REVIEW: Current Status of Extenders and Cryoprotectants on Fish Spermatozoa Cryopreservation

An important component of many studies of cryopreservation of fish spermatozoa is the type of extenders and cryoprotectants. The suitability of extenders and cryoprotectants differs from one fish to another. There are many studies have been done in cryopreservation of fish spermatozoa. However, there are few review have been done. This review reveals some aspects of cryopreservation especially the role of extender and cryoprotectant in fish sperm cryopreservation. Fish produce high viscosity of sperm and in some cases only small volume is produced. Before cryopreserved in liquid nitrogen, sperm have to dilute with extenders and for long-term cryopreservation, cryoprotectants are needed to protect the sperm cell from cold and hot shock treatments and prevent cell dehydration during pre-freezing, freezing and post thawed. The suitability of extenders and cryoprotectants differs from one fish to another. Over the last decade, studies on the cryopreservation of mammalian sperm, animal husbandry sperm and human sperm have progressed significantly but studies on fish sperm is still confined to some aquatic

Step by step optimization of a sperm cryopreservation protocol for spotted wolffish (Anarhichas minor Olafsen, 1772)

Spotted wolffish Anarhichas minor reproduction in captivity is dependent on in vitro fertilization. However, low sperm volume with relatively low cell concentration and the lack of gametes synchronization (simultaneous availability of mature eggs and sperm) represent a challenge for the industry. Thus, the development of protocols for sperm storage are crucial. Four sequential experiments were conducted to optimize a sperm cryopreservation protocol for this species. First, three different cryoprotectants (DMSO; 1, 2-propanediol; and methanol) at different concentrations (5, 10, and 20%) were tested for their toxicity. No significant differences (p > 0.05) were detected between the control samples and cryoprotectants at concentration up to 10% DMSO, 10% propanediol, and 20% methanol in terms of motility parameters. Second, using the highest non-toxic concentrations of cryoprotectants, sperm was cryopreserved in 0.5 mL straws, at different distances from the liquid nitrogen (1.5, 2.5, 4.5, and 7.5 cm) that correspond to different freezing rates. Motility parameters after freezing/thawing decreased for all the cryoprotectants (p  0.05) between the two thawing rates. The best results were obtained using 10% DMSO. Finally, the fertilization capacity of cryopreserved sperm (10% DMSO and thawed at 5 °C for 1 min) was tested against fresh sperm using two spermatozoa:egg ratios and 4 h gametes contact time. The ratio of eggs with normal cell cleavage, abnormal cleavage or undeveloped were counted at the 2-4 cell stage. Cryopreserved sperm showed lower fertilization capacity at a concentration of 5 × 104 spermatozoa:egg compared with fresh sperm (p  0.05). To cryopreserve spotted wolffish sperm it is recommended to use 10% DMSO, loaded in 0.5 mL straws, freeze at a height between 4.5 (-14.05 °C/min) and 7.5 cm (-5.9 °C/min) from liquid nitrogen for 10 min and thaw for 1 min at 5 °C (177.9 °C/min). In vitro fertilization with cryopreserved sperm should be performed with a concentration of at least 5 × 105 spermatozoa per egg.WOLFSTORE project (AF0078) supported by the MABIT program from Norway. JS was supported by a Cost action FA1205 AQUAGAMETE and an ERASMUS grant.info:eu-repo/semantics/publishedVersio

Development and standardization of a protocol for sperm cryopreservation of two important commercial oyster species

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015Aquaculture activities have a huge contribution for the world food production and their development is extremely necessary to answer to the lack of resources, especially to the demand for seafood. Bivalve production, especially Crassostrea angulata (Portuguese oyster) has been practiced from long ago, and although its production suffered several constraints, in recent years it has been increasing the interests in recovering production and in preserving nature populations. In this sense, new research needs to guarantee an efficient and economically viable production, contributing to a relatively new environmental concern: wild population restoration. Nowadays, pure wild populations of Crassostrea angulata are rare to find due to multiple factors that affected this oyster industry. Cryopreservation technology could promote alternative techniques to contribute for the resource management efficiency of the Portuguese oyster and associated economic activity. In this sense, standardization of procedures is important for Crassostrea genus. At the present there are no cryopreservation reports on Crassostrea angulata sperm, and therefore, one of the objectives of this work is to design a cryopreservation protocol for this species, testing the more adequate cryoprotectant solution, its ideal concentration, different freezing rates and types of containers. In parallel, this stablished protocol was applied in Crassostrea gigas and compared to other previously published for this species. Analysis of motility, viability, agglutination and fertilizations were used as guides for the establishment of the protocol in C. angulata. Moreover, ATP content, DNA fragmentation and lipid peroxidation were done in order to standardize the same protocol for both species. Movement analysis were assessed by CASA system, viability through common staining techniques and flow cytometer, agglutination was quantified according to the scale developed by Dong et al., (2007), ATP content determined by bioluminescence, Comet assay was performed to quantify the DNA fragmentation and lipid peroxidation determined spectrophotometrically by measuring the absorbance of the malondialdehyde (MDA). Significant differences were observed (p<0.05) for lipid peroxidation and fertilization trials whereas ATP content and fragmentation of DNA of the cryopreserved samples did not differ significantly from the control. In C. gigas, the same analysis were performed and did not reveal post-thaw quality differences in the samples cryopreserved with 10% DMSO. The established protocol revealed to be effective and with a low degree of cellular damage on C. angulata sperm and, at the same time, viable to apply in other species, such as Crassostrea gigas

