Effect of the association of IGF-I, IGF-II, bFGF, TGF-β1, GM-CSF, and LIF on the development of bovine embryos produced in vitro

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-β1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-β1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; P < 0.05) on Day 8 after in vitro fertilization and similar results to use of SOF + 10% fetal calf serum (38% and 16%, at the same stages, respectively). The averages of total cells, inner cell mass cells, and trophectoderm cells of exclusively in vitro Day-8 blastocysts for pooled GF-CYK treatments were higher than those for SOF and similar to those for fetal calf serum. The presence of these growth factors and cytokines in the embryo culture medium therefore has a combined stimulatory action on embryonic development; in particular through an increase in hatching rate and in the number of cells of both the inner cell mass and trophoblast. These results are the first to demonstrate that use of a combination of recombinant growth factors and cytokine, as IGF-I, IGF-II, bFGF, TGF-β1, LIF, and GM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer.     

Assessment of the role of meta-topolins on in vitro produced phenolics and acclimatization competence of micropropagated ‘Williams’ banana

The effects of five topolins (meta-Topolin, mT; meta-Topolin riboside, mTR; meta-Methoxy topolin, MemT; meta-Methoxy topolin riboside, MemTR and meta-Methoxy topolin 9-tetrahydropyran-2-yl, MemTTHP) on the phenolic content and subsequent acclimatization potential of micropropagated ??Williams?? bananas were compared to benzyladenine (BA). Sterile shoot-tip explants were cultured on modified Murashige and Skoog (MS) media containing 10, 20 or 30 ??M of the above aromatic cytokinins (CKs) for 42?days. Phenolic contents were quantified spectrophotometrically. Growth parameters and photosynthetic pigments of the greenhouse-acclimatized plants were determined after 5?months. Total phenolic levels were highest in 10 ??M mT-treated plantlets within the aerial parts and 30 ??M MemTTHP for the underground parts. In the underground parts, 10 ??M mT resulted in the production of the highest amount of proanthocyanidins which was approximately five-fold higher than in the control plants. Furthermore, 10 ??M MemTTHP-treated plantlets had significantly higher total flavonoids (30.1?±?0.24 mg CE/g DW) within the aerial parts. Plantlets regenerated using MemT, MemTR and MemTTHP had significantly longer roots and better shoot/root ratios compared to the control and BA-treated plants. In terms of root fresh weight, it was significantly higher in MemT-treated plantlets than in the control and BA treatments. Chlorophyll a/b ratio was significantly improved with the use of MemT, mTR and mT compared to control. Current findings indicate the potential of topolins in stimulating the accumulation of phenolic compounds in micropropagated plantlets. In view of the importance of plant secondary metabolites, their substantial accumulation probably enhanced the acclimatization and subsequent ex vitro survival of the micropropagated plantlets. Topolins, particularly, the new derivative MemTTHP could be an alternative CK for the micropropagation of plant species based on their stimulatory effect on ex vitro rooting that inevitably enhances acclimatization competence. Furthermore, topolins are demonstrated as potential elicitors in micropropagation.     

α-Tocopherol and l-ascorbic acid increase the in vitro development of IVM/IVF swamp buffalo ( Bubalus bubalis) embryos

This study was conducted to investigate the effects of capacitating agents added at in vitro fertilization (IVF) and antioxidants supplemented during in vitro culture (IVC) on the development of buffalo embryos. In experiment I, in vitro embryo development of buffalo embryos was compared when the IVF medium was supplemented with heparin, caffeine and calcium ionophore A23187 either alone or in combination. There was no significant difference (P > 0.05) in the cleavage rates of oocytes among the treatment groups but the development rate to the blastocyst stage and the cell numbers of blastocyst in the heparin-treated group were significantly higher (P < 0.05) than that of other treatments. In experiment II, in vitro embryo development of buffalo embryos was compared when IVC medium was supplemented with either α-tocopherol (250 and 500 μM) or l-ascorbic acid (250 and 500 μM). The rate of development to the blastocyst stage of embryos cultured in medium supplemented with 250 μM α-tocopherol (33%, 41/123) and 250 μM l-ascorbic acid (31%, 38/123) was significantly higher (P < 0.05) than that of those cultured in medium alone (19%, 20/108) but not significantly different (P < 0.05) from medium supplemented with either 500 μM α-tocopherol (24%, 30/123) or 500 μM l-ascorbic acid (25%, 33/133). These results suggest that buffalo spermatozoa treated with heparin were suitable for IVF and that α-tocopherol and l-ascorbic acid added during IVC increased the rate of buffalo embryo development.     

