{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,10]],"date-time":"2026-10-10T04:28:26Z","timestamp":1791606506965,"version":"4.3.4"},"reference-count":42,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2022,3,19]],"date-time":"2022-03-19T00:00:00Z","timestamp":1647648000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["21H00357"],"award-info":[{"award-number":["21H00357"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>The Tsushima leopard cat (Prionailurus bengalensis euptilurus) is an endangered wild felid that lives solely on Tsushima Island, Nagasaki, Japan. Japan\u2019s Ministry of the Environment is working with the Japanese Association of Zoo and Aquariums for ex situ conservation of this species. However, considering genetic diversity, it is difficult to conduct captive breeding programs by natural breeding alone; hence, assisted reproductive technologies are required. This study aimed to breed Tsushima leopard cats, which otherwise cannot be paired, using laparoscopic oviductal artificial insemination (AI). Female Tsushima leopard cats (female 1, aged 7 years; female 2, aged 6 years) were treated with 200 IU equine chorionic gonadotropin, followed by administration of 1000 IU porcine luteinizing hormone (pLH) after 96 h to induce ovulation. Laparoscopic AI was performed 32 h post-pLH administration. Females 1 and 2 were inseminated in the oviduct with 2.4 \u00d7 10\u2076 and 3.3 \u00d7 10\u2076 motile spermatozoa, respectively, collected from two males. Pregnancy was confirmed by radiography 45 and 51 days post-AI in females 1 and 2, respectively; one fetus was found in female 2. Moreover, female 2 had spontaneous delivery of a female kitten 66 days post-AI. This is the first successful case of AI in a Tsushima leopard cat.<\/jats:p>","DOI":"10.3390\/ani12060777","type":"journal-article","created":{"date-parts":[[2022,3,20]],"date-time":"2022-03-20T21:25:00Z","timestamp":1647811500000},"page":"777","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Successful Laparoscopic Oviductal Artificial Insemination in the Endangered Tsushima Leopard Cat (Prionailurus bengalensis euptilurus)"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1193-1663","authenticated-orcid":false,"given":"Akinori","family":"Azumano","sequence":"first","affiliation":[{"name":"Yokohama Zoological Gardens, Yokohama 241-0001, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miya","family":"Ueda","sequence":"additional","affiliation":[{"name":"Yokohama Zoological Gardens, Yokohama 241-0001, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mika","family":"Nomura","sequence":"additional","affiliation":[{"name":"Yokohama Zoological Gardens, Yokohama 241-0001, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masashi","family":"Usui","sequence":"additional","affiliation":[{"name":"Yokohama Zoological Gardens, Yokohama 241-0001, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Midori","family":"Ichinose","sequence":"additional","affiliation":[{"name":"Preservation and Research Center, Yokohama 241-0804, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7625-8492","authenticated-orcid":false,"given":"Yojiro","family":"Yanagawa","sequence":"additional","affiliation":[{"name":"Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3451-1939","authenticated-orcid":false,"given":"Satoshi","family":"Kusuda","sequence":"additional","affiliation":[{"name":"Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9143-3498","authenticated-orcid":false,"given":"Yuki","family":"Matsumoto","sequence":"additional","affiliation":[{"name":"Anicom Specialty Medical Institute Inc., Yokohama 231-0033, Japan"},{"name":"Anicom Insurance, Inc., Tokyo 171-0033, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koichi","family":"Murata","sequence":"additional","affiliation":[{"name":"Yokohama Zoological Gardens, Yokohama 241-0001, Japan"},{"name":"College of Bioresource Science, Nihon University, Fujisawa 252-0880, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,3,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"655","DOI":"10.2108\/zsj.12.655","article-title":"Two Japanese Wildcats, the Tsushima Cat and the Iriomote Cat, Show the Same Mitochondrial DNA Lineage as the Leopard Cat Felis bengalensis","volume":"12","author":"Masuda","year":"1995","journal-title":"Zool. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1884","DOI":"10.1016\/j.biocon.2009.05.005","article-title":"Ecology and Conservation of Two Endangered Subspecies of the Leopard Cat (Prionailurus bengalensis) on Japanese Islands","volume":"142","author":"Izawa","year":"2009","journal-title":"Biol. Conserv."},{"key":"ref_3","first-page":"94","article-title":"Husbandry and Breeding of the Tsushima Leopard Cat, Felis bengalensis euptilura","volume":"43","author":"Machii","year":"2002","journal-title":"J Jpn. Assoc. Zool. Aquat."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1326","DOI":"10.1016\/j.theriogenology.2007.07.022","article-title":"Pregnancy in the Domestic Cat After Vaginal or Transcervical Insemination with Fresh and Frozen Semen","volume":"68","author":"Chatdarong","year":"2007","journal-title":"Theriogenology"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1002\/zoo.1430010106","article-title":"Induction of ovulation and successful artificial insemination in a Persian leopard (Panthera pardus saxicolor)","volume":"1","author":"Dresser","year":"1982","journal-title":"Zoo Biol."},{"key":"ref_6","first-page":"288","article-title":"Successful induction of ovarian activity and laparoscopic intrauterine artificial insemination in the cheetah (Acinonyx jubatus)","volume":"23","author":"Howard","year":"1992","journal-title":"J. Zoo Wildl. Med."