Cryoprotective Agents Research Articles

Ice formation and its elimination in cryopreservation of oocytes

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming was examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions—consistent with crystallization of most free water—during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-warming oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.

Cryopreservation of goat spermatozoa in the Mekong Delta region of Vietnam: evaluating cryoprotectant effectiveness

Conserving and improving the quality of breeds is crucial for enhancing small ruminant production in Vietnam. To support this purpose, cryopreservation was offered as a promising method for semen preservation and maintaining genetic diversity. Moreover, many small-scale farmers in Mekong Delta rely on goats for both income and nutrition, making them an important part of farming activities. This study aimed to develop a sperm cryopreservation protocol and identify the optimal cryoprotectant for goat sperm in the Mekong Delta region of Vietnam. Semen samples were diluted with TCG-EggYolk freezing medium containing cryoprotectant agent (CPA) in a 1:1 ratio. The samples were then placed in 0.5 mL French straws, cooled to 15 °C and 5 °C, and subjected to nitrogen vapor before being immersed in liquid nitrogen at -196 °C. Experiment 1 utilized Glycerol as the CPA in concentrations of 0 %, 5 %, 8 %, and 11 %. Experiment 2 employed dimethyl sulfoxide (DMSO) as the CPA with the same concentrations. Thawed samples were evaluated for sperm quality. Results indicated that in Experiment 1, increasing the Glycerol concentration improved the overall motility, viability, and membrane integrity of sperm. The best outcomes were achieved with 8 % Glycerol, resulting in progressive motility of 57.5 % (P<0.05). In Experiment 2, the extender containing 8 % DMSO demonstrated significantly better performance in terms of overall motility, viability, and membrane integrity compared to 5 % or 11 % DMSO. Progressive motility was 53.5 % (P<0.05). In conclusion, the study determined that using 8 % Glycerol or 8 % DMSO is the optimal choice for freezing goat sperm in the Mekong Delta region of Vietnam.

L-Proline and GelMA hydrogel complex：An efficient antifreeze system for cell cryopreservation

Cryopreservation of biological samples is an important technology for expanding their applications in the biomedical field. However, the quality and functionality of samples after rewarming are limited by the toxicity of commonly used cryoprotectant agents (CPAs). Here, we developed a novel preservation system by combining the natural amino acid l-proline (L-Pro) with gelatin methacryloyl (GelMA) hydrogels. Compared with dimethyl sulfoxide (DMSO), L-Pro and GelMA demonstrated excellent biocompatibility when co-culturing with cells. Cryopreservation procedures were optimized using 3T3 as model cells. The results showed that rapid cooling was the most suitable cooling procedure for L-Pro and GelMA among the three cooling procedures. Co-culturing with cells for 3 h before cryopreservation, 6 % L-Pro +7 % GelMA had the highest survival rate, reaching up to 80 %. Differential Scanning Calorimetry (DSC) analysis showed that 6 % L-Pro + 7 % GelMA lowered the freezing point of the solution to −4.2 °C and increased the unfrozen water content to 20 %. To the best of our knowledge, this is the first report of cell cryopreservation using a combination of L-Pro and GelMA hydrogels, which provides a new strategy for improving cell cryopreservation.

Cryopreservation of neuroectoderm on a pillar plate and in situ differentiation into human brain organoids.

Cryopreservation in cryovials extends cell storage at low temperatures, and advances in organoid cryopreservation improve reproducibility and reduce generation time. However, cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles, we developed a cryopreservation technique using a pillar plate platform. To illustrate cryopreservation application to human brain organoids (HBOs), early-stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NEs) in an ultralow atachement (ULA) 384-well plate. These NEs were transferred and encapsulated in Matrigel on the pillar plate. The early-stage HBOs on the pillar plate were exposed to four commercially available CPAs, including PSC cryopreservation kit, CryoStor CS10, 3dGRO, and 10% DMSO, before being frozen overnight at -80°C and subsequently stored in a liquid nitrogen dewar. We examined the impact of CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of early-stage HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving these HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability post-cryopreservation than larger ones. An incubation period of 80 minutes with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400 - 600 μm. These cryopreserved early-stage HBOs successfully matured over 30 days, exhibiting gene expression patterns akin to non-cryopreserved HBOs. The cryopreserved early-stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids, by integrating cryopreservation, thawing, culturing, staining, rinsing, and imaging processes within a single system, thereby preserving the 3D structure of the organoids.

High throughput method for simultaneous screening of membrane permeability and toxicity for discovery of new cryoprotective agents.

