Table 2.
Comparison of various protocols for freezing of ram semen.
| Freezing step | Diluents | Equilibration time | Freezing rate | Concentration/ dilution | Reference |
|---|---|---|---|---|---|
| Three | Tris/tes/glucose (TTG) solution | 5°C for 3 h | 40°C/min (+5°C to −35°C) | 100 × 106 sperm/mL | [70] |
| 17°C/min (−35°C to −65°C) | |||||
| 3°C/min (−65°C to −85°C) and finally −196°C | |||||
| Three | Tris/tes/glucose (TTG) solution | 5°C for 3 h | 4°C/min (+5°C to −5°C) | 100 × 106 sperm/mL | [70] |
| 25°C/min (−5°C to −110°C) | |||||
| 35°C/min (−110°C to −140°C) and finally −196°C | |||||
| Two | Tris/tes/glucose (TTG) solution | 5°C for 3 h | 5°C/min (+5°C to −10°C) | 100 × 106 sperm/mL | [70] |
| 60°C/min (−10°C to −130°C) and finally −196°C | |||||
| One | Tris-citrate-fructose-egg yolk and Triladyl® | 5°C for 4 h | −15.26°C/min (+5°C to −140°C) and finally −196°C | 800 × 106 sperm/ml | [71] |
| Two | Tris-citrate-fructose-egg yolk and Triladyl® | 5°C for 4 h | −30°C/min (+5°C to −80°C) | 800 × 106 sperm/ml | [71] |
| −11.33°C/min (−80°C to −140°C) and finally −196°C | |||||
| Three | Tris-citrate-fructose-egg yolk and Triladyl® | 5°C for 4 h | −11.33°C/min (+5°C to −80°C) | 800 × 106 sperm/ml | [71] |
| −26.66°C/min (−80°C to −120°C) | |||||
| −13.33°C/min (−120°C to −140°C) & finally −196°C | |||||
| One | Tris-citrate-fructose media | 4°C for 2 h | Vapor freezing 5 cm above the LN2 for 12 min, then immersed directly into liquid nitrogen at − 196°C | 4 × 108 sperm/ml | [72] |
| Two | Tris-citrate-fructose media | 4°C for 2 h | 3°C/min (+5°C to −8°C) | 4 × 108 sperm/ml | [73] |
| 15°C/min (−8°C to −120°C) & finally −196°C | |||||
| Two | Tris-citrate-fructose-egg yolk | 5°C for 120 min | 3°C/min (+5 to −8°C) | 1 : 2 (semen/extender) | [69] |
| 15°C/min (−8 to −120°C) & finally −196°C | |||||
| One | Tris-fructose-egg yolk media | 5°C for 2 h | −20 ◦C/min (5°C to −100°C) & finally -196 °C | 100 × 106 sperm/ml | [74] |
| One | Tris-citrate-fructose media | 4°C for 2 h | Vapor freezing 5 cm above the LN2 for 12 min, then immersed directly into liquid nitrogen at −196°C | 350 × 106 sperm/ml | [75] |
| Two | Tris-citrate-fructose-egg yolk | 5°C for 80 min | −0.3°C/min (5°C to −10°C) | 4 × 108 sperm/ml | [76] |
| −25°C/min (−10°C to −150°C) & finally −196°C | |||||
| One | Tris-citrate-fructose-egg yolk | 5°C for 3 h | Vapor freezing 4 cm above the LN2 for 15 min, then immersed directly into liquid nitrogen at −196°C | 4 × 108 sperm/ml | [76] |
| One | Tris-citrate-fructose-egg yolk | Vapor freezing 4 cm above the LN2 for 10 min, then immersed directly into liquid nitrogen at −196°C | 80 × 106 sperm/ml | [77] | |
| One | Steridyl® (Minitube, Germany) | 5°C for 2 h | Vapor freezing 7 cm above the LN2 for 15 min, then immersed directly into liquid nitrogen at −196°C | 100 × 106 sperm/ml | [78] |
| Two | Hepes–glucose buffer | −5°C/min (+5°C to −5°C) | 20 × 106 sperm/ml | [79] | |
| −50°C/min (−5°C to −50°C) & finally −196°C | |||||
| Two | TRIS-based extender | 5°C for 4 h | 3°C/min (+5°C to −8°C) | 1 : 1 (semen/extender) | [7] |
| 25°C/min (−8°C to −120°C) & finally −196°C | |||||
| Two | Skim milk and egg yolk | 5°C for 90 min | −5°C/min (5°C to −25°C) | 800 × 106 sperm/ml | [80] |
| −50°C/min (−25°C to −130°C) & finally −196°C | |||||
| One | Tris, fructose, egg yolk or Triladyl® | 4°C for 4 h | Vapor freezing 4 cm above the LN2 for 6 min, then immersed directly into liquid nitrogen at −196°C | 400 × 106 sperm/mL | [9] |
| One | Tris or milk-based diluent | Vapor freezing at −75°C for 7 min, then immersed directly into liquid nitrogen at −196°C | 50, 100, 200, 400, 500, or 800 × 106 sperm/mL | [97] | |
