Maternal undernutrition in pregnancy results in low birth-weights and impaired postnatal survival in sheep. Largely anecdotal evidence suggests that the expression of appropriate maternal and neonate behaviours may also be disrupted by undernutrition. In the present study, we investigated the effect of a moderate (35 %) reduction in ewe nutritional intake in pregnancy on the expression of ewe–lamb bonding behaviours in primiparous Scottish Blackface ewes. Low-intake (L) ewes had significantly higher plasma progesterone than high-intake (H) ewes from mid-gestation onwards (e.g. plasma progesterone at 20 weeks (n/l): H 15·72, L 22·38, SED 1·80, P<0·001), AND A LOWER OESTRADIOL: PROGESTERONE VALUE THAN H EWES AT DELIVERY (H 0·46, L 0·35, sed 0·05, P<0·05). Lamb birth-weight was reduced in the L lambs compared with H lambs (mean body weight (kg): H 3·31, L 3·00, sed 0·14, P<0·05), but the incidence of malpresentation at delivery was greater in L lambs. L ewes spent significantly less time licking their lambs than H ewes after delivery (time grooming in 2 h after birth (%): H 56·12, L 48·17, sed 2·639, P<0·01) and were more aggressive towards the lambs. Lamb behaviours were not directly affected by maternal nutritional treatment, but lamb birth-weight had a significant effect on neonatal developmental progress. Low-birth-weight lambs were slower than heavier lambs to stand and sucked less frequently. In tests of maternal attachment to the lamb, H ewes received higher scores than L ewes at both 24 h after birth (ewes receiving high scores (%): H 41·3, L 21·4, P<0·05) and at 3 d postnatal. We conclude that even a moderate level of undernutrition impairs the attachment between ewes and lambs by affecting maternal behaviours expressed at birth. In addition, the results suggest that levels of nutrition resulting in a decrease in birth weight will affect neonatal lamb behavioural progress.

The determination of whether increased dietary protein can positively affect health outcomes is hindered by the absence of prospective, randomized trials directly addressing this issue in which all pertinent variables are controlled. Consequently, we can only address the question deductively by considering the support for the rationale underlying the notion of a beneficial effect of increased dietary protein intake. With regard to health outcomes, we have focused on older individuals. Muscle mass and function are progressively lost with aging, so that by the age of 60 many individuals have reached a threshold where function begins to be affected. An association between reduced muscle mass and strength and unfavourable health outcomes is more likely to be revealed in individuals who have significant decrements in muscle mass and strength. In this article support for the rationale underlying the notion of a beneficial effect of increased dietary protein intake is considered. Dietary protein intake, and the resulting increased availability of plasma amino acids, stimulates muscle protein synthesis. If all other variables are controlled, increased muscle protein synthesis leads to improved muscle mass, strength and function over time. Increased muscle mass, strength and function are related to improved health outcomes in older individuals. Since adverse effects of reasonable increases in protein intake above the recommended dietary allowance (RDA) of 0·8 g protein/kg/day have not been reported, it is reasonable to conclude that the optimal protein intake for an older individual is greater than the RDA.

Fe absorption was measured in adult human subjects consuming different cereal foods fortified with radiolabelled FeSO4, ferrous fumarate or NaFeEDTA, or with radiolabelled FeSO4 or ferric pyrophosphate in combination with different concentrations of Na2EDTA. Mean Fe absorption from wheat, wheat–soyabean and quinoa (Chenopodium quinoa) infant cereals fortified with FeSO4 or ferrous fumarate ranged from 0·6 to 2·2 %. For each infant cereal, mean Fe absorption from ferrous fumarate was similar to that from FeSO4 (absorption ratio 0·91–1·28). Mean Fe absorption from FeSO4-fortified bread rolls was 1·0 % when made from high-extraction wheat flour and 5·7 % when made from low-extraction wheat flour. Fe absorption from infant cereals and bread rolls fortified with NaFeEDTA was 1·9–3·9 times greater than when the same product was fortified with FeSO4. Both high phytate content and consumption of tea decreased Fe absorption from the NaFeEDTA-fortified rolls. When Na2EDTA up to a 1:1 molar ratio (EDTA:Fe) was added to FeSO4-fortified wheat cereal and wheat–soyabean cereal mean Fe absorption from the wheat cereal increased from 1·0 % to a maximum of 5·7 % at a molar ratio of 0·67:1, and from the wheat–soyabean cereal from 0·7 % to a maximum of 2·9 % at a molar ratio of 1:1. Adding Na2EDTA to ferric pyrophosphate-fortified wheat cereal did not significantly increase absorption (P>0·05). We conclude that Fe absorption is higher from cereal foods fortified with NaFeEDTA than when fortified with FeSO4 or ferrous fumarate, and that Na2EDTA can be added to cereal foods to enhance absorption of soluble Fe-fortification compounds such as FeSO4.

