Alteração da membrana espermática de suínos, bovinos e eqüinos na qualidade do sêmen

• Ano de defesa: 2008
• Autor(a) principal: Amorim, Elenice Andrade Moraes e
• Orientador(a): Torres, Ciro Alexandre Alves
• Banca de defesa: Costa, Eduardo Paulino da , Murgas, Luis David Solis
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Zootecnia
• Departamento: Genética e Melhoramento de Animais Domésticos; Nutrição e Alimentação Animal; Pastagens e Forragicul
• País: BR
• Palavras-chave em Português: Características seminais; Criopreservação; Espermatozóides; Nutrição
• Palavras-chave em Inglês: Seminal characteristics; Cryopreservation; Spermatozoa; Nutrition
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::ZOOTECNIA::PRODUCAO ANIMAL
• Link de acesso: http://locus.ufv.br/handle/123456789/1689

This study was realized with the objective to study the effects of the plasmatic membrane alteration of boar, bull and stallion spermatozoa on the sperm quality. Three studied, with different species, were developed. First study, twenty-four mature Dalboard 85 boars, of proven fertility and in routine semen production for artificial insemination, randomly divided, in factorial arrangement 2 X 3, with two oil sources (soy and salmon) and three levels of antioxidants (150, 300 and 450 vitamin E mg/kg). This study was divided in four experiments (E), in E1 the objective was to evaluate the adding oil sources and dietary supplementation of the vitamin E on the fatty acid composition of boar sperm. The dietary supplementation with salmon oil reduced (P<0.05) the proportion of fatty acid &#969;6, specifically of docosapentaenoic acid (22:5&#969;6; DPA) and it increased (P<0.05) the proportion of docosahexanoic acid (22:6&#969;3; DHA). Except for these fatty acids, no other difference was observed (P>0.05). The increase of the proportion of the DHA (P<0.05) in the sperm of salmon oil treated was from 34.67 to 45.5%, and DPA decreased from 23.3 to 11.4%, from first to 10 week. The proportion of vitamin E in the boar sperm no changed (P>0.05) for all treatments. In E2, the objective was to evaluate the adding oil sources and dietary supplementation of the vitamin E on the fatty acid composition of raw boar sperm. The volume, total motile sperm, HOST, percentage of live cells and morphology not differ (P<0.05) between all treatments. There was effect of the salmon oil (P<0.05) on the sperm vigor. The level vitamin E added in the diet changed (P<0.05) the sperm concentration, however, no difference was observed between the oil sources (P>0.05). Salmon oil treatment showed the smallest (P<0.05) concentration of total antioxidants in the semen. Vitamin E and concentration of total antioxidant presented linear effect (P<0.05). In E3, the objective was to evaluate the adding oil sources and dietary supplementation of the vitamin E on seminal characteristics of boar sperm cooled at 17 and 5 oC. The motility and HOST of the animals sperm treated with salmon oil at 17 and 5oC was superior (P<0.05) that animals treated with soy oil, after 24, 48 and 72 hours. The treatment with salmon oil increased (P<0.05) the sperm vigor in both temperatures evaluated after 24 and 48 hours. The motility, vigor and HOST of sperm at 17 and 5 oC differed (P<0.05) during all period that were preserved, where 24>48>72 hours. The total abnormal morphology increased (P<0.05) in the semen of the animals treated with soy oil and cooled at 17 oC while the semen at 5 oC no difference was presented (P>0.05) among the all treatments. The abnormal morphology was bigger (P<0.05) in the semen at 5 oC that at 17 oC, after 24, 48 and 72 hours. The semen at 5 oC presented viability parameters (motility, morphology and Host) inferior (P<0.05) for the semen at 17 oC. And E4, the objective was to evaluate the adding oil sources and dietary supplementation of the vitamin E on the fatty acid composition of boar sperm. The frozen process of semen reduced (P<0.05) the sperm quality when compared this results with fresh sperm, independent of treatment. After thawing, the semen of the animals treated with soy oil in the diet reduced (P<0.05) the total motile sperm, sperm vigor, percentage of live sperm and HOST. The motility of the semen after dilution and during cryopreservation process was 85 and 84% for all animals treated with salmon and soy oil, respectively. Therefore thawing, the motility of the animals treated with salmon and soy oil was 30 and 24%, respectively (P<0.05). For the percentage of sperm died and morphology, the sperm of the animals treated with soy oil presented higher values (P<0.05) that animals treated with salmon oil in the diet. In second study, the objective was realized to compare the