Teste hiposmótico para avaliação da viabilidade do sêmen eqüino resfriado com diferentes diluidores / Hypoosmotic test to predict viability of equine chilled semen in different extenders

Utilizou-se o teste hiposmótico (HO) para estudar a capacidade de preservação da membrana plasmática do sêmen eqüino resfriado em diferentes meios. Estimou-se a correlação entre os resultados do teste HO e os exames de rotina aplicados ao sêmen, usando-se sêmen de sete garanhões. Cada ejaculado foi diluído em três meios, Kenney (K), Baken com 3% de gema (B3) e Baken com 10% de gema de ovo (B 10), e resfriado a 5°e. Avaliaram-se a motilidade total (MT), a motilidade progressiva (MP), o vigor espermático (V), a porcentagem de espermatozóides morfologicamente normais (NOR) e os resultados do teste HO no sêmen fresco e a cada 24 horas pós-resfriamento. A longevidade espermática foi considerada como o tempo de manutenção da motilidade espermática progressiva superior a 10% do sêmen diluído e resfriado. Maior longevidade espermática foi obtida nas amostras diluídas em meio B3. Os resultados do teste HO sugerem que os diluidores à base de gema de ovo preservaram melhor a membrana plasmática. Foram obtidos valores de correlação (P<0,05) entre o teste HO e a motilidade espermática (MT=0,57; MP=0,59), e correlação baixa entre HO e NOR (0,16).

Effect of 17beta-estradiol on the in vitro maturation of bovine oocytes.

Although 1 microg/ml of 17beta-estradiol (E2) is often used in routine in vitro maturation (IVM) and in vitro fertilization (IVF), its effect remains controversial. The objective of our study was to investigate the effects of E2 on bovine oocyte IVM and subsequent embryo development, using a defined medium. Bovine cumulus oocyte complexes (COCs), aspirated from 2 to 8 mm follicles of slaughterhouse ovaries, were matured in TCM199 in the presence of 1 microg/ml E2 with or without 0.05 IU/ml recombinant hFSH. Cultures without E2, FSH or both served as controls. COCs were matured for 22 h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. To investigate the effect of E2 with and without FSH on nuclear maturation, COCs were fixed after maturation and the nuclear stage was assessed following DAPI staining. Similarly, denuded oocytes (DO) were matured in the presence of E2 and the nuclear stage assessed after 22 h. To investigate the effect of E2 with and without FSH during IVM on subsequent embryo development, in vitro matured COCs were fertilized in vitro and after removal of the cumulus cells, the presumed zygotes were cocultured on BRL monolayer for 11 days. At Day 4, the number of cleaved embryos, and at Days 9 and 11, the number of blastocysts, were assessed. Addition of 1 microg/ml E2 to TCM199 significantly decreased the percentage of Metaphase II (MII) compared to control (56.3 and 74.0%, respectively), and increased the percentage of nuclear aberrations compared to control (13.3 and 2.1%, respectively). The negative effect of E2 on nuclear maturation was stronger when DO were matured; 25.1 and 60.0% of the oocytes reached MII stage for the E2 and control groups, respectively. When COCs were matured in TCM199 supplemented with FSH, the addition of 1 microg/ml E2 did not influence the proportion of MII oocytes, although a higher percentage of nuclear aberrations as compared to control was observed. Presence of E2 during IVM also decreased the blastocyst rate (14.4 and 10.0% for control and E2 groups, respectively). However, when FSH was present, the addition of E2 had no effect on the cleavage rate and blastocyst formation (20.3 and 21.7% for control and E2 groups, respectively). In conclusion, supplementation of 1 microg/ml E2 to a serum free maturation medium negatively affects bovine oocyte nuclear maturation and subsequent embryo development. Although these effects are attenuated in the presence of FSH, we strongly suggest omission of E2 in routine maturation protocols of bovine oocytes.