Storage and reproductive strategy of the carpet-shell clam,Ruditapes decussatus in the Gulf of Gabès (Tunisia)

In this study, the variations of glycogen concentrations in Ruditapes decussatus from Sfax coasts (Tunisia) were described in relation to the reproductive cycle. Separate analyses were made of gonad, adductor muscle and ‘remainder’. The timing of gametogenic development and spawning was evaluated using qualitative histology associated with image analysis including (1) the estimation of the gonadal occupation index (GOI), (2) surface area occupied by reserve tissues and (3) variation in oocyte diameter. The reproductive cycle of R. decussatus exhibited partial asynchronization between sexes, the major difference being observed in the duration of the spawning period. Contrary to previous studies, we observed continuous partial spawning (e.g. 50% of ripe oocytes still subsisted at the partial spawning stage). During the gametogenic cycle, GOI varied significantly in males and in females. Most oocytes were ripe and ready to spawn when their diameter reached or exceeded 45?µm. The expansion of gonad was inversely proportional to the development of the foot tissue. Glycogen concentration showed significant temporal variations between tissues, indicating the clear differentiation in energy storage between the clam organs.     

First partial cranium of Togocetus from Kpogamé (Togo) and the protocetid diversity in the Togolese phosphate basin

Earliest cetaceans (whales) originated from the early Eocene of Indo-Pakistan, but the group dispersed through most of the oceans of the planet by the late middle to late Eocene. This late Eocene global distribution indicates that important dispersal events took place during the middle Eocene (Lutetian), a globally undersampled time interval that is well documented in the Togolese phosphate series. We report here the first discovery of a partial cetacean cranium from middle Eocene deposits of Togo (West Africa). A 3D model of the cranium and teeth was reconstructed in order to reveal hidden anatomical features. The dental and cranial characteristics of the Togolese specimen recall those of protocetid taxa described in Africa, Asia, and North America, but also display significant differences. In particular, we show that the new specimen shares a number of morphological features with the Togolese taxon Togocetus. Such a hypothesis is further supported by a cladistic analysis including 45 taxa and 167 morphological characters, which recovers the new specimen close to Togocetus as the first offshoot of protocetids. Phylogenetic analysis including all the protocetids remains of Kpogamé confirms the singular diversity of the Togolese phosphate basin, and enables to examine potential connections with faunas from contemporaneous localities in Africa.     

In vitro–in vivo study on the effects of plant compounds on rumen fermentation,microbial abundances and methane emissions in goats

Two in vitro and one in vivo experiments were conducted to investigate the effects of a selection of plant compounds on rumen fermentation, microbial concentration and methane emissions in goats. Treatments were: control (no additive), carvacrol (CAR), cinnamaldehyde (CIN), eugenol (EUG), propyl propane thiosulfinate (PTS), propyl propane thiosulfonate (PTSO), diallyl disulfide (DDS), a mixture (40 : 60) of PTS and PTSO (PTS+PTSO), and bromochloromethane (BCM) as positive control with proven antimethanogenic effectiveness. Four doses (40, 80, 160 and 320 µl/l) of the different compounds were incubated in vitro for 24 h in diluted rumen fluid from goats using two diets differing in starch and protein source within the concentrate (Experiment 1).The total gas production was linearly decreased (P<0.012) by all compounds, with the exception of EUG and PTS+PTSO (P⩾0.366). Total volatile fatty-acid (VFA) concentration decreased (P⩽0.018) only with PTS, PTSO and CAR, whereas the acetate:propionate ratio decreased (P⩽0.002) with PTS, PTSO and BCM, and a tendency (P=0.064) was observed for DDS. On the basis of results from Experiment 1, two doses of PTS, CAR, CIN, BCM (160 and 320 µl/l), PTSO (40 and 160 µl/l) and DDS (80 and 320 µl/l) were further tested in vitro for 72 h (Experiment 2). The gas production kinetics were affected (P⩽0.045) by all compounds, and digested NDF (DNDF) after 72 h of incubation was only linearly decreased (P⩽0.004) by CAR and PTS. The addition of all compounds linearly decreased (P⩽0.009) methane production, although the greatest reductions were observed for PTS (up to 96%), DDS (62%) and BCM (95%). No diet–dose interaction was observed. To further test the results obtained in vitro, two groups of 16 adult non-pregnant goats were used to study in vivo the effect of adding PTS (50, 100 and 200 mg/l rumen content per day) and BCM (50, 100 and 160 mg/l rumen content per day) during the 9 days on methane emissions (Experiment 3). The addition of PTS and BCM resulted in linear reductions (33% and 64%, respectively, P⩽0.002) of methane production per unit of dry matter intake, which were lower than the maximum inhibition observed in vitro (87% and 96%, respectively). We conclude that applying the same doses in vivo as in vitro resulted in a proportional lower extent of methane decrease, and that PTS at 200 mg/l rumen content per day has the potential to reduce methane emissions in goats. Whether the reduction in methane emission observed in vivo persists over longer periods of treatments and improves feed conversion efficiency requires further research.     

