The DNA methylation status of the serotonin metabolic pathway associated with reproductive inactivation induced by long-light exposure in Magang geese.

BACKGROUND: Domestic geese are seasonal breeders and have the lowest reproductive capacity among all poultry species. Magang geese is a topical short-day breeder, short photoperiod exposure stimulates its reproductive activity while long photoperiod inhibits. To explore epigenetic change that could influence reproductive activity, we performed whole genome bisulfite sequencing and transcriptome sequencing in the hypothalamus at three reproductive stages during long-light exposure in male Magang geese. RESULTS: A total number of 10,602 differentially methylated regions (DMRs) were identified among three comparison groups. We observed that the vast majority of DMRs were enriched in intron regions. By integrating the BS-sequencing and RNA-seq data, the correlation between methylation changes of CG DMRs and expression changes of their associated genes was significant only for genes containing CG DMRs in their intron. A total of 278 DMR-associated DEGs were obtained among the three stages. KEGG analysis revealed that the DMR-associated DEGs were mainly involved in 11 pathways. Among them, the neuroactive ligand-receptor interaction pathway was significantly enriched in both two comparisons (RA vs.RD and RD vs.RI); the Wnt signaling pathway, apelin signaling pathway, melanogenesis, calcium signaling pathway, focal adhesion, and adherens junction were significantly enriched in the RA vs. RI comparison. In addition, the expression level of two serotonin-metabolic genes was significantly altered during reproductive axis inactivation by the methylation status of their promoter region (TPH2) and intron region (SLC18A2), respectively. These results were confirmed by Bisulfite sequencing PCR (BSP), pyrosequencing, and real-time qPCR, indicating that serotonin metabolic signaling may play a key role in decreasing the reproductive activity of Magang geese induced by long-light exposure. Furthermore, we performed a metabolomics approach to investigate the concentration of neurotransmitters among the three stages, and found that 5-HIAA, the last product of the serotonin metabolic pathway, was significantly decreased in the hypothalamus during RI. CONCLUSIONS: Our study reveals that the methylation status of the serotonin metabolic pathway in the hypothalamus is associated with reproductive inactivation, and provided new insight into the effect of DNA methylation on the reproductive regulation of the hypothalamus in Magang geese.

miR-100-5p Regulates Skeletal Muscle Myogenesis through the Trib2/mTOR/S6K Signaling Pathway.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial regulatory roles in many biological processes, including the growth and development of skeletal muscle. miRNA-100-5p is often associated with tumor cell proliferation and migration. This study aimed to uncover the regulatory mechanism of miRNA-100-5p in myogenesis. In our study, we found that the miRNA-100-5p expression level was significantly higher in muscle tissue than in other tissues in pigs. Functionally, this study shows that miR-100-5p overexpression significantly promotes the proliferation and inhibits the differentiation of C2C12 myoblasts, whereas miR-100-5p inhibition results in the opposite effects. Bioinformatic analysis predicted that Trib2 has potential binding sites for miR-100-5p at the 3'UTR region. A dual-luciferase assay, qRT-qPCR, and Western blot confirmed that Trib2 is a target gene of miR-100-5p. We further explored the function of Trib2 in myogenesis and found that Trib2 knockdown markedly facilitated proliferation but suppressed the differentiation of C2C12 myoblasts, which is contrary to the effects of miR-100-5p. In addition, co-transfection experiments demonstrated that Trib2 knockdown could attenuate the effects of miR-100-5p inhibition on C2C12 myoblasts differentiation. In terms of the molecular mechanism, miR-100-5p suppressed C2C12 myoblasts differentiation by inactivating the mTOR/S6K signaling pathway. Taken together, our study results indicate that miR-100-5p regulates skeletal muscle myogenesis through the Trib2/mTOR/S6K signaling pathway.

Circular Intronic RNA circTTN Inhibits Host Gene Transcription and Myogenesis by Recruiting PURB Proteins to form Heterotypic Complexes.

