Semen Preservation Dr Hany Lotfi Faculty of veterinary medicine zagazig uinversity

Transcription

1 Semen Preservation Dr Hany Lotfi Faculty of veterinary medicine zagazig uinversity

2 Aim Prolonged the life span of the sperm cell without reducing its fertilizing capacity Idea Induce reversible controllable reduction of the metabolic activity Methods 1- Reducing the PH 2- lowering the temperature

3 I- Preservation of semen sample at room temperature 1- Ilinoy Variable Temperature medium (IVT) Sod. Citrate Sod. Bicarbonate K Cl Glucose Antibiotic 20 gm 2.1 gm 0.4 gm 3 gm 1 L + Co2 Carbonic acid Egg yolk is added at optimum concentration. The semen is diluted and I ml of semen is stored in glass ampules and stored in the dark at room temperatures (18oC to 24oC).

4 2- Preservation of semen sample blow body temperature (Liquid semen) 1- Preparation of the diluents 2- Collection of semen sample 3- Semen evaluation

5 4- Calculation of the dilution rate - The number of motile sperm present in each one ml of the ejaculate - The number of the sperm cells required in each inseminating does Inseminating dose The minimum number of the sperm that can achieve maximum fertilization rate ( ) - The volume of the inseminating dose.

6 Dilution rate= Number of motile sperm in 1ml (Spcc/ml X motility %) Number of sperm in inseminating dose x Volume of inseminating dose 10 9 X 80 X 1 = Total volume of the diluent = Dilution rate X Volume of the ejaculate

7 5- Technique of dilution Added what to what and How??????????

8 6- Cooling

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10 Boar is the most sensitive Bull, ram, and stallion are very sensitive; Dog and cat are somewhat sensitive Rabbit, human, and rooster are less sensitive to cold shock.

11 7- Packaging and label Name of AI center Name of bull Date of collection

12 8- Storage and re-evaluation

13 Transportation:

14 Disadvantages of the liquid semen: 1.Short life span of the sperm cells, about 72 hours. 2. Can be transported only for short distance. 3. Many sperm cells may be exposed to cold shock 4. Some micro-organisms can be established at 5 o C. Advantages of the liquid semen: the same ejaculate can be used to produce higher number of inseminating doses in liquid semen than in frozen semen.

15 III- Preservation of semen on frozen state

16 Package of frozen semen

17 I- Straw technique Cotton plug Mini Medium Polyvinyl alcohol powder Length Diameter 133mm 2mm 133mm 2.8mm Volume 0.25 ml 0.5 ml

18 Time Freezing and vitrification Vitreous Crystallain solid Tf Tg Temperature

19 Extra cellular ice Crystal Concentrated solute Intracellular water Intra cellular ice Crystal shrinkage

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21 Methods 1- Preparation of the diluent 1 packet of Laiciphos 271 (dried skimmed milk+ sugar + antibiotic) Dissolved in 400 ml of glass bidistilled water at 40 o C 50 ml egg yolk disolved in 50 ml glass bidistalwater Laiciphos % egg yolk Portion A Laiciphos % egg yolk 3% glycerol Portion B Laiciphos % egg yolk 11% glycerol Water path 37 o c Cold cabinet 4 o C

22 Portion A Laiciphos % egg yolk 3% glycerol Portion B Laiciphos % egg yolk 11% glycerol Water path 37 o c Cold cabinet 4 o C

23 2- Semen collection 3- Semen evaluation 4- Semen dilution Dilution rate Number of motile sperm in 1ml (Spcc/ml X motility %) Number of sperm in inseminating dose x Volume of inseminating dose Mini straw Medium straw Number of sperm in inseminating dose x x 10 6 Volume of inseminating dose 0.25ml 0.5ml

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25 Technique Dilution rate Portion A Half diluted semen

26 4- Cooling half diluted Semen 5- Achieve final dilution of semen sample

27 6- Equilibration and packaging: Printing or labeling

28 Filling and sealing

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30 Manual filling Semen Negative pressure

31 Clamping

32 Filling

33 Creating air space

34 Sealing and cleaning

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36 8- Freezing

37 Medium straw At -150 o C for 7 min Mini straw At -120 o C for 9 min

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39 Computer Controlled freezing system with integrated PC.

40 9- Storage and re- evaluation

41 10- Distribution and storage

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44 Goblet Rack Canister

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46 Semen inventory

47 Liquid nitrogen levels

48 Advantages of straw technique - Easy labeled. - No chance for bacterial contamination. - Need less storage space - Easy thawed. - Easy insemination - The straw permits more uniform control of the freezing and thawing process which has led to improved sperm cell recovery

49 3- Pellet technique 1- Preparation of the diluent Lactose 11% Egg yolk Glycerol ml 4.3 ml Water path 37 o C 2- Semen collection 3- Semen evaluation

50 4- Dilution Dilution rate Number of motile sperm in 1ml (Spcc/ml X motility %) Number of sperm in inseminating dose x Volume of inseminating dose Number of sperm in inseminating dose x 10 6 Volume of inseminating dose 0.2 ml Method of dilution

51 5- Cooling 7- Equilibration time 8- Freezing

52 9- Evaluation 10- Storage Movie..\pellet.wmv

53 Disadvantages: - It needs special diluent - It needs different media for freezing, storage - Unsanitary technique - Difficult to identify Advantages: - Simple technique - Low coast needed equipments - Require low storage space - Great life sperms recovery after thawing

54 2- Ampoule technique 1- Preparation of the diluent Portion A Egg yolk sod. Citrate Portion B Egg yolk sod. Citrate 14% glycerol Water path 37 o c Cold cabinet 4 o C 2- Semen collection 3- Semen evaluation

55 4- Dilution Dilution rate Number of motile sperm in 1ml (Spcc/ml X motility %) Number of sperm in inseminating dose x Volume of inseminating dose Number of sperm in inseminating dose 30 x 10 6 Volume of inseminating dose 1ml Method of dilution

56 5- Cooling half diluted Semen 6- Achieve final dilution of semen sample 7- Equilibration time and packaging 8- Freezing First stage From 4 to -15 o C 1 o C/m Second stage From -15 to -79 o C As quickly as possible 8- Storage

57 Advantages: - Easy labeled. - It is more familiar than straws. - Sanitary condition. - Can be stored easy in dry ice or liquid nitrogen Disadvantages: - Improper sealing may result in expulsion during freezing. - Need more storage space - Need more time for thawing. - Large number of sperm cells were lost during thawing.