Effects of Cryoprotective Medium Composition, Dilution Ratio, and Freezing Rates on Spotted Halibut (Verasper variegatus) Sperm Cryopreservation

The spotted halibut is species that has a high potential market value in Korea, but the supply of seed is unstable because of the limited milt production of males. The objective of this research was to explore different aspects, such as CPAs, diluents, dilution ratio, and freezing rates, to develop an optimal sperm cryopreservation. The parameters assessed were movable sperm ratio, sperm activity index, survival rate, and DNA damage. The CPAs tested in this research were propylene glycol, dimethyl sulfoxide (DMSO), methanol, ethylene glycol, and glycerol. Different diluents, including 300 mM sucrose, 300 mM glucose, Stain’s solution, and Ringer’s solution, were investigated. The previous experiment showed that the optimal CPA for cryopreservation was DMSO with a concentration of 15% with 300 mM as diluent. To determine the effect of the dilution ratio, sperm was diluted to 1:1, 1:2, 1:10, 1:100, and 1:1000 with 300 mM sucrose containing DMSO at a final concentration of 15%. Lastly, the optimal freezing rate of the sperm was evaluated with four different freezing rates (−1, −5, −10, and −20 °C/min). Post-thaw sperm motility was higher with a dilution ratio lower than 1:2, and the freezing rate was less than −5 °C/min. In conclusion, these findings represent the development of a cryopreservation protocol for spotted halibut.

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Improvement of cryopreservation protocol in both purebred horses including Spanish horses

Spanish Journal of Agricultural Research ◽

10.5424/sjar/2018164-13677 ◽

2019 ◽

Vol 16 (4) ◽

pp. e0406

Author(s):

Jordi Miró ◽

Marion Papas

Keyword(s):

Sperm Quality ◽

Sperm Concentration ◽

Observation Point ◽

Sperm Cells ◽

Sperm Viability ◽

Dilution Ratio ◽

Sperm Cryopreservation ◽

Morphological Abnormalities ◽

Cryopreservation Protocol

There is a widely held belief that the semen of Purebred Spanish Horses (PRE) is of generally poorer quality than that of other breeds, and survives cryopreservation less well. To determine whether this is the case, sperm concentration, viability and morphological abnormalities were examined in a total 610 fresh ejaculates from 64 healthy PRE (N=47) and non-PRE stallions (N=17). Sperm concentration and viability were then re-examined after pre-freezing centrifugation, and once again after freezing-thawing. No differences were observed between the PRE and non-PRE stallions in terms of any sperm quality variable at any observation point. When considering all PRE and non-PRE samples together, differences in sperm viability were observed between fresh and fresh-centrifuged sperm viability (70.1±12.5% compared to 76.3±10.9%; p<0.01). After centrifugation the samples were also more homogeneous in terms of the total number of recovered sperm cells. Centrifugation also improved frozen-thawed sperm viability, reducing differences in sperm quality between individual stallions. For all centrifugations, a sperm:extender ratio of 1:5 was used. This would appear to provide better final results than those reported in the literature for the 1:1 ratio commonly used for PRE stallion sperm cryopreservation. In conclusion, obtained results show that the quality and frozen/thawed results of PRE stallion sperm are not lower than that of non-PRE breeds. In addition, using a 1:5 sperm:extender dilution ratio when selecting sperms by centrifugation prior to freezing, seems to provide better results than those usually reported when using a 1:1 ratio.

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The development of a sperm cryopreservation protocol for winter flounder Pseudopleuronectes americanus (Walbaum): evaluation of cryoprotectants and diluents

Aquaculture Research ◽

10.1046/j.1365-2109.2003.00879.x ◽

2003 ◽

Vol 34 (8) ◽

pp. 653-659 ◽

Cited By ~ 32

Author(s):

R M Rideout ◽

M K Litvak ◽

E A Trippel

Keyword(s):

Winter Flounder ◽

Sperm Cryopreservation ◽

Pseudopleuronectes Americanus ◽

Cryopreservation Protocol

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27 Comparison of cryoprotectants and their combinations in the optimisation of a sperm cryopreservation protocol in the Argentine black and white tegu (Salvator merianae)