Muscle cell growth plays an important role in skeletal muscle development. Circular RNAs (circRNAs) have been proven to be involved in the regulation of skeletal muscle growth and development. In this study, we explored the effect of circTTN on myoblast growth and its possible molecular mechanism. Using C2C12 cells as a functional model, the authenticity of circTTN was confirmed by RNase R digestion and Sanger sequencing. Previous functional studies have showed that the overexpression of circTTN inhibits myoblast proliferation and differentiation. Mechanistically, circTTN recruits the PURB protein on the Titin (TTN) promoter to inhibit the expression of the TTN gene. Moreover, PURB inhibits myoblast proliferation and differentiation, which is consistent with circTTN function. In summary, our results indicate that circTTN inhibits the transcription and myogenesis of the host gene TTN by recruiting PURB proteins to form heterotypic complexes. This work may act as a reference for further research on the role of circRNA in skeletal muscle growth and development.

Integrated analysis of muscle transcriptome, miRNA, and proteome of Chinese indigenous breed Ningxiang pig in three developmental stages.

The Ningxiang pig, a distinguished local breed in China, is recognized for its good meat quality traits. This study examines the proteomics of Ningxiang pigs at three developmental stages and delves into the upstream transcriptomics of these proteomics. Such an analysis facilitates a deeper understanding of the molecular interplay between proteins and transcriptomes in the Ningxiang pig muscle, influencing muscle growth and development. In this research, we analyzed the muscles of Ningxiang pigs at three developmental stages: 30 days in weaned piglets, 90 days in nursery pigs, and 210 days in late fattening pigs. There a total of 16 differentially co-expressed miRNAs (ssc-miRNA-1, ssc-miRNA-378, ssc-miRNA-143, ssc-miRNA-30e, etc.), 74 differentially co-expressed mRNA (PLIN3, CPT2, IGF2 and HSP90AB1, etc.) have been identified in the three stages. 572 differentially abundant proteins (DAPs) (APOC3, NDUFA2, HSPD1, ATP5E, PDHA1, etc.) were readily identified by comparing different time periods. According to the KEGG enrich pathway results that DAPs most enriched in growth and development pathways, immune mechanism pathways and maintaining functions of physical. Through short time-series expression miner (STEM) association analysis, a total of 571 negative miRNA-mRNA interaction pairs and 2 negative miRNA-mRNA-protein (Chr05_11955-Pig.17268.1-ATP5F1B, ssc-miR-194a-3p-Pig.15802.1-ACY1) interaction pairs were found. Our study provides a theoretical basis on molecular mechanism for the study of IMF deposition, muscle growth and immunity in Ningxiang pig breed.

Effects of stocking density on the homeostasis of uric acid and related liver and kidney functions in ducks.

OBJECTIVE: Stocking density (SD) is an important issue in the poultry industry, which is related to the production performance, intestinal health and immune status. In the present study, the effects of SD on the metabolism and homeostasis of uric acid as well as the related functions of the liver and kidney in ducks were examined. METHODS: A total of 360 healthy 56-day-old Shan-ma ducks were randomly divided into the low stocking density (n = 60, density = 5 birds/m2), medium stocking density (n = 120, density = 10 birds/m2) and high stocking density groups (HSD; n = 180, density = 15 birds/m2). Samples were collected in the 3rd, 6th, and 9th weeks of the experiment for analysis. RESULTS: The serum levels of uric acid, lipopolysaccharide and inflammatory cytokines (interleukin-1ß [IL-1ß], IL-8, and tumor necrosis factor-α [TNF-α]) were increased significantly in the HSD group. Serious histopathological lesions could be seen in both the livers and kidneys in the HSD group in the 9th week. The mRNA expression levels of inflammatory cytokines (IL-8 and TNF-α) and related pathway components (toll-like receptor 4, myeloid differentiation primary response gene 88, and nuclear factor-κB) were increased significantly in both the livers and kidneys in the HSD group. The mRNA expression levels of enzymes (adenosine deaminase, xanthine oxidase, phosphoribosyl pyrophosphate amidotransferase, and phosphoribosyl pyrophosphate synthetase 1) related to the synthesis of uric acid increased significantly in the livers in the HSD group. However, the mRNA expression level of solute carrier family 2 member 9, which plays an important role in the excretion of uric acid by the kidney, was decreased significantly in the kidneys in the HSD group. CONCLUSION: These results indicated that a higher SD could cause tissue inflammatory lesions in the liver and kidney and subsequently affect the metabolism and homeostasis of uric acid, and is helpful for guiding decisions related to the breeding and production of ducks.

