Storage of sexed boar spermatozoa: Limits and perspectives.

Despite the great potential application of sex-sorted spermatozoa in swine, the technology is not practiced in the pig industry because of technical factors and species-specific issues. The susceptibility of boar spermatozoa to stresses induced by the sorting procedure, the relative slowness of the sex-sorting process together with the high sperm numbers required for routine artificial insemination in pig are some of the main factors limiting the commercial application of this technology in pigs. This review briefly describes the damage to spermatozoa during sex sorting, focusing on an additional limiting factor: increased susceptibility of sexed boar spermatozoa to injuries induced by liquid storage and cryopreservation that, in turn, impairs sperm quality leading to unsatisfactory results in vivo. Strategies to extend the lifespan of sex-sorted boar spermatozoa and to improve their fertilizing ability after liquid storage or cryopreservation need to be implemented before this technology can be used in pig farms. In this regard, encapsulation in barium alginate membranes could be a promising technique to optimize the in vivo use of sexed boar spermatozoa, by protecting, targeting, and controlling the release of sperm into the female genital tract.

Sperm Encapsulation from 1985 to Date: Technology Evolution and New Challenges in Swine Reproduction.

In the last 30 years, encapsulation technology has been applied to different species to minimize the loss of spermatozoa after artificial insemination. In particular, the vehiculation of boar sperm cells in barium alginate membrane has proved a valid strategy to reduce the risk of polyspermy and optimize in vivo fertilizing yields. Controlled release of male gametes into the female genital tract has reduced the minimum fertilizing dose of spermatozoa. Notwithstanding these results, encapsulation has not yet reached commercial application, largely due to the additional costs of production. However, encapsulation could be useful in advanced reproductive technology, such as sex sorting, to store sorted boar semen. The controlled release of flow cytometrically sorted spermatozoa could be a promising strategy to reduce the number of cells necessary for each insemination and hence allow the widescale use of sex sorting in this species.

Formulation and characterization of silk fibroin films as a scaffold for adipose-derived stem cells in skin tissue engineering.

Skin substitutes are epidermal, dermal or complete bilayered constructs, composed by natural or synthetic scaffolds and by adherent cells such as fibroblasts, keratinocytes or mesenchymal stem cells. Silk fibroin is a promising polymer to realize scaffolds, since it is biocompatible, biodegradable, and exhibits excellent mechanical properties in terms of tensile strength. Moreover, fibroin can be added of others components in order to modify the biomaterial properties for the purpose. The aim of this work is to prepare silk fibroin films for adipose-derived stem cell (ADSCs) culture as a novel feeder layer for skin tissue engineering. Pectin has been added to promote the protein conformational transition and construct strength, while glycerol as plasticizer, providing biomaterial flexibility. Eighteen formulations were prepared by casting method using fibroin, pectin (range 1-10% w/w), and glycerol (range 0-20% w/w); films were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry assay, to select the optimal composition. A stable fibroin conformation was obtained using 6% w/w pectin, and the best mechanical properties were obtained using 12% w/w glycerol. Films were sterilized, and human ADSCs were seeded and cultured for 15 days. Cells adhere to the support assuming a fibroblastic-like shape and reaching confluence. The ultrastructural analysis evidences typical active-cell features and adhesion structures that promote cell anchorage to the film, thus developing a multilayered cell structure. This construct could be advantageously employed in cutaneous wound healing or where the use of ADSCs scaffold is indicated either in human or veterinary field.

Boar sperm changes after sorting and encapsulation in barium alginate membranes.

A routine use of boar-sexed semen is limited by the long sorting time necessary to obtain an adequate number of sexed spermatozoa for artificial insemination and by the high susceptibility of spermatozoa of this species to damages induced by sorting procedure and subsequent cryopreservation. The aim of this work was to study the impact of encapsulation in barium alginate membrane on sorted boar spermatozoa by evaluating membrane integrity, chlortetracycline staining patterns, protein tyrosine phosphorylation, and Hsp70 immunolocalization during storage over 72 hours in liquid or encapsulated form. The encapsulation procedure significantly (P < 0.05) decreased the overall membrane integrity of control unsorted semen (81.8 vs. 57.4, CTR vs. CPS), but did not negatively affect the overall viability and the chlortetracycline staining patterns of sorted encapsulated cells. Moreover, encapsulation significantly decreased (P < 0.05) the overall phosphotyrosin A pattern cell percentage in unsorted (98.4 vs. 92.6, CTR vs. CPS) but not in sorted semen (64.0 vs. 74.2; SORT CTR vs. SORT CPS). As for Hsp70, the overall percentage of cells displaying the different patterns was significantly influenced (P < 0.05) by treatment but not by storage time. The sorting procedure seems to induce the major changes, whereas encapsulation tends to exert a protective effect on sorted semen by increasing the percentage of spermatozoa displaying the T pattern (2.8 vs. 24.3; SORT CTR vs. SORT CPS). In conclusion, our data confirm that the damaging impact of the encapsulation in barium alginate capsules seems to be limited when compared with that of the sorting procedure and, moreover, the association of the two procedures does not result in an algebraic sum of the negative effects. These results suggest the possibility of a future utilization of the encapsulation technology in order to store sorted spermatozoa and permit their controlled release in the female genital tract.

Dimensional analysis of milk fat globules in sow milk: effects of the lactation stage and fat content and comparison with vaccine milk.

To assess the differences in the granulometry of milk fat globules between swine and bovine species, milk samples from 30 lactating sows were analyzed for fat globule dimensions and compared with cow milk samples. Results showed differences between the fat globules: sow milk presents reduced globule diameters compared with cow milk (volume-weighted diameter 2.62 vs. 3.27 microm, p < 0.001) and reduced interglobular distance. A positive relationship was observed between milk fat content and globule diameter, while a slight, insignificant inverse trend was detected between the day of lactation and fat globule diameter. These complex interactions between milk lipids, globule membrane proteins, and globule dimensions provide a better understanding of digestion/absorption phenomena in the design of milk replacers.

Boar sperm encapsulation reduces in vitro polyspermy.

A boar sperm encapsulation technology in barium alginate has been developed to enhance reproductive performances and spermatozoa preservation time; aim of this work was to evaluate the effect of in vitro sperm encapsulation on polyspermy as a function of storage time at 18 degrees C. A total number of 40 in vitro fertilization (IVF) tests were performed using encapsulated or diluted spermatozoa (20 IVF each treatment). Overall, 1288 in vitro matured oocytes were fertilized with spermatozoa stored at 24, 48 or 72 h at 18 degrees C for both treatments polyspermy and normospermy, and the non-penetration rates were assessed by optical microscopy. Results indicate a significant reduction in risk of polyspermic oocytes when spermatozoa are preserved in barium alginate membranes (incidence risk ratio: 0.766 with respect to diluted); such enhancement could be explained by lesser damage of sperm membranes achieved by encapsulation technology.

Semen controlled-release capsules allow a single artificial insemination in sows.

Controlled-release capsules containing boar spermatozoa were developed to extend the preservation time of spermatozoa and maximize the efficiency of a single artificial insemination. A large trial (4245 sows) was performed with these capsules using double/triple conventional artificial insemination as a control. The effect of treatment on pregnancy diagnosis, delivery, and born piglets was investigated, with allowance being made for considering season, spermatozoa amount, and the weaning-to-estrus interval as confounding variables. The same pregnancy rate and prolificacy were obtained by two insemination techniques, and a higher parturition frequency was reached with capsules. The reproductive performance in pigs has therefore been optimized by a single instrumental insemination with controlled-release capsules.