Successful Cryopreservation Protocol Research Articles

Modulation of stress factors for cryopreservation of Paracentrotus lividus (Lamarck 1816) larvae

In the present work we modulated two stress factors salinity and temperature, whose ranges have been previously determined by bioassays using six pre-treatments (18 °C-29.5‰; 18 °C-35‰; 18 °C-39‰ and 20 °C-29.5‰; 20 °C-35‰; 20 °C-39‰), in order to obtain a successful cryopreservation protocol for pluteus larvae of the sea urchin P. lividus (Lamarck 1816). Toxicity tests were performed with different cryoprotectants in a range of 0.5–3 M. Best results pointed out to METH and Me2SO as those more suitable for cryopreservation. First an exploratory cryopreservation experiment with Me2SO supplemented with 0.04 M trehalose (TRE) was tested following the protocol for cryopreservation of embryos (8-h blastula) of Bellas and Paredes, 2011, which did not give satisfactory results. A cryopreservation experiment was performed with both cryoprotectants supplemented with 0.04 M trehalose on 4-arm pluteus larvae (48 h-old) developed in these pre-treatment conditions, followed by a simpler and shorter protocol with a cooling rate of 1 °C/min to −35 °C, achieving for the first time the successful cryopreservation of P. lividus larvae. When larvae were incubated in low salinity or low temperature pre-treatments, they showed delayed larval development and abnormalities. In contrast, pretreatments with high temperature and salinity showed good results. Dimethyl sulfoxide with trehalose proved to be the only effective cryoprotectant for successful cryopreservation of P. lividus larvae. The success of dimethyl sulfoxide is consistent with that described for other cases in previous literature, where dimethyl sulfoxide, although not the least toxic compound, gave the best cryopreservation result.

Co-supplementation of freezing media with trehalose and vitamin C on cell viability and apoptotic gene expression in ovine spermatogonial stem cells

The purpose of this research was to investigate the trehalose and vitamin C (Vit C) co-supplementation of freezing media to create a successful cryopreservation protocol for conservation of ovine spermatogonial stem cells (SSCs). SSCs were cryopreserved and cultured with an essential freezing medium containing 200 mM trehalose, 40 µg/mL Vit C, and a combination of both for 3 weeks. Cell viability, colony number and diameter and mRNA levels of Bax, and Bcl-2 genes were evaluated before and after cryopreservation with quantitative real-time PCR. The results showed that cells cryopreserved in freezing medium containing 200 mM of trehalose plus 40 µg/mL Vit C had considerably greater cell viability than the control group (P<0.0001). Up to the 3rd week of cell culture, supplementation of freezing medium with 200 mM trehalose resulted in significantly lower colonies diameters than in the control group. No significant differences were observed during the 1st to 2nd weeks in colony diameter and number of colonies between cells cryopreserved in the freezing medium containing either Vit C or trehalose compared with the control groups. Following thawing, the mRNA level of Bax in the Vit C + trehalose group was drastically lower than in those treated with trehalose or Vit C only (P<0.0001). High expression of Bcl-2 in the 40 µg/mL Vit C group was recorded in the thawed cells compared to the control group (P<0.0001). These findings indicate that the concomitant use of antioxidants and sugar in the freezing medium can improve the quality and viabi­lity of SSCs after freezing via different mechanisms. Further studies are needed to clarify apoptosis and cell biomarkers in SSCs during freezing and thawing.

Development of Sperm Cryopreservation Protocols for Sharks and Rays: New Tools for Elasmobranch Conservation