Variation in breeding parameters of Eleonora’s falcon (Falco eleonorae) and factors affecting its reproductive performance

We gathered data for a four-year period (2004?C2007) on the nesting ecology and reproductive performance of Eleonora??s falcon in the Aegean Sea. We investigated in an indirect way the relation between clutch size and pre-laying food availability, the significance of site and pair quality on productivity, and the effects of habitat and intraspecific competition on breeding success. Overall, the species selected nest sites sheltered from sun exposure but not from the prevailing wind. Hatching, fledging, and breeding success rates were estimated at 64, 72%, and 60%, respectively. Fledglings per breeding pair ranged from 1.19 to 1.75, and fledglings per successful pair from 1.84 to 2.0, between the years. Productivity depended on parental care rather than nest-site quality. Breeding parameters varied significantly between the years, exhibiting a strong spatial yet local effect. Low success rates were recorded in specific colonies, which were attributed to adverse weather conditions and habitat degradation. Insect food availability prior to egg-laying, estimated via plant biomass, was positively correlated to clutch size. Meanwhile, low wind strengths in August, large distances from the mainland, and population size in the vicinity of the colonies had negative effects on breeding success. Considering data at the Mediterranean scale, a longitudinal trend is observed across the breeding range of this species, with breeding parameters slightly decreasing from west to east.     

Low genetic diversity and recovery implications of the vulnerable Bankoualé Palm Livistona carinensis (Arecaceae), from North-eastern Africa and the Southern Arabian Peninsula

The Bankoualé Palm, Livistona carinensis is the only known species of Livistona occurring in Africa and is currently classified as vulnerable (IUCN 2004). This extreme outlier species of the genus is restricted to Yemen, Somalia and Djibouti, where all populations are in rapid decline. In Djibouti the palm is confined to three valley systems within the upland plateau of the Goda Massif. This study used microsatellite markers to investigate the genetic diversity and relationships within the species. At the species level L. carinensis contained very low genetic diversity. Most variation was due to the variation between the samples from Yemen and Somalia compared with those in Djibouti. The Djibouti populations were almost monomorphic across the nine loci tested. Interestingly, and despite the small sample sizes, the individuals from botanic gardens collections of the Yemen and Somalia populations were more genetically diverse than the Djibouti populations. This study indicates that the populations in Yemen and Somalia are highly significant for the conservation of the species genetic diversity. Given the lack of genetic diversity both within and among L. carinensis populations in Djibouti, plants could be cultivated for in-situ population enhancement from any seed that is available from within Djibouti with no significant genetic impacts of provenance mismatch. Clearly the populations from Djibouti, Somalia and Yemen are different genetic provenances raising some issues for the conservation and recovery of L. carinensis.     

