Tissue cryobanking for conservation programs: effect of tissue type and storage time after death

In this study, we investigated the temporal post-mortem limits, within which there will be guarantees of obtaining living cells from several tissues of sheep and cattle and the effect of vitrification on the ability of cells from tissue stored at different times. Muscle tissue and auricular cartilage were stored at 4°C for 5, 48, 72, 96 and 216 h post-mortem (hpm). Tissue samples were sorted into two groups: one group was in vitro cultured immediately after storage and the other was vitrified after storage and then in vitro cultured. In cattle and sheep, no differences in subconfluence rates were observed between the two experimental groups. At the same time, no significant differences were observed in the number of days required in culture to reach confluence between non-vitrified and vitrified groups when tissues were stored at 4°C for different times. In sheep, while the population doubling times (PDT) were similar in cartilage cells from vitrified and non-vitrified tissues and stored at 4°C for 5 and 216 hpm, PDT of muscle cells were longer in 216 hpm stored groups than in 5 hpm stored groups. In bovine, although the PDT of muscle cells were similar for 5 and 216 hpm and both vitrified and non-vitrified tissues and the PDT were longer in cartilage cells from vitrified than from non-vitrified tissues. In conclusion, although storage times and vitrification have different effects on tissues from cattle and sheep, this study showed that living cells could be obtained from all groups. Therefore, cartilage and muscle tissues can be stored at 4°C for 216 hpm and used for cyrobanking.     

Biochemical investigations into the absence of rigor mortis in the Norway lobster Nephrops norvegicus

It was found that the striated muscle of the Norway lobster (Nephrops norvegicus) does not exhibit the rigor mortis state otherwise typical for this type of muscle. This absence of rigor was investigated, concentrating on changes in the structure, ultrastructure and post-mortem biochemistry of the muscle. Samples were initially fixed for light and electron microscopy at the time of death and at different times post-mortem (3, 6, 12 and 24 h). Protein extracts were obtained in parallel to compare the banding patterns of the myofibrillar proteins using SDS-PAGE. A Western blot was applied to elucidate if myosin - a representative major myofibrillar protein - was degraded post-mortem. And finally, ATP levels in the muscle were analyzed using HPLC. Using TEM imaging it was found that between 12 and 24 h post-mortem at a storage temperature of 10 °C, when rigor mortis should set in (according to the muscular ATP concentrations), an extensive, but rather specific breakdown of myofibrillar proteins occurred. The Z-disks were degraded and the myofibrillar structure was lost. SDS-PAGE and Western blot clearly demonstrated the post-mortem breakdown of myosin. The nature of the observed protein breakdown seems to impede rigor mortis in some way by the activation of at least one of the several proteolytic systems (cathepsins, calpains and others) found in vertebrates and invertebrates. It is speculated that the proteolysis simply overtakes the rigor-inducing post-mortem changes.     

Post-mortem elemental redistribution in rat studied by X-ray microanalysis and electron microscopy

Summary Post-mortem elemental redistribution in various tissues from rat was studied by means of electron probe X-ray microanalysis, and correlated with morphological changes in these tissues. Pancreas, liver and cardiac muscle were removed from the animal either immediately, or after some hours after death. Elemental distribution at the cellular level was studied by X-ray microanalysis of thick cryosections. Calcium redistribution at the subcellular level was studied using tissue fixed with glutaraldehyde/oxalate. In all tissues, post-mortem redistribution of electrolytes had taken place within 2 h. The cellular concentrations of Na, Cl and Ca increased markedly, those of Mg and K decreased; no significant changes were found in the concentrations of P and S. The number of oxalate precipitates (indicating the presence of calcium) increased both in the mitochondria and in the cytoplasm and endoplasmic reticulum, reaching a maximum at 2 h. Morphological changes included mitochondrial swelling and vesiculation of the endoplasmic reticulum. Since the post-mortem ion shifts are similar to those encountered in some diseases and types of cell injury, great care has to be taken in the interpretation of X-ray microanalytical results from autopsy material.     

