Suplementação com óleos ricos em ácidos graxos poli-insaturados na dieta de leitões na fase de creche: efeitos no desempenho, na resposta inflamatória, no perfil lipídico e no "status" oxidativo / Supplementation with oils rich in polyunsaturated fatty acids in the diet of pigs in the nursery phase: effects on performance, inflammatory response, lipid profile and oxidative "status"

Nos sistemas de produção, os produtos naturais vêm se destacando na substituição a produtos sintéticos, dentre eles podemos ressaltar os óleos vegetais ricos em ácido graxos poli-insaturados (PUFA), que são conhecidos, popularmente, por seus efeitos benéficos. Dessa forma, o objetivo com este trabalho foi avaliar os efeitos da suplementação dos óleos de pequi (Caryocar brasiliense) e girassol (Helianthus annus) sobre parâmetros fisiológicos em leitões na fase de creche. Foram utilizados 180 leitões alojados em granja comercial, distribuídos em três grupos (n=60) conforme suplementação alimentar: óleo de pequi, óleo de girassol e controle negativo. Amostras de sangue e o peso dos animais foram obtidos nos intervalos de quatro trocas de rações da fase de creche para avaliação do desempenho, da resposta inflamatória, do perfil lipídico e do "status" oxidativo. Os parâmetros fisiológicos mensurados demonstraram que os óleos interferiram positivamente na resposta inflamatória sistêmica, por meio dos leucócitos totais e da relação neutrófilo/linfócito (P<0,05); no equilíbrio oxidante-antioxidante, por mensuração de óxido nítrico e do malondialdeído (P<0,05); e no metabolismo lipídico, com a avaliação de colesterol total e triglicérides (P<0,05). Por outro lado, esses óleos vegetais interferiram no ganho de peso e no consumo de ração (P<0,05). Assim, concluí-se que a suplementação com óleos de pequi e girassol melhora a saúde dos animais, mas tem impacto negativo no desempenho zootécnico de leitões na fase de creche.(AU)

In production systems, natural products have been outstanding in replacing synthetic products, among them, we can highlight the vegetable oils rich in polyunsaturated fatty acids (PUFA) that are popularly known for their beneficial effects. Thus, the objective of this study was to evaluate the effects of pequi (Caryocar brasiliense) and sunflower (Helianthus annus) oil supplementation on nursery physiological parameters. We used 180 piglets housed in a commercial farm, distributed in 3 groups (n=60) according to food supplementation: pequi oil, sunflower oil and negative control. Blood samples and animal weight were obtained at intervals of four nursery phase rations to evaluate performance, inflammatory response, lipid profile and oxidative status. The physiological parameters measured showed that the oils positively interfered in the systemic inflammatory response through total leukocytes and neutrophil / lymphocyte ratio (P<0.05), in the oxidant-antioxidant balance by measuring nitric oxide and malondialdehyde (P<0.05). and lipid metabolism with the assessment of total cholesterol and triglycerides (P<0.05). On the other hand, these vegetable oils interfered with weight gain and feed intake (P<0.05). Thus, it can be concluded that supplementation with pequi and sunflower oils improves animal health, but has a negative impact on the piglet's performance in the nursery phase.(AU)

Avaliação do estresse e do desempenho de suínos na fase de creche, empregando-se técnicas de enriquecimento ambiental / Evaluation of stress and performance of pigs during the nursery phase, using environmental enrichment techniques

O enriquecimento ambiental é uma ferramenta importante dentro dos sistemas de produção, a fim de promover o bem-estar e favorecer a saúde dos animais. Portanto, o objetivo deste trabalho foi avaliar o efeito do enriquecimento ambiental sobre o estresse de suínos na fase de creche. Foram utilizados 32 leitões, alojados em granja experimental, distribuídos em quatro grupos (n= 8): corda, corrente, garrafa PET e controle negativo. Amostras de sangue foram coletadas no início e no final do experimento para contagem de leucócitos circulantes e determinação de antioxidantes não enzimáticos, óxido nítrico, malondialdeído, e de saliva para avaliação do cortisol. Foi aplicado etograma e fez-a ganho médio de peso diário e a conversão alimentar. Os parâmetros avaliados no primeiro dia do experimento não variaram entre os grupos (P>0,05). No último dia do experimento, os valores de neutrófilos e da relação neutrófilo/linfócito foram mais elevados nos leitões do grupo corrente, assim como os valores de cortisol salivar (P<0,05). O ácido úrico apresentou-se mais elevado nos leitões do grupo corrente e o malondialdeído (MDA) nos do grupo garrafa (P< 0,05). Os enriquecimentos ambientais estimularam comportamentos positivos nos leitões, tendo a corda se destacado como o mais atrativo. Por outro lado, a corrente apresentou efeito negativo sobre a fisiologia dos animais, gerando estresse, assim como a garrafa, que induziu a peroxidação lipídica e um menor ganho de peso nos leitões.(AU)

