Introduction
The use of feed enzymes as dietary supplements has become common practice in poultry
farming. Multi-enzyme blends of xylanases, amylases, lipases, and proteases have shown
notable benefits[1,2,3], chiefly by trapping nutrients and
improving gut health and digestive function[4,5,6]. Whereas
exogenous multi-enzymes have shown a considerable interaction with the nutrient density of
diets, and to a lesser extent age[7] and sex[8], the impact of breeding-stock age on these interactions
has not been explored.
Breeder hen age influences prenatal nutritional and hormonal conditions, thereby affecting
offspring metabolism. Compared with the offspring of young breeder hens, those of older hens
consume 70–600 g more feed over a growth period of 35–44 days, despite exhibiting a similar
gain in weight[9,10,11]. In general, older breeders may
allocate varying amounts or ratios of nutrients to their eggs, which can cause offspring to
consume more feed to boost growth[12,13]. However, excessive consumption leads to a decrease
in feed conversion efficiency[14,15,16]. One
approach to mitigate this decline in nutrient utilization could rely on the use of exogenous
enzymes; although their effect on gut health and nutrient absorption remains to be
determined[1].
Research on broiler chickens has revealed synergistic interactions between carbohydrase and
lipase enzymes[17], and between carbohydrase and
protease enzymes[3,4]. This relationship becomes more important during the finishing phase, when
metabolic and physiological capacities are surpassed by excessive intake[15]. The present study aimed to determine whether breeder
age affected the ability of broilers to digest lipids or proteins, in addition to
carbohydrates, upon exogenous enzyme supplementation.
Offspring from older breeding stocks have been proposed to exhibit a stronger response to
multi-enzyme supplementation (MES), implying a link between broiler breeder hen age (BBA)
and MES that influences growth, nutrient digestion, and absorption[11,14,15,16]. Using a factorial design,
broiler chicks from breeders aged 39 and 60 weeks were fed a practical diet with or without
exogenous enzyme mixtures during four distinct growth stages. Growth performance, nutrient
digestibility, pancreatic enzyme activity, development of the gastrointestinal tract, and
composition of fecal microbiota were evaluated. The findings could inform breeders on the
most effective dietary interventions for rearing broilers produced by differently aged
hens.
Materials and Methods
Experimental design
A 2 × 3 factorial arrangement of treatments included two BBA (39 and 60 weeks of age) and
three MES (no supplementation or supplemented with multi-enzyme, including lipase or
protease) variables. In total, 780 chicks were randomly assigned to six treatments, each
with five replicates of 26 birds. A pen was used as the experimental unit.
Birds and management
Straight-run Ross 308 broiler chicks (n = 780), sexed by feather type, were obtained from
a local commercial hatchery (Ege Tav, İzmir, Turkey) on the day of hatching. Upon arrival,
the birds were weighed and randomly allocated to 30 floor pens (150 × 100 cm, length ×
width) with 13 males and 13 females per pen. Chicks produced by young breeders (39 weeks
of age) had a mean initial body weight (BW) of 43.10 ± 1.06 g per bird; those produced by
older breeders (60 weeks of age) had a weight of 47.68 ± 1.29 g per bird. The birds were
bedded in fresh, 6-cm-long pine wood shavings on a concrete floor. Each pen was equipped
with four nipple drinkers, one tubular feeder, and an electrical heater. The photoperiod
was set at 23/1 h light/dark for the first 5 days post-hatch, 21/3 h light/dark from days
6 to 11, and 18/6 h light/dark for the remainder of the study. House temperature was set
at 33 °C during placement and until day 7, 30 °C from 8 to 13 days, 28 °C from 14 to 21
days, and 24 °C from 21 to 42 days. The chicks had free access to water and food
throughout the experiment, which lasted 42 days: 1–14 days for the starter phase, 15–28
days for the grower phase, and 29–42 days for the finisher phase.
Preparation of diets
Diets based on corn, wheat, and soybean meal were provided to the broilers. Ground maize
and wheat used in this study were obtained from the same lot. Whole corn and wheat were
ground in a hammer mill (MÜNCH-Edelstahl GmbH, Hilden Germany) to pass through a screen of
6.0 mm. After grinding the main cereals, all components of the mash diet were mixed in a
horizontal mixer. Birds were fed following a three-phase feeding program: starter (days
1–14), growers (days 15–28), and finishers (days 29–42). Each phase was formulated to meet
the birds’ nutritional requirements, as outlined in the Ross 308 broiler nutrition
specification handbook[18]. The experimental diets
contained no anticoccidial, antibiotic, or growth enhancer feed additives and were
provided ad libitum throughout the experiment. The chemical composition was determined
according to the AOAC[19]. All feed samples were
analyzed for dry matter (934.01), ash (942.05), nitrogen (Kjeldahl procedure: 988.05),
ether extract (920.39), crude fiber (962.09), starch (996.11), and sugar (982.14).
Composition and nutrient content of the diets for each growth stage are presented in
Table 1. Before chemical analysis, excreta
samples were dried at 57 °C for 72 h. Dried excreta were milled (0.75-mm mesh) and
analyzed for dry matter, crude protein, starch, and ether extract as previously
described[19].
