Cell dynamics during cocoon secretion in the aquatic leech, Theromyzon tessulatum (Annelida: Clitellata: Glossiphoniidae).

One distinguishing feature of clitellate annelids is the presence of specialized segments comprising the clitellum, whose primary function is to secrete a cocoon. Using histological analyses, we have documented cell types (I-V) and cellular processes associated with cocoon secretion in the aquatic leech, Theromyzon tessulatum. Our data indicate that the bulk of the cocoon's biomass arises from precursor cells of a single type that hypertrophy and proliferate approximately 1 week prior to egg laying, and then differentiate into either of two cell types (i.e., Type II or Type III) depending on their position within the clitellum. Type II cells are concentrated along the lateral edges and venter of the clitellum and secrete alcian blue-staining granules that form opercula (i.e., glue-like material that seals both cocoon ends), while Type III cells populate the dorsal midline and secrete azocarmine-staining granules that build the cocoon wall. Both cell types occupy spaces between deep muscle layers and extend long-neck tubules to the surface epithelium as they fill with granules a few days prior to egg laying. Other cell types appear to make minor contributions to the cocoon (e.g., Type I, Type IV) or have supporting or signaling roles (e.g., Type V). Our observations suggest that post-translational modification (i.e., glycosylation) of the same core protein(s) distinguishes the granules of Type II/III cells, and that the default state of the Type II/III precursor may be evolutionarily linked to secretory cells in basal polychaetes.

Morphologic characterization of the ice worm Mesenchytraeus solifugus.

Ice worms occupy a unique position in metazoan phylogeny in that they are the only known annelid that completes its life cycle in ice. The mechanism(s) associated with this adaptation are likely to occur at different levels, ranging from modification of their metabolism to changes in morphology. In this study, we examined specimens of Mesenchytraeus solifugus by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in an effort to identify morphologic structures that may aid in its glacial habitation. We report that M. solifugus contains an elongated head pore at the tip of its prostomium, numerous sensory structures, and differentially oriented setae that curve abruptly at their distal end.

Segmentation of the central nervous system in leech.

Central nervous system (CNS) in leech comprises segmentally iterated progeny derived from five embryonic lineages (M, N, O, P and Q). Segmentation of the leech CNS is characterized by the formation of a series of transverse fissures that subdivide initially continuous columns of segmental founder cells in the N lineage into distinct ganglionic primordia. We have examined the relationship between the N lineage cells that separate to form the fissures and lateral ectodermal and mesodermal derivatives by differentially labeling cells with intracellular lineage tracers and antibodies. Although subsets of both lateral ectoderm and muscle fibers contact N lineage cells at or near the time of fissure formation, ablation experiments suggest that these contacts are not required for initiating fissure formation. It appears, therefore, that this aspect of segmentation occurs autonomously within the N lineage. To support this idea, we present evidence that fundamental differences exist between alternating ganglionic precursor cells (nf and ns primary blast cells) within the N lineage. Specifically, ablation of an nf primary blast cell sometimes resulted in the fusion of ipsilateral hemi-ganglia, while ablation of an ns primary blast cell often caused a 'slippage' of blast cells posterior to the lesion. Also, differences in cell behavior were observed in biochemically arrested nf and ns primary blast cells. Collectively, these results lead to a model of segmentation in the leech CNS that is based upon differences in cell adhesion and/or cell motility between the alternating nf and ns primary blast cells. We note that the segmentation processes described here occur well prior to the expression of the leech engrailed-class gene in the N lineage.

Evaluation of nitrogen and organic matter balance in the feedlot as affected by level and source of dietary fiber.

