Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant.

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an ever-increasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg/L BAP, 0.3 mg/L GA3, and 0.3 mg/L ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity. https://doi.org/10.54680/fr24210110412.

Strip pulled straw: cost effective alternative cryodevice for the vitrification of oocytes.

BACKGROUND: Increased cooling and warming rates by using a suitable cryodevice allows the use of lower cryoprotectant concentration and reduces cryoinjuries as a result of the rapid passage through the 'dangerous' temperature zone. OBJECTIVE: To evaluate the effectiveness of newly customized strip pulled straw (SPS) with respect to post warming quality, viability, and in vitro maturation for immature oocytes post-vitrification of. MATERIALS AND METHODS: SPS was prepared using conventional French mini straw to combine the merits of OPS and the Cryotop system. Immature sheep oocytes were treated in 15% EG + 15% DMSO, loaded on SPS and plunged into liquid nitrogen (LN). Post thaw quality, viability, and maturation rates of oocytes were determined after 1 week storage in LN. RESULTS: SPS achieved a post-thaw morphological survival of 90.9% with 9.0% morphological defects, 96.4% viability and 51% in vitro maturation. In comparison to OPS, SPS had higher post thaw survival (86.5% vs 67.9%) and maturation rate (57.7% vs 50.5%) with lower morphological defects (13.5% vs 32.1%). Cumulus cell loss was the highest among morphological abnormalities of post warm oocytes in SPS (40.9%) and OPS (44.1%). The data showed acceptable post thaw survival, viability and in vitro maturation rate of immature ovine oocytes using SPS as compared to traditional OPS. CONCLUSION: SPS can be used as a cost effective alternative device for oocyte vitrification. Doi: 10.54680/fr23410110212.

Assessment of the stallion sperm acrosome in two different extenders: Spermac stain in comparison with PNA/PSA/PI triple-staining.

Since the stallion acrosome is very small compared to other species and cannot be properly assessed without additional staining, several labelling techniques were developed to facilitate its assessment. The aim of this study was to compare the Spermac stain (Minitüb GmbH) and a PNA/PSA/PI triple-staining detected by flow cytometry with regard to method agreement for detecting non-intact acrosomes within two different extenders. For this purpose, eighteen stallion ejaculates were split in half and diluted with the semen extenders EquiPlus or Gent (Minitüb GmbH) to a final concentration of 50 × 106 sperm/mL, respectively. Subsequently, 126 semen samples were stained with both methods between 4 and 240 h (mean: 63.8 ± 48.9 h) after semen collection. Calculated Intraclass correlation coefficients revealed excellent correlations between both methods for EquiPlus (r = .77, p < .001) and fair correlations for Gent (r = .49, p < .001). Interestingly, flow cytometry detected more non-intact acrosomes in EquiPlus than in Gent (p < .001), whereas the Spermac stain showed no differences (p = .902) between extenders. The poorer method agreement in Gent could be caused by egg yolk artefacts, which made interpretation difficult, so flow cytometry might be preferred. The differences in detected non-intact acrosomes between extenders highlighted the importance of establishing adapted laboratory protocols for different extender types in order to generate comparable results.

Improved cryopreservation media formulation reduces costs of maintenance while preserving function of genetically modified insect cells.

Insect cell lines are an invaluable resource that facilitate various fundamental and applied research programs. Genetically engineered insect cell lines, in particular, serve as a platform through which the function of heterologously expressed proteins can be studied. However, a barrier to more widespread utilization and distribution of insect cell lines, genetically modified or not, is the technical and operational challenge associated with traditional cryopreservation methods, including their dependence on the use of liquid nitrogen facilities, animal or human serum products, and relatively high concentrations of permeating cryoprotectants (e.g., DMSO). Recent innovations in cryopreservation technologies have produced reagents with improved abilities to effectively preserve mammalian cell lines for long periods in regular laboratory deep freezers without using serum products, but their effectiveness in preserving genetically engineered insect cell lines has not yet been evaluated. In this study, we engineered Sf9 cells to express a dopamine receptor and used them as a model for evaluating the efficacy of a novel cryopreservation medium product, C80EZ®-INSECT, in not only preserving cell viability and proliferation efficiency but also maintaining the insect cell line's "functionality" after storage at -80°C. We found that the engineered Sf9 cells frozen using C80EZ®-INSECT with 5% DMSO alone and stored at -80°C for 6 mo displayed higher viability and growth rates than cells frozen using traditional fetal bovine serum (FBS)-based cryopreservation media with 10% DMSO that were stored at -80°C or in liquid nitrogen for the same period of time. We also found that after 6 mo of storage at -80°C or in liquid nitrogen the cells retained a responsiveness to dopamine comparable to that of the initial cell line, regardless of the cryopreservation reagent used. These results suggest that, due to the unique characteristics of C80EZ®-INSECT in preventing ice recrystallization and reducing ice crystal size and cellular apoptosis during cryostorage procedures, it is an effective cryopreservation reagent for genetically engineered Sf9 cells, and it practically eliminates the need for liquid nitrogen-based storage facilities and FBS-based cryopreservation formulations, as well as reduces the use of permeating cryoprotectants.

