{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T02:45:46Z","timestamp":1781232346031,"version":"3.54.1"},"reference-count":64,"publisher":"The Royal Society","issue":"3","license":[{"start":{"date-parts":[[2021,3,1]],"date-time":"2021-03-01T00:00:00Z","timestamp":1614556800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/royalsociety.org\/journals\/ethics-policies\/data-sharing-mining\/"}],"funder":[{"DOI":"10.13039\/501100008530","name":"European Regional Development Fund","doi-asserted-by":"crossref","award":["Portugal 2020, COMPETE 2020, 038433 - REARLING"],"award-info":[{"award-number":["Portugal 2020, COMPETE 2020, 038433 - REARLING"]}],"id":[{"id":"10.13039\/501100008530","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003030","name":"Ag\u00e8ncia de Gesti\u00f3 d'Ajuts Universitaris i de Recerca","doi-asserted-by":"crossref","award":["PhD Grant"],"award-info":[{"award-number":["PhD Grant"]}],"id":[{"id":"10.13039\/501100003030","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003141","name":"Consejo Nacional de Ciencia y Tecnolog\u00eda","doi-asserted-by":"crossref","award":["PhD Grant"],"award-info":[{"award-number":["PhD Grant"]}],"id":[{"id":"10.13039\/501100003141","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Instituto Nacional de Investigaci\u00f3n y Tecnolog\u00eda Agraria y Alimentaci\u00f3n","award":["RTA2014-0048"],"award-info":[{"award-number":["RTA2014-0048"]}]}],"content-domain":{"domain":["royalsocietypublishing.org"],"crossmark-restriction":true},"short-container-title":["R. Soc. open sci."],"published-print":{"date-parts":[[2021,3]]},"abstract":"<jats:p>\n                    Cultured Senegalese sole (\n                    <jats:italic>Solea senegalensis<\/jats:italic>\n                    ) breeders fail to spawn fertilized eggs. The implantation of large-scale\n                    <jats:italic>in vitro<\/jats:italic>\n                    fertilization protocols, to solve this problem, has been frustrated by low production of poor quality sperm. Cultured females were induced to ovulate with a 5 \u00b5g kg\n                    <jats:sup>\u22121<\/jats:sup>\n                    single injection of gonadotropin-releasing hormone agonist (GnRHa) and viable eggs (82.6 \u00b1 9.2% fertilization) were stripped 41:57 \u00b1 1:46 h after the injection. Sperm was collected from cultured males, diluted in modified Leibovitz and used fresh to fertilize the eggs. Males were not treated with hormones. A nonlinear regression, an exponential rise to a maximum (\n                    <jats:italic>R<\/jats:italic>\n                    = 0.93,\n                    <jats:italic>p<\/jats:italic>\n                    &lt; 0.0001) described the number of motile spermatozoa required to fertilize a viable egg and 1617 motile spermatozoa were sufficient to fertilize 99 \u00b1 12% (\u00b195% CI) of viable eggs. Similar, spermatozoa egg\n                    <jats:sup>\u22121<\/jats:sup>\n                    ratios of 592 \u00b1 611 motile spermatozoa egg\n                    <jats:sup>\u22121<\/jats:sup>\n                    were used in large-scale\n                    <jats:italic>in vitro<\/jats:italic>\n                    fertilizations (190 512 \u00b1 38 471 eggs). The sperm from a single male (145 \u00b1 50 \u00b5l or 8.0 \u00b1 6.8 \u00d7 10\n                    <jats:sup>8<\/jats:sup>\n                    spermatozoa) was used to fertilize the eggs. The mean hatching rate was 70 \u00b1 14% to provide 131 540 \u00b1 34 448 larvae per fertilization. The viability of unfertilized eggs stored at room temperature decreased gradually, and the sooner eggs were fertilized after stripping, the higher the viability of the eggs. The collection of sperm directly into a syringe containing modified Leibovitz significantly increased the percentage of motile spermatozoa (33.4 \u00b1 12.2%) compared with other collection methods. The spz egg\n                    <jats:sup>\u22121<\/jats:sup>\n                    ratios for Senegalese sole were at the lower end of ratios required for fish. Senegalese sole have a pair-spawning reproductive behaviour characterized by gamete fertilization in close proximity with no sperm competition. The provision of a large-scale\n                    <jats:italic>in vitro<\/jats:italic>\n                    fertilization protocol (200 \u00b5l of sperm per 100 ml of eggs) will enable the industry to operate sustainably and implement breeding programmes to improve production.