Optimal Incubation Conditions Research Articles

Brood Care Strategies by Newly Mated Monogyne <I>Solenopsis invicta</I> (Hymenoptera: Formicidae) Queens During Colony Founding

Abstract For most species of ants, newly mated queens found new colonies from stored body reserves, and without the assistance of workers. Consequently, until the first batch of colony members hatches, newly mated queens face a potential shortage of labor for brood care. The purpose of this study was to describe the brood rearing strategies of newly mated monogyne Solenopsis invicta queens, under controlled laboratory conditions, to determine how they compensated for the lack of worker labor in rearing the first batch of colony members. Newly mated S. invicta queens were collected (n = 60) and reared individually in artificial nests in the laboratory under optimal incubation conditions. Using video-technology, brood care activity by newly mated queens was quantified and compared with brood care activity of mature queens who were incubated with workers. In addition, newly mated queens and their offspring (i.e., the incipient colony) were censused biweekly for 6 mo to determine the ontogeny of the adult:lar...

HPLC analysis of 4-chlorophenyl methyl sulphide and diphenyl sulphide and their corresponding sulphoxides and sulphones in rat liver microsomes.

Simple high performance liquid chromatography (HPLC) methods for the analysis of 4-chlorophenyl methyl sulphide (CPMS), diphenyl sulphide (DPS) and their corresponding sulphoxide and sulphone metabolites in rat liver microsomes are described. The assay methods are based on a reversed phase HPLC column (Spherisorb(R) 5 ODS, 15 x 0.46 cm) using a mixture of water and tetrahydrofuran (THF) as mobile phase at a flow rate of 0.5 ml/min and ultraviolet detection at 260 nm. The compounds were extracted into diethyl ether (2 x 5 ml) from rat liver microsomal incubation mixture (2 ml) and the recoveries were more than 80%. The calibration curves for determining the sulphoxide and sulphone of CPMS or DPS were linear (r > or =0.995) in the range of 0-50 microg/ml and the assays were reproducible with low inter- and intra-assay variation of less than 13.5%. The lower limit of quantitation (LOQ) was 0.1 microg/ml for CPMSO and 0.025 microg/ml for CPMSO(2), diphenyl sulphoxide (DPSO) and diphenyl sulphone (DPSO(2)). The HPLC methods were successfully applied to measure enzymically formed CPMSO, CPMSO(2), DPSO and DPSO(2) in rat liver microsomes and to characterise the Michaelis-Menten kinetics associated with the metabolism of CPMS and DPS and their corresponding sulphoxides. About 20% of the initial CPMS (0.5 mM) concentration in the incubation was converted to the sulphoxide although the sulphone was not detected under these optimum incubation conditions. Similarly, about 15-20% of DPS was converted to the sulphoxide while less than 0.1% of DPS was converted to DPSO(2). Eadie-Hofstee plot of CPMS sulphoxidation was biphasic. This suggests that the sulphoxidation of CPMS is a consequence of at least two enzyme systems, one characterized by low affinity and high capacity (K(m)=0.1 mM; V(max)=2.1 nmoles/mg protein/min) and the other by high affinity and low capacity (K(m)=0.05 mM; V(max)=1.5 nmoles/mg protein/min). On the other hand, the Eadie-Hofstee plot of DPS sulphoxidation was monophasic with an apparent V(max) and K(m) of 1.8 nmoles/mg protein/min and 0.036 mM, respectively.

Relationship Between Broiler Breeder's Age and Egg Weight Loss and Embryonic Mortality During Incubation in Large-Scale Conditions

The age of broiler breeders is an important parameter to be taken into account by the hatchery manager. Eggs produced by young or old breeders do not hatch as well as the eggs from the breeders of 40 to 42 wks of age. Field reports indicate that there are quadratic relationships between age of breeders and absolute egg weight loss during incubation, age of breeders and hatchability, and age of breeders and embryonic mortality. There is a trend of optimum relative egg weight loss on Day 18 of incubation for the highest hatchability. These results indicate that the eggs that incubate best are produced by the hens of 40 to 42 wk of age. It is suggested that the egg characteristics and the age of breeders have to be taken into account during incubation in order to design optimal incubation conditions and to improve hatchability. However, because the relative egg weight loss is not linked to the age of breeders, regulation of relative humidity during incubation to achieve optimum weight loss may take into account only the eggs characteristics.