Quantitative assessment of intracellular/extracellular dimethyl sulfoxide concentrations during freezing with low-temperature confocal Raman micro-spectroscopy.

Although cryopreservation plays an indispensable role in the clinical application of cell therapy, the research on the osmotic behavior of cells during freezing is still at the level of theoretical models, and quantitative experimental data are still lacking. Therefore, the Raman spectra of dimethyl sulfoxide (DMSO) solutions with different standard concentrations (5%-80% v/v) were recorded experimentally to establish a quantitative evaluation method with the intensity ratio of different labeled peaks to the hydrogen bonding peak (as the internal standard) of water molecules in relation to different DMSO concentrations. By using this method, the characteristics of quantitative changes in intra- and extracellular concentrations under three different freezing methods were explored, including direct freezing, ice seeding freezing and vitrification. It was found that the intracellular concentration (@ -50 °C) after the ice seeding (@ -7 °C) freezing (1 °C min-1) method could reach 41.6%-49.2%, significantly higher than that of the direct freezing method (1 °C min-1 to -50 °C) of 32.4%-39.1%. Moreover, it is worth noting that the quantitative values of concentrations (@ -50 °C) of the ice seeding freezing are more consistent with the primary saturation curve of the DMSO solution. Thus, for the first time, it was revealed from the experimental data that the fundamental reason for the improvement of cell survival after ice seeding operation was pre-dehydration, higher concentration and smaller osmotic pressure difference between the inside and outside of the cell. These results also confirmed the validity of the famous two-factor hypothesis and more work will be carried out in depth.

Real-time computer assisted measurement of oocyte and embryo volume for assessment of transport parameters.

Cryopreservation of gametes has revolutionized both animal agriculture and human reproductive medicine. Although many new technologies have tremendously improved the cryopreservation of oocytes and embryos, osmotic stress encountered during the equilibration process can cause their loss of function. Rational cryoprotective agent (CPA) equilibration strategies can be used to minimize this stress but require trained personnel to monitor the process in individual oocytes or embryos or require the use of suboptimal average transport parameter values in mathematically guided protocols. To enable individually optimized equilibration of CPAs in individual cells, here we establish experimental and computational techniques to track the osmotic behavior of individual bovine oocytes and embryos during CPA equilibration in real time. We designed a microfluidic device to provide a controlled flow of CPA and modified standard image analysis techniques to estimate real-time cell volume changes. In particular, we used a level-set method to define a boundary within a contour plot which could automate the image analysis process. A colour based level set algorithm coupled with contour smoothing not only provided the best fit but also reduced the segmentation time to well under a second per image. The accuracy of the automated method was comparable to human segmented images for both oocytes and embryos. This technology should enable both rapid evaluation of key biophysical parameters in oocytes and embryos undergoing CPA equilibration and the development of real-time feedback-control of CPA equilibration, enabling individual oocyte- and embryo-specific optimal protocols.

Cryopreservation of six Symbiodiniaceae genera and assessment of fatty acid profiles in response to increased salinity treatments.

