Increasing the stability of immobilizedLactococcus lactis cultures stored at 4 °C

Summary Immobilized cell technology was used to prepare concentrated cultures ofLactococcus lactis that lost only 22% of viability over a 30-day storage period at 4°C. Concentrated cultures ofL lactis CRA-1 were immobilized in calcium alginate beads and added to glycerol, NaCl or sucrose-NaCl solutions in order to obtain a_w readings ranging from 0.91 to 0.97. The suspensions were subsequently placed at 4°C and viability (CFU g^–1 of bead) was followed during storage. Viability losses were high at a_w readings of 0.95 and 0.97 and pH dropped significantly (up to one unit) in the unbuffered solutions. Addition of 1% soytone or glycerophosphate helphed stabilize pH, and a beneficial effect on viability during storage was observed in the glycerol-soytone mix when the beads were added to the conservation solutions immediately following immobilization. When beads were added to the conservation solution immediately following immobilization, a 70% drop in cell counts occurred during the first 5 days of incubation. Dipping theL lactis-carrying beads in milk for 2h before mixing with the glycerolsoytone 0.93 a_w solution reduced this initial 5-day viability loss. Cultures grown in the alginate beads also had good stability in the 0.93 a_w glycerol-soytone solution, where 78% of the population was viable after 30 days at 4°C. The process could be used to store immobilized cells at a processing plant, or by suppliers of lactic starters who wish to ship cultures without freezing or drying.     

Zinc-binding proteins from boar seminal plasma -- isolation, biochemical characteristics and influence on spermatozoa stored at 4°C

Affinity chromatography on Chelating Sepharose Fast Flow Gel-Zn(2+) was used for fractionation of boar seminal plasma proteins. Approximately 30% of total boar seminal plasma proteins showed affinity for zinc ions (ZnBP fraction). Native electrophoresis (PAGE) of ZnBP revealed six protein fractions which separated into 27 bands under denaturing conditions (SDS/PAGE). Two-dimensional electrophoresis (2D PAGE) showed 148 polypeptides with isoelectric points mostly in the basic and neutral pH range. The zinc-binding proteins comprise mainly 10-20 kDa polypeptides which are probably members of the spermadhesin family. ZnBP present in the incubation mixture of spermatozoa stored for 1 or 24 h at 4 °C allowed preservation of a higher percentage of cells exhibiting linear motility in comparison to a control sample stored in PBS. Presented results indicate that proteins binding Zn(2+) ions have a shielding effect on the sperm plasma membrane and acrosome of spermatozoa, protecting these structures against consequences of cold shock.     

Ram seminal plasma improves pregnancy rates in ewes cervically inseminated with ram semen stored at 5 °C for 24 hours

In this study, we compared pregnancy rates obtained using ram semen stored at 5 °C for 24 h, with ram or bull seminal plasma (SP) added to TRIS-egg yolk extender. During the breeding period, 670 adult Corriedale ewes were cervically inseminated with semen (2 × 10⁸ sperm in a volume of 0.2 mL) from eight adult Corriedale rams. Ejaculates, obtained using an artificial vagina, were split into three aliquots and diluted with the following: TRIS-egg yolk based extender (T), T + 30% ram SP (R), or T + 30% bull SP (B). Samples were refrigerated and stored at 5 °C for 24 h until used for AI. Pregnancy was assessed by ultrasonography 35 to 40 d after AI. Pregnancy rate was not affected by ram (P = 0.77) or breeding period (P = 0.43), and there were no interactions between extender and ram (P = 0.94), or extender and breeding period (P = 0.24). However, there was an effect of extender (P = 0.0009) on pregnancy rates; ram SP, but not bull SP, increased pregnancy rates compared with extender without SP (49.7, 38.1, and 31.1%, for R, B, and T respectively). In conclusion, ram SP added to TRIS-egg yolk extender had a beneficial effect on the pregnancy rate of ram sperm stored at 5 °C for 24 h and used for cervical insemination of ewes.     

