Search

Foi avaliada a influência da citocalasina B na vitrificação de oócitos e embriões bovinos produzidos in vitro. No Experimento I, 956 oócitos foram maturados por 22h, sendo imediatamente vitrificados (tratamento Vitri) ou expostos por 15 a 20 minutos, à solução com 7,5μg/mL (tratamento CB7,5Vitri) ou 45μg/mL (tratamento CB45Vitri) de citocalasina B, antes da vitrificação. Após 30 segundos de exposição à SV1 [400μl TCM-Hepes com 10% soro fetal (SF), 50μl etilenoglicol (EG) e 50μl DMSO], e 20 segundos à SV2 (300μl trealose 1,0M + 20% SF, 100μl EG e 100μl DMSO), os oócitos foram vitrificados em palhetas estiradas (OPS). O reaquecimento foi realizado a 37-38ºC em duas etapas de 5 minutos cada, em trealose 0,3M e 0,15M. Não houve diferenças (P>0,05) nos percentuais de clivagem e desenvolvimento embrionário entre os tratamentos Vitri, Cito7,5Vitri e Cito45Vitri, os quais foram inferiores (P0,05) no percentual de re-expansão e eclosão entre os grupos Vitri e CB45Vitri, os quais foram inferiores (P0.05) in the cleavage and embryo rates between Vitri, Cito7.5Vitri and Cito45Vitri treatments, which were inferior to control group (P0.05), which were lower than those observed in the Control group (P

In vitro produced bovine embryos were individually cultured from D7 to D9 to observe their development. Forty-nine blastocysts (early blastocyst, blastocyst and expanded blastocyst) were individually cultured, from D7 to D9 (D0= fertilization), in 50ml SOF medium plus 5% OCS. Between D7 and D8, blastocysts were cultured in straws (SC) or in plates (PC) and from D8 up to D9, they were cultured on plates. The ratio of early blastocyst, blastocyst, and expanded blastocyst advancing at least one stage of development were, respectively, 71%, 37%, 44% in PC and 100%, 66%, 36% in SC (P>;0.05). Overall development rates on D8 were not significantly different (P>;0.05) for PC (50%) and SC (60%). At D9, both culture systems produced similar (P>;0.05) hatching rates (29% and 24% for PC and SC, respectively). After fluorescent nuclei staining, the average number of cells counted in the hatched (182.7 vs. 202.8) and expanding (94.5 vs. 88.0) blastocysts were similar (P>;0.05) for PC and SC, respectively. In vitro produced bovine blastocysts can be individually cultured from D7 to D9, and the culture system employed (dishes or straws) has no effect on hatching rates and cell number of developed blastocysts. Embriões bovinos produzidos in vitro foram cultivados individualmente do D7 ao D9, com o intuito de avaliar o seu desenvolvimento posterior. Quarenta e nove embriões, nos estágios de blastocisto inicial, blastocisto e blastocisto expandido foram cultivados, individualmente, em 50ml de meio SOF + 5% SVE, do D7 ao D9 (D0=fecundação). Entre o D7 e D8, os blastocistos foram cultivados em palhetas (TcP) ou em placas (CP) e, entre o D8 e D9, foram cultivados apenas em placas. O índice de blastocistos que avançaram pelo menos um estágio de desenvolvimento, entre D7 e D8, foi de 71%, 37% e 44% no CP e de 100%, 66% e 36% no TcP, respectivamente para blastocistos iniciais, blastocistos e blastocistos expandidos. O percentual total de embriões que evoluíram do D7 para D8 foi de 50% (12/24) para o CP e de 60% (15/25) para o TcP, os quais não foram significativamente diferentes (P>;0,05). Na avaliação efetuada no D9, não foram constatadas diferenças (P>;0,05) no percentual de blastocistos eclodidos, entre os dois sistemas de cultivo (29% e 24% para CP e TcP, respectivamente). Após coloração fluorescente dos núcleos, não foi observada diferença (P>;0,05) entre o número médio de células dos blastocistos eclodidos (182,66 vs. 202,8) e expandidos (94,5 vs. 88), para o CP e TcP, respectivamente. Blastocistos bovinos produzidos in vitro podem ser cultivados individualmente do D7 ao D9, não havendo efeito do sistema de cultivo empregado (placas ou palhetas) sobre a taxa de eclosão e o número de células.