Environmental enrichment is an important tool within production systems to promote welfare and animal health. The aim of this study was to evaluate the effect of enrichment objects on stress of piglets in nursery phase. 32 piglets housed in experimental farm were distributed in 4 groups (n= 8): rope, chain, pet bottle and negative control. Blood samples were collected at the beginning and end of the experiment to count circulating leukocytes and determine non-enzymatic antioxidants, nitric oxide, malondialdehyde, and saliva to evaluate cortisol. At the same time, an etogram and evaluation of mean daily gain and feed conversion were applied. The evaluated parameters on first day of the experiment did not vary between groups (P> 0.05). On the last day, neutrophil and neutrophil /lymphocyte ratios were higher in chain group piglets, as were salivary cortisol values (P< 0.05). Uric acid was higher in chain group and MDA in bottle group (P< 0.05). Environmental enrichment stimulated positive behaviors in piglets, where the rope stood out as the most attractive. On the other hand, chain had a negative effect on animal physiology, generating stress, as well as the bottle that induced lipid peroxidation and a lower weight gain in piglets.(AU)

Role of lipid polymorphism in pulmonary surfactant.

The development of artificial surfactants for the treatment of respiratory distress syndrome (RDS) requires lipid systems that can spread rapidly from solution to the air-water interface. Because hydration-repulsion forces stabilize liposomal bilayers and oppose spreading, liposome systems that undergo geometric rearrangement from the bilayer (lamellar) phase to the hexagonal II (HII) phase could hasten lipid transfer to the air-water interface through unstable transition intermediates. A liposome system containing dipalmitoylphosphatidylcholine was designed; the system is stable at 23 degrees C but undergoes transformation to the HII phase as the temperature increases to 37 degrees C. The spreading of lipid from this system to the air-water interface was rapid at 37 degrees C but slow at 23 degrees C. When tested in vivo in a neonatal rabbit model, such systems elicited an onset of action equal to that of native human surfactant. These findings suggest that lipid polymorphic phase behavior may have a crucial role in the effective functioning of pulmonary surfactant.

Pore-forming peptides induce rapid phospholipid flip-flop in membranes.

A kinetic model for pore-mediated and perturbation-mediated flip-flop is presented and used to characterize the mechanism of peptide-induced phospholipid flip-flop in bilayers. The model assumes that certain peptides can bind to and aggregate within the membrane. When the aggregate attains a critical size (M peptides), a channel is created that results in a fast flip-flop of phospholipids. In addition, certain peptides induce flip-flop through perturbation of the membrane without forming a pore. Donor phospholipid vesicles with an asymmetrical distribution of the fluorescent phospholipid 1-oleoyl-2-[12-[(7-nitro-1,2,3-benzoxadiazol-4- yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) were used to measure the extent of flip-flop by quantitating the decrease in fluorescence as the NBD-PC exchanged from the donor vesicles to acceptor vesicles that contained a quencher of the NBD fluorescence. Flip-flop curves generated at lipid/peptide ratios ranging from 30/1 to 300000/1 could be well-simulated by the model. Pore-forming peptides, such as melittin or the synthetic peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA), induce rapid phospholipid flip-flop with half-times for flip-flop of seconds at low peptide/vesicle ratios. The deduced pore sizes are M = 10 +/- 2 for GALA and M = 2 - 4 for melittin. The synthetic peptide LAGA (WEAALAEAEALALAEHEALALAEAELALAA) can catalyze flip-flop via bilayer perturbation. In contrast, hydrophobic peptides such as gramicidin A and valinomycin intercalate into the membrane, but induce little flip-flop. Modeling of the kinetics of phospholipid translocation supports pore formation as the key factor in accelerating phospholipid flip-flop. Thus, amphipathic segments from membrane proteins may account for non-energy-dependent phospholipid flip-flop in biological membranes.