1. A relationship was found between selenium concentrations and glutathione peroxidase (EC 1.11.1.9) activities in whole blood of 264 New Zealand residents (r 0.71, P < 0.001).

2. New Zealand residents returning from visits overseas of 7 months to 3 years had elevated blood Se, but normal GSH-Px activities, whereas for some new settlers in New Zealand both Se and GSH-Px activities were high.

1. Previous publications by the author and his colleagues have provided extensive records, and additional ruminating behaviour of cows receiving a wide range of diets. These recoreds, and additional unpublished records, have been used to justify a proposed index of the physical property of fibrousness, or roughage, in diets for ruminants.

2. It is proposed that the total time spent by ruminants in chewing their food, during eating and during ruminating, should be the basis of the basis of the roughage index

3. Experssion of the roughage index as total time spent chewing per kg dietary dry matter largely eliminates differences resulting from variation in the amount of food consumed, and differences resulting from the time of access to the food.

4. Examples of the proposed roughasge index are given. The values range from 145 to 191 min/Kg dry matter for oat straw ot under 20 min/kg dry matter for diets of concentrates or of finely ground herbages; hays and silages tend to fall in the region of 90–110 min/kg dry matter. Values for the indx increase with dietary proportaions of hay or straw.

1. Concentrations of nucleic acid nitrogen (NA-N) and other nitrogenous constituents were estimated in samples of rumen fluid taken from calves and cows which were either given stall diets of varying nitrogen content or were allowed to graze pasture. Concentrations of NA-N ranged from 1.5 to 27.5 mg/100 g water.

2. Ratios of RNA: DNA in rumen fluid were similar to those in rumen bacteria and were not related to those in the diets. Pure nucleic acids added to the rumen were rapidly degraded. It was therefore concluded that NA-N in rumen fluid was largely of microbial origin and provided an index of total microbial nitrogen.

3. In most experiments, with an individual animal consuming diets of various nitrogen contents, NA-N formed a fairly constant percentage (8-15 for different animals) of the total non-ammonia nitrogen in rumen fluid. This suggested that nitrogen entering the rumen fluid limited microbial growth. Consumption of a diet containing extracted decorticated groundnut meal (diet B), however, led to lower values for this percentage than did the other diets. Diet B was also exceptional in leading to marked diurnal variations in NA-N concentrations in rumen fluid, suggesting a cyclic fluctuation in the size of the microbial population.

4. Comparison of NA-N:total nitrogen ratios in rumen fluid and bacteria suggested that, for all the diets except diet B, 55–80 and 40–50% of the non-ammonia nitrogen in rumen fluid was of microbial origin for the calves and cows respectively.

First promulgated in 1959, the 3Rs of Replacement, Reduction and Refinement have evolved as fundamental principles underlying the use of animals and alternatives in science throughout the modern world. This review describes a contemporary approach to delivering the 3Rs through acknowledging the contribution of new technologies and emphasising that applying the 3Rs can be beneficial to good science as well as to animal welfare. This science-led approach moves the concept of the 3Rs out of an ethical silo where they were often considered by scientists to be an inconvenient obligation. On the contrary, relevant examples demonstrate the opportunity to practise better science using 3Rs technologies which deliver faster, more reproducible and more cost-effective results. Indeed, methods harnessing Replacement approaches may permit discoveries which are simply not feasible using animals and frequently are more flexible and agile since compliance with regulatory oversight requirements is simplified. Although the necessity for rigorous oversight is well recognised, it is important that the associated bureaucracy is not allowed to become prohibitive, causing scientists to avoid pursuing justifiable and important research involving animals. Public support for research is conditional – animals should not suffer unnecessarily and sufficient potential benefit should accrue from the research. However, society also actively seeks pioneering medical and scientific advances which can only be achieved through research. Therefore, a balance must be struck between safeguarding animal welfare whilst enabling high-quality science. It is this balance which promotes and sustains public confidence that animal based research is acceptable and being appropriately managed.

