Research Article

 

Dose-Response Effect of Exogenous Enzymes Treatment of Tomato and Watermelon Crop Byproducts on In vitro Nutrient Degradability and Rumen Fermentation Kinetics

 

Mohamed M. Abd-Elkerem, Sabry M. Bassiony, Sabry A. Shehata, Adham A. Al-Sagheer*

Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt.

 

Received | October 14, 2020; Accepted | October 22, 2020; Published | January 01, 2021

*Correspondence | Adham Al-Sagheer, Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt; Email: [email protected]

Citation | Abd-Elkerem MM, Bassiony SM, Shehata SA, Al-Sagheer AA (2021). Dose-response effect of exogenous enzymes treatment of tomato and watermelon crop byproducts on in vitro nutrient degradability and rumen fermentation kinetics Adv. Anim. Vet. Sci. 9(2): 194-202.

DOI | http://dx.doi.org/10.17582/journal.aavs/2021/9.2.194.202

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright © 2021 Al-Sagheer et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

 

INTRODUCTION

 

Byproducts from agriculture may be of importance not only for reducing nutrition costs but also to reduce environmental problems associated with byproduct accumulation (Al-Sagheer et al., 2019). Most of these biomaterials are not used and end up in municipal sites, where microbial breakdown and leachate production produce severe environmental challenges. Thus, the use of agricultural byproducts in ruminant nutrition has been virtually adopted as an approach for minimizing the expense of feeding and reducing the need to recycle wastes, which its disposal is costly (Omer and Abdel-Magid, 2015). Fibrous feeds contain high cellulose and hemicellulose content, making a complex with lignin and carbohydrates to decrease the carbohydrates’ digestibility and decrease the utilization of ruminants from forages. Improvement of fibrous feeds digestibility is generally very desirable and leads to better animal productivity. Degradation of plant cell walls of high fiber feed in the rumen is possible principally because of the produced enzymes of rumen microflora (Krause et al., 2003). Numerous studies have focused on enhancing the efficiency of fibrous feeds degradation in the rumen using physical, chemical, and biological treatment such as yeast products, exogenous fiber degrading enzymes, and inoculants (Adesogan et al., 2019).

 

As an effective treatment strategy, exogenous enzymes have attracted worldwide growing interest and proved to be a useful tool to improve ruminant production (Beauchemin et al., 2019). The enhancement in the nutritive value of fibrous feeds by exogenous fiber enzymes treatment may be due to increasing attachment by rumen bacteria (Wang et al., 2001). Moreover, the enzymes act synergistically with the rumen microbes, which increases their ability to hydrolytic the fiber in the rumen (Beauchemin et al., 2004). ZAD® is a commercial product prepared from anaerobic bacteria, which by particular enzymes convert polysaccharides into monosaccharides (Salem et al., 2013). The ZADO® enzyme mixture contains α-amylase, xylanase, endoglucanase, and protease. This enzyme mixture has been used in previous studies to improve microbial crude protein synthesis, ruminal fermentation parameters, nutrient digestibility, and milk production (Gado et al., 2009, López et al., 2013, Salem et al., 2013).

 

Therefore, this in vitro study aimed to evaluate the ZADO® enzyme mixture’s dose-response effect on the gas production, nutrient degradability, and rumen fermentation characteristics of two crop residues of tomato and watermelon. In particular, no study, to our knowledge, has considered the nutritional assessment of two highly available crops byproducts treated with exogenous enzyme mixture.

 

MATERIALS AND METHODS

 

The current study was accompanied by the Animal Nutrition Laboratory, Animal Production Department, Faculty of Agriculture, Zagazig University, Zagazig, Egypt.

 

Plant byproducts and chemical analysis

Samples of tomato (Solanum Lycopersicum) and watermelon (Citrullus lanatus) crop byproducts (TCB and WCB, respectively) were manually collected from a private farm in Zagazig, Egypt. Plant byproducts were weighed, wholly cut, dried at 65 °C to about 90 % dry matter (DM), and finely powdered (1 mm). These dried samples were used for chemical analysis and in vitro experiment. The sample was analyzed for organic matter (OM), DM, crude fiber (CF), ash, ether extract, and crude protein (CP) according to the AOAC (2006). Estimate the concentration of elements in the sample by atomic absorption device (iCE 3000 Series AAS), according to Elmer and Conn (1982). Estimate the concentration of phosphorus element by element appreciation chromatography device, according to Marker (1992). The chemical and mineral composition of TCB and WCB compared with berseem hay are listed in Table 1.

 

Enzyme mixture and additive dose

The used exogenous enzyme (ZADO®) was kindly provided by Dr. Hany Gado, Professor of Animal Nutrition, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. ZADO® is a multi-enzyme powder (Patent No.: 22155, Cairo, Egypt), commercially available, manufactured by the Academy of Scientific Research and Technology in Egypt from Ruminococcus flavefaciens bacteria. The enzyme mixture has been examined for enzymatic activities and was found to contain (per g of enzyme) 61.5 units of α-amylase, 7.1 units of endoglucanase, 29.2 units of protease, and 2.3 units of xylanase activity. Four doses of exogenous enzymes (0, 6, 12, and 24 mg per g DM) were added with the substrate (TCB and WCB) inside the incubation tubes. Berseem hay substrate was used as a positive control.

