Insect response to cold stress is often associated with adaptive strategies and chemical variation. However, low-temperature domestication to promote the cold tolerance potential of Bactrocera dorsalis and transformation of main internal substances are not clear. Here, we use a series of low-temperature exposure experiments, supercooling point (SCP) measurement, physiological substances and cryoprotectants detection to reveal that pre-cooling with milder low temperatures (5 and 10°C) for several hours (rapid cold hardening) and days (cold acclimation) can dramatically improve the survival rate of adults and pupae under an extremely low temperature (−6.5°C). Besides, the effect of rapid cold hardening for adults could be maintained even 4 h later with 25°C exposures, and SCP was significantly declined after cold acclimation. Furthermore, content of water, fat, protein, glycogen, sorbitol, glycerol and trehalose in bodies were measured. Results showed that water content was reduced and increased content of proteins, glycogen, glycerol and trehalose after two cold domestications. Our findings suggest that rapid cold hardening and cold acclimation could enhance cold tolerance of B. dorsalis by increasing proteins, glycerol, trehalose and decreasing water content. Conclusively, identifying a physiological variation will be useful for predicting the occurrence and migration trend of B. dorsalis populations.

Cryopreservation of domestic cat semen is mainly performed as a model for the establishment of endangered wild feline protocols. The supplementation of antifreeze protein type I (AFP I) to cryopreservation medium has shown improvement in frozen–thawed sperm quality in other species, but its effect on cat semen has not yet been tested. This study aimed to assess the addition of AFP I to cryopreservation medium in domestic cats. Sperm was obtained from the cauda epididymis of orchiectomized cats; sperm was then pooled in Tris buffer and allocated into three treatments, according to AFP I final concentration: 0 (control), 0.1, and 0.5 µg/ml. Nine replicates were cryopreserved in a two-step protocol and subsequently thawed at 37°C for 30 s. There was no difference (P > 0.05) among the control, 0.1 and 0.5 µg/ml groups for parameters such as motility, vitality, functional membrane integrity, mature chromatin, normal morphology, and sperm binding to egg perivitelline membrane. In the 0.5 μg/ml group only, percentages of live sperm with intact acrosome and of sperm with most inactive mitochondria (DAB III) showed a significant reduction, along with a tendency (P = 0.053) to an increase in the percentage of sperm with most active mitochondria (DAB II). In conclusion, the supplementation of 0.1 and 0.5 µg/ml of AFP I did not promote consistent beneficial effects on the overall sperm cryotolerance in domestic cats.

The experiment aimed to compare conventional freezing and different vitrification protocols for cryopreservation of caprine embryos at morphological, ultrastructural, and functional levels. Caprine embryos produced in vivo were allocated randomly to three groups: (1) conventional freezing with ethylene glycol (EG); (2) dimethyl sulfoxide + EG (DMSO/EG) vitrification; and (3) dimethylformamide + EG (DMF/EG) vitrification. All groups were scored for cell viability (propidium iodide staining and ultrastructural levels) and re-expansion rate after thawing or warming. Embryos subjected to DMSO/EG vitrification showed higher cell viability (73.33%), compared with DMF/EG vitrification and conventional freezing group embryos (40.00 and 66.66%, respectively). The ultrastructural study revealed that vitrified embryos had greater preservation of cellular structure than embryos from conventional freezing with EG. DMSO/EG vitrification resulted in higher rates of re-expansion in vitro (47.36%) than DMF/EG vitrification (31.58%), and conventional freezing (25.00%). In conclusion, caprine embryos produced in vivo are better cryopreserved after vitrification than conventional freezing, therefore we conclude that DMSO/EG vitrification is the most effective protocol for cryopreservation.

Morphological studies of helminths by scanning electron microscopy are generally limited to the external topography of the organisms. In this work, we present a simple technique using ethanol as a cryoprotectant without postfixation in osmium tetroxide that allows for observation of the inner organization of helminths and preserves cellular structures. We tested the technique in three helminths: Echinostoma paraensei, Cruzia tentaculata, and Hassalstrongylus epsilon. The results show that this technique could potentially be applied to study the morphology, ultrastructure, and taxonomy of helminths.

