cells in vitro under near infrared laser irradiation. To ensure the safety for medical applications, the bio-effects of Fe3O4@Au NCs on RAW264.7 cells were carefully assessed, in terms of cell viability, oxidative stress and apoptosis. We have demonstrated that Fe3O4@Au NCs had good biocompatibility in RAW264.7 cells and no significant cytotoxicity was found. Therefore, the Fe3O4@Au NCs synthesized in this study have great potential as an ideal candidate for CT/MR imaging and photothermal therapy.

Development and imprinted gene expression in uniparental preimplantation mouse embryos in vitro.

Increasing numbers of reports show that imprinted genes play a crucial role in fetal development, and uniparental embryos, which possess two paternally or two maternally derived pronuclei, are excellent tools for investigating the biological significance of imprinted genes. In the present study, to examine the in vitro developmental ability and expression pattern of eight imprinted genes in uniparental embryos, we produced androgenones, gynogenones, and parthenogenones using enucleation. Our data confirmed the previously observed restriction in haploid androgenetic development potential and first indicated that diploid androgenetic embryos were arrested in the 3/4-cell stage. Some imprinted genes were expressed in androgenetic, gynogenetic, and parthenogenetic blastocysts, suggesting that they were unable to maintain their imprinted expression status in uniparental embryos and that both paternal and maternal alleles are required for the specific expression of some imprinted genes.

[The progress of sperm functional proteins regulating the process of fertilization].

The process of mammalian fertilization involves a series of sperm functional activities, including the sperm transportation, hyperactivation and capacitation, acrosome reaction and sperm-egg fusion, etc. The semen proteins play indispensable roles in these processes, and they are closely associated with the fecundity of males. So these proteins can be biomarkers to evaluate the fertilization capacity of mammalian semen. In this review, we mainly introduce some semen proteins relevant to spermatozoa functions and illustrate their important regulatory roles on the fertilization processes, involving spermatozoa motility, capacitation, acrosome reaction, penetrating the zona pellucida and sperm-egg fusion, etc. Moreover, their potential applications in the evaluation of heredity in livestock are also summarized.

Potential contribution of alpha-fetoprotein level to biomarker of pregnancy outcome in Asian elephants.

Alpha-fetoprotein (AFP) is a structural serum glycoprotein that plays vital roles in reproduction and mammalian development. Analysis of serum prolactin (PRL) is considered one of the useful methods for diagnosing pregnancy in Asian elephants. However, the expression profiles of AFP in pregnant and nonpregnant Asian elephants remain unclear, nor is the relationship with PRL. In this study, serum seven gonadal hormones and AFP in three pregnant and seven nonpregnant Asian elephants were analysed by via radioimmunoassay (RIA) and enzyme-linked immunosorbent (ELISA) assay. We found that the mean (±SD) concentration of prolactin (PRL) in pregnant (136.782 ± 30.987 ng/mL) elephants was significantly higher than that in nonpregnant elephants (52.803 ± 21.070 ng/mL; p ≤ 0.0005). The mean (±SD) concentration of AFP in pregnant elephants (11.598 ± 0.824 ng/mL) was significantly higher than that in nonpregnant elephants (7.200 ± 2.283 ng/mL; p ≤ 0.05). Furthermore, the AFP concentration was positively correlated with the PRL concentration in the 10 Asian elephants studied. In conclusion, our findings suggest that serum AFP concentration is a potential biomarker of pregnancy outcomes in Asian elephants.

PF-05231023 reduces lipid deposition in apolipoprotein E-deficient mice by inhibiting the expression of lipid synthesis genes.

Fibroblast growth factor 21 (FGF21) is a peptide hormone that is primarily expressed and secreted by the liver. The hormone is crucial for regulation of glucose homeostasis, lipid metabolism, and energy balance. Compared with natural FGF21, FGF21 analogs have become drug candidates for the treatment of cardiovascular and metabolic diseases owing to their long half-life and greater stability in vitro. Apolipoprotein E (Apoe)-knockout (Apoe -/-) mice exhibit progressive disruptions in lipid metabolism in vivo and develop further atherosclerosis pathological features owing to Apoe deletion. Therefore, this study used an Apoe -/- mouse model to investigate the effects of a long-acting FGF21 analog (PF-05231023) on lipid metabolism and related parameters. Eighteen Apoe -/- female mice were fed a Western diet equivalent for 12 weeks, and then randomly assigned to intraperitoneally receive either physiological saline (the control group) or 10 mg/kg PF-05231023 (the treatment group) three times a week for seven consecutive weeks. Body composition, glucose tolerance, blood and liver cholesterol, triglyceride levels, liver vacuolization levels, peri-ovarian white adipocyte hypertrophy, aortic atherosclerotic plaque formation, and the expression of genes related to lipid metabolism in adipose tissue were subsequently assessed before and after treatment. The aortic atherosclerotic plaque area was reduced in mice in the PF-05231023 treatment group compared with that in the saline group. Although the effect of PF-05231023 on the plasma biochemical indexes of mice was small, it significantly reduced lipid levels and lipid droplet accumulation in the liver, and reduced adipocyte hypertrophy in white adipose tissue. Transcriptome analysis of adipose tissue showed that PF-05231023 treatment downregulated the expression of lipid synthesis-related genes and inhibited the sterol regulatory element binding transcription factor 1 gene, thereby improving lipid deposition. PF-05231023 effectively improved the lipid metabolism of Apoe -/- mice, demonstrating an anti-atherosclerotic effect and providing a scientific basis and experimental foundation for the clinical treatment of cardiovascular diseases by using long-acting FGF21 analogs.

