Ice Crystal Damage Research Articles

Damage to Mitochondria During the Cryopreservation, Causing ROS Leakage, Leading to Oxidative Stress and Decreased Quality of Ram Sperm.

Semen cryopreservation can achieve long-term preservation of sperm. Ice crystal damage, as well as oxidative stress, result in mitochondrial dysfunction and a reduction in sperm motility after thawing. However, limited information exists regarding the impact of reactive oxygen species (ROS) and mitochondria on the cryopreservation of ram sperm. The primary objective of this study was to investigate the relationship between ROS and mitochondria concerning sperm quality during the cryopreservation of ram sperm. This investigation assessed sperm motility, kinematic characteristics, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, expression of mitochondrial respiratory genes (NDUFV2, SDHA, CYC1, and COXIV), ROS levels, malondialdehyde (MDA) content, phosphatidylserine externalisation rate, sperm ultrastructure, mtDNA copy number, expression of apoptosis-related genes (Bax, Caspase-3, and Caspase-8), Cytochrome C, and Caspase-3 content. The results showed the cryopreservation significantly (p < 0.05) decreased motility, kinetic parameters, membrane integrity, acrosome integrity, MMP, ATP, mRNA expression levels of mitochondrial respiratory-related genes, and significantly (p < 0.05) increased ROS levels, MDA content, phosphatidylserine externalisation rate, damage of sperm ultrastructure, mtDNA copy number, mRNA expression levels of apoptosis-related genes, Cytochrome C and Caspase-3 content compared to the fresh semen group. In conclusion, the cryopreservation causes damage to mitochondria, leading to increased ROS and subsequent oxidative stress. This process also initiates mitochondrial dysfunction and interferes with the electron transport chain, ultimately resulting in decreased MMP and ATP production. Furthermore, the liberation of Cytochrome C prompted the increase in Caspase-3 expression and subsequent sperm apoptosis occurred, ultimately leading to a deterioration in sperm quality after thawing.

TaTPS11 enhances wheat cold resistance by regulating source-sink factor

The presence of sugar in plant tissue can lead to an increase in the osmotic pressure within cells, a decrease in the freezing point of plants, and protection against ice crystal damage to the tissue. Trehalose is closely related to sucrose, which comprises the largest proportion of sugar and has become a hot topic of research in recent years. Our previous studies have confirmed that a key trehalose synthesis gene, TaTPS11, from the cold-resistant winter wheat DM1, could enhance the cold resistance of plants by increasing sugar content. However, the underlying mechanism behind this phenomenon remains unclear. In this study, we cloned TaTPS11-6D, edited TaTPS11-6D using CRISPR/Cas9 technology and transformed ‘Fielder’ to obtain T2 generation plants. We screened out OE3-3 and OE8-7 lines with significantly higher cold resistance than that of ‘Fielder’ and Cri 4–3 edited lines with significantly lower cold resistance than that of ‘Fielder’. Low temperature storage limiting factors were measured for OE3-3, OE8-7 and Cri 4–3 treated at different temperatures.The results showed that TaTPS11-6D significantly increased the content of sugar in plants and the transfer of sugar from source to storage organs under cold conditions. The TaTPS11-6D significantly increased the levels of salicylic, jasmonic, and abscisic acids while also significantly decreasing the level of gibberellic acid. Our research improves the model of low temperature storage capacity limiting factor.

Viable Vitreous Grafts of Whole Porcine Menisci for Transplant in the Knee and Temporomandibular Joints.

The shortage of suitable donor meniscus grafts from the knee and temporomandibular joint (TMJ) impedes treatments for millions of patients. Vitrification offers a promising solution by transitioning these tissues into a vitreous state at cryogenic temperatures, protecting them from ice crystal damage using high concentrations of cryoprotectant agents (CPAs). However, vitrification's success is hindered for larger tissues (>3 mL) due to challenges in CPA penetration. Dense avascular meniscus tissues require extended CPA exposure for adequate penetration; however, prolonged exposure becomes cytotoxic. Balancing penetration and reducing cell toxicity is required. To overcome this hurdle, a simulation-based optimization approach is developed by combining computational modeling with microcomputed tomography (µCT) imaging to predict 3D CPA distributions within tissues over time accurately. This approach minimizes CPA exposure time, resulting in 85% viability in 4-mL meniscal specimens, 70% in 10-mL whole knee menisci, and 85% in 15-mL whole TMJ menisci (i.e., TMJ disc) post-vitrification, outperforming slow-freezing methods (20%-40%), in a pig model. The extracellular matrix (ECM) structure and biomechanical strength of vitreous tissues remain largely intact. Vitreous meniscus grafts demonstrate clinical-level viability (≥70%), closely resembling the material properties of native tissues, with long-term availability for transplantation. The enhanced vitrification technology opens new possibilities for other avascular grafts.

