Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: II—Mathematical prediction and experimental validation of optimal cryopreservation protocols

In Part I, we documented differences in cryopreservation success measured by membrane integrity in four mouse embryonic stem cell (mESC) lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1), and we demonstrated a potential biophysical basis for these differences through a comparative study characterizing the membrane permeability characteristics and osmotic tolerance limits of each cell line. Here we use these values to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures. We subsequently verified these predictions experimentally for their effects on post-thaw recovery. From this study, we determined that a cryopreservation protocol utilizing 1 M propylene glycol, a cooling rate of 1 °C/minute, and plunging into liquid nitrogen at −41 °C, combined with subsequent warming in a 22 °C water bath with agitation, significantly improved post-thaw recovery for three of the four mESC lines, and did not diminish post-thaw recovery for our single exception. It is proposed that this protocol can be successfully applied to most mESC lines beyond those included within this study once the effect of propylene glycol on mESC gene expression, growth characteristics, and germ-line transmission has been determined. Mouse ESC lines with poor survival using current standard cryopreservation protocols or our proposed protocol can be optimized on a case-by-case basis using the method we have outlined over two papers. For our single exception, the CBA cell line, a cooling rate of 5 °C/minute in the presence of 1.0 M dimethyl sulfoxide or 1.0 M propylene glycol, combined with plunge temperature of −80 °C was optimal.     

Cooling rate optimization for zebrafish sperm cryopreservation using a cryomicroscope coupled with SYBR14/PI dual staining

The Zebrafish has gained increased popularity as an aquatic model species in various research fields, and its widespread use has led to numerous mutant strains and transgenic lines. This creates the need to store these important genetic materials as frozen gametes. Sperm cryopreservation in zebrafish has been shown to yield very low post-thaw survival and many protocols suffer from great variability and poor reproducibility. The present study was intended to develop a freezing protocol that can be reliably used to cryopreserve zebrafish sperm with high post-thaw survival. In particular, our study focused on cooling protocol optimization with the aid of cryomicroscopy. Specifically, sperm suspended in 8% DMSO or 4% MeOH were first incubated with live/dead fluorescent dyes (SYBR14/PI) and then cooled at various rates from 4 °C to different intermediate stopping temperatures such as −10, −20, −30 and −80 °C before rewarming to 35 °C at the rate of 100 °C/min. %PI-positive (dead) cells were monitored throughout the cooling process and this screening yielded an optimal rate of 25 °C/min for this initial phase of freezing. We then tested the optimal cooling rate for the second phase of freezing from various intermediate stopping temperatures to −80 °C before plunging into liquid nitrogen. Our finding yielded an optimal intermediate stopping temperature of −30 °C and an optimal rate of 5 °C/min for this second phase of freezing. When we further applied this two-step cooling protocol to the conventional controlled-rate freezer, the average post-thaw motility measured by CASA was 46.8 ± 6.40% across 11 males, indicating high post-thaw survival and consistent results among different individuals. Our study indicates that cryomiscroscopy is a powerful tool to devise the optimal cooling conditions for species with sperm that are very sensitive to cryodamage.     

Cryopreservation of red snapper (Lutjanus argentimaculatus) sperm: Effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity

Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120 min at room temperature (25 °C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 °C/min from an initial temperature of 25 °C to final temperatures of −40 or −80 °C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 °C/min to a final temperature of −80 °C had the highest motility (91.1 ± 2.2%) and viability (92.7 ± 2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4 ± 2.4%) was not different (P > 0.05) from that of fresh sperm (75.5 ± 2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm.     

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me₂SO), N,N-dimethylacetamide (DMA), N,N-dimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 °C; (2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me₂SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 °C/min) and were compared to Me₂SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ? 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ?10% which was significantly lower than that of methanol and ME. With Me₂SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ? 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.     

Improved cryopreservation protocol for Blanca-Celtibérica buck semen collected by electroejaculation

The collection of sperm samples by electroejaculation (EE) leads to an increase of the production of seminal plasma which could modify the tolerance of spermatozoa to the cryopreservation procedure. This study aims to compare a standard sperm cryopreservation protocol for samples collected by artificial vagina (AV) with the same protocol and modifications to this for samples obtained by EE. Semen from six males of Blanca-Celtibérica goat breed was collected by AV (control) and EE, and three experiments were conducted. In Experiment 1, it was examined the effects of egg yolk concentration contained in freezing extender (0%, 1.5%, 10% and 20% of egg yolk); in Experiment 2, it was evaluated the cooling rate from 30 to 5 °C (fast: 10 min and slow: 90 min) and the temperature of glycerol addition (30 and 5 °C); and in Experiment 3, it was examined the time of equilibration at 5 °C (0, 1, 2 or 3 h). A heterologous in vitro fertilization test was carried out in order to compare the fertility of control samples with that resulting from the EE protocol which showed the highest sperm quality. Results showed greater sperm motility parameters after thawing for control samples cryopreserved in standard conditions in the three experiments. For samples collected by EE, extender with 20% egg yolk, a slow cooling rate and a longer equilibration time (3 h) provided higher sperm quality, and no differences were observed between temperatures of glycerol addition. Samples collected by EE and cryopreserved with the protocol which yielded the best sperm quality after thawing showed higher fertility compared to AV.     