Table 1. Composition and nutrient content (g/kg on a fed basis) of the diets given to
chickens through the different growth stages, from 1 to 42 days of age.
|
Ingredients (g/kg)
|
Starter |
Grower |
Finisher |
| Corn |
270.61 |
279.33 |
334.80 |
| Soybean meal |
310.21 |
257.82 |
197.22 |
| Full fat soybean |
60.00 |
100.00 |
100.00 |
| Wheat |
300.00 |
300.00 |
300.00 |
| Soy oil |
18.30 |
28.60 |
36.30 |
| Dicalcium phosphate |
17.60 |
15.46 |
13.85 |
| Limestone |
9.00 |
7.48 |
7.33 |
| Vitamin-mineral premix1 |
3.50 |
3.50 |
3.50 |
| NaCl |
1.99 |
2.01 |
2.04 |
| D-L Methionine |
3.89 |
3.00 |
2.22 |
| L-Lysine HCL |
3.00 |
1.90 |
1.71 |
| L-Threonine |
0.86 |
0.40 |
0.53 |
| Choline chloride |
0.50 |
0.50 |
0.50 |
| Total |
1000 |
1000 |
1000 |
| Analyzed nutrient content (g/kg; unless
otherwise indicated) |
| Dry matter |
880.9 |
890.9 |
893.2 |
| Crude protein |
227.8 |
213.9 |
191.6 |
| Crude protein2 |
224.0 |
209.0 |
189.0 |
| Ether extract |
56.9 |
70.0 |
77.3 |
| Starch |
350.4 |
369.2 |
378.8 |
| Sugar |
23.2 |
25.1 |
28.3 |
| Crude fiber |
38.9 |
41.2 |
4.04 |
| Crude ash |
6.74 |
6.57 |
58.2 |
| Contents by calculation (g/kg; unless
otherwise indicated) |
| Methionine |
7.08 |
6.18 |
5.63 |
| Lysine |
14.4 |
12.9 |
12.5 |
| Methionine+Cysteine |
10.8 |
9.86 |
9.03 |
| Arginine |
15.0 |
13.8 |
12.2 |
| Threonine |
9.64 |
8.28 |
7.37 |
| Valine |
11.3 |
9.92 |
8.71 |
| Linoleic acid |
27.6 |
34.1 |
38.6 |
| Na |
16.3 |
16.5 |
16.1 |
| Ca |
10.6 |
9.58 |
8.86 |
| P (total) |
7.10 |
6.83 |
6.14 |
| P (available) |
4.74 |
4.55 |
3.97 |
| DEB (mEq/kg diet)3 |
232 |
229 |
201 |
| ME (Kcal/kg) |
2962 |
3101 |
3139 |
| ME (Kcal/kg)2 |
2930 |
3030 |
3100 |
1Supplied per kg of diet: vitamin A (trans-retinylacetate), 4.12 mg;
vitamin D3 (cholecalciferol), 66 µg; vitamin E
(all-rac-tocopherol-acetate), 50 mg; vitamin B1, 2 mg;
vitamin B2, 6 mg; vitamin B6, 2.4 mg; vitamin B12
(cyanocobalamin), 0.020 mg; vitamin K3 (bisulphatemenadione complex), 4.5
mg; nicotinic acid, 40 mg; pantothenic acid (D-calciumpantothenate), 12 mg; folic
acid, 0.6 mg; D-biotin; zinc (ZnO),100 mg; manganese (MnO), 120 mg; iron
(FeSO4), 40 mg; copper (CuSO4·5H2O), 16 mg; cobalt
(CoCo3), 0.1 mg; iodine (CaI), 0.4 mg; selenium
(Na2SeO3), 0.25 mg. 2Value formulated in accordance
with the intention. 3Dietary electrolyte balance = [(Na × 434.98) + (K ×
255.74) − (Cl × 282.06)].
The chickens were fed three experimental diets: a control diet without enzyme preparation
(CNT), 250 g/ton of a commercial enzyme cocktail (AVZ; Avizyme 1500®; Danisco
Animal Nutrition, Marlborough, Wiltshire, UK), and a laboratory-formulated multi-enzyme
preparation (LTE; Molkim Endüstriyel Ürünler Sanayi ve Ticaret Anonim Şirketi, İzmir,
Turkey). Avizyme 1500® is a multimicrobial enzyme produced by
Trichoderma and Bacillus containing at least 1,000
units of xylanase/g, 4,000 units of protease/g, and 2,000 units of α-amylase/g (activity
determined by the manufacturer). The LTE cocktail is the final product of a
government-funded biotechnology project (TÜBİTAK) containing at least 1,250 units of
xylanase/g, 750 units of β-glucanase/g, 1,700 units of α-amylase/g, and 2,000 units of
lipase (activity determined by the manufacturer) secreted by Fusarium sp.
and Bacillus sp. Exogenous enzyme preparations were top-dressed by mixing
with approximately 500 g of finely ground corn before being added to the main batch of the
basal diet, which was further mixed to ensure homogeneous distribution within the diet.
The feeds were prepared in 250-kg batches and mixed for 3.5 min. The feed mixer was
thoroughly cleaned by sequential treatment to remove residual enzymes from previously
prepared feed. No phytase was included in the feed to prevent any potential dietary
contributions or synergistic effects with other enzymes[4].
Data collection
The chicks were weighed on a pen basis on days 0, 14, 28, and 42 to determine BW and body
weight gain (BWG). Feed intake (FI) within each subgroup was calculated over the same
experimental period. The feed conversion ratio (FCR) was calculated as the ratio of FI to
BWG (g feed/g gain). Mortality was recorded daily and was expressed as the percentage of
the initial number of chicks. Growth performance traits, including BW, BWG, FI, and FCR,
were adjusted for mortality and calculated on a pen basis. The coefficient of variation
(CV%) was calculated based on the individual BW of the birds in each pen on days 1, 14,
28, and 42. The average BW was divided by the standard deviation of the pen’s BW and
multiplied by 100. This is described as uniformity of body weight (UBW). On days 9 and 42,
two birds (one male and one female) per experimental unit, whose BW was close to the group
mean, were randomly selected and euthanized for further determination of carcass yield,
relative weight of digestive organs, length of the intestines, pancreatic enzyme activity,
and microbial composition of the cecum.
The initial experimental measurements in this study, except for growth performance
traits, were conducted at 9 d of age based on the scientific evidence outlined below.
Chicks, particularly during the first 7–10 d post hatching, have immature digestive
systems and are still establishing their intestinal microbiota[20,21,22]. Consequently, they are particularly sensitive to supplemental
exogenous enzymes[4]. Poor digestion and absorption
of lipids in broilers during early development, associated with low production of bile
acids and pancreatic lipase, can be improved via exogenous lipase supplements[23,24,25]. The second measurement was taken on day 42 to
assess changes in the enzymatic response of adult broilers at slaughter age. This timing
was chosen because, as the digestive system develops and reaches peak functionality, the
magnitude of the positive enzymatic response diminishes[7,26,27].