The introduction of more specialised and productive dairy breeds into commercial herds in recent decades has caused a gradual loss of interest in dual-purpose cattle breeds, such as the Flamenga. Because of its importance to biodiversity, the high risk of loss of this small Brazilian herd, located in one geographic region in Southern Brazil, justifies the need for and efforts in genetic conservation of the Flamenga breed. The ovum pickup (OPU) procedure is a potential tool for such conservation purposes. Thus, the aim of this study was to evaluate the oocyte and embryo yield potential of the Flamenga breed, as compared with Holstein control counterparts, after in vivo oocyte retrieval followed by in vitro embryo production (IVP). Four Flamenga and 4 Holstein multiparous milking cows from the same herd were subjected to weekly ultrasound-guided OPU sections for 10 consecutive weeks. The number of visible follicles per ovary per female was recorded before the aspiration of ≥3-mm follicles. Retrieved cumulus–oocyte complexes (COC) were morphologically graded according to the method of (Leibfried and First 1979 J. Anim. Sci. 48, 76–86), and viable COC (Grades I, II, and III) from both breeds and from slaughterhouse ovaries (IVP controls) were used for the IVP of embryos, based on our established procedures (Vieira et al. 2002 Cryobiology 45, 91–94). Frozen semen from the same Flamenga breed bull was used in all groups. Quantitative (visible and aspirated follicles, collected COC), and qualitative (COC recovery efficiency, cleavage and blastocyst rates) data were compared between breeds and IVP controls (when pertinent) by Student’s t-test and chi-square test, respectively, for P

The pig is an important animal model for the study of human diseases. An important step for better use of this species in biomedical research is to obtain genetically identical individuals by procedures such as somatic cell nuclear transfer (SCNT). As the in vitro culture environment is usually sub-optimal for embryo development, the oviductal transfer of cloned embryos at the 1-cell stage may be more efficient for the establishment of pregnancies. However, the transfer at such an early stage usually requires the presence of zona pellucida or agar embedding to protect embryos from the recipient’s immune system (Loi et al. 1999 Livest. Prod. Sci. 60, 281-294). This study aimed to evaluate the developmental viability of 1-cell-stage porcine handmade cloned embryos directly transferred to the oviduct of female recipients without the zona pellucida or agar embedding. After 40 h of IVM in TCM-199 +10% follicular fluid, COCs obtained from sows were denuded, selected for the presence of a polar body (459/689), and submitted to a 0.2% pronase solution in 25% fetal bovine serum (FBS) for partial zona removal, followed by rinses in manipulation medium and pure FBS. Subsequent to oocyte splitting by manual bisection in a 5 μg mL-1 cytochalasin B solution (CCB), hemi-oocytes (87.1%) were screened under fluorescent microscopy using Hoechst 33 342 stain, resulting in 57.6% enucleated halves (461/800). A somatic cell culture established from a fetal clone pig biopsy (Adam et al. 2007 Oncogene 26, 1038-1045) at passage 4 was used for embryo reconstruction, which was done in a 0.05% phytohemagglutinin (PHA) solution, by sticking 2 cytoplasts and a somatic cell in a linear orientation. Reconstructed couplets, rinsed in calcium- and magnesium-free fusion medium, were electrofused in a fusion chamber after exposure to a 30-V AC pulse for 20 s for alignment, followed by two 24-μs-long DC fusion pulses of 1.3 kV cm-1. Fused couplets (154/214) were exposed for 10 min to a solution containing 5 μg mL-1 CCB and 10 μg mL-1 cycloheximide, followed by electrical activation (two 24-μs-long DC pulses of 0.9 kV cm-1) in fusion medium containing calcium and magnesium. Activated embryos were cultured in vitro for 12 h in 500 μL of PZM-3 medium in the well of the well (WOW) system, in a plastic bag filled with gas mixture (90% N2, 5% O2, 5% CO2), at 38.5°C. Then, a total of 70 and 80 non-agar-embedded, zona-free 1-cell-stage cloned porcine embryos were transferred directly to the oviducts of a sow and a gilt, respectively, both synchronous at approximately 12 h before ovulation. The recipient sow was diagnosed pregnant by ultrasonography on Day 66 of gestation. Although the sow was diagnosed open on Day 72, this study demonstrates that the transfer of 1-cell-stage zona-free embryos directly to the oviduct of a synchronous sow can result in pregnancy.

