Effect of elevated temperature on the morphology of a chemostat culture of Candida utilis yeasts and on their content of nucleic acids

The effect of elevated temperature (40 degrees C) on a chemostat culture of Candida utilis was studied at different rates of dilution, D = 0.1 and 0.3 hr-1. The cells in the fermenter being in the stationary state at the optimum temperature of 30 degrees C were gradually washed-out 5 hours after the action of this temperature, and the population consisted of non-divided cells. In the majority of such "double" cells, the nucleus was contained in both the parent and the daughter parts. The content of RNA decreased by 31%, that of DNA, by 20%, and that of protein, by 13% (per 1 mg of biomass).     

Reversible effects of glycerol on the metabolism of platelets kept at room temperature

The effects of the addition and removal of glycerol on the metabolic activities of human platelets were studied. Platelet concentrates (PC) with 20 ml plasma were stored with 3-7% (v/w) glycerol in 150-ml polyvinylchloride plastic bags for 2 days at 22 degrees C with constant agitation. Incubation of glycerol with platelets produced a dose-dependent inhibition of oxygen consumption. The inhibitions of glucose utilization and lactate production had reached the plateau level at 3% glycerol. The rate of adenosine triphosphate (ATP) generation of control platelets was 9.8 nmol/min/10(9) platelets, in which over 90% ATP generation was derived from oxidative phosphorylation. There was a dose-dependent decrease (up to 20%) by glycerol in the rate of platelet ATP generation. Glycerol inhibited glycolysis more than oxidative phosphorylation. However, the inhibition potency diminished with increasing concentrations of glycerol. The energy metabolism of platelets after removal of 5% glycerol was examined. Deglycerolized platelets after 1 hr incubation facilitated energy metabolism more strongly than that of 24 hr incubation. The platelet aggregation response to collagen was not impaired by a cycle of the addition and removal of glycerol. The results indicate that glycerol lowered the rate of ATP generation of platelets stored at 22 degrees C. However, the removal of glycerol reversed the decreased energy metabolism.     

Phenotypic modifications in amino acid profiles of cell residues of Candida utilis and Enterobacter aerogenes

Glucose-limited chemostat cultures of Candida utilis were cultivated at various pH levels (3.0-7.5), temperatures (15-37.5 degrees C), dilution rates (0.006-0.42 hr-1), and with one of two nitrogen sources (NH+4 or NO-3). Enterobacter aerogenes was also cultivated in the chemostat under nitrogen and phosphorus limitations. The amino acid profile of total cell protein is expressed as the content of each amino acid relative to the sum of all amino acids recovered ater acid hydrolysis. Cell residues obtained after hot trichloracetic acid extraction display small variations in amino acid profile. Some of these variations correlate with the growth rate at satisfactory levels of statistical significance. In C. utilis, the correlations cover increased levels of lysine, arginine, and leucine and decreased levels of serine and glutamic acid with increased "reduced dilution rate" (D/Dc). In E. aerogenes, increased levels of lysine and arginine and a decreased level of glutamic acid correlate with increased dilution rate. The directions of most of these correlations and the extents of those pertaining to lysine and arginine are consistent with the change predicted to occur simultaneously in the relative level of the ribosomal protein group.     

Influence of the alternative respiratory pathway on the adenylate pools in heterotrophic Euglena gracilis

Etiolated Euglena gracilis Pringsheim, strain Z, were cultured in a lactate medium either in the presence of 2 μ M antimycin A for cells adapted to this inhibitor, or in the absence of antimycin A for controls. The adenylates (ATP, ADP and AMP) and the energy charge (EC) were followed during the growth of both types of cells. The effects of KCN, salicylhydroxamic acid (SHAM) and rotenone on the respiration and the adenylate pool, were investigated during the exponental and stationary phases. EC values of controls and antimycin-adapted cells were not significantly different during culture. In the logarithmic phase, EC of controls was unaffected by 3 m M SHAM, an inhibitor of the alternative pathway, but markedly decreased by 0.3 m M KCN, which inhibits the cytochrome pathway. In contrast, in antimycin-adapted Euglena , in which the cytochrome pathway was blocked, ATP content and EC were markedly lowered in the presence of SHAM but slightly increased by 0.3 m M KCN. The combination of the preceeding treatments, as well as 15 m M KCN alone, were deleterious for both types of cells, in the logarithmic and the late stationary phases. The data indicate that the energy level in Euglena was dependent on the alternative pathway when the cytochrome pathway was blocked. Such dependence could be explained by the engagement of the first rotenone-sensitive site of phosphorylation. Indeed, 50 μ M rotenone caused a similar relative decrease of oxygen consumption and ATP content in controls and in antimycin-adapted Euglena . In the absence of cytochrome respiration, the alternative pathway allowed electrons to flow through this first segment of the respiratory chain, and ATP production by the first site of phosphorylation.     