Effect of different equilibration times and extenders on deep freezing of buffalo semen

Thirty semen collections from 3 Murrah buffalo bulls were frozen in Tris yolk glycerol (TY-G) and Citric acid whey glycerol (CAW-G) extenders using 2, 4 and 6 hours equilibration times and 7 percent glycerol level. Sperm motility after freezing was studied at an interval of 15 minutes 7 days and 30 days storage in liquid nitrogen. Sperm survivability was found to be better at all the stages of deep-freezing using 4 hours equilibration time. Significant differences (P 0.01 ) were observed between extenders and equilibration times.     

Proteome analysis of early post-mortem changes in two bovine muscle types: M. longissimus dorsi and M. semitendinosis

To study early post-mortem changes in muscle tissues from bull calves, cytosole proteins from two muscles: M. longissimus dorsi (LD) and M. semitendinosis (ST) at 0 and 24 h after slaughter were analysed by 2-DE. Principal component analysis (PCA) and rotation testing were used to analyse the protein patterns in the two muscles in order to select protein spots that were significantly different at the two time-points. Selected proteins were identified by MALDI-TOF/TOF. Five proteins, namely cofilin, lactoylglutathione lyase, substrate protein of mitochondrial ATP-dependent proteinase SP-22, HSP 27 and HSP20, were changed in both LD and ST muscles during post-mortem storage. Fifteen additional protein changes were observed in either LD or ST muscles, and some of these changes have not previously been observed to change during post-mortem storage of bovine muscles. Further studies will reveal the relevance of these biomarkers for meat quality.     

Subcellular localization of cadmium in the root cells of<Emphasis Type="Italic">Allium cepa</Emphasis> by electron energy loss spectroscopy and cytochemistry

The ultrastructural investigation of the root cells ofAllium cepa L. exposed to 1 mM and 10 mM cadmium (Cd) for 48 and 72 h was carried out. The results indicated that Cd induced several obvious ultrastructural changes such as increased vacuolation, condensed cytoplasm with increased density of the matrix, reduction of mitochondrial cristae, severe plasmolysis and highly condensed nuclear chromatin. Electron dense granules appeared between the cell wall and plasmalemma. In vacuoles, electron dense granules encircled by the membrane were aggregated and formed into larger precipitates, which increase in number and volume as a consequence of excessive Cd exposure. Data from electron energy loss spectroscopy (EELS) confirmed that these granules contained Cd and showed that significantly higher level of Cd in vacuoles existed in the vacuolar precipitates of meristematic or cortical parenchyma cells of the differentiating and mature roots treated with 1 mM and 10 mM Cd. High levels of Cd were also observed in the crowded electron dense granules of nucleoli. However, no Cd was found in cell walls or in cells of the vascular cylinder. A positive Gomori-Swift reaction showed that small metallic silver grains were abundantly localized in the vesicles, which were distributed in the cytoplasm along the cell wall.     

Uptake and metabolism of [3H]norepinephrine in the cerebral hemispheres of chick embryos

Abstract— The uptake and metabolism of [³H]norepinephrine were studied in slices of cerebral hemispheres removed from chick embryos at 10, 15 and 20 days of embryonic age, as well as from 90-day-old hens. Brain tissue from all age groups concentrated [³H]norepinephrine to much greater levels at 37°C than at 0°C. There was a marked increase in the rate of accumulation of [³H]norepinephrine in tissues from 10 to 15 days of embryonic age, with no further increase in the rate observed from 15 to 20 days of embryonic age. Tissue slices were incubated for 20 min with [³H]norepinephrine, and the deaminated metabolites of norepinephrine were separated by paper chromatography. In tissues from all age groups, the neutral metabolites were produced in greater amounts than the acid metabolites. A significant increase in the amounts of deaminated metabolites formed was observed in the period from 10 to 15 days of embryonic age and a significant decrease in the amounts formed was observed in the period from 15 to 20 days of embryonic age. The deamination at 20 days was very similar to that observed in the adult. A significant decrease in the level of the deaminated metabolites was noted in all age groups in response to cocaine (an inhibitor of neuronal uptake mechanisms), an observation suggesting that mechanisms for neuronal uptake of NE are functional by 10 days of embryonic development in the chick. However, a significant increase in the level of deaminated metabolites in response to reserpine (an inhibitor of uptake of NE into storage granules) was observed only in slices taken from 20-day embryos and from the 90-day-old hen. The effect in the hen was more prominent than in the 20-day embryo. These results were interpreted to indicate that mechanisms for the uptake of NE develop at an earlier embryonic age than mechanisms for the storage of NE and that mechanisms for storage continue to develop after hatching.     