Reproduction Fertility and Development ◽

10.1071/rdv34n2ab27 ◽

2022 ◽

Vol 34 (2) ◽

pp. 247

Author(s):

C. Young ◽

N. Ravida ◽

M. Curtis ◽

F. Mazzotti ◽

B. Durrant

Keyword(s):

Sperm Cryopreservation ◽

Black And White ◽

Cryopreservation Protocol

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Sperm cryopreservation of lane snapper Lutjanus synagris(Linnaeus, 1758)

Brazilian Journal of Biology ◽

10.1590/1519-6984.20613 ◽

2015 ◽

Vol 75 (3) ◽

pp. 662-669 ◽

Cited By ~ 2

Author(s):

EG Sanches ◽

IR Oliveira ◽

PCS Serralheiro ◽

VR Cerqueira

Keyword(s):

Sperm Motility ◽

Sperm Quality ◽

Equilibration Time ◽

Dilution Ratio ◽

Sperm Cryopreservation ◽

Ph Values ◽

Cryopreserved Sperm ◽

Time Density ◽

Motility Rate

AbstractThis study aims developing and evaluate a protocol of semen cryopreservation of the lane snapper Lutjanus synagris. Firstly, sperm motility rate, motility time, density and spermatocrit were appraised to characterize the sperm quality of the lane snapper. The effect of three extenders with distinct ionic compositions and pH values combined with seven concentrations of cryoprotector dimethylsulfoxide (0; 2.5; 5.0; 7.5; 10.0; 12.5 e 15.0%), five cooling rates (110, 90, 60, 45 e 30°C –min), nine equilibration time (1; 2,5; 5; 10; 15; 20; 25; 30 e 60 minutes) e five dilutions ratio (1:1; 1:3; 1:6; 1:10 e 1:20) on the sperm motility rate and motility time were analyzed. Fertilization test was accomplished to evaluate the viability of the cryopreserved sperm. The higher sperm motility rate and motility time (P<0.05) was achieved by combining extender with pH 8.2 with 10% concentration of dimethylsulfoxide and cooling rate 60°C –min, 1 minute of equilibration time and 1:3 (v/v) dilution ratio. The use of cryopreserved sperm presented fertilization rates >60% validating the present protocol for lane snapper. The cryoconserved sperm of lane snapper is a viable alternative, being possible to maintain appropriate sperm viability.

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Acrosomal integrity and capacitation are not influenced by sperm cryopreservation in the giant panda

Reproduction ◽

10.1530/rep.1.00034 ◽

2004 ◽

Vol 127 (5) ◽

pp. 547-556 ◽

Cited By ~ 26

Author(s):

R E Spindler ◽

Y Huang ◽

J G Howard ◽

P Wang ◽

H Zhang ◽

...

Keyword(s):

