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Home / Equine Research Bank / J Assist Reprod Genet / Use of confocal microscopy and intracytoplasmic sperm inject...
Journal of assisted reproduction and genetics2023; 40(11); 2565-2576; doi: 10.1007/s10815-023-02935-4

Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification.

Abstract: The impact of vitrification on oocyte developmental competence as a function of donor age remains an important issue in assisted reproductive technologies (ARTs). Methods: Equine germinal vesicle (GV) or metaphase II (M(II) oocytes were vitrified using the Cryotop® method. Spindle organization and chromosome alignment were evaluated from confocal imaging data sets of in vivo (IVO) or in vitro (IVM) matured oocytes subjected to vitrification or not. Intracytoplasmic sperm injection (ICSI) from the same groups was used to assess developmental potential. Results: An increase in chromosome misalignment was observed in spindles from older mares when compared to those of younger mares (P < 0.05). When MII oocytes subjected to vitrification were examined following warming, there was no difference in the percentage of oocytes displaying chromosome misalignment. Next, GV oocytes, collected from the ovaries of younger and older mares, were compared between fresh IVM and IVM following vitrification and warming. For nonvitrified samples, an age difference was again noted for spindle organization and chromosome alignment, with a higher (P < 0.05) percentage of normal bipolar meiotic spindles with aligned chromosomes observed in nonvitrified oocytes from young versus older mares. Vitrification led to a reduction of spindle length (P < 0.05) for oocytes from old mares, whether vitrified at GV or MII stages, whereas this effect was not observed in oocytes from young mares except those vitrified at GV and subjected to IVM. Oocyte developmental potential after vitrification was evaluated after ICSI of vitrified and warmed MII or GV oocytes from young mares. From 25 MII oocytes, 18 oocytes were injected with sperm, and six blastocysts were produced, which, upon transfer to mares' uteri, resulted in four pregnancies. Immature (GV) oocytes collected from live mares were also vitrified, warmed, and matured in vitro before ICSI. In this group, nonvitrified, control, and vitrified oocytes did not differ (P > 0.05) with respect to the incidence of maturation to MII, cleavage after ICSI, or blastocyst development. Conclusions: These findings demonstrate an effect of maternal age in an equine model at the level of meiotic spindle integrity and chromosome positioning that is influenced by both the meiotic stage at which oocytes are vitrified and whether meiotic maturation occurred in vivo or in vitro.
© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
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Publication Date: 2023-09-19 PubMed ID: 37725179PubMed Central: PMC10643763DOI: 10.1007/s10815-023-02935-4Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article focuses on understanding the effect of vitrification on the viability of equine oocytes – female reproductive cells – taken from both young and old mares. Using techniques of confocal microscopy and intracytoplasmic sperm injection (ICSI), the study found that maternal age had an impact on the integrity of meiotic spindles and chromosome positioning in the oocytes.

Objective of the Study

  • The primary objective of this study was to explore the impact of vitrification, a rapid freezing technique used in assisted reproductive technologies (ARTs), on the competency of equine oocytes derived from mares of different age groups. The research also aimed to compare the spindle organization and chromosome alignment of both in vivo (occurred naturally) and in vitro (occurred artificially) matured oocytes.

Methodology

  • The researchers used equine germinal vesicle (GV) or metaphase II (M(II) oocytes and subjected them to vitrification using the Cryotop® method.
  • Spindle organization and chromosome alignment were evaluated using confocal imaging, comparing in vivo (IVO) or in vitro (IVM) matured oocytes.
  • Intracytoplasmic sperm injection (ICSI) was performed to gauge the developmental potential of these oocytes.

Findings

  • The analyses showed a significant increase in chromosome misalignment in older mares compared to younger ones.
  • However, when MII oocytes subjected to vitrification were examined after warming, no difference was observed in chromosome misalignment, regardless of the age of the donor mares.
  • Vitrification caused a reduction of spindle length in oocytes from older mares, whether vitrified at GV or MII stages. This effect, however, was not observed in the oocytes from young mares.