Post-mortem changes in structure and function of ox muscle mitochondria. 1. Electron microscopic and polarographic investigations

Electron microscopy shows that intact mitochondria can be isolated from neck-muscle stored at 144h post-mortem at 4°. Isolated mitochondria, all in the condensed configuration, have clearly defined outer and inner membranes, outer compartments and intracristal spaces; a larger proportion of swollen ones was isolated from the 144h than from the 120 h post-mortem tissue.Mitochondria from 96 h tissue still retained the following % of the initial values for the ADP/O ratio, respiratory control index (RCI) and state 3 respiratory rate observed for 0–5h tissue: malate+pyruvate, 100, 72 and 53; succinate, 80, 30 and 74; ascorbate+ tetramethyl-p-phenylencdiamine (TMPD), 92, 88 and 72.Both the succinate and ascorbate-TMPD oxidase systems appear to have a critical storage time of about 70 h, whereas the malate+pyruvate system has one of about 96 h. Asharp decline of the ADP/O ratio, RCI and the state 3 respiratory rate occurred after this time, but these three parameters were better preserved in the ascorbate-TMPD oxidase system.The oxidation of the citric acid cycle intermediates in the neck-muscle mitochondria thus shows a higher sensitivity to post-mortem ageing with respect to cytochrome oxidase activity. This is probably due to post-mortem muscle acidification.     

Cytochemical Localization of Calcium in Prefusion Myoblasts from the Chick Embryo Myotome

Myoblast fusion is a Ca²⁺-dependent process. The aim of this report was to study the localization of Ca²⁺ in prefusion myoblasts from the brachial somites of chick embryos (51–108h of incubation), using the potassium pyroantimonate cytochemical method. When observed under a transmission electron microscope, electron-dense precipitates of Ca²⁺-antimonate were found in the basement membrane of the myotome, which separates the myotome from the adjacent mesenchyma. Within myoblasts, triads and sarcoplasmic reticulum associated with the first newly formed sarcomeres were observed, but a T-tubule network was not found. Moreover, Ca²⁺-antimonate precipitates were not observed in structures resembling T-tubules or sarcoplasmic reticulum. The results suggest that sarcomerogenesis and sarcoplasmic reticulum development occur simultaneously and that prefusion myoblasts have neither a T-tubule network nor Ca²⁺ deposits on sarcoplasmic reticulum. Small Ca²⁺ pools were found in the myoblast nuclei, cytoplasmic vesicles and mitochondrias. Ca²⁺-antimonate precipitates periodically distributed at the cell periphery, close to the cell membrane, were observed. These precipitates could represent internal Ca²⁺ stores located in the peripheral couplings and it is proposed that these pools of Ca²⁺ could be mobilized before fusion, leading to the increase in free intracellular Ca²⁺ that precedes myoblast fusion.     

Preparation and characterization of synthetic models for the dense bodies of human platelets

Calcium-pyrophosphate-nucleotide precipitates have been formed $\text{in}$$\text{vitro}$ with a composition which is essentially identical to that reported for the dense bodies (storage vesicles) of human platelets. The solution composition and effect of pH on precipitate formation, as well as the dissolution behavior and degree of protonation of the various species, were determined. The precipitates have a similar composition and form equally well at pH 5.7 or 7.4, or with various monovalent counter ions in the starting solutions. In all cases, the adenine nucleotides appear to be associated with fewer protons than the corresponding species in free solution, and all precipitate components can exchange or dissolve rapidly unless the precipitate is maintained in a solution relatively rich in calcium and adenine nucleotides. These precipitates may serve as useful models for examining the relative contributions of the dense-body membrane and core constituents to the uptake and storage of biogenic amines by human platelets.     

