Canine and Feline Testicular Preservation

The increased interest in breeding dogs and cats and their use as models for other canids and felids demand research to improve reproductive techniques. Among them, testicular cryopreservation stands out. Testicular cryopreservation enables the maintenance of reproductive capacity and allows the establishment of germplasm banks for several species of commercial value or at risk of extinction. Furthermore, it enables the transport of genetic material among different regions. It is noteworthy that this biotechnology represents the only possibility of preserving the fertility of prepubertal animals that have died, so it has great importance in the propagation of the genetic material of animals. The spermatogonia present in the testes can be cultivated in vitro and the sperm obtained can be used in artificial reproduction programs. Although advances have been achieved with the use of testicular fragments to obtain viable and functional germ cells, the establishment of protocols that can be used in clinical routine have not been concluded yet. The testicular cryopreservation process can be carried out through techniques such as slow freezing, fast freezing and vitrification. However, the protocols used for the canine and feline species are still in the experimental phase. Given the importance of the topic, the aim of this review is to draw a profile of the subject approaching the main works on testicular cryopreservation in dogs and cats.

Biochemical composition of fresh, dried and frozen berries different varieties of sea buckthorn (Hippophae rhanoides L.)

Purpose. Determine the formation of physico-chemical quality indicators (berry weight, protein, carbohydrates, fat, dietary fiber, organic acids) of fresh, dried and frozen sea buckthorn fruits of different varieties. Methods. Laboratory, mathematical and statistical, physicochemical. Results. The water content in sea buckthorn berries was the highest, but also varied significantly depending on the variety. The varieties of sea buckthorn ‘Haleryt’ and ‘Yelyzaveta’ had the highest content – 90%, by 2 and 3% less in the varieties ‘Burshtynove namysto’ and ‘Altaiska’, in the varieties ‘Sonechko’ and ‘Uliublena’ – 85%. The lowest water content was in the berries of ‘Ryzhyk’, ‘Podruha’, ‘Diuimovochka’, ‘Chechek’ varieties – 75–78%. Dried fruits contained water from 22 to 16%. In the varieties ‘Uliublena’, ‘Yelyzaveta’, ‘Altaiska’ – 22%, ‘Haleryt’, ‘Diuimovochka’, ‘Sonechko’, ‘Burshtynove Namysto’ – 20%, ‘Avhustyna’, ‘Ryzhyk’, ‘Podruha’ – 18%, ‘Veleten’ and ‘Chechek’ – 17 and 16%, respectively. The content of dietary fiber was 4.5–6.2% depending on the variety. The lowest content of dietary fiber was found in the varieties ‘Uliublena’ and ‘Altaiska’ – 4.5–4.7% in fresh and 2.4–2.5% in dried berries. The highest rates were in the varieties ‘Avhustyna’, ‘Sonechko’, ‘Burshtynove Namysto’ in fresh varieties – 6.0, 6.2 and 5.9%, and in dried varieties – 5.5%. The ‘Chechek’ and ‘Veleten’ varieties contained 5.7% in fresh berries and 5.0 and 4.5% in dried berries. The content of fat in fresh berries was in the range of 5.0–5.7%, depending on the variety. In dried berries it increased to 6.0–6.7% or 18–20% depending on the variety of sea buckthorn. In fast-freezing berries, this figure decreased to 4.5–5.2% or 9–10%. The content of organic acids in fresh fruits was the highest – 1.5–2.0%, depending on the sea buckthorn variety. Their content in dried berries decreased to 1.3–1.7% except for the varieties ‘Avhustyna’, ‘Sonechko’ and ‘Burshtynove namysto’, and in the frozen ones to 0.3–0.9%. It should be noted that a similar trend is observed in frozen berries of ‘Avhustyna’, ‘Sonechko’ and ‘Burshtynove namysto’ varieties. Conclusions. It is established that physicochemical quality indicators of fresh, dried and frozen sea buckthorn berries vary depending on the variety. The varieties ‘Avhustyna’, ‘Sonechko’, ‘Burshtynove Namysto’ have the largest weight of one berry 1.4–1.5 g. 8–6.0%), fat (5.5–5.7%), protein (1.5%), organic acids (2.0%) and dietary fiber (5.9–6.2%) in fresh berries.

