Effects of mint,thyme, and curcumin extract nanoformulations on the sperm quality,apoptosis, chromatin decondensation,enzyme activity,and oxidative status of cryopreserved goat semen

Goat semen cryopreservation is a challenging process as it results in reduced motility, vitality, and fertility of spermatozoa after freezing. In this study, we evaluated the effects of different herbal extract nanoformulations (NFs) [mint (MENFs), thyme (TENFs), and curcumin (CENFs)], supplemented at either 50 or 100 μg into Tris-extender on the cryopreserved goat semen quality. The hydrothermal squeezing method was used for the preparation of the NFs extracts. The morphological evaluation of the NFs extracts was conducted by transmission electron microscopy. All NFs supplements improved (p < 0.05) the progressive motility, vitality, and plasma membrane integrity of sperm compared with the control extender after equilibration (5 °C for 2 h) and thawing (37 °C for 30 s), but had no effect on sperm abnormality and acrosome integrity. All NFs supplements decreased (p < 0.05) the apoptosis, malondialdehyde level, and chromatin decondensation of sperm cells, while increased (p < 0.05) the total antioxidant capacity and catalase activity in the frozen/thawed extender. Particularly, CENFs at a level of 100 μg showed improvement of sperm parameters and antioxidant status during cryopreservation of goat semen more than TENFs and MENFs. The CENFs improved the quality of goat spermatozoa in post-thawed semen in terms of preventing cryodamage and promoting the cryotolerance of spermatozoa when compared with TENFs and MENFs. Therefore, supplementation of Tris-extender with CENFs could enhance goat semen processing during cryopreservation.     

Raman spectroscopy evidence of lipid separation in domestic cat oocytes during freezing

Although lipid droplets are believed to play an important role in cryopreservation of mammalian embryos and oocytes, the effect of low temperatures on lipid droplets and related mechanisms of cryodamage are still obscure. Here, we provide Raman spectroscopy evidence of lipid separation inside the lipid droplets in domestic cat oocytes during slow freezing. It was shown that at −25 °C lipids coexist in two separated phase states inside lipid droplets. The scale of detected domains was a few micrometers size. We also found that under certain conditions these areas have a specific spatial distribution. Lipids with high melting temperatures are distributed near the surface of lipid droplets while fusible lipids are located deep inside. Raman spectroscopy was found to be a prospective approach to study inhomogeneity of lipid phase transition in cells and to reveal effects of this inhomogeneity on cryopreservation of biological cells.     

Cryopreservation of Panax ginseng cells

The impacts of cryoprotectants (CP) and cell status during the growth cycle on Panax ginseng cell viability during cryopreservation were investigated. The ginseng cells used had a 5–7 times proliferation rate (compared with inoculum) in 2–3 weeks and were subcultured at 2- and 4-week intervals in liquid and on solid media, respectively. After testing various CP solutions of glycerol, dimethylsulphoxide, ethylene glycol and sucrose, a combination of 10% (v/v) glycerol and 4% (w/v) sucrose was selected for its least cytotoxicity and highest cell viability after thawing. With this CP solution, cells throughout the growth cycle exhibited a ‘U’-shaped fluctuation of post-thaw cell viability. The highest viability (86.5%) occurred during the lag phase from cells already maintained in suspension culture and then in the late exponential phase (61.7%); the lowest level of 15.4% was in the mid-exponential phase. Callus freshly transferred to liquid medium showed a less obvious fluctuation pattern. The recovered cells were brown-to-reddish at first and gradually returned to a light yellow colour after several subcultures. Received: 1 October 1998 / Revision received: 6 January 2000 / Accepted: 11 January 2000     

Successful ultrarapid cryopreservation of wild Iberian ibex (Capra pyrenaica) spermatozoa