Overall dietary patterns have been associated with health and longevity. We used principal component (PC) and cluster analyses to identify the prevailing dietary patterns of 99 744 participants, aged 60 years or older, living in nine European countries and participating in the European Prospective Investigation into Cancer and Nutrition (EPIC-Elderly cohort) and to examine their socio-demographic and lifestyle correlates. Two PC were identified: PC1 reflects a ‘vegetable-based’ diet with an emphasis on foods of plant origin, rice, pasta and other grain rather than on margarine, potatoes and non-alcoholic beverages. PC2 indicates a ‘sweet- and fat-dominated’ diet with a preference for sweets, added fat and dairy products but not meat, alcohol, bread and eggs. PC1 was associated with a younger age, a higher level of education, physical activity, a higher BMI, a lower waist:hip ratio and never and past smoking. PC2 was associated with older age, less education, never having smoked, a lower BMI and waist:hip ratio and lower levels of physical activity. Elderly individuals in southern Europe scored positively on PC1 and about zero on PC2, whereas the elderly in northern Europe scored negatively on PC1 and variably on PC2. The results of cluster analysis were compatible with the indicated dietary patterns. ‘Vegetable-based’ and a ‘sweet- and fat-dominated’ diets are prevalent among the elderly across Europe, and there is a north–south gradient regarding their dietary choices. Our study contributes to the identification of groups of elderly who are likely to have different prospects for long-term disease occurrence and survival.

The aim of the study was to examine the association of dietary carbohydrates and glycaemic load with the risk of type 2 diabetes among an urban adult Asian Indian population. Adult subjects aged >20 years (n 1843) were randomly selected from the Chennai Urban Rural Epidemiology Study, in Chennai city in southern India. Dietary carbohydrates, glycaemic load and food groups were assessed using FFQ. Oral glucose tolerance tests were performed using 75 g glucose in all subjects. Diagnosis of diabetes was based on WHO Consulting Group criteria. OR for newly detected diabetes were calculated for carbohydrates, glycaemic load and specific food groups comparing subjects in the highest with those in the lowest quartiles, after adjustment for potential confounders such as age, sex, BMI, family history of diabetes, physical activity, current smoking, alcohol consumption and relevant dietary factors. We identified 156 (8·5 %) newly diagnosed cases of type 2 diabetes. Refined grain intake was positively associated with the risk of type 2 diabetes (OR 5·31 (95 % CI 2·98, 9·45); P < 0·001). In the multivariate model, after adjustment for potential confounders, total carbohydrate (OR 4·98 (95 % CI 2·69, 9·19), P < 0·001), glycaemic load (OR 4·25 (95 % CI 2·33, 7·77); P < 0·001) and glycaemic index (OR 2·51 (95 % CI 1·42, 4·43); P = 0·006) were associated with type 2 diabetes. Dietary fibre intake was inversely associated with diabetes (OR 0·31 (95 % CI 0·15, 0·62); P < 0·001). In urban south Indians, total dietary carbohydrate and glycaemic load are associated with increased, and dietary fibre with decreased, risk of type 2 diabetes.

Using data from the National Health and Nutrition Examination Survey (NHANES) 2003–2012, we investigated the prevalence and characteristics of under-reporting and over-reporting of energy intake (EI) among 19 693 US adults ≥20 years of age. For the assessment of EI, two 24-h dietary recalls were conducted using the US Department of Agriculture Automated Multiple-Pass Method. Under-reporters, acceptable reporters and over-reporters of EI were identified by two methods based on the 95 % confidence limits: (1) for agreement between the ratio of EI to BMR and a physical activity level for sedentary lifestyle (1·55) and (2) of the expected ratio of EI to estimated energy requirement (EER) of 1·0. BMR was calculated using Schofield’s equations. EER was calculated using equations from the US Dietary Reference Intakes, assuming ‘low active’ level of physical activity. The risk of being an under-reporter or over-reporter compared with an acceptable reporter was analysed using multiple logistic regression. Percentages of under-reporters, acceptable reporters and over-reporters were 25·1, 73·5 and 1·4 %, respectively, based on EI:BMR, and 25·7, 71·8 and 2·5 %, respectively, based on EI:EER. Under-reporting was associated with female sex, older age, non-Hispanic blacks (compared with non-Hispanic whites), lower education, lower family poverty income ratio and overweight and obesity. Over-reporting was associated with male sex, younger age, lower family poverty income ratio, current smoking (compared with never smoking) and underweight. Similar findings were obtained when analysing only the first 24-h recall data from NHANES 1999–2012 (n 28 794). In conclusion, we found that misreporting of EI, particularly under-reporting, remains prevalent and differential in US adults.

