Innovative reproductive technologies for in vitro preservation and utilization of female gametes from domestic birds (review)

The swift reduction in the biodiversity of farm birds and their wild relatives necessitates the development of effective in vitro preservation methods. Creation of cryobanks for long-term storage of avian reproductive cells represents a relevant strategic solution to this problem. However, to date, effective protocols for cryogenic preservation have been developed for bird spermatozoa only. Egg cryopreservation in birds remains impossible, which leads to loss of genetic information, namely nuclear and mitochondrial DNA of female birds. The utilization of the latest advances in production and cultivation of primordial germ cells (PGSs) and embryonic gonadal germ cells (GGCs) is a viable alternative for preserving the gene pool of birds. The advantage of protocols using GGCs is the early determination of the donor’s sex and targeted in vitro preservation of female reproductive material. Furthermore, there is a possibility of low-temperature intraovarian preservation of female bird gametes. The use of frozen-thawed ovarian tissues of both embryonic and postnatal origin as future transplants is being actively developed. The success of using donor reproductive material largely depends on the recipient preparation (sterilization). The world’s leading laboratories have achieved impressive results in producing offspring of both sexes using donor reproductive material. This review highlights the progress made in the developing technologies for the in vitro preservation and use of avian reproductive material: embryonic cells, donor ovarian tissues, and tissue transplantation to recipients. The development of biotechnological methods for manipulation with donor material of female birds will contribute to achieving the goal of the world scientific community, which is to prevent the loss of biodiversity of domestical birds, and will be further applied in the restoration of avian breeds and species that are currently at risk of extinction. An additional argument for the importance of developing these technologies is the significant economic advantage in reconstructing endangered breeds, compared to using only cryopreserved semen when conducting multiple backcrosses with a breed close in phenotype.

1. Gambini A., Ortiz I., Hidalgo M. [et al.], Selection and Conservation of Gametes and Embryos for Improving Assisted Reproductive Technologies, Animals, 2023, Vol. 13, pp. 2761, DOI: 10.3390/ani13172761.

2. Stanishevskaya O., Silyukova Y., Pleshanov N. [et al.], Effects of saccharides supplementation in the extender of cryopreservedrooster (Gallus domesticus) semen on the fertility of frozen/thawed spermatozoa, Anim, 2021, Vol. 11(1), pp. 1–9.

3. Stanishevskaya O., Silyukova Y., Pleshanov N. [et al.], Role of mono-and disaccharide combination in cryoprotective medium for rooster semen to ensure cryoresistance of spermatozoa, Molecules, 2021, Vol. 26(19), pp. 5920, DOI: 10.3390/molecules26195920.

4. Silversides F.G., Purdy P.H., Blackburn H.D., Comparative costs of programmes to conserve chicken genetic variation based on maintaining living populations or storing cryopreserved material, Brit Poult Sci, 2012, Vol. 53(5), pp. 599– 607, DOI: 10.1080/00071668.2012.727383.

5. Johnson A.L., The avian ovary and follicle development: some comparative and practical insights, Turk J Vet Anim Sci, 2014, Vol. 38 (6), pp. 660–669, DOI:10.3906/vet-1405-6.

6. González-Morán M.G., Histological and Stereological Changes in Growing and Regressing Chicken Ovaries During Development, Anatomical Record, 2011, Vol. 294(5), pp. 893–904, DOI: 10.1002/ar.21364.

7. Perinek O.Y., Volkova N.A., Gagieva Z.V., Histological examination of the ovary in a universal chicken breed at different ages, Ptisevodstvo, 2025, Vol. 1, pp. 49–55, DOI: 10.33845/0033-3239-2025-74-1-49-55.

8. Gupta A., Kapoor K., Avian ovary: a unique dynamic reproductive organ, Int J Avian & Wildlife Biol, 2024, Vol. 8(2), pp. 77–78, DOI: 10.15406/ijawb.2024.08.00217.

9. Johnson A.L., Woods D.C., Ovarian dynamics and follicle development. In: Jamieson B.G.M., editor., Reprod Biol Phyl Birds, Vol. 6A, Science Publishers; Enfield, NH, USA: 2007, pp. 243–277.

