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Growth performance, caecal microbiome profile, short-chain fatty acids, and litter characteristics in response to placement on reused litter and combined threonine, arginine and glutamine supplementation to juvenile male broiler chickens

Growth performance, caecal microbiome profile, short-chain fatty acids, and litter... Background Exposure of broilers to litter microbiome may increase specific amino acid (AA) requirements towards activated immune responses. This may challenge the generality of the ideal protein (IP) concept, in which dietary essential AA to lysine ratios aimed to mimic presumably constant AA to lysine ratios in whole bird requirements. Therefore, we tested the effect of threonine, arginine and glutamine ( TAG) supplementation to IP-based control diets (C) on performance, caecal microbiome composition, short-chain fatty acids and litter characteristics of broiler chick- ens placed on reused litter. Results Thirty-two pens with ten male broiler chickens each were used in a 2 × 2 factorial arrangement of two diet treatments (with or without TAG supplementation) and two litter treatments (placement on clean or reused litter) for 21 days (n = 8). Caecal contents were analysed for microbiome profile using percent guanine + cytosine (%G + C profile) method and short chain fatty acids. TAG-supplemented birds underperformed compared to C birds (P = 0.002), whereas birds placed on reused litter outperformed those on clean litter (P = 0.047). Diet, reused litter and their interaction impacted the %G + C profile at different ranges. Whilst TAG supplementation reduced bacterial abundance at %G + C 51–56 (P < 0.05), reused litter placement tended to reduce %G + C 23–31 and increase %G + C 56–59 (P < 0.10). However, TAG supplementation reduced bacterial abundance at %G + C 47–51 (P < 0.05) and increased caecal branched chain fatty acids on clean litter only (P = 0.025). Greater levels of propionic acid were observed for C birds placed on reused litter only (P = 0.008). Litter pH was greater for reused litter pens than clean litter pens at day 21 (P < 0.001). In addition, litter moisture content was less for TAG birds and reused litter pens compared to C birds (P = 0.041) and clean litter pens (P < 0.001), respectively. Conclusions These data support the view that irrespective of performance benefits arising from bird placement on reused litter, TAG supplementation to IP-formulated baseline rations impaired growth, supported by the lowered *Correspondence: Marwa A. Hussein marwa.hussein@sruc.ac.uk Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Hussein et al. Animal Microbiome (2023) 5:18 Page 2 of 15 abundance of caecal bacteria known to dominate in well-performing birds and greater levels of caecal branched chain fatty acids. Keywords Amino acids, Reused litter, Ideal protein, Broilers, Growth performance, Caecal microbiome, Litter characteristics Introduction enhanced growth performance during sub-clinical Dietary protein requirement should represent the sum challenges upon Gln supplementation has been shown to of a balanced level of essential amino acids (AA) and concur with improved gut morphology, i.e., increased sufficient non-essential AA that fulfils age-dependant villus height and lowered crypt depth, which improves AA requirement for optimal performance. This view is absorptive capacity [30, 31]. Wu et  al. [14] further the basis for the ideal protein (IP) concept that has been reported that Gln supplementation may ameliorate introduced into diet formulation for poultry to optimise detrimental effects of Salmonella enteritidis infection on AA supply and, thus, nitrogen utilisation [1–3]. The IP intestinal immune barrier functions and lymphoid organ concept represents dietary essential AA (eAA) to lysine weights (i.e., bursa of Fabricius, spleen and thymus). ratios as in whole bird AA requirements for maintenance Host responses to specific AA supplementation may be and production, leading to an ideal AA profile in sensitive to the level of microbiota exposure, as immune which all eAA are equally limiting [4, 5]. Several studies and possible pathological responses could result in reported a positive impact of AA supplementation on increased whole bird AA requirements that deviate from performance, e.g. threonine (Thr) [6–8], arginine (Arg) the ideal AA profile. In the current study, placement [9–11], but also the non-essential amino acid glutamine on reused litter, which may impact caecal microbiome (Gln) [12–14]. However, AA supplementation to an IP characteristics [32], was used to create two contrasting formulated baseline would not be expected to improve levels of microbiota exposure, under which the AA performance but result in excess AA intake. The latter supplementation was tested. We hypothesised that the would be expected to facilitate proteolytic activity of the effect of Thr, Arg and Gln (TAG) supplementation to IP hindgut microbiome resulting in changes in composition formulated basal rations on broiler growth performance, and/or metabolite production, deteriorated litter caecal microbiome parameters, and litter characteris- quality and potentially reduced performance [15]. However, tics (pH and moisture content) is sensitive to reused birds under external microbial and other pathogen litter exposure. To our knowledge, this is the first time that exposure would be expected to require different dietary the effects of AA supplementation to IP-based rations AA ratios for the combined optimal performance and are assessed on caecal microbiome composition and enhanced immune responses, arising from competition fermentation metabolites of broilers placed on reused litter. for limiting AA between these functions [16–19], which may thus challenge the generality of the IP concept. Results Each of Thr, Arg and Gln has been implicated in host Diet analysis and growth performance responses to viral, bacterial and/ or parasitic exposure. Although relative to the TAG diets, the non-TAG AA For instance, Thr plays a vital role in the maintenance of levels of the C diets were on average 4% greater and 3% intestinal barrier integrity and mucin synthesis [8, 20] smaller for the starter and grower phase, respectively, and is a major component of gamma globulin [21, 22]. overall, the analysed nutrient and individual AA compo- Supplementation with Thr has been shown to increase sition of the experimental diets were within the expected hemagglutination titres of birds infected with the New- range (Table  1). Since ~ 98.5% of the diets were derived castle disease virus [23]. In addition, Thr supplemen - from a common basal, and the part that varied consisted tation has been found to improve gut health during of starch or TAG only, observed variation likely reflects salmonellosis in broilers [24]. The supplementation with variation in analysis rather than an actual chemical com- Arg, which is a precursor for nitric oxide, polyamines, position. The effect of TAG supplementation and litter and creatine [25], has been shown to improve intestinal treatments on growth performance during the entire morphology [26] during sub-clinical enteric challenges growth phase (days 0–21) is shown in Table 2. There were as it can ameliorate coccidiosis-induced intestinal vil- no significant interactions between diet and litter treat - lus damage and goblet cell depletion [27]. As the main ment on performance data. However, diet treatment energy source for immune and intestinal epithelial cells, impacted growth performance measurements, i.e., body Gln could become limiting during elevated Gln require- weight gain (BWG), feed intake (FI) and crude protein ments arising from enteric challenges [28, 29]. Indeed, conversion (CPC), as TAG birds had smaller BWG, FI Hussein  et al. Animal Microbiome (2023) 5:18 Page 3 of 15 Table 1 Analysed chemical composition, gross energy, and total Table 2 Growth performance of broilers fed C or TAG diets and amino acid content of the experimental starter (0 to 11 days) and placed as day-old on either clean or reused litter over 21 days grower (11 to 21 days) rations Litter Diet BWG, g FI, g FCR, g/g CPC* Starter rations Grower rations Clean C 738 1044 1.447 0.298 C TAG C TAG TAG 663 914 1.417 0.319 Reused C 779 1028 1.349 0.278 Chemical composition TAG 708 947 1.371 0.308 DM (%) 88.00 89.00 88.30 88.30 SED 28.8 21.7 0.051 0.011 Crude ash (%) 6.20 10.40 5.70 5.30 Means for main effect of litter CP (%) 22.82 24.12 21.14 23.21 Clean 700 979 1.432 0.308 NDF (%) 7.80 7.90 7.70 7.50 Reused 743 988 1.360 0.293 ADF (%) 4.15 3.88 3.53 3.48 SED 20.4 15.4 0.036 0.008 Sucrose (%) 5.36 3.95 4.17 4.44 Means for main effect of diet Total starch (%) 35.80 34.10 39.80 36.00 b b a C 758 1036 1.398 0.288 EE (%) 3.83 4.21 4.80 4.53 a a b TAG 685 931 1.394 0.313 AHEE (%) 4.74 5.02 5.74 5.24 SED 20.4 15.4 0.036 0.008 GE (MJ/kg) 16.45 16.63 16.77 16.77 P-values for main effects and interaction Amino acids composition (%) Litter 0.047 0.581 0.056 0.068 Methionine 0.55 0.50 0.46 0.50 Diet 0.002 < 0.001 0.902 0.004 Cysteine 0.36 0.36 0.35 0.35 Litter × diet 0.924 0.134 0.483 0.544 Methionine + cysteine 0.90 0.86 0.81 0.85 Lysine 1.45 1.31 1.26 1.35 BWG Body weight gain; FI Feed intake; FCR Feed conversion ratio; CPC* Crude protein conversion = FI (kg) × CP content diet (g/kg)/BWG (g); C Control diets; Threonine 0.98 1.16 0.87 1.10 TAG Threonine, arginine and glutamine supplemented diets; SED Standard error Arginine 1.48 1.76 1.36 1.68 of difference; Means within the same column with different superscripts differ at P < 0.05; Simple means represent 8 pens of 10 birds per pen Isoleucine 0.95 0.90 0.88 0.90 Leucine 1.55 1.49 1.47 1.50 Valine 1.10 1.02 0.99 1.02 as a response to diet or litter treatment and thus enables Histidine 0.52 0.50 0.50 0.51 the detection of any putative alterations at the commu- Phenylalanine 1.02 0.98 0.97 1.00 nity level. Diet and litter treatment interacted for caecal Glycine 0.87 0.85 0.84 0.84 %G + C 47–51 (P < 0.01) as birds fed the test diet (TAG) Serine 1.04 1.02 0.99 1.01 compared to birds fed the control diet (C) showed a lower Proline 1.33 1.33 1.27 1.33 abundance over that range but only when placed on clean Alanine 0.88 0.85 0.84 0.86 litter (Fig. 1). However, diet treatment affected %G + C at Aspartic acid 2.04 1.95 1.91 1.97 a higher range, as TAG birds displayed a significant shift Glutamic acid 4.31 5.19 4.17 5.16 towards a lower abundance of bacteria at %G + C 51–56 than C birds (Fig. 2). In contrast, litter treatment did not C Control; TAG Threonine, arginine and glutamine supplemented diets; DM Dry matter; CP Crude protein; EE Ether extract; AHEE Ether extract preceded by significantly affect %G + C profile; the consistently lower acid hydrolysis; NDF Neutral detergent fibre; ADF Acid detergent fibre; GE Gross %G + C 23–31 and greater %G + C 56–59 for birds on energy reused litter compared to those on clean litter averaged at P = 0.112 and P = 0.099, respectively (Fig. 3). and larger CPC than C birds. Furthermore, birds