Solid Surface Vitrification Research Articles

Investigating cryopreservation techniques for maintaining morphology and in vitro viability of cartilage and skin from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831) for conservation through biobanks.

Conservation of the genetic diversity through skin and cartilage biobanks represents an essential strategy for maintaining biodiversity. Biobanks for the wild species of the order Rodentia have been little studied. Considering that the cryopreservation technique has specific relationships with the tissue and species of interest, we propose investigating different techniques for preserving tissue integrity and cell viability after cartilage and skin culture from Spix's yellow-toothed cavies. Subsequently, two techniques [solid-surface vitrification (SSV) vs. slow freezing (SF)] were used for cartilage and skin cryopreservation. Tissues not subjected to cryopreservation were used as controls. All tissues were evaluated for morphology and proliferation by histological techniques. Moreover, fragments were cultured, and cells were evaluated for viability, proliferation, metabolism, and apoptosis. Regardless of the cryopreservation technique, no differences were observed for the thickness of the epidermis, dermis, skin, spinous and basal layers, fibroblasts, and proliferative activity regarding the number of nucleolar organizer regions (NOR). SSV ensured better maintenance of epidermal cells, normal chondrocytes, filled gaps, collagen fibers, proliferative activity by NOR area/cell, and reduced perinuclear halos and empty gaps compared to SF. SF ensured the conservation of corneum thickness compared to the control. Although both techniques promoted cell recovery after culture, cells from SF resulted in better subconfluence time and day with cell growth around fragments compared to SSV. In conclusion, both cryopreservation techniques resulted in viable cells after culture. However, SSV promoted better maintenance of tissue morphological integrity, and SF ensured the preservation of all cell quality parameters in Spix's yellow-toothed cavies.

Boric Acid Improved Cryopreserved Mouse Embryo Development.

Boric acid (BA) is an essential trace element that is required to support the metabolic pathways in plants, humans, and animals. The present study investigates the in vitro development and quality of single-cell mouse embryos in a BA-added culture medium after cryopreservation using the solid-surface vitrification method. For this purpose, the pronuclear-stage embryos derived from superovulated C57Bl/6j mouse strains and the one-cell embryos were then cryopreserved using the solid-surface vitrification (SSV) method. After thawing, the embryos were cultured in a BA-added medium at 37 °C in a 5% CO2 environment until the blastocyst stage. The resulting in vitro development rates of the embryos in the control group, SSV group, and SSV + 1.62 × 10-4 μM BA group were 68.11% (36/59), 40.16% (16/48), and 64.92% (28/48) respectively, indicating that the BA supported the in vitro development of the embryos cryopreserved using the SSV method. Our results suggest that the addition of boric acid to the culture media increased the development rate of the embryos that were vitrified using the SSV method.

Influence of cryopreservation techniques and low concentrations of permeating cryoprotectants on the conservation of ear cartilage and skin derived from six-banded armadillos (Euphractus sexcinctus Linnaeus, 1758)

Considering the importance of efficiently obtaining somatic resource banks as an ex-situ conservation strategy for wild mammals, we evaluated two techniques (slow freezing - SF and solid-surface vitrification - SSV) for the cryopreservation of ear cartilage and skin from six-banded armadillos. Additionally, we analyzed the effects of two combinations of intracellular cryoprotectants (3.0 M or 6.0 M ethylene glycol - EG and dimethyl sulfoxide - Me2SO) on SSV. Tissues not subjected to cryopreservation were used as controls. All samples were evaluated for morphological analysis and cell ability during culture. The thickness of the basal layer was similar to the control only for tissues derived from SF, while SSV ensured the preservation of the cartilage thickness. Moreover, fragments derived from SF and SSV, especially in the 3.0 M EG-Me2SO group, resulted in dermis thickness and total skin similar to the control. All cryopreserved techniques maintained normal patterns of the fibroblasts, epidermal cells, and melanocytes. While only SF-derived fragments maintained the number of degenerated chondrocytes similar to the control, no difference was observed between groups for normal chondrocytes and lacunae. Moreover, SSV maintained the collagen fibers percentage of the tissues even after warming. After culture, SF and SSV techniques were efficient for the recovery of the somatic cells in all parameters evaluated, except the day of subconfluence, which was greater for the SSV group with 6.0 M EG-Me2SO. In summary, SSV, especially with 3 M EG-Me2SO, was as efficient as SF in preserving ear skin and cartilage derived from six-banded armadillos.

