Effect of cryopreservation protocol on postthaw characteristics of stallion sperm

Three ejaculates from each of eight stallions were subjected to cryopreservation in a milk/egg yolk-based freezing extender or an egg yolk-based freezing extender. Semen was exposed to a fast prefreeze cooling rate (FAST; semen immediately subjected to cryopreservation) or a slow prefreeze cooling rate (SLOW; semen pre-cooled at a controlled rate for 80 min prior to cryopreservation). Postthaw semen was diluted in initial freezing medium (FM) or INRA 96 (IMV Technologies, L'Aigle, France) prior to analysis of 10 experimental end points: total motility (MOT; %), progressive motility (PMOT; %), curvilinear velocity (VCL; μm/s), linearity (LIN; %), intact acrosomal and plasma membranes (AIMI; %), intact acrosomal membranes (AI; %), intact plasma membranes (MI; %), and DNA quality. Eight of 10 experimental endpoints (MOT, PMOT, average-path velocity [VAP], mean straight-line velocity [VSL], LIN AIMI, AI, and MI) were affected by extender type, with egg yolk-based extender yielding higher values than milk/egg yolk-based extender (P < 0.05). Exposure of extended semen to a slow prefreeze cooling period resulted in increased values for six of eight endpoints (MOT, PMOT, VCL, AIMI, AI, and MI), as compared with a fast prefreeze cooling period (P < 0.05). As a postthaw diluent, INRA 96 yielded higher mean values than FM for MOT, PMOT, VCL, average-path velocity, and mean straight-line velocity (P < 0.05). Treatment group FM yielded slightly higher values than INRA 96 for LIN and MI (P < 0.05). In conclusion, a slow prefreeze cooling rate was superior to a fast prefreeze cooling rate, regardless of freezing extender used, and INRA 96 served as a satisfactory postthaw diluent prior to semen analysis.     

Influence of alternating temperature preculture on cryopreservation results for potato shoot tips

Cryopreservation is the most suitable long-term storage method for genetic resources of vegetatively maintained crops like potato. In the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) the DMSO droplet method is applied, and so far more than 1000 accessions are cryopreserved with an average regeneration rate of 58%. New experiments with four potato accessions using alternating temperatures (22/8 degrees C day/night temperature, 8 h photoperiod, 7 d) prior to cryopreservation showed improved regeneration. The influence of this preculture on the shoot tips was studied for two wild, frost resistant species Solanum acaule and S. demissum and for two cultivated, frost sensitive potatoes S. tuberosum 'Désirée' and 'King Edward'. Comparison of liquid and solid media after cryopreservation showed improved regeneration on solid media with higher regeneration percentages, less callus formation and better plantlet structure. In comparative analyses biochemical factors like soluble sugars, starch, and amino acid concentrations were measured. Shoot tips after constant and after alternating temperature preculture were analyzed. Total concentrations of soluble sugars (glucose, fructose, and sucrose) were higher for all accessions after the alternating temperature preculture, which could be the reason for improved cryopreservation results.     

Effect of preculture on the slime production inChlamydomonas humicola Lucksch fo

Summary Batch-culturedCblamydomonas bumicola Lucksch fo. showed seasonal oscillations of slime production even in constant conditions. These alterations were found to be correlated to the natural day length during the preculture of the inocula. After preculture in short day conditions production and excretion of mucopolysaccharides started earlier than after preculture in long day conditions. Dry weight production and carbohydrate content were not correlated to preculture.     

Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws

Experiments were conducted to evaluate two extenders (egg-yolk Tris and egg-yolk lactose), varying concentrations of two cryopreservatives (glycerol and dimethyl sulfoxide), and rates for cooling to 5 degrees C, cooling from 5 to -100 degrees C, and warming for canine spermatozoa packaged in 0.5-ml French straws. At optimal concentrations of glycerol, egg-yolk Tris extender was superior to egg-yolk lactose in preserving spermatozoal motility. Addition of dimethyl sulfoxide, alone or in combination with glycerol in either extender, was not beneficial to spermatozoal survival after thawing. Canine spermatozoa withstood a range of cooling and equilibration times with no detrimental effect on spermatozoal motility prior to freezing. However, there were differences in spermatozoal motility immediately after thawing; these differences were variable, resulting in a cooling time by equilibration time interaction. Spermatozoal motility after thawing was best preserved by freezing in egg-yolk Tris extender containing 2-4% glycerol, using a moderate rate of cooling from 5 to -100 degrees C (-5 degrees C/min from 5 to -15 degrees C, then -20 degrees C/min from -15 to -100 degrees C). Three of 12 bitches inseminated intravaginally with semen frozen using this protocol became pregnant.     

Effects of preculture variability on clavulanic acid fermentation.