Vitrification is the most promising method for cryopreservation of complex structures such as organs and tissue constructs. However, this method requires multimolar concentrations of cell-permeant cryoprotective agents (CPAs), which can be toxic at such elevated levels. The selection of CPAs for organ vitrification has been limited to a few chemicals; however, there are numerous chemicals with properties similar to commonly used CPAs. In this study, we developed a high-throughput method that significantly increases the speed of cell membrane permeability measurement, enabling ~100 times faster permeability measurement than previous methods. The method also allows assessment of CPA toxicity using the same 96-well plate. We tested five commonly used CPAs and 22 less common ones at both 4 °C and room temperature, with 23 of them passing the screening process based on their favorable toxicity and permeability properties. Considering its advantages such as high throughput measurement of membrane permeability along with simultaneous toxicity assessment, the presented method holds promise as an effective initial screening tool to identify new CPAs for cryopreservation.

Analysis of cryopreservation media thermophysical characteristics after ultra-rapid cooling through differential scanning calorimetry

Cryoprotective agents play a critical role in minimizing cell damage caused by ice formation during cryopreservation. However, high concentrations of CPAs are toxic to cells and tissues. Required concentrations of CPAs can be reduced by utilizing higher cooling and warming rates, but insight into the thermophysical properties of biological solutions in the vitrification method is necessary for the development of cryopreservation protocols. Most studies on thermophysical properties under ultra-rapid cooling conditions have been qualitatively based on visualization. Differential scanning calorimetry methods are ideal for studying the behavior of biomaterials in various freezing conditions quantitatively and accurately, though previous studies have been predominantly restricted to slower cooling rates. Here, we developed an ultra-rapid cooling method for DSC that can achieve minimal cooling rates exceeding 2000 °C/min. We investigated the thermophysical vitrification behavior of ternary solutions of phosphate buffer saline (1X), dimethyl sulfoxide or glycerol and ice blocking polymers (X-1000 or Z-1000). We quantified the impact of solute concentration on ice crystal formation during rapid cooling. Our findings support the expectation that increasing the solute concentration reduces the amount of ice formation, including devitrification. Devitrification increases from 0 % to 40 % (v/v) Me2SO and then reduces significantly. The relative amounts of devitrification to the total ice formation are 0 %, 60 %, 0 % in 20 %, 40 %, 60 % (v/v) Me2SO, and 2 %, 48 %, 49 % in 20 %, 40 %, 60 % (v/v) glycerol, respectively. The results suggest that at low concentrations, such as below 20 % (v/v) for Me2SO or glycerol, increasing the warming rate after ultra-rapid freezing is not essential to eliminate devitrification. Furthermore, ice blocking polymers do not reduce ice formation substantially and cannot eliminate devitrification under ultra-rapid cooling conditions. In conclusion, our results provide insights into the impact of solute concentration on ice formation and devitrification during rapid cooling, which can be practical for optimizing cryopreservation protocols.

L-Proline as a Cryoprotective Agent for the Preservation of Galea Spixii Skin Fibroblasts.

Somatic cell biobanking is a promising strategy for developing reproductive techniques. Although cryopreservation, a technique used for creating biobanks, has been performed on Galea spixii, structural and physiological damage to its cells highlight the need to optimize the cryoprotective solution being used. Therefore, the osmoprotective activity of 5 mM L-proline was evaluated as an alternative cryoprotectant for G. spixii fibroblast conservation. The concentration was defined based on previous studies conducted on mammalian cells. Cells derived from the skin of six individuals were cultured until the fifth passage were cryopreserved under the following treatments: (i) control (non-cryopreserved); (ii) a solution with 10% dimethyl sulfoxide (Me2SO), 10% fetal bovine serum (FBS), and 0.2 M sucrose; (iii) a solution with 10% Me2SO, 10% FBS, and 5 mM L-proline; and (iv) a solution with 10% Me2SO, 10% FBS, 0.2 M sucrose, and 5 mM L-proline. Tests were conducted to analyze cell morphology, viability, metabolism, proliferation, and apoptosis; reactive oxygen species (ROS) levels; and mitochondrial membrane activity (ΔΨm). A reduction in the number of viable cells (72.3% ± 1.2%) was observed in the sucrose-containing group compared to the control (86.7% ± 2.0%) and L-proline (88.4% ± 1.8% and 87.8% ± 2.1%) groups. After apoptotic analysis, a reduction in the number of viable cells was observed in the group with sucrose alone (74.6% ± 4.1%) compared to the control group (88.2% ± 1.1%). The ROS levels (1.03 ± 0.5 and 1.07 ± 0.5, respectively) and ΔΨm values (0.99 ± 0.42 and 1.22 ± 0.73, respectively) observed in the groups with L-proline were similar to that observed in the control group (1.00 ± 0.5 and 1.00 ± 0.4, respectively). Moreover, no difference was observed between groups for cell morphology, metabolism, or proliferation. Thus, L-proline is a cryoprotectant agent that can be used during G. spixii fibroblast cryopreservation, alone or with sucrose. In addition, we developed an adequate biobank for G. spixii, whereby stored cells could be used for reproductive techniques.