Elasmobranchs are one of the most endangered vertebrate groups on the planet, but despite this situation the use of reproductive techniques in elasmobranch conservation strategies has been scarce. Among these techniques, sperm preservation is a potential tool forex situconservation and aquaria sustainability. However, there are no widespread preservation protocols for elasmobranch sperm, and shark sperm cryopreservation has never been achieved before. Here we present the establishment of successful cryopreservation protocols for elasmobranch sperm, tested in several species. We have formulated a sperm extender that can be used for different elasmobranch species, capable of maintaining sperm motility for several weeks. Additionally, we achieved the cryopreservation of sperm by previously diluting it in our extender and supplementing it with different combinations of cryoprotectants. The effects of methanol and dimethyl sulfoxide as permeating cryoprotectants were evaluated, as well egg yolk as a non-permeating cryoprotectant. Sperm quality was assessed by studying the motility and membrane integrity post-thawing, demonstrating its effectiveness in the 10 species tested, including two which are considered Critically Endangered. This is the first time that shark sperm cryopreservation has been reported, broadening our knowledge of the reproductive techniques that can be applied to elasmobranchs and laying the foundations for the first cryobanks for shark and ray sperm. Outcomes from this study will be useful forex situconservation efforts developed by public aquaria. A regular supply of frozen sperm will reduce the problems that result from the transport of specimens, inbreeding or lack of synchronized reproductive cycles in captivity.

Osmometric Measurements of Cryoprotective Agent Permeation into Tissues.

Quantification of the amount of cryoprotective agent (CPA)in a tissue is an essential step in the design of successful cryopreservation protocols. This chapter details two inexpensive methods to measure cryoprotective agent permeation into tissues as functions of time. One of the methods to measure the CPA permeation is to permeate a series of tissue samples from a surrounding solution at a specified concentration of CPA, each sample for a different amount of time, and then to quantitate the amount of CPA that was taken up in the tissue during that time period. The quantification is performed by equilibrating the permeated tissue with a surrounding solution and then measuring the osmolality of the solution to determine the amounts of CPAs that have come out of each tissue sample corresponding to each permeation time. An alternative method to measuring the CPA permeation as a function of time, which requires fewertissue samples, is to measure the CPA efflux as a function of time. In the efflux method, a CPA-permeated tissue sample is placed in a surrounding solution, and solution samples are taken at different time points throughout the efflux to quantitate how much CPA has left the tissue by each time point.

The role of microRNAs in recovery rates of Arabidopsis thaliana after short term cryo-storage

Cryopreservation is a safe and cost-effective long-term storage technique which helps to conserve plant germplasm in liquid nitrogen (LN). However, up to now, the molecular regulatory mechanisms that effect survival after cryopreservation have not been satisfactorily elucidated in plants. Thus, the aim of the study concerned to reveal out microRNA (miRNA)-based post-transcriptional regulations during different stages of cryopreservation in 48-h and 72-h in vitro germinated Arabidopsis thaliana since miRNAs not only regulate gene expression in developmental process of plants, but also control transcription in stress conditions by post-transcriptional degradation. Among the screened 10 miRNAs and their selected target genes, differential expression levels were detected in two tested germination time according to exposure of cryopreservation stages, as 6 miRNAs (miR393, miR397, miR398, miR159, miR169 and miR172) showed a mixed pattern in 48-h germinated seedlings while 6 miRNAs (miR397, miR398, miR159, miR169, miR172 and miR408) were significantly down-regulated in OD (Osmoprotection-Dehydration) and LD (Liquid nitrogen-Dilution) samples in comparison to control in 72-h germinated ones. The most striking expression change was obtained in miR393 as it was up-regulated 15-fold in LN treated 48-h seedlings while relatively lower amount induction (twofold) was recorded in cryopreserved 72-h seedlings. The concominant down-regulation of its target TIR1 and HOS1 expression and up-regulation of CBF1 and CBF3 and their downstream genes (COR15 and COR47) were also observed in cryopreserved 48-h germinated seedlings. Thus, the alteration of expression levels of cold-induced genes related-miRNAs plays a crucial role for successful cryopreservation. The alteration of expression levels of cold- and oxidative stress-induced genes related-miRNAs play a crucial role in development of successful cryopreservation protocols.

