In this study the effects of DLM and MHA-FA supplementation in protein reduced diets on the performance, footpad health and liver health of fattening turkeys were evaluated and the results obtained for the several parameters will be discussed in separate sections.
4.1. Performance
There were no complications or mortality observed during the experimental period. On arrival at the experimental facility, female turkeys had an average BW of 4.03 kg ± 0.355 and were therefore 15% below breeder recommendations of 4.73 kg for B.U.T. 6 females at day 63 [
37]. However, the BW achieved at day 98 particularly regarding with standard protein levels exceeded 4640 g/bird which was suggested by the breeder as an efficient amount of feed. However, the average BW of turkeys at day 98 was 8.69 kg/bird and thus still 8% below 9.45 kg as suggested by the breeder. It should be noted that the FCR observed in the present trial ranged between 2.60 and 2.79 kg/kg and substantially exceeded the performance goals of the breeding company [
37]. Therefore, the experimental feeds were basically well utilised. This is remarkable because specifications for the amino acid levels and applied profile are for heavy female turkeys 98 to 111 days-old, whereas recommendations for 70 to 97 days-old females would be somewhat higher [
38].
To reduce nitrogen emissions, a protein reduction in the diets is necessary [
16]. This is only possible by balancing in particular the essential amino acids in the feed using free amino acid supplements. In the diets of all experimental groups, the essential amino acids lysine, Met, threonine, tryptophan, arginine, valine and isoleucine were optimised on a digestible amino acid basis to achieve similar levels. This was achieved by individually adding crystalline amino acids (with the exception of MHA-FA which is liquid). Basically, analyses of diets confirmed successful feed production as targeted levels were recovered. However, protein reduction in diets containing 15% protein was strong (3% points) and showed lower contents of some amino acids than the diets containing 18% protein. In this context, the lower content of the essential amino acids leucine, histidine and phenylalanine and of the semi-essential amino acids serine and glycine in the protein reduced diets has to be noted. This is in line with observations by Lemme et al. [
39] who reduced crude protein more moderately by only 1% point and who achieved a significant reduction in Gly-equivalents (Gly + 0.714 * Ser) in the protein-reduced feeds. Moreover, the researchers reported a significantly lower intake of Gly-equivalents at protein reduction.
Despite balancing for six essential amino acids using commercially available products in the experiment, all performance parameters (
Table 4 and
Table 5) were negatively affected by the protein-reduced diets. In contrast to Lemme et al. [
39,
40], we formulated the experimental feed on a standardised ileal digestible amino acid basis using digestibility coefficient as proposed by Lemme et al. [
39,
40,
41]. However, these digestibility coefficients were determined with broilers. It has been reported that digestibility coefficients differ between turkeys and broilers which might have interfered with protein reduction, as proportions of ingredients substantially changed [
42,
43,
44]. Nonetheless, Kluth and Rodehutscord [
44] did not report any substantial differences for amino acid digestibility with respect to soybean meal, but Kozslowski et al. [
43] suggested differences. Probably more of importance are the amino acids which were not considered for balancing. As mentioned above, the degree of protein reduction was high in this trial, this having tremendous impact on levels of not considered essential amino acids as well as conditionally essential and non-essential amino acids. According to feed analysis, the total levels of lysine, methionine + cysteine, threonine, tryptophane, arginine, valine and isoleucine were 3–7% lower in low protein diets compared to standard protein, while on a digestible level, they were very similar. However, leucine, histidine and phenylalanine as well as glycine and serine were 20–25% lower in low protein feeds. While there is no information about requirements for turkeys for these amino acids, it is assumed that one or more of them were below the requirements limiting protein synthesis and, therefore, resulted in lower BW gain and higher PER and FCR. Lemme et al. [
40] were not successful in protein reduction as performance dropped in any case. Nevertheless, their research indicated that balancing amino acids beyond lysine, methionine + cysteine, threonine and tryptophan were needed to achieve the same FCR and breast meat deposition as with protein reduction including the entire range of essential amino acids. Indeed, Lemme et al. [
6] were able to at least maintain BW gain and FCR when reducing the protein level of commercial type diets by 1% point and balancing for lysine, methionine + cysteine, threonine, arginine, valine and isoleucine. However, meat deposition was slightly affected. They calculated the intake of essential amino acids as well as glycine-equivalents and concluded that a significantly lower glycine-equivalent intake was responsible for reduced meat deposition. The content of the semi-essential amino acids serine and glycine must be noted. The synthesis of glycine and serine can be limited at a high BW gain intensity, in this case either glycine or serine have to be supplemented to the diet [
45]. Dean et al. [
45] reported that glycine and serine first had a limiting effect on BW gain and FCR of broilers when protein content of diets was reduced from 22% to 19% and all essential amino acids were supplemented. Thus, there is a likelihood that the intake of certain amino acids in this trial was insufficient for maintaining the performance at the level of the standard protein treatments.
