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This study tested the hypothesis that different sward structures, which were constructed by varying the pre- and post-grazing sward heights of annual ryegrass pasture ( Lolium multiflorum Lam.) in southern Brazil, affect the short-term intake rate ( STIR) by dairy cows. Treatments consisted of four sward-management strategies defined by a combination of two pre- (25 and 15 cm) and two post-grazing sward heights (10 and 5 cm): 15-05, 15-10, 25-05 and 25-10. A completely randomized block design with four replicates was used for the experimental design. The STIR was determined by the double-weighing technique. Jaw movements were evaluated using automatic recorders ( IGER Behaviour Recorder). The results showed that treatment 25-10 allowed the animals to collect more herbage with a greater bite mass and thus resulted in a greater STIR. Treatments in which sward height was reduced to 10 cm generally favoured the ingestion process than treatments with a post-grazing sward height of 5 cm. [ABSTRACT FROM AUTHOR]

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 vitroculture 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-1cytochalasin 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-1CCB and 10 μg mL-1cycloheximide, 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 vitrofor 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. [ABSTRACT FROM AUTHOR]

Despite the rather successful and widespread use of cloning in various species, distinct cell types from the same species and even the same genotype display differences in blastocyst yield. Moreover, variations in the protocol for embryo production can influence development to the blastocyst stage and subsequent fetal development. The aim of this study was to evaluate the effect of 2 cell types and 2 embryo pre-activation protocols with or without the presence of FCS in the in vitroculture medium on development of handmade pig cloned embryos to the blastocyst stage. Cumulus-oocyte complexes recovered from sow ovaries were in vitro-matured for 38 to 40 h. Denuded matured oocytes selected by the presence of a polar body had the zona pellucida removed in a 0.2% protease HEPES-buffered solution +25% FCS, followed by manual bisection and UV screening of enucleated halves using Hoechst stain. Clone embryo reconstruction was performed using a phytohemoagglutinin solution to adhere 2 cytoplasts and a somatic cell. Adipocyte-derived mesenchymal stem cells (ADMSC) from a Yorkshire pig or granulosa cells (GC) from an Ossabaw pig were used as nuclear donors. Following electrical fusion, couplets were pretreated with a brief exposure to cytochalasin B (CB) or cytochalasin B + cycloheximide (CB+CX) in the presence of serum before the electrical activation (Naruse et al.2007 Theriogenology 68, 709-716; Du et al.2009 Reprod. Fertil. Dev. 21, 114). Activated embryos were in vitro-cultured in the well of the well (WOW) system, with 2 embryos per microwell, for 7 days in PZM-3 medium +0.3% BSA in the presence (FBS+) or absence (FBS-) of 10% FCS. Cleavage (Day 2, chi-square test) and blastocyst (Day 7, Fisher test) rates, on a per WOW basis, were compared for a level of significance of 5%. Our preliminary data indicate that the presence of serum in the IVC affected cleavage and blastocyst yield in a cell-type-dependent manner. The presence of serum enhanced the blastocyst yield for ADMSC, whereas for GC, only the absence of serum allowed any blastocyst development. The cell type and the pre-activation protocol did not appear to affect cleavage and embryo development to the blastocyst stage. Despite the low number of replications, our results reinforce the importance of optimizing the embryo production system taking into consideration the individual requirements for distinct cell types, procedures, and culture conditions. Table 1.Effects of cell type, pre-activation process and in vitroculture (IVC) medium on development of handmade pig cloned embryos [ABSTRACT FROM AUTHOR]

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 vitroculture 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 [ABSTRACT FROM AUTHOR]

