Lysine efficiency in piglets fed diets with a phytogenic feed additive and conclusion of lysine requirement data

Two experiments were conducted to examine effects o f a commercial phytogenic feed additive (PFA), containing inul in , an essential o i l m i x (carvacrol, thymol) , and chest nut meal (polyphenols) on nutrient digestibility, protein ut i l izat ion, and lysine efficiency in piglets. Each experiment ut i l ized sixteen piglets (male castrated, 8 w k ) and four experimental groups. Experiment 1 studied graded levels o f the PFA (0, 0.05, 0 .1 , 0.15%) in lysine l imi ted diets (wheat, barley, soyabean meal, fish meal). Experiment 2 examined 0 . 1 % o f the PFA and two dietary levels o f lysine. The results indicated that apparent nutrient digestibility, protein uti l izat ion, and lysine efficiency were not significantly affected by the PFA (P>0.05). Observed daily lysine requirements (7.3, 9.9 and 13.1 g for 76, 100, 124 g daily protein deposition) in growing barrows (20-30 kg body weight) were in line w i t h recommendations and contribute to the l imi ted database for model l ing o f lysine requirements in piglets. K E Y W O R D S : piglets, protein ut i l izat ion, feed additives, lysine INTRODUCTION Mixtures of herbs, spices and essential oils are currently, applied to achieve feed intake, growth and health promoting effects in several animal species. However, mode of action and potential of phytogenic feed additives (PFA) in vivo 1 Corresponding author: e-mail: flieber@gwdg.de 50 PHYTOGENIC FEED ADDITIVE AND LYS EFFICIENCY IN PIGLETS is mostly unknown. Currently, contradictory observations do not withstand an objective and critical judgement. Additionally, the diversity of PFA mixtures allows no general conclusion (Jeroch et al., 2008). Earlier studies with a commercial PFA examined zootechnical data, parameters of gut microflora, digestive enzyme activity and unspecific immune response in weaned piglets (Muhl and Liebert, 2007a,b). The current study investigated parameters of digestibility, protein utilization and dietary lysine efficiency. A validated procedure (Liebert, 2008; Liebert and Wecke, 2008; Wecke and Liebert, 2009) for modelling of protein utilization depending on observed dietary amino acid efficiency was applied for conclusion of lysine requirement data for piglets. MATERIAL AND METHODS N-balance experiments were conducted making use of a commercial PFA containing the fructopolysaccharide inulin (53%) from Jerusalem artichoke, an essential oil mix from oregano oil (8%), tannins from chest nut meal (3%) and cellulose powder (36%), respectively. An external gas chromatographic analysis of the PFA yielded 6.0% carvacrol and 0.14% thymol.


INTRODUCTION
Mixtures of herbs, spices and essential oils are currently, applied to achieve feed intake, growth and health promoting effects in several animal species.However, mode of action and potential of phytogenic feed additives (PFA) in vivo is mostly unknown.Currently, contradictory observations do not withstand an objective and critical judgement.Additionally, the diversity of PFA mixtures allows no general conclusion (Jeroch et al., 2008).Earlier studies with a commercial PFA examined zootechnical data, parameters of gut microflora, digestive enzyme activity and unspecific immune response in weaned piglets (Muhl and Liebert, 2007a,b).The current study investigated parameters of digestibility, protein utilization and dietary lysine efficiency.A validated procedure (Liebert, 2008;Liebert and Wecke, 2008;Wecke and Liebert, 2009) for modelling of protein utilization depending on observed dietary amino acid efficiency was applied for conclusion of lysine requirement data for piglets.

MATERIAL AND METHODS
N-balance experiments were conducted making use of a commercial PFA containing the fructopolysaccharide inulin (53%) from Jerusalem artichoke, an essential oil mix from oregano oil (8%), tannins from chest nut meal (3%) and cellulose powder (36%), respectively.An external gas chromatographic analysis of the PFA yielded 6.0% carvacrol and 0.14% thymol.

