A review of the use of isotopic and nuclear techniques in animal production

Isotopic and nuclear techniques play an important role in food and agriculture, health and industry. This paper discusses the use of these techniques and highlights potential for their use in the area of Animal Production. These techniques are discussed in two parts: (1) Isotopic methods and (2) non-isotopic nuclear methods. The isotopic techniques discussed are: stable- (¹⁵N) and radio-isotope (³⁵S or ³²P) incorporation methods for measuring microbial mass in vitro and in vivo; ¹²⁵I-labelled bovine serum albumin and ¹⁴C-labelled polyethylene glycol assays for measuring tannin in feeds; a method based on the feeding of isotope-labelled protein (¹⁵N or ¹²⁵I) complexed with tannin for ranking different tannins for their abilities to release protein for digestion in vivo; ¹⁴C-uric acid and ¹⁴C-allantoin infusion methods for development of models describing excretion of purine derivatives in urine and microbial protein supply to ruminants, which permit assessment of nutritional status of animals and determination of nutritional quality of feed resources; a ¹⁵N isotope dilution technique using ¹⁵N-leucine to distinguish feed and endogenous secretions at the ileum, for determination of true digestibility of protein-rich tree leaves and aquatic plants in pigs; progesterone radioimmunoassay (RIA) for enhancing reproductive efficiency of ruminants, and RIA based leptin and insulin like growth factor assays for assessing the nutritional status of animals; feeding of ¹⁵N enriched plant material to generate ¹⁵N-labelled excreta for research on the fate of excreta N in the environment; ¹⁵N, ¹³C and ³⁴S isotopic methods for nutrient budgeting and for following the nutrient pathways in the soil–plant–animal continuum; ³²P- or ³³P-labelled fertilizers for estimating the efficiency of P utilization in legume leaf production used for livestock feeding; double labelled water (¹⁸O and ²H labelled) method for estimating energy expenditures of grazing animals, body composition, basal metabolic rate, and milk output in cows with calves; NaH¹³CO₃/NaH¹⁴CO₃ infusion for estimation of the carbon dioxide production, which in turn is used to estimate energy expenditure in free-ranging animals; ³H- or ¹⁴C-labelled methane and ¹⁴C-labelled volatile fatty acids dilution technique for direct and indirect (using stoichiometry of carbohydrate fermentation) for determination of methane emission from livestock; ¹⁵N dilution technique requiring labeling the soil with ¹⁵N fertilizer (¹⁵N-ammonium sulphate or ¹⁵N-urea) for estimation of nitrogen fixation by leguminous trees and pastures.

The non-isotopic nuclear techniques that have been used or have the potential for use are: dual energy X-ray absorptionmetry and computer tomography for body composition determination; nuclear magnetic resonance techniques, fast atom bombardment mass spectroscopy, and mass ionisation spectroscopy for identification and structure determination of bioactive moieties of plant origin having potential for rumen manipulation or controlling internal parasites; gamma irradiation for inactivating antinutrients such as protease inhibitors, lectin, phytic acid, non-starch polysaccharides and oligosaccharides in feeds; induced mutations with gamma radiation, electron beam and fast neutrons for producing useful mutants of forage plants with improved yield, nutrient profiles and uptake.     

Feeding of Achelia echinata Hodge (Pycnogonida) on marine algae

Field observations of Achelia echinata Hodge in Southampton Water suggested that it may be feeding on Griffithsia and Enteromorpha, although this pycnogonid was not known to be algivorous. This possibility was experimentally tested by maintaining the pycnogonid on ¹⁴C-labelled weeds, whence they were found to take up activity in proportion to the concentration of ¹⁴C in the weed. The experimental design precluded sea water, bacteria or any passive uptake as a source of the ¹⁴C in the pycnogonids, and comparisons between Enteromorpha and Griffithsia eliminated epiphytes as the source. It is concluded that Achelia echinata actively feeds on these two species of seaweed, possibly more readily on Enteromorpha. The feeding behaviour and its significance are discussed.     