The coordination of the cell cycle of the donor nucleus and the recipient cytoplasm is thought to be one of the major essential factors needed for successful development of cloned embryos and offspring from somatic cell populations. Cell cycle synchronization protocols used for somatic cell nuclear transfer (SCNT) vary in preference among groups, with the confluence inhibition by contact appearing to be one of the most widely used methods today. However, the relationship between the level of cell confluence in a culture dish at or near the plateau phase of growth and blastocyst yield following cloning by SCNT still needs to be better characterized. The aim of this study was to examine the effect of 3 distinct cell culture confluences before nuclear transfer and embryo aggregation on in vitro development of clone bovine embryos. In vitro-matured bovine COC were used for the production of clone embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells 11). Oocytes were manually bisected following cumulus and zona removal. After selection of hemi-cytoplasts by DNA staining, 1 (50%) or 2 (100%) enucleated hemi-cytoplasts were paired with an adult Nelore skin somatic cell and then electrofused (15 V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs). Cells were selected from 1 out of 3 distinct culture confluences: (1) 70 to 80%; (2) 80 to 90%; and (3) >90%; assessed by morphological evaluation before the SCNT procedure. Reconstructed clone embryos and groups of zona-intact oocytes (parthenote controls) were activated in ionomycin and 6-DMAP. Clone embryos (100%) and hemi-embryos (50%) reconstructed from the 3 groups underwent IVC in the well of the well (WOW) system for 7 days, allocating 1 embryo (1 × 100%) or aggregating two hemi-embryos (2 × 50%) per WOW. After 11 replications, cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, were compared using the chi-square test. Results are summarized in Table 1. Cleavage rates were similar for all groups. The aggregation scheme did not appear to have influenced, either positively or negatively, the developmental outcome to the blastocyst stage. However, blastocyst rates increased nonlinearly (7.0, 17.5, and 29.4%) with the increase in cell confluence. A highly confluent cell culture has already been shown to have a greater proportion of cells in G0/G1 than cycling cells at the log phase (>91% v. 59%; Sun et al. 2008 Zygote 16, 111-116). However, blastocyst development in this study was lower than anticipated for cells in the early plateau phase (70 to 80%), when predicting such development based on the expected G0/G1 proportion in that cell population. Table 1.In vitro development of bovine cloned embryos from distinct cell culture confluences and aggregation scheme This study was supported by FAPESP and CAPES/Brazil.