Malnutrition, secondary to deficiency in intake of proteins, minerals or vitamins, consistently results in changes in the thymus. This organ undergoes a severe atrophy due to apoptosis-induced thymocyte depletion, particularly affecting the immature CD4+CD8+ cells, as well as a decrease in cell proliferation. This feature is apparently linked to a hormonal imbalance, involving a decrease in leptin and consequent increase in glucocorticoid hormone levels in the serum. The thymic microenvironment is also affected in malnutrition: morphological changes in thymic epithelial cells have been found, together with a decrease of thymic hormone production by these cells. Additionally, intrathymic contents of extracellular proteins, such as fibronectin, laminin and collagens, are increased in thymuses from malnourished children. Taken together, these data clearly point to the notion that the thymus is significantly affected in malnutrition. Similar patterns of thymic changes occur in acute infectious diseases, including a severe atrophy of the organ, mainly due to the apoptosis-related depletion of immature CD4+CD8+ thymocytes. Additionally, thymocyte proliferation is compromised in acutely-infected subjects. The microenvironmental compartment of the thymus is also affected in acute infections, with an increased density of the epithelial network and an increase in the deposition of extracellular matrix. In conclusion, it seems clear that the thymus is targeted in malnutrition as well as in acute infections. These changes are related to the impaired peripheral immune response seen in malnourished and infected individuals. Thus, strategies inducing thymus replenishment should be considered in therapeutic approaches, in both malnutrition and acute infectious diseases.

Inulin is extracted from the chicory root. It is a set of fructans with its monomers (n=2–65) linked by means of β(2–1) bonds. This linkage cannot be hydrolysed by either pancreatic or by brush border digestive enzymes in the upper intestinal tract of humans. As such the carbohydrates arrive in the colon, where they are fermented by bifidobacteria and other lactic acid producing bacteria, thus enhancing their relative populations in the gut. Recent research in experimental animal models revealed that inulin has significant anticarcinogenic properties. It acts chemopreventively by reducing the incidence of azoxymethane (AOM) — induced aberrant crypt foci and tumours in the colon. These effects may be due to the stimulation of bifidobacteria, which themselves have been shown to act as antigenotoxic in the colon and to reduce AOM-induced tumours. Also fermentation products, including the short-chain fatty acid butyrate, could contribute to the protective effects. In this case a mechanism may be the induction of apoptosis of already transformed cells. The experimental evidence from animal studies and from studies elucidating potential mechanisms strongly supports the possibility that inulin will contribute to reducing risks for colon cancer in humans. In order to obtain more insight into this possibility, human dietary intervention studies relating biomarkers of reduced risk to inulin consumption are needed.

The aim of the present study was to assess the relationship between the disappearance of dietary fibre sugars and the production of individual short-chain fatty acids (SCFA). The bacterial degradation of five dietary fibres whose sugars were quantified was investigated in vitro using a human faecal inoculum. Involvement of the main fibre sugars in SCFA production was evaluated by a stepwise multiple linear regression. The results show first that the nature and chiefly the associations between the fibre sugars were key variables in the fermentability. Second, the nature and the amounts of SCFA produced were closely related to the in vitro fermentation of the main sugars available: uronic acids seemed to be principally involved in the production of acetic acid whereas the production of propionic acid could be promoted by the fermentation of glucose and, to a lesser extent, by that of xylose and arabinose. Xylose tended to have a greater impact than uronic acids and glucose on the production of butyric acid. Thus, it would be possible to predict which SCFA could be specifically produced during the fermentation of a fibre, as far as the chemical composition and structure of this fibre are known.