Recombinase-aid amplification combined with lateral flow detection assay for sex identification of the great white pelican (Pelecanus onocrotalus).

Sex identification in avian species is essential for biodiversity conservation and ecological studies. However, the sex of nearly half of the birds could not be identified based on their external appearance. It is difficult to visually identify sex to monitor the ecology and conservation of wild populations. In this study, we designed primer pairs for large white pelican using recombinase-based isothermal amplification combined with a lateral flow dipstick (RAA-LFD) assay for chromo-helicase-DNA binding protein (CHD) genes mapped to W chromosomes and an ultra-conserved element (UCE) located on chromosome 6, respectively. Our result showed that the raaW4-RAA-LFD can detect up to 0.1 ng of genomic DNA (gDNA) templates of female pelicans in 30 min at 39 ℃ and accurately distinguish female from male without any cross reactivity. RaaUCE2-RAA-LFD can amplify both male and female pelicans with a detection limit of 25 pg. To further evaluate the assay, 15 white pelicans of unknown sex were tested using the RAA-LFD assay and conventional polymerase chain reaction (PCR). The results of the raaW4-RAA-LFD assay were consistent with those of the conventional PCR. The developed RAA-LFD assay is equipped with field-deployable instruments and offers a field platform for rapid and reliable sex identification in pelicans.

Tert-Butylhydroquinone Mitigates T-2-Toxin-Induced Testicular Dysfunction by Targeting Oxidative Stress, Inflammation, and Apoptosis in Rats.

Tert-butylhydroquinone (tBHQ) has emerged as a promising candidate for mitigating the adverse effects of T-2-induced reproductive toxicity. The protective effects of tBHQ on rat sperm quality, testicular injury, apoptosis, and inflammation induced by T-2 toxin exposure were investigated. Histopathological examination of testicular tissues revealed severe damage in the T-2-treated group, characterized by disorganized germ cell arrangement, thinning of the convoluted seminiferous tubule walls, and significant cellular necrosis. However, tBHQ administration, either as a preventive or therapeutic measure, mitigated this structural damage. Image analysis confirmed an increase in the cross-sectional area and height of the convoluted seminiferous tubules in the tBHQ-treated groups compared to the T-2-treated group (p < 0.05), indicating tBHQ's efficacy in alleviating testicular damage. Additionally, tBHQ treatment significantly inhibited T-2-induced apoptosis of testicular tissue cells, as evidenced by the results showing reduced apoptotic cell counts and downregulation of the BAX/BCL2 ratio and caspase-3 expression (p < 0.05). tBHQ significantly increased the concentrations of the antioxidant factors SOD, CAT, TAC, and GSH-PX. Furthermore, tBHQ attenuated the inflammatory response induced by T-2 exposure, as indicated by the decreased mRNA expression of the proinflammatory cytokines Tnf, Il1, and Il10 in testicular tissue (p < 0.05). Additionally, tBHQ treatment alleviated the decline in serum testosterone induced by the T-2 and promoted testosterone synthesis gene expression, including for the genes 17ß-HSD and Cyp11a1, in rat testes (p < 0.05). These findings underscore tBHQ's role as a therapeutic agent combatting T-2-induced reproductive toxicity, highlighting its antioxidative, anti-apoptotic, and anti-inflammatory properties. Further elucidation of tBHQ's mechanisms of action may offer novel strategies for preventing and treating reproductive disorders induced by environmental toxins.

Cloprostenol sodium improves reproductive performance of multiparous sows during lactation.