Long term survival of cryopreserved mussel larvae (Mytilus galloprovinciallis)

Due to the economic importance of the mussel Mytilus galloprovincialis in the Spanish aquaculture, there is a growing interest in developing alternative methods to ensure the supply of good-quality biological material and to minimize the uncertainty and variability that might be a source of economic risk for this sector. The aim of this work was to develop a successful cryopreservation protocol for Mytilus galloprovincialis larvae and study long-term effects by assessing survival and development post cryobanking. We evaluated the effect of previously studied cryoprotecting agent, Ethylene glycol (EG) for cryopreservation of trochophore larvae and D-veliger larvae 48 h and 72 h old, following an existing preliminary protocol for M. galloprovincialis mussel trochophores. The protocol selected for cryopreservation consisted on holding at 4 °C for 2 min, then cooling at 1 °C/min to −12 °C, holding for 5 min, then cooling at −35 °C with a rate of 1 °C/min, then plunging into liquid nitrogen for storage. Thawing took place by immersion in 35 °C water bath. With this protocol the initial percentage of short-term developed a D-larvae (48 h incubation post-thaw) was 48.9 ± 7.6% with 10% EG + 0.2 M Trehalose (TRE). This initial test only studied the trochophore larvae until they reached the D-stage (48 h old); in the present experiment experiments, we replicated this study but also carried out a long-term larval rearing with cryopreserved trochophore larvae in comparison to fresh larvae. This experiment allowed a comparative post-freezing analysis of both larval development and survival. Larval settlement was also characterized after 13 days. The data revealed that until settling, the survival of the cryopreserved larvae was slightly lower than the 2.8% reported in the larval rearing for Greenshell™ mussel. Over time, there was an initial difference in size of cryopreserved larvae when compared to controls, but from day 17 onwards the size between both types of larvae began to stabilize. Attending to settlement, we obtained a 64% of success of cryopreserved larvae respect to the control.

Evaluation of five additives to mitigate toxicity of cryoprotective agents on porcine chondrocytes

Cryoprotective agents (CPAs) are used in cryopreservation protocols to achieve vitrification. However, the high CPA concentrations required to vitrify a tissue such as articular cartilage are a major drawback due to their cellular toxicity. Oxidation is one factor related to CPA toxicity to cells and tissues. Addition of antioxidants has proven to be beneficial to cell survival and cellular functions after cryopreservation. Investigation of additives for mitigating cellular CPA toxicity will aid in developing successful cryopreservation protocols. The current work shows that antioxidant additives can reduce the toxic effect of CPAs on porcine chondrocytes. Our findings showed that chondroitin sulphate, glucosamine, 2,3,5,6-tetramethylpyrazine and ascorbic acid improved chondrocyte cell survival after exposure to high concentrations of CPAs according to a live-dead cell viability assay. In addition, similar results were seen when additives were added during CPA removal and articular cartilage sample incubation post CPA exposure. Furthermore, we found that incubation of articular cartilage in the presence of additives for 2 days improved chondrocyte recovery compared with those incubated for 4 days. The current results indicated that the inclusion of antioxidant additives during exposure to high concentrations of CPAs is beneficial to chondrocyte survival and recovery in porcine articular cartilage and provided knowledge to improve vitrification protocols for tissue banking of articular cartilage.

Cryopreservation of tropical recalcitrant-seeded species – perspectives

Cryopreservation is an alternative strategy for the long-term conservation of tropical recalcitrant-seeded species. Embryonic axes/embryos or shoot tips are possible explants for this purpose as the seeds are sensitive to cold temperature and they are not tolerant to desiccation to a low moisture content required for seed banking. Shoot tips are used for species with large axes or those without clear differentiated embryonic axes in their seeds. Though not strictly, desiccation and vitrification are usually used for embryonic axes and shoot tips, respectively. Several species under study provide examples, including species in the genus Garcinia. Cryopreservation of shoot tips of tropical recalcitrant species faces many challenges. Amongst these are the lack of tolerance to ultra-low temperature; osmotic stress and chemical toxicity (to the vitrification chemicals used) and the availability of experimental materials throughout the year. With a systematic approach in addressing each problem/challenge, a successful cryopreservation protocol could possibly be obtained. Differential scanning calorimetry and ultrastructural changes observed during each step of the protocol are helpful in understanding successes or failures of the cryopreservation technique used.