Despite the apparent success of cloning by somatic cell nuclear transfer (SCNT), the efficiency in development to term remains low, with a high rate of losses occurring throughout pregnancy due to faulty reprogramming and conceptus abnormalities. As the ideal fusion-activation interval for optimal nuclear reprogramming after cloning is still ill-defined, the aim of this study was to determine the effect of 2 distinct fusion-activation intervals and embryo aggregation on in vitro development of cloned bovine embryos. Bovine COCs from slaughterhouse ovaries were used after IVM for the production of cloned embryos by handmade cloning, according to our established procedures (Ribeiro et al. 2009 Cloning Stem Cells, in press). Following cumulus and zona removal, oocytes were manually bisected, with hemi-cytoplasts selected by DNA staining. Two hemi-cytoplasts and an adult skin somatic cell were attached and fused with a 15V AC pre-pulse for 5 s, followed by a double 1.2 kV cm-1 DC pulse for 20 μs. Reconstructed embryos were activated in ionomycin exactly at 2 or 4 h post-fusion (2 hpf or 4 hpf), followed by an incubation in 6-DMAP for 4 h. Cloned embryos from both fusion-activation intervals were in vitro-cultured in the well of the well (WOW) system for 7 days, allocating one (1 × 100%) or two (2 × 100%) cloned embryos per WOW. Grade 1 Day-7 blastocysts were transferred to synchronous recipients. Cleavage (Day 2) and blastocyst (Day 7) rates, on a per WOW basis, and pregnancy (Days 30 and 150) rates were compared using the chi-square or the Fisher test, with results from 9 replications summarized in Table 1. Increasing the fusion-activation interval to 4 h decreased cleavage but not blastocyst rates in 1 × 100% embryos. Also, blastocyst rates were lower in 1 × 100% embryos activated 2 h post-fusion. In general, cleavage and blastocysts rates for 2 × 100% embryos (91.5 and 46.0%) were higher than for 1 × 100% embryo counterparts (74.4 and 31.3%), respectively, regardless of the activation time. In addition, blastocyst rates for 4 hpf-activated embryos (50.3%), based on cleavage, were higher than for 2 hpf-activated embryos (38.3%), irrespective of the aggregation scheme. Nonetheless, despite differences in in vitro development, pregnancy rates and conceptus development in the first half of pregnancy were similar between groups. A longer fusion-activation interval (4 hpf) or embryo aggregation (2 × 100%) increased blastocyst yield but did not improve in vivo development and pregnancy maintenance following the transfer to female recipients in cattle. Table 1.In vitro and in vivo development of cloned bovine embryos This study was supported by FAPESP and CAPES, Brazil.

Exposing bovine embryos and porcine oocytes to hydrostatic pressure has been shown to increase cryosurvival, possibly by a resulting expression of stress tolerance proteins. This study aimed to evaluate the effect of the negative pressure stress condition (a 5-min-long embryo exposure to a negative pressure) and the interval between vacuum exposure and vitrification (40 min or 2 h) on survival of bovine in vitro-produced (IVP) embryos. The negative pressure was achieved with the same apparatus used previously for the cryopreservation of embryos (Nitrocooler; Mezzalira et al. 2009 Reprod. Fert. Dev. (1) 134), in which a negative pressure (vacuum) is applied to liquid nitrogen to increase the cooling rate through the slush phenomenon, except that in this study, the vacuum was applied to the chamber without liquid nitrogen, at room temperature. Grades 1 and 2 bovine IVP expanded blastocysts were allocated to 1 of 5 experimental groups: embryos in vitro-cultured as fresh (control) or after vitrification (Vitri); and embryos subjected to the negative pressure for 5 min and then in vitro-cultured as fresh (NP-fresh) or after vitrification performed 40 min (NP-Vitri-40 min) or 2 h (NP-Vitri-2h) following the vacuum exposure. Embryos were vitrified in pulled glass micropipettes in a solution with 20% ethylene glycol + 20% dimethylsulfoxide + 20% fetal bovine serum and rewarmed in decreasing sucrose