Simultaneous sewage sludge digestion and metal leaching — effect of temperature

The effect of temperature on the simultaneous sewage sludge digestion and metal leaching process was studied in laboratory bioreactors of 20 l working volume. The results thus obtained showed that the process can be employed efficiently for metal solubilization, elimination of indicator microorganisms and sewage sludge stabilization at temperatures between 10°C and 30°C. Rates of pH reduction, sulfur oxidation, growth of thiobacilli, elimination of indicator microorganisms and solids degradation were found to decrease with temperature. Low metal solubilization efficiency was observed at 10°C; however, metals were solubilized to below the recommended level. The solubilization of organic matter and nutritive elements (N, P and K) was not significantly affected by the variation in temperature. The fertilizer value of sludge after leaching and digestion did not change significantly and remained the same irrespective of temperature. *** DIRECT SUPPORT *** AG903078 00005     

Processes of liquefaction/solubilization of Spanish coals by microorganisms

Several fundamental aspects of microbial coal liquefaction/solubilization were studied. The liquefied/solubilized products from coal by microorganisms were analysed. The liquid products analysed by IR titration and UV/visible spectrometry showed some alterations with regard to the original coal. Humic acids extracted from the liquefied lignite showed a reduction in the average molecular weight and a increase in the condensation index, probably due to depolymerization caused by microorganisms. The mechanisms implicated in coal biosolubilization by two fungal strains, M2 (Trichoderma sp.) and M4 (Penicillium sp.) were also studied. Extracellular peroxidase, esterase and phenoloxidase enzymes appear to be involved in coal solubilization. Received: 15 June 1998 / Received revision: 23 November 1998 / Accepted: 29 November 1998     

Solubilization of coal by an extracellular product fromStreptomyces setonii 75Vi2

Summary Several low-ranked coals were solubilized when placed on the surface of agar cultures ofStreptomyces viridosporous T7A andS. setonii 75Vi2. When grown in submerged cultureS. setonii 75Vi2 produced an extracellular component that was capable of solubilizing coals. The extracellular coal solubilizing component had a molecular weight of <10000 and was heat stable since, after 1h at 121°C, only 30–40% of the activity was lost. Treatment with any of three proteases also appeared to be ineffective in decreasing activity. These results suggest that coal solubilization byS. setonii 75Vi2 is nonenzymatic.Research supported by the Fossil Energy Advances Research and Technology Program, managed by the Pittsburg Energy Technology Center, U.S. Department of Energy, under Contract No. DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.     

Cold-induced hemolysis in a hypertonic milieu

Summary Suspension of human erythrocytes at 37° C in an environment made hypertonic by increasing concentrations of sodium chloride and sucrose was followed by hemolysis when the temperature was lowered to 0° C. Two distinct stages were involved in this hemolytic phenomenon, the first being incubation with hypertonic solute at some temperature above 20° C with an increasing effect up to 45° C, and the second stage consisting of lowering the temperature below 15° C with increasing hemolysis down to 0° C. The rate of cooling was not an important factor, but the presence of ions reduced the extent of cold-induced hemolysis in hypertonic sucrose. No significant release of membrane phospholipid and cholesterol accompanied this hemolysis. The solubilization of membrane protein components was investigated, with some differences appearing on sodium dodecyl sulfate polyacrylamide gel electrophoresis between hypertonic and isotonic supernatants. Spectrin could not be identified in solubilized form. Correlation of the temperatures of note in these studies with results from the literature on other biological effects of temperature-induced phase transitions in membrane lipids strongly points to the conclusion that such transitions are involved in the mechanism of cold-induced hypertonic hemolysis. It is postulated that the hypertonic milieu has resulted in membrane-protein alteration damage which prevents normal adaption to the new physical state of the membrane lipids during cooling.     