},{"key":"ref_7","first-page":"185","article-title":"Birth of a Siberian tiger cub (Panthera tigris altaica) following laparoscopic intrauterine artificial insemination","volume":"23","author":"Donoghue","year":"1993","journal-title":"J. Zoo Wildl. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1530\/jrf.0.1010103","article-title":"Gonadotrophin dose and timing of anaesthesia for laparoscopic artificial insemination in the puma (Felis concolor)","volume":"101","author":"Barone","year":"1994","journal-title":"Reproduction"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1530\/jrf.0.1060087","article-title":"Responsiveness of ovaries to exogenous gonadotrophins and laparoscopic artificial insemination with frozen\u2013thawed spermatozoa in ocelots (Felis pardalis)","volume":"106","author":"Swanson","year":"1996","journal-title":"Reproduction"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1530\/jrf.0.1070053","article-title":"Timing of ovulation after gonadotrophin induction and its importance to successful intrauterine insemination in the tiger (Panthera tigris)","volume":"107","author":"Donoghue","year":"1996","journal-title":"Reproduction"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1002\/(SICI)1098-2361(1996)15:1<55::AID-ZOO6>3.0.CO;2-B","article-title":"Successful ovulation induction and laparoscopic intrauterine artificial insemination in the clouded leopard (Neofelis nebulosa)","volume":"15","author":"Howard","year":"1996","journal-title":"Zoo Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1071\/R96048","article-title":"Seasonal effects on ovarian responsiveness to exogenous gonadotrophins and successful artificial insemination in the snow leopard (Uncia uncia)","volume":"9","author":"Roth","year":"1997","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1095\/biolreprod56.4.1059","article-title":"Sensitivity to exogenous gonadotropins for ovulation induction and laparoscopic artificial insemination in the cheetah and clouded leopard","volume":"56","author":"Howard","year":"1997","journal-title":"Biol. Reprod."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.theriogenology.2008.09.046","article-title":"Approaches and Efficacy of Artificial Insemination in Felids and Mustelids","volume":"71","author":"Howard","year":"2009","journal-title":"Theriogenology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"372","DOI":"10.1638\/2013-0269R.1","article-title":"Successful Nonsurgical Artificial Insemination and Hormonal Monitoring in an Asiatic Golden Cat (Catopuma temmincki)","volume":"45","author":"Lueders","year":"2014","journal-title":"J. Zoo Wildl. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.theriogenology.2019.07.022","article-title":"Non-Surgical Artificial Insemination Using a GnRH Analogue for Ovulation Induction During Natural Oestrus in African Lions (Panthera leo)","volume":"139","author":"Callealta","year":"2019","journal-title":"Theriogenology"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1095\/biolreprod.112.105353","article-title":"Laparoscopic oviductal artificial insemination improves pregnancy success in exogenous gonadotropin-treated domestic cats as a model for endangered felids","volume":"89","author":"Conforti","year":"2013","journal-title":"Biol. Reprod."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1071\/RDv26n1Ab3","article-title":"3 Application of Laparoscopic Oviductal Artificial Insemination for Conservation Management of Brazilian Ocelots and Amur Tigers","volume":"26","author":"Lambo","year":"2014","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1071\/RDv28n2Ab121","article-title":"121 Fixed-Time Laparoscopic Oviductal Artificial Insemination (LO-AI) with Frozen\u2013Thawed Semen in Pallas\u2019 Cats (Otocolobus manul)","volume":"28","author":"Swanson","year":"2016","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1071\/RD18350","article-title":"Practical Application of Laparoscopic Oviductal Artificial Insemination for the Propagation of Domestic Cats and Wild Felids","volume":"31","author":"Swanson","year":"2018","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1499","DOI":"10.1016\/j.theriogenology.2010.04.031","article-title":"Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus)","volume":"74","author":"Adachi","year":"2010","journal-title":"Theriogenology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1117","DOI":"10.1016\/S0093-691X(00)00420-9","article-title":"Chorionic gonadotropin administration in domestic cats causes an abnormal endocrine environment that disrupts oviductal embryo transport","volume":"54","author":"Graham","year":"2000","journal-title":"Theriogenology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"204","DOI":"10.1002\/j.1939-4640.1990.tb03229.x","article-title":"Teratospermic and Normospermic Domestic Cats: Ejaculate Traits, Pituitary-Gonadal Hormones, and Improvement of Spermatozoal Motility and Morphology After Swim-Up Processing","volume":"11","author":"Howard","year":"1990","journal-title":"J. Androl."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1016\/j.theriogenology.2007.10.019","article-title":"Quality and In Vitro Fertilizing Ability of Cryopreserved Cat Spermatozoa Obtained by Urethral Catheterization After Medetomidine Administration","volume":"69","author":"Zambelli","year":"2008","journal-title":"Theriogenology"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1093\/humrep\/deg199","article-title":"Evaluation of the One-Step Eosin-Nigrosin Staining Technique for Human Sperm Vitality Assessment","volume":"18","author":"Kvist","year":"2003","journal-title":"Hum. Reprod."