Thermal Analyses of a Human Kidney and a Rabbit Kidney During Cryopreservation by Vitrification.

This study focuses on thermal analysis of the problem of scaling up from the vitrification of rabbit kidneys to the vitrification of human kidneys, where vitrification is the preservation of biological material in the glassy state. The basis for this study is a successful cryopreservation protocol for a rabbit kidney model, based on using a proprietary vitrification solution known as M22. Using the finite element analysis (FEA) commercial code ANSYS, heat transfer simulations suggest that indeed the rabbit kidney unquestionably cools rapidly enough to be vitrified based on known intrarenal concentrations of M22. Scaling up 21-fold, computer simulations suggest less favorable conditions for human kidney vitrification. In this case, cooling rates below -100 °C are sometimes slower than 1 °C/min, a rate that provides a clear-cut margin of safety at all temperatures based on the stability of rabbit kidneys in past studies. Nevertheless, it is concluded in this study that vitrifying human kidneys is possible without significant ice damage, assuming that human kidneys can be perfused with M22 as effectively as rabbit kidneys. The thermal analysis suggests that cooling rates can be further increased by a careful design of the cryogenic protocol and by tailoring the container to the shape of the kidney, in contrast to the present cylindrical container. This study demonstrates the critical need for the thermal analysis of experimental cryopreservation and highlights the unmet need for measuring the thermophysical properties of cryoprotective solutions under conditions relevant to realistic thermal histories.

First study in cryopreserved Crassostrea angulata sperm

Sperm cryopreservation is a widely employed technique that promotes alternative techniques to contribute to broodstock management or restoration programs for species of commercial interest, endangered species or species with an interesting genotype. The preservation of genetic material from improved stocks or from the original population is extremely important for the oyster aquaculture industry to prevent the potential impacts of epidemic diseases and natural disasters. The Portuguese oyster, Crassostrea angulata, was the most important species commercialized by the shellfish industry. However, inadequate management of this industry and pathology occurrences resulted in a significant decrease in natural populations. For this reason, in this work a successful sperm cryopreservation protocol for this important species has been developed for the first time. Different internal cryoprotectants (DMSO, ethylene glycol, polyethylene glycol and methanol) at several concentrations (5, 10, 20%), containers (straws vs cryovials) and freezing rates (slow and fast rates) were tested. Cryoprotectant toxicity tests corroborated that this assay did not take into account the following steps of cryopreservation protocol as sperm agglutination. A fast freezing rate of cells diluted in10% DMSO and the use of straws as containers were the best cryopreservation conditions for Portuguese oyster sperm. Finally, fertilization assays confirmed the efficiency of the cryopreservation protocol in oyster sperm. These results demonstrated that different susceptibilities have been detected concerning sperm cryopreservation depending on oyster species or genetic material composition.

Damage to fetal bovine ovarian tissue caused by cryoprotectant exposure and vitrification is mitigated during tissue culture.

The objective of this study is to characterize the impact of exposure to cryoprotectants followed by vitrification on primordial follicle survival and activation using a fetal bovine model. In the first study, fetal bovine cortical pieces were exposed to cryoprotectants with or without sucrose and cultured up to 7 days in the presence or absence of insulin. In the second study, cortical pieces were exposed to cryoprotectants with or without sucrose, vitrified, and cultured up to 7 days after warming in the presence or absence of insulin. Viability and morphology of follicles, as well as proliferation and/or DNA repair in ovarian tissue were analyzed. When compared to non-exposed controls, normal follicular morphology was affected in groups exposed to cryoprotectants only immediately post-exposure and after 1 day of culture, but improved by day 3 and did not significantly differ by day 7. Similarly, normal follicular morphology was compromised in vitrified groups after warming and on day 1 compared to controls, but improved by days 3 and 7. Proliferation and/or DNA repair in ovarian tissue was not affected by vitrification in this model. Cryoprotectant exposure and vitrification of ovarian tissue did not impair the activation of primordial follicles in response to insulin, although activation was delayed relative to non-exposed controls. Interestingly, sucrose had no noticeable protective effect. Vitrified fetal bovine ovarian tissue has the intrinsic capacity to mitigate the immediate damage to primordial follicles' morphology and retains the capacity to activate. These findings provide a basis for a successful cryopreservation protocol for ovarian cortical tissue in other species including humans.

