Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.     

Browsing preference and ecological carrying capacity of sambar deer (Cervus unicolor brookei) on secondary vegetation in forest plantation

The browsing preference and ecological carrying capacity (ECC) of sambar deer (Cervus unicolor brookei) in acacia plantations for management and conservation of the ecosystem were investigated at Sabal Forest Reserve in Sarawak, Malaysia. The identification of the species browsed by the sambar deer was based on an observation of the plant parts consumed. ECC estimation was based on body weight (BW) and the physiological stages of animals browsed in six fenced 4‐ha paddocks. Sambar deer were found foraging on only 29 out of 42 species of secondary vegetation in the acacia plantation. The remaining species are too high for the deer to reach. Planted species, Shorea macrophylla are not palatable to the deer. This augurs well for the integration of sambar deer into shorea plantations. The most frequently exploited plants were Ficus spp. Sambar deer preferred woody species more than non‐woody species and they are browser animals. By producing metabolizable energy of 19 000 to 27 000 MJ/ha, the ECC was five head/ha to 5.25 head/ha. Given its contribution to the conservation of wildlife and its capacity to sustain the ecosystem, the sambar deer integrated farming system offers a promising strategy for the future of tropical forestry management.     

Production of nuclear transfer embryos by using somatic cells isolated from milk in buffalo (Bubalus bubalis)

Somatic cells in milk are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important in animals that are susceptible to risks of bacterial infection on biopsy collection. In this study, a minimum of 10 milk samples were collected from each of the three buffaloes representing Murrah breed. All the samples were processed immediately and cell colonies were obtained. Cell colonies from one buffalo (MU-442) survived beyond 10 passages and were evaluated by fluorescence microscopy and used in nuclear transfer experiments. In culture, these cells expressed vimentin, indicating they were of fibroblast origin similar to ear cells. We compared the effectiveness of cloning using those milk-derived fibroblast (MDF) cells and fibroblast cells derived from the ear derived fibroblast (EDF). Fusion and cleavage rates of MDF-NT and EDF-NT embryos were found to be similar (92.43 ± 1.28% vs 94.98 ± 1.24%, and 80.27 ± 1.75% vs 84.56 ± 3.73%, respectively; p > 0.01); however, development to blastocyst stage and total cell number was higher for EDF-NT embryos (50.24 ± 2.54%, 227.14 ± 13.04, respectively, p < 0.01), than for MDF-NT embryos (16.44 ± 0.75%, 170.57 ± 4.50 respectively). We conclude that somatic cells from milk can be cultured effectively and used as nucleus donor to produce cloned blastocyst-stage embryos.     

Geographical Location has Greater Impact on Carotenoid Content and Bioaccessibility from Tomatoes than Variety

The suggested health benefits of consuming tomatoes and tomato-based products have been attributed, in part, to the carotenoids present in these foods. Therefore, the objectives of the present study were to (i) analyse carotenoid content and bioaccessibility from different tomato (Lycopersicon esculentum L.) types namely cherry, plum, round, and certain tomatoes-on-the-vine; and (ii) determine if geographical location (Ireland vs Spain) influenced the content and bioaccessibility of carotenoids in tomatoes of the same variety. Carotenoid bioaccessibility is defined as the amount of ingested carotenoids that, after digestion, are available for absorption by intestinal cells. Differences were seen in carotenoid content and bioaccessibility between the different tomato types tested. For instance, Irish round high-lycopene tomatoes contained the greatest amounts of lycopene and lutein but lowest levels of β-carotene compared with the other Irish tomatoes. Furthermore, the content and bioaccessibility of carotenoids that were sourced from Ireland and Spain also varied greatly. Spanish tomatoes were generally superior in the content, bioaccessibility, and micelle content of carotenoids. To conclude, our findings suggest that geographical location, rather than the type of tomato, seems to have a more pronounced effect on carotenoid bioaccessibility from tomatoes.     

Expression of nonacclimated freezing tolerance and cold acclimation capacity in somatic hybrids between hardy wild Solanum species and cultivated potatoes

The expression of freezing tolerance was characterized in interspecific somatic hybrids between S. tuberosum (tbr) and two cold-hardy wild species, S. brevidens (brd) and S. commersonii (cmm). The nonacclimated freezing tolerance (NA) and acclimation capacity (ACC, increase in freezing tolerance in response to low nonfreezing temperature), two main genetic components of freezing tolerance, were evaluated separately. In contrast to cmm, which exhibited excellent NA and ACC, the freezing tolerance of brd was mainly due to ACC. However, the ACC of brd was only moderately expressed in the somatic hybrids. The NA of cmm was also suppressed in combination with tbr genomes. However, with acclimation, some of the tbr (+) cmm somatic hybrids achieved freezing tolerance comparable to pure hardy species such as brd used in this study. Analysis of chloroplast DNA type by RFLP markers revealed no significant difference in ACC between somatic hybrids carrying chloroplasts from either tbr or cmm. The reasons for the reduced expression of freezing tolerance from either the brd or cmm parent and the utilization of these somatic hybrids in breeding programs are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Screening tuber-bearingSolanum (potato) germplasm for efficient accumulation of tuber calcium

Resistance to several pathological and physiological tuber defects has been correlated with calcium level in the peel. In this study, a representative sample of potato species (including cultivated), was grown in the greenhouse. Plants were watered with low and high calcium solutions, and the resulting tubers analyzed. Thus, potato germplasm was screened for ability to accumulate tuber calcium when a control level of calcium is available, and ability to respond when higher levels are supplied. Some wild species significantly exceeded cultivated materials in one or both of these measures. Such species may provide a resource for breeding varieties which are more efficient calcium accumulators.     