Effect of Oocyte Vitrification Before and After in Vitro Maturation Towards Bcl-2, Bax and Bcl-2/Bax Ratio Expression

Objectives: to compare the expression of Bcl-2, Bax and Bcl-2/Bax ratio in cumulus cell and oocyte between vitrified oocyte pre and post in vitro maturation.Materials and Methods: Maturation was operated in medium TC 100 µl for 24 hours. Vitrification begins with washing oocyte in PBS basic medium supplemented of 20% serum for 1-2 minutes, followed by equilibration medium PBS + 20% serum + 10% ethylene glycol for 10-14 minutes, then transferred to 20% serum + PBS + 0.5 M sucrose + 15% ethylene glycol + PROH 15% for 25-30 seconds. Thawing is processed by submerging the oocytes in the media: 1). PBS + 20% serum + 0.5 M sucrose, 2). PBS + 20% serum + 0.25 M sucrose, and 3). PBS + 20% serum + 0.1 M sucrose. Imunocytochemistry observed the expression of Bcl-2, bax and Bcl-2/bax ratio.Results: Bcl-2 expression on oocyte in control group differed significantly with treatment group, Bcl-2 expression on cumulus in control group differed significantly with treatment 1 group. Bax expression on oocyte in control group differed significantly with treatment group. Bax expression on cumulus in control group differed significantly with treatment group. Bcl-2/Bax expression ratio on oocyte and cumulus did not differ significantly in all groupConclusion: No difference Bcl-2/Bax expression ratio on oocyte and cumulus between vitrified oocyte pre and post in vitro maturation

Human oocytes and zygotes are ready for ultra-fast vitrification after 2 minutes of exposure to standard CPA solutions

Vitrification of human oocytes and embryos in different stages of development is a key element of daily clinical practice of in vitro fertilization treatments. Despite the cooling and warming of the cells is ultra-fast, the procedure as a whole is time consuming. Most of the duration is employed in a long (8–15 minutes), gradual or direct exposure to a non-vitrifying cryoprotectant solution, which is followed by a short exposure to a more concentrated vitrifying solution. A reduction in the duration of the protocols is desirable to improve the workflow in the IVF setting and reduce the time of exposure to suboptimal temperature and osmolarity, as well as potentially toxic cryoprotectants. In this work it is shown that this reduction is feasible. In silico (MatLab program using two-parameter permeability model) and in vitro observations of the oocytes’ osmotic behaviour indicate that the dehydration upon exposure to standard cryoprotectant solutions occurs very fast: the point of minimum volume of the shrink-swell curve is reached within 60 seconds. At that point, intracellular water ejection is complete, which coupled with the permeation of low molecular weight cryoprotectants results in similar intracellular and extracellular solute concentrations. This shows that prolonging the exposure to the cryoprotectant solutions does not improve the cytosolic glass forming tendency and could be avoided. To test this finding, human oocytes and zygotes that were donated for research were subjected to a shortened, dehydration-based protocol, consisting of two consecutive exposures of one-minute to two standard cryoprotectant solutions, containing ethylene glycol, dimethyl sulfoxide and sucrose. At the end of this two-minute dehydration protocol, the critical intracellular solute concentration necessary for successful vitrification was attained, confirmed by the post-warming survival and ability to resume cytokinesis of the cells. Further studies of the developmental competency of oocytes and embryos would be necessary to determine the suitability of this specific dehydration protocol for clinical practice, but based on our results, short times of exposure to increasingly hypertonic solutions could be a more time-efficient strategy to prepare human oocytes and embryos for vitrification

Viability of subitaneous eggs of the copepod, Acartia tonsa (Dana), following exposure to various cryoprotectants and hypersaline water