Symbiodiniaceae are a diverse group of dinoflagellates, the majority of which are free-living and/or associated with a variety of protists and other invertebrate hosts. Maintenance of isolated cultures is labour-intensive and expensive, and cryopreservation provides an excellent avenue for their long-term storage. We aimed to cryopreserve 15 cultured isolates from six Symbiodiniaceae genera using dimethyl sulfoxide (DMSO) as the cryoprotectant agent (CPA). Under 15% DMSO, 10 isolates were successfully cryopreserved using either rapid freezing or controlled-rate freezing. Cultures that failed or had low survival, were subjected to (1) a reduction of CPA to 10%, or (2) increased salinity treatment before freezing. At 10% DMSO, three further isolates were successfully cryopreserved. At 15% DMSO there were high cell viabilities in Symbiodinium pilosum treated with 44 parts per thousand (ppt) and 54 ppt culture medium. An isolate of Fugacium sp. successfully cryopreserved after salinity treatments of 54 ppt and 64 ppt. Fatty acid (FA) analyses of S. pilosum after 54 ppt salinity treatment showed increased saturated FA levels, whereas Fugacium sp. had low poly-unsaturated FAs compared to normal salinity (34 ppt). Understanding the effects of salinity and roles of FAs in cryopreservation will help in developing protocols for these ecologically important taxa.

Assessment of larval quality of two bivalve species, Crassostrea angulata and Chamelea gallina, exposed and cryopreserved with different cryoprotectant solutions.

Marine bivalves are valuable resources, however, some shellfish populations are endangered due to factors such as anthropogenic pressure, pathologies or lack of reproduction synchrony. Portuguese oyster (Crassostrea angulata) and striped venus clam (Chamelea gallina) have high socio-economic value and their endangered natural populations require rehabilitation. Cryopreservation is a valuable method for the preservation and management of genetic resources for aquaculture and restocking. Larvae cryopreservation is particularly valuable since diploid organisms are obtained upon thawing. The objective of this work was the establishment of C. angulata and C. gallina D-larvae cryopreservation through the selection of permeant cryoprotectant in the freezing solution, namely ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). Cryoprotectants exposure showed that, in C. angulata, Me2SO promoted significantly higher incidence of abnormalities and enhanced glutathione reductase activity when compared to control (larvae without cryoprotectant exposure) or even to EG treatment. However, for both species, EG significantly reduced D-larvae average path velocity (VAP). In C. angulata post-thaw D-larvae, EG treatment promoted significantly lower motility and velocity when compared to control and Me2SO treatment. Superoxide dismutase (SOD) activity showed a reduction in C. angulata post-thaw D-larvae when compared to control, which was compensated by the enhancement of glutathione peroxidase (GPX) activity. In C. gallina post-thaw D-larvae, only motility, velocity and SOD activity were significantly lower than control. Therefore, the best treatment to cryopreserve C. angulata D-larvae was EG while for C. gallina Me2SO produced better results. This work established for the first time D-larvae cryopreservation protocols for C. angulata and C. gallina.

Assessment of thawed sperm quality from feline species: Ocelot (Leopardus pardalis) and oncilla (Leopardus gutullus).

This study aimed to evaluate the cryopreservation effects on the semen of oncilla (Leopardus guttulus, n = 5, 15 ejaculates) and ocelot (Leopardus pardalis, n = 5, 17 ejaculates) and compare two extenders (commercial and non-commercial extender). An andrological exam was conducted (testicle measurements and penis evaluation), including semen collection by electroejaculation. After collection, the semen was assessed to volume, color, pH, sperm motility, vigor, sperm number in the ejaculate, viability, membrane integrity, and sperm morphology. Samples were centrifuged (300 g for 10 min) and pellet diluted in two extenders (TRIS/glucose/egg yolk and BotuCRIO®), packed into 0.25 mL French straws (20 × 106 spermatozoa/mL), equilibrated at 5 °C for 1 h (<0.5 °C/min), freezing in nitrogen vapor for 20 min. Thawing was achieved at 46 °C for 15 min. Thawed samples were evaluated to the same characteristics and ultrastructural analysis. There is no difference for extenders, but in ocelot the spermatozoa maintained higher quality after thawing. Major defects were increased in thawed samples, especially acrosome injuries, in both species. Semen contamination by urine was remarkable to oncilla (53% of the ejaculates) which can have reduced sperm cryoresistance of this species. Ultrastructural analysis endorsed morphological analysis under light microscopy and identified cells with acrosome vesiculation. In conclusion, the spermatozoa of ocelot were more cryoresistent and the extender commercial and non-commercial were suitable for their cryopreservation. Other extenders should be investigated for oncilla.

The Assessment of Various Factors Affecting the Postwarming Viability and Developmental Capability of Goat Metaphase II Oocytes Vitrified by Cryotop.