Inhibition of enzymatic browning in freeze-thawed apricot fruit by combined chlorogenic acid and osmotic dehydration treatments

In this study, the inhibitors of apricot browning were analyzed by investigating the effects of osmotic dehydration (OD) pretreatment combined with chlorogenic acid (CA) on cell disruption, loss of phenolic substrates, and changes in key enzymes during freeze-thawing. Both OD and OD-CA mitigated the deterioration caused by ice crystal damage during freeze-thawing. OD and OD-CA significantly reduces the BI of apricots by 43.27 and 59.55 compared to CK. The PPO and POD activity in OD-CA apricots are only 61.11% and 42.13% of those in OD. OD-CA could also protect SOD and CAT activity, with their activity in the OD-CA being 1.41 and 1.14 times that of OD group. This indicated that OD-CA treatment protected against the activities of ROS-scavenging enzymes. Compared with OD, OD-CA could better protect the bioactive substance of apricot. The contents of ascorbic acid, total phenols, and total flavonoids in OD-CA were 1.32 times, 1.11 times, and 1.05 times higher than those in OD. OD-CA also effectively protected chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, catechins, and rutin. In summary, OD-CA primarily reduced the browning by protecting the integrity of cell structures, minimizing the potential for exposure to air, enzymes, and phenolic substrates, and inhibiting browning enzymes activity.

Quantitative Analysis of Ice Crystal Growth During Freezing of Dimethyl Sulfoxide Solutions Under Alternating Current Electric Fields.

During cryopreservation, the growth of ice crystals can cause mechanical damage to samples, which is one of the important factors limiting the quality of preserved samples. To enhance the preservation quality of biological samples, scholars have tried various engineering methods. Among them, an electric field is an essential factor affecting solution freezing. Dimethyl sulfoxide, as a commonly used cryoprotectant, can cause mechanical damage to cells due to ice crystals even when freezing at the optimal freezing rate. Water is a strongly polar dielectric material, and the applied alternating current (AC) electric field will affect the water freezing performance. Therefore, a quantitative study of ice crystal nucleation and growth during freezing of dimethyl sulfoxide solutions under different AC electric field conditions is needed to try to reduce ice crystal damage. We created a liquid-film device to approximate the ice crystal growth process as a two-dimensional image. The frequency of the AC voltage was set from 0 to 50 kHz. We measured the supercooling of the dimethyl sulfoxide solution under AC electric field conditions. As an objective and accurate quantitative analysis of the ice crystal growth process, we propose a Dilated Convolutional Segmentation Transformer for semantic segmentation of ice crystal images. It is concluded that the average area and the growth rate of single ice crystals decrease with increasing electric field frequency at a certain concentration of dimethyl sulfoxide solution. Lower concentrations of dimethyl sulfoxide solution in combination with an AC electric field can achieve similar ice suppression effects as when higher concentrations of dimethyl sulfoxide solution act alone. We believe that AC electric fields are expected to be an aid to cryopreservation and provide some theoretical basis and experimental foundation for its development.

Effects of Xanthan gum and Potassium carbonate on the quality and flavor properties of frozen Jiuniang doughs

Chinese Jiuniang (CJ) is a flavorful and nutritious food, but underutilized in frozen dough (FD) production. In addition, frozen storage can harm FD's gluten structure and degrade quality and flavor. Therefore, the impacts of two excellent protective agents (XG-Xanthan Gum; PC-Potassium Carbonate) on frozen Jiuniang dough (F-JD) quality and flavor during dynamic freezing were investigated. The results suggested that adding XG conferred F-JD with good processing stability, maintained the bound water levels, stabilized rheological properties, diminished ice crystal damage to the protein structure, and inhibited the increase in frozen water content during the freezing process. In contrast, although PC reduced free water production during freezing, it increased dough hardness and offered less protein protection than XG. Additionally, GC-QTOF/MS analysis showed that adding XG during freezing increased the relative content of pleasant flavor compounds like Phenylethyl Alcohol and decreased undesirable ones like Hexanal. Moreover, PC lowered the relative content of undesirable flavor substances (Formic acid) but reduced the relative content of beneficial flavor compounds (1-Hexanol). Importantly, the study confirmed that XG maintained the new F-JD product's storage quality during dynamic freezing. In conclusion, this study broadens CJ's application possibilities and provides new insights into mechanisms for preserving F-JD's quality and flavor.

