Ram and Goat Semen Immunosexed and Diluted in Powdered Coconut Water-Based Preservation Medium (ACP101/102c).
DOI:
https://doi.org/10.22456/1679-9216.114066Abstract
Background: Sperm sexing aims to separate sperm populations in carriers of the “X” or “Y” chromosome. Currently, flow cytometry is a technique that allows greater accuracy; however, it causes structural changes in sperm, reduces viability, and has a high cost. As a result, other methods have been researched, including immunosexing, which uses monoclonal antibodies to detect sex-specific surface antigens. Thus, the objective of this study was to evaluate the immunosexing technique using a monoclonal antibody against sex-specific protein (HY) in the conservation of ram and goat semen in ACP101/102c.
Materials, Methods & Results: Ejaculates from 5 rams and 5 goats were collected with the aid of an artificial vagina; they were evaluated and submitted to the immunosexing protocol, according to the manufacturer's recommendations, using the Monoclonal Antibody Kit specific for mammalian sperm with “Y” chromosomes (HY; HY Biotechnology, Rio de Janeiro, RJ, Brazil). After sexing, the supernatant was resuspended in the cryopreservation diluent: ACP ram (ACP101/102c + 20% egg yolk + 7% glycerol) and ACP goat (ACP101/102c + 2.5% egg yolk + 7% glycerol), packaged in 0.25 mL straws, refrigerated at 4°C, stabilized for 30 min, frozen in liquid nitrogen vapor (-60°C) for 15 min, immersed in liquid nitrogen, and stored in cryogenic cylinders. The samples were evaluated in natura (T1), after immunosexing (T2) and after thawing (T3) for sperm motility subjectively using conventional microscopy (40x). Plasma membrane integrity (IMP) and sperm cell morphology were evaluated by the smear staining technique using eosin-nigrosine dye, and the percentages of healthy and morphologically defect spermatozoa were determined. In the evaluation of ram semen regarding sperm motility and IMP, no statistically significant differences were observed between treatments after sexing in the evaluation of absolute data (P > 0.05), with the difference being observed only between T1 and T2, and T3 (P < 0.05). Regarding the relative percentage and sperm morphology, no statistically significant differences were observed (P > 0.05). Regarding the evaluation of goat semen samples, the motility parameters were consistent with the technique submitted; however, the IMP data did not appear as expected, requiring further evaluation for a better assessment of the technique for this species.
Discussion: The data obtained from ram semen submitted to the immunosexing protocol, regarding the absolute evaluation of motility and IMP, demonstrated that the non-sexed semen (T1) was superior to the sexed treatments (T2 and T3); however, it is noteworthy that freezing started with approximately 50% of the cells, since the immunosexing technique results in a loss of viability of approximately 50% of the sperm, which corresponds to the ratio of sperm carrying the X chromosome. In addition, when the data in this study were transformed into relative values, no statistical differences were observed, indicating that the immunosexing protocol, as well as the freezing protocol, did not significantly affect the quality of ram sperm cells. In relation to the immunosexing of goat semen, future studies should be conducted in vitro to define a more appropriate protocol for the species and, in addition, in vivo studies should be performed to prove the quality of the technique. It was concluded that the immunosexing process using a monoclonal antibody against sex-specific protein (HY) associated with the use of powdered coconut water diluent (ACP101/102c) in the cryopreservation of semen proved to be efficient in the in vitro evaluation of ovine species.
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Alves F.S.F., Holanda Júnior E.V. & Lopes R.S. 2009. Produção Integrada no Brasil: Agropecuária Sustentável Alimentos Seguros. Brasília: MAPA 2009. 1008p. Disponível em: <https://ainfo.cnptia.embrapa.br/digital/bitstream/CNPAT-2010/11427/1/CL09007.pdf>. [Accessed online in July 2018].
Carvalho J.O., Sartori R. & Dode M.A.N. 2014. Different ways to evaluate bovine sexed sperm in vitro. Animal Reproduction. 11(3): 199-206.
Carvalho J.O., Sartori R., Machado G.M., Mourão G.B. & Dode M.A. 2010. Quality assessment of bovine cryopreserved sperm after sexing by flow cytometry and their use in in vitro embryo production. Theriogenology. 74(9): 1521-1530.