Secondary structure and orientation of the amphipathic peptide GALA in lipid structures. An infrared-spectroscopic approach.

GALA, a synthetic, amphipathic 30-amino-acid peptide, based upon a Glu-Ala-Leu-Ala motive, was designed to mimic the behavior of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH, and becomes an amphipathic alpha helix as the pH is lowered to 5, where it interacts with phospholipid bilayers. Attenuated total-reflection infrared spectroscopy, using polarized light, provides information on the structure and orientation of the peptide and the lipids, which is not subject to artifacts due to light scattering with large particles. H/2H-exchange rate of the amide N-H group and analysis of the shape of the amide I' by Fourier self-deconvolution and curve fitting indicate that the alpha-helical content increases from 19% to 69%, on lowering the pH. A further increase to 100% alpha helix is observed after interaction with palmitoyloleoylglycerophosphocholine (PamOleGroPCho) vesicles. Dichroism data obtained with oriented bilayers of the PamOleGroPCho-GALA complex demonstrate that PamOleGroPCho hydrocarbon chains and the peptide alpha helical axis are essentially perpendicular (+/- 15) to the membrane plane. At neutral pH, in the presence of dimyristoylglycerophosphocholine (Myr2GroPCho), GALA is known to form discoidal structures similar to those formed under the same conditions by apolipoproteins AI and AII. In these discoidal complexes, the alpha-helical content was estimated to be 65%, with the rest of the structure being essentially unordered. No significant modification of the all-trans conformation of the hydrocarbon chain of Myr2GroPCho was detected upon disc formation. Dichroism measurements show that the alpha-helical axis is essentially parallel to the hydrocarbon chains. These data support a model in which a discoidal patch of the bilayer is surrounded by amphipathic helices which shield the hydrophobic region of the bilayer from the aqueous environment. The infrared spectrum of GALA in this complex was found to be very similar to those of apolipoproteins AI and AII which form discoidal complexes with Myr2GroPCho, but the spectrum is quite different from that of apolipoprotein B100 in low-density lipoproteins, which does not form discoidal complexes.

Association of a pH-sensitive peptide with membrane vesicles: role of amino acid sequence.

The solution properties and bilayer association of two synthetic 30 amino acid peptides, GALA and LAGA, have been investigated at pH 5 and 7.5. These peptides have the same amino acid composition and differ only in the positioning of glutamic acid and leucine residues which together compose 47% of each peptide. Both peptides undergo a similar coil to helix transition as the pH is lowered from 7.5 to 5.0. However, GALA forms an amphipathic alpha-helix whereas LAGA does not. As a result, GALA partitions into membranes to a greater extent than LAGA and can initiate leakage of vesicle contents and membrane fusion which LAGA cannot (Subbarao et al., 1987; Parente et al., 1988). Membrane association of the peptides has been studied in detail with large phosphatidylcholine vesicles. Direct binding measurements show a strong association of the peptide GALA to vesicles at pH 5 with an apparent Ka around 10(6). The single tryptophan residue in each peptide can be exploited to probe peptide motion and positioning within lipid bilayers. Anisotropy changes and the quenching of tryptophan fluorescence by brominated lipids in the presence of vesicles also indicate that GALA can interact with uncharged vesicles in a pH-dependent manner. By comparison to the peptide LAGA, the membrane association of GALA is shown to be due to the amphipathic nature of its alpha-helical conformation at pH 5.

Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA.

