1. Introduction
The world population has been steadily growing in recent decades, and by 2050 a figure of 9.7 billion people is forecasted, coinciding with a 70% increase in the demand for food [
1]. With changing consumption patterns, meat shares an increasing part of this growing demand for food [
2]. World meat production is expected to increase from 320 million tonnes in 2016 to 360 million tonnes in 2025, with pork meat accounting for 36% of world meat production [
3] with a growth rate of 12%. Accordingly, large quantities of feed protein will be needed, and as it currently stands soybean products are the main protein ingredients in pig diets. Therefore, they act as a reference in the hunt for alternative sources to meet future protein demands. However, suitable feed alternatives are still needed [
4,
5]. Algae production could also be an acceptable alternative, because this type of aquatic biomass grows adequately with only marginal land use [
4]. Additionally, insects could be an alternative protein source because they can be reared on grain and the larvae grow rapidly. The microalga
Spirulina platensis is a prokaryotic multicellular cyanobacterium. Spirulina alga belongs to the photosynthetic organisms and grows only in warm climates with high light intensity. Natural environmental conditions are alkaline salt lakes as well as basic fresh water bodies. The black soldier fly
Hermetia illucens is a widespread fly and belongs to the family of Stratiomyidae, which is a member of the order Diptera. The larvae of Hermetia are able to utilize a wide range of nutrient sources and develop rapidly between 20 °C and 30 °C. More details about the alternative proteins are presented earlier [
6,
7,
8,
9]. Generally, both of the alternative proteins have the potential for being included as a protein source in animal feeds. Currently, there are only a few studies about Spirulina in piglet and pig diets, mostly dealing with health-promoting aspects of the microalga [
10,
11]. Earlier studies [
12,
13] examined the growth response of piglets with higher inclusion rates of Spirulina meal (8% to 12%) without negative effects. Hermetia also appears to be an adequate protein source; Newton et al. [
14] demonstrated the acceptance of diets containing up to 33% full-fat meal from Hermetia larvae as the main protein source in pig diets. Dankwa et al. [
15] substituted 100% of the fishmeal content in weaned piglet diets by non-defatted housefly larvae meal without a reduction in growth. Experiments by Ji et al. [
16] on early weaned piglets showed that 5% of plasma proteins in diets can be replaced by various insect meals (mealworm (
Tenebrio molitor), house fly larvae (
Musca domestica), and large mealworm (
Zophobas morio)) and induce no significant deficiencies in feed intake and growth performance. As a part of the multidisciplinary project “Sustainability Transitions in the Food Chain” (supported by the Lower Saxony Ministry of Science and Culture) the current study aimed to measure the protein quality of piglet and fattening pig diets based on N balance studies with a high inclusion of partly defatted Hermetia meal (HM) or Spirulina meal (SM) with a graded extent of AA supplementation.
3. Results
The results of the piglet N balance trials are summarized in
Table 5. Between the four experimental diets no significant differences in body weight (BW) (
p = 0.736), feed intake (
p = 0.224) and N intake (
p = 0.257) were observed. Fecal N excretions were enhanced (
p = 0.004) in the diets HM (A) (717 ± 60), SM (A) (841 ± 132) and SM (AA) (745 ± 123). Superior urine N excretion was observed both with diets HM (A) (1087 ± 118) and SM (A) (958 ± 121), which did not have an extended AA supplementation. Total N excretion was lowest with diet HM (AA) (1463 ± 146), but was not significantly different from diet SM (AA) (1584 ± 285). Daily N balance data were also the highest with diet HM (AA) (2182 ± 325), but again were only numerically different between diets (
p = 0.053). According to the observed fecal N excretion, HM diets yielded superior apparent N digestibility. As compared to diet HM (A), the extended AA supplementation in diet HM (AA) responded significantly (
p = 0.004) in dietary protein quality (NPU
std). This effect was less pronounced between the SM diets.
The observed bc
-1 values (
Table 5) are a reflection of the individual AA efficiency and need to be compared between the diets, while contextualized with the degree of limiting position as related to the other AAs under study (
Table 3 and
Table 4). Between piglet diets, the highest Lys efficiency (35 ± 3) was observed in diet SM (A), but this was only significantly different (
p = 0.032) from diet SM (AA) (30 ± 4) with its Lys supply according to the recommendations. Superior Met efficiency (137 ± 6) was achieved with diet HM (A) having the lowest Met supply, which indicated that Met was the most limiting AA in this diet. A significant difference in Thr efficiency between diets was not observed, but the observed trends require discussion. The extended degree of AA supplementation in diet HM (AA) provided superior Leu efficiency (37 ± 5) indicating the strong limiting position of Leu in this diet. Accordingly, SM diets yielded a significantly (
p < 0.001) higher His efficiency (118 ± 9 and 125 ± 18) as compared to HM (73 ± 3 and 91 ± 12) diets, supporting the strong His limitation as calculated (
Table 3) in both of the SM diets.