 

In vitro incubations

The ruminal liquid was collected from a slaughtered cow from a slaughterhouse located at Zagazig, Sharkia Governorate, Egypt. Rumen liquids were immediately transported to the laboratory in pre-warmed (39 °C) isolate flasks and kept under anaerobic environments. The rumen fluid was filtered through four layers of cheesecloth and incubated at 39 °C in a water bath, and it was saturated with CO2 until inoculation.

 

The buffer incubation medium (MB9) contained CaCl2 (0.1g/l), NaCl (2.8g/l), MgSO4.7H2O (0.1g/l), KH2PO4.H2O (2g/l), and Na2HPO4 (6g/l). The rumen fluid was mixed with the MB9 media at the MB9 media ratio to rumen fluid of 2:1 (v/v). Thirty millimeters of mixed ruminal fluid was introduced into calibrated glass tubes, containing 200 mg of the byproducts + dose of ZADO® (ENZ), quickly closed by a gas release rubber stopper fitted with a tri-way valve coupled with a calibrated plastic syringe to measure the gas production. The volume of gas production was measured at 3, 6, 12, 24, 36, 48, and 72 h of incubation. The total gas volume was corrected from a blank tube.  Three runs of gas production were conducted for all substrates. In each run as four blank bottles (without substrate) and six bottles for each treatment. Gas production kinetics were calculated following the model of Ørskov and McDonald (1979).

 

The contents of three tubes of each treatment were used for determining truly degraded dry matter (DMD) after 72 h of incubation by adding 30 mL of neutral detergent solution to each bottle and placed at 105 °C for three h. Then, each sample was filtered through pre-weighed Gooch crucibles, dried at 105°C for three h, and the residual DM weight was estimated (Blümmel et al., 1997). After that, it was used to estimate crude protein and crude fiber degradability (CPD and CFD), according to AOAC (2006). The concentration of NH3-N, total volatile fatty acids (TVFA), and protozoa count were determined in the contents of another three tubes of each treatment. The protozoa count

 

Table 1: Analyzed nutrient and minerals contents of tested substrates.

 

	Berseem hay	Watermelon (whole plant byproduct)	Tomato (whole plant byproduct)
Chemical composition (% on DM basis)
Crude protein	15.70	10.08	8.55
Crude fiber	25.18	36.39	26.90
Ether extract	4.15	2.40	2.04
Organic matter	88.30	79.09	82.18
Ash	11.70	20.91	18.82
Nitrogen free extract	43.27	30.22	43.69
Mineral composition (on DM basis)
Calcium, %	1.45	1.02	0.073
Potassium, %	2.32	0.112	0.111
Phosphorus, %	0.262	0.008	0.006
Magnesium, %	0.290	0.032	0.024
Copper, ppm	15.18	0.822	0.646
Zinc, ppm	20.25	2.939	1.3416
Iron, ppm	178.2	42.69	17.99
Chromium, ppm	0.520	0.6154	0.4103

 

Table 2: Effect of various exogenous enzyme (ENZ) doses addition to watermelon crop byproducts (WCB) on cumulative gas production kinetics, predicted values, and fermentation parameter compared with berseem hay as a positive control

 