Comprehensive analysis of transcriptomic and metabolomic profiles uncovered the age-induced dynamic development pattern of subcutaneous fat in Ningxiang pig.

Enhancing meat production and quality is the eternal theme for pig breeding industries. Fat deposition has always been the focus of research in practical production because it is closely linked to pig production efficiency and pork quality. In the current study, multi-omics techniques were performed to explore the modulatory mechanisms of backfat (BF) accumulation at three core developmental stages for Ningxiang pigs. Our results identified that 15 differentially expressed genes (DEGs) and 9 significantly changed metabolites (SCMs) contributed to the BF development via the cAMP signaling pathway, regulation of lipolysis in adipocytes, and biosynthesis of unsaturated fatty acids. Herein, we found a series of candidate genes such as adrenoceptor beta 1 (ADRB1), adenylate cyclase 5 (ADCY5), ATPase Na+/K+ transporting subunit beta 1 (ATP1B1), ATPase plasma membrane Ca2+ transporting 3 (ATP2B3), ATPase Na+/K+ transporting subunit alpha 2 (ATP1A2), perilipin 1 (PLIN1), patatin like phospholipase domain containing 3 (PNPLA3), ELOVL fatty acid elongase 5 (ELOVL5) and metabolites like epinephrine, cAMP, arachidonic acid, oleic acid, linoleic acid, and docosahexaenoic acid existed age-specificeffects and played important roles in lipolysis, fat accumulation, and fatty acid composition. Our findings provide a reference for molecular mechanisms in BF tissue development and the optimization of carcass quality.

Integrated analysis of circRNA, lncRNA, miRNA and mRNA to reveal the ceRNA regulatory network of postnatal skeletal muscle development in Ningxiang pig.

Introduction: The development of skeletal muscle is regulated by regulatory factors of genes and non-coding RNAs (ncRNAs). Methods: The objective of this study was to understand the transformation of muscle fiber type in the longissimus dorsi muscle of male Ningxiang pigs at four different growth stages (30, 90, 150, and 210 days after birth, n = 3) by histological analysis and whole transcriptome sequencing. Additionally, the study investigated the expression patterns of various RNAs involved in muscle fiber transformation and constructed a regulatory network for competing endogenous RNA (ceRNA) that includes circular RNA (circRNA)/long non-coding RNA (lncRNA)-microRNA (miRNA)-messenger RNA (mRNA). Results: Histomorphology analysis showed that the diameter of muscle fiber reached its maximum at 150 days after birth. The slow muscle fiber transformation showed a pattern of initial decrease followed by an increase. 29,963 circRNAs, 2,683 lncRNAs, 986 miRNAs and 22,411 mRNAs with expression level ≥0 were identified by whole transcriptome sequencing. Furthermore, 642 differentially expressed circRNAs (DEc), 505 differentially expressed lncRNAs (DEl), 316 differentially expressed miRNAs (DEmi) and 6,090 differentially expressed mRNAs (DEm) were identified by differential expression analysis. Functions of differentially expressed mRNA were identified by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). GO enrichment analysis indicates that 40 known genes and 6 new genes are associated with skeletal muscle development. Additionally, KEGG analysis shows that these genes regulate skeletal muscle development via MAPK, FoxO, Hedgehog, PI3K-Akt, Notch, VEGF and other signaling pathways. Through protein-protein interaction (PPI) and transcription factor prediction (TFP), the action mode of skeletal muscle-related genes was explored. PPI analysis showed that there were stable interactions among 19 proteins, meanwhile, TFP analysis predicted 22 transcription factors such as HMG20B, MYF6, MYOD1 and MYOG, and 12 of the 19 interacting proteins were transcription factors. The regulatory network of ceRNA related to skeletal muscle development was constructed based on the correlation of various RNA expression levels and the targeted binding characteristics with miRNA. The regulatory network included 31 DEms, 59 miRNAs, 667 circRNAs and 224 lncRNAs. conclusion: Overall, the study revealed the role of ceRNA regulatory network in the transformation of skeletal muscle fiber types in Ningxiang pigs, which contributes to the understanding of ceRNA regulatory network in Ningxiang pigs during the skeletal muscle development period.

Transcriptome Analysis Reveals the Age-Related Developmental Dynamics Pattern of the Longissimus Dorsi Muscle in Ningxiang Pigs.