Conclusion

  • The results suggest that maternal age has considerable effects on spindle integrity and chromosome positioning in equine oocytes. This impact is influenced both by the meiotic stage at which oocytes are vitrified and whether meiotic maturation occurred in vivo or in vitro.

Cite This Article

APA
Maclellan LJ, Albertini DF, Stokes JE, Carnevale EM. (2023). Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification. J Assist Reprod Genet, 40(11), 2565-2576. https://doi.org/10.1007/s10815-023-02935-4

Publication

Journal of assisted reproduction and genetics
ISSN: 1573-7330
NlmUniqueID: 9206495
Country: Netherlands
Language: English
Volume: 40
Issue: 11
Pages: 2565-2576

Researcher Affiliations

Maclellan, Lisa J
  • Department of Biomedical Sciences, Colorado State University, 1601 Campus Delivery, Fort Collins, Colorado, 80523, USA.
  • Seven Creeks Equine Reproduction, Euroa, 3666, Australia.
Albertini, David F
  • Bedford Research Foundation, Bedford, MA, 01730, USA.
Stokes, Joanne E
  • Department of Biomedical Sciences, Colorado State University, 1601 Campus Delivery, Fort Collins, Colorado, 80523, USA.
Carnevale, Elaine M
  • Department of Biomedical Sciences, Colorado State University, 1601 Campus Delivery, Fort Collins, Colorado, 80523, USA. Elaine.Carnevale@colostate.edu.

MeSH Terms

  • Animals
  • Horses
  • Male
  • Female
  • Vitrification
  • Sperm Injections, Intracytoplasmic / veterinary
  • Cryopreservation / veterinary
  • Cryopreservation / methods
  • Semen
  • Oocytes
  • Microscopy, Confocal

Conflict of Interest Statement

The authors declare no competing interests.

