1. Introduction
In semi-arid and arid regions, improvement of utilization of available feed resources and search of alternative feeds for ruminants are required due to the shortage of green fodders. Egypt and many other countries lack adequate availability of animal-feed ingredients, causing the utilization of unconventional feeds and secondary agricultural products as a premium approach to feed animals. However, most of the unconventional feeds have a limited nutritive value and some improvements should be considered before feeding to ruminants to obtain optimum production performance.
Date palm (
Phoenix dactilifera) is one of the main crops in Egypt and many semiarid and arid regions of the world. In Egypt, there are around 650,000 tons of leaves’ dry matter (DM) available from date palms annually [
1], but without significant utilization. The main problem with the date-palm leaves (DPL) is the high fiber and low crude protein (CP) content and low nutritive value and digestibility causing its limited utilization as a feed for ruminants. The CP content in DPL ranges from 42 to 165 g/kg DM [
1,
2]. Fiber content in DPL is high, ranging from 430 to 730 g/kg DM of neutral detergent fiber (NDF) [
1,
2]. Therefore, improvement of its nutritive value is recommended before feeding to ruminants.
Ensiling is a good approach to improve the nutritive value of poor-quality forages [
3] and reduces the negative effects of some antinutritional factors present in feeds [
1]. To improve the ensiling process, some inoculants and other feed additives are included during the ensiling process to improve the anerobic conditions and fermentation of silage [
4]. Multi-species probiotics (MSP) such as lactic-acid bacteria (LAB) [
5,
6], and fibrolytic enzymes [
3] are good examples of these feed additives. Inoculating silages with MSP improves silage characteristics and prohibits the growth of undesirable bacteria and other spoilages, and increases the initial LAB growth in silages [
4]. Kaewpila et al. [
6] stated that inoculating forage sorghum mixture silage with LAB could promote ensiling characteristics (e.g., lowering pH and increasing lactic-acid contents) and nutritive value (increased in vitro DM degradability and total gas production, and decreased methane production). Lactic-acid bacteria also improve gut health, immunity and productive performance of animals [
7,
8], which will be added to advantages besides improving silage fermentation [
8]. In the study by Hamdon et al. [
7], Farafra lambs fed DPL-based diets supplemented with MSP showed higher growth performance, feed intake and feed efficiency. In another experiment, Maake et al. [
9] reported that feeding MSP to South African goats had no effect on feed intake, but increased average weight gain.
The administration of fibrolytic enzymes during ensiling was reported to improve nutritive value of feeds, especially those with low nutritive value such as agricultural byproducts [
3,
10]. Mixing the enzymes into the diet prior to feeding is the most effective way to maximize their nutritive potential [
11]. Administration of enzymes prior to feeding (e.g., during ensiling) allows enzymes to attach to the target nutrients (especially those related to fiber components) before consumption, causing a reduction in the lag time between consumption and ruminal degradation. Additionally, administration of fibrolytic enzymes in solutions before ensiling improves the enzyme function due to the hydrolysis of soluble sugars (i.e., glucose) from a complex polymer (such as cellulose). This process of hydrolysis involves the addition of water to specific bonds within a complex carbohydrate, and can be limited if there is insufficient water in the environment [
11]. Fibrolytic enzymes also alter ruminal fermentation characteristics and increase fiber digestibility through solubilizing dietary fiber components [
11]. Moreover, fibrolytic enzymes increase the supply of readily fermentable nutrients to ruminal microorganisms, and increase the microbial enzyme activities and microbial attachment to feed particles in the rumen [
12]. Recently, Abid et al. [
10] evaluated the enzymatic treatment of olive-mill waste containing a high lignocellulose content and high concentrations of anti-nutritional factors with exogenous fibrolytic enzymes produced from
Trichoderma longibrachiatum as a feed for ruminants. They observed that enzymatic treatments increased degradation of cellulose and hemicellulose and increased the amounts and rate of gas production as well as the microbial crude protein production. Azzaz et al. [
13] observed that feeding lactating goats on diets supplemented with fibrolytic enzymes improved feed utilization, milk production, and composition and feed efficiency.
We hypothesized that inoculating DPL during silage-making with fibrolytic enzymes would help in degrading the rigid structure of fibers in DPL, making the nutrients available for animals. Moreover, we hypothesized that inoculating silage with MSP containing LAB, along with other probiotic bacteria such as Bacillus subtilis and Bacillus lichenifomis would facilitate maintenance of the ensiling conditions and improve the nutritive value of ensiled DPL before feeding. The present study aimed to evaluate the effects of inoculating DPL with MSP or fibrolytic enzymes during ensiling on its nutritive value as a feed for lactating Farafra ewes under the arid conditions in the New Valley area in Egypt.
