Metabolisable protein supply and diurnal rumen nitrogen management and use in beef steers grazing fodder beet : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

The winter feeding period in New Zealand presents a significant challenge for beef farmers to provide adequate feed for stock to maintain live weight (LWT) gains. The recent development of the fodder beet (Beta vulgaris subsp vulgaris L.) grazing system has enabled high LWT gains over this period. Commercial beef finishing operations have repeatedly reported ~1kg LWT gain per day, with dietary CP of <13%, and high sucrose and water content. Experiments were developed to investigate rumen and plasma nitrogen use and movement in the fodder beet diet, to better understand the mechanisms enabling high daily LWT gains. Experiment 1 of Chapter 3 quantified the daily and diurnal (1, 3, 6 and 24-hour) dry matter (DM) and nitrogen intake of steers grazing industry standard fodder beet diets (90% fodder beet, 10% supplement), to match intakes with LWT gain of the steers, on a commercial farm. A single group of 69 steers (LWT 352kg) grazed fodder beet in situ for >120 days, with average LWT gain 1.0kg per day. Measured DM intakes and in sacco derived coefficients of rumen nitrogen disappearance were used to estimate metabolisable protein supply, compared with the estimated metabolisable protein demand, and then altered to reduce rumen nitrogen losses and increase bypass nitrogen, to provide new estimates of metabolisable protein supply. These data were used to model rumen nitrogen release across the diurnal period. The diurnal DMI was non-uniform, and with a greater proportional intake of leaf and pasture components in the early grazing period. The diurnal nitrogen intake was then strongly non-uniform, with 47, 66 and 77% of daily nitrogen intake by 1, 3 and 6-hours. The feed nitrogen release was also strongly non-uniform, with 28% released in the first hour, then progressively declining to 24-hours. There was a 2.8kg WSC intake in the first 6-hours, and 0.97kg of WSC intake in the final 18-hours, with reduced nitrogen supply. It was concluded this diet was markedly asynchronous in rumen supply of energy and nitrogen. Despite a dietary CP content of 9.4%, the estimated metabolisable protein supply was adequate to meet the estimated metabolisable protein demand according to the AFRC feeding reference standard, but not for the NRC, for the observed LWT gain. Altering quickly degradable protein losses maximally from 0.8 to 0.95 increased the estimated metabolisable protein supply by 7%, and maximally altering undegraded dietary protein by 12%. It was concluded that the non-uniform pattern of nitrogen intake and asynchronous supply of energy and nitrogen to the rumen did not significantly adversely reduce production in steers grazing this diet, as high LWT gains were observed. The strongly non-uniform rumen nitrogen release and high LWT gains suggested nitrogen recycling would be required. Finally, it was therefore concluded that there may be additional nitrogen management strategies in operation, as well as the traditional rumen and whole-animal nitrogen use in steers fed this diet, perhaps due to the novel characteristics of the diet, evidenced by the high LWT gains observed. The strongly non-uniform, asynchronous intakes and high LWT gains observed in Experiment 1 led to the development of Experiment 2 in Chapter 4. This compared the DM and nitrogen intakes, LWT gains, rumen and plasma urea and ammonia concentrations, and plasma amino acids of steers on a fodder beet diet or a synchronous total mixed ration (TMR) on a commercial farm. A total of 938 steers in six mobs of ~150 steers, mobs grazed a diet of ad libitum fodder beet in situ or an ad libitum TMR. The diurnal DM and nitrogen intake of steers on the fodder beet and TMR diet treatments were measured at 1, 3, 6 and 24-hours, and rumen and plasma samples were obtained at two times during the grazing period. The LWT gains were similar (0.98kg per day; P>0.05) between treatment groups, despite the different dietary characteristics. The fodder beet diet treatment displayed a greater non-uniform pattern of DM and nitrogen intake than the TMR diet treatment (P<0.05), with 47 and 75% DMI, and 45 and 78% nitrogen intake, at 1 and 6-hours, respectively, and the TMR 20 and 45% DM and nitrogen intake at 1 and 6-hours, respectively. The fodder beet diet treatment had lower mean rumen ammonia concentrations (86mg /L) and higher mean rumen urea concentrations (992mg /L) (P<0.05) than the TMR diet. Plasma ammonia concentrations were 2.13 and 2.46mg /L at the Mid-Winter (P=0.091) and 1.98 and 2.41mg /L at the Late Winter (P=0.022) sampling for the fodder beet and TMR diet treatments, respectively. Plasma urea concentrations were 75.9 and 254.8mg /L at the Mid-Winter (P<0.001) and 180.6 and 318.92mg /L at the Late Winter (P<0.001) sampling for the fodder beet and TMR diet treatments, respectively. Plasma amino acid concentrations were similar (P>0.05) between the two diet treatments, with no apparent differences in glucogenic or ketogenic amino acid groups. While the TMR diet treatment estimated metabolisable protein supply was higher, both diet treatments supplied adequate metabolisable protein to meet demand despite the lower CP content of the fodder beet diet treatment (11.6 vs. 14.1%). It was concluded that there were important differences in rumen and whole-animal nitrogen use between the two diet treatments, despite the observed LWT gains being similar. Experiment 3 in Chapter 5 was designed to investigate rumen nitrogen use and export across the diurnal period in fodder beet diets. A comparison of fodder beet and pasture feeding in treatment groups of eight pen-fed sheep (LWT: 34.1 and 39.3kg) with total urinary collection was used. Rumen