Screening and identification of resistance to bacterial soft rot in Brassica rapa

Mist-chamber, field, and detached leaf inoculation procedures identified plants resistant to bacterial soft rot [Erwinia carotovora subsp. carotovora, (Ecc)] in Brassica rapa and related species. The mist-chamber seedling inoculation provided the best correlation of mean disease severity ratings with the field plant inoculation (r = 0.67**) and was used to identify resistant materials. The optimum mist-chamber incubation conditions to distinguish the resistance of accessions were 23 °C and 100% RH and were used for primary screening. A total of 752 accessions of B. rapa and related Cruciferae were screened. In general, accessions of B. oleracea were more resistant than accessions of B. rapa. Within B. rapa, subspecies pekinensis and chinensis were more susceptible than other subspecies. No completely resistant material was found. In species of B. rapa, only 7% of accessions showed some degree of resistance with plant-to-plant variation within the accessions. G30444, G30449, and AVRDC2837 were identified as the most resistant materials in B. rapa by both mist-chamber and field inoculations. The resistance was correlated between an USA isolate (Geneva-1) and two Chinese isolates (RL4-1 and RL-19) of Ecc.

Multisite reproducibility of Etest for susceptibility testing of Mycobacterium abscessus, Mycobacterium chelonae, and Mycobacterium fortuitum.

A multicenter study was conducted to assess the inter- and intralaboratory reproducibility of the Etest for susceptibility testing of the rapidly growing mycobacteria. The accuracy also was evaluated by comparing Etest results to those obtained by broth microdilution. Ten isolates (four of the Mycobacterium fortuitum group, three of Mycobacterium abscessus, and three of Mycobacterium chelonae) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, and trimethoprim-sulfamethoxazole in each of four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) using common lots of media and Etest strips. Interlaboratory agreement among MICs (i.e., mode +/- 1 twofold dilution) varied for the different drug-isolate combinations and overall was best for trimethoprim-sulfamethoxazole (75% for one isolate and 100% for all others), followed by doxycycline and ciprofloxacin. Interlaboratory agreement based on interpretive category also varied and overall was best for doxycycline (100% for all isolates), followed by trimethoprim-sulfamethoxazole and ciprofloxacin. Interlaboratory reproducibility among MICs was most variable for imipenem, and agreement by interpretive category was lowest for imipenem and amikacin. Modal Etest MICs agreed with those by broth microdilution only for doxycycline and the sulfonamides. For all other drugs, the modal MICs by the two methods differed by more than +/- 1 twofold dilution for one or more isolates. In all cases, the Etest MIC was higher and would have caused reports of false resistance. In summary, the Etest in this evaluation did not perform as well as broth microdilution for susceptibility testing of the rapidly growing mycobacteria. It was problematic for most species and drugs, primarily because of a trailing endpoint and/or high MICs compared to broth. Its use will necessitate further investigation, including determination of the optimal medium and incubation conditions and clarification of endpoint interpretation.

LIVE/DEAD ®BacLight™: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water

A rapid epifluorescence staining method using the LIVE/DEAD ® Bacterial Viability Kit ( BacLight™) was applied to estimate both viable and total counts of bacteria in drinking water. BacLight is composed of two nucleic acid-binding stains: SYTO 9™ and propidium iodide. SYTO 9™ penetrates all bacterial membranes and stains the cells green, while propidium iodide only penetrates cells with damaged membranes, and the combination of the two stains produces red fluorescing cells. Optimal incubation conditions were found to be 15 to 20 min, at room temperature in the dark. Total (red+green) and viable (green) cells can hence be counted simultaneously. Factors affecting the staining procedure were tested (addition of glutaraldehyde, staining time, chlorine impact). In the absence of stress, BacLight viable counts were comparable and to 5-cyano-2,3-ditolyl tetrazolium (CTC) counts. BacLight total counts were comparable to acridine orange counts (differing by <0.1 log/ml). However, the increase in environmental stresses (chlorine, growth rate or temperature) induced a decrease in viability that was more pronounced for CTC and plate counts than for BacLight viable counts.