The production profile of clavulanic acid by Streptomyces clavuligerus was shown to be strongly dependent on inoculum activity. Two sets of fermentations (A and B) were investigated at industrial pilot-plant scale using complex media. Type A fermentations were inoculated using late exponential growth phase mycelia. Type B fermentations were inoculated using mycelia harvested at stationary phase. Productivities throughout type A fermentations were consistently higher than type B, reaching a maximum at about 70 h and then decaying to the same final productivities at 140 h of type B runs. Several scheduling alternatives, based on combinations of the two inocula types and different fermentation lengths, were compared in terms of the overall process economics (fermentation and downstream). An increase of ca. 22% on the overall process profit is predicted using late exponential growth phase inocula and a fermentation duration of only 96 h. A new operating strategy was thus proposed for inoculum production based on the control of preculture activity using off-gas analysis. This method ensures higher productivity and better batch-to-batch reproducibility of clavulanic acid fermentations than traditional methods based on constant age inocula.     

Oxygen consumption characteristics of porcine hepatocytes

Oxygen uptake rate (OUR) of hepatocytes is an important parameter for the design of bioartificial liver assist (BAL) devices. Porcine hepatocytes were cultured in a specially constructed measurement chamber with an incorporated mixing system and a Clark polarographic oxygen electrode. Signal noise associated with conventional Clark electrode implementations was circumvented by the combination of real time digital numerical averaging and subsequent finite impulse response (FIR) spectral filtering. Additional software allowed for the automated generation of cellular oxygen consumption coefficients, namely, Vmax and K0.5, adding a high degree of objectivity to parameter determination. Optimization of the above numerical techniques identified a 0.1 Hz/200 data point sample size and a 0.004 Hz cutoff frequency as ideal parameters. Vmax values obtained for porcine hepatocytes during the first two weeks of culture showed a maximal consumption of 0.9 nmole/sec/10(6) cells occurring on Day 4 post seeding, and a gradual decrease to 0.31 nmole/sec/10(6) cells by Day 15. K0.5 values increased from 2 mm Hg on Day 2 to 8 mm Hg by Day 8, with gradual subsequent decrease to 4 mm Hg by Day 15. The Vmax and K0.5 values measured for porcine cells were higher than maximal values for rat hepatocytes (Vmax: 0.43 nmole/sec/10(6) cells, K0.5: 5.6 mmHg) and thus may necessitate significantly altered BAL device design conditions to ensure no oxygen limitations. Finally, these results highlight the need for species specific characterization of cellular function for optimal BAL device implementations.     

Influence of 2-chloroadenosine on the nucleotide content of isolated rat hepatocytes

2-Chloroadenosine is presumably a non-metabolizable analogue of adenosine; however, this compound induced an increase in the enzymatically measured nucleotide content of isolated rat hepatocytes. HPLC separation and spectral analysis of the peaks showed that this increase may be related to the formation of 2-chloro nucleotides and that the 2-chloro nucleotides appeared in the first minutes of the incubation period. These results demonstrate that 2-chloroadenosine may be metabolized by phosphorylation in rat liver cells.     

Influence of D-galactosamine on the synthesis of sugar nucleotides and glycoconjugates in rat hepatocytes

Rat hepatocytes were incubated in the presence of a high concentrationof the hepatopathogenic agent D-galactosamine (GalN), and theeffect on the cellular concentrations of pyrimidine nucleotidesand nucleotide sugars was determined. The UTP pool became depleted.The pools of UMP and CMP in RNA decreased to 72%, indicativefor an inhibition of RNA synthesis. UDP-HexNAc (where HexNAcis GlcNAc + GalNAc) and UDP-HexN (where HexN is GlcN + GalN)levels increased, and those of UDP-hexose and UDP-GlcA (whereGlcA is glucuronic acid) decreased. The cellular concentrationof CTP did not change, whereas that of CMP-NeuAc (where NeuAcis N-acetylneuraminic add) showed a 2-fold increase. Labellingwith [¹⁴C]orotic acid and [³H]cytidine showed that the metabolicflow via the de novo pathway was not changed. The depletionof the so-called overflow pool of UTP [Pels Rijcken et al, Biochem.J., 293, 207–213, 1993] caused a release of the feedbackinhibition by UTP and thus an increased flow through the salvagepathway. Finally, it appeared that GalN, when added to hepatocytes,gives rise to a pool of UDP-GlcNAc (where GlcNAc is N-acetylglueosamine)that is separate from the pool of UDP-GlcNAc that is derivedfrom GlcN. D-galactosamine glycosylation sugar nucleotide biosynthesis     