placed Caecal SCFA concentration and composition on reused litter had greater BWG and tended to have The total short chain fatty acids (SCFA) concentration in better feed conversion ratio (FCR) and CPC than birds the caecal content and its composition were determined placed on clean litter. Mortality was low at 0.3% (1 out of as an indicator of the fermentative activity of the micro- 320 birds placed). This 11-day-old bird was culled due to bial population and are presented in Table 3. Total SCFA hunched posture. The post-mortem reported that there concentration did not significantly differ between treat - was a large yolk sac remnant with necrotic content. ments. However, a significant interaction between diet and litter treatments was observed for the percentage of Caecal %G + C profile propionic acid and branched-chain fatty acids (BCFA); The percent guanine + cytosine (%G + C) profile of the the latter consisted of iso-butyric acid only, as the other total chromosomal DNA was determined to illustrate the two BCFA (2-methyl-butyric acid and iso-valeric acid) relative abundance of the entire microbial community Hussein et al. Animal Microbiome (2023) 5:18 Page 4 of 15 Diet ×Litter interaction effect 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 -1 %G + C C+Clean TAG+Clean C+Reused TAG+Reused P- values from ANOVA 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 %G + C Diet × Litter 0.05 Fig. 1 Diet and Litter interaction effects on the %G + C profile of caecal bacteria from broilers aged 21 days. In the upper panel, the solid blue line represents the mean %G + C profile of birds fed C diets and placed on clean litter, the solid red line represents the mean %G + C profile of birds fed TAG diets and placed on clean litter, the solid green line shows the mean %G + C of birds fed C diets and placed on reused litter and the solid purple line illustrates the mean %G + C of birds fed TAG diets and placed on reused litter (n = 8). In the lower panel, the solid blue line shows the results from ANOVA and the solid red line marks the threshold of P = 0.05 Relative abundance, A 280 nm P- value Hussein  et al. Animal Microbiome (2023) 5:18 Page 5 of 15 Main effect of Diet 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 -1 %G + C C TAG P-values from ANOVA 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 %G + C Diet 0.05 Fig. 2 The main effect of Diet treatment on the %G + C profile of caecal bacteria from broilers aged 21 days. In the upper panel, the solid blue line represents the mean %G + C profile of birds fed C diets and the solid red line shows the mean %G + C profile of birds fed TAG diets (n = 16). In the lower panel, the solid blue line shows the results from ANOVA and the solid red line marks the threshold of P = 0.05 Relative abundance, A 280 nm P-value Hussein et al. Animal Microbiome (2023) 5:18 Page 6 of 15 Main effect of Litter 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 -1 %G + C Clean Reused P- values from ANOVA 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 %G + C Litter 0.05 Fig. 3 The main effect of Litter treatment on the %G + C profile of caecal bacteria from broilers aged 21 days. In the upper panel, the solid blue line represents the mean %G + C profile of birds placed on clean litter and the solid red line represents the mean %G + C profile of birds placed on reused litter (n = 16). In the lower panel, the solid blue line shows the results from ANOVA and the solid red line marks the threshold of P = 0.05 Relative abundance, A 280 nm P- value Hussein  et al. Animal Microbiome (2023) 5:18 Page 7 of 15 Table 3 Short chain fatty acids in the ceca of 21-day old broilers fed C or TAG diets and placed on either clean or reused litter Litter Diet Total SCFA (mM) %Acetic acid %Propionic acid %Butyric acid %Lactic acid %Iso- butyric acid a a Clean C 105.10 72.45 3.98 13.34 10.07 0.10 ab b TAG 97.60 71.32 5.67 12.88 9.79 0.32 b a Reused C 92.20 73.99 6.35 10.45 9.08 0.12 a a TAG 92.40 72.26 3.65 11.79 12.14 0.16 SED 8.15 2.06 1.05 1.55 1.80 0.05 Means for main effect of litter Clean 101.40 71.88 4.83 13.11 9.93 0.21 Reused 92.30 73.13 5.00 11.12 10.61 0.14 SED 5.77 1.46 0.74 1.09 1.27 0.04 Means for main effect of diet C 98.60 73.22 5.16 11.89 9.57 0.11 TAG 95.00 71.79 4.66 12.33 10.97 0.24 SED 5.77 1.46 0.74 1.09 1.27 0.04 P values for main effects and interaction Litter 0.129 0.404 0.819 0.083 0.597 0.064 Diet 0.536 0.337 0.508 0.690 0.286 0.003 Litter × diet 0.515 0.838 0.008 0.418 0.204 0.025 SCFA Short chain fatty acids; C Control diets; TAG Threonine, arginine and glutamine supplemented diets; SED Standard error of difference; Means within the same column with different superscripts differ at P < 0.05; Simple means represent 8 pens of pooled sampled from 2 birds sampled per pen were below the detection limit. C birds had a greater Table 4 pH and moisture content of litter samples of 21-day-old broilers fed C or TAG diets and placed on either clean or reused percentage of propionic acid than TAG birds on reused litter at day-old litter only. In addition, whilst TAG birds had a greater proportion of iso-butyric acid than C birds across litter Litter Diet pH Moisture, % treatments, the interaction indicated this was most pro- Clean C 6.67 30.00 nounced on clean litter. TAG 6.57 27.36 Reused C 7.35 25.10 Litter characteristics TAG 7.40 22.78 At day 0, polymerase chain reaction (PCR) screens did SED 0.074 1.674 not detect Salmonella spp., Clostridium perfringens, Means for main effect of litter Eimeria tenella and E. maxima in either clean or reused a b Clean 6.62 28.68 litter. However, the 16S rRNA gene copy numbers of b a Reused 7.38 23.94 total bacteria per g of chicken litter were 2.08 and 8.13 SED 0.052 1.184 copies per g of clean and reused litter, respectively. The Means for main effect of diet initial pH of clean and reused litter was 5.76 and 8.09, C 7.01 27.55 respectively, whilst their moisture content was 10.79 and TAG 6.99 25.07 12.49%, respectively. SED 0.052 1.184 The diet and litter treatment effects on final litter pH P values for main effects and interaction and moisture content are presented in Table  4. There Litter < 0.001 < 0.001 were no significant interactions between diet and litter Diet 0.707 0.041 treatments for both parameters. Whilst diet treatment Litter × Diet 0.172 0.894 did not impact litter pH, placement of birds on reused C Control; TAG Threonine, arginine, and glutamine supplemented diets; Means litter resulted in significantly higher litter pH than their within the same column with different superscripts differ at P < 0.05; SED, clean litter counterparts. In addition, both diet and litter standard error of difference; Simple means represent 8 pens per treatment treatments independently impacted litter moisture con- tent, as the TAG and reused litter treatments reduced moisture content compared to the C and clean litter treatments, respectively. Hussein et al. Animal Microbiome (2023) 5:18 Page 8 of 15 Discussion deposition [44, 45]. This suggestion is supported by the This study investigated the effects of Thr, Arg and Gln reduced FI of TAG birds over C birds, as FI depression is supplementation to IP formulated diets on perfor- one of the first manifestations of dietary AA imbalance in mance, caecal microbiome and litter characteristics in broilers [46, 47]. In addition, it has also been suggested the absence and presence of placement at reused litter to that AA supplementation may reduce FI arising from create two contrasting conditions in terms of microbiota the amino static hypothesis, in which free AA in plasma exposure. We hypothesised that reused litter exposure at serve as a signal to an appetite-controlling mechanism placement would affect the growth performance response [48–50]. to TAG supplementation. However, this hypothesis was The %G + C profile is used to indicate the relative rejected as TAG supplementation reduced performance abundance of bacteria with different DNA base compo - for both clean and reused litter treatments. Supported by sitions and hence allows detecting any putative altera- effects on caecal microbiome composition and SCFAs, tions at the community level [51]. The most abundant collectively the data indicated that TAG supplementation bacteria observed in our study represent species with may have resulted in excess protein over the IP-basis, %G + C 40–55, such as Lachnospiraceae (Clostridial which disadvantaged bird performance [33–35]. cluster IV) and Lactobacilli, which are known to domi- Here, birds placed on the reused litter had 6% greater nate caecal microbiome composition of well-performing BWG than those on clean litter. The use of reused litter birds [52]. However, under-performing birds often have has been shown to result in variable outcomes on perfor- two peaks at < ~ 37% and > ~ 60 of %G + C instead of one mance, as it has been associated with penalised [36–39], peak at ~ 45%G + C [52]. Here, TAG supplementation similar [40, 41] or improved [42] performance relative resulted in a lower abundance of bacteria with %G + C to birds placed on clean litter. Such variable outcomes 47–56, which indeed concurred with reduced growth of using reused litter can be expected to arise from dif- performance over C birds, though there was no signifi - ferences in litter characteristics, which are typically not cant increase in the %G + C 20–30, which is often asso- reported in studies using reused litter, and include micro- ciated with the presence of pathogenic bacteria [53]. bial composition, pH, moisture, and level of recycled This suggests that a possible microbiological basis of nutrients from the previous flock. An early exposure of the reduction in performance on TAG diets was most newly hatched chicks to reused litter facilitates the colo- likely metabolic rather than pathogenic. Although birds nisation and cycling of microbiomes between gut and on reused litter performed better and showed a greater litter which accords with a probiotic or direct-fed micro- proportion of propionic acid in their SCFA pool than bial approach to improve intestinal microbiota and thus those on clean litter, this did not concur with significant improved performance [32, 42]. In support of the positive changes in microbial profile. Whilst there was some indi - effect of reused litter exposure on performance reported cation that the reused litter treatment indeed lowered here, pathogens such as C. perfringens, E. tenella and E. bacterial abundance associated with %G + C 23–31 and maxima were not detected in the tested litter samples. increased bacterial abundance at %G + C 56–59 (Fig.  3), The reduced performance upon TAG supplementation these did not reach statistical significance in this study. may have a multi-factorial basis. Firstly, since diets were Caecal SCFA analysed include the volatile fatty formulated to be isoenergetic, TAG supplementation acids (VFA) acetate, propionate and butyrate, but also as expected increased CP content and thus reduced the the non-volatile lactate, produced by gut microbiota