Artificial insemination with cryopreserved sperm from feline epididymides stored at 4 °C

Recovering and storing sperm from the epididymides of males of rare felidae is useful for preserving the species. The objective of the present study was to determine pregnancy rates following artificial insemination (AI) of frozen-thawed epididymal sperm, which were cryopreserved following low-temperature storage of the epididymides. In this study, these sperm were used for unilateral intrauterine AI (UIUAI) or unilateral intratubal AI (UITAI) using 40 × 10⁶ and 10 × 10⁶ sperm, respectively. The caudal epididymides of 17 cats were stored at 4 °C for 24 h after castration. Artificial insemination of seven female cats was performed on Days 3 or 4 (start of estrus = Day 1) by UIUAI, 20 h after injection of 100 IU hCG to induce ovulation. Furthermore, UITAI at 24 h (UITAI-24) or 30 h (UITAI-30) after hCG were also done (five cats per group). It was noteworthy that AI by UIUAI and UITAI-24 was performed before ovulation, whereas AI by UITAI-30 was performed after ovulation. Pregnancy rates were 28.6% (2/7) by UIUAI, 80% (4/5) by UITAI-24, and 20% (1/5) by UITAI-30. Litter size was one or two by UIUAI, and one to four by UITAI. Spontaneous abortion occurred on Days 25-30 of pregnancy in one of the two female cats pregnant following UIUAI, and in two of five female cats pregnant following UITAI. Based on the high pregnancy rate obtained with 10 × 10⁶ sperm in the UITAI-24 group (AI performed before ovulation), we concluded that this was the most appropriate method for AI with frozen-thawed epididymal sperm after initial low-temperature storage of epididymides.     

A method to maintain mammalian cells for days alive at 4 °C

This paper describes a method for the temporary storage of cultured cells. Cells from recently completed cell monolayers were trypsinized and then centrifuged. After centrifugation, the supernatant and pellet were kept at 4 °C for one week. After storage, the supernatant was discarded, the cells were resuspended and used for seeding new flasks and for titration of virus. The cells not only remained viable, but also rapidly formed new monolayers and allowed immediate infection and growth of viruses. We conclude that this method can be a helpful asset to cell culture experiments.     

Biosynthetic capacity of hepatocytes after storage at 4°C

Capacity to synthesize glucose, urea, and ketone bodies is well maintained in hepatocytes after storage for at least 24 h at 4 degrees C. Substrates and albumin are the only requirements.     

Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 °C in presence of several antioxidants

Antioxidants may be useful for supplementing sperm extenders. We have tested dehydroascorbic acid (DHA), TEMPOL, N-acetyl-cysteine (NAC) and rutin on epididymal spermatozoa from red deer, during incubation at 37 °C. Cryopreserved spermatozoa were thawed, washed and incubated with 1 mm or 0.1 mm of each antioxidant, including oxidative stress (Fe²⁺/ascorbate). Motility (CASA and clustering of subpopulations), viability, mitochondrial membrane potential, and acrosomal status were assessed at 2 and 4 h. Lipoperoxidation, intracellular reactive oxygen species (ROS) and DNA damage (DNA) status (TUNEL) were checked at 4 h. Oxidative stress increased ROS, lipoperoxidation and DNA damage. Overall, antioxidants negatively affected motility and physiological parameters. Only DHA 1 mm protected motility, increasing the fast and progressive subpopulation. However, it had a detrimental effect on acrosomal and DNA status, in absence of oxidative stress. Tempol and rutin efficiently reduced lipoperoxidation, ROS, and DNA damage in presence of oxidative stress. NAC was not as efficient as TEMPOL or rutin reducing lipoperoxidation or protecting DNA, and did not reduce ROS, but its negative effects were lower than the other antioxidants when used at 1 mm, increasing the subpopulation of hyperactivated-like spermatozoa at 2 h. Our results show that these antioxidants have mixed effects when spermatozoa are incubated at physiological temperatures. DHA may not be suitable because of prooxidant effects, but TEMPOL, NAC and rutin may be considered for cryopreservation trials. In general, exposure of red deer spermatozoa to these antioxidants should be limited to low temperatures, when only protective effects may develop.     

A Fast Universal Immobilization of Immunoglobulin G at 4°C for the Development of Array-based Immunoassays

To maintain the antibody activity and enhance performance of array-based immunoassays, protein G was used to allow a shorter duration of immunoglobulin G immobilization at 4°C, with the antibody placed in the appropriate orientation. The multiplexed detection of six pain-related message molecules (PRMMs) was used as examples for the development of array-based immunoassays: substance P, calcitonin gene-related peptide, nerve growth factor, brain-derived neurotrophic factor, tumor necrosis factor-α, and β-endorphin. Protein G- and non-protein G-coated slides were tested. Compared to non-protein G immunoassays, protein G shortened the antibody immobilization time at 4°C from overnight to 2 hours. Only protein G-facilitated immunoassays succeeded in simultaneously detecting all six PRMMs with high specificity. Dose-response curves showed that the limits of detection of the protein G-multiplexed immunoassays for the PRMMs was approximately 164, 167, 120, 60, 80, and 92 pg/ml, respectively. Thus, protein G effectively shortens the duration of antibody immobilization at 4°C, allowing the use of sensitive array-based immunoassays for the simultaneous detection of PRMMs.     

