Lupinus angustifolius seed meal supplemented to dairy cow diet improves fatty acid composition in milk and mitigates methane production

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Animal Feed Science and Technology

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Lupinus angustifolius seed meal supplemented to dairy cow diet improves fatty acid composition in milk and mitigates methane production

Magdalena Bryszak

^a

, Malgorzata Szumacher-Strabel

^a

, Haihao Huang

^a

, Piotr Pawlak

^b

, Dorota Lechniak

^b

, Paweł Kołodziejski

^c

, Yulianri Rizki Yanza

^a

, Amlan Kumar Patra

^d

, Zora Váradyová

^e

, Adam Cieslak

^a,

*

aDepartment of Animal Nutrition, Poznan University of Life Sciences, Wolynska 33, 60-637 Poznan, Poland

bDepartment of Animal Physiology and Biochemistry, Poznan University of Life Sciences, Wolynska 35, 60-637 Poznan, Poland

cDepartment of Genetics and Animal Breeding, Poznan University of Life Sciences, Wolynska 33, 60-637 Poznan, Poland

dDepartment of Animal Nutrition, West Bengal University of Animal and Fishery Sciences, Belgachia, K.B. Sarani 37, Kolkata 700037, India

eInstitute of Animal Physiology, Centre of Biosciences of Slovak Academy of Sciences, Šoltésovej 4-6, 040 01 Košice, Slovak Republic

A R T I C L E I N F O Keywords:

Blue lupine Ruminal fermentation Methane

Milk yield Fatty acid profile

A B S T R A C T

Recently, the narrow-leafed, blue lupine (Lupinus angustifolius) seeds, including the Tango variety, have been used as a potential feed of livestock animals. However, there is still a limited knowledge of possible action of the Tango lupine seed meal (LSM) on ruminal fermentation, enteric methane production, milk yield and composition in dairy cattle. Thus, the present study aimed to investigate the effect of LSM supplementation to dairy cows on ruminal enteric methane production, microbial population, ruminal biohydrogenation and fatty acid (FA) proportion in the ruminal fluid and milk. Anin vitrotrial comprising of control and five experimental groups (supplementation of LSM at 20, 40, 60, 80, and 100 g/kg diet) was conducted to determine an effective dose level for anin vivoexperiment. Thein vitrotrial was followed by anin vivoex- periment conducted on 60 high-yielding Polish Holstein Friesian dairy cows assigned into two groups: the control (n = 30) and the experimental LSM group (n = 30; 2 kg LSM/day/cow).

Supplementation of LSM decreased methane emission and archaeal abundancesin vitroandin vivoand increased ammonia levelin vivo. Ruminal total volatile fatty acid concentration and acetate proportion were reduced by LSM supplementation bothin vitroandin vivo. Milk yield and its basic composition were not influenced by the LSM. However, the LSM increased the un- saturated FA content and decreased n6 to n3 FA ratio in milk. Supplementation of LSM upre- gulated the mRNA expressions ofFASNandELOVL5genes and downregulated theLPLgene expression in milk somatic cells. The daily cost of LSM diet and the cost of 1 kg of milk production

https://doi.org/10.1016/j.anifeedsci.2020.114590

Received 10 October 2019; Received in revised form 9 June 2020; Accepted 10 June 2020

Abbreviations: ACACA, acetyl-CoA carboxylase 1; BCS, body condition score; BH, biohydrogenation; CLA, conjugated linoleic acid; CP, crude protein; DI, desaturation index; DM, dry matter; DMI, dry matter intake; dNTP, deoxyribonucleotide triphosphate; ECM, energy corrected milk; EE, ether extract;ELOVL5, fatty acid elongase 5 (elongase 5);FADS1, fatty acid desaturase 1 (Δ5-desaturase); FA, fatty acids; FAME, fatty acids methyl ester;FASN, fatty acid synthase; FISH, fluorescencein situhybridization; GC, gas chromatograph; IVDMD,in vitrodry matter digestibility; IVOMD,in vitroorganic matter digestibility; LA, linoleic acid; LCFA, long chain fatty acids;LPL, lipoprotein lipase; LSM, lupine seeds meal; MCFA, medium chain fatty acids; MUFA, monounsaturated fatty acids; NDF, neutral detergent fiber; NSP, non-starch polysaccharide; OM, organic matter; PCR, polymerase chain reaction; PUFA, polyunsaturated fatty acids; RA, rumenic acid; RNA, ribonucleic acid; RT, reverse transcription; SA, stearic acid;