The synthetic, amphipathic peptide GALA undergoes a pH-dependent conformational change and induces leakage of contents from large unilamellar phosphatidylcholine vesicles when in a helical conformation. The kinetics of this process have been investigated over a wide range of pH and lipid and peptide concentrations. Leakage from lipid vesicles is rapidly initiated (within 2 s) when the pH is lowered below 6 and is rapidly terminated when the pH is raised to 7.5. The leakage shows a selectivity to the size of the entrapped molecules and occurs by an all or none mechanism; vesicles either leak or retain all of their contents. Using this experimental data, we have developed a mathematical description of the kinetics of leakage induced by GALA. This model assumes that GALA becomes incorporated into the vesicle bilayer and aggregates to form a pore. Leakage occurs when a critical number of peptides assemble into a supramolecular aggregate in the bilayer. Leakage curves generated at lipid/peptide ratios ranging from 500/1 to 30000/1 can be well described by this formalism. On the basis of the results and the model, we suggest that GALA forms a transbilayer channel composed of 8-12 monomers. The channel diameter ranges from 5 to 10 A. To the best of our knowledge, this is the first model that can predict the leakage kinetics of solutes entrapped in lipid vesicles induced by a pore-forming peptide. The analysis may be of general use in defining the kinetics and state of aggregation of similarly acting peptides and proteins which form multimeric assemblies in membranes.

pH-dependent fusion of phosphatidylcholine small vesicles. Induction by a synthetic amphipathic peptide.

A synthetic, amphipathic 30-amino acid peptide with the major repeat unit Glu-Ala-Leu-Ala (GALA) was designed to mimic the behavior of the fusogenic sequences of viral fusion proteins. GALA is a water-soluble peptide with an aperiodic conformation at neutral pH and becomes an amphipathic alpha-helix as the pH is lowered to 5.0 where it interacts with bilayers. Fluorescence energy transfer measurements indicated that GALA induced lipid mixing between phosphatidylcholine small unilamellar vesicles but not large unilamellar vesicles. This lipid mixing occurred only at pH 5.0 and not at neutral pH. Concomitant with lipid mixing, the vesicles increased in diameter from 500 to 750 to 1000 A as measured by dynamic light scattering and internal volume determination. GALA induced leakage of small molecules (Mr 450) at pH 5.0 was too rapid to permit detection of contents mixing. However, retention of larger molecules (Mr 4100) under the same conditions suggests that vesicle fusion is occurring. For a 100/1 lipid/peptide ratio all vesicles fused just once, whereas for a 50/1 ratio higher order fusion products formed. A mass action model gives good simulation of the kinetics of increase in fluorescence intensity and yields rate constants of aggregation and fusion. As the lipid to peptide ratio decreases from 100/1 to 50/1 both rate constants of aggregation and fusion increase, indicating that GALA is a genuine inducer of vesicle fusion. The presence of divalent cations which can alter GALAs conformation at pH 7.5 had little effect on its lipid mixing activity. GALA was modified by altering the sequence while keeping the amino acid composition constant or by shortening the sequence. These peptides did not have any lipid mixing activity nor did they induce an increase in vesicle size. Together, these results indicate that fusion of phosphatidylcholine small unilamellar vesicles induced by GALA requires both a peptide length greater than 16 amino acids as well as a defined topology of the hydrophobic residues.

pH-dependent bilayer destabilization by an amphipathic peptide.

A 30-residue amphipathic peptide was designed to interact with uncharged bilayers in a pH-dependent fashion. This was achieved by a pH-induced random coil-alpha-helical transition, exposing a hydrophobic face in the peptide. The repeat unit of the peptide, glutamic acid-alanine-leucine-alanine (GALA), positioned glutamic acid residues on the same face of the helix, and at pH 7.5, charge repulsion between aligned Glu destabilized the helix. A tryptophan was included at the N-terminal as a fluorescence probe. The rate and extent of peptide-induced leakage of contents from large, unilamellar vesicles composed of egg phosphatidylcholine were dependent on pH. At pH 5.0 with a lipid/peptide mole ratio of 500/1, 100% leakage of vesicle contents occurred within 1 min. However, no leakage of vesicle contents occurred at pH 7.5. Circular dichroism measurements indicated that the molar ellipticity at 222 nm changed from about -4000 deg cm2 dmol-1 at pH 7.6 to -11,500 deg cm2 dmol-1 at pH 5.1, indicating a substantial increase in helical content as the pH was reduced. Changes in molar ellipticity were most significant over the same pH range where a maximum change in the extent and rate of leakage occurred. The tryptophan fluorescence emission spectra and the circular dichroism spectra of the peptide, in the presence of lipid, suggest that GALA did not associate with the bilayer at neutral pH. A change in the circular dichroism spectrum and a blue shift of the maximum of the tryptophan fluorescence emission spectra at pH 5.0, in the presence of lipid, indicated an association of GALA with the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Rate and extent of poly(ethylene glycol)-induced large vesicle fusion monitored by bilayer and internal contents mixing.

