| Juvenile in vitro embryo transfer (JIVET) in sheep has become one of research hotspots in the field oflivestock breeding, but because the lambs have not reached sexual maturity and physical maturity, undernatural conditions, oocytes can not be collected. At a particular stage of lambs by exogenous hormoneinduced, then recovered oocytes as experimental material. Based on culture condition for adult sheepocctyes, this study evaluated the effect of exogenous hormone on lamb follicular development and theeffect of addition of BCB and PDE3on maturation of lamb Octcye in vitro. The aim of this study was toimprove the development ability of lamb oocyte and the experiments include four parts as following:Section1Effect of different diameters of follicles on developmental competence of Sheep oocytes in vitrocultureSheep oaries were obtained from abattoir and the occytes from different diameter follicles werecollected to evaluate proportion of different diameter follicles and the effect on ooctyes diameter, thecontent of GSH and the developmental competence after in vitro culture. In this study, oocytes werecollected from different size follicles by aspiration, and they were divided into three groups according tothe size of the the follicles: small group（＜2mm）, middle group（2mm～5mm）, big group（＞5mm）.The collecting number of oocytes had no significant difference among each groups（P＞0.05. The resultsshowed that the proportion of different diameter follicle was significantly different（P＜0.01), the diameterof oocytes obtained from small group was significantly lower than the other groups（P＜0.01）. After24h invitro maturation, the maturation rate and content of GSH of oocytes obtained from middle group or biggroup were significantly higher then the small follicles group（P＜0.01）. After parthenogenetic acitationand in vitro cultivation, the blastocyst formation rate of oocytes obtained from middle size follicles(2mm～5mm）or big size follicles (＞5mm）were significantly higher then the small follicles group（P＜0.01）. The results indicated that oocytes from bigger follicles (＞2mm）were more suitable for in vitromaturation and embryo production.Section2The Screening by BCB staining on sheep oocytes with different follicle diameterThe aim of this paper was to study the effects of BCB staining on the efficiency of parthenogeneticactivation(PA) with oocytes of different follicle diameter in sheep, comparison of post-developmentcapability from different different groups of oocytes. The results showed that oocytes from folliclesdiameter larger than5mm group positive rate were significantly higher than below5mm group(P＜0.01).After Oocytes were PA, and oocytes from follicles diameter larger than5mm BCB+group, the effect ofthe developmental competence were significantly higher than others in vitro culture (P＜0.05). The resultsshowed that oocytes from different experimental groups after PA obtained the total number of blastocystcells, more than2mm group was significantly higher than below2mm group(P＜0.01), there was nosignificant difference in all BCB-groups(P＜0.05). In unstained oocytes, more than2mm group wassignificantly higher than below2mm group(P＜0.05). The results showed that oocytes from folliclesdiameter larger than2mm were appropriate for cultivation in vitro by BCB staining, and more than2mmBCB+group oocytes have better developmental potential.Section3Research lamb follicular development induced by exogenous hormonesIn order to optimize and improve the efficiency of follicle development on lambs, the experiment wasdivided into five aspects, FSH dose, age, follicle diameter, weight and breed. The results showed that theeffect of follicle development,160IU groups were significantly higher than320IU(P＜0.05),6-week-oldlambs were significantly higher than4～5weeks and7～8weeks,(P＜0.05),8～12Kg lambs weresignificantly higher than above12Kg(P＜0.05),6-week-old lambs were injected the same dose FSH, theeffect of follicle development from Suffolk and Merino were significantly higher than Texel(P <0.05). Therefore,6-week-old weight about8～12Kg Suffolk lambs were treated with160IU FSH, folliculardevelopment was better.Section4Application of BCB and Milrinone in in-vitro culture of lambs oocytesThe present study was to investigate the effects of BCB staining on lambs oocytes and the effect ofmilrinone on BCB-oocytes, with the aim to improve the efficiency of blastocyst production in vitro. Theresults shown13μM BCB+oocytes were no significant difference with26μM in vitro culture efficiency(P＞0.05). The diameter and content of GSH from BCB+oocytes were significantly higher compared withBCB-group, but was no significant difference with adult sheep（P＞0.05）. In addition, the rate of embryodevelopment in vitro could be significantly increased by inhibiting the BCB-oocytes for6h with milrinonethan BCB-oocytes and control (P＜0.05), but was no significant difference with adult sheep（P＞0.05）.Therefore BCB combined with milrinone improved the efficiency of lambs oocytes culture in vitro. |