SCD, stearoyl-CoA desaturase (Δ9-desaturase); SFA, saturated fatty acids; SNF, solid non-fat; UFA, unsaturated fatty acids; TMR, total mixed ration;

VA, vaccenic acid; VFA, volatile fatty acids

⁎Corresponding author.

E-mail address:adam.cieslak@up.poznan.pl(A. Cieslak).

Available online 15 June 2020

0377-8401/ © 2020 Published by Elsevier B.V.

T

were lower compared with the control. It is concluded that LSM supplementation may improve milk quality without affecting milk yield and has the potential to decrease methane production in dairy cows.

1. Introduction

Methane (CH4) production is responsible for significant proportion of energy losses of total dietary energy that could affect ruminant productivity such as milk production. Moreover, the contribution of enteric CH₄emission from the ruminants has been estimated at about 17 % of the global greenhouse gas outputs (Knapp et al., 2014). Therefore, dietary intervention is required to mitigate methane production and improve the livestock production.

Poland is the highest lupine producer in the European region and the second highest lupine producer in the world after Australia (FAO, 2018). Lupine is a rich source of protein and -also contains non-starch polysaccharides (NSP), fats and several alkaloids (van Barneveld, 1999;Stanek et al., 2015). Due to its high protein content, lupine can improve productivity of ruminants (Ephrem et al., 2015). A few lupine species (e.g. Lupinus albus,Lupinus luteus, andLupinus angustifolius) were commonly used in the animal diets. In recent years, narrow- leaved blue lupine (L. angustifolius) has been widely introduced as a potential feed source for livestock animals. Blue lupine seeds contain 330 g/kg DM of protein and 400 g/kg DM of NSP (Gdala and Buraczewska, 1996;Księżak et al., 2018). Studies on lupine as a source of protein and NSP are of growing interest due to the positive effect on production and biological value of animal products (van Barneveld, 1999;Księżak et al., 2018). Up to date, the dietary use of lupine in ruminants has been mainly investigated for its protein content and supplement (Valentine and Bartsch, 1986;May et al., 1993). Along with milk production performance, investigation on milk quality and environmental assessment (for example, enteric CH4 production) is pertinent for sustainable dairy production. Milk fatty acid (FA) composition containing greater proportions of polyunsaturated FA (PUFA), especially n-3 FA, is beneficial for human health (Nguyen et al., 2019). However, there are only few studies on the effect of lupine,L. albusvariety, on methane production and FA content in animal products (Masucci, et al., 2006;Staerfl et al., 2012).L. angustifoliusseeds contain higher amount of NSP (non-starch polysaccharide) composed mostly of non-cellulosic polymer and pectic polysaccharides compared with the others lupine varieties (van Barneveld, 1999;

Table 1

The feed ingredients, chemical composition, and fatty acid content in the experimental diet^a.