Influence of the environmental temperature on the metabolic use of dietary energy and nitrogen in the growing rat

Sixty-seven male rats of the Wistar CF strain were used in two trials to study the effect of ambient temperature on nitrogen and energy balances (experiment 1) and on the variations of certain biochemical parameters of metabolism (experiment 2). In both cases, the rats, housed individually in metal cages and fed ad libitum, were kept under three different temperatures : 21 degrees C (control), 3 degrees C (cold), 31 degrees C (hot). Compared to the controls, the rats at 3 degrees C showed a significant increase (P less than 0.01) in food intake and a significant decrease (P less than 0.01) in daily growth rate and feed efficiency ratio; body fat content was unchanged. Compared to the controls, the rats at 31 degrees C showed a decrease (P less than 0.01) in food intake and growth rate, a better feed efficiency ratio (P less than 0.01) and a significant increase in carcass lipid content. When compared to rats at 21 degrees C, those at 3 degrees C had a much lower nitrogen retention; at 31 degrees C this retention was only slightly lower. The urinary nitrogen excretion, increased by the cold temperature, reflected higher nitrogen catabolism, while its increase at a high temperature was probably due to a decrease in protein synthesis, and a subsequent degradation of excess ingested nitrogen. Energy retention increased (P less than 0.01) as the ambient temperature increased from 3 to 31 degrees C, and wide variations in the amounts of fixed energy in the form of proteins or lipids were observed. The balance results obtained have been discussed in relation to the variations of some biochemical parameters of metabolism such as free fatty acids, free glycerol, lipoprotein-lipase activity and in vitro utilization of labelled glucose.     

Regulation of phosphoenolpyruvate carboxylase of Zea mays by metabolites

Crude preparations of phosphoenolpyruvate carboxylase obtained from aetiolated seedlings of Zea mays are unstable but can be stabilized with glycerol. At the pH optimum of 8.3, the K(m) value for phosphoenolpyruvate is 80mum. When assayed at 30 degrees C, the enzyme shows normal Michaelis-Menten kinetics, but when assayed at 45 degrees C sigmoid kinetics are exhibited. At pH7.0 the enzyme is inhibited by a number of dicarboxylic acids and by glutamate and aspartate. d and l forms of the hydroxy acids and amino acids are inhibitory and the kinetics approximate to simple non-competitive inhibition. The same compounds produce less inhibition at pH7.6 than at pH7.0 and the kinetics of inhibition are more complex. The enzyme is activated by P(i), by SO(4) (2-) and by a number of sugar phosphates. Maximum activation occurs at acid pH values, where enzyme activity is lowest. The enzyme is activated by AMP and inhibited by ADP and ATP so that the response to energy charge is of the R type and is thus at variance with Atkinson's (1968) concept of energy charge. The physiological significance of the response to metabolites is discussed.     

Thermoregulation in the meerkat (Suricata suricatta Schreber, 1776)

Tre of the suricates exhibits a marked diurnal rhythm (mean Tre at night 36.3 +/- 0.6 degrees C and 38.3 +/- 0.5 degrees C during the day). Oxygen consumption is lowest at Ta 30-32.5 degrees C (mean 0.365 +/- 0.022 ml O2 g-1 hr-1); this is 42% below the value expected from body mass. At Ta below the TNZ, oxygen uptake rises rapidly, minimal thermal conductance (0.040 ml O2 g-1 h-1 degrees C-1) being 18% above the mass-specific level. Lowest heart rates occur at Ta 30 degrees C (mean 109.6 +/- 9.8 beats min-1) and oxygen pulse is minimal at Ta 30-35 degrees C with 40-45 microliter O2 beat-1. At Ta 15-32.5 degrees C total evaporative water loss is between 0.46-0.63 ml H2O kg-1 hr-1 and increases markedly during heat stress (to a mean of 5.35 ml H2O kg-1 hr-1 at Ta 40 degrees C). This rise of TEWL is mainly attributable to the onset of panting at Ta above 35 degrees C.     