A method for cryopreserving wild ibex sperm at high cooling rates was developed. To design a freezing solution based on Tris, citric acid, and glucose (TCG), two preliminary experiments were performed using glycerol (GLY) and dimethyl sulfoxide (DMSO) at different concentrations (5%, 10%, 20%). The 10% GLY + 10% DMSO combination reduced (P < 0.05) frozen-thawed sperm motility, which reached a minimum when 20% GLY + 20% DMSO was used. In the second experiment, sperm tolerance to three sucrose concentrations was evaluated (100-mM sucrose, 300-mM sucrose, 500-mM sucrose). Frozen-thawed sperm motility and sperm viability decreased (P < 0.05) at concentrations above 300 mM. The ultrarapid cooling procedure finally used involved a TCG egg yolk (ey)–based extender with 100-mM sucrose, either alone or with 5% GLY with or without BSA. Two warming procedures (37 °C vs. 60 °C) were also evaluated. The TCG ey with 100-mM sucrose but without GLY/BSA returned the best sperm quality variables. Slow warming at 37 °C strongly affected (P < 0.05) sperm motility and viability in all groups. Sperm selection by density gradient centrifugation produced no motile sperm when slow warming was performed. In contrast, when fast warming was used, sperm selection increased (P < 0.05) percentage of motility, viability, and the percentage of sperms with intact acrosomes. Heterologous in vivo fertilization involving domestic goats was performed to evaluate the in vivo fertilization capacity of the ultrarapidly cooled cryopreserved sperm (in TCG-ey + 100 mM sucrose), with warming undertaken at 60 °C. Inseminations of domestic goats resulted in three pregnancies (3 of 16, 18.7% fertility). In conclusion, ibex spermatozoa are strongly sensitive to high concentrations of permeable cryoprotectants and sucrose. However, the combination of ultrarapid cooling, using TCG-ey + 100-mM sucrose, and fast warming at 60 °C, followed by sperm selection by density gradient centrifugation to collect the motile sperm, has a positive effect on sperm viability.     

A decision-based key to determine the most appropriate protocol for the preservation of fungi

The long-term preservation of valuable fungal cultures can be achieved in several ways and the choice of methodology can be problematical. Firstly, there is the decision whether to use a public service culture collection or `in-house' facilities. Secondly, the wide variety of preservation methods available often leads to confusion about which protocol(s) are best suited for specific fungi. No method can be universally applied to all fungi. Some species are notoriously difficult to preserve, whilst other fungi can be preserved by almost any method. A decision-based key has been devised, which uses questions related to fungal characters and user facilities and economics to determine the most appropriate method for long-term preservation of cultures. This key should facilitate the decisions of microbiologists when considering preservation of important fungal cultures.     

Structural, metabolic and developmental evaluation of ovulated rabbit oocytes before and after cryopreservation by vitrification and slow freezing

The cryopreservation of oocytes is valuable for the preservation of women's fertility and might also be an interesting tool to preserve animal genetic biodiversity but it is not often used because of the very poor fertility recovered after thawing, especially in rabbit species. The objective of our study was to evaluate the effect of slow-freezing and vitrification on the structural integrity of ovulated rabbit oocytes, their ATP contents, and their developmental competence. Results show that, whatever the method is used, cryopreservation has a dramatic effect on the metabolic integrity, the structural integrity, and the developmental ability of the oocytes. Vitrification and slow freezing both impair the rabbit oocytes viability after thawing but the processes act differently. Further studies are needed to improve the cryopreservation techniques in rabbit species. Moreover, we underlined that morphology and maintenance of the structural integrity of the oocytes are not suitable enough to assess the potential for further development of cryopreserved M_II oocytes. The assessment of ATP metabolism allows efficient evaluation of the viability of the frozen or vitrified oocytes. It should be used in addition to parthenogenesis to better assess the potential for further development.     

