Cryopreservation Techniques for Ram Sperm

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 × 10^6 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 × 10^6 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 × 10^6 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 × 10^6 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 × 10^6 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 × 10^6 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 × 10^8 sperm/ml	[72]
Two	Tris-citrate-fructose media	4°C for 2 h	3°C/min (+5°C to −8°C)	4 × 10^8 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 × 10^6 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 × 10^6 sperm/ml	[75]
Two	Tris-citrate-fructose-egg yolk	5°C for 80 min	−0.3°C/min (5°C to −10°C)	4 × 10^8 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 × 10^8 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 × 10^6 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 × 10^6 sperm/ml	[78]
Two	Hepes–glucose buffer		−5°C/min (+5°C to −5°C)	20 × 10^6 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 × 10^6 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 × 10^6 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 × 10^6 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 × 10^9 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 × 10^9 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 × 10^6 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 × 10^6 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 × 10^6 sperm/ml	[86]
Three	Tris-citrate-fructose-egg yolk	4–5°C for 4 h	−5°C/min (+4°C to −10°C)	150 × 10^6 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 × 10^9 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 × 10^6 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 10^8 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 × 10^6 sperm/ml	[91]
One			Vapor freezing 5 cm above the LN2, then immersed directly into liquid nitrogen at −196°C	100–200 × 10^6 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 × 10^6 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 × 10^8 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 × 10^8 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 × 10^8 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 × 10^6 sperm/ml	[12]
One	Tris-egg yolk extender	5°C for 90 min	12.5°C/min (5 to −120°C)	100 × 10^6 sperm/mL	[85]
Two		5°C for 4 h	20°C/min to −100°C	200 × 10^6 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 × 10^6 sperm/ml	[102]
Two	Milk-based extender	60–90 min at 5°C	5°C/min (5°C to −10°C)	400 × 10^6 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 × 10^6 sperm/mL	[104]
Two	Milk-based extender	5°C for 90 to 120 minutes	5°C/min (5°C to −10°C)	1000 × 10^6 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 10^9 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 × 10^6 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 × 10^6 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 × 10^6 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 × 10^6 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 × 10^8 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 × 10^6 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 × 10^6 sperm/ml	[113]
One	TEST-yolk-glycerol extender	5°C for 2 h	−25°C/min (5 to −25°C) finally −196°C	1000 × 10^6 sperm/ml	[114]
One	TEST-yolk-glycerol extender	5°C for 2 h	−25°C/min (5 to −75°C) finally −196°C	1000 × 10^6 sperm/ml	[135]
One	TEST-yolk-glycerol extender	5°C for 2 h	−25°C/min (5 to −125°C) finally −196°C	1000 × 10^6 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 × 10^6 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 × 10^9 sperm/ml	[118]