MOLECULAR SEXING OF EQUINE CONCEPTUS SAMPLE COLLECTED VIA VAGINAL FORNIX
DOI:
https://doi.org/10.56238/revgeov17n3-144Keywords:
Equine, Molecular Sexing, Conceptus, Transvaginal RouteAbstract
The determination of the sex of the embryo is of great importance and has been improved over time, especially in cattle, due to its greater adequacy to existing techniques and methodologies. In horses, the techniques are not as precise, and can only be performed in short periods during gestation. In humans, as well as in cattle, amniocentesis is successfully performed, since the samples obtained make it possible to perform genotyping and sexing of fetuses with precision. In this work, a technique of transvaginal puncture of yolk fluid (occurs up to day 30 of the embryonic phase) and/or allantoic fluid from embryos and fetuses was tested, in order to obtain samples of genetic material for molecular sexing and to determine the most suitable period for puncture, making it a safe and efficient technique. . The genotyping of the conceptus, as well as of the adult horses, was successful in characterizing the profiles of the individuals and distinguishing them, especially in cases where the conceptus was female, proving that the genetic profile obtained from the DNA extracted from the punctured sample was distinct from the genetic profile obtained from the DNA of the blood sample of the pregnant mare. It was proven that the puncture was performed correctly and that the collection method was effective in bringing DNA samples from the conceptus both in quantity and quality, making it possible to perform genetic tests of sexing and genotyping efficiently.
Downloads
References
ABD-ELNAEIM MMM, LEISER R, WILSHER S, ALLEN WR. Structural and haemovascular aspects of placental growth throughout gestation in young and aged mares. Placenta, v. 27, p. 1103-1113, 2006. Disponível em: https://www.sciencedirect.com/science/article/pii/S0143400405002912, Acesso em: 14/02/2025.
AKOLEKAR, R,; FARKAS, D.H.; VANAGTMAEL, A.L.; BOMBARD, A.T.; NICOLAIDES, K.H. Foetal sex determination using circulating cell-free foetal DNA (ccffDNA) at 11 to 13 weeks of gestation. Prenat Diagn., v. 30(10), p. 918-923, 2010. Disponível em: https://pubmed.ncbi.nlm.nih.gov/20721878/, Acesso em 02/03/2025.
ALLEN WR. The Physiology of Early Pregnancy in the Mare. In: American Association of Equine Practitioners. Proceedings…, 46: 338-354, 2000.
BUCCA S. Equine foetal gender determination from mid- to advanced-gestation by ultrasound. Theriogenology, v. 64(3), p. 568-571, 2005. Disponível em: https://www.sciencedirect.com/science/article/pii/S0093691X05001731, Acesso em: 12/03/2025. doi:10.1016/j.theriogenology.2005.05.013.
BUTLER, J.M. Forensic DNA typing: biology, technology, and genetics of STR markers. Amsterdam; Boston, Elsevier Academic Press, 2005. Disponível em: https://books.google.com.br/books/about/Forensic_DNA_Typing.html?id=gwDyBq2xLjIC&redir_esc=y, Acesso em 04/40/2024.
BYDLOWSKI, S. P.; LEYTON J. A tecnologia do DNA; A Revista Oficial da ABCCMM, n 36, 1998.
CARMO, M.T.; OLIVEIRA, J.V.; ALMEIDA, M.T.; ALVARENGA, M.A. Avaliação ultrassonográfica da gônada fetal em equinos: uma nova alternativa para sexagem. In: Conferência Anual da Abraveq e Congresso Internacional de Medicina Veterinária, 9, 2008, São Paulo. Anais eletrônicos... São Paulo, SP: Abraveq, 2008. Disponível em: http://www.itarget.com.br/newclients/abraveq2012/down/2012/ix_conf_08%20Sexagem_fetal_ABRAVEQ.pdf, Acesso em: 10/11/2024.
CHOI, Y.H.; GUSTAFSON-SEABURY, A.; VELEZ, I.C.; HARTMAN, D.L.; BLISS, S.; RIERA, F.L.; ROLDÁN, J.E.; CHOWDHARY, B.; HINRICHS, K. Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis. Reproduction, v. 140, p. 893-902, 2010. Disponível em: https://pubmed.ncbi.nlm.nih.gov/20843896/, Acesso em: 04/09/2023.