The Ca<Superscript>2+</Superscript>-ATPase of the Scallop Sarcoplasmic Reticulum Is of a Cold-adapted Type

At 0 to 20°C, the Ca²⁺-ATPase activity of the scallop sarcoplasmic reticulum (SR) was observed to be 7–60% of the peak activity at 30°C, while the ATPase activity of the rabbit SR was 0–7% of its peak at 55°C. The relative rabbit ATPase activity (0.7–7.0%) at 7–20°C became higher (6–15 times) and lower (1/4–1/2), respectively, by the solubilization of the rabbit ATPase with a detergent, dodecyloctaethylenglycol monoether, and by the reconstitution of the ATPase with asolectin (soybean lecithin). No activity at 0°C remained irrespective of these treatments. The relative scallop ATPase activity at 0–20°C was, however, scarcely affected by such solubilization and reconstitution. In contrast to the rabbit ATPase, the scallop ATPase seems to be capable of operating independently without the help of the membrane lipid at low temperature.     

Detergent solubilization of mammalian cardiac and hepatic beta-adrenergic receptors.

The solubilization of canine cardiac and hepatic β-adrenergic receptors was characterized with 19 different ionic and nonionic surfactants and surfactant combinations. The effects of alterations in the hydrophobic and hydrophilic moieties of the nonpolar detergents were examined in relation to their efficacy in solubilizing these receptor molecules. Within this group of surfactants the more effective agents contained an average of 9–10 polyoxyethylene units per molecule. The best degree of β-receptor solubilization for both heart and liver was obtained with 1% Brij 96 or a combination of 1% digitonin with 0.25% Triton X-100. Hepatic but not cardiac β-receptors were solubilized by polyoxyethylene ether W-1 or by Triton X-100. Solubilization time courses indicated that the maximum degree of receptor solubilization occurred within 5 min at 0–4 °C. Solubilization temperatures were varied from 0 to 37 °C. Temperatures up to 30 °C increased numbers of cardiac receptors solubilized by 30% over those obtained at 0 °C. The same temperature changes had no significant effects on liver β-receptor solubilization. Increasing the solubilization temperature to 37 °C decreased the detectable number of β-receptors by 91 (liver) and 72% (heart). β-Receptors solubilized in the absence of receptor ligand were extremely labile with a half-life on the order of 90 min at 4 °C for both heart and liver. Occupation of the receptors by [¹²⁵I]-iodohydroxybenzylpindolol prior to solubilization conferred a certain degree of stability on the receptors.     

Brain lysosomal hydrolases: I. Solubilization and electrophoretic behavior of acid hydrolases in nerve-ending and mitochondrial-lysosomal fractions from rat brain. Effects of autolysis,neuraminidase, and storage

In solubility studies of 7 acid hydrolases, the extent of solubilization by sonic disruption varied with the enzyme species and increased with increasing pH and Triton X-100 concentration of the suspension medium. Hydrolases in the nerve-ending (NE) fraction were more resistant to solubilization than those in the mitochondrial-lysosomal (M-L) fraction, but nearly quantitative solubilization was attained by sonication in an alkaline buffer containing 0,5% Triton X-100. Polyacrylamide gel electrophoresis of extracts revealed multiple components of acid phosphatase, acid esterase, arylsulfatase,-glucuronidase, and-N-acetyl-hexosaminidase. The enzyme patterns varied with the subcellular fraction and the composition of the medium. In general, the acidic (anodic) forms of these hydrolases were more readily solubilized by sonication in acidic buffer, alkaline pH and Triton X-100 being required to solubilize the basic (cationic) components. The acidic forms of these enzymes were converted to less anodic or cathodic forms, or both, during autolysis at pH 6 at 0 and 37°C, and during storage at –20°C.     

Solubilization of Jerusalem artichoke tuber chromatin by 2,4-dichlorophenoxyacetic acid

Chromatin from the tuber of the Jerusalem artichoke (Helianthus tuberosus) was solubilized in 2,4-dichlorophenoxyacetic acid (2,4-D) solution (100 mM) at pH 7.0. This solubilization was much affected by the pH; below 6.0 less chromatin was solubilized. The elution pattern of the products on gel filtration with Sepharose 4B showed that the solubilization was caused by the dissociation of the DNA and associated proteins. The pattern of polyacrylamide gel electrophoresis of histones extracted from the chromatin solubilized by 2,4-D was quite different from those of histones extracted from the original chromatin or from NaCl (2.0M) solubilized chromatin. The F1 and F3 fractions seemed to be little affected by 2,4-D, but the F2a1, F2a2 and F2b fractions were greatly decreased. In addition, the ratios of histones and non-histone proteins to DNA changed considerably in 2,4-D solubilized chromatin in an inverse manner. None of these changes were observed with NaCl. which suggests that the behaviour of 2,4-D for the solubilization of chromatin differs substantially from that of NaCl.     

