Animal Reproduction (AR)
Animal Reproduction (AR)

Cryopreservation of jaguar (Panthera onca) sperm cells using different cryoprotectants and different thawing temperatures

Pedro Nacib Jorge-Neto; Thiago Cavalheri Luczinski; Gediendson Ribeiro de Araújo; Letícia Alecho Requena; Rogério Silva de Jesus; Larissa Schneider Brandão Souza; Ricardo Zanella; Eliane Vianna da Costa e Silva; Thyara de Deco-Souza; Cristiane Schilbach Pizzutto

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The cryopreservation of jaguar semen must be improved to produce high-quality biobanking doses. Until now, the rare studies of semen freezing in the species have only evaluated glycerol, always with a significant reduction in sperm quality in thawed semen. The purpose of this study was to assess the efficacy of three cryoprotectants, dimethylsulfoxide (DMSO), glycerol (GLY), and methanol (MET), in the cryopreservation of jaguar semen in an LDL-based extender, as well as the effect of thawing temperature on dosage quality. Five mature males with a history of reproduction were used. On the males, an infrared thermal image (IRT) was captured, the spicules and testes were analyzed, and the CASA system was used to evaluate the quality of fresh and thawed sperm. The superficial IRT was 4.6 ± 1.2 °C cooler than the anal sphincter, and the semen measured between 27.3 and 28.7 °C shortly after exiting the urethra. The total motility of fresh sperm was 55.3 ± 22.6%, and progressive motility was 36.3 ± 18%. The total motility of thawed sperm was 5.28 ± 2.51%, 4.49 ± %2.49, and 0.51 ± 0.62% for DMSO, GLY, and MET, respectively. DMSO and GLY performed better than MET, and there was no difference in thawing temperature (37°C 30 s vs. 50°C 12 s). All animals exhibit a considerable level of morphological changes in sperm. Low amounts of total and progressive motility were found in the thawed sperm. Males with a high level of sperm morphological changes were found to be fertile, but the lone male with normospermia was infertile. Thus, we contest the applicability of the commonly used morphological classification for bovines to felid species.


biobank, feline, infrared thermography, penile spines, reproduction


Araujo G, Deco-Souza T, Bergo LCF, Silva LC, Morato RG, Jorge-Neto PN, Silva MCC, Macedo GG, Paula TAR. Field friendly method for wild feline semen cryopreservation. J Threat Taxa. 2020a;12(5):15557-64.

Araújo G, Jorge-Neto P, Csermak Jr AC, Pizzutto CS, Luczinski TC, Deco-Souza T. Avanços na andrologia de grandes felinos neotropicais. Rev Bras Reprod Anim. 2021a;45(4):219-28.

Araujo G, Jorge-Neto P, Salmão-Jr JA, Zanella R, Silva MCC, Csermak Jr AC, Francisco FM, Deco-Souza T, Pizzutto CS. Pharmacological semen collection in giant anteaters (Myrmecophaga tridactyla): a feasible option for captive and free-living animals. Theriogenology Wild. 2023;2. In press.

Araujo GR, Deco‐Souza T, Morato RG, Crawshaw PG Jr, Silva LC, Jorge‐Neto PN, Csermak AC Jr, Bergo LCF, Kantek DLZ, Miyazaki SS, Beisiegel BM, Tortato FR, May-Junior JA, Silva MCC, Leuzinger L, Salomão JÁ Jr, Paula TAR. Use of foot snares to capture large felids. Methods Ecol Evol. 2021b;12(2):322-7.

Araujo GR, Paula TAR, Deco-Souza T, Garay RM, Bergo LCF, Silva LC, Csermak AC Jr, Ferrer JBS, Barros JBG. Criptorquidismo em jaguatirica de vida livre capturada no Parque Estadual do Rio Doce, Brasil. Arq Bras Med Vet Zootec. 2013;65(1):1-5.