026 Cryoconservation: Successful sperm cryopreservation and develop-mental outcomes using endangered North American amphibians

In response to the dramatic global decline of amphibian species, captive assurance colonies are being established to safeguard genetic diversity; however, many of these founder populations fail to reproduce. Therefore, assisted reproductive technologies such as gamete cryopreservation and artificial fertilisation are valuable conservation tools in the genetic management and breeding of these non-reproducing populations. The following study aimed to develop a successful sperm cryopreservation protocol for two endangered amphibian species maintained in captivity: the threatened boreal toad (Anaxyrus boreas boreas) and the critically endangered Mississippi gopher frog (Lithobates sevosa). Spermiation was induced by exogenous hormone administration of human chorionic gonadotropin at 10IU/g for A. b. boreas and by a cocktail of 500 IU hCG and 15 μg luteinizing hormone-releasing hormone for L. sevosa. Fresh sperm quality was assessed using the following parameters: (1) sperm concentration (2) percentage motile sperm, and (3) percentage sperm moving progressively forward. Sperm samples were subsequently extended 1:1 in chilled cryoprotectant containing 10% (v/v) N-N-dimethylformamide and 10% (w/v) trehalose. The chilled sperm suspensions were held at 4 °C for an acclimation period of 10 min before being loaded into 0.25 mL plastic freezing straws and frozen in a step-wise slow-rate freezing process. Straws were thawed in a 40 °C water bath before a 1:10 dilution with aged tap water to activate the sperm for re-evaluation. Exogenous hormone induction stimulated spermiation in A. b. boreas and L. sevosa, producing mean fresh sperm concentrations of 4.6 × 106/mL and 2.4 × 106/mL respectively, with high rates of motility for both A. b. boreas (86 ± 4%) and L. sevosa (92 ± 2%) (mean ± SE). Upon activation, motility recovery from cryopreserved sperm suspensions was high for A. b. boreas (62 ± 2%) and L. sevosa (70 ± 3%) and sperm forward progression was maintained for both species (57 ± 3% and 29 ± 6%, for A. b. boreas and L. sevosa, respectively). Furthermore, frozen-thawed spermatozoa were used for fertilisation trials and developmental stages analysis. Preliminary investigations using cryopreserved L. sevosa sperm indicated that 14% of fertilised oocytes developed to 2-cell stage embryos with 6% continuing to tadpole stage. The results of this study demonstrate that sperm from both A. b. boreas and L. sevosa can be successfully cryopreserved and maintain high rates of sperm motility and forward progressive movement recovery. Additionally, fertilisation can be achieved in the critically endangered Mississippi gopher frog with subsequent tadpole production. The ability to successfully cryopreserve sperm from endangered amphibian species has enormous potential in augmenting declining populations by facilitating gene flow, both between breeding facilities and between in situ and ex situ populations, without removing source animals. Moreover, this methodology may be directly applicable to a wide range of amphibian taxa, affording a viable option for securing the remaining genetic diversity of endangered species in a global genetic resource bank. Source of funding: Institute of Museum and Library Services. Conflict of interest: None declared. cjl224@msstate.edu

Effects of short-term storage on the motility, oxidative stress, and ATP content of Persian sturgeon (Acipenser persicus) sperm

The effective short-term storage of semen is essential when processing multiple sperm samples and when semen must be transported from collection sites to hatcheries for the fertilization of ova, or to laboratories for cryopreservation. In the present study, the spermatozoa of Persian sturgeon (Acipenser persicus) were used to evaluate the effects of short-term storage on quality parameters (the percentage of motile cells and the total period of sperm motility), oxidative stress indices, and the ATP content. Spermatozoa cells exhibited >50% motility during 6 days of storage where the average total duration of sperm motility varied from 376.42±80.86s initially to 19.28±10.96s after 6 days. No motile spermatozoa were recorded after 9 days of storage. The levels of oxidative stress indices (TBARS and CP) and antioxidant activity (SOD) increased significantly with the storage time. The ATP content also decreased significantly after 2 days of storage. The results of this study may facilitate successful reproduction management and cryopreservation protocols for this endangered fish.