Cryoprotective Effects of Carrageenan Oligosaccharides on Crayfish (Procambarus clarkii) during Superchilling.

Cryoprotectants are widely used to protect muscle tissue from ice crystal damage during the aquatic products freezing process, but traditional phosphate cryoprotectants may cause an imbalance in the calcium-to-phosphorus ratio for the human body. This study evaluated the effects of carrageenan oligosaccharides (CRGO) on quality deterioration and protein hydrolysis of crayfish (Procambarus clarkii) during superchilling. The physical-chemical analyses showed that CRGO treatments could significantly (p < 0.05) inhibit the increase of pH values, TVB-N, total viable counts, and thawing loss, and improve the water holding capacity and the proportion of immobilized water, which indicated that CRGO treatment effectively delayed the quality deterioration of crayfish. The myofibrillar protein structural results demonstrated that the increase of the disulfide bond, carbonyl content, S0-ANS, and the decrease of total sulfhydryl content were suppressed significantly (p < 0.05) in CRGO treatment groups. Furthermore, SDS-PAGE results showed that the band intensity of myosin heavy chain and actin in CRGO treatment groups were stronger than in the control. Overall, the application of CRGO to crayfish might maintain better quality and stable protein structure during the superchilling process, and CRGO has the potential to replace phosphate as a novel cryoprotectant for aquatic products.

Application of interval arithmetic in numerical modeling of cryopreservation process during cryoprotectant loading to microchamber

This article presents numerical modeling of the heat and mass exchange occurring in a two-dimensional axially symmetric articular cartilage sample submerged in a microchamber into CPTes2 bathing solution undergoing cryopreservation. In the considered mathematical model, the parameters defined as interval numbers were assumed. The heat transfer process was described by the interval Fourier equation, the transport of cryoprotectant (CPA) through the cell membrane was given by a two-parameter model including the simulation of H2O volume in chondrocytes and the change of DMSO concentration in time. In addition, the transport of CPA via microchannels to the sample in the microchamber was descripted by the Navier-Stokes equation. The model uses the liquidus tracking (LT) protocol introduced by Pegg et al. and refined by Yu et al. This approach regulates both the temperature and the concentration of CPA in the bathing solution. The protocol protects chondrocytes from ice crystal, osmotic stress, and electrolyte damage. The entire cryopreservation process considered is divided into 7 periods in the cooling and CPA supply phase and 6 periods in the heating and CPA removal phase, which allows precise regulation of the temperature and cryoprotectant (CPA) concentration in the bath solutions. In the final part of the article the results of numerical simulations are presented.

Numerical Study of Heat and Mass Transfer during Cryopreservation Process with Application of Directed Interval Arithmetic.

In the present paper, numerical modelling of heat and mass transfer proceeding in a two-dimensional axially symmetrical articular cartilage sample subjected to a cryopreservation process is presented. In the model under consideration, interval parameters were assumed. The heat transfer process is described using the Fourier interval equation, while the cryoprotectant transport (DMSO) across the cell membrane is analyzed using a two-parameter model taking into account the simulation of the water volume in the chondrocytes and the change in DMSO concentration over time. The liquidus tracking (LT) protocol introduced by Pegg et al. was used to model the cryopreservation process. This procedure divides the heating and cooling phases into eight and seven steps, respectively, allowing precise regulation of temperature and cryoprotectant (CPA) concentration of bathing solutions. This protocol protects chondrocytes from ice crystal, osmotic stress, and electrolyte damage. The obtained interval concentrations of cryoprotectant in chondrocytes were compared with previous simulations obtained using the deterministic model and they are mostly in agreement with the simulation data.

Ovarian tissue cryopreservation and novel bioengineering approaches for fertility preservation.