DAMIANI, D.; DICHTCHEKENIAN, V.; SETIAN, N. O Enigma da Determinação Gonadal, Arq Bras Endocrinol Metab, v.44 n.3, 2000. Disponível em: https://www.scielo.br/j/abem/a/gKSzZGBkZc6BKZBz3gWJbzz/, Acesso em: 11/12/2024.
DYCE, K.M.; SACK, W.O.; WENSING, C.J.G. Textbook of veterinary anatomy. 3.ed. Philadelphia: WB Saunders, 2002. 258p.
GERSTENBERG, C.; ALLEN, W.R.; STEWART, F. Cell proliferation patterns during development of the equine placenta. Journals of Reproduction and Fertility, 117: 143-152, 1999. Disponível em: https://www.researchgate.net/publication/12669810 _Cell_proliferation_patterns_during_development_of_the_equine_placenta, Acesso em: 04/05/2025.
GINTHER, O.J. Reproductive biology of the mare: basic and applied aspects. 2.ed. Cross Plains: Equiservices, 642p, 1992.
HAFEZ, B.; HAFEZ, E.S.E. Reprodução Animal. 7ª Ed. Monole, São Paulo, 513 p., 2004.
HAN, S.H.; YANG, B.C.; KO, M.S.; OH, H.S.; LEE, S.S. Length difference between equine ZFX and ZFY genes and its application for molecular sex determination. J Assist Reprod Genet., v. 27(12), p. 725-728, 2010. Disponível em: https://pubmed.ncbi.nlm.nih.gov/20809415/, Acesso em: 23/05/2025.
HASEGAWA, T.; SATO, F.; ISHIDA, N.; FUKUSHIMA, Y.; MUKOYAMA, H. Sex determination by simultaneous amplification of equine SRY and amelogenin genes. J Vet Med Sci., v. 62(10), p. 1109-1110, 2000. Disponível em: https://pubmed.ncbi.nlm.nih.gov/11073085/, Acesso em 23/05/2025.
JAUNIAUX, E.; CIRIGLIANO, V.; ADINOLFI, M. Very early prenatal diagnosis on coelomic cells using quantitative fluorescent polymerase chain reaction. Reprod Biomed online, 6(4): 494-498, 2003. Disponível em: https://www.sciencedirect.com/science/article/pii/S1472648310621736, Acesso em: 12/05/2025.
KADIVAR, A.; HASSANPOUR, H.; MIRSHOKRAEI, P.; AZARI, M.; GHOLAMHOSSEINI, K.; KARAMI, A. Detection and quantification of cell-free foetal DNA in ovine maternal plasma; use it to predict foetal sex. Theriogenology, v. 79(6), p. 995-1000, 2013. Disponível em: https://pubmed.ncbi.nlm.nih.gov/23434357/, Acessoe em 05/05/2025.
KADIVAR, A.; RASHIDZADEH, H.; DAVOODIAN, N.; NAZARI, H.; DEHGHANI TAFTI, R.; HEIDARI KHOEI, H. Evaluation of the efficiency of TaqMan duplex real-time PCR assay for non-invasive pre-natal assessment of foetal sex in equine. Reprod Domest Anim., v. 56(2), p. 287-291, 2021. doi:10.1111/rda.13831. Disponível em: https://pubmed.ncbi.nlm.nih.gov/32978850/, Acesso em: 23/04/2025.
KAMIMURA, S.; NISHIYAMA, L. N.; OOKUTSU, I. S.; GOTO, K.; HAMANAL, K. Determination of bovine fetal sex by PCR using fetal fluid aspirated by transvaginal ultrasound-guided amniocentesis, Kagoshima, Japan. Elsevier, 1996.
LEON, P.M.M.; CAMPOS, V.F.; DELLAGOSTIN, O.A.; DESCHAMPS, J.C.; SEIXAS, F.K.; COLLARES, T. Equine foetal sex determination using circulating cell-free foetal DNA (ccffDNA). Theriogenology, v. 77(4), p. 694-698, 2012. Disponível em: https://pubmed.ncbi.nlm.nih.gov/22000028/, Acesso em: 17/02/2025.