A trial was conducted to determine the effect of level and source of dietary fiber on N and OM excretion by cattle on finishing diets. One hundred twenty steers were stratified by weight and allotted to one of the following treatments: 7.5% roughage (7.5% R), wet corn gluten feed (WCGF; 41.5% of dietary DM), and all-concentrate (All Con) diet. Cattle were fed for 87 d during the summer with 23.7 m2 of pen area per animal. Steers fed the WCGF diet had heavier final weights, greater DMI, and higher ADG (P < .01) than the 7.5% R and All Con treatments. Steers fed All Con had lower (P < .01) DMI than the other two treatments. Nitrogen and OM mass balances in the feedlot were quantified. Main components were nutrient input, retention, and excretion. Nitrogen and OM intake of steers fed WCGF were greater (P < .05) than those of steers fed the other treatments. The WCGF treatment had a greater percentage of fecal N output (P < .05). The All Con treatment had a greater (P < .01) percentage of urinary N than WCGF and 7.5% R diets. Steers fed the WCGF treatment excreted more (P < .01) OM compared with the other treatments, which led to more N and OM being removed in manure at cleaning. The All Con treatment had more (P < .01) N and OM in runoff than the other treatments. Nutrition can change site of fermentation, which affects the composition of excreted material; however, total amount of N excreted may be more important than route of excretion in decreasing N losses to the environment and maximizing recovery in manure.

The effect of forage source and particle size on finishing yearling steer performance and ruminal metabolism.

Two finishing trials and a metabolism trial were conducted to evaluate the effect of forage source and particle size in dry-rolled corn finishing diets. In Exp. 1, 224 crossbred yearling steers (BW = 342+/-11 kg) were used in a randomized complete block design consisting of seven treatments. Treatments were an all-concentrate diet or diets containing equal NDF levels provided by alfalfa hay or wheat straw (three treatments each) with each forage source ground to pass through a .95-, 7.6-, or 12.7-cm screen. Steers fed diets containing forage had greater (P < .05) DMI than steers fed an all-concentrate diet. Steers fed alfalfa diets gained faster (P < .05) with a greater (P < .05) concentrate efficiency than steers fed either all-concentrate or straw diets. In Exp. 2, 120 crossbred yearling steers (BW = 307+/-2 kg) were used in a completely randomized design and fed dry-rolled corn diets containing 10% alfalfa ground to pass through either a .95- or 7.6-cm screen. Alfalfa particle size had no effect on performance or carcass measurements. In Exp. 3, six ruminally fistulated steers (BW = 508+/-34 kg) were used in a 6 x 6 Latin square design and fed an all-concentrate diet or diets containing equal NDF levels provided by alfalfa hay, wheat straw, or ground corncobs with alfalfa and straw ground to pass through either a 2.54- or 12.7-cm screen. Steers fed straw diets spent more time (P < .10) chewing than those receiving the other diets. In conclusion, forage particle size had no effect on finishing cattle performance or ruminal metabolism data. However, cattle consuming different forage sources in dry-rolled corn finishing diets may not respond similarly in animal performance.

Gangliogenesis in leech: morphogenetic processes leading to segmentation in the central nervous system.

Using intracellular lineage tracers to study the main neurogenic lineage (N lineage) of the glossiphoniid leech embryo, we have characterized events leading from continuous columns of segmental founder cells (nf and ns primary blast cells) to discrete, segmentally iterated ganglia. The separation between prospective ganglia was first evident as a fissure between the posterior boundary of nf- and the anterior boundary of ns-derived progeny. We also identified the sublineages of nf-derived cells that contribute parallel stripes of cells to each segment. These stripes of cells project ventrolaterally from the dorsolateral margin of each nascent ganglion to the ventral body wall. The position and orientation of the stripes suggests that they play a role in forming the posterior segmental nerve; they are not coincident with the ganglionic boundary, and they form well after the separation of ganglionic primordia. Previous work has shown that cells in the anterior stripe express the leech engrailed-class gene. Thus, in contrast to the role of cells expressing engrailed in Drosophila, the stripes of N-derived cells expressing an engrailed-class gene in leech do not seem to play a direct role in segmentation or segment polarity.

Effect of energy source and escape protein on receiving and finishing performance and health of calves.