| One | TEST buffer | 5°C for 4 h | Vapor freezing 5–7 cm above the LN2 for 10 min, then immersed directly into liquid nitrogen at −196°C | 1 : 4 (semen/extender) | [82] |
| One | Tris-citrate modified solution | 5°C for 2 h | Vapor freezing at −100°C, then immersed directly into liquid nitrogen at −196°C | 1 × 109 sperm/mL | [83] |
| One | Tris-citrate-fructose-egg yolk | 4°C for 2 to 3 h | Liquid nitrogen vapor for 12 min, then immersed directly into liquid nitrogen at −196°C | 1 × 109 sperm/mL | [52] |
| One | Tris-citrate-glucose-egg yolk-glycerol media | 5°C for 3 h | Vapor freezing 3 cm above the LN2 for 15 min, then immersed directly into liquid nitrogen at −196°C | 100 × 106 sperm/mL | [67] |
| Two | Bioxel | 5°C for 3 h | 5°C/min (5°C to −20°C) | 1 : 5 (semen/extender) | [84] |
| 25°C/min (−20 to −120°C) & finally −196°C | |||||
| One | TRIS-egg yolk | 5°C for 4 h | Vapor freezing at −110°C for 10 min, then immersed directly into liquid nitrogen at −196°C | 1 : 1 (semen/extender) | [54] |
| One | Tris-egg yolk-glycerol | 5°C for 3 h | −15°C/min (+5°C to −120°C) finally −196°C | 240 × 106 sperm/mL | [140] |
| One | Tris-citrate-fructose-egg yolk | 4°C for 140 min | Freezing on dry ice, then immersed directly into liquid nitrogen at −196°C | 400 × 106 sperm/ml | [86] |
| Three | Tris-citrate-fructose-egg yolk | 4–5°C for 4 h | −5°C/min (+4°C to −10°C) | 150 × 106 per straw | [87] |
| −40°C/min (−10°C to −100°C) | |||||
| −20°C/min (−100°C to −140°C) & finally −196°C | |||||
| One | Tris-citrate-fructose-egg yolk- glycerol media | Vapor freezing at −100°C, then immersed directly into liquid nitrogen at − 196 °C | 1 × 109 cells/ml | [88] | |
| One | Tris-citrate-fructose-egg yolk-glycerol media | 5°C for 4 h | Vapor freezing 5 cm above the LN2 for 10 min, then immersed directly into liquid nitrogen at −196°C | 200 × 106 sperm/ml | [89] |
| One | Tris-citrate-fructose-egg yolk | 5°C for 2 h | Vapor freezing, then immersed directly into liquid nitrogen at −196°C | 4 x 108 sperm/ml | [90] |
| One | Tris-citrate-glucose-egg yolk-glycerol media | Frozen in pellets on dry ice (−79°C), then immersed directly into liquid nitrogen at −196°C | 400 × 106 sperm/ml | [91] | |
| One | Vapor freezing 5 cm above the LN2, then immersed directly into liquid nitrogen at −196°C | 100–200 × 106 spz/ml | [92] | ||
| One | Tris-citrate-fructose-egg-yolk-glycerol media and AndroMed | 4°C for 2–3 h | Vapor freezing (–125°C to –130°C) for 3–4 min, then immersed directly into liquid nitrogen at −196°C | 250 × 106 per ml | [58] |
| One | Tris-citrate-fructose-egg yolk-glycerol media | 5°C for 3 h | Vapor freezing 4 cm above the LN2 for 15 min, then immersed directly into liquid nitrogen at −196°C | 4 × 108 sperm/ml | [93] |
| One | Tris-citrate-fructose-egg yolk-glycerol media | 5°C for 2 h | Vapor freezing 4.5 cm above the LN2 for 15 min, then immersed directly into liquid nitrogen at −196°C | 4 × 108 sperm/ml | [94] |
| One | Tris-citrate-fructose-egg yolk-media | Vapor freezing (−100°C to −120°C), then immersed directly into liquid nitrogen at −196°C | 4 × 108 sperm/ml | [95] | |
| One | Tris-citrate extender | Vapor freezing 5 cm above the LN2 for 10 min, then immersed directly into liquid nitrogen at −196°C | 100 × 106 sperm/ml | [12] | |
| One | Tris-egg yolk extender | 5°C for 90 min | 12.5°C/min (5 to −120°C) | 100 × 106 sperm/mL | [85] |
| Two | 5°C for 4 h | 20°C/min to −100°C | 200 × 106 sperm/ml | [99] | |
| 10°C/min (−100°C to −140°C) | |||||
| One | Tris-fructose-citrate-egg yolk | 4°C for 4 h | Vapor freezing 5–6 cm above the surface of the liquid nitrogen for 5–6 (temperature −80°C) minutes, then immersed directly into liquid nitrogen at −196°C | [100] | |