Item TMR LSM TMR + LSMin vivo

Ingredient composition, g/kg DM

Grass silage 325 – 317

Maize silage 227 – 221

Beet pulp 104 – 102

Brewer’s grains 60 – 58

Lupine seed meal – – 94

Wheat 117 – 91

Extracted rapeseed meal 95 – 70

Extracted soybean meal 72 – 47

Chemical composition, g/kg DM

Organic matter 916 965 916

Ash 83.7 35 84.1

Crude protein 189 345 185

Neutral detergent fiber (amylase treated) 355 202 381

Ether extract 31.5 40 26.6

Fatty acids (FA), g/100 g FA

C14:0 1.75 0.73 1.42

C16:0 25.4 11.2 26.1

C18:0 5.2 4.24 4.4

C18:1,cis-9 14.2 26.5 14.3

C18:2,cis-9,cis-12 25.8 31.8 26.3

C18:3,cis-9,cis-12,cis-15 4.16 6.58 4.02

Other FA^b 23.8 19.0 23.5

SFA 35.9 20.0 35.2

UFA 64.1 80.0 64.8

MUFA 30.4 37.4 30.4

PUFA 33.7 42.6 34.4

n-6 FA 31.5 39.6 32.5

n-3 FA 28.6 37.1 28.7

a TMR: total mixed ration; LSM: lupine seed meal; DM: dry matter; SFA: saturated fatty acids; UFA: unsaturated fatty acids; MUFA: mono- unsaturated fatty acids; PUFA: polyunsaturated fatty acids; LNA: α-linolenic acid; LA: linoleic acid; MCFA: medium chain fatty acids; LCFA: long chain fatty acids.

b Other FA include C12:0, C14:1, C16:1, C18:1cis-11, C18:1cis-15, and C20:3n-6.

Petterson et al., 1997). There is no literature data regarding the direct effect of NSP on methanogenesis in ruminants. It was, however, demonstrated that lupine seed supplementation reduced ruminal protozoa populations in dairy cows (Hynd et al., 1985), which may indirectly decrease methanogenesis by lowering protozoa-associated methanogens and their activities (Patra et al., 2017). We hypothe- sized that feeding ofL. angustifoliusTango seeds as a partial protein supplement may also reduce enteric methane production and modulate the fatty acid proportions in the ruminal fluid and milk of dairy cows altering ruminal microbial populations due to the presence of NSP.

Thus, the present study was conducted to investigate the effect of a diet supplemented withL. angustifoliusTango seed meal on the ruminal methane production, microbial population as well as milk production and the fatty acids proportion in dairy cows.

2. Materials and methods 2.1. Diets

The diet was composed of hay silage, maize silage, beet pulp, brewery, wheat, rapeseed meal, and soybean meal as main in- gredients of total mixed ration (TMR;Table 1). TheL. angustifoliusTango seeds were collected from the Department of Production and Seed Breeder, Smigiel city, Koscian province, Poland. The dry lupine seeds were crushed and loaded into an extractor to obtain pelleted lupine seeds meal (LSM). The LSM then was analyzed for the chemical composition (Table 1).

The carbohydrate content (g/kg dry matter) inL. angustifolius Tango seed was as follows: non-starch polysaccharide (321), Rhamnose (2.26), Arabinose (37.0), Xylose (28.7), Mannose (6.60), Galactose (103), Glucose (104), Uronic acids (40.0).

2.2. Experimental design and treatments 2.2.1. In vitro experiment

The study was carried out using anin vitrobatch culture system where the LSM was included as a partial substrate in the diet. Six treatment groups were used in the experiment: a control group with total mixed ration (TMR), and five experimental groups in which the TMR was replaced with 20, 40, 60, 80 and 100 g/kg of LSM (DM basis). Thein vitroexperiment had 5 repetitions in each group (6 groups × 5 bottles) and was completed in 3 consecutive runs (in different days) with 24 h of incubation for each runs.