metabolisable energy to CP ratio. The latter may directly as fermentation products from undigested nutrients reduce the AA pool for protein deposition and uric acid [54]. The SCFA play a role in intestinal health, includ - synthesis as well as the supply of fat and carbohydrate to ing the promotion of mucin production, blood flow, meet the energetic requirements of the birds [43]. Sec- enterocytes growth and proliferation [55]. The VFAs ondly, the AA availability in TAG supplemented birds mentioned are a valuable energy source for the host, might be lower than expected for growth due to increased especially butyrate being the preferred energy source proteolytic fermentation from excess AA, which has for epithelial cells [56]. Furthermore, the increased pro- been shown to result in poorer intestinal health [33]. portion of propionic acid in the SCFA pool of birds on Around half of the undigested and unabsorbed protein reused litter and fed C diets could indicate the presence is fermented by putrefactive caecal bacteria producing of beneficial bacteria such as Lactobacillus spp., which toxic compounds, i.e., amines, indoles, phenols, cresol are known to have bacteriostatic or bactericidal proper- and ammonia, which may impede performance [34, 35]. ties against pathogenic microbes [52, 57]. This accords Thirdly, the AA imbalance arising from surplus AA in with the improved performance observed for birds on TAG supplemented birds might decrease the efficiency reused litter and fed C diet. The BCFA (iso-butyric, of utilisation of limiting AA for maintenance and protein 2-methyl-butyric and iso-valeric) within the SCFA pool Hussein  et al. Animal Microbiome (2023) 5:18 Page 9 of 15 can only be produced from fermenting branched-chain Conclusions AA, i.e., valine, leucine, and isoleucine. As such, vari- In this study, Thr, Arg and Gln supplementation to IP- ation in caecal BCFA levels may indicate variation in based diets altered caecal microbial composition and protein fermentation activity but also the flow of undi - enhanced proteolytic fermentation, indicative of excess gested protein into the caecum. Thus, elevated caecal protein leading to impaired performance. However, this BCFA could be indicative of reduced ileal crude protein study also supports the view that reused litter, particu- (CP) digestibility, which would result in poorer growth larly as assessed here in the absence of pathogenic bac- performance [35, 58]. This is consistent with the ele - teria, might benefit bird performance. The use of such vated levels of iso-butyric acid in TAG- supplemented litter accords with a probiotic or direct-fed microbial birds, being most pronounced on clean litter, and the approach, combined with being a source of recycled reduced performance observed for those birds. nutrients. Litter pH and moisture content are some of the major determinants implicated in the survival and growth Materials and methods of litter pathogens [59]. Generally, litter pH ranges Bird management and experimental design between 6.5 and 8.5, with negligible ammonia produc- A total of 320 male Ross 308 broiler chickens were tion below pH 7 [60, 61]. In the current study, diet treat- used in a 21-day experiment. Upon arrival (day 0), the ment did not have a significant impact on the final litter birds were allocated to 32 floor pens (1.47 m × 0.94  m), pH, though reused litter pens had greater final pH levels separated through plastic-sheeted panels, with 10 birds than clean litter pens. However, temporal effects need to per pen in a randomised complete block design. The be considered, as pH for the clean litter pens increased temperature was set to 32  °C for the first 3  days and from 5.76 at day 0 to 6.62 at day 21, whilst for the reused then was gradually reduced over a week until 25  °C litter pens, pH decreased from 8.09 to 7.38. Both the was reached and maintained until day 21 as per breed difference at day 0 between clean and reused litter and guidelines. The light was provided for 23  h per day for the increase in pH for the clean litter pens over time can the first week and then reduced to 18  h of light per be attributed to the accumulation of excreta during the day. Birds were provided ad libitium access to feed and grow-out period, with elevated pH arising from protein water throughout the experiment, with feed offered degradation and ammonia production [62, 63]. How- as a meal. Birds were fed wheat-soyabean meal-based ever, whilst accumulation of excreta would also have starter (0–11 days of age) and grower diets (11–21 days occurred for the reused litter pens, the net reduction in of age) with the control diets (see below) formulated to pH overtime for these pens may be the consequence of meet Ross 308 nutrient recommendations [68]. continuing composting activity in  situ as litter pH was The experimental set up consisted of a 2 × 2 facto- observed to reduce over a period of 28  days for stored rial arrangement of two diet treatments and two lit- litter [64]. These data suggest that temporal rather than ter treatments (see below) with 8 pens per treatment current variation in litter pH may better inform varia- combination within a complete randomised block tion in performance, where the latter was greater for arrangement. birds on reused litter compared to those on clean litter. The final litter moisture for the TAG treatment was Diet treatments lower than that for the C treatment. This could be related The control (C) diet was formulated on an IP basis and to the reduced FI for the TAG birds, which would have supplemented with the synthetic AA to meet all eAA concurred with reduced water intake as well as reduced requirements on a digestible AA basis. The TAG treat - total metabolites elimination with the excreta [65], both ment consisted of feeding the C diet with additional contributing to reduced water spillage and excretion. Thr and Arg at 25% above requirements and 1% Gln, However, at similar levels of FI, reused litter pens also as informed by previous studies [16, 69, 70]. For each had lower final moisture content compared with clean lit - phase, a common basal diet was prepared by including ter pens. This suggests other reasons might also explain a 3% of corn starch for the C diets, against which the the variation in litter moisture and accords with previ- tested AA were included for the TAG diets. The TAG ous studies [66, 67], where lower moisture content in lit- diets were therefore calculated to be isoenergetic but ter used for multiple grow-outs (reused litter) was also with varying CP levels. The ingredients and the calcu - observed. Reused litter has been found to have lower lated chemical compositions of the starter and grower water activity and faster rate of excreta drying than clean diets are presented in Table 5. litter, which might detriment the survival and growth of litter pathogens [67], and benefit performance. Hussein et al. Animal Microbiome (2023) 5:18 Page 10 of 15 Litter treatments Table 5 Feed ingredients and calculated chemical compositions (%) of the experimental starter (0–11 days) and grower (11– For each diet treatment, half of the pens were supplied 21 days) rations with all new wood shavings (clean litter) and the other half had 100% reused wood shavings litter (reused lit- Starter rations Grower rations ter). The reused litter was derived from a previous C TAG C TAG 1152-bird broiler study (Ross 308) with no history of clinical diseases. The duration between litter collec - Ingredients tion and its reuse at the start of the current trial was Corn starch 3.00 1.44 3.00 1.51 28 days, during which litter was untreated and stored in Threonine 0.23 0.45 0.18 0.37 bags in an empty unheated shed. Arginine 0.10 0.44 0.05 0.35 Glutamine 0.00 1.00 0.00 1.00 Wheat 58.22 58.22 60.56 60.56 Sampling and data collection Soybean meal 31.59 31.59 28.50 28.50 Chemical analysis of diets Soya oil 2.20 2.20 3.50 3.50 Experimental diets were analysed for dry matter (DM), Salt 0.05 0.05 0.05 0.05 neutral detergent fibre (NDF), acid detergent fibre (ADF), Limestone 0.95 0.95 0.87 0.87 ether extract (EE), ether extract preceded by acid hydrol- Dicalcium phosphate 1.85 1.85 1.65 1.65 ysis (AHEE), ash, starch, and total sugar (as sucrose) at Sodium bicarbonate 0.50 0.50 0.50 0.50 Sciantec Analytical Services Ltd. (Cawood, UK) using Lysine HCl 0.39 0.39 0.32 0.32 standard protocols based upon Commission Regula- Methionine 0.24 0.24 0.21 0.21 tion (EC) No. 152/2009. Analysis of CP and AA content, Valine 0.09 0.09 0.06 0.06 including tryptophan, were performed at Evonik Nutri- Tryptophan 0.14 0.14 0.12 0.12 tion & Care GmbH (Hanau-Wolfgang, Germany). The CP Isoleucine 0.05 0.05 0.03 0.03 was estimated using the Dumas method, and AA analysis Vitamin & mineral premix 0.40 0.40 0.40 0.40 was done by standard procedures [71] using an AA ana- Calculated chemical composition lyser (Biochrom 30 + , Cambridge, UK). Tryptophan was Crude protein % 22.47 24.51 21.01 22.95 determined by high-performance liquid chromatography AME MJ/kg 12.51 12.51 12.94 12.94 following preparation by hydrolysis. Gross energy (GE) Calcium % 0.96 0.96 0.87 0.87 was determined through an isoperibol bomb calorimeter Phosphorous % 0.72 0.72 0.67 0.67 system using benzoic acid as an internal standard (model Available phosphorous % 0.48 0.48 0.44 0.44 6200, Parr Instruments, Moline, Illinois, USA). Salt % 0.19 0.19 0.16 0.16 Sodium % 0.19 0.19 0.19 0.19 Growth performance Chloride % 0.16 0.16 0.15 0.15 Growth performance parameters, i.e., BWG, FI, and FCR, Digestible essential amino acids % were calculated from mean body weights (BWT) through Threonine 0.86 1.08 0.77 0.96 bulk weighing and bird counting at pen level, weights of Arginine 1.37 1.71 1.23 1.53 feed offered on days 0 and 11, and weights of feed refusals Histidine 0.48 0.48 0.45 0.45 on days 11 and 21. The resulting BWG, FI and FCR were Isoleucine 0.86 0.86 0.78 0.78 calculated for the entire growth period of days 0 to 21. Leucine 1.40 1.40 1.31 1.31 Birds that were found dead or were culled were recorded Lysine 1.28 1.28 1.15 1.15 for date, weighed and sent for post-mortem examination. Methionine 0.51 0.51 0.47 0.47 BWG and total pen FI were corrected for mortality. FCR Cysteine 0.31 0.31 0.30 0.30 was calculated by dividing the average feed consumed per Tryptophan 0.20 0.20 0.19 0.19 pen by the average weight gain of birds per pen. CPC was Valine 0.96 0.96 0.87 0.87 calculated by multiplying the average feed consumed by Methionine + cysteine 0.82 0.82 0.77 0.77 the dietary CP content and divided by the average weight Phenylalanine + tyrosine 1.58 1.58 1.48 1.48 gain of birds per pen as CPC = FI (kg) × CP content diet (g/kg)/BWG (g). AME Apparent metabolisable energy; C Control diets; TAG Threonine, arginine and glutamine supplemented diets; Vitamin and mineral premix provided −1 (units kg diets): Vit A, 16,000 IU; Vit D3, 3,000 IU; Vit E, 75 IU; Vit B1, 3 mg; Caecal microbiome profile and short chain fatty acid analysis Vit B2, 10 mg; Vit B6, 3 mg; Vit B12, 15 µg; Vit K3, 5 mg; Nicotinic acid 