	Berseem hay	ENZ levels added to WCB substrate				SEM	P-value
		0 g/kg-1	6 g/kg-1	12 g/kg-1	24 g/kg-1		T	L	Q
Gas production, ml/g DM
3 h	37.00ab	30.17b	32.07b	39.06ab	48.13a	1.58	0.0001	0.0001	0.200
6 h	59.25ab	42.75c	47.21bc	53.56abc	65.56a	2.02	0.0001	0.0001	0.236
12 h	86.38a	59.50b	69.43ab	77.83ab	86.50a	2.64	0.0001	0.0001	0.870
24 h	110.38ab	95.54b	102.79ab	107.44ab	119.13a	2.42	0.009	0.0001	0.536
36 h	127.88ab	119.88b	128.00ab	131.78ab	143.38a	2.45	0.015	0.0001	0.631
48 h	141.38ab	132.88b	137.00b	140.94ab	157.69a	2.87	0.039	0.001	0.001
72 h	151.63ab	143.71b	147.00b	148.89b	168.44a	2.85	0.032	0.002	0.108
a	18.63ab	14.06b	14.24b	21.20ab	32.89a	1.88	0.003	0.0001	0.079
b	139.11	143.66	141.19	135.72	146.76	3.00	0.813	0.992	0.227
c	0.05	0.04	0.04	0.05	0.04	0.002	0.124	0.160	0.015
a+b	157.74	157.72	155.44	156.92	179.65	3.20	0.079	0.025	0.042
Predicted values
SCFA, mmol /0.2g DM	0.62a	0.42c	0.45bc	0.47bc	0.51b	0.011	<0.001	0.001	0.978
ME (MJ/kg DM)	5.54a	4.14c	4.39bc	4.52bc	4.75b	0.080	<0.001	0.001	0.978
NEL (MJ/kg DM)	3.18a	2.56c	2.69bc	2.84bc	2.99b	0.059	<0.001	0.001	0.978
OMD, %	47.83a	36.46c	37.85bc	38.58bc	39.89b	0.554	<0.001	0.001	0.978
MCP (mg/g DM)	751.38a	605.34b	638.61b	665.80b	660.47b	11.87	<0.001	0.024	0.936
PF (mg TDOM/ml gas)	1.71b	1.92a	1.84ab	1.80ab	1.76ab	0.019	<0.001	0.001	0.658
Fermentation parameters
pH (24 h)	6.21	6.20	5.89	5.81	5.80	0.07	0.092	0.041	0.244
pH (48 h)	6.21	6.21	6.01	5.95	5.88	0.05	0.085	0.009	0.371
pH (72 h)	5.40	5.50	5.60	5.30	5.30	1.65	0.098	0.521	0.106
Ammonia-N, mg/dL	41.07	34.16	36.55	36.40	33.25	1.15	0.327	0.862	0.305
TVFA, ml eq./L	167.67	154.60	203.75	218.50	193.00	12.09	0.442	0.270	0.208
Protozoa count, 103/mL	164.83b	198.00ab	235.00a	233.75a	198.75ab	8.19	0.038	0.855	0.037

Means in the same row bearing different letters differ significantly (P < 0.05); SEM indicates standard error of the mean; Probability of main effects of treatment (T), linear (L), and quadratic (Q); a = the gas production from the immediately soluble fraction (ml); b = the gas production from the insoluble fraction (ml); c = the gas production rate constant for the insoluble fraction b (h); a+b = potential gas production (ml); ME, metabolizable energy; NEL, net energy lactation; OMD, organic matter degradability; PF, partitioning factor at 72 h of incubation; SCFA, short-chain fatty acids; MCP, microbial crude protein production; TVFA is the total volatile fatty acids.

 

was estimated microscopically after sample preparation in line with the method of Kamra et al. (1991). The TVFA concentration was estimated by the steam distillation method, according to Warner (1964). Ruminal NH3-N concentration was detected according to the method described by Conway (1957). At 24 h of incubation, the partitioning factor (PF) was calculated as the ratio of OM (mg) degradability to the volume of gas production (in mL after 24 h) (Blümmel et al., 1997). Net energy lactation (NEL, MJ/kg DM) and metabolizable energy (ME, MJ/kg DM) were calculated according to Menke and Steingass (1988). The calculation of the in vitro organic matter digestibility (OMD %) was accomplished according to Menke et al. (1979) equation. Short chain fatty acid concentrations (SCFA) were calculated according to Getachew et al. (2002). Microbial CP biomass production was estimated, according to Blümmel et al. (1997).

 

Statistical analysis

The statistical analysis of the in vitro results was conducted using the general linear model procedure (GLM) using SPSS 21 (Chicago, IL) software. The orthogonal polynomial contrast was applied to identify the linear, quadratic, and cubic effects of increasing exogenous enzyme levels. Tukey’s test was used to test the significant differences (at P < 0.05) between the means.

 

RESULTS

 

Watermelon crop byproducts

Data presented in Table 2 exhibited no significant differences in gas production kinetics, TVFA, NH3-N, pH, and protozoal count between untreated WCB and berseem hay. In contrast, a significant reduction in predicted NE, SCFA, ME, MCP, and OMD, but a significant increase in PF, were observed in the untreated WCB group compared to the berseem hay (Table 2). The addition of ENZ led to a linear increase (P<0.001) in cumulative gas production, gas production from a soluble fraction (a), and the predicted value of ME, NE, SCFA, and OMD with increasing dietary ENZ levels. The highest values were observed in diet fortified with ENZ at 24 g/ kg compared with un-supplemented WCB. All concentrations of the tested ENZ did not alter the ruminal NH3-N, TVFA, pH, and protozoal count (Table 2).

 

At all incubation periods, DMD, CFD, and CPD for untreated WCB were significantly lower (P<0.001) than that for berseem hay (Table 3). However, the application of ENZ at 24 g/kg improved (P<0.01) the degradation of CF and CP after 72 h of incubation compared with untreated WCB (Table 2). Also, the highest (P=0.001) value of DMD was detected in WCB treated with 12 and 24 g/kg of ENZ after 24h but not at 48 and 72h of incubation (Table 3).