References

This article includes 57 references
  1. Altermatt JL, Suh TK, Stokes JE, Carnevale EM. Effects of age and eFSH on collection and viability of equine oocytes assessed by morphology and developmental competency after ICSI. Reprod Fertil Dev 2009;21(4):615–623.
    doi: 10.1071/RD08210pubmed: 19383268google scholar: lookup
  2. Barrett SL, Albertini DF. Allocation of gamma-tubulin between oocyte cortex and meiotic spindle influences asymmetric cytokinesis in the mouse oocyte. Biol Reprod 2007;76(6):9–957.
    doi: 10.1095/biolreprod.106.057141pubmed: 17287496google scholar: lookup
  3. Battaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod 1996;11(10):2217–2222.
    doi: 10.1093/oxfordjournals.humrep.a019080pubmed: 8943533google scholar: lookup
  4. Bogliolo L, Murrone O, Piccinini M, Ariu F, Ledda S, Tilocca S, Albertini DF. Evaluation of the impact of vitrification on the actin cytoskeleton of in vitro matured ovine oocytes by means of Raman microspectroscopy. J Assist Reprod Genet 2015;32(2):185–189.
    doi: 10.1007/s10815-014-0389-7pmc: PMC4354177pubmed: 25399064google scholar: lookup
  5. Boiso I, Marti M, Santalo J, Ponsa M, Barri PN, Veiga A. A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle and metaphase II stage. Hum Reprod 2002;17:1885–1891.
    doi: 10.1093/humrep/17.7.1885pubmed: 12093855google scholar: lookup
  6. Bromfield JJ, Coticchio G, Hutt K, Sciajno R, Borini A, Albertini DF. Meiotic spindle dynamics in human oocytes following slow-cooling cryopreservation. Hum Reprod 2009;24(9):2114–2123.
    doi: 10.1093/humrep/dep182pubmed: 19465461google scholar: lookup
  7. Canesin HS, Brom-de-Luna JG, Choi YH, Ortiz I, Diaw M, Hinrichs K. Blastocyst development after intracytoplasmic sperm injection of equine oocytes vitrified at the germinal vesicle stage. Cryobiology 2017;75(2017):52–59.
    doi: 10.1016/j.cryobiol.2017.02.004pubmed: 28209499google scholar: lookup
  8. Canesin HS, Brom-de-Luna JG, Choi Y-H, Hinrichs K. Toxicity of a vitrification protocol for equine immature oocytes. J Equine Vet Sci 2016;47:79–80.
    doi: 10.1016/j.jevs.2016.04.079google scholar: lookup
  9. Carboni S, Rosati I, Maclellan LJ, Ariu F, Bogliolo L, Zedda MT, Pau S, Carnevale EM, Ledda S. Vitrification of GV and IVM horse oocytes with two different equilibration methods. Proceedings of 10th Congress of Italian Society of Animal Reproduction (SIRA 2012) 2012; pp. 12–13.
  10. Carnevale EM. The mare model for follicular maturation and reproductive aging in the woman. Theriogenology 2008;69:23–30.
    doi: 10.1016/j.theriogenology.2007.09.011pubmed: 17976712google scholar: lookup
  11. Carnevale EM. The mare as an animal model for reproductive aging in the woman. Animal Models and Human Reproduction (H Schatten and GM Constantinescu) Hoboken: Wiley-Blackwell 2017; pp. 235–245.
  12. Carnevale EM, Ginther OJ. Defective oocytes as a cause of subfertility in old mares. Biol Reprod 1995;1(Equine Reproduction VI):209–214.
    doi: 10.1093/biolreprod/52.monograph_series1.209google scholar: lookup
  13. Carnevale EM, Uson M, Bozzola JJ, King SS, Schmitt SJ, Gates HD. Comparison of oocytes from young and old mares with light and electron microscopy. Theriogenology 1999;51:299.
    doi: 10.1016/S0093-691X(99)91858-7google scholar: lookup
  14. Carnevale EM, Maclellan LJ, Coutinho da Silva MA, Scott TJ, Squires EL. Comparison of culture and insemination techniques for equine oocyte transfer. Theriogenology 2000;54(6):981–987.
    doi: 10.1016/S0093-691X(00)00406-4pubmed: 11097049google scholar: lookup
  15. Carnevale EM, Maclellan LJ, Countinho da Silva MA, Squires EL. Pregnancies after collection and transfer of oocytes from ovaries of five euthanized mares. J Am Vet Med Assoc 2003;222(1):60–62.