60 mg; Pantothenic acid 14.5 mg; Folic acid 1.5 mg; Biotin 275 µg; Choline chloride At day 21, caecal digesta was collected in a sterile petri- 250 mg; Iron 20 mg; Copper 10 mg; Manganese 100 mg; Cobalt 1 mg; Zinc dish from two randomly selected broilers per pen after 82 mg; Iodine 1 mg; Selenium 0.2 mg; Molybdenum 0.5 mg Hussein  et al. Animal Microbiome (2023) 5:18 Page 11 of 15 being individually weighed, electrically stunned, and Table 7 Positive controls of the pathogens used in this study exsanguinated. Approximately 1  g of the pooled cae- Positive strain Source cal content of the two birds was immediately preserved Salmonella enterica subsp. SRUC Veterinary Services using BioFreeze sampling kits (Alimetrics Diagnostics enterica serotype Poona Ltd., Espoo, Finland) following their recommended pro- C. perfringens type A isolate SRUC Veterinary Services tocol pending analysis of total microbial community and MPRL 4739 SCFA using their in-house optimised and validated pro- E. tenella RNA from E. tenella infected tissue [79] tocols [53]. E. maxima RNA from, E. maxima infected tissue [79] The total microbial community was analysed using a culture-independent DNA-based method that was employed to determine the %G + C profile as described using DNeasy PowerSoil Kit (Qiagen, United Kingdom) by [72]. The SCFA, which include acetic acid, propionic as per manufacturer instruction. The yield and quality of acid, butyric acid, the sum of the BCFA and lactic acid, the DNA extracts were checked by NanoDrop 1000 spec- were analysed using gas chromatography (Agilent Tech- trophotometer (Thermo Scientific, UK) at 260 nm. nologies, Santa Clara, CA, USA) as previously described DNA extracted from litter samples was used in PCR [53]. to test for the presence of Salmonella spp [74], C. per- fringens [75–77], and E. tenella and E. maxima [78]. The Assessment of pathogens in reused litter specific genes, primer sequences, conditions, expected Representative litter samples for both clean and reused size of each amplicon, and PCR references are shown litter treatments were collected and analysed in tripli- in Table  6. PCR reactions for amplification of the target cates at day 0 using sterilised gloves in self-sealed sterile genes were carried out in a final volume of 25 µL contain - plastic bags and kept at − 80 °C prior to analysis. ing 1 × Q5 Hot Start High-Fidelity Master Mix (New Litter samples were prepared for DNA extraction as England Biolabs, UK), 200  nM of each primer (Table  6), previously described [73]. Briefly, 5 g of the collected lit - 10  ng of DNA template and nuclease free water. The ter sample was suspended in 30  mL of phosphate-buff - PCR cycling program consisted of an initial denatura- ered saline and then mixed for 5  min with an incubator tion step at 98  °C (30  s), followed by 30 cycles of a 10  s shaker set at the maximum speed. Debris was removed denaturation step at 98  °C, a 30  s optimized annealing by low-speed centrifugation (50 × g for 15  min at 4  °C), step at respective temperature (Table  6) and a 30 s elon- and the supernatant was collected in a sterile falcon tube. gation step at 72 °C, and a final extension step at 72 °C for The bacteria were pelleted by high-speed centrifugation 2 min before a 4 °C hold. The expected PCR amplification (3650 × g for 15 min at 4 °C) and resuspended in 1 mL of products were confirmed by agarose gel (1.5%) electro - phosphate-buffered saline, whereas DNA was extracted phoresis. Negative control template and positive control Table 6 Primers for PCR and qPCR with PCR conditions Pathogen Target Tm* (°C) Amplicon size (bp) Ref** Primer and probe sequences (5′–3) Salmonella ttr-4F:AGC TCA GAC CAA AAG TGA CCATC 66 94 [74] R:CTC ACC AGG AGA TTA CAA CATGG C. perfringens 16S rRNAF:GGG GGT TTC AAC ACC TCC 63 170 [75] R:GCA AGG GAT GTC AAG TGT CPαF:GCT AAT GTT ACT GCC GTT GA 60 324 [76] R:CCT CTG ATA CAT CGT GTA AG NetBF:GCT GGT GCT GGA ATA AAT GC 65 383 [77] R:TCG CCA TTG AGT AGT TTC CC E. tenella ITS1F:AAT TTA GTC CAT CGC AAC CCTTG 65 279 [78] R:CGA GCG CTC TGC ATA CGA CA E. maxima ITS1F:GTG GGA CTG TGG TGA TGG GG 65 205 [78] R:ACC AGC ATG CGC TCA CAA CCC Total bacteria 16S rRNAF:ACT CCT ACG GGA GGC AGC AGT 60 194 [80] R:TAT TAC CGC GGC TGC TGG C Probe:CGC GTG ACC CTT ATT GCT CCACA Tm*, optimised annealing temperature; Ref**, references Hussein et al. Animal Microbiome (2023) 5:18 Page 12 of 15 samples were included in each PCR screening. Positive 24  h. Samples were removed from the oven, weighed, controls for the bacteria targeted were prepared by isolat- returned to the oven for 1  h and weighed again to con- ing total DNA from pure cultures (Table 7). Positive con- firm no further weight loss. Litter moisture was then trols for the Eimeria spp. were obtained isolating RNA calculated from the difference in sample start and end from infected tissue as previously described (Table  7) weight. [79]. Quantification of the 16S rRNA gene was included Statistical analysis as a proxy of total bacterial load and absolute quanti- Data were subjected to analysis of variance (ANOVA) fication of the target was carried out based on Taqman using a GenStat 16 statistical software package (IACR, probe chemistry as described previously [80]. Briefly, Rothamstead, Hertfordshire, UK). The data were ana - qPCR mixtures reactions were prepared in a final volume lysed through a 2 × 2 factorial analysis of variance for of 20  µL, containing a final concentration of ~ 7  ng per diet treatments (C vs. TAG), litter treatments (clean vs. reaction of DNA template, 1X of Brilliant III Ultra-Fast reused) and their interaction, using pen location as a qPCR Mastermix (Agilent Technologies, United States), block, day 0 BWT as a covariate for day 21 BWT and the containing 30 nM of freshly prepared reference dye (Agi- pen of 10 chickens as the experimental unit. Data were lent Technologies, United States) and 100  nM of each checked for normality by examining residuals, histo- primer/ probe. Each reaction was carried out in tripli- grams and box plots, and none required transformation cate in a 96-well plate, including non-template control prior to statistical analysis. Effects at P < 0.05 and P < 0.10 and the standard curve. The latter was prepared via serial were considered significant and trends, respectively. 7 1 tenfold dilutions (10 to 10 gene copy numbers/reaction) Means were separated using Tukey’s honest significance of plasmid DNA containing the same target of this qPCR test. as an insert. Cycling conditions were set in a Stratagene MX3005P qPCR System (Agilent Technologies, United Abbreviations Kingdom) and were 95 °C (5 min), followed by 40 cycles AA Amino acids of amplification at 95 °C (15 s) then 60 °C (30 s). IP Ideal protein Thr Threonine Absolute quantification was performed using the Strat - Arg Arginine agene MxPro Software (Agilent Technologies, United Gln Glutamine Kingdom) through fitting a linear regression model with CP Crude protein BWT Body weight log standard copy number [x] and standard threshold BWG Body w eight gain (CT) (y). The quality of the reactions was verified by ana - FI Feed intake lysing the slope of the standard curve regression R and FCR Feed conversion ratio CPC Crude protein conversion efficiency calculation. The copy number calculated from SCFA Short chain fatty acids the standard curve represented copies per µL of DNA BCFA Branched-chain fatty acids extract. These values were log -transformed and multi- %G + C % Guanine + cytosine plied by 20 to obtain the 16S rRNA gene copy numbers Acknowledgements per g of chicken litter [80]. The authors acknowledge the staff and students at the Monogastric Science Research Centre, Auchincruive, UK, for all their help and support. The authors also acknowledge Evonik Nutrition & Care GmbH for providing amino acids Litter pH and moisture analysis and their help with feed analysis. Moreover, the authors acknowledge Dr At day 0, representative litter samples for both clean Sarah Brocklehurst (Biomathematics and Statistics Scotland) for her guidance on %G+C profile data analysis. and reused litter treatments were collected using sterile gloves and self-sealed plastic bags. At day 21, representa- Author contributions tive litter samples were collected from each pen from the MAH, FK, LV, SA and JGMH contributed to experimental design. MAH con- ducted the experiment, collecting samples, data analysis, writing-original four pen corners and the middle (around the feeders and draft, and presentation of the published work. FK and JGMH contributed to the drinkers) using sterile gloves and self-sealed plastic the provision of study materials. FK, LV, SA and JGMH contributed to review, bags. The collected litter samples were kept at − 80  °C editing and supervision. JGMH contributed to project administration and funding acquisition. All authors read and approved the final version of the freezer prior to analysis. Litter pH was determined by manuscript and approved publication. placing 10 g of each litter sample into 90 mL of distilled water and mixing for 10–15  min. pH was then meas- Funding Marwa A. Hussein is a recipient of PhD scholarship programme under the ured using a pH meter (Fisher Scientific accumet AE150 Newton-Mosharafa fund from the Egyptian Cultural Affairs and Missions pH Benchtop Meter) after calibration with pH 4, 7 and Sector and the British Council. SRUC received support from Scottish Govern- 10 buffers. Litter moisture content was also analysed in ment [RESAS]. Lonneke Vervelde received funding from a Biotechnology and Biological Sciences Research Council Institute Strategic Programmes Grant to duplicates by placing 10 g of each litter sample onto tared The Roslin Institute [BBS/E/D30002276]. aluminium drying dishes in a drying oven at 100  °C for Hussein  et al. Animal Microbiome (2023) 5:18 Page 13 of 15 Availability of data and materials 12. Nassiri Moghaddam H, Alizadeh-Ghamsari AH. Improved perfor- The datasets generated during and/or analysed during the current study are mance and small intestinal development of broiler chickens by dietary available from the corresponding author upon reasonable request. L-glutamine supplementation. J Appl Anim Res. 2013;41:1–7. 13. Ribeiro V Jr, Albino LF, Rostagno HS, Hannas MI, Ribeiro CL, Vieira RA, de Araújo WA, Pessoa GB, Messias RK, da Silva DL. Eec ff ts of dietary Declarations L-glutamine or L-glutamine plus L-glutamic acid supplementation pro- grams on the performance and breast meat yield uniformity of 42-d-old Ethics approval and consent to participate broilers. Braz J Poult Sci. 2015;17:93–8. All the experimental animal procedures in the current study were carried out 14. Wu QJ, Jiao C, Liu ZH, Cheng BY, Liao JH, Zhu DD, Ma Y, Li YX, Li W. 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The impact of group medication on AMR gene dynamics during commercial pig production. bioRxiv. 2019;1:659771. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Animal Microbiome Springer Journals

