Effect of tree leaves with or without urea as a feed supplement on nutrient digestion and nitrogen balance in sheep

Two nitrogen balance experiments in a 4 × 4 Latin square design were conducted to compare leaves of Grewia oppositifolia and Ziziphus mauritiana containing condensed tannins (CT) 0.08 g and 34.7 g per kg DM, respectively as supplement on feed intake, nutrients digestibility and nitrogen (N) retention in adult wethers and to examine the benefits of adding urea to Z. mauritiana leaves containing higher level of CT on N utilization in the animals. In experiment 1, the basal diet of oat hay was fed to sheep either un-supplemented or supplemented with 320 g dried leaves of Z. mauritiana or 320 g dried leaves of G. oppositifolia or 160 g dried leaves each of Z. mauritiana and G. oppositifolia. In vivo dry matter (DM) digestibility was not different among the four diets while N digestibility remained significantly lower (P<0.05) in Z. mauritiana supplemented diets. Daily intake of oat hay reduced (P<0.05) and that of total diet DM increased (P<0.001) with feeding of the leave supplements. Mean N retention on control diet was 4.39 g/d and increased to 7.51 g/d with inclusion of G. oppositifolia leaves but did not change with the other two supplements. In experiment 2, a basal diet of sorghum hay was fed ad libitum without any supplement (control diet) and the other three diets were supplemented with 320 g dried leaves of Z. mauritiana with no urea, 5 g urea/d or 10 g urea/d. Daily intake (g/d) of sorghum hay or total DM intake did not respond to inclusion of urea in the diets. Supplementation of Z. mauritiana with or without urea did not affect digestibility of DM, organic matter and acid detergent fiber. Nitrogen retention increased (P<0.05) from 0.57 g/d on control diet to 3.72 g/d with supplementation of Z. mauritiana leaves. Addition of urea 5 g/d did not further increase the N retention (4.78 g/d) but was significantly increased to 7.16 g/d in response to 10 g urea/d in the diet. It was concluded that response to urea feeding in the presence of tannin rich Z. mauritiana leaves was dose-dependant and that feeding 10 g urea/d increased the capacity of sheep to consume more feed and retain more N in the body.     

Effect of suplemental nitrogen source and feeding frequency on nutrient supply to lambs fed a kikuyu grass (Pennisetum clandestinum) hay-based diet

Eight castrated male lambs (35 ± 4 kg live weight), fed a basal diet of kikuyu grass hay, were used in a replicated 4 × 4 Latin Square experiment with a 2 × 2 factorial arrangement of treatments to evaluate the effect of supplemental feeding frequency and source of rumen degradable N on intake, digestibility, ruminal fermentation, and microbial protein yield. Treatments were supplementation with cassava meal plus calcium caseinate or cassava meal plus urea offered at a rate of 7 g/kg live weight daily in one or two meals per day. Lambs were fed twice daily in such manner to allow ad libitum comsumption of forage. There was significant feeding frequency by N source interaction on variables of intake. In general, intake of feed components was higher (P ≤ 0.05) by lambs offered the caseinate-supplement twice daily over intake observed in lambs given the others diet treatments. Digestibility of feed components was neither affected by supplemental N source (DM, P = 0.541; OM, P = 0.585; NDF, P = 0.828) nor by feeding frequency (DM, P = 0.122; OM, P = 0.175; NDF, P = 0.591). Urinary excretion of N increased (P ≤ 0.05) in lambs supplemented twice daily whereas N retention was similar for all treatments (N source, P = 0.748; feeding frequency, P = 0.418). Microbial protein entering into the small intestine was affected by the interaction between feeding frequency and N source such as an increasing (P < 0.10) in this variable was observed when lambs received the caseinate but not the urea supplement twice daily. Efficiency of microbial protein synthesis, however, was not affected by treatments (N source, P = 0.588; feeding frequency, P = 0.334). Rumen pH averaged 6.70 and it was neither affected by N source (P = 0.827) nor by feeding frequency (P = 0.740). Ruminal concentration of ammonia N was not affected by feeding frequency (P = 0.144) while it increased (P < 0.05) when urea rather than caseinate was the supplemental N source (mean of 7.61 mg/dl vs. 6.00 mg/dl). Concentration of sugars in rumen fluid was higher (P ≤ 0.05) in lambs supplemented once a day compared to twice daily (mean of 49.4 mg/dl vs. 34.4 mg/dl) for both N sources. A significant (P ≤ 0.05) N source by feeding frequency interaction effect was observed for ruminal concentrations of α-amino N compounds. In urea treatment α-amino N concentration increased (P ≤ 0.05) in lambs receiving the supplement twice daily compared to once a day (mean of 4.59 mg/dl vs. 3.70 mg/dl) while in caseinate treatment it was higher (P ≤ 0.05) in lambs offered the supplement in one meal per day compared to twice daily (mean of 5.29 mg/dl vs. 4.07 mg/dl). In conclusion, for ruminants fed a tropical grass-based diet, starch-rich supplement containing non-protein N as N source may be offered only once a day whereas the supply of nutrients may be improved if degradable true protein is included as N source and supplement is offered in two meals per day.     

