Production of horse foals via direct injection of roscovitine-treated donor cells and activation by injection of sperm extract

We evaluated the effects of different donor cell treatments and activation methods on production of blastocysts after equine nuclear transfer. Nuclear transfer was performed by direct injection of donor cells, using a piezo drill, and standard activation was by injection of sperm factor followed by culture with 6-dimethylaminopurine. There was no difference in blastocyst development between embryos produced with roscovitine-treated or confluent donor cells (3.6% for either treatment). Addition of injection of roscovitine or culture with cycloheximide at the time of activation did not affect blastocyst development. Overall, transfer of eight blastocysts produced using roscovitine-treated donor cells and our standard activation protocol yielded three pregnancies, of which two (25% of transferred embryos) resulted in delivery of viable foals. Flow cytometric evaluation showed that roscovitine treatment significantly increased the proportion of cells classified as small, in comparison to growth to confluence or serum deprivation, but did not significantly affect the proportion of cells in G0/G1 (2N DNA content). Transfer of one blastocyst produced using roscovitine-treated donor cells, with addition of roscovitine injection at activation, yielded one pregnancy which was lost before 114 days' gestation. Transfer to recipients of two blastocysts produced using confluent donor cells with addition of cycloheximide at activation gave no resulting pregnancies. We conclude that roscovitine treatment of donor cells yields equivalent blastocyst production after nuclear transfer to that for confluent donor cells, and that direct injection of roscovitine-treated donor cells, followed by activation using sperm extract, is compatible with efficient production of viable cloned foals.     

Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse

Work with lyophilized sperm helps delineate the factors required for successful fertilization. We investigated the use of lyophilized sperm in equine embryo production. In Experiment 1, sperm DNA fragmentation index was not affected by three freeze/thaw or lyophilization cycles. In Experiment 2, oocytes injected with lyophilized sperm or with sperm from a treatment in which lyophilized sperm were suspended in sperm cytoplasmic extract (SE) yielded blastocyst development rates of 0 and 28% respectively (P < 0.05). In Experiment 3, blastocyst development rate was 6-11% after injection of sperm lyophilized from fresh or frozen-thawed semen, suspended in SE. In Experiment 4, sperm lyophilized 3.5 months or 1 week previously, suspended in SE, yielded similar blastocyst rates (6 and 3% respectively). Rates of normal pregnancy after transfer were 7/10 and 5/7 for embryos from control and lyophilized sperm treatments respectively. Three pregnancies from the lyophilized sperm treatments were not terminated, resulting in two healthy foals. Parentage testing determined that one foal originated from the lyophilized sperm; the other was the offspring of the stallion providing the sperm extract. Further testing indicated that two of five additional embryos in the lyophilized sperm treatment originated from the stallion providing the sperm extract. We conclude that both lyophilized stallion sperm and stallion sperm processed by multiple unprotected freeze-thaw cycles (as for sperm extract) can support production of viable foals. To the best of our knowledge, this is the first report on production of live offspring by fertilization with lyophilized sperm in a non-laboratory animal species.     

Intracellular calcium oscillations and activation in horse oocytes injected with stallion sperm extracts or spermatozoa

In oocytes from all mammalian species studied to date, fertilization by a spermatozoon induces intracellular calcium ([Ca(2+)](i)) oscillations that are crucial for appropriate oocyte activation and embryonic development. Such patterns are species-specific and have not yet been elucidated in horses; it is also not known whether equine oocytes respond with transient [Ca(2+)](i) oscillations when fertilized or treated with parthenogenetic agents. Therefore, the aims of this study were: (i) to characterize the activity of equine sperm extracts microinjected into mouse oocytes; (ii) to ascertain in horse oocytes the [Ca(2+)](i)-releasing activity and activating capacity of equine sperm extracts corresponding to the activity present in a single stallion spermatozoon; and (iii) to determine whether equine oocytes respond with [Ca(2+)](i) transients and activation when fertilized using the intracytoplasmic sperm injection (ICSI) procedure. The results of this study indicate that equine sperm extracts are able to induce [Ca(2+)](i) oscillations, activation and embryo development in mouse oocytes. Furthermore, in horse oocytes, injection of sperm extracts induced persistent [Ca(2+)](i) oscillations that lasted for >60 min and initiated oocyte activation. Nevertheless, injection of a single stallion spermatozoon did not consistently initiate [Ca(2+)](i) oscillations in horse oocytes. It is concluded that stallion sperm extracts can efficiently induce [Ca(2+)](i) responses and parthenogenesis in horse oocytes, and can be used to elucidate the signalling mechanism of fertilization in horses. Conversely, the inconsistent [Ca(2+)](i) responses obtained with sperm injection in horse oocytes may explain, at least in part, the low developmental success obtained using ICSI in large animal species.     