Growth performance, caecal microbiome profile, short-chain fatty acids, and litter characteristics in response to placement on reused litter and combined threonine, arginine and glutamine supplementation to juvenile male broiler chickens

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Springer Journals
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10.1186/s42523-023-00240-0
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Abstract

Background Exposure of broilers to litter microbiome may increase specific amino acid (AA) requirements towards activated immune responses. This may challenge the generality of the ideal protein (IP) concept, in which dietary essential AA to lysine ratios aimed to mimic presumably constant AA to lysine ratios in whole bird requirements. Therefore, we tested the effect of threonine, arginine and glutamine ( TAG) supplementation to IP-based control diets (C) on performance, caecal microbiome composition, short-chain fatty acids and litter characteristics of broiler chick- ens placed on reused litter. Results Thirty-two pens with ten male broiler chickens each were used in a 2 × 2 factorial arrangement of two diet treatments (with or without TAG supplementation) and two litter treatments (placement on clean or reused litter) for 21 days (n = 8). Caecal contents were analysed for microbiome profile using percent guanine + cytosine (%G + C profile) method and short chain fatty acids. TAG-supplemented birds underperformed compared to C birds (P = 0.002), whereas birds placed on reused litter outperformed those on clean litter (P = 0.047). Diet, reused litter and their interaction impacted the %G + C profile at different ranges. Whilst TAG supplementation reduced bacterial abundance at %G + C 51–56 (P < 0.05), reused litter placement tended to reduce %G + C 23–31 and increase %G + C 56–59 (P < 0.10). However, TAG supplementation reduced bacterial abundance at %G + C 47–51 (P < 0.05) and increased caecal branched chain fatty acids on clean litter only (P = 0.025). Greater levels of propionic acid were observed for C birds placed on reused litter only (P = 0.008). Litter pH was greater for reused litter pens than clean litter pens at day 21 (P < 0.001). In addition, litter moisture content was less for TAG birds and reused litter pens compared to C birds (P = 0.041) and clean litter pens (P < 0.001), respectively. Conclusions These data support the view that irrespective of performance benefits arising from bird placement on reused litter, TAG supplementation to IP-formulated baseline rations impaired growth, supported by the lowered *Correspondence: Marwa A. Hussein marwa.hussein@sruc.ac.uk Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Hussein et al. Animal Microbiome (2023) 5:18 Page 2 of 15 abundance of caecal bacteria known to dominate in well-performing birds and greater levels of caecal branched chain fatty acids. Keywords Amino acids, Reused litter, Ideal protein, Broilers, Growth performance, Caecal microbiome, Litter characteristics Introduction enhanced growth performance during sub-clinical Dietary protein requirement should represent the sum challenges upon Gln supplementation has been shown to of a balanced level of essential amino acids (AA) and concur with improved gut morphology, i.e., increased sufficient non-essential AA that fulfils age-dependant villus height and lowered crypt depth, which improves AA requirement for optimal performance. This view is absorptive capacity [30, 31]. Wu et  al. [14] further the basis for the ideal protein (IP) concept that has been reported that Gln supplementation may ameliorate introduced into diet formulation for poultry to optimise detrimental effects of Salmonella enteritidis infection on AA supply and, thus, nitrogen utilisation [1–3]. The IP intestinal immune barrier functions and lymphoid organ concept represents dietary essential AA (eAA) to lysine weights (i.e., bursa of Fabricius, spleen and thymus). ratios as in whole bird AA requirements for maintenance Host responses to specific AA supplementation may be and production, leading to an ideal AA profile in sensitive to the level of microbiota exposure, as immune which all eAA are equally limiting [4, 5]. Several studies and possible pathological responses could result in reported a positive impact of AA supplementation on increased whole bird AA requirements that deviate from performance, e.g. threonine (Thr) [6–8], arginine (Arg) the ideal AA profile. In the current study, placement [9–11], but also the non-essential amino acid glutamine on reused litter, which may impact caecal microbiome (Gln) [12–14]. However, AA supplementation to an IP characteristics [32], was used to create two contrasting formulated baseline would not be expected to improve levels of microbiota exposure, under which the AA performance but result in excess AA intake. The latter supplementation was tested. We hypothesised that the would be expected to facilitate proteolytic activity of the effect of Thr, Arg and Gln (TAG) supplementation to IP hindgut microbiome resulting in changes in composition formulated basal rations on broiler growth performance, and/or metabolite production, deteriorated litter caecal microbiome parameters, and litter characteris- quality and potentially reduced performance [15]. However, tics (pH and moisture content) is sensitive to reused birds under external microbial and other pathogen litter exposure. To our knowledge, this is the first time that exposure would be expected to require different dietary the effects of AA supplementation to IP-based rations AA ratios for the combined optimal performance and are assessed on caecal microbiome composition and enhanced immune responses, arising from competition fermentation metabolites of broilers placed on reused litter. for limiting AA between these functions [16–19], which may thus challenge the generality of the IP concept. Results Each of Thr, Arg and Gln has been implicated in host Diet analysis and growth performance responses to viral, bacterial and/ or parasitic exposure. Although relative to the TAG diets, the non-TAG AA For instance, Thr plays a vital role in the maintenance of levels of the C diets were on average 4% greater and 3% intestinal barrier integrity and mucin synthesis [8, 20] smaller for the starter and grower phase, respectively, and is a major component of gamma globulin [21, 22]. overall, the analysed nutrient and individual AA compo- Supplementation with Thr has been shown to increase sition of the experimental diets were within the expected hemagglutination titres of birds infected with the New- range (Table  1). Since ~ 98.5% of the diets were derived castle disease virus [23]. In addition, Thr supplemen - from a common basal, and the part that varied consisted tation has been found to improve gut health during of starch or TAG only, observed variation likely reflects salmonellosis in broilers [24]. The supplementation with variation in analysis rather than an actual chemical com- Arg, which is a precursor for nitric oxide, polyamines, position. The effect of TAG supplementation and litter and creatine [25], has been shown to improve intestinal treatments on growth performance during the entire morphology [26] during sub-clinical enteric challenges growth phase (days 0–21) is shown in Table 2. There were as it can ameliorate coccidiosis-induced intestinal vil- no significant interactions between diet and litter treat - lus damage and goblet cell depletion [27]. As the main ment on performance data. However, diet treatment energy source for immune and intestinal epithelial cells, impacted growth performance measurements, i.e., body Gln could become limiting during elevated Gln require- weight gain (BWG), feed intake (FI) and crude protein ments arising from enteric challenges [28, 29]. Indeed, conversion (CPC), as TAG birds had smaller BWG, FI Hussein  et al. Animal Microbiome (2023) 5:18 Page 3 of 15 Table 1 Analysed chemical composition, gross energy, and total Table 2 Growth performance of broilers fed C or TAG diets and amino acid content of the experimental starter (0 to 11 days) and placed as day-old on either clean or reused litter over 21 days grower (11 to 21 days) rations Litter Diet BWG, g FI, g FCR, g/g CPC* Starter rations Grower rations Clean C 738 1044 1.447 0.298 C TAG C TAG TAG 663 914 1.417 0.319 Reused C 779 1028 1.349 0.278 Chemical composition TAG 708 947 1.371 0.308 DM (%) 88.00 89.00 88.30 88.30 SED 28.8 21.7 0.051 0.011 Crude ash (%) 6.20 10.40 5.70 5.30 Means for main effect of litter CP (%) 22.82 24.12 21.14 23.21 Clean 700 979 1.432 0.308 NDF (%) 7.80 7.90 7.70 7.50 Reused 743 988 1.360 0.293 ADF (%) 4.15 3.88 3.53 3.48 SED 20.4 15.4 0.036 0.008 Sucrose (%) 5.36 3.95 4.17 4.44 Means for main effect of diet Total starch (%) 35.80 34.10 39.80 36.00 b b a C 758 1036 1.398 0.288 EE (%) 3.83 4.21 4.80 4.53 a a b TAG 685 931 1.394 0.313 AHEE (%) 4.74 5.02 5.74 5.24 SED 20.4 15.4 0.036 0.008 GE (MJ/kg) 16.45 16.63 16.77 16.77 P-values for main effects and interaction Amino acids composition (%) Litter 0.047 0.581 0.056 0.068 Methionine 0.55 0.50 0.46 0.50 Diet 0.002 < 0.001 0.902 0.004 Cysteine 0.36 0.36 0.35 0.35 Litter × diet 0.924 0.134 0.483 0.544 Methionine + cysteine 0.90 0.86 0.81 0.85 Lysine 1.45 1.31 1.26 1.35 BWG Body weight gain; FI Feed intake; FCR Feed conversion ratio; CPC* Crude protein conversion = FI (kg) × CP content diet (g/kg)/BWG (g); C Control diets; Threonine 0.98 1.16 0.87 1.10 TAG Threonine, arginine and glutamine supplemented diets; SED Standard error Arginine 1.48 1.76 1.36 1.68 of difference; Means within the same column with different superscripts differ at P < 0.05; Simple means represent 8 pens of 10 birds per pen Isoleucine 0.95 0.90 0.88 0.90 Leucine 1.55 1.49 1.47 1.50 Valine 1.10 1.02 0.99 1.02 as a response to diet or litter treatment and thus enables Histidine 0.52 0.50 0.50 0.51 the detection of any putative alterations at the commu- Phenylalanine 1.02 0.98 0.97 1.00 nity level. Diet and litter treatment interacted for caecal Glycine 0.87 0.85 0.84 0.84 %G + C 47–51 (P < 0.01) as birds fed the test diet (TAG) Serine 1.04 1.02 0.99 1.01 compared to birds fed the control diet (C) showed a lower Proline 1.33 1.33 1.27 1.33 abundance over that range but only when placed on clean Alanine 0.88 0.85 0.84 0.86 litter (Fig. 1). However, diet treatment affected %G + C at Aspartic acid 2.04 1.95 1.91 1.97 a higher range, as TAG birds displayed a significant shift Glutamic acid 4.31 5.19 4.17 5.16 towards a lower abundance of bacteria at %G + C 51–56 than C birds (Fig. 2). In contrast, litter treatment did not C Control; TAG Threonine, arginine and glutamine supplemented diets; DM Dry matter; CP Crude protein; EE Ether extract; AHEE Ether extract preceded by significantly affect %G + C profile; the consistently lower acid hydrolysis; NDF Neutral detergent fibre; ADF Acid detergent fibre; GE Gross %G + C 23–31 and greater %G + C 56–59 for birds on energy reused litter compared to those on clean litter averaged at P = 0.112 and P = 0.099, respectively (Fig. 3). and larger CPC than C birds. Furthermore, birds