Cristiano Feltrin

Calves Born after Direct Transfer of Vitrified Bovine In Vitro-produced Blastocysts Derived from Vitrified Immature Oocytes

Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super-cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non-vitrified control group, (ii) vitrified in normal (-196 degrees C) liquid nitrogen (LN(2)) and (iii) vitrified in super-cooled LN(2) (< or =-200 degrees C). Open-pulled glass micropipettes were used as vitrification containers. Immature oocytes were in vitro-matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN(2) state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super-cooled LN(2), resulted in viable blastocysts and live calves following in vitro embryo production.

Preliminary Study in Immature Canine Oocytes Stained with Brilliant Cresyl Blue and Obtained From Bitches with Low and High Progesterone Serum Profiles

This study was conducted: (i) to observe the features and levels of blue colour impregnation in morphologically selected immature canine cumulus oocyte complexes (COCs) stained with the brilliant cresyl blue (BCB) dye, as indicators of quality, and integrity of nuclear oocyte chromatin configuration before in vitro maturation (IVM); (ii) to observe the relationship between the influence of serum progesterone (SP) concentrations from ovary donors and BCB staining of immature dog oocytes. The results showed that out of 138 canine COCs, germinal vesicle (GV) stage prevailed in BCB+ oocytes at percentages of 67.4% (60/89), which were statistically higher than those observed in BCB+/- (52.2%; 23/44) and BCB- (20%; 1/5) oocytes (p = 0.023). Oocytes BCB+ were interpreted as those having completed their growth and therefore possessing the capacity to mature and develop in vitro. Ooplasm and cumulus cells (CCs) of canine oocytes were BCB staining independent. Ooplasm blue colour staining reaction varied between grown oocytes, revealing different levels of glucose-6-phosphate dehydrogenase activity among and within oocytes. Additionally, SP profile of ovary donors was not a relevant indicator for selection of oocytes screened with the BCB stain. Similar numbers of high quality oocytes were observed to be BCB+, BCB+/- and BCB- between groups of females with SP varying from 0 to 2.5 ng/ml (n = 5), and those with SP varying from 2.6 to 16.7 ng/ml (n = 4) (p = 0.680). It may be inferred that bitches with low and high SP profiles have grown oocytes in their ovaries, as determined by the BCB absorbance in their ooplasms.

Intoxicação por Baccharis megapotamica var. weiriiem ovinos

As intoxicacoes por Baccharis coridifolia afetam especialmente animais de fazenda famintos ou curiosos e que nao haviam tido contato previo com a planta. B. coridifolia ocorre usualmente em terrenos secos de coxilhas no Rio Grande do Sul e estados ou paises vizinhos. Uma forma indistinguivel e esporadica da doenca tem sido associada com a ingestao de Baccharis megapotamica que ocorre em areas umidas. Relata-se a intoxicacao natural de quatro cordeiros apos ingestao de Baccharis megapotamica var. weirii. A doenca foi observada em uma propriedade localizada em Barra do Ribeiro, Rio Grande do Sul. O rebanho era composto por 220 ovinos, os quais eram mantidos em area de pastagem nativa sem qualquer suplementacao. Uma rapida doenca clinica caracterizada por anorexia, colica, diarreia e desidratacao causou a morte de tres cordeiros em um periodo de 8 a 24 horas, o outro foi encontrado morto. A necropsia revelou alteracoes significativas no rumen, no qual havia edema de serosa e hemorragias equimoticas na submucosa. Microscopicamente, o rumen apresentou edema de submucosa, alem de edema, tumefacao, vacuolizacao e necrose de mucosa. O diagnostico foi fundamentado nos achados clinicos, patologicos e epidemiologicos.

