ABSTRACT
Exogenous enzymes are added to diets to improve nutrient utilization and feed efficiency. A study was conducted to evaluate the effects of dietary exogenous enzyme products with amylolytic (Amaize, Alltech) and proteolytic (Vegpro, Alltech) activity on performance, excretion of purine derivatives, and ruminal fermentation of dairy cows. A total of 24 Holstein cows, 4 of which were ruminally cannulated (161 ± 88 d in milk, 681 ± 96 body weight, and 35.2 ± 5.2 kg/d of milk yield), were blocked by milk yield, days in milk, and body weight, and then distributed in a replicated 4 × 4 Latin square design. Experimental periods lasted 21 d, of which the first 14 d were allowed for treatment adaptation and the last 7 d were used for data collection. Treatments were as follows: (1) control (CON) with no feed additives, (2) amylolytic enzyme product added at 0.5 g/kg diet dry matter (DM; AML), (3) amylolytic enzyme product at 0.5 g/kg of diet DM and proteolytic enzyme product at 0.2 g/kg of diet DM (low level; APL), and (4) amylolytic enzyme products added at 0.5 g/kg diet DM and proteolytic enzyme product at 0.4 g/kg of diet DM (high level; APH). Data were analyzed using the mixed procedure of SAS (version 9.4; SAS Institute Inc.). Differences between treatments were analyzed by orthogonal contrasts: CON versus all enzyme groups (ENZ); AML versus APL+APH; and APL versus APH. Dry matter intake was not affected by treatments. Sorting index for feed particles with size <4 mm was lower for ENZ group than for CON. Total-tract apparent digestibility of DM and nutrients (organic matter, starch, neutral detergent fiber, crude protein, and ether extract) were similar between CON and ENZ. Starch digestibility was greater in cows fed APL and APH treatments (86.3%) compared with those in the AML group (83.6%). Neutral detergent fiber digestibility was greater in APH cows compared with those in the APL group (58.1 and 55.2%, respectively). Ruminal pH and NH3-N concentration were not affected by treatments. Molar percentage of propionate tended to be greater in cows fed ENZ treatments than in those fed CON. Molar percentage of propionate was greater in cows fed AML than those fed the blends of amylase and protease (19.2 and 18.5%, respectively). Purine derivative excretions in urine and milk were similar in cows fed ENZ and CON. Uric acid excretion tended to be greater in cows consuming APL and APH than in those in the AML group. Serum urea N concentration tended to be greater in cows fed ENZ than in those fed CON. Milk yield was greater in cows fed ENZ treatments compared with CON (32.0, 33.1, 33.1, and 33.3 kg/d for CON, AML, APL, and APH, respectively). Fat-corrected milk and lactose yields were higher when feeding ENZ. Feed efficiency tended to be greater in cows fed ENZ than in those fed CON. Feeding ENZ benefited cows' performance, whereas the effects on nutrient digestibility were more pronounced when the combination of amylase and protease was fed at the highest dose.
Key words
INTRODUCTION
Enzymes are chemically defined as proteins that catalyze biological processes in a specific biochemical reaction, degrading a restricted range of substrates in specific reaction sites (
Ravindran, 2013
). In ruminant nutrition, exogenous enzymes (ENZ), particularly those derived from fungal populations, have been fed to improve performance by increasing the hydrolytic activity in the rumen and total-tract digestibility of nutrients in cattle (Meale et al., 2014
). Exogenous enzymes break down complex carbohydrates in different sites and generate different oligosaccharides compared with those that enzymes from ruminal microbiota would normally produce (Tricarico et al., 2008
). In addition, ENZ can increase the adherence of bacteria to the substrates while working synergistically with enzymes from the ruminal microbes to increase hydrolytic potential within the rumen (Morgavi et al., 2000
; Beauchemin et al., 2003
). Because ENZ are “generally recognized as safe” (Sewalt et al., 2016
) and the costs of production have decreased, the interest of researchers and ruminant nutritionists in evaluating ENZ has increased in the last decades.Several studies have evaluated a wide variety of feed additives with amylolytic enzymes on performance of dairy cows (
Takiya et al., 2017
; Silva et al., 2018
; Zilio et al., 2019
). Interestingly, the most common observation was an increase in total-tract fiber digestibility, possibly linked with conditions in the ruminal environment that favor fiber degradation (Klingerman et al., 2009
; Gencoglu et al., 2010
; Silvestre et al., 2022
). Responses to amylase supplementation on starch utilization were not commonly observed because few studies (Nozière et al., 2014
) have evaluated starch degradability in the rumen and because post-ruminal compensation of starch digestibility may occur (Owens et al., 1986
). However, Nozière et al., 2014
found an increase in ruminal digestibility of starch when feeding amylase to primiparous cows. A few studies have evaluated proteases in vitro (Colombatto et al., 2003b
; Eun and Beauchemin, 2007
; Colombatto and Beauchemin, 2009
) or in vivo (Eun and Beauchemin, 2005
). Feeding a protease enzyme product, particularly when associated with low-forage diets, increased ruminal fluid activity of xylanase and improved total-tract digestibility of starch and acid detergent fiber, milk yield, and feed efficiency in dairy cows (Eun and Beauchemin, 2005
).In tropical conditions, corn hybrids used are generally of flint-type endosperm, which are more resistant to enzymatic attack when compared with dent-type hybrids (
Kaczmarek et al., 2014
). Flint-type endosperm is positively associated with grain hardness, density, and percentage of zein proteins involving starch granules, which can impair starch degradation (Giuberti et al., 2013
). Exogenous proteases may be an option to increase proteolysis of zein proteins and thus improve starch digestibility and energy availability. The combination of different ENZ may allow the breakdown of cross-linkages between structural proteins in feed (Beauchemin et al., 2003
). Thus, we hypothesized that feeding amylase and protease enzymes would have synergistic effects compared with feeding individual enzymes. We conducted an experiment to evaluate the effects of the combination of feed additives with amylolytic and proteolytic activities on nutrient intake and total-tract apparent digestibility, feed sorting index, ruminal fermentation parameters, purine derivative excretion, serum concentrations of glucose and urea N, and milk yield and composition in dairy cows. We hypothesized that feeding amylolytic enzymes would improve performance by increasing total-tract apparent digestibility of nutrients and that the addition of proteases would amplify that response.MATERIALS AND METHODS
This study was carried out between September and November 2020 at Laboratório de Pesquisa em Bovinos de Leite – LPBL (Laboratory on Dairy Cattle Research; Pirassununga, Brazil) under the approval of the Ethics Committee on Animal Use from the School of Veterinary Medicine and Animal Sciences, University of Sao Paulo (protocol no. 9274250920).