concentrations (Mezzalira et al. 1999 Acta Scientiae Veterinariae 27 262-262). In vitro culture was carried out in all treatments for 72 h for the assessment of re-expansion and hatching rates (Table 1), which were analyzed by the chi-square test, for P < 0.05. No differences in re-expansion rates were observed between groups. However, the vitrification of embryos after 2 h of exposure to a 5-min-long negative pressure (NP-Vitri-2h) improved embryo survival expressed by a higher hatching rate than for embryos vitrified without vacuum exposure (Vitri) or after 40 min following the 5-min-long exposure to vacuum. In addition, hatching rates in group NP-Vitri-2h were similar to those for fresh embryos (control and NP-fresh). Our results indicated that a short exposure of embryos to a negative pressure can improve cryotolerance following vitrification, which is dependent on the time interval between NP exposure and cryopreservation. Bovine IVP embryos should be allowed to recover for at least 2 h after NP exposure before the increase in cryotolerance is achieved. Table 1.Effect of negative pressure on re-expansion and hatching rates of fresh and vitrified

As the recipient cytoplast plays a key role in nuclear reprogramming after somatic cell nuclear transfer (SCNT), the aim of this study was to compare the type of cytoplast/karyoplast [metaphase II (MII) oocyte, early zygote, somatic cells] and the chemical (CA) or sperm-mediated/spontaneous activation (SA) on in vitro development of bovine SCNT embryos produced by handmade cloning (HMC). After 17 h of in vitro maturation, a group of cumulus–oocyte complexes (COCs, n = 945) was manually bisected following zona removal and segregated as enucleated (MII hemi-Cyt) or non-enucleated (MII hemi-Kar). Another group of COCs was in vitro-fertilized, and, 4 h after the onset of IVF, zona-free zygotes with 2 polar bodies (n = 490) were manually bisected under fluorescent light to obtain IVF hemi-Cyt and IVF hemi-Kar. A somatic cell (SC) culture from an adult cow was used for HMC procedures (SC Kar). In 5 replications, experimental groups were composed of: zona-intact MII oocytes (parthenote control, PG); zona-intact zygotes (IVF control); MII Cyt + MII Cyt + SC Kar (SCNT control); IVF Cyt + MII Cyt + SC Kar (G1); MII Cyt + IVF Kar (G2); IVF Cyt + IVF Kar (G3); IVF Cyt + IVF Cyt + SC Kar (G4); and MII Cyt + MII Kar (G5). Following reconstruction and electrofusion, groups G1 to G5 were further divided into 2 sub-groups each, 1 being chemically activated (ionomycin/6-DMAP) along with the control groups PG and SCNT, whereas the others were cultured to verify sperm-mediated (G1 to G4) or spontaneous (G5) activation. Embryos were in vitro-cultured in the WOW system for 7 days. Cleavage (Day 2) and blastocyst (Day 7) rates were compared by the chi-square and Fisher tests, respectively. Cleavage rates in G1-SA, G2-SA, and G3-SA were lower than in their CA counterparts, which were similar to controls (Table 1). Such decrease in cleavage in G1-SA and G2-SA may be caused by the manipulation process rather than by sperm-mediation, since the observed rates were very similar to the G5-SA group. Cleavage in G3 and G4 were also similar to controls, most likely due to the fusion of 2 sperm-activated IVF hemi-Cyt. Blastocyst rates were generally higher in CA than in SA sub-groups except for G4, for which SA benefited from 2 sperm-activated cytoplasts. The lower blastocyst yield in SA sub-groups may reflect at least 2 possible mechanisms: an increased level of heteroplasmy (G1 and G2), potentially caused by an insufficient sperm-activated IVF hemi-Cyt or by a blocking effect imposed by the M-phase-derived hemi-Cyt, and/or a disruption in karyokinetic events caused by the manipulation in sperm-activated IVF hemi-Kar (G2 and G3). In G4, both mechanisms were probably attenuated by the use of 2 sperm-activated IVF hemi-Cyt and a SC-kar, analogous to conditions in the SCNT and G5 groups. Table 1.Effect of cytoplast type and activation process on in vitro development of bovine SCNT embryos This study was supported by a grant from CAPES/Brazil.