Effect of storage method and extender osmolality in the quality of cryopreserved epididymal ram spermatozoa

Post-mortem sperm recovery and cryopreservation could be a complement to germplasm banking in sheep, especially for endangered breeds. This study is an attempt to identify factors for improving the success of cryopreserving ram epididymal spermatozoa, considering the decrease of sperm quality with post-mortem time. Epididymal spermatozoa from 9 rams were kept at 5°C using three storage methods: within the epididymes, undiluted sperm mass, and diluted in extenders of different osmolality (TES-Tris-fructose at 320, 370 or 420 mOsm/kg, 20% egg yolk, 8% glycerol). At 0, 24, 48 and 72h, spermatozoa were cryopreserved using each extender. Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability and acrosomal status). Post-mortem time decreased pre-freezing and post-thawing sperm quality. Some storage x extender combinations improved the effect of post-mortem time on sperm quality. Both epididymis storage combined with the 420 extender, and storing the spermatozoa diluted in the 320 extender improved post-thawing quality, especially at long post-mortem times. Storing the spermatozoa diluted in the 370 extender was detrimental for the acrosomal status. These findings have practical applications. The simplest storage method (within the epididymes) seems to be adequate if hyperosmotic extenders were used for freezing. An alternative method could be storing the spermatozoa diluted in a hypoosmotic extender. These recommendations are limited to the osmolalities tested in this study (420 mOsm/kg and 320 mOsm/kg); other osmolalities should be tested.     

Tracer studies of food absorption in the digestive tract of Nautilus pompilius (Cephalopoda, Tetrabranchiata)

In Nautilus pompilius, tracer experiments with 14C-labelled food show that the midgut gland, caecum and crop are involved in absorption of nutrients. According to liquid scintillation and light- and electron-microscopic autoradiography, the midgut gland exhibits the highest activity, followed by the caecum and crop. The density of silver precipitates is highest in the terminal alveoli of the midgut gland. Precipitates are also seen in the main cells of the caecal epithelium. Few precipitates are found in the lamina epithelialis mucosae of the crop, indicating that, in addition to food storage, digestive processes begin in this organ. These results have been confirmed by injection of the protein ferritin into the buccal cavity. The largest amount of ferritin is seen in the dense bodies of the main cells of the midgut gland, whereas those of the main cells of the caecum and crop contain less ferritin.     

Ability of Shiga toxin-producing Escherichia coli and Salmonella spp. to survive in a desiccation model system and in dry foods

In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35 degrees C for 24 h in paper disks. At an inoculum level of 10(7) CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 10(3) to 10(4) CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 10(2) CFU/disk). After 22 to 24 months of subsequent storage at 4 degrees C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (10(3) to 10(4) CFU/disk). In contrast to the case for storage at 4 degrees C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25 degrees C and 35 degrees C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70 degrees C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25 degrees C.     

POST-MORTEM CHANGES IN HIGH AFFINITY CHOLINE UPTAKE

Abstract— When animals were killed by decapitation and the heads kept refrigerated at 2–4°C. high affinity choline uptake was maintained up to 3 days post-mortem. At 5 days post-mortem, there was a significant reduction in uptake. In tissues kept at 2–4°C for 1 day, the ionic dependence, drug sensitivity and kinetic parameters of uptake were identical to that of control tissues. At 3 days post-mortem, intact synaptosomal profiles, although with features characteristic of degenerating neuronal tissues, were observed in electronmicroscopic studies. In tissues maintained at room temperature, however, the uptake activity was nearly completely gone by 1 day. It is concluded that high affinity choline uptake is maintained for days, a surprisingly long time, in tissues kept in the cold immediately after death.
When pentylenetetrazol or pentobarbital were administered to rats in order to activate or depress the choline uptake, it was found that the activity-related changes in choline uptake undergo a reversal in post-mortem tissues. The changes in uptake were significantly lost by 10min post-mortem and totally absent by 30min post-mortem. In vitro studies with whole hippocampi indicate that the postmortem reversal in activity-related changes in uptake is temperature-dependent. It is concluded that because of post-mortem reversals in activity-related states of uptake the true magnitude of these activity-related changes in uptake may be underestimated by existing methods of assay. Acetylcholine levels in synaptosomal preparations did not clearly correlate with levels of choline uptake.     

