Animal Reproduction (AR)
Animal Reproduction (AR)
Original Article

Rescue of caprine fetal ovaries, vitrification and follicular development after xenotransplantation in two immunodeficient mice models

Muriel Magda Lustosa Pimentel; Fernanda Araujo dos Santos; Luã Barbalho de Macêdo; Parmênedes Dias de Brito; Gabriela Liberalino Lima; Raimundo Alves Barreto Junior; Marcelo Barbosa Bezerra

Downloads: 0
Views: 249


Abstract: Domestic and wild goats are very susceptible animals to predation, specially when pregnancy occurs. This study aimed to evaluate the use of goat fetal ovarian tissue for vitrification followed by xenotransplantation and fresh xenotransplantation in two immunosuppressed mice models (C57BL/6 SCID and Balb-C NUDE). Goat fetus ovaries were collected in slaughterhouses, divided into small cortical pieces and were destined for fresh xenotransplantation (FX) and cryopreservation followed by xenotransplantation (CX). Five recipients from each lineage were used for FX and 10 animals from each lineage for CX. The mice were euthanized after 65 postoperative days, and the transplants were collected for microscopic assessment. The blood plasma was collected for estradiol measurement. Independently of mice strain, all recipients presented complete estrus cycle in FX and 80% after CX groups. Follicles were observed at all development stages without morphological changes. The volume density and total vessel surface observed in the transplants were different (p <0.01) between groups. The estradiol levels in the recipients did not differ (p <0.05) among the treatments. Thus, it is possible to activate the preantral follicles in the ovaries of fetuses by optimizing germplasm utilization and conservation of domestic and endangered wild goats that are in predatory situations, undesirable drowning or accidental death, since provided conditions for xenotransplantation are performed.


cryopreservation, assisted reproductive technology, livestock


Aerts JM, Martinez-Madrid B, Leroy JL, Van Aelst S, Bols PE. Xenotransplantation by injection of a suspension of isolated preantral ovarian follicles and stroma cells under the kidney capsule of nude mice. Fertil Steril. 2010;94(2):708-14. PMid:19423102.

Ayuandari S, Winkler-Crepaz K, Paulitsch M, Wagner C, Zavadil C, Manzl C, Ziehr SC, Wildt L, Hofer-Tollinger S. Follicular growth after xenotransplantation of cryopreserved/thawed human ovarian tissue in SCID mice: dynamics and molecular aspects. J Assist Reprod Genet. 2016;33(12):1585-93. PMid:27465301.

Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overström EW, Echelard Y. Production of goats by somatic cell nuclear transfer. Nat Biotechnol. 1999;17(5):456-61. PMid:10331804.

Barros FFPC, Teixeira PPM, Silva MAM, Silva ASL, Medeiros RM, Coelho CMM, Vicente WRR. Xenotransplante em camundongos imunossuprimidos coletado por meio de ovariectomia unilateral total videolaparoscópica em ovelha. Rev Bras Reprod Anim. 2014;38:121-6.

Bartoskova A, Ondrackova P, Leva L, Vitasek R, Novotny R, Janosovska M, Faldyna M. The effects of in vitro exposure to progesterone and estradiol-17β on the activity of canine neutrophils. Vet Med (Praha). 2014;59(4):202-9.

Bentov Y, Jurisicova A, Kenigsberg S, Casper RF. What maintains the high intra-follicular estradiol concentration in pre-ovulatory follicles? J Assist Reprod Genet. 2016;33(1):85-94. PMid:26552664.

Bertolini LR, Meade H, Lazzarotto CR, Martins LT, Tavares KC, Bertolini M, Murray JD. The transgenic animal platform for biopharmaceutical production. Transgenic Res. 2016;25(3):329-43. PMid:26820414.

Bezerra MB, Rondina D, Lima AKF, Oliveira LC, Cecchi R, Lucci CM, De Figueiredo JR. Aspectos quantitativos e qualitativos da foliculogênese pré-natal na espécie caprina. Ciênc Anim. 1998;8(2):47-56.

