Oxygen Concentration and Cysteamine Supplementation During In vitro Production of Buffalo (Bubalus bubalis) Embryos Affect mRNA Expression of BCL‐2, BCL‐XL,MCL‐1, BAX and BID

This study examined the effects of O₂ concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm , respectively; IVM, IVF and IVC carried out in 20% O₂), on blastocyst rate and relative mRNA abundance of some apoptosis‐related genes measured by real‐time qPCR in immature and in vitro‐matured buffalo oocytes and in embryos at 2‐, 4‐, 8‐ to 16‐cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL‐positive cells was significantly lower (p < 0.05) under 5% O₂ than that under 20% O₂. The mRNA expression of anti‐apoptotic genes BCL‐2 and MCL‐1 was significantly higher (p < 0.05) and that of pro‐apoptotic genes BAX and BID was lower (p < 0.05) under 5% O₂ than that under 20% O₂ concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL‐XL and MCL‐1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O₂ groups and in cysteamine supplemented vs controls. At the 8‐ to 16‐cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL‐2 and MCL‐1 was highest under 5% O₂ concentration and that of BAX and BID was highest (p < 0.05) under 20% O₂ concentration. These results suggest that one of the mechanisms through which beneficial effects of low O₂ concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti‐apoptotic and a decrease in the expression of pro‐apoptotic genes.     

Dimensionality-driven insulator-to-metal transition in the bechgaard salts

Optical experiments were conducted on a series of organic linear chain conductors with different values of the interchain single-electron transfer integral tb, which quantifies the degree of anisotropy. Electron-electron interactions together with Umklapp scattering resulted in a correlation gap and an insulating state for small tb. An insulator-to-metal transition was observed when tb exceeded a critical value, on the order of the correlation gap Egap. The absence of a plasma edge on the insulator side of the transition for polarization perpendicular to the chains suggests that the electrons are confined to the chains. The optical features of the metallic state, when contrasted with the magnetic properties, are suggestive of spin-charge separation.     

Mapping agricultural responses to water supply shocks in large irrigation systems, southern India

Irrigated agriculture experienced a water supply shock during a drought in southern India in 2002-2003. In this paper, hotspots of agricultural change were mapped and put in the context of hydrology and water management. Time series of MODIS imagery taken every eight days before (2001-2002) and during (2002-2003) the supply shock were combined with agricultural census data to document changes in cropping patterns in four large irrigation projects in the downstream sections of the Krishna and Godavari River basins (total command area 18,287 km²). The area cropped in rice in the four irrigated command areas decreased by 32% during the drought year, and rice production in the two districts that experienced the largest flow reductions fell below production levels of 1980. The irrigation project that showed the largest change in double cropped area (−90%) was upstream of the Krishna Delta. In the Krishna Delta, large areas changed from rice-rice to rice-gram double cropping. Historical water management contributed to the vulnerability of rice production to drought: the main reservoir in the system was drained to dead storage levels by the end of each growing season over 1968-2000, with little carryover storage. The land cover change maps suggested that the lower Krishna Basin has experienced a “hard landing” during basin closure, and revised management strategies that account for the new flow regime will be required to maintain agricultural production during droughts.     

Amplification of Bovine Viral Diarrhoea Virus Introduced into an In Vitro Embryo Production System Via Oocytes from Persistently Infected Cattle

The purpose of this study was to determine whether or not embryos derived from in vitro fertilization of oocytes from persistently infected (PI) cattle would contain infectious virus. Three in vitro embryo production treatment groups were assessed: 1) oocytes and uterine tubal cells (UTC) free of bovine viral diarrhoea virus (BVDV) (negative control), 2) oocytes free of BVDV fertilized and cultured in media containing UTC obtained from PI heifers, and 3) oocytes from PI heifers fertilized and cultured in media containing UTC free of BVDV. The developmental media, UTC and embryos (individual or groups of five) were assayed for virus. Virus was not isolated from any samples in treatment group 1. As shown in previous studies, a proportion of embryo samples were positive for BVDV in treatment group 2. In treatment group 3, the virus associated with the oocytes contaminated the developmental media and infected susceptible co-culture cells used during fertilization and culture. In addition, 65% (11/17) of the degenerated ova from treatment group 3 had infectious virus associated with them. While none of the ova developed into transferable embryos, the study did confirm that use of oocytes from PI cows could lead to amplification of BVDV and cross contamination during in vitro embryo production.     

