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Proteins administered orally must pass through the gastric environment in order to reach their site of absorption in the intestine. How to protect these exogenously administered proteins from the damaging effects of gastric acid and pepsin proteolytic activity, which often induce irreversible structural and functional alterations to the molecules, is an intriguing challenge. Another problem is the physical and chemical instability of proteins during some technological processes, which often involve the use of organic solvents or high temperatures. In this study we investigated the use of alginate microparticles containing one of two enzymes, an enteric polymer and a lyoprotectant for the intestinal delivery of proteins. The two enzymes tested in this protein delivery system were lactate dehydrogenase and alpha-amylase: the former was chosen because of its sensitivity to denaturation, the latter for its relevance in nutrition and medicine. A sodium alginate aqueous solution containing the enteric polymer, a lyoprotectant and the enzyme was either extruded or sprayed into a calcium chloride solution, with the resultant formation of beads and microspheres which were freeze-dried. About 90% of the enzyme activity was maintained during the process of loading the proteins into the microparticles and the subsequent freeze-drying process. The stability of the encapsulated enzyme in an acid medium and the enzymatic activity in an intestinal environment were then investigated by a dissolution test. This consisted of exposing the microparticles to simulated gastric fluid (pH 1.2) for 2 hours and to simulated intestinal fluid (pH 7.5+/-0.1) for 1 hour. The morphology of the microparticles did not change in the acid environment, whereas they completely dissolved within 3 min in the simulated intestinal fluid. Residual enzymatic activity after the test remained satisfactory for both enzymes. In conclusion, these microparticle systems offer promise for applications in human and veterinary medicine as well as in human and animal nutrition.

Objective To verify the hypothesis that a three-dimensional, follicle-mimicking structure enhances in vitro maturation yields without hormonal supplementation in an in vitro maturation program. Design Feasibility study; 204 anonymous denuded germinal vesicles retrieved from gonadotropin-treated women were cultured for 48 hours without hormonal supplementation in microdrop culture or in a three-dimensional coculture with granulosa cells in a barium alginate membrane. Setting An assisted reproduction center in Italy. Patient(s) One hundred twenty-two informed women. Intervention(s) Germinal vesicles retrieved after ovarian stimulation were denuded and cultured without hormonal supplementation in microdrop culture or in a three-dimensional coculture with granulosa cells enclosed in the core of a barium alginate capsule. Main Outcome Measure(s) Oocyte maturation and morphological investigation of follicle-mimicking structures. Result(s) Statistically significantly higher oocyte maturation yields were obtained by using the three-dimensional coculture system enclosed in a barium alginate membrane (after 48 hours: coculture, 90.3%; microdrop, 52.0%; odds ratio, 8.51). Morphological investigation indicated that after 48 hours of coculture, the decumulated oocyte appeared to be surrounded by a pseudocumulus structure that lies on the inner surface of the alginate membrane and protrudes toward the capsule's lumen. Conclusion(s) The maturation yield of cocultured oocytes is eightfold higher than that achieved with a microdrop maturation technique, making a higher number of gametes available for IVF programs. Stereomicroscopic morphological survey indicates a reassembly of granulosa cells surrounding oocyte zona, mimicking an antral cumulus oophorus.

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.

Summary Platelet count (PLT) mean platelet volume (MPV) and plateletcrit (PCT) were determined for 117 Landrace × Large White piglets aged 3–21 days; counts were performed with an automated blood cell counter (ABX Pentra 120). Reference values were estimated following the guidelines of the International Federation of Clinical Chemistry (IFCC) and the International Committee for Standardization in Haematology (ICSH). The calculated central 95% reference limits for PLT was 49.9–516.2 × 109/l, for MPV 6.71–9.91 fl and for PCT 0.009–0.395%. When observations are divided into three age groups (about 1 week each) there is an increase in mean PLT count and PCT in 2-week piglets, and a decrease in MPV from the first to the third week of life. These reference values provide guidelines for interpreting for experimental and clinical observations, as well as for monitoring of the health status of similar aged piglets determined using automated impedance-light focusing methodology.

Mare milk is taken into account as a substitute for cow milk in case of allergy, especially in infant nutrition due to a composition similar to human milk. The aim of the work is to define some mare milk characteristics during lactation, from foaling to the 4 th month of lactation. All the parameters considered to describe fat globule dimensions vary significantly during the lactation period. Fatty acid composition generally changes within the first days of lactation and after 42-56 days, with different behavior when considering medium or higher carbon chains. The ROS-scavenging activity expresses a positive trend and reaches the zenith at day 84. Finally there is a number of correlations between fat globules dimensional parameters and fatty acid composition and between long chain unsaturated fatty acids and ROS-scavenging activity, while dimensional parameters and ROS-scavenging activity are not statistically related. Results could be useful in veterinary and human field as milk substitutes.

Munari, E., Asti, A., Colombani, C., Benzoni, E., Faustini, M., Torre, M.L., Riccardi, A., Villani, S., Sesso, L., Conte, U. and Vigo, D., 2007. Competence of swine oocytes matured by three-dimensional gonadotropin-free co-culture. Veterinary Research Communications, 31(Suppl. 1), 181–184

Granulosa cells from bovine and porcine ovaries were cultured either in monolayer or in follicle-like barium alginate capsules for 6 days. Morphological investigation by electron scanning microscopy indicated that culture in a three-dimensional (3D) system allows self-organization of spherical-polyhedral shape cells. The luteinization index (progesterone:17beta-estradiol ratio) was significantly higher for monolayer cells than for the 3D cell culture system, confirming the results of morphological analysis and indicating more physiological growth. The encapsulated 3D culture system appears to be a promising way of obtaining in vitro maturation and development of follicles and oocytes.

Summary Blood samples were taken between February and April from 105 healthy Stamboek pre-pubertal gilts, aged 1–3 months, which were housed at a modern pig farm in northern Italy. The blood was analysed for nine haematological and nine selected haematochemical variables by means of automated and semi-automated blood analysis apparatus. After detection and rejection of outliers, the data were submitted to reference limits evaluation, also taking into account the limits for the red blood cell volume histogram as the anisocytosis index. Some haematological reference values deal with previously published data; in the haematochemical parameters, several discrepancies between evaluated limits and existing reference limits were noted, mainly for aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase levels and total protein concentration. The results confirm the relevance of age in determining blood reference intervals and that ‘normal’ values should be determined by each laboratory, taking into account the age of subjects, the sample size and methods of analysis.

Encapsulation in a barium alginate membrane is a promising strategy to obtain a three dimensional culture of living cells: membrane properties are crucial for a realistic clinical application. A one-step encapsulation technique, recently developed for controlled release of boar semen, was employed to prepare barium alginate and protamine-alginate membranes: permeability to two model molecules (haemoglobin and glucose) was evaluated. Capsules were evaluated for technological properties and scanning electron microscopy was used to examine the external morphology of the capsules and the 3D distribution of the cells within the core. The results indicate that 3D arrangement and cell shape are maintained, capsule dimensions and mechanical properties can be modulated, as well as their permeability to model molecules such as haemoglobin and glucose.