Droplet freeze-thawing system based on solid surface vitrification and laser rewarming

Ultra-rapid cooling and rewarming rate is a critical technical approach to achieve ice-free cells during the freezing and melting process. A set of ultra-rapid solid surface freeze-thaw visualization system was developed based on a sapphire flim, and experiments on droplet freeze-thaw were carried out under different cryoprotectant components, volumes and laser energies. The results showed that the cooling rate of 1 μL mixed cryoprotectant [1.5 mol/L propylene glycol (PG) + 1.5 mol/L ethylene glycol (EG) + 0.5 mol/L trehalose (TRE)] could be 9.2×10 3 °C/min. The volume range of 1-8 μL droplets could be vitrified. After comparing the proportions of multiple cryoprotectants, the combination of equal proportion mixed permeability protectant and trehalose had the best vitrification freezing effect and more uniform crystallization characteristics. During the rewarming operation, the heating curve of glassy droplets containing gold nanoparticles was measured for the first time under the action of 400-1 200 W laser power, and the rewarming rate was up to the order of 10 6 °C/min. According to the droplet images of different power rewarming processes, the laser power range for ice-free rewarming with micron-level resolution was clarified to be 1 400-1 600 W. The work of this paper simultaneously realizes the ultra-high-speed temperature ramp-up, transient visual observation and temperature measurement of droplets, providing technical means for judging the ice free droplets during the freeze-thaw process. It is conducive to promoting the development of ultra-rapid freeze-thaw technology for biological cells and tissues.

Effect of Different Vitrification Techniques on Viability and Apoptotic Index of Domestic Cat Testicular Tissue Cells.

Vitrification is essential for successful tissue cryopreservation and biobanking in wild cats. This study aimed to compare different methods of vitrification (Ovarian Tissue Cryosystem-OTC, Straws-STW, and Solid Surface vitrification-SSV) for testicular fragment vitrification in tom cats. Testicular fragments were recovered from five adult tom cats and subjected to equilibrium vitrification using different cryovials and methods under the same conditions of vitrification solutions and cryoprotectants. The efficiencies of the methods were evaluated using histological analysis of spermatogonia and Sertoli cell nuclei, seminiferous tubular basement membrane detachment, and the gonadal epithelium shrinkage score scale. Cell viability was assessed using Hoechst PI and Terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay. The results showed that OTC is an effective vitrification method for maintaining the distinction between spermatogonia and Sertoli cells. OTC was similar to the control for basal membrane detachment parameters (p = 0.05). Epithelial shrinkage was low in the SSV group, which showed the highest percentage of viable cells among the vitrified groups (p = 0.0023). The OTC and SSV vitrification methods were statistically similar in terms of the percentage of TUNEL-positive cells (p = 0.05). Therefore, OTC and SSV provide favorable conditions for maintaining viable cat testicular tissue cells after vitrification.

The initial steps toward the formation of somatic tissue banks and cell cultures derived from captive Antillean manatee (Trichechus manatus manatus) skin biopsies.

The declining population of the Antillean manatee caused by ecosystem degradation and rising pollution has prompted interest in developing conservation strategies for this species. Given this scenario, somatic tissue banks are important tools for acquiring knowledge about the species, as well as for obtaining somatic cells for biotechnological and ecotoxicological applications. Therefore, we aimed to assess the effects of slow freezing (SF) and solid-surface vitrification (SSV) of the dermis of captive Antillean manatees on the histology and ultrastructure of the tissue and cell viability in culture. While the SSV did not change the dermis thickness, the SF maintained the tissue proliferative potential, assessed by the nucleolar organizer region area, similar to noncryopreserved tissues. Moreover, both techniques reduced the number of fibroblasts and increased the percentage of collagen fibers. Nevertheless, only tissues cryopreserved with SF and noncryopreserved tissues were able to produce cells after in vitro culture. Although SF did not alter cell viability and proliferative activity, cells derived from cryopreserved tissues showed decreased metabolism, altered apoptosis, increased levels of reactive oxygen species, and mitochondrial membrane potential compared to cells from noncryopreserved tissues. In summary, we demonstrated for the first time that Antillean manatee somatic tissues can be cryopreserved by SF, and cells can be obtained after in vitro culture. Improvements in cryopreservation conditions, especially vitrification, of somatic samples are needed to increase the quality of somatic tissue banks in this species.