Production of nonproteinaceous amino acids using recombinant Escherichia coli cells expressing cysteine synthase and related enzymes with or without the secretion of O-acetyl-L-serine

Beta-(pyrazol-1-yl)-L-alanine (beta-PA), a model nonproteinaceous amino acid, was specifically synthesized by two methods using recombinant Escherichia coli cells that express cysteine synthase, comprising serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS-A) and related enzymes from E. coli. In the first method (method A), recombinant cells that express wild-type SAT, OASS-A, acetate kinase (AK), and phosphotransacetylase (PTA) showed the highest beta-PA production. beta-PA was produced at 140 mM from 200 mM L-serine and 200 mM pyrazole under optimum conditions. Using the cells expressing SATDeltaC20 (truncated SAT), OASS-A, AK, and PTA, beta-PA was produced at a level of only 80 mM, whereas O-acetyl-serine (OAS) was found to be secreted into the broth. Under optimum conditions, OAS accumulated at levels of around 105 mM from 300 mM L-serine. Thus, in the second method (method B), the secreted OAS was used as the substrate for the syntheses of beta-PA and beta-(triazol-1-yl)-L-alanine (beta-TA). The OAS that accumulated in the broth was efficiently converted to beta-PA and beta-TA at levels of around 90 mM from 105 mM OAS using free OASS-A. In both methods A and B, the addition of glucose was essential for the efficient production of beta-PA and OAS, respectively.     

Production of ethanol from liquefied cassava starch using co-immobilized cells of Zymomonas mobilis and Saccharomyces diastaticus

Co-immobilized cells of Saccharomyces diastaticus and Zymomonas mobilis produced a high ethanol concentration compared to immobilized cells of S. diastaticus during batch fermentation of liquefied cassava starch. The co-immobilized cells produced 46.7 g/l ethanol from 150 g/l liquefied cassava starch, while immobilized cells of yeast S. diastaticus produced 37.5 g/l ethanol. The concentration of ethanol produced by immobilized cells was higher than that by free cells of S. diastaticus and Z. mobilis in mixed-culture fermentation. In repeated-batch fermentation using co-immobilized cells, the ethanol concentration increased to 53.5 g/l. The co-immobilized gel beads were stable up to seven successive batches. Continuous fermentation using co-immobilized cells in a packed bed column reactor operated at a flow rate of 15 ml/h (residence time, 4 h) exhibited a maximum ethanol productivity of 8.9 g/l/h.     

Fining of red wines with gluten or yeast extract protein

Due to a growing interest in alternatives to substitute gelatin as clarifying agent, this work investigates the use of yeast extracts and glutens. Physico‐chemical characteristics of proteins were analysed. Fining experiments were carried out on young red wines. Turbidity reduction, lees volume and effects on polyphenolic content and colour characteristics were determined. The results indicated that glutens and yeast extracts turbidity reduction is comparable to gelatin. Lees production was considerably reduced by non‐animal proteins: yeast extracts produced 44%, wheat glutens 60% and maize gluten 92% less than gelatin. Lees volume is highly correlated (R = ?0.720) with the absolute superficial charge density value (SCD) and with the pI (0.936) when clarifier was used in combination with bentonite. Treatments with glutens reduced less the polyphenolic content (between 0.6 and 8.7 units) than gelatin did. There were slight differences on the modification of colour intensity.     

Production of high energy density fermented uji using a commercial alpha-amylase or by single-screw extrusion

The effects of alpha-amylase and extrusion on the viscosity and energy density of uji, a spontaneously fermented thin porridge from different combinations of maize, finger millet, sorghum and cassava, were investigated. Fermentation alone was not able to reduce the viscosity of uji, but addition of 0.1-2.1 ml/100 ml alpha-amylase to the fermented slurry or extrusion of the fermented and dried flour at 150-180°C and a screw speed of 200 rpm reduced the viscosity of 20 g/100 ml uji from 6000-7000 to 1000-2000 cP, measured at 40°C and a shear rate of 50 s⁻¹. The amount of flour required to make uji could thus be increased by a factor of 2.0-2.5 and consequently it was possible to produce uji with acceptable energy densities (0.6-0.8 kcal/g) for child feeding.     