Survival of Bacteria on Respirator Filters

The survival and potential growth of two different kinds of bacteria were investigated on NIOSH-certified polypropylene respirator filters. Bacillus subtilis var niger (B. subtilis) spores represented environmentally resistant bacteria, whereas Pseudomonas fluorescens (P. fluorescens) vegetative cells represented stress-sensitive bacteria. The bacteria were aerosolized and loaded on the respirator filters under three nutritional conditions: water, saliva, and tryptic soy broth (TSB). The loaded filters were incubated for 0-13 days and analyzed for culturable and total bacteria count. The analysis was optimized through the evaluation of three methods for eluting bacteria from respirator filter samples: low frequency shaking, vortexing, and ultrasonic vibrating. Vortexing was found to be the most effective with the highest total and culturable count and was therefore used for the analysis of bacterial survival and growth. Neither of the test bacteria was able to grow on the respirator filters even under optimal nutrition and incubation conditions. It therefore appears that reuse of a polypropylene respirator poses minimal risk of bacterial growth, provided the respirator has been carefully handled and stored. The data on respirator survival show that sensitive P. fluorescens cells lost their viability in less than three days, whereas resistant B. subtilis spores remained viable on the filter for over thirteen days of testing. It appears that resistant bacteria may pose risk of infection to the people near the respirator wearer if the bacteria are reaerosolized from the respirator. However, reaerosolization of a very small fraction of collected bacteria can occur only under extreme conditions of violent sneezing or coughing.

Microbial dechlorination of hexachlorobenzene in anaerobic sewage sludge

From this study of the dechlorination of hexachlorobenzene (HCB) in municipal sewage sludge by an anaerobic mixed culture, we determined the dechlorination rate to be 0.29 mg/L/day under the optimal incubation conditions of pH M, 30 C temperature, and a concentration of total solids at 26.8 g/L. In addition, we found that dechlorination did not occur during agitation. Biotransformation occurred along a pathway of HCB → pentachlorobenzene (QCB) → 1,2,3,4tetrachlorobenzene (TecB) + 1,2,3,5-TeCB → 1,2,4-techlorobenzene (TCB) + 1,2,3-TCB + 1,3,5-TCB → 1,2-(dichlorobenzenc (DCB) + 1,4-DCB. Sequential dechlorination was observed within a substrate concentration range of 2–50 mg/L, but slowed down significantly at concentrations of 50 mg/L or higher. High-to-low dechlorination rates under three reducing conditions were identified as methanogenic conditions (0.30 mg/L/day) > sulfate-reducing conditions (0.12 mg/L/day) > denitrifying conditions (0.08 mg/L/day). 01998 Elsevier Science Ltd.

Regeneration and transformation via Agrobacterium tumefaciens of the strawberry cultivar Chandler

The effects of growth regulator balance and culture conditions on the morphogenetic response of leaf disks from greenhouse grown plants of the strawberry cultivar Chandler, have been studied. Best results were obtained in the presence of 2.46 μM IBA and 8.88 μM BA, where 47% of the cultures regenerated after 16 weeks with 2.9 shoot colonies per regenerating leaf disk. Optimum incubation conditions included two weeks in the dark with subsequent transfer to light (40 μmol m-2 s-1, 16 h). The regeneration protocol was also valuable when leaf disks from in vitro grown plants were used as explants. Transformation was attempted using Agrobacterium tumefaciens carrying the plasmid pBI121. Leaf disks from in vitro cultures proliferating in the presence of 2.21 μM kinetin were best explants for transformation. A 4.22% of inoculated explants showed kanamycin resistance after 16 weeks in a medium containing 25 mg l-1 of this antibiotic. The transgenic nature of several shoots was also confirmed by the GUS assay and PCR analysis.

Kinetics of formation of GlcNAc-GlcNAc-P-P-dolichol by microsomes from the retina of the embryonic chick.