In vivo and in situ measurements of the digestive characteristics of sainfoin in comparison with lucerne fed to sheep as fresh forages at two growth stages and as hay

In vivo and in situ digestive characteristics of sainfoin (Onobrychis viciifolia L., a tannin-rich forage) and lucerne (Medicago sativa L., a tannin-free forage) were compared to evaluate the effects of condensed tannins (CT) and growth stage (vegetative v. early flowering) in experiment 1. In experiment 2, the hays of the two forages, harvested at early flowering, were compared. Ingestibility, organic matter digestibility (OMD) and nitrogen (N) retention were measured in sheep fed sainfoin and lucerne fresh forages and hays. The loss of dry matter (DM) and N from polyester bags suspended in the rumen, abomasum and small intestine was also measured using rumen fistulated sheep and other intestine fistulated sheep. Nitrogen content was lower in sainfoin than in lucerne. Content of CT in sainfoin decreased with growth stage (3.5 to 2.5 g CT/kg DM) and was lower for sainfoin hay (0.6 g CT/kg DM). Ingestibility and OMD did not differ between fresh-fed forage species. Total N tract digestibility in vivo was much lower for sainfoin than for lucerne fresh forages (mean value 0.540 v. 0.721, P < 0.001) and for sainfoin hay than lucerne hay (0.464 v. 0.683, P < 0.001). In both species, N digestibility was not altered by growth stage. The rumen degradation of N was lower in sainfoin than in lucerne, resulting in a lower proportion of N intake excreted in urine. The intestinal digestibility of sainfoin was also lower than that of lucerne, resulting in a higher N excretion in faeces. Hence the efficiency of N utilisation by sheep (ENr) was similar (mean value 0.205 and 0.199 g N retained/g N intake for fresh sainfoin and lucerne, respectively). The coefficient of N retention by the animal was higher for sainfoin at the vegetative stage than for all the other forages. Nitrogen degradability in the rumen determined by the nylon bag technique (DegN) was lower for sainfoin than for lucerne when forages were studied both fresh (mean value 0.608 and 0.818, respectively) and as hays (0.631 and 0.767). The efficiency of forage N digestion (ENd) was higher for sainfoin at the vegetative stage. Compared with lucerne, sainfoin greatly increased the in situ estimate of forage N escaping the rumen but decreased its intestinal digestibility.     

Growth and embryo formation in wild-carrot suspension cultures with ammonium ion as a sole nitrogen source

Summary Wild-carrot (Daucus carota L.) suspension cultures grew and produced embryos on ammonium ion as a sole source of nitrogen in the absence of any exogenous Kreb's cycle acid when the pH of the medium was controlled by continuous titration with KOH or KHCO₃.     