Alternative cryoprotective agent for corneal stroma-derived mesenchymal stromal cells for clinical applications

Cryopreservation of human corneal stroma-derived mesenchymal stromal cells (hCS-MSCs) with dimethylsulfoxide (DMSO) as a cryoprotective agent (CPA) has not been previously compared to that with glycerol under standard conditions. The hCS-MSCs were hereby cryopreserved with both compounds using a freezing rate of 1 °C/minute. The CPAs were tested by different concentrations in complete Minimum Essential Medium (MEM) approved for good manufacturing practice, and a medium frequently used in cell laboratory culturing—Dulbecco’s modified eagle serum. The hCS-MSCs were isolated from cadaveric human corneas obtained from the Norwegian Eye Bank, and immunophenotypically characterized by flow cytometry before and after cryopreservation. The survival rate, the cellular adhesion, proliferation and cell surface coverage after cryopreservation of hCS-MSCs has been studied. The hCS-MSCs were immunofluorescent stained and examined for their morphology microscopically. The results showed that cryopreservation of hCS-MSCs in MEM with 10% glycerol gives a higher proliferation rate compared to other cryopreserving media tested. Based on the results, hCS-MSCs can safely be cryopreserved using glycerol instead of the traditional use of DMSO.

Disaccharide-assisted inkjet freezing for improved cell viability

Non-permeable disaccharides are widely used as cryoprotectant agents due to their low cytotoxicity, but their protective effect is insufficient when the disaccharides are present only extracellularly. On the other hand, cryoprotectant agent (CPA)-free cryopreservation has been recently achieved by instantaneously inkjet-freezing cells as tiny droplets. However, CPA-free cryopreservation requires skilled handling operations due to instability of the vitreous water without the CPA. In this study, the effectiveness of separately adding two types of disaccharides in inkjet freezing of 3T3 cells was evaluated and the following results were obtained. First, trehalose showed the highest effect at 0.57 M, twice the plasma osmolarity, with a maximum cell viability of over 90 % when freezing 70 pL droplets. However, higher concentrations of trehalose decreased cell viability due to damage caused by dehydration. Similarly, sucrose gave cell viability close to 90 % at 0.57 M with 70 pL droplets, and higher concentrations decreased cell viability. Next, the relationship between minimum trehalose concentrations to prevent intracellular and extracellular ice crystal formation and droplet size was analyzed. The results indicated that trehalose of less than 0.57 M was able to inhibit intracellular ice crystal formation even in the largest droplet used in this study, 450 pL, while trehalose of nearly 0.57 M was required to inhibit extracellular ice crystal formation in the smallest droplet, 70 pL. In other words, the suppression of extracellular ice crystals by the addition of CPA was shown to be crucial in improving the viability of inkjet superflash freezing.

The Importance of Cryoprotective Agents (CPAS) in the Cryopreservation Process for the Preservation of Plant Resources

The Importance of Cryoprotective Agents (CPAS) in the Cryopreservation Process for the Preservation of Plant Resources

Potential of fructans as natural cryoprotectant agents in plant cryopreservation: concept validation on Arabidopsis thaliana L.