Results of the N balance study with growing pigs (
Table 6) also indicate that the diets with basic AA supplementation are inferior as compared to the diets with extended AA fortification. In addition, both of the HM diets created less fecal N excretion (
p < 0.001) and in consequence have significantly higher (
p < 0.001) N digestibility compared to the SM based diets. The urine N losses were significantly decreased (
p < 0.001) both with diet SM
1(AA) (971 ± 111) and SM
2(AA) (967 ± 50). Resulting N balance data were similar in diets HM (A) (1718 ± 241), SM (A) (1847 ± 135), HM (AA) (2054 ± 272), and SM
1(AA) (1915 ± 126). However, due to the addition of L-His, diet SM
2(AA) created a significantly improved (
p < 0.001) daily N balance (2411 ± 55). Looking more closely at the NPU
std, an enhanced protein quality was found with diets on the extended degree of AA supplementation, and the superior protein quality (
p < 0.001) was achieved in diet SM
2(AA) (71.8 ± 1.3) due to the compensated His deficiency. Excluding the consideration of the preliminary results in diet SM
2(AA), diet SM
1(AA) yielded superior His efficiency (
p < 0.001), indicating the validated limiting position of His in this diet.
Lys efficiency did not significantly differ amongst the other diets, but the observed trend between diet HM (A) (60 ± 11) and HM (AA) (68 ± 19) was unexpected. Regarding the Met efficiency, the highest bc−1 (285 ± 78) value for diet HM (AA) could indicate that Met supply was still nearly limiting in the AA balanced diet HM (AA), but both the observed Thr efficiency and Leu efficiency could reflect a co-limiting situation.
4. Discussion
In the current studies, no significant effects were observed for average body weight of piglets or growing pigs. In addition, we found no significant influence on feed intake for piglets, but did observe an effect for growing pigs. However, these parameters were only measured within the time schedule of N balance studies and should not be over-interpreted, but were eliminated for protein quality evaluation by the applied modelling procedure (Goettingen approach). A few previous studies have already demonstrated that SM could be a good protein source in pig diets [
10,
12,
13,
43]. Grinstead et al. [
10] examined graded contents of SM (2–20 g/kg) in diets for weaned piglets. Pigs that received 20 g Spirulina per kg feed had significantly higher feed intake and higher daily gains than the control group. Pig diets with 12% SM [
12], 9% SM [
13] or at least half of the protein from soybean meal substituted by algae meal [
43] created no adverse effects on growth performance. Other studies have also reported that pig diets containing insect meals are well accepted [
14,
16,
44]. Pig diets with 5% insect meals (
Tenebrio molitor,
Musca domestica larvae, and
Zophobas morio) substituting 5% plasma protein powder [
44] or 6% mealworm meal instead of SBM [
16] yielded no significant effects on growth performance.
Regarding the apparent N digestibility observed in our studies (piglets and pig), HM diets achieved superior N digestibility as compared to the SM diets. This supports the conclusion that, in particular, the algae meal of
Spirulina platensis is more poorly digestible than the insect larvae meal. The level of AA supplementation in the experimental diets was not a factor of significant influence on the observed N digestibility. Nonetheless, Février and Sève [
12] demonstrated that digestibility of feed mixtures was somewhat inferior when Spirulina meal was incorporated. Other reports from Martinavičius [
45] with a low level of added SM (2 g/d) showed numerically higher daily gains as well as enhanced digestibility of fat, organic matter, and protein. The very low supplementation of Spirulina in this diet could explain the effects on protein digestibility. To our present knowledge, further relevant studies assessing the digestibility of micro algae meals in pig diets are not available. Other aspects influencing the digestibility of SM are discussed elsewhere [
7,
8,
9]. As for the inclusion of insect meals, Newton et al. [
14] studied the digestibility of piglet diets with
Hermetia illucens when substituting 100% of the SBM by a dried larvae meal of
Hermetia illucens. The reported apparent digestibility of dry matter, nitrogen, ether extract, crude fiber, ash, NFE, calcium, and phosphorus for the larvae meal based diet were 77.5, 76.0, 83.5, 53.8, 45.2, 84.7, 38.9, and 23.0, respectively. The corresponding data for the soybean meal-based diet were 85.3, 77.2, 73.0, 49.2, 61.6, 91.3, 39.3, and 51.3. The observed digestibility of dry matter, nitrogen, ash, and NFE was higher in the plant-based diet. Further aspects about the digestibility of HM are discussed elsewhere [
7,
8,
9].
