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Intake, growth and carcass composition of defaunated and faunated lambs consuming oaten chaff based diet supplemented with nitrate as a source of non-protein nitrogen

Son Hung Nguyen and R S Hegarty1

Faculty of Animal Science, Vietnam National University of Agriculture, Hanoi, Vietnam
1 School of Environmental and Rural Science, University of New England, Armidale, NSW 2351, Australia


The effects of the presence or absence of rumen protozoa (defaunation) and of dietary nitrate (NO3) supplementation on feed intake, growth rate and carcass composition of Merino ewe lambs consuming a protein deficient diet were studied. Twenty ewe lambs were randomly allocated in a 2×2 factorial experiment (NO3 supplementation at 0% or 1.9% in dry matter basis and protozoa status either defaunated or faunated). Defaunation did not affect dry matter intake (DMI), liveweight (LW), average daily gain (ADG), carcass bone weight (CBW), carcass lean weight (CLW) or estimated carcass weight (CW) but significantly decreased carcass fat weight (CFW) and increased carcass lean percentage (CL%; p < 0.05). In contrast, NO3 supplementation significantly increased DMI, LW and ADG (p < 0.05). Further, NO3 -supplemented lambs showed greater CFW, CBW and CLW, but a lower CL% ( p < 0.01). There was no significant interaction between defaunation treatment and NO3 supplementation on DMI and ADG parameters (p > 0.05), but there was an additive interaction for faunated lambs supplemented with NO3 to have higher CW and CBW (p < 0.05).

Keywords: coconut oil distillate, fat, methaemoglobin, protozoa


Supplementation with NO3 has recently been confirmed as an effective method to mitigate enteric methane emissions of ruminants (Lee and Beauchemin 2014; Villar et al 2020) and also provides a non-protein nitrogen (NPN) source for ruminal microbial growth (de Raphélis-Soissan et al 2014; Hegarty et al 2016). Previous studies have largely focused on NO3 to replace urea in a protein sufficient diet as a mean to reduce methane production. Little is known about the effects of NO3 supplementation of protein deficient diets or in ruminants without ciliate protozoa, such as animal growth carcass weight and composition.

Defaunation of ruminants was reported to increase average daily gain (ADG) because the absence of rumen protozoa allowed a compensatory increase in bacterial populations, leading to more microbial protein outflow in defaunated compared to faunated animals (Newbold et al 2015). However, there is little data on carcass weight and its composition consequences of defaunation with mixed responses reported (Hegarty et al 2000). This experiment was conducted to examine the growth and carcass composition of defaunated and faunated lambs on either NO3 supplemented or non-supplemented diets of protein deficient oaten chaff. A previous report of the fermentation parameters in these sheep has already being published (Nguyen et al 2016a).

Materials and methods

Experimental procedure

All protocols for care and treatment of the experimental animals were approved by the University of New England Animal Ethics Committee (AEC 14-083). A 2×2 factorial design study was conducted with twenty Merino ewe lambs evaluating the factors of dietary NO3 inclusion (0% or 1.9% in dry matter basis) and protozoa status (either defaunated or faunated). Defaunated and faunated lambs were allocated to dietary nitrogen (N) levels by stratified randomisation based on liveweight (LW). The diet with calcium nitrate (NO3 as 5Ca(NO3)2.NH 4NO3.10H2O, Bolifor CNF, Yara, Oslo, Norway) was prepared by sprinkling a dilute solution of the NO3 salt onto oaten chaff while the chaff was tossed in a ribbon mixer (+NO3; Table 1). Another diet (control) was only oaten chaff (-NO3; Table 1).

Table 1. Chemical composition of oaten chaff with and without nitrate supplementation as fed to growing lambs

Component (% in DM)

Oaten chaff (-NO3)

Oaten chaff (+NO3)

Dry matter (% as fed)



Dry matter digestibility



Digestible organic matter



Inorganic ash



Organic matter



Neutral detergent fibre



Acid detergent fibre



Crude protein



Metabolisable energy (MJ/kg)



Nitrate-nitrogen (mg/kg)






# Nitrate was included as calcium nitrate (Bolifor CNF: 5Ca(NO3)2.NH4NO3.10H2O) with a nitrate content of 63.12%

Feed sampling and chemical analyses

Feed samples (~100 g) were collected before and after each mix of feed and stored at -20oC. All samples were pooled and sub-samples were taken to analyze chemical composition (Table 1). Feed samples were analyzed by the NSW DPI Feed Quality Service, Wagga Wagga Agriculture Institute, NSW, Australia. Feed dry matter (DM), acid detergent fibre (ADF), neutral detergent fibre (NDF), and inorganic ash were determined by wet chemistry. Feed dry matter digestibility (DMD) and digestible organic matter were determined by near-infrared spectroscopy. Feed crude protein (CP) was determined by the DUMAS combustion method (AOAC 990.03). The calculation of metabolisable energy (ME) was based on AFIA method (2-2R) and nitrate-N was determined by FIA SPAC10 (AFIA, 2014).