Production of lactose-hydrolyzed milk using ethanol permeabilized yeast cells

To overcome the problem of enzyme extraction and poor permeability of cell membrane to lactose, experimentation was carried to permeabilize Kluyveromyces marxianus NCIM 3465 cells for their subsequent use for the production of lactose-hydrolyzed milk. Different process parameters, such as biomass load, temperature, agitation and treatment time, were optimized for maximum lactose hydrolysis in skim milk using these cells. The ethanol-permeabilized yeast cells gave 89% hydrolysis of milk lactose under optimized conditions.     

Production of Restructured Meat using Microbial Transglutaminase without Salt or Cooking

To produce restructured meat, microbial transglutaminase (MTGase) was evaluated for its ability to introduce covalent crosslinks between protein molecules. Pork muscle cubes were mixed with MTGase and held at 5°C for 2 hr for the enzyme reaction. Restructured products were analyzed for binding strength without cooking. MTGase treatment resulted in effective binding of meat pieces provided there was addition of salt (NaCl). To ensure proper binding without NaCl, some food proteins with MTGase were also investigated. Meat cubes in combination with MTGase and sodium caseinate showed acceptable bind, and sodium caseinate appeared to be a superior substrate for the crosslinking to meat proteins than soy protein, whey protein, or gelatin. These results suggest a useful method for producing restructured meat which can be distributed in the raw, chilled state.     

Physical and antimicrobial properties of Gelidium corneum/nano-clay composite film containing grapefruit seed extract or thymol

To improve the physical properties of Gelidium corneum (GC) film, Cloisite Na⁺ and 30B were incorporated into the preparation of the film. X-ray diffraction patterns of the films indicated that a degree of exfoliation and intercalation are formed depending on the type of nano-clays and its concentration and the film structure affects the physical properties of the GC/nano-composite films. Tensile strength (TS) of the GC film was increased by the addition of nano-clays. GC film had a TS of 19.59 MPa for the control, while the GC film having 3% Cloisite Na⁺ or 30B had TS of 27.37 and 26.40 MPa, respectively. Elongation at break and water vapor permeability were also improved by the addition of 3% nano-clays. The physical properties of the film were not improved by the addition of GSE or thymol, but the additions did inhibit the growth of Escherichia coli O157:H7 and Listeria monocytogenes. The results suggest that the nano-composite films containing GSE or thymol may extend the shelf life of food.     

Production of electricity from acetate or butyrate using a single-chamber microbial fuel cell

Hydrogen can be recovered by fermentation of organic material rich in carbohydrates, but much of the organic matter remains in the form of acetate and butyrate. An alternative to methane production from this organic matter is the direct generation of electricity in a microbial fuel cell (MFC). Electricity generation using a single-chambered MFC was examined using acetate or butyrate. Power generated with acetate (800 mg/L) (506 mW/m2 or 12.7 mW/ L) was up to 66% higher than that fed with butyrate (1000 mg/L) (305 mW/m2 or 7.6 mW/L), demonstrating that acetate is a preferred aqueous substrate for electricity generation in MFCs. Power output as a function of substrate concentration was well described by saturation kinetics, although maximum power densities varied with the circuit load. Maximum power densities and half-saturation constants were Pmax = 661 mW/m2 and Ks = 141 mg/L for acetate (218 ohms) and Pmax = 349 mW/m2 and Ks = 93 mg/L for butyrate (1000 ohms). Similar open circuit potentials were obtained in using acetate (798 mV) or butyrate (795 mV). Current densities measured for stable power outputwere higher for acetate (2.2 A/m2) than those measured in MFCs using butyrate (0.77 A/m2). Cyclic voltammograms suggested that the main mechanism of power production in these batch tests was by direct transfer of electrons to the electrode by bacteria growing on the electrode and not by bacteria-produced mediators. Coulombic efficiencies and overall energy recovery were 10-31 and 3-7% for acetate and 8-15 and 2-5% for butyrate, indicating substantial electron and energy losses to processes other than electricity generation. These results demonstrate that electricity generation is possible from soluble fermentation end products such as acetate and butyrate, but energy recoveries should be increased to improve the overall process performance.     