Determination of the relative size of carcass and digestive organs
The chickens were anesthetized using an electrical shock. The stunning device was
operated at an alternating current of 50 Hz, 42 V, and 100 mA for 6 s. Following
anesthesia, the birds were sacrificed by severing the jugular vein, immersed in hot water
(62 °C for 60 s), mechanically defeathered, and manually eviscerated. Whole carcasses were
weighed, and carcass yield (carcass weight as a percentage of final BW) was determined.
The digestive tract (from the beginning of the crop to the cloaca, including digesta
content), liver, and pancreas were removed aseptically. Subsequently, the proventriculus
and gizzard were removed from the digesta, cleaned, dried with desiccant paper, and
weighed. The small intestine and cecum, along with their contents and abdominal fat
(consisting of fat surrounding the gizzard and abdominal cavity), were removed and
weighed. The weights were expressed relative to live BW (g/100 g). The lengths of the
duodenum (from the gizzard to the pancreaticobiliary ducts), jejunum (from the
pancreaticobiliary ducts to Meckel’s diverticulum), ileum (from Meckel’s diverticulum to
the ileocecal junction), and the two ceca (from the ostium to the tip of the right and
left ceca) were measured on a glass surface using a flexible tape with a precision of 1 mm
and expressed relative to live BW (cm/100 g). The average value of the two measurements
was used to determine the cecum length.
Determination of digestive enzyme activities and amount of protein
The pancreas was harvested from each bird within 5 min after death, placed in aluminum
foil, snap frozen in liquid nitrogen, and stored at −80 °C.
Amylase activity (EC 3.2.1.1) was determined using a modified version of the
3,5-dinitrosalicylic acid method, as described previously[28]. Briefly, 0.5 mL of enzyme was mixed with 1.0% (w/v) soluble starch solution
prepared in 0.1 M phosphate buffer (pH 7.0). The mixture was incubated at 37 °C for 10
min. To terminate the reaction, 1.0 mL of dinitrosalicylic acid reagent was added, and the
mixture was boiled for 5 min. After cooling to room temperature, the reaction mixture was
diluted with 10.0 mL of distilled water. The absorbance of the resulting colored product
was measured at 540 nm using a spectrophotometer. A calibration curve was constructed
using maltose standards to quantify the amount of released reducing sugars. One unit of
amylase activity was defined as the amount of enzyme that released 1.0 µmol of maltose per
minute under assay conditions.
Lipase activity was determined according to a method described previouslyof[29] with slight modification. Briefly, 0.25 mL of
enzyme preparation was mixed with 0.65 mL of potassium phosphate buffer (50 mM, pH 7.2)
and 0.1 mL of p-nitrophenyl laurate (10 mM in ethanol). The reaction mixture was incubated
for 30 min at 40 °C and then mixed with 0.25 mL of Na2CO3 (0.1 M).
After centrifugation at 10,000 × g for 20 min, absorbance of the
supernatant was measured at 410 nm using a spectrophotometer. One unit of lipase activity
was determined as the amount of enzyme that caused the release of 1.0 µmol of
p-nitrophenol per minute under the experimental conditions.
Protease activity was determined as described previously[30] with slight modifications. Briefly, 0.1 mL of the enzyme preparation was
mixed with 0.5 mL of 0.6% casein in potassium phosphate buffer (50 mM, pH 7.0). The
mixture was incubated at 37 °C for 10 min. To stop the reaction, 10% trichloroacetic acid
(0.5 mL) was added, mixed gently, and left for 30 min at room temperature. After
incubation, the reaction mixture was centrifuged at 10,000 × g for 7 min.
Following centrifugation, 25 µL of the supernatant was aliquoted to a different tube and
mixed with 125 µL of 0.5 M Na2CO3 and 25 µL of 1 N Folin-Ciocalteu’s
phenol reagent. The mixture was incubated in the dark for 30 min at room temperature.
Absorbance of the supernatant was measured at 660 nm using a spectrophotometer. One unit
of protease activity was defined as the amount of enzyme that released 1.0 µmol of
tyrosine per minute under trial conditions.
Protein concentration was determined using the Bradford method[31] with slight modifications. Briefly, 0.1 mL of sample was mixed with
1.0 mL of Bradford reagent and incubated for 10 min at room temperature. Absorbance of the
resulting solution was measured at 595 nm using a spectrophotometer. Protein concentration
was determined by comparing the absorbance of the sample with a standard curve prepared
using bovine serum albumin. All enzyme activities and protein amounts were measured
spectrophotometrically (SPEKTROstar Nano; BMG Labtech, Offenburg, Germany).
Apparent total tract digestibility coefficients of nutrients
The apparent total tract digestibility (ATTD) coefficients of nutrients were determined
by adding Cr2O3 as an indigestible analytical marker. At 38 days of
age, two birds with a BW similar to the average of the corresponding pen were selected and
transferred to battery cages (two birds per cage and replicate) with a wire mesh bottom
and plastic excreta collection trays. Each cage (60 × 40 × 46 cm, length × width × height)
was equipped with a feeder and nipple drinkers were placed outside the cage. The
experimental diets were identical to those used during the finisher period except that
Cr2O3 was added at 3 g/kg. The digestibility experiment was
preceded by 3 days of adaptation and was followed by 2 days for collection. During the
latter (from days 40 to 42), excreta from each cage were collected twice daily (i.e., from
10:00 AM to 12:00 AM and from 6:00 PM to 8:00 PM) and stored in sealed bags at -20 °C.
Leftover feed and feathers were carefully removed from the excreta trays. Excreta
collected during the 3-day period were pooled per cage and represented one replicate,
resulting in five samples for each of the six groups. The following equation was used to
calculate the ATTD of the nutrients:
ATTD (%) = 100 × [(diet
Cr2O3/excreta Cr2O3) × (nutrient in
excreta/nutrient in diet)] ×100.