Treatments and Experimental Design
A total of 24 Holstein cows (4 primiparous and 20 multiparous, 161 ± 88 DIM, 681 ± 96 kg BW, and 35.2 ± 5.2 kg/d of milk yield, at the start of the study), 4 (multiparous) of which were rumen cannulated, were enrolled in this study. Cows were used in a replicated 4 × 4 Latin square experimental design with squares blocked by parity, milk yield, DIM, and BW. Treatment sequence distribution was balanced for carry-over effects (
Seo et al., 2018
). Experimental periods lasted 21 d, of which the first 14 d were allowed for treatment adaptation (Machado et al., 2016
) and the last 7 d were used for data collection and sampling. The treatments were as follows: (1) control (CON) with no feed additives, (2) amylolytic enzyme product added at 0.5 g/kg of diet DM (AML), (3) amylolytic enzyme product at 0.5 g/kg of diet DM and proteolytic enzyme product at 0.2 g/kg of diet DM (low level; APL), and (4) amylolytic enzyme products added at 0.5 g/kg of diet DM and proteolytic enzyme product at 0.4 g/kg of diet DM (high level; APH).The enzymes were mixed with minerals in the concentrate feed. The enzyme products were weighed for a concentrate batch of 250 kg, manually mixed with the mineral premix, and then added to the mixer of the feed mill to be homogenized with other concentrate feeds. After concentrate ingredients were mixed, concentrate was packaged in bags of 40 kg and stored in the barns until consumption (no longer than 9 d). The barn air temperature during the trial averaged 24.8°C (39.9°C max and 12.3°C min), whereas the average air relative humidity was 56.0%. Air temperature and air relative humidity were measured by 2 data loggers (TagTemp Stick, Novus) placed at a height of 2 m in the barn. The commercial products considered as amylolytic enzyme and proteolytic enzymes were Amaize (Alltech) and VegPro (Alltech), respectively. According to manufacturer's information, Amaize is composed of inactivated yeast and dry product derived from Aspergillus oryzae (IMICC 507151) fermentation, containing a minimum of 600 U of amylase activity per gram (1 U is the equivalent of the amount of enzyme that dextrinizes 1 g of soluble starch per min at 4.8 pH and 30°C). VegPro comprises calcium carbonate, sodium chloride, derived products from Aspergillus niger (NCIMB 30289) fermentation, and derived products from Trichoderma longibrachiatum (NCIMB 302545). VegPro contains a minimum of 7,500 U of protease activity per gram and 45 U of cellulase activity per gram. One unit of protease activity is equivalent to the amount of enzyme that produces a hydrolysate of which the absorbance at 275 nm is equal to the absorbance of a solution with 1.10 µg/mL of tyrosine in 0.006 N hydrochloric acid. Enzyme products were manufactured in July 2020 and expired after 18 mo. Doses of proteolytic enzymes were chosen based on results of in vitro kinetic parameters and gas production in
da Freiria et al., 2018
. Briefly, da Freiria et al., 2018
reported a linear increase in gas production when incubating a grass forage with proteases at doses ranging from 0.05 to 0.20 mg/mL. Furthermore, the authors found a marginal increase in OM degradability at the highest dose of protease in the same study (da Freiria et al., 2018
). The dose of amylolytic enzyme was selected based on positive results on milk yield described in previous studies (Tricarico et al., 2005
; Harrison and Tricarico, 2007
).Cows were housed in a barn with individual pens (area 17 m2), sand beds, fans, and free access to water. Cows were individually fed twice daily (0700 and 1300 h in equal amounts). Total mixed ration was prepared in the feed bunk by hand-mixing the corn silage and concentrate individually weighed for each cow. Feeding rate was adjusted to allow refusals between 5 and 10% on as-fed basis. Corn silage DM content was monitored thrice a week by drying samples in oven at 105°C overnight to make dietary adjustments when necessary. The experimental diet (Table 1) was formulated according to the
NRC (National Research Council), 2001
to meet or exceed nutrient requirement estimates of cows producing 35 kg/d milk, 3.2% protein, and 3.8% fat; DMI was estimated at 24.3 kg/d by the software.Table 1Ingredients and chemical composition of basal diet (average ± SD)
| Item | Diet |
|---|---|
| Ingredient, % DM | |
| Corn silage | 48.0 |
| Ground corn | 18.7 |
| Soybean meal | 11.8 |
| Citrus pulp | 8.20 |
| Whole raw soybean | 6.42 |
| Bypass soybean meal | 4.01 |
| Minerals and vitamins | 1.50 |
| Sodium bicarbonate | 0.80 |
| Urea | 0.15 |
| Limestone | 0.20 |
| Salt | 0.20 |
| Particle size distribution, % as fed | |
| >19 mm | 16.7 |
| 19–8 mm | 39.0 |
| 8–4 mm | 15.7 |
| <4 mm | 28.5 |
| Chemical composition, % DM | |
| DM, % as fed | 47.8 ± 1.83 |
| NDF | 39.7 ± 3.08 |
| Forage NDF | 27.3 ± 1.85 |
| Acid detergent fiber | 21.8 ± 2.65 |
| Lignin | 8.97 ± 0.91 |
| Starch | 27.5 ± 1.04 |
| CP | 16.1 ± 0.99 |
| Neutral detergent insoluble protein | 2.84 ± 0.71 |
| Acid detergent insoluble protein | 1.64 ± 0.18 |
| Ether extract | 3.56 ± 0.39 |
| Ash | 6.87 ± 0.35 |
1 Chemical composition (% DM): 31.7 DM (% as fed), 96.4 OM, 56.9 NDF, 31.6 ADF, 25.6 starch, 2.16 ether extract, 7.93 CP, and 4.26 lignin.