Briefly, the fresh ruminal fluid was collected from a slaughter house from 9 Polish Friesian Holstein dairy cows (three cows in each run). The cows were fed the same TMR that was used in thein vitrotrial (Table 1). The animals were fed with a total of 21 kg of DM every day and the diets were served twice a day at 06:00 and 18:00 h. Collected fresh ruminal fluid was taken from different parts of the rumen and squeezed through a four-layer cheesecloth into two Schott Duran bottles maintained at 39 °C with the anaerobic condition and immediately transported to the laboratory. Ruminal fluid was then put into the water bath at 39 °C. The equal volumes of ruminal fluid from each cow were mixed in a beaker and diluted with an artificial buffer solution according toSzumacher-Strabel et al. (2011). The buffered ruminal fluid was then transferred (40 mL) into 125-mL glass incubation bottles (Midland Scientific Inc., Omaha, NE) containing 400 mg of the substrate (DM basis). The substrate was ground to 1 mm (Retsch ZM 200, Haan, Germany). The bottles were then filled with CO2, and closed with rubber stoppers, incubated in an incubator (Galaxy 170R, Eppendorf North America Inc., Hauppauge, NY) for 24 h at a temperature of 39 °C in an anaerobic condition and periodically mixed. After 24 h ofin vitrofermentation, total gas production from each bottle was measured by a calibrated Hohenheim 100-mL gas-tight syringe (Häberle LABORTECHNIK GmbH + Co., Lonsee-Ettlenschieß, Germany) fitted with a needle to the rubber stopper of the fermentation bottle. For methane concentrationin vitro, at the same time, 500 μL (GASTIGHT Syringes, Hamilton Bonaduz AG, Switzerland) of gas sample was collected from the headspace of each bottle.

2.2.2. In vivo experiment

Thein vivoexperiment was designed based on thein vitrofindings, mainly on the results demonstrating reduction of the methane production and higher concentration of unsaturated fatty acid (UFA) in buffered rumen fluid after 24 h of incubation. For this reason, the highest concentration of LSM (100 g/kg diet) was selected for furtherin vivostudies.In vivoexperiment was carried out for 60 days from January to March 2016 (54 days of adaptation and 6 days of sampling). A total of 60 multiparous Polish Friesian-Holstein lactating dairy cows (580 ± 40 kg of body weight; 5−6-month of lactation) were randomly allocated to 2 dietary groups (control versusLSM; 30 animals in each group). The same TMR diet was used as in the Experiment 1, however, in the LSM group, 1 kg of wheat, 0.5 kg of rapeseed meal, and 0.5 kg of soybean meal were replaced with 2 kg/d of LSM. The composition of the diet used in the experiment is presented inTable 1. Samples of fresh TMR and LSM were collected 4 times a week and immediately delivered to the laboratory to perform analyses. In order to avoid the confounding effects of DM intake (DMI), the feeding schedule was designed as described byvan Zijderveld et al. (2011)with some modifications. Briefly, fresh TMR was offered daily as equal meals at 06:00 and 18:00 h. During the first 30 days of the experiment, the cows were fed TMRad libitum. From day 30 onwards, the cows were provided TMR restricted to 95 % of the average daily voluntary DMI based on day 20–30 of the cows assigned to each treatment. The daily DMI was maintained at 21.0 ± 0.4 kg/animal in both groups. Drinking water was offeredad libitum. Cows were milked twice daily at 05:30 and 17:30 h and daily milk production was measured using a milk meter (WB Ezi-Test Meter 33 kg; Tru-Test Limited, Manukau, New Zealand). Milk samples were taken at the 54–59 days of the experiment. Milk samples were collected at each milking, and daily morning and evening milk samples were pooled for each cow. The milk samples were stored at -20 °C until analysis. Ruminal fluid was collected at the last day (60th) of the experiment using rumenocentesis technique during the routine monitoring procedure (acidosis) carried out on the farm. Ruminal fluid samples were collected from the ventral sack, as described byNordlund and Garrett (1994). The pyrogen-free needles (2.0 × 120 mm) and 50-mL sterile syringes were used for ruminal fluid collection, 3 h after morning feeding. Fifteen cows from each group were randomly selected to monitor acidosis. Body condition score (BCS) was

determined on the last two days of the experiment (59th and 60th) for all 60 cows. Determination of BCS was done separately for each day by the same person experienced in conducting this assessment following the protocol prepared byEdmonson et al. (1989).

From 1 (thin) to 5 (fat) points scale system with increments of 0.25 units was used for BCS determination.

2.3. Data collection and laboratory analysis 2.3.1. Chemical composition of feeds

The chemical composition of samples of TMR and LSM was analyzed following the procedures ofAOAC (2007)method no. 934.01 for dry matter (DM), method no. 942.05 for ash, method no. 976.05 for crude protein (CP) using Kjel-Foss Automatic 16,210 analyser (Foss Electric, Hillerod Denmark), and method no. 973.18 for ether extract (EE) using a Soxhlet System HT analyser (Tecator, Hoganas, Sweden).