    doi: 10.2460/javma.2003.222.60pubmed: 12523482google scholar: lookup
  16. Carnevale EM. Advances in collection, transport and maturation of equine oocytes for assisted reproductive techniques. Vet Clin North Am Equine Pract 2016;32(3):379–399.
    doi: 10.1016/j.cveq.2016.07.002pubmed: 27726987google scholar: lookup
  17. Cheng J, Jia B, Wu T, Zhou G, Hou Y, Fu X, Zhu S. Effects of vitrification for germinal vesicle and metaphase II oocytes on subsequent centromere cohesion and chromosome aneuploidy in mice. Theriogenology 2014;82(3):495–500.
    doi: 10.1016/j.theriogenology.2014.05.009pubmed: 24930605google scholar: lookup
  18. Chen SU, Lien YL, Chen HF, Chao KH, Ho HN, Yang YS. Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod 2000;15:2598–2603.
    doi: 10.1093/humrep/15.12.2598pubmed: 11098033google scholar: lookup
  19. Chen CK, Wang CW, Tsai WJ, Hsieh LL, Wang HS, Soong YK. Evaluation of meiotic spindles in thawed oocytes after vitrification using polarized light microscopy. Fertil Steril 2004;82(3):666–672.
    doi: 10.1016/j.fertnstert.2003.12.053pubmed: 15374712google scholar: lookup
  20. Clerico G, Taminelli G, Veronesi JC, Polola J, Pagura N, Pinto C, Sansinena M. Mitochondrial function, blastocyst development and live foals born after ICSI of immature vitrified/warmed equine oocytes matured with or without melatonin. Theriogenology 2021;160:40–49.
    doi: 10.1016/j.theriogenology.2020.10.036pubmed: 33171351google scholar: lookup
  21. Cobo A, Bellver J, Domingo J, Pérez S, Crespo J, Pellicer A, Remohí J. New options in assisted reproduction technology: the Cryotop method of oocyte vitrification. Reprod Biomed Online 2008;17(1):68–72.
    doi: 10.1016/S1472-6483(10)60295-7pubmed: 18616893google scholar: lookup
  22. Coticchio G, Bromfield JJ, Sciajno R, Gambardella A, Scaravelli G, Borini A, Albertini DF. Vitrification may increase the rate of chromosome misalignment in the metaphase II spindle of human mature oocytes. Reprod Biomed Online 2009;19(Suppl 3):29–34.
    doi: 10.1016/S1472-6483(10)60281-7pubmed: 20034421google scholar: lookup
  23. de Leon PM, Campos VF, Corcini CD, Santos EC, Rambo G, Lucia T Jr, Deschamps JC, Collares T. Cryopreservation of immature equine oocytes, comparing a solid surface vitrification process with open pulled straws and the use of a synthetic ice blocker. Theriogenology 2012;77(1):21–27.
    doi: 10.1016/j.theriogenology.2011.07.008pubmed: 21835449google scholar: lookup
  24. Ducheyne KD, Rizzo M, Beitsma M, Deelan C, Daels PF, Stout TAE, de Ruijter-Viallani M. Vitrifying equine oocytes at the germinal vesicle stage disturbs spindle morphology and chromosome alignment. J Equine Vet 2018;66:178.
    doi: 10.1016/j.jevs.2018.05.070google scholar: lookup
  25. Ducheyne KD, Rizzo M, Daels PF, Stout TAE, de Ruijter-Villani M. Vitrifying immature equine oocytes impairs their ability to correctly align the chromosomes on the MII spindle. Reprod Fertil Dev 2019;31(8):1330–1338.
    doi: 10.1071/RD18276pubmed: 30967171google scholar: lookup
  26. Foss R, Ortis H, Hinrichs K. Effect of potential oocyte transport protocols on blastocyst rates after intracytoplasmic sperm injection in the horse. Equine Vet J 2013;45(Suppl. 45):39–43.
    doi: 10.1111/evj.12159pubmed: 24304402google scholar: lookup
  27. Ginther OJ. The mare: a 1000-pound guinea pig for study of the ovulatory follicular wave in women. Theriogenology 2012;77:18–828.
    doi: 10.1016/j.theriogenology.2011.09.025pubmed: 22115815google scholar: lookup
  28. Ginther OJ. Reproductive biology of the mare: basic and applied aspects. 2. Cross Plains: Equiservices; 1992.
  29. Hinrichs K, Schmidt AL, Friedman PP, Selgrath JP, Martin MG. In vitro maturation of horse oocytes: characterization of chromatin configuration using fluorescence microscopy. Biol Reprod 1993;48(2):363–370.