placed Caecal SCFA concentration and composition on reused litter had greater BWG and tended to have The total short chain fatty acids (SCFA) concentration in better feed conversion ratio (FCR) and CPC than birds the caecal content and its composition were determined placed on clean litter. Mortality was low at 0.3% (1 out of as an indicator of the fermentative activity of the micro- 320 birds placed). This 11-day-old bird was culled due to bial population and are presented in Table 3. Total SCFA hunched posture. The post-mortem reported that there concentration did not significantly differ between treat - was a large yolk sac remnant with necrotic content. ments. However, a significant interaction between diet and litter treatments was observed for the percentage of Caecal %G + C profile propionic acid and branched-chain fatty acids (BCFA); The percent guanine + cytosine (%G + C) profile of the the latter consisted of iso-butyric acid only, as the other total chromosomal DNA was determined to illustrate the two BCFA (2-methyl-butyric acid and iso-valeric acid) relative abundance of the entire microbial community Hussein et al. Animal Microbiome (2023) 5:18 Page 4 of 15 Diet ×Litter interaction effect 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 -1 %G + C C+Clean TAG+Clean C+Reused TAG+Reused P- values from ANOVA 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 %G + C Diet × Litter 0.05 Fig. 1 Diet and Litter interaction effects on the %G + C profile of caecal bacteria from broilers aged 21 days. In the upper panel, the solid blue line represents the mean %G + C profile of birds fed C diets and placed on clean litter, the solid red line represents the mean %G + C profile of birds fed TAG diets and placed on clean litter, the solid green line shows the mean %G + C of birds fed C diets and placed on reused litter and the solid purple line illustrates the mean %G + C of birds fed TAG diets and placed on reused litter (n = 8). In the lower panel, the solid blue line shows the results from ANOVA and the solid red line marks the threshold of P = 0.05 Relative abundance, A 280 nm P- value Hussein  et al. Animal Microbiome (2023) 5:18 Page 5 of 15 Main effect of Diet 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 -1 %G + C C TAG P-values from ANOVA 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 %G + C Diet 0.05 Fig. 2 The main effect of Diet treatment on the %G + C profile of caecal bacteria from broilers aged 21 days. In the upper panel, the solid blue line represents the mean %G + C profile of birds fed C diets and the solid red line shows the mean %G + C profile of birds fed TAG diets (n = 16). In the lower panel, the solid blue line shows the results from ANOVA and the solid red line marks the threshold of P = 0.05 Relative abundance, A 280 nm P-value Hussein et al. Animal Microbiome (2023) 5:18 Page 6 of 15 Main effect of Litter 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 -1 %G + C Clean Reused P- values from ANOVA 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 %G + C Litter 0.05 Fig. 3 The main effect of Litter treatment on the %G + C profile of caecal bacteria from broilers aged 21 days. In the upper panel, the solid blue line represents the mean %G + C profile of birds placed on clean litter and the solid red line represents the mean %G + C profile of birds placed on reused litter (n = 16). In the lower panel, the solid blue line shows the results from ANOVA and the solid red line marks the threshold of P = 0.05 Relative abundance, A 280 nm P- value Hussein  et al. Animal Microbiome (2023) 5:18 Page 7 of 15 Table 3 Short chain fatty acids in the ceca of 21-day old broilers fed C or TAG diets and placed on either clean or reused litter Litter Diet Total SCFA (mM) %Acetic acid %Propionic acid %Butyric acid %Lactic acid %Iso- butyric acid a a Clean C 105.10 72.45 3.98 13.34 10.07 0.10 ab b TAG 97.60 71.32 5.67 12.88 9.79 0.32 b a Reused C 92.20 73.99 6.35 10.45 9.08 0.12 a a TAG 92.40 72.26 3.65 11.79 12.14 0.16 SED 8.15 2.06 1.05 1.55 1.80 0.05 Means for main effect of litter Clean 101.40 71.88 4.83 13.11 9.93 0.21 Reused 92.30 73.13 5.00 11.12 10.61 0.14 SED 5.77 1.46 0.74 1.09 1.27 0.04 Means for main effect of diet C 98.60 73.22 5.16 11.89 9.57 0.11 TAG 95.00 71.79 4.66 12.33 10.97 0.24 SED 5.77 1.46 0.74 1.09 1.27 0.04 P values for main effects and interaction Litter 0.129 0.404 0.819 0.083 0.597 0.064 Diet 0.536 0.337 0.508 0.690 0.286 0.003 Litter × diet 0.515 0.838 0.008 0.418 0.204 0.025 SCFA Short chain fatty acids; C Control diets; TAG Threonine, arginine and glutamine supplemented diets; SED Standard error of difference; Means within the same column with different superscripts differ at P < 0.05; Simple means represent 8 pens of pooled sampled from 2 birds sampled per pen were below the detection limit. C birds had a greater Table 4 pH and moisture content of litter samples of 21-day-old broilers fed C or TAG diets and placed on either clean or reused percentage of propionic acid than TAG birds on reused litter at day-old litter only. In addition, whilst TAG birds had a greater proportion of iso-butyric acid than C birds across litter Litter Diet pH Moisture, % treatments, the interaction indicated this was most pro- Clean C 6.67 30.00 nounced on clean litter. TAG 6.57 27.36 Reused C 7.35 25.10 Litter characteristics TAG 7.40 22.78 At day 0, polymerase chain reaction (PCR) screens did SED 0.074 1.674 not detect Salmonella spp., Clostridium perfringens, Means for main effect of litter Eimeria tenella and E. maxima in either clean or reused a b Clean 6.62 28.68 litter. However, the 16S rRNA gene copy numbers of b a Reused 7.38 23.94 total bacteria per g of chicken litter were 2.08 and 8.13 SED 0.052 1.184 copies per g of clean and reused litter, respectively. The Means for main effect of diet initial pH of clean and reused litter was 5.76 and 8.09, C 7.01 27.55 respectively, whilst their moisture content was 10.79 and TAG 6.99 25.07 12.49%, respectively. SED 0.052 1.184 The diet and litter treatment effects on final litter pH P values for main effects and interaction and moisture content are presented in Table  4. There Litter < 0.001 < 0.001 were no significant interactions between diet and litter Diet 0.707 0.041 treatments for both parameters. Whilst diet treatment Litter × Diet 0.172 0.894 did not impact litter pH, placement of birds on reused C Control; TAG Threonine, arginine, and glutamine supplemented diets; Means litter resulted in significantly higher litter pH than their within the same column with different superscripts differ at P < 0.05; SED, clean litter counterparts. In addition, both diet and litter standard error of difference; Simple means represent 8 pens per treatment treatments independently impacted litter moisture con- tent, as the TAG and reused litter treatments reduced moisture content compared to the C and clean litter treatments, respectively. Hussein et al. Animal Microbiome (2023) 5:18 Page 8 of 15 Discussion deposition [44, 45]. This suggestion is supported by the This study investigated the effects of Thr, Arg and Gln reduced FI of TAG birds over C birds, as FI depression is supplementation to IP formulated diets on perfor- one of the first manifestations of dietary AA imbalance in mance, caecal microbiome and litter characteristics in broilers [46, 47]. In addition, it has also been suggested the absence and presence of placement at reused litter to that AA supplementation may reduce FI arising from create two contrasting conditions in terms of microbiota the amino static hypothesis, in which free AA in plasma exposure. We hypothesised that reused litter exposure at serve as a signal to an appetite-controlling mechanism placement would affect the growth performance response [48–50]. to TAG supplementation. However, this hypothesis was The %G + C profile is used to indicate the relative rejected as TAG supplementation reduced performance abundance of bacteria with different DNA base compo - for both clean and reused litter treatments. Supported by sitions and hence allows detecting any putative altera- effects on caecal microbiome composition and SCFAs, tions at the community level [51]. The most abundant collectively the data indicated that TAG supplementation bacteria observed in our study represent species with may have resulted in excess protein over the IP-basis, %G + C 40–55, such as Lachnospiraceae (Clostridial which disadvantaged bird performance [33–35]. cluster IV) and Lactobacilli, which are known to domi- Here, birds placed on the reused litter had 6% greater nate caecal microbiome composition of well-performing BWG than those on clean litter. The use of reused litter birds [52]. However, under-performing birds often have has been shown to result in variable outcomes on perfor- two peaks at < ~ 37% and > ~ 60 of %G + C instead of one mance, as it has been associated with penalised [36–39], peak at ~ 45%G + C [52]. Here, TAG supplementation similar [40, 41] or improved [42] performance relative resulted in a lower abundance of bacteria with %G + C to birds placed on clean litter. Such variable outcomes 47–56, which indeed concurred with reduced growth of using reused litter can be expected to arise from dif- performance over C birds, though there was no signifi - ferences in litter characteristics, which are typically not cant increase in the %G + C 20–30, which is often asso- reported in studies using reused litter, and include micro- ciated with the presence of pathogenic bacteria [53]. bial composition, pH, moisture, and level of recycled This suggests that a possible microbiological basis of nutrients from the previous flock. An early exposure of the reduction in performance on TAG diets was most newly hatched chicks to reused litter facilitates the colo- likely metabolic rather than pathogenic. Although birds nisation and cycling of microbiomes between gut and on reused litter performed better and showed a greater litter which accords with a probiotic or direct-fed micro- proportion of propionic acid in their SCFA pool than bial approach to improve intestinal microbiota and thus those on clean litter, this did not concur with significant improved performance [32, 42]. In support of the positive changes in microbial profile. Whilst there was some indi - effect of reused litter exposure on performance reported cation that the reused litter treatment indeed lowered here, pathogens such as C. perfringens, E. tenella and E. bacterial abundance associated with %G + C 23–31 and maxima were not detected in the tested litter samples. increased bacterial abundance at %G + C 56–59 (Fig.  3), The reduced performance upon TAG supplementation these did not reach statistical significance in this study. may have a multi-factorial basis. Firstly, since diets were Caecal SCFA analysed include the volatile fatty formulated to be isoenergetic, TAG supplementation acids (VFA) acetate, propionate and butyrate, but also as expected increased CP content and thus reduced the the non-volatile lactate, produced by gut