Effect of supplementing sheep diets with macroalgae species on in vivo nutrient digestibility, rumen fermentation and blood amino acid profile.

In this study, a brown macroalgae species, Saccharina latissima, processed to increase its protein concentration, and a red macroalgae species, Porphyra spp., were used to evaluate their in vivo digestibility, rumen fermentation and blood amino acid concentrations. Four castrated rams were used, whose diets were supplemented with a protein-rich fraction of S. latissima, a commercial Porphyra spp. and soybean meal (SBM). Our results show that the protein digestibility of a diet with S. latissima extract was lower (0.55) than those with Porphyra spp. (0.64) and SBM (0.66). In spite of the higher nitrogen (N) intake of diets containing Porphyra spp. and SBM (20.9 and 19.8 g N/day, respectively) than that with S. latissima (18.6 g N/day), the ratio of N excreted in faeces to total N intake was significantly higher in the diet with S. latissima than those with Porphyra spp. and SBM. This reflects that the utilization of protein in S. latissima was impaired, possibly due to reduced microbial activity. The latter statement is corroborated by lower volatile fatty acid composition (25.6, 54.8 and 100 mmol/l for S. latissima, Porphyra spp. and SBM, respectively) and a non-significant tendency for lower ammonia concentration observed in diets with S. latissima and Porphyra spp. compared to SBM. It is important to note that the S. latissima used in this trial was rinsed during processing to remove salt. This process potentially also removes other water-soluble compounds, such as free amino acids, and may have increased the relative fraction of protein resistant to rumen degradation and intestinal absorption. Furthermore, the phlorotannins present in macroalgae may have formed complexes with protein and fibre, further limiting their degradability in rumen and absorption in small intestines. We recommend that further studies explore the extent to which processing of macroalgae affects its nutritive properties and rumen degradability, in addition to studies to measure the intestinal absorption of these macroalgae species.

Implications

The protein concentration of seaweed varies greatly from species to species. Those with low-to-medium protein level require processing to remove the salt and to increase protein concentration. This process applied to Saccharina latissima species appeared to have reduced the nutrient availability, reflecting lower protein digestibility than that of Porphyra spp. and soybean meal. Both rumen fermentation and intestinal absorption were impaired in animals fed S. latissima. If the limitations in processing can be fathomed, S. latissima may present a potential biomass source that is abundant in nature; however, the challenges in its palatability and chemical composition should be addressed.

Introduction

Future projections estimate an increase in the import of soybean in Norway by 35% in 2050 compared to 1961 to 1990 levels, due to increasing demand for food and protein feed (Özkan Gülzari et al., 2017). This threatens the competitiveness of ruminant production systems and urges the need for alternative protein sources with local origin. For this purpose, macroalgae (also known as seaweed) present a significant and yet potential biomass source. Currently, the brown macroalgae Saccharina latissima constitutes the major cultivated species in Europe. Despite its relatively low protein concentration (50–150 g/kg DM), large-scale cultivation may provide significant amounts of protein for use in animal feed. Red macroalgae contain a higher protein concentration than brown macroalgae, but cultivation technology does not yet exist for native, Northern European species. However, red macroalgae (e.g. Porphyra spp.) are the largest source of food among macroalgae, for example, nori in Japanese sushi (Makkar et al., 2016), and contain up to 347 g/kg DM protein (Tayyab et al., 2016).

A number of studies have investigated the digestibility of brown and red macroalgae. An in vitro trial measuring the organic matter (OM) digestibility of S. latissima reported digestibility as high as 0.97 in sheep (Makkar et al., 2016). A previous study by Ramin et al. (2017) found that increasing the proportion of a protein-enriched fraction of S. latissima in an in vitro trial increased both the OM digestibility and utilizable protein concentration. Porphyra spp. were tested by Tayyab et al. (2016) in situ, and their crude protein concentration was found to be comparable to oil-seed by-products such as sunflower meal and rapeseed meal. However, given that the aforementioned studies are either in vitro or in situ, there seems to be a lack of studies investigating the in vivo digestibility of macroalgae, and the extent to which they may provide comparable nutrient characteristics to soybean meal (SBM) for ruminants. To fill this gap, in this study we evaluated the in vivo digestibility, rumen fermentation parameters and amino acid composition in plasma of a diet supplemented with S. latissima protein extract or Porphyra spp., and compared them with a diet containing SBM. Due to its high salt and iodine-concentration, the biomass of S. latissima was exposed to a simple processing by which salt concentration was removed and protein concentration was enriched. The Porphyra spp, a commercial product produced in large quantities in Asia, were included for comparison; and an extracted SBM was used as reference.