Adenine nucleotide contents in callus-forming potato tuber discs with different respiratory characteristics

ATP content (per g fresh weight) and energy charge were higher during incubation at 8°C than at 28°C in the early stages of callus induction of potato tuber discs ( Solanum luberosum L. cv. Bintje). After a transfer from 28°C to 8°C, ATP content increased while a sharp decline in ATP content was observed after a transfer from 8°C to 28°C. ADP and AMP pools did not increase correspondingly. When the callus discs had entered the logarithmic growth phase, the energy charge was maintained within relatively narrow limits (0.77–0.80) at all culture temperatures.     

Phosphofructokinase type 1 kinetics, isoform expression, and gene polymorphisms in cancer cells

Kinetic analysis of PFK-1 from rodent AS-30D, and human HeLa and MCF-7 carcinomas revealed sigmoidal [fructose 6-phosphate, Fru6P]-rate curves with different V(m) values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH(4)(+), or K(+). The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non-essential hyperbolic activation. PFK-1 from rat liver and heart also exhibited the mixed cooperative-hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased V(m) and increased content of inactive (T) enzyme forms. High K(+) prompted increased (Fru6P) or decreased (activators) affinities; increased V(m); and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK-1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF-7 cells, of L>M in AS-30D cells, and of C in HeLa cells. PFK-1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK-1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK-1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK-1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux-control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect.     

杜氏盐藻渗透调节过程中的甘油代谢途径

杜氏盐藻在适应外界盐浓度变化的过程中,甘油是其主要的渗透调节物质。低渗处理提高藻细胞的呼吸速率６０％以上;高渗处理对呼吸无明显影响,但大大刺激光合放氧速率。呼吸链的细胞色素电子传递链抑制剂ＫＣＮ和交替氧化酶抑制剂ＳＨＡＭ对杜氏藻渗透调节过程中的呼吸．胞内甘油、ＡＴＰ、淀粉会量的变化有不同的抑制效果。低渗情况下,胞内甘油转化为淀粉,所需能量由正常呼吸链和交替氧化酶途径同时提供;高渗情况下．淀粉则降解为甘油,光下甘油合成的能量主要由光合电子链提供,暗中则由正常呼吸链提供。     

Effects of carbon source and growth rate on cell wall composition of Bacillus subtilis subsp. niger.

A study was made to determine whether factors other than the availability of phosphorus were involved in the regulation of synthesis of teichoic and teichuronic acids in Bacillus subtilis subsp. niger WM. First, the nature of the carbon source was varied while the dilution rate was maintained at about 0.3 h-1. Irrespective of whether the carbon source was glucose, glycerol, galactose, or malate, teichoic acid was the main anionic wall polymer whenever phosphorus was present in excess of the growth requirement, and teichuronic acid predominated in the walls of phosphate-limited cells. The effect of growth rate was studied by varying the dilution rate. However, only under phosphate limitation did the wall composition change with the growth rate: walls prepared from cells grown at dilution rates above 0.5 h-1 contained teichoic as well as teichuronic acid, despite the culture still being phosphate limited. The wall content of the cells did not vary with the nature of the growth limitation, but a correlation was observed between the growth rate and wall content. No indications were obtained that the composition of the peptidoglycan of B. subtilis subsp. niger WM was phenotypically variable.     

Role of the adenylate system and glycolytic flux in the control of protein synthesis in isolated rat lung cells

(1) Glucose stimulates the incorporation of amino acids into protein in lung cells isolated by digestion of the lung stroma with collagenase. This effect reflects mainly an increase in protein synthesis since no effect of glucose had been found to the uptake of amino acid precursors and, although glucose decreases the rate of intracellular proteolysis by 15%, this effect cannot account for the increased incorporation of radioactivity into proteins. Furthermore, glucose did not induce any significant change in the intracellular content of valine. (2) For glucose to act on protein synthesis, it must be glycolyzed since its stereoisomer, L-glucose, which is not metabolized by lung cells, has no effect. (3) The mechanism of glucose action does not seem to be related simply to variations of cellular ATP content or energy charge. The following arguments seem to support this conclusion: (i) glucose does not bring about significant variations in the concentration of reactants of the adenylate system; (ii) the increase in protein synthesis induced by glucose in energy-depleted cells correlates with a rise in ATP content and energy charge; however, adenosine, which increases ATP levels in a form quantitatively similar to glucose, is unable to affect protein synthesis: (iii) glucose also accelerates the incorporation of amino acids into proteins in adenosine-treated lung cells in which the ATP concentration was almost double that of the control and the energy charge was considerably elevated, ruling out the possibility that a rise in the steady-state concentration of ATP and/or energy charge alone could be responsible for the acceleration of protein synthesis. (4) It can be concluded that the effect of glucose in increasing protein synthesis in lung cells is dependent on some signal arising from its breakdown and not to variations in the concentration of reactants or energy charge of the adenylate system.     

