Higher incorporation of heterologous chicken immunoglobulin Y compared with homologous quail immunoglobulin Y into egg yolks of Japanese quail (Coturnix japonica).

In avian species, blood IgY is selectively incorporated into the yolks of maturing oocytes, although the precise mechanism is poorly understood. Our previous study showed that 22% of i.v.-injected heterologous chicken IgY (cIgY) was incorporated into egg yolks of Japanese quail (Coturnix japonica). However, it is not known whether homologous quail IgY (qIgY) can be more efficiently incorporated into quail egg yolks than cIgY. Therefore, we compared the uptakes of qIgY and cIgY i.v. administered into quail egg yolks and further characterized the uptakes of these 2 antibodies into quail ovarian follicles. Quail IgY and cIgY purified from the blood of the respective bird were labeled with digoxigenin, and their uptakes into quail egg yolks were determined by ELISA. Unexpectedly, total incorporation of the injected qIgY was only one-third of that of cIgY, although much more qIgY was left in blood compared with cIgY, suggesting that qIgY is the less preferable antibody as a transport ligand into quail egg yolks. On the other hand, deposition of the qIgY into heart, lung, liver, spleen, kidney, and ovarian follicular membrane was markedly higher than that of cIgY. Amino acid sequence analysis of 3 peptides derived from the trypsin-digested qIgY heavy chain revealed low homology between qIgY and cIgY. In conclusion, our results show that heterologous cIgY is more efficiently incorporated into quail egg yolks than homologous qIgY, possibly due to a distinctive antibody transport system existing in oocytes. The present results also may provide a new strategy for delivering useful proteinaceous substances into egg yolks in an attempt to produce designer eggs.

An improved method to illuminate and record a phytase assay in SDS-PAGE zymogram gels.

Experimentation at the Lethbridge Research Center in Alberta, Canada using cross-polarized transmitted light to photographically record a staining technique on zymograms has proved to be successful with both color and black and white films. It has been possible to obtain the desired visible contrast without compromising the intensity of the enzyme activity bands. Increasing numbers of such PAGE gels are being submitted for photographic recording when it is believed that the image will be used for records, publication, scientific posters or AV presentations.

Localization of phytase in Selenomonas ruminantium and Mitsuokella multiacidus by transmission electron microscopy.

The localization of phytase (myo-inositol-hexaphosphate phosphohydrolase) in the ruminal bacteria, Selenomonas ruminantium JY35 and Mitsuokella multiacidus 46/5(2), was determined with transmission electron microscopy. Phosphate produced from the enzymatic dephosphorylation of the calcium salt of phytic acid is precipitated as calcium phosphate. The calcium is then replaced with lead to produce electron-dense lead phosphate. This deposition of lead phosphate localized phytase in S. ruminantium JY35 and M. multiacidus 46/5(2) to the outer membrane, and confirmed intracellular expression of the enzyme in Escherichia coli pSrP.2, the recombinant clone which possesses the gene (phyA) encoding phytase (phyA) in S. ruminantium.

A novel staining method for detecting phytase activity.

Differential agar media for the detection of microbial phytase activity use the disappearance of precipitated calcium or sodium phytate as an indication of enzyme activity. When this technique was applied to the study of ruminal bacteria, it became apparent that the method was unable to differentiate between phytase activity and acid production. Strong positive reactions (zones of clearing around microbial colonies) observed for acid producing, anaerobic bacteria, such as Streptococcus bovis, were not corroborated by subsequent quantitative assays. Experimentation revealed that acidic solutions generated false positive results on the selected differential medium. Empirical studies undertaken to find a solution to this limitation determined the false positive results could be eliminated through a two step counterstaining treatment (cobalt chloride and ammonium molybdate/ammonium vanadate) which reprecipitates acid solubilized phytate. This report discusses the application of the developed two step counterstaining treatment for the screening of phytase producing ruminal bacteria as well as its use in phytase zymogram assays.

Phytase activity of anaerobic ruminal bacteria.

Phytase catalyses the release of phosphate from phytate (myo-inositol hexakisphosphate), the predominant form of phosphorus in cereal grains, oilseeds and legumes. The presence of phytase activity was investigated in 334 strains of 22 species of obligately anaerobic ruminal bacteria. Measurable activities were demonstrated in strains of Selenomonas ruminantium, Megasphaera elsdenii, Prevotella ruminicola, Mitsuokella multiacidus and Treponema spp. Strains isolated from fermentations with cereal grains proved to have high activity, and activity was particularly prevalent in S. ruminantium, with over 96% of the tested strains being positive. The measured phytase activity was found exclusively associated with the bacterial cells and was produced in the presence of approximately 14 mM phosphate. The most highly active strains were all S. ruminantium, with the exception of the one Mitsuokella multiacidus strain examined. Phytase activity varied greatly among positive strains but activities as high as 703 nmol phosphate released (ml culture)-1 were measured for a S. ruminantium strain and 387 nmol phosphate released (ml culture)-1 for the Mitsuokella multiacidus strain.

Microbial attachment and feed digestion in the rumen.

