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The objective of this study was to compare the effect of two culture media: modified synthetic oviductal fluid (mSOF) and G1.2/G2.2, on the developmental competence of bovine somatic cell-cloned embryos. Cloned embryos were produced by transferring adult skin fibroblasts into enucleated MII oocytes. After activation, the reconstructed embryos were randomly allotted to either mSOF or G1.2/G2.2 for culture (the embryos were transferred from G1.2 to G2.2 on days 3 of culture). The development competence of cloned embryos in these two culture systems was compared in terms of cleavage rate, blastocyst formation rate and apoptosis cell number in day 7 blastocyts. To investigate the in vivo developmental competence of cloned embryos in the two culture systems, a total of 87 and 104 blastocysts derived from mSOF and G1.2/G2.2 medium groups were transferred individually to recipient Angus cows, respectively. No differences were observed in terms of cleavage rate, day 7 blastocyst rate and blastocyst cell number between these two culture systems. However, the day 6 blastocyst formation rate was significantly higher in G1.2/G2.2 than that in mSOF. In addition, blastocysts cultured in mSOF have a higher percentage of apoptotic blastomeres compared to those in G1.2/G2.2 (8.5 ± 1.2 vs 16.8 ± 1.5, p<0.05). Although difference in pregnancy rate was not observed 40 days after embryo transfer, significantly higher pregnancy rate was observed in G1.2/G2.2 group after 90 days of embryo transfer (12.4% vs 37.5%, p<0.05). Moreover, calving rate was significantly improved in G1.2/G2.2 group compared to mSOF group (27.9% vs 6.7%, p<0.05). In conclusion, our results indicate that G1.2/G2.2 can improve developmental competence of bovine SCNT embryos both in vitro and in vivo, which is more suitable for culture of bovine SCNT embryos than mSOF medium.

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To expand the breeding flock of Poll Dorset sheep in China, multiple ovulation and embryo transfer breeding program was applied to the limited number of imported Australian Poll Dorset sheep. This study investigated the effects of FSH from three different manufacturers, parity (nulliparous vs multiparous), repeated superovulation, oestrus induction, corpus luteum regression and oestrus delay on Poll Dorset superovulation. The results showed that gonadotropin FSH from Canada Folltropin-V (Ca-FSH) was successfully used for superovulatory treatment with 160 mg-200 mg dosage per ewe and recovered 12.91 ± 7.80 embryos. Multiparous ewes for superovulation treatment were significantly better nulliparous ewes (p<0.05). The successive superovalution treatment reduced significantly embryo collection but did not affect transferable embryo number. Ewes with natural oestrus resulted in significantly higher number of embryos (13.83 ± 4.64) and of transferable embryos (12.00 ± 5.76) than ewes with induced oestrus (7.00 ± 4.92; 4.22 ± 3.42) and unknown oestrus cycle (5.94 ± 3.38; 3.19 ± 2.56, p<0.05). The delayed oestrus ewes at 24 h after superovulatory treatment produced significantly fewer embryos and transferable embryos (0.92 ± 1.51 vs 0.42 ± 0.90) than those with normal oestrus (p<0.01). Furthermore, the more transferable embryos were recovered from ewes with normal corpus luteum than those with corpus luteum regression (5.88 ± 5.09 vs 3.59 ± 4.30 and 8.83 ± 5.75 vs. 6.66 ± 5.41, p<0.01). These results suggest that in our farm practice, a comprehensive treatment method by using the Canadian FSH (Folltropin-V), plus choosing multiparous and natural oestrus ewes with normal corpus luteum might obtain an optimum embryo collection and embryos transfer in sheep.

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Cadmium (Cd) is considered a possible etiological factor in neurodegenerative diseases. However, the exact mechanism by which Cd induces neurotoxicity is not well elucidated. In this study, Neuro-2a cells were treated with 0, 10, 20, and 40 μM cadmium chloride for 24 hours to investigate the effects of Cd on the cytoskeleton of nerve cells. MTT assay and ELISA assay were used to examine cell viability and release of lactate dehydrogenase (LDH) from cells, respectively. Results showed that Cd reduced cell viability and increased the release of LDH in a dose-dependent manner (P < 0.05). The morphology of treated cell was damaged as indicated by cell collapse and dimensionality reduction. Moreover, the axonal spines and normal features of Cd-treated neurons disappeared. We checked the ultrastructure of Neuro-2a cells and found that Cd-induced swelling, membrane damage, overflow of cytoplasm contents, and cell fragmentation. Damaged mitochondria, expanded endoplasmic reticulum, and abnormal microfilaments were found in Cd-treated cells rather than in untreated cells. Compared with the control group, the relative release of glutamate in the supernatant after Cd treatment was reduced, indicating that Cd exposure could reduce the release of glutamate by inhibiting the function of nerve-2a cells. Cd decreased the mRNA and protein expression levels of cytoskeletal proteins including DBN, SYP, and TAU, which might promote cytoskeleton alterations in Cd-treated cells. In conclusion, Cd-induced actin cytoskeleton alterations and dysfunction of cultured neurons. The results of the present study provide new insights for the investigation of Cd-induced neurotoxicity.

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The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the M II chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female Jining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5 mumol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no significant differences in development in vitro between the cloned embryos derived from donor cells precooled at 4 degrees C for 24 h and nonprecooled donor cells. The cleavage rates, 4-cell development, and blastocyst development of reconstructed embryos were 72.48% (79/109), 53.16% (42/79), and 19.05% (8/42) in precooled group; 68.5% (211/308), 59.72% (126/211), and 17.46% (22/126) in nonprecooled group, respectively. Eighteen cloned 4-cell embryos derived from precooled donor cells were transferred and one cloned kid was born. Eighty-four cloned 4-cell embryos derived from nonprecooled donor cells were transferred and no offspring were produced. Of 18 cloned morale from nonprecooled donor cells transferred, one kid was born. The results of microsatellite DNA analyses indicated that the two cloned kids were from the same donor fibroblast cell line derived from an adult goat ear skin.