Physical treatment and protease or probiotic supplementation and feather meal digestibility by broilers
Y. M. Sun
A * , X. Li A , D. Zhang A and W. L. Bryden A B
A
B
Abstract
Improving the utilisation of alternative protein ingredients in livestock production will reduce feeding costs and improve industry sustainability. Feather meal (FM) is an abundant, alternative protein source with a high protein content but poor amino acid (AA) digestibility.
This study evaluated strategies for improving AA digestibility of FM.
Experiment 1 examined the effects of physical treatment with ultrasound, microwave and autoclaving on FM AA profile and digestibility. Experiment 2 evaluated the dietary addition of a protease (Ronozyme ProAct, 200 and 600 mg/kg; RPA) and a probiotic (BioPlus 400, 1500 mg/kg) on FM AA digestibility. Apparent ileal digestibility was determined by feeding each treatment to four replicate groups of six birds in Experiment 1 and five replicate groups of seven birds in Experiment 2, and then collecting the contents of the lower half of the ileum.
None of the physical treatments improved (P > 0.05) the AA profile or ileal AA digestibility of FM. Dietary supplementation with RPA at 200 mg/kg or BioPlus 400 at 1500 mg/kg did not significantly (P > 0.05) influence the apparent ileal AA digestibility of FM. However, the higher concentration of RPA (600 mg/kg) significantly (P < 0.05) increased the apparent ileal AA digestibility of FM.
The increased digestibility of FM by the protease and numerical increase (P < 0.1) by the probiotic (1500 mg/kg) presumably reflects keratinase activity of both feed supplements.
The results of this study indicated that there is scope for further improvement in the nutritive value of FM for broilers.
Keywords: amino acid digestibility, autoclave, broilers, feather meal, microwave, probiotic, protease, ultrasound.
Introduction
Hydrolysed feather meal (FM) is a major by-product of the poultry industry and is potentially a valuable source of crude protein and amino acids (AAs; El Boushy et al. 1990; Polin 1992). Attempts have been made in Australia for many years to increase the use of FM in both broiler (MacAlpine and Payne 1977) and layer (Luong and Payne 1977) diets. However, the major constraint is keratin, a feather protein, containing disulfide bonds, which form a tight network structure that hinders the proteolytic degradation of keratin by intestinal proteases, thus reducing FM digestibility (Parry and North 1998; Kreplak et al. 2004). Feather meal is considered a low-quality protein for broilers because of low digestibility, reduced feed intake, and an imbalance of nutrients, which result in poor utilisation (Cherry et al. 1975; Onifade et al. 1998; Ravindran et al. 1999).
To improve the feeding value of FM, many technologies (physical, chemical, and biological) have been reported and each process has pros and cons in terms of feather degradation and industrial application (Latshaw et al. 1994; Grazziotin et al. 2006; Lee et al. 2016; Li 2019). Physical technologies that have been shown to improve the efficiency of protein hydrolysis include microwave, ultrasound and autoclave (Li et al. 2010; Zoccola et al. 2012; Dai et al. 2020). Processing with microwaves has been used as a novel physical method to increase protein digestibility of feed ingredients (Chen et al. 2014; Sun et al. 2020). Sun et al. (2020) reported that microwave cooking of pigeon pea flour for 3 min resulted in a significant increase of 17.2% in protein hydrolysis compared with the raw sample. Lee et al. (2016) reported that microwave heating could denature keratin by disrupting both the secondary and tertiary structures of keratin and Rodríguez-Clavel et al. (2019) demonstrated that microwave or ultrasound irradiation could induce the recrystallisation stage of feather keratin. Moreover, the effect of microwave or ultrasound irradiation on modifying the texture and morphology of materials depends on the exposure time and power level (Rodríguez-Clavel et al. 2019). Ultrasonic waves change protein conformation, which exposes more hydrophobic groups, thus increasing protein solubility (Patist and Bates 2008). However, we are not aware of any reports that compare these different physical treatments on FM utilisation in poultry.