GANGLIOSIDES IN NERVOUS TISSUE CULTURES AND BINDING OF 125I-LABELLED TETANUS TOXIN, A NEURONAL MARKER

Abstract— —Continuous cell lines, primary cell cultures derived from embryonic CNS, and homogenates made from adult and embryonic CNS were compared with respect to their lipid pattern and their ability to bind ¹²⁵I-labelled tetanus toxin. In parallel experiments de novo synthesis of gangliosides in the cell lines was studied, using [¹⁴C]glucosamine as precursor. Of the total lipid only gangliosides were specifically labelled by [¹⁴C]glucosamine. The patterns of the de novo synthesized gangliosides corresponded to those present in the respective cells.
Pronounced binding of ¹²⁵I-labelled toxin was only detectable in tissues containing long-chain gangliosides (ganglioside C which represents GDIb and GTI).
Accordingly, hybrid (neuroblastoma x glioma) cells, due to their lack of long-chain gangliosides, bound just-discernible amounts of labelled toxin. When previously exposed to gangliosides, their binding of tetanus toxin tremendously increased.
It was concluded that only the long-chain gangliosides in the neuronal cells are functionally involved in the binding of the tetanus toxin and that these acceptors of tetanus toxin can be transplanted.     

The effect of exogenous gibberellic acid on gibberellin biosynthesis by Gibberella fujikuroi

The addition of gibberellic acid and some other gibberellins to cultures of Gibberella fujikuroi suppresses the incorporation of [2-¹⁴C]MVA and ¹⁴C-labelled ent-kaurene into the gibberellin metabolises.     

The digestion of dietary protein bound by condensed tannins in the gastro-intestinal tract of sheep

The digestion of dietary protein bound by condensed tannins (CTs) in ruminants was investigated by determining the extent of dissociation of insoluble ¹²⁵I-BSA + CT complexes administered to abomasally and intestinally fistulated sheep. The extent of dissociation was registered as the true digestibility of iodinated bovine serum albumin (¹²⁵I-BSA). The true digestibility of ¹²⁵I-BSA originally bound to Leucaena pallida CT (0.721) was lower (P<0.05) than that of ¹²⁵I-BSA originally bound to L. leucocephala CT (0.880) between the abomasum and terminal ileum. These results indicate that differences in the ability of CT to inhibit ¹²⁵I-BSA digestion in vivo matched the relative abilities of the same CT to bind BSA in vitro, indicating that the in vitro BSA-binding assay for ranking CT behaviour was biologically relevant in vivo. Furthermore, the true digestibility of CT-bound ¹²⁵I-BSA between the mouth and faeces permitted the prediction of the quantitative contribution that CT-bound dietary proteins make to improved nitrogen supply to the small intestines.     

Increased urinary excretion of labelled deoxyribonucleosides in rats with stable pre-label led dna treated with methyl methanesulphonate or nitrogen mustard

After the DNA of newborn female rats had been labelled by repeated injections of [¹⁴C]orotate (totalling 36 μCi) during the first 3 weeks of life, approximately 1 000 000 dpm were found in the DNA of the liver, lungs, kidneys, gut, brain, heart and spleen of 8-week-old rats. Methyl methanesulphonate (MMS) (80 mg/kg) and di-(2-chloroethyl)methylamine (HN2) (5 mg/kg) injection increased the amount of ¹⁴C-labelled DNA pyrimidine nucleosides excreted in the urine to 5000 dpm from 350 dpm before injection. The effect on RNA products was much less marked.     

Localization of retinal dehydrogenase type 1 in the stomach and intestine

Rats were injected with liposomes containing iodixanol (CTP10 Injection; 100 mg iodine per kg body weight) followed by a second injection of 125I-tyramine-cellobiose-albumin microspheres. The amounts of phagocytosed and degraded labelled albumin in liver were measured. A reduced uptake and degradation of albumin microspheres was observed when the labelled microspheres were injected 2 h or 24 h after the liposomes compared with that obtained in control animals receiving saline. No effect on the uptake and degradation of labelled microspheres was observed when the time lag between the injection of liposomes and labelled microspheres was 1 week. The data show that the uptake and degradation of 125I-tyramine-cellobiose-albumin microspheres can be used as indicators of Kupffer cell phagocytotic function following drug uptake by these cells.     

