Fertility after post-cervical artificial insemination with cryopreserved sperm from boar ejaculates of good and poor freezability

This study compared the field fertility outcomes in frozen–thawed (FT) sperm from boar ejaculates with different freezability (good, GFE/poor, PFE) while testing the reliability of the post-cervical artificial insemination (post-CAI) in FT sperm. The assay was conducted over eight months with 86 weaned sows being inseminated by post-CAI. Every ejaculate in a total of 26 from 15 Piétrain boars was divided into a refrigerated semen portion (FS; control treatment) and a cryopreserved portion (FT sperm), and the ejaculates were in turn classified as GFE or PFE in function of the sperm progressive motility and viability at 240 min post-thaw. As result, one of four possible treatments was randomly given to each sow: FS-GFE, FS-PFE, FT-GFE and FT-PFE. The number of pregnant and farrowing sows in FT-GFE did not significantly differ from those of FS control treatments. Contrarily, the probabilities of pregnancy were two times lower after inseminations with FT-PFE (P < 0.05) compared to FT-GFE, which indicates that ejaculates with high post-thaw sperm progressive motility and viability are more likely to result in pregnancies than those with poor in vitro sperm function. There were no differences in litter size or the risk of backflow among treatments. Further trials are required to determine the optimal volume and concentration of FT sperm in post-CAI to obtain a more reliable method for farmers interested in cryopreserved sperm.     

Overexpression of cytosolic group IVA phospholipase A2 protects cells from Ca2+-dependent death

The calcium ionophore ionomycin induces apoptosis-like events in the human embryonic kidney cell line at early times. Plasma membrane blebbing, mitochondrial depolarization, externalization of phosphatidylserine, and nuclear permeability changes can all be observed within 15 min of treatment. However, there is no activation of caspases or chromatin condensation. Expression of a fusion protein containing the enhanced green fluorescent protein (EGFP) and human cytosolic Group IVA phospholipase A(2)alpha (EGFP-cPLA(2)alpha) in these cells prevents ionomycin-induced phosphatidylserine externalization and death. Cells expressing the cPLA(2)alpha mutant D43N, which does not bind calcium, retain their susceptibility to ionomycin-induced cell death. Both nonexpressing and EGFP-D43N-cPLA(2)alpha-expressing human embryonic kidney cells can be spared from ionomycin-induced cell death by pretreating them with exogenous arachidonic acid. Moreover, during calcium overload, mitochondrial depolarization is significantly lower in the EGFP-cPLA(2)alpha-expressing cells than in cells expressing normal amounts of cPLA(2)alpha. These results suggest that early cell death events promoted by an overload of calcium can be prevented by the presence of high levels of arachidonic acid.     

Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophila melanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.     

Net P-removal deterioration in enriched PAO sludge subjected to permanent aerobic conditions

Recently, some research in the field of enhanced biological phosphorus removal (EBPR) has been focused on studying systems where the electron donor (substrate) and the electron acceptor (nitrate or oxygen) are present simultaneously. This can occur, for example, in a full scale wastewater treatment plant during heavy rainfall periods when the anaerobic hydraulic retention time is temporarily shortened. To study this situation that could induce EBPR failure, the operation of a sequencing batch reactor (SBR) working under alternating anaerobic-aerobic conditions with an enriched EBPR population (50% Candidatus Accumulibacter phosphatis and less than 1% Candidatus Competibacter phosphatis) was shifted to strict aerobic operation. Seven cycle studies were performed during the 11 days of aerobic operation. Net P-removal was observed in this aerobic SBR during the first 4 days of operation but the system could not achieve net-P removal after this period, although the microbial composition, in terms of percentage of Accumulibacter and Competibacter, did not change significantly. The observed changes in the different compounds analysed (phosphorus, acetate, glycogen and PHB) as well as in the OUR profile indicate that metabolic changes are produced for the adaptation of PAO to aerobic conditions.     

Structural and ultrastructural features of boar bulbourethral glands

The morphological features of boar bulbourethral glands were examined by light and transmission microscopy. Bulbourethral glands are compound tubuloalveolar glands surrounded by a capsule of dense connective tissue and arranged in multiple lobules formed by endpieces and excretory ducts. Endpieces and excretory ducts are both lined by a single epithelium of mucous cells with a basal nucleus. Epithelial cells accumulate secretory granules containing neutral and carboxylated acid mucosubstances and a small amount of sulphated acid mucosubstances. The ultrastructure of epithelial cells varies according to the secretory cycle. In initial stages, the cells show a columnar shape and secretory granules unevenly distributed in the cytoplasm. As the synthesis of mucosubstances progresses, the amount of the secretory granules increases and the cellular shape becomes pyramidal. Secretory granules can contain inclusions and present differences among them according to their different phases of formation. In pyramidal cells, secretory products are released into the lumen by a merocrine mechanism.     

Inhibitors of sphingolipid metabolism enzymes

Sphingolipids are a family of lipids that play essential roles both as structural cell membrane components and in cell signalling. The cellular contents of the various sphingolipid species are controlled by enzymes involved in their metabolic pathways. In this context, the discovery of small chemical entities able to modify these enzyme activities in a potent and selective way should offer new pharmacological tools and therapeutic agents.     

Inhibitors of sphingolipid metabolism enzymes

Sphingolipids are a family of lipids that play essential roles both as structural cell membrane components and in cell signalling. The cellular contents of the various sphingolipid species are controlled by enzymes involved in their metabolic pathways. In this context, the discovery of small chemical entities able to modify these enzyme activities in a potent and selective way should offer new pharmacological tools and therapeutic agents.     

Crystalline structure of poly(hexamethylene adipate). Study on the morphology and the enzymatic degradation of single crystals

The crystalline structure of polyester 6 6 was studied by means of X-ray and electron diffraction and real-space electron microscopy. An orthorhombic unit cell containing eight chain segments with a quasi planar zigzag conformation was derived. The chain axis projection could be defined by a small rectangular cell containing only two molecular segments. Simulation of electron diffraction patterns indicates that molecular segments were arranged with azimuthal angles close to +/-46 degrees . X-ray diffraction patterns suggested that the large dimensions of the unit cell were a consequence of a slight shift between neighboring chains that improved the electrostatic interactions. Chain-folded lamellar crystals were obtained by isothermal crystallization of dilute n-hexanol or n-octanol solutions. The crystalline habit was studied, and the influence of temperature was evaluated. A regular folding surface was observed by using polyethylene decoration techniques. Lamellar crystals were easily degraded with different lipases. A preferential enzymatic attack was, in some cases, observed to occur in the crystal edges, giving rise to highly irregular borders with a fringed texture.