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Effects of extenders containing low density lipoproteins (LDL) and cholesterol, and their interactions with equilibration time, on bovine semen cryopreservation

Grant number: 12/18277-6
Support Opportunities:Scholarships in Brazil - Post-Doctoral
Effective date (Start): December 01, 2012
Effective date (End): November 30, 2015
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal Investigator:Rubens Paes de Arruda
Grantee:Ticiano Guimarães Leite
Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil


This project aims to evaluate the effects of association of Cholesterol Loaded Cyclodextrins (CLC) and extenders based on Low Density Lipoproteins (LDL) purified from egg yolk, and possible interactions between these cryoprotective agents and equilibration time during the process of bovine semen cryopreservation on sperm motility, integrity, lipid peroxidation, function and lipid composition of sperm membranes after thawing. For this, two experiments will be performed. In experiment 1, ejaculates from 15 bulls will be collected. Each ejaculate will be divided in four aliquots, each one will be diluted with different extenders, using the same sperm concentration (30x106 sperm/mL): 1) Control (Tris-Egg yolk-glycerol), 2) Cholesterol (containing only CLC) 3) LDL (containing only LDL), 4) Cholesterol + LDL (LDL + CLC association). Each diluted aliquot will be packaged in 0.5 mL straws and subdivided among three automated freezing machines, which will perform the same cooling and freezing rates, varying only the equilibration times at 5 ºC: 0, 4 and 6 h. (totaling 12 treatments). In experiment 2, ejaculates from 6 bulls will be collected; each ejaculate will be subjected to the same treatments as in experiment 1, but using the concentration of 60x106 sperm/mL. For experiment 1, the assessment of each sample will be determined by Computer-Assisted Semen Analysis of motility (CASA), and flow cytometry analyses of plasma and acrosomal membrane integrity, capacitation status, lipid peroxidation and degree of protein tyrosine phosphorylation in sperm surface. In experiment 2 will be carried out analyses of integrity and lipid composition of sperm membranes after thawing, in order to find correlations between these variables. The variables will be submitted to analysis of variance, using the software SAS (1998). The means will be compared by SNK test at a 5% level of significance, and Pearson's correlations between variables will be performed.

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
DE ANDRADE, ANDRE F. C.; ARRUDA, RUBENS P.; TORRES, MARIANA A.; PIERI, NAIRA C. G.; LEITE, TICIANO G.; CELEGHINI, ENEIVA CARLA C.; OLIVEIRA, LETICIA Z.; GARDES, THAYNA P.; BUSSIERE, MARIA CLARA C.; SILVA, DANIELA F.. Nitric oxide in frozen-thawed equine sperm: Effects on motility, membrane integrity and sperm capacitation. Animal Reproduction Science, v. 195, p. 176-184, . (12/18277-6, 09/54906-5)
AFFONSO, FERNANDA J.; CARVALHO, HENRIQUE F.; LANCONI, RENATA; LEMES, KLEBER M.; LEITE, TICIANO G.; OLIVEIRA, LETICIA Z.; CELEGHINI, ENEIVA C. C.; DE ARRUDA, RUBENS P.. Addition of Antioxidants Myoinositol, Ferulic Acid, and Melatonin and Their Effects on Sperm Motility, Membrane Integrity, and Reactive Oxygen Species Production in Cooled Equine Semen. Journal of Equine Veterinary Science, v. 59, p. 57-63, . (12/18277-6)

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