The growth and development of the Longissimus Dorsi muscle are complex, playing an important role in the determination of pork quality. The study of the Longissimus Dorsi muscle at the mRNA level is particularly crucial for finding molecular approaches to improving meat quality in pig breeding. The current study utilized transcriptome technology to explore the regulatory mechanisms of muscle growth and intramuscular fat (IMF) deposition in the Longissimus Dorsi muscle at three core developmental stages (natal stage on day 1, growing stage on day 60, and finishing stage on day 210) in Ningxiang pigs. Our results revealed 441 differentially expressed genes (DEGs) in common for day 1 vs. day 60 and day 60 vs. day 210, and GO (Gene Ontology) analysis showed that candidate genes RIPOR2, MEGF10, KLHL40, PLEC, TBX3, FBP2, and HOMER1 may be closely related to muscle growth and development, while KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that DEGs (UBC, SLC27A5, RXRG, PRKCQ, PRKAG2, PPARGC1A, PLIN5, PLIN4, IRS2, and CPT1B) involved the PPAR (Peroxisome Proliferator-Activated Receptor) signaling pathway and adipocytokine signaling pathway, which might play a pivotal role in the regulation of IMF deposition. PPI (Protein-Protein Interaction Networks) analysis found that the STAT1 gene was the top hub gene. Taken together, our results provide evidence for the molecular mechanisms of growth and development and IMF deposition in Longissimus Dorsi muscle to optimize carcass mass.

Molecular Mechanism of MYL4 Regulation of Skeletal Muscle Development in Pigs.

The processes of muscle growth and development, including myoblast proliferation, migration, differentiation, and fusion, are modified by a variety of regulatory factors. MYL4 plays an important role in atrial development, atrial cardiomyopathy, muscle-fiber size, and muscle development. The structural variation (SV) of MYL4 was found via the de novo sequencing of Ningxiang pigs, and the existence of SV was verified in the experiments. The genotype distribution of Ningxiang pigs and Large White pigs was detected, and it was found that Ningxiang pigs were mainly of the BB genotype and that Large White pigs were mainly of the AB genotype. However, the molecular mechanisms behind the MYL4-mediated regulation of skeletal muscle development need to be deeply explored. Therefore, RT-qPCR, 3'RACE, CCK8, EdU, Western blot, immunofluorescence, flow cytometry, and bioinformation analysis were used to explore the function of MYL4 in myoblast development. The cDNA of MYL4 was successfully cloned from Ningxiang pigs, and its physicochemical properties were predicted. The expression profiles in six tissues and four stages of Ningxiang pigs and Large White pigs were found to be the highest in the lungs and 30 days after birth. The expression of MYL4 increased gradually with the extension of the myogenic differentiation time. The myoblast function test showed that the overexpression of MYL4 inhibited proliferation and promoted apoptosis and differentiation. The knockdown of MYL4 showed the opposite result. These results enhance our understanding of the molecular mechanisms of muscle development and provide a solid theoretical foundation for further exploring the role of the MYL4 gene in muscle development.

OPN5 Regulating Mechanism of Follicle Development Through the TSH-DIO2/DIO3 Pathway in Mountain Ducks Under Different Photoperiods.

Photoperiod is an important environmental factor that influence seasonal reproduction behavior in bird. Birds translates photoperiodic information into neuroendocrine signals through deep brain photoreceptors (DBPs). OPN5 has been considered as candidate DBPs involving in regulation of seasonal reproduction in birds. However, little is known about the effect of OPN5 in non-seasonal breeding birds. Thus, we pondered on whether OPN5 regulating follicular development through TSH-DIO2/DIO3 system responds to different photoperiods in non-seasonal laying ducks. As an ideal non-seasonal breeding bird, a total of 120 mountain ducks were randomly divided into three groups and treated respectively to a different photoperiod: group S (8 L:16D), group C (17 L:7D), and group L (24 L:0D). The ducks were caged in a fully enclosed shelter with the same feeding conditions for each group, free water and limited feeding (150 g per duck each day). Samples were collected from each group at d 0, d 5, d 8, d 20, and d 35 (n = 8). The ducks in 24 h photoperiod had the highest laying rate and the lowest feed-to-egg ratio, while the ducks in 8 h photoperiod had the lowest laying rate and the highest feed-to-egg ratio. Long-day photoperiod for 24 h significantly increased the ovarian index and GnRH, LH, E2, and P4 levels in serum; short-day photoperiod for 8 h increased testosterone levels in serum. Compared with 8 h photoperiod, long-day photoperiod significantly or highly significantly increased the mRNA level and protein expression of OPN5 in the hypothalamus of long-day photoperiod on d 35 (p < 0.05). The gene or protein expression patterns of GnRH, TRH, TSHß, DIO2, THRß, VIP, and PRL were positively correlated with OPN5, whereas the gene expression patterns of GnIH and DI O 3 were negatively correlated with OPN5. The results revealed that OPN5 mediated the effect of light on follicular development through the TSH-DIO2/DIO3 pathway, the expression of OPN5 increased with light duration and improved the efficiency of the HPG axis to promote follicular development in mountain ducks.