Growth and embryo formation in wild-carrot suspension cultures with ammonium ion as a sole nitrogen source.

Wild-carrot (Daucus carota L.) suspension cultures grew and produced embryos on ammonium ion as a sole source of nitrogen in the absence of any exogenous Kreb's cycle acid when the pH of the medium was controlled by continuous titration with with KOH or KHCO3.     

In situ estimates of annual net nitrogen mineralization and nitrification in a subarctic watershed

Summary Annual estimates of surface soil nitrogen transformations were determined using an in situ method in four different subarctic vegetation types within a watershed in southwestern Alaska. The net nitrogen mineralization estimates were 22.5, 0.5, 4.7, and 2.7 kg-N ha^-1 yr^-1 for the alder, dry tundra, moist tundra, and white spruce sites, respectively. Only the soil from the alder site showed net nitrification (about 10 kg-N ha^-1 yr^-1). Annual inogranic nitrogen flux from the overlying organic layer to the mineral soil was almost seven times greater than net N production in the surface mineral soil in the alder site, indicating that the alder forest floor is potentially a substantial source for plant-available N. Rates of mobilization of N from the surface organic layers of the other sites were similar to net N production rates in surface mineral soils. In situ rates of N transformations showed a similar trend among sites as did laboratory estimates conducted in a previous study, suggesting a strong substrate control of N transformations in these soils.     

Isolation and characterization of a bacterium which utilizes polyester polyurethane as a sole carbon and nitrogen source

Abstract Various soil samples were screened for the presence of microorganisms which have the ability to degrade polyurethane compounds. Two strains with good polyurethane degrading activity were isolated. The more active strain was tentatively identified as Comamonas acidovorans . This strain could utilize polyester-type polyurethanes but not the polyether-type polyurethanes as sole carbon and nitrogen sources. Adipic acid and diethylene glycol were probably the main degradation products when polyurethane was supplied as a sole carbon and nitrogen source. When ammonium nitrate was used as nitrogen source, only diethylene glycol was detected after growth on polyurethane.     

In vitro and in vivo release of albumin using a biodegradable MPEG-PCL diblock copolymer as an in situ gel-forming carrier

An MPEG-PCL diblock copolymer was synthesized as an in situ gel carrier, and its phase transition behavior in aqueous solutions was examined. For comparison, aqueous solutions of Pluronic F-127, a widely used injectable gel-forming solution, were also studied. Both MPEG-PCL copolymer and Pluronic aqueous solutions were sols at room temperature. As the temperature was increased above room temperature, the diblock copolymer and Pluronic solutions underwent a sol-to-gel phase transition, which manifested as an increase in viscosity indicative of the formation of a gel. All of the copolymer solutions became gels at body temperature, although the gel viscosity increased with the increasing concentration of the MPEG-PCL diblock copolymer in the solution. In in vitro experiments, in which the gels were exposed to PBS, the MPEG-PCL gels maintained their structural integrity for more than 28 days, whereas the Pluronic gel disappeared within 2 days. The same results were observed when the polymer solutions were subcutaneously injected into rats. The MPEG-PCL gels maintained their structural integrity longer than 30 days, while the Pluronic gel could not be observed after 2 days. The ability of the gels as drug carriers was studied by measuring the release of fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) from MPEG-PCL diblock copolymer gels in vitro as well as in vivo. In vitro, BSA release was sustained above 20 days, with a greater release at lower diblock copolymer concentration; by contrast, Pluronic gels exhibited almost complete release of BSA-FITC within 1 day. When the BSA-FITC-loaded diblock copolymer and Pluronic solutions were subcutaneously injected into rats, they immediately transformed into a gel. In vivo, sustained release of BSA-FITC over 30 days was observed from the MPEG-PCL gel, whereas BSA-FITC release from the Pluronic gel ceased within 3 days. Collectively, the present findings show that MPEG-PCL diblock copolymer solutions are thermo-responsive and maintain their structural integrity under physiological conditions, indicating that they are suitable for use as injectable drug carriers.     