Instantaneous benthic response to different organic matter quality: In situ experiments in the Benguela Upwelling System

The benthic community in continental slope and deep-sea sediments of the Benguela Upwelling System was supplied with ¹³C-labelled organic matter (OM) of two different qualities using a benthic chamber lander. Freeze-dried cultures of Skeletonema costatum served as 'fresh' OM. 'Altered' OM of the same material had been additionally dialysed to remove low-molecular weight compounds. In order to investigate the benthic response pattern, mineralization of labelled OM, uptake by macrofauna and incorporation into bacteria were followed over 18-36 h. Total oxygen uptake was not affected beyond natural variation by the OM addition. Mineralization dominated the ¹³C-labelled phytodetritus processing, constituting 71-95% of the total processed OM. Bacterial incorporation of phytodetrital carbon exceeded macrofaunal uptake at all stations. Stations situated in a major centre of OM deposition showed phytodetritus processing rates on average twice as high as outside the depocentre. Phytodetritus processing was 1.5, 2.5 and 4.3 times higher for fresh than for altered OM at 605, 1019 and 1335 m water depth, respectively. Our observations clearly indicate the importance of OM quality on mineralization rates.     

Stoichiometry of the oligomycin-sensitivity-conferring protein (OSCP) in the mitochondrial F0F1-ATPase determined by an immunoelectrotransfer blot technique

The ratio between the amount of oligomycin-sensitivity-conferring protein (OSCP) and the amount of the and β subunits of F₁-ATPase in the mitochondria has been determined by a method combining electrophoresis, electrotransfer and immunotitration with monoclonal antibodies. The peptides separated in SDS-polyacrylamide gel electrophoresis were blotted to nitrocellulose sheets by electrotransfer. The nitrocellulose sheets were incubated with ¹²⁵I-labelled purified monoclonal antibodies specific to various peptides. The ¹²⁵I-labelled immune complexes were located by immunodecoration using peroxidase-conjugated second antibodies and the blotted peptides were revealed with H₂O₂ and -naphthol. The amount of immune complex present on the nitrocellulose was determined by counting the radioactivity present on the spots. The amount of peptide blotted is directly proportional to the amount of protein loaded on the electrophoresis. By comparing standard curves made with the isolated proteins to the values obtained in the presence of various amounts of the membrane-protein complex, one can calculate the content of this peptide in the membrane. It was found that the mitochondrial membrane contains 2 mol of OSCP per mol of F₁.     

Vasoactive intestinal polypeptide binds with high affinity to non-small cell lung cancer cells and elevates cyclic AMP levels

Vasoactive intestinal polypeptide (VIP) receptors were characterized on non-small cell lung cancer (NSCLC) cells. ¹²⁵I-VIP bound specifically to membranes derived from 6 NSCLC cell lines. Specific ¹²⁵I-VIP was time dependent and a linear function of EPLC-65H membrane concentration. ¹²⁵I-VIP bound with high (K_d=0.2 nM) and moderate (K_d=39 nM) affinity to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP > rGHRH > PHI = helodermin > secretin > glucagon. Also VIP elevated the cAMP levels 10-fold using cell line ADLC-5M2. These data indicate that functional VIP receptors are present on NSCLC cells.     

[I]Iodonaphtylazide labeling selectively a cysteine residue in the F0 of the ATP-synthase from E. coli is unsuitable for topographic studies of membrane proteins

The ATP synthase from E. coli was reacted with the hydrophobic photolabel [¹²⁵I]iodonaphtylazide. Subunit b in the F₀-part was selectively labelled. Label was traced back to the single cysteine₂₁ in subunit b. Thus the reactive intermediate of INA generated by photolysis had a high preference for nucleophiles. Due to this high selectivity the detection of membrane spanning peptide segments by labelling with INA is not reliable.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of ¹⁴C into several subcellular fractions of adult rat brain was studied as a function of time, following intracerebral injection of [2-¹⁴C]mevalonic acid. As expected from previous studies, the microsomal fraction was indicated as the site of sterol biosynthesis. The myelin fraction showed a marked and early uptake of ^I4C-labelled, digitonin-precipitable material. This was assumed to be a non-enzymic uptake of sterol intermediates. The mitochondrial fraction exhibited a rapid uptake of ¹⁴C-labelled, nonsaponifiable material, but a very slow accumulation of ¹⁴C-labelled, digitonin-precipitable product. Examination of the nonsaponifiable ¹⁴C-fractions by TLC showed a rapid appearance of labelled 4-desmethyl sterols in the microsomal fraction. The myelin fraction selectively retained 4,4'-dimethyl sterol but seemed to release this with time, possibly to be further metabolized by the microsomes. Examination of [¹⁴C]digitonin-precipitable material by the dibromide method showed that although labelled 4-desmethyl sterol appeared quite early, cholesterol itself was formed slowly in all fractions.     

