Journal of Reproduction and Development最新文献

{"title":"Factors influencing in vivo embryo production in Japanese Black donors: the role of Anti-Müllerian hormone and inflammation parameters.","authors":"Hiroaki Okawa, Norihiro Yukiyama, Osamu Yamato, Akira Goto, Oky Setyo Widodo, Yasuo Fushimi, Mitsuhiro Takagi","doi":"10.1262/jrd.2024-092","DOIUrl":"https://doi.org/10.1262/jrd.2024-092","url":null,"abstract":"<p><p>In Japanese Black (JB) cattle, the number of transferable embryos produced after superovulation is crucial for the economic success of embryo production for both farmers and veterinarians. Anti-Müllerian hormone (AMH) has emerged as a key reproductive marker for predicting the number of embryos produced in vivo and oocytes retrieved through transvaginal pickup. This study investigated the relationship between AMH, inflammatory markers, including serum amyloid A (SAA) and albumin/globulin (A/G) ratio, and the number of embryos recovered and transferable after superovulation in JB cows. A total of 96 JB donor cows underwent artificial insemination after superovulation, and embryo retrieval was performed 7 days later. Embryos retrieved were classified based on the International Embryo Technology Society criteria, wherein \"transferable embryos\" included those with codes 1 or 2, while \"total embryos\" included transferable embryos as well as those with codes 3 and 4. Blood samples collected during embryo recovery were used to measure serum AMH, SAA, and A/G ratios. When grouped by AMH quartiles, the high-AMH and middle-high-AMH groups produced significantly more total embryos compared to the low-AMH group. The total number of embryos increased with higher AMH levels (r = 0.3336, P = 0.0009). Correlation analysis revealed associations between AMH, α₁-globulin and SAA. Additionally, a significant positive correlation was observed between total and transferable embryos (r = 0.6339, P < 0.0001) and between AMH and the yield ratio (r = 0.25583, P = 0.0119). These findings confirm that AMH concentration is a valuable reproductive marker for predicting the total and transferable embryos produced by JB donor cows.</p>","PeriodicalId":16942,"journal":{"name":"Journal of Reproduction and Development","volume":" ","pages":""},"PeriodicalIF":1.9,"publicationDate":"2025-01-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143080473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Involvement of nuclear receptor corepressor 2 (NCOR2) in estrogen-induced repression of arcuate Kiss1 expression in female rats.","authors":"Marina Takizawa, Sae Miyazaki, Hitomi Tsuchida, Mayuko Nagae, Shunsuke Seki, Masumi Hirabayashi, Fumitaka Osakada, Naoko Inoue, Hiroko Tsukamura, Yoshihisa Uenoyama","doi":"10.1262/jrd.2024-100","DOIUrl":"https://doi.org/10.1262/jrd.2024-100","url":null,"abstract":"<p><p>Hypothalamic arcuate (ARC) kisspeptin neurons are considered the gonadotropin-releasing hormone pulse generator in rats. In virgin rats, the expression of the ARC kisspeptin gene (Kiss1) is repressed by proestrous levels of estradiol-17β (high E2) but not by diestrous levels of E2 (low E2). In lactating rats, ARC Kiss1 expression is repressed by low E2 during late lactation. This study aimed to investigate whether nuclear receptor corepressor 2 (NCOR2, encoded by Ncor2), an estrogen receptor α corepressor, is involved in the estrogen-induced repression of ARC Kiss1 expression in rats. Double in situ hybridization for Kiss1 and Ncor2 revealed that approximately 80% of ARC Kiss1-expressing cells co-expressed Ncor2 in ovariectomized (OVX) + low E2 virgin rats, while approximately 90% of ARC Kiss1-expressing cells co-expressed Ncor2 in OVX + low E2 lactating rats. To further examine the role of Ncor2, we studied the effects of Kiss1-dependent Ncor2 knockdown on ARC Kiss1 expression and luteinizing hormone (LH) pulses. An adeno-associated virus vector carrying Cre-activated short hairpin RNA (shRNA) for Ncor2 was administered to the ARC in two Kiss1-Cre rat models: OVX + high E2 Kiss1-Cre virgin rats and OVX + low E2 Kiss1-Cre lactating rats. Ncor2-shRNA treatment significantly increased the number of ARC Kiss1-expressing cells and the intensity of Kiss1 signals in OVX + high E2 virgin rats but failed to fully restore low E2-induced Kiss1 repression in lactating rats. The Ncor2-shRNA treatment failed to affect LH pulses in both models. These findings suggest that NCOR2 in ARC kisspeptin neurons mediates high E2-induced repression of ARC Kiss1 expression in virgin rats.