BACKGROUND: Today, synthetic chemicals are used in vitrification solutions for cryopreservation studies to mimic natural cryoprotectants that supply tolerance to organisms in nature against freezing stress. In the case of plants, PVS2, containing glycerol, dimethyl sulfoxide (Me2SO), ethylene glycol and sucrose, is considered as the golden standard for successful cryopreservation. However, Me2SO can generally cause toxicity to certain plant cells, adversely affecting viability after freezing and/or thawing. Hence, the replacement (or substantial reduction) of Me2SO by cheap, non-toxic and natural cryoprotectants became a matter of high priority to vitrification solutions or reducing their content gained escalating importance for the cryopreservation of plants. Fructans, sucrose derivatives mainly consisting of fructose residues, are candidate cryoprotectants. OBJECTIVE: Inspired by their protective role in nature, we here explored, for the first time, the potential of an array of 8 structurally different fructans as cryoprotectants in plant cryopreservation. MATERIALS AND METHODS: Arabidopsis thaliana L. seedlings were used as a model system with a one-step vitrification method. PVS2 solutions with different Me2SO and fructan contents were evaluated. RESULTS: It was found that branched low DP graminan, extracted from milky stage wheat kernels, led to the highest recovery (85%) among tested fructans with 12.5% Me2SO after cryopreservation, which was remarkably close to the viability (90%) observed with the original PVS2 containing 15% Me2SO. Moreover, its protective efficacy could be further optimized by addition of vitamin C acting as an antioxidant. CONCLUSION: Such novel formulations offer great perspectives for cryopreservation of various crop species.

Effects of chilling and cryoprotectants on glycans in shrimp embryos

Glycans are carbohydrates present in every organism that bind to specific molecules such as lectins, a diverse group of proteins. Glycans are vital to cell proliferation and protein trafficking. In addition, embryogenesis is a critical phase in the development of marine organisms. This study investigated the effects of chilling and cryoprotective agents (CPAs) on glycans in the embryos of Stenopus hispidus. The glycan profiles of embryos of S. hispidus at the heartbeat stage were analyzed using lectin arrays. The results of analyses revealed that mannose was the most abundant glycan in the S. hispidus embryos; mannose is crucial to cell proliferation, providing the energy required for embryonic growth. Additionally, the results reveled that chilling altered the content of several glycans, including fucose and Gla-GlcNAc. Chilling may promote monosaccharide accumulation, facilitating osmotic regulation of cells and signal molecules to aid S. hispidus embryos in adapting to cold conditions. Changes were also observed in the lectins NPA, orysata, PALa, ASA, discoidin II, discoidin I, UDA, PA-IIL, and PHA-P after the samples were treated with different CPAs. DMSO may minimize cell damage during exposure to chilling by preserving cell structures, membrane properties, and functions. The present study is the first to investigate the profiles and functions of glycans in shrimp embryos subjected to low-temperature injuries. This study enhances the understanding of cell reproduction during embryogenesis and provides valuable information for the study of glycans in embryos.

Primordial germ cells of Astyanax altiparanae, isolated and recovered intact after vitrification: A preliminary study for potential cryopreservation of Neotropical fish germplasm

Primordial germ cells (PGCs) constitute an important cell lineage that directly impacts genetic dissemination and species conservation through the creation of cryobanks. In order to advance the field of animal genetic cryopreservation, this work aimed to recover intact PGCs cryopreserved in embryonic tissues during the segmentation phase for subsequent in vitro maintenance, using the yellow-tailed tetra (Astyanax altiparanae) as a model organism. For this, a total of 202 embryos were distributed in two experiments. In the first experiment, embryos in the segmentation phase were dissociated, and isolated PGCs were maintained in vitro. They were visualized using gfp-Pm-ddx4 3′UTR labeling. The second experiment aimed to vitrify PGCs using 3 cryoprotective agents or CPAs (dimethyl sulfoxide, ethylene glycol, and 1,2 propanediol) at 3 molarities (2, 3, and 4 M). The toxicity, somatic cell viability, and recovery of intact PGCs were evaluated. After cryopreservation and thawing, 2 M ethylene glycol produced intact PGCs and somatic cells (44 ± 11.52 % and 42.35 ± 0.33 %, respectively) post-thaw. The recovery of PGCs from frozen embryonic tissues was not possible without the use of CPAs. Thus, the vitrification of PGCs from an important developmental model and Neotropical species such as A. altiparanae was achieved, and the process of isolating and maintaining PGCs in a culture medium was successful. Therefore, to ensure the maintenance of genetic diversity, PGCs obtained during embryonic development in the segmentation phase between 25 and 28 somites were stored through vitrification for future applications in the reconstitution of species through germinal chimerism.

Enhancing Vascularized Composite Allograft Supercooling Preservation: A Multifaceted Approach with CPA Optimization, Thermal Tracking, and Stepwise Loading Techniques.

