1. Introduction
One of the main challenges faced by animal nutritionists is the scarcity and high cost of concentrates, which forces scientists to explore suitable alternatives for ruminant and nonruminant animals [
1,
2]. Use of multipurpose tree leaves (e.g.,
Moringa oleifera) and protein-rich microalgae (e.g.,
Chlorella vulgaris) in animal feeding has gained increasing interest, with mixed results [
3,
4,
5,
6]. Usually, supplementation of foliages in low-quality forage diets at low levels (20 to 40% of the total diet) is beneficial for ruminant performance and nutrient utilization due to better ruminal microbial activities. The multipurpose tree leaves are important low-cost feed resources for smallholder farmers in the low-income countries.
M. oleifera is a rapid-growing softwood tree that grows in all tropical and subtropical areas and can adapt to different environmental and soil conditions [
2], making it available throughout the year. The proximate analysis revealed that
M. oleifera leaves contain crude protein (CP) content (23.0–29.4%), fat (4.03–9.51%), mainly α-linolenic acid, fiber (6.00–9.60%), and ash (8.05–10.38%). Additionally,
M. oleifera leaves contain vitamin C (188–279 mg/100 g), Ca (1.32–2.65%), P (0.152–0.304 g/100 g), and K (1.32–2.03 g/100 g) [
2,
7]. Moreover, protein in the
M. oleifera leaves has about 47% rumen bypass protein [
8] with a good amino acid profile [
9]. A recent experiment partially replaced protein feeds (e.g., soybean and sesame meals) in the diets of ruminants with unconventional alternatives (e.g., plant leaves rich in protein) and observed increased nutrient intake and digestibility and altered ruminal fermentation (e.g., increased concentrations of ruminal acetic, propionic, and total volatile fatty acids), and improved final body weight, and daily weight gain [
10]. Replacement of berseem clover with
M. oleifera leaves in the diets of lactating goats improved feed efficiency and milk production [
6].
C. vulgaris is a fresh-water, unicellular microalga, with a high concentration of CP (about 600 g CP/kg DM) containing all essential amino acids [
11,
12]. The main amino acids in
C. vulgaris are glutamic acid and leucine with relatively high concentrations of lysine and methionine, which are the first two limiting amino acids in animal nutrition [
2].
C. vulgaris also contains other biological active components such as antioxidants, provitamins, vitamins, pigments, a growth phytonutrient known as the
C. vulgaris growth factor (CGF), unsaturated fatty acids (UFA), glycoproteins and carotenoids [
11]. Experiments [
3,
13] showed that
C. vulgaris improved ruminal bacterial growth and altered ruminal biohydrogenation of monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) due to their effects on ruminal microbes, especially those involved in the metabolism and biohydrogenation of fatty acids in the rumen [
11,
14]. Recently, Kholif et al. [
3] observed that the inclusion of
C. vulgaris in the diets of lactating Boer goats improved nutrient intake and digestibility, ruminal fermentation, lactational performance and milk nutritive value; however, other researchers [
15] observed weak effects on feed intake, digestibility or daily milk production when feeding
C. vulgaris to lactating Finnish Ayrshire cows.
In an in vitro study, we evaluated three levels (1%, 2% and 3%) of C. vulgaris and twelve levels (0 to 100%) of M. oleifera silage to decide optimum levels of these two ingredients in diets and we noted that 1% of C. vulgaris and up to 40% of M. oleifera in the diets were the best to improve ruminal fermentation. However, there is limited information on the synergic effects of C. vulgaris microalgae and M. oleifera as concentrate feeds on feed utilization and lactation performance of lactating goats. Based on the literature on C. vulgaris and M. oleifera as feed ingredients in ruminant diets, we hypothesized that a combination of these two ingredients could enhance milk production performance in goats. Accordingly, this experiment aimed to evaluate two replacement levels of concentrates with M. oleifera leaves silage in the presence of C. vulgaris microalgae at 1% of total diet (DM basis) on nutrient utilization, ruminal fermentation, biochemical blood parameters and milk production in lactating Damascus goats.
2. Materials and Methods
2.1. Study Location
This study was carried out in the experimental farm at Gemmeiza Station of the Animal Production Research Institute, Egypt. Management of the does was in accordance with the 3rd edition (2010) of the guide of Agricultural Research and Teaching of the Federation of Animal Science Societies, Champaign, IL, USA and approved by the Institutional Animal Care and Use Committee of the Animal Production Research Institute, Egypt.
