The maintenance of respiratory control at 25 degrees C by mitochondria from various animal and plant sources.

1. Mitochondria from four different animal and five different plant sources exhibit a wide variation in their capacity to maintain respiratory control at 25 degrees C. 2. Beef heart mitochondria, and pear and avocado fruit mitochondria exhibit the longest retention of respiratory control extending to periods of approximately 80 and 40 hr, respectively.     

Survival of bovine embryos stored at 4 degrees C

These experiments were designed to test the efficacy of storing bovine embryos at 4 degrees C. Of particular interest were the age of embryo at which maximum post-storage survival could be achieved and longevity at 4 degrees C. A greater proportion of day 8 blastocysts developed in vitro at 37 degrees C following refrigeration for 48 hr than did embryos collected 2, 4 or 6 days after estrus (P<0.01). Survival of blastocysts stored at 4 degrees C for 48 hr was similar to that of nonstored blastocysts. In a subsequent experiment, day 8 blastocysts were recovered nonsurgically and assigned to one of the following treatments: (a) immediate transfer; (b) culture at 37 degrees C; or (c) storage at 4 degrees C for 1, 2, 3 or 5 days. Post-storage viability was assessed by either development in culture at 37 degrees C or embryo survival following nonsurgical transfer to synchronized recipients. In vitro survival of nonstored embryos and embryos stored 1 day did not differ. Survival decreased after storage for 2 days (P<0.10) or longer (P<0.05). Similar results were observed for survival after transfer, but embryo viability decreased even more rapidly with increasing duration of storage. In vitro survival was approximately 50% for blastocysts stored for 3 and 5 days, but few pregnancies resulted from transfer of embryos stored for these periods. In another experiment survival after transfer of blastocysts stored at 4 degrees C for up to 2 days was similar to that of nonstored blastocysts.     

Studies on rat liver mitochondria: 4. Enzyme activities in mitochondria preserved at 0-4 degrees C

Rat liver mitochondria, stored with the energy-linked functions preserved or in aging conditions, were used to assay the activity of various enzymes during five days. The preservation of energy-linked functions was monitored by the respiratory control coefficient. ATPase, cytochrome oxidase and NADH dehydrogenase showed increased activity when the energy-linked functions were preserved. In aging conditions, cytochrome oxidase, NADH dehydrogenase and ATPase showed decreased activity. The ATPase activity increased only when mitochondria were stored in the presence of inhibitors of the electron transport chain. The activity of NADH oxidase did not change, and succinate oxidase and succinate dehydrogenase showed a small decrease in their activity. The enzymes of the matrix, alpha-ketoglutarate dehydrogenase, malate dehydrogenase and aspartate aminotransferase showed little decrease in activity under either of the conditions of storage. The total protein content decreased slightly under both conditions of storage. These results show that the activity of the enzymes analysed was maintained at reasonable levels, when the energy-linked functions of isolated mitochondria were preserved.     

Survival of ovine embryos stored at 4 degrees C for 24 hours

This study was conducted to ascertain if sheep embryos collected for transfer can be stored for short periods without freezing to allow for international transport. Of twelve Finnish Landrace ewes treated with equine follicle stimulating hormone (FSH), eleven ewes ovulated with a mean of 9.1 +/- 4.3 (SD) corpora lutea. Recovery rate from the nine ewes with normal corpora lutea was 68 +/- 27%, providing 61 morulae which were then cooled to 4 C and stored for 24 h while transporting them from Scotland to France. Romanov recipients received either 4 (n = 14) or 5 (n = 1) of these morulae. Fourteen of the recipients lambed, with a mean lambing rate of 2.1 +/- 0.8, representing 48.3% of embryos transferred. Cooling of embryos to 4 C and storing them in ovum culture medium for 24 h at 4 C may be a valuable technique for the handling and short-term storage of embryos.     

Survival of Entamoeba and related Amoebae at low temperature. I. Viability of Entamoeba cysts at 4 degrees C

The time of survival at 4°C of mature cysts of six species of Entamoeba was studied. The length of time cysts generally survived storage was E. histolytica 12 weeks, E. moshkovskii 36 weeks, E. terrapinae and E. knowlesi 24 weeks, E. ranarum 25 weeks and E. invadens 7 weeks.     

Studies on rat liver mitochondria. 6. The effect of contaminating particles in mitochondria stored at 0-4 degrees C

Rat liver mitochondria were stored at 0-4 degrees C for several days using an appropriate medium and energy source. The elimination of the majority of microsomes and lysosomes, that normally contaminate isolated mitochondria, had a positive effect in preservation of respiratory control, P:O ratio, and monoamine oxidase activity during long term storage.     