Influence of rat strain on P-glycoprotein expression in cultured hepatocytes

The amount and activity of the multi-drug transport P-glycoprotein (Pgp) have been measured in cultured hepatocytes derived from different rat strains. A marked increase in Pgp, as revealed by Western blotting, occurred 48 h after seeding in hepatocytes from Sprague-Dawley, Wistar and Fischer 344 rats, the last showing the highest value. The addition of dexamethasone (DEX) to culture medium delayed Pgp overexpression in all the strains, proportionally to the protein amount in the absence of hormone. The R-123 functional test for Pgp showed that Fischer 344 hepatocytes had the lowest ability to extrude the fluorescent dye as compared with Sprague-Dawley or Wistar rats. These results suggest that the Fischer 344 rat is more prone than other strains to culture stressing conditions, leading to an overexpression of Pgp that is not necessarily functional.Abbreviations DEX dexamethasone - FCS fetal calf serum - HRP horseradish peroxide - Pgp P-glycoprotein     

Influence of oxalate on the rate of the tricarboxylic acid cycle in rat hepatocytes

In hepatocytes isolated from fed rats, physiological concentrations of oxalate lower the flux through the tricarboxylic acid cycle (-48%) and reduce the steady-state levels of oxaloacetate and other Krebs cycle intermediates. All the metabolic modifications observed are explained by pyruvate carboxylase inhibition, since oxalate hardly modifies the flux through pyruvate dehydrogenase.     

Influence of preculture treatment and types of explants on shoot growth and in vitro flowering of feathered amaranth (Celosia argentea var. plumose)

The shoots developed from both the shoot tip and nodal explants of feathered amaranth (Celosia argentea var. plumosa—feathered cockscomb or plumed cockscomb) after 8 weeks of culture in the presence of either paclobutrazol or benzyladenine (BA) were shorter than those developed on basal Murashige and Skoog (MS) medium (Physiol Plant, 15:473–497, 1962) alone. However, this retarding effect was more pronounced in the nodal explant culture. Shoot tip explants from 2-week-old seedlings were more adversely affected by 0.85 or 1.7 μM paclobutrazol than those from older seedlings. In contrast, regardless of preculture duration investigated nodal explants did not exhibit different response to three different concentrations of paclobutazol. The response to 2.2 or 4.4 μM BA appeared to be largely independent of the age of the shoot tip explants or preculture treatment of nodal explants. Shoots developed from nodal explants produced a higher number of terminal inflorescence than those from shoot tip explants. Moreover, only lateral shoots from nodal explant culture formed inflorescence. Increased preculture duration on basal MS medium could generally lessen the inhibitory effect of lower concentrations of paclobutazol or BA on terminal or lateral inflorescence formation in nodal explant culture.     

Growth characteristics of Tyzzer''s organism in cultured mouse hepatocytes

The growth characteristics of Tyzzer's organisms in cultured mouse hepatocytes were examined by direct bacterial counting and plaque assay. The viability of propagated bacteria and time required for the maximum titers depended on incubation time and inoculum dose, respectively. Both infectious titers and bacterial counts at optimum harvests were much higher than those obtained from infected mouse livers. Immunofluorescence revealed, after a lag phase of about 2 h, a linear multiplication during a 6 h period.     

Influence of gastrointestinal peptides on bile acid transport by isolated rat hepatocytes

While numerous effects of gut peptides on gastric, pancreatic, and intestinal secretion have been described, there has been little investigation of the influence of these peptides on hepatic function. In the present studies, effects of vasoactive intestinal peptide (VIP), somatostatin, thyrotropin-releasing hormone (TRH), and bombesin on taurocholate transport by isolated rat hepatocytes have been examined. Somatostatin, TRH, and bombesin in incubation media produced no change from control incubations with regard to either uptake of taurocholate by hepatocytes or efflux of bile acid from preloaded cells. However, incubation of hepatocytes with VIP produced a significant decrease in taurocholate uptake (1.34 +/- 0.13 versus 1.73 +/- 0.16 nmole.min-1.10(6) cells-1, P less than 0.001). Studies with verapamil, a calcium-channel blocking agent, and theophylline, an inhibitor of cAMP catabolism, failed to provide evidence for transmembrane Ca2+ flux or alteration in intracellular levels of cAMP, respectively, as mechanisms for the observed inhibition of hepatocyte taurocholate uptake by VIP. These data, coupled with both clinical and other basic observations, suggest that VIP may play a significant role in the regulation of hepatic bile secretion.     