Two receiving and two finishing trials evaluated energy source and escape protein supplementation for calves. In receiving Trial 1, 398 calves (257 +/- 24 kg BW) were used in a 2 x 2 factorial arrangement of treatments. Energy sources were dry-rolled corn (DRC) and wet corn gluten feed (WCGF); each was fed without or with supplemental escape protein (EP). Calves fed WCGF gained slower (P < .05) and consumed less DM (P < .01) than calves fed DRC. Feed efficiency improved (P < .10) with EP supplementation. In finishing Trial 1, 240 calves (305 +/- 21 kg BW) were used. The arrangement of treatments was the same as in receiving Trial 1. Calves fed DRC/WCGF tended (P = .15) to be more efficient and consumed less DM (P < .05) than calves fed DRC. In receiving Trial 2, 315 calves (252 +/- 23 kg BW) were fed diets similar to those fed in receiving Trial 1. Calves fed WCGF consumed less DM (P < .01), gained similarly (P > .15), and were more efficient (P < .10) than calves fed DRC. In finishing Trial 2, 320 calves (298 +/- 23 kg BW) were fed diets containing DRC, DRC/WCGF, high-moisture corn (HMC), HMC/WCGF, and DRC/HMC; each was fed without or with supplemental EP. An energy source x protein supplement interaction was detected for gain (P < .05) and efficiency (P < .01). Results suggest that WCGF has a NEg greater than DRC in receiving diets and a NEg similar to that of DRC but lower than that of HMC in finishing diets.

Transcription initiation from a poly(dA) tract.

The mammalian ME1 gene encodes a non-tissue-specific, helix-loop-helix transcription factor that is enriched in morphogenetically active regions during development. Regulation of mouse ME1 gene expression is controlled by a novel initiator (ME1 Inr) that promotes transcription from the center of a 13 bp poly(dA) tract. We show here that the ME1 Inr autonomously directs initiation from the poly(dA) tract both in vitro and in vivo. This transcription was dependent upon two protein complexes; MBPalpha, which associated directly with the poly(dA) tract, and MBPbeta, which introduced an approximately 60 degrees bend immediately downstream of the poly(dA) tract. The MBPalpha and MBPbeta binding sites were strikingly conserved in homologous DNA from several mammalian species and the frog Xenopus laevis. These results suggest that the ME1 Inr constitutes a robust nucleation site that promotes transcription initiation in the absence of conventional promoter elements.

Effect of wet corn gluten feed, supplemental protein, and tallow on steer finishing performance.

Two trials evaluated the effects of level of wet corn gluten feed (WCGF), type of supplemental protein, and supplemental tallow on steer finishing performance. In Trial 1, WCGF was fed at 0, 25 (two diets), or 50% of the dietary DM replacing dry-rolled corn (DRC), molasses, and a portion of the supplement. The DRC control diet and one 25% WCGF diet were supplemented with a combination of protein sources. The second 25% WCGF diet was supplemented with urea alone. The 50% WCGF diet contained no additional protein supplementation. No differences in DMI (P > .10) were observed. Calves fed 25% WCGF plus a combination of protein sources or 50% WCGF gained faster (P < .10) and more efficiently (P < .10) than calves fed the DRC control. Calves fed 25% WCGF plus urea gained faster (P < .10) and tended (P = .14) to be more efficient than calves fed the DRC control. In Trial 2, WCGF was fed to replace 0 or 50% of the DRC and the molasses-urea supplement (DM basis). Both diets were fed with or without 3% tallow. Steers fed WCGF gained faster (P < .01) and more efficiently (P < .01) than steers fed DRC. Inclusion of 3% tallow increased gain (P < .05) and improved efficiency (P < .05). Feed efficiency is improved by the addition of WCGF or tallow to DRC finishing diets.

Effect of degradable intake protein level on finishing cattle performance and ruminal metabolism.