Microbial desulfurization of solubilized coal

Microbial desulfurization of low rank coal by Rhodococcus rhodochrous IGTS8 was investigated using three different pretreated coal samples. Solubilized coal was desulfurized more efficiently than hard coal and more sulfur was extracted from biologically solubilized coal than from chemically solubilized coal. Microbial desulfurization combined with biological solubilization removed 75% of the total sufur while the microbial desulfurization combined with chemical solubilization removed 63%.     

A simple procedure for solubilizing 3beta-hydroxysteroid dehydrogenase from microsomes of rat adrenals and testis interstitial cells

A simple procedure is described for solubilizing microsomal 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Microsomes from rat adrenals or from testicular interstitial cells were incubated for 1 or 2 h at 0 C in a buffer containing NaCl followed by overnight storage at -20 C. Maximum solubilization of 3beta-hydroxy-5beta-androstan-17-one-HSD (androstane-3beta-HSD) was obtained by incubating adrenal microsomes with 1 M NaCl and interstitial cell microsomes with 2 M NaCl. Incubation with NaCl for 1 or 2 h resulted in maximum solubilization; incubation with NaCl for 4, 8 or 24 h did not change the amount of enzyme solubilized. From adrenal microsomes incubated with 1 M NaCl, up to 80% (105.7 millimicron/mg microsomes) of the total androstane-3beta-HSD activity was recovered in the supernatant following centrifugation at 130,000 x g for 1 h. The maximum amount of androstane-3beta-HSD solubilized from interstitial cell microsomes was 56% (29.5 millimicron/mg microsomes) at 2 M NaCl. The "solubilized" androstane-3beta-HSD was retarded when chromatographed on a Sephadex G-200 column and it did not pellet out when centrifuged at 130,000 x g for 15 h. KCL appeared to be equally effective in solubilizing androstane-3beta-HSD from microsomes. Other steroid dehydrogenase activities such as pregnanolone-HSD and 3beta-hydroxy-5alpha-androstan-17-one-HSD were also found in the 130,000 x g supernatant.     

In vitro glycoxidation of insoluble fibrous type I collagen: solubilization and advanced glycation end products

The deleterious effects of glycoxidation are dependent on the half-life of proteins. Collagen, the main component of extracellular matrices, is a long live protein and thus may be sensitive to the glycoxidation process. We incubated calf skin fibrous type I collagen in PBS at 37 degrees C with glucose. The fibrous type I collagen was solubilized and an increase in the amount of advanced glycation end products of the solubilized fraction was observed. As there was no bacterial contamination and no proteolytic activities in the incubation medium, the solubilization of fibrous type I collagen is probably due to the speculative production of the free radicals in our experimental conditions. To test this hypothesis, fibrous type I collagen was incubated in PBS with AAPH (2,2'azo-bis 2-aminodinopropane) a free radicals generator. AAPH induced a dramatic and dose dependent solubilization of fibrous type I collagen.     

Definition and characterization of enzymes for maximal biocatalytic solubilization of prebiotic polysaccharides from potato pulp

Potato pulp is a high-volume co-processing product resulting from industrial potato starch manufacturing. Potato pulp is particularly rich in pectin, notably galactan branched rhamnogalacturonan I polysaccharides, which are highly bifidogenic when solubilized. The objective of the present study was to characterize and compare four homogalacturonan degrading enzymes capable of catalyzing the required solubilization of these pectinaceous polysaccharides from potato pulp in a 1 min reaction. An additional purpose was to assess the influence of the pH and the potential buffer chelating effects on the release of these polysaccharides from the potato pulp. The pH and temperature optima of two selected pectin lyases from Emericella nidulans (formerly known as Aspergillus nidulans) and Aspergillus niger were determined to 8.6 and 4.0, respectively, at ≥100 °C within 1 min of reaction. The optima for the two selected polygalacturonases from E. nidulans and Aspergillus aculeatus were determined to pH 4.4 and 46 °C, and pH 3.7 and ≥80 °C, respectively. The polygalacturonase from A. aculeatus was 4-42 times more heat-resistant at 50 °C than the other enzymes. The difference in pH optima of the pectin lyases and the exceptional thermal stabilities of some of the enzymes are proposed to be related to specific amino acid substitutions, stabilizing hydrogen bonding and structural traits of the enzymes. The K_M and V_max values ranged from 0.3-0.6 g/L and 0.5-250.5 U/mg protein, respectively. Phosphate buffer induced release of a higher amount of dry matter than Tris-acetate buffer at pH 6, indicating a chelating effect of the phosphate. Moreover, the phosphate had a higher chelating effect at pH 6 than at pH 4. The optimal conditions for a high yield of polysaccharides from potato pulp were therefore: 1% (w/w) potato pulp treated with 1% (w/w) enzyme/substrate (E/S) pectin lyase from E. nidulans and 1% (w/w) E/S polygalacturonase from A. aculeatus at pH 6.0 and 60 °C for 1 min.     