2. Materials and Methods
2.1. Study Location
This experiment was carried out at the experimental farm of the Department of Animal Production, Faculty of Agriculture of New Valley, Al Kharga, Egypt (25°26′ N and 30°32′ E). The chemical analyses were performed at the laboratory of Dairy Animal Production, National Research Centre, Cairo, Egypt. Animals were managed and cared for in accordance with the 3rd edition (2010) of the guide of the Agricultural Research and Teaching of Federation of Animal Science Societies, Champaign, IL, USA. The protocol of the experiment was revised and approved by the Institutional Animal Care and Use Committee of the Faculty of Agriculture, New Valley University, New Valley, Egypt.
2.2. Date-Palm Leaves
Fresh DPL were collected from different sites in the New Valley Governorate (Egypt). Materials were sun-dried for 10 days [
7]. Date-palm leaves were ensiled under anaerobic conditions for 45 days using tightly closed polythene bags. Briefly, the chopped DPL were spread with a solution containing clean water and solid urea (40 g/L solution) and crude liquid molasses (40 g/L solution). Before ensiling, moisture content in DPL was increased to reach about 35–40% with the urea-molasses solution. Three types of DPL were prepared: DPL ensiled without fibrolytic enzymes or MSP and DPL ensiled with fibrolytic enzymes (Polyzyme, Zeus Biotech, Mysuru, India) at 4 g/kg DM or MSP (ProAct, Bengaluru, India). The materials were packed into polythene-bag silos (40 × 70 cm) and compressed manually to create an anaerobic environment. The enzyme product contained (per kg): 4 × 10
6 IU xylanase, 4 × 10
5 IU cellulase, 2.4 × 10
5 IU pectinase, 2 × 10
5 IU β-glucanase, 21.5 × 10
6 IU amylase, 7.5 × 10
5 IU protease, 4 × 10
5 IU galactosidase, 2 × 10
5 IU mannanase, 5 × 10
4 IU phytase and 4 × 10
4 IU lipase along with fermented rice bran. In addition to some species of LAB, the MSP contained 1.75 × 10
12 CFU
B. subtilis and 1.75 × 10
12 CFU
B. lichenifomis per gram product and dextrose monohydrate as a filler.
For assessment of the ensiling process, 200 g (fresh weight of silage was mixed with 800 mL distilled water, homogenized for 3 min with a blender and filtered through 4-layer cheesecloth. The filtrate was collected for measurement of pH using a digital pH meter, ammonia-N (NH
3-N) according to AOAC [
14], and volatile fatty acids (VFA) according to AOAC [
14]. Aflatoxin F
1 concentration was measured in silage with the use of a Fluorometer, Series-4 (Vicam, Milford, MA, USA) based on the methods described by AOAC [
14].
2.3. Ewes and Management
Two weeks before expected parturition, fifty lactating Farafra ewes (mean ± standard deviation: 2 ± 1.2 parity; 33.3 ± 3.04 kg body weight; 24 ± 3.3 months of age; 550 ± 10/4 g/d of previous milk production) were assigned randomly to five dietary treatments (
n = 10 ewes/treatment). Ewes were randomly stratified to treatments in a completely randomized design. Ewes were individually kept in semi-opened concrete floor pens (1.5 m
2/sheep) with free choice fresh water. Sheep were fed a diet comprising (per kg DM) 600 g of a concentrate feed mixture and 400 g of DPL ensiled without additives in the control treatment. In the other four diet treatments, ensiled (without fibrolytic enzymes of MSP) DPL of the control treatment diet was replaced with DPL ensiled with fibrolytic enzymes (ENZ50 and ENZ100) or MSP (MSP50 and MSP100) at 50 or 100% level, respectively. Ewes were first offered the allotted amounts of concentrate feed mixture in the feeder, followed by DPL after the consumption of concentrate feed. Diets were prepared to meet nutrient requirements for milk production of ewes according to NRC [
15] recommendations. To ensure orts collection, feeds were offered 1.10 times above the NRC recommendations. The experiment lasted for 90 d. Individual animals were weighed at monthly intervals.
Table 1 shows the chemical compositions of ingredient and experimental diets. The daily samples of diets were composited weekly and dried at 60 °C in a forced-air oven for 48 h [
14] (method 930.15) before storing for chemical analyses.
2.4. Feed Intake and Nutrient Apparent Digestibility
Three digestibility trials were conducted during the last 10 d of each month using acid-insoluble ash as an internal indigestibility marker. The equations of Ferret et al. [
16] were used to calculate the coefficients of apparent digestion. Feed intake was calculated as the difference between feed offered and orts from the previous day’s feeding. Individual fecal grab samples were collected twice daily during the collection period at 07:00 and 15:00 h, dried at 60 °C in a forced-air oven for 48 h, and pooled per ewe.