 

Tomato crop byproducts

As shown in Table 4, cumulative gas production (12-72h of incubation) was significantly (P<0.001) reduced in untreated TCB compared with berseem hay substrate. Also, a significant decrease in the predicted value of ME, NE, SCFA, MCP, and OMD, but a significant increase in PF, were detected in the un-supplemented WCB substrate compared to those in berseem hay. Incubation of TCB substrate with ENZ resulted in a linear increase in cumulative gas production (3-72h of incubation) with a significant effect at the level of 24 g/kg (Table 4). Predicted values of ME, NE, SCFA, MCP, and OMD showed a linear increase at the addition of ENZ to the incubation media containing TCB relative to the untreated one. Notably, the highest significant effect on the earlier parameters was evident at the level of 24 g/kg. Nevertheless, a linear decrease was recorded in the PF when the incubation media containing TCB was fortified with ENZ with a significant effect at 12 and 24 g/kg. The values of pH, NH3-N, TVFA, and protozoa were not significantly altered by the ENZ treatment (Table 4).

 

A significant reduction in DMD (after 48 and 72h), CFD

 

Table 3: Effect of various exogenous enzyme (ENZ) doses addition to watermelon crop byproducts (WCB) on nutrient degradability after 24, 48, and 72 hours of incubation compared with berseem hay as a positive control.

 

	Berseem hay	ENZ levels added to WCB substrate				SEM	P-value
		0 g/kg-1	6 g/kg-1	12 g/kg-1	24 g/kg-1		T	L	Q
Dry matter degradability (%) after
24 h	39.00a	32.97c	34.48bc	37.37ab	37.42ab	0.64	0.001	0.001	0.313
48 h	55.18a	40.15b	40.07b	42.22b	42.92b	1.64	<0.001	0.911	0.217
72 h	59.96a	44.89b	47.83b	50.68b	50.98b	1.21	<0.001	0.015	0.372
Crude fiber degradability (%) after
24 h	21.14a	6.21c	6.75bc	7.53bc	9.83b	1.51	<0.001	0.011	0.082
48 h	32.66a	9.24c	13.48bc	18.77bc	20.20b	2.25	<0.001	0.004	0.525
72 h	36.98a	10.30d	20.2c	27.70bc	30.85ab	2.20	<0.001	0.000	0.143
Crude protein degradability (%) after
24 h	44.00ab	38.88b	43.35ab	43.68ab	46.48a	0.88	0.041	0.007	0.579
48 h	58.41ab	45.93c	53.01bc	58.04ab	62.77a	1.70	0.001	0.000	0.468
72 h	68.09a	57.50c	59.03bc	59.80bc	64.49ab	1.04	0.001	0.093	0.103

Means in the same row bearing different letters differ significantly (P < 0.05); SEM indicates standard error of the mean; Probability of main effects of treatment (T), linear (L), and quadratic (Q).

 

Table 4: Effect of various exogenous enzyme (ENZ) doses addition to tomato crop byproduct (TCB) on cumulative gas production kinetics, predicted values, and fermentation parameter compared with berseem hay as a positive control

 

	Berseem hay	ENZ levels added to TCB substrate				SEM	P-value
		0 g/kg-1	6 g/kg-1	12 g/kg-1	24 g/kg-1		T	L	Q
Gas production, ml/g DM
3 h	46.38b	36.88b	51.75ab	51.06b	66.83a	2.16	<0.001	<0.001	0.978
6 h	68.25ab	51.44b	65.80b	63.72b	85.25a	2.51	<0.001	<0.001	0.472
12 h	102.63ab	71.06c	85.70bc	84.11bc	109.50a	2.85	<0.001	<0.001	0.254
24 h	140.50a	107.31b	119.85ab	119.33ab	137.75a	2.89	0.001	0.001	0.646
36 h	163.63a	128.38b	137.95b	141.44ab	162.92a	3.18	0.001	0.001	0.328
48 h	178.00a	138.44c	147.25bc	155.78abc	170.50ab	3.57	0.004	0.003	0.674
72 h	184.50a	151.13b	160.50ab	167.50ab	183.92a	3.49	0.010	0.003	0.627
a	21.14b	19.95b	35.67ab	36.19ab	50.98a	2.70	0.001	<0.000	0.792
b	157.77	140.70	133.05	146.27	143.25	3.49	0.358	0.492	0.647
c	0.05	0.04	0.04	0.04	0.04	0.002	0.542	0.987	0.632
a+b	178.91	160.65	168.72	182.45	194.23	4.26	0.113	0.006	0.806
Predicted values
SCFA, mmol /0.2 g DM	0.62a	0.46b	0.54ab	0.55a	0.61a	0.01	<0.001	<0.001	0.434
ME (MJ/kg DM)	5.52a	4.36c	4.91bc	5.00ab	5.39ab	0.09	<0.001	<0.001	0.434
NEL (MJ/kg DM)	2.90a	2.05c	2.45bc	2.52ab	2.81ab	0.07	<0.001	<0.001	0.434
OMD, %	47.74a	39.42c	42.53bc	43.07b	45.27ab	0.55	<0.001	<0.001	0.434
MCP (mg/g DM)	759.66a	632.24b	721.75a	710.14a	716.99a	10.10	<0.001	0.010	0.073
PF (mg TDOM/ml gas)	1.71b	1.89a	1.75ab	1.72 b	1.65b	0.02	<0.001	<0.001	0.243
Fermentation parameters
pH (24 h)	6.43a	5.80b	5.80b	5.85b	5.77b	0.06	<0.001	0.877	0.369
pH (48 h)	6.42a	6.10ab	5.96b	5.96b	6.04b	0.05	0.008	0.480	0.074
pH (72 h)	5.48	5.59	5.56	5.65	5.54	0.033	0.722	0.870	0.625
Ammonia-N, mg/dL	40.73b	46.58ab	46.90ab	43.30ab	52.80a	1.39	0.041	0.165	0.102
TVFA, ml eq./L	165.00	223.25	218.25	208.25	246.60	10.39	0.196	0.496	0.347
Protozoa count, 103/mL	170.67b	177.38ab	241.88a	164.13b	170.90ab	9.18	0.021	0.189	0.115