In situ proteomics with imaging mass spectrometry and principal component analysis in the Scrapper-knockout mouse brain

Imaging MS is emerging as a useful tool for proteomic analysis. We utilized this technique to analyze gene knockout (KO) mice in addition to traditional 2-DE analysis. The Scrapper-knockout (SCR-KO) mouse brain showed two types of neurodegenerative pathologies, the spongiform neurodegeneration and shrinkage of neuronal cells. 2-DE analysis of the whole brain lysates of SCR-KO mice indicated slight changes in annexin A6, Rap1 GTPase, and glyoxalase domain containing four spots while most of the main components did not show significant changes. By imaging MS analysis based on principal component analysis (PCA), we could find numerous alterations in the KO mouse brain. Furthermore, we could also know the information on the position of altered substances all together. PCA provides information about which molecules in tissue microdomains have altered and is helpful in analyzing large dataset of imaging MS, while exact identification of each molecule from peaks in MALDI imaging MS may require additional analyses such as MS/MS. Direct imaging with PCA is a powerful tool to perform in situ proteomics and will lead to novel findings. Our study shows that imaging MS yields information complementary to conventional 2-DE analysis.     

In vitro and early in vivo development of sheep gynogenones and putative androgenones

Genomic imprinting, where only one of the two parental genes is expressed, occurs in many phyla. In mammals, however, this phenomenon has been primarily studied in mice, and to a lesser extent, in humans. To understand how genomic imprinting may affect development in other species, particularly those with a different mode of placental development from mice and humans, 339 sheep zygotes were micromanipulated to contain either 2 large (presumptive male) or 2 small (presumptive female) pronuclei. One hundred and twenty-seven of these embryos and 86 manipulated and nonmanipulated control embryos were transferred to recipient ewes over 3 breeding seasons. Twenty-one control and 7 experimental conceptuses were recovered on day 21. Four of these conceptuses derived from zygotes with 2 small pronuclei were identified by karyotyping to be gynogenones (maternal-derived genome). While the gross morphology of the embryos appeared no different to those of normal controls, the extra-embryonic tissue from the conceptuses showed some hypertrophy and hypervascularization. Preliminary Northern blots of mRNA from allantoic and trophoblast tissue showed an overexpression of H19 and an underexpression of IGF2. Although the sheep gynogenetic phenotype contrasts with that seen in mice, these two genes appear to be similarly differentially expressed. Mol. Reprod. Dev. 50:154–162, 1998. © 1998 Wiley-Liss, Inc.     

Effect of ambient temperature on nutrient digestibility and nitrogen balance in sheep fed brown-midrib maize silage

The aim of the experiment was to determine the impact of heat stress on nutrient digestibility and nitrogen balance in sheep fed silages differing in fibre quality. The digestibility trial was conducted at three different ambient temperatures (15°C, 25°C and 35°C for 24 h/d). The tested brown-midrib maize (Bm) silage had a higher nutrient digestibility, except for ether extract (EE) and a higher metabolisable energy (ME) content than the control maize (Con) silage. Nitrogen (N) excretion with faeces was higher but N excretion with urine was lower for sheep fed Bm silage, subsequently N balance did not differ between the two silages. Temperature had no effect on nutrient digestibility, except for crude protein (CP), but N excretion with urine was lower at elevated temperatures. A diet by temperature interaction was found for dry matter (DM) and organic matter (OM) digestibility. When the ambient temperature increased from 15°C to 25°C, the DM and OM digestibility increased in animals fed Con silage, but decreased in animals fed Bm silage. Concomitantly, ME estimated from digestible nutrients was higher for Bm than for Con at 15°C, but no differences were found at 25°C and 35°C. Effects of diet by temperature interaction, furthermore, were observed for EE and CP digestibility. Therefore, forage quality has to be considered when feeding heat-stressed animals.     