Composited samples of dried feeds, orts and feces were ground to pass through a 1mm screen using a mill and analyzed for DM, ash, nitrogen, and ether extract (EE) according to AOAC [
14] official methods. Neutral detergent fiber and lignin contents were determined according to Van Soest et al. [
17]. Acid detergent fiber (ADF) content was analyzed according to AOAC [
14] and expressed exclusive of residual ash. Non-structural carbohydrates, cellulose, hemicellulose, and organic matter (OM) concentrations were calculated.
2.5. Sampling and Analysis of Rumen Fluid
On d 30, d 60 and d 90 of the experiment, ruminal fluid samples were collected from all animals in the morning at 3 h post feeding to analyze fermentation variables (VFA and NH
3). About 100 mL of ruminal fluid was collected from each ewe and strained through 4 layers of cheesecloth for NH
3-N analysis [
14] and VFA [
18] determination. The collected samples were preserved at −20 °C pending analyses. Concentration of VFA and its individual molar proportions were determined using a gas chromatograph (Thermo Fisher Scientific, Inc., TRACE1300, Rodano, Milan, Italy) fitted with an AS3800 autosampler and equipped with a capillary column HP-FFAP (19091F-112; 0.320 mm o.d., 0.50 μm i.d., and 25 m length; J & W Agilent Technologies Inc., Palo Alto, CA, USA). A mixture of known concentrations of individual short-chain fatty acids was used as an external standard (Sigma Chemie GmbH, Steinheim, Germany) to calibrate the integrator.
2.6. Sampling and Analysis of Blood Serum
On d 30, d 60 and d 90 of the experiment, blood samples (10 mL) were collected at 4 h post feeding from the jugular vein of each ewe into clean dry tubes without anticoagulants. Collected samples were centrifuged at 4000× g for 20 min at 4 °C, and serum was decanted into 2-mL Eppendorf tubes and frozen at −20 °C pending analysis using specific kits (Stanbio Laboratory, Boerne, TX, USA) according to manufacturer instructions. Globulin concentration was calculated (total protein—albumin).
2.7. Milk Sampling and Composition
Ewes were hand-milked during the last 10 d of each experimental period at 09:00 and 21:00 h, and 10% of recorded milk yield samples was taken at each milking and composited daily for the analysis of milk components (fat, lactose, total solids, and protein) using infrared spectrophotometry (Lactostar Dairy Analyzer, Funke Gerber, Berlin, Germany).
Fatty acids in milk were determined using methyl esters prepared by base-catalyzed methanolysis of the glycerides (potassium hydroxide in methanol) according to International Standards (International Standard ISO 15884-IDF 182. 2002, Brussels, Belgium: International Dairy Federation) on a Perkin-Elmer chromatograph (model 8420, Beaconsfield, Perkin Elmer, Beaconsfield, UK) equipped with a Cp-Sil 88 fused-silica capillary column (100 m × 0.25 mm internal diameter × 0.2 µm film thickness; Chrompack, Middelburg, Netherlands) and a flame ionization detector (Perkin Elmer, Beaconsfield, UK). Atherogenic index (AI) was calculated according to Ulbricht and Southgate [
19].
Gross energy content in milk, fat-corrected milk (4% FCM, kg/day) and energy-corrected milk (ECM, kg/day) were calculated according to Tyrrell and Reid [
20]. Feed efficiency was calculated and expressed as milk yield, FCM, and ECM per unit of DM intake.
2.8. Statistical Analyses
The Shapiro-Wilk test was used to test the normal distribution of data. For the small number of variables that showed significance for the Shapiro-Wilk test, data transformation (e.g., natural log, inverse of the natural log, square root, or inverse of the square root) was applied before statistical analysis. Data were analyzed using a completely randomized design with repeated measurements in time, in which each ewe was an experimental unit using PROC MIXED of SAS (Online Version, SAS
® On Demand for Academics, SAS Inst., Inc., Cary, NC, USA). The following model was used as:
where Y
ijkl expressed each observation of the
jth ewe in the
kth sampling time given
ith diet, T
i expressed the diet’s effect, A(T)
ji expressed the ewe within each diet, P
k expressed the sampling week effect, (T × P)
ik expressed the interaction between the diets and sampling period, and E
ijkl expressed the experimental error. Polynomial (linear and quadratic) contrasts were used to examine level responses to increasing the level of DPL separately for enzyme or MSP effect. Additionally, contrast between enzyme vs. MSP treatments was applied. The period and diet × period interactions were non-significant (i.e.,
p > 0.05) for most of the measurements; thus, only the main effects of diets were reported. Significance was declared at a level of
p < 0.05.