Increasing the cooling rate is a common strategy to enhance vitrification efficiency of bovine oocytes and in vitro-produced (IVP) embryos. Under vacuum conditions, liquid N2 (LN2) temperature decreases, increasing the cooling rate during the vitrification procedure. However, commercially available brands of equipment to supercool nitrogen are expensive. The aim of this study was to verify the effectiveness of a low-cost homemade nitrogen supercooling apparatus (Nitrocooler) for vitrification of bovine oocytes and IVP embryos. The device consists of a vacuum pump coupled to a close-tight-lidded flask with a styrofoam cup filled with 300 mL of LN2. After 5 to 7 min of vacuum pumping, LN2 goes through the slushing phenomenon, turning solid. After the lid is opened, the N2 turns liquid again, but in a stable, supercooled physical state lasting for approximately 10 min, boiling off when objects are plunged into it. Nitrocooler was tested for vitrified oocytes (Experiment I), vitrified oocytes in different containers (Experiment II), and embryos in 2 different vitrification solutions (Experiment III). In Experiment I (Ciência Rural, 2006 36, 1501–1506), immature (IM, n = 172) or in vitro-matured (IVM, n = 174) oocytes were exposed to 10% ethylene glycol (EG) + 10% DMSO for 30 s, followed by 20% EG + 20% DMSO + 0.5 m sucrose for 20 s, loaded in open-pulled straws (OPS), and plunged into LN2 or Nitrocooler-pumped LN2. For both IM (8.8%) and IVM (10.6%) oocytes, Nitrocooler-pumped LN2 increased blastocyst rates (Bonferroni, P < 0.05). In Experiment II, 1454 oocytes were vitrified using Nitrocooler after loading in different containers: stainless steel straws (SSS), beveled-tip straws (BTS), open-OPS, and glass-pulled straws (GPS). Greater blastocyst rates were obtained in the SSS (10.2%) and the GPS (8.0%) treatments, both being greater than the BTS (6.1%) and OPS (6.1%) containers (Tukey, P < 0.05). In Experiment III (Acta Sci. Vet. 2006 34, 77–82), 195 IVP bovine blastocysts were vitrified with or without Nitrocooler under 2 different cryoprotectant solutions: 10% EG + 10% DMSO or 10% EG + 10% propylene glycol (PROP). Hatching rates using Nitrocooler for EG + DMSO (50.2%) or EG + PROP (54.0%) were similar (ANOVA, P > 0.05) to normal atmosphere rates using EG + DMSO (50.1%) or EG + PROP (56.0%). Under these experimental conditions, our results suggest that the greater cooling rates obtained favored an increase in oocyte viability after vitrification, whereas no such beneficial effects were detected for vitrified IVP embryos. In conclusion, Nitrocooler was proven effective as a low-cost device to supercool LN2, increasing viability after vitrification of bovine oocytes, but not of IVP embryos. Table 1.Effect of Nitrocooler apparatus on cryopreservation of bovine oocytes and embryos

The control of oxidative processes in in vitro production (IVP) systems by the use of additives may be an alternative approach to improve embryo cryotolerance. The aim of this study was to verify the effect of β-mercaptoethanol (βME) on the cryotolerance of bovine IVP embryos. In 7 replications, and following IVM-IVF, presumptive zygotes (n = 3735) were in vitro-cultured in SOF medium supplemented or not with 100 μm βME (IVC treatment), at 38.5°C and high humidity. The initial 24 h of IVC was performed in 5% CO2 in air, with the remaining 6 days of IVC carried out in 5% CO2, 5% O2, and 90% N2. On Day 7, resulting blastocysts and expanded blastocysts were vitrified in glass micropipettes in a solution with 20% ethylene glycol + 20% propylene glycol. After warming, embryos were randomly allocated to 1 of 2 sub-groups for an additional 72 h of IVC to the hatching blastocyst (HBL) stage, in fresh SOF medium supplemented or not with 100 μm βME (PVC treatment), at 38.5°C, high humidity and 5% CO2. Experimental groups were as follows: G1 (βME-free medium during IVC and PVC); G2 (βME only during PVC); G3 (βME only during IVC); and G4 (βME during IVC and PVC). Cleavage (Day 2) and blastocyst (Day 7) rates in the IVC treatment