Nutritional ecology of thalassinidean shrimps constructing burrows with debris chambers: The distribution and use of macronutrients and micronutrients

Four different approaches were combined to determine the nutritional relevance of debris chambers in the burrows of two thalassinidean shrimps: (1) the natural abundance of carbon and nitrogen stable isotopes in potential food sources, (2) their nutritional value based on the content and composition of essential nutrients, (3) a dual labelling experiment with shrimp in aquaria employing ¹⁵N- and ¹³C-labelled seagrass debris and (4) ration estimates using the acquisition rate of plant debris by the shrimps. The results of the four approaches confirmed the use of plant debris as a food source. Based on the natural abundance of stable isotopes, Corallianassa longiventris apparently relies on the chamber content and the burrow wall as sources of carbon and nitrogen, whereas Pestarella tyrrhena probably relies on ambient debris and on benthic foraminiferans and microphytobenthos in the surface sediment. Corallianassa longiventris obtains its essential nutrients predominantly from chamber debris and to a lesser extent from its burrow wall, P. tyrrhena from chamber debris, the burrow wall and the surface sediment. Among the essential nutrients, those amino acids commonly deficient to deposit feeders were particularly enriched in the burrow environments of the two shrimps. Highly unsaturated fatty acids (HUFAs) were lacking in all of C. longiventris potential food sources; this species may either be able to synthesize them de novo from linolic acid or may use another unknown source. For P. tyrrhena, surface sediment and chamber debris represent potential HUFA sources. The most probable thiamine and β-carotene supplier for C. longiventris is the chamber debris, for P. tyrrhena again the surface sediment. In both species, the rate of debris introduction into the burrow is sufficient to meet the nutritional demand.     

Effects of 15N application frequency on nitrogen uptake efficiency in citrus trees

Two irrigation systems were used to compare nitrogen uptake efficiency in citrus trees and to evaluate the NO₃⁻ runoff in «Navelina» orange trees [Citrus sinensis (L.) Osbeck] on Carrizo citrange rootstock (Citrus sinensis × Poncirus trifoliata Raf.). These were fertilized with 125 g N as labelled K¹⁵NO₃ and grown outdoors in containers filled with a sand-loamy soil. Two groups of 3 trees received this N dose either in five equally split applications by a flooding irrigation system or in 66 applications by drip. Trees were harvested at the end of the vegetative cycle (December) and the isotopic ratios of ¹⁵N/¹⁴N were measured in the soil-plant system. The N uptake efficiency of the whole tree was higher with drip irrigation (75 percnt;) than with flooding system (64 percnt;). In the 0-90 cm soil profile, the N immobilized in the organic fraction was similar for both irrigation methods (around 13 percnt;), whereas the N retained as NO₃⁻ was 1 percnt; of the N applied under drip and 10 percnt; under flooding. In the last case, most of NO₃⁻ remained under root system and it could be lost to leaching either by heavy rainfalls or excessive water applications. These results showed that a drip irrigation system was more efficient for improving water use and N uptake from fertilizer, in addition to potentially reduced leaching losses.     