There has been much interest in supplementation of poultry diets with exogenous feed enzymes to improve digestibility (Bedford and Morgan 1996; Ravindran 2013). It has been shown that proteases can improve protein digestibility and reduce the need for supplementation of poultry diets with first limiting AAs (Zheng et al. 2023). Furthermore, protease is critical for optimising the use of alternative protein sources (Cowieson and Roos 2016), including poultry by-products (Lindberg et al. 2021). Keratinase, which is a specific protease that degrades insoluble keratin substrates, especially disulfite bonds, has attracted much attention as a potential way to improve the nutritional value of FM (Onifade et al. 1998; Brandelli et al. 2010). The use of keratinases with FM not only improves nutritive value in terms of digestibility but also the biochemical composition (Grazziotin et al. 2006). Recently, Ronozyme® ProAct (RPA) was introduced for broiler feed supplementation as a novel pure serine protease expressed in Bacillus licheniformis that has shown promising results when tested in vitro and in some in vivo studies (Glitsø et al. 2012; Navone and Speight 2018).
The ability of probiotics to enhance nutrient digestibility and growth performance of poultry has been shown in many studies (Saleh et al. 2020a; Zaghari et al. 2020; Bajagai et al. 2023) but the effect of probiotics in FM-based poultry diets has not been reported. Bacillus is one of the major probiotic genera used in poultry nutrition (Shini and Bryden 2022). BioPlus 2B® is a Bacillus probiotic (contains B. subtilis and B. licheniformis spores), and several studies using it have shown improved growth, nutrient utilisation and carcass characteristics of broilers (Šabatková et al. 2008; Bai et al. 2013). Bacillus licheniformis and B. subtilis produce enzymes in the intestine such as amylase, protease, and lipase. Some proteases have the ability to hydrolyse keratin (Brandelli 2008; Daroit et al. 2010; Kaewtapee et al. 2017). BioPlus 400 is an in-feed probiotic containing B. subtilis and B. licheniformis, as does BioPlus® 2B (Liu et al. 2012). Interestingly, most commercial keratinase enzymes are produced from B. licheniformis (Navone and Speight 2018). It is possible therefore, that BioPlus 400 has the potential to hydrolyse feather proteins and improve the nutritional value of FM. Moreover, the keratinous substrates in FM may induce keratinase production by B. licheniformis (Mazotto et al. 2011).
The aims of the current in vivo studies in broiler chickens were to (1) determine the effect of different physical treatments on AA digestibility of FM, and (2) evaluate the dietary addition of a protease and a probiotic on AA digestibility of FM. The findings will provide guidance for further studies to improve FM utilisation by broilers.
Materials and methods
Physical treatment of feather meal
The hydrolysed FM used in this study was supplied by CSF Proteins Melbourne (1–9 Merino Street, Laverton, Victoria, Australia). After the physical treatment (ultrasound, microwave and autoclave) as described below, the FM samples were oven-dried at 60°C and then ground to a particle size of 2 mm in a Wiley Mill (Thomas Scientific, USA). The FM samples were then stored in a cool room at 14°C and subsampled for protein and AA analysis.
The ultrasound treatment was conducted in an ultrasonic cleaner FXP14 (Unisonics®, Australia) with a frequency of 40 kHz in pulse mode. Feather meal samples were mixed with deionised water at a solid:liquid ratio of 1:2 in glass bottles with screw caps and sonicated for 30 min or 120 min.
Feather meal samples were added to deionised water at a solid:liquid ratio of 1:2 in a microwave container and subjected to microwaves at residence times of either 2 min or 10 min. Microwave treatment of FM was conducted using a domestic microwave oven (Anko, P11034AL-B6), with an operating frequency of 2450 MHz and power level of 1100 W.
Birds
Day-old, male broiler chicks (Ross 308) were obtained from a local hatchery. The chicks were reared in an environmentally controlled room where the temperature was maintained at 32 ± 1°C during the first week and gradually decreased to ~23°C by the end of the third week. The birds received a commercial broiler starter diet (230 g protein/kg) from Day 1 to 20 and a commercial broiler finisher diet (180 g crude protein/kg) from Day 21. The experimental procedures involving birds were approved by the Animal Care and Ethics Committee of the University of Queensland and complied with the Australia Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC 2013).