Little quantitative information is available concerning individual reactions of the dolichol pathway. We have investigated the kinetics of the GlcNAc-transferase that catalyzes the biosynthesis of GlcNAc-GlcNAc-P-P-dolichol using chemically synthesized GlcNAc-P-P-dolichol as the substrate. Using microsomal preparations from the retina of the embryonic chick as enzyme source, optimal incubation conditions of pH, metal ion and detergent concentrations were established, after which apparent kinetic constants (Km and Vmax) were determined under initial rate conditions for GlcNAc-P-P-dolichol and UDP-GlcNAc. These studies provide the first quantitative description of the kinetics of this reaction.

Factors influencing bacterial attachment to the ruminal epithelium in vitro.

Some factors influencing bacterial attachment to the rumen epithelium were studied in vitro using mixed rumen bacteria (upper- or lower-layer bacteria formed at bacterial sediment by centrifugation), isolated from steers fed a roughage diet, and rumen epithelial cells collected from beef cattle given low-concentrate (50%; LC) and high-concentrate (90%; HC) diets. Optimal incubation conditions for bacterial attachment to rumen epithelial cells were 39 degrees C for 30 min. The bacteria isolated from the upper layer had a higher attaching activity to the LC epithelial cells than those of the lower layer. A higher degree of bacterial attachment was observed using the rumen epithelium from steers fed the LC diet rather than the HC diet (p<0.01). Ethylenediamine dihydroiodide (EDDI) added at 10 through 40 &mgr;g/ml increased bacterial attachment to the HC epithelial cells. Ammonia at 50 through 100 &mgr;g/ml positively affected bacterial attachment to both LC and HC epithelial cells. Bacterial attachment to the HC epithelial cells was enhanced (p<0.01) by the addition of a reducing agent (L-cysteine.HCl) but no increase was noted with LC cells. L- or D-lactate, volatile and unsaturated fatty acids markedly decreased bacterial attachment to rumen epithelial cells.

Rat renal cortical slices: Maintenance of viability and use in in vitro nephrotoxicity testing

The majority of in vitro toxicology studies employing precision-cut rat renal slices are carried out using simple incubation systems over relatively short periods of time (usually up to 8 hr). We aimed to develop a system for longer-term (up to 24 hr) culture of rat renal slices. The viability of precision-cut rat renal cortical slices was therefore investigated under a variety of incubation conditions. Slices cultured using a dynamic organ culture system were found to be more viable than those cultured in shaken multiwell dishes. This improved viability was evident after only 2 hr of culture (as assessed by the measurement of LDH leakage). An oxygen enriched gaseous environment was found to be important for the maintenance of slice viability using the dynamic organ culture system and conditions were established which permitted the culture of viable slices for periods up to 24 hr. Using these optimized incubation conditions, less than 8% LDH leakage was observed at 24 hr and slices retained 84% of fresh ATP values. Histologically, few signs of slice degeneration were noted until 20 hr of culture. The renal slice system was shown to discriminate between the most toxic and least toxic of a series of cephalosporin antibiotics, based on measurements of slice ATP content.

Recherche de bacteries thermophiles resistantes aux antibiotiques dans les bains traditionnels

Three thermophilic bacteria strains, designated strain BS1, BS2 and BS3, resistant to β-lactam antibiotics, and leaving at an optimal temperature for growth of about 50°C, were isolated from traditional baths in Meknes-city in Morocco. Physiological and biochemical studies showed that these organisms belong to Gram positive Bacilli. They could not be identified with the Bergey's Manuel of Systematic Bacteriology (1986). The dosage of β-lactamase during the exponential growth phase has revealed that the strain BS3 produces a maximal amount of this enzyme. Studies aimed at determining the optimal conditions for incubation and growth have been performed in order to optimize the excretion of β-lactamase by BS3 cells and thus facilitate the purification and and characterization of this enzyme.