Dark-recovery of Gamma- and UV-irradiated Saccharomyces carlsbergensis at 4° C

Experiments were carried out to find the effect of dark-holding in distilled water at 4° C on recovery of Gamma- and UV-irradiated cells of a haploid strain ofSaccharomyces carlsbergensis. It was found that there was an appreciable increase in survival of the irradiated cells following 4 to 24 hours' holding while no increase in the number of control cells was observed following similar treatment. It is suggested that some common type of damage induced by both Gamma- and UV-radiations inS. carlsbergensis may be repaired efficiently under metabolic conditions associated with growth at 4° C in distilled water.     

The effects of different sugars on motility, morphology and DNA damage during the liquid storage of rat epididymal sperm at 4°C

This study evaluated the protective effects of supplementation with three different sugars on the motility, morphology and DNA integrity of rat epididymal sperm chilled and stored at 4°C Epididymides were obtained from each donor. Rat epididymal sperm was diluted in Ham's F10 plus raffinose, Ham's F10 plus trehalose, Ham's F10 plus fructose, and Ham's F10 medium for control purposes. Thereafter, the extended sperm were chilled and stored in liquid form at 4°C. Sperm motility, morphological abnormalities and DNA damage were determined at 0 and 12h after chilling. No significant difference was observed in any of the parameters evaluated at 0h, before storage (P>0.05). After 12h of storage, all sugar additives led to statistically higher motility, normal sperm morphology and DNA integrity in comparison to the control group. Raffinose gave the best motility percentages (32.86±1.84%) after 12h of storage at 4°C, compared to the other groups (P<0.001). In conclusion, Raffinose, trehalose and fructose provided a better protection of sperm functional parameters against chilling injury, in comparison to the control group.     

Methionine supplementation improves ram sperm parameters during liquid storage at 5 °C

The aim of this study was to investigate the effects of methionine and lipoic acid on ram sperm parameters during liquid storage (5 °C). Ejaculates collected from five Merino rams were pooled at 37 °C. Each pooled ejaculate was divided into five equal aliquots and diluted (37 °C) with five extenders, one of which was without additives, two of which contained methionine at two different doses, and the other two of which contained lipoic acid at two different doses. Sperm parameters were determined at 0, 24, 48, 72 and 96 h of liquid storage at 5 °C.The extenders containing 2 and 4 mM of methionine resulted in higher motility percentages, in comparison to the control, up to 96 h of storage. Methionine at doses of 2 and 4 mM led to higher viability and sperm mitochondrial activity percentages, when compared to the controls during 48, 72 and 96 h of liquid storage (P < 0.05). The findings of this study showed that methionine was of greater benefit to ram sperm parameters during liquid storage.     

Changes in sperm membrane and ROS following cryopreservation of liquid boar semen stored at 15 °C

Boar semen is occasionally transferred to different locations in liquid form at 15 °C for cryopreservation. However, the use of frozen boar semen is limited due to the high susceptibility of boar sperm to cold shock. The aim of this study was to help improve the quality of frozen boar semen by determining the changes in sperm membrane and ROS during the cryopreservation processes of 15 °C-stored boar semen. Semen was collected from ten Duroc boars and transferred to our laboratory in liquid form stored at 15 °C. After cooling to 5 °C and freezing-thawing, conventional sperm parameters (total motility, progressive motility, and normal morphology), plasma membrane integrity, acrosomal membrane status, and intracellular ROS were evaluated. Sperm function, as assessed by conventional parameters, was unaffected by cooling but was decreased by freezing-thawing (P<0.05). However, the cooling and freezing-thawing processes led to damages in the sperm plasma membrane, and the cooling process caused increase in mean PNA (peanut agglutinin)-fluorescence intensity in viable acrosome-intact sperm (P<0.05). In ROS evaluation, the cooling process decreased intracellular (·)O(2) and H(2)O(2) in viable sperm (P<0.05), while the freezing-thawing process increased intracellular H(2)O(2) (P<0.05) without change in intracellular (·)O(2) in viable sperm. Our results suggest that, in liquid boar semen stored at 15 °C, cooling may be primarily responsible for the destabilization of sperm membranes in viable sperm, while freezing-thawing may induce reductions in sperm function with increase in membrane damage and H(2)O(2).     