Araujo GR, Paula TAR, Deco-Souza T, Morato RG, Bergo LCF, dSilva LC, Costa DS, Braud C. Comparison of semen samples collected from wild and captive jaguars (Panthera onca) by urethral catheterization after pharmacological induction. Anim Reprod Sci. 2018;195:1-7. PMid:29935916.

Araujo GR, Paula TAR, Deco-Souza T, Morato RG, Bergo LCF, Silva LC, Jorge-Neto PN, Sampaio BFB. Pharmacological semen collection in cougars (Puma concolor: Mammalia:Carnivora: Felidae). Arq Bras Med Vet Zootec. 2020b;72:641-6.

Baqir S, Al-Zeheimi N, Orabah AB, Al-Shakaili Y, Al-Rasbi K, Gartley CJ, Mastromonaco GF. Semen evaluation in an aged critically endangered captive Arabian leopard (Panthera pardus nimr): a case study. Rev Med Vet. 2015;166:244-52.

Barnabe RC, Guimarães MABV, Oliveira CA, Barnabe AH. Analysis of some normal parameters of the spermiogramof captive capuchin monkeys (Cebus apella Linnaeus, 1758). Braz J Vet Res Anim Sci. 2002;39(6):331-3.

Bencharif D, Amirat L, Anton M, Schmitt E, Desherces S, Delhomme G, Langlois ML, Barrière P, Larrat M, Tainturier D. The advantages of LDL (low density lipoproteins) in the cryopreservation of canine semen. Theriogenology. 2008;70(9):1478-88. PMid:18817963.

Broekhuijse MLWJ, Šoštarić E, Feitsma H, Gadella BM. Additional value of computer assisted semen analysis (CASA) compared to conventional motility assessments in pig artificial insemination. Theriogenology. 2011;76(8):1473-86. PMid:21872316.

Buranaamnuay K. Effect of different permeable cryoprotectants on the quality of cat epididymal spermatozoa. Cryo Letters. 2020;41(4):237-44. PMid:33988653.

Buranaamnuay K. Protocols for sperm cryopreservation in the domestic cat: a review. Anim Reprod Sci. 2017;183:56-65. PMid:28629655.

Cavalero TMS, Papa FO, Schmith RA, Scheeren VFC, Canuto LEF, Gobato MLM, Rodrigues LT, Freitas-Dell’aqua CP. Protocols using detomidine and oxytocin induce ex copula ejaculation in stallions. Theriogenology. 2019;140:93-8. PMid:31454723.

Cochran JD, Amann RP, Froman DP, Pickett BW. Effects of centrifugation, glycerol level, cooling to 5°C, freezing rate and thawing rate on the post-thaw motility of equine sperm. Theriogenology. 1984;22(1):25-38. PMid:16725933.

Comizzoli P. Biobanking and fertility preservation for rare and endangered species. Anim Reprod. 2017;14(1):30-3.

Curry E, Roth TL. 118 a rapid, minimally invasive method of collecting semen from polar bears. Reprod Fertil Dev. 2016;28(2):189.

Deco-Souza T, Paula TAR, Costa DS, Costa EP, Barros JBG, Araujo GR, Carreta-Jr M. Comparação entre duas concentrações de glicerol para a criopreservação de sêmen de suçuarana (Puma concolor). Pesq Vet Bras. 2013;33(4):512-6.

Dhami AJ, Sahni KL, Mohan G. Effect of various cooling rates (from 30°C to 5°C) and thawing temperatures on the deep-freezing of Bos Taurus and Bos Bubalis semen. Theriogenology. 1992;38(3):565-74. PMid:16727159.

Franklin AD, Waddell WT, Goodrowe KL. Red wolf (Canis rufus) sperm quality and quantity is affected by semen collection method, extender components, and post-thaw holding temperature. Theriogenology. 2018;116:41-8. PMid:29777963.

Gonzalez SJ, Howard JG, Brown J, Grajales H, Pinzón J, Monsalve H, Moreno MA, Escobar CJ. Reproductive analysis of male and female captive jaguars (Panthera onca) in a Colombian zoological park. Theriogenology. 2017;89:192-200. PMid:28043351.