A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles

Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25ml plastic straw, vitrification block and fibreplug™). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9±18.4% when fibreplug and V16 (1.5M methanol+4.5M propylene glycol) solution were employed. When vitrified in V2 (1.5M methanol+5.5M Me2SO) the membrane integrity decreased to 42.0±21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance 2h post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes.

Cryopreservation of Cymbidium eburneum Lindl. and C. hookerianum Rchb. f., two threatened and vulnerable orchids via encapsulation–dehydration

A successful cryopreservation protocol for the long-term conservation of protocorms of two threatened and vulnerable orchids, Cymbidium eburneum Lindl. and Cymbidium hookerianum Rchb. f., was developed using encapsulation–dehydration. Protocorms were osmoprotected in liquid Murashige and Skoog medium (MS) containing 0.7 M sucrose for 20 h at 25 ± 2°C on a rotary shaker, and incorporated into an encapsulation matrix [consisting of 3% (w/v) sodium alginate and 100 mM CaCl2]. The encapsulated protocorms, which were desiccated in a laminar airflow cabinet for 6 h, were able to withstand cryostorage in liquid nitrogen. Maximum regeneration into complete plantlets (72% for C. eburneum and 70% for C. hookerianum) of the cryostored, encapsulated protocorms was obtained using MS medium containing 3% sucrose and 0.8% agar. Using this protocol of cryopreservation, long-term preservation for ex situ conservation of these two threatened orchids can be accomplished.

Effect of short-term storage on quality parameters, DNA integrity, and oxidative stress in Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeon sperm

The sperm of Russian sturgeon (A. gueldenstaedtii) and Siberian sturgeon (A. baerii) was used to evaluate the effects of short-term (liquid) storage on functional parameters (spermatozoa motility and velocity), DNA integrity and oxidative stress indices. Spermatozoa showed >50% motility during 6 days of storage with an average velocity of 133.12±15.4 to 87.9±11.23μm s−1 in both species. No motile spermatozoa were recorded after nine days of storage. Analysis of Russian sturgeon sperm showed no significant differences in DNA damage expressed as percent tail DNA and Olive Tail Moment for first three days of storage. In Siberian sturgeon significant differences in DNA damage were detected after two days of storage. The level of oxidative stress indices (TBARS, CP) and antioxidant activity (SOD) increased significantly with storage time in both species. Results of this study can be utilized for successful reproduction management and cryopreservation protocols of these endangered species.

The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification

This report highlights the first successful cryopreservation protocol for shoot tips of Garcinia mangostana L. achieved by using vitrification technique. We investigated the effects of different temperatures and exposure periods to a plant vitrification solution 2 (PVS2), sucrose concentrations and preculture periods, and unloading treatments in steps of the vitrification protocol on the survival of G. mangostana shoot tips after cryopreservation. Exposure to PVS2 for 25 min gave beneficial effects with 10.4 ± 1.8 % survival at 0 °C with average water content of 1.1 ± 0.3 g g−1 dry mass. Survival was 13.7 ± 5.5 % when using preculture medium with full-strength Murashige and Skoog (MS) medium supplemented with 0.6 M sucrose for 2 days. A significant difference was observed in survival of shoot tips when treated with various sucrose concentrations in preculture which strengthens their importance towards enhancing survival of shoot tips after cryopreservation. MS with 0.4 M sucrose and 2 M glycerol applied as an unloading solution increased the survival of shoot tips to 44.1 ± 6.5 %. Experiments on the effect of ascorbic acid were also conducted for each step of vitrification. Our results showed higher survival of 45.8 ± 3.8 % but there were no significant effects compared with the control (without ascorbic acid). Further study on the recovery dark/light period was conducted. Survival of shoot tips significantly increased to 50.0 ± 16.7 % when subjected to 7 days in the dark before transferring to 16 h/8 h light/dark photoperiod. These studies strengthen suggestions that cryopreservation through vitrification is possible for ex situ conservation of germplasm of this tropical recalcitrant species.