    doi: 10.1095/biolreprod48.2.363pubmed: 8439626google scholar: lookup
  30. Hinrichs K, Schmidt AL. Meiotic competence in horse oocytes: interactions among chromatin configuration, follicle size, cumulus morphology, and season. Biol Reprod 2000;62:1402–1408.
    doi: 10.1095/biolreprod62.5.1402pubmed: 10775193google scholar: lookup
  31. Hinrichs K, Choi Y-H, Norris JD, Love LB, Bedford-Guaus SJ, Hartman DL, Velez IC. Evaluation of foal production following intracytoplasmic sperm injection and blastocyst culture of oocytes from ovaries collected immediately before euthanasia or after death of mares under field conditions. J Amer Vet Med Assoc 2012;241(8):1070–1074.
    doi: 10.2460/javma.241.8.1070pubmed: 23039983google scholar: lookup
  32. Hochi S, Kozawa M, Fujimoto T, Hondo E, Yamada J, Oguri N. In vitro maturation and transmission electron microscopic observation of horse oocytes after vitrification. Cryobiology 1996;33(3):300–310.
    doi: 10.1006/cryo.1996.0030pubmed: 8689887google scholar: lookup
  33. Hurtt AE, Landim-Alvarenga F, Seidel GE Jr, Squires EL. Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws. Theriogenology 2000;54:119–128.
    doi: 10.1016/S0093-691X(00)00330-7pubmed: 10990353google scholar: lookup
  34. Kuwayama M, Vajta G, Leda S, Kato O. Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination. Reprod Biomed Online 2005;11(5):608–614.
    doi: 10.1016/S1472-6483(10)61169-8pubmed: 16409712google scholar: lookup
  35. Kuwayama M, Vatja G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005;11(3):300–308.
    doi: 10.1016/S1472-6483(10)60837-1pubmed: 16176668google scholar: lookup
  36. Maclellan LJ, Carnevale EM, Coutinho da Silva MA, Scoggin CF, Bruemmer JE, Squires EL. Pregnancies from vitrified equine oocytes collected from super-stimulated and non-stimulated mares. Theriogenology 2002;58(5):911–919.
    doi: 10.1016/S0093-691X(02)00920-2pubmed: 12212891google scholar: lookup
  37. Maclellan LJ, Stokes JE, Preis KA, McCue PM, Carnevale EM. Vitrification, warming, ICSI and transfer of equine oocytes matured in vivo. Anim Reprod Sci 2010;121:S260–S261.
    doi: 10.1016/j.anireprosci.2010.04.157google scholar: lookup
  38. Madill S. Reproductive considerations: mare and stallion. Veterinary Clinics Equine 2002;18:591–619.
    doi: 10.1016/S0749-0739(02)00030-5pubmed: 12516936google scholar: lookup
  39. Messinger SM, Albertini DF. Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. J Cell Sci 1991;100(Pt 2):289–298.
    doi: 10.1242/jcs.100.2.289pubmed: 1721916google scholar: lookup
  40. Ortiz-Escribano N, Bogado Pascottini O, Woelders H, Vandenberghe L, De Schauwer C, Govaere J, Van den Abbeel E, Vullers T, Ververs C, Roels K, Van De Velde M, Van Soom A, Smits K. An improved vitrification protocol for equine immature oocytes, resulting in a first live foal. Equine Vet J 2018;50(3):391–397.
    doi: 10.1111/evj.12747pubmed: 28833413google scholar: lookup
  41. Park SE, Son WY, Lee SH, Lee KA, Ko JJ, Cha KY. Chromosome and spindle configurations of human oocytes matured in vitro after cryopreservation at the germinal vesicle stage. Fertil Steril 1997;68:920–926.
    doi: 10.1016/S0015-0282(97)00365-8pubmed: 9389826google scholar: lookup
  42. Parks JE, Ruffing NA. Factors affecting low temperature survival of mammalian oocytes. Theriogenology 1992;37:59–73.
    doi: 10.1016/0093-691X(92)90247-Ogoogle scholar: lookup
  43. Rader K, Choi YH, Hinrichs K. Intracytoplasmic sperm injection, embryo culture, and transfer of in vitro–produced blastocysts. Vet Clin Equine 2016;32:401–413.
    doi: 10.1016/j.cveq.2016.07.003pubmed: 27726990google scholar: lookup