2 Bypass soybean meal (Soypass, Cargill).
3 Content per kilogram of product: 215 g of Ca, 15 g of Co, 700 mg of Cu, 10 mg of Cr, 20 g of S, 600 mg of F, 40 mg of I, 20 g of Mg, 1,600 mg of Mn, 20 mg of Se, 70 g of Na, 2,500 mg of Zn, 200,000 IU of vitamin A, 50,000 IU of vitamin D3, 1,500 IU of vitamin E.
Samples of corn silage were collected during the last 7 d of each experimental period and pooled on a wet basis to form a composite sample per period. Other feed ingredients were collected during the preparation of concentrate at the feed mill during the sampling period. Samples were analyzed for contents of DM (method 930.15; ), ash (method 942.05; ), OM (DM − ash), CP (N × 6.25; Kjeldahl method 984.13; ), and ether extract (method 920.39; ). Neutral detergent fiber (
Van Soest et al., 1991
) was analyzed using α-amylase and sodium sulfite (TE-149 fiber analyzer; Tecnal Equipamentos para Laboratório), and acid detergent fiber and lignin (method 973.18) were analyzed according to . Feed ingredients were also analyzed for contents of starch using an enzymatic degradation method (Amyloglicosidase, Novozymes Latin America Ltda.) and absorbances measured by spectrophotometer (SBA-200, Celm) according to Hendrix, 1993
.Nutrient Intake, Sorting Index, and Total-Tract Apparent Digestibility
Feed offered and refusals were recorded daily to determine feed intake. Refusals were sampled during the last 7 d of each experimental period, pooled by cow per period, and frozen for further chemical analysis according to the methods described earlier. Samples of TMR and refusals were collected for 2 consecutive days (d 15 and 16 of each period) during the collection period for determination of particle size distribution (
The intake corresponding to each sieve was expressed as the percentage of the total estimated intake, where PTMR is the TMR particle size, and Prefusals is the particle size distribution of refusals. Sorting index equal to 1 means no sorting, <1 indicates sorting against the particular particle size, and values >1 shows that cows sorted for a specific particle size.
Maulfair and Heinrichs, 2012
) and feed sorting index with particles stratified on as-fed basis (Silveira et al., 2007
). Feed particles were stratified using the Penn State particle size separator to the following fractions: long (>19 mm), medium (19–8 mm), short (8–4 mm), and fine (<4 mm) particles. The sorting index was calculated using the following equations:
Expected intake (kg/d) = intake [(kg as-fed)/d] × PTMR (kg/kg);
The intake corresponding to each sieve was expressed as the percentage of the total estimated intake, where PTMR is the TMR particle size, and Prefusals is the particle size distribution of refusals. Sorting index equal to 1 means no sorting, <1 indicates sorting against the particular particle size, and values >1 shows that cows sorted for a specific particle size.
Undegradable NDF (uNDF) contents in feeds, refusals, and feces were used to estimate fecal excretion of DM. Fecal samples were collected directly from the rectum of cows every 9 h during 3 consecutive days (0600, 1500, and 2400 h on d 15; 0900 and 1800 h on d 16; and 0300, 1200, and 2100 h on d 17, to represent every 3 h over a 24-h period) and pooled per cow per period for analyses. For uNDF analysis, ground samples (2 mm) of feeds, refusals, and feces were placed in nonwoven fabric bags (5 × 5 cm at 20 mg DM/cm2) and incubated in the rumen of 2 cannulated lactating cows receiving the same basal diet of this study for 288 h (
Huhtanen et al., 1994
; Casali et al., 2008
). After removal from the rumen, bags were washed in running tap water and dried in a forced-ventilation oven at 55°C for 3 d, and NDF content was determined. Digestibility of DM and nutrients were calculated using the following equations:
Ruminal Fermentation
Ruminal fluid samples were collected from cannulated cows on the last day of each period, before the morning feeding (0 h) and at 2, 4, 6, 8, 10, 12, 14, and 16 h after feeding. Digesta were collected from different sites within the rumen (dorso-cranial, ventral-cranial, ventral, caudo-ventral, and caudo-dorsal regions) and strained through 4 layers of cheesecloth to extract ruminal fluid (250 mL). Ruminal fluid pH was measured using a glass electrode and a reference electrode (MB-10, Marte Científica). Ruminal fluid samples were centrifuged (2,000 × g for 15 min at room temperature), and 1.8 mL of the supernatant was pipetted into a centrifuge tube containing 400 μL of orthophosphoric acid solution (1 N) for further VFA analysis. Peaks of VFA were measured on a gas chromatograph (Shimadzu GC-2010 Plus, Shimadzu) equipped with an automatic injector (AOC-20i, Stabilwax-DA, 30-m capillary column, 0.25-mm i.d., 0.25-μm film thickness; Restek) and a flame ionization detector, as described by
Del Valle et al., 2018
. Another aliquot from the supernatant (800 μL) was mixed with 400 μL of 1 N sulfuric acid solution for NH3-N determination using the phenol-hypochlorite method (Broderick and Kang, 1980
). and absorbances were measured on a microplate reader (Biochrom Asys, Biochrom).Excretion of Purine Derivatives
Urine samples (20 mL) were collected at the same time points as feces. Urine samples were diluted (1:4 ratio, vol/vol) into a sulfuric acid solution at 0.036 N to preserve purine derivatives (
Chen et al., 1995
, Chen and Gomes, 1992
). Samples were stored frozen for total N, allantoin, uric acid, and creatinine analyses. Daily urine volume was estimated considering a daily creatinine excretion of 29 mg/kg BW (Valadares et al., 1999
). Body weights were measured (2 consecutive days) after the morning milking and before the morning feeding, at the beginning of experiment and during the last 2 d of each experimental period. Urine creatinine concentration was assessed using commercial kits (Kinetic Creatinine, catalog no. K-067, Bioclin) and absorbances measured with a spectrophotometer (SBA 200, Celm). Allantoin concentrations in urine and milk were assessed by a colorimetric method according to Chen and Gomes, 1992
, with absorbances measured using a microplate reader (Biochrom Asys UVM 340, Biochrom Asys). Uric acid concentration in urine was analyzed using a commercial kit (Uric Acid Stable Liquid, catalog no. K-052, Bioclin).Serum Metabolites and Milk Yield and Composition
Blood samples were collected on d 16 of each period, 4 h after the morning feeding in vacuum tubes (10 mL; Becton Dickinson) by puncture of coccygeal vessels. Blood samples were centrifuged after clotting (15 min at 2,000 × g at room temperature), and serum was harvested and stored frozen (−20°C) for further glucose and urea analyses. Blood glucose and urea were determined using colorimetric commercial kits (urea, catalog #K056; glucose, catalog #K082; Bioclin), and absorbances were measured using a spectrophotometer (SBA-200, Celm). Blood urea N was calculated by multiplying the urea concentration by 0.4667.