We hypothesise that the protein digestibility and utilization of diets containing extracted S. latissima and purchased Porphyra spp. are similar to that of SBM, and better than the diet without any additional protein source.

Materials and methods

Experimental design and animals

An in vivo digestibility trial was run with four wethers of Norwegian White Sheep, 30 months of age and 80 to 88 kg live body weight, using four diets in a 4×4 Latin square design. The four diets were a control, a diet with protein enriched fraction of S. latissima (SW1), a diet with Porphyra spp. (SW2) (CoDo International Limited, Qingdao, China) and a diet with extracted SBM (Champion Soyapellets, Felleskjøpet, Lillestrøm, Norway). The trial was run for four periods from October to November 2017. Each period consisted of 8 days adaptation in individual pens followed by 7 days in individual metabolism crates for daily collection of urine, faeces and feed refusal, if any.

Feeding

Animals were stalled and fed a control diet containing hay, oats and mineral/vitamin pellets in individual pens for 10 days before the experiment started. Body weight was measured every other day to adjust the maintenance requirements for hay. The DM intake was restricted during the trial and corresponded to maintenance requirements of adult rams in energy as calculated according to the body weight at pre-trial period. Diets with protein enrichment (SW1, SW2 and SBM) were planned to be isocaloric and isonitrogenous (Jarrige, 1988). Adjustments were made to the pre-trial rations where oats and mineral/vitamin pellets were removed to balance the diet. In addition, to increase the palatability of those who refused to eat the macroalgae, the protein feed was mixed with a fixed amount of sugar cane molasses (Felleskjøpet Agri, Sandnessjøen, Norway) at the time of feeding. The control group also received the same amount of molasses. The chemical composition of feed ingredients and the formulation and nutritive value of the individual feed ingredients are presented in Tables 1 and 2, respectively (also see Özkan Gülzari et al., 2018a). The SW1 included 3.8 g/kg DM phlorotannins as phloroglucinol, which was not analysed for other feed ingredients. Note that even though the diets were designed to be isonitrogenous, chemical analyses showed that some variation in nitrogen (N) concentration existed.

Table 1

Chemical composition of individual feed ingredients fed to wethers (g/kg DM unless specified otherwise)

	Feed ingredients
Item	Hay		Molasses		SW1		SW2		SBM
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
Ash	43	(0.5)	132	(0.4)	221	(2.7)	87	(3.7)	77	(19.1)
Kjeldahl-Nitrogen	13.3	(0.33)	10.9	(0.09)	35.8	(0.66)	60.9	(0.50)	82.1	(3.18)
Amino acids	57	(6.1)	20	(1.9)	163	(4.0)	282	(2.4)	468	(25.5)
Crude fat	10.5	(0.79)	2.0	(0.34)	18.5	(3.32)	2.5	(1.06)	16.3	(4.3)
aNDFom1	615	(8.6)	0	–	408	(25.1)	431	(13.5)	182	(24.0)
ADF2	364	(5.0)	0	–	352	(8.5)	66	(7.0)	90	(5.7)
Crude fibre	308	(4.4)	0	–	152	(2.1)	36	(6.1)	64	(3.9)
Gross energy3, MJ/kg DM4	19.0	(0.08)	15.7	(0.03)	15.7	(0.05)	18.8	(0.07)	19.5	(0.51)
Iodine, mg/kg DM	–		–		1230		1.5		–

SW1=processed S. latissima; SW2=Porphyra spp.; SBM=soybean meal.