Harcourt AH, Purvis A, Liles L. Sperm competition: mating system, not breeding season, affects testes size of primates. Funct Ecol. 1995;9(3):468.

Iaffaldano N, Iorio M, Miranda M, Zaniboni L, Manchisi A, Cerolini S. Cryopreserving turkey semen in straws and nitrogen vapour using DMSO or DMA: effects of cryoprotectant concentration, freezing rate and thawing rate on post-thaw semen quality. Br Poult Sci. 2016;57(2):264-70. PMid:26872498.

Jeong DH, Kim JH, Na KJ. Characterization and cryopreservation of Amur leopard cats (Prionailurus bengalensis euptilurus) semen collected by urethral catheterization. Theriogenology. 2018;119:91-5. PMid:29982141.

Jeong DH, Yang JJ, Seo MK, Lee AN, Lim YK. Effectiveness of urethral catheterization under ultrasound guidance for semen collection from Asiatic black bears (Ursus thibetanus). Theriogenology. 2019;129:154-9. PMid:30851479.

Johnson AEM, Freeman EW, Wildt DE, Songsasen N. Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide. Cryobiology. 2014;68(3):361-70. PMid:24731851.

Johnson L, Petty CS, Neaves WB. A new approach to quantification of spermatogenesis and its application to germinal cell attrition during human spermiogenesis. Biol Reprod. 1981;25(1):217-26. PMid:6793101.

Jorge-Neto PN, Araujo GR, Deco-Souza T, Bittencourt RF, Csermak AC Jr, Pizzutto CS, Silva MCC, Salomão JA Jr, Madrigal-Valverde M, Curvelo VP, Gomes MC, Paula TAR. Pharmacological semen collection of Brazilian wild felids. Rev Bras Reprod Anim. 2019;43:704.

Jorge-Neto PN, Araújo GR, Silva MCC, Salomão JA Jr, Csermak AC Jr, Pizutto CS, Deco-Souza T, Camus AD. Description of the CASA system configuration setup for jaguar (Panthera onca). Anim Reprod Sci. 2020a;220:106436.

Jorge-Neto PN, Luczinski TC, Araújo GR, Salomão JÁ Jr. Can jaguar (Panthera onca) ovulate without copulation? Theriogenology. 2020b;147:57-61. PMid:32092606.

Jorge-Neto PN, Pizzutto CS, Araújo GR, Deco-Souza T, Silva LC, Salomão JA Jr, Baldassarre H. Copulatory behavior of the Jaguar Panthera onca (Mammalia: Carnivora: Felidae). J Threat Taxa. 2018;10(15):12933-9.

Jorge-Neto PN, Silva MCC, Csermak-Júnior AC, Salmão-Júnior JA, Araújo GR, Oliveira G, Leuzinger L, Pizzutto CS, Deco-Souza T. Cryptorchidism in free-living jaguar (Panthera onca): first case report. Anim Reprod. 2020c;17(4):e20200555. PMid:33791034.

Kheirkhah MS, Sisakht MM, Mohammadsadegh M, Moslemi HR. Sperm evaluation of jungle cat (Felis chaus) obtained by urethral catheterization (CT) after medetomidine administration. Theriogenology. 2017;91:17-20. PMid:28215681.

Lambo CA, Grahn RA, Lyons LA, Bateman HL, Newsom J, Swanson WF. Comparative fertility of freshly collected vs frozen-thawed semen with laparoscopic oviductal artificial insemination in domestic cats. Reprod Domest Anim. 2012;47(Suppl 6):284-8. PMid:23279520.

Laurenson MK. High juvenile mortality in cheetahs (Acinonyx jubatus) and its consequences for maternal care. J Zool. 1994;234(3):387-408.

Lorton SP. Andrology laboratory: equipment and techniques - measurement of ejaculate volume, sperm concentration, extenders, and cryopreservation, the microscope, and sperm motility evaluation. In: Chenoweth PJ, Lorton SP, editors. Manual of animal andrology. Oxfordshire: CABI; 2022. p. 19-44.