Chloroethene Biodegradation in Sediments at 4°C

Microbial reductive dechlorination of [1,2-¹⁴C]trichloroethene to [¹⁴C]cis-dichloroethene and [¹⁴C]vinyl chloride was observed at 4°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 viable cells from rabbit skin biopsies after storage at ?20°C for up to 10?days

Frozen animal tissues are thought to be appropriate for use as a donor for somatic cell nuclear transfer. This makes the freezing for long term storage a valuable tool for breeders needing to protect an animal population that is endangered by sanitary problems or for cryobanking of genetic resources. We report the successful cryopreservation of explants of skin derived from small biopsies from rabbit ear biopsies by using a protocol that can be easily performed by usual breeders, which are not equipped with cooling devices. By optimizing the procedure, we show that small biopsies can be kept at -20°C in a physiological solution containing 10% DMSO for up to 20 days before being deeply frozen in liquid nitrogen for long-term storage. After 10 days of storage at -20°C, the rate of viability of biopsies was similar to the control one (86 and 82% respectively). After 20 days of storage at -20°C, the rate of viability was dramatically lowered (39%), but it still allows to recover a significant population of viable cells from the preserved sample. Being appropriate to places lacking specific device, such a very simple technique may contribute to facilitate genome banking policies dedicated to the management of genetic resources in wild and domestic animals.     

In vitro culture of fibroblast-like cells from postmortem skin of Katahdin sheep stored at 4��C for different time intervals

Live animals have been produced recently from animal tissues preserved for decades at frozen temperatures with or without cryoprotectants. However, the tissues in these studies were cryopreserved within few hours of animal death to obtain culturable live cells as nuclear donors. How long the tissues can be left unfrozen after animal death, without losing the viability and potential to in vitro culture with comparable morphology and proliferative rate as the fresh tissues, is not completely understood. To understand this phenomenon, ear skin samples from individual sheep (n=3) were procured from slaughter plant and stored at 4 °C. After various intervals (2, 8, 24, 32, 48, and 56 h after slaughter), 2-3 mm(2) pieces (n=10) of skin samples were cultured for 12 d on two dishes (60 mm) for each sheep. Outgrowth of fibroblast-like cells was observed as early as day 4 of culture and was visible on dishes of all time points including 56 h by day 10. The number of outgrowing cells decreased with increasing time interval between animal slaughter and culture initiation. Secondary cultures were successfully established for all the time points. All cultures proliferated well and were apparently normal. Passage 2 cultures of 2 h and 56 h interval for one of the three sheep were compared for their growth pattern and proliferation rates. The population doubling time of 2 h and 56 h intervals was 33.12 and 34.8 h, respectively, and both the lines exhibited similar cell morphology and an "S"-shaped growth curve. These results suggest that skin tissues of sheep and perhaps other animal species with superior traits are effectively preserved at cellular level at least for 56 h at normal refrigerating conditions, without need of complicated cryopreservatives/cryotanks that are usually not available at small farms.     

Artificial insemination of sheep: Effects on fertility of number of spermatozoa inseminated and of storage of diluted semen for up to 18 hours at 5°C

Ram semen was prepared in a buffered glucose-saline solution containing 3% (v/v) egg yolk so that insemination doses of 25 or 100 million spermatozoa in volumes of 50 or 250 μl could be given per ewe at artificial insemination (AI). Fertility was significantly reduced by dilution and, within the treatments of diluted semen, significantly higher lambing rates followed the use of doses of 100 million spermatozoa. The volume of the AI dose had no significant effect on fertility.Of 945 inseminations performed using diluted semen, 388 were with samples that had been cooled to 5°C and stored chilled for 5 or 18 hr. The mean lambing result of 40% for freshly diluted semen was significantly higher than 31.6% and 30.2% for samples stored chilled for 5 and 18 hr respectively. Ewes inseminated with doses of chilled semen containing 25 million spermatozoa had a low lambing rate of 21.3%. The presence of 7.5% glycerol (v/v) in the diluent did not significantly affect the fertility of chilled semen.     

The growth of dermatophytes at 4° C and 37° C; The relation of this character to others

Summary The ability of the generaEpidermophyton, Microsporon andTrichopyton to grow on some media at 4° C and 37° C was studied. It has been shown that specific differences exist among these fungi in the capability or rapidity of the growth at extreme temperatures.There is high positive correlation among perfect state production, isolation from the soil and growth at 4° C (group of characters A) and between pathogenicity and growth at 37° C (group of characters B). Between the groups A and B of characters exists a slighter negative correlation. Some prognosis about the five characters by certain species of dermatophytes may be given.