nitrogen loss and microbial protein (MCP) outflow via sub-diurnal urinary nitrogen and purine excretion patterns were quantified. Measurements of total DMI and total urine production were undertaken at periods of 1, 3, 6 and 24-hours, to correspond with measurements conducted in Chapters 3 and 4. At 1 and 3-hours, the fodder beet diet treatment had 2-fold higher (P<0.001) nitrogen intakes (5.38 and 5.73g vs 2.28 and 2.92g, respectively). The urinary nitrogen excretion at 1 and 3-hours was not different between the two diet treatments (P>0.05), with 0.36 and 0.71g nitrogen excretion for the fodder beet diet treatment, and 0.23g and 0.47g nitrogen excretion for the pasture diet treatment. The urinary purine excretion was also similar between the two diet treatments at 1, 3 and 6-hours (P>0.05). The low DM content and heterogeneous nature of the fodder beet diet resulted in greater non-uniform diurnal intake of feed water for the fodder beet diet treatment. The feed water intake was ~3-fold and ~2-fold higher for the fodder beet diet treatment at 1 and 3-hours, respectively, and 48% higher for the fodder beet diet treatment at 6-hours (P=0.001, P=0.001, P=0.002). While urine production was higher for the fodder beet diet treatment at 1, 3 and 6-hours (P=0.001, P=0.004, P=0.017), this was not proportionate to the increase in feed water intake compared to the pasture diet treatment. From the comparatively greater water intake against urinary output in the early (1 and 3-hour) feeding period for the fodder beet diet treatment it was concluded that there was a net water retention, most likely an increased GIT volume as rumen digesta volume. It was concluded the proportionately lower rumen export of nitrogen in the fodder beet than the pasture diet treatment in the early (1 and 3-hour) period of the diurnal cycle to urinary nitrogen or to purines was likely due to greater nitrogen retention in the rumen in the early (1 and 3-hour) feeding period, and this may be a mechanism of nitrogen use and movement in the fodder beet diets that may explain the strong production observed with comparatively low CP content. The results of Experiment 3 suggesting reduced rumen nitrogen export in the early feeding period and with this a net water retention were further examined in Chapter 6 in two experiments. Experiment 4a was designed to quantify the proportion of rumen nitrogen exported from the rumen, or recycled to the rumen, in ad libitum fodder beet fed steers, using infusions of isotopically labelled 15N15N urea to either the plasma or the rumen. Experiment 4b quantified the diurnal rumen volume fluctuations in fistulated rising-one-year-old beef steers fed fodder beet or pasture. Four rising-one-year-old steers in metabolism crates (Experiment 4a) had feed intake measured and total faecal and urinary collection, in a 2 x 2 crossover experimental design. In each of two 76-hour periods separated by an 18-day washout, two steers were infused with the stable isotope of 15N15N urea into the rumen, and two steers were infused with 15N15N urea into the jugular and blood and rumen digesta samples were then obtained from steers at 4-hour intervals for 24-hours to determine abundance of 15N in rumen fluid and plasma, and also in daily urine and faeces. Experiment 4b used complete rumen evacuations every 16-hours for 48-hours to determine rumen digesta volume across the diurnal cycle (0, 8, 24-hours after feeding) in steers grazing ad libitum pasture or fodder beet. Diurnal rumen fluid abundance of 15N for the rumen infusion treatment displayed substantial diurnal variation (P<0.001), with the highest abundance observed (488 δ15N ‰) at morning feeding time, but reaching a nadir (223 δ15N ‰) 12-hours after feeding. The jugular infusion treatment did not vary across the diurnal cycle for rumen fluid 15N abundance (P=0.137), with <20 δ15N ‰ difference from the nadir at 12-hours post-feeding. The rumen and jugular infusion treatment abundances were different at 0, 4 and 20-hours after feeding (P<0.05). Plasma 15N abundance for the jugular infusion treatment did not vary across the diurnal cycle (P=0.182), whereas, the rumen infusion treatment did vary (P=0.006). However, the two infusion treatments were not different in plasma 15N abundance (P>0.05) at any measurement period throughout the diurnal cycle. Mean daily urinary excretion of 15N was 3701 for the rumen 15N infusion compared with 9842 for the jugular 15N infusion (P=0.004). The mean abundance of 15N in faeces was higher (P=0.037) for the rumen (238 δ15N ‰) than the jugular infusion (137 δ15N ‰) treatment. These results indicated low transfer of rumen nitrogen to the plasma, with little diurnal variation in this transfer, and that rumen import of nitrogen was more substantial, and greater at diurnal phases associated with higher rumen activity. Experiment 4b demonstrated that rumen volume increases at 8-hours were greater in fodder beet fed steers, compared to pasture fed steers, and the rumen urea pool was significantly greater at 8 and 16-hours after feeding (P<0.05) for the fodder beet diet treatment, and the rumen ammonia pool significantly greater for the pasture diet treatment, across the entire diurnal cycle (P<0.05). From the results of this series of experiments, it was concluded that, in addition to nitrogen recycling strategies previously described, rumen retention of nitrogen in the early post-prandial period, by these several mechanisms identified in this study, was a possible novel rumen nitrogen management approach which may have influenced the high LWT gains observed with this diet by reducing rumen nitrogen losses. The role of rumen adaptation to a diet of low CP and NDF, but high WSC and water content, in any rumen nitrogen management mechanism should be an important consideration in further research.