Please use this identifier to cite or link to this item: https://www.arca.fiocruz.br/handle/icict/28816
Title: Low Density Lipoproteins at 2% Concentration Can Replace Whole Egg Yolk in TES-Tris-Milk Extender for Freezing Buffalo Sperm Cells
Authors: Brito, Mayara Ferreira
Neves, Beatriz Parzewski
Andrade, Guilherme de Oliveira
Gouvêa, Rafael Ruas
Almeida, Jaci
Morais, Camila Leite
Martins Filho, Olindo Assis
Araújo, Márcio Sobreira Silva
Melo, Maria Isabel Vaz de
Henry, Marc
Affilliation: Universidade Federal de Minas Gerais. Escola de Veterinária. Departamento de Clinica e Cirurgia Veterinária. Belo Horizonte, MG, Brazil.
Universidade Federal de Minas Gerais. Escola de Veterinária. Departamento de Clinica e Cirurgia Veterinária. Belo Horizonte, MG, Brazil.
Universidade Federal de Minas Gerais. Escola de Veterinária. Departamento de Clinica e Cirurgia Veterinária. Belo Horizonte, MG, Brazil.
Universidade Federal de Minas Gerais. Escola de Veterinária. Departamento de Clinica e Cirurgia Veterinária. Belo Horizonte, MG, Brazil.
Universidade Federal de Minas Gerais. Escola de Veterinária. Departamento de Clinica e Cirurgia Veterinária. Belo Horizonte, MG, Brazil.
Universidade Federal de Minas Gerais. Escola de Veterinária. Departamento de Clinica e Cirurgia Veterinária. Belo Horizonte, MG, Brazil.
Fundação Oswaldo Cruz. Instituto René Rachou. Laboratório de Biomarcadores de Diagnóstico e Monitoração. Belo Horizonte, MG, Brazil.
Fundação Oswaldo Cruz. Instituto René Rachou. Laboratório de Biomarcadores de Diagnóstico e Monitoração. Belo Horizonte, MG, Brazil.
Pontifícia Universidade Católica de Minas Gerais. Curso de Veterinária. Betim, MG. Brazil.
Universidade Federal de Minas Gerais. Escola de Veterinária. Departamento de Clinica e Cirurgia Veterinária. Belo Horizonte, MG, Brazil.
Abstract: Background: Over the years, the most commonly used extenders for semen cryopreservation contain egg yolk as cryopro¬tectant. However, more recent studies have used the low density lipoproteins, extract of hen egg yolk which is responsible for the cryoprotective effect. Nevertheless, little was known about its required minimum concentration as well as its inter-action with other extra cellular cryoprotectants, like skimmed milk. The present study aimed at investigating the effect of replacing whole egg yolk by adding low density lipoproteins at low concentrations, in TES-Tris-skim milk based extender, on the post-thaw quality of buffalo bull sperm. Materials, Methods & Results: Eighteen ejaculates were collected from six buffalo bulls and diluted with TES-Tris-skim milk based extender containing LDL, extracted from hen egg yolks, at the concentrations of 2%, 4%, 8% and 14%, against a control extender containing 20% fresh egg yolk. After semen collection, analyses of subjective motility, vigor, force tour¬billon, sperm concentration (Neubauer chamber) and sperm morphology (phase contrast microscopy) were performed. The diluted semen was packaged in 0.25 mL straws, and cooling was performed on computerized machine (TK 4000®), using a cooling rate of -0.25°C/min to 5°C. Semen was kept in balance at 5°C for 4 h. The straws were frozen in an ice chest, kept at 5 cm from the surface of liquid nitrogen for 20 min and then immersed in liquid nitrogen. The samples were kept in cryogenic container until thawing. Post-thaw kinetic parameters during incubation at 37°C (CASA), sperm membrane integrity (SYBR-14/PI), membrane functionality (hypo-osmotic swelling test) and DNA fragmentation (%DFI - SCSA) were evaluated after thawing. Immediately post-thaw, total motility was higher in the control (56.53 ± 9.73) than in the tested extenders; however, after 30 min the difference was no longer detected. All other kinetic parameters presented sig¬nificantly better results in extenders containing 2%, 4% and 8% LDL, compared with the control. There was no difference between treatments regarding the integrity of membranes or fragmentation of the DNA after freezing/thawing procedures. Discussion: The molecules of low density lipoproteins of egg yolk have the known action of sequestering BSP ( binder of sperm proteins) protein, due to the chemical affinity of their main components, phosphatidylcholine and phosphatidyl¬ethanolamine. The BSP are responsible for removing cholesterol from the plasma membrane of the sperm, preparing it for the sperm capacitation phase. Low density lipoproteins by inhibiting the action of the BSP increase the stability of the plasma membrane during the freeze-thaw process. The milk caseins micelles have a similar ability to bind the BSP, but with lower affinity. The present study has shown that the use of purified low density lipoproteins has an advantage over the use of whole egg yolk, especially with regard to the kinetic parameters of the spermatozoa after thawing. Furthermore, it was observed that low concentrations of low density lipoproteins, such as 2%, in extenders containing skim milk, could preserve the sperm cell during the freeze-thawing process like that higher LDL concentrations and whole egg yolk. Further studies are needed to determine if the cryoprotectant action found in this study was a result of the synergistic action of skim milk with lipoproteins or even at low concentrations like 2% the lipoproteins can provide protection to the buffalo spermatozoa during freezing, as it has been reproduced in other domestic species. In conclusion, our results indicated that the extender TES-Tris-skim milk containing 2% LDL extracted from egg yolk could be used successfully in the cryopreservation of buffalo sperm cells.
Keywords: LDL
cryopreservation
Bubalus bubalis
flow cytometry
sperm kinetic parameters
parâmetros cinéticos do esperma
keywords: LDL
criopreservação
Bubalus bubalis
citometria de fluxo
Issue Date: 2018
Publisher: Universidade Federal do Rio Grande do Sul
Citation: BRITO, Mayara Ferreira et al. Low Density Lipoproteins at 2% Concentration Can Replace Whole Egg Yolk in TES-Tris-Milk Extender for Freezing Buffalo Sperm Cells. Acta Scientiae Veterinariae, 46, 1530, 2018.
DOI: 10.22456/1679-9216.81815
ISSN: 1679-9216
Copyright: open access
Appears in Collections:MG - IRR - Artigos de Periódicos

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