Organic matter (OM) content was calculated subtracting ash concentration from DM content. Ash-free neutral detergent fiber (aNDFom) content was determined following theVan Soest et al. (1991)method with the addition of α-amylase and sodium sulfite without residual ash. Non-starch polysaccharide content was determined by gas-liquid chromatography (component-neutral sugars) and colorimetrically (uronic acids). The procedure for determination of neutral sugar content was described bySlominski and Campbell (1990).

2.3.2. Determination of ruminal fermentation characteristic

In both experiments, pH was measured immediately after sample collection using a pH meter (Type CP-104, Elmetron, Zabrze, Poland).

The ammonia concentration was measured according to Nessler calorimetric method as described bySzumacher-Strabel et al. (2011). For determination ofin vitrodry matter digestibility (IVDMD), the contents of the incubation flasks were transferred to previously weighed crucibles at the end of incubation. The residues were washed with distilled water thoroughly and dried at 105 °C for 3 d. The loss in weight of the feed DM in microbial ruminal fermentation process was expressed as IVDMD.In vitroorganic matter digestibility (IVOMD) was determined by the loss in weight of the incubated fed OM. The volatile fatty acid (VFA) concentrations were determined following: the 3.6 mL of ruminal fluid samples were preserved immediately after collection with 0.4 mL of HgCl2solution (46 mM) and stored at −20 °C until analysis. VFA samples were analyzed using gas chromatograph (GC Varian CP 3380, Sugarland, TX, USA) equipped with a capillary column (30 m × 0.25 mm; Agilent HP-Innowax, 19091N-133, Agilent Technologies, Santa Clara, CA) and a flame ionization detector. The concentrations of VFA were determined individually using an external VFA standard prepared by mixing individual VFA (Sigma-Aldrich, St. Louis, MO, USA). Methane and hydrogen concentrations in thein vitroexperiment were measured by a gas chromatograph (GC; SRI Peak Simple model 310 Alltech, PA, USA) using an appropriate gas standard (mix gases of 5.63 % CO₂, 5.56 % CH₄, 5.0/d10 % H₂and rest N2(Multax, Zielonki-Parcela, Poland) using PeakSimple ver. 3.29 (Szumacher-Strabel et al., 2011). Nitrogen was used as the carrier gas at a constant flow of 30.0 mL/min. The oven temperature was programmed as follows: initially 180 °C for 1.5 min, then increasing at 20 °C/

min to 220 °C. The GC was equipped with a thermal conductivity detector and a Carboxen 1000 column (mesh side 60/80, 15 FT × 1.8 INS.S, Supelco, Bellefonte, USA) (Szumacher-Strabel et al., 2011). Methane production in thein vivostudy was calculated based on stoichiometric relationships between VFA composition and methane production proposed byMoss et al. (2000)considering 90 % of hydrogen recovery. Stoichiometry model for estimating CH4from VFA composition was as follow:

CH4(mmol) = 0.45 acetate (mmol) – 0.275 propionate (mmol) + 0.40 butyrate (mmol).

2.3.3. Fatty acid analysis

The FA concentrations in milk and ruminal fluid were determined using a gas chromatograph (Szczechowiak et al., 2016). Briefly, 3 mL of 2 M NaOH solution was added to 2.500, 100, and 500 mg of ruminal fluid, feed, and milk samples, respectively, in screw-cap Teflon-stoppered glass tubes (15 mL) for hydrolysis of fats. Hydrolyzed FA samples were incubated on a block heater at 90 °C for 40 min.