Variations in the Adenylate Energy Charge During Phased Growth (Cell Cycle) of Candida utilis Under Energy Excess and Energy-Limiting Growth Conditions

The variations in the levels of adenine nucleotides during the phased growth (cell cycle) of the yeast Candida utilis growing under nitrogen, sulfate, or iron limitation with glycerol as carbon source have been determined. Synchronous cultures were obtained by the continuous phasing technique, and the results were compared with those of chemostat cultures growing at similar growth rates and under the same types of nutrient limitation. Whereas the chemostat experiments indicated only the average energy status of cultures growing at random, results from phased cultures showed that the adenylate energy charge, defined as (ATP + (1/2)ADP)/(ATP + ADP + AMP) (where ATP, ADP, and AMP signify adenosine 5'-triphosphate, -diphosphate, and -monophosphate, respectively), varied during the phased growth of the yeast. These variations were related to the stage of development of the cells and to the type of nutrient limitation. In every case the energy charge dropped to a low value during the first half of the phasing cycle (cell cycle). Whereas the energy charge was maintained at relatively high levels (ranging from 0.78 to 0.94), for sulfate- or nitrogen-limited cultures, it was very low when iron was the growth-limiting nutrient (0.44 to 0.78). In spite of the low energy charge, the yeast continued to grow under iron limitation. The main component of the adenylate pool of the iron-limited culture was ADP and not ATP as observed with other types of nutrient limitation. It is concluded that under iron limitation the growth of the organism is limited by energy and that under energy-limited growth the energy charge of a growing organism is maintained at low levels. The reason for maintaining a low energy charge in an energy-limited culture is discussed.     

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.     

持续低温弱光对黄瓜叶片气体交换、叶绿素荧光猝灭和吸收光能分配的影响

持续常温弱光(25℃/18℃,l00umol m-2 s-1)、低温弱光(12℃/12℃,100 umol m-2 s-1和7℃/7℃,l00μmolm-2s-1)均导致黄瓜生长减慢或停滞、叶绿素含量、气孔导度和净光合速率、光合电子传递速率下降以及胞间CO2浓度上升.常温弱光和12℃弱光处理对光系统II的最大光化学效率Fv/Fm无显著影响,而7℃弱光处理导致Fv/Fm的可逆性下降.常温弱光和7℃、12℃弱光处理均导致了光化学反应速率的降低以及天线热耗散和反应中心过剩能量的增加.在胁迫后,12℃弱光0比7℃弱光更有利于植株光合功能的恢复.     

Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma

The specific monoclonal antibody productivity (q(Mab)) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33 degrees C to 39 degrees C. q(Mab) was constant at each temperature and was independent of the phase of culture. The q(Mab) increased 97% at 39 degrees C and decreased by 21% at 33 degrees C compared with controls at 37 degrees C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, Y(Lac/Llc') was constant for 33 degrees C to 39 degrees C and Y(Amm/Gin) was constant for 37 degrees C to 39 degrees C. Y(Amm/Gin) at 33 degrees C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U-ratio (UTP/UDP-GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP-/N acetyl galactosamine, UDP-N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP-sugars) ratio, purine/pyrimidine, ATP/GTP, and U-ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of q(Mab) observed at the higher temperatures. (c) 1994 John Wiley & Sons, Inc.     

Physiology and morphology of Legionella pneumophila in continuous culture at low oxygen concentration.