In vivo leukocyte tropism of bovine leukemia virus in sheep and cattle.

Bovine leukemia virus (BLV), an oncovirus related to human T-cell leukemia virus type I, causes a B-cell lymphoproliferative syndrome in cattle, leading to an inversion of the T-cell/B-cell ratio and, more rarely, to a B-cell lymphosarcoma. Sheep are highly sensitive to BLV experimental infection and develop B-cell pathologies similar to those in cattle in 90% of the cases. BLV tropism for B cells has been well documented, but the infection of other cell populations may also be involved in the BLV-induced lymphoproliferative syndrome. We thus looked for BLV provirus in other leukocyte populations in sheep and cattle by using PCR. We found that while B cells harbor the highest proviral load, CD8+ T cells, monocytes, and granulocytes, but not CD4+ T cells, also bear BLV provirus. As previously described, we found that persistent lymphocytosis in cows is characterized by an expansion of the CD5+ B-cell subpopulation but we did not confirm this observation in sheep in which the expanded B-cell population expressed the CD11b marker. Nevertheless, BLV could be detected both in bovine CD5+ and CD5- B cells and in sheep CD11b+ and CD11b- B cells, indicating that the restricted BLV tropism for a specific B-cell subpopulation cannot explain its expansion encountered in BLV infection. Altogether, this work shows that BLV tropism in leukocytes is wider than previously thought. These results lead the way to further studies of cellular interactions among B cells and other leukocytes that may intervene in the development of the lymphoproliferative syndrome induced by BLV infection.     

In situ determination of nitrogen fixation in Antarctica using a high sensitivity portable gas chromatograph

A portable gas chromatograph was employed in the Vestfold Hills, Antarctica, during the austral summer of 1979-80 for determining nitrogenase activity of the blue-green alga Nostoc commune Vaucher by the acetylene reduction assay. Acetylene reduction was measured in samples taken along a transect where the vegetation changed with respect to differing topography and water availability. Submerged colonies of Nostoc recorded the highest fixation rates (6.89 nmol C₂H₄. cm^-2 h^-1). Damp mosscyanophyte associations growing on shallow slopes showed moderate rates of acetylene reduction (1.99 nmol C₂H₄. cm^-2 h^-1) whilst the drier vegetation of the steeper terrain was the least active (0.19 nmol C₂H₄. cm^-2 h^-1. The employment of a high sensitivity portable gas chromatograph provided an accurate and reliable method of measuring acetylene reduction.     

Nitrate as a sole nitrogen source forMethanococcus thermolithotrophicus and its effect on growth of several methanogenic bacteria

Methanococcus (Mc.) thermolithotrophicus can use nitrate as the sole source of nitrogen, but four other species of methanogens cannot. The growth rate was similar on both nitrate and ammonium, but yields were 20–25% lower on nitrate.Mc. thermolithotrophicus, Methanobacterium thermoautotrophicum, andMethanobrevibacterium smithii were not inhibited by 20 mM nitrate, butMethanospirillum hungatei was inhibited 35%, andMethanosarcina barkeri was completely inhibited by 20 mM nitrate. WhenMc. thermolithotrophicus was growing with nitrate as the sole source of nitrogen, growth was dependent on either molybdenum or tungsten, and the presence of both gave the best growth response; vanadium or chromium did not replace the requirement for these metals. Growth on ammonium could not be strictly demonstrated to require either of these metals, but both molybdenum and tungsten stimulated growth.     