Quality of chilled and cold-stored (5 °C) canine spermatozoa submitted to different rapid cooling rates

The aim of this study was to evaluate the sperm quality in chilled canine semen using di?erent cooling rates from room temperature (23 °C) to 5 °C and subsequently cold-stored at 5 °C for up to 96 hours. In experiment 1, semen samples from five dogs were pooled, diluted in Tris-fructose-citrate extender with 20% egg yolk and split into four aliquots that were chilled to 5 °C using di?erent cooling rates of 2.25, 0.9, 0.45, and 0.2 (control) °C/min. In experiment 2, semen from five dogs was processed individually as described above and split into two aliquots that were chilled to 5 °C using rates of either 2.25 °C/min or 0.2 °C/min. In both experiments, the sperm quality (i.e., sperm motility and viability) was evaluated before cooling and after 0, 24, 48, 72, and 96 hours of storage at 5 °C. The total motility, progressive motility, and quality of movement parameters were assessed using computer-assisted analysis system, and the percentage of viable spermatozoa was determined using ?ow cytometry (H-42/PI//FITC-PNA). The cooling rate did not in?uence the sperm quality parameters at any of the evaluation times. All evaluated males showed the same response to chilling semen at a rapid cooling rate. Storage time negatively in?uenced (P < 0.05) sperm motility, regardless of the cooling rate used. In conclusion, canine sperm could be chilled and stored for 96 hours at 5 °C in a Tris-fructose extender with 20% egg yolk using rapid cooling rates, with values for sperm quality similar to those from a conventional protocol.     

Cryopreservation of sperm in farmed Australian greenlip abalone Haliotis laevigata

This study investigated factors important to the development of the liquid nitrogen (LN) vapor sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata, including (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature (height above LN surface); (3) thawing temperature; (4) sperm to egg ratio; and (5) sugar supplementation, using sperm motility, fertilization rate or integrity/potential of sperm components and organelles as quality assessment indicators. Results suggested that among the single CPAs evaluated 6% dimethyl sulfoxide (Me2SO) would be the most suitable for sperm cryopreservation in this species. The highest post-thaw sperm motility was achieved with the sperm that had been exposed to LN vapor for 10 min at 5.2 cm above the LN surface, thawed and recovered in 60 and 18 °C seawater bathes, respectively after at least 2 h storage in LN. The highest fertilization rates were achieved at a sperm to egg ratio of 10,000:1 or 15,000:1. Addition of 1% glucose or 2% sucrose produced significantly higher post-thaw sperm motility than 6% Me2SO alone. Among the three cryoprotectant solutions further trialled, 6% Me2SO + 1% glucose produced the highest fertilization rate of 83.6 ± 3.7%. Evaluation of sperm has shown that the addition of glucose could significantly improve the sperm plasma membrane integrity and mitochondrial membrane potential. These results demonstrated a positive role of glucose in the improvement of sperm cryopreservation in farmed greenlip abalone.     

Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification

There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to −196 °C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250 °C/min to −196 °C and for each cooling rate, subjecting them to five warming rates back above 0 °C at rates ranging from 610 to 118,000 °C/min. In samples warmed at the highest rate (118,000 °C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610 °C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000 °C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.     

Extreme rapid warming yields high functional survivals of vitrified 8-cell mouse embryos even when suspended in a half-strength vitrification solution and cooled at moderate rates to −196 °C

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to do so is to subject them to procedures that convert cell water into a non-crystalline glass. Current belief is that to achieve this vitrification, cells must be suspended in very high concentrations of glass-inducing solutes (i.e., ?6 molal) and cooled at very high rates (i.e., ?1000 °C/min). We report here that both these beliefs are incorrect with respect to the vitrification of 8-cell mouse embryos. In this study, precompaction 8-cell embryos were vitrified in several dilutions of EAFS10/10 using various cooling rates and warming rates. Survival was based on morphology, osmotic functionality, and on the ability to develop to expanded blastocysts. With a warming rate of 117,500 °C/min, the percentages of embryos vitrified in 1×, 0.75×, and 0.5× EAFS that developed to blastocysts were 93%, 92%, and 83%, respectively. And the percentages of morphological survivors that developed to expanded blastocysts were 100%, 92%, and 97%, respectively. Even when the solute concentration of the EAFS was reduced to 33% of normal, we obtained 40% functional survival of these 8-cell embryos.     