Looking more closely at the standardized NPU (NPU
std,) as an N intake independent measure of the feed protein quality, during the piglet period a significantly higher protein quality was observed for the HM (AA) diet compared to diets HM (A) and SM (A). In growing pigs, diets SM
2(AA) and HM (AA) created superior protein quality. However, the achieved dietary AA balance following extended the AA supplementation was an important factor of influence. Our results have clearly demonstrated that diets composed of the alternative proteins under study with an extended AA supplementation level yielded superior parameters of feed protein quality. This observation is also supported by current reports with chickens from Neumann et al. [
6,
7] and Velten et al. [
9]. The actual AA supplementation aimed to meet the current ideal amino acid (IAAR) assumption [
17]. In piglets, the extended AA supplementation with diet HM (AA) yielded a significantly higher protein quality, indicating that an enlarged range of AA supplementation (Lys, Met, Thr) is required. In contrast, diet SM (AA) with extended AA supplementation did not significantly achieve improved feed protein quality compared to diet SM (A). However, the dietary His supply remained 30% below the recommendation of NRC [
18] and BSAS [
19] and this fact meant that His was the limiting AA. In growing pigs, the extended AA supplementation with diet HM (AA) achieved only a numerical increase in feed protein quality. However, the additional supplementation of His in diet SM
2(AA) yielded a significant improvement in feed protein quality, indicating that His was indeed the limiting AA in diet SM
1(AA). Accordingly, the elevated level of Lys in diet SM
1(AA) could not improve the dietary protein quality, but the supplementation of His in diet SM
2(AA) provided significantly higher feed protein quality.
A large variation in the recommended optimal amount of His supply can be observed throughout the literature. The GfE [
17] recommendations for piglets are substantially higher (+15%) in comparison to NRC [
18] and BSAS [
19]. Also, for growing pigs, higher in-feed concentrations (+17–26%) were recommended in the German requirement standards [
17]. As a consequence, diet SM
2(AA) containing 3.68 gHis/kg (Ratio Lys:His = 100:35) supplied 12% more His as recommended by NRC [
18] and BSAS [
19], but taking into account the German recommendation (Ratio Lys:His = 100:47) His could still be considered the limiting AA. As demonstrated both by the very high level of His efficiency (bc
−1His) and insignificant difference between diets SM
1(AA) and SM
2(AA), a further limitation of His in the His supplemented diet was plausible. This is the first validated measure of His efficiency under an approved His limitation in the diet according to the ‘Goettingen approach’. However, the His supplementation unexpectedly affected protein quality leading to significantly improved AA efficiency of each of the AAs under study. As a consequence, the Lys efficiency in diet SM
2(AA) also exceeded the results of the other diets, but cannot be explained from a physiological point of view and we rank these results as preliminary. Therefore, the recommendation of NRC [
18] and BSAS [
19] for optimal His supply could be set too low. However, the limited number of repetitions with this diet is only indicating in this direction, but should not be taken as a validating conclusion, yet.
Continuing the discussion on the effect that diet has on AA efficiency, it has to be pointed out that effects only within the age periods and for relative AA supply below 100% are of interest. The methodical background is reported in more detail elsewhere [
24,
25]. Accordingly, only the most important observations will be discussed further. In piglet diets, the Lys efficiency differed only between the SM diets, indicating that the elevated Lys supply with diet SM (AA) impaired the Lys efficiency due to the remaining low level of His in both of these diets. Accordingly, the His efficiency was superior in SM diets. The high level of Met efficiency in diet HM (A) (exceeding the data of the other diets), clearly indicates Met as the limiting AA in diet HM (A). Otherwise, the lowest Leu supply created a significantly improved Leu efficiency with diet HM (AA). The observed Thr efficiency was not significantly different between diets and in agreement with the fact that this AA was not in a clear first limiting position in any of the diets under study.
In growing pigs, significantly superior data of AA efficiency were generally observed in diet SM2(AA), but this result should not be over-interpreted according to the former discussion related to His efficiency. Lys efficiency was only numerically different between the other diets, indicating that a limiting position of Lys was not verified. Accordingly, Met efficiency did not significantly differ between diets, with the exception of diet SM2(AA). In addition, diet HM (AA) produced superior efficiency of both Met and Thr, but the effect was not significant. In the case of Leu efficiency, the response with HM (AA) was more distinct but only significantly different to diet SM (A); although the same relative level of Leu was supplied in both of the HM diets. In summary, the high level of His efficiency as found with the His limiting SM diets in growing pigs is of further interest to generate the first AA efficiency data under conditions of a validated limiting position for His using the ‘Goettingen approach’.
Finally, further investigations are required to optimize the dietary AA balance in pig diets with an elevated inclusion level of the alternative feed proteins under study. However, up to now it can be ascertained that both protein sources are useful from the viewpoint of dietary protein quality when an appropriate AA supplementation is applied. Actually, insect-based meals are still not authorized for pig feeds in the European Union [
46]. Nonetheless, the EU legislative barriers are expected to be overcome in the near future according to the permission for aquafeed [
47]. However, the profitability of the two alternative protein sources needs to be improved.
The general conclusion of this study is that partly defatted insect meal from Hermetia illucens (HM) larvae or the microalgae Spirulina platensis (SM) at inclusion rates of 21% (piglets) and 13% (pigs) are useful protein alternatives in diets for piglets and growing pigs. The high inclusion rate of SM and HM was applied to demonstrate their potential; however, limitations were uncovered when the dietary AA balance is not sufficiently supplemented. In this context, we aimed to quantify these responses on dietary protein quality by means of N balance studies.