| Four | BullXcell® AndroMed® | 2 h at 4°C | Vapor freezing (1st phase, distance 15 cm, 4 min; 2 nd phase, distance 9.5 cm, 5 min; 3rd phase, distance 5 cm, 6 min; 4th phase, distance 1.5 cm, 8 min) | [101] | |
| One | Tris-citrate-glucose-egg yolk- glycerol media | 5°C for 2 h | Vapor freezing 4.5 cm above the LN2 for 13 min, then immersed directly into liquid nitrogen at −196°C | 600 × 106 sperm/ml | [102] |
| Two | Milk-based extender | 60–90 min at 5°C | 5°C/min (5°C to −10°C) | 400 × 106 sperm/ml | [103] |
| 60°C/ min (−10°C to −130°C) & finally −196°C | |||||
| One | Tris-citrate-egg yolk-glycerol media | 5°C for 120 min | 20°C/min (5 to −120°C) | 100 × 106 sperm/mL | [104] |
| Two | Milk-based extender | 5°C for 90 to 120 minutes | 5°C/min (5°C to −10°C) | 1000 × 106 sperm/mL | [105] |
| 60°C/min (−10°C to −130°C) & finally −196°C | |||||
| One | Tris-citrate-glucose-egg yolk media | 5°C for 90 min | Vapor freezing −100°C for 10 min, then immersed directly into liquid nitrogen at −196°C | 1 x 109 sperm/mL | [106] |
| One | Tris-based extender | 20°C/min (4°C to −100°C) finally −196°C | 1 : 3 or 1 : 6 (semen/extender) | [3] | |
| One | Tris-based extender | Vapor freezing 5 cm above the LN2 level (−150°C) for 20 min., then immersed directly into liquid nitrogen at −196°C | 1 : 3 or 1 : 6 (semen/extender) | [3] | |
| One | Tris-egg yolk-glycerol | 5°C for 3 h | Vapor freezing from 5 to −120°C in 15 min, then immersed directly into liquid nitrogen at −196°C | 400 × 106 sperm/mL | [66] |
| Three | Tris-citrate-fructose egg yolk media | 5°C for 4 h | 11.33°C/min (+5°C to −80°C) | 400 or 800 × 106 sperm/mL | [107] |
| 26.66°C/min (−80°C to −120°C) | |||||
| 13.33°C/min (−120°C to −140°C) & finally −196°C | |||||
| One | Tris-citrate-fructose egg yolk media | 4°C for 2-3 h | Vapor freezing (−125 to −130°C) for 3-4 min, then immersed directly into liquid nitrogen at −196°C | [108] | |
| One | Tris-based extender | 5°C for 2.5 h | Vapor freezing 5 cm above the LN2 for 15 min, then immersed directly into liquid nitrogen at −196°C | 800 × 106 sperm/ml | [109] |
| One | Tris-citrate—fructose-egg yolk-glycerol media | 4°C for 4-5 h | Vapor freezing for 10 min, then immersed directly into liquid nitrogen at −196°C | 400 × 106 sperm/ml | [110] |
| One | Tris-citrate—glucose-egg yolk-glycerol media | 5°C for 2.5 h | Vapor freezing 12 cm and 4 cm above liquid nitrogen, then immersed directly into liquid nitrogen at −196 °C | 4 × 108 sperm/ml | [112] |
| One | Soybean lecithin-based semen extender | 4°C for 2 h | Vapor freezing 5 cm above the LN2 for 12 min, then immersed directly into liquid nitrogen at −196°C | 350 × 106 sperm/ml | [75] |
| One | Tris—fructose-egg yolk-glycerol media | 5°C for 3, 10 and 22 h | −25°C/min (5 to −125°C) finally −196°C | 800 × 106 sperm/ml | [113] |
| One | TEST-yolk-glycerol extender | 5°C for 2 h | −25°C/min (5 to −25°C) finally −196°C | 1000 × 106 sperm/ml | [114] |
| One | TEST-yolk-glycerol extender | 5°C for 2 h | −25°C/min (5 to −75°C) finally −196°C | 1000 × 106 sperm/ml | [135] |
| One | TEST-yolk-glycerol extender | 5°C for 2 h | −25°C/min (5 to −125°C) finally −196°C | 1000 × 106 sperm/ml | [114] |
| One | Article | 5°C for 2 h | 25°C/min (5°C to −125°C) finally −196°C | 1 x 109/ml sperms/ml | [115] |
| One | Tris—citrate-fructose-egg yolk-media | 5°C for 2 h | 30°C/min (5°C to −150°C) finally −196°C | 400 × 106 sperm/ml | [117] |
| One | Tris–citric acid–fructose–yolk extender | 5°C for 3.5 h | Vapor freezing 4–5 cm above the LN2 for 10 min, then immersed directly into liquid nitrogen at −196°C | 1 × 109 sperm/ml | [118] |