Byers SL, Wiles MV, Dunn SL, Taft RA. Mouse estrous cycle identification tool and images. PLoS One. 2012;7(4):e35538. PMid:22514749.

Carvalho AA, Faustino LR, Silva CMG, Castro SV, Luz HKM, Rossetto R, Lopes CAP, Campello CC, Figueiredo JR, Rodrigues APR, Costa APR. Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue. Theriogenology. 2011;76(5):933-41. PMid:21719087.

Christie BA, Bjorling DE. Kidney. In: Slatter DH, editor. Textbook of small animal surgery. Philadelphia: Elsevier; 2003. Vol. 1, p. 1698-713.

Comizzoli P. Biobanking efforts and new advances in male fertility preservation for rare and endangered species. Asian J Androl. 2015;17(4):640-5. PMid:25966625.

Comizzoli P, Wildt DE. Mammalian fertility preservation through cryobiology: value of classical comparative studies and the need for new preservation options. Reprod Fertil Dev. 2013;26(1):91-8. PMid:24305181.

Commin L, Buff S, Rosset E, Galet C, Allard A, Bruyere P, Joly T, Guérin P, Neto V. Follicle development in cryopreserved bitch ovarian tissue grafted to immunodeficient mouse. Reprod Fertil Dev. 2012;24(3):461-71. PMid:22401278.

Cooper R. Monitoring of estrous cycle in the laboratory rodent by vaginal lavage. In: Chapin RE, Heindel J, editors. Methods in toxicology. Orlando: Academic Press; 1993. Vol. 3, Part B: female reproductive toxicology, pp. 45-56.

Donfack NJ, Alves KA, Alves BG, Rocha RMP, Bruno JB, Lima LF, Lobo CH, Santos RR, Domingues SFS, Bertolini M, Smitz J, Rodrigues APR. In vivo and in vitro strategies to support caprine preantral follicle development after ovarian tissue vitrification. Reprod Fertil Dev. 2018;30(8):1055-65. PMid:29332622.

Durawo C, Zindove TJ, Chimonyo M. Influence of genotype and topography on the goat predation challenge under communal production systems. Small Rumin Res. 2017;14:115-20.

Fonseca JF, Souza-Fabjan JMG, Oliveira MEF, Leite CR, Nascimento-Penido PMP, Brandão FZ, Lehloenya KC. Nonsurgical embryo recovery and transfer in sheep and goats. Theriogenology. 2016;86(1):144-51. PMid:27177961.

Gastal GDA, Hamilton A, Alves BG, de Tarso SGS, Feugang JM, Banz WJ, Apgar GA, Nielsen CK, Gastal EL. Ovarian features in white-tailed deer (Odocoileus virginianus) fawns and does. PLoS One. 2017;12(5):e0177357. PMid:28542265.

González C, Boada M, Devesa M, Veiga A. Concise review: fertility preservation: an update. Stem Cells Transl Med. 2012;1(9):668-72. PMid:23197873.

Gosden RG, Boulton MI, Grant K, Webb R. Follicular development from ovarian xenografts in SCID mice. J Reprod Fertil. 1994;101(3):619-23. PMid:7966017.

Hajian M, Hosseini SM, Ostadhosseini S, Nasr-Esfahani MH. Comparative stepwise pattern of reactive oxygen species production during in vitro development of fertilized and nuclear transferred goat embryos. Int J Fertil Steril. 2017;11(2):93-8. PMid:28670426.

Hernandez-Fonseca HJ, Bosch P, Miller DM, Wininger JD, Massey JB, Brackett BG. Time course of follicular development after bovine ovarian tissue transplantation in male non-obese diabetic severe combined immunodeficient mice. Fertil Steril. 2005;83(4, Suppl 1):1180-7. PMid:15831291.