Enumeration of cecal microflora
After aseptic removal, the intestines were divided into sections (i.e., ileum, cecum, and
colon), and the cecum was ligated with a silk catgut before separating it from the small
intestine. Cecum samples were immediately frozen at −80 °C, sealed in sterile bags filled
with 50 mL ice-cold cryo-protective broth[32] and
immediately stored at −80 °C until subsequent analyses.
Cecal digesta contents were then aseptically emptied in a new sterile bag, immediately
diluted tenfold (i.e., 10% w/v) with sterile ice-cold anoxic phosphate-buffered saline
(0.1 M, pH 7.0), and homogenized for 3 min in a stomacher (Bagmixer® 100
Minimix; Interscience, Arpents, France). Each cecal digesta homogenate was serially
diluted from 10−1 to 10−7. The dilutions were subsequently plated in
duplicate on selective agar medium to enumerate the target bacterial groups.
Cecal microflora composition was determined as reported by Mountzouris et al.[32] with slight modifications. Total aerobes,
coliforms, sulfite-reducing anaerobes, Clostridium spp., and
Lactobacillus spp. were counted using plate count agar, violet red bile
agar, iron sulfite agar, tryptose sulfite cycloserine agar, and De Man–Rogosa–Sharpe agar,
respectively. Plates were incubated at 39 °C for 24–72 h aerobically (aerobes and
coliforms) or 48–120 h anaerobically (other microorganisms) and colonies were counted.
Anaerobic incubation was achieved using appropriate catalysts (AnaeroGen®;
Oxoid, Basingstoke, Hampshire, UK) in sealed anaerobic jars (Oxoid). The results are
expressed as log10 CFU/g of cecal digesta.
Statistical analysis
The experimental design was completely randomized, with six treatments arranged
factorially with two BBA (39 or 60 weeks of age) and three MES (non-supplemented,
supplemented with LTE, or AVZ) variables. The experimental unit consisted of pens for BW,
BWG, FI, FCR, and UBW. For carcass yield, relative weight of digestive organs,
proportional weight and length of intestines, pancreatic enzyme activity, total tract
digestibility of nutrients, and cecal microbial composition, the experimental unit
consisted of two birds chosen randomly from each replicate. Pooled means from two bird
replicates were used for the analysis. Data were checked using the Shapiro–Wilk test to
ensure that normality assumptions were met. Statistical comparisons across treatment
groups were performed in SAS software using a general linear model with a factorial
design[33]. The model evaluated the main effects
of BBA and MES treatments, as well as their interactions, on the dependent variables. The
arcus sinus transformation was applied to the percentage values before testing for
differences. When the model was significant, Tukey’s honest significant difference
multiple comparison test was used to separate the treatment means. Results are presented
as the mean ± standard error of the mean, and differences between treatment means were
considered significant at P < 0.05.
Results
No significant effect of the interaction between MES and BBA was found for any of the
performance traits studied; therefore, only the main effects are presented here.
Growth performance traits
The effects of MES and BBA on the BW, BWG, FI, and FCR of chickens during the starter,
grower, and finisher phases, as well as throughout the experimental period, are presented
in Table 2. Data on UBW measured on days 1,
14, 28, and 42 are reported in Table 2.
Table 2. Effect of multi-enzyme supplementation and breeder age on body weight (BW) body
weight gain (BWG), feed intake (FI), feed conversion ratio (FCR), and uniformity body
weight (UBW) in broiler chickens.
|
Diet |
Breeder age |
SEM6 |
P7 |
|
CNT1 |
LTE2 |
AVZ3 |
Young4 |
Old5 |
Diet |
Breeder age |
Diet × Age |
|
0 to14 days
|
|
|
|
|
|
|
|
|
|
| Hatch weight |
45.25 |
45.36 |
45.54 |
43.10b |
47.68a |
0.082 |
0.2237 |
0.0001 |
0.6472 |
| BW |
494 |
493 |
501 |
494 |
498 |
4.953 |
0.3238 |
0.3736 |
0.6027 |
| BWG |
449 |
448 |
456 |
451 |
451 |
4.968 |
0.3400 |
0.9741 |
0.6043 |
| FI |
486 |
485 |
494 |
488 |
490 |
6.075 |
0.4051 |
0.7199 |
0.5041 |
| FCR |
1.082 |
1.082 |
1.083 |
1.082 |
1.086 |
0.013 |
0.9803 |
0.7263 |
0.8025 |
|
14 to 28 days
|
|
|
|
|
|
|
|
|
|
| BW |
1635 |
1653 |
1655 |
1646 |
1649 |
20.24 |
0.5544 |
0.8481 |
0.5606 |
| BWG |
1146 |
1160 |
1155 |
1152 |
1155 |
20.98 |
0.7924 |
0.8686 |
0.8339 |
| FI |
1753 |
1772 |
1765 |
1761 |
1768 |
38.14 |
0.8834 |
0.8455 |
0.6183 |
| FCR |
1.535 |
1.528 |
1.529 |
1.528 |
1.532 |
0.032 |
0.9726 |
0.8764 |
0.8716 |
|
28 to 42 days
|
|
|
|
|
|
|
|
|
|
| BW |
2981 |
3019 |
2987 |
2981 |
3011 |
29.25 |
0.3910 |
0.2202 |
0.7208 |
| BWG |
1347 |
1366 |
1332 |
1335 |
1362 |
29.85 |
0.5198 |
0.2801 |
0.9980 |
| FI |
2476 |
2471 |
2436 |
2417b |
2505a |
26.66 |
0.2860 |
0.0005 |
0.9003 |
| FCR |
1.842 |
1.811 |
1.832 |
1.810 |
1.839 |
0.0304 |
0.5958 |
0.2233 |
0.9876 |
|
0 to 42 days
|
|
|
|
|
|
|
|
|
|
| BW |
2981 |
3019 |
2987 |
2981 |
3011 |
29.25 |
0.3910 |
0.2202 |
0.7208 |
| BWG |
2936 |
2974 |
2942 |
2937 |
2964 |
29.25 |
0.3898 |
0.2848 |
0.7227 |
| FI |
4715 |
4728 |
4695 |
4666 |
4763 |
59.63 |
0.9399 |
0.0801 |
0.7320 |
| FCR |
1.605 |
1.589 |
1.595 |
1.588 |
1.607 |
0.015 |
0.5760 |
0.2004 |
0.8973 |
|
UBW8
|
|
|
|
|
|
|
|
|
|
| 1 d |
7.56 |
7.55 |
7.60 |
7.50 |
7.64 |
0.266 |
0.856 |
0.658 |
0.771 |
| 14 d |
8.92 |
8.71 |
8.69 |
8.70 |
8.84 |
0.302 |
0.404 |
0.699 |
0.544 |
| 28 d |
10.38 |
10.13 |
10.22 |
10.15 |
10.33 |
0.296 |
0.664 |
0.867 |
0.841 |
| 42 d |
11.45 |
11.34 |
11.30 |
11.26 |
11.47 |
0.346 |
0.801 |
0.704 |
0.799 |
The dietary treatments were: 1CNT, a control diet without enzyme
preparation; 2LTE, a laboratory type enzyme cocktail (Molkim/250 g/ton
diet) containing at least 1,250 units of xylanase/g, 750 units of β-glucanase/g, 1,700
units of α-amylase/g, and 2,000 units of lipase; 3AVZ, a commercial enzyme
cocktail (Avizyme® 1500/ 250 g/ton diet) containing at least 1,000 units of
xylanase/g, 4,000 units of protease/g, and 2,000 units of α-amylase/g.