Flower or spikelet? Understanding the morphology and development of reproductive structures in Exocarya (Cyperaceae, Mapanioideae, Chrysitricheae)

Fundamental questions of floral morphology remain unresolved in the grasslike monocots in order Poales, including what constitutes a flower and what constitutes a spikelet. The mapaniid sedges have particularly complex spikeletlike structures, variously interpreted as clusters of flowers or spikelets. Recent phylogenetic studies of Cyperaceae have identified the mapaniid clade as sister to the rest of the family, but the homology of mapaniid reproductive units (RUs) and spikeletlike units (SLUs) to other sedge flowers and spikelets is unclear. We examined reproductive development in the mapaniid Exocarya sclerioides. Inflorescence branches terminated in a SLU with bracts and 1-4 RUs. RUs had four small leaflike structures (LLSs): two lateral LLSs, each associated with a stamen, an abaxial LLS associated with a stamen, and an adaxial LLS. The gynoecium terminated the RU. All RUs were axillary to bracts, and unexpanded bracts and RUs were produced beyond expanded RUs, so SLUs were racemose. RUs developed from a single primordium that initiated two lateral LLSs, then two lateral stamens, then the gynoecium. Initiation of the abaxial LLS and stamen and the adaxial LLS followed. We hypothesize that the RU is a sympodial branch that terminates in a hermaphroditic flower with two stamens and a gynoecium; the two lateral LLSs are halves of a deeply divided prophyll.     

Geographical variation in the genetic diversity and composition of the endangered queen conch Strombus gigas (Mesogastropoda: Strombidae) from Yucatán, México

In Mexico and elsewhere in the Caribbean, the queen conch Strombus gigas is an endangered species. Understanding the genetic connectivity of their populations will support management strategies for long-term conservation of the species. Genetic diversity and population differentiation was assessed from samples collected at Banco Chinchorro and Isla Cozumel in the Mexican Caribbean and at Arrecife Alacranes in the Gulf of Mexico. Samples were obtained from the commercial capture at Banco Chinchorro (n = 50) and Isla Cozumel (n = 40) on March 2004. On November 2004, a non-invasive method for the Arrecife Alacranes sampling was applied, taking the hemolymph of live animals (n = 65) and releasing them to the wild. The mitochondrial DNA variation at two genes (COI and Cyt-b) was analyzed. Genetic diversity at the three locations ranged between 0.55-0.65 in COI and 0.87-0.94 in Cyt-b, showing no bottleneck evidences. A non-significant fixation index (F(ST) = 0.019, p = 0.161) and a Maximum Parsimony Network tree that did not show particular clades associated with any of the geographical locations, suggested a lack of statistically significant genetic differentiation among populations. Nevertheless, the cline patterns observed in both genetic diversity and haplotypic frequencies from Banco Chinchorro through Arrecife Alacranes, and the larger genetic distance between these locations from those between Isla Cozumel, Banco Chinchorro and Arrecife Alacranes, suggest the possibility of a pattern of isolation-by distance. The role of the main current systems over the potential genetic differences in S. gigas populations along the Mexican Caribbean, and the conservation management of S. gigas at these locations as discrete units is discussed.     

Anti-inflammatory activities of inotilone from Phellinus linteus through the inhibition of MMP-9, NF-κB, and MAPK activation in vitro and in vivo

Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK)?[extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4(th) and the 5(th) h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5(th) h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5(th) h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5(th) h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT, SOD, and GPx in the paw edema through the suppression of TNF-α and NO. This study presents the potential utilization of inotilone, as a lead for the development of anti-inflammatory drugs.     

Genetic variation and structure in natural and reintroduced populations of the endangered legume,Pyne’s ground plum (Astragalus bibullatus)

Conservation Genetics - Rare species with limited geographic distributions and small census populations are particularly susceptible to genetic drift and inbreeding. Assessing genetic variation...     