Cows were milked twice daily (0600 and 1700 h), and production was electronically recorded (Alpro, DeLaval). Data from the last 7 d of milk yield were used for statistical analysis. Milk samples (300 mL) were collected during 3 consecutive days during each sampling period to assess concentrations of CP, fat, and lactose using the mid-infrared method (Lactoscan, Entelbra). The 3.5% FCM was calculated according to
Sklan et al., 1992
, where 3.5% FCM = (0.432 + 0.165 × milk fat %) × milk yield (kg/d). Milk samples were deproteinized with a trichloroacetic acid solution (25%; 2:1 vol/vol; Shahani and Sommer, 1951
) and stored at −20°C for allantoin and MUN analyses. The MUN was determined using commercial kits (catalog no. K-056; Bioclin).Statistical Analysis
Data were submitted to ANOVA after testing for normal distribution of residuals (Shapiro-Wilk test, using the univariate procedure of SAS version 9.4; SAS Institute Inc.). ANOVA was performed using the mixed procedure of SAS according to the following statistical model (except for ruminal fermentation data):
where Yijkl is the value observed of response variable; μ is the overall mean; Si is the fixed effect of square (i = 1 to 6); aj:i is the random effect of animal within square (j = 1 to 24); Tk is the fixed effect of treatment (k = 1 to 4); Pl is the fixed effect of period (l = 1 to 4); and eijkl is the residual.
Yijkl = μ + Si + aj:i + Tk + Pl + eijkl,
where Yijkl is the value observed of response variable; μ is the overall mean; Si is the fixed effect of square (i = 1 to 6); aj:i is the random effect of animal within square (j = 1 to 24); Tk is the fixed effect of treatment (k = 1 to 4); Pl is the fixed effect of period (l = 1 to 4); and eijkl is the residual.
Ruminal fermentation data were analyzed as repeated measures according to the following model:
where Yijklm is the value observed of response variable; μ is the overall mean; aj is the random effect of animal (j = 1 to 4); Tk is the fixed effect of treatment (k = 1 to 4); Pl is the fixed effect of period (l = 1 to 4); ωijkl is the residual error associated with cows; STm is the fixed effect of sampling time point (m = 1 to 9); T × STkm is the fixed interaction effect between treatment and sampling time point; and eijklm is the residual. Several covariance matrices structures (compound symmetry, autoregressive, heterogeneous autoregressive, homogeneous Toeplitz, heterogeneous Toeplitz, unstructured, ante-dependence, and no diagonal factor analytic) were tested and chosen based on the lowest Bayesian information criteria values.
Yijklm = μ + aj + Tk + Pl + ωijkl + STm + T × STkm + eijklm,
where Yijklm is the value observed of response variable; μ is the overall mean; aj is the random effect of animal (j = 1 to 4); Tk is the fixed effect of treatment (k = 1 to 4); Pl is the fixed effect of period (l = 1 to 4); ωijkl is the residual error associated with cows; STm is the fixed effect of sampling time point (m = 1 to 9); T × STkm is the fixed interaction effect between treatment and sampling time point; and eijklm is the residual. Several covariance matrices structures (compound symmetry, autoregressive, heterogeneous autoregressive, homogeneous Toeplitz, heterogeneous Toeplitz, unstructured, ante-dependence, and no diagonal factor analytic) were tested and chosen based on the lowest Bayesian information criteria values.
Differences between treatments were evaluated by orthogonal contrasts to test the effects of feeding enzyme (CON vs. all ENZ); the effects of combining amylase with protease (AML vs. APL+APH); and the effects of different doses of protease (APL vs. APH). The significance level was set at P ≤ 0.05, and tendencies were described when 0.05 < P ≤ 0.10.
RESULTS
Dry matter and nutrient intakes were similar among treatments (Table 2). Feed sorting index for small feed particles (<4 mm) was lower (P = 0.04) in cows fed ENZ than in those fed CON. Feeding the blends of amylase and protease (APL+APH) resulted in greater (P = 0.02) feed sorting index for long particles (>19 mm) and lower (P = 0.04) feed sorting index for small particles (<4 mm) than feeding amylase alone (AML). No differences in sorting index were detected when comparing the APL and APH treatment groups. Total-tract apparent digestibilities of DM and nutrients were similar between CON and ENZ. Starch digestibility was greater (P < 0.01) in cows fed both amylase and protease than in those fed only amylase. Starch digestibility tended (P = 0.06) to be greater in cows consuming APH than in those consuming APL. Neutral detergent fiber digestibility was greater (P = 0.02) in APH cows compared with those in the APL group. Crude protein digestibility was similar among groups, regardless of the concentration of the proteolytic enzyme.
