{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,11]],"date-time":"2026-02-11T12:58:30Z","timestamp":1770814710005,"version":"3.50.1"},"reference-count":47,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,2,10]],"date-time":"2026-02-10T00:00:00Z","timestamp":1770681600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>In Italy, the breeding of alpacas (Vicugna pacos) is on the rise, driven by the value of their precious wool and their docile temperament. This growth is, however, severely limited by the species\u2019 reproductive inefficiency. This study aimed to investigate the specific role of the neuropeptide orexin A (OxA) and its receptor OX1R in modulating testicular steroidogenesis in the alpaca, testing the hypothesis that the OxA\/OX1R pathway stimulates testosterone production. Using testicular tissue from two groups of adult male alpacas (five- and seven-year-olds, n = 6 per group), we combined immunohistochemistry with ex vivo functional assays. Immunohistochemical analysis confirmed the expression of both OxA and OX1R in Leydig cells. Functional studies on testicular fragments demonstrated that OxA enhanced testosterone synthesis via OX1R activation. Furthermore, OxA reduced estradiol levels by suppressing aromatase activity and counteracted the inhibitory effect of M\u00fcllerian inhibiting substance (MIS) on testosterone secretion. These results provide the first evidence that the peripheral OX1R pathway is functionally involved in promoting a testosterone-dominant steroidogenic milieu in alpaca testes. This preliminary study identifies the orexin system as a potential target for developing strategies to improve reproductive management in this species.<\/jats:p>","DOI":"10.3390\/ani16040545","type":"journal-article","created":{"date-parts":[[2026,2,11]],"date-time":"2026-02-11T09:16:08Z","timestamp":1770801368000},"page":"545","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Preliminary Study on the Role of Orexin A in Leydig Cell Steroidogenesis and Its Implications for Fertility in Alpacas (Vicugna pacos)"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9600-6401","authenticated-orcid":false,"given":"Fabio","family":"Castagna","sequence":"first","affiliation":[{"name":"Department of Health Sciences, University of Catanzaro Magna Graecia, 88100 Catanzaro, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4173-8584","authenticated-orcid":false,"given":"Roberto","family":"Bava","sequence":"additional","affiliation":[{"name":"Department of Health Sciences, University of Catanzaro Magna Graecia, 88100 Catanzaro, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-0401-4027","authenticated-orcid":false,"given":"Stefano","family":"Ruga","sequence":"additional","affiliation":[{"name":"Department of Health Sciences, University of Catanzaro Magna Graecia, 88100 Catanzaro, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6628-2459","authenticated-orcid":false,"given":"Emilia","family":"Langella","sequence":"additional","affiliation":[{"name":"School of Agricultural, Forestry, Food and Environmental Sciences (SAFE), University of Basilicata, 85100 Potenza, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna","family":"Costagliola","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine and Animal Production, University of Naples Federico II, 80137 Naples, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-7759-2141","authenticated-orcid":false,"given":"Renato","family":"Lombardi","sequence":"additional","affiliation":[{"name":"Local Health Authority (ASL), 71121 Foggia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicola","family":"Mirabella","sequence":"additional","affiliation":[{"name":"Department of Veterinary Medicine and Animal Production, University of Naples Federico II, 80137 Naples, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3031-345X","authenticated-orcid":false,"given":"Giovanna","family":"Liguori","sequence":"additional","affiliation":[{"name":"Local Health Authority (ASL), 71121 Foggia, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,10]]},"reference":[{"key":"ref_1","first-page":"97","article-title":"Fibre Quality of South American Camelids in Argentina: A Review","volume":"56","author":"Mueller","year":"2015","journal-title":"Anim. Genet. Resour. Resour. G\u00e9n\u00e9tiques Anim. Recur. Gen\u00e9ticos Anim."