Means in the same row bearing different letters differ significantly (P < 0.05); SEM indicates standard error of the mean; Probability of main effects of treatment (T), linear (L), and quadratic (Q); a = the gas production from the immediately soluble fraction (ml); b = the gas production from the insoluble fraction (ml); c = the gas production rate constant for the insoluble fraction b (h); a+b = potential gas production (ml); ME, metabolizable energy; NEL, net energy lactation; OMD, organic matter degradability; PF, partitioning factor at 72 h of incubation; SCFA, short-chain fatty acids; MCP, microbial crude protein production; TVFA is the total volatile fatty acids.

 

Table 5: Effect of various exogenous enzyme (ENZ) doses addition to tomato crop byproduct (TCB) on nutrient degradability after 24, 48, and 72 hours of incubation compared with berseem hay as a positive control.

 

	Berseem hay	ENZ levels added to TCB substrate				SEM	P-value
		0 g/kg-1	6 g/kg-1	12 g/kg-1	24 g/kg-1		T	L	Q
Dry matter degradability (%) after,
24 h	40.37	36.73	37.83	38.44	38.28	0.86	0.833	0.565	0.756
48 h	54.30a	46.61c	49.89bc	50.95ab	54.19a	0.77	<0.001	<0.001	0.985
72 h	60.30a	47.69b	56.32a	56.36a	57.67a	1.02	<0.001	0.019	0.117
Crude fiber degradability (%) after
24 h	25.80a	14.37b	20.71a	22.20a	24.97a	1.08	<0.001	<0.001	0.007
48 h	34.32ab	25.54b	31.49ab	33.81ab	35.88a	1.21	0.031	<0.001	0.244
72 h	36.86ab	30.36b	34.23b	36.07b	47.67a	1.66	0.003	0.001	0.351
Crude protein degradability (%) after
24 h	46.75	46.34	49.19	50.54	53.37	0.90	0.057	0.012	0.099
48 h	60.41ab	49.77b	56.32ab	59.12ab	63.64a	1.65	0.004	<0.001	0.844
72 h	67.26a	55.35b	64.15a	64.15a	65.84a	1.04	<0.001	0.008	0.055

Means in the same row bearing different letters differ significantly (P < 0.05); SEM indicates standard error of the mean; Probability of main effects of treatment (T), linear (L), and quadratic (Q).

 

(after 24h), and CPD (after 72h) were evident in untreated TCB compared to berseem hay (Table 5). Treatment of TCB with ENZ displayed a linear increment in the DMD, CFD, and CPD. In particular, after 72h of incubation, a significant effect was apparent on the degradability of both DM and CP at 6, 12, and 24 g/kg of ENZ, while CFD was significantly affected at 24 g/kg (Table 5).

 

DISCUSSION

 

For enhancement of livestock productivity with minimal environmental impact, several nutritional strategies have been developed in recent years, like the use of plant byproduct and natural feed additives (Alsaht et al., 2014, Al-Sagheer et al., 2017, Ayyat et al., 2018). Herein, two types of crop residues treated with exogenous enzyme were assessed for the efficiency in enhancing feed degradability in ruminants. The results of decreased gas production of untreated TCB (12-72h of incubation) and WCB (6-12 h of incubation) is in line with the findings of Sallam et al. (2007). These authors found a significant decrease in gas production from linseed straw and rice straw compared to berseem hay. This finding might be related to the low feeding value of these crop residues. Haddi et al. (2003) noted a significant negative correlation between the rate of gas production and fiber contents (NDF and ADF) of the plant. The negative influence of fiber content on gas production might be related to the decreased ruminal microbes’ activity through the lack of suitable environmental conditions for fermentation as incubation time progresses (Bakhashwain et al., 2010).

 

Our observations of increased gas production with increasing ENZ levels at all the incubation times in both crop residues are in harmony with numerous studies (Nsereko et al., 2002, López et al., 2013). This response might be due to the Ruminococcus flavefaciens content of the ENZ that stimulate ruminal microbial fermentation. Also, increased gas production up to 72 h of incubation period due to ENZ supplementation may reflect the increase in microbial numbers, and hence, the degradation efficiency of the ruminal microbes (López et al., 2013).