Characterization of glucagon-like peptide-I(7–36)amide receptors of rat lung membranes by covalent cross-linking

¹²⁵I-labelled GLP-I(7–36)amide was cross-linked to a specific binding protein in rat lung membranes using disuccinimidyl suberate. A single radio-labelled band at M_r 66000 was identified by SDS-PAGE after solubilization of the ligand-binding protein complex which is consistent with the presence of a single class of binding sites on rat lung membranes. The band was undetectable when 1 μmol/1 GLP-I(7–36)amide was included in the binding assay. No change in the mobility of the band was observed under reducing conditions suggesting that the binding protein in the receptor is not part of a larger disulphide-liked protein. The intensity of the radiolabelled protein band was reduced when the incubation with ¹²⁵I-labelled GLP-I(7–36)amide was carried out in the presence of guanine nucleotides suggesting that the GLP-I(7–36)amide receptor is coupled to the adenylate cyclase system.     

An immunoradiometric assay for the measurement of neuropeptide Y in plasma

The development of an immunoradiometric assay (IRMA) for the direct measurement of neuropeptide Y (NPY) concentrations in plasma is reported. The assay employs simultaneous addition of ¹²⁵I-labelled affinity purified sheep anti-(NPY 31–36) immunoglobulin (IgG) and a rabbit anti-NPY serum to 0.25 ml volumes of standard or unknown. After 16 hr incubation at 4°C NPY-bound labelled IgG is precipitated using sheep anti-(rabbit IgG Fc region) IgG coupled to Dynospheres solid phase. Precipitated counts are proportional to the NPY concentration in samples. Using this methodology it is possible to measure basal levels in normal human subjects (range 1–5 fmol/ml). Technical difficulties encountered in raising “site-specific” antisera to NPY during the establishment of this assay are outlined.     

Receptor inactivation by dye-neuropeptide conjugates. 3. Comparative binding of dye-neuropeptide conjugates to FMRFamide receptors of Helix aspersa and Loligo pealei

Three neuropeptide analogues of FMRFamide (FMRFa) were covalently attached to a tethered derivative of methylene blue to form dye-neuropeptide conjugates. The comparative binding of the latter to FMRFa receptors was subsequently examined in both Helix aspersa (circumesophageal ganglia) and squid (optic lobe membrane). In Helix, the FMRFa analogue CFMRFamide (CFMRFa) inhibited the specific binding of the FMRFa ligand [¹²⁵I]daYFnLRFa in a dose-dependent manner. Az-CFMRFa, one of the dye-neuropeptide conjugates, also dose-dependently inhibited the specific binding of [¹²⁵I]daYFnLRFa. Moreover, their potencies equaled or exceeded that of FMRFamide. In squid, the binding of CFMRFa and FMRFa was similar. However, the dye-neuropeptide conjugate (IC₅₀ of 14 nM) was about 44-fold less potent than FMRFa. The conjugates were synthesized as part of a study seeking to target and inactivate preselected receptors with heretofore unattainable selectivity and permanence.     

Photosynthetic CO2 fixation by chloroplast populations isolated by a polymer two-phase technique

Preparations of spinach chloroplasts, essentially free from contamination by other cellular material or whole cells, incorporated ¹⁴C almost entirely into glycolate, a polyglucan (probably starch) and intermediates of the Calvin cycle and starch synthesis. About 70% of the ¹⁴C was found in dihydroxyacetone phosphate, 3-phosphoglycerate and glycolate. Only small amounts were found in sucrose and amino acids.

On the other hand, preparations consisting of particles containing chloroplasts surrounded by a membrane-bound cytoplasmic layer including mitochondria and microbodies, gave a much broader spectrum of ¹⁴C-labelled products. Much less of the ¹⁴C was found in dihydroxyacetone phosphate and 3-phosphoglycerate. Instead, sucrose, malate, aspartate, alanine and some other amino acids contained about 40% of the ¹⁴C incorporated. It is concluded that sucrose is synthesized by cooperation between the chloroplast and the surrounding layer.     

Characterization of amylin binding sites in a human hepatoblastoma cell line

Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. ¹²⁵I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (K_d = 0.11 ± 0.04 nM) and low (K_d = 1.3 ± 0.4 μM) affinity binding sites for ¹²⁵I-amylin in HepG2 cells. The dissociation experiments also showed that ¹²⁵I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing ¹²⁵I-amylin bound to HepG2 cells. Nonhomologous peptides did not displace ¹²⁵I-amylin. Rat amylin was, however, less potent than rat CGRP in displacing ¹²⁵I[Tyr⁰]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of ¹²⁵I-amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8–37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.