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

The birth of cloned goats has been well documented, but the overall goat cloning efficiency by somatic cell nuclear transfer procedures is still low, which may be further intensified in extreme environments. The aim of this study was to produce cloned goats under the conditions of the Brazilian SemiArid region, in a transgenic program for the expression of human lysozyme in the milk to target childhood diarrhea and malnutrition, comparing the effects of oocyte source, cell type, and embryo reconstruction procedures on in vitro and in vivo embryo survival after cloning by micromanipulation or by handmade cloning. The use of in vitro-matured oocytes resulted in more viable embryos after cloning than in vivo-matured cytoplasts, but no differences in pregnancy rates on day 23 were seen between oocyte sources (77.5 vs. 77.8%, respectively). The presence or absence of the zona pellucida for embryo reconstruction (78.8 vs. 76.0%, respectively) did not affect pregnancy outcome after transfer. However, pregnancy rate on day 23 was higher for embryos chemically activated by a conventional than a modified protocol (88.1 vs. 50.0%), and for embryos reconstructed with mesenchymal stem cells and fetal fibroblasts (100.0 and 93.3%) than with adult fibroblasts (64.7%). Although most pregnancies were lost, the birth of a cloned female was obtained from embryos reconstructed by micromanipulation using non-transgenic control cells and in vitro-matured oocytes with intact zona pellucida, after conventional activation and transfer at the 1-cell stage.

29 EVALUATION OF DIFFERENT FUSION PARAMETERS IN THE RECONSTRUCTION OF SWINE HANDMADE CLONING EMBRYOS

The goal of this experiment was to compare different fusion parameters in the handmade cloning technique to produce cloned swine embryos. After in vitro maturation of 618 oocytes, 431 (69.8%) presented a visible polar body and were used in the experiment. The next step was the removal of the cumulus oophorus cells and the digestion of the zona pellucida using pronase (5 mg mL–1) in HEPES TCM199. Oocytes were then exposed to a medium containing cytochalasin B (5 µg mL–1) for 15 min before being bisected with a hand-held blade. The bisected oocytes (cytoplasts) were then placed in medium supplemented with Hoechst 33342 and exposed to UV light to select cytoplasts without metaphase II plates. Next, two cytoplasts and a mesenchymal stem cell (nucleus donor) were pushed together in a phytohemagglutinin (550 µg mL–1) solution. Once adhered, these structures were divided into 3 groups (G) to be fused using different parameters: (G1) 2 pulses (DC) of 0.6 kV cm–1 for 30 µs, (G2) 2 pulses (DC) of 0.9 kV cm–1 for 30 µs, and (G3) 2 pulses (DC) of 1.2 kV cm–1 for 30 µs. For all three groups, 0.3 m of mannitol solution (without calcium) was used in the fusion chamber, and an initial pre-pulse (AC) of 10V for 15 s was performed to permit the alignment of 100% of the cytoplast-donor cell structures. After fusion, reconstructed embryos were activated in 0.3 m mannitol and 0.1 mm calcium in the fusion chamber using 2 pulses of 0.9 kV cm–1 for 30 µs followed by incubation in 10 µg mL–1 of cycloheximide solution for 4 h. Afterwards, the reconstructed embryos were transferred to NCSU23 medium supplemented with amino acids (nonessential and essential) and 0.4% bovine serum albumin. The embryos were cultured at 39°C in a 100% humidified atmosphere containing 5% CO2, 5% O2, and 90% N2. Cleavage rates were evaluated after 48 h of culture. For G1, the fusion rate was 43% (25/58) with 72% cleavage (18/25), the G2 fusion rate was 87% (56/64) with 80% cleavage (45/56), and the G3 fusion rate was 79% (53/67) with 69% cleavage (37/53). Statistical analysis was performed using the chi-square test. There were no significant differences in fusion rates between groups G2 and G3, but the fusion rate of these groups was significantly different from that of G1 (P < 0.05). No significant differences in cleavage rate were observed among the three groups. In conclusion, fusion using 2 pulses at either 0.9 or 1.2 kV cm–1 for 30 µs was more efficient for embryo reconstruction in the handmade cloning technique compared to that using 2 pulses at 0.6 kV cm–1 for 30 µs. Further studies need to be performed to improve cleavage rates and assess development to the blastocyst stage.