</p>","PeriodicalId":16942,"journal":{"name":"Journal of Reproduction and Development","volume":" ","pages":""},"PeriodicalIF":1.9,"publicationDate":"2025-01-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143047062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Influences of 5-hydroxytriptamine on sperm hyperactivation and in vitro fertility in rats.","authors":"Yuki Koyano, Masakatsu Fujinoki","doi":"10.1262/jrd.2024-078","DOIUrl":"https://doi.org/10.1262/jrd.2024-078","url":null,"abstract":"<p><p>The neurotransmitter, 5-hydroxytriptamine (5-HT), is well known. Furthermore, it enhances the acrosome reaction, hyperactivation, and in vitro fertilization (IVF) success in hamsters and mice. In the present study, we examined whether 5-HT enhances hyperactivation and increases IVF success in rats. When rat sperm was exposed to 5-HT, hyperactivation was significantly enhanced. Only the 5-HT₄ receptor agonists significantly enhanced hyperactivation. Additionally, both 5-HT and 5-HT₄ receptor agonists significantly increase the success of IVF. These results suggested that 5-HT increases IVF success by enhancing hyperactivation and effects of 5-HT are associated with the 5-HT₄ receptor. Therefore, in rats, 5-HT enhances capacitation and the 5-HT₄ receptor is the key molecule for capacitation.</p>","PeriodicalId":16942,"journal":{"name":"Journal of Reproduction and Development","volume":" ","pages":""},"PeriodicalIF":1.9,"publicationDate":"2025-01-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143047053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Can thermoregulatory response to heat stress be improved in lactating dairy cows? Insights from counter-current heat transfer systems impacting reproduction.","authors":"Fernando López-Gatius","doi":"10.1262/jrd.2024-101","DOIUrl":"https://doi.org/10.1262/jrd.2024-101","url":null,"abstract":"<p><p>Climate change has caused heat stress (HS) to become an increasingly severe problem for high-producing dairy herds. Although cooling systems allow milk production to remain nearly constant throughout the year, fertility decreases during summer. Physiological counter-current heat transfer mechanisms maintaining brain/hypothalamic and reproductive functions in cattle are vulnerable to HS. In this study, I propose strategies to improve cooling systems, particularly in zones with the highest risk of increased body temperature, such as milking areas. In addition, heat transfer mechanisms to protect the brain-hypothalamus axis from hyperthermia must be considered when implementing measures to reduce HS-related problems.</p>","PeriodicalId":16942,"journal":{"name":"Journal of Reproduction and Development","volume":" ","pages":""},"PeriodicalIF":1.9,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143007327","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.","authors":"Qingyi Lin, Koki Takebayashi, Nanaka Torigoe, Bin Liu, Zhao Namula, Maki Hirata, Fuminori Tanihara, Megumi Nagahara, Takeshige Otoi","doi":"10.1262/jrd.2024-054","DOIUrl":"10.1262/jrd.2024-054","url":null,"abstract":"<p><p>CRISPR/Cas9-based multiplex genome editing via electroporation is relatively efficient; however, lipofection is versatile because of its ease of use and low cost. Here, we aimed to determine the efficiency of lipofection in CRISPR/Cas9-based multiplex genome editing using growth hormone receptor (GHR) and glycoprotein alpha-galactosyltransferase 1 (GGTA1)-targeting guide RNAs (gRNAs) in pig zygotes. Zona pellucida-free zygotes were collected 10 h after in vitro fertilization and incubated with Cas9, gRNAs, and Lipofectamine 2000 (LP2000) for 5 h. In Experiment 1, we evaluated the mutation efficiency of gRNAs targeting either GHR or GGTA1 in zygotes transfected using LP2000 and cultured in 4-well plates. In Experiment 2, we examined the effects of the culture method on the development, mutation rate, and mutation efficiency of zygotes with simultaneously double-edited GHR and GGTA1, cultured using 4-well (group culture) and 25-well plates (individual culture). In Experiment 3, we assessed the effect of additional GHR-targeted lipofection before and after simultaneous double gRNA-targeted lipofection on the mutation efficiency of edited embryos cultured in 25-well plates. No significant differences in mutation rates were observed between the zygotes edited with either gRNA. Moreover, the formation rate of blastocysts derived from GHR and GGTA1 double-edited zygotes was significantly increased in the 25-well plate culture compared to that in the 4-well plate culture. However, mutations were only observed in GGTA1 when zygotes were transfected with both gRNAs, irrespective of the culture method used. GHR mutations were detected only in blastocysts derived from zygotes subjected to GHR-targeted lipofection before simultaneous double gRNA-targeted lipofection. Overall, our results suggest that additional lipofection before simultaneous double gRNA-targeted lipofection induces additional mutations in the zygotes.