Direct microscopic examination of the rumen and its contents shows microbial populations largely attached to feed particles in the digesta. Most feeds contain a surface layer that is resistant to attachment and therefore to digestion. Infiltration of these recalcitrant epidermal layers through damage sites or through focused enzymatic attack is essential for initiation of the digestive process. Proliferation of primary colonizing cells produces glycocalyx-enclosed microcolonies. Secondary colonizers from the ruminal fluid associate with microcolonies, resulting in the formation of multispecies microbial biofilms. These metabolically related organisms associate with their preferred substrates and produce the myriad of enzymes necessary for the digestion of chemically and structurally complex plant tissues. Upon accessing the internal, enzyme-susceptible tissues, microbial "digestive consortia" attach to a variety of nutrients, including protein, cellulose, and starch and digest insoluble feed materials from the inside out. Substances that prevent microbial attachment or promote detachment (e.g., condensed tannins, methylcellulose) can completely inhibit cellulose digestion. As the microbial consortium matures and adapts to a particular type of feed, it becomes inherently stable and its participant microorganisms are notoriously difficult to manipulate due to the impenetrable nature of biofilms. Properties of feed that place constraints on microbial attachment and biofilm formation can have a profound effect on both the rate and extent of feed digestion in the rumen. Developments in feed processing (i.e., chemical and physical), plant breeding, and genetic engineering (both of ruminal microorganisms and plants) that overcome these constraints through the promotion of microbial attachment and biofilm formation could substantially benefit ruminant production.

Effect of condensed tannins from birdsfoot trefoil on endoglucanase activity and the digestion of cellulose filter paper by ruminal fungi.

The ruminal fungi Neocallimastix frontalis RE1, Neocallimastix patriciarum 27, Piromyces communis 22, and Orpinomyces joyonii 19-2 were examined for their ability to digest filter paper in the presence of condensed tannins from birdsfoot trefoil (Lotus corniculatus L.). For all four fungi, inhibition of endoglucanases was evident at 100 micrograms condensed tannins.mL-1 with nearly complete inhibition at 300 micrograms condensed tannins.mL-1. At 100 and 200 micrograms condensed tannins.mL-1, the endoglucanase activity of N. frontalis RE1 was greater (P < 0.01) than that of the other three fungal species. Exposure to 100 micrograms condensed tannins.mL-1 did not affect the ability of N. frontalis RE1 or N. patriciarum 27 to digest filter paper, and although digestion was reduced, N. frontalis RE1 and P. communis 22 solubilized more than 20% of the filter paper at 500 micrograms condensed tannins.mL-1. In contrast, O. joyonii 19-2 was virtually unable to digest filter paper at 300 micrograms condensed tannins.L-1. Mycelia of fungi grown with condensed tannins were covered by filamentous material, which may have arisen from the formation of condensed tannin-protein complexes. Less than 86% of the condensed tannins (as measured by the H2SO4 method) were recovered after 120 h of incubation with N. frontalis RE1, P. communis 22, and N. patriciarum 27. The need for detailed studies to examine the ability of ruminal fungi to metabolize condensed tannins is evident.

Comparison of amylolytic and proteolytic activities of ruminal fungi grown on cereal grains.

Strains of the ruminal fungi Neocallimastix patriciarum, Orpinomyces joyonii, and Piromyces communis were grown on cellobiose and on cereal grains and then examined for proteolytic and amylolytic activities. On cellobiose all three fungi displayed similar activities, with the exception of little amylolytic activity in the cell-associated fraction of N. patriciarum. Growth on the cereal grains barley, corn, and wheat showed differences in proteolytic and amylolytic activities amongst the ruminal fungi and between the cereal grains. The data suggest that while these fungi are capable of fermenting the cereal grains the mode of enzymatic attack varies both with the particular fungus and the type of cereal grain.

Effects of Condensed Tannins on Endoglucanase Activity and Filter Paper Digestion by Fibrobacter succinogenes S85.

The effect of condensed tannins from birdsfoot trefoil (Lotus corniculatus L.) on the cellulolytic rumen bacterium Fibrobacter succinogenes S85 was examined. Condensed tannins inhibited endoglucanase activity in the extracellular culture fluid, at concentrations as low as 25 mug ml. In contrast, cell-associated endoglucanase activity increased in concentrations of condensed tannins between 100 and 300 mug ml. Inhibition of endoglucanase activity in both the extracellular and the cell-associated fractions was virtually complete at 400 mug of condensed tannins ml. Despite the sharp decline in extracellular endoglucanase activity with increasing concentrations of condensed tannins, filter paper digestion declined only moderately between 0 and 200 mug of condensed tannins ml. However, at 300 mug ml, filter paper digestion was dramatically reduced and at 400 mug ml, almost no filter paper was digested. F. succinogenes S85 was seen to form digestive grooves on the surface of cellulose, and at 200 mug ml, digestive pits were formed which penetrated into the interior of cellulose fibers. Cells grown with condensed tannins (100 to 300 mug ml) possessed large amounts of surface material, and although this material may have been capsular carbohydrate, its osmiophilic nature suggested that it had arisen from the formation of tannin-protein complexes on the cell surface. The presence of electron-dense extracellular material suggested that similar complexes were formed with extracellular protein.

Cereal grain digestion by selected strains of ruminal fungi.

The ruminal fungi Orpinomyces joyonii strain 19-2, Neocallimastix patriciarum strain 27, and Piromyces communis strain 22 were examined for their ability to digest cereal starch. All strains digested corn starch more readily than barley or wheat starch. Orpinomyces joyonii 19-2 exhibited the greatest propensity to digest starch in wheat and barley, whereas the digestion of these starches by N. patriciarum 27 and P. communis 22 was limited. Media ammonia concentrations were lower when fungal growth was evident, suggesting that all strains assimilate ammonia. Fungi formed extensive rhizoidal systems on the endosperm of corn, but O. joyonii 19-2 was the only strain to form such systems on the endosperm of wheat and barley. All strains penetrated the protein matrix of corn but did not penetrate starch granules. Starch granules from all three cereals were pitted, evidence of extensive digestion by extracellular amylases produced by O. joyonii 19-2. Similar pitting was observed on the surface of corn starch granules digested by N. patriciarum 27 and P. communis 22, but not on wheat and barley starch granules. The ability of ruminal fungi to digest cereal grains depends on both the strain of fungus and the type of grain. The extent to which fungi digest cereal grain in the rumen remains to be determined.