This study aimed to determine the effect of prostaglandin F2α (PGF2α) analog (D-cloprostenol sodium and DL-cloprostenol sodium) administration on the milk yield of multiparous sows (MS) and piglet growth performance. In total, 320 Landrace×Yorkshire parturient MS were randomly divided into three groups on day 115 of pregnancy: without treatment (N = 50), with 75 µg D-cloprostenol sodium (N = 137), and with 200 µg DL-cloprostenol sodium (N = 133). After delivery, the sows treated with D-cloprostenol sodium and DL-cloprostenol sodium were randomly allocated into three subgroups, respectively: (i) no additional treatment after farrowing; (ii) administration of cloprostenol sodium at 3 h and 5 days after farrowing; and (iii) administration of cloprostenol sodium at 3 h, 5 days, and 10 days after farrowing. Cloprostenol sodium effectively induced sows to synchronize parturition approximately 23 h after administration and increased the daytime delivery rates (p < 0.05). Compared with DL-cloprostenol sodium, D-cloprostenol sodium shortened the farrowing duration and birth interval of sows for inducing farrowing (p < 0.05). Moreover, we observed that a single administration of both D-cloprostenol sodium and DL-cloprostenol sodium a day before delivery significantly reduced the rates of stillborn piglets type II in MS (p < 0.05). Compared to no treatment and single treatment with cloprostenol sodium, quartic treatments with cloprostenol sodium significantly increased the daily feed intake of MS, litter weight after weaning, and average daily gain of piglets (p < 0.05). Cloprostenol sodium improved the 21-day milk yield, with D-cloprostenol sodium showing the best effect, which increased lactation ability by 30.30% (176.72 kg vs. 135.63 kg) (p < 0.05). DL-cloprostenol sodium followed closely, increasing lactation ability by approximately 25.00% (169.71 kg vs. 135.63 kg) (p < 0.05). During lactation, sows administered with D-cloprostenol sodium observed increased serum prolactin levels. Compared to untreated sows, the sows administered with D-cloprostenol sodium and multiple DL-cloprostenol sodium visibly shortened the weaning-to-estrus interval (WEI) and weaning-to-service interval (WSI) (p < 0.05). Furthermore, quartic injections of D-cloprostenol sodium resulted in an 18 percentage point increase in the pregnancy rate of breeding sows compared to controls (82.61% vs. 64.58%) (p > 0.05). In summary, cloprostenol sodium could enhance the reproductive performance of MS, particularly in terms of lactation performance. Additionally, the effect of quartic injections of D-cloprostenol sodium was the most pronounced.

[The effect of microRNAs on the regulatory network of pluripotency in embryonic stem cells].

Embryonic stem cells (ESCs) are pluripotent stem cells characterized by their ability to self-renew and their pluripotency to differentiate into all cell types. MicroRNA (miRNA) is a small non-coding RNA molecule which can regulate transcriptional and post-transcriptional gene expression, and may also play significant roles in regulating proliferation and differentiation of ESCs. The maintenance of pluripotency in ESCs may involve a regulatory network of many factors and pathways regulated by miRNA, which includes ESCs transcription factors, cell cycle regulation, epigenetic modifications as well as intracelluar signal transduction. This review mainly elaborates the biogenesis of miRNA, the miRNA families regulating the pluripotency of ESCs, and the effect of miRNA on the regulatory network of pluripotency in ESCs.

Quantifying the contribution of ecological water replenishment on aquifer recovery using a refined groundwater model.

Due to its independent control and directly easy operation, ecological water replenishment (EWR) has been an important measure for restoring river ecosystems. However, the positive and negative contribution of the EWR activities to aquifer system are not fully understood under the combined influences of climate change and human activities across time scales. A refined groundwater flow model integrating an open channel flow at daily time scales is developed in a part of Northern China Plain to reproduce the dynamic process of groundwater level changes. After model calibration with groundwater level and runoff data, the changes of simulated groundwater level and river runoff have the Nash-Sutcliffe efficiency coefficient of 0.98 and 0.60, respectively. Results clearly demonstrate that the impulse response of aquifer recovery to runoff in three centralized EWRs. By using with and without EWR method, the simulated maximum contribution of EWR near river to aquifer recovery may be over 70 %. Scenario analysis method considering different precipitation, groundwater exploitation reduction and EWR activities are applied to evaluate the total quantities of aquifer recovery. The prediction of nine-year EWR activities under multiple scenarios shows that the increased groundwater level generally varies from 4.08 to 8.57 m, and the contribution of EWR accounts for 7.88 %-36.59 %. It is also noticed that 14 out of the 18 informal landfill sites will face potential groundwater pollution risks, indicating the negative influences of long-term EWR activities. This study can provide a method for quantifying the influences and contribution of EWR on aquifer recovery and can be referred to as a guideline for EWR evaluation with similar hydrogeological conditions.