Digestibility bioassay
The bioassay procedure, used in both experiments, has been described by Ravindran et al. (1999). Briefly, on Day 35, birds with bodyweights close to the mean were selected and randomly allocated into cages and fed experimental diets. These diets were based on the basal control diet, shown in Table 1, where FM was the sole source of dietary protein in all experimental diets. Celite® (Celite Corporation, Lompoc, CA, USA), a source of acid insoluble ash (AIA), was added (20 g/kg) to all diets as an indigestible marker. The fibre component of the diet was satisfied by adding cellulose (Solkafloc®, James River Co., New Jersey, USA). After 5 days of feeding, all birds were euthanased by cervical dislocation and digesta from the lower half of the ileum was collected by gently flushing with distilled water. The digesta from all birds in the same pen were pooled and stored at −20°C prior to freeze-drying and grinding to pass a 0.5 mm sieve.
| Ingredient | Basal diet | |
|---|---|---|
| Feather meal | 285.7 | |
| Dextrose | 563.3 | |
| Canola oil | 60 | |
| Celite | 20 | |
| Dicalcium P | 19 | |
| Limestone | 10 | |
| Choline chloride | 3 | |
| Salt | 2 | |
| Solkafloc | 30 | |
| Premix A | 7 | |
| Total | 1000 |
In Experiment 1, each experimental diet was fed to four cages of six birds per cage for 5 days. The six experimental dietary treatments involved feeding the physically treated FM as described in Table 2.
| Diet/treatment | Treatment method | |
|---|---|---|
| 1. Control | n.a. | |
| 2. U30 | Ultrasonic treatment for 30 min | |
| 3. U120 | Ultrasonic treatment for 120 min | |
| 4. M2 | Microwave treatment for 2 min | |
| 5. M10 | Microwave treatment for 10 min | |
| 6. Auto | Autoclaved treatment |
In Experiment 2, each experimental diet was fed to five cages of seven birds per cage for 5 days. The four experimental dietary treatments examined in Experiment 2 are shown in Table 3. The basal or control diet was Diet 1, and Diets 2 and 3 consisted of the control diet supplemented with two protease, Ronozyme® ProAct (DSM, Switzerland) at two concentrations of 200 mg/kg and 600 mg/kg respectively. Diet 4 consisted of the control diet supplemented with the probiotic BioPlus 400® (Chr. Hansen, Denmark) at 1500 mg/kg.
| Diet/treatment | Basal diet + additive | Supplement or species | Spore number per kilogram of feed | |
|---|---|---|---|---|
| 1. Control | Diet 1* | n.a. | n.a. | |
| 2. Enzyme 1 | Diet 1 + RPA 200 | Ronozyme® ProAct | n.a. | |
| 3. Enzyme 2 | Diet 1 + RPA 600 | Ronozyme® ProAct | n.a. | |
| 4. Probiotic | Diet 1 + BioPlus® | B. subtilis B. licheniformis | 3.2 × 105 3.2 × 105 |
*Diet 1 composition see Table 1.
Chemical analysis
The AA contents of FM and ileal digesta were determined as described by Cai et al (2022). Briefly, the protein sample (50 mg) was hydrolysed under nitrogen with 6 mol/L hydrochloric acid containing 5 mL dithiothreitol (DTT; 20 mM) at 110°C overnight. DTT is an unusually strong reducing agent and used to is reduce the disulfide bonds of proteins and to prevent intramolecular and intermolecular disulfide bonds from forming among cysteine residues of proteins. Hydrolysates were evaporated to dryness with a rotary evaporator (BUCHI Rotavapor® R-300) to remove residual HCl. The dried sample was dissolved in 5 mL 0.1% formic acid and collected in a syringe and filtered through a 0.22-μm pore nylon filter membrane (Alltech, Baulkam Hills, NSW, Australia) into injection vials.