Defaunation of lambs

The defaunation procedure of lambs was described by Nguyen et al (2016a). Briefly, ten lambs were offered lucerne cereal mix supplemented with coconut oil distillate (COD) with initial inclusion of 3% to 5% of COD over 7 days to supress rumen protozoa. After 7 days feeding COD, lambs were fasted for 24h and orally dosed with sodium 1-(2-sulfonatooxyethoxy) dodecane (Empicol ESB/70AV, Albright and Wilson Australia Ltd, Melbourne) administered at 10 g/day in a 10% v/v solution for three consecutive days to remove protozoa. Feed was withheld during the three-day dosing period. Defaunated lambs required 12 days to recover to their pre-treatment voluntary intake and the 3-day dosing with Empicol was then repeated. Defaunated lambs were given 14 days after the second drenching program to recover from the defaunation treatment. Fourteen days after the completion of defaunation treatment, defaunated and faunated lambs were given ad libitum access to oaten chaff for 7 days before day 0. During the defaunation period, the 10 faunated lambs were restricted fed at their maintenance requirement (Freer et al 2007) to prevent divergence in LW while the defaunated group was being prepared. All lambs were held in individual pens throughout the entire preparation and experimental program.

Dry matter intake and liveweight

At commencement (21 days after completion of defaunation), lambs were gradually adapted to NO3-supplemented oaten chaff from day 0 to day 15 from an initial inclusion of NO3 of 1% up to 1.9% in DM basis in the diet, with the dose of calcium NO3 increased incrementally every two days. After this period of NO3 adaptation, lambs were given ad libitum access to NO3 -supplemented oaten chaff or oaten chaff alone, twice daily in two equal portions at 0930 and 1500 hours. Feed refusals were removed and weighed every morning before feeding, all samples were pooled each week and stored at -200C for later analysis. Drinking water was available at all times. Lambs were weighed at 0800 prior to feeding on days 0, 15, 21, 30, 65 and 93 to monitor LW changes and determine ADG over the experiment.

Estimation of carcass weight and its composition

On day 93 after weighing, 16 lambs were selected based on initial LW to place in a Computed Tomography (CT) scanner. A whole body scan from 3rd - 4th thoracic vertebrae and 1st - 2 nd caudal vertebrae with 5 mm thickness, 10 mm spacing and 480 mm field of view was performed using a Picker UltraZ 2000 CT scanner, Philips (Philips Medical Imaging Australia, Sydney, NSW) as described by Kvame and Vangen (2007). After scanning, each CT image was edited using the software program OsiriX (Rosset et al 2004) to estimate reticulorumen (RR) volume and then remove non-carcass tissues from each image, leaving carcass fat, carcass lean and carcass bone for estimation of carcass weight and composition. These images were further divided by greyscale into tissue areas of fat (-194 to -23 HU), lean (-22 to 146 HU) and bone (147 to 1024 HU) as defined by Houndsfield Units (HU) using the ImageJ software program, which was developed on methods similar to those described by Thompson and Kinghorn (1992) and these were corrected for tissue density to provide estimates of tissue weights of carcass fat weight (CFW), carcass bone weight (CBW) and carcass lean weight (CLW) based on the relationship between HU and density (Fullerton 1980).

Statistical analyses

All data were subject to analysis of variance in General Linear Model using Minitab 16; factors being protozoa, NO3 and protozoa × NO3 interaction. Data for the time intervals of 0-21d; 21-65d; 65-93d and 0-93d were analysed separately. For all analyses, means were analysed using the least squares means and compared in the pairwise comparisons in the Tukey method. A probability of error of less than 5% was considered to be statistically significant.