},{"key":"ref_2","first-page":"551","article-title":"From the Andes to the Outback: Acclimatising Alpacas in the British Empire","volume":"45","author":"Cowie","year":"2017","journal-title":"J. Imp. Commonw. Hist."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"407","DOI":"10.17221\/8415-VETMED","article-title":"The Influence of Sex, Age and Season on the Haematological Profile of Alpacas (Vicugna pacos) in Central Europe","volume":"60","author":"Pavlata","year":"2015","journal-title":"Vet. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1196","DOI":"10.1080\/1828051X.2023.2273982","article-title":"Body Measures, Growth Curves and Body Weight Prevision of Alpacas (Vicugna pacos) Reared in Italy","volume":"22","author":"Ablondi","year":"2023","journal-title":"Ital. J. Anim. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"106445","DOI":"10.1016\/j.tvjl.2025.106445","article-title":"Helminth Infections in Alpacas (Vicugna pacos), Husbandry and Worm Control Practices in South American Camelids in Italy","volume":"314","author":"Castaldo","year":"2025","journal-title":"Vet. J."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Suba-Bokodi, \u00c9., Nagy, I., and Moln\u00e1r, M. (2024). Unconventional Animal Species Participation in Animal-Assisted Interventions and Methods for Measuring Their Experienced Stress. Animals, 14.","DOI":"10.3390\/ani14202935"},{"key":"ref_7","unstructured":"Bonanno, S., Burr, E., and Vogel, N. (2022). Animal-Assisted Activities with Alpacas May Facilitate a Reduction in Anxiety-Related Symptoms Among College Students, JMU Libraries."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"215","DOI":"10.12775\/JEHS.2023.45.01.015","article-title":"Therapeutic Role of Animals: A Comprehensive Literature Review on the Prevalent Forms and Species in Animal-Assisted Interventions","volume":"45","author":"Szewczyk","year":"2023","journal-title":"J. Educ. Health Sport"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/0378-4320(93)90121-7","article-title":"Manipulation of Reproductive Functions in Male and Female New World Camelids","volume":"33","year":"1993","journal-title":"Anim. Reprod. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1016\/0093-691X(94)90169-J","article-title":"Reproduction in Llamas and Alpacas: A Review","volume":"41","author":"Smith","year":"1994","journal-title":"Theriogenology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1016\/0378-4320(96)01538-2","article-title":"Reproduction in Llamas and Alpacas","volume":"42","author":"Sumar","year":"1996","journal-title":"Anim. Reprod. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Niehaus, A.J. (2022). Reproduction and the Reproductive System. Medicine and Surgery of Camelids, Wiley.","DOI":"10.1002\/9781119583295"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1071\/RD12050","article-title":"Alpaca Semen Quality in Relation to Different Diets","volume":"25","author":"Juyena","year":"2013","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Valdivia, M., Bravo, Z., Reyes, J., and Gonzales, G.F. (2021). Rescue and Conservation of Male Adult Alpacas (Vicugna pacos) Based on Spermatogonial Stem Cell Biotechnology Using Atomized Black Maca as a Supplement of Cryopreservation Medium. Front. Vet. Sci., 8.","DOI":"10.3389\/fvets.2021.597964"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1111\/rda.12907","article-title":"Assessment of Spermatozoa in Fertile Alpaca (Vicugna pacos) Males: Study of Sperm Head Morphometry Using a Nonautomated Digital Method and Sperm Morphology Based on Strict Criteria","volume":"52","author":"Valdivia","year":"2017","journal-title":"Reprod. Domest. Anim."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"13668","DOI":"10.3389\/past.2024.13668","article-title":"Social Sustainability and Genetic Biodiversity in Peruvian Alpaca Production: A Review","volume":"14","author":"Pilares","year":"2024","journal-title":"Pastor. Res. Policy Pract."},{"key":"ref_17","unstructured":"Reyna, J. (2005). Artificial Insemination in Alpacas, New Zealand Alpaca."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Fowler, M. (2011). Medicine and Surgery of Camelids, John Wiley & Sons.","DOI":"10.1002\/9781118785706"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Costagliola, A., Montano, L., Langella, E., Lombardi, R., Squillacioti, C., Mirabella, N., and Liguori, G. (2024). A Comparative Analysis of Orexins in the Physio-Pathological Processes of the Male Genital Tract: New Challenges? A Review. Vet. Sci., 11.","DOI":"10.3390\/vetsci11030131"},{"key":"ref_20","unstructured":"van Hasselt, S., Martinez-Gonzalez, D., Mekenkamp, G.