A Shimadzu LC-MS 8050 analyser (Shimazu Co., Kyoto, Japan) was used for AA analysis. The samples were diluted in 0.1% formic acid in MS-grade water and injected onto a F5 column (Sigma-Aldrich, Australia). Ions were detected in positive mode and monitored using targeted multiple-reaction monitoring (MRM) approach, that is also known as selective reaction monitoring. It is a highly specific and sensitive mass spectrometry technique that can selectively quantify compounds within complex mixtures. Spectra results obtained from mass spectrometry were processed using the software package Skyline (v20.2.1.286, AB Sciex). Each amino acid was quantified using a standard curve generated from known standards. Amino acid analyses were conducted in duplicate.
Dry matter (DM) was determined by drying samples at 105°C for 48 h. Nitrogen content was determined by using a FP-428 nitrogen analyser (LECO®, St Joseph, MI, USA) (Sweeney 1989). The AIA contents of diet and digesta samples were determined after ashing the samples and boiling the ash with 4 M hydrochloric acid (Li et al. 2006).
Calculations
Ileal digestibility of protein and AAs was calculated using AIA as an indigestible marker, as follows:
where diet inclusion level = the level of FM in complete diet, Ning = nutrient concentration in the FM, and Nileal = nutrient concentration in the ileal digesta; AIAd = acid insoluble in the diet, and AIAileal = acid insoluble in ileal digesta.
Percentage DM was calculated as
Statistical analyses
All data were subjected to ANOVA by using Minitab Software ver. Pl18 (The University of Queensland). Treatment means were separated using Tukeys’ pair-wise comparison procedure. Means were compared and considered significant when P ≤ 0.05, although probability values up to P < 0.10 were considered if the data suggest a trend.
Results
Composition of FM before and after physical treatment
As shown in Table 4, the DM content was highest after autoclaving in (6-Auto, 93.2%) and lowest following ultrasound (2-U30, 89.5% and 3-U120, 89.9%). Crude protein content was highest (P < 0.05) after autoclaving (2-U30, 91.1%), while the lowest value was recorded after microwaving (4-M2, 89.5%).
| Nutrient | Dietary treatment A | s.e.m. | P-value | ||||||
|---|---|---|---|---|---|---|---|---|---|
| 1-FM | 2-U30 | 3-U120 | 4-M2 | 5-M10 | 6-Auto | ||||
| Dry matter | 913b | 895c | 899c | 914b | 910b | 932a | 1.50 | 0.000 | |
| Crude protein (N × 6.25) | 897ab | 911a | 907ab | 895b | 895ab | 902ab | 2.43 | 0.031 | |
| Arginine | 49.1 | 54.9 | 56.8 | 51.4 | 53.2 | 53.3 | 2.28 | 0.362 | |
| Alanine | 17.5 | 20.5 | 20.7 | 18.9 | 18.0 | 18.1 | 1.17 | 0.415 | |
| Aspartic acid | 47.5 | 53.6 | 56.2 | 48.5 | 52.4 | 51.3 | 2.95 | 0.437 | |
| Glutamic acid | 129.0 | 151.5 | 155.6 | 136.4 | 144.2 | 146.9 | 7.58 | 0.295 | |
| Glycine | 55.0 | 69.8 | 68.9 | 61.5 | 62.7 | 68.8 | 3.55 | 0.186 | |
| Histidine | 8.1ab | 8.3a | 7.9ab | 5.5c | 5.5c | 6.0abc | 0.35 | 0.01 | |
| Isoleucine | 32.6 | 35.9 | 37.9 | 34.0 | 34.4 | 35.5 | 1.76 | 0.437 | |
| Leucine | 60.7 | 65.0 | 67.8 | 58.9 | 63.2 | 62.6 | 3.78 | 0.58 | |
| Lysine | 19.8 | 21.5 | 22.4 | 17.7 | 17.8 | 19.1 | 0.88 | 0.064 | |
| Methionine | 6.8 | 7.3 | 7.5 | 6.6 | 6.7 | 6.8 | 0.26 | 0.25 | |
| Phenylalanine | 34.8 | 37.4 | 38.4 | 32.5 | 36.4 | 36.8 | 2.25 | 0.584 | |
| Proline | 69.0 | 82.2 | 82.3 | 80.4 | 76.5 | 77.3 | 3.92 | 0.385 | |
| Serine | 109.1 | 117.5 | 120.2 | 112.1 | 119.3 | 122.2 | 6.6 | 0.775 | |
| Threonine | 35.7 | 39.1 | 39.8 | 36.3 | 36.7 | 37.5 | 1.71 | 0.544 | |
| Tyrosine | 21.5 | 23.0 | 24.1 | 20.9 | 22.8 | 23.3 | 0.93 | 0.411 | |
| Valine | 51.2 | 63.9 | 65.0 | 61.3 | 50.4 | 52.2 | 2.83 | 0.065 | |
| Total | 785.5 | 890.9 | 909.4 | 820.0 | 841.4 | 862.8 | 38.28 | 0.345 | |
The AA profile of the six FM samples after physical treatment are shown in Table 4. There was no statistical difference between FM samples after treatment although it is worth noting that all physical treatments numerically increased total AA concentration of all AAs and this increase ranged from 4% to 12%. Leucine was the most abundant essential AA, whereas glutamic acid was the most abundant non-essential one.