Influences of glycine supplementation during vitrification on the developmental potential of vitrified/warmed immature dromedary camel oocytes.

Oocytes experience detrimental osmotic stress during vitrification and warming procedures because of the osmolality imbalance between the vitrification-warming fluids and the intracellular environment. Cellular osmotic homeostasis can be preserved by glycine, a powerful osmolyte with antioxidant properties. We aimed to examine the influences of supplementing glycine to the vitrification solutions (VS) on the developmental potential of vitrified/warmed immature dromedary camel oocytes following IVM/IVF and in vitro embryo culture (IVC). Cumulus oocyte complexes (COCs) were collected from dromedary camel ovaries and randomly allocated into two groups namely control (oocytes subjected directly to IVM) and vitrified (COCs were vitrified into VS supplemented with 0.0, 0.5, 1.0 or 2.0 mM glycine). For vitrification, COCs were equilibrated for 3 min in 12.5% ethylene glycol; EG plus 12.5% dimethyl sulfoxide; DMS and then they were vitrified for 60 s in VS composed of 25% EG + 25% DMSO using solid surface vitrification (SSV). Warming of vitrified oocytes was conducted in decreasing concentrations of trehalose solution. Following vitrification and warming, the morphologically viable oocytes were subjected to IVM for 36 h. Matured oocytes were then fertilized in vitro by epididymal spermatozoa and cultured for seven days. The results showed that the percentage of viable oocytes assessed by trypan blue stain was significantly higher (p ≤ .05) in the 1.0 mM glycine-supplemented group than 0.0- and 2.0-mM glycine-supplemented ones (90.0 % vs. 80.0% and 76.6%, respectively). However, no significant difference was observed between 0.5 mM glycine and other vitrified groups. Nuclear maturation rates, cleavage (48-h post-insemination; pi) and blastocyst rate (7-days pi) were significantly lower in vitrified groups than control ones (p ≤ .05). Among vitrified groups, these parameters were the highest in the 1.0 mM glycine-supplemented group. Taken together, supplementation of vitrification solutions with 1.0 mM glycine could enhance the developmental potential of vitrified/warmed immature dromedary camel oocytes.

Droplet Generation, Vitrification, and Warming for Cell Cryopreservation: A Review.

Cryopreservation is currently a key step in translational medicine that could provide new ideas for clinical applications in reproductive medicine, regenerative medicine, and cell therapy. With the advantages of a low concentration of cryoprotectant, fast cooling rate, and easy operation, droplet-based printing for vitrification has received wide attention in the field of cryopreservation. This review summarizes the droplet generation, vitrification, and warming method. Droplet generation techniques such as inkjet printing, microvalve printing, and acoustic printing have been applied in the field of cryopreservation. Droplet vitrification includes direct contact with liquid nitrogen vitrification and droplet solid surface vitrification. The limitations of droplet vitrification (liquid nitrogen contamination, droplet evaporation, gas film inhibition of heat transfer, frosting) and solutions are discussed. Furthermore, a comparison of the external physical field warming method with the conventional water bath method revealed that better applications can be achieved in automated rapid warming of microdroplets. The combination of droplet vitrification technology and external physical field warming technology is expected to enable high-throughput and automated cryopreservation, which has a promising future in biomedicine and regenerative medicine.

Effects of Vitrification Techniques on the Formation of Skin Cryobank of the Ocelot (Leopardus Pardalis)

BACKGROUND: Skin cryobanks represent important tools for the conservation of the maximum genetic representation of a population, especially those with a certain degree of threat to extinction, such as the ocelot. A relevant step towards the proper establishment of these banks is the definition of adequate cryopreservation techniques for the conservation of the skin. OBJECTIVE: We evaluated the effects of two different techniques [direct vitrification in cryovials (DVC) and solid-surface vitrification (SSV)] for the preservation of ear skin derived from ocelot. MATERIALS & METHODS: For both techniques, we vitrified the ear skin using Dulbecco's modified Eagle's medium with 3.0 M dimethyl sulfoxide, 0.25 M sucrose, and 10% fetal bovine serum. Non-cryopreserved tissues were used as control (control group). All tissues were analyzed for their morphometric characteristics by conventional histology and morphological/functional analysis by cell ability during the culture. RESULTS: While tissues cryopreserved by DVC showed similar values for dermis thickness and number of perinuclear halos to the control, tissues cryopreserved by SSV showed similarities to the control regarding the number of melanocytes, percentage of collagen fibers, and numbers of viable cells by apoptosis analysis. Additionally, none of the vitrification techniques affected stratum corneum thickness, number of keratinocytes, tissue proliferative activity, cell viability, or metabolism. CONCLUSION: Both vitrification techniques (DVC and SSV) can be used for the conservation of ocelot skin; however, SSV guarantees a higher cellular quality after in vitro tissue culture in most of the parameters evaluated, such as viability, metabolism, and apoptosis analysis.