Response of potatoes (cv russet burbank) to supplemental calcium applications under field conditions: Tuber calcium, yield, and incidence of internal brown spot

Potato tuber tissue is low in calcium as compared to leaves and stems. Several recent studies have provided evidence linking tuber quality with tuber tissue Ca concentration. The purpose of the present study was to determine the influence of supplemental Ca and N fertilization on tuber Ca concentration and internal brown spot of potato. Three soluble products (calcium nitrate, calcium chloride, and N-Plus; 9N:0K:0P:11Ca) were used as Ca sources with and without gypsum. In addition, a combination of calcium chloride, urea, and calcium nitrate (CUC) was also used without gypsum. Each treatment consisted of five replications, which received the same total amount of N (252 kg ha^?1) supplied either alone or in various combinations of ammonium nitrate, urea, calcium nitrate, calcium chloride, and N-Plus. The soluble sources of Ca were added at 68.8 to 168.0 kg ha^?1 while gypsum supplied Ca at 49 kg ha^?1. The application of all N by hilling (non-split) was compared to split application of N and Ca at hilling and 2, 4, and 6 wk after hilling. Tubers were mechanically harvested and graded according to industry standards. About 100 U.S. No. 1 tubers from each replication were cut in half along the longitudinal axis and visually inspected for internal defects. From each replication, non-periderm tissue from 15 tubers was combined for Ca analysis. There was no treatment effect on tuber yield in both years. Application of gypsum, either alone or in combination with the soluble sources, was not effective in reducing incidence of internal brown spot (IBS). Furthermore, with one exception, application of gypsum either alone or in combination with soluble sources of Ca was not effective at increasing tuber tissue Ca concentration. Combined analyses of data from 2 years showed that the application of soluble sources of Ca without gypsum increased tuber Ca concentration and decreased the incidence of IBS. Furthermore, split application of N had similar beneficial effects as compared to non-split N. Data from 1997 (a year with high IBS) gave a significant inverse relationship between tuber Ca and IBS, suggesting that an increase in tuber Ca can reduce IBS incidence.     

Buffalo (Bubalus bubalis) Embryonic Stem Cell‐Like Cells and Preimplantation Embryos Exhibit Comparable Expression of Pluripotency‐Related Antigens

In this study, inner cell mass (ICM) cells were isolated from in vitro produced buffalo blastocysts and were cultured on mitomycin‐C treated buffalo foetal fibroblast feeder layer for producing embryonic stem (ES) cells. Among different sources (hatched vs expanded blastocysts) or methods (enzymatic vs mechanical), mechanical isolation of ICM from hatched blastocysts resulted in the highest primary colony formation rate and the maximum passage number up to which ES cells survived. Putative ES cells expressed alkaline phosphatase and exhibited a normal karyotype up to passage 7. Putative ES cells and embryos at 2‐ to 4‐cell, 8‐ to 16‐cell, morula and blastocyst stages strongly expressed stage‐specific embryonic antigen (SSEA)‐4 but lacked expressions of SSEA‐1 and SSEA‐3. Putative ES cells also expressed tumour rejection antigen (TRA)‐1‐60, TRA‐1‐81 and Oct4. Whereas in all early embryonic stages, TRA‐1‐60 was observed only in the periplasmic space, and TRA‐1‐81 expression was observed as small spots at a few places inside the embryos, both these markers were expressed by ICM. Oct4 expression, which was observed at all the embryonic stages and also in the trophectoderm, was the strongest in the ICM. Buffalo putative ES cells possess a unique pluripotency‐related surface antigen phenotype, which resembles that of the ICM.     

Seasonal differences in freezing stress resistance of needles of Pinus nigra and Pinus resinosa: evaluation of the electrolyte leakage method

Seasonal changes in freezing stress resistance of needles of red pine (Pinus resinosa Ait.) and Austrian pine (Pinus nigra Arnold) trees were measured by an electrolyte leakage method and by visual observation. During most of the year, freezing stress resistance determined by the two methods gave similar results. The electrolyte leakage method provided a good estimate of seasonal changes in freezing stress resistance except for red pine needles in their most winter-hardy state. To obtain a reliable estimate of freezing stress resistance in winter-hardy red pine needles it was necessary to combine the electrolyte leakage method with visual observations. When red pine needles survived exposure to -80 degrees C or lower, electrolyte leakage was never more than 30% even when the needles were exposed to a slow freeze-thaw stress of -196 degrees C. However, rapid freezing of red pine needles to -196 degrees C resulted in electrolyte leakage of over 80%. Red pine needles attained a much higher freezing stress resistance during the winter than Austrian pine. Red pine needles also acclimated and deacclimated faster than Austrian pine needles. An index of injury was developed based on the electrolyte leakage method ((R(2) + R(1))/2, where R(1) is the minimum % electrolyte leakage from noninjured tissue and R(2) is the maximum % electrolyte leakage at the highest injury) that reliably predicted freezing stress resistance of pine needles for most of the year. Important aspects for developing a successful index of injury for pine needles are: use of cut needles, vacuum infiltration and shaking during incubation in water.We conclude that: (1) during cold acclimation the cell wall properties of the pine needles changed and these changes, which appeared to differ in the two species, might explain the very low leakage of electrolytes from winter-hardy needles of red pine; (2) pine needles survive winter by developing the ability to tolerate extracellular ice formation, because after rapid freezing the needles were severely injured; and (3) red pine is adapted to a shorter growing season and colder winters than Austrian pine.