engleski

Influence of cysteamine supplementation during in vitro culture of bovine embryos

The objective of this study was to evaluate the effect of cysteamine addition to the in vitro culture on bovine embryo quality. Bovine oocytes collected from ovaries of slaughtered donors were matured in vitro in medium 199 supplemented with 10% FCS, FSH/LH (Pergonal, 75/75 IU, Serono), 1 µ ; ; g/ml estradiol-17ß and 100 µ ; ; M cysteamine. Matured oocytes were fertilized with modified Tyrode's solution supplemented with 10 µ ; ; g/ml heparine, 0, 5 µ ; ; g/ml hypotaurine, 0, 5 µ ; ; g/ml epinephrine and 6 mg/ml BSA. Fertilization in vitro was performed with frozen/thawed bull semen prepared using BoviPure® ; ; separation protocol. Following fertilization embryos were cultured in SOFaaBSA without glucose. After 48 h embryos were transfered to SOFaaBSA with 1, 5 mM glucose (SOF g) with or without cysteamine (Cys). Embryos that were developed to the ≥ 4 cell stage after 48 h of culture were alocated in four different groups: 1) 0 Cys ; 2) 50 µ ; ; ; M Cys ; 3) 100 µ ; ; ; M Cys and 4) 200 µ ; ; ; M. Cys. After 48 h of IVC in those media the embryos were transfered in SOF g without cysteamine and cultured until Day 9. Total of 958 oocytes were used in seven repetitions. The number of cleaved embryos on Day 2, the total number of morulas (M) and blastocysts (Bl) on Day 7 and the numbers of hatched blastocysts (hBl) on Day 9 were registered. Differential staining of the inner cell mass (ICM) and trophectoderm (TE) cells were performed on Day 9 blastocysts. The average values of the IVF results were: 83% cleaved on Day 2, 26, 2% of M+Bl on Day 7 and 11, 3% of hBl on Day 9 of development, without significant differences between treatments (p>0.05), although the differences in the percentage of morulas and blastocysts on Day 7 between 0 Cys group and 100 µ ; ; ; M Cys group were over 10% (21.9% vs 33.3%). Number of total and inner cell mass cells (mean± ; ; ; SEM) in Day 9 Bl Group N total cell no. ICM cell no. Proportion (%) 0 Cys 11 125.9± ; ; ; 11.5 a 33.4± ; ; ; 4.1b 26.6± ; ; ; 1.6b 50µ ; ; ; M Cys 12 129.9± ; ; ; 10.9 a 37.9± ; ; ; 3.9 ab 29.6± ; ; ; 1.5ab 100µ ; ; ; M Cys 15 141.6± ; ; ; 9.8a 48.7± ; ; ; 3.6a 33.7± ; ; ; 1.3a 200µ ; ; ; M Cys 7 85.0± ; ; ; 14.4b 22.6± ; ; ; 5.2b 26.5± ; ; ; 1.9b Values with different superscripts within the same columns differ significantly (P<0.05 ; ANOVA) The embryos cultured in the presence of 100 µ ; ; ; M cysteamine had significantly higher number of ICM cells and the proportion of ICM cells which demonstrated that cysteamine supplementation during in vitro culture improves embryo quality but the addition of 200 μ M cysteamine is proven to have deleterious effect on embryo quality.

cattle; embryo; IVC; embryo quality

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