Table 2Nutrient intake, sorting index, and total-tract apparent digestibility in lactating cows fed amylase combined or not with protease
| Item | Treatment 1 Control (CON) = no feed additives; amylase (AML) = amylolytic enzyme (Amaize, Alltech) added at 0.5 g/kg diet DM; amylase and protease (APL) = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme (VegPro, Alltech) added at 0.2 g/kg diet DM; and APH = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme added at 0.4 g/kg diet DM. | SEM | P-value | |||||
|---|---|---|---|---|---|---|---|---|
| CON | AML | APL | APH | C1 | C2 | C3 | ||
| Intake, kg/d | ||||||||
| DM | 28.3 | 28.5 | 28.5 | 28.0 | 0.93 | 0.99 | 0.56 | 0.43 |
| OM | 26.3 | 26.5 | 26.5 | 26.1 | 0.87 | 0.99 | 0.52 | 0.44 |
| Starch | 8.43 | 8.48 | 8.44 | 8.33 | 0.27 | 0.92 | 0.46 | 0.47 |
| NDF | 10.8 | 10.8 | 10.8 | 10.7 | 0.37 | 0.99 | 0.61 | 0.62 |
| CP | 4.62 | 4.65 | 4.67 | 4.57 | 0.15 | 0.95 | 0.72 | 0.22 |
| Ether extract | 1.03 | 1.05 | 1.05 | 1.02 | 0.04 | 0.79 | 0.63 | 0.22 |
| Intake, % BW | ||||||||
| DM | 4.22 | 4.22 | 4.20 | 4.17 | 0.16 | 0.74 | 0.62 | 0.70 |
| NDF | 1.60 | 1.61 | 1.60 | 1.59 | 0.06 | 0.94 | 0.57 | 0.91 |
| BW, kg | 691 | 696 | 698 | 690 | 15.6 | 0.23 | 0.56 | 0.06 |
| Feed sorting index | ||||||||
| >19 mm | 0.976 | 0.970 | 0.995 | 1.00 | 0.011 | 0.30 | 0.02 | 0.65 |
| 19–8 mm | 0.986 | 0.988 | 0.989 | 0.984 | 0.003 | 0.89 | 0.59 | 0.32 |
| 8–4 mm | 0.994 | 0.995 | 0.999 | 1.01 | 0.004 | 0.33 | 0.27 | 0.40 |
| <4 mm | 1.03 | 1.03 | 1.02 | 1.01 | 0.005 | 0.04 | 0.04 | 0.60 |
| Total-tract apparent digestibility, % | ||||||||
| DM | 71.8 | 71.3 | 71.8 | 72.8 | 0.66 | 0.82 | 0.12 | 0.23 |
| OM | 72.9 | 72.3 | 72.9 | 73.9 | 0.69 | 0.85 | 0.14 | 0.22 |
| Starch | 84.1 | 83.6 | 85.2 | 87.3 | 0.94 | 0.19 | <0.01 | 0.06 |
| NDF | 56.1 | 55.5 | 55.2 | 58.1 | 0.96 | 0.82 | 0.27 | 0.02 |
| CP | 71.5 | 71.9 | 71.5 | 72.8 | 1.00 | 0.53 | 0.85 | 0.26 |
| Ether extract | 79.5 | 78.3 | 77.9 | 78.9 | 1.40 | 0.42 | 0.95 | 0.62 |
1 Control (CON) = no feed additives; amylase (AML) = amylolytic enzyme (Amaize, Alltech) added at 0.5 g/kg diet DM; amylase and protease (APL) = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme (VegPro, Alltech) added at 0.2 g/kg diet DM; and APH = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme added at 0.4 g/kg diet DM.
2 Orthogonal contrasts: C1 = CON versus enzyme treatments; C2 = AML vs. APL+APH; C3 = APL vs. APH.
3 No sorting = 1; values <1 indicate sorting against, and values >1 indicate sorting for particles on the particular particle size range. Sorting index was calculated according to
Silveira et al., 2007
on as-fed basis.Neither ruminal pH nor concentrations of NH3-N were altered by treatments (Table 3; Supplemental Figure S1, https://doi.org/10.6084/m9.figshare.21681836.v1;
Takiya, 2022
). Molar percentage of propionate tended to be greater (P = 0.08) in cows fed ENZ treatments than CON. No differences were observed in molar percentages of acetate, butyrate, iso-valerate, iso-butyrate, or acetate to propionate ratio when comparing ENZ treated with CON-cows. Molar percentage of propionate was greater (P = 0.04) in cows fed AML than those fed the blends of amylase and protease. Molar percentage of propionate tended to be greater (P = 0.09) in cows fed APH than those in APL group.Table 3Ruminal fermentation in ruminally cannulated (n = 4) cows fed amylase combined or not with protease
| Item | Treatment 1 Control (CON) = no feed additives; amylase (AML) = amylolytic enzyme (Amaize, Alltech) added at 0.5 g/kg diet DM; amylase and protease (APL) = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme (VegPro, Alltech) added at 0.2 g/kg diet DM; and APH = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme added at 0.4 g/kg diet DM. | SEM | P-value | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| CON | AML | APL | APH | C1 | C2 | C3 | Trt | Time | Trt × Time | ||
| pH | 6.21 | 6.20 | 6.25 | 6.27 | 0.118 | 0.80 | 0.62 | 0.86 | 0.95 | <0.01 | 0.82 |
| NH3-N, mg/dL | 13.0 | 12.0 | 12.8 | 11.6 | 0.799 | 0.32 | 0.87 | 0.30 | 0.55 | <0.01 | 0.30 |
| VFA, % | |||||||||||
| Acetate | 66.1 | 66.0 | 65.8 | 65.1 | 0.807 | 0.56 | 0.52 | 0.40 | 0.67 | 0.13 | 0.30 |
| Propionate | 18.2 | 19.2 | 18.2 | 18.8 | 0.754 | 0.08 | 0.04 | 0.09 | 0.03 | <0.01 | 0.55 |
| Butyrate | 12.1 | 11.7 | 12.3 | 12.1 | 0.673 | 0.95 | 0.33 | 0.78 | 0.78 | 0.05 | 0.48 |
| Iso-valerate | 1.58 | 1.54 | 1.54 | 1.55 | 0.090 | 0.58 | 0.92 | 0.87 | 0.95 | <0.01 | 0.74 |
| Valerate | 1.21 | 1.33 | 1.30 | 1.53 | 0.119 | 0.22 | 0.59 | 0.19 | 0.33 | 0.16 | 0.46 |
| Iso-butyrate | 0.834 | 0.780 | 0.778 | 0.826 | 0.044 | 0.25 | 0.52 | 0.25 | 0.38 | <0.01 | <0.01 |
| Branched-chain fatty acids | 3.63 | 3.65 | 3.61 | 3.90 | 0.167 | 0.70 | 0.68 | 0.35 | 0.73 | <0.01 | 0.35 |
| Acetate-to-propionate ratio | 3.67 | 3.49 | 3.64 | 3.51 | 0.186 | 0.37 | 0.56 | 0.43 | 0.59 | <0.01 | 0.42 |
| Total VFA, mmol/L | 66.9 | 69.8 | 80.8 | 60.7 | 9.49 | 0.74 | 0.93 | 0.15 | 0.48 | <0.01 | 0.05 |
1 Control (CON) = no feed additives; amylase (AML) = amylolytic enzyme (Amaize, Alltech) added at 0.5 g/kg diet DM; amylase and protease (APL) = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme (VegPro, Alltech) added at 0.2 g/kg diet DM; and APH = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme added at 0.4 g/kg diet DM.