Effects of stocking density on ovarian development and maturation during the rearing period in Shan-ma ducks.

Stocking density critically affects the growth and subsequent performance of animals in modern poultry production. This study investigated the effects of stocking density on ovarian development, ovarian maturation, and the mRNA expression of key genes in the reproductive axis during the rearing period of Shan-ma ducks. The experiments involved 180 healthy 7-wk-old Shan-ma ducks and randomly divided into low stocking density (LSD; n = 30, density = 5 birds/m2), medium stocking density (MSD; n = 60, density = 10 birds/m2) and high stocking density groups (HSD; n = 90, density = 15 birds/m2), for rearing. After examining ovarian development and measuring hormone levels in the plasma and expression levels of key regulatory genes in the reproductive axis at 19 wk of rearing, analysis of the gonad index analysis, reflecting stocking density, uncovered statistically significant differences. The gonad index of the LSD group was significantly higher than those of the MSD and HSD groups (P < 0.01), while no significant difference was observed between the MSD and HSD groups. pre-ovulatory follicles (POFs) and small yellow follicles (SYFs) development was only apparent in the LSD group, with the large white follicles (LWFs) number of this group being significantly higher than that of the MSD group (P < 0.05). The blood levels of E2 (estradiol), P4 (progesterone), and T (testosterone) were significantly higher in the LSD group than in the MSD and HSD groups (P < 0.05 or 0.01). Also, the levels of both P4 and T were significantly higher in the MSD group than in the HSD group (P < 0.01). The gene expression levels of GnRHR, FSH, AMHR, and FSHR were significantly increased in the LSD group compared to the MSD and HSD groups (P < 0.05 or 0.01), while the expression levels of GnIHR and GDF9 were significantly decreased in the LSD and MSD groups compared to the HSD group (P < 0.05 or 0.01). Steroid biosynthesis pathway genes such as StAR, CYP11A1, 3ß-HSD, CYP19A1, and BMP15 were significantly downregulated at greater stocking densities (P < 0.05 or 0.01). Likewise, the protein expression of StAR, 3ß-HSD, and CYP19A1 was also significantly decreased (P < 0.05 or 0.01). These results demonstrate that both medium and high stocking densities suppressed the expression of the key reproduction-promoting factors, while the expression level of the key reproductive inhibitory factors was enhanced. Therefore, rates of ovarian development and maturation could be reduced by a high stocking density leading to a delay in reproduction performance during the rearing period of Shan-ma ducks.

Photoperiodic Changes in Both Hypothalamus Neurotransmitters and Circulating Gonadal Steroids Metabolomic Profiles in Relation to Seasonal Reproduction in Male Quail.