Two finishing trials and a metabolism trial were conducted to evaluate level of supplemental degradable intake (crude) protein (DIP) in finishing diets on cattle performance, carcass characteristics, and ruminal metabolism. Finishing trials were conducted in two consecutive years using 128 crossbred yearling steers (BW = 343+/-5 kg, Trial 1) and 176 crossbred yearling steers (BW = 375+/-4 kg, Trial 2) in a randomized complete block design. Steers were fed dry-rolled corn diets containing urea at 0, .88, 1.34, or 1.96% (DM basis). No differences in DMI, daily gain, or feed efficiency were noted among steers receiving diets containing supplemental urea. However, steers fed diets supplemented with urea were 5.4% more efficient (P < .01) and gained 6.6% faster (P < .01) than steers receiving no supplemental urea. Metabolizable protein (MP) content of all diets exceeded the steers' requirements. However, diets containing no urea were deficient in DIP. In the metabolism trial, four ruminally fistulated steers (BW = 380+/-22 kg) were used in a 4 x 4 Latin square design and fed (ad libitum) diets similar to those used in the finishing trials. Nitrogen intake and ruminal ammonia N concentration increased linearly (P < .05) with increasing level of urea supplementation. Diets containing no supplemental urea were calculated to be deficient in DIP, resulting in reduced bacterial synthesis. Results indicate that dry-rolled corn finishing diets containing no supplemental N are deficient in ruminally degradable N. Supplementing these diets with an inexpensive source of ruminally degradable N improved animal performance. However, supplementation with urea above .88% was not beneficial.

Selective conservation of an E-protein gene promoter during vertebrate evolution.

The murine E-protein gene ME1 encodes a non-tissue-specific, helix-loop-helix transcription factor that is associated with morphological development. ME1 gene expression is regulated by a TATA-less promoter that contains multiple Sp1 consensus elements, E-boxes, and a novel transcription initiation site. In this study, we compared DNA homologous to the ME1 promoter from vertebrate species ranging from frog to human. A region of striking sequence similarity was identified in a region corresponding to the ME1 transcription initiation site (ME1 Inr). Within this region, a poly d(A) tract and a 9-bp inverted repeat (5'-GTCCGCCTG) were highly conserved in all species that were examined. Protein complexes that recognized these DNA elements were present among distant vertebrates (frog, chick, monkey and human), and were able to bend the ME1 Inr to a similar extent (approximately 60 degrees) as the previously described murine MBP alpha and MBP beta proteins. Collectively, these results suggest that an ME1 Inr-like element and its associated proteins functioned in an ancestral vertebrate more than 350 million years ago.

Embryonic expression and evolution of duplicated E-protein genes in Xenopus laevis: parallels with ancestral E-protein genes.

E-proteins comprise a subfamily of helix-loop-helix transcription factors that have been identified in arthropods and several chordate taxa. In mammals, there are three classes of E-protein genes (E2A, E2-2, and HEB) that encode related, and often interchangeable, gene products. We have determined that the clawed frog Xenopus laevis contains twice the number of transcriptionally active E-protein genes when compared with other vertebrate species. Based upon genomic Southern blots and nucleotide sequence comparisons, it is likely that the additional X. laevis genes arose from tetraploidization. During embryogenesis, XE2A (homologue of mammalian E2A) transcripts were broadly expressed in anterior and posterior regions of the embryo while homologues of E2-2 (XE2.2) and HEB (XE1.2) appeared in vertebrate-specific structures including the pineal gland, olfactory bulb, and brachial arches. A phylogenetic analysis of these genes and other known metazoan E-proteins suggests that there were two periods of marked E-protein gene expansion; one that predated the radiation of vertebrates, and the other that coincided with Xenopus tetraploidization. Both of these periods were characterized by the rapid evolution of E2-2 and HEB-class genes, but not of E2A. We propose that the former genes acquired new or specialized roles during early chordate evolution and also more recently in Xenopus, as reflected by the stereotypic expression patterns of these genes during X. laevis development.

Evaluation of wet distillers composite for finishing ruminants.