Planktonic and Biochemical Composition of Periphyton Grown on Some Biodegradable and Non-Degradable Substrates

ABSTRACT

Ten locally available substrates, five biodegradable and five non-degradable, were evaluated for their potential to harbor periphyton in cement tanks fertilized with poultry manure. The tanks were fertilized regularly and the periphyton was allowed to grow for 70 days. Weekly samples of periphyton and plankton were collected for enumeration and biochemical analyses. Among the substrates, earthen tiles harbored negligible amount of periphyton. The phytoperiphyton genera encountered on the substrate belonged mainly to Chlorophyceae (14 genera), followed by Cyanophyceae (2 genera), Chrysophyceae (1 genus), Bacillariophyceae (1 genus), and Dinophyceae (1 genus). Nauplius, Keratella, Diaptomus, Cyclops, Moina, Chironomus and insect eggs were the zooplankton encountered on substrates. Phytoplankton density was higher on tyre (86,426 cells or colonies/cm²) and palm leaf (85,808 cells or colonies/cm²) and lowest on ceramic tile (21,081 cells or colonies/cm²). Glass plates harbored the highest number of zooplankton species per unit area (1050 cells or colonies/cm²), while arecanut leaf-sheath had the lowest (210 cells or colonies/cm²). All five families of phytoplankton present on the substrates were also present in tank water. While periphyton contained 26 genera, tank water had only 24. Periphytic dry matter, ash, ash-free dry matter, plankton density on substrates and water showed a general increase with respect to time. Polyvinyl chloride (PVC) (0.972 mg/cm²), glass (0.913 mg/cm²), and bamboo (0.897 mg/cm²) had higher periphytic dry matter and ceramic tile (0.262 mg/cm²) the lowest. All the proximate composition parameters of periphyton, except nitrogen free extract (NFE) varied significantly (P < 0.05) between the substrates. The moisture content of periphyton ranged from 85.58% (bamboo) to 95.27% (arecanut leaf-sheath). Crude protein was high in periphyton from bamboo (3.77%) and tyre (3.66%) and low in that from arecanut leaf-sheath (0.99%).     

Further studies on the use of mixed feeding schedules with plant- and animal-based diets for common carp Cyprinus carpio (Linnaeus)

A growth trial was conducted on common carp Cyprinus carpio (L.) fry in cement tanks for 100 days in order to test the efficacy of mixed feeding schedules. The diets tested were a fishmeal‐based diet (diet D with 30.9% protein) and three Colocasia esculenta‐based diets (diet A with 16.7% protein, diet B with 19.7% protein and diet C with 25.8% protein) separately and in three mixed feeding combinations of diet A, B and C with D on alternate 2 days (2A/2D, 2B/2D and 2C/2D, where the numeral indicates the number of days that the particular diet was offered continuously). The results revealed no difference in growth of common carp fed the plant protein‐based diets A and B (P > 0.05). Although the fish fed on diets C and D and mixed feeding combinations 2A/2D and 2B/2D performed comparably and higher than those on diets A and B, the schedule 2C/2D resulted in the highest final weight. Specific growth rate, feed conversion ratio and RNA/DNA ratios were the highest in the 2C/2D schedule. Among the mixed feeding schedules, the highest saving in protein and cost was recorded with the 2A/2D schedule, followed by 2B/2D and 2C/2D. An increase in dietary protein led to decreased protein and increased fat deposition in the carcass. An increasing trend in the protease and amylase activities was recorded with increased dietary protein level. The study highlighted the efficient utilization of plant proteins by common carp under mixed feeding schedules.     

Effect of Physiologically Relevant Heat Shock on Development,Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.     

Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.