Carvalho J.O., Sartori R., Rodello L., Mourão G.B., Bicudo S.D. & Dode M.A.N. 2018. Flow cytometry sex sorting affects bull sperm longevity and compromises their capacity to bind to oviductal cells. Livestock Science. 207(2018): 30-37.
Celeghini E.C.C., Arruda R.P., Andrade A.F.C., Nascimento J. & Raphael C.F. 2007. Practical techniques for bovine sperm simultaneous fluorimetric assessment of plasma, acrosomal and mitochondrial membranes. Reproduction in Domestic Animals. 42(5): 479-488.
Chemineau P., Caignie Y., Guerin Y. & Orgeus P. 1991. Training Manual on Artificial Insemination in Sheep and Goats. Rome: Food and Agriculture Organization of the United Nations, 223p.
Colégio Brasileiro de Reprodução Animal (CBRA). 2013. Ovino, Caprino. In: Colégio Brasileiro de Reprodução Animal. Manual Para Exame Andrológico e Avaliação do Sêmen Animal. Belo Horizonte: CBRA, pp.35-42.
Frijters A.C.J., Mullaart E., Roelofs R.M.G., Van Hoorne R.P., Moreno J.F., Moreno O. & Merton J.S. 2009. What affects fertility of sexed bull semen more, low sperm dosage or the sorting process? Theriogenology. 71(1): 64-67.
Garner D.L. 2006. Flow cytometric sexing of mammalian sperm. Theriogenology. 65(5): 943-957.
Garner D.L., Evans K.M. & Seidel G.E. 2013. Sex-sorting sperm using flow cytometry/cell sorting. Methods in Molecular Biology. 927: 279-295.
Hollinshead F.K., Gillan L., O’Brien J.K., Evans G. & Maxwell W.M. 2003. In vitro and in vivo assessment of functional capacity of flow cytometrically sorted ram spermatozoa after freezing and thawing. Reproduction, Fertility, and Development. 15(6): 351-359.
Liu X., Hu T., Sun W., Hao H., Liu Y., Zhao X., Zhu H. & Du W. 2015. Comparison of the developmental competence and quality of bovine embryos obtained by in vitro fertilization with sex-sorted and unsorted semen from seven bulls. Livestock Science. 181: 263-270.
Machado R., Oliveira J.A.M., Simplício A.A. & Zagatto L.C.A.G. 1996. Custo de produção de sêmen caprino na central de Inseminação artificial da Embrapa-CNPC. Brasília: Embrapa Caprinos e Ovinos, 3p. Disponível em: <http://www.alice.cnptia.embrapa.br/bitstream/doc/43419/1/PROCIRM1996.00040.pdf>. [Accessed online in November 2019].
Maia L.C.P., Brito B.F., Santos B.M.B., Cabral L.A.R., Salgueri C.C.M., Matta M.F.R. & Nunes J.F. 2020. Aspectos econômicos e avaliação do período de viabilidade de espermatozoides ovino imunossexados e diluídos em meio de conservação à base de água de coco em pó (ACP-102c). Acta Scientiae Veterinariae. 48: 1740.
Matta C.G.F. 2003. Desenvolvimento de uma metodologia para sexagem de espermatozóides de bovinos utilizando anticorpos monoclonais e complemento. 55f. Goytacazes, RJ. Tese (Doutorado em Produção Animal) - Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense.
Matta M.F.R. 2016. A method of enriching spermatozoa of mammals bearing X-chromosome or Y-chromosome. European Patent Office. Paris. Patente N/Ref.: EP2402757B1.
Santos B.M.B. 2020. Bioprocesso de imunossexagem espermática em bovinos e ovinos de corte. 91f. Fortaleza, CE. Tese (Doutorado em Biotecnologia) - Rede Nordeste em Biotecnologia, Universidade Estadual do Ceará.
Souza C.J.P., Matta M.F.Rd, Cruz G.M., Alves E.W., Kanashiro M.M. & Silva J.F.S. 1999. Monoclonal antibody against male-specific protein of 19 KDa from bovine spermatozoa: A successful methodology for imunosexing. Revista Brasileira de Zootecnia. 28(1): 74-78.
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