\n                  <\/jats:p>","DOI":"10.1098\/rsos.201718","type":"journal-article","created":{"date-parts":[[2021,3,10]],"date-time":"2021-03-10T03:06:56Z","timestamp":1615345616000},"update-policy":"https:\/\/doi.org\/10.1098\/crossmark-policy","source":"Crossref","is-referenced-by-count":8,"title":["Low sperm to egg ratio required for successful\n                    <i>in vitro<\/i>\n                    fertilization in a pair-spawning teleost, Senegalese sole (\n                    <i>Solea senegalensis<\/i>\n                    )"],"prefix":"10.1098","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4242-5894","authenticated-orcid":true,"given":"Sandra","family":"Ramos-J\u00fadez","sequence":"first","affiliation":[{"name":"IRTA, Sant Carles de la R\u00e0pita, C. Poble Nou km. 5.5, 43540 Sant Carles de la R\u00e0pita, Tarragona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4378-9123","authenticated-orcid":true,"given":"Wendy \u00c1ngela","family":"Gonz\u00e1lez-L\u00f3pez","sequence":"additional","affiliation":[{"name":"IRTA, Sant Carles de la R\u00e0pita, C. Poble Nou km. 5.5, 43540 Sant Carles de la R\u00e0pita, Tarragona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5983-0557","authenticated-orcid":true,"given":"Jhons","family":"Huayanay Ostos","sequence":"additional","affiliation":[{"name":"IRTA, Sant Carles de la R\u00e0pita, C. Poble Nou km. 5.5, 43540 Sant Carles de la R\u00e0pita, Tarragona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8944-8159","authenticated-orcid":true,"given":"Noem\u00ed","family":"Cota Mamani","sequence":"additional","affiliation":[{"name":"Direcci\u00f3n General de Investigaciones en Acuicultura, Instituto del Mar del Per\u00fa (IMARPE), Lima, Peru"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5726-6710","authenticated-orcid":true,"given":"Carlos","family":"Marrero Alem\u00e1n","sequence":"additional","affiliation":[{"name":"IRTA, Sant Carles de la R\u00e0pita, C. Poble Nou km. 5.5, 43540 Sant Carles de la R\u00e0pita, Tarragona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4444-7563","authenticated-orcid":true,"given":"Jos\u00e9","family":"Beir\u00e3o","sequence":"additional","affiliation":[{"name":"Faculty of Biosciences and Aquaculture, Nord University, 8049 Bod\u00f8, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5456-9217","authenticated-orcid":true,"given":"Neil","family":"Duncan","sequence":"additional","affiliation":[{"name":"IRTA, Sant Carles de la R\u00e0pita, C. Poble Nou km. 5.5, 43540 Sant Carles de la R\u00e0pita, Tarragona, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"175","published-online":{"date-parts":[[2021,3,10]]},"reference":[{"key":"e_1_3_7_2_2","unstructured":"APROMAR. 2019 La acuicultura en Espa\u00f1a 2019 Asociaci\u00f3n Empresarial de Acuicultura de Espa\u00f1a (APROMAR) Cadiz Spain 1\u201390. See http:\/\/apromar.es\/content\/informes-anuales."},{"key":"e_1_3_7_3_2","doi-asserted-by":"publisher","DOI":"10.1111\/raq.12091"},{"key":"e_1_3_7_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2004.09.026"},{"key":"e_1_3_7_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2013.12.042"},{"key":"e_1_3_7_6_2","volume-title":"Biology of sole","author":"Duncan N","year":"2019","unstructured":"Duncan N, Carazo I, Chereguini O, Ma\u00f1an\u00f3s E. 2019 Mating behaviour. In Biology of sole (eds J Munoz-Cueto, E Ma\u00f1an\u00f3s-S\u00e1nchez, J S\u00e1nchez-V\u00e1zquez). Boca Raton, FL: CRC Press."},{"key":"e_1_3_7_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2020.734922"},{"key":"e_1_3_7_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ygcen.2008.02.002"},{"key":"e_1_3_7_9_2","doi-asserted-by":"publisher","DOI":"10.5424\/sjar\/2019174-14953"},{"key":"e_1_3_7_10_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cbpa.2010.11.016"},{"key":"e_1_3_7_11_2","doi-asserted-by":"publisher","DOI":"10.1071\/RD18453"},{"key":"e_1_3_7_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ygcen.2013.06.006"},{"key":"e_1_3_7_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2012.10.035"},{"key":"e_1_3_7_14_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2012.08.016"},{"key":"e_1_3_7_15_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ygcen.2012.04.009"},{"key":"e_1_3_7_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2012.05.018"},{"key":"e_1_3_7_17_2","doi-asserted-by":"publisher","DOI":"10.1242\/jeb.150318"},{"key":"e_1_3_7_18_2","first-page":"3","volume-title":"Methods