</p>","PeriodicalId":16942,"journal":{"name":"Journal of Reproduction and Development","volume":" ","pages":"356-361"},"PeriodicalIF":1.9,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11658923/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142108615","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MiR-145-5p regulates granulosa cell proliferation by targeting the <i>SET</i> gene in KGN cells.","authors":"Gao Lingling, Yang Qingxing, Xu Jianbo, Wang Weijie, Lu Dan","doi":"10.1262/jrd.2024-053","DOIUrl":"10.1262/jrd.2024-053","url":null,"abstract":"<p><p>MiR-145-5p has been implicated in the development and progression of various disorders, and it is primarily recognized as a tumor suppressor in numerous cancers types. Its expression has been reported to decrease in the granulosa cells of patients with polycystic ovarian syndrome (PCOS). This study aimed to investigate whether miR-145-5p plays a role in granulosa cell proliferation and to shed light on the underlying pathological mechanisms of follicular development in patients with PCOS. Follicular fluid samples were collected from patients with PCOS and healthy individuals. The Cell Counting Kit-8 and bromodeoxyuridine assays were performed to assess KGN cell proliferation. The expression of miR-145-5p was significantly decreased in PCOS granulosa cells than in control cells, whereas the expression of SET was increased. Furthermore, miR-145-5p suppressed the proliferation of KGN cells. SET was identified as a direct target of miR-145-5p. Additionally, SET promoted the proliferation of KGN cells, and knockdown of SET counteracted the effect of the miR-145-5p inhibitor. Therefore, miR-145-5p regulates granulosa cell proliferation by targeting the SET in KGN cells; this process may be a potential pathological pathway that contributes to follicular developmental disorders in PCOS.</p>","PeriodicalId":16942,"journal":{"name":"Journal of Reproduction and Development","volume":" ","pages":"372-378"},"PeriodicalIF":1.9,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11658921/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142307991","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Change in the ability of bovine granulosa cells to elongate transzonal projections and their transcriptome changes during follicle development.","authors":"Mihoko Fushii, Hirohisa Kyogoku, Jibak Lee, Takashi Miyano","doi":"10.1262/jrd.2024-016","DOIUrl":"10.1262/jrd.2024-016","url":null,"abstract":"<p><p>Granulosa cells (GCs) in secondary follicles differentiate into cumulus cells (CCs) and mural granulosa cells (MGCs) in the antral follicle. Only CCs maintain direct connections with oocytes through transzonal projections (TZPs) and support oocyte growth. Here, we examined whether granulosa cells (GCs) from secondary follicles and MGCs from early and late antral follicles were able to reconstruct complexes with TZP-free denuded oocytes (DOs) and regenerate TZPs. Furthermore, to confirm that the regenerated TZPs were functional, the development of the reconstructed complexes and oocyte growth in the complexes were evaluated. After coculture, GCs and MGCs from early antral follicles reconstructed the complexes with DOs and regenerated TZPs. Furthermore, the oocytes in the integrally reconstructed complexes grew fully and acquired meiotic competence, suggesting that the regenerated TZPs were functional. In contrast, MGCs from the late antral follicles lost their ability to elongate TZPs. As the ability to regenerate TZPs differed among cells, we analyzed the transcriptomes of GCs, CCs, and MGCs collected from follicles of different sizes. The characteristics of TZP generation coincided with the transcriptome changes in two directions: from GCs to CCs and MGCs. In conclusion, until the early antral follicle stage, bovine GCs, CCs, and MGCs have common characteristics to elongate TZPs and form antrum-like structures that support oocyte growth in vitro. Furthermore, as the follicle develops, MGCs lose the ability to elongate TZPs.</p>","PeriodicalId":16942,"journal":{"name":"Journal of Reproduction and Development","volume":" ","pages":"362-371"},"PeriodicalIF":1.9,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11658919/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142468412","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"<i>Ccdc152</i> is not necessary for male fertility, but contributes to maintaining sperm morphology.","authors":"Ryua Harima, Takahiro Sasaki, Takayuki Kaneko, Fuka Aso, Hayato Takashima, Takashi Toyama, Kenshiro Hara, Kentaro Tanemura, Yoshiro Saito","doi":"10.1262/jrd.2024-058","DOIUrl":"10.1262/jrd.2024-058","url":null,"abstract":"<p><p>Selenoprotein P (SeP) is synthesized in the liver and plays a vital role in maintaining selenium homeostasis via transport throughout the body. Previous studies have shown that SeP-deficient mice have severely reduced expression of selenoproteins essential for testicular function, leading to male infertility. We previously reported that the high expression of Ccdc152 in hepatocytes acts as a lncRNA, suppressing SeP expression in the liver. Ccdc152 reduces SeP translation by binding to SeP mRNA and decreasing its interaction with SECIS-binding protein 2. Although Ccdc152 is highly expressed in testes, its function remains unclear. Therefore, this study aimed to elucidate the role of Ccdc152 in the testes. Using the CRISPR/Cas9 system, we generated mice lacking all exons of Ccdc152 and found that SeP expression levels in the liver and plasma, as well as overall selenium homeostasis, remained unchanged. No significant differences were observed in the expression of glutathione peroxidase 1/4 or level of selenium in the testes. Subsequent investigation of the impact on male reproductive function revealed no abnormalities in sperm motility or Mendelian ratios of the offspring. However, a slight decrease in testicular weight and an increased rate of sperm malformations in the epididymis were observed. RNA-seq and pathway analyses identified the reduced expression of multiple genes related to kinesin and reproductive pathways. Based on these findings, Ccdc152 may not be essential for male reproductive function, but it may enhance reproductive capabilities by maintaining the expression of genes necessary for reproduction.