  44. Rienzi L, Gracia C, Maggiulli R, LaBarbera AR, Kaser DJ, Ubaldi FM, Vanderpoel S, Racowsky C. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review of meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Hum Reprod Update 2017;23(2):139–155.
    pmc: PMC5850862pubmed: 27827818
  45. Rizzo M, Kops GJPL, Deelan C, Beitsma M, Cristarella S, Stout TAE, de Ruijter-Viallani M. Compromised spindle assembly checkpoint function in oocytes from aged mares impairs correct chromosome alignment. J Equine Vet 2018;66:177.
    doi: 10.1016/j.jevs.2018.05.069google scholar: lookup
  46. Rojas C, Palomo MJ, Albarracín JL, Mogas T. Vitrification of immature and in vitro matured pig oocytes: study of distribution of chromosomes, microtubules, and actin microfilaments. Cryobiology 2004;49(3):211–220.
    doi: 10.1016/j.cryobiol.2004.07.002pubmed: 15615607google scholar: lookup
  47. Rosati I, Maclellan LJ, Ariu F, Bogliolo L, Zedda MT, Pau S, Carnevale EM, Ledda S. Vitrification of GV and IVM horse oocytes with two different equilibration methods. Reprod Domest Anim 2012;47(Suppl. 4):508.
  48. Ruggeri E, DeLuca KF, Galli C, Lazzari G, DeLuca JG, Stokes JE, Carnevale EM. Use of confocal microscopy to evaluate equine zygote development after sperm injection of oocytes matured in vivo or in vitro. Microsc Microanal 2017; 10.1017/S1431927617012740. published online: 06 Dec 2017 ahead of print.
    pmc: PMC5976488pubmed: 29208065doi: 10.1017/s1431927617012740google scholar: lookup
  49. Saunders KM, Parks JE. Effects of cryopreservation procedures on the cytology and fertilization rate of in vitro-matured bovine oocytes. Biol Reprod 1999;61:178–187.
    doi: 10.1095/biolreprod61.1.178pubmed: 10377047google scholar: lookup
  50. Schatten H, Sun QY. Centrosome dynamics during mammalian oocyte maturation with a focus on meiotic spindle formation. Mol Reprod Dev 2011;78:757–768.
    doi: 10.1002/mrd.21380pubmed: 21887720google scholar: lookup
  51. Scoggin CF. Not just a number: effect of age on fertility, pregnancy and offspring vigour in thoroughbred brood-mares. Reprod Fertil Dev 2015;27:872–879.
    doi: 10.1071/RD14390pubmed: 25786411google scholar: lookup
  52. Tharasanit T, Colleoni S, Galli C, Colenbrander B, Stout TA. Protective effects of the cumulus-corona radiata complex during vitrification of horse oocytes. Reproduction 2009;137(3):391–401.
    doi: 10.1530/REP-08-0333pubmed: 19073713google scholar: lookup
  53. Tharasanit T, Colenbrander B, Stout TAE. Effect of maturation stage at cryopreservation on post-thaw cytoskeleton quality and fertilizability of equine oocytes. Mol Reprod Dev 2006;73:627–637.
    doi: 10.1002/mrd.20432pubmed: 16477611google scholar: lookup
  54. Tharasanit T, Colleoni S, Lazzari G, Colenbrander B, Galli C, Stout TA. Effect of cumulus morphology and maturation stage on the cryopreservability of equine oocytes. Reproduction 2006;132(5):759–769.
    doi: 10.1530/rep.1.01156pubmed: 17071777google scholar: lookup
  55. Tremoleda JL, Van Haeften T, Stout TA, Colenbrander B, Bevers MM. Cytoskeleton and chromatin reorganization in horse oocytes following intracytoplasmic sperm injection: patterns associated with normal and defective fertilization. Biol Reprod 2003;69(1):186–194.
    doi: 10.1095/biolreprod.102.012823pubmed: 12646492google scholar: lookup
  56. Vajta G, Kuwayama M. Improving cryopreservation systems. Theriogenology 2006;65(1):236–244.
    doi: 10.1016/j.theriogenology.2005.09.026pubmed: 16289262google scholar: lookup
  57. Vajta G, Holm P, Kuwayama M, Booth PJ, Jacobsen H, Greve T, Callesen H. Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998;51(1):53–58.
    doi: 10.1002/(SICI)1098-2795(199809)51:1<53::AID-MRD6>3.0.CO;2-Vpubmed: 9712317google scholar: lookup
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