in reproductive aquaculture","author":"Ma\u00f1an\u00f3s E","year":"2008","unstructured":"Ma\u00f1an\u00f3s E, Duncan N, Mylonas C. 2008 Reproduction and control of ovulation, spermiation and spawning in cultured fish. In Methods in reproductive aquaculture (eds E Cabrita, V Robles, P Herr\u00e1ez), pp. 3-80. Boca Raton, FL: CRC Press. (doi:10.1201\/9780849380549.sec1)"},{"key":"e_1_3_7_19_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.anireprosci.2011.04.009"},{"key":"e_1_3_7_20_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.theriogenology.2009.07.021"},{"key":"e_1_3_7_21_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.theriogenology.2010.07.003"},{"key":"e_1_3_7_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2006.08.020"},{"key":"e_1_3_7_23_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2019.734649"},{"key":"e_1_3_7_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2013.02.011"},{"key":"e_1_3_7_25_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2011.11.021"},{"key":"e_1_3_7_26_2","first-page":"10","article-title":"Large scale artificial reproduction and rearing of Senegal sole, Solea senegalensis Kaup","volume":"29","author":"Liu X","year":"2008","unstructured":"Liu X, Liu X, Lian J, Wang Y, Zhang F, Yu H, Ma A, Liu S, Zhai J. 2008 Large scale artificial reproduction and rearing of Senegal sole, Solea senegalensis Kaup. Marine Fisheries Research 29, 10-16.","journal-title":"Marine Fisheries Research"},{"key":"e_1_3_7_27_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.theriogenology.2019.05.004"},{"key":"e_1_3_7_28_2","doi-asserted-by":"publisher","DOI":"10.3354\/ab00439"},{"key":"e_1_3_7_29_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2109.1999.00326.x"},{"key":"e_1_3_7_30_2","doi-asserted-by":"publisher","DOI":"10.1016\/0044-8486(94)00395-5"},{"key":"e_1_3_7_31_2","doi-asserted-by":"publisher","DOI":"10.5424\/sjar\/2016144-9108"},{"key":"e_1_3_7_32_2","doi-asserted-by":"publisher","DOI":"10.1002\/9780470995259"},{"key":"e_1_3_7_33_2","doi-asserted-by":"publisher","DOI":"10.1111\/ele.13431"},{"key":"e_1_3_7_34_2","doi-asserted-by":"crossref","first-page":"897","DOI":"10.1111\/j.1469-185X.2010.00140.x","article-title":"Sperm competition and ejaculate economics","volume":"85","author":"Parker GA","year":"2010","unstructured":"Parker GA, Pizzari T. 2010 Sperm competition and ejaculate economics. Biol. Rev. 85, 897-934. (doi:10.1111\/j.1469-185X.2010.00140.x)","journal-title":"Biol. Rev."},{"key":"e_1_3_7_35_2","doi-asserted-by":"publisher","DOI":"10.1086\/286031"},{"key":"e_1_3_7_36_2","first-page":"1179","article-title":"Male reproductive system in Senegalese sole Solea senegalensis (Kaup): anatomy, histology and histochemistry","volume":"20","author":"Garc\u00eda-L\u00f3pez \u00c1","year":"2005","unstructured":"Garc\u00eda-L\u00f3pez \u00c1, Mart\u00ednez-Rodr\u00edguez G, Sarasquete C. 2005 Male reproductive system in Senegalese sole Solea senegalensis (Kaup): anatomy, histology and histochemistry. Histol. Histopathol. 20, 1179-1189. (doi:10.14670\/HH-20.1179)","journal-title":"Histol. Histopathol."},{"key":"e_1_3_7_37_2","unstructured":"Marrero-Alem\u00e1n C Gonz\u00e1lez-L\u00f3pez W Ramos-J\u00fadez S Navarro I Duncan N. 2019 Artificial fertilisation in Senegalese sole ( Solea senegalensis ): induction with GnRHa and determination of egg quality. In Aquaculture Europe 19 Abstracts Berlin Germany."},{"key":"e_1_3_7_38_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2006.02.001"},{"key":"e_1_3_7_39_2","volume-title":"NIST\/SEMATECH e-engineering statistics handbook","author":"Prins J","year":"2012","unstructured":"Prins J. 2012 Product and process comparisons. In NIST\/SEMATECH e-engineering statistics handbook (eds C Croarkin, P Tobias)."},{"key":"e_1_3_7_40_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2008.09.005"},{"key":"e_1_3_7_41_2","first-page":"103","volume-title":"Intensive fish farming","author":"Bromage N","year":"1992","unstructured":"Bromage N. 1992 Propagation and stock improvement. In Intensive fish farming (eds N Bromage, J Shepherd), pp. 103-153. Oxford, UK: Blackwell Science."