Animals and diets
Each of the experiments was carried out on sixteen male castrated eight week old piglets [Pietrain x (Large White x German Landrace)].Piglet mash diets were based on the main ingredients: wheat, soyabean meal, barley and fish meal, respectively.
Experiment 1 examined three graded levels of the PFA under study (Table 1) for investigation of effects of the additive on dietary lysine efficiency.To adjust lysine in the first limiting position, the dietary lysine supply was approximately 80%) of the NRC (1998) recommendation (Table 2).Experiment 2 utilized only one level of PFA addition, but supplemented crystalline lysine (Table 1, diets C, D) to approve the limiting position of lysine and to examine the PFA effect at optimal lysine supply.
Balance studies were conducted with individual metabolism cages making use of an adaptation period of five days and two consecutive collecting periods (five days each).For standardization of feed intake during the collecting periods, piglets were restrictive fed twice a day.

Sampling
Faeces were quantitatively collected twice a day during morning and afternoon feeding and stored at -18°C for further analyses.In order to minimize ammonia losses, the urine was acidified (pH below 2) with 60 g of sulphuric acid (30%).Individual aliquots of the daily urine sampling were stored at 4°C, carefully mixed and further analysed.15.9 MJ 16.0 MJ 1 calculated according to Kirchgessner and Roth (1983) All experimental protocols were approved by the Animal Welfare Committee of the Agricultural Faculty of Goettingen University.

Chemical analysis
Diets were analysed for dry matter (DM), crude protein (CP), crude fibre (CF), ether extract (EE), ash, starch and sugar.Nitrogen content of feed and faeces was estimated by Dumas-method (LECO® FP-2000), nitrogen content of urine with the Micro-Kjeldahl-Apparatus (BUECHI 315).CP was calculated from nitrogen content by factor 6.25.Crude fat analyses were conducted following HCl-hydrolysis.Analyses of ingredients, mixed diets and excreta were according to the German VDLUFA standards.Amino acid analyses run by ion-exchange chromatography (LC 3000, Biotronik) using acid hydrolysis with and without oxidation step for quantification of sulphur containing amino acids.

Data analysis
Apparent faecal digestibility of crude nutrients was established.Analysis of N-balance data and modelling procedure was conducted according to Liebert and Wecke (2008).Additional current applications of the N-utilization model are reported elsewhere (Thong and Liebert, 2004a,b,c;Liebert, 2008;Wecke and Liebert, 2009).
The basic function of the model is given in equation ( 1): NR-NR T(l-e bN1 ) (1) where: NR -daily N-retention (mg/BW kg °67 ) = Daily N-deposition + NMR; NMR -daily N-maintenance requirement (mg/BW kg 067 ); NR max T -theoretical maximum for daily N-retention (mg/BW kg 0 67 ); b -model parameter for the slope of the exponential function, depending on the dietary protein quality; NI -daily N-intake (mg/BW kg °67 ); e -basic number of natural logarithm (In).
Model parameters NMR and NR T were taken from earlier studies with Net protein utilization (NPU) data were standardized for equal daily N-intake (3650 mg/BW kg °67 ) according to Thong and Liebert (2004a).The applied procedure for modelling lysine requirements was according to earlier reports (Thong and Liebert, 2004b,c).
Equation (3) summarises the derived requirement dependent on NR and observed dietary amino acid efficiency (be 1 ): where: LAAI -required daily intake of the limiting amino acid for a given NR, mg/BW kg °67 ; be 1 -model parameter for the dietary amino acid efficiency.

Statistical analysis
Statistical data analysis utilized ANOVA (P<0.05 or higher) within the programme SPSS 12.0 for Windows.Data were subjected to a verification of variance homogeneity according to Levene test, following LSD in case of homogeneity of variance.Following in-homogeneity of variance, the Games-Howell post-hoc test was applied.

RESULTS
Apparent nutrient digestibility.Digestibility of crude nutrients (Table 3) did not significantly respond to the added PFA.The dietary lysine supply was not a significant factor for the observed results of the digestibility study.Nitrogen balance studies.Daily N-balance, N-utilization and dietary lysine efficiency were not significantly affected by the added PFA (Table 4).Supplementation of crystalline lysine in diets C and D (Experiment 2) improved N-utilization significantly.Consequently, lysine was confirmed to be in first limiting position in all of the diets not supplemented with crystalline lysine.Modelling of lysine requirement data.Conclusion of lysine requirement data (Table 5) was not in first focus of the conducted experiments.However, approved limiting position of lysine provided an additional database to improve information about quantitative lysine requirements for piglets.Requirements were derived for 40, 50 and 60% of the growth potential (NR max T), according to graded daily protein deposition (76, 100 and 124 g daily protein gain at 24.2 kg average BW).Assuming 17.0% crude protein in daily gain (GRRS 2006(GRRS . 2008), these protein deposition data were equal with 447, 588 and 729 g daily gain of BW.Additionally, according to the applied modelling procedure the requirements were also derived for graded dietary efficiency of lysine (Table 6).Based on predictions for the daily feed intake of piglets which were in line with feed intake pattern observed in the current experiments, recommendations for the optimal dietary lysine supply (as % of diet) were concluded.