Monitoring mammals in the Caxiuanã National Forest,Brazil – First results from the Tropical Ecology,Assessment and Monitoring (TEAM) program

The need for long-term biodiversity monitoring using standardized protocols led to the creation of the Tropical Ecology, Assessment and Monitoring (TEAM) initiative. At some 50 field stations in tropical forests around the world, TEAM will monitor various taxa such as ants, birds, butterflies, medium and large terrestrial mammals, primates, litter fall, and trees, as well as landscape change in nine tropical biodiversity hotspots and three tropical wilderness areas. The TEAM terrestrial mammal program calls for using a grid of camera phototraps to monitor long-term trends in densities and occupancy rates of species that can or cannot be uniquely identified, respectively. We describe the TEAM camera phototrapping program and provide results for the first TEAM site–Caxiuanã National Forest in northern Brazil. An intensive one year camera trapping effort was carried out to determine which months were most suitable for long-term monitoring. Fifteen species of medium and large terrestrial mammals and two large birds were recorded, including three xenarthrans, five carnivores, one perissodactyle, three artiodactyles, two rodents, and one marsupial. The medium and large terrestrial mammal diversity was well represented during two consecutive wet and dry months, respectively. We also recorded activity patterns for all species photographed by our camera traps more than 10 times.     

Reaction specificities of the ε-ionone-forming lycopene cyclase from rice (Oryza sativa) elucidated in vitro

Lycopene cyclases responsible for the formation of ε-ionone rings (LCYe) mark a plant-specific bifurcation of carotenogenesis. We investigated purified rice LCYe (OsLCYe) in a liposome-based biphasic assay system. OsLCYe depends on reduced flavin cofactors stabilizing a transient state formed during the non-redox cyclization reaction. In contrast to OsLCYb, OsLCYe produces predominantly monocyclic products and monocyclic carotene intermediates are not suitable substrates. Determination of the OsLCYe reaction specificities and the combined use of OsLCYb allow the characterization of the reaction sequence leading to heterocyclic carotenoids. It was also found that 5-cis-lycopene, which was thought to be decisive for ε-cyclization, was not involved in the reaction, with OsLCYe acting as an exclusion filter for this naturally occurring isomer.     

The first record of anguine lizards (Anguimorpha,Anguidae) from the early Miocene locality Ulm – Westtangente in Germany

Abstract

The first fossil anguine material from the lower Miocene (MN 2) locality Ulm – Westtangente in Germany is described. The parietal and compound bone of the lower jaw can be attributed to Ophisaurus holeci, previously known only from younger age (MN 3–MN 7). Moreover, the parietal represents the largest parietal of this species. In other disarticulated material, such as frontal, maxilla, dentary and osteoderms, alpha taxonomy is not possible and these elements cannot be allocated at the species level. Despite a limited data source, a phylogenetic analysis was done (16 taxa, 36 characters) producing four equally parsimonious trees. The analysis shows a close relationship of O. holeci and the Eocene Ophisauriscus quadrupes. These two taxa form a monophyletic clade, a sister-clade to Ophisaurus + Anguis. However, more complete skeletal material of O. holeci is needed to support such a statement. We used our phylogenetic analysis to analyze trace character history for one frontal and three parietal characters. The palaeoenvironmental conditions of the locality Ulm – Westtangente bring further support of the previous hypothesis that O. holeci was adapted to environments with high ground water levels – environments around lakes or rivers.     

γ-Aminobutyric acid (GABA) and other amino acids in tissues of the tick,Amblyomma hebraeum (Acari: Ixodidae) throughout the feeding and reproductive periods

We assayed a variety of tick (Amblyomma hebraeum Koch; Acari, Ixodidae) tissues for a number of amino acids throughout the feeding and early reproductive periods. Our HPLC assay could detect as little as 2–5 pmol per sample of the following: GABA, glycine, serine, glutamine, alanine, taurine, glutamate and aspartate. All of these amino acids could be detected in the salivary gland, synganglion (=total CNS in acarines), haemolymph, Gené's organ, seminal receptacle and ovary. GABA reached high levels in the salivary gland of freshly engorged ticks (685 nmol g^-1) and in the synganglion it exceeded 1000 nmol g^-1 throughout most of the feeding cycle and the first week post-engorgement. GABA also reached a peak titre in the haemolymph of 40 nmol ml^-1. Taurine levels peaked at 1065 nmol g^-1 in the salivary gland from large partially fed ticks. Glutamate and aspartate were likewise found in the salivary gland and synganglion at high concentrations. For most of the amino acids there is insufficient information to correlate these titres (and fluctuations of titres) to neuromodulatory functions. It is possible, however, that the high GABA titre in the salivary gland of engorged ticks is correlated with an augmented level of fluid secretion.Deceased: Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9.