With special regard to methionine:cysteine ratio, literature data reporting the optimal methionine:cysteine ratio in poultry diets differ markedly [
46]. One important influencing factor is that the metabolic degradation of the indispensable methionine may yield cysteine [
47,
48,
49]. Consequently, an appropriate amount of cysteine leads to increases the availability of methionine [
50]. However, the close metabolic link between methionine and cysteine makes it more difficult to assess both the animals’ requirement for total sulfur containing amino acids and the optimal dietary methionine:cysteine [
46]. However, Wheeler and Latshaw [
51] also found an age-dependent effect on the optimal methionine:cysteine.
Dietary protein reduction significantly reduced water intake. This is an often described effect in the context of protein reduction and is assigned to decreased soybean meal use [
4,
16,
52,
53]. Soybean meal contributes relatively high levels of potassium to the feed, stimulating water intake [
54]. In our trial, inclusion of soybean meal was reduced by 46%. In addition, less nitrogen has to be excreted which reduces the water requirement [
54]. Assuming 33 g nitrogen/kg weight gain and analysed protein levels of the experimental feeds, average nitrogen depositions of 149, 140, 165, and 160 g/bird and nitrogen intakes of 314, 295, 390 and 372 g/bird can be estimated for birds of treatments LP + DLM, LP + MHA-FA, HP + DLM and HP + MHA-FA, respectively, for the experimental period [
6]. Accordingly, dietary protein reduction lowered nitrogen excretion by 27% and thus increased nitrogen utilisation from about 42.6% to 47.5%. Protein reduction by 1% point over the entire production cycle reduced N-excretion by 8%, which is a similar magnitude of response when related to 1% point protein reduction. It is remarkable that reduced water intake due to protein reduction was not reflected in dry matter content of litter in the current trial [
6]. Also, Lemme et al. [
6] were unable to observe a clear relationship between dietary protein level and litter DM content although they reported markedly improved foot pad health. In the current trial, water intake was not necessarily only influenced by dietary protein per se but also by feed intake. There was a high correlation between water intake and feed intake (
r = 0.94) and, accordingly, also feed intake was reduced by protein reduction (
p < 0.05). Still, W:F intake ratio would confirm the relationship with soybean meal and related potassium intake as well as with reduced N-excretion. However, the above discussed strong impact on the dietary amino acid profile and respective match with amino acid requirements negatively affected the feed intake. D’Mello [
55] described in his review that amino acid imbalances result in reduced or even depressed feed intake in monogastrics including poultry, and the 20–25% lower levels of those amino acid not considered in feed optimisation may indicate an imbalance. Moreover, Schutte and Pack [
56] reported that the increase in methionine levels (i.e., increase in availability and absorption) increased feed intake in broilers. It is well known that The DLM are actively absorbed (transported against a concentration gradient), while MHA-FA is absorbed by the H+ dependent system which is slower than the Na+ system in DLM [
57]. According to the literature, the average relative bioavailability of MHA-FA products compared to DLM is approximately 75–80% on an equimolar basis [
23,
58]. Thus, MHA-FA may be less efficiently absorbed and utilised by birds than DLM and may consequently led to reduce feed intake.
