Development of non-egg yolk based (Nano-encapsulated drug delivery based liposome systems) semen extender to increase the shelf life and post-thaw motility in stallion semen.

Background:

Cryopreservation of semen is widely used for conservation of animal genetic resources and exploitation of genetically superior sires through artificial insemination. Mammalian sperm undergo thermal shocks during initial cooling from room to refrigerator temperature (supra-zero phase) and then during deep freezing (sub-zero phase). Since the discovery of egg yolk as an ingredient of bull semen extender, it has been extensively used in mammalian semen cryopreservation to protect sperm against initial cold shock. Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. Egg yolk has biosecurity risks. The artificial insemination industry has been facing biosecurity risks through the use of animal products (egg yolk and milk) in semen cryopreservation procedure. Egg yolk is a component of animal origin and represents a potential risk of contamination of artificial insemination doses with bacteria. Indeed, such contamination is a possible source of endotoxins capable of damaging the fertility potential of spermatozoa. Egg yolk is not practical, because it is not ready to use. Indeed, before adding egg yolk to the extender the user has to break eggs manually, to carefully isolate the yolk to eliminate traces of albumen-a “cooking exercise” which is time-consuming. Egg yolk is also not practical because it can interfere with microscopic observations or biochemical assays as it contains granular material of the same size and shape as spermatozoa. Egg yolk is an extremely complex product, whose composition may be extremely variable and may differ between batches. Its lipid composition may change with the hens’ diet. Moreover egg yolk may contain components with beneficial, as well as detrimental, effects on spermatozoa. The presence has been reported of substances in egg yolk that inhibit respiration of spermatozoa or reduce their motility. Considering these disadvantages, there is an urgent need to replace whole egg yolk by its cryoprotective fraction.

Technology Details:

Keeping the above facts, we at Equine production Campus, ICAR-NRCE have developed novel liposome based nano-encapsulated drug delivery systems containing various antioxidants and agents that can protect the spermatozoa from the cold shock and oxidative stress. For the development of novel non- egg yolk semen extender we used nano-liposomes encapsulated with Vit E, Selenium, Cholesterol and Albumin in varying concentrations. Vitamin E used in liposome preparation of this invention provides the protection from lipid per-oxidation or oxidative stress during stallion semen preservation at 4ºC temperature. Phospholipids, cholesterol and vitamin E are the lipids and not soluble in water. The liposome provides the better water solubility and bioavailability of phospholipids, cholesterol and vitamin E in water. Bovine serum albumin (BSA) fraction V provides physical stability to liposome during long term storage and it has cryoprotective effect on sperm during semen preservation. In present invention, the mass ratio of the lipid ingredient for liposome preparation including lecithin, cholesterol and vitamin E is 9:2:1. The technical scheme for manufacturing the liposome of the present invention consisted by five steps. The steps are as follows: Step 1- Mixing of preselected lipids, Step 2 - Formation of thin lipid film, Step 3 - Hydration of thin lipid film, Step 4 – Sonication, Step 5 – Filtration. The 5-10 % (v/v) liposome concentration in semen extender base solution is beneficial and protects from cold shock to stallion sperm and it also maintains the total motility (TM), progressive motility (PM), straight-line velocity (VSL), average path velocity(VAP) and curvilinear velocity (VCL) of stallion spermatozoa during the liquid preservation of stallion semen at 4ºC temperature for 10 days. The best combination of these were tested initially and the best combination was selected further for the use as extender. All the preparations of nano-liposomes were prepared at NRCE only in customised methods.