Two trials evaluated the effect of a composite of feed ingredients formulated to be similar in nutrient composition to wet distillers byproducts on finishing performance of sheep and cattle. Trial 1 used 60 crossbred lambs (31 kg) assigned to one of four treatments: dry-rolled corn (DRC) control, dried distillers grains plus solubles, wet corn gluten feed (WCGF), and wet distillers grains composite (COMP1). The COMP1 consisted (DM basis) of 47.5 WCGF, 11.9% condensed distillers solubles, 30.5% corn gluten meal, 9.7% tallow, and .4% dicalcium phosphate and was fed at 40% of the diet DM. Lambs fed the COMP1 diet were 27% more efficient (P < .10) than lambs fed WCGF and 12% more efficient (P > .10) than lambs fed DRC. In Trial 2, 60 yearling crossbred steers (272 kg) were assigned to one of five treatments: DRC control, WCGF, wet distillers grains composite (COMP2), COMP2 minus tallow (-FAT), or COMP2 minus corn gluten meal (-CGM). the COMP2 consisted (DM basis) of 65.7% WCGF, 26.3% corn gluten meal, and 8.0% tallow and was fed at 40% of the diet DM. Steers fed COMP2 were more efficient (P < .10) than steers fed DRC or WCGF, and the steers fed -FAT and -CGM were intermediate to these three dietary treatments. A composite diet of WCGF, condensed distillers solubles, corn gluten meal, and tallow, formulated to be similar in nutrient composition to wet distillers byproducts, may improve feed efficiency compared with WCGF or DRC.

Evaluation of corn and sorghum distillers byproducts.

Two trials were conducted to determine the feeding value of sorghum distillers byproducts. Trial 1, a finishing trial, used 160 yearling steers (327 kg). Treatments consisted of dry-rolled corn (DRC) control, sorghum wet distillers grains (SWDG), sorghum wet distillers grains plus solubles (SWDGS), and sorghum dried distillers grain plus solubles (SDDGS). Distillers byproducts were fed at 40% of the diet DM. Cattle fed diets containing SWDG, SWDGS, or DRC were similar in efficiency of gain (P > .10); cattle fed SDDGS were less efficient (P < .10) than all other treatments. Sorghum wet distillers grains, SWDGS, and SDDGS contained 96, 102, and 80% relative NEg of corn, respectively. In Trial 2, 16 crossbred lambs (55 kg) were used to determine the digestibility of sorghum and corn distillers byproducts. Byproducts were fed at 80% of the diet DM and treatments consisted of corn wet distillers grains (CWDG), corn dried distillers grains plus solubles (CDDGS), SWDG, and SDDGS. Neutral detergent fiber digestibility was not different among treatments (P > .10). Corn wet distillers grains were higher in true nitrogen (P < .001), apparent nitrogen (P < .01), and organic matter digestibility (P < .05) than SWDG. Wet distillers byproducts were higher (P < .01) in apparent organic matter and nitrogen digestibility than dried distillers byproducts. Digestibility of distillers byproducts and subsequent energy values are influenced by type of grain used in the fermentation process and drying of the finished byproduct.

Limiting amino acids in meat and bone and poultry by-product meals.

In situ, digestion, and growth studies were conducted to evaluate four meat and bone meals and six poultry by-product meals as sources of escape protein and to predict the first-limiting amino acid for growing calves. Escape protein values, determined by 12-h in situ incubation, ranged from 41.7 to 51.0% of CP for meat and bone meals; poultry by-product meals ranged from 32.0 to 39.8%. True protein digestion in the gastrointestinal tract of lambs differed among protein sources (P < .05), ranging from 79 to 95%. In each of three growth trials, 60 steers (258 +/- 24, 241 +/- 23, and 230 +/- 16 kg for Trials 1, 2, and 3, respectively) were supplemented with 4 of the 10 protein sources along with a urea supplement. Protein sources were fed at 30, 40, 50, and 60% of the supplemental CP, with urea supplying the remainder. Protein efficiency differed among treatments ( P < .10), ranging from .61 to 1.55. Amino acid composition was determined for each protein source, and the individual metabolizable amino acids were regressed on the protein efficiency values. Escape protein values were correlated (R2 = .75) with protein efficiency but had a negative slope. Metabolizable methionine was the only amino acid moderately correlated (R2 = .40, slope = 1.9) to protein efficiency, whereas other amino acids either correlated poorly or had negative slopes. These data indicate that the protein value of meat and bone meal and poultry by-product meal is limited by the amount of metabolizable methionine they contain.