-J., Vyssotski, A.L., Verhulst, S., Rattenborg, N.C., and Peter, M. (2022, January 27\u201330). Sleep Homeostasis in the European Jackdaw (Coloeus monedula): Sleep Deprivation Increases NREM Sleep Time and EEG Power while Reducing Hemispheric Asymmetry. Proceedings of the 26th Congress of the European Sleep Research Society (ESRS), Athens, Greece."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Inutsuka, A., and Yamanaka, A. (2013). The Physiological Role of Orexin\/Hypocretin Neurons in the Regulation of Sleep\/Wakefulness and Neuroendocrine Functions. Front. Endocrinol., 4.","DOI":"10.3389\/fendo.2013.00018"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"987","DOI":"10.1097\/00001756-200206120-00001","article-title":"Roles of Orexins in Regulation of Feeding and Wakefulness","volume":"13","author":"Sakurai","year":"2002","journal-title":"NeuroReport"},{"key":"ref_23","first-page":"e34009","article-title":"Physiological Role of Orexin\/Hypocretin in the Human Body in Motivated Behavior: A Comprehensive Review","volume":"15","author":"Singh","year":"2023","journal-title":"Cureus"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1146\/annurev.neuro.24.1.429","article-title":"To Eat or to Sleep? Orexin in the Regulation of Feeding and Wakefulness","volume":"24","author":"Willie","year":"2001","journal-title":"Annu. Rev. Neurosci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1016\/j.ygcen.2012.07.020","article-title":"Presence, Distribution and Steroidogenic Effect of the Peptides Orexin A and Receptor 1 for Orexins in the Testis of the South American Camelid Alpaca (Vicugna pacos)","volume":"179","author":"Liguori","year":"2012","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Liguori, G., Squillacioti, C., Assisi, L., Pelagalli, A., Vittoria, A., Costagliola, A., and Mirabella, N. (2018). Potential Role of Orexin A Binding the Receptor 1 for Orexins in Normal and Cryptorchid Dogs. BMC Vet. Res., 14.","DOI":"10.1186\/s12917-018-1375-6"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Assisi, L., Pelagalli, A., Squillacioti, C., Liguori, G., Annunziata, C., and Mirabella, N. (2019). Orexin A-Mediated Modulation of Reproductive Activities in Testis of Normal and Cryptorchid Dogs: Possible Model for Studying Relationships between Energy Metabolism and Reproductive Control. Front. Endocrinol., 10.","DOI":"10.3389\/fendo.2019.00816"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1016\/j.anireprosci.2018.03.032","article-title":"Effects of Orexins on 17\u03b2-Estradiol Synthesis and P450 Aromatase Modulation in the Testis of Alpaca (Vicugna pacos)","volume":"192","author":"Liguori","year":"2018","journal-title":"Anim. Reprod. Sci."},{"key":"ref_29","unstructured":"Fowler, M.E. (1989). Medicine and Surgery of South American Camelids: Llama, Alpaca, Vicu\u00f1a, Guanaco, Iowa State University Press."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1346","DOI":"10.1016\/j.bbrc.2017.07.026","article-title":"Decreased Expression of Orexin 1 Receptor in Adult Mice Testes during Alloxan-Induced Diabetes Mellitus Perturbs Testicular Steroidogenesis and Glucose Homeostasis","volume":"490","author":"Joshi","year":"2017","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Squillacioti, C., Pelagalli, A., Assisi, L., Costagliola, A., Van Nassauw, L., Mirabella, N., and Liguori, G. (2022). Does Orexin B-Binding Receptor 2 for Orexins Regulate Testicular and Epididymal Functions in Normal and Cryptorchid Dogs?. Front. Vet. Sci., 9.","DOI":"10.3389\/fvets.2022.880022"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1957","DOI":"10.1210\/jc.2003-031778","article-title":"Expression of Human Prepro-Orexin and Signaling Characteristics of Orexin Receptors in the Male Reproductive System","volume":"89","author":"Karteris","year":"2004","journal-title":"J. Clin. Endocrinol. Metab."