Optimizing the conventional method of sperm freezing in liquid nitrogen vapour for Wallachian sheep conservation program

The aim of the present study was to optimize the conventional method of sperm freezing in liquid nitrogen (LN₂) vapour for successful cryopreservation of Wallachian ram sperm, the genetic resources of the Czech Republic. Sperm in straws were frozen using the conventional freezing method via a static exposure of sperm doses to LN₂ vapour, or by four different modified freezing methods. Under modified freezing, straws were frozen by a discontinuous, time-dependent decremental change in the distance between the straws and the surface of LN₂. The viability of sperm was evaluated by flow cytometry after sperm equilibration, and immediately after thawing. Besides the observed inter-sire and daily variation, the obtained results suggest the methodological weakness of the conventional freezing method via the static exposure of sperm doses to LN₂ vapour. With the use of the optimized freezing procedure, all parameters of thawed sperm were significantly (P < 0.05) improved in comparison with the conventional method: percentage of thawed sperm viability increased up to 48.3%, percentage of sperm with plasma membrane damage after thawing decreased to 6.58%, percentage of sperm with acrosome damage decreased to 24.4%, and percentage of sperm with deteriorated mitochondrial activity decreased to 6.28%. In conclusion, our results suggest that an optimized freezing procedure should be routinely used instead of the conventional method to cryopreserve Wallachian ram sperm.

Cellulose Nanofibrils/Xyloglucan Bio-Based Aerogels with Shape Recovery

Bio-based aerogels containing cellulose nanofibrils (CNFs) are promising materials due to the inherent physical properties of CNF. The high affinity of cellulose to plant hemicelluloses (xyloglucan, xylan, pectin) is also an opportunity to develop biomaterials with new properties. Here, we prepared aerogels from gelled dispersions of CNFs and xyloglucan (XG) at different ratios by using a freeze-casting procedure in unidirectional (UD) and non-directional (ND) manners. As showed by rheology analysis, CNF and CNF/XG dispersions behave as true gels. We investigated the impact of the freezing procedure and the gel’s composition on the microstructure and the water absorption properties. The introduction of XG greatly affects the microstructure of the aerogel from lamellar to cellular morphology. Bio-based aerogels showed high water absorption capacity with shape recovery after compression. The relation between morphology and aerogel compositions is discussed.

Effect of N-Methylacetamide Concentration and Thawing Rate on Chicken Sperm Quality after Cryopreservation

In seeking alternative cryoprotectants to glycerol for a reference chicken semen freezing procedure, the aim of the present study was to compare the effect of two concentrations of N-Methylacetamide (MA) and two thawing rates on the quality of frozen-thawed semen. Semen samples were diluted in Lake pre-freezing extender, including 0.1 M trehalose in presence of 6% or 9% MA, loaded into straws, frozen in nitrogen vapors, and stored in liquid nitrogen. The following thawing treatments were used: 5 °C for 100 s and 38 °C for 30 s. Sperm quality (cell membrane integrity, motility and kinetic parameters) was assessed before and after cryopreservation. The decrease of MA concentration from 9 to 6% improved sperm quality after freezing/thawing and this effect was dependent on thawing temperature. Decreasing the MA concentration from 9 to 6% improved the proportion of undamaged membrane, motile, and progressive motile sperm recovered after thawing at 5 °C for 100 s; in contrast, no effect of the MA concentration was observed thawing at 38 °C for 30 s. Therefore, the treatment with 6% MA and thawing at 5 °C for 100 s has given the best cryoprotective action. These results contribute to improve the efficacy of the current chicken semen cryopreservation procedures.

Semen donation and establishment of an open canine semen bank: a novel tool to prevent inbreeding in pedigree dogs

At present, only 5% of pedigree dogs is being used for breeding. To increase the number of breeding dogs, one solution could be to start a canine semen bank based on the principle of semen donation, like in humans. Many dog owners have no desire to become dog breeders but are willing to preserve the genetic material of their dog, if offered this possibility. However, not all canine ejaculates are suitable for cryopreservation as the initial quality may differ and the resistance of sperm cells to survive the freezing procedure is highly variable. In order to freeze the semen of as many male dogs as possible, it is important to optimize and individualize the cryopreservation protocol per ejaculate. Practically, frozen semen can be stored in the CanIfreeze-semen bank or in veterinary practices adjacent to the owner of the bitch and can be used for insemination at a later time.

Whether the right of pursuit is constitutional property interest protectible against the state II part

The author, in this paper, is looking for Ariadne’s thread, which would help us to find the way through the legal labyrinth composed of justified reaction of the state in the war against organized crime, from one side and what is considered an appropriate protection of secured creditor’s subjective rights, from the other side. He points out that in resolving a dilemma, does the mortgagee have the right to enforce its debt against the Republic of Serbia (right of pursuit), in case if the extended confiscation of property (proceeds of crime) includes subject-matter of mortgage, the court should take into account does the mortgagee know or should know about criminal origins of encumbered assets. The court should evaluate a good faith of the mortgagee, bearing in mind all the circumstances of the case, especially: 1) The moment of a mortgage establishment, i.e. whether the registration of mortgage is prior in time to the initiation of a property freezing procedure; 2) Overall business and other relations between mortgagee and mortgagor, i.e. are these parties associated in any other way? 3) Objective changes in the economic position of mortgagee and mortgagor, i.e.is the security agreement true or simulated, in particular, whether the value of the secured claim actually enhanced the property of the mortgagor and at what consideration.

Cryopreservation of Mycobacterium bovis isolates

Research, development of new biotechnological methods, diagnostic tests, confirmation of results, and reinvestigations are possible because of the availability of well-preserved living organisms maintained without any changes. Cryopreservation is a simpler, more reliable and long-term stable method for culture maintenance. Storage temperature and composition of the suspending vehicle are factors that affect the viability of mycobacterial strains. Three vehicles and three storage temperatures were evaluated to define a suitable cryoprotective medium for the preservation of Mycobacterium bovis strains. Colonies of sixteen M. bovis isolates were used to prepare the suspensions, which were then added to three vehicles: sterile 0.85% saline solution (SS), Middlebrook 7H9 broth (7H9), and Middlebrook 7H9 broth with sodium pyruvate (7H9p) replacing glycerol. Aliquots of these suspensions were frozen by three different methods, directly in the -20°C freezer, directly in the -80°C freezer, and at -196°C by immersion in liquid nitrogen (LN). The frozen aliquots were thawed at room temperature after 45, 90 and 120 days. Mycobacterial viability was assessed by counting the living cells on plates of Stonebrink medium before and after the freezing procedure. Storage at -20°C exhibited a lower recovery of M. bovis compared to storage at -80°C (Dunn’s test, p=0.0018) and LN (Dunn’s test, p=0.0352). There was no statistically significant difference between storage at -80°C and in LN (Dunn’s test, p=0.1403), yet -80°C showed better results than LN. All three suspending vehicles showed no statistically significant difference in terms of viability (Friedman’s test, p=0.7765). Given the low loss proportion of 5% during storage at -20°C and the high cost equipment required for storage at -80°C and LN, we recommend storage at -20°C or -80°C, when this is available, for preservation of M. bovis field strains.