10. Apperson K.D., Bird K.E., Cherian G., Löhr C.V., Histology of the Ovary of the Laying Hen (Gallus domesticus), Vet Sci, 2017, Vol. 4(4), pp. 66, DOI: 10.3390/vetsci4040066.

11. Ecker A., Lázár B., Tóth R.I.[et al.], The Effects of Freezing Media on the Characteristics of Male and Female Chicken Primordial Germ Cell Lines, Life, 2023, Vol. 13, pp. 867, DOI: 10.3390/life13040867.

12. Nagai H., Sezaki M., Kakiguchi K. [et al.], Cellular analysis of cleavage-stage chick embryos reveals hidden conservation in vertebrate early development, Development, 2015, Vol. 42(7), pp. 1279–1286, DOI: 10.1242/dev.118604.

13. Nandi S., Whyte J., Taylor L. [et al.], Cryopreservation of specialized chicken lines using cultured primordial germ cells, Poult Sci, 2016, Vol. 95(8), pp. 1905–1911, DOI: 10.3382/ps/pew133.

14. Nakamura Y., Usui F., Ono T. [et al.], Germline Replacement by Transfer of Primordial Germ Cells into Partially Sterilized Embryos in the Chicken, Biol Reprod, 2010, Vol. 83(1), pp. 130–137, DOI: 10.1095/biolreprod.110.083923.

15. Hamai N., Koide C., Tansho Y. [et al.], Development of cryopreservation media for the slow-freezing of cultured primordial germ cells in chicken, J Reprod Dev, 2023, Vol. 69(2), pp. 109–117, DOI: 10.1262/jrd.2022-123.

16. Tonus C., Connan D., Waroux O. [et al.], Cryopreservation of chicken primordial germ cells by vitrification and slow freezing: A comparative study, Theriogenology, 2017, Vol. 88, pp. 197–206, DOI: 10.1016/j.theriogenology.2016.09.022.

17. Silversides F.G., Robertson M.C., Liu J., Cryoconservation of avian gonads in Canada, Poult Sci, 2013, Vol. 92(10), pp. 2613–2617, DOI: 10.3382/ps.2013-03185.

18. Mucksová J., Reinišová M., Kalina J. [et al.], Conservation of chicken male germline by orthotopic transplantation of primordial germ cells from genetically distant donors, Biol Reprod, 2019, Vol. 101(1), pp. 200–207, DOI: 10.1093/biolre/ioz064.

19. Chaipipat S., Prukudom S., Sritabtim K. [et al.], Primordial germ cells isolated from individual embryos of red junglefowl and indigenous pheasants of Thailand, Theriogenology, 2021, Vol. 165, pp. 59–68, DOI: 10.1016/j.theriogenology.2021.02.010.

20. Yang S.Y., Lee H.J., Lee H.C. [et al.], The dynamic development of germ cells during chicken embryogenesis, Poult Sci, 2018, Vol. 97, pp. 650–657, DOI: 10.3382/ps/pex316.

21. Hu T., Purdy P.H., Blank M.H. [et al.], Direct in vitro propagation of avian germ cells from an embryonic gonad biorepository, Poult Sci, 2024, Vol. 103(11), pp. 104260, DOI: 10.1016/j.psj.2024.104260.

22. Hu T., Taylor L., Sherman A. [et al.], A low-tech, cost-effective and efficient method for safeguarding genetic diversity by direct cryopreservation of poultry embryonic reproductive cells, eLife, 2022, Vol. 11, pp. e74036, DOI: 10.7554/eLife.74036.

23. Song Y., Silversides F.G., The technique of orthotopic ovarian transplantation in the chicken, Poult Sci, 2006, Vol. 85(6), pp. 1104–1106, DOI: 10.1093/ps/85.6.1104.

24. Song Y., Silversides F.G., Transplantation of ovaries in Japanese quail (Coturnix japonica), Anim Reprod Sci, 2008, Vol. 105(3–4), pp. 430–437, DOI: 10.1016/j.anireprosci.2007.12.024.