Subitaneous eggs were obtained from monocultures of the calanoid copepod Acartia tonsa (Dana), Gulf of Mexico strain. Eggs were exposed to methanol, ethylene glycol, propylene glycol, glycerine, and DMSO at 0.0, 0.1, 0.5, 1.0, 2.0, and 5.0 M and hypersaline water at 50, 75, 100, 150, and 200 g/L. Treatments were evaluated after 10 and 20 min of exposure and at 4 and 26 °C. Viability (percent hatched) was determined after 24 h of incubation in 35 g/L saltwater at 26 °C. Methanol, ethylene glycol, and glycerine had high viability up to 2M, and all experienced large decreases at 5M when the exposure temperature was 26 °C compared to 4 °C. Eggs exposed to propylene glycol had lower mean viability with greater variability at the lower concentrations although viability was greater than 81.4% at 2 M. Significant decreases in viability were observed at 5 M, and the decreases were much greater at an exposure temperature of 26 °C versus 4 °C. DMSO exposed at 26 °C produced high viability up to 1 M before significant decreases occurred, while an exposure temperature of 4 °C produced high viability up to 2 M. Viability of eggs exposed to hypersaline water of 50, 75, and 100 g/L were not significantly different from controls for all treatment combinations except the 26 °C temperature exposed for 20 min, which was significantly lower at 100 g/L. Concentrations of 150 and 200 g/L produced very few to no viable eggs. These results indicate further research is justified to investigate if viability of A. tonsa eggs can be protected by these cryoprotectants and hypersaline water after exposure to cryopreservation conditions

Effects of the slow cooling during cryopreservation on the survival and morphology of Taiwan shoveljaw carp (Varicorhinus barbatulus) spermatozoa

Over the past decades, pollution, overfishing, and habitat degradation have driven the population size of Taiwan shoveljaw carp down markedly in Taiwan. Cryopreservation is a useful tool which could be used to maintain genetic resources to protect and preserve this endemic species. Four cryoprotectants [dimethyl sulphoxide (DMSO), dimethylacetamide (DMA), glycerol and methanol] and six freezing rates (0.5, 1, 2, 4, 8, 16 °C min-1) were tested in order to develop an optimal controlled slow-freezing protocol for Taiwan shoveljaw carp spermatozoa. Samples were subsequently examined under the scanning electron microscope to reveal whether cryopreservation had affected their ultrastructural morphology. The highest survival rate (50.1 ± 2.0%) was observed with a freezing rate of 8 °C min-1 in 1M DMSO, using SYBR-14 + PI staining. Fertility and hatching rate results using frozen-thawed spermatozoa (90.2 ± 2.2% and 22.3 ± 2.5%, respectively) were not significantly different from results with fresh spermatozoa. After cryopreservation, 21.0 ± 1.6% of frozen-thawed spermatozoa had mid-piece swelling and rupture of the head. Cryopreservation might, therefore, slightly affect Taiwan shoveljaw carp spermatozoa in terms of morphological change. However, these alterations could be compensated by using large enough numbers of normally functioning frozen-thawed spermatozoa to achieve a standard equal to fresh spermatozoa. This is the first report of successful cryopreservation of Taiwan shoveljaw carp spermatozoa using a controlled slow-cooling method

Cryopreservation of common carp sperm

Experiments were carried out to investigate the effect of five extenders (sucrose, glucose, fructose, KCl and a saline carp sperm extender) and two cryoprotectants (dimethyl-sulfoxide (DMSO) and methanol) on the cryopreservation of common carp sperm. Freezing of sperm using glucose extender and methanol as cryoprotectant resulted in the highest post-thaw motility, fertilization as well as hatching rates (63 ± 9%, 74 ± 15% and 67 ± 17% vs. 87 ± 5%, 84 ± 14% and 69 ± 14% using fresh sperm, respectively). In general, sugar-based extenders combined with methanol as cryoprotectant yielded higher motility, fertilization and hatching rates than ionic extenders in combination with DMSO. The jelly-like agglutination observed after thawing in samples frozen with sugar-based extenders did not reduce fertilization and hatching rates. Frozen–thawed sperm samples were able to successfully fertilize 10 g (8000) eggs

Combinatorial biomaterials discovery strategy to identify new macromolecular cryoprotectants

Cryoprotective agents (CPAs) are typically solvents or small molecules, but there is a need for innovative CPAs to reduce toxicity and increase cell yield, for the banking and transport of cells. Here we use a photochemical high-throughput discovery platform to identify macromolecular cryoprotectants, as rational design approaches are currently limited by the lack of structure–property relationships. Using liquid handling systems, 120 unique polyampholytes were synthesized using photopolymerization with RAFT agents. Cryopreservation screening identified “hit” polymers and nonlinear trends between composition and function, highlighting the requirement for screening, with polymer aggregation being a key factor. The most active polymers reduced the volume of dimethyl sulfoxide (DMSO) required to cryopreserve a nucleated cell line, demonstrating the potential of this approach to identify materials for cell storage and transport