2 Orthogonal contrasts: C1 = CON versus enzyme treatments; C2 = AML vs. APL+APH; C3 = APL vs. APH. Trt = treatment.
Purine derivative excretions in urine and milk were measured as a proxy of ruminal microbial protein supply (
Chen and Gomes, 1992
), and they were similar in cows fed ENZ and CON treatments (Table 4). Urinary allantoin excretion, however, was greater (P = 0.02) in cows fed only amylase than in those fed both amylase and protease. Uric acid excretion tended to be greater (P = 0.10) in cows consuming both amylase and protease than in those fed only amylase. Purine derivative excretion was not different between treatment groups fed different doses of the proteolytic enzyme (APL vs. APH). Serum glucose concentrations were similar among treatment groups. Serum urea N concentration tended to be greater (P = 0.08) in cows fed ENZ than in those fed CON.Table 4Purine derivative excretion, serum metabolites, and milk yield and composition in cows fed amylase combined or not with protease
| Item | Treatment 1 Control (CON) = no feed additives; amylase (AML) = amylolytic enzyme (Amaize, Alltech) added at 0.5 g/kg diet DM; amylase and protease (APL) = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme (VegPro, Alltech) added at 0.2 g/kg diet DM; and APH = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme added at 0.4 g/kg diet DM. | SEM | P-value | |||||
|---|---|---|---|---|---|---|---|---|
| CON | AML | APL | APH | C1 | C2 | C3 | ||
| Purine derivatives, mmol/d | ||||||||
| Urinary allantoin | 178 | 221 | 152 | 169 | 20.9 | 0.90 | 0.02 | 0.54 |
| Urinary uric acid | 38.8 | 37.1 | 42.3 | 49.6 | 4.98 | 0.42 | 0.10 | 0.25 |
| Milk allantoin | 83.5 | 66.1 | 62.0 | 60.6 | 17.31 | 0.23 | 0.79 | 0.95 |
| Total | 312 | 324 | 263 | 299 | 27.0 | 0.54 | 0.14 | 0.29 |
| Serum metabolite, mg/dL | ||||||||
| Glucose | 75.8 | 76.6 | 75.5 | 74.7 | 2.32 | 0.94 | 0.56 | 0.76 |
| Urea N | 16.7 | 17.2 | 18.0 | 18.3 | 1.59 | 0.08 | 0.17 | 0.77 |
| Yield, kg/d | ||||||||
| Milk | 32.0 | 33.1 | 33.1 | 33.3 | 0.66 | <0.01 | 0.81 | 0.65 |
| FCM | 33.3 | 34.2 | 34.5 | 34.6 | 0.85 | 0.03 | 0.50 | 0.86 |
| Fat | 1.20 | 1.22 | 1.24 | 1.24 | 0.04 | 0.10 | 0.44 | 0.99 |
| Protein | 1.02 | 1.05 | 1.05 | 1.05 | 0.02 | 0.07 | 0.95 | 0.98 |
| Lactose | 1.52 | 1.57 | 1.57 | 1.58 | 0.03 | 0.01 | 0.81 | 0.63 |
| Composition, % | ||||||||
| Fat | 3.76 | 3.73 | 3.77 | 3.76 | 0.081 | 0.78 | 0.53 | 0.93 |
| Protein | 3.16 | 3.15 | 3.16 | 3.15 | 0.015 | 0.53 | 0.78 | 0.45 |
| Lactose | 4.75 | 4.75 | 4.74 | 4.74 | 0.023 | 0.70 | 0.79 | 0.98 |
| MUN, mg/dL | 9.19 | 9.52 | 10.6 | 10.1 | 0.31 | <0.01 | <0.01 | 0.13 |
| Efficiency | ||||||||
| Milk yield ÷ DMI | 1.17 | 1.20 | 1.19 | 1.22 | 0.039 | 0.06 | 0.61 | 0.18 |
| FCM ÷ DMI | 1.21 | 1.23 | 1.24 | 1.26 | 0.042 | 0.14 | 0.40 | 0.33 |
1 Control (CON) = no feed additives; amylase (AML) = amylolytic enzyme (Amaize, Alltech) added at 0.5 g/kg diet DM; amylase and protease (APL) = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme (VegPro, Alltech) added at 0.2 g/kg diet DM; and APH = amylolytic enzyme added at 0.5 g/kg diet DM and proteolytic enzyme added at 0.4 g/kg diet DM.