25. Song Y., Silversides F.G., Long-term production of donor-derived offspring from chicken ovarian transplants, Poult Sci, 2008, Vol. 87(9), pp. 1818–1822, DOI: 10.3382/ps.2008-00103.

26. Liptoi K., Horvath G., Gal J. [et al.], Preliminary results of the application of gonadal tissue transfer in various chicken breeds in the poultry gene conservation, Anim Reprod Sci, 2013, Vol. 141(1–2), pp. 86–89, DOI: 10.1016/j.anireprosci.2013.06.016.

27. Hall G.B., Long J.A., Wood B.J., Bedecarrats G.Y., In ovo culturing of turkey (Meleagris gallopavo) ovarian tissue to assess graft viability and maturation of prefollicular germ cells and follicles, Poult Sci, 2020, Vol. 99(12), pp. 7109– 7121, DOI: 10.1016/j.psj.2020.09.014.

28. Hall G.B., Beeler-Marfisi J., Long J.A. [et al.], Cyclosporin A Prevents Ovarian Graft Rejection, and Permits Normal Germ Cell Maturation Within the First 5 Weeks Post-transplantation, in the Domestic Turkey (Meleagris gallopavo), Front Vet Sci, 2022, Vol. 9, pp. 855164, DOI: 10.3389/fvets.2022.855164.

29. Hall G.B., Long J.A., Susta L. [et al.], Turkey ovarian tissue transplantation: effects of surgical technique on graft attachment and immunological status of the grafts, 6 days post-surgery, Poult Sci, 2022, Vol. 101(3), pp. 101648, DOI: 10.1016/j.psj.2021.101648.

30. Quach C.C., Fulton J.E., Benson J.D. [et al.], Major Histocompatibility Complex-B haplotype and ovarian graft response, Poult Sci, 2023, Vol. 102(9), pp. 102850, DOI: 10.1016/j.psj.2023.102850.

31. Nakamura Y., Yamamoto Y., Usu F., [et al.], Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens, Reprod Fertil Dev, 2008, Vol. 20(8), pp. 900–907, DOI: 10.1071/rd08138.

32. Lee H., Kim S., Park T. [et al.], Compensatory proliferation of endogenous chicken primordial germ cells after elimination by busulfan treatment, Stem Cell Res Ther, 2013, Vol. 4(6), pp. 136, DOI: 10.1186/scrt347.

33. Liu J., Cheng K.M., Silversides F.G., Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica), Anim Reprod Sci, 2012, Vol. 134(3–4), pp. 197–202.

34. Liu J., Cheng K.M., Purdy P.H., Silversides F.G., A simple vitrification method for cryobanking avian testicular tissue, Poult Sci, 2012, Vol. 91(12), pp. 3209–13, DOI: 10.3382/ps.2012-02454.

35. FAO, 2012. FAOSTAT. Food and Agriculture Organization of the United Nations: https://www.fao.org/4/i3028e/i3028e.pdf.

36. Nateghi R., Alizadeh A., Jafari Ahangari Y. [et al.], Ethylene glycol and dimethyl sulfoxide combination reduces cryoinjuries and apoptotic gene expression in vitrified laying hen ovary, Biopreserv Biobank, 2017, Vol. 15(6), pp. 519– 528, DOI: 10.1089/bio.2017.0078.

37. Liptoi K., Buda K., Rohn E. [et al.], Improvement of the application of gonadal tissue allotransplantation in the in vitro conservation of chicken genetic lines, Anim Reprod Sci, 2020, Vol. 213, pp. 106280, DOI: 10.1016/j.anireprosci.2020.106280.

38. Fujihara M., Shiraishi J.-i., Onuma M. [et al.], Cryopreservation Competence of Chicken Oocytes as a Model of Endangered Wild Birds: Effects of Storage Time and Temperature on the Ovarian Follicle Survival, Animals, 2022, Vol. 12, pp. 1434, DOI: 10.3390/ani12111434.

39. Buda K., Vegi B., Lehoczky I. [et al.], Orthotopic Transplantation of Native and Cryopreserved Ovarian Tissue in Day-Old Geese, Vet Sci, 2025, Vol. 12, pp. 169, DOI: 10.3390/vetsci12020169.