Tyrosine phosphorylation of cytochrome c as a signaling event in frozen thawed buffalo spermatozoa at the cross-roads of capacitation and apoptosis

Considering the importance of cytochrome c in both life and death, it was of significant interest to investigate the expression of cytochrome c, its tyrosine phosphorylation status and immunolocalization patterns in a frozen-thawed buffalo sperm cell in comparison to in vitro capacitated [heparin (10 μg/ml) induced, for 4 h] and stress [apoptotic (10 μM staurosporine), oxidative (25 μM H₂O₂) and osmotic (180 mM NaCl) for 4 h] induced conditions. Proteins were subjected to immunoblotting and probed by using monoclonal anti-phosphotyrosine antibodies. A significant (p < 0.05) increase in expression of tyrosine phosphorylated cytochrome c was observed in capacitated buffalo sperm in comparison to frozen-thawed samples. cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent tyrosine phosphorylation of cytochrome c was found during in vitro capacitation of buffalo spermatozoa. Localized increase in cytochrome c and tyrosine phosphorylated proteins were observed in frozen thawed and capacitated sperm. The information generated in this study can be used to understand the molecular mechanism of regulation of an apoptotic protein (cytochrome c) by tyrosine phosphorylation (a capacitation marker) in a frozen thawed sperm cell which could be a good target to combat apoptosis.     

Cryostorage of silver barb (Barbodes gonionotus) semen in dry shipper: Efficacy,risk of bacterial cross-contamination and effects of Aeromonas hydrophila on post-thaw sperm quality

This study was conducted to investigate the efficacy of dry shipper for the cryostorage of silver barb (Barbodes gonionotus) sperm, the subsequent risk of bacterial cross-contamination, and the effects of Aeromonas hydrophila on post-thaw sperm. Semen was diluted with calcium-free Hank's balanced salt solution containing 10% ME₂SO, frozen at −8 °C/min and stored for 14 d in a dry shipper. A significant decline (P < 0.05) in the post-thaw sperm motility and viability of samples kept in the dry shipper for 14 d showed a reverse correlation (P < 0.05) with a slight increase in temperature within the dry shipper. The levels of contaminated bacteria in the compartments of the dry shipper were significantly (P < 0.05) lower than those detected in the liquid nitrogen tank. Bacteria from the atmosphere could recontaminate the chambers of the dry shipper and liquid nitrogen tank after 14 d. Bacillus was the most common bacteria isolated from the dry shipper, liquid nitrogen tank, circulating air, bench surface and outer surface of straws. There was no cross-contamination of A. hydrophila from contaminated straws to pathogen-free straws kept in either cryogenic tank. Post-thaw sperm motility and sperm viability significantly (P < 0.05) declined during cryostorage in the dry shipper and liquid nitrogen tank due to the introduction of A. hydrophila and the interaction effect of A. hydrophila and freezing. This study reports, for the first time, the efficacy of a dry shipper for the cryostorage of fish sperm for at least 14 d without a risk of bacterial cross-contamination.     

Morphological and biochemical analysis of human cardiac valve allografts after an increment of the cryostorage temperature

Cryopreserved human cardiac valve allografts could suffer lethal damages if the temperature is elevated during cryostorage. This work describes the functional and morphological alterations suffered by human cardiac valve allografts after a gradual increment of the cryostorage temperature from −147 °C to −47 °C due to a technical failure. Three experimental groups of human pulmonary and aortic allografts were compared: fresh, cryopreserved (−147 °C) and cryopreserved with temperature changes from −147 °C up to −47 °C and back to −147 °C. Fibroblast functionality was studied to asses the degree of valvular damages. Collagen network was also analyzed with bright light field and polarized microscopy; an immunohistochemistry for procollagen I was performed and the MTT colorimetric assay was used to evaluate fibroblast mitochondrial enzymatic activity. Porcine heart grafts valves were used to set the MTT colorimetric assay.With bright light field microscopy, disorganized collagen network was seen together with interstitial edema in cryopreserved groups. Polarized microscopy showed that fresh allografts had abundant collagen type I and III, cryopreserved group had less amount of collagen type I and in allografts that suffered cryopreservation temperature elevation collagen type I synthesis could not been demonstrated. Procollagen I was present in fibroblast cytoplasm of fresh group, but it was diminished in cryopreserved group and was absent in the group that suffered temperature elevation.Temperature changes during the cryopreservation period of human cardiac valve allografts induced fibroblast activity reduction. When the cryopreservation temperature is elevated during cryostorage, fibroblasts lost their functionality and the allografts may be not suitable for transplant.