Neutral detergent fibre assayed with a heat stable amylase and expressed exclusive of residual ash.

Acid detergent fibre expressed inclusive of residual ash.

Gross energy calculated from gross energy determined with bomb calorimetry of diet ingredients. MJ: megajoule.

Dry matter.

Table 2

Formulation, chemical analysis and nutritive value of experimental diets fed to wethers averaged over four periods

Item	Experimental diets
	Control	SW1	SW2	SBM
Ingredients (g/kg DM1)
Hay	961	799	854	888
Protein feed1	0	162	108	72
Molasses	39	39	38	40
Sum	1000	1000	1000	1000
Chemical analysis (g/kg DM)
Organic matter	953	925	949	951
Kjeldahl-Nitrogen	13.2	16.8	18.3	18.1
Amino acids	56	73	80	85
aNDFom2	591	557	571	559
ADF3	349	347	318	330
Crude fibre	296	271	267	278
Gross energy (MJ/kg DM)4	18.9	18.3	18.8	18.9
Iodine (mg/kg DM)	0.04	180	0.20	0.04

SW1=diet containing processed S. latissima; SW2=diet containing Porphyra spp.; SBM=diet containing soybean meal.

Dry matter.

Neutral detergent fibre assayed with a heat stable amylase and expressed exclusive of residual ash.

Acid detergent fibre expressed inclusive of residual ash.

Gross energy calculated from gross energy determined with bomb calorimetry of diet ingredients. MJ: megajoule.

Animals were fed their daily allowance in two equal portions at 0800 and 1600 h. Water was freely accessible through individual drinkers in each pen and metabolism crate. Blood and rumen fluid samples were taken on the last day of each collection period. Blood samples were taken before feeding in the morning, and again 3–4 h after morning feeding. Rumen fluid samples were taken 3–4 h after morning feeding. After sampling for blood at noon, animals were handled back to the stable and were offered their adaptation diet of next period in the afternoon feeding. Any feed refusal was collected every morning during the collection period and weighed, and 10% of the total waste was stored at −18°C until further analyses.

Preparation and chemical analyses of seaweed species

Porphyra spp. were purchased in the form of powder (dried and milled) and was used without further processing. The product was stored in a clean and dry place until use. Cultivated S. latissima was harvested at the coast of Sør-Trøndelag County, Norway, in May–June 2016. Seawater was drained, and small stones and other impurities were removed manually. The biomass was stored in plastic bags at −20°C until further processing. To produce the protein-enriched product, 2×375 kg biomass wet weight, 750 kg in total, was milled and washed in water (60°C–70°C) to reduce the salt concentration. Alginate lyase was added for a partial degradation of alginate, to facilitate the subsequent separation by centrifugation. The solid phase (‘sludge’) after centrifugation was dried in a pilot-scale Forberg® Dryer (Forberg International AS, Oslo, Norway) at 40°C (product temperature during drying). The salt concentration in the processed biomass (SW1) was reduced from 440 to 180 g/kg DM and the iodine concentration from 5.9 to 1.2 g/kg DM. The protein concentration as total amino acids was increased from 89 to 186 g/kg DM.

Collection and storage of samples

Feed samples were collected during the adaptation and collection periods for 15 days in each period by taking a handful of each feed ingredient every day and storing them in plastic boxes, except for hay which was stored in a cardboard box to prevent humidification. At the end of each collection period, 2×100 g samples of SW1, SW2, SBM and 2×200 g samples of hay were prepared. Molasses samples were taken as 2×100 g at the end of each collection period. All feed samples were stored at −18°C until further processing.

Faeces and urine were collected and weighed from each animal on a 24-h basis after feeding each morning and stored at 2°C–3°C until the end of the collection period (7th day). Urine was collected in individual buckets. To each collection bucket for urine, 100 ml sulphuric acid was added before collection started, in order to avoid loss of ammonia. A pH indicator strip non-bleeding pH 0–6.0 (Teststrips, pH, MColorpHast™, product number: 1.09531.0001, VWR International, Merck KGaA, Darmstadt, Germany) was used to measure the acidity of urine every day. After 7 days of collection period, pooled samples of urine and faeces were weighed again as control and 10% of the control weight was taken as a subsample and stored at −18°C until further processing. The rest of the urine and faeces samples were discarded.