Although the sperm cryopreservation of freshwater and marine teleosts has been feasible for years, the cryopreservation of some fish embryos still remains elusive. Thus, the objective of this experiment was to analyze the embryo morphology after freezing and thawing 40 embryos of Piaractus mesopotamicus immersed into methanol and ethylene glycol, both at 7, 10 and 13% plus 0.1 M sucrose for 10 min. Soon after thawing, three embryos were treated with historesin, stained with hematoxylin–eosin and analyzed under an optical microscope. From every treatment, one palette containing embryos was thawed and incubated, but none of the eggs hatched. Samples containing two embryos were immersed into 10% methanol or 10% ethylene glycol both in association with sucrose, and embryos immersed into only water or sucrose solution were frozen, processed and analyzed using scanning electron microscopy (SEM). In both cases, the control group was immersed into only water. Although the embryos had the chorion, vitello, yolk syncytial layer and blastoderm, all of them were found altered under the optical microscope and by SEM. The chorion was irregular and injured; there was no individuality in the yolk granules; the yolk syncytial layer had an irregular shape, thickness and size; the blastoderm showed injuries in the nucleus shape and sometimes was absent; the blastoderm was located in atypical areas and absent in some embryos. In conclusion, no treatment was effective in preserving the embryos, and none of the embryos avoided injury from intracellular ice formation. These morphological injuries during the freezing process made the P. mesopotamicus embryos unfeasible for hatching.

Cryopreservation of mammal embryos has been technically feasible for many years, but morphological injuries still persist in fish embryos during cryopreservation. Thus, the objective of the present study was to describe these freezing injuries in Piaractus mesopotamicus embryos. Two hundred and twenty-five embryos were collected at the post-gastrula stage and assigned into four treatments of sucrose at 8.5, 17.0, 25.0 or 34.0% plus 9.0% methanol. The control was prepared with distilled water only. The gradual decrease in the temperature was 0.5°C/min. After the seeding stage, the fish embryos were stored in liquid nitrogen at −33°C. Thereafter, they were thawed for evaluating per cent hatching, and the samples collected from every treatment were submitted to scanning electron microscopy for morphological analysis. The micrographic images showed that there was substantial alterations in embryo morphology under the highest concentrations of sucrose. These solutions did not prevent the formation of ice crystals, which lead to deformities and killed the embryos, but the observed reduced level of morphological structure in these embryos when treated with 17.0% sucrose plus 9.0% methanol is a compelling argument for additional studies.

With the development of embryo technologies, such as in vitro fertilization, cloning and transgenesis, cryopreservation of mammalian gametes and embryos has acquired a particular interest. Despite a certain success, various cryopreservation techniques often cause significant morphological and biochemical alterations, which lead to the disruption of cell organelles, cytoskeleton damages, cell death and loss of embryo viability. Ultrastructural studies confirm high sensitivity of the cell membrane and organelle membrane to freezing and thawing. It was found that many substances with low molecular weights have a protective action against cold-induced damage. In this concern, an anti-freeze protein (AFP) and anti-freeze glycoproteins (AFGPs), which occur at extremely high concentrations in fish that live in Arctic waters and protect them against freezing, may be of potential interest for cryostorage of animal embryos at ultra-low temperatures. This mini-review briefly describes several models of AFP/AFGP action to preserve cells against chilling-induced damages and indicates several ways to improve post-thaw developmental potential of the embryo.

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and elethylene glycol as an uncompetitve inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.

With the aim of establishing cryopreservation protocols for oyster (Crassostrea gigas) embryos, toxicity of single or combined cryoprotectants to oyster embryos was investigated. In experiments on the toxicity tolerance of oyster embryos, four conventional cryoprotectants, namely, acetamide (A), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG), with concentrations from 1 to 5 M were used to test the toxicity tolerance of oyster embryos at eight different developemental stages. On a molar-equivalent basis, DMSO appeared to be less toxic to PG, EG and acetamide in general. Oyster embryos were tolerant to low concentrations of all cryoprotectants tested in the range from 1 M to 2 M for all developmental stages. Early stage embryos were more vulnerable to high concentration (4 M and 5 M) cryoprotectants tested than late stage embryos. Experiments were subsequently performed to study the combined effects of DMSO and acetamide adding trehalose or glucose in reducing toxicity to 4 hour oyster embryos. The use of trehalose or glucose reduced toxicity of high cryoprotectant concentrations. Survival was higher when DMSO was combined with acetamide than when using DMSO or acetamide alone at high concentration (5 M). By adding trehalose to combined 5 M DMSO and acetamide (total concentration) an average survival rate of 67 ± 7% was obtained in contrast to 8 ± 5% or 2 ± 1% survival when testing with 5 M DMSO or 5 M acetamide, respectively.