microbiota metabolisable energy to CP ratio. The latter may directly as fermentation products from undigested nutrients reduce the AA pool for protein deposition and uric acid [54]. The SCFA play a role in intestinal health, includ - synthesis as well as the supply of fat and carbohydrate to ing the promotion of mucin production, blood flow, meet the energetic requirements of the birds [43]. Sec- enterocytes growth and proliferation [55]. The VFAs ondly, the AA availability in TAG supplemented birds mentioned are a valuable energy source for the host, might be lower than expected for growth due to increased especially butyrate being the preferred energy source proteolytic fermentation from excess AA, which has for epithelial cells [56]. Furthermore, the increased pro- been shown to result in poorer intestinal health [33]. portion of propionic acid in the SCFA pool of birds on Around half of the undigested and unabsorbed protein reused litter and fed C diets could indicate the presence is fermented by putrefactive caecal bacteria producing of beneficial bacteria such as Lactobacillus spp., which toxic compounds, i.e., amines, indoles, phenols, cresol are known to have bacteriostatic or bactericidal proper- and ammonia, which may impede performance [34, 35]. ties against pathogenic microbes [52, 57]. This accords Thirdly, the AA imbalance arising from surplus AA in with the improved performance observed for birds on TAG supplemented birds might decrease the efficiency reused litter and fed C diet. The BCFA (iso-butyric, of utilisation of limiting AA for maintenance and protein 2-methyl-butyric and iso-valeric) within the SCFA pool Hussein  et al. Animal Microbiome (2023) 5:18 Page 9 of 15 can only be produced from fermenting branched-chain Conclusions AA, i.e., valine, leucine, and isoleucine. As such, vari- In this study, Thr, Arg and Gln supplementation to IP- ation in caecal BCFA levels may indicate variation in based diets altered caecal microbial composition and protein fermentation activity but also the flow of undi - enhanced proteolytic fermentation, indicative of excess gested protein into the caecum. Thus, elevated caecal protein leading to impaired performance. However, this BCFA could be indicative of reduced ileal crude protein study also supports the view that reused litter, particu- (CP) digestibility, which would result in poorer growth larly as assessed here in the absence of pathogenic bac- performance [35, 58]. This is consistent with the ele - teria, might benefit bird performance. The use of such vated levels of iso-butyric acid in TAG- supplemented litter accords with a probiotic or direct-fed microbial birds, being most pronounced on clean litter, and the approach, combined with being a source of recycled reduced performance observed for those birds. nutrients. Litter pH and moisture content are some of the major determinants implicated in the survival and growth Materials and methods of litter pathogens [59]. Generally, litter pH ranges Bird management and experimental design between 6.5 and 8.5, with negligible ammonia produc- A total of 320 male Ross 308 broiler chickens were tion below pH 7 [60, 61]. In the current study, diet treat- used in a 21-day experiment. Upon arrival (day 0), the ment did not have a significant impact on the final litter birds were allocated to 32 floor pens (1.47 m × 0.94  m), pH, though reused litter pens had greater final pH levels separated through plastic-sheeted panels, with 10 birds than clean litter pens. However, temporal effects need to per pen in a randomised complete block design. The be considered, as pH for the clean litter pens increased temperature was set to 32  °C for the first 3  days and from 5.76 at day 0 to 6.62 at day 21, whilst for the reused then was gradually reduced over a week until 25  °C litter pens, pH decreased from 8.09 to 7.38. Both the was reached and maintained until day 21 as per breed difference at day 0 between clean and reused litter and guidelines. The light was provided for 23  h per day for the increase in pH for the clean litter pens over time can the first week and then reduced to 18  h of light per be attributed to the accumulation of excreta during the day. Birds were provided ad libitium access to feed and grow-out period, with elevated pH arising from protein water throughout the experiment, with feed offered degradation and ammonia production [62, 63]. How- as a meal. Birds were fed wheat-soyabean meal-based ever, whilst accumulation of excreta would also have starter (0–11 days of age) and grower diets (11–21 days occurred for the reused litter pens, the net reduction in of age) with the control diets (see below) formulated to pH overtime for these pens may be the consequence of meet Ross 308 nutrient recommendations [68]. continuing composting activity in  situ as litter pH was The experimental set up consisted of a 2 × 2 facto- observed to reduce over a period of 28  days for stored rial arrangement of two diet treatments and two lit- litter [64]. These data suggest that temporal rather than ter treatments (see below) with 8 pens per treatment current variation in litter pH may better inform varia- combination within a complete randomised block tion in performance, where the latter was greater for arrangement. birds on reused litter compared to those on clean litter. The final litter moisture for the TAG treatment was Diet treatments lower than that for the C treatment. This could be related The control (C) diet was formulated on an IP basis and to the reduced FI for the TAG birds, which would have supplemented with the synthetic AA to meet all eAA concurred with reduced water intake as well as reduced requirements on a digestible AA basis. The TAG treat - total metabolites elimination with the excreta [65], both ment consisted of feeding the C diet with additional contributing to reduced water spillage and excretion. Thr and Arg at 25% above requirements and 1% Gln, However, at similar levels of FI, reused litter pens also as informed by previous studies [16, 69, 70]. For each had lower final moisture content compared with clean lit - phase, a common basal diet was prepared by including ter pens. This suggests other reasons might also explain a 3% of corn starch for the C diets, against which the the variation in litter moisture and accords with previ- tested AA were included for the TAG diets. The TAG ous studies [66, 67], where lower moisture content in lit- diets were therefore calculated to be isoenergetic but ter used for multiple grow-outs (reused litter) was also with varying CP levels. The ingredients and the calcu - observed. Reused litter has been found to have lower lated chemical compositions of the starter and grower water activity and faster rate of excreta drying than clean diets are presented in Table 5. litter, which might detriment the survival and growth of litter pathogens [67], and benefit performance. Hussein et al. Animal Microbiome (2023) 5:18 Page 10 of 15 Litter treatments Table 5 Feed ingredients and calculated chemical compositions (%) of the experimental starter (0–11 days) and grower (11– For each diet treatment, half of the pens were supplied 21 days) rations with all new wood shavings (clean litter) and the other half had 100% reused wood shavings litter (reused lit- Starter rations Grower rations ter). The reused litter was derived from a previous C TAG C TAG 1152-bird broiler study (Ross 308) with no history of clinical diseases. The duration between litter collec - Ingredients tion and its reuse at the start of the current trial was Corn starch 3.00 1.44 3.00 1.51 28 days, during which litter was untreated and stored in Threonine 0.23 0.45 0.18 0.37 bags in an empty unheated shed. Arginine 0.10 0.44 0.05 0.35 Glutamine 0.00 1.00 0.00 1.00 Wheat 58.22 58.22 60.56 60.56 Sampling and data collection Soybean meal 31.59 31.59 28.50 28.50 Chemical analysis of diets Soya oil 2.20 2.20 3.50 3.50 Experimental diets were analysed for dry matter (DM), Salt 0.05 0.05 0.05 0.05 neutral detergent fibre (NDF), acid detergent fibre (ADF), Limestone 0.95 0.95 0.87 0.87 ether extract (EE), ether extract preceded by acid hydrol- Dicalcium phosphate 1.85 1.85 1.65 1.65 ysis (AHEE), ash, starch, and total sugar (as sucrose) at Sodium bicarbonate 0.50 0.50 0.50 0.50 Sciantec Analytical Services Ltd. (Cawood, UK) using Lysine HCl 0.39 0.39 0.32 0.32 standard protocols based upon Commission Regula- Methionine 0.24 0.24 0.21 0.21 tion (EC) No. 152/2009. Analysis of CP and AA content, Valine 0.09 0.09 0.06 0.06 including tryptophan, were performed at Evonik Nutri- Tryptophan 0.14 0.14 0.12 0.12 tion & Care GmbH (Hanau-Wolfgang, Germany). The CP Isoleucine 0.05 0.05 0.03 0.03 was estimated using the Dumas method, and AA analysis Vitamin & mineral premix 0.40 0.40 0.40 0.40 was done by standard procedures [71] using an AA ana- Calculated chemical composition lyser (Biochrom 30 + , Cambridge, UK). Tryptophan was Crude protein % 22.47 24.51 21.01 22.95 determined by high-performance liquid chromatography AME MJ/kg 12.51 12.51 12.94 12.94 following preparation by hydrolysis. Gross energy (GE) Calcium % 0.96 0.96 0.87 0.87 was determined through an isoperibol bomb calorimeter Phosphorous % 0.72 0.72 0.67 0.67 system using benzoic acid as an internal standard (model Available phosphorous % 0.48 0.48 0.44 0.44 6200, Parr Instruments, Moline, Illinois, USA). Salt % 0.19 0.19 0.16 0.16 Sodium % 0.19 0.19 0.19 0.19 Growth performance Chloride % 0.16 0.16 0.15 0.15 Growth performance parameters, i.e., BWG, FI, and FCR, Digestible essential amino acids % were calculated from mean body weights (BWT) through Threonine 0.86 1.08 0.77 0.96 bulk weighing and bird counting at pen level, weights of Arginine 1.37 1.71 1.23 1.53 feed offered on days 0 and 11, and weights of feed refusals Histidine 0.48 0.48 0.45 0.45 on days 11 and 21. The resulting BWG, FI and FCR were Isoleucine 0.86 0.86 0.78 0.78 calculated for the entire growth period of days 0 to 21. Leucine 1.40 1.40 1.31 1.31 Birds that were found dead or were culled were recorded Lysine 1.28 1.28 1.15 1.15 for date, weighed and sent for post-mortem examination. Methionine 0.51 0.51 0.47 0.47 BWG and total pen FI were corrected for mortality. FCR Cysteine 0.31 0.31 0.30 0.30 was calculated by dividing the average feed consumed per Tryptophan 0.20 0.20 0.19 0.19 pen by the average weight gain of birds per pen. CPC was Valine 0.96 0.96 0.87 0.87 calculated by multiplying the average feed consumed by Methionine + cysteine 0.82 0.82 0.77 0.77 the dietary CP content and divided by the average weight Phenylalanine + tyrosine 1.58 1.58 1.48 1.48 gain of birds per pen as CPC = FI (kg) × CP content diet (g/kg)/BWG (g). AME Apparent metabolisable energy; C Control diets; TAG Threonine, arginine and glutamine supplemented diets; Vitamin and mineral premix provided −1 (units kg diets): Vit A, 16,000 IU; Vit D3, 3,000 IU; Vit E, 75 IU; Vit B1, 3 mg; Caecal microbiome profile and short chain fatty acid analysis Vit B2, 10 mg; Vit B6, 3 mg; Vit B12, 15 µg; Vit K3, 5 