Physicochemical properties and tenderness of meat samples using proteolytic extract from Calotropis procera latex

This study was conducted in order to tenderise muscle foods (pork, beef and chicken) by using crude enzyme extract from Calotropis procera latex. Chunks of knuckle muscle from pork and beef as well as of breast muscle from chicken were marinated with distiled water (control) and 0.05%, 0.1%, 0.2%, 0.3% and 0.5% (w/w) of crude enzyme extract powder for 60 min at 4 °C. The marinated samples were then subjected to various physical and chemical property determinations. A decrease in moisture content was observed when the crude enzyme extract was added. Firmness and toughness of the muscle samples significantly decreased with the increased addition of crude enzyme extract (p < 0.05). The water holding capacity and cooking yield of the treated samples showed no significant difference throughout the crude enzyme extract addition (p > 0.05). Crude enzyme extract had no effect on the pH of the pork sample, but it slightly increased the pH in the beef and chicken. An increase in protein solubility and TCA-soluble peptides content was observed in all of the treated samples. The electrophoresis pattern of the muscle treated samples also revealed extensive proteolysis occurring in each muscle type. From the results, it is determined that latex from Calotropis procera can be used as an alternative source of proteolytic enzymes for the effective tenderising of meat.     

Conjugated linoleic acid production by cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus with additions of different fatty acids

The objective of the study was to determine the effect of fatty acid additions to the cells and enzyme extract of Lactobacillus delbrueckii ssp. bulgaricus (CCRC14009) on CLA production. Washed cells of L. delbrueckii ssp. bulgaricus, obtained by cultivation in a MRS broth, were mixed with BSA and each of the three fatty acids: linoleic, oleic, and linolenic acids in sodium phosphate buffer at pH 6.5. After incubation at 37 °C for 108 h, CLA concentration was analyzed by HPLC. Enzyme extract from the culture was also reacted with each fatty acid at 50 °C for 10 min at pH 5 to test for CLA production. Results showed that linoleic acid addition to the culture improved CLA production, indicating the presence of linoleic acid isomerase activity in the culture. The crude enzyme extract from the culture was observed to be capable of oleic and linolenic acid conversions into CLA, demonstrating the possible presence of desaturase activity in the enzyme extract.     

Effect of diet therapy using horse meat on liver function of patients with metabolic-alimentary obesity

The data are provided for the first time about the use of the national food, horse meat, in dietetic management of patients with metabolic-alimentary obesity. Horse meat is characterized by low caloricity, considerable content of full-value protein with an optimally balanced amino acid composition, high content of essential polyunsaturated fatty acids, vitamins, mineral substances. Dietetic management with the use of horse meat, a product that possesses lipotropic and choleretic properties, made in possible to reduce markedly the patients' body mass, with the reduction amounting to 54% of the excess body weight. Furthermore, such a management led to a considerable improvement of liver function. The research data permit the authors to recommend the inclusion of horse meat, a product having a high biological value, into the diets of patients suffering from metabolic-alimentary obesity.     

Production performance and milk composition of dairy cows fed whole or ground flaxseed with or without monensin

Eight multiparous Holstein cows averaging 570 ± 43 kg of body weight and 60 ± 20 d in milk were used in a double Latin square design with four 21-d experimental periods to determine the effects of feeding ground or whole flaxseed with or without monensin supplementation (0.02% on a dry matter basis) on milk production and composition, feed intake, digestion, blood composition, and fatty acid profile of milk. Intake of dry matter was similar among treatments. Cows fed whole flaxseed had higher digestibility of acid detergent fiber but lower digestibilities of crude protein and ether extract than those fed ground flaxseed; monensin had no effect on digestibility. Milk production tended to be greater for cows fed ground flaxseed (22.8 kg/d) compared with those fed whole flaxseed (21.4 kg/d). Processing of flaxseed had no effect on 4% fat-corrected milk yield and milk protein and lactose concentrations. Monensin supplementation had no effect on milk production but decreased 4% fat-corrected milk yield as a result of a decrease in milk fat concentration. Feeding ground compared with whole flaxseed decreased concentrations of 16:0, 17:0, and cis6-20:4 and increased those of cis6-18:2, cis9, trans11-18:2, and cis3-18:3 in milk fat. As a result, there was a decrease in concentrations of medium-chain and saturated fatty acids and a trend for higher concentrations of long-chain fatty acids in milk fat when feeding ground compared with whole flaxseed. Monensin supplementation increased concentrations of cis9 and trans11-18:2 and decreased concentrations of saturated fatty acids in milk fat. There was an interaction between flaxseed processing and monensin supplementation, with higher milk fat concentration of trans11-18:1 for cows fed ground flaxseed with monensin than for those fed the other diets. Flaxseed processing and monensin supplementation successfully modified the fatty acid composition of milk fat that might favor nutritional value for consumers.     