4Young breeders were 39 weeks old. 5Old breeders were 60 weeks
old. 6SEM: pooled standard error of the mean (data are the means of five
replicate pens with 26 birds each per treatment). 7Source of variation
(probability values). 8Uniformity of body weight (described as coefficient
of variation). a,bMeans within lines with different superscripts differ at
P < 0.05.
During the experiment, none of the growth performance variables studied were affected by
MES or BBA; except for FI, which showed a significant response to BBA treatment.
Specifically, FI was 88 g higher (P = 0.0005) in broilers from older breeders than in
those from younger breeders during the finisher phase (days 28–42). Over the entire
experimental period (days 1–42), birds from aged breeders tended to have higher FI (97 g)
than those from young progeny (P = 0.0801). The overall mortality rate was low, (2.31%,
and was not associated with any treatment. The mortality counts were 6, 5, 7, and 18 on
days 1–14, 15–28, 29–42, and 1–42, respectively.
Carcass yield and gastrointestinal tract development
The carcass yield and relative weights of the proventriculus, liver, and small intestine,
as well as the length of the small intestine, were not affected by BBA or MES (Table 3). However, both LTE and AVZ lowered
pancreas weight on day 9 (P = 0.0215) and gizzard weight on day 42 (P = 0.0014).
Furthermore, on day 42, MES increased the percentage weight of the abdominal fat pad (P =
0.0445).
Table 3. Influence of exogenous enzymes and breeder age on the relative weight (g/100 g
body weight) of carcass, digestive organs, abdominal fat, and relative length (cm/100
g body weight) of small intestines in broilers at d 9 and 42 days.
|
Live weight (g) |
Proventriculus |
Carcass yield |
Gizzard |
Pancreas |
Liver |
Small intestines weight8 |
Small intestines lenghth9 |
Abdominal fat |
| Day 9 |
|
Diet
|
|
|
|
|
|
|
|
|
|
| CNT1 |
218.5 |
0.75 |
57.63 |
5.83 |
0.54a |
3.14 |
8.01 |
50.7 |
1.74 |
| LTE2 |
236.8 |
0.74 |
57.84 |
5.56 |
0.47b |
3.14 |
8.41 |
48.5 |
1.77 |
| AVZ3 |
219.8 |
0.77 |
58.09 |
5.61 |
0.49b |
3.24 |
8.44 |
50.0 |
1.86 |
|
Breeder age
|
|
|
|
|
|
|
|
|
|
| Young4 |
224.4 |
0.75 |
57.71 |
5.74 |
0.50 |
3.17 |
8.34 |
49.1 |
1.85 |
| Old5 |
225.7 |
0.76 |
57.99 |
5.59 |
0.50 |
3.17 |
8.24 |
50.2 |
1.73 |
|
SEM6
|
9.398 |
0.025 |
0.271 |
0.181 |
0.020 |
0.106 |
0.368 |
1.898 |
0.064 |
| P7 |
|
|
|
|
|
|
|
|
|
| Diet |
0.1066 |
0.6313 |
0.2774 |
0.2860 |
0.0215 |
0.5767 |
0.4367 |
0.3907 |
0.4310 |
| Breeder age |
0.8714 |
0.7002 |
0.3945 |
0.3420 |
0.7802 |
0.9617 |
0.7254 |
0.4552 |
0.3965 |
| Diet × Age |
0.3135 |
0.1920 |
0.7458 |
0.1810 |
0.9380 |
0.0728 |
0.5231 |
0.2013 |
0.2983 |
| Day 42 |
|
Diet
|
|
|
|
|
|
|
|
|
|
| CNT1 |
2951 |
75.53 |
0.343 |
2.38a |
0.22 |
2.05 |
5.11 |
8.66 |
1.54b |
| LTE2 |
3008 |
76.06 |
0.324 |
2.12b |
0.21 |
1.98 |
5.10 |
8.40 |
1.85a |
| AVZ3 |
2961 |
76.19 |
0.339 |
2.20b |
0.23 |
2.10 |
4.97 |
8.46 |
1.89a |
|
Breeder age
|
|
|
|
|
|
|
|
|
|
| Young4 |
2946 |
75.80 |
0.324 |
2.28 |
0.22 |
2.01 |
5.08 |
8.64 |
1.82 |
| Old5 |
3001 |
76.04 |
0.347 |
2.19 |
0.22 |
2.08 |
5.04 |
8.32 |
1.70 |
|
SEM6
|
59.84 |
0.369 |
0.014 |
0.058 |
0.008 |
0.067 |
0.178 |
0,209 |
0.045 |
| P7 |
|
|
|
|
|
|
|
|
|
| Diet |
0.2420 |
0.3492 |
0.3634 |
0.0014 |
0.2792 |
0.1923 |
0.7041 |
0.6860 |
0.044 |
| Breeder age |
0.2114 |
0.7332 |
0.0533 |
0.0949 |
0.6696 |
0.1919 |
0.7667 |
0.0590 |
0.4968 |
| Diet × Age |
0.9404 |
0.5842 |
0.8089 |
0.0625 |
0.4021 |
0.4979 |
0.2736 |
0.4700 |
0.2985 |
The dietary treatments were: 1CNT, a control diet without enzyme
preparation; 2LTE, a laboratory type enzyme cocktail (Molkim/250 g/ton
diet) containing at least 1,250 units of xylanase/g, 750 units of β-glucanase/g, 1,700
units of α-amylase/g, and 2,000 units of lipase; 3AVZ, a commercial enzyme
cocktail (Avizyme® 1500/ 250 g/ton diet) containing at least 1,000 units of
xylanase/g, 4,000 units of protease/g, and 2,000 units of α-amylase/g.