Enhancement of the sensitivity of a chemiluminescent immunoassay for estradiol based on hapten heterology

Objective: To develop a highly sensitive chemiluminescent immunoassay (CLIA) that measures the serum estradiol (E2) levels in postmenopausal women. Methods: The previously developed competitive CLIA for E2 consisted of an immobilized antigen and a labeled antibody with an N-functionalized acridinium ester that was modified to enhance its sensitivity. The modifications were changing the immobilized antigen from E2 to its analogues with its expected effect on hapten heterology and selecting optimal incubation conditions. Results: The hapten heterology in which estriol was used as the immobilized antigen instead of E2 enhanced the sensitivity of the CLIA about 3-fold. A low incubation temperature and a long incubation time also effectively increased the sensitivity of the CLIA. The combination of these modifications enhanced sensitivity about 10-fold. The proposed CLIA with a 16 pmol/L detection limit, was about 5-fold more sensitive than commercially available immunoassay kits for E2. Conclusion: The proposed CLIA is sensitive enough to measure serum E2 levels in postmenopausal women.

Study of phenformin metabolism in rat liver microsomes by HPLC, CE and on-line HPLC-electrospray ionization mass spectrometry.

Methods for the analysis of phenformin and its metabolite by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLC-ESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4-hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and K(m) was 4 microM.

Luminol-and lucigenin-amplified chemiluminescence with rat liver microsomes: Kinetics and influence of ascorbic acid, glutathione, dimethylsulfoxide, N-t-butyl-a-phenyl-nitrone, copper-ions and a copper complex, catalase, superoxide dismutase, hexobarbital and aniline

For the investigation of luminol (LM)-and lucigenin (LC)-amplified chemiluminescence (CL) in rat liver microsomes using both a liquid-scintillation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basic kinetics have been established. Whereas calibration curves for both LM- and LC-CL are performed with hydrogenperoxide (LC quantum yield is 6.25 fold higher as that of LM), distinct differences were revealed with microsomes, indicating that different reactive oxygen species (ROS) are determined: Both LM- and LC-CL follow the kinetics of enzymatic reactions in terms of dependence on protein and NADPH or NADH concentration, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper ions and copper bound in a complex abolish CL, LC-CL being much more sensitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of pheno-barbital treated rats alone proved to be inactive in LM-and LC-CL production, whereas te combination 1:1 without and with addition of lipid was highly active in both LM-and LC-CL. Ascorbic acid and glutathione as scavengers diminish both LM- and LC-CL in concentrations higher then 10(5). Dimethyl-sulfoxide (DMSO) was ineffective in LM-CL up to concentrations of 0.2 M, the very high concentration of 2 M diminished LM-CL only to 1/3. LC-CL was diminished starting at concentrations of 100 mM and at 2 M only 10% of maximum LC-CL was observed. The trap substance N-t-butyl-a-phenylnitrone (BNP) also diminished LC-CL more effectively than LM-CL. Clearcut differences were revealed by the addition of catalase and superoxide dismutase: both enzymes diminished LM-CL only, without any influence on LC-CL. Hexobarbital, a potent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barely influenced. Aniline (without uncoupling capability) decreased both LM-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predominantly superoxide anion radicals, whereas LC-CL is mainly a measure for microsomal hydroxyl radical formation or of reactive organic radicals. With microsomes of phenobarbital and beta-naphthoflavone treated rats CL was much higher but in principle the same kinetic characteristics could be shown. All results on microsomes were obtained uniformly with the liquid scintillation counter and the Berthold luminometer, the letter being much more effective and more sensitive.

Somatic Embryogenesis from the Etiolated Petiole of Cyclamen(Cyclamen persicum Mill.).

The induction of somatic embryos from etiolated petioles was examined in five commercial cyclamen (Cyclamen persicum Mill.) cultivars. Somatic embryogenesis was only observed in ‘Anneke’. The optimal medium was Murashige and Skoog medium with 5.0μM 2, 4-D and 0.5μM kinetin, and the optimal incubation condition was complete dark at 25°C. A much higher percentage of explants forming embryoids and also larger number of embryoids were recorded in the explants from a third distal end of 5-10cm petioles than from other explants. This study suggests that somatic embryogenesis from etiolated petiole tissues was effectively utilized for the micropropagation of cyclamen.