Influence of antimonite, selenite, and mercury on the toxicity of arsenite in primary rat hepatocytes

The long-term toxicity of arsenic (As) as a result of exposure to contaminated drinking water might be modified by coinciding exposures to elements like selenium, antimony, or mercury. In this study the influence of tetravalent selenite, trivalent antimonite, and divalent mercury was investigated in vitro using cultured primary rat hepatocytes. The cell vitality was assessed in the 3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] (MTT), assay with concurrent exposures of the cells to up to 50 microM sodium arsenite(III) and a potential modifier [50 microM sodium(IV) selenite, 10 microM antimony(III) chloride, 25 microM mercuric(II) chloride], which indicated an additive increase in the combined cytotoxicity. Sodium arsenite was tested for genotoxicity in the micronucleus test in a concentration range of 0.25 up to 7.5 microM. In this range, the MTT conversion was at least 80%, indicating high cell viability. Adose-dependent induction of micronuclei was observed. The lowest concentration causing a significantly elevated frequency of micronuclei was 1 microM As (p < 0.05). A significant influence (i.e., reduction of the combined genotoxicity as a result of the presence of a potential modifier) was only observed for 10 and 25 microM antimony chloride (p < 0.05, Fisher's exact test). The metabolic methylation of arsenite was not affected by concurrent incubation with any of the potential modifiers.     

Influence of ornithine on albumin synthesis by fetal and neonatal hepatocytes maintained in culture

Fetal rat as well as fetal and neonatal human hepatocytes were cultured in an arginine-free medium with or without L-ornithine. This amino acid was found greatly to improve cell survival of both rat and human parenchymal cells and to favour their proliferation. In addition, L-ornithine appeared to modulate the expression of various functions and particularly to increase albumin secretion. Since a correlated increase of the corresponding mRNA was observed, it may be postulated that L-ornithine acts at a pre-translational level.     

Influence of bacterial density during preculture on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of tomato

An improved bacterial preculture protocol for Agrobacterium-mediated genetic transformation was developed for an economic tomato cultivar (Solanum lycopersicum L. cv. Zhongshu No. 4). Frequencies of transient gene expression and stable transformation were influenced by the density of Agrobacterium preculture and not the density of Agrobacterium used for infection. The improved protocol presented in this study depends on the use of an overnight-grown Agrobacterium preculture density of OD_600 nm = 1.0, diluted 1/10th with Luria-Bertani (LB) liquid medium, and grown for an additional 4 h. Cultures are collected and resuspended in a liquid cocultivation medium-I, adjusted to OD_600 nm = 0.1. Using this modified Agrobacterium preparation, transient β-glucuronidase expression was higher than 90%, and transformation efficiency reached 44.7%. This improved transformation is simple, repeatable, does not require a feeder layer, and most notably, the transformation frequency is stable and highly efficient.     

The effect of preculture conditions on the culturability and viability of Aeromonas hydrophila when exposed to oligotrophic and cold stresses

The aim of this study is to assess the relationship between pre-culture conditions and loss of culturability of Aeromonas hydrophila exposed to a nutrient deprivation and low temperature (4°C). The present results show that the behavior of A. hydrophila, when exposed to nutritional deprivation and low temperature, depends on the pre-culture conditions. After a 60-days period of incubation in Filtered Sterilized Distilled Water (FSDW) at 4°C no culturable cells were observed for cells with a pre-culture in a liquid culture medium whereas a cell density of 1.8 log CFU/ml was observed for the bacterium with a pre-culture on solid medium with cells fixed on cellulose nitrate membrane. Colony count of A. hydrophila cells with anaerobic pre-culture declined to 0 CFU/ml within 16 days of incubation in FSDW at 4°C showing that the cells under these conditions are more sensitive to nutritional deprivation and low temperature than cells with a pre-culture under aerobic conditions. Our findings showed also no culturable cells, after 8 days of incubation in FSDW, for A. hydrophila with a pre-culture at 4°C. Moreover the present study showed no recovered cells from non culturable A. hydrophila when the FSDW was supplemented by catalase or sodium pyruvate. However, the addition of oxyrase, to non culturable A. hydrophila cells, was found to allow the recovery of non culturable cells.     

Enhancement of in vitro organogenetic capacity of rose by preculture of donor shoots on the medium with thidiazuron

Rose genotypes can be recalcitrant in in vitro regeneration. A lack of competence for regeneration results in unsatisfactory numbers of adventitious shoots, which diminishes achievements in use of genetic engineering in rose modern breeding. Among the factors which can partially overcome this problem is the use of thidiazuron (TDZ) in the induction phase of regeneration. We studied the use of TDZ in concentrations 0.05 and 1.0 mg l⁻¹, in the medium for the culture of donor shoots with the aim of increasing the regeneration competence of five rose cultivars differing in their regeneration ability. All the tested genotypes increased their regeneration potential when the donor shoots were cultured on TDZ media for 15 or 30 days. The strongest effect, doubling to quadrupling the number of shoots, was obtained in the cultivars characterized by the lowest regeneration potential. This enhanced regeneration ability was maintained through two or three subsequent 4-week long regeneration passages.