4Young breeders are 39 weeks old. 5Old breeders are 60 weeks
old. 6SEM: pooled standard error of the mean (data are the means of five
replicate pens with 26 birds each per treatment). 7Source of variation
(probability values). 8g/100 g body weight. 9cm/100 g body
weight. a,bMeans within columns with different superscripts differ at P
< 0.05.
Pancreatic enzyme activities in chickens measured on days 9 and 42 showed no differences
between BBA treatments; although MES had a significant effect on day 9 (Table 4). Pancreatic amylase secretion was
significantly lower (P = 0.0001) in chicks fed diets supplemented with LTE and AVZ than in
those fed the CNT diet; similar values were observed in the LTE and AVZ groups. Lipase
production was significantly lower (P = 0.0001) in chicks fed the LTE diet than in those
fed the CNT and AVZ diets, with no significant differences between the latter two.
Pancreatic protease activity showed no response to MES, regardless of bird age.
Table 4. Influence of multi-enzyme supplementation and breeder age on pancreatic enzyme
activities (μmol/min) in the chickens at 9 and 42 d of age.
|
Day 9 |
Day 42 |
|
Diet
|
Lipase |
Amylase |
Protease |
Lipase |
Amylase |
Protease |
| CNT1 |
0.73a |
2.59a |
19.5 |
13.6 |
2.53 |
34.7 |
| LTE2 |
0.50b |
1.96b |
19.1 |
13.6 |
2.24 |
33.8 |
| AVZ3 |
0.70a |
2.00b |
17.0 |
14.3 |
2.41 |
32.5 |
|
Breeder age
|
|
|
|
|
|
|
| Young4 |
0.62 |
2.19 |
18.4 |
14.6 |
2.35 |
33.7 |
| Old5 |
0.67 |
2.17 |
18.7 |
13.1 |
2.44 |
33.6 |
|
SEM6
|
0.037 |
0.119 |
1.374 |
1.274 |
0.145 |
3.441 |
| P7 |
|
|
|
|
|
|
| Diet |
0.0001 |
0.0001 |
0.1526 |
0.7944 |
0.1381 |
0.8134 |
| Breeder age |
0.0595 |
0.8281 |
0.7939 |
0.1397 |
0.4146 |
0.9784 |
| Diet × Age |
0.6233 |
0.4255 |
0.9547 |
0.8563 |
0.3777 |
09926 |
The dietary treatments were: 1CNT, a control diet without enzyme
preparation; 2LTE, a laboratory type enzyme cocktail (Molkim/ 250 g/ton
diet) containing at least 1,250 units of xylanase/g, 750 units of β-glucanase/g, 1,700
units of α-amylase/g, and 2,000 units of lipase; 3AVZ, a commercial enzyme
cocktail (Avizyme® 1500/ 250 g/ton diet) containing at least 1,000 units of
xylanase/g, 4,000 units of protease/g, and 2,000 units of α-amylase/g.
4Young breeders are 39 weeks old. 5Old breeders are 60 weeks
old. 6SEM: pooled standard error of the mean (data are the means of five
replicate pens with 26 birds each per treatment). 7Source of variation
(probability values). a,bMeans within columns with different superscripts
differ at P < 0.05.
As shown in Table 5, the ATTD of dry matter,
crude protein, and starch was not significantly affected by BBA or MES. However, compared
with CNT and AVZ dietary treatments, chickens fed the LTE-supplemented diet exhibited a
higher digestibility coefficient of ether extract at both 9 (P = 0.0001) and 42 (P =
0.0176) days of age.
Table 5. Influence of multi-enzyme supplementation and breeder age on total tract
percentage of digestibility of nutrients in chickens at 9 and 42 days of age.