Despite all the biotic and abiotic factors affecting foraging by ruminants, there is a common and fundamental process, which is bite gathering. We hypothesised that because the mechanics of bite formation dominate the foraging process, changes in short-term bite mass are reflected in longer-term animal performance across a wide range of sward conditions. We focus at the meal level of foraging, using experiments in which the effect of abiotic factors and digestive constrains are minimised, making intake rate the main currency. We estimated bite mass across a wide range of structural challenges to large-herbivore foraging in a long-term experiment with heterogeneous native grasslands. A conceptual model was developed for average daily gain, where energy gain and energy costs were proximate causal variables. Energy gain was a function of diet quality and components of daily intake rate, where bite mass was the main component estimated. In turn, components of intake rate were determined by sward structure and bodyweight. Energy costs were a function of bodyweight and abiotic conditions. Finally, sward structure, bodyweight and abiotic conditions were determined by experimental treatments, seasons and years. Then, the conceptual model was translated into statistical models that included variables measured or estimated, and coefficients representing all links in the conceptual model. Weight gain was a function of bite mass, forage characteristics, and animal and abiotic conditions. Models were set up to test whether forage and stocking conditions affected monthly gain beyond the effects through bite mass, after correcting for abiotic factors. Forage mass, height and disappearance did help predict monthly gain after bite mass was included in the model, which supported our hypothesis. However, stocking treatments and season had significant effects not incorporated in bite mass. Although the model explained 77.9% of liveweight gain variation, only 35.2% was due to fixed effects, with 10.8% accounted by bite mass and its interactions. Concomitant experiments showed that sward structure (first with sward height and the second with tussock cover) does determine bite mass and short-term intake rate in the complex native grasslands we studied. Yet, other temporal varying components of monthly gain not correlated with bite mass, temperature or wind, added most of the observed variation in monthly animal performance. Part of the model failure to account for variation in performance may be related to a significant and temporally variable grazing of tussocks. We used a bite mass model that assumed no tussock grazing. In light of these results and a parallel experiment, we conclude that tussock grazing must be incorporated in future versions of the model. We investigated whether longer-term animal performance can be explained by changes in short-term grazing processes in a heterogeneous native grassland. Although bite mass and its interactions accounted for 10.8% of the variation in average daily gain, stocking treatments and season had significant effects not incorporated in bite mass. We conclude that a large portion of the variation in performance may be related to temporally variable grazing of tussocks and changes in diet quality not contemplated in the model. [ABSTRACT FROM AUTHOR]

To maximize herbage DMI, pregrazing sward surface height (SSH) and level of herbage depletion (HD) must be such that variables determining short-term herbage intake such as bite mass (BM) and bite rate (BR) are optimized. The objective of this study was to determine a SSH target and the level of HD as a proportion of the SSH that optimizes BM and BR of beef heifers grazing Sorghum bicolor swards. Two experiments were conducted using 2 S. bicolor swards and 4 beef heifers (25 mo old; 322 kg BW). Experiment 1 compared the effect of 6 pregrazing SSH, 30, 40, 50, 60, 70, and 80 cm, on BM, BR, and jaw movements. Experiment 2 assessed the effect of HD level as a proportion of SSH (0.17, 0.34, 0.50, 0.67 and 0.84) on BM, BR, and jaw movements using the optimal pregrazing SSH defined in Exp. 1. Short-term herbage DMI was estimated using a double-weighing technique and corrected for insensible BW loss. Herbage DMI was subsequently used to calculate the BM. Net eating time and jaw movements for apprehension and manipulation + mastication during grazing as well as total jaw movements were determined using the IGER (Institute of Grassland and Envoronmental Research) behavior recorders. Bite rate and the number of total jaw movements per gram herbage DMI were derived from jaw movement count and measurements of herbage DMI. The results of Exp. 1 showed low and high SSH constraint the ease of herbage harvesting. Greater BM are maintained until a SSH of 50 cm is reached (P < 0.05) and then decline at greater SSH due to herbage dispersion. The nonbiting jaw movement rate increased at greater SSH whereas BR decreased (P < 0.05). For both variables, the turning point was close to a SSH of 50 cm. Experiment 2 showed that such an optimization of BM and BR was maintained until an HD level of 0.34 was reached (P < 0.05). There was a linear increase in both the total jaw movements per unit herbage DMI and the nonbiting jaw movements rate (manipulation + mastication) subsequent to levels of HD greater than 0.34 (P < 0.05). These studies provide, for the first time, sward feature targets to manage grazing and optimize BM and BR, aiming to maximize the shortterm herbage DMI of cattle grazing S. bicolor swards. [ABSTRACT FROM AUTHOR]

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 vitrodevelopment 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-1DC 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 vitrodevelopment, 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 vivodevelopment and pregnancy maintenance following the transfer to female recipients in cattle. Table 1.In vitroand in vivodevelopment of cloned bovine embryosThis study was supported by FAPESP and CAPES, Brazil. [ABSTRACT FROM AUTHOR]

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 vitrodevelopment 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-1DC 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/G1than 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/G1proportion in that cell population. Table 1.In vitrodevelopment of bovine cloned embryos from distinct cell culture confluences and aggregation schemeThis study was supported by FAPESP and CAPES/Brazil. [ABSTRACT FROM AUTHOR]

Grazing intensity is the key component of the interrelations between plants and their environment in pastoral ecosystems. In this way, the natural structural and floristic heterogeneity that is a response to soil variability can be modified by grazing. But the degree of such alterations must to be controlled in order to maintain ecosystems services and at same time to offers an adequate nutritional environment to the herbivores. In this paper we summarize some results from a series of experiments conducted in natural pastures from south Brazil, to demonstrate how grazing control can modified structural pasture characteristics, enhancing animal performance, soil quality, water infiltration, and producing a meat with particular fatty acids composition. [ABSTRACT FROM AUTHOR]