Storage of equine semen

2. Storage of equine semen

3. Gamal A. A. El Sisy Presented by Professor of animal reproduction and artificial insemination Veterinary division National Research Centre Gelsisy@yahoo.com Egypt

4. Historical Background

5. Egypt is a member of the WAHO Al Zahraa Arab Horse Stud In Egypt There are About 8000 purebred Arabian horses 403 private studs 458 horses kept in the El-Zahraa stud

6. AbbasHelmy Ahmad Kamal

7. Ali sherif Mohamed Ali

8. Prince Omar tosson

9. Egyptian Arabian horse has always been an icon of beauty and utter elegance

12. Horse artificial insemination • Horse was the first animal for artificial insemination practice • In 1322 an Arab Sheikh applied AI in horse • In 1891, Sir Walter Heape inseminated mares with fresh semen. • In 1950 Horse's semen cryo preservation has been started • In 1957; the first foal born from AI with frozen semen

13. Advantages of AI in stallion • Long term semen storage for unlimited time (Semen international distribution and exchange) • Enhanced rates of genetic improvement • Controls the venereal diseases • Insurance against unanticipated loss of a stallion

14. Challenges of AI and semen storage in stallion • Stallion spermatozoa sensitive to temperature changes and osmotic stress • Low fertility of frozen semen • 30% of all stallions are ‘poor freezers’ • Breeds and individual variability to cooling and freezing • Difficulty to get one customized freezing protocol and extender for all horses

15. Research has revealed that: • Sperm of bulls are resistant to the effects of cryopreservation. • This phenomenon is probably attributed to years of careful selection of bulls with sperm capable of surviving the freezing and thawing processes, • This practice not followed in horse breeding circles.

16. Nature of semen used in AI program

17. Fresh Semen • Inhibiting sperm metabolism • Decreasing the production of lethal metabolites and maintenance of ATP • No needs for cryoprotectants • Additives are antioxidants, nutritional substance, and osmoregulators

18. Cooled Semen • Commonly used technique for the storage and transport of stallion semen • Maintain sperm for up to 72 hours. • Stallion spermatozoa are vulnerable to cold shock • 44% conception rates

20. Frozen Semen • practical for long term storage. • Accepted registration of foals • Many stallions are available only through frozen semen • Low conception rate

23. What happens during cryopreservation ofspermatozoa? • Alterations in sperm membrane • Ice crystal formation • Drastic changes in temperature • Cryo-capacitation • Oxidative damage • Alteration in antioxidant and DNA damage • Mechanical stress on cell membranes due to osmotic stress and toxicity of CPs

24. Aim of state of the lecture Throws light on updates of the currently used cryoprotectants and additives for improving efficiency of semen storage In stallion with emphasis on their possible effects in order to make critical decision before inclusion in diluents.

25. Strategies to Improve Efficiency of Semen Preservatbilty in Stallion This improvements can be done by • Dietary supplementations • Inclusions of various CPs and additives in the basic extenders

26. Dietary supplementations

27. Most common dietary supplements • Polyunsaturated fatty acids (linseed oil, rice oil, omega-3) • L Carnitine • Ascorbic acid, tocopherol, zinc and selenium • Folic acid • Herbs, malt, honey and orange juice

28. Functions of dietary additives • Improvement of sperm membrane quality and lipid composition • Improved antioxidative capacity of semen and counteracting of oxidative stress

29. Minerals and/or vitamins Vitamin C, E, L-Carnitine, zinc, selenium and antioxidants resulted in: • improvements in fresh, cooled and frozen semen quality • Reduction in ROS and elevation of the antioxidant status • Dietary supplement is more effective than exogenous addition to semen diluent

30. Inclusions of various CPs and additives in the basic extenders

44. Cooling and freezing extenders

45. Extenders composition Extenders includes: • A non-penetrating CPs (egg yolk, milk, or soybean lecithin) • A penetrating CPs (glycerol, ethylene glycol, or dimethylsulfoxide, etc) • Ionic or nonionic substances to maintain osmotic pressure and pH (buffer such as Tris, sodium citrate, citric acid, etc) • One or more sugars (glucose, lactose, raffinose, saccharose, or trehalose) as energy source substrate • Antibiotics (penicillin, streptomycin, etc) • Other additives

46. Permeating CPs CPAs were categorized: • Alcohols (glycerol; ethylene glycol, etc.) • Amides (methyl–formamide, or dimethyl–formamide, etc). • Ultimate CPs must possess • Low molecular weight, • Higher water solubility • Least toxicity

47. Glycerol • Gly was discovered by Polgeet al 1949 • Is widely accepted CP • Replaces water inside the sperm and decreases ice crystalization • 2 and 5% in extender • Gly toxicity necessitated searching for alternative CPs

48. Amides • Amides (MF and DMF) are similar or more efficient than glycerol • Have lesser molecular weights, highly permeable • Cause little osmotic stress • DMF as the CP of choice sex-sorted sperm • Combinations of different CPAs provide better protection

49. Non- permeating CPs • Egg yolk and egg yolk Alternatives LDL, yolk plasma and lyophilized,yolk, Liposomes, CLCs and BHT) • Milk and milk substitutes (Casein, lactose, B-lactoglobulin and lactoferrin) • Plant-origin cryoprotectants(Soybean lecithin, Coconut water, Date palm pollen grain)

50. Egg yolk and egg yolk substitutes • Egg yolk used (2–20%) in extenders • LDL is the cryoprotective fraction of egg yolk which: • Adhere to sperm membrane • Forming a protective film on the sperm surface • providing various lipids to spermatozoa • LDL seizes the deleterious proteins present in seminal plasma