Digestibility of FM after different physical treatments
Results of the effects of the physical treatments on amino acid digestibility are shown in Table 5. No statistical difference was found in amino acid digestibility among the six treatments (P > 0.05). Numerically, ultrasonic- or microwave-treated FMs had higher ileal amino acid digestibility in broilers than did untreated FM, but autoclaved FM had lower ileal amino acid digestibility than did untreated feather meal.
| Nutrient | Dietary treatment A | s.e.m. | P-value | ||||||
|---|---|---|---|---|---|---|---|---|---|
| 1-FM | 2-U30 | 3-U120 | 4-M2 | 5-M10 | 6-Auto | ||||
| Alanine | 72.7 | 75.7 | 71.8 | 76.0 | 70.7 | 69.4 | 2.77 | 0.377 | |
| Arginine | 74.4 | 75.0 | 72.0 | 76.7 | 72.4 | 70.6 | 2.79 | 0.557 | |
| Aspartic acid | 39.2 | 40.7 | 38.1 | 45.4 | 39.8 | 37.0 | 5.26 | 0.844 | |
| Glutamic acid | 60.0 | 64.0 | 59.4 | 64.8 | 59.9 | 58.1 | 3.75 | 0.533 | |
| Glycine | 65.4 | 73.4 | 69.4 | 71.0 | 69.1 | 66.8 | 3.86 | 0.356 | |
| Histidine | 71.7 | 67.7 | 62.4 | 63.3 | 60.2 | 56.1 | 4.32 | 0.326 | |
| Isoleucine | 79.6 | 80.1 | 77.9 | 81.9 | 76.9 | 75.9 | 2.4 | 0.478 | |
| Leucine | 76.0 | 74.1 | 71.8 | 75.9 | 72.1 | 69.9 | 2.73 | 0.754 | |
| Lysine | 67.6 | 64.4 | 63.8 | 66.7 | 60.0 | 61.5 | 2.91 | 0.656 | |
| Methionine | 61.6 | 62.8 | 59.1 | 63.6 | 58.9 | 57.7 | 2.89 | 0.659 | |
| Phenylalanine | 79.0 | 76.9 | 74.9 | 77.9 | 77.4 | 72.7 | 2.56 | 0.755 | |
| Proline | 56.1 | 63.7 | 57.2 | 64.8 | 58.1 | 56.0 | 3.74 | 0.193 | |
| Serine | 66.6 | 67.8 | 63.9 | 70.0 | 66.1 | 64.9 | 3.46 | 0.629 | |
| Threonine | 59.6 | 61.4 | 58.0 | 63.6 | 58.3 | 57.6 | 3.75 | 0.729 | |
| Tyrosine | 71.4 | 70.3 | 68.4 | 73.1 | 69.6 | 67.3 | 2.98 | 0.772 | |
| Valine | 73.4 | 78.5 | 75.6 | 79.9 | 72.2 | 70.5 | 2.43 | 0.076 | |
| Overall mean | 64.5 | 66.8 | 65.0 | 66.3 | 65.8 | 62.2 | 3.43 | 0.749 | |
Protease and probiotic supplementation and digestibility of feather meal
The digestibility coefficient of crude protein was increased following supplementation with both a protease and a probiotic (Table 6). The effect was significantly (P < 0.05) greater with the higher protease concentration of 600 mg/kg. The overall digestibility of amino acids was about 10% lower than for crude protein, but the average values were numerically improved in all experimental treatments (Table 6). Digestibility values for histidine were considered unreliable and have not been included for this experiment. The greatest increase in amino acid digestibility occurred following supplementation with RAP at 600 mg/kg, followed by supplementation with the probiotic, BioPlus 400. The increases in AA digestibility were minimal in diet 2 (200 mg/kg RPA). For individual amino acids, significant (P < 0.05) improvements were observed in the digestibility of arginine, isoleucine, phenylalanine, and valine. glutamic acid, leucine, lysine and serine all had notable (P < 0.10) increases in digestibility (Table 6).