37 Survival rate of immature oocytes postcryopreservation by solid surface vitrification, conventional straw vitrification, or conventional slow-freezing method

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Influence of Intracellular Cryoprotectants on the Conservation of Dermal Somatic Tissues Derived from Antillean Manatees (Trichechus manatus manatus Linnaeus, 1758).

Cryopreservation of somatic tissue has been studied as a tool for the knowledge and conservation of endangered species, such as Antillean manatees. The use of vitrification protocols is an important step in the establishment of biological banks. To decrease the damage caused by this technique, a reduction in the concentration of cryoprotectants has been proposed. Therefore, we aimed to evaluate combinations and concentrations of intracellular cryoprotectants for the conservation of somatic tissues derived from Antillean manatees. Dulbecco's modified Eagle's medium, F-12 composed of 10% fetal bovine serum and 0.25 M sucrose, was supplemented with 3.0 M ethylene glycol (EG) plus 3.0 M dimethyl sulfoxide (DMSO), or 1.5 M EG plus 1.5 M DMSO or 3.0 M EG or 3.0 M DMSO, to produce four solutions for solid-surface vitrification. Noncryopreserved tissues were used as the controls. After warming, tissues derived from four Antillean manatees were evaluated for ultrastructure, histology, and in vitro culture. No differences were observed among the cryopreserved and noncryopreserved tissues in terms of ultrastructure. The dermis thickness of the cryopreserved fragments in solutions containing 3.0 M EG plus 3.0 M DMSO, 3.0 M EG, and 3.0 DMSO was similar to that of the control. Moreover, cryopreservation with 3.0 M EG plus 3.0 M DMSO maintained tissue proliferative capacity potential evaluated by quantification of nucleolar organizing regions. Nevertheless, none of the cryopreserved fragments were able to maintain the number of fibroblasts and the collagen percentage as compared with that of the noncryopreserved fragments. Also, none of the cryopreserved fragments in the different solutions were able to produce cells in vitro. In summary, even reducing the concentration of intracellular cryoprotectants as well as their association did not guarantee the maintenance of cells after in vitro culture. Further studies are needed to optimize the cryopreservation protocols in Antillean manatee somatic tissues.

Effects of L-carnitine and cryodevices on the vitrification and developmental competence of invitro fertilized buffalo oocytes

In the current, study the effect of the addition of L-carnitine (LC) in in vitro maturation (IVM) medium for buffalo oocytes and different cryo-devices on developmental competence. They were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of LC and vitrified by using either straw (ST), open pulled-straw (OPS), solid surface vitrification (SSV). The effects of LC during invitro fertilization and invitro culture on the developmental potential were examined. ST showed a higher recovery rate when using 0.6 mg/ml; viability rate (VR) when using 0.3mg/ml and lower zona pellucida (ZP) and cytoplasmic abnormalities when using 1.2 mg/ml LC (P<0.05). OPS showed a higher recovery and viability rates when using 0.3 mg (P<0.05). SSD showed a higher recovery and VR when using 0.6 mg/ml LC (P<0.05). Maturation and fertilization rates were increased when using 0.3 mg LC and OPS (P<0.05). Cleavage was increased in 0.3 (OPS) and 0.6 mg LC (SSD). The blastocyst rate was increased in 0.3 (OPS) and 0.6 mg/ml (SSD) (P< 0.05). In conclusion, 0.3 mg/ml LC and OPS gives a higher recovery and viability rates but 0.6 mg/ml LC and of both SSD and ST gives a higher recovery and viability rates.