New device for the vitrification and in-straw warming of in vitro produced bovine embryos

Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1, 0.5 and 0 M sucrose; protocol W3) or directly in 0.5 M (protocol W1/0.5) or 0 M sucrose (protocol W1/0). Similar survival rates were recorded 24 h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24 h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5 M sucrose) dilution/warming and different warming temperatures (45, 50, 60 and 70 °C) were tested. When warming solution passed through the new vitrification/warming device at 45 °C, 61.5% of blastocysts were fully re-expanded or hatched at 24 h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24 h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45 °C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions.     

Suprazero cooling rate,rather than freezing rate,determines post thaw quality of rhesus macaque sperm

Sperm become most sensitive to cold shock when cooled from 37 °C to 5 °C at rates that are too fast or too slow; cold shock increases the susceptibility to oxidative damage owing to its influence on reactive oxygen species (ROS) production, which are significant stress factors generated during cooling and low temperature storage. In addition, ROS may be a main cause of decreased motility and fertility upon warming. They have been shown to change cellular function through the disruption of the sperm plasma membrane and through damage to proteins and DNA. The objective of this study was to determine which cryopreservation rates result in the lowest degree of oxidative damage and greatest sperm quality. In the rhesus model, it has not been determined whether suprazero cooling or subzero freezing rates causes a significant amount of ROS damage to sperm. Semen samples were collected from male rhesus macaques, washed, and resuspended in TEST-yolk cryopreservation buffer to 100 × 10⁶ sperm/mL. Sperm were frozen in 0.5-mL straws at four different combinations of suprazero and subzero rates. Three different suprazero rates were used between 22 °C and 0 °C: 0.5 °C/min (slow), 45 °C/min (medium), and 93 °C/min (fast). These suprazero rates were used in combination with two different subzero rates for temperatures 0 °C to −110 °C: 42 °C/min (medium) and 87 °C/min (fast). The different freezing groups were as follows: slow-med (SM), slow-fast (SF), med-med (MM), and fast-fast (FF). Flow cytometry was used to detect lipid peroxidation (LPO), a result of ROS generation. Motility was evaluated using a computer assisted sperm motion analyzer. The MM and FF treated sperm had less viable (P < 0.0001) and motile sperm (P < 0.001) than the SM, SF, or fresh sperm. Sperm exposed to MM and FF treatments demonstrated significantly higher oxidative damage than SM, SF, or fresh sperm (P < 0.05). The SM- and SF-treated sperm showed decreased motility, membrane integrity, and LPO compared with fresh semen (P < 0.001). Slow cooling from room temperature promotes higher membrane integrity and motility post thaw, compared with medium or fast cooling rates. Cells exposed to similar cooling rates with differing freezing rates were not different in motility and membrane integrity, whereas comparison of cells exposed to differing cooling rates with similar freezing rates indicated significant differences in motility, membrane integrity, and LPO. These data suggest that sperm quality seems to be more sensitive to the cooling, rather than freezing rate and highlight the role of the suprazero cooling rate in post thaw sperm quality.     

Directional freezing as an alternative method for cryopreserving rhesus macaque (Macaca mulatta) sperm

The objective was to develop a freezing protocol using a directional freezing (DF) technique for cryopreservation of rhesus macaque sperm and achieve a survival rate comparable to that achieved with a conventional freezing (CF) technique. Rhesus macaque sperm frozen with a DF technique, with cooling rates of 12 or 16 °C/min, had higher post-thaw motility (P < 0.05) than those cooled at 7 °C/min (59.3, 61.1, and 50.3%, respectively). Furthermore, sperm cryopreserved with 5% glycerol and a DF technique had similar frozen-thawed sperm motility to those cryopreserved by a CF technique (63.7 vs. 53.9%, P > 0.05). The function of sperm cryopreserved at the optimized cooling rate using a DF technique was evaluated by in vitro fertilization of oocytes collected from gonadotropin-stimulated rhesus macaques. Of the 38 mature oocytes collected, 78.9% were fertilized and 71.1, 47.4, and 42.1% of the oocytes developed to the 2-cell, morulae, and blastocyst stages, respectively. In conclusion, rhesus macaque sperm was effectively cryopreserved using a DF technique, providing a new and effective method for genetic preservation in this important species.     