The effects of the equilibration time, the vitrification procedure, and the warming procedure on the quality of goat oocytes vitrified by Cryotop were assessed. In the first part of the study, oocytes were exposed to 10% dimethyl sulfoxide (DMSO) and 10% ethylene glycol (EG) for 1, 3, 5, or 10 minutes, respectively, followed by vitrification. In the second part, after equilibration in 7.5% DMSO +7.5% EG for 3 minutes, 10% DMSO +10% EG for 3 minutes, or 4% EG for 10 minutes, oocytes were equilibrated in 15% DMSO +15% EG, 20% DMSO +20% EG, or 35% EG for 30 seconds before vitrification. The vitrification procedures were designated as first vitrification procedure (VPI), second vitrification procedure (VPII), and third vitrification procedure (VPIII), respectively. In the third part, oocytes vitrified using VPIII were warmed by the three procedures (first warming procedure [TPI], second warming procedure [TPII], or third warming procedure [TPIII]) containing different concentrations of trehalose. The results showed that after equilibration for 1 or 3 minutes in 10% DMSO and 10% EG, the viability and developmental capability of vitrified oocytes were significantly superior to the groups after equilibration for over 5 minutes (p < 0.05). With the VPIII procedure, the frequencies with normal morphology, cleavage, and blastocyst formation of vitrified oocytes were 91.87% ± 4.14%, 76.51% ± 4.37%, and 39.84% ± 2.91%, respectively, demonstrating a significant increase compared to the VPI or VPII group (p < 0.05). The rates of vitrified oocytes with normal morphology and cleavage in the TPI group were higher than the TPII or TPIII group (p < 0.05). In conclusion, equilibration in 10% DMSO and 10% EG for <3 minutes benefits the viability of vitrified oocytes. EG may be more efficient for vitrification of goat oocytes compared to DMSO. Higher concentrations (more than 1 M) of trehalose enhance cryosurvival of goat oocytes when warming.

Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts.

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

Assessment of the cytoprotective effect of the homeopathic compound Canova® on African green monkey kidney (VERO) cell line exposed to the drug dipyrone sodium.

Dipyrone or metamizole is one of the most frequently used analgesic worldwide. Despite its widespread use, this drug may exert genotoxic and cytotoxic effects on lymphocytes. Therefore, studies with therapeutic agents that may provide protection against these effects are important. The homeopathic compound Canova® (CA) appears to be a beneficial candidate for preventing DNA damage and cellular lethality, since this compound acts as an immunomodulator associated with cytoprotective actions. Hence, the aim of the present investigation was to determine the potential cytoprotective effects of CA using cell line VERO as a model. VERO cells were incubated with sodium dipyrone and subsequently subject to the comet, apoptosis and immunocytochemistry assays. Data demonstrated that sodium dipyrone induced an increase in DNA damage index (DI) employing the comet assay. However, when VERO cells were co-treated with CA at the three concentrations studied, a significant reduction in DI was observed, indicating an antigenotoxic effect attributed to CA. Further dipyrone induced an elevation in %apoptosis at 24 and 48 hr. However, when dipyrone was co-incubated with CA, a significant reduction in %apoptosis was noted at the three concentrations of CA employed. Results from immunocytochemical analysis showed a rise in the expression of caspase 8 and cytochrome C when cells were exposed to dipyrone. In contrast, co-treatment of dipyrone and CA significantly reduced the effect of dipyrone. Therefore, evidence indicated that CA acted as an anticytotoxic and antigenotoxic agent counteracting damage induced by dipyrone.

PERSPECTIVE: Rooster Spermatozoa Cryopreservation and Quality Assessment.

Unsuccessful rooster fertility following cryopreservation may be linked to specific changes in spermatozoa quality, which can be determined using various methods. These determinations also facilitate the design of improved freezing and thawing processes. Here, we update the current state of methodologies available for the assessment of rooster semen quality after cryopreservation. Computer-assisted sperm analyses (CASA) is one of the main systems used to analyse motion parameters of spermatozoa (total motility, progressive motility and motion parameters). Moreover, fluorescent techniques and flow cytometry can improve the assessment of various aspects of semen quality (viability, acrosome status, mitochondrial potential, lipid peroxidation, DNA damage, lipid peroxidation and cell debris removal) using specific fluorescent markers such as ethidium bromide, Yo-Pro-1, Annexin V, propidium iodide, SYBR-14, PNA, JC-1, BODIPY, acridine orange and DRAQ5. Transmission electron microscopy also yields valuable information on spermatozoa ultrastructure. The application of these techniques to rooster spermatozoa is reviewed in relation to specific freezing techniques, the effects of cryoprotective agents (CPAs) and extenders, and the determination of spermatozoa quality after cryopreservation.