Utilization of chymotrypsin as a sole carbon and (or) nitrogen source by Escherichia coli.

alpha-Chymotrypsin serves as a sole carbon source, sole nitrogen source, and as sole carbon plus nitrogen source for wild-type Escherichia coli in a totally defined medium. Hence, a mammalian host for E. coli may supply the necessary carbon and nitrogen nutrients for the microorganism. Growth is most rapid when chymotrypsin is a sole nitrogen source and least rapid with chymotrypsin as a carbon source. The approximate doubling times for E. coli utilizing chymotrypsin as a nitrogen source, carbon plus nitrogen source, and carbon source are 1.6, 4.6, and 11.3 h, respectively. The activity of the residual enzyme in the culture supernates falls off asymptotically over the source of time, as followed by cleavage of glutaryl-L-phenylalanine-p-nitroanilide. Chymotrypsin hydrolyzes succinyl-L-ala-L-ala-p-nitroanilide, the elastase substrate, to some extent. Peptidases do not appear to be secreted that hydrolyze such model substrates as benzoyl-DL-arginine-p-nitroanilide, the tryptic and cathepsin B substrate, L-leucine-p-nitroanilide, the leucine amino-peptidase substrate, or L-lysine-p-nitroanilide, the aminopeptidase B substrate. Growth of E. coli is generally directly related to the loss of chymotryptic activity in the medium. Hence, autolysis of chymotrypsin, i.e., self-degradation, is an important factor for the availability of degradation products of the enzyme to the bacterium for growth purposes. Accordingly, the degradation of a host protein by autolysis presents an opportunity for E. coli to survive during periods of host nutritional crisis by utilization of the degradation peptides that are produced during autolysis.     

In vitro and in vivo developmental capacity of oocytes from prepubertal and adult sheep

Development to the blastocyst stage and survival following embryo transfer were assessed for oocytes obtained from prepubertal and adult sheep matured and fertilized in vitro. The rates of maturation, fertilization and cleavage in vitro did not differ significantly between oocytes from prepubertal and adult sheep. The proportion of cleaved zygotes reaching the blastocyst stage was significantly lower for oocytes derived from prepubertal than for those from adult sheep (15.4% and 34.1% respectively). There were no differences in the pregnancy rate and number of lambs born following transfer of blastocyst stage embryos derived from prepubertal and adult sheep to adult recipients. These data show that embryos derived from prepubertal lamb oocytes have reduced developmental potential in vitro but, of those which do reach the blastocyst stage, they have equal capacity to develop to term as embryos derived from adult sheep.     

Effect of supplementing sheep diets with macroalgae species on in vivo nutrient digestibility,rumen fermentation and blood amino acid profile

In this study, a brown macroalgae species, Saccharina latissima, processed to increase its protein concentration, and a red macroalgae species, Porphyra spp., were used to evaluate their in vivo digestibility, rumen fermentation and blood amino acid concentrations. Four castrated rams were used, whose diets were supplemented with a protein-rich fraction of S. latissima, a commercial Porphyra spp. and soybean meal (SBM). Our results show that the protein digestibility of a diet with S. latissima extract was lower (0.55) than those with Porphyra spp. (0.64) and SBM (0.66). In spite of the higher nitrogen (N) intake of diets containing Porphyra spp. and SBM (20.9 and 19.8 g N/day, respectively) than that with S. latissima (18.6 g N/day), the ratio of N excreted in faeces to total N intake was significantly higher in the diet with S. latissima than those with Porphyra spp. and SBM. This reflects that the utilization of protein in S. latissima was impaired, possibly due to reduced microbial activity. The latter statement is corroborated by lower volatile fatty acid composition (25.6, 54.8 and 100 mmol/l for S. latissima, Porphyra spp. and SBM, respectively) and a non-significant tendency for lower ammonia concentration observed in diets with S. latissima and Porphyra spp. compared to SBM. It is important to note that the S. latissima used in this trial was rinsed during processing to remove salt. This process potentially also removes other water-soluble compounds, such as free amino acids, and may have increased the relative fraction of protein resistant to rumen degradation and intestinal absorption. Furthermore, the phlorotannins present in macroalgae may have formed complexes with protein and fibre, further limiting their degradability in rumen and absorption in small intestines. We recommend that further studies explore the extent to which processing of macroalgae affects its nutritive properties and rumen degradability, in addition to studies to measure the intestinal absorption of these macroalgae species.