At the end of each collection period (7th day), blood and rumen fluid samples were taken from each animal. Blood samples were collected from the jugular vein before (at approximately 0800–0830 h) and approximately 4 h after morning feeding (at approximately 1300–1330 h) to Vacuette® EDTA tubes (product number: 454021, Greiner Bio-One, GmbH, Kremsmünster, Austria). Plasma was separated by centrifugation at 2000 rpm for 20 min, after which aliquots of two samples were removed and pooled into a glass vial to give one sample per wether per period. Plasma was stored at −80°C until further chemical analyses.

Rumen fluid samples were taken within 3–4 h after morning feeding at approximately 1200–1300 h via the esophagus, using two flexible polyvinyl chloride tubes, with diameters of around 3 and 1 cm, respectively, of which the latter was connected to a vacuum pump and sucked approximately 50 ml rumen fluid from each animal. Collected samples of rumen fluid were filtered through an absorbent gauze and pH was measured after filtering using a pH indicator strip non-bleeding pH 7.5–14 (Teststrips, pH, MColorpHast™, product number: 1.09532.0001, VWR International, Merck KGaA, Darmstadt, Germany). Three subsamples of each rumen fluid sample were made for analysing ammonia, lactic acid and volatile fatty acids (VFAs) by mixing 4 ml rumen fluid with 4 ml hydrochloric acid (HCl) 0.5M; 4 ml rumen fluid with 0.5 ml solution A; and 0.8 ml rumen fluid with 0.5 ml solution A, respectively. Solution A included 20 g/l metaphosphoric and 4 g/l crotonic acids in 0.5M HCl for deprotonization of samples whilst HCl was used to acidify the medium. Any leftover rumen fluid was transferred to glass vials and stored as reserve. Both subsamples and the reserve rumen fluid samples were stored at −80°C until further processing.

Processing and chemical analyses of samples

Feed samples, except for molasses, were ground to pass 1-mm screen using a Tecator Cyclotec Sample Mill® (Foss Analytical Co., Ltd., Suzhou, China), and were analysed for amino acids and iodine (as described below), and feed composition (Norwegian University of Life Sciences: LabTek, NMBU, Ås, Norway). Feed refusal was oven dried for 48 h, weighed and reweighed after storage at ambient temperature for 24 h. Frozen faeces samples were course-ground and freeze-dried for 48 h, using a Labconco FreeZone 4.5 Plus® (Kansas City, Missouri, USA) freeze-drier at a temperature and vacuum ranging between −80°C and −86°C, and 0.52 and 0.97 mbar, respectively. The freeze-dried samples were weighed immediately and again after 24 h storage in ambient temperature. The samples were then ground to pass 1-mm screen using a Tecator Cyclotec Sample Mill® (Foss Analytical Co., Ltd., Suzhou, China). The final weight of the samples was measured, and subsamples were taken and stored in plastic zipped bags at −18°C until further chemical analyses at LabTek, NMBU, for composition.

Feed, feed refusal and faeces samples were analysed for DM (103°C for at least 4 h to constant weight), ash (550°C for at least 4 h), Kjeldahl-N (KjeltecTM 8400; Foss Electric, Hillerød, Denmark), crude fat (accelerated solvent extraction, ASETM 350 Accelerated Solvent Extractor, Dionex, USA) and gross energy (PARR 1281 Bomb Calorimeter, Moline, Illinois, USA). Neutral detergent fibre was determined with an ANKOM200 fibre analyser (ANKOM Technology, Fairport, New York, USA) according to Mertens (2002) using sodium sulphite, alpha amylase and ash correction (aNDFom), and acid detergent fibre (ADF) was determined according to Method 973.18 (AOAC, 2000) with the modification that the samples were not washed with acetone and were corrected for ash. Iodine in macroalgae was determined according to Roleda et al. (2018) who used the HPLC method to extract iodine by dry alkaline incineration (Nitschke and Stengel, 2015). The polyphenolic concentration in the S. latissima extract was determined according to Roleda et al. (2019) using phloroglucinol as standard reference.