Breast cancer patients who cannot delay treatment or for whom hormone stimulation and egg retrieval are contraindicated require alternative methods of fertility preservation prior to gonadotoxic treatment. Ovarian tissue cryopreservation is an alternative approach that may offer patients the opportunity to preserve fertility and carry biologically-related children later in life. Various experimental approaches are being explored to obtain mature gametes from cryopreserved and thawed ovarian tissue for fertilization and implantation using biomimetic tissue culture in vitro. Here we review the most recent developments in ovarian tissue cryopreservation and exciting advances in bioengineering approaches to in vitro tissue and ovarian follicle culture. Slow freezing is the most widely accepted method for ovarian tissue cryopreservation, but efforts have been made to modify vitrification for this application as well. Numerous approaches to in vitro tissue and follicle culture are in development, most prominently two-step culture systems for ovarian cortical tissue and encapsulation of ovarian follicles in biomimetic matrices for in vitro culture. Refinements to slow freeze and vitrification protocols continue to address challenges associated with cryopreservation, such as ice crystal formation and damage to the stroma. Similarly, improvements to in vitro tissue and follicle culture show promise for utilizing patients' cryopreserved tissues to obtain mature gametes after disease treatment and remission. Development of an effective and reproducible culture system for human ovarian follicles will serve as a broad assisted reproductive technology for cancer survivors who cryopreserved tissue prior to treatment.

Quality Retention of Fresh Tuna Stored Using Supercooling Technology.

The present study was focused on the investigation of physiochemical changes in tuna subjected to a novel supercooling preservation, which was assisted using a combination of pulsed electric fields (PEF) and oscillating magnetic fields (OMF). Fresh tuna fillets were stored without freezing at −3.2 °C for 8 days. The electrochemical impedance spectroscopy (EIS) parameter indicated that there was a significant difference between the frozen-thawed samples (36.3%) and fresh (46.6%) and supercooled (45.9%) samples, indicating that cell damage from ice crystal growth did not occur in the supercooled tuna sample. The microstructure observation and drip loss measurement further confirmed that the ice crystal damage was present in frozen tuna, whereas no cellular damage was found in the supercooled samples. The EIS proved its ability to distinguish between tuna samples that were frozen or chilled (i.e., refrigerated and supercooled) during storage; however, it was less sensitive in detecting the extent of spoilage. Instead, the K-value was used to evaluate tuna freshness, and the measured K-values of the refrigerated, supercooled, and frozen tuna samples after 8 days of storage were 74.3%, 26.4%, and 19.9%, respectively, suggesting that the supercooling treatment significantly preserved the tuna fillets fresh with the improved shelf-life when compared to conventional refrigeration.

Cryobanking of Fish and Shellfish Egg, Embryos and Larvae: An Overview

Number of attempts has been made to cryopreserve fish and shellfish gametes and success has been achieved to establish only sperm banks in case of some commercially important fish. In shellfish also particularly in shrimps though the sperm cryopreservation was successful, no attempts were made to develop sperm banks. As far as cryobanking of egg and embryos of fish and shellfish is concerned, less research efforts were made that too with limited success. Number of reasons has been given for the failure of egg /embryo cryopreservation and the main barriers speculated are low membrane permeability in the eggs, and the large yolk mass of the oocyte and the presence of compartments in early developing embryos. Both these factors result in ice crystal formation during the freezing process. In addition, the oocytes and embryos are prone to chilling injuries unrelated to ice crystal damage. There are number of other problems reported by several workers in the egg/embryo cryobanking protocols which are elaborately discussed in the present paper. There is an urgent need to develop a viable cryobanking technology for fish egg/embryos to enhance fish production in captive condition. Attempt to cryopreserve larvae of aquatic animals is another challenge coming up in recent past. The purpose of the present review is to collect comprehensive information on the efforts so far made on fish and shellfish egg and embryo cryobanking, assess the challenges in the development of viable technology and plan for future research for making this technology viable and cost effective.