Slow and steady phosphate solubilization by a psychrotolerant strain of <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">hepiali</Emphasis> (MTCC 9621)

A psychrotolerant phosphate solubilizing fungus has been isolated from the rock soil of a cold desert site in Indian Himalaya. The fungus grows from 4 to 35°C (optimum 21°C), and from 2 to 13.5 pH (optimum 9) under laboratory conditions. Based on phenotypic characters and 26S rDNA analysis, the fungus is identified as Paecilomyces hepiali. In quantitative estimation that was carried out at 9, 14, and 21°C, the fungus solubilized maximum phosphate at 14°C. In view of the slow growth and persistence of the desired activity at low temperature, the estimation was carried out for a longer period, i.e., up to 6 weeks. The suboptimal conditions for growth and biomass production were found to be optimal for phosphate solubilization by the fungus. At 14 and 9°C, the solubilization touched its maximum on day 42. Decline in pH was found to be significantly correlated with the phosphate solubilization at all the temperatures, under consideration. The acid phosphatase activity was found to be more prominent than alkaline phosphatase in culture filtrate. High performance thin layer chromatography (HPTLC) analysis showed production of six organic acids, gluconic and α-keto glutaric acid being in maximum amount in the culture filtrate. The study has ecological significance in view of the nutrient cycling under low temperature environment, prevalent in Himalayan region.     

A preliminary evaluation of the use of microbial culture filtrates for the control of contaminants in plant tissue culture systems

The partitioning of carbon between reserve polysaccharide and alkaloid secondary products was investigated in batch cultures of transformed roots of Datura stramonium grown in media in which the carbon substrate concentration was held constant and the level of mineral nutrients was varied. The growth and accumulation of starch and hyoscyamine was examined in roots grown at temperatures of 20°C, 25°C or 30°C in media containing 5% sucrose and levels of mineral nutrients varying from 1/4 to twice the standard level of Gamborg's B5 salts. The dry matter content was highest (up to 15% w/w) in roots grown at either 20°C or 25°C in medium of the lowest ionic strenth (1/4 B5 salts) and decreased as the ionic strength was raised (down to 7% w/w with 2 B5 salts). Up to half of this decrease could be accounted for by loss of starch from the roots. At 20°C and 25°C, the starch content of the roots grown in medium of the lowest ionic strength (1/4 B5) was 40 mg g^-1 and 22 mg g^-1 fresh weight respectively but decreased to less than 1 mg g^-1 weight at either temperature when the ionic strength of the medium was raised to 2 B5. At 30°C, starch accumulation was severely inhibited in all media. In contrast, varying either the temperature or the ionic strength of the medium had only a small effect on hyoscyamine accumulation which remained at between 0.4–0.6 mg g^-1 fresh weight. Although increases in the level of mineral salts had little effect on the hyoscyamine content of the roots, total yields however, increased due to stimulation of growth. Time course experiments showed that cultures grown at either 20°C or 25°C continued to accumulate both starch and hyoscyamine into late stationary phase.     