The effects of type III antifreeze protein and human heat shock protein 70 added to the vitrification medium of mouse embryos on in vitro embryonic development rates

The effect of antifreeze protein type III (AFPIII) and human heat shock protein 70 (HHSP70), added to the vitrification medium of mouse embryos, on post-freeze/thaw in vitro embryonic growth rates and cell numbers were investigated. In total 20 female mice were synchronized. After synchronization, 2 females and 1 male were mated in the same cage. Twenty-four h after mating, the embryos were collected at the pronuclear stage. In total 8 groups were established, including a positive control group (C+), a negative control group (C-), and treatment groups that were vitrified in a medium added with 200, 400 and 800 ng/ml of AFPIII (AFPIII200, AFPIII400, AFPIII800), and 1, 2 and 4 µg/ml of HHSP70 (HHSP70-1, HHSP70-2, HHSP70-4). Solid surface vitrification (SSV) medium was prepared for the vitrification of the embryos. Once thawed, in vitro development rates of embryos were followed at 24, 48, 72 and 96 h. Four embryos, which progressed to the full blastocyst stage, were taken from each group and stained with the Hoeschst 33258 and propidium iodine (PI) dyes to determine the inner cell mass (ICM), trophectoderm (TE) and total cell numbers. The groups showed statistically significant difference for in vitro embryonic development rates at 48, 72 and 96 h (p

Cryopreservation of Testicular Tissue from Adult Red-Rumped Agoutis (Dasyprocta leporina Linnaeus, 1758).

Simple SummaryTesticular tissues are composed of many types of germ cells, including early stages which can be grown up to fully formed spermatozoa following grafting or in vitro culture. The systematic banking of testicular tissues at freezing temperatures is useful for future use in assisted reproduction and to improve the reproductive management of rare mammalian species. The present study explored testicular tissue cryopreservation in the red-rumped agouti by slow freezing or vitrification methods, using different combinations of cryoprotectants. Solid-surface vitrification using the combination of dimethyl sulfoxide and ethylene glycol was the most effective protocol to preserve testicular cell morphology and proliferative potential.This study measured the effects of different freezing techniques and permeating cryoprotectants on the preservation of testicular tissues from adult red-rumped agoutis. Tissue biopsies (3.0 mm3) from five individuals were allocated to different experimental groups: control (non-cryopreserved); slow freezing (SF), solid-surface vitrification (SSV), and conventional vitrification (CV). Each method used dimethyl sulfoxide (DMSO), ethylene glycol (EG), or a DMSO + EG combination. Morphology, viability, mitochondrial activity, and proliferative potential were assessed in fresh and frozen tissue samples. Testicular morphology was better using SSV with a combination of DMSO and EG. Across the different cryopreservation approaches, as well as cryoprotectant combinations, cell viability was comparable. Regarding mitochondrial activity, DMSO + EG/SSV or CV, and DMSO + EG/CV were similar to the EG/SF group, which was the best group that provided values similar to fresh control groups. Adequate preservation of the proliferative potential of spermatogonia, Leydig cells, and Sertoli cells was obtained using SSV with DMSO + EG. Overall, the use of SSV with DMSO + EG was the best protocol for the preservation of testicular tissues from adult red-rumped agoutis.

Solid surface vitrification of goat testicular cell suspension enriched for spermatogonial stem cells

This study reports solid surface vitrification (SSV) of goat testicular cell suspensions (TCS) enriched for spermatogonial stem cells (SSCs). The TCS was isolated from pre-pubertal goat testis by enzymatic digestion, enriched for SSCs by filtration and differential plating, and were vitrified-warmed by SSV. The study showed that SSV could successfully vitrify goat TCS although the percentage of live cells in the vitrified-warmed group was lower (74.8 ± 4.1%) than in non-vitrified control (80.6 ± 6.27%). The vitrified-warmed TCS formed putative SSC colonies upon their in vitro culture, but the colony size of vitrified-warmed cells (24.3 ± 1.8 μm) was smaller than those of non-vitrified warmed cells (58.4 ± 2.5 μm). Mitochondrial activity (0.40 vs. 0.38 A U.), population doubling time (33.45 ± 1.25 h vs. 31.86 ± 1.90 h), and the cell proliferation rate (0.72 ± 0.10 vs. 0.75 ± 0.11 per day) of total cells (including putative SSCs and other somatic cells) did not differ (p > 0.05) between control and SSV vitrified-warmed groups. However, during in vitro culture for 96 h, vitrified-warmed cells showed significantly lower (0.75 vs. 1.33 A U.; p < 0.05) mitochondrial activity than non-vitrified controls. The DCFDA assay showed that ROS activity was significantly (p < 0.05) higher in vitrified-warmed cells (52.8 ± 4.1 A U) than non-vitrified control cells (32.8 ± 2.1 AU). In conclusion, our results suggest that SSC-enriched goat TCS could be successfully cryopreserved by SSV. However, ROS-induced damages to cell cytoplasmic components reduce their cellular proliferation and require further improvement in the protocol. To the best of our knowledge, this study is the first report on the SSV of SSC-enriched goat TCS.