The Effect of Aging/Freezing Sequence and Freezing Rate on Quality Attributes of Beef Loins (M. longissimus lumborum)

The objective of this study was to determine the effect of different aging/freezing sequences combined with different freezing rates on quality attributes of beef loins (M. longissimus lumborum). Loins from 1 side of 8 beef carcasses (USDA Low Choice grade) were obtained at 2 d postmortem, cut into 10 steaks (2.54 cm), and randomly assigned into 5 treatments, where factorial combinations of 2 aging/freezing sequences (aging first then freezing/thawing [AFT] and freezing first then thawing/aging [FTA]) and 2 freezing rates (fast freezing [FF] and slow freezing [SF]) were used and compared to an aged only (AO) treatment as a control. Samples frozen using the FF condition were found to have a critical freezing time (Tc) of 21.5 min while the SF counterpart had a Tc of 175 min. Histological observation showed more visually enlarged gaps between muscle fibers in the SF samples, regardless of aging/freezing sequence. The SF-FTA samples had a significantly higher purge/ thaw loss followed by FF-FTA, while the loss from FF-AFT, SF-AFT, and AO were not different from each other (P &gt; 0.05), suggesting that freezing rate effect might be more profound when applied in FTA. Cook loss was higher in AO compared to both FTA treatments (P &lt; 0.05); however, it was not different when compared to AFT (P &gt; 0.05). A lower shear force value and higher troponin-T protein degradation were found in both AFT and FTA compared to AO (P &lt; 0.05), which would be attributed to the ice-crystal damage due to freezing. These results suggest that aging/freezing sequence could be an overriding factor in determining major meat quality attributes of frozen/thawed meat over freezing rate. However, fast freezing could negate some quality defects associated with the FTA process.

Effect of butylated hydroxytoluene supplementation on ultrastructure of crossbred bovine spermatozoa

The present study was planned to observe the effect of Butylated hydroxytoluene (BHT) on ultra structure changes in bovine crossbred spermatozoa. The sperm plasma membrane is the primary site of damage induced by cryopreservation. There are many harmful conditions such as cold shock, osmotic stress, ice crystal formation or oxidative damage etc which cause sperm cryo-injury and loss of sperm viability and fertility during cryopreservation. At the time of cryopreservation there were considerable ultra structure changes to the acrosomes and middle piece studied by transmission electron microscopy. Semen from two crossbred bulls were collected and extended with egg yolk tris dilutor along with addition of different concentrations of BHT i.e. 0.0mM (Control), 0.5mM (T 1 ) and 1.0mM (T 2 ), respectively. The effect of BHT on ultrastructural alterations was revealed by Transmission electron microscopy during post dilution (Stage I), post equilibration (Stage II) and post thaw (Stage III), respectively. TEM results of spermatozoa at Stage I, II and III showed that major ultrastrucural alterations occur in plasma membrane and outer acrosomal membrane. Group T 1 and T 2 showed significantly lower number of ultrastructural defects compared to control at Stage I, II and III. The study indicated that 1.0mM BHT increases the cryosurvival of sperm as revealed by post thaw lower ultrastructural defects of bovine bull spermatozoa

Current issues in plant cryopreservation and importance for ex situ conservation of threatened Australian native species

An alarming proportion of Australia’s unique plant biodiversity is under siege from a variety of environmental threats. Options for in situ conservation are becoming increasingly compromised as encroaching land use, climate change and introduced diseases are highly likely to erode sanctuaries regardless of best intentions. Ex situ conservation is currently limited to botanic garden living collections and seed banking, with in vitro and cryopreservation technologies still being developed to address ex situ conservation of species not amenable to conventional storage. Cryopreservation (storage in liquid nitrogen) has been used successfully for long-term biosecure storage of shoot tips of several species of threatened Australian plants. We present a case for building on this research and fostering further development and utilisation of cryopreservation as the best means of capturing critical germplasm collections of Australian species with special storage requirements (e.g. recalcitrant-seeded taxa and species with short-lived seeds) that currently cannot be preserved effectively by other means. This review highlights the major issues in cryopreservation that can limit survival including ice crystal damage and desiccation, toxicity of cryoprotective agents, membrane damage, oxidative stress and mitochondrial function. Progress in understanding and mitigating these stresses is vital for advancing cryopreservation for conservation purposes.

Effects of heating processes on changes in ice crystal formation, water holding capacity, and physical properties of surimi gels during frozen storage

The effects of heating processes on changes in ice crystal formation, water holding capacity, and physical properties of surimi gels with different freezing conditions were evaluated. Heated surimi gels were frozen by quick or slow freezing and stored at −40 °C, −20 °C, or −10 °C for 12 weeks. Results showed that ice crystal size and structural damage increased after frozen storage, and it was greater in two-step heated gels than in direct heated gels. Drip loss increased after frozen storage, with higher drip loss in two-step heated gels. The disulfide bond content decreased after frozen storage, and these changes were more notable in two-step heated gels. In direct heated gels, the breaking force increased with the frozen storage period, but in two-step heated gels, breaking force decreased in the first two weeks and then increased. Moreover, the degree of quality deterioration increased with slower freezing rates, higher storage temperatures, and longer storage periods. From the above, it was concluded that changes in ice crystal formation, water holding capacity, and physical properties of frozen surimi gels were affected by the heating processes, and the gel properties could maintain to a major extent by quick freezing and low temperature storage.

