Effects of different diluents, cryoprotective agents, and freezing rates on sperm cryopreservation in Epinephelus akaara

In human sperm cryopreservation, test yolk buffer and human serum albumin have been used as permeating macromolecular-weight cryoprotectants. In clinical reproductive medicine, human serum albumin is frequently used because of low risks of zoonoses and allergic reactions. However, the risk of allogeneic infectious diseases exists, and the supply may be unstable because human serum albumin is derived from human blood. Therefore, the development of xeno-free human sperm cryopreservative reagents that could overcome the aforementioned problems is warranted. We succeeded in developing a new xeno-free and defined sperm cryopreservation reagent containing glycerol, carboxylated poly-l-lysine, and raffinose. The cryopreservation reagent was not significantly different in terms of sperm motility, viability, and DNA fragmentation and was comparable in performance to a commercial cryopreservation reagent containing human serum albumin. Moreover, the addition of saccharides was essential for its long-term storage. These results may help elucidate the unknown function of macromolecular-weight permeating cryoprotective agents.

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Structures Formed by Cryoprotective Agents Used in Freezc-Etching

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100066292 ◽

1971 ◽

Vol 29 ◽

pp. 448-449

Author(s):

G. G. Cocks ◽

C. E. Cluthe

Keyword(s):

Aqueous Solution ◽

Polyethylene Oxide ◽

Liquid Nitrogen Temperature ◽

Ice Crystals ◽

Etching Technique ◽

Structural Constituent ◽

Cryoprotective Agents ◽

Quick Freezing ◽

Freeze Etching ◽

Fractured Surface

The freeze etching technique is potentially useful for examining dilute solutions or suspensions of macromolecular materials. Quick freezing of aqueous solutions in Freon or propane at or near liquid nitrogen temperature produces relatively large ice crystals and these crystals may damage the structures to be examined. Cryoprotective agents may reduce damage to the specimem, hut their use often results in the formation of a different set of specimem artifacts.In a study of the structure of polyethylene oxide gels glycerol and sucrose were used as cryoprotective agents. The experiments reported here show some of the structures which can appear when these cryoprotective agents are used.Figure 1 shows a fractured surface of a frozen 25% aqueous solution of sucrose. The branches of dendritic ice crystals surrounded hy ice-sucrose eutectic can be seen. When this fractured surface is etched the ice in the dendrites sublimes giving the type of structure shown in Figure 2. The ice-sucrose eutectic etches much more slowly. It is the smooth continuous structural constituent surrounding the branches of the dendrites.

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Diurnal Rhythm of Feeding Activity and Estimation of Daily Ration of the Larval Red Spotted Grouper Epinephelus akaara.

NIPPON SUISAN GAKKAISHI ◽

10.2331/suisan.62.399 ◽

1996 ◽

Vol 62 (3) ◽

pp. 399-405 ◽

Cited By ~ 4

Author(s):

Syozo Yamamoto

Keyword(s):

Diurnal Rhythm ◽

Feeding Activity ◽

Daily Ration ◽

Epinephelus Akaara

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Opportunities and challenges related to the implementation of sperm cryopreservation into breeding of salmonid fishes

Theriogenology ◽

10.1016/j.theriogenology.2019.03.022 ◽

2019 ◽

Vol 132 ◽

pp. 12-21 ◽

Cited By ~ 11

Author(s):

Sylwia Judycka ◽

Joanna Nynca ◽

Andrzej Ciereszko

Keyword(s):

Salmonid Fishes ◽

Sperm Cryopreservation

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Sperm Cryopreservation in American Flamingo (Phoenicopterus Ruber): Influence of Cryoprotectants and Seminal Plasma Removal

Animals ◽

10.3390/ani11010203 ◽

2021 ◽

Vol 11 (1) ◽

pp. 203

Author(s):

María Gemma Millán de la Blanca ◽

Eva Martínez-Nevado ◽

Cristina Castaño ◽

Juncal García ◽

Berenice Bernal ◽

...

Keyword(s):

Seminal Plasma ◽

Genetic Material ◽

First Year ◽

Sperm Cryopreservation ◽

Straight Line ◽

The Family ◽

Second Year ◽

American Flamingo ◽

Phoenicopterus Ruber

The American flamingo is a useful model for the development of successful semen cryopreservation procedures to be applied to threatened related species from the family Phoenicopteridae, and to permit genetic material banking. Current study sought to develop effective sperm cryopreservation protocols through examining the influences of two permeating cryoprotectants and the seminal plasma removal. During two consecutive years (April), semen samples were collected and frozen from American flamingos. In the first year, the effect of two permeating cryoprotectants, DMA (dimethylacetamide) (6%) or Me2SO (dimethylsulphoxide) (8%), on frozen–thawed sperm variables were compared in 21 males. No differences were seen between DMA and Me2SO for sperm motility, sperm viability, and DNA fragmentation after thawing. In the second year, the role of seminal plasma on sperm cryoresistance was investigated in 31 flamingos. Sperm samples were cryopreserved with and without seminal plasma, using Me2SO (8%) as a cryoprotectant. The results showed that samples with seminal plasma had higher values than samples without seminal plasma for the following sperm variables: Straight line velocity (22.40 µm/s vs. 16.64 µm/s), wobble (75.83% vs. 69.40%), (p < 0.05), linearity (62.73% vs. 52.01%) and straightness (82.38% vs. 73.79%) (p < 0.01); but acrosome integrity was lower (55.56% vs. 66.88%) (p < 0.05). The cryoresistance ratio (CR) was greater in samples frozen with seminal plasma than without seminal plasma for CR-progressive motility (138.72 vs. 54.59), CR-curvilinear velocity (105.98 vs. 89.32), CR-straight line velocity (152.77 vs. 112.58), CR-average path velocity (122.48 vs. 98.12), CR-wobble (111.75 vs. 102.04) (p < 0.05), CR-linearity (139.41 vs. 113.18), and CR-straightness (124.02 vs. 109.97) (p < 0.01). This research demonstrated that there were not differences between Me2SO and DMA to successful freezing sperm of flamingos; seminal plasma removal did not provide a benefit for sperm cryopreservation.

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

Animals ◽

10.3390/ani10112153 ◽

2020 ◽

Vol 10 (11) ◽

pp. 2153

Author(s):

Irfan Zidni ◽

Yun Ho Lee ◽

Jung Yeol Park ◽

Hyo Bin Lee ◽

Jun Wook Hur ◽

...

Keyword(s):

Previous Experiment ◽

Activity Index ◽

Medium Composition ◽

Freezing Rate ◽

Dilution Ratio ◽

Sperm Cryopreservation ◽

Ringer’S Solution ◽

Potential Market ◽

Spotted Halibut ◽

Cryopreservation Protocol

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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