Because no interaction between defaunation treatment and NO3 supplementation was observed for DMI and ADG parameters, only the main effects of defaunation and NO3 supplementation are reported. The presence or absence of rumen protozoa did not affect DMI in any of the time intervals of 0-21d; 21-65d; 65-93d and 0-93d during the feeding trial ( p > 0.05; Table 2). Lambs without rumen protozoa exhibited higher ADG in the first interval of 0-21d (P < 0.05), but this effect diminished after d21 to the end of the experiment. Lambs supplemented with NO3 consistently consumed more DM compared to lambs without NO3 supplementation (p < 0.05; Table 2). The ADG of lambs was increased by NO3 in the time intervals of 0-21d; 21-65d and 0-93d, but was not significantly increased in the interval of 65-93d.

Table 2. Progressive dry matter intake (DMI) and average daily gain (ADG) of faunated (+P) and defaunted (-P) lambs with (+NO3) or without (-NO3) supplementation

Experimental intervals


DMI (g/d)

ADG (g/d)

0-21 days

- P






- NO3









21-65 days

- P






- NO3









65-93 days

- P






- NO3









0-93 days

- P






- NO3









Significant difference between (+P) v (-P) or (+NO3) v (-NO3):*P < 0.05; **P < 0.01; ***P < 0.001

The 16% increase in the final LW (p = 0.01; Table 3) by NO 3-supplemented lambs was probably due to increased DMI while defaunation did not increase the final LW of defaunated lambs. Neither defaunation nor NO3 supplementation affected RR volume, RR weight (including tissue and content) or the proportion of LW present as RR (p > 0.05). Defaunation did not increase estimated CW of defaunated lambs on the protein deficient diet, but NO3 supplementation increased CW of lambs by 22% (p < 0.001). There was a positive correlation between LW and CW across all data (CW = 0.32LW + 6.69; R2 = 0.47; p <0.01; Fig. 1). There was no effect of defaunation on CLW or CBW in the lambs, but CFW and, CF% was less while CL% was greater in defaunated compared to faunated lambs (p < 0.01). Nitrate inclusion significantly increased CFW, CBW and CLW, but CL% was significantly decreased by NO3. Further, an additive interaction for faunated lambs supplemented with NO3 to have higher CW and CBW (p < 0.05) were observed. There was a positive correlation between LW and CLW across all data (CLW = 0.21LW + 4.92; R2 = 0.63; p < 0.001; Fig. 2).

Table 3. Reticulorumen volume, reticulorumen weight, liveweight, carcass weight and weight and percentage of fat, bone and lean in the carcass of faunated (+P) and defaunted (-P) lambs with (+NO3) or without (-NO3) supplementation





-P (n=8)

+P (n=8)



P × NO3





Initial LW









Final LW









RR volume (cm3)









RR weight (g)









RR/LW ratio (%)








































































Reticulorumen: RR; Liveweight: LW; Carcass weight (kg): CW; Carcass lean weight (kg): CLW; Carcass fat weight (kg): CFW; Carcass bone weight (kg): CBW; Carcass lean percentage: CL%; Carcass fat percentage: CF%; Carcass bone percentage: CB%

Figure 1. Relationship between liveweight and carcass weight
estimated by Computed Tomography
Figure 2. Relationship between liveweight and carcass lean weight
estimated by Computed Tomography


Nitrate feeding has been widely studied as a mean to mitigate enteric methane emissions (van Zijderveld et al 2011; Lee and Beauchemin 2014; Velazco et al 2014) and potentially replace urea as a NPN supplement for protein deficient forages (Villar et al 2020). Defaunation has also been widely shown to increase amino acid supply to the intestine for a given feed intake and so enhance animal performance on low-protein diets while reducing enteric methane emissions (Newbold et al 2015). It was therefore, hypothesised that removal of rumen protozoa and/or introducing NO3 supplementation with this protein-deficient diet would improve the productivity of lambs.

Intake and growth performance

The growing lambs in this study were offered oaten chaff based diets which were characterised as protein-deficient diets, providing only 41 g CP per kg DM (control diet) and 71 g CP per kg DM (NO3-supplemented diet). Both diets provided less rumen degradable N than would be required for optimum rumen fermentation (Freer et al 2007) and this reflected in lower rumen NH3 concentrations [14 (-P) and 21 (+P) mgN/L; Nguyen et al (2016a)] than the 50mgN/L required. In this situation, rumen fill is likely to constrain feed intake (Weston, 1985) so the lack of difference in DMI with defaunation (735 v 783 g DM/lamb/day) was surprising considering the higher rumen NH3 concentration in refaunated lambs. A negative effect of defaunation on ruminal fibre degradation, due to the loss of protozoal fibrolytic activity (Eugène et al 2004; Newbold et al 2015) may have tempered any response to increased NH3 availability. Santra and Karim (2002) reported greater polysaccharidase enzyme activity in the rumen of faunated animals, leading to a positive effect of protozoa on ruminal digestion.