Visualizing the three-step freezing process and three-phase reaction not predicted by the (NH4) 2SO4/H2O phase diagram

According to the conventional phase diagrams, aqueous solutions freeze at the liquidus and are frozen/solid below the eutectic solidus. Herein, using differential scanning calorimetry (DSC) and optical cryo-microscopy (OC-M), we demonstrate that hy-poeutectic, eutectic 40 wt% (NH4)2SO4 and hypereutectic (NH4)2SO4/H2O remain liquid well below the eutectic solidus before freezing in three steps: fast-slow-fast. The first fast freezing produces a ramified ice microstructure (IM) and freeze-concentrated solution (FCS) containing up to ~70 wt% (NH4)2SO4. As temperature decreases further, the slow freezing of FCS precedes its fast freezing, which produces a striped IM and (NH4)2SO4 microcrystals. Videos recorded upon warming of frozen (NH4)2SO4/H2O reveal a new three-phase reaction, which is the recrystallization of ice and (NH4)2SO4 microcrystals into the lamellar eutectic ice-(NH4)2SO4 superlattice. This work demonstrates limitations of the (NH4)2SO4/H2O phase diagram and pro-poses an effective strategy for studying other deeply supercooled solutions whose behavior is not predicted by the phase dia-gram.

3D Characterization of Sponge Cake as Affected by Freezing Conditions Using Synchrotron X-ray Microtomography at Negative Temperature

In this study, the microstructural evolution of a non-reactive porous model food (sponge cake) during freezing was investigated. Sponge cake samples were frozen at two different rates: slow freezing (0.3 °C min−1) and fast freezing (17.2 °C min−1). Synchrotron X-ray microtomography (µ-CT) and cryo-scanning electron microscopy (Cryo-SEM) were used to visualize and analyze the microstructure features. The samples were scanned before and after freezing using a specific thermostated cell (CellStat) combined with the synchrotron beamline. Cryo-SEM and 3D µ-CT image visualization allowed a qualitative analysis of the ice formation and location in the porous structure. An image analysis method based on grey level was used to segment the three phases of the frozen samples: air, ice and starch. Volume fractions of each phase, ice local thickness and shape characterization were determined and discussed according to the freezing rates.

Effects of freezing rate on structural changes in l-lactate dehydrogenase during the freezing process

Freezing is a common method for improving enzyme storage stability. During the freezing process, the freezing rate is an important parameter that can affect protein stability. However, there is limited information on the denaturation mechanisms and protein conformational changes associated with the freezing rate. In this study, the effects of freezing rate on activity loss and conformational changes in a model enzyme, l-lactate dehydrogenase, were evaluated. Enzyme solutions were frozen at various rates, from 0.2 to 70.6 °C/min, and ice seeding was conducted to reduce supercooling. The results demonstrated that fast freezing results in activity loss, structural changes, and aggregation. The residual activities at freezing rates of 0.2, 12.8, and 70.6 °C/min were 77.6 ± 0.9%, 64.1 ± 0.4%, and 44.8 ± 2.0%, respectively. As the freezing rate increased, the degree of dissociation and unfolding increased significantly, as determined using blue native-polyacrylamide gel electrophoresis and fluorescence spectroscopy. Moreover, a large number of amyloid aggregates were detected in samples frozen at a fast freezing rate (70.6 °C/min). The enzyme inactivation mechanism induced by fast freezing was proposed in terms of increased dehydration at the enzyme surface and an ice/unfroze solution interface, which could be helpful to establish a common understanding of enzyme inactivation during the freezing process.

Development of temperature regime of storage of frozen black currants

The process of ice formation in the pericarp of black currant depends on the pomological variety, the degree of ripeness of the fruit, and the method of freezing. During the slow and fast freezing of black currant, four ranges of fruit cooling temperatures are distinguished: 1) from the temperature of the fruit to the temperature of initiation of ice formation; 2) from the temperature of the front (initiation) of ice formation to the lowest possible temperature of the fetal mesocarp; 3) from the lowest possible temperature of the mesocarp to the lowest temperature of the fetal endocarp; 4) from the lowest possible endocarp temperature to fetal freezing temperature. Fast freezing boosts cooling, freezing and freezing from 37 min. (slow) up to 5.6 min. due to a halving of the temperature of initiation of ice formation, an increase of 1.3 times in the rate of heat extraction and an increase in the freezing temperature from –22...–24 °С (slow) to –20.8 °С. It is scientifically substantiated that the temperatures of freezing fruits significantly change the general existing recommendations (not higher than –18 °С) regarding the storage conditions of black currant fruits: with quick freezing, not higher than –21 °С, with slow freezing, not higher than –24 °С. The formation of the properties of black currant occurs during the growing season under various agro-climatic conditions and affects the parameters of ice formation indicators. The marketable condition, quality and organoleptic characteristics of black currant fruits depend on the method of freezing. The advantages of fast freezing of black currant fruits in a quick-freezing chamber with forced air circulation at a speed of 1.5–2.5 m/s at a temperature of –30...–32 °С in comparison with slow freezing in freezers at a temperature of –20 have been established. ..– 22 °С.