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"286","DOI":"10.1016\/j.acthis.2013.04.012","article-title":"Orexin 1 Receptor in the Seminiferous Tubules of Boar Testis: An Immunohistochemical Study","volume":"116","author":"Russo","year":"2014","journal-title":"Acta Histochem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"112312","DOI":"10.1016\/j.mce.2024.112312","article-title":"Knockdown of Type 2 Orexin Receptor in Adult Mouse Testis Potentiates Testosterone Production and Germ Cell Proliferation","volume":"592","author":"Sahoo","year":"2024","journal-title":"Mol. Cell. Endocrinol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"170979","DOI":"10.1016\/j.peptides.2023.170979","article-title":"Localization and Expression of Orexin B (OXB) and Its Type 2 Receptor (OX2R) in Mouse Testis during Postnatal Development","volume":"164","author":"Yadav","year":"2023","journal-title":"Peptides"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/j.biochi.2018.06.021","article-title":"The Hypothalamic Neuropeptide Orexin A\u2013a Possible Regulator in Glucose Homeostasis and Germ Cell Kinetics in Adult Mice Testes","volume":"152","author":"Joshi","year":"2018","journal-title":"Biochimie"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"5164","DOI":"10.1210\/en.2005-0455","article-title":"Pattern of Orexin Expression and Direct Biological Actions of Orexin-a in Rat Testis","volume":"146","author":"Barreiro","year":"2005","journal-title":"Endocrinology"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3324","DOI":"10.1210\/endo.142.8.8299","article-title":"Prepro-Orexin and Orexin Receptor mRNAs Are Differentially Expressed in Peripheral Tissues of Male and Female Rats","volume":"142","author":"Neidert","year":"2001","journal-title":"Endocrinology"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1007\/s00441-012-1394-8","article-title":"Expression and Role of Receptor 1 for Orexins in Seminiferous Tubules of Rat Testis","volume":"348","author":"Assisi","year":"2012","journal-title":"Cell Tissue Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1038\/nrurol.2012.77","article-title":"Metabolic Regulation Is Important for Spermatogenesis","volume":"9","author":"Rato","year":"2012","journal-title":"Nat. Rev. Urol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1677\/joe.0.1790063","article-title":"Expression and Regulation of Glucose Transporter 8 in Rat Leydig Cells","volume":"179","author":"Chen","year":"2003","journal-title":"J. Endocrinol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"9030","DOI":"10.1038\/sj.onc.1207116","article-title":"Control of Apoptosis by P53","volume":"22","author":"Fridman","year":"2003","journal-title":"Oncogene"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2932","DOI":"10.1002\/jnr.21739","article-title":"Signaling Pathways of Bisphenol A\u2013Induced Apoptosis in Hippocampal Neuronal Cells: Role of Calcium-induced Reactive Oxygen Species, Mitogen-activated Protein Kinases, and Nuclear Factor\u2014\u03baB","volume":"86","author":"Lee","year":"2008","journal-title":"J. Neurosci. Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1111\/andr.12475","article-title":"The Neuropeptide Orexin A\u2014Search for Its Possible Role in Regulation of Steroidogenesis in Adult Mice Testes","volume":"6","author":"Joshi","year":"2018","journal-title":"Andrology"},{"key":"ref_45","first-page":"387","article-title":"Fertility in Male Sheep: Modulators of the Acute Effects of Nutrition on the Reproductive Axis of Male Sheep","volume":"61","author":"Blache","year":"2019","journal-title":"Biosci. Proc."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/S0167-0115(01)00359-7","article-title":"Cellular Localization of Orexin Receptors in Human Adrenal Gland, Adrenocortical Adenomas and Pheochromocytomas","volume":"104","author":"Blanco","year":"2002","journal-title":"Regul. Pept."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.regpep.2010.06.011","article-title":"Novel Localization of Orexin A in the Tubular Cytotypes of the Rat Testis","volume":"164","author":"Tafuri","year":"2010","journal-title":"Regul. Pept."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/4\/545\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,11]],"date-time":"2026-02-11T09:42:35Z","timestamp":1770802955000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/4\/545"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,10]]},"references-count":47,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["ani16040545"],"URL":"https:\/\/doi.org\/10.3390\/ani16040545","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,10]]}}}