Hoff J. Methods of blood collection in the mouse [Internet]. Labome; 2000 [cited 2018 May 13]. Available from:

Ingberg E, Theodorsson A, Theodorsson E, Strom JO. Methods for long-term 17β-estradiol administration to mice. Gen Comp Endocrinol. 2012;175(1):188-93. PMid:22137913.

Khan MZ, Khan B, Awan MS, Begum F. Livestock depredation by large predators and its implications for conservation and livelihoods in the Karakoram Mountains of Pakistan. Oryx. 2018;52(3):519-25.

Kumari AG, Rooh UA, Sadasiva RK. Morphometric and histological characterization of goat fetal ovaries. Dairy and Vet Sci J. 2017;3(1):555605.

Laschke MW, Menger MD, Vollmar B. Cryopreservation does not affect neovascularization of freely transplanted ovarian follicles. Fertil Steril. 2003;79(6):1458-60. PMid:12798902.

Leal ÉSS, Vieira LA, Sá NAR, Silva GM, Lunardi FO, Ferreira ACA, Campello CC, Alves BG, Cibin FWS, Smitz J, Figueiredo JR, Rodrigues APR. In vitro growth and development of isolated secondary follicles from vitrified caprine ovarian cortex. Reprod Fertil Dev. 2018;30(2):359-70. PMid:28768567.

Lee J, Kim SK, Youm HW, Kim HJ, Lee JR, Suh CS, Kim SH. Effects of three different types of antifreeze proteins on mouse ovarian tissue cryopreservation and transplantation. PLoS One. 2015;10(5):e0126252. PMid:25938445.

Liu L, Wood GA, Morikawa L, Ayearst R, Fleming C, McKerlie C. Restoration of fertility by orthotopic transplantation of frozen adult mouse ovaries. Hum Reprod. 2008;23(1):122-8. PMid:17993475.

Magalhães DM, Duarte ABG, Araújo VR, Brito IR, Soares TG, Lima IMT, Lopes CAP, Campello CC, Rodrigues APR, Figueiredo JR. In vitro production of a caprine embryo from a preantral follicle cultured in media supplemented with growth hormone. Theriogenology. 2011;75(1):182-8. PMid:20875671.

Mofarahe ZS, Novin MG, Salehnia M. Folliculogenesis-associated genes expression in human vitrified ovarian tissue after xenotransplantation in γ-irradiated mice. Cell J. 2020;22(3):253-400.

Mohammadi Z, Hayati Roodbari N, Parivar K, Salehnia M. Supplementation of culture media with lysophosphatidic acid improves the follicular development of human ovarian tissue after xenotransplantaion into the back muscle of γ-irradiated mice. Cell J. 2020;22(3):358-66. PMid:31863662.

Noorafshan A, Ahmadi M, Mesbah SF, Karbalay-Doust S. Stereological study of the effects of letrozole and estradiol valerate treatment on the ovary of rats. Clin Exp Reprod Med. 2013;40(3):115-21. PMid:24179868.

Pellicer-Rubio MT, Boissard K, Forgerit Y, Pougnard JL, Bonné JL, Leboeuf B. Evaluation of hormone-free protocols based on the “male effect” for artificial insemination in lactating goats during seasonal anestrus. Theriogenology. 2016;85(5):960-9. PMid:26707385.

Pimentel MM, Santos FA, Teixeira AC, Izzo RG, Lima MA, Macedo MF, Bezerra MB. Biochemical, thermographic, and follicular responses of murine models of hormone-treated bovine ovarian renal capsule xenografts. Pesq Vet Bras. 2017;37(5):425-31.

Praxedes ÉCG, Lima GL, Bezerra LGP, Santos FA, Bezerra MB, Guerreiro DD, Rodrigues APR, Domingues SFS, Silva AR. Development of fresh and vitrified agouti ovarian tissue after xenografting to ovariectomised severe combined immunodeficiency (SCID) mice. Reprod Fertil Dev. 2018;30(3):459-68. PMid:28784201.