Microbiological load and preantral follicle preservation using different systems for ovarian tissue vitrification in the red-rumped agouti

We evaluated the effect of open and closed systems used for ovarian tissue vitrification on the microbiological load and preservation of preantral follicles (PAFs) in the red-rumped agoutis. The ovaries from eight females were recovered and fragmented, with four cortexes fragments immediately fixed and evaluated (fresh group). The other fragments were processed for the solid-surface vitrification method (SSV) or an ovarian tissue cryosystem (OTC) using fetal calf serum, ethylene glycol, and sucrose as cryoprotectants, stored for two weeks, and rewarmed. Subsequently, fragments were subjected to a 24-h in vitro culture and assessed for microbiological load, PAF morphology, and DNA integrity. There was no fungal contamination; however, the vitrified samples from two individuals showed bacterial contamination of 79 200 colony forming units per milliliter (CFU)/mL for SSV and 3120 CFU/mL for OTC. From those samples, a total of eight different types of bacterial colonies were isolated and identified as coagulase-negative Staphylococci and Gram-positive bacilli. Regarding PAF morphology, both systems provided adequate preservation, with values higher than 70% normal follicles observed before and after culture. The TUNEL assay revealed that both SSV (52.39%) and OTC (41.67%) could preserve DNA integrity after vitrification and after 24 h of culture. In summary, both open and closed systems were equally efficient in preserving agouti ovarian tissues, especially concerning the preantral follicle morphology and DNA integrity; however, the OTC seems to provide a less adequate environment for bacterial proliferation.

“Comparison of two closed vitrification methods for vitrifying dromedary camel (Camelus dromedarius) embryos"

A total of 184 dromedary camel embryos were vitrified using a novel vitrification kit specifically developed for camel embryos. These embryos were vitrified using a 3-step process by exposing them to vitrification solutions (VS) containing 20% foetal calf serum (FCS) with (+) or without (−) the addition of bovine serum albumin (BSA). Embryos were then further divided into two groups (<or ≥ 500 μm), vitrified using a closed vitrification system and stored on a Cryolock® embryo storage device or vitrified using solid surface vitrification of the Cryologic Vitrification Method (CVM)™ and stored on a Fibreplug™ embryo storage device. Embryos were then warmed and transferred in pairs, of approximately equal size, into recipient camels on Day 6 after ovulation. A total of 92 embryos were vitrified using the Cryolock; 86 embryos (C-BSA, n = 46; C + BSA, n = 40) survived warming, and were then transferred into 43 recipients. The pregnancy rate (PR) per recipient was 17% (C-BSA: 4/23) and 45% (C + BSA: 9/20) at 20 days after transfer but reduced to 9% and 30% at 60 days of gestation, respectively. For the Fibreplug group, a total of 92 embryos were vitrified, 86 embryos (F-BSA, n = 44; F + BSA, n = 42) survived warming, and were transferred into 43 recipients. The PR per recipient was 73% (F-BSA: 16/22) and 48% (F + BSA: 10/21) at 20 days after transfer but reduced to 50% (11/22) and 33% (7/21) at 60 days of gestation, respectively. Of the 39 pregnancies at 20 days after transfer, 77% (30/39) resulted from embryos with a diameter of 300–450 μm and 23% (9/39) were from embryos with a diameter of 500–850 μm. In conclusion, 1) significantly higher pregnancy rates were achieved when embryos were vitrified using the Fibreplug than using the Cryolock; 2) the effect of BSA on embryo vitrification is dependent on embryo storage device type and embryo diameter; 3) smaller vitrified embryos (<500 μm) tend to produce higher pregnancy rates than larger embryos (≥500 μm), however, the negative effect of embryo size on embryo vitrification is reduced when embryos are frozen using the original novel vitrification kit (no BSA) solid surface vitrification using the CVM.