2.2. Moringa Oleifera and Chlorella Vulgaris Microalgae Cultivation
M. oleifera seeds were planted at a density of 100,000–150,000 seeds per ha. The field was irrigated with 900 m
3 water/ha biweekly without any fertilizer. When plant height reached to 65–70 cm, a first uniformity cutting was carried out at 5–7 cm cutting height 65 days after seeding. This cut was used for feeding other animals, not for the animals used in the present experiment. A second cut of
M. oleifera (45 days after the first cut) biomass, composed of leaves and small twigs was harvested and large twigs were removed. Usually,
M. oleifera results nine harvests per year and yielding 70–80 tons of fresh biomass/ha/year (∼23 tons DM/ha/year). The material (about 1 ton) was left on the field for 1 h and then chopped and used to prepare silage used in the present experiment. Molasses was mixed at 5% of fresh weight. About 40 kg fresh materials per bag was packed into a polythene silo bag (40 × 70 cm) and compressed manually for quick creation of anaerobic conditions. The bags were sealed and stored indoors on a dry concrete floor for 45 days. Samples of ensiled materials were collected from five different bags (1 kg/bag), dried and kept for silage evaluation and chemical analysis. Silage pH, ammonia-N (NH
3-N) and volatile fatty acids (VFA) were analyzed as quality indicators of silage according to AOAC [
16]. Aflatoxin F
1 concentration was determined in silage with the use of a Fluorometer, Series-4 (Vicam, Milford, MA, USA) based on the method described by AOAC [
16]. Tannin [
17] and total phenolic concentration [
18] in
M. oleifera silage were determined following standard protocols.
Laboratory production of
C. vulgaris was performed using 5 L glass flasks containing 3 L algal growth medium. Pure strain of
C. vulgaris H1957 was obtained from the Marine Toxins laboratory, National Research Centre, Egypt. The culture media for cultivation of
C. vulgaris was BG-11 medium containing (/L) 1.5 g NaNO
3, 0.04 g K
2HPO
4, 0.075 g MgSO
4.7H
2O, 0.036 g CaCl
2.2H
2O, 0.006 g citric acid, 0.006 g ferric ammonium citrate, 0.001 g EDTA, 0.02 g Na
2CO
3, and 1 mL trace-metal mix A5 [
19]. After autoclaving and cooling, the pH of the medium was adjusted to 7.1.
C. vulgaris was cultivated under continuous illumination with white fluorescent lamps at room temperature and aeration was performed using an air compressor linked with polyethylene tubes (3 mm). After 25 days,
C. vulgaris in their late exponential phase was transferred at 1:10 into 1000 L polyethylene tanks (
n = 5) containing 600 L culture media and linked with an aeration system.
C. vulgaris biomass was harvested using the continuous separating centrifuge apparatus (Westevalia Separator centrifuge at 15,000 L/h) and drained water was recycled to the ponds. The harvested biomass (0.75 kg microalgae per day) was re-washed three times with tap water to remove any residues of salts from the culture media. Biomass was then partially dried using an air-drying oven at 45 °C for 2–4 h.
2.3. Goats, Feeding and Management
Fifteen lactating Damascus does (mean ± SD: 2 ± 0.5 parity, 41.0 ± 1.5 kg body weight, 24 ± 4.1 months of age, 850 ± 30.5 g/d of previous milk production, twin birth, normal suckling) in the first week of lactation were randomly assigned to three experimental treatments in a quintuplicate 3 × 3 Latin square design. The experimental design had three treatments, three periods and five does per treatment within each period, resulting in 15 replicates per treatment. The three experimental treatments were assigned randomly to the three groups in the first period, after which a predetermined sequence was followed that allowed each doe to receive each treatment.
Does were individually housed in semi-opened concrete floor pens (1.5 m
2/goat) under shade, without bedding and with free access to water. Kids were kept with their mothers throughout the experimental period, with the exception of days when feed intake and nutrient digestibility were determined. Does were offered the experimental diets to meet their minimum CP and net energy requirements according to NRC [
20] recommendations plus 10% extra allowance.
The basal diet fed to the goats (control treatment) contained rice straw and a concentrate feed mixture at 40:60 (DM basis). In the other experimental diets, a mixture of
M. oleifera silage and
C. vulgaris microalgae (at 10 g/kg DM), produced as previously mentioned, replaced the concentrate mixture at 20% (MA20 treatment) or 40% (MA40 treatment) on DM basis. The replacement levels were recommended by an in vitro experiment (unpublished data). Does were offered the allotted amounts of concentrate feed mixture mixed with
C. vulgaris, followed by
M. oleifera silage and then rice straw. The ingredients and chemical composition of the diets are presented in
Table 1.
2.4. Feed Intake and Apparent Nutrient Digestibility
Diets were offered to the does individually at 08:00 and 16:00 h in two equal amounts. Each experimental period lasted 30 days: 20 days of adaptation to the new diet, and 10 days for measurements (feed intake and milk yield) and sample collection (sampling of feed and orts, feces, ruminal fluid, blood and milk). Three digestibility trials were conducted during the last 10 d of each experimental period (d 20–30, d 50–60 and d 80–90) to determine apparent total tract nutrient digestibility by a marker method. In each day, the offered feeds and orts amounts were recorded individually for each goat. Daily orts of individual feeds (concentrate feed mixture mixed with
C. vulgaris,
M. oleifera silage and rice straw) from the two times of feeding were individually collected and pooled for each doe before sampling. During sample collection periods, daily feed intake was measured as the difference between feed offered and orts from the previous day’s feeding. During collection periods, individual fecal samples from all does were collected twice daily at 07:00 and 15:00 h, dried at 60 °C in a forced-air oven for 48 h, and pooled per doe. Nutrient intake was calculated by multiplying the total intake by nutrient concentration in the feed. Acid-insoluble ash was used as an internal indigestibility marker, and coefficients of digestion were calculated according to Ferret et al. [
23]. Goats were weighed monthly on a digital multi-purpose platform scale. Diets were sampled daily, composited weekly, dried at 60 °C in a forced-air oven for 48 h [
16] (method 930.15), and stored pending chemical analyses.