Sperm Motility ◽

Giant Panda ◽

Calcium Ionophore ◽

Sperm Cryopreservation ◽

Management Tools ◽

Giant Pandas ◽

Before And After ◽

Cryopreservation Protocol ◽

Acrosomal Integrity

Sperm cryopreservation and artificial insemination are important management tools for giant panda breeding and the preservation of extant genetic diversity. This study examined the influence of freeze–thawing on sperm function, specifically capacitation. Sperm from nine giant pandas were assessed before and after rapid (− 40 and − 100 °C/min) cryopreservation by incubation in HEPES-buffered Ham’s F10 medium with and without the capacitation accelerators, 3-isobutyl-1-methylxanthine (IBMX) and dibutyryl cyclic AMP (dbcAMP). At 0, 3 and 6 h of exposure, aliquots were assessed for sperm motility traits and capacitation, defined as the proportion of sperm with intact acrosomes following exposure to solubilised zonae pellucidae (ursid or felid) or calcium ionophore subtracted from the proportion of sperm with intact acrosomes before exposure. Although mean±s.e.m. sperm motility post-thaw (56.1 ± 3.9% at 0 h) was less (P < 0.05) than pre-freeze (71.7 ± 6.0%), there was no difference (P > 0.05) in the proportion of acrosome-intact sperm (fresh, 93.0 ± 1.7% versus cryopreserved–thawed, 81.7 ± 4.7% at 0 h). Incidence of capacitation was greater (P < 0.05) in fresh sperm incubated with capacitation accelerators IBMX and dbcAMP (9 h: 50.9 ± 1.1) compared with fresh sperm incubated without accelerators (9 h: 41.2 ± 1.1%). Frozen–thawed sperm preincubated without accelerators underwent capacitation (49.6 ± 1.1%) to a greater extent (P < 0.05) compared with these fresh counterparts. Thawed samples with (9 h: 45.9 ± 1.4%) and without accelerators (9 h: 41.2 ± 1.1%) did not differ (P > 0.05) during the 9-h incubation. We conclude that giant panda spermatozoa (1) undergo capacitation in vitro with or without chemical accelerators and (2) withstand a rapid cryopreservation protocol, including retaining normal acrosomal integrity and functional capacitation ability.

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Development of a sperm cryopreservation protocol for the Argentine black and white tegu (Tupinambis merianae)

Theriogenology ◽

10.1016/j.theriogenology.2016.08.006 ◽

2017 ◽

Vol 87 ◽

pp. 55-63 ◽

Cited By ~ 6

Author(s):

Carly Young ◽

Nicole Ravida ◽

Michelle Curtis ◽

Frank Mazzotti ◽

Barbara Durrant

Keyword(s):

Sperm Cryopreservation ◽

Black And White ◽

Tupinambis Merianae ◽

Cryopreservation Protocol

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Dimethylacetamide can be used as an alternative to glycerol for the successful cryopreservation of koala (Phascolarctos cinereus) spermatozoa

Reproduction Fertility and Development ◽

10.1071/rd08036 ◽

2008 ◽

Vol 20 (6) ◽

pp. 724 ◽

Cited By ~ 21

Author(s):

Yeng Peng Zee ◽

William V. Holt ◽

Jaime Gosalvez ◽

Camryn D. Allen ◽

Vere Nicolson ◽

...

Keyword(s):

Disulfide Bonds ◽

Membrane Integrity ◽

Sperm Nucleus ◽

Sperm Chromatin ◽

Similar Degree ◽

Sperm Cryopreservation ◽

Phascolarctos Cinereus ◽

Degree Of Protection ◽

Fish Sperm ◽

Cryopreservation Protocol

Swelling of koala sperm chromatin following cryopreservation has largely been attributed to the absence of intermolecular disulfide cross-linkages in the marsupial sperm nucleus. Fish spermatozoa also lack disulfide bonds within their chromatin, but have been successfully cryopreserved. The present study examined the hypothesis that the cryoprotectants used for fish sperm cryopreservation would confer a similar degree of protection on koala spermatozoa. Three concentrations each of five cryoprotectants (dimethyl sulfoxide, methanol, propylene glycol, ethylene glycol and dimethylacetamide (DMA)) were evaluated. Each treatment was compared against an established koala sperm cryopreservation protocol that uses 14% glycerol. Post-thaw assessment of progressive motility, plasma membrane integrity and mitochondrial membrane potential (MMP) revealed that protocols using 15% DMA achieved 62.2 ± 3.6% (P < 0.05) sperm survival, of which 79% (P < 0.05) had high MMP, an improvement of 32% and 40%, respectively, over sperm frozen in 14% glycerol. The percentage of spermatozoa with swollen nuclei was also lowest when frozen in 15% DMA, both immediately after thawing (18.0 ± 3.5%; P < 0.05) and after 2 h incubation at 35°C (35.8 ± 4.4%; P < 0.05). A second study was conducted to determine the optimal concentration of DMA for use in the cryopreservation of koala spermatozoa. High DMA concentrations (17.5% and 20%) resulted in significantly lower proportions of live spermatozoa showing high MMP immediately after thawing compared with spermatozoa frozen in the lower concentrations. The percentage of koala spermatozoa with swollen chromatin following cryopreservation was not affected by DMA concentration.

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