Epididymal tail solid-surface vitrification as an effective method for domestic cat sperm cryobanking.

This study aimed to describe the viability of domestic feline spermatozoa after epididymal tail vitrification. For this, 10 pairs of testis-epididymis complexes were used. The epididymal tails were vitrified using the solid-surface vitrification (SSV) method, in which two vitrification media containing ethylene glycol (EG) 40% or glycerol (GLY) 40% were tested. Vitrification with the presence of EG resulted in better results for all sperm motility parameters (motility, vigour and CASA) compared with GLY (P < 0.05). There were no statistical differences for sperm viability and acrosome integrity, plasma membrane integrity, or overall health of morphologically normal sperm before or after vitrification among experimental groups. In conclusion, epididymal tail vitrification appears to be a suitable method for long-term storage of cat sperm, especially if the procedure is performed with EG as the cryoprotectant.

Influence of freezing techniques and glycerol-based cryoprotectant combinations on the survival of testicular tissues from adult collared peccaries

The objective of the study was to evaluate the effects of different cryopreservation techniques including glycerol-based cryoprotectant combinations on the structure and viability of testicular tissues from adult collared peccaries. Tissue biopsies (3.0 mm³) from 5 different individuals were allocated to 10 different groups: fresh control; slow freezing (SF), conventional vitrification (CV), or solid-surface vitrification (SSV); each of them using three different combinations of cryoprotectants [dimethyl sulfoxide (DMSO) + ethylene glycol (EG); DMSO + Glycerol; and EG + Glycerol]. After thawing/warming, samples were evaluated for histomorphology, viability, proliferative capacity potential, and DNA integrity. Most effective preservation of testicular histomorphology was achieved using SF and CV with DMSO + EG. However, the use of glycerol-based cryoprotectant combinations increased the occurrence of tubular cell swelling, tubular cell loss and shrinkage from the basal membrane. Cell viability was comparable among cryopreservation methods and cryoprotectant combinations. Regarding cell proliferative capacity, the use of SF with EG + Glycerol and SSV with DMSO + Glycerol impaired the conservation of spermatogonia proliferative potential compared to other treatments. Moreover, CV with DMSO + EG was better than SF with EG + Glycerol for Sertoli cell proliferation potential. Regarding DNA integrity, less damage occurred when using SF with DMSO + EG while more fragmentations were observed when using CV with EG + Glycerol or DMSO + Glycerol as well as SSV with EG + Glycerol or DMSO + Glycerol. In sum, SF and CV appeared to be the most suitable methods for the cryopreservation of adult peccary testicular tissues. Additionally, the use of glycerol-based cryoprotectant combinations did not improve testicular tissues preservation with DMSO + EG being the most efficient option.

Comparison of two culture methods during in vitro spermatogenesis of vitrified-warmed testis tissue: Organ culture vs. hanging drop culture

Solid surface vitrification (SSV) is a cost effective and simple method for testis tissue preservation. Vitrified-warmed testis tissue was successfully cultured using various organ culture methods. In this study, we compared two culture methods viz. hanging drop (HD) and organ culture (OC) methods for in vitro spermatogenesis of goat testis tissue vitrified-warmed by SSV. It was observed that OC method was superior (p < 0.05) to HD method in terms of post-warming metabolic activity of testicular tissue, as measured by MTT assay on Day 7 and Day 14 of culture, respectively. The size of the tissue also played an important role in post-warming metabolic activity and viability (4 mm3: 72.7 ± 1.2% vs. 9 mm3: 62.7 ± 1.3% vs. 16 mm3: 40.5 ± 1.7%) of vitrified tissues with smaller tissue resulting in better result. The vitrification-induced ROS activity significantly decreased during their in vitro culture. Histology and scanning electron microscopy (SEM) showed the rupture of basal membrane, surface morphology and, cell loss due to vitrification. However, histology and immunohistochemistry showed the progression of in vitro spermatogenesis and formation of elongated spermatozoa in both fresh and vitrified-warmed testis tissue cultured by OC method. Taken together, our results suggest that OC method is superior to HD method for culturing goat testis tissue vitrified-warmed by SSV.