Giovanni Maffeo

Boar semen controlled delivery system: storage and in vitro spermatozoa release

Swine spermatozoa were encapsulated in barium alginate and protamine-barium alginate membranes to lengthen their preservation time and to provide a means of controlling their release. Precocious acrosome reactions and secondary anomalies were measured as indices of semen quality. These characteristics were observed for two forms of encapsulated spermatozoa when stored at 18 and 38 degrees C for 24 h and for semen diluted in a classical extender at both temperatures. The results indicate that encapsulation enhances semen preservation, providing protection against membrane damage upon dilution. The effect is even more evident at the higher temperature (38 degrees C), where cell metabolism is higher. An in vitro release test of spermatozoa showed a massive cell delivery from barium alginate capsules within 6 h, and a slow release from protamine-barium alginate capsules. The properties of spermatozoa 24 h after release did not differ from the semen stored at the same temperature in capsules, indicating that the release process does not impair semen quality.

Boar semen controlled-delivery system: morphological investigation and in vitro fertilization test

A technology for encapsulation of swine semen in barium alginate and protamine alginate has recently been proposed for the controlled release of the spermatozoa, thus reducing the number of instrumental inseminations required. Controlled-release capsules containing swine spermatozoa were prepared by adding saturated BaCl2 solution to ejaculate and dropping the resulting suspension into a sodium alginate solution, leading to the formation of barium alginate capsules. A second type of capsule was obtained by cross-linking the barium alginate with protamine sulfate. Two types of membrane were thus obtained: barium alginate gel and a protamine cross-linked alginate membrane. Morphological (scanning electron microscopy and transmission electron microscopy), functional (motility, membrane integrity and in vitro fertilization test) and technological (capsule structure and weight) approaches were used to characterize the encapsulated spermatozoa and the controlled-delivery system. No differences in terms of morphological and functional characteristics (acrosome integrity and spermatozoa motility) between free and encapsulated semen were found. The technological process did not compromise in vitro fertilization potency of the spermatazoa, although seasonal variability was found. The capsule weight was related to either the pH of the semen or the season. This study represents the starting point for the development of further investigations into the storage and release kinetics of cells from the capsules and for the development of an in vivo fertilization protocol.

Barium alginate cell-delivery systems: correlation between technological parameters

The most recent trends for the development of several in vitro cell cultures have been oriented towards the cell immobilisation in 3-dimensional scaffolds and cell encapsulation. In fact, an important requirement of cell survival is self-assembly in functional communities, in the presence of an artificial extracellular matrix. In our research, a previously described technique for spermatozoa encapsulation was applied to obtain capsules loaded with an opaque agent as a model, and to perform a formulative study. A process variable, barium ion concentration, was correlated to some capsule properties, such as weight, gel thickness, total and core diameter. Ion concentration can be modified to obtain capsules with predictable characteristics.

Two-dimensional polyacrylamide gel electrophoresis map of bovine ovarian fluid proteins.

Molecular mechanisms underlying the cystic degeneration of ovarian follicles in the dairy cow have not been clarified yet. A useful approach for complementing endocrinological and clinical studies could be represented by the systematic analysis of the protein patterns in follicular and cystic fluid. With this aim, a two‐dimensional polyacrylamide gel electrophoresis map of proteins contained in fluid from bovine ovarian follicles at different stages of development and from bovine ovarian cyst has been obtained. About 200 spots were detected after silver staining. Polypeptides from nine spots or series of spots have been identified by N‐terminal sequencing, and further analysis of the map has been performed by gel comparison. Alpha‐1‐antitrypsin, albumin, serotransferrin and apolipoprotein A‐I and A‐IV were located on the map. Comparison between protein patterns revealed the differential expression of some spots among follicles of smaller diameter, follicles of larger diameter, and cysts. This could represent the first step toward the identification of proteins differentially expressed and associated with ovarian cyst development.

Boar semen controlled delivery system: analysis of batch seasonal variability

A new encapsulation technology of swine semen is proposed to improve the quality of stored spermatozoa and to obtain the controlled release of viable cells, reducing the number of instrumental inseminations. This technology has been employed to produce barium alginate gel capsules in different seasons; an analysis of batch reproducibility was performed, and total capsule diameter, gel thickness and weight of capsules were determined as indices of batch properties. A seasonal variability was found but each batch was substantially homogeneous. The variability could be related to the biological variability of the semen employed as raw material as well as to the technological process.