Luczinski TC, Araújo GR, Silveira MF, Kirnew MD, Navarrete RA, Salomão JA Jr, Requena LA, Santos JAM, Koshiyama MH, Pizzutto CS, Jorge-Neto PN. Medetomidine may cause heart murmur in cougars and jaguars: case report. J Threat Taxa. 2020;12(14):17000-2.

Luczinski TC, Jorge-Neto PN, Ribeiro RM, Jesus RS, Pizzutto CS, Deco-Souza T, Araújo GR, Morgado TO, Corrêa SHR, Peixer MAS, Malard PF, Bortolotto G, Fernandes KVB, Polizelle SR, Morato RG. One conservation concept in practice. Theriogenology Wild. 2023;2:100024.

Lueders I, Luther I, Scheepers G, van der Horst G. Improved semen collection method for wild felids: urethral catheterization yields high sperm quality in African lions (Panthera leo). Theriogenology. 2012;78(3):696-701. PMid:22538007.

Machado LC, Orlandin JR, Karam RG, Rós FA, Martins DS, Costa GM, Ambrósio CE. Morphology of male and female reproductive tract of the ocelot (Leopardus pardalis). Anim Reprod. 2020;17(2):e20200010. PMid:32714460.

Madrigal-Valverde M, Bittencourt RF, Ribeiro AD Fo, Barbosa VF, Vieira CA, Romão EA, Carneiro IB, Azevedo MC, Araujo GR. Quality of domestic cat semen collected by urethral catheterization after the use of different alpha 2-adrenergic agonists. J Feline Med Surg. 2021;23(8):745-50. PMid:33206029.

Morais RN, Mucciolo RG, Gomes MLF, Lacerda O, Moraes W, Moreira N, Graham LH, Swanson WF, Brown JL. Seasonal analysis of semen characteristics, serum testosterone and fecal androgens in the ocelot (Leopardus pardalis), margay (L. wiedii) and tigrina (L. tigrinus). Theriogenology. 2002;57(8):2027-41. PMid:12066863.

Morato RG, Conforti VA, Azevedo FC, Jacomo AT, Silveira L, Sana D, Nunes AL, Guimarães MA, Barnabe RC. Comparative analyses of semen and endocrine characteristics of free-living versus captive jaguars (Panthera onca). Reproduction. 2001;122(5):745-51. PMid:11690535.

Mulia BH, Widianti A, Manansang J, Setiadi DR, Yoelinda VT, Nugraha TP, Karja NWK, Arifiantini RI. Establishment of semen collection technique using electroejaculator and semen cryopreservation of Javan leopard (Panthera pardus melas Cuvier, 1809). Vet World. 2021;14(12):3156-63. PMid:35153407.

Paz RCR, Züge RM, Barnabe VH, Morato RG, Felippe PAN, Barnabe RC. Avaliação da capacidade de penetração de sêmen congelado de onça pintada (Panthera onca) em oócitos heterólogos. Braz J Vet Res Anim Sci. 2000;37:462-6.

Paz RCR, Züge RM, Barnabe VH. Frozen jaguar (Panthera onca) sperm capacitation and ability to penetrate zona free hamster oocytes. Braz J Vet Res Anim Sci. 2007;44:337-44.

Pizzutto CS, Colbachini H, Jorge-Neto PN. One conservation: the integrated view of biodiversity conservation. Anim Reprod. 2021;18(2):e20210024. PMid:34122656.

R Development Core Team. R: a language and environment for statistical computing. Viena: The R Foundation for Statistical Computing; 2011.

Rouillon C, Gavin-Plagne L, Schmitt E, Camus A. Where is the true evaluation of semen quality? Anim Reprod Sci. 2020;220:106434.

Ruiz-Olmo J, Pinyol C, Sánchez D, Such-Sanz À. Breeding pattern of wildcat Felis silvestris (Schreber, 1777) studied in captivity in the Iberian Peninsula. Hystrix. 2018;29:202-10.