 

Herein, the untreated WCB and TCB showed significantly lower SCFA, MCP, OMD, DMD, CFD, CPD, and energy content, but higher PF compared with berseem hay. This observed decrease was probably because of low CP and high NDF, and ADL contents of tested crop byproduct (Bakhashwain et al., 2010). Parissi et al. (2005) reported a positive link between ME or nutrient degradability and CP content. Also, there is a negative relationship between lignin, phenolics, and NDF with digestibility (Ammar et al., 2005). Similarly, the cell-wall content and assessed indicators like NE, OMD, SCFA, MP, and ME were considered negatively correlated, as reported by Parissi et al. (2005). The high protein lignification (nearly half protein content is associated with ADF) might clarify the low CP degradability (Ventura et al., 2009). Besides, Fondevila et al. (1994) showed that tomato byproducts have poorly degradable CP. Owing to the poor CP degradability and the great NDF lignification, the most degradable OM was non-structural carbohydrates (Ventura et al., 2009).

 

Our findings showed that ENZ addition to TCB and WCB resulted in a significant linear increase in SCFA, ME, NE, MCP, and OMD. Similarly, Gado et al. (2009) and Salem et al. (2013) have also shown that ENZ addition amplified SCFA amounts. Also, these results are comparable to those of Omer et al. (2009), who reported that ENZ supplementation to calves diets enhanced nutrient digestibility and rumen SCFA concentrations. Beauchemin et al. (2003) stated that ENZ supplementation augmented the digestible energy intake of ruminants fed diets rich in fiber content, and energy was the controlling nutrient in the diet. The linear increase of OMD, ME, MCP, and SCFA in TCB and WCB with the ENZ addition may have been because of amplified CFD and modify fermentation in the rumen (Nsereko et al., 2002). Additionally, the enhanced predicted parameters might also be due to increasing rumen microorganisms colonization to the cell wall of the plant (Wang et al., 2001, Nsereko et al., 2002). Also, the synergism between the ENZ and ruminal enzymes could be a potential ENZ mode of action (Morgavi et al., 2001).

 

The enhanced DMD, CPD, and CFD due to ENZ addition to the incubation media containing TCB and WCB are in line with earlier studies that evaluated the same mixture of enzymes (Gado et al., 2009, Salem et al., 2013). This effect can be attributed to ENZ’ ability to degrade the lignocellulose substrate complex into simple compounds that could alter its surface structure or weaken the chemical bond between lignocelluloses, making it easier to access ruminal microbial degradation or to promote ruminal microbial colonization and fermentation efficacy (López et al., 2013). Also, several modes of action are suggested, like the ability of ENZ to increase colonization of ruminal microbe on the feed particles surface (Yang et al., 2000). Also, ENZ capacity to augment colonization and enhance the entrance to the matrix surface via ruminal microbes to hasten digestion rate has also been suggested as a possible mode of action (Jalilvand et al., 2008). Moreover, ENZ has been reported to enhance the ruminal microbes hydrolytic potency because of enzyme activities or augmented synergism with enzymes of ruminal microbes (Morgavi et al., 2000). The obtained results suggest that the tested ENZ of Ruminococcus flavefaciens could have the potency to enhance ruminants’ feed utilization efficiency, as evidenced by better gas production, in vitro DM, CP, and CF degradability.

 

CONCLUSION

 

This study is an attempt to improve the nutritional quality of two crop wastes (TCB and WCB) as feed for ruminants using a multi-enzyme feed additive resulted from R. flavefaciens. The low nutritional content of both crop residues resulted in dwindling in the in vitro gas production, nutrient degradability, and the energy content (ME and NE). However, WCB and TCB treatment with ENZ, especially at 24 mg/g DM, enhanced gas production and energy content and DM, CP, and CF degradation compared with untreated ones. Nevertheless, further studies are needed to be carried out to apply these results in vivo.

 

CONFLICT OF INTEREST

 

The authors declare no conflict of interest.

 

AUTHORS CONTRIBUTION

 

SMB, SAS, AAA conceived and designed the experiments. MMA, AAA performed the experiments, analyzed the data, and drafted the manuscript. SMB, SAS, AAA reviewed the manuscript and performed the final check. All authors read and approved the final manuscript.