|
Day 9 |
Day 42 |
| Dry matter |
Protein |
Ether extract |
Starch |
Dry matter |
Protein |
Ether extract |
Starch |
|
Diet
|
|
|
|
|
|
|
|
|
| CNT1 |
74.7 |
66.1 |
57.3b |
93.9 |
75.1 |
65.8 |
68.3b |
96.2 |
| LTE2 |
75.0 |
66.3 |
67.6a |
95.0 |
75.5 |
66.2 |
72.7a |
97.0 |
| AVZ3 |
74.6 |
67.1 |
58.1b |
95.1 |
75.7 |
66.8 |
69.1b |
97.1 |
|
Breeder age
|
|
|
|
|
|
|
|
|
| Young4 |
74.8 |
67.1 |
61.8 |
95.0 |
75.6 |
66.8 |
70.8 |
96.9 |
| Old5 |
74.8 |
65.9 |
60.2 |
94.3 |
75.2 |
65.8 |
69.2 |
96.3 |
|
SEM6
|
0.593 |
1.121 |
1.710 |
1.133 |
0.718 |
1.152 |
1.838 |
1.008 |
| P7 |
|
|
|
|
|
|
|
|
| Diet |
0.8408 |
0.6545 |
0.0001 |
0.4713 |
0.7039 |
0.6873 |
0.0176 |
0.5108 |
| Breeder age |
0.9749 |
0.2237 |
0.2126 |
0.5348 |
0.5457 |
0.2721 |
0.2327 |
0.4719 |
| Diet × Age |
0.8356 |
0.9217 |
0.9368 |
0.9993 |
0.9526 |
0.9354 |
0.9128 |
0.9910 |
The dietary treatments were: 1CNT, a control diet without enzyme
preparation; 2LTE, a laboratory type enzyme cocktail (Molkim/250 g/ton
diet) containing at least 1,250 units of xylanase/g, 750 units of β-glucanase/g, 1,700
units of α-amylase/g, and 2,000 units of lipase; 3AVZ, a commercial enzyme
cocktail (Avizyme® 1500/ 250 g/ton diet) containing at least 1,000 units of
xylanase/g, 4,000 units of protease/g, and 2,000 units of α-amylase/g.
4Young breeders are 39 weeks old. 5Old breeders are 60 weeks
old. 6SEM: pooled standard error of the mean (data are the means of five
replicate pens with 26 birds each per treatment). 7Source of variation
(probability values). a,bMeans within columns with different superscripts
differ at P < 0.05.
No significant differences in total aerobes, coliforms, and
Lactobacillus spp. in the cecal microflora were observed between
treatments at any time point (Table 6).
Clostridium spp. counts were below detectable limits (<1 log CFU/g)
and, therefore, are not presented. On day 9, sulfite-reducing anaerobes were fewer (P =
0.0013) in chicks fed diets supplemented with LTE and AVZ than in the CNT treatment.
Table 6. Influence of multi-enzyme supplementation and breeder age on cecal microbial
composition (Log CFU/mL) in chickens at 9 and 42 days of age.
|
Day 9 |
Day 42 |
|
Total aerobes |
Coliform |
Sulfite-reducing anaerobes |
Lactobacillus spp |
Total aerobes |
Coliform |
Sulfite-reducing anaerobes |
Lactobacillus spp |
|
Diet
|
|
|
|
|
|
|
|
|
| CNT1 |
8.26 |
4.67 |
2.53a |
7.95 |
7.62 |
6.45 |
4.04 |
7.42 |
| LTE2 |
8.37 |
4.72 |
2.08b |
8.11 |
7.62 |
6.37 |
4.08 |
7.44 |
| AVZ3 |
8.32 |
4.64 |
2.19b |
8.19 |
7.51 |
6.33 |
3.92 |
7.41 |
| Breeder age |
|
|
|
|
|
|
|
|
| Young4 |
8.37 |
4.64 |
2.26 |
8.03 |
7.48 |
6.28 |
3.99 |
7.32 |
| Old5 |
8.26 |
4.72 |
2.27 |
8.13 |
7.69 |
6.48 |
4.02 |
7.52 |
|
SEM6
|
0.177 |
0.180 |
0.118 |
0.152 |
0.169 |
0.257 |
0.144 |
0.160 |
| P7 |
|
|
|
|
|
|
|
|
| Diet |
0.8169 |
0.9237 |
0.0013 |
0.2727 |
0.7674 |
0.8948 |
0.5672 |
0.9828 |
| Breeder age |
0.4497 |
0.5632 |
0.8976 |
0.4371 |
0.1720 |
0.3310 |
0.9465 |
0.1396 |
| Diet × Age |
0.9571 |
0.9085 |
0.7439 |
0.9315 |
0.9809 |
0.9113 |
0.8322 |
0.8830 |
The dietary treatments were: 1CNT, a control diet without enzyme
preparation; 2LTE, a laboratory type enzyme cocktail (Molkim/250 g/ton
diet) containing at least 1,250 units of xylanase/g, 750 units of β-glucanase/g, 1,700
units of α-amylase/g, and 2,000 units of lipase; 3AVZ, a commercial enzyme
cocktail (Avizyme 1500®/ 250 g/ton diet) containing at least 1,000 units of
xylanase/g, 4,000 units of protease/g, and 2,000 units of α-amylase/g.
4Young breeders are 39 weeks old. 5Old breeders are 60 weeks
old. 6SEM: pooled standard error of the mean (data are the means of five
replicate pens with 26 birds each per treatment). 7Source of variation
(probability values). a,bMeans within columns with different superscripts
differ at P < 0.05.
Discussion
To our knowledge, the efficacy of nutrient utilization remains poorly studied in broiler
chicken breeders of varying ages[7,12], as well as in chicks produced by layered breeder
hens[13]. The few existing studies involving
broiler chickens[34,35] report that yolk testosterone levels likely affect progeny growth. In these
intervention studies, testosterone was experimentally injected into the yolk sac; however,
they did not demonstrate that natural variations in yolk testosterone with age accounted for
the subsequent BW under different conditions. Earlier studies reported that the advanced age
of broiler breeders resulted in higher FI and less efficient nutrient utilization in the
progeny[9,11,14,15], in spite of unchanged BWG[14,15,16]. Therefore,
it is necessary to determine the potential effects of egg yolk testosterone concentrations
on the growth rate and FI of modern broiler strains. Similarly, dietary interventions that
may help counteract the decline in feed conversion efficiency in chicks caused by aging
breeding stocks deserve further investigation. The present study is the first to investigate
the potential of multi-enzyme preparations in addressing this challenge.
In contrast to our findings, most studies on MES in chickens have shown that exogenous
carbohydrases combined with proteases[3,4] and lipases[17]
are more effective in diets with low nutrient densities, such as those low in energy or
protein, than in diets formulated according to the recommendations of broiler strain
breeders. The discrepancies between this and previous studies arise from the nutrient
density of the applied diets. The present findings suggest that broilers fed a nutritionally
adequate or nearly adequate diet may not respond to enzyme supplementation to the expected
extent.