| Nutrient | Dietary treatment | s.e.m. | P-value | ||||
|---|---|---|---|---|---|---|---|
| 1-Control | 2-RAP (200 mg/kg) | 3-RAP (600 mg/kg) | 4-Bioplus 400 (1500 mg/kg) | ||||
| Crude protein | 74.1b | 77.5ab | 80.5a | 78.4ab | 1.11 | 0.038 | |
| Alanine | 69.5 | 70.7 | 75.0 | 71.1 | 1.75 | 0.154 | |
| Arginine | 70.8b | 72.2b | 77.5a | 74.9ab | 1.00 | 0.009 | |
| Aspartic acid | 43.7 | 42.9 | 48.4 | 39.2 | 3.18 | 0.133 | |
| Glutamic acid | 61.6 | 61.7 | 67.9 | 62.7 | 1.85 | 0.069 | |
| Glycine | 66.6 | 68.8 | 73.0 | 70.6 | 1.47 | 0.16 | |
| Isoleucine | 74.6b | 75.4b | 81.6a | 80.9a | 0.91 | 0.006 | |
| Leucine | 69.2 | 69.0 | 74.1 | 72.6 | 1.24 | 0.076 | |
| Lysine | 70.5 | 68.5 | 71.4 | 65.3 | 1.51 | 0.097 | |
| Methionine | 79.5 | 81.0 | 82.4 | 78.4 | 1.81 | 0.287 | |
| Phenylalanine | 72.7b | 75.1ab | 79.9a | 78.0ab | 1.51 | 0.021 | |
| Proline | 61.5 | 62.7 | 68.4 | 63.8 | 1.81 | 0.149 | |
| Serine | 65.1 | 67.6 | 71.8 | 68.9 | 1.67 | 0.083 | |
| Threonine | 60.1 | 61.6 | 65.1 | 61.1 | 1.55 | 0.232 | |
| Tyrosine | 70.5 | 71.3 | 75.1 | 72.9 | 1.69 | 0.241 | |
| Valine | 68.2ab | 67.4b | 74.1a | 71.3ab | 1.05 | 0.03 | |
| Overall mean | 64.8 | 65.8 | 70.8 | 67.4 | 1.48 | 0.055 | |
Values in the same row with different letters are significantly different (at P = 0.05).
RPA, Ronozyme® ProAct.
Discussion
The AA profiles of FMs determined in the present experiments were within the ranges reported previously (Papadopoulos et al. 1985; Papadopoulos et al. 1986; Han and Parsons 1991; Latshaw et al. 1994; Moritz and Latshaw 2001; Ravindran et al. 2005; Bandegan et al. 2010). Methionine and histidine had the lowest concentrations of essential AAs, whereas leucine and valine had the highest concentrations. No significant differences were observed among physically treated FMs in total AA concentrations and apparent ileal AA digestibility; demonstrating that AA profiles of FM were not affected by the physical treatments applied in these studies. This was a disappointing result because physical treatments are recognised as green processes because the use of toxic chemicals is avoided (Zoccola et al. 2012). It was unexpected because physical treatments including ultrasonic, microwave and autoclave treatments were thought to be practical methods that would improve the utilisation of FM protein. It may be that the hydrolysis of raw feathers to create FM leaves little scope for further improvement with additional physical processing. Nevertheless, a number of studies have shown that autoclaving (Papadopoulos et al. 1986), microwaving (Chen et al. 2015) and ultrasound (Qin et al. 2023) improve FM quality. However, the major difference between these studies and the current study is the starting material used. We used hydrolysed feathers or FM, whereas the other studies used raw feathers and focused on improving keratin solubility.