Hyaluronic acid delays boar sperm capacitation after 3 days of storage at 15 degrees C

The present study was undertaken to determine the effects of the addition of hyaluronic acid (HA), ranged from 12.5 to 200 microg/ml, on boar sperm capacitation status during a storage time (up to 3 days) at 15 degrees C in Beltsville thawing solution (BTS). The raw extender was the negative control whereas different concentrations of caffeine (CAF), ranged from 0.25 to 8mM, served as positive controls. Sperm viability, motility, morphology, and osmotic resistance were also determined before and after assessing the treatments. Samples were obtained from 28 healthy and post-pubertal Piétrain boars and sperm parameters were tested immediately after the addition of treatments and after 1, 2 and 3 days of refrigeration at 15 degrees C. Sperm capacitation status was determined by chlortetracycline (CTC) staining and sperm viability by means of a multiple fluorochrome-staining test. Sperm motility and morphology were assessed using phase-contrast microscopy accompanied by a computer assisted sperm analysis system (CASA). Whereas HA delayed sperm capacitation, CAF increased the frequency of capacitated spermatozoa after 2 days of cooling. Moreover, HA did not modify other sperm parameters, such as sperm velocity, whereas CAF increased progressive motility during the first 2 days of cooling and then decreased. It can be concluded that the addition of HA at 50 and 100 microg/ml to the BTS extender may delay sperm capacitation after 3 days of cooling.     

In vitro survival of Echinococcus granulosus protoscolices in several media, at +4 degrees C and +37 degrees C

As a first step towards hydatid disease control, the in vitro survival of protoscolices of Echinococcus granulosus was investigated for various media and temperatures. Higher percentage survival was obtained than previously reported: at 4 degrees C, 100% survival was obtained for 20 days in medium 199 (GIBCO) and for 25 days in hydatid fluid from the host of origin. Maximal survival was 30% at 55 days in these conditions. Flame cell activity was the criterion of choice for viability. At 37 degrees C survival rates were lower and morphological changes in protoscolices were observed.     

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.     

Effect of dihydroxyacetone on metabolic parameters and survival of resuspended erythrocytes after storage at 25 degrees and 4 degrees]

Erythrocyte concentrates from CPD blood were resuspended after separating the leukocyte-thrombocyte layer and stored at 4 degrees C and 25 degrees C. The solutions for resuspension differed in the content of substrate, pH, condition of sterilization and the resuspension volume used, 1 or 4 volumes of cell concentrate to 1 volume of resuspension solution. At 4 degrees C, there was no effect of DHA on the the 2.3 bisphosphoglycerate content (P2G content). The decreased activity of triokinase at low temperature, phosphate deficiency and a partial disintegration of DHA during the autoclave as well as the difference of the commercial DHA preparations are discussed as underlying causes. At 25 degrees C DHA delayed the P2G decrease during storage. The enhanced triokinase activity above 15 degrees C is principally considered to be the cause for it. High rates of haemolysis, rapid ATP decrease, survival rates of 56% after a storage of three weeks for erythrocyte resuspensions with DHA in used compositions render a clinical application impossible at present. It could be well suitable as a substrate for resynthesizing P2G. An addition of ascorbate + nicotinamid + adenin at a storage temperature of 4 degrees C had no influence on the P2G content. A resuspension solution on the basis of xylitol with additions of sorbitol, bicarbonate, inorganic phosphate, pyruvate, adenine and guanosine showed the best properties in the content of P2G and ATP, lactate formation and survival rate during a storage at 4 degrees C.     

Chloroethene biodegradation in sediments at 4 degrees C

Microbial reductive dechlorination of [1,2-14C]trichloroethene to [14C]cis-dichloroethene and [14C]vinyl chloride was observed at 4 degrees C in anoxic microcosms prepared with cold temperature-adapted aquifer and river sediments from Alaska. Microbial anaerobic oxidation of [1,2-14C]cis-dichloroethene and [1,2-14C]vinyl chloride to 14CO2 also was observed under these conditions.     