 

REFERENCES

 

• Adesogan AT, Arriola KG, Jiang Y, Oyebade A, Paula EM, Pech-Cervantes AA, Romero JJ, Ferraretto LF, Vyas D (2019). Symposium review: Technologies for improving fiber utilization. J. Dairy Sci. 102: 5726-5755. https://doi.org/10.3168/jds.2018-15334

• Al-Sagheer AA, Daader AH, Gabr HA, Abd El-Moniem EA (2017). Palliative effects of extra virgin olive oil, gallic acid, and lemongrass oil dietary supplementation on growth performance, digestibility, carcass traits, and antioxidant status of heat-stressed growing New Zealand White rabbits. Environ. Sci. Pollut. Res. 24: 6807-6818. https://doi.org/10.1007/s11356-017-8396-8

• Al-Sagheer AA, El-Hack A, Mohamed E, Alagawany M, Naiel MA, Mahgoub SA, Badr MM, Hussein EO, Alowaimer AN, Swelum AA (2019). Paulownia leaves as a new feed resource: Chemical composition and effects on growth, carcasses, digestibility, blood biochemistry, and intestinal bacterial populations of growing rabbits. Animals. 9: 95. https://doi.org/10.3390/ani9030095

• Alsaht AA, Bassiony SM, Abdel-Rahman GA, Shehata SA (2014). Effect of cinnamaldehyde thymol mixture on growth performance and some ruminal and blood constituents in growing lambs fed high concentrate diet. Life Sci. J. 11: 240-248.

• Ammar H, Lopez S, Gonzalez J (2005). Assessment of the digestibility of some Mediterranean shrubs by in vitro techniques. Anim. Feed Sci. Technol. 119: 323-331. https://doi.org/10.1016/j.anifeedsci.2004.12.013

• AOAC (2006). Official methods of analysis, 18th edn. USA, Washington, DC.

• Ayyat MS, Al-Sagheer AA, Abd El-Latif KM, Khalil BA (2018). Organic Selenium, Probiotics, and Prebiotics Effects on Growth, Blood Biochemistry, and Carcass Traits of Growing Rabbits During Summer and Winter Seasons. Biol. Trace Elem. Res. 186: 162-173. https://doi.org/10.1007/s12011-018-1293-2

• Bakhashwain A, Sallam S, Allam A (2010). Nutritive value assessment of some Saudi Arabian foliages by gas production technique in vitro. JKAU: Met., Env. Arid Land Agric. Sci 142: 1-31. https://doi.org/10.4197/met.21-1.5

• Beauchemin K, Colombatto D, Morgavi D, Yang W (2003). Use of exogenous fibrolytic enzymes to improve feed utilization by ruminants. J. Anim Sci. 81: E37-E47.

• Beauchemin K, Colombatto D, Morgavi D, Yang W, Rode L (2004). Mode of action of exogenous cell wall degrading enzymes for ruminants. Can. J. Anim. Sci. 84, 13-22. https://doi.org/10.4141/A02-102

• Beauchemin KA, Ribeiro GO, Ran T, Marami Milani MR, Yang W, Khanaki H, Gruninger R, Tsang A, McAllister TA (2019). Recombinant fibrolytic feed enzymes and ammonia fibre expansion (AFEX) pretreatment of crop residues to improve fibre degradability in cattle. Anim. Feed Sci. Technol. 256, 114260. https://doi.org/10.1016/j.anifeedsci.2019.114260

• Blümmel M, Steingaβ H, Becker K (1997). The relationship between in vitro gas production, in vitro microbial biomass yield and 15 N incorporation and its implications for the prediction of voluntary feed intake of roughages. Br. J. Nutr. 77, 911-921. https://doi.org/10.1079/BJN19970089

• Conway E (1957). Micro-diffusion Analysis and Volumetric Error, 4th edn. London Crossby, Lockwood and Sons Ltd. In University Press, Glasgow.

• Elmer P, Conn N (1982). Analytical methods for atomic absorption spectrophotometry. Perkin Elmer, Norwalk, CT.

• Fondevila M, Guada J, Gasa J, Castrillo C (1994). Tomato pomace as a protein supplement for growing lambs. Small Rumin. Res. 13, 117-126. https://doi.org/10.1016/0921-4488(94)90086-8

• Gado H, Salem A, Robinson P, Hassan M (2009). Influence of exogenous enzymes on nutrient digestibility, extent of ruminal fermentation as well as milk production and composition in dairy cows. Anim. Feed Sci. Technol. 154, 36-46. https://doi.org/10.1016/j.anifeedsci.2009.07.006

• Getachew G, Makkar H, Becker K (2002). Tropical browses: contents of phenolic compounds, in vitro gas production and stoichiometric relationship between short chain fatty acid and in vitro gas production. J. Agric. Sci. 139, 341-352 https://doi.org/10.1017/S0021859602002393.