Both the protease and probiotic evaluated in Experiment 2 improved ileal AA digestibility of FM, but in both instances, this effect was observed when a dose that exceeded the commercially recommended dose was fed. The protease chosen for this study had been shown in vitro to outperform other commercial proteases in the degradation of feathers and hair (Navone and Speight 2018). Although not marketed as a keratinase, RAP has keratinolytic activity, especially when added to diet at a concentration of 600 mg/kg. However, Bertechini et al. (2020) found that RAP at the recommended commercial concentration of 200 mg/kg enhanced AA digestibility of FM for broilers when incorporated in a corn–soybean basal diet and fed for 7 days. Although the two studies are not directly comparable because many factors can influence both AA digestibility and the protease value in poultry diets (Cowieson and Roos 2013; Romero and Plumstead 2013), they demonstrate the keratinolytic activity of RPA. Other studies (Angel et al. 2011; Fru-Nji et al. 2011; Saleh et al. 2020b) using the recommended dose, achieved satisfactory improvements (4–8%) in protein digestibility, but the diets fed did not include a refractory protein such as keratin found in FM.
There are a number of possible mechanisms through which probiotics may enhance bird performance (see Shini and Bryden 2022). It was our hypothesis in this study that the Bacillus licheniformis component of BioPlus 440 produces a keratinase and that ‘super dosing’ with the probiotic at 1500 mg/kg or three times the recommended dose would allow the expression of the keratinolytic activity of B. licheniformis. The results of the study support the hypothesis. Moreover, Mazotto et al. (2011) reported that FM was a great substrate for induction of the activity of a keratinase and peptidase in B. subtilis 1273. Such a mechanism may have contributed to the results observed in Experiment 2, following the addition of BioPlus 400. Furthermore, the proportion of Bacillus licheniformis and Bacillus subtilis in broiler diets is also a factor that influences their effect on broilers. Yang et al. (2017) indicated that 6.6 × 105:3.3 × 105 was the optimal proportion for Partridge Shank chicks to significantly improve the small intestine morphology. However, the optimal ratio for Ross 308 broilers has not been reported and this might be an important aspect in the effect of BioPlus 400 in the current experiment.
It is likely that the addition of protease or probiotic in the current study would improve nutrient digestibility of diets by modifying the morphology of the small intestine of broilers. Probiotics have been shown to improve the small intestinal morphology of broilers, including proteome changes and increases to villous length and width, crypt depth and epithelial thickness (Luo et al. 2014; Shini et al. 2021). This would increase nutrient utilisation because of an increased intestinal surface area for absorption (Awad et al. 2008). Kamel et al. (2015) added RPA at a rate of 200 ppm in corn–soybean meal diets for broilers and observed a significant increase in villus:crypt ratio. The ability of both feed additives to maintain or improve gut integrity would contribute to the results observed.
In conclusion, the quest for technologies to improve the utilisation of FM continues. The current studies indicated that there is scope for further improvement in the nutritive value of FM for broilers. Both the protease and Bacillus sp. probiotic demonstrated an ability to improve the protein digestibility of FM as a protein source for broilers. How this was achieved was unclear and further delineation of the modes of action of both the protease and probiotic would facilitate their strategic application.
Conflicts of interest
WLB is a member of the Editorial Board of Animal Production Science but was not involved in the review and editorial process for this paper. The authors have no further conflicts of interest to declare.
Declaration of funding
This study was supported by the Australian Government Department of Agriculture, Water and the Environment as part of its Rural R&D for Profit program and the School of Agriculture and Food Sustainability, the University of Queensland.
Acknowledgements
We appreciate the assistance of postgraduate students who helped with the conduct of the bioassays and Dr G. Cai is thanked for assistance with amino acid analysis.
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