Both hypothalamic neurotransmitters and serum steroid hormones are impacted by photoperiod and have effects on physiology and seasonal reproductive. However, the relationship between circulating gonadal steroids and hypothalamic neurotransmitters underlying different photoperiod is still unclear. To further understand the crosstalk of neurotransmitters and steroids in seasonal reproduction, metabolic changes of 27 neurotransmitters concentrated in hypothalamus tissues and 42 steroids hormones in serum were assessed during two artificial photoperiodic programs. The results showed that photoperiod induce testicular atrophy and recrudescence. In L-to-S groups, significantly decreased levels of testosterone concentration were found in serum (P < 0.001) and increased 11-Dehydrocorticosterone (P < 0.05); Testosterone were almost undetectable at SD_14d. In addition, the hypothalamus exhibited significantly increased arginine and 4-aminobutyric acid (GABA) concentration and decreased serotonin and epinephrine content (P < 0.01 or P < 0.05). Accordingly, serum testosterone and androstenedione became detectable at LD_3d in the S-to-L group and were markedly increase at LD_7d. Furthermore, Serum androstenedione showed a significant increase with long light expose (P < 0.01). Additionally, the hypothalamus exhibited both significantly increased L.Tryptophan and phenylalanine concentration, as well as decreased L-glutamine and L-glutamine.acid content (P < 0.01 or P < 0.05). Serotonin metabolism showed significant differences between L-to-S group and S-to-L group. Furthermore, in the correlation analysis, serum testosterone had a positive correlation with 5-Hydroxyindole-3-acetic acid (5-HIAA), while Androstenedione was significantly negative with L.Tryptophan in L-to-S (P < 0.05). However, in S-to-L group, serum testosterone showed strong negative correlation with both serotonin and 5-HIAA (P < 0.05), but positive correlation with L.Tryptophan (P < 0.01), while Androstenedione was significantly negative correlation with both serotonin (P < 0.05) and L-Glutamine (P < 0.01). Photoperiod also had significant effects on the mRNA expression. We found significant differences in gene expression patterns of both serotonin signaling and steroid biosynthesis, while MAOB, NR5A1, and 3ß-HSD showed an opposite tendency between two groups. Taken together, our results revealed that circulating gonadal steroids and hypothalamic neurotransmitters were significantly impact quail's seasonal reproduction. Circulating gonadal steroids have different effects on neurotransmitter at different photoperiodism, which may coordinately influence the seasonal reproduction of quails.

Long-day photoperiods affect expression of OPN5 and the TSH-DIO2/DIO3 pathway in Magang goose ganders.

This study sought to understand the regulation mechanism of OPN5 through the TSH-DIO2/DIO3 pathway mediated photoperiod on the breeding activity of short-day breeding birds. In this study, the reproductive activity of Magang goose was regulated by artificial light, and the reproductive activity of the ganders were determined according to the daily laying rate of female geese. The testicular development and the serum reproductive hormone concentrations of ganders were measured during the reproductive period (d 0), the reproductive degeneration period (d 13 and 27) and the resting period (d 45). The mRNA and protein expression patterns of OPN5, the HPG axis reproductive genes, and TSH-DIO2/DIO3 pathway related genes were examined. Results showed that the laying rate of geese and the gonadal indices (GSI) decreased gradually after the photoperiod increased. Histological observation found that the spermatogenic function of the testis was normal on d 0 and 13, while degeneration occurred by d 27 and 45. Serum testosterone, FSH, and LH concentration showed a slight increase on d 13, followed by a sharp decrease on d 27 and 45 (P < 0.01), while PRL concentrations were low on d 0 and 13, and increased rapidly on d 27 and 45 (P < 0.01).The expression pattern of GnRH, FSH, LH, and THRß mRNA were similar, with high levels on d 0 and 13 and a decreasing trend on d 27 and 45 (P < 0.05 or P < 0.01); and GnRHR mRNA levels were higher on d 13 (P < 0.05), but then had decreased by d 27 and 45 (P < 0.01). The expression pattern of GnIH and GnIHR was similar, which was opposite to that of GnRHR. VIP, PRL, and PRLR increased gradually and peaked on d 45 (P < 0.01). The expression trend of TRH, TSHß, and DIO2 was similar to that of GnRHR, and the expression abundance increased on d 13, and then decreased on d 27 and 45. GnRH protein expression was significantly higher than during the other 3 periods (P < 0.01) while the GnIH protein levels were extremely low on d 0, had gradually increased by d 13, and significantly increased by d 27 and 45 (P < 0.01). The protein expression trends of THR and DIO2 were similar to that of GNIH. DIO3 protein expression was low on d 0 and 13, and increased by d 27 and 45. These results suggest that when the photoperiod increased, the hypothalamus OPN5 gene and protein were upregulated and the pituitary TSHß, TSHR, and hypothalamus THRß, TRH, and DIO2 were downregulated, and thus the reproductive activity of geese was inhibited.