Vascularized composite allografts (VCAs) present unique challenges in transplant medicine, owing to their complex structure and vulnerability to ischemic injury. Innovative preservation techniques are crucial for extending the viability of these grafts, from procurement to transplantation. This study addresses these challenges by integrating cryoprotectant agent (CPA) optimization, advanced thermal tracking, and stepwise CPA loading strategies within an ex vivo rodent model. CPA optimization focused on various combinations, identifying those that effectively suppress ice nucleation while mitigating cytotoxicity. Thermal dynamics were monitored using invasive thermocouples and non-invasive FLIR imaging, yielding detailed temperature profiles crucial for managing warm ischemia time and optimizing cooling rates. The efficacy of stepwise CPA loading versus conventional flush protocols demonstrated that stepwise (un)loading significantly improved arterial resistance and weight change outcomes. In summary, this study presents comprehensive advancements in VCA preservation strategies, combining CPA optimization, precise thermal monitoring, and stepwise loading techniques. These findings hold potential implications for refining transplantation protocols and improving graft viability in VCA transplantation.

Conditioned media and DMSO enhance the cryopreservation of bovine adipose tissue-derived mesenchymal stem cells

Mesenchymal stem cells derived from adipose tissue (AD-MSCs) show great potential for repro ductive biotechnology in the livestock sector. However, enzyme-based isolation of MSCs is expensive and time-consuming, so it is still rarely done, especially for applications in the livestock sector in de veloping countries. So, MSCs must be cryopreserved with an efficient cryoprotective agent to be stored and reproduced in various laboratories after isolation. This study was aimed to optimize the cryopreser vation media for adipose-derived MSCs in cattle. This study evaluated the viability, proliferation, and morphology of AD-MSCs. The results of this study indicate that a combination of 10% DMSO, 45% DMEM, and 45% conditioned media significantly improves post-thaw viability, proliferation, and sur vival as compared to other mediums. Furthermore, AD-MSCs cryopreserved in this medium exhibit similar morphology as fresh cells. These findings suggest that the optimized cryopreservation medium can enhance the quality and safety of AD-MSCs for clinical applications in the livestock industry.

Impact of cryoprotectant-free sperm vitrification in pulled-glass capillary on sperm parameters and DNA integrity: A lab trial study.

Vitrification is a recently introduced yet widely applied assisted reproduction technique. So far, the effects of the chemicals and devices in vitrification on sperm motility and DNA integrity are still unclear. This study aimed to examine sperm quality, as determined by semen analysis and sperm DNA integrity when vitrified with or without cryoprotectant agents (CPAs) using pulled-glass capillaries. Between February and June 2020, 50 infertile men from the Hue Center for Reproductive Endocrinology and Infertility, Hue University of Medicine and Pharmacy, Vietnam, were enrolled. Sperm samples, prepared using the swim-up technique, were divided into 2 groups: vitrification with CPAs (group 1) and without CPAs (group 2). Vitrified sperm samples were preserved in 10 µL pulled-glass capillaries. Motility, sperm membrane integrity, and the DNA fragmentation index were tested. Sperm motility in vitrified media with CPAs (54.4 11%) was statistically higher than in media without CPAs (51.14 10.6%, p 0.05). CPAs did not affect sperm membrane integrity or large halo ratio (71.34 8.47 vs. 70.38 8.11 and 50.84 18.92 vs. 51.98 19.44, respectively). Group 2 exhibited a lower DNA fragmentation index than group 1 after vitrification (14.2 8.47 vs. 12.60 9.03, p = 0.021). Using a pulled-glass capillary for sperm vitrification, the presence of CPAs in the vitrification medium resulted in higher progressive motility and lower DNA fragmentation index than the medium without CPAs.

VCA supercooling in a swine partial hindlimb model

Vascularized composite allotransplantations are complex procedures with substantial functional impact on patients. Extended preservation of VCAs is of major importance in advancing this field. It would result in improved donor-recipient matching as well as the potential for ex vivo manipulation with gene and cell therapies. Moreover, it would make logistically feasible immune tolerance induction protocols through mixed chimerism. Supercooling techniques have shown promising results in multi-day liver preservation. It consists of reaching sub-zero temperatures while preventing ice formation within the graft by using various cryoprotective agents. By drastically decreasing the cell metabolism and need for oxygen and nutrients, supercooling allows extended preservation and recovery with lower ischemia–reperfusion injuries. This study is the first to demonstrate the supercooling of a large animal model of VCA. Porcine hindlimbs underwent 48 h of preservation at − 5 °C followed by recovery and normothermic machine perfusion assessment, with no issues in ice formation and favorable levels of injury markers. Our findings provide valuable preliminary results, suggesting a promising future for extended VCA preservation.