Protozoa-free lambs in this study showed higher ADG compared to lambs with protozoa during the first 21d, but did not exhibit a greater ADG at any time subsequently. This was consistent with a previous study by van Nevel et al (1985). Bird and Leng (1978) reported that defaunated cattle increased growth rates by 43% when supplemented with 240g of protein pellets compared with faunated cattle. This largely degradable protein significantly stimulated ruminal microbial growth in response to defaunation. In an extensive review by Newbold et al (2015) showed that defaunated animals had a greater efficiency of microbial protein synthesis by 27%, leading to a greater ADG by 9%. These positive effects of defaunation were observed with poor quality diets in which fermantable carbohydrate and rumen degradatble protein were low (Williams and Coleman 1992).

The more substantial and significant rise in rumen NH3 concentration with NO3 supplementation [29 v 9 mgN/L; Nguyen et al (2016a)] may have underpinned a faster rate of ruminal DM fermentation and thus the greater DMI in NO3 supplemented compared to unsupplemented lambs (896 v 623 g DM/lamb/day). The greater feed intake would itself have supported the increased ADG (52 v -17 g/lamb/day) achieved by NO3 supplementation. This confirms the role of NO 3 as a potentially valuable NPN source for rumen microbiota, though the risk of NO3 poisoning is recognised (Nolan et al 2016). Dietary NO3 can replace urea as a NPN source to reduce enteric methane emissions (Leng and Preston 2010; Nolan et al 2010; van Zijderveld et al 2011; Lee and Beauchemin 2014). The review by Lee and Beauchemin (2014) reported that supplementation with NO3 or urea supported similar growth rates of ruminants but there are few previous studies on low-protein roughages.

Reticulorumen volume and carcass composition

Defaunation did not alter RR volume, RR weight or the ratio of RR to LW in defaunated lambs compared to faunated lambs. This was inconsistent with Orpin and Letcher (1984) who reported a 30% increased rumen volume associated with defaunation. Jouany et al (1988) reported the change in rumen volume following defaunation resulted from changes in ruminal digestion. The increased weight of ruminal contents after defaunation was probably due to longer particle retention of ruminal digesta associated with the rumen fill effect of lower DMD (Eugène et al 2004). The similar DMI of defaunated and faunated lambs in this study may be a reason why no effect of defaunation treatment on RR volume or RR weight were observed.

There is little data on carcass composition consequences of defaunation in lambs and NO3 supplementation. Nguyen et al (2016b) reported that defaunation tended to increase RR weight, but did not affect CW or its composition. The current study found that CFW and CF% of defaunated lambs were lower while CL% was higher in defaunated compared to faunated lambs. This was consistent with Demeyer et al (1982) and van Nevel et al (1985) who slaughtered defaunated animals and reported that defaunation tended to have more carcass lean and less fat percentage in the carcass. Greater C-site fat depth and tissue thickness at the GR site of lambs close to finishing weights were also reported in a flock of defaunated lambs relative to their faunated cohort (Hegarty et al 2000). In contrast, NO 3-supplemented lambs significantly increased CW, CFW and CLW of the growing lambs fed protein deficient diets. These positive effects of NO 3, however were not observed when feedlot beef cattle consumed protein sufficient diets supplemented with NO3 or urea as NPN sources (Hegarty et al 2016). Nitrate, therefore is confirmed not only acting as a H2 sink to mitigate enteric methane emissions but also providing necessary NPN sources for rumen microbes.

All NO3-supplemented lambs were measured for whole-blood methaemoglobin (MetHb) on days 0, 15, 50 and 85 between 2.5-3h after feeding with the average of [3.52% (-P) and 0.89% (+P); Nguyen et al (2016a)]. Defaunated lambs showed increased blood MetHb after 85 days of feeding NO3 which was consistent with a finding by Lin et al (2011), suggesting that rumen ciliate protozoa may have an important role in the reduction of NO3 into NH3 in the rumen. However, levels of measured blood MetHb of defaunated and faunated lambs on NO3 were far below a toxicity level suggested by Bruning-Fann and Kaneene (1993) and all NO3-supplemented lambs were healthy and had no clinical sign of NO3 toxicity.



This research was funded by Australian Department of Agriculture, National Livestock Methane Program and Meat and Livestock Australia. The authors are grateful for the technical support from Mr Graeme Bremner and Mr Andrew Blakely at the University of New England, Australia.


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