Drug release, stability and efficiency of vitamin e loaded in liposomes for bovine sperm protection in cryopreservation medium.

BACKGROUND: It is known that a considerable number of drugs in clinical use or under development are water-insoluble drugs with poor bioavailability. The liposomal delivery system has drawn attention as one of the noteworthy approaches to increase both dissolution and absorption because of its biocompatibility and ability to encapsulate hydrophobic molecules in the lipid domain. However, several drawbacks have been reported, the most common is liposome structural instability. OBJECTIVE: To encapsulate alpha tocopherol into liposomes, to determine the new formulation stability and to study the drug-release of alpha tocopherol into the sperm cryopreservation medium. MATERIALS AND METHODS: The liposomes prepared by an ethanol injection method were characterized for size stability, alpha tocopherol release and sperm motility tests. RESULTS: The prepared unilamellar vesicles had both narrow size distribution (around 99 nm) and a good physical and chemical stability at 4°C during 12 months. The liposomes did not release the vitamin E immediately, but retained the protectant for 24 hours, probably due to the rigidity of the liposomal fence which was reinforced by adding cholesterol. Then, all vitamin E molecules were released by 48 hours. Release was potentially by Fickian diffusion probably by the creation of mini-ducts due to both agitation and fence hydration. Moreover, semen motility treated with vitamin E liposome preparations was significantly improved compared to all other treatments (including commonly used sperm conservation media). CONCLUSION: The stable vitamin E liposomes formulated in this work are a promising alternative for semen cryopreservation protection.

Rosmarinus officinalis Essential Oil Preloaded in ß-Cyclodextrin: Effect on Ram Spermatozoa Motility, Membrane Integrity and Oxidative Status During 4°C Storage.

BACKGROUND: Rosmarinus officinalis essential oil (Rom) has been reported recently to be of interest for use in sperm cryopreservation. However, related to its lipophilic characteristics, encapsulation in cyclodextrin could enhance Rom positive effects by increasing its solubility in sperm extenders. OBJECTIVE: To compare the effect of Rom preloaded in hydroxypropyl-ß-cyclodextrin (Rom-cd) to Rom alone (Rom) on ram sperm conserved at 4°C. MATERIALS AND METHODS: Ram epididymal sperm was collected from six testes. Each collected sperm was split into four equal aliquots. The control aliquot was diluted with Tris extender (Tris + citric acid + fructose + penicillin), two aliquots were treated with Rom at 0.5µl ml-1 and 1µl ml-1 respectively, and one aliquot treated with Rom-cd at 1µl ml-1. All sperm aliquots were analyzed for motility after 0, 2, 4, 24 and 48 h of storage at 4°C using a Computer Aided Semen Analysis (CASA). Membrane integrity and oxidative stress status were measured after 48 h of storage. RESULTS: The results indicated that motility parameters were best preserved in the extender containing Rom-cd compared to the groups treated by Rom without cyclodextrin. Rom alone resulted to higher sperm motility than the control group. Lower oxidative stress and more cell membrane protection were observed in Rom treated samples, especially when using Rom-cd. CONCLUSION: The ability of Rom to protect ram sperm against cryopreservation damages was improved after encapsulation in hydroxypropyl-ß-cyclodextrin (HPßCD).

The Complementary Effect of Cholesterol and Vitamin E Preloaded in Cyclodextrins on Frozen Bovine Semen: Motility Parameters, Membrane Integrity and Lipid Peroxidation.

BACKGROUND: During cryopreservation sperm cells suffer from two major deleterious impacts: oxidative stress and cold shock. OBJECTIVE: To investigate in bovine species the benefit of cholesterol and vitamin E, both loaded in cyclodextrins, as a double protection against oxidative stress and cold shock. MATERIALS AND METHODS: Semen was collected from nine mature bulls using an artificial vagina and each ejaculate was split into four equal aliquots. The control aliquot was diluted with Fraction A (Tris+citric acid+fructose+penicillin) without further supplementation; the treated samples were diluted in Fraction A supplemented with cholesterol-loaded cyclodextrins (CD-CHL), vitamin E-loaded cyclodextrins (CD-Vit E) or CD-CHL in association with CD-Vit E (CD-CHL-VitE). After incubation at 22°C for 15 min and addition of Fraction B (Fraction A+egg yolk+glycerol), all aliquots were frozen in 0.25 ml straws. Straws were then thawed individually at 37C for 30 seconds in a water bath and immediately analyzed for motility, using Computer Aided Semen Analysis (CASA), membrane integrity and oxidative stress status. RESULTS: The results showed that samples treated with CD-CHL and CD-Vit E were protected against the deleterious impact of freezing thawing process. However, the optimal protection was observed when the two complexes CD-CHL and CD-Vit E were simultaneously used. All analysed semen parameters including motility, membrane integrity and oxidative stress status were significantly improved in CD-CHL-Vit E compared to all other treatments. CONCLUSION: Cholesterol and vitamin E, both preloaded in cyclodextrins to increase their solubility, appeared as a powerful protection in cryopreserved bovine semen to fight simultaneously cold shock and oxidative stress.