},{"key":"e_1_3_7_42_2","volume-title":"Practical flatfish culture and stock enhancement","author":"Brown N","year":"2010","unstructured":"Brown N. 2010 Halibut aquaculture in North America. In Practical flatfish culture and stock enhancement (eds H Daniels, W Watanabe). Ames, IA: Blackwell Publishing."},{"key":"e_1_3_7_43_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2016.04.021"},{"key":"e_1_3_7_44_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2019.05.033"},{"key":"e_1_3_7_45_2","first-page":"12 309","article-title":"Effects of storage duration and storage temperature on viability of stored ova of kutum (Rutilus frisii kutum) in ovarian fluid","volume":"10","author":"Samarin AM","year":"2011","unstructured":"Samarin AM, Amiri BM, Soltani M, Mohammad R, Kamali A, Naghavi MR. 2011 Effects of storage duration and storage temperature on viability of stored ova of kutum (Rutilus frisii kutum) in ovarian fluid. Afr. J. Biotechnol. 10, 12 309-12 314. (doi:10.5897\/AJB11.919)","journal-title":"Afr. J. Biotechnol."},{"key":"e_1_3_7_46_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0093-691X(03)00108-0"},{"key":"e_1_3_7_47_2","doi-asserted-by":"publisher","DOI":"10.2478\/v10020-010-0026-6"},{"key":"e_1_3_7_48_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1439-0426.2003.00470.x"},{"key":"e_1_3_7_49_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0044-8486(97)00301-3"},{"key":"e_1_3_7_50_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.theriogenology.2006.06.003"},{"key":"e_1_3_7_51_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2015.06.040"},{"key":"e_1_3_7_52_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.theriogenology.2013.01.019"},{"key":"e_1_3_7_53_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cbpa.2018.06.022"},{"key":"e_1_3_7_54_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0174387"},{"key":"e_1_3_7_55_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2009.03.024"},{"key":"e_1_3_7_56_2","doi-asserted-by":"publisher","DOI":"10.1590\/S1516-35982013000100001"},{"key":"e_1_3_7_57_2","doi-asserted-by":"publisher","DOI":"10.1590\/1982-0224-20140158"},{"key":"e_1_3_7_58_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.aquaculture.2013.08.035"},{"key":"e_1_3_7_59_2","doi-asserted-by":"publisher","DOI":"10.1016\/0093-691X(87)90061-6"},{"key":"e_1_3_7_60_2","doi-asserted-by":"publisher","DOI":"10.1139\/f06-108"},{"key":"e_1_3_7_61_2","doi-asserted-by":"publisher","DOI":"10.1093\/biolre\/iox015"},{"key":"e_1_3_7_62_2","first-page":"698","article-title":"Tropical reef fish spawning aggregations: defined and reviewed","volume":"60","author":"Domeier ML","year":"1997","unstructured":"Domeier ML, Colin PL. 1997 Tropical reef fish spawning aggregations: defined and reviewed. Bull. Mar. Sci. 60, 698-726.","journal-title":"Bull. Mar. Sci."},{"key":"e_1_3_7_63_2","doi-asserted-by":"publisher","DOI":"10.5424\/sjar\/2015131-6750"},{"key":"e_1_3_7_64_2","doi-asserted-by":"publisher","DOI":"10.1643\/0045-8511(2006)6[735:GMITCC]2.0.CO;2"},{"key":"e_1_3_7_65_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0809990106"}],"container-title":["Royal Society Open Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/royalsocietypublishing.org\/doi\/pdf\/10.1098\/rsos.201718","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/royalsocietypublishing.org\/doi\/full-xml\/10.1098\/rsos.201718","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/royalsocietypublishing.org\/doi\/pdf\/10.1098\/rsos.201718","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,8,25]],"date-time":"2024-08-25T15:20:10Z","timestamp":1724599210000},"score":1,"resource":{"primary":{"URL":"https:\/\/royalsocietypublishing.org\/doi\/10.1098\/rsos.201718"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3]]},"references-count":64,"journal-issue":{"issue":"3","published-print":{"date-parts":[[2021,3]]}},"alternative-id":["10.1098\/rsos.201718"],"URL":"https:\/\/doi.org\/10.1098\/rsos.201718","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2020.08.26.267740","asserted-by":"object"}]},"ISSN":["2054-5703"],"issn-type":[{"value":"2054-5703","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3]]},"assertion":[{"value":"2020-09-24","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2021-02-10","order":1,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2021-03-10","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"rsos.201718"}}