and hatching rates (Days 7 to 9) for the PVC treatment were analyzed by the chi-square test, for P < 0.05. Total cell number (TCN) estimated by fluorescence staining in HBL derived from vitrified and nonvitrified embryos was analyzed by ANOVA. The use of βME during IVC did not affect cleavage rates (βME-free, 1491/1858, 80.2% v. βME, 1522/1877, 81.1%), but negatively affected development to the blastocyst stage (βME-free, 813/1858, 43.8% v. βME, 525/1877, 28.0%). Following vitrification, however, βME supplementation during PVC improved hatching rates (G2, 58.1% and G4, 63.8%) compared with groups without the additive (G1, 36.6% and G3, 42.0%). In addition, the presence of βME either during IVC or PVC, or during both culture periods, increased TCN in HBL from vitrified embryos (Table 1). The use of βME during IVC, irrespective of the presence of βME during the PCV period, caused an increase in TCN in HBL in G3 + G4, with no effects on hatching rates (Table 1b), whereas the addition of βME during PVC, irrespective of the presence of βME during the IVC period, resulted in greater hatching rates and TCN in HBL in G2 + G4 than in G1 + G3 (Table 1). In conclusion, the addition of βME during the IVC period did not affect cleavage, but reduced blastocyst yield. Despite that, βME supplementation during the IVC period appeared to have increased the cryotolerance of the resulting blastocysts, expressed by greater TCN in HBL, whereas βME supplementation during the PVC period also improved embryo survival to the vitrification process, manifested by greater hatching rates and TCN in HBL. Table 1.Effect of βME on the cryotolerance of bovine IVP embryos This study was supported by a grant from CNPq/Brazil.

RESUMO A fim de estabelecer um parâmetro para a avaliação da viabilidade após o descongelamento, os resultados do cultivo de embriões frescos de camundongos, no meio PBS Modificado sem a adição de C02, foram comparados aos resultados obtidos após o cultivo no meio de Whitten Modificado em atmosfera com 5% de C02. Foram coletadas 1458 mórulas provenientes de fêmeas Mus musculus da cepa Suíço Albina CF 1, previamente superovuladas com 6-8UI de eCG e 6-8UI de HCG, com intervalo de 48 horas. Destas, 1051 foram cultivadas em PBS Modificado acrescido de 20% de SFB, a 37°C e 95% de umidade. As restantes 407 foram cultivadas no meio de Whitten Modificado, acrescido de 3% de BSA, a 37°C, 95% de umidade e 5% de C02. As avaliações foram realizadas com 24h de cultivo, sendo considerados os embriões que atingiram os estágios de blastocisto inicial (Bi), blastocisto (Bl), blastocisto expandido (Bx), blastocisto em eclosão (Bh) ou blastocisto eclodido (Be), e após 48h de cultivo os que atingiram pelo menos o estágio de blastocisto expandido. Os resultados obtidos após 24h (87,9 e 89,4%) e após 48h de cultivo (95,1 e 93,8%) em PBS e Whitten respectivamente, não apresentaram diferenças estatisticamente significativas (p < 0,05). SUMMARY The development of fresh mouse embryos in Modified PBS without adding C02 was compared to the development in Modified Whitten supplemented with 5% de C02 in order to establish a parameter to assess the viability after thawing. One thousand and four hundred and fifty eight morulae were collected from Strain CF1 Swiss Albino Mus musculus females which had been previously superovulated with 6-8UI of PMSG and 6-8UI of HCG 48h apart. One thousand and fifty one morulae were cultured in Modified PBS + 20% FCS, at 37°C and 95% of humidity. The others 407 were cultured in Modified Whitten's medium + 3% BSA, at 37 °C, 95% of humidity and 5% of C02. At the evaluation, performed after 24h of culture, were considered developed the embryos that reached early blastocyst, expanded blastocyst, hatching blastocyst and hatched blastocyts stages. After 48h of culture were considered developed the embryos that reached at least the expanded blastocyst stage. The results obtained after 24h (87.9 and 89.4%) and 48h of culture (95.2 and 93.8%) in PBS and Whitten, respectively, were similar (p < 0,05).