Recovery of motile,membrane-intact spermatozoa from canine epididymides stored for 8 days at 4 degrees C

The purpose of this study was to determine how long canine spermatozoa remain motile and with intact membranes when maintained within epididymides stored at 4 degrees C, and to determine whether such stored spermatozoa are able to bind to canine zonae pellucidae. Testes with attached epididymides, obtained from 32 dogs (26 purebred; six mixed breeds) at orchiectomy, were refrigerated at 4 degrees C, and spermatozoa were collected from caudae epididymides at nine time intervals ranging from 5 to 192 h. The effects on spermatozoa that had been refrigerated within epididymides for various times were determined by assaying sperm motility, integrity of plasma membranes and of acrosomes, and measuring binding of membrane-intact spermatozoa to canine zonae pellucidae. Membrane integrity was assessed using a double fluorescent dye, and acrosome integrity by staining with Pisum sativum agglutinin. For the zona-binding assay at various refrigeration time points, duplicate sets of six oocytes each, isolated from ovaries retrieved at elective ovariohysterectomy, were placed into 100 microl droplets of sperm capacitation medium containing 5 x 10(6) spermatozoa/ml. One minute later, oocytes were rinsed vigorously by pipetting, and then incubated for 1 h at 38.5 degrees C in a humidified atmosphere of 5% CO2 in air; the number of membrane-intact spermatozoa bound to zonae were counted. There was no significant decrease in membrane integrity and acrosome integrity of spermatozoa recovered from epididymides stored at 4 degrees C within the first 48 h of refrigeration. In contrast, sperm motility decreased significantly within the first 5 h of refrigeration (P < 0.05), but then declined more gradually thereafter. Some spermatozoa recovered from epididymides that had been refrigerated for 192 h retained their capability to bind to zonae pellucidae, although the mean number of refrigerated spermatozoa (0.4) bound to zonae was less than that of fresh samples (9.0). Membrane integrity of spermatozoa recovered from epididymides refrigerated for various times was highly correlated (r = 0.88) with sperm motility. Even after storage for 192 h (8 days) at 4 degrees C, motile spermatozoa could be recovered from the epididymides, and such refrigerated spermatozoa were capable of binding to zonae. We interpreted these data to indicate that it might be possible to recover functional spermatozoa from postmortem specimens of domestic and nondomestic canids.     

Rapid-cooling and storage of plant nematodes at -140 degrees C

Low temperatures can assure the long-term or even indefinite preservation of important biological specimens. Nematode cryopreservation allows for the availability of large numbers of living nematodes at any one time, especially for experimental purposes. New isolates of Bursaphelenchus have recently been collected, including Bursaphelenchus eremus (Rühm) Goodey. This species was identified in north-central Italy on dying oak trees and from the bark beetle Scolytus intricatus Ratzeburg as dauer larvae. We therefore, sought to develop a cryopreservation technique for the long-term storage of all available Bursaphelenchus spp. The technique consists of a rapid-cooling protocol involving immersion in a liquid nitrogen bath before storage of the frozen samples in a mechanical freezer at -140 degrees C. The survival of nematodes subjected to this rapid-cooling protocol was higher than previously reported using slow-cooling methods and is suitable for several species of Bursaphelenchus and other phytoparasitic nematodes.     

Sub-lethal damage of Listeria monocytogenes after long-term chilled storage at 4 degrees C

The possibility that long term in vitro chilled storage may result in sub-lethal damage to Listeria monocytogenes cells was investigated by comparing growth of chill-stored (starvation at 4 degrees C) and fresh cultures on selective and non-selective media. Growth of freshly grown cells was minimally (3-8%) affected by selective LSAMM agar compared with non-selective Brain Heart Infusion agar. In contrast, numbers of chill-stored strains were reduced by greater than 99% after direct plating on the same selective and non-selective media. Furthermore, chill-stored strains were able to grow in standard selective broth (Listeria Selective broth and Fraser broth) only if undiluted inocula (approximately 10(5)-10(6) cfu ml-1) were used, whereas they were capable of growth in Brain Heart Infusion broth even when the lowest dilutions were used (approximately 10(1) cfu ml-1). The potential public health consequences of this finding for the isolation of Listeria monocytogenes from foods is considered.     

Relationship between dead cells and DNA fragmentation in bovine embryos produced in vitro and stored at 4 degrees C.

DNA fragmentation and its relationship with dead cells were examined in bovine blastocysts produced in vitro and stored at 4 degrees C for 1-5 days. Survival and development to the hatching and hatched blastocyst stage decreased with increasing storage time. Both were significantly lower at 72 hr than at 48 hr. None of the embryos stored for 120 hr developed to the hatching or hatched blastocyst stage. The proportion of dead cells per embryo increased progressively as the time of storage increased, until 69% of embryonic cells were dead after 120 hr of storage. There was no significant difference between the proportions of DNA fragmentation per embryo stored for 0 and 24 hr (12% vs 16%). However, the proportion of DNA fragmentation in embryos stored for longer than 48 hr was significantly greater than that in embryos stored for less than 24 hr. There were no significant differences among those stored for longer than 48 hr (28-33%). These results suggest that the reduced developmental competence of bovine embryos stored at 4 degrees C is characterized by necrotic change rather than apoptotic change.