Sankhala KS. Breeding behaviour of the tiger Panthera tigris in Rajasthan. Int Zoo Yearb. 1967;7(1):133-47.

Santos MVO, Silva HVR, Bezerra LGP, Oliveira LRM, Oliveira MF, Alves ND, Silva LDM, Silva AR, Pereira AF. Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders. Anim Reprod. 2022;19(1):e20210093. PMid:35371300.

Schepper M. A comparative study in morphological defects of semen from African lions (Panthera leo) and caracal (Caracal caracal): collected by urethral catheterization and electro-ejaculation [thesis]. Ghent: Ghent University; 2016.

Silinski S, Walzer C, Schwarzenberger F, Slotta-Bachmayr L, Stolla R. Pharmacological methods of enhancing penile erection for ex-copula semen collection in standing white rhinoceros (Ceratotherium simum simum). In: 4th Scientific Meeting, Joint with the Annual Meeting of the European Wildlife Disease Association (EWDA); 2002 May 8-12; Heidelberg. Emmen: European Association of Zoo and Wildlife Veterinarians; 2002. p. 1-4.

Silva HVR, Nunes TGP, Brito BF, Campos LB, Silva AM, Silva AR, Comizzoli P, Silva LMD. Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca). Cryobiology. 2020;92:53-61. PMid:31704199.

Silva HVR, Nunes TGP, Ribeiro LR, Freitas LA, Oliveira MF, Assis AC No, Silva AR, Silva LDM. Morphology, morphometry, ultrastructure, and mitochondrial activity of jaguar (Panthera onca) sperm. Anim Reprod Sci. 2019a;203:84-93. PMid:30853119.

Silva HVR, Silva AR, Silva LDM, Comizzoli P. Semen cryopreservation and banking for the conservation of neotropical carnivores. Biopreserv Biobank. 2019b;17(2):183-8. PMid:30589562.

Silva MCC, Jorge-Neto PN, Miranda GM, Csermak AC Jr, Zanella R, Pizzutto CS, Colbachini H, Silva AR, Araújo GR, Deco-Souza T. Reproductive parameters of male crab-eating foxes (Cerdocyon thous) subjected to pharmacological semen collection by urethral catheterization. Theriogenology Wild. 2022;1:100004.

Silva MCC, Ullony KM, Araújo GR, Jorge-Neto PN, Albuquerque VB, Caramalac SM, Oliveira AR, Zanella R, Marques MG, Csemark AC Jr, Luczinski TC, Frazílio FO, Silva EVDCE, Deco-Souza T. Can detomidine replace medetomidine for pharmacological semen collection in domestic cats? Anim Reprod. 2021;18(2):e20210017. PMid:34249155.

Silvatti B, Granato TM, Jorge-Neto PN, Luppi MMCP, Reisfeld LC, Henrique PC, Padilha FLA, Leite RF, Losano JDA, Kawai GKV, Nichi M, Pizzutto CS. Sperm evaluation and morphological description of male genitalia of meerkats (Suricata suricatta). Anim Reprod Sci. 2020;221:106585. PMid:32889408.

Stander H. Sperm cryopreservation, in vitro fertilization and faecal oestrogen enzyme immunoassay validation in the African lion (Panthera leo) [dissertation]. Bloemfontein: University of the Free State; 2004.

Swanson WF, Roth TL, Blumer E, Citino SB, Kenny D, Wildt DE. Comparative cryopreservation and functionality of spermatozoa from the normospermic jaguar (Panthera onca) and teratospermic cheetah (Acinonyx jubatus). Theriogenology. 1996;45(1):241.

Veronesi MC, Fusi J. Feline neonatology: from birth to commencement of weaning: what to know for successful management. J Feline Med Surg. 2022;24(3):232-42. PMid:35209772.

Zambelli D, Prati F, Cunto M, Iacono E, Merlo B. Quality and in vitro fertilizing ability of cryopreserved cat spermatozoa obtained by urethral catheterization after medetomidine administration. Theriogenology. 2008;69(4):485-90. PMid:18082880.

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