In vitro Assessment of Tris Egg Yolk and Soybean Lecithin Based Extenders for Cryopreservation of Crossbred Ram Semen.

BACKGROUND: The replacement of egg yolk with alternative plant-derived soybean lecithin is gaining interest in both animal and human sperm cryopreservation owing to biosecurity issues with egg yolk based extenders. OBJECTIVE: To evaluate the comparative effect of egg yolk and soyabean lecithin based extenders on the quality of cryopreserved crossbred ram semen. METHODS: Pooled ejaculates (total ejaculates = 36) were divided into two aliquots and extended with Tris egg yolk extender (Tris extender) and soybean lecithin based commercial extender (Ovixcell) RESULTS: Among the two extenders, Ovixcell showed better sperm quality both at the pre-freeze (Sperm motility) and post-thaw stages. Lower malondialdehyde (MDA) level (nmol/mL) was observed in Ovixcell as compared to Tris extender. Both sperm quality and MDA level decreased significantly (P < 0.05) from pre-freeze to post-thaw in both the extenders. CONCLUSION: The findings of the present study indicate that Ovixcell is a comparable alternative to Tris extender for the cryopreservation of crossbred ram semen.

The efficacy of ice recrystallization inhibitors in rat lung cryopreservation using a low cost technique for ex vivo subnormothermic lung perfusion.

Although lung transplant remains the only option for patients with end-stage lung failure, short preservation times result in an inability to meet patient demand. Successful cryopreservation may ameliorate this problem; however, very little research has been performed on lung cryopreservation due to the inability to prevent ice nucleation or growth. Therefore, this research sought to characterize the efficacy of a small-molecule ice recrystallization inhibitor (IRI) for lung cryopreservation given its well-documented ability to control ice growth. Sprague-Dawley heart-lung blocks were perfused at room temperature using a syringe-pump. Cytotoxicity of the IRI was assessed through the subsequent perfusion with 0.4% (w/v) trypan blue followed by formalin-fixation. Ice control was assessed by freezing at a chamber rate of -5 °C/min to -20 °C and cryofixation using a low-temperature fixative. Post-thaw cell survival was determined by freezing at a chamber rate of -5 °C/min to -20 °C and thawing in a 37 °C water bath before formalin-fixation. In all cases, samples were paraffin-embedded, sliced, and stained with eosin. The IRI studied was found to be non-toxic, as cell membrane integrity following perfusion was not significantly different than controls (p = 0.9292). Alveolar ice grain size was significantly reduced by the addition of this IRI (p = 0.0096), and the addition of the IRI to DMSO significantly improved post-thaw cell membrane integrity when compared to controls treated with DMSO alone (p = 0.0034). The techniques described here provide a low-cost solution for rat ex vivo lung perfusion which demonstrated that the ice control and improved post-thaw cell survival afforded by IRI-use warrants further study.

Development of a fast and user-friendly cryopreservation protocol for sweet potato genetic resources.

Sweet potato (Ipomoea batatas) is one of the ten most important staple crops and provides a livelihood for many people around the globe. To adapt to ever-changing circumstances farmers and breeders need to have access to a broad diversity of germplasm. This study focuses on the development of a cryopreservation protocol that allows the long term storage of different sweet potato cultivars. For this, a droplet vitrification protocol was optimized, comparing several parameters; preculture method (0.3 M sucrose vs no preculture); meristem position (axillary vs apical); plant age (3 to 9 weeks); regeneration medium (MS + 2.22 µM BA, Hirai and MS); and length of loading solution treatment (20 to 360 min). Two months after cryopreservation, the regeneration rates of the meristems were compared, which resulted in significant differences for the preculture method, meristem position and loading solution. With these new insights an optimized droplet vitrification protocol was developed with the following parameters: use of 3-9 week old axillary meristems, no preculture phase, 20 min LS treatment, 30 min PVS2 treatment, exposure to liquid nitrogen by droplet vitrification, warming treatment in RS for 15 min, 1 day 0.3 M sucrose recuperation culture, 1 month MS + 2.22 µM BA followed by 1 month of MS cultures. This protocol was subsequently tested on 10 representative accessions resulting in a post cryopreservation regeneration rate of more than 40% for 70% of the tested cultivars, showing that this protocol could be implemented for a large portion of existing sweet potato collections.