Poly(ethylene glycol) (PEG) of average molecular weight 8000 was used to mediate the fusion of large unilamellar vesicles composed of dipalmitoylphosphatidylcholine. Fusion was monitored by fluorescence assays of lipid mixing and aqueous contents mixing. The extent of lipid mixing, as monitored by DPHpPC fluorescence lifetime, indicated that large unilamellar vesicles underwent a single fusion cycle when incubated with PEG and subsequently diluted into buffer. The ANTS/DPX assays for contents mixing and leakage indicated that, while addition and dilution of PEG were accompanied by extensive contents leakage, this occurred on a much different time scale as compared to contents mixing. Both the lipid-mixing and contents-mixing assays gave comparable estimates for the number of rounds of fusion that occurred in a given time following PEG addition, although the contents-mixing assay always yielded an estimate 10-15% larger than the lipid-mixing assay. These assays were used to evaluate several factors purported to influence PEG-induced fusion. First, the initial rate of fusion was found to be dependent on PEG concentration in the range of 0-35 wt %, while the extent of fusion was not. In addition, a substantial rate enhancement occurred when vesicles were incubated with greater than 26% PEG. Second, the creation of an osmotic gradient upon dilution of vesicle-PEG mixtures was shown to have no effect on either the extent or the initial rate of fusion. Consistent with this observation, both contents and lipid mixing were found to occur prior to and independent of the dilution of the PEG-vesicle suspension. Third, impurities, either present in our commercially available PEG or added to vesicle-PEG mixtures, also had no effect on the rate or extent of fusion. Fourth, another dehydrating polymer, dextran (average mol wt 9000), was capable of promoting fusion, though at a much lower rate than PEG. These results suggest that even partial bilayer dehydration accompanied by vesicle collapse and close interbilayer contact may be sufficient to induce vesicle fusion.

Fusion and phase separation monitored by lifetime changes of a fluorescent phospholipid probe.

The sensitivity of the fluorescence lifetime of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]- 3-sn-phosphatidylcholine (DPHpPC) to its local concentration in lipid bilayers was used to monitor both lipid mixing and phase separation occurring during membrane vesicle fusion. Vesicles containing 2 mol % DPHpPC were mixed with a 10-fold excess of vesicles devoid of probe. Upon addition of a fusogen, mixing of bilayer lipids associated with fusion was followed as an increase in the fluorescence lifetime of DPHpPC. Ca2+-induced fusion of phosphatidylserine vesicles served to test the method and was shown to have an exponential half-time of 7 s. Phase separation (between the phosphatidylserine head groups of bulk lipid and the phosphatidylcholine head groups of the probe) was monitored by DPHpPC under the same conditions used to follow lipid mixing due to fusion. Phase separation was not significant until 10 min after Ca2+ addition and was completely reversible by disodium ethylenediaminetetraacetate addition. Vesicle aggregation induced by Ca2+ addition to mixed phosphatidylserine/phosphatidylcholine vesicles did not alter the DPHpPC lifetime, indicating that close association of vesicles did not promote intervesicular exchange of the probe. In addition, we have investigated the effects of CA2+ on the fluorescence properties of this probe and of the head-group-labeled fluorescent probes N-(4-nitro-2,1,3-benzoxadiazolyl)phosphatidylethanolamine and N-(lissamine Rhodamine B sulfonyl)dioleoyl-phosphatidylethanolamine, which are used in the fluorescence energy transfer assay of Struck et al.(ABSTRACT TRUNCATED AT 250 WORDS)

Advantages and limitations of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3- sn-phosphatidylcholine as a fluorescent membrane probe.