Impact of cryoprotective agents on human gut microbes and invitro stabilized artificial gut microbiota communities.

The availability of microbial biobanks for the storage of individual gut microbiota members or their derived and artificially assembled consortia has become fundamental for invitro investigation of the molecular mechanisms behind microbe-microbe and/or microbe-host interactions. However, to preserve bacterial viability, adequate storage and processing technologies are required. In this study, the effects on cell viability of seven different combinations of cryoprotective agents were evaluated by flow cytometry for 53 bacterial species representing key members of the human gut microbiota after one and 3 months of cryopreservation at -80°C. The obtained results highlighted that no universal cryoprotectant was identified capable of guaranteeing effective recovery of intact cells after cryopreservation for all tested bacteria. However, the presence of inulin or skimmed milk provided high levels of viability protection during cryoexposure. These results were further corroborated by cryopreserving 10 artificial gut microbiota produced through invitro continuous fermentation system technology. Indeed, in this case, the inclusion of inulin or skimmed milk resulted in a high recovery of viable cells, while also allowing consistent and reliable preservation of the artificial gut microbiota biodiversity. Overall, these results suggest that, although the efficacy of various cryoprotective agents is species-specific, some cryoprotectants based on glycerol and the addition of inulin or skimmed milk are preferable to retain viability and biodiversity for both single bacterial species and artificial gut microbiota.

Various Saccharides Addition of Extender Ram Efficiency to Improve Cryopreservation in Semen Etawah Crossbreed Bucks with Seminal Plasma Replacement

Phospholipase in buck seminal plasma will trigger a coagulation incident resulting in the demise of the spermatozoa during liquid semen processing using milk or egg yolk substrates diluent. A plasma replacement process is performed to avoid this possibility. The cryopreservation process leads to sperm cell damage due to the freezing process, therefore cryoprotectant agents such as saccharides are required as protective agents. This study aimed to investigate the effect of various types of saccharides on the quality of frozen semen of etawah crossbreed (PE) bucks during cryopreservation and thawing with plasma replacement using Priangan ram semen plasma. Semen was collected using an artificial vagina once a week. Fresh PE bucks semen centrifuged at 3,000 RPM for 30 min. The supernatant (seminal plasma) was removed and replaced in equal volume with Priangan ram seminal plasma. Semen was divided into four treatments: goat semen with sheep semen plasma in Tris diluent (control); control with 0.6% dextrose added (monosaccharides); control with 0.6% sucrose added (disaccharides); control with 0.6% raffinose added (trisaccharides). Semen quality including percentages of motile spermatozoa, live spermatozoa, and intact plasma membrane (IPM) were evaluated after diluted, equilibrated, and thawing, respectively. Results of this study showed that viability and IPM of monosaccharides, disaccharides, and trisaccharides were significantly (P<0.05) higher than control (54.40 and 51.40; 55.00 and 53.60; 55.60 and 52.20 vs 48.40 and 52.20, respectively). The motility of disaccharides was significantly (P<0.05) higher compared to control and other diluent (47.00 vs 41.00, respectively). In conclusion, different types of saccharides were effective in maintaining the quality of etawah crossbreed buck frozen semen. The addition of disaccharides seems more effective compared to monosaccharides and trisaccharides in tris extender on the quality of etawah crossbreed buck frozen semen.

Cryoprotective Agents to Improve the Quality of Tambaqui Surimi (Colossoma macropomum)

One major method in fish processing to maximize utilization is through surimi preparation, extensively used in various products. To ensure desired organoleptic qualities, cryoprotective agents are crucial. This study aimed to evaluate the production potential of Amazon fish surimi prepared with different blends of cryoprotectants: CF (no cryoprotectant), S1 (2% sodium chloride, 1% sucrose), S2 (8% sorbitol, 0.5% sodium tripolyphosphate), and S3 (8% sucrose, 8% sorbitol, 0.5% sodium tripolyphosphate). Proximal composition and techno-functional properties were assessed. Moisture content (<79%) indicated excellent quality across all treatments. S2 had high protein (17.91%) and low lipid content, second only to S3. Increasing cryoprotectant concentration led to higher carbohydrate content and energy value. Color differences were insignificant except for luminosity (L*) influenced by sucrose in S1. Cryoprotectant treatments showed no significant texture or water retention differences, displaying a bending profile well-suited for the product. Univariate statistics and Principal Component Analysis concluded S2 blend as most suitable for tambaqui surimi (Colossoma macropomum) production regarding final nutritional and techno-functional gel characteristics.