Composited samples of dried feeds, orts of each feed and feces were ground to pass through a 1-mm screen using a Wiley mill, and analyzed for different components (nitrogen, ether extract and ash) according to AOAC [
16] official methods. Neutral detergent fiber (NDF) content was determined according to Van Soest et al. [
24] with use of alpha amylase and sodium sulfite and expressed without residual ash. Acid detergent fiber (ADF) [
16] and lignin [
24] contents were determined. Concentrations of non-structural carbohydrates (NSC), cellulose, hemicellulose, and organic matter (OM = 1000–ash) were calculated. Total digestible nutrient and energy content of diets were estimated according to NRC [
21] and INRA [
22] equations.
2.5. Sampling and Analysis of Rumen Fluid
On the last day of each experimental period, ruminal contents were sampled 3 h after the morning feeding to determine the pH and concentration of fermentation end-products. After initial discarding of 50 mL ruminal fluid, 100 mL ruminal fluid were collected by using a stomach tube, and the samples taken from each doe were strained through four layers of cheesecloth. Ruminal fluid pH was measured immediately using a pH meter (HI98127 pHep®4 pH/Temperature Tester, Hanna® Instruments, Villafranca Padovana PD, Italy).
A subsample of 5 mL ruminal fluid was preserved with 5 mL of 0.2
M HCl for ammonia-N analysis [
16], and 0.8 mL of ruminal fluid was mixed with 0.2 mL of a solution containing 250 g of metaphosphoric acid/L for total volatile fatty acids (VFA) analysis. 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 (acetate, propionate and butyrate) was used as an external standard (Sigma Chemie GmbH, Steinheim, Germany) to calibrate the integrator.
2.6. Sampling and Analysis of Blood Serum
On the last day of each experimental period, blood samples (10 mL) were collected 4 h after feeding from the jugular vein of each doe into a clean dry tube without anticoagulants. Blood samples were centrifuged at 4000× g at 4 °C for 20 min. Serum was separated into 2-mL clean dried Eppendorf tubes and frozen at −20 °C until analysis. Concentrations of blood parameters were enzymatically analyzed in blood serum samples using specific kits (Stanbio Laboratory, Boerne, Texas, USA), following manufacturer instructions.
2.7. Milk Sampling and Composition
During the last 10 days of each experimental period, does were milked by hand twice daily at 09:00 and 21:00 h, amount of milk yield was measured in a weighing balance, and milk samples (10% of recorded milk yield) were collected at each milking. A mixed sample of morning and evening milk was taken daily. Milk samples were analyzed for different components using infrared spectrophotometry (Lactostar Dairy Analyzer, Funke Gerber, Berlin, Germany).
Fatty acid contents in milk were determined in fatty acid methyl esters prepared by base-catalyzed methanolysis of the glycerides (potassium hydroxide in methanol) according to international standards (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 length × 0.25 mm internal diameter × 0.2 µm film thickness; Chrompack, Middelburg, Netherlands) and a flame ionization detector (HP, Little Falls, DE, USA). The atherogenic index (AI) was calculated according to Ulbricht and Southgate [
25].
Average yield (g/d) of each milk component was calculated by multiplying milk yield by the component content (g/kg). Gross energy content in milk was calculated according to Tyrrell and Reid [
26]. Milk energy output (MJ/d) was calculated as milk energy (MJ/kg) × milk yield (kg/d). Fat-corrected milk (FCM, kg/day) and energy-corrected milk (ECM, kg/day) were calculated according to Tyrrell and Reid [
26]. Feed efficiency was calculated and expressed as milk yield, FCM, and ECM per unit of DM intake. Feed efficiency was calculated as milk: intake, ECM: intake and FCM: intake ratios.
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 quintuplicate 3 × 3 Latin square design, with three periods and three treatments. The statistical model included the fixed effect of square and treatment, and the random effects of period and goat nested within square: Yijkl = μ + Si + Tj + Pk + Gl(Si) + Eijkl, where Yijkl is each individual observation for a given variable, μ is the overall mean, Si is the square effect, Tj is the treatment effect, Pk is the period effect, Gl(Si) is the effect of goat within square and Eijkl is the residual error. Statistical analyses were performed using PROC MIXED of SAS (Online Version, SAS® OnDemand for Academics, SAS Inst. Inc. Cary, NC, USA). When the treatment F-test was significant at p < 0.05, means were then compared by applying the probability of difference option of the least squares means statement. The contrast between control versus silage treatments was used to test for differences between control diet versus both M. oleifera leaves silage and C. vulgaris microalgae diets. Significance was declared at a level of p < 0.05.