Semen of Beluga, Huso huso: Ionic Content and Osmolality of Seminal Plasma and Their Physiological Correlation with Sperm Motility Indices

The ionic contents and osmolality of seminal plasma of beluga spermatozoa to determine and their physiological relationship with sperm motility characteristics (total duration of sperm motility and percentage of motile cell) were studied. The results obtained show that Na+ (82.54 ± 5.46 mM L-1), Cl- (15.95 ± 0.72 mM L-1) and K+ (3.57 ± 0.15 mM L-1) were predominant ions in the seminal plasma. The osmolality of seminal plasma ranged from 57 to 93 mOsm kg-1. Significant positive correlations were observed between osmolality vs. Na+ (p<0.01) and Cl- (P<0.05) and Na+ vs. Cl- (P<0.05). No significant correlations were found between sperm motility and others composition of the seminal plasma. Probably, the Na+ and Cl- are the main electrolytes playing a key role in maintaining the osmolality of the seminal plasma and the viability of the spermatozoa in vivo. However analysis of ionic contents of the seminal plasma revealed some species-specific characteristics especially for K+ which is important because of its inhibitory effect on sperm motility. Results of the present study provide new data on beluga spermatozoa quality and can be utilized for successful reproduction management and cryopreservation protocols of this endangered species.

Refinement of a vitrification protocol for protocorm-like bodies of Dendrobium sonia-28

The hybrid Dendrobium sonia-28 is an important ornamental orchid in the Malaysian flower industry. However, it faces the risk of producing heterozygous progenies through conventional seed propagation or somaclonal variation. Cryopreservation is a favoured long-term storage method in the preservation and maintenance of orchids with specific desired traits. This paper presents a successful cryopreservation protocol for protocorm-like bodies (PLBs) of Dendrobium sonia-28 through the vitrification technique and the use of ascorbic acid and charcoal. Survival assessments were conducted through growth evaluations and visual and spectrophotometric 2,3,5-triphenyltetrazolium chloride assays. Results of this study, an improvement over the previous protocol applied to this orchid hybrid, showed that the addition of ascorbic acid to the preculture, loading, dehydration, unloading, and regeneration media, coupled with the addition of charcoal to the regeneration medium, helped boost recovery rates of cryopreserved PLBs of Dendrobium sonia-28.

Physico-chemical properties and protein profiles of sperm from three freshwater chondrostean species: a comparative study among Siberian sturgeon (Acipenser baerii), sterlet (Acipenser ruthenus) and paddlefish (Polyodon spathula)

Summary Physico-chemical properties (spermatozoa motility, spermatozoa concentration, seminal plasma protein concentration, osmolality, pH, ions) and sperm protein profiles were evaluated in three Acipenseriforme species [Siberian sturgeon (Acipenser baerii), sterlet (Acipenser ruthenus) and paddlefish (Polyodon spathula)]. The spermatozoa velocity was observed significantly highest in sterlet and lowest in Siberian sturgeon (P < 0.05). Spermatozoa velocity was positively correlated to pH and the concentration of Na+ in seminal plasma in all species examined. Other tested parameters, in particular the concentration of K+, expressed species specific patterns, which could be linked to taxonomical status of species. The protein profiles of seminal plasma indicated a protein band with a molecular weight of 32.6 kDa exclusively detected in paddlefish. In spermatozoa, 95 protein spots were detected in Siberian sturgeon and sterlet, respectively, whereas only 70 spots were observed in paddlefish. Each spermatozoa protein map expressed a strong species specific pattern, which offers a basic benchmark to trace molecular differences among Acipenseriformes. It can be concluded that results presented in this study could be applied for successful reproduction management and cryopreservation protocols of these endangered species.