Backscattered electron imaging of high pressure frozen soybean root nodules visualizes formation of symbiosome membranes.

High-pressure frozen soybean root nodules were fractured and backscattered electron images were obtained from uncoated samples in a low vacuum scanning electron microscope equipped with a cryo-transfer system. Structures of infected cells were well preserved: numerous symbiosomes, as well as nuclei, plastids and mitochondria were observed without ice crystal damage. After appropriate sublimation of water, bacteria included in symbiosomes were visualized. Membrane accumulation near nuclei, and vesicles and tubular membranes, which possibly contribute to symbiosome membrane formation, could be observed in a near native state. The method promises to be widely applicable to visualize interaction between membranes in various biological systems.

Natural deep eutectic systems as alternative nontoxic cryoprotective agents

Natural deep eutectic systems (NADES) are mostly composed of natural primary metabolites such as sugars, sugar alcohols, organic acids, amino acids and amines. These simple molecules have been identified in animals living in environments with extreme temperature amplitudes, being responsible for their survival at negative temperatures during winter. Herein, we report for the first time the use of NADES based on trehalose (Treh) and glycerol (Gly) in cryopreservation, as cryoprotective agents (CPA). The evaluation of the thermal behaviour of these eutectic systems, showed that NADES have a strong effect on the water crystallization/freezing and melting process, being able to reduce the number of ice crystals and hence ice crystal damage in cells, which is a crucial parameter for their survival, upon freezing. Using this NADES as CPA, it is possible to achieve similar or even better cellular performance when compared with the gold standard for cryopreservation dimethyl sulfoxide (DMSO). In this sense, this work relates the physical properties of the NADES with their biological performance in cryopreservation. Our comprehensive strategy results in the demonstration of NADES as a promising nontoxic green alternative to the conventional CPA's used in cryopreservation methods.

Advances in understanding the fundamental aspects required for successful cryopreservation of Australian flora

Australia is host to an amazing diversity of species, many of which require conservation efforts. In vitro culture provides a tool for not only conserving these threatened species but allows for their propagation from limited starting material. Cryopreservation provides the greatest long-term storage option for in vitro cultures and as a conservation tool for other germplasm. However, while cryopreservation has proven capable of delivering viable long-term storage with some plant taxa, the process of deriving protocols is still largely an incremental process. The key to faster and more intuitive optimising of cryopreservation protocols lies with continuing to develop a better understanding of key factors, including issues with plant physiology (such as genetic stability, the composition of the proteome and metabolome, cell membrane characteristics, and antioxidant defences) and how the stresses imposed by cryopreservation (such as the excision damage, desiccation, cryoprotective agent toxicity, ice crystal damage, and cooling to cryogenic temperatures) interact and contribute to the cryocapability of a species. This review focuses on the advances that have been made towards understanding cryogenic stress and how this has led to improved cryopreservation protocols, in the context of cryopreserving Australian flora.

Effect of compressibility on plasma-based transition control for a wing with leading-edge excrescence

ABSTRACTCompressibility effects were numerically investigated for use of plasma-based flow control, which was applied to delay transition generated by excrescence on the leading edge of a wing. The wing airfoil section incorporates a geometry that is representative of modern reconnaissance air vehicles, and has an appreciable region of laminar flow at design conditions. Modification of the leading edge can be caused by the accumulation of debris, insect impacts, microscopic ice crystal formation, damage, or structural fatigue, resulting in premature transition and an increase in drag. A dielectric barrier discharge (DBD) plasma actuator, located downstream of the excrescence, was employed to delay transition, mitigate the effects of turbulence, decrease drag, and increase energy efficiency. Solutions were obtained for several Mach numbers, up to the transonic range. The effect of compressibility on transitional behaviour was explored, and the effectiveness of plasma-based control to delay transition with increasing Mach number was determined.