Lignocellulose solubilization and conversion by extremely thermophilic Caldicellulosiruptor bescii improves by maintaining metabolic activity

The extreme thermophile Caldicellulosiruptor bescii solubilizes and metabolizes the carbohydrate content of lignocellulose, a process that ultimately ceases because of biomass recalcitrance, accumulation of fermentation products, inhibition by lignin moieties, and reduction of metabolic activity. Deconstruction of low loadings of lignocellulose (5 g/L), either natural or transgenic, whether unpretreated or subjected to hydrothermal processing, by C. bescii typically results in less than 40% carbohydrate solubilization. Mild alkali pretreatment (up to 0.09 g NaOH/g biomass) improved switchgrass carbohydrate solubilization by C. bescii to over 70% compared to less than 30% for no pretreatment, with two-thirds of the carbohydrate content in the treated switchgrass converted to acetate and lactate. C. bescii grown on high loadings of unpretreated switchgrass (50 g/L) retained in a pH-controlled bioreactor slowly purged (τ = 80 hr) with growth media without a carbon source improved carbohydrate solubilization to over 40% compared to batch culture at 29%. But more significant was the doubling of solubilized carbohydrate conversion to fermentation products, which increased from 40% in batch to over 80% in the purged system, an improvement attributed to maintaining the bioreactor culture in a metabolically active state. This strategy should be considered for optimizing solubilization and conversion of lignocellulose by C. bescii and other lignocellulolytic microorganisms.     

High-Melting Lipid Mixtures and the Origin of Detergent-Resistant Membranes Studied with Temperature-Solubilization Diagrams

The origin of resistance to detergent solubilization in certain membranes, or membrane components, is not clearly understood. We have studied the solubilization by Triton X-100 of binary mixtures composed of egg sphingomyelin (SM) and either ceramide, diacylglycerol, or cholesterol. Solubilization has been assayed in the 4–50°C range, and the results are summarized in a novel, to our knowledge, form of plots, that we have called temperature-solubilization diagrams. Despite using a large detergent excess (lipid/detergent 1:20 mol ratio) and extended solubilization times (24–48 h) certain mixtures were not amenable to Triton X-100 solubilization at one or more temperatures. DSC of all the lipid mixtures, and of all the lipid + detergent mixtures revealed that detergent resistance was associated with the presence of gel domains at the assay temperature. Once the system melted down, solubilization could occur. In general adding high-melting lipids limited the solubilization, whereas the addition of low-melting lipids promoted it. Lipidomic analysis of Madin-Darby canine kidney cell membranes and of the corresponding detergent-resistant fraction indicated a large enrichment of the nonsolubilized components in saturated diacylglycerol and ceramide. SM-cholesterol mixtures were special in that detergent solubilization was accompanied, for certain temperatures and compositions, by an independent phenomenon of reassembly of the partially solubilized lipid bilayers. The temperature at which lysis and reassembly prevailed was ∼25°C, thus for some SM-cholesterol mixtures solubilization occurred both above and below 25°C, but not at that temperature. These observations can be at the origin of the detergent resistance effects observed with cell membranes, and they also mean that cholesterol-containing detergent-resistant membrane remnants cannot correspond to structures existing in the native membrane before detergent addition.     

Growth promotion of wheat seedlings by <Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis> 1P (MTCC 8707) a cold tolerant bacterial strain from the Uttarakhand Himalayas

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a gram-positive, rod-shaped, yellow pigmented bacterium isolated from soil on nutrient agar plates at 4°C. The identity of the bacterium was arrived on the basis of the biochemical characterization, BIOLOG sugar utilization pattern and sequencing of the 16S rRNA gene. It grew at temperatures ranging from 4 to 42°C, with temperature optima at 30°C. It expressed multiple plant growth promotion attributes such as phosphate solubilization, indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN) production, differentially at suboptimal growth temperatures (15 and 4°C). At 15°C it solubilized phosphate (21.1 μg of P ml⁻¹ day⁻¹), and produced IAA (14.9 μg ml⁻¹ day⁻¹) in tryptophan amended media. Qualitative detection of siderophore production and HCN were possible at 15°C. At 4°C it retained all the plant growth promotion attributes. Seed bacterization with the isolate, positively influenced the growth and nutrient uptake parameters of wheat seedlings in glass house studies at suboptimal cold growing temperatures.     