Estrogen Receptors in the Stingray (Dasyatis akajei) Ultimobranchial Gland

On the basis of our previous observation that estrogen stimulates calcitonin secretion by the ultimobranchial gland in the stingray,Dasyatis akajei,experiments were conducted to examine the presence of estrogen receptor (ER) and its mRNA in the gland, employing exchange assay and Northern blot analysis, respectively. The optimal incubation conditions of the exchange assay for cytosolic ER were found to be 25° for 2 hr. Scatchard analysis of cytosolic ER revealed two components with dissociation constants (Kd) of 0.090 ± 0.015 and 17.780 ± 1.910 nMand with a maximum number of binding sites (Bmax) of 62.2 ± 7.9 and 2290 ± 180 fmol/mg protein, respectively. Furthermore, Sephadex G-100 filtration of the cytosol and immunoblot analysis using antiserum against a recombinant rat ER confirmed the presence of two specific binding components for estrogen (41 and 57 kDa). Total RNA extracted from the ultimobranchial gland was subjected to Northern blot analysis using a rat ER cDNA as a probe. Two positive signals were detected at 2.5 and 2.0 kb. Thus, the presence of ER was confirmed for the first time in nonmammalian calcitonin-secreting cells.

A method of culturing coronary artery explants for measuring vascular smooth muscle proliferation in rainbow trout

A standardized method of in vitro [3H]thymidine incorporation during the S phase of cell division was developed to study vascular smooth muscle proliferation in the coronary artery of rainbow trout (Oncorhynchus mykiss). We established a reliable medium system, an optimum [3H]thymidine dose, and optimum incubation conditions. Incorporation of the radiolabel into vascular smooth muscle nuclei was confirmed with autoradiography. To test the sensitivity of the assay system, the coronary artery was surgically exposed and gently rubbed in anesthetized rainbow trout. Following either a 1-, 2-, or 3-day recovery period, fish were sacrificed and the coronary artery explants were cultured with [3H]thymidine. Gentle rubbing of the coronary artery in vivo resulted in a significant increase in [3H]thymidine incorporation into the coronary artery explant compared with sham-operated and untreated control groups of fish. Peak incorporation of [3H]thymidine occurred at day 2 in the treated group, when incorporation was three times that in the sham-operated group. This technique has potential application in the study of coronary arteriosclerosis in salmonids, where vascular smooth muscle proliferation is a primary event.

Investigation of dry matter degradation and acidotic effect of some feedstuffs by means of in sacco and in vitro incubations

The dry matter degradation in the rumen and pH decrease of nine primary feedstuffs, three compounds feeds and three combinations of each two primary feedstuffs, were evaluated by means of in sacco and in vitro incubations. The ingredients included barley, wheat, manioc, maize, sorghum, sugar beet pulp, soya-bean meal, soya-bean hulls and maize gluten feed. The compound feeds differed in content and composition of the starch and sugars fraction, as well as in fibre content. The three combinations each consisted of a rapidly and slowly degradable feedstuff. The feedstuffs were incubated for different times to examine optimal incubation conditions for both estimating methods. For the in sacco technique, best predictions were obtained after 3 h of incubation. In this way, ingredients were ranked in order of decreasing dry matter degradation: manioc, wheat, barley, maize gluten feed, beet pulp, soya-bean meal, maize, sorghum, soya-bean hulls. With the in vitro method, better results were observed when rumen fluid was taken after rather than before feeding. After 5 h of incubation, ingredients were ranked in order of declining pH decrease: manioc, wheat, beet pulp, maize gluten feed, barley, maize, soyabean meal, sorghum, soya-bean hulls. Except for beet pulp, in vitro ranking agreed fairly well with in sacco results. The validity of these techniques was examined by limited results of in vivo experiments, where the effect of the nature of the compound feed on rumen fermentation was investigated. In vivo, a clear difference in risk for rumen disturbances could be observed between the three compound feeds. In sacco and in vitro, however, the compound feed with the high content of slowly degradable starch, could hardly be distinguished from the crude fibre rich compound feed. As only a limited amount of in vivo material was available, the estimating value of these methods require confirmation.