In vitro assessment of egg yolk-, soya bean lecithin- and liposome-based extenders for cryopreservation of dairy bull semen.

The study was conducted to compare the effect of four commercially available extenders (Triladyl®- egg yolk-based; Andromed® and Bioxcell®-plant based and Optixcell®-liposome-based) on post-thaw sperm quality and functionality variables evaluated using computer-assisted sperm analysis and flow cytometry. A total of 30 ejaculates from five bulls were analysed. With use of Triladyl®, sperm had a greater post-thaw total motility than with use of Bioxell® and Optixcell® but there was no difference as compared with use of Andromed® with the greatest (P < 0.05) percentage of progressively motile cells. With use of Optixcell®, there was a greater (P < 0.05) percentage of sperm with an intact membrane than with use of Triladyl® and Bioxcell®, but values were similar with use of Andromed®. Acrosome damage in semen preserved with use of Optixcell® was less than with use of Bioxcell® and Andromed®. With use of Optixcell®, there was a greater percentage of viable spermatozoa with a lesser lipid disruption (P < 0.05) when compared with the other extenders. Production of peroxides was greater for sperm cryopreserved with use of Triladyl® and Optixcell® while less superoxide was produced in the samples cryopreserved with the egg yolk-based extender. Optixcell® appears to be a promising alternative to replace traditional egg yolk extenders. With use of Optixcell®, however, there were greater peroxide concentrations after thawing. With use of Andromed®, there were similar results as with use of Optixcell®, therefore, it could be an effective substitute for egg-yolk based media due to the greater proportion of highly and progressively motile spermatozoa at thawing.

Safety assessment of poly(N-vinylcaprolactam) as a potential drug carrier in extenders for boar sperm cryopreservation.

Polymers may be used to deliver compounds in freezing extenders to minimize injuries in spermatozoa during cryopreservation, although their activity and toxicity for boar sperm are unknown. This study investigated the effects of the polymer (N-vinylcaprolactam) (PNVCL), when included in extenders for boar sperm cryopreservation. In Experiment 1, sperm was exposed to PNVCL at: 0 (control); 39.1; 78.1; 156.3; and 312.5 µg/mL. Spermatozoa structure, kinetics and biochemical functions were unaltered in contact with PNVCL at 38 °C (P > .05) but declined with prolonged exposure (10, 60 and 120 min) in all treatments (P > .05). In Experiment 2, after inclusion of PNVCL in the freezing extender at the same concentrations, post-thawing sperm quality did not differ compared to the control (P > .05). Lipid peroxidation and the production of reactive oxygen species were the only parameters of sperm quality that were unaffected in both experiments, even after contact with PNVCL for 120 min (P > .05). As no negative effects were observed in post-thawing boar sperm quality, PNVCL did not incur in cytotoxicity and may be a potential carrier for antioxidants in freezing extenders.

Assessment of a germplasm bank for the autochthonous cattle breed Asturiana de la Montaña: Extender (Biociphos vs. BIOXCell) affected sperm quality but not field fertility.

Semen banking is critical to preserving rare and autochthonous breeds. However, protocols can change with time, leaving heterogeneous semen batches. The objective of this study was to assess differences in sperm quality and field fertility. We report differences between batches frozen with the Biociphos and BIOXCell extenders in the Asturiana de la Montaña cryobank (autochthonous and endangered breed, Northern Spain). Doses from 48 bulls were analysed by CASA and flow cytometry. The 85-days non-return rates from AI records were used to assess the fertility of 23,853 AI. BIOXCell showed higher quality post-thawing. Differences increased after a 5-hr incubation at 37°C, and Biociphos yielded doses with lower resilience. Field fertility did not differ between extenders (Biociphos: 57.4% ± 1.2; BIOXCell: 56.6% ± 3.0), possibly because of AI protocols compensating for differences in quality. However, this needs to be confirmed by controlled intervention studies. In conclusion, batches frozen with Biociphos may require specific strategies for compensating for the lower sperm quality. Regular surveys and evaluation of cryobank procedures may be useful to characterizing stored batches and defining strategies to guaranteeing success in their future use.

On the assessment of the stability of vitrified cryo-media by differential scanning calorimetry: A new tool for biobanks to derive standard operating procedures for storage, access and transport.

When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me2SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4 wt% changed the devitrification time with a factor of 342 and 271 for Me2SO and EG, respectively. Concentration changes in EG/Me2SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.