Effect of cooling rate and cryoprotectant concentration on intracellular ice formation of small abalone (Haliotis diversicolor) eggs

The intracellular ice formation (IIF) behavior of Haliotis diversicolor (small abalone) eggs is investigated in this study, in relation to controlling the cooling rate and the concentration of dimethyl sulfoxide (DMSO). The IIF phenomena are monitored under a self-developed thermoelectric cooling (TEC) cryomicroscope system which can achieve accurate temperature control without the use of liquid nitrogen. The accuracy of the isothermal and ramp control is within ±0.5 °C. The IIF results indicate that the IIF of small abalone eggs is well suppressed at cooling rates of 1.5, 3, 7 and 12 °C/min with 2.0, 2.5, 3.0 and 4.0 M DMSO in sea water. As 2.0 M DMSO in sea water is the minimum concentration that has sufficient IIF suppression, it is selected as the suspension solution for the cryopreservation of small abalone eggs in order to consider the solution’s toxicity effect. Moreover, IIF characteristics of the cumulative probability of IIF temperature distribution are shown to be well fitted by the Weibull probabilistic distribution. According to our IIF results and the Weibull distribution parameters, we conclude that cooling at 1.5 °C/min from 20 to −50 °C with 2.0 M DMSO in sea water is more feasible than other combinations of cooling rates and DMSO concentrations in our experiments. Applying this protocol and observing the subsequent osmotic activity, 48.8% of small abalone eggs are osmotically active after thawing. In addition, the higher the cooling rate, the less chance of osmotically active eggs. A separate fertility test experiment, with a cryopreservation protocol of 1.5 °C/min cooling rate and 2.0 M DMSO in sea water, achieves a hatching rate of 23.7%. This study is the first to characterize the IIF behavior of small abalone eggs in regard to the cooling rate and the DMSO concentration. The Weibull probabilistic model fitting in this study is an approach that can be applied by other researchers for effective cryopreservation variability estimation and analysis.     

The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm

Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10 to 12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4 °C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150 °C by using various cooling rates (10, 40, 70, and 100 °C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37 °C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P < 0.05). Sperm motility increased as cooling rate was increased for both strains (P < 0.05). Highest cryosurvival was achieved when 100 °C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1 M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70 °C/min and 100 °C/min cooling rate improved post-thaw motility of rat sperm.     

Cryopreservation of Greenshell™ mussel (Perna canaliculus) trochophore larvae

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16–20 h old). Toxicity tests showed that levels of 10–15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40–60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min⁻¹ to −10 °C, holding for a further 5 min, then cooling at 0.5 °C min⁻¹ to −35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

Cryopreservation of Acropora digitifera sperm with use of sucrose and methanol based solution

Coral biodiversity has recently been considered an important topic in environmental studies. Biodiversity could be preserved with successful cryopreservation of endangered species gametes or embryos. Herein, we developed cryopreservation protocols for Acropora digitifera sperm with use of sucrose and methanol based extender. We studied cryopreservation of A. digitifera sperm with floating frames, allowing the placement of 250 μl French straws 4 cm above the liquid nitrogen surface, resulting in a 40 °C/min freezing rate. This method enabled the successful cryopreservation of sperm in 0.9 M sucrose supplemented with 20% methanol. In this protocol, we used a 1:3 (sperm:extender) dilution ratio. The fertilization ratios of freezing:thawed sperm were similar to the control and reached 63%. This method might be a valuable option in the formation of A. digitifera gene banking. Further studies are needed to explore possibilities of using this method in cryopreservation of other coral’s sperm.     

Development of cryopreservation protocols for early stage zebrafish (Danio rerio) ovarian follicles using controlled slow cooling

Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, cryopreservation of early stage zebrafish ovarian follicles was studied for the first time using controlled slow freezing. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. Stages I and II ovarian follicles were frozen in 4 M methanol and 3 M DMSO in either L-15 medium or KCl buffer. Ovarian follicle viability was assessed using trypan blue, FDA + PI staining and ADP/ATP assay. The results showed that KCl buffer was more beneficial than L-15 medium, methanol was more effective than DMSO, optimum cooling rates were 2-4 °C/min, stepwise removal of cryoprotectant improved ovarian follicle viability significantly and stage I ovarian follicles were more sensitive to freezing. The results also showed that FDA + PI staining and ADP/ATP assay were more sensitive than TB staining. The highest follicle viabilities after post-thaw incubation for 2 h obtained with FDA + PI staining were 50.7 ± 4.0% although ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell damage. Studies are currently being carried out on in vitro maturation of these cryopreserved ovarian follicles.     

Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time

Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 10⁶ spermatozoa/mL in a TES–Tris–Fructose–based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at −196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin–fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.     

Survival of Pacific oyster, Crassostrea gigas, oocytes in relation to intracellular ice formation

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to −35 °C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to −35 or −60 °C at 0.1 or 0.3 °C min⁻¹ and holding for 0 or 30 min at −35 or −60 °C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to −60 °C was significantly lower than that of oocytes cooled to −35 °C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.