The intended decrease in protein and energy content in the three-phase diets (as detailed
in Table 1) was only partial. This limitation
probably prevented exogenous enzymes from having a significant effect when birds consumed
easily digestible maize- and wheat-based diets. As a result, AVZ and LTE were unable to
perform their specific functions despite the physiological changes observed in chickens.
Reducing dietary metabolic energy by 90 kcal/kg and lysine by 0.10% can stimulate a positive
reaction to AVZ, thereby improving energy and protein use in birds grown for over 42
days[36]. Hence, enzyme supplementation is
effective only when the contribution of the nutrient matrix is preserved.
A decrease in pancreatic weight and the release of amylase and lipase on day 9 following
AVZ and LTE supplementation may indicate an early physiological adjustment in endogenous
digestive enzyme production. This likely reflects a reduced pancreatic demand owing to the
contribution of exogenous enzymes, suggesting that the pancreas lowers its own exocrine
output because part of the digestive workload is supported by supplemented enzymes. However,
this interpretation remains inferential and requires further mechanistic confirmation, such
as pancreatic histology, circulating enzyme markers, and time-course data, to verify true
compensation rather than a simple reduction in organ size. Nevertheless, the findings from
this study align with those reported by Zhu et al.[37] and Brenes et al.[38] in broiler
chickens, which indicated a significant reduction in pancreas weight following
supplementation of carbohydrase enzyme blends of amylase, β-glucanase, and xylanase.
The results of this study align with the existing literature, indicating that lipase
enhances fat digestibility in young chicks, particularly during the first 7–10 days after
hatching[23,39]. This is because chicks have an underdeveloped digestive system that responds
poorly to dietary enzymes[21]. The improvement
observed with LTE in this study may also be linked to the combined effect of carbohydrase
and lipase enzymes, a synergy previously noted in broilers[17,40]. Here, despite improved fat
utilization, growth performance remained stable in broilers at either 9 or 42 days of age.
These findings are further supported by the observation that this effect occurs when lipase
is applied to low-energy diets[41].
Excessive abdominal fat in fast-growing modern broiler chickens has several negative
consequences, including the inefficient use of dietary metabolizable energy and reduced
carcass yield. Additionally, it raises environmental concerns owing to increased waste
production and higher water consumption in slaughterhouses[42]. The additional contribution of lipase, along with carbohydrase enzymes, has
been shown to elicit increased abdominal fat accumulation in finishing broiler chickens,
thereby increasing the dietary energy value[43]. The
increase in abdominal fat mass associated with MES in this study may have resulted from the
redistribution of fat towards adipose tissue, diverting it from physiological needs and
muscle development. This is an unwanted consequence of the excess energy released by
enzymes, which is stored as body fat when chickens are fed diets that are almost
nutritionally adequate. Similar findings have been reported in previous studies involving
avizymes[44,45].
A possible explanation for unchanged pancreatic protease production in response to the
enzyme preparation containing AVZ could be the high-protein, nearly nutritionally complete
diet provided to the chickens[46].
The disparate effect of MES on intestinal size is linked to nutrient content and
composition of the diet[47]. Here, intestinal length
and weight remained unchanged, likely because of the highly digestible basal-phase diets,
which were similar to those based on maize and wheat. These diets reduced the potential for
enzymes to influence gut morphology, a result consistent with the findings reported by
Mohammadigheisar et al. and Alqhtani et al.[48,49], who administered MES to broilers.
The impact of MES on liver weight or carcass yield remains controversial, with some studies
reporting increases[50]; whereas others, including
ours, showing no significant changes[51]. The ability
of enzymes to release trapped nutrients and allocate them towards muscle growth are clearly
affected by several dietary factors[2]. Consequently,
because of the differences in dietary approaches used in multi-enzyme research, it cannot be
assumed that enhanced nutrient utilization will always lead to increased meat yields in
broiler chickens. The finding that AVZ and LTE reduced gizzard weight by 9% in finishing
broilers is difficult to explain[48,49] because only the physical structure of the feed has
been associated with increased gizzard weight[52,53].
The results of this study show no improvement in apparent total tract nutrient retention.
In contrast, previous research has reported that the digestibility of dry matter, starch,
fat, energy, and nitrogen in broiler chickens improved with dietary supplementation of
multi-enzyme blends—such as xylanase, amylase, β-glucanase, β-mannanase and protease—across
different growth stages throughout the production period[3,49]. Interestingly, the methodology used
in this study did not involve a reduction in nutrient content. In addition, the
metabolizable energy content of the growth and finishing diets was approximately 80 kcal/kg
higher than that reported previously. This highlights how the energy levels of the corn-,
wheat-, and soybean-based diets fed to finishing birds may have been too high to elicit a
multi-enzyme response and improve energy use by Ross 308 broiler chickens.
From the perspective of gut health, MES showed a limited ability to alter the bacterial
community in the cecum. This could be attributed to the composition of the diets, which
consisted of highly digestible corn, wheat, and soybeans. Such a diet likely provides fewer
substrates for enzymatic activity in the digesta, making carbohydrase enzymes less effective
than with diets higher in non-starch polysaccharides[2,54] and slows the growth of beneficial
bacteria[6,55]. Nevertheless, on day 9, there was a slight but significant decrease in the
number of sulfite-reducing anaerobic bacteria in birds that received AVZ and LTE
supplements. Overall, MES helped support intestinal health in young chicks by inhibiting the
growth of harmful bacteria, especially sulfite-reducing anaerobic bacteria[56].
In conclusion, the anticipated benefits of MES were not observed in broiler chickens raised
over a 42-day period. This is likely because the birds were fed a nearly nutritionally
complete diet, which may have resulted in a lack of interaction between BBA and MES.
Accordingly, the hypothesis that MES effectively enhanced nutrient utilization in progeny
from older breeders was not supported, as these broiler chickens exhibited feed conversion
efficiency comparable to that of birds from younger breeders. The present results suggest
that parental age is not an important factor affecting the overall productive performance of
the offspring, thus providing useful insights for the broiler industry.