Two strains of Legionella pneumophila serogroup 1 monoclonal subgroup Pontiac were grown for the first time in continuous culture using a chemically defined medium. The influence of temperature on physiology and morphology was investigated by fixing the growth rate (equal to the dilution rate, D) at 0.08 h-1 and controlling the pH and dissolved oxygen concentration of the culture. Serine provided the principal source of carbon and energy but growth was limited by tyrosine. The bacterium behaved as a microaerophile in this medium, with maximal growth occurring at 0.31 (mg O2)I-1 (equivalent to a dissolved oxygen tension of 4% (v/v) air saturation at 30 degrees C). The cultures consisted of flagellated, short rods at 24 degrees C, but exhibited an increased level of pleomorphism and the loss of flagella as the temperature was increased to 37 degrees C. The presence of intracellular granules was noted, and their abundance was temperature-dependent. Polyhydroxybutyrate was present in L. pneumophila, and the proportion of the cell dry weight that it accounted for varied with temperature, being maximal at 24 degrees C. The ratio of saturated to unsaturated fatty acids in the cells decreased as the temperature was reduced towards 24 degrees C, so as to maintain membrane fluidity at low growth temperature.     

Energy Metabolism of Rickettsia typhi: Pools of Adenine Nucleotides and Energy Charge in the Presence and Absence of Glutamate

The obligate intracellular bacterium Rickettsia typhi was examined for its ability to generate and maintain an adenylate energy charge in an extracellular environment. Freshly purified organisms were incubated, at 34 degrees C and pH 7.4, with or without glutamate and various other metabolites, and the levels of ATP, ADP, and AMP were determined. Of the metabolites tested, glutamate and glutamine were the most effective for the generation of ATP. In the presence of glutamate, there was a rapid increase in the level of ATP, followed by a moderate decrease during 150 min of incubation. The energy charge increased from a level of 0.2 to 0.5 to about 0.7 to 0.75, and then slowly declined to about 0.45 to 0.6. In the absence of glutamate, after an occasional initial surge in ATP level as the temperature was changed from 4 to 34 degrees C, there was a sharp decline in both ATP and energy charge (to 0.1 and sometimes to 0.01). The rickettsiae maintained their ability to regenerate their energy charge upon the addition of glutamate for about 30 min, but this ability declined with further incubation. In contrast to Escherichia coli, the decline in ATP in R. typhi was accompanied by a sharp increase in the level of AMP and the total adenylate pool. No adenine or adenosine was recovered from rickettsiae incubated with labeled AMP, ADP, or ATP. From these experiments and the demonstration reported elsewhere that rickettsiae transport the adenine nucleotides, it can be concluded that the adenylate energy charge in R. typhi is governed by the salvage of the adenine nucleotides rather than their unphosphorylated precursors. Thus, R. typhi undergoes greater shifts in energy charge than other bacteria, a phenomenon which may account for their instability in an extracellular environment. Under optimal conditions the adenylate energy charge of R. typhi approaches levels that border on those generally regarded as adequate for growth.     

Genome‐derived minimal metabolic models for Escherichia coli MG1655 with estimated in vivo respiratory ATP stoichiometry

Metabolic network models describing growth of Escherichia coli on glucose, glycerol and acetate were derived from a genome scale model of E. coli. One of the uncertainties in the metabolic networks is the exact stoichiometry of energy generating and consuming processes. Accurate estimation of biomass and product yields requires correct information on the ATP stoichiometry. The unknown ATP stoichiometry parameters of the constructed E. coli network were estimated from experimental data of eight different aerobic chemostat experiments carried out with E. coli MG1655, grown at different dilution rates (0.025, 0.05, 0.1, and 0.3 h^?1) and on different carbon substrates (glucose, glycerol, and acetate). Proper estimation of the ATP stoichiometry requires proper information on the biomass composition of the organism as well as accurate assessment of net conversion rates under well‐defined conditions. For this purpose a growth rate dependent biomass composition was derived, based on measurements and literature data. After incorporation of the growth rate dependent biomass composition in a metabolic network model, an effective P/O ratio of 1.49 ± 0.26 mol of ATP/mol of O, K_X (growth dependent maintenance) of 0.46 ± 0.27 mol of ATP/C‐mol of biomass and m_ATP (growth independent maintenance) of 0.075 ± 0.015 mol of ATP/C‐mol of biomass/h were estimated using a newly developed Comprehensive Data Reconciliation (CDR) method, assuming that the three energetic parameters were independent of the growth rate and the used substrate. The resulting metabolic network model only requires the specific rate of growth, µ, as an input in order to accurately predict all other fluxes and yields. Biotechnol. Bioeng. 2010;107: 369–381. © 2010 Wiley Periodicals, Inc.     

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws

The interaction of glycerol concentrations of 0-10% and cooling rates from 1 to 1,500 degrees C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30 degrees C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0-1% glycerol after cooling at 30 degrees C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30 degrees C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1 degrees C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500 degrees C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30 degrees C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.