The present study investigates the benefits of the dietary intake of chia seed (Salvia hispanica L.) rich in α-linolenic acid and fibre upon dyslipidaemia and insulin resistance (IR), induced by intake of a sucrose-rich (62·5 %) diet (SRD). To achieve these goals two sets of experiments were designed: (i) to study the prevention of onset of dyslipidaemia and IR in Wistar rats fed during 3 weeks with a SRD in which chia seed was the dietary source of fat; (ii) to analyse the effectiveness of chia seed in improving or reversing the metabolic abnormalities described above. Rats were fed a SRD during 3 months; by the end of this period, stable dyslipidaemia and IR were present in the animals. From months 3–5, half the animals continued with the SRD and the other half were fed a SRD in which the source of fat was substituted by chia seed (SRD+chia). The control group received a diet in which sucrose was replaced by maize starch. The results showed that: (i) dietary chia seed prevented the onset of dyslipidaemia and IR in the rats fed the SRD for 3 weeks – glycaemia did not change; (ii) dyslipidaemia and IR in the long-term SRD-fed rats were normalised without changes in insulinaemia when chia seed provided the dietary fat during the last 2 months of the feeding period. Dietary chia seed reduced the visceral adiposity present in the SRD rats. The present study provides new data regarding the beneficial effect of chia seed upon lipid and glucose homeostasis in an experimental model of dislipidaemia and IR.

The effects of incremental exercise on appetite, energy intake (EI), expenditure (EE) and balance (EB) in lean men and women were examined. Six men (age 29·7 (sd 5·9) years, weight 75·2 (sd 15·3) kg, height 1·75 (sd 0·11) m) and six women (age 24·7 (sd 5·9) years, weight 66·7 (sd 9·10) kg, height 1·70 (sd 0·09) m) were each studied three times during a 16 d protocol, corresponding to no additional exercise (Nex), moderate-intensity exercise (Mex; 1·5–2·0 MJ/d) and high-intensity exercise (Hex; 3·0–4·0 MJ/d) regimens. Subjects were fed to EB during days 1–2, and during days 3–16 they fed ad libitum from a medium-fat diet of constant composition. Daily EE, assessed using the doubly labelled water method, was 9·2, 11·6 and 13·7 MJ/d (P < 0·001; sed 0·45) for the women and 12·2, 14·0 and 16·7 MJ/d (P = 0·007; sed 1·11) for the men on the Nex, Mex and Hex treatments, respectively. EI was 8·3, 8·6 and 9·9 MJ/d (P = 0·118; sed 0·72) for the women and 10·6, 11·6 and 12·0 MJ/d (P = 0·031; sed 0·47) for the men, respectively. On average, subjects compensated for about 30 % of the exercise-induced energy deficit. However, the degree of compensation varied considerably among individuals. The present study captured the initial compensation in EI for exercise-induced energy deficits. Total compensation would take a matter of weeks.

The aim of this experiment was to determine CH4 production and energy partition for a range of diets fed to Bos indicus cattle. Six Brahman cattle were fed on three different diets in a replicated Latin square experiment over three periods. The diets were (1) long-chopped Angleton grass (Dicanthium aristatum) hay ad libitum (DM digestibility (DMD) 41 (se 2)%; 4 g N/kg), (2) long-chopped Rhodes grass (Chloris gayana) hay ad libitum (DMD 60 (se 1)%; 14 g N/kg) or (3) 2 kg long-chopped lucerne (Medicago sativa) hay/d plus a high-grain diet (ad libitum) (DMD 70 (se 1)%; 31 g N/kg). CH4 production was measured using confinement-type respiration chambers. Metabolizable energy intake (MJ/d) of cattle fed on Angleton grass (18·4 (se 2·0)) was lower (P< 0·01) than that for Rhodes grass (54·9 (se 2·1)), which was lower (P< 0·01) than that for the high-grain diet (76·7 (se 5·8)). CH4 production (g/d) for cattle fed on Rhodes grass (257 (se 14)) was higher (P< 0·01) than that for cattle fed on both the high-grain diet (160 (se 24)) and Angleton grass (113 (se 16)). CH4 conversion rate (MJ CH4 produced per 100 MJ gross energy intake) was not significantly different between cattle fed on Angleton (10·4 (se 1·1)) and Rhodes (11·4 (se 0·3)) grass, but was higher (P< 0·01) than for cattle fed on the high-grain diet (6·7 (se 0·7)). CH4 production (g/kg live-weight gain) was associated (P< 0·001) with both live-weight gain and feed: gain ratio. We conclude that the relationships between CH4 production, energy utilization and live-weight change of cattle fed on tropical forages differ from those of cattle fed on diets based on temperate forages.