Supplementation of MHA-FA followed the recommendation of Hoehler and Hooge [
24] and Hoehler et al. [
25]. Accordingly, a relative bioavailability of 65% is assigned to MHA-FA compared to DLM. This suggests that by replacing MHA-FA with DLM in a weight-to-weight ratio of 100:65 in feed, animal performance is not affected. Indeed, the results of the current study basically confirm no detrimental effect of the relatively low inclusion levels of DLM on any performance parameter. Rather the contrary is the case as the BW at the end of the experiments tended to be higher with DLM reflected in the BW gain. Moreover, the apparently lower weight gain can be explained by significantly lower feed intake of MHA-FA fed turkeys, which due to the strong correlation between feed and water intake, was also mirrored in a significantly lower water intake. It is emphasised that neither W:F intake ratio nor PER and FCR were affected by Met sources. Höhler et al. [
25] also tested DLM and MHA-FA in a 65:100 ratio at three different supplementation levels in one of the reported trials and described the same growth performance and FCR. Hoehler and Hooge [
24] confirmed this observation, as four commercial type trials with >5000 turkeys per treatment replacement of MHA-FA with DLM in a 100:65 ratio resulted in the same performance. Agostini et al. [
59] fed turkeys over an entire production cycle and while final BW and FCR were not affected by the Met sources supplemented in a 65:100 ratio, they reported a significantly lower breast meat yield in the case of MHA-FA feeding. Lemme and Meyer [
38] applying the same experimental design as Agostini et al. [
59], also found significantly lower breast meat yield in MHA-FA fed turkeys compared to DLM fed turkeys. When correcting for mortality, FCR tended to be higher with MHA-FA (
p = 0.54) in the latter study. When summarising literature findings and the current study, it can be concluded that replacing MHA-FA with DLM in turkey feed at a weight-to-weight ratio of 100:65 results in the same performance. Few results even indicated a slight advantage for DLM. There is quite a lot research on these Met sources with respect to physiology and metabolism providing evidence for the lower bioavailability of MHA-FA compared to DLM. Research performed by Maenz and Engele-Schaan [
57], Drew et al. [
60] and Malik et al. [
61] suggested that a substantial portion of ingested MHA-FA is degraded by intestinal microbes and thus, not available to the host. DLM is absorbed by sodium- driven transport with high affinity of the transporters to DLM and a relatively high velocity, whereas MHA-FA is absorbed like short chain fatty acids with proton-driven transporters with relatively low affinity to MHA-FA and relatively low velocity [
57]. Moreover, the latter study suggests that diffusion through tight junctions of the intestinal wall does not play a role. Finally, absorption studies by Mitchell and Lemme [
62] suggest that especially the short chains of liquid MHA-FA are poorly absorbed. This finding is confirmed by Rhone Poulenc [
63] and Van Weerden et al. [
64] who conducted simultaneous dose-response studies with DLM, MHA-FA and only short MHA-FA chains, consistently reporting a lower relative bioavailability of MHA-FA oligomers compared to the commercial MHA-FA product which contains about 65% monomers, 23% monomers and 12% water.
4.2. Footpad Health
Impaired footpad health is often related to increased water intake, increased water excretion and, as a consequence, increased litter moisture. Indeed, we could not find a clear effect of protein reduction on litter moisture nor could Lemme et al. [
6], but Lemme et al. [
6] found a clear improvement in footpad health, as the average lesion score could be reduced especially by increased proportions of grade 0 and grade 1 scores. The same was observed in the current study, as footpad scoring numbers were significantly reduced with protein reduction. This effect was measurable after four weeks of feeding the diets. In their review on low protein nutrition of broilers, Greenhalgh et al. [
54] clearly indicated the relationship with improved footpad health. At day 98, the evaluation of the influence of the Met source showed tendentiously lower FPD scores for DLM compared to MHA-FA (
p < 0.10). This is in line with the positive effect of Met supplementation on footpad health which has been described many times [
10,
12,
13,
65,
66]. Deficiency in Met intake has a significant negative impact on animals such as growth inhibition via interruption in protein synthesis (i.e., affect skin structure), the induction of metabolic disorder, and the reduction of disease defensive potential [
67]. Abd El-Wahab et al. [
10] found that at almost identical litter DM contents (measured weekly), the young turkeys fed high level of Met in diet had lower FPD scores than those fed diet with low Met content. It means that level of dietary Met plays an important role for health of skin rather than moisture content in the litter. Thus, it seems that Met has a specific function regards foot pad health (as known for skin and feathers) as mentioned by Abd El-Wahab et al. [
14] via protein synthesis and continuous production of keratin. Consequently, low availability of Met could affect protein synthesis negatively and affect skin of foot pad.
Chavez and Kratzer [
13] compared the influence of DLM and MHA-FA on the footpad health of white fattening turkeys. They found that supplementing a basal diet with 4.00 g DLM/kg diet resulted in an improvement in footpad health that was not achieved to the same extent by supplementing 4.03 g of MHA-FA/kg diet. When interpreting the studies of Chavez and Kratzer [
13], it must be noted that the comparing 4 g DLM with 4.03 g MHA-FA, an equivalence of both substances was assumed regarding the biological activity, whereas the authors of other studies assumed a lower biological activity of MHA-FA [
10]. The tendentiously lower FPD scores of the animals whose diets contained DLM compared to those with equivalent supplementation of MHA-FA (based on 65% biological activity) corresponds to the observations of Chavez and Kratzer [
13] and Abd El Wahab et al. [
68].