• Haddi M-L, Filacorda S, Meniai K, Rollin F, Susmel P (2003). In vitro fermentation kinetics of some halophyte shrubs sampled at three stages of maturity. Anim. Feed Sci. Technol. 104, 215-225. https://doi.org/10.1016/S0377-8401(02)00323-1

• Jalilvand G, Odongo N, López S, Naserian A, Valizadeh R, Shahrodi FE, Kebreab E, France J (2008). Effects of different levels of an enzyme mixture on in vitro gas production parameters of contrasting forages. Anim. Feed Sci. Technol. 146, 289-301. https://doi.org/10.1016/j.anifeedsci.2008.01.007

• Kamra D, Sawal R, Pathak N, Kewalramani N, Agarwal N (1991). Diurnal variation in ciliate protozoa in the rumen of black buck (Antilope cervicapra) fed green forage. Lett. Appl. Microbiol. 13, 165-167. https://doi.org/10.1111/j.1472-765X.1991.tb00598.x

• Krause DO, Denman SE, Mackie RI, Morrison M, Rae AL, Attwood GT, McSweeney CS (2003). Opportunities to improve fiber degradation in the rumen: microbiology, ecology, and genomics. FEMS Microbiol. Rev. 27, 663-693 https://doi.org/10.1016/S0168-6445(03)00072-X.

• López D, Elghandour M, Salem A, Vázquez-Armijo J, Salazar M, Gado H (2013). Influence of exogenous enzymes on in vitro gas production kinetics and dry matter degradability of a high concentrate diet. Anim. Feed Sci. Technol. 13, 527-536.

• Marker TB (1992). Multi-element analysis in plant materials In: ADR IANO DC. Biogeochemistry of trace metals. Lewis Publishers: Boca Raton, pp. 401-428, 1992. In Lewis Publishers: Boca Raton, FL.

• Menke K, Raab L, Salewski A, Steingass H, Fritz D, Schneider W (1979). The estimation of the digestibility and metabolizable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor in vitro. J. Agric. Sci. 93, 217-222. https://doi.org/10.1017/S0021859600086305

• Menke KH, Steingass H (1988). Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev. 28, 7-55.

• Morgavi D, Beauchemin K, Nsereko V, Rode L, Iwaasa A, Yang W, McAllister T, Wang Y (2000). Synergy between ruminal fibrolytic enzymes and enzymes from Trichoderma longibrachiatum. J. Dairy Sci. 83, 1310-1321. https://doi.org/10.3168/jds.S0022-0302(00)74997-6

• Morgavi D, Beauchemin K, Nsereko V, Rode L, McAllister T, Iwaasa A, Wang Y, Yang W (2001). Resistance of feed enzymes to proteolytic inactivation by rumen microorganisms and gastrointestinal proteases. J. Anim. Sci. 79, 1621-1630. https://doi.org/10.2527/2001.7961621x

• Nsereko V, Beauchemin K, Morgavi D, Rode L, Furtado A, McAllister T, Iwaasa A, Yang W, Wang Y (2002). Effect of a fibrolytic enzyme preparation from Trichoderma longibrachiatum on the rumen microbial population of dairy cows. Can. J. Microbiol. 48, 14-20. https://doi.org/10.1139/w01-131

• Omer H, Abdel-Magid SS (2015). Incorporation of dried tomato pomace in growing sheep rations. Glob. Vet 14, 1-16.

• Omer H, El-Rahman H, Salman FM, Abdel-Magid SS, Mohamed M, Awadalla I (2009). Response of growing calves to diets containing different levels of exogenous enzymes mixture. Egypt J. Nutr.& Feeds 12, 385-401.

• Ørskov E, McDonald I (1979). The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agri. Sci. 92, 499-503. https://doi.org/10.1017/S0021859600063048

• Parissi Z, Papachristou T, Nastis A (2005). Effect of drying method on estimated nutritive value of browse species using an in vitro gas production technique. Anim. Feed Sci. Technol. 123, 119-128. https://doi.org/10.1016/j.anifeedsci.2005.04.046

• Salem A, Gado H, Colombatto D, Elghandour M (2013). Effects of exogenous enzymes on nutrient digestibility, ruminal fermentation and growth performance in beef steers. Livest. Sci. 154, 69-73. https://doi.org/10.1016/j.livsci.2013.02.014

• Sallam S, Nasser M, El-Waziry A, Bueno I, Abdalla A (2007). Use of an in vitro rumen gas production technique to evaluate some ruminant feedstuffs. J. Appl. Sci. Res 3, 34-41 https://doi.org/10.1017/S1752756200021219.

• Ventura MR, Pieltain M, Castanon J (2009). Evaluation of tomato crop by-products as feed for goats. Anim. Feed Sci. Technol. 154, 271-275. https://doi.org/10.1016/j.anifeedsci.2009.09.004

• Wang Y, McAllister T, Rode L, Beauchemin K, Morgavi D, Nsereko V, Iwaasa A, Yang W (2001). Effects of an exogenous enzyme preparation on microbial protein synthesis, enzyme activity and attachment to feed in the Rumen Simulation Technique (Rusitec). Br. J. Nutr. 85, 325-332. https://doi.org/10.1079/BJN2000277

• Warner A (1964). Production of volatile fatty acids in the rumen: methods of measurement. Nutr. Abstr. Rev. 34, 339.

• Yang W, Beauchemin K, Rode L (2000). A comparison of methods of adding fibrolytic enzymes to lactating cow diets. J. Dairy Sci. 83, 2512-2520. https://doi.org/10.3168/jds.S0022-0302(00)75143-5