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"858","DOI":"10.1095\/biolreprod.106.058065","article-title":"Toward a Feline-Optimized Culture Medium: Impact of Ions, Carbohydrates, Essential Amino Acids, Vitamins, and Serum on Development and Metabolism of In Vitro Fertilization-Derived Feline Embryos Relative to Embryos Grown In Vivo","volume":"76","author":"Herrick","year":"2007","journal-title":"Biol. Reprod."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e7297","DOI":"10.7717\/peerj.7297","article-title":"The Tsushima Leopard Cat Exhibits Extremely Low Genetic Diversity Compared with the Korean Amur Leopard Cat: Implications for Conservation","volume":"7","author":"Ito","year":"2019","journal-title":"PeerJ"},{"key":"ref_28","unstructured":"Babraham Institute (2019). Trim Galore, Version 0.6.5, Babraham Institute. Available online: https:\/\/www.bioinformatics.babraham.ac.uk\/projects\/trim_galore\/."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Buckley, R.M., Davis, B.W., Brashear, W.A., Farias, F.H.G., Kuroki, K., Graves, T., Hillier, L.W., Kremitzki, M., Li, G., and Middleton, R.P. (2020). A New Domestic Cat Genome Assembly Based on Long Sequence Reads Empowers Feline Genomic Medicine and Identifies a Novel Gene for Dwarfism. PLoS Genet., 16.","DOI":"10.1101\/2020.01.06.896258"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1186\/s13742-015-0047-8","article-title":"Second-Generation PLINK: Rising to the Challenge of Larger and Richer Datasets","volume":"4","author":"Chang","year":"2015","journal-title":"GigaScience"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2867","DOI":"10.1093\/bioinformatics\/btq559","article-title":"Robust Relationship Inference in Genome-Wide Association Studies","volume":"26","author":"Manichaikul","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.theriogenology.2006.03.024","article-title":"Application of Assisted Reproduction for Population Management in Felids: The Potential and Reality for Conservation of Small Cats","volume":"66","author":"Swanson","year":"2006","journal-title":"Theriogenology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1030","DOI":"10.1111\/j.1365-294X.2008.04062.x","article-title":"Cryobanking of Viable Biomaterials: Implementation of New Strategies for Conservation Purposes","volume":"18","author":"Lermen","year":"2009","journal-title":"Mol. Ecol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"677","DOI":"10.1292\/jvms1939.51.677","article-title":"Induced Ovulation in Cats Using Porcine Pituitary Gland Preparation During the Non-Breeding Season","volume":"51","author":"Tsutsui","year":"1989","journal-title":"Jpn. J. Vet. Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1292\/jvms.15-0683","article-title":"Intrauterine Insemination with Fresh Semen in Amur Leopard Cat (Pionailurus bengalensis eutilura) During Non-Breeding Season","volume":"79","author":"Tajima","year":"2017","journal-title":"J. Vet. Med. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1095\/biolreprod53.1.93","article-title":"Natural Versus Chorionic Gonadotropin-Induced Ovarian Responses in the Clouded Leopard (Neofelis nebulosa) Assessed by Fecal Steroid Analysis","volume":"53","author":"Brown","year":"1995","journal-title":"Biol. Reprod."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"804","DOI":"10.1638\/2016-0287.1","article-title":"Successful Laparoscopic Oviductal Artificial Insemination in the Clouded Leopard (Neofelis nebulosa) in Thailand","volume":"48","author":"Tipkantha","year":"2017","journal-title":"J. Zoo Wildl. Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.theriogenology.2018.06.004","article-title":"Characterization and Cryopreservation of Amur Leopard Cats (Prionailurus bengalensis euptilurus) Semen Collected by Urethral Catheterization","volume":"119","author":"Jeong","year":"2018","journal-title":"Theriogenology"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1163","DOI":"10.1292\/jvms.62.1163","article-title":"Artificial Intravaginal Insemination Using Fresh Semen in Cats","volume":"62","author":"Tanaka","year":"2000","journal-title":"J. Vet. Med. Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1241","DOI":"10.1292\/jvms.62.1241","article-title":"Unilateral Intrauterine Horn Insemination of Fresh Semen in Cats","volume":"62","author":"Tsutsui","year":"2000","journal-title":"J. Vet. Med. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1071\/RD05052","article-title":"Semen Characteristics and Sperm Morphology in the Arabian Leopard (Panthera pardus nimr) and How These Vary with Age and Season","volume":"17","author":"Strick","year":"2005","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1002\/zoo.10043","article-title":"Reproductive Endocrine Responses to Photoperiod and Exogenous Gonadotropins in the Pallas\u2019 Cat (Otocolobus manul)","volume":"21","author":"Brown","year":"2002","journal-title":"Zoo Biol."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/12\/6\/777\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:39:30Z","timestamp":1760135970000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/12\/6\/777"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,3,19]]},"references-count":42,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2022,3]]}},"alternative-id":["ani12060777"],"URL":"https:\/\/doi.org\/10.3390\/ani12060777","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,3,19]]}}}