Cloning by somatic cell nuclear transfer has been associated with developmental abnormalities, with the level of heteroplasmy imposed by cell fusion being one of many potential determining factors. As the cytoplast exerts a key role in nuclear reprogramming, embryo aggregation is an alternative to minimize such negative effects during cloning. The aim of this study was to determine the effect of fusion of hemi-cytoplasts or aggregation of hemi-embryos on in vitro development and cell number of clone and parthenote embryos. Bovine cumulus–oocyte complexes (COCs) from slaughterhouse ovaries, after 17 h of IVM, were used for the production of parthenotes by chemical activation, and clone embryos by handmade cloning (HMC) (Vajta et al. 2003 Biol. Reprod. 68, 571–578). Following cumulus and zona removal, oocytes were manually bisected, followed by segregation of nucleated and enucleated hemi-cytoplasts by fluorescence using Hoechst stain. One or two enucleated hemi-cytoplasts were paired with an adult skin somatic cell from primary cultures (>90% confluence) and fused using a 25V AC pre-pulse, followed by a single 1.2 kV cm–1 DC pulse for 10 μs. Reconstructed clone structures and groups of zona-intact oocytes and nucleated hemi-cytoplasts were chemically activated in ionomycin and 6-DMAP. Clone and parthenote structures were in vitro-cultured in the WOW system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) for 7 days, as follows: (G1) clone embryos reconstructed by aggregation of two hemi-embryos per WOW; or (G2) one embryo (two hemi-cytoplasts + cell) perWOW; and parthenote embryos composed of (G3) zona-intact oocytes cultured in wells; or aggregation of one (G4), two (G5), three (G6), or four (G7) nucleated hemi-cytoplasts per WOW. Fusion, cleavage (Day 2), and blastocyst (Day 7) rates, evaluated on a per WOW basis, were compared by the chi-square test (8 replications). Total cell number estimated by fluorescence (Hoechst stain) in blastocysts was analyzed by the Student t-test. Fusion rates of one hemi-cytoplast + cell (G1; 275/592, 46.5%) were lower than for two hemi-cytoplasts + cell (G2; 264/337, 78.3%). Cleavage rates were lower in G1 and G4 and higher in G6 and G7 than G2 and G3. A significant linear increase in blastocyst rates was observed in G5, G6, and G7. Total cell numbers were lower in parthenotes than in clones, except in G6 and G7. The lower fusion and cleavage rates after the aggregation of two clone hemi-embryos (G1) caused nearly a 50% reduction in the overall cloning efficiency. In addition, the aggregation of parthenogenetic hemi-embryos increased cleavage and blastocyst rates and cell number. However, aggregation of hemi structures did not improve blastocyst yield or cell number on a hemi-cytoplast basis. Table 1. In vitro development of parthenote or clone bovine embryos This work was supported by funding from CAPES/Brazil.

Made available in DSpace on 2022-04-28T18:56:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-09-01 Castration is used to improve the management of water buffalo beef males raised under extensive conditions. However, as buffalo are considered robust animals, their welfare is often neglected, which, among other implications, may compromise their productivity. The aim of this study was to determine the effects of different castration methods on the stress level and weight gain of water buffalo males. Two experiments were performed with three treatments each. In experiment 1, serum cortisol concentrations were used as stress indicators for non-castrated (control group) or castrated males, either surgically or by burdizzo clamp. In experiment 2, blood levels of fibrinogen were used as stress indicators for males in the control group compared to those castrated by either burdizzo clamp or intratesticular injection of calcium chloride (chemical castration). In both experiments, clinical parameters and the mean daily weight gain were measured for all males. Surgical castration and chemical castration caused higher stress than castration with burdizzo, with no differences observed in weight gain among castration methods. In conclusion, for water buffalo males, castration with burdizzo clamp is preferable to surgical and chemical methods because it causes the lowest stress level in the animals. © 2011 Elsevier B.V. Agro-veterinary Research Center Universidade do Estado de Santa Catarina, Lages, Santa Catarina Veterinary and Animal Science College Universidade do Estado São Paulo, São Paulo Veterinary Faculty Universidade Federal de Pelotas, Capão do Leão, Rio Grande do Sul