Ability of Shiga Toxin-Producing Escherichia coli and Salmonella spp. To Survive in a Desiccation Model System and in Dry Foods

In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35°C for 24 h in paper disks. At an inoculum level of 10⁷ CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 10³ to 10⁴ CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 10² CFU/disk). After 22 to 24 months of subsequent storage at 4°C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (10³ to 10⁴ CFU/disk). In contrast to the case for storage at 4°C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25°C and 35°C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70°C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25°C.     

Characterization and short-term storage of Tasmanian devil sperm collected post-mortem

The Tasmanian devil is suffering from a severe population decline due to the fatal Devil Facial Tumour Disease (DFTD). The development of assisted reproductive technologies such as AI and long-term sperm storage could facilitate genetic management of captive insurance populations. The aim of this study was to characterise semen samples collected post-mortem, and to develop a suitable diluent for short-term preservation of devil sperm. Low numbers of sperm (1.33 ± 0.85 × 10⁶ sperm per male) were extracted from the epididymides of 17 males. Devil sperm sample characteristics such as concentration and morphology were similar to other dasyurids. The most commonly observed morphological abnormalities were midpiece separation, tail curling, and tail twisting (on the axial plane). Changes in motility occurred throughout the regions of the epididymis with (mean ± SD) 29.4 ± 16.8, 46.8 ± 13.6 and 29.4 ± 18.1% of sperm exhibiting motility, and 88.9 ± 11.4, 32.0 ± 24.3 and 0.1 ± 0.2% of motile sperm exhibiting forward progressive motility in the cauda, corpus and caput, respectively. Sperm from the cauda and corpus epididymis maintained 31.7 ± 26.6 and 80.6 ± 85.9%, respectively, of initial motility after 12 h at 15 °C in a TEST yolk buffer diluent. These findings provided new information regarding devil sperm biology and short-term sperm storage; such information is necessary for future development of long-term sperm preservation methods in the Tasmanian devil.     

QUALITY OF POWDERED BLACK PEPPER (PIPER NIGRUM L.) DURING STORAGE. I. SENSORY AND PHYSICOCHEMICAL ANALYSES

Black pepper powder (60 mesh) was stored in consumer unit packs of 100g capacity in low density polyethylene (LDPE) films of 100, 300, and 500 gauge at 27°C and 65% RH. Analyses for sensory quality (odor and flavor), volatile oil, oleoresin, piperine content, and TLC were carried out at 15, 30, 45 and 80 days of storage. Significant loss of “top notes”, volatile oil, and hydrocarbons were seen after 15 days of storage itself while the “basic notes” and oxygenated compounds were retained up to 45 days. There was no loss of piperine up to the end of the study. The black pepper powder was not fit for table use after 15 days, though it could be used for other culinary purposes up to 80 days of storage.     

Stability of RNA isolated from human trabecular bone at post-mortem and surgery

To determine the reliability of gene expression studies in human post-mortem bone, it is important to evaluate the stability of RNA isolated from such tissues as a function of the post-mortem interval. The stability of total RNA and bone-specific mRNA species was examined in bone samples obtained from routine autopsies and at surgery. The optimal temperature for any storage and transport of the bone before RNA isolation was shown to be 4 degrees C, and RT-PCR analysis is the preferred technique for the analysis of gene expression in post-mortem bone as it tolerates partial RNA degradation. For gene expression studies in bone, post-mortem cases, with a post-mortem interval of less than 48 h, should be selected, and the time that bone is stored after retrieval at autopsy or surgery should be kept to a minimum. Overall, our findings indicate that with appropriate storage and handling, RNA can be reliably isolated from human bone obtained at post-mortem and surgery to study ex vivo the pattern of gene expression in healthy individuals and in patients with musculoskeletal diseases such as osteoporosis and osteoarthritis.     

Ultrastructural demonstration of amine granules in the adrenal medullary cells of the rat using acid permanganate fixation.

Potassium permanganate fixative is usually employed at pH 7.0. At this pH the amines in the granules of the adrenal medullary cells do not react with permanganate. When the pH was adjusted to 5.0, electron dense precipitates were seen in the amine granules of part of the medullary cells, probably noradrenalin containing cells.