Roshnath R, Sethu D, Gokul D, Arjun CP. Concerns about Wild Dog (Cuon alpinus) Predation on Livestock in the Fringes of Aralam Wildlife Sanctuary, Kannur, Kerala. Indian For. 2017;143(10):1037-41.

Santos FA, Pimentel MML, Teixeira ACG, Cosmo ÍC, Lima MA, Brito PD, Bezerra MB. Follicular right shift: xenografting queen’s ovarian tissue into severe combined imunnodeficiency mice and its responses to exogenous gonadotropin. Theriogenology. 2016;86(7):1811-7. PMid:27371971.

Santos RR, Amorim C, Cecconi S, Fassbender M, Imhof M, Lornage J, Paris M, Schoenfeldt V, Martinez-Madrid B. Cryopreservation of ovarian tissue: an emerging technology for female germline preservation of endangered species and breeds. Anim Reprod Sci. 2010;122(3-4):151-63. PMid:20832203.

Santos RR, Knijn HM, Vos PL, Oei CH, Van Loon T, Colenbrander B, Gadella BM, van den Hurk R, Roelen BA. Complete follicular development and recovery of ovarian function of frozen-thawed, autotransplanted caprine ovarian cortex. Fertil Steril. 2009;91(4, Suppl):1455-8. PMid:18722611.

Saraiva MV, Rossetto R, Brito IR, Celestino JJ, Silva CM, Faustino LR, Almeida AP, Bruno JB, Magalhães DM, Matos MH, Campello CC, Figueiredo JR. Dynamic medium produces caprine embryo from preantral follicles grown in vitro. Reprod Sci. 2010;17(12):1135-43. PMid:20926838.

Senbon S, Ishii K, Fukumi Y, Miyano T. Fertilization and development of bovine oocytes grown in female SCID mice. Zygote. 2005;13(4):309-15. PMid:16388699.

Soleimani R, Heytens E, Oktay K. Enhancement of neoangiogenesis and follicle survival by sphingosine-1-phosphate in human ovarian tissue xenotransplants. PLoS One. 2011;6(4):e19475. PMid:21559342.

Tahaei LS, Eimani H, Hajmusa G, Fathi R, Valojerdi MR, Shahverdi A, Eftekhari-Yazdi P. Follicle development of xenotransplanted sheep ovarian tissue into male and female immunodeficient rats. Int J Fertil Steril. 2015;9(3):354-60. PMid:26644859.

Van Eyck AS, Bouzin C, Feron O, Romeu L, Van Langendonckt A, Donnez J, Dolmans MM. Both host and graft vessels contribute to revascularization of xenografted human ovarian tissue in a murine model. Fertil Steril. 2010;93(5):1676-85. PMid:19539913.

Wiedemann C, Hribal R, Ringleb J, Bertelsen MF, Rasmusen K, Andersen CY, Kristensen SG, Jewgenow K. Preservation of primordial follicles from lions by slow freezing and xenotransplantation of ovarian cortex into an immunodeficient mouse. Reprod Domest Anim. 2012;47(Suppl 6):300-4. PMid:23279524.

Wolvekamp MCJ, Cleary ML, Cox SL, Shaw JM, Jenkin G, Trounson AO. Follicular development in cryopreserved common wombat ovarian tissue xenografted to nude rats. Anim Reprod Sci. 2001;65(1-2):135-47. PMid:11182515.

Yang M, Hall J, Fan Z, Regouski M, Meng Q, Rutigliano HM, Stott R, Rood KA, Panter KE, Polejaeva IA. Oocytes from small and large follicles exhibit similar development competence following goat cloning despite their differences in meiotic and cytoplasmic maturation. Theriogenology. 2016;86(9):2302-11. PMid:27650944.

Youm HW, Lee JR, Lee J, Jee BC, Suh CS, Kim SH. Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified–warmed ovarian tissue survival. Hum Reprod. 2014;29(4):720-30. PMid:24365801.

5f0473380e8825da1cbb8295 animreprod Articles
Links & Downloads

Anim Reprod

Share this page
Page Sections