effect of adding other cholesterol conjugates, which should incorporate into and increase membrane fluidity at low temperatures thereby increasing cryosurvival. In this study ejaculates from four bulls were divided in three experiments (E). Ejaculates from each of four bulls were diluted to 120 million cells in a Tris diluent. In E1 and E2, the sperm diluted were subdivided into four treatments: No additive (control); 1.5 mg CLC/120 million sperm (positive control); and 1.5 mg/120 million sperm of cyclodextrin preloaded with cholestanol or desmosterol. In E3, the sperm diluted were subdivided into 10 treatments: T1: no additive (control); T2: 0.75 mg and T3: 1.5 mg (Heptanoate); T4: 0.75 mg and T5: 1.5 mg (Palmitate); T6: 0.75 mg and T7: 1.5 mg (Pelargonate); T8: 0.75 mg and T9: 1.5 mg (stearate), and T10: 1.5 mg CLC/mL (positive control). For all experiments, the sperm treated were incubated for 15 min at 22 °C to allow for incorporation of cholesteryl conjugates. After this period, in E1 samples from each treatment were used to determine the motility and osmotic tolerance and, ability of fresh sperm to bind to the zona pellucida (ZP) using a Hamilton Thorne Motility Analyser (CASA), and chicken egg perivitelline membrane (CEPM) using a epifluorescence microscopy. And in E2 and E3, the sperm were diluted 1:1 (v:v) in Tris with 20% Egg Yolk EY) and cooled to 5 oC. After dilution 1:1 (v:v) with Tris containing 10% EY and 16% glycerol for final concentration 30 million sperm and allowed to equilibrate for 15 minutes before packaging into 0.5 mL French straws, freezing in static liquid nitrogen vapor for 20 minutes and plunging into liquid nitrogen for storage. Samples were thawed to determine the motility (E2 and E3), ability of sperm to bind to the ZP (E3) and the CEPM (E2), and viability (E3) using Epics V Flow Cytometer. Treatment differences for all parameters were determined using analysis of variance. For E1, treating fresh sperm with CLC resulted in more binding to the ZP and compared to all other treatments (P<0.05). No differences were observed between ZP and CEPM binding (P>0.05). The percentages of motile sperm were higher for fresh samples treated with cholesterol, cholestanol or desmosterol loaded cyclodextrin than control cells (P<0.05) when sperm were exposed to anismotic conditions, and then returned to isosmolality. For E2, after cryopreservation the percentages of motile sperm and number of sperm binding to each CEPM were similar for sperm treated with CLC and cholestanol compared to sperm treated with desmosterol (P>0.05). All treatments provided higher motility and binding efficiency than control sperm (P<0.05). And for E3, higher percentages of motile sperm and viable cells were maintained after thawing sperm treated with CLC and cholesteryl-pelargonate compared to all other treatments (P<0.05). The percentage of motile sperm and number sperm binding to CEPM was higher for CLCtreated cells (P<0.05). Therefore, adding cholesterol or cholesteryl- pelargonate or cholestanol to bull sperm membranes improved cell cryosurvival. Third study, the objective was to compare the effect of adding cholesterol and cholestanol loaded cyclodextrins in stallion sperm prior to cryopreservation to optimize sperm cryosurvival. Ejaculates from each of 8 stallions were diluted to 120 million cells in a STALP diluent. The sperm diluted were sub-divided into three treatments: No additive (control); 1.5 mg CLC/120 million sperm (positive control); and 1.5 mg/120 million sperm of cyclodextrin pre- loaded with cholestanol. For all experiments, the sperm treated were incubated for 15 min at 22 °C to allow for incorporation of cholesteryl conjugates. After this period, the semen was diluted 1:5 (v/v) with Lactose-Egg Yolk diluent and cooled to 5 oC over a 2 hours period. Loaded into 0.25 mL polyvinylchloride straws, frozen in liquid nitrogen vapor for 10 min, and then plunged into liquid nitrogen until further use. Treatment differences for all parameters were determined using analysis of variance. Higher percentages of motile sperm were maintained after thawing when 1.5 mg CLC was added to sperm from stallions whose sperm do not survive freezing well, compared to cholestanol and control sperm from those same stallions (P<0.05). Addition of CLC also resulted in more sperm binding to the zona pellucida of bovine oocytes after cryopreservation than cholestanol and control sperm (P<0.05). In conclusion, CLC improved the percentage of post-thaw motility in equine sperm as well as increased the number of sperm that bind to zona pellucida. Addition of CLC to stallion sperm prior to cryopreservation is a simple procedure that increases the cryosurvival of cells.