LIVINI, M. Determination of fetal gender by transrectal ultrasound examination: field´s experience. In: American Association of Equine Practitioners Annual Convention, 56, 2010, Baltimore. Anais eletrônicos…Baltimore: Maryland, 2010. Disponível em: http://www.cabi.org/cabdirect/FullTextPDF/2011/20113042293.pdf, Acesso em: 30 jun. 2012.
LO, Y.M.; CORBETTA N, C.P.F.; RAI, V.; SARGENT, I.L.; REDMAN, C.W.; WAINSCOAT, J.S. Presence of fetal DNA in maternal plasma and serum. Lancet, v. 350, p. 485-487, 1997. Disponível em: https://pubmed.ncbi.nlm.nih.gov/9274585/, Acesso em: 23/07/2024.
PEIPPO, J.; HUHTINEN, M.; KOTILAINEN, T. Sex diagnosis of equine preimplantation embryos using the polymerase chain reaction. Theriogenology, v.44, p.619-627, 1995. Disponível em: https://www.sciencedirect.com/science/article/ pii/0093691X9500242Z, Acesso em: 06/08/2023.
PIPREK, R,P. Molecular and cellular machinery of gonadal differentiation in mammals. Int J Dev Biol, v. 54(5-6), p. 779-86, 2010. Disponível em: https://pubmed.ncbi.nlm.nih.gov/20446275/, Acesso em: 17/03/2025.
MOURIJAVAGAR, D.; MALARMATHI, M.; RAGHAVENDRAN, V.B. Early foetal sexing in the equines by detection of cfDNA in maternal serum, International Journal of Veterinary Sciences and Animal Husbandry, v. 10(2), p. 23-25, 2025. Disponível em: https://www.veterinarypaper.com/pdf/2025/vol10issue2/PartA/10-2-7-971.pdf, Acesso em: 11/03/2025.
MACHADO, L.V. Genotipagem, sexagem molecular e verificação de pedigree em equinos por PCR quantitativo fluorescente, Centro de biociências e biotecnologia -NUDIM- Campos dos Goytacazes, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 2007.
NAZARETH, H. R. S.; PINTO JR, W.; ANDRADE, J. A. D. Diagnóstico pré-natal de aberrações cromossômicas. Primeira experiência brasileira. Revista Brasileira de Genética, v. (3), p. 459-470, 1981. Disponível em: https://nepas.emnuvens.com.br/amabc/article/view/613, Acesso em: 23/04/2025.
OLIVEIRA, R.A.; YAMIM, R.S.; PIVATO, I.; RAMOS, A.F. Sexagem fetal em equinos, Rev. Bras. Reprod. Anim, v. 38, n.1, p.3 7-42, 2014. Disponível em www.cbra.org.br, Acesso em: 12/04/2024.
SHERLOCK, J.; CIRIGLIANO, V.; PETROU, M. Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells. Ann. Hum. Genet., v. 62, p. 9–23, 1998. Disponível em: https://pubmed.ncbi.nlm.nih.gov/9659974/, Acesso em: 19/03/2025.
TONEKABONI, F.R.; NARENJISANI, R.; STAJI, H.; AHMADI-HAMEDANI, M. Comparison of cell-free foetal DNA plasma content used to sex determination between three trimesters of pregnancy in Torkaman pregnant mare. J Equine Vet Sci., v. 95, p. 103273, 2020. Disponível em: https://www.sciencedirect.com/science/article/pii /S0737080620303646, Acesso em: 12/03/2025.
TOZAKI, T.; KAKOI, H.; MASHIMA, S.; HIROTA, K.; HASEGAWA, T.; ISHIDA, N.; MIURA, N.; CHOI-MIURA, N. H.; TOMITA, M. Population study and validation of paternity testing for Thoroughbred horses by 15 microsatellite loci. J Vet Med Sci., v. 63: 1191-1197, 2001. Disponível em: https://pubmed.ncbi.nlm.nih.gov/11767052/, Acesso em: 03/04/2025.
WACHTEL, S.S. H−Y Antigen and the Biology of Sex Determination (Grune & Stratton, New York, 1983.