mg; Nicotinic acid 60 mg; Pantothenic acid 14.5 mg; Folic acid 1.5 mg; Biotin 275 µg; Choline chloride At day 21, caecal digesta was collected in a sterile petri- 250 mg; Iron 20 mg; Copper 10 mg; Manganese 100 mg; Cobalt 1 mg; Zinc dish from two randomly selected broilers per pen after 82 mg; Iodine 1 mg; Selenium 0.2 mg; Molybdenum 0.5 mg Hussein  et al. Animal Microbiome (2023) 5:18 Page 11 of 15 being individually weighed, electrically stunned, and Table 7 Positive controls of the pathogens used in this study exsanguinated. Approximately 1  g of the pooled cae- Positive strain Source cal content of the two birds was immediately preserved Salmonella enterica subsp. SRUC Veterinary Services using BioFreeze sampling kits (Alimetrics Diagnostics enterica serotype Poona Ltd., Espoo, Finland) following their recommended pro- C. perfringens type A isolate SRUC Veterinary Services tocol pending analysis of total microbial community and MPRL 4739 SCFA using their in-house optimised and validated pro- E. tenella RNA from E. tenella infected tissue [79] tocols [53]. E. maxima RNA from, E. maxima infected tissue [79] The total microbial community was analysed using a culture-independent DNA-based method that was employed to determine the %G + C profile as described using DNeasy PowerSoil Kit (Qiagen, United Kingdom) by [72]. The SCFA, which include acetic acid, propionic as per manufacturer instruction. The yield and quality of acid, butyric acid, the sum of the BCFA and lactic acid, the DNA extracts were checked by NanoDrop 1000 spec- were analysed using gas chromatography (Agilent Tech- trophotometer (Thermo Scientific, UK) at 260 nm. nologies, Santa Clara, CA, USA) as previously described DNA extracted from litter samples was used in PCR [53]. to test for the presence of Salmonella spp [74], C. per- fringens [75–77], and E. tenella and E. maxima [78]. The Assessment of pathogens in reused litter specific genes, primer sequences, conditions, expected Representative litter samples for both clean and reused size of each amplicon, and PCR references are shown litter treatments were collected and analysed in tripli- in Table  6. PCR reactions for amplification of the target cates at day 0 using sterilised gloves in self-sealed sterile genes were carried out in a final volume of 25 µL contain - plastic bags and kept at − 80 °C prior to analysis. ing 1 × Q5 Hot Start High-Fidelity Master Mix (New Litter samples were prepared for DNA extraction as England Biolabs, UK), 200  nM of each primer (Table  6), previously described [73]. Briefly, 5 g of the collected lit - 10  ng of DNA template and nuclease free water. The ter sample was suspended in 30  mL of phosphate-buff - PCR cycling program consisted of an initial denatura- ered saline and then mixed for 5  min with an incubator tion step at 98  °C (30  s), followed by 30 cycles of a 10  s shaker set at the maximum speed. Debris was removed denaturation step at 98  °C, a 30  s optimized annealing by low-speed centrifugation (50 × g for 15  min at 4  °C), step at respective temperature (Table  6) and a 30 s elon- and the supernatant was collected in a sterile falcon tube. gation step at 72 °C, and a final extension step at 72 °C for The bacteria were pelleted by high-speed centrifugation 2 min before a 4 °C hold. The expected PCR amplification (3650 × g for 15 min at 4 °C) and resuspended in 1 mL of products were confirmed by agarose gel (1.5%) electro - phosphate-buffered saline, whereas DNA was extracted phoresis. Negative control template and positive control Table 6 Primers for PCR and qPCR with PCR conditions Pathogen Target Tm* (°C) Amplicon size (bp) Ref** Primer and probe sequences (5′–3) Salmonella ttr-4F:AGC TCA GAC CAA AAG TGA CCATC 66 94 [74] R:CTC ACC AGG AGA TTA CAA CATGG C. perfringens 16S rRNAF:GGG GGT TTC AAC ACC TCC 63 170 [75] R:GCA AGG GAT GTC AAG TGT CPαF:GCT AAT GTT ACT GCC GTT GA 60 324 [76] R:CCT CTG ATA CAT CGT GTA AG NetBF:GCT GGT GCT GGA ATA AAT GC 65 383 [77] R:TCG CCA TTG AGT AGT TTC CC E. tenella ITS1F:AAT TTA GTC CAT CGC AAC CCTTG 65 279 [78] R:CGA GCG CTC TGC ATA CGA CA E. maxima ITS1F:GTG GGA CTG TGG TGA TGG GG 65 205 [78] R:ACC AGC ATG CGC TCA CAA CCC Total bacteria 16S rRNAF:ACT CCT ACG GGA GGC AGC AGT 60 194 [80] R:TAT TAC CGC GGC TGC TGG C Probe:CGC GTG ACC CTT ATT GCT CCACA Tm*, optimised annealing temperature; Ref**, references Hussein et al. Animal Microbiome (2023) 5:18 Page 12 of 15 samples were included in each PCR screening. Positive 24  h. Samples were removed from the oven, weighed, controls for the bacteria targeted were prepared by isolat- returned to the oven for 1  h and weighed again to con- ing total DNA from pure cultures (Table 7). Positive con- firm no further weight loss. Litter moisture was then trols for the Eimeria spp. were obtained isolating RNA calculated from the difference in sample start and end from infected tissue as previously described (Table  7) weight. [79]. Quantification of the 16S rRNA gene was included Statistical analysis as a proxy of total bacterial load and absolute quanti- Data were subjected to analysis of variance (ANOVA) fication of the target was carried out based on Taqman using a GenStat 16 statistical software package (IACR, probe chemistry as described previously [80]. Briefly, Rothamstead, Hertfordshire, UK). The data were ana - qPCR mixtures reactions were prepared in a final volume lysed through a 2 × 2 factorial analysis of variance for of 20  µL, containing a final concentration of ~ 7  ng per diet treatments (C vs. TAG), litter treatments (clean vs. reaction of DNA template, 1X of Brilliant III Ultra-Fast reused) and their interaction, using pen location as a qPCR Mastermix (Agilent Technologies, United States), block, day 0 BWT as a covariate for day 21 BWT and the containing 30 nM of freshly prepared reference dye (Agi- pen of 10 chickens as the experimental unit. Data were lent Technologies, United States) and 100  nM of each checked for normality by examining residuals, histo- primer/ probe. Each reaction was carried out in tripli- grams and box plots, and none required transformation cate in a 96-well plate, including non-template control prior to statistical analysis. Effects at P < 0.05 and P < 0.10 and the standard curve. The latter was prepared via serial were considered significant and trends, respectively. 7 1 tenfold dilutions (10 to 10 gene copy numbers/reaction) Means were separated using Tukey’s honest significance of plasmid DNA containing the same target of this qPCR test. as an insert. Cycling conditions were set in a Stratagene MX3005P qPCR System (Agilent Technologies, United Abbreviations Kingdom) and were 95 °C (5 min), followed by 40 cycles AA Amino acids of amplification at 95 °C (15 s) then 60 °C (30 s). IP Ideal protein Thr Threonine Absolute quantification was performed using the Strat - Arg Arginine agene MxPro Software (Agilent Technologies, United Gln Glutamine Kingdom) through fitting a linear regression model with CP Crude protein BWT Body weight log standard copy number [x] and standard threshold BWG Body w eight gain (CT) (y). The quality of the reactions was verified by ana - FI Feed intake lysing the slope of the standard curve regression R and FCR Feed conversion ratio CPC Crude protein conversion efficiency calculation. The copy number calculated from SCFA Short chain fatty acids the standard curve represented copies per µL of DNA BCFA Branched-chain fatty acids extract. These values were log -transformed and multi- %G + C % Guanine + cytosine plied by 20 to obtain the 16S rRNA gene copy numbers Acknowledgements per g of chicken litter [80]. The authors acknowledge the staff and students at the Monogastric Science Research Centre, Auchincruive, UK, for all their help and support. The authors also acknowledge Evonik Nutrition & Care GmbH for providing amino acids Litter pH and moisture analysis and their help with feed analysis. Moreover, the authors acknowledge Dr At day 0, representative litter samples for both clean Sarah Brocklehurst (Biomathematics and Statistics Scotland) for her guidance on %G+C profile data analysis. and reused litter treatments were collected using sterile gloves and self-sealed plastic bags. At day 21, representa- Author contributions tive litter samples were collected from each pen from the MAH, FK, LV, SA and JGMH contributed to experimental design. MAH con- ducted the experiment, collecting samples, data analysis, writing-original four pen corners and the middle (around the feeders and draft, and presentation of the published work. FK and JGMH contributed to the drinkers) using sterile gloves and self-sealed plastic the provision of study materials. FK, LV, SA and JGMH contributed to review, bags. The collected litter samples were kept at − 80  °C editing and supervision. JGMH contributed to project administration and funding acquisition. All authors read and approved the final version of the freezer prior to analysis. Litter pH was determined by manuscript and approved publication. placing 10 g of each litter sample into 90 mL of distilled water and mixing for 10–15  min. pH was then meas- Funding Marwa A. Hussein is a recipient of PhD scholarship programme under the ured using a pH meter (Fisher Scientific accumet AE150 Newton-Mosharafa fund from the Egyptian Cultural Affairs and Missions pH Benchtop Meter) after calibration with pH 4, 7 and Sector and the British Council. SRUC received support from Scottish Govern- 10 buffers. Litter moisture content was also analysed in ment [RESAS]. Lonneke Vervelde received funding from a Biotechnology and Biological Sciences Research Council Institute Strategic Programmes Grant to duplicates by placing 10 g of each litter sample onto tared The Roslin Institute [BBS/E/D30002276]. aluminium drying dishes in a drying oven at 100  °C for Hussein  et al. Animal Microbiome (2023) 5:18 Page 13 of 15 Availability of data and materials 12. Nassiri Moghaddam H, Alizadeh-Ghamsari AH. Improved perfor- The datasets generated during and/or analysed during the current study are mance and small intestinal development of broiler chickens by dietary available from the corresponding author upon reasonable request. L-glutamine supplementation. J Appl Anim Res. 2013;41:1–7. 13. Ribeiro V Jr, Albino LF, Rostagno HS, Hannas MI, Ribeiro CL, Vieira RA, de Araújo WA, Pessoa GB, Messias RK, da Silva DL. Eec ff ts of dietary Declarations L-glutamine or L-glutamine plus L-glutamic acid supplementation pro- grams on the performance and breast meat yield uniformity of 42-d-old Ethics approval and consent to participate broilers. Braz J Poult Sci. 2015;17:93–8. All the experimental animal procedures in the current study were carried out 14. Wu QJ, Jiao C, Liu ZH, Cheng BY, Liao JH, Zhu DD, Ma Y, Li YX, Li W. 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Journal

Animal Microbiome – Springer Journals

Published: Mar 22, 2023

Keywords: Amino acids; Reused litter; Ideal protein; Broilers; Growth performance; Caecal microbiome; Litter characteristics

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