Amino acid concentration in feed ingredients was analysed by an HPLC system (Agilent Infinity 1260, Agilent Technologies) coupled to an online post-column derivatization module (Pinnacle PCX, Pickering laboratories, Mountain View, California, USA), using ninhydrin (Trione) as a derivatizing reagent and Na+-ion exchange column (4.6×110 mm, 5 µm). Eighteen standard amino acids, ammonia and taurin were quantified from standard curves measured with amino acid standards (Pickering Laboratories, Mountain View, California, USA) (Table 3).

Table 3

Amino acid concentration in experimental rations fed to wethers

Item	Experimental diets
	Control	SW1	SW2	SBM
Essential AA1 (g/kg DM2)
Histidine	1.1	1.5	1.6	2.0
Isoleucine	2.8	3.6	3.7	4.3
Leucine	5.0	6.5	6.9	7.3
Lysine	3.3	4.3	4.8	5.4
Methionine	0.6	1.0	0.8	0.8
Phenylalanine	3.3	4.4	4.5	5.1
Threonine	2.2	2.9	3.2	2.7
Tryptophan	0.6	0.5	0.8	0.5
Valine	3.9	5.1	5.9	5.3
Non-essential AA (g/kg DM)
Alanine	4.5	6.1	7.9	5.8
Arginine	2.8	3.9	4.6	4.9
Aspartic acid	5.4	6.6	6.9	8.7
Cystine	0.6	1.2	2.0	0.8
Glutamic acid	6.7	8.5	8.7	12.3
Glycine	3.3	4.6	4.5	4.4
Proline	0.6	1.2	2.0	0.8
Serine	3.9	5.1	5.6	6.0
Tyrosine	1.7	2.2	2.4	2.6

SW1=diet containing processed S. latissima; SW2=diet containing Porphyra spp.; SBM=diet containing soybean meal.

Amino acid(s).

Dry matter.

The frozen urine samples were thawed and subsamples were stored at 4°C until further chemical analyses at Vitas AS (Oslo, Norway) for iodine, and at LabTek, NMBU, for Kjeldahl N (Kjeltec 2460; Foss Electric, Hillerød, Denmark). Urine was diluted and homogenized before the samples were analysed for their iodine concentration by inductively coupled plasma mass spectrometry (Agilent 7900 ICP-MS, Japan). Unknowns were calibrated against known standards from Sigma-Aldrich.

Plasma samples were sent without further processing to University of Helsinki for amino acid analysis. Plasma amino acid concentrations were determined as described by Puhakka et al. (2016). Briefly, plasma samples were precipitated using 10% sulphosalisylic acid and analysed by ultra-performance liquid chromatography, equipped with an Ethylene Bridged Hybrid C18 column, and a photodiode array detector to detect amino acids.

The three subsamples of rumen fluid per animal and period were sent without further processing to University of Helsinki for ammonia and VFA analyses. Ammonia and VFA from rumen fluid samples were analysed according to McCullough (1967) and Lamminen et al. (2017), respectively.

Statistical analyses

Data were analysed using Proc MIXED in SAS (Statistical Analysis System Institute Inc., 2011) for a 4×4 Latin square design according to the following model: where Yijkl is the dependent variable, µ the overall mean, Pi the effect of period i, Tj the effect of diet j, sk the effect of sheep k and Eijk the residual error. Period and diet were considered fixed effects, using the ‘Repeated’ statement to account for within-animal time-dependent correlations. The optimal covariance structure was assessed for each dependent variable with attention to the corrected Akaike’s information criterion. Degrees of freedom were estimated by using the formula of Satterthwaite. In two out of four periods, the animals refused to eat some of the SW1 supplement. The data from these sheep were excluded from the data analysis (n = 14). Differences between least squares means of response variables were estimated with Tukey’s test. Significance was declared at P ≤ 0.05 and trends at 0.05