2 Orthogonal contrasts: C1 = CON versus enzyme treatments; C2 = AML versus APL+APH; C3 = APL versus APH.
3 3.5% Fat-corrected milk (FCM) = (0.432 + 0.165 × milk fat %) × milk yield, kg/d.
Milk, FCM, and lactose yields were greater (P ≤ 0.03) in cows under ENZ treatments compared with CON. Fat and protein yields tended to be greater (P ≤ 0.10) in ENZ-fed cows than in the CON group. Other treatment comparisons did not reveal differences in yields of milk or solids. Milk concentration of solids (fat, protein, and lactose) were similar between treatments. Milk urea nitrogen concentration was greater (P < 0.01) in cows fed ENZ, whereas cows fed treatments containing protease had higher (P < 0.01) MUN concentrations than those in the AML group. Feed efficiency (milk yield ÷ DMI) tended to be greater (P = 0.06) in cows fed ENZ than in those fed CON.
DISCUSSION
It was postulated that feeding ENZ would result in better performance of cows by supporting greater ruminal NDF and starch digestibility, and that the combination of different enzyme activities would have a positive synergistic effect. Feeding ENZ, indeed, resulted in higher milk and FCM yields, although without altering total-tract apparent digestibility in comparison with CON. Note that AML cows presented the highest value of ruminal propionate molar percentage among treatment groups, which may support the greater milk yield in cows fed only amylase in comparison with CON. Although we did not observe positive synergistic effects on milk yield, combining amylase with protease influenced starch digestibility compared with feeding amylase alone. The reasons for greater milk yields in cows fed ENZ are likely related to the marginally greater molar percentages in ruminal propionate and greater starch digestibility, especially when combining amylase and protease at the highest level. We speculate that the combination of ENZ might have enhanced ruminal degradation of starch, favoring the production of propionate. Shifting the site of starch digestibility allows greater total digestibility of starch and enhanced microbial protein synthesis and, thus, ammonia utilization in the rumen (
Huntington, 1997
). Literature, however, is scarce of experiments that evaluated ruminal degradability of nutrients when feeding ENZ (Nozière et al., 2014
). Nozière et al., 2014
reported an increase in ruminal degradability of starch and molar percentage of propionate when feeding amylase (from Bacillus licheniformis) to primiparous cows, especially when a high-starch diet (30% diet DM) was fed. Nozière et al., 2014
, however, did not report amylase effects on total-tract digestibility of nutrients and milk yield. Contrasting with the results from this experiment, responses in molar percentages of propionate to amylase supplementation using the same product of the current study have been nonsignificant (DeFrain et al., 2005
; Takiya et al., 2017
; Zilio et al., 2019
) or negative (Tricarico et al., 2005
). The greater digestibility of NDF and starch when feeding the combination of enzymes might be partially linked to the greater feed sorting index for long feed particles. Long feed particles are rich in forage components, which may slow the feed passage rate, increase rumination, and favor ruminal degradation of nutrients such as starch and NDF due to the time that feed is retained in the rumen and greater pH (Greter and DeVries, 2011
; Miller-Cushon and DeVries, 2017
). Studies in literature have supplemented amylase in diets with most of the starch derived from ground corn (Tricarico and Dawson, 2005
; Ferraretto et al., 2011
; Nozière et al., 2014
) whereas only 1 study evaluated amylase addition in diets with rehydrated and ensiled corn (Andreazzi et al., 2018
), which is a more ruminally degradable source of starch than ground corn (Arcari et al., 2016
). Positive results in milk yield seem to not be related to intrinsic digestibility of the starch source, as studies with either coarsely ground corn or rehydrated ensiled corn reported greater milk yields when feeding amylase to dairy cows (Tricarico and Dawson, 2005
; Andreazzi et al., 2018
). To the best of our knowledge, no study has evaluated the interaction effect between starch source (e.g., corn, wheat, barley) and exogenous amylase in lactation diets.Because supplementing ENZ can potentially remove structural barriers of starch granules and increase accessibility of microbial α-amylases to corn starch (
Ferraretto et al., 2015
), we expected that proteolytic enzymes would increase the propionate molar percentage in the rumen. Exogenous proteases have been used as additives during the ensiling of high-moisture corn or whole-plant corn with positive effects in corn starch digestibility due to the breakdown of the protein matrix surrounding the starch granules (Ferraretto et al., 2015
; Der Bedrosian and Kung, 2019
). Differing from what we expected, combining proteolytic enzyme with amylolytic enzyme resulted in a lower propionate molar percentage in relation to feeding amylolytic enzyme alone. Most studies that fed additives with amylase activity have not reported differences in ruminal propionate molar percentage in lactating cows even when milk production was increased (Tricarico et al., 2005
; Gado et al., 2009
; Andreazzi et al., 2018
). Recently, a meta-analysis evaluating the effects of feeding amylase preparations reported positive but not statistically significant differences on milk yield and ruminal propionate percentage in dairy cows (Pech-Cervantes et al., 2022
). The reduction in molar percentage of propionate observed when feeding protease may indicate a modulatory effect of combined ENZ on the microbial population, which is supported by the reduction in urinary allantoin excretion (an index of ruminal microbial protein supply; Chen and Gomes, 1992
) and the increased MUN concentration. The literature, however, lacks studies evaluating the effects of combining amylase and protease on ruminal microorganisms in dairy cows. Feeding proteolytic enzymes (from B. licheniformis) alone did not alter propionate molar proportion in lactating cows (Eun and Beauchemin, 2005
). During in vitro continuous cultures, exogenous proteolytic enzymes (from Bacillus subtilis) did not affect propionate proportions (Vera et al., 2012
). In addition, studies have reported no or incremental effects of feeding proteases on urinary allantoin excretion (Eun and Beauchemin, 2005
).Total-tract apparent digestibility of starch was greater when feeding proteolytic enzymes, and the highest digestibility value was observed when the highest dose of proteolytic enzyme was supplied. In vitro ruminal degradability (96 h) of OM and NDF have been found not to be altered by protease supplementation (
da Freiria et al., 2018