Exploring detergent insolubility in bovine hippocampal membranes: a critical assessment of the requirement for cholesterol

The phenomenon of detergent insolubility of bovine hippocampal membranes in Triton X-100 was monitored by estimating the presence of phospholipids in the insoluble pellet. This represents a convenient and unambiguous assay and reports the dependence of the extent of phospholipid solubilization on detergent concentration. The advantage of this approach is its ability to accurately determine the extent of detergent insolubility in natural membranes. Importantly, our results show that when suboptimal concentrations of Triton X-100 are used for solubilization, interpretations of the mechanism and extent of detergent insolubility should be made with adequate caution. At concentrations of Triton X-100 that leads to no further solubilization, ∼44% of phospholipids are left insoluble at 4 °C in bovine hippocampal membranes. Cholesterol depletion using methyl-β-cyclodextrin enhanced phospholipid solubilization at low detergent concentrations but produced no significant change in the amount of insoluble phospholipids at saturating detergent concentration. Progressive solubilization by the detergent resulted in insoluble membranes that contained lipids with higher fatty acyl chain order as reported by fluorescence polarization studies using 1,6-diphenyl-1,3,5-hexatriene (DPH). These results suggest that it is the presence of such lipids rather than their association with cholesterol that determines detergent insolubility in membranes.     

UDP-Glucose: (1,3)-beta-Glucan Synthase from Daucus carota L. : Characterization, Photoaffinity Labeling, and Solubilization

The membrane-bound UDP-glucose-β-(1,3)-glucan synthase from Daucus carota L. was characterized and a solubilization procedure was developed. The enzyme exhibited maximal activity in the presence of 0.75 millimolar Ca²⁺, 0.5 millimolar EGTA, and 5 millimolar cellobiose at pH 7.5 and 30°C at 1 millimolar UDPG. Reaction products were confirmed to be (1,3)-linked glucan. Polypeptides of 150, 57, and 43 kilodaltons were labeled with the photoactivatible affinity label 5-azido-uridine 5′-β-[³²P] diphosphateglucose. Labeling of the 150 and 57 kilodalton polypeptides was completely protected against by 1 millimolar non-radioactive UDPG suggesting that one or both of these polypeptides may represent the UDPG binding subunit of glucan synthase. Carrot glucan synthase was solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in the absence of divalent cations and chelators; however, the percentage of enzyme which could be solubilized showed variability with membrane source. With microsomal membranes, up to 80% of the enzyme was released with 0.7% CHAPS. Solubilized enzyme was stable for at least 9 hours at 4°C. When more highly purified membrane fractions were isolated from sucrose step gradients a slightly different picture emerged. Activity from the 20/30% interface (Golgi and tonoplast enriched) was readily solubilized and expressed. Activity from the 30/40% interface (plasma membrane enriched) was also solubilized; however, it was necessary to add heat inactivated microsomes to assay mixtures for full activity to be expressed. A requirement for endogenous activators is suggested.     

Effect of holding medium, temperature and time on structural integrity of equine ovarian follicles during the non-breeding season

The objective was to evaluate the efficiency of phosphate-buffered saline (PBS) and Minimum Essential Medium (MEM) during the transport of equine preantral and antral follicles at various temperatures and incubation interval. Equine ovaries (n = 10) from an abattoir were cut into 19 fragments; one was immediately fixed in Bouin's solution (control) and the other fragments were placed in PBS or MEM solution at 4, 20, or 39 °C for 4, 12, or 24 h. After the respective incubation periods, all fragments were fixed in Bouin's solution for 24 h and then submitted to standard histologic analysis. In total, 2567 ovarian follicles were analyzed, including 1752 primordial, 764 primary, 34 secondary and seven antral follicles. Relative to the control group, the transport of equine ovarian fragments in both solutions significantly reduced the percentage of morphologically normal follicles with increasing time and temperature. At 4 °C for 4 h, considering primordial and developing follicles, PBS had a higher (P < 0.05) rate (98.9%) of morphologically normal follicles than MEM, 48.7%. At 39 °C for 12 h, all follicles in both solutions were degenerated. Regarding the stage of follicular development, primordial follicles were less (P < 0.05) affected by preservation than primary and secondary follicles in all media, times and temperatures tested, except at 4 °C for 12 h in PBS, in which the primary and secondary follicles were less (P < 0.05) affected. Overall, 43% of antral follicles were morphologically normal when maintained in MEM at 4 °C for 4 h. In conclusion, equine follicles were successfully preserved in ovarian fragments at 4 °C in phosphate-buffered saline for up to 4 h.