Sodium dodecyl sulfate (SDS)-based whole-mount in situ hybridization of Xenopus laevis embryos.

We describe a streamlined whole-mount in situ hybridization protocol that utilizes high concentrations of the detergent sodium dodecyl sulfate (SDS). Our results suggest that SDS is an effective blocking agent in Xenopus laevis embryos which permeabilizes membranes without disrupting morphology. Consequently, riboprobes appeared to disperse uniformly within the embryo and several arduous and/or laborious steps of conventional procedures could be eliminated without compromising the technique.

Characterization of a single-locus minisatellite DNA in Xenopus laevis.

We have cloned a minisatellite tandem array (XTA) from Xenopus laevis that contains approximately 200 copies of the 20-bp repeat 5'-CCAACAGCCTGCCCATCCAT-3'. The XTA sequence is present only once per haploid genome and is polymorphic with respect to repeat number and location of flanking restriction endonuclease sites. Although the 20-bp repeat has not previously been described, flanking sequences suggest that it lies proximal to coding regions in the Xenopus genome.

Identification of non-tissue-specific helix-loop-helix genes in Xenopus laevis.

We have isolated four non-tissue-specific helix-loop-helix (HLH) transcription factors from Xenopus laevis (Xl). While some are clearly orthologous to known mammalian HLH proteins, others have dramatic amino-acid differences in otherwise highly conserved protein domains. We propose that these changes arose following the tetraploidization of Xl approx. 30 Myr ago.

Cloning and embryonic expression of Xenopus laevis GAP-43 (XGAP-43).

Xenopus laevis GAP-43 (XGAP-43) is highly related to other vertebrate GAP-43 proteins in its N-terminal region which contains a membrane-targeting sequence, serine phosphorylation site, and calmodulin binding domain. Unlike other species examined, however, there appear to be two GAP-43-class genes in X. laevis which resulted from the genome duplication in Xenopus approximately 30 million years ago. During embryogenesis, XGAP-43 is expressed in a complex spatiotemporal pattern that is consistent with its putative role in neuronal growth and development.

Influence of grain type, tallow level, and tallow feeding system on feedlot cattle performance.

Two experiments were conducted to evaluate the effects of grain type, tallow level, and tallow feeding system on finishing steer performance. Experiment 1 involved 256 yearling steers (359 kg) in a 4 x 2 factorial arrangement of treatments. Steers were assigned randomly to one of four tallow feeding systems: 1) 0% tallow fed throughout the experiment; 2) 4% tallow fed throughout the experiment; 3) 0% tallow fed d 1 through 33 and then 4% tallow fed until slaughter; and 4) 4% tallow fed d 1 through 33 and then 0% tallow fed until slaughter. Tallow treatments were applied to diets containing either dry-rolled corn (DRC) of high-moisture corn (HMC). No fat treatment x grain type interaction (P > .10) was observed. Steers fed 4% tallow throughout the experiment, only during d 1 through 33, or only during d 34 until slaughter were more (P < .10) efficient than steers fed 0% tallow. No differences in DMI or ADG were observed (P > .10). In Exp. 2, 120 large-framed steer calves (286 kg) were blocked by weight and allotted randomly within block to one of three treatments consisting of the addition fo 0, 2, or 4% tallow added d 1 and fed for 197 d. Feed efficiency of calves increased linearly (P < .05) with increasing tallow level. Daily gain was not different (P > .10), but DMI decreased linearly (P < .05) with increasing tallow level. This research indicates that tallow added during or after grain adaptation to DRC- or HMC-based diets fed to yearling steers will result in similar improvement in feed efficiency, and that including up to 4% tallow to diets fed to large-framed calves can significantly improve feed efficiency.