). Thus, it is possible that proteases might have acted at the post-ruminal level or, at least, might have facilitated post-ruminal nutrient digestion. One would expect that proteases could increase the deamination rate and, thus, increase the NH3-N content in the rumen (Eun and Beauchemin, 2005
). Although not observed in the current experiment, other authors have reported tendencies for increased ruminal NH3-N content when feeding protease (Eun and Beauchemin, 2005
), amylase (Takiya et al., 2017
), or a mixture of amylase and protease to lactating cows (Gado et al., 2009
).As stated in our hypothesis, it was postulated that feeding ENZ would augment NDF digestibility or degradability, as previously reported in several studies (
Klingerman et al., 2009
; Gencoglu et al., 2010
; Weiss et al., 2011
), or as reported in studies using proteases in vitro (Colombatto et al., 2003a
; Eun and Beauchemin, 2005
) and in vivo (Eun and Beauchemin, 2005
). Although we did not detect differences when comparing CON and ENZ treatments, the APH group had the greatest NDF digestibility (2 units' increase in relation to CON), demonstrating a potential synergism in degrading fiber. Oba and Allen, 1999
found that a 1-unit increase in NDF digestibility was associated with a 0.25-kg increase in FCM, which may partially explain the differences in milk yield observed in this study. The reasons for improvements in fiber digestibility when feeding enzymes that do not target their primary substrate are not clear but has been related to cross-feeding mechanisms (Tricarico et al., 2008
) and the breakdown of cross-linkages between structural proteins in forage (Colombatto et al., 2003b
; Colombatto and Beauchemin, 2009
). Tricarico et al., 2008
suggested that exogenous enzymes hydrolyze complex carbohydrates into different oligosaccharides (malto-, cello-, and xylo-oligosaccharides), which would support the growth of fibrolytic microorganisms. Colombatto et al., 2003b
hypothesized that alkaline serine proteases in ruminant diets would remove structural barriers, allowing ruminal microorganisms access to digestible nutrients. These barriers would consist of lignified middle lamella or primary walls, which could prevent or delay microbial access (Jung et al., 2000
).In the current study, a lower sorting index for feeds with small particle size (<4 mm) in cows fed ENZ, especially in those fed the combination of amylase and protease, was observed. It is important to highlight that TMR was prepared individually for each cow; thus the mixing procedure might have interfered in feed sorting. In addition, the feed sorting index was calculated based on sieving wet material (i.e., without adjusting for DM), which has been highlighted as a methodology limitation by
NASEM (National Academies of Science, Engineering, and Medicine), 2021
. Several studies have observed differences in feed sorting when feeding exogenous amylase to lactating cows (Andreazzi et al., 2018
; Zilio et al., 2019
). Interestingly, in this study, starch total-tract digestibility was the highest in groups that sorted for feed with longer particle size. Total mixed ration particle size distribution revealed a relatively large amount of feed particles in the top screen (>19 mm, 16.7%), which might have caused a bias in the feed separation score. Lactating cows present the typical pattern of sorting against longer forage particles, resulting in greater intake of highly fermentable carbohydrates and lesser intake of physically effective fiber (Miller-Cushon and DeVries, 2017
). Dairy cows would increase their dietary preference for a feed with longer particle size when they present low ruminal pH (Keunen et al., 2002
). However, we did not detect differences in ruminal pH, and cows did not experience pH <5.8 throughout our sampling periods. Sorting for feeds with longer particle size did not result in major differences in the nutrient composition of the consumed diet. For instance, the mean composition of feed consumed by ENZ cows was 38.0% NDF, 29.7% starch, and 16.3% CP, which is comparable to the chemical composition of the fed TMR. Thus, feed sorting in this study likely had no meaningful influence on performance of cows.Despite the differences observed in urine allantoin and urinary uric acid excretion, total purine derivatives were similar among treatments, suggesting no differences in ruminal microbial protein supply. No study has evaluated whether feeding protease to dairy cows can alter RUP/RDP proportions or MP supply. In general, feeding amylase to lactating cows did not alter estimated rumen microbial protein supply or duodenal microbial N flow (
Nozière et al., 2014
; Silva et al., 2018
; Zilio et al., 2019
). Serum urea N concentration tended to be greater in cows fed ENZ, which was not expected, considering that ruminally cannulated cows did not exhibit differences in ruminal ammonia concentration. Aligning with higher serum urea N concentration results, MUN concentration was greater in cows fed ENZ than in those fed CON. Because ammonia concentration in the rumen depends on ruminal pool or volume as well as the production and absorption rates, it is difficult to ascertain the potential effects of ENZ, especially of proteases, in enhancing deamination, and thus promoting urea concentration in the rumen. Along these lines, authors have reported an increase of xylanase and protease activities in the ruminal fluid of cows fed proteolytic enzymes and low-forage TMR (Eun and Beauchemin, 2005
). However, neither blood urea nitrogen nor ruminal concentration of urea in dairy cows have been altered by feeding amylase (Nozière et al., 2014
; Takiya et al., 2017
; Zilio et al., 2019
; Silvestre et al., 2022
) or protease (Eun and Beauchemin, 2005
).CONCLUSIONS
In summary, this study demonstrated that ENZ likely contributed to enhanced milk yield due to improvements in fiber and starch digestibility and apparently minor effects on ruminal fermentation. The expected positive synergism of combining amylase and protease was more evident in total-tract digestibility of nutrients and depended on the protease dose, but combining enzymes did not result in enhanced milk production performance. Further studies should explore the effects of different concentrations of enzymes (amylase and protease) on ruminal degradability of fiber and starch.
ACKNOWLEDGMENTS
The authors appreciate the support of staff of the Dairy Cattle Research Laboratory (Pirassununga, Brazil) for animal care and management. The authors thank Alltech Inc. for funding. The authors have not stated any other conflicts of interest.
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Publication history
Published online: March 10, 2023
Accepted:
December 8,
2022
Received:
August 1,
2022
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