Fatty acids are known to play diverse roles in immune cells. They are important as a source of energy, as structural components of cell membranes, as signaling molecules and as precursors for the synthesis of eicosanoids and similar mediators. Recent research has suggested that the localization and organisation of fatty acids into distinct cellular pools has a direct influence on the behaviour of a number of proteins involved in immune cell activation, including those associated with T cell responses, antigen presentation and fatty acid-derived inflammatory mediator production. This article reviews these studies and places them in the context of existing literature in the field. These studies indicate the existence of several novel mechanisms by which altered fatty acid availability can modulate immune responses and impact upon clinical outcomes.

The intestinal anti-inflammatory effects of two probiotics isolated from breast milk, Lactobacillus reuteri and L. fermentum, were evaluated and compared in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis. Colitis was induced in rats by intracolonic administration of 10 mg TNBS dissolved in 50 % ethanol (0·25 ml). Either L. reuteri or L. fermentum was daily administered orally (5 × 108 colony-forming units suspended in 0·5 ml skimmed milk) to each group of rats (n 10) for 3 weeks, starting 2 weeks before colitis induction. Colonic damage was evaluated histologically and biochemically, and the colonic luminal contents were used for bacterial studies and for SCFA production. Both probiotics showed intestinal anti-inflammatory effects in this model of experimental colitis, as evidenced histologically and by a significant reduction of colonic myeloperoxidase activity (P < 0·05). L. fermentum significantly counteracted the colonic glutathione depletion induced by the inflammatory process. In addition, both probiotics lowered colonic TNFα levels (P < 0·01) and inducible NO synthase expression when compared with non-treated rats; however, the decrease in colonic cyclo-oxygenase-2 expression was only achieved with L. fermentum administration. Finally, the two probiotics induced the growth of Lactobacilli species in comparison with control colitic rats, but the production of SCFA in colonic contents was only increased when L. fermentum was given. In conclusion, L. fermentum can exert beneficial immunomodulatory properties in inflammatory bowel disease, being more effective than L. reuteri, a probiotic with reputed efficacy in promoting beneficial effects on human health.

Probiotic agents have been shown to have significant clinical beneficial effects in the prevention and management of gastrointestinal and non-gastrointestinal conditions. These observations have led to work demonstrating that an important mechanism of these agents is their close interaction with the gut associated lymphoid tissue (GALT) and suggested immunomodulatory effects on systemic immune response. Studies on the possibility that prebiotic agents might directly or indirectly induce similar immunomodulation have only recently begun. The preliminary findings of several recent human clinical trials reviewed in this article indicate that prebiotics may indeed prove to be a clinically beneficial dietary supplement, in the context of novel nutritional strategies for the management of gastrointestinal and systemic conditions.

1. Three sheep, each fitted with a ruminal cannula and duodenal re-entrant cannulas were given three isonitrogenous, isoenergetic diets in a Latin-Square design. Each diet contained (/kg) approximately 400 g N as white fish meal, soya-bean meal or urea and approximately 600 g dry matter (dm) was barley grain. The diets were fed continuously and supplied about 28 g N/d.

2. Total duodenal digesta was collected manually for 72 h and the proportions of microbial N in that digesta were simultaneously estimated for all sheep using RNA, radioactive sulphur (35S), diaminopimelic acid (DAPA) and aminoethylphosphonic acid (AEPA) as markers.

3. Three of the estimation methods showed that the variable source of dietary N had the greatest (RNA P < 0.05, 35S P < 0.005, DAPA P < 0.1) effect on the proportions of microbial N in duodenal digesta, though differences between sheep accounted for some variation.

4. These methods also ranked the diets in the order: urea > soya-bean meal > fish meal with respect to the proportions of digesta N that were microbial in origin; the respective mean values for these diets with the different markers were: RNA 0.98, 0.70, 0.56; 35S 0.92, 0.64, 0.54; DAPA 0.80, 0.47, 0.42.

5. AEPA was found to be present in substantial quantities not only in isolated rumen protozoa, but also in dietary and bacterial material; an observation that invalidated its further use as a protozoal marker.

6. Calculations using values obtained from the 35S procedure showed that the proportions of dietary N degraded within the rumen were 0.38, 0.43 and 0.89 for the white fish meal, soya-bean meal and barley respectively.

7. The marker methods are compared and their advantages and disadvantages (real and apparent) are discussed. It is concluded that where microbial N estimates of a more general and comparative nature are required, the use of RNA as a marker is probably adequate. Where information for more exacting purposes is required, the use of 35S appears to be more appropriate.