We have investigated the behavior of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn -phosphatidylcholine (DPHpPC) in synthetic, multilamellar phosphatidylcholine vesicles. This fluorescent phospholipid has photophysical properties similar to its parent fluorophore, diphenylhexatriene (DPH). DPHpPC preferentially partitioned into fluid phase lipid (Kf/s = 3.3) and reported a lower phase transition temperature as detected by fluorescence anisotropy than that observed by differential scanning calorimetry. Calorimetric measurements of the bilayer phase transition in samples having different phospholipid to probe ratios demonstrated very slight changes in membrane phase transition temperature (0.1-0.2 degree C) and showed no measurable change in transition width. Nonetheless, measurements of probe fluorescence properties suggested that DPHpPC disrupts its local environment in the membrane and may even induce perturbed probe-rich local domains below the phospholipid phase transition. Temperature profiles of steady-state fluorescence anisotropy, limiting anisotropy, differential tangent, and rotational rate were similar to those of DPH below the main lipid phase transition but indicated more restricted rotational motion above the lipid phase transition temperature. As for DPH, the fluorescence decay of DPHpPC could be described by either a single or double exponential both above and below the DPPC phase transition. The choice seemed dependent on the treatment of the sample. The intensity-weighted average lifetime of DPHpPC was roughly 1.5 ns shorter than that of DPH. In summary, the measured properties of DPHpPC and its lipid-like structure make it a powerful probe of membrane structure and dynamics.

Morphology and phase behavior of two types of unilamellar vesicles prepared from synthetic phosphatidylcholines studied by freeze-fracture electron microscopy and calorimetry.

Differential scanning calorimetry and freeze-fracture electron microscopy have been used to characterize the phase behavior and morphology of two types of unilamellar vesicles composed of synthetic phosphatidylcholines. The first type displayed an average diameter of roughly 100 nm and was formed by slow dilution and dialysis of octylglucoside-solubilized lipid. These large, unilamellar vesicles were termed dialyzed, octylglucoside vesicles and could be obtained as a fairly well defined and uniform population of vesicles. The second vesicle type was prepared by a unique procedure involving dialysis of deoxycholate-solubilized lipid at its pre-transition temperature. This procedure produced a much more heterogeneous distribution of vesicle sizes (500 to 4000 nm in diameter) and left some dilamellar and oligolamellar species which could not be conveniently separated from the giant, unilamellar vesicles constituting the major portion of the sample. Both populations of vesicles displayed phase behavior similar, but not identical to that of large, multilamellar vesicles (LMV). Fracture-face morphology of the gel phase was also observed to differ between the two unilamellar and the multilamellar species. LMV have previously been shown to have clear undulated or banded fracture-faces in the P beta phase, while octylglucoside vesicles are shown here to have facetted fracture-faces. Giant, unilamellar vesicles displayed a faint banded morphology similar to but less distinct than that of the LMV P beta phase. These results have demonstrated that bilayer apposition is not required to support the banded fracture-face morphology characteristic of the P beta phase but that a limiting curvature is necessary.

Phase behavior of large unilamellar vesicles composed of synthetic phospholipids.

Large unilamellar vesicles (LUV) have been prepared by three procedures from several synthetic and natural phosphatidylcholines. Reverse-phase evaporation vesicles (REV) and fusion vesicles were prepared by established procedures. A published procedure for the preparation of dialyzed octyl glucoside vesicles (DOV) was modified to allow its use with synthetic phospholipids. Negative-staining and freeze--fracture electron microscopy was used to determine the vesicle size distribution (mean diameters 800-1000 A) and extent of oligolamellar contamination in DOV preparations. Trapping of 6-carboxyfluorescein yielded measurements of the internal volume (2.6 +/- 0.3 microL/mumol of Pi) consistent with the size distributions determined by electron microscopy. An upper limit of less than 3 mol % oligolamellar vesicle contamination was indicated by calorimetric heat capacity profiles. The phase behaviors of large multilamellar vesicles and all three types of LUV were compared by using high-sensitivity differential scanning calorimetry and fluorescence depolarization of the membrane probe diphenylhexatriene. The most remarkable feature was the increased breadth of the main transition of DOV and of REV relative to the multilamellar species and to fusion vesicles. Both the main transition and the pretransition occurred at nearly the same temperatures in unilamellar and multilamellar species, but the unilamellar pretransition involved less than half the enthalpy observed in the multilamellar transition. Additional experiments indicated that the broadened main phase transition associated with DOV and REV reflected bilayer impurities resulting from preparation. It is concluded that LUV prepared by procedures that avoid impurities undergo a highly cooperative phase transition, as demonstrated here for fusion vesicles.