</p>","PeriodicalId":16942,"journal":{"name":"Journal of Reproduction and Development","volume":" ","pages":"396-404"},"PeriodicalIF":1.9,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11658925/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preovulatory follicular dynamics and ovulatory events following the use of GnRH 84 h after medroxyprogesterone acetate sponge removal in postpartum buffaloes.","authors":"Muhammad Usman Mehmood, Ghazanfar Ali Chishti, Muhammad Waseem, Burhan E Azam, Zahid Naseer, Muhammad Saadullah, Kehuan Lu, Yangqing Lu","doi":"10.1262/jrd.2024-040","DOIUrl":"10.1262/jrd.2024-040","url":null,"abstract":"<p><p>Herein, we evaluated the effects of gonadotropin hormone-releasing hormone (GnRH) administration 84 h after medroxyprogesterone acetate (MAP) sponge removal on follicular growth, ovulation timing, and pregnancy per artificial insemination (AI) in cosynchronized postpartum Nili Ravi buffaloes. In this study, 58 Nili Ravi postpartum buffaloes (DIM = 103 ± 1.64) were randomly divided into two treatment groups (n = 29/treatment): GnRH-TAI-84 and TAI-84. All buffaloes were administered a MAP sponge for seven days. Upon MAP sponge removal, all the subjects received prostaglandin F<inf>2α</inf> (PGF<inf>2α</inf>) and Timed AI (TAI) was performed 84 h after sponge removal. In the GnRH-TAI-84 group, the buffaloes received GnRH alongside insemination, whereas in the TAI-84 group, the buffaloes were inseminated without GnRH administration. Follicle diameter and blood estradiol levels were measured every 6 h from 72-108 h after MAP sponge removal. The animals were checked for pregnancy using ultrasonography 40 days after AI. Animals subjected to the GnRH-TAI-84 protocol had a higher follicular growth rate and preovulatory follicle size than those in the TAI-84 group. The follicular diameter was also larger in animals that received GnRH-TAI-84 than in those that received TAI-84 90 and 96 h after MAP sponge removal. Buffaloes in the GnRH-TAI-84 group had lower estradiol concentrations at 90, 96, 102, and 108 h than those in the TAI-84 group. Ovulation in GnRH-TAI-84 buffaloes occurred 11 h earlier than that in buffaloes from the TAI-84 group. A shorter interval between AI and ovulation in GnRH-TAI-84 buffaloes (14 h vs. 25 h) led to greater pregnancies per AI (62% vs. 17%) compared to buffaloes from the TAI-84 group.</p>","PeriodicalId":16942,"journal":{"name":"Journal of Reproduction and Development","volume":" ","pages":"349-355"},"PeriodicalIF":1.9,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11658920/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142055917","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Serum concentrations of anti-Müllerian hormone modulate ovarian response to different doses of follicle-stimulating hormone in Japanese Black donors.","authors":"Hiroaki Okawa, Norihiro Yukiyama, Oky Setyo Widodo, Kanae Niimi, Yuta Chuman, Yasuo Fushimi, Mitsuhiro Takagi","doi":"10.1262/jrd.2024-067","DOIUrl":"10.1262/jrd.2024-067","url":null,"abstract":"<p><p>Anti-Müllerian hormone (AMH) is a marker for predicting embryo production in cows subjected to superovulation; however, it remains to be established as a reliable predictor of reproductive performance. We hypothesized that the serum AMH concentration of donors affects the ovarian response to follicle-stimulating hormone (FSH) treatment during superovulation. Herein, we retrospectively investigated the association between different FSH doses and AMH concentrations in donor Japanese Black cows in a superovulation program and analyzed the number of total and transferable embryos recovered. The number of transferable embryos recovered from donors with high AMH levels was significantly higher than that recovered from donors with low AMH levels. Additionally, it increased further with a reduction in the FSH dose. These results illustrate that the AMH concentration is a useful marker for predicting embryo production after superovulation, and donors with high AMH levels produce more transferable embryos at low FSH doses than at high doses.</p>","PeriodicalId":16942,"journal":{"name":"Journal of Reproduction and Development","volume":" ","pages":"418-422"},"PeriodicalIF":1.9,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11658917/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142622345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}