de Souza Joanna Maria Gonçalves, Duffard Nicolas, Bertoldo Michael J, Locatelli Yann, Corbin Emilie, Fatet Alice, Freitas Vicente José de Figueiredo, Mermillod Pascal
INRA, UMR7247, Physiologie de la Reproduction et des Comportements, INRA, CNRS, Université de Tours, Haras Nationaux. 37380 Nouzilly, France; Laboratory of Physiology and Control of Reproduction, Veterinary School, State University of Ceará, Av. Dedé Brasil, 1700, 60740-90, Fortaleza, CE, Brazil.
INRA, UMR7247, Physiologie de la Reproduction et des Comportements, INRA, CNRS, Université de Tours, Haras Nationaux. 37380 Nouzilly, France; Museum National d'Histoire Naturelle, Réserve de la Haute Touche, 36290, Obterre, France.
Anim Reprod Sci. 2013 Apr;138(1-2):82-9. doi: 10.1016/j.anireprosci.2013.02.012. Epub 2013 Feb 28.
Considerable research has been focused on in vitro production (IVP) of goat embryos to improve its efficiency. In Experiment 1, the effect of the cumulus cells by comparing slaughterhouse-oocytes denuded on purpose (DOP) prior to IVF to intact COC, and the effect of heparin during IVF were assessed. In Experiment 2, oocytes that were already denuded at collection (DOC), DOP and intact COC were studied. Three treatments used oocytes denuded at collection: DOC oocytes were cultured alone for both IVM and IVF; DOC and COC were cultured together for both IVM and IVF or DOC were IVM alone and then mixed with COC for IVF. In other treatments, COC were allocated to four IVF treatments: Intact COC; COC were denuded prior to IVF; COC were denuded and IVF with added cumulus cells; COC were denuded and IVF mixed with intact COC giving two sub-treatments: Denuded oocytes that were IVF with COC; and COC that were IVF with denuded oocytes. After fertilization, all presumptive zygotes were cultured for 8 days. In Experiment 1, the yield of blastocysts as a proportion of total oocytes was greater (P<0.05) for COC that were IVF in the presence of heparin (54%) than without heparin (42%) or oocytes already denuded at collection that were IVF with or without heparin (41%; 38%; respectively). In Experiment 2, the developmental potential of oocytes denuded at collection was reduced (cleavage and blastocyst rates calculated from total oocytes: 34%; 11%, respectively) as compared to COC (77%; 59%, P<0.05). However, when equal numbers of both were mixed at the start of IVM, the rates were not significantly different to COC alone (68%; 45%), but when both were mixed equally only for IVF, the rates were reduced (57%; 40%, P<0.05). Denuded oocytes co-cultured with cumulus cells were not significantly different to intact COC (76%; 55%). The effect of adding COC during IVF to oocytes denuded after IVM was similar to adding cumulus cells to the same type of oocytes. In conclusion, both the use of heparin and the association of oocytes with cumulus cells, either detached or in intimate contact, during IVM and/or IVF significantly improve IVP of goat embryos. Furthermore, some oocytes that are already denuded at collection will develop satisfactorily to blastocysts when matured and fertilized with intact COC.
为提高山羊胚胎体外生产(IVP)效率,已有大量研究聚焦于此。在实验1中,通过比较体外受精(IVF)前特意去除卵丘细胞的屠宰场采集卵母细胞(DOP)与完整卵丘-卵母细胞复合体(COC),评估了卵丘细胞的作用以及IVF过程中肝素的作用。在实验2中,研究了采集时已去除卵丘细胞的卵母细胞(DOC)、DOP和完整COC。三种处理方式使用了采集时已去除卵丘细胞的卵母细胞:DOC卵母细胞在体外成熟(IVM)和IVF过程中单独培养;DOC和COC在IVM和IVF过程中共同培养,或者DOC单独进行IVM,然后与COC混合进行IVF。在其他处理中,COC被分配到四种IVF处理方式:完整COC;IVF前去除卵丘细胞的COC;去除卵丘细胞并在IVF时添加卵丘细胞;去除卵丘细胞并在IVF时与完整COC混合,产生两种子处理方式:去除卵丘细胞的卵母细胞与COC进行IVF;COC与去除卵丘细胞的卵母细胞进行IVF。受精后,所有假定的合子培养8天。在实验1中,在肝素存在下进行IVF的COC(54%),其囊胚产量占总卵母细胞的比例高于无肝素组(42%),也高于采集时已去除卵丘细胞且有无肝素情况下进行IVF的卵母细胞(分别为41%和38%)(P<0.05)。在实验2中,与COC相比(分别为77%和59%,P<0.05),采集时已去除卵丘细胞的卵母细胞发育潜能降低(根据总卵母细胞计算的卵裂率和囊胚率分别为34%和11%)。然而,当在IVM开始时将两者等量混合时,其比率与单独培养的COC无显著差异(68%和45%),但仅在IVF时将两者等量混合时,比率降低(57%和40%,P<0.05)。与卵丘细胞共培养的去除卵丘细胞的卵母细胞与完整COC无显著差异(76%和55%)。IVM后去除卵丘细胞的卵母细胞在IVF时添加COC的效果与向同类型卵母细胞添加卵丘细胞的效果相似。总之,在IVM和/或IVF过程中使用肝素以及卵母细胞与卵丘细胞的结合,无论是分离的还是紧密接触的,都能显著提高山羊胚胎的IVP。此外,一些采集时已去除卵丘细胞的卵母细胞,当与完整COC一起成熟和受精时,将令人满意地发育成囊胚。