DISCUSSION
Commercial phytogenic feed additives are varying in supply of individual bioactive substances.Consequently, several studies observed improved nutrient digestibility by supplementation of essential oils, but other reports failed to yield significant effects (Moller, 2001).Wald (2002) summarized that positive effects of essential oils are only caused by their antimicrobial action which could contribute to improve nutrient and energy utilization in the host animal.However, according to Wald (2002) our previous studies (Muhl and Liebert, 2007a,b) did not yield significant growth effects.Enhanced activity of digestive enzymes (Platel et al., 2002) was not supported by results of Muhl and Liebert (2007b).
According to Moller (2001), presented N-balance data failed to show significant effects of the PFA under study.According to Branner et al. (2004), no effect on total N-excretion, N-retention and N-excretion via urine was attributed to supplementation of inulin.However, due to supplied native prebiotic compounds in pig feeds effects of low quantity of added prebiotics is questionable.Furthermore, masking or dilution by dietary oligosaccharides from cereals is also discussed (Gabert et al., 1995).However, experimental data for inulin effects on availability and retention of nutrients are scarce (Verdonk et al., 2005).In the current study, the very low supply of added inulin from the PFA under study was not sufficient to induce any significant effect.Mosenthin and Zimmermann (2000) concluded that healthy and well-kept animals not crucial exposed to pathogenic bacteria and stress factors will scarcely respond to this type of feed additives.
The insignificant effects of the PFA on observed dietary lysine efficiency provided data for modelling of lysine requirements.In growing barrows (20-30 kg BW) 7.3, 9.9 and 13.1 g lysine per day were required for 76, 100 and 124 g daily protein deposition.These data were in line with NRC (1998) recommendations (10-20 kg BW: 11.5 g/d; 20-50 kg BW: 17.5 g/d).In terms of total lysine, earlier German recommendations vary between 7.2 and 12.3 g lysine per day (20-25 kg BW, 300-600 g daily gain).Actual German recommendations (GRRS, 2006(GRRS, , 2008) ) advise 10.1 and 12.1 g lysine per day for 500 and 600 g daily gain (25 kg BW) in terms of standardized praecaecal digestible lysine.Assuming 85% as an average of ileal lysine digestibility, these recommendations are in the scope of presented requirement data for 100 g daily protein deposition.Our reported lysine requirement data for piglets are also in line with several other studies (Gatel et al., 1992;Whittemore et al., 2003;Wecke and Liebert, 2009).For feed formulation, Bertolo et al. (2005) recommended 0.91% ileal digestible lysine (24.1 kg BW; 982 g daily feed intake), which are in line with our data (85% predicted ileal lysine digestibility) and several other studies (Martinez and Knabe, 1990;Coma et al., 1995).In contrast, Fu et al. (2004) recommended 1.32% true digestible lysine for late nursery pig diets (29 kg BW).Generally, the applied procedure makes use of benchmarks for modelling amino acid requirements which can be adapted to different needs, like genotype, individual BW and the aimed daily protein deposition as important factors of influence (Martinez and Knabe, 1990;Cromwell et al., 1993;Thong and Liebert, 2004a,b,c).

CONCLUSIONS
According to our previous studies, no significant response was observed due to application of a commercial phytogenic feed additive (PFA) in piglet diets, but more investigations are needed to clarify mode of action and efficacy of PFA mixtures under several environmental circumstances.The applied modelling procedure was confirmed as a reliable tool to improve database for lysine recommendations in piglets.

Table 4 .
Results of the N-balance study (n=8; mean BW: experiment I = 23.2kg, experiment II =

Table 5 .
Model calculation of lysine requirement for piglets (20 to 30 kg) depending on daily CP-deposition, observed efficiency of dietary lysine utilization and predicted feed intake max BW = 24.2kg; 4 BW = 24.2kg

Table 6 .
Model calculation of lysine requirement for piglets (20 to 30 kg) with varying dietary