Binder of sperm 1 and epididymal sperm binding protein 1 are associated with different bull sperm subpopulations

Previously, we showed that binder of sperm 1 (BSP1) and epididymal sperm binding protein 1 (ELSPBP1) proteins are more abundant in the immotile bovine sperm subpopulation following cryopreservation. In this study, we investigated the association of BSP1 and ELSPBP1 with sperm in relation to their ability to survive the cryopreservation process. Fresh and cryopreserved semen samples from the same ejaculate collected from nine Holstein bulls were incubated with a fixable viability probe, fixed and permeabilised and then immunolabelled with rabbit anti-BSP1, rabbit anti-ELSPBP1 or rabbit IgG as negative control. Spermatozoa were then incubated with Alexa 488-conjugated secondary antibody and Hoechst 33342. For each sample, 10?000 'Hoechst positive' events were analysed by flow cytometry. Alternatively, sperm populations were obtained by fluorescence-activated cell sorting. In freshly ejaculated live sperm, two distinct BSP1 detection patterns were revealed: a first population where BSP1 is present along the flagellar region (P1 subpopulation) and a second population where BSP1 is localised on both the flagellar and the acrosomal regions (P3 subpopulation). The dead population presented a BSP1 distribution similar to P3 but with a more intense fluorescence signal (P4 subpopulation). In the corresponding cryopreserved samples, all sperm in the P3 subpopulation were dead while only a small proportion of the P1 subpopulation was dead (P2 subpopulation). ELSPBP1 was detected only in dead spermatozoa and in comparable proportions in both freshly ejaculated and cryopreserved semen. These results show that the presence of BSP1 over the acrosomal region characterises spermatozoa sensitive to cryopreservation and that ELSPBP1 characterises spermatozoa that are already dead at ejaculation.     

Reduction of lipid accumulation in white adipose tissues by Cassia tora (Leguminosae) seed extract is associated with AMPK activation

Natural herbal medications may be one answer to the worldwide epidemic of obesity. This study examines the effects of Cassia seed ethanol extract (CSEE) upon lipid accumulation in white adipose tissue (WAT). CSEE exhibited a significant concentration-dependent decrease in the intracellular accumulation of trigycerides in 3T3-L1 adipocytes. After being fed a high-fat diet (HFD) for 2 weeks, rats were fed CSEE (100, 200 or 300 mg/kg) once daily for 8 weeks. CSEE caused dose-related reductions in body weight gain (as well as plasma lipid levels and epididymal WAT sizes in HFD-fed rats). CSEE enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and its primary downstream targeting enzyme, acetyl-CoA carboxylase, up-regulated gene expression of carnitine palmitoyl transferase 1, and down-regulated sterol regulatory element-binding protein 1 and fatty acid synthase protein levels in epididymal WAT of HFD-fed rats. CSEE could attenuate lipid accumulation in WAT via AMPK signaling pathway activation.     

Nutritional implications and flour functionality of popped/expanded horse gram

Utilization of horse gram and its flour in legume composite flours and products is limited due to the presence of antinutritional components, poor functional and expansion properties. Enzymatic treatment was used to improve the expansion and functional properties of horse gram to facilitate its use as an ingredient in food processing. Xylanase-mediated depolymerization of cell wall polysaccharides of horse gram lead to the development of a new expanded/popped horse gram. Expansion process of enzyme treated horse gram resulted in increased length (5.3–6.8 mm) and higher yield of expanded grains (63–98%). The expanded horse gram had lower bulk density, higher protein digestibility and more resistant starch compared to the control raw grains. Dietary fibre content of raw and processed horse gram was in the range of 14.57–16.14%. High temperature short time (HTST) conditions used during expansion process lowered the levels of phytic acid, tannins and protease inhibitors by 46%, 61% and 92%, respectively. The flour obtained from xylanase treated and expanded horse gram had higher water (204.3 g/100 g) and oil absorption capacities (98.4 g/100 g) than unprocessed flour, which had 135.8 g/100 g and 74.6 g/100 g, respectively at ambient conditions. There was a decrease in foaming capacity and foam stability in expanded gram flour. However, emulsion stability increased significantly in the processed samples. Thus, the study indicated that nutritional value and flour functionality of horse gram could be improved by processing it into a new expanded product that can be used as an ingredient in food processing.