Samples were then extracted and trans-esterified using 0.5 M NaOH in methanol boron tri-fluoride (Fluka, Sigma-Aldrich, Darmstadt, Germany) to fatty acid methyl esters (FAME). Analysis of FAME was performed on a gas chromatograph (GC Bruker 456-GC, Billerica, MA, USA) equipped with a capillary column (100-m fused-silica, 0.25 mm i.d., 0.25 μm film thickness; Chrompack CP7420, Agilent HP, Santa Clara, CA, USA) and a flame ionization detector using 1 μL of sample injected into the column. Temperatures in injector and detector were maintained at 200 and 250 °C, respectively. The oven temperature was initially set at 120 °C for 7 min, then increased at a rate of 7 °C/min to 140 °C, held for 10 min, and then increased at a rate of 4 °C/min to 240 °C. Hydrogen gas was used as a carrier gas at a flow rate of 1.3 mL/min. Fatty acids were identified and quantified with an appropriate FAME standard (37 FAME Mix, Sigma-Aldrich, Darmstadt, Germany). The concentrations of conjugated linoleic acid (CLA) were determined using a CLA standard (a mixture ofcis- and trans9,11 and 10,12-octadecadienoic acid methyl esters; Sigma-Aldrich) using a Galaxie Work Station 10.1 (Varian, Walnut Creek, CA USA). The desaturase index, atherogenic index, thrombogenic index, were calculated as described byBryszak et al. (2019)and energy values of milk were estimated using the appropriate equations of energy corrected milk (Nielsen et al., 2009).

2.3.4. Gene expression analysis

For gene expression in milk somatic cells, RNA was isolated from 10 m L-milk samples frozen in liquid nitrogen. Gene expression was analyzed only in thein vivoexperiment and was based on relative transcript abundance measured by real time PCR (polymerase chain reaction). After thawing at 4 °C, the samples were centrifuged in 15 mL tubes for 10 min at 3000g. The supernatant was discarded, and the pellet was dissolved in 1 mL of TriPure reagent (Roche Diagnostics, Basel, Switzerland) following the standard procedure provided by the manufacturer. The RNA was resuspended in DEPC treated water. A reverse transcription reaction (RT) was performed using a

Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s protocol. Each sample contained equal concentrations of RNA measured on Nanodrop c2000 (Thermo Fisher, Sunnyvale, CA, USA). Briefly, RNA (500 ng), random hexameters, oligodT (60 μM and 2.5 mM respectively) were denatured at 65 °C for 10 min. Next, reverse transcriptase, RNAse inhibitor, dNTP, and buffer were mixed and added to the RNA to a final volume of 20 μL. The RT conditions were as follows: 25

°C for 5 min, followed by 42 °C for 45 min and 85 °C for 5 min. mRNA expressions of six genes encoding enzymes regulating FA metabolism [Acetyl-CoA carboxylase 1 (ACACA), fatty acid synthase (FASN), lipoprotein lipase (LPL), stearoyl-CoA desaturase (SCD), fatty acid desaturase 1 (FADS1) and fatty acid elongase 5 (ELOVL5)] were measured in milk somatic cells using previously published primer pairs (Szczechowiak et al., 2016). Relative mRNA expression was calculated using two reference genes and standard curves prepared for every examined gene. Each gene was analyzed in technical duplicates in a Light Cycler 480 instrument (Roche Diagnostics, Basel, Switzerland) using a Light Cycler 480 Sybr Green I Master (Roche Roche Diagnostics, Basel, Switzerland).

2.3.5. Microbial quantification and analysis

The samples for microbial quantification and analysis were collected after 24 hin vitroincubation and 3 h after morning feedingin vivo. Protozoa population of each sample was quantified in a drop where 1 mL rumen fluid was mixed with 6 mL of 4 % formalin (Michalowski et al., 1986) using the Zeiss Primo Star no. 5 (Zeiss, Jena, Germany), under 100× magnification. The size of the drops (10 μL for the entodiniomorphids and 100 μL for holotrichs) depended on the type of protozoa counted according to the methodology described byWilliams and Coleman (1997). The obtained results were then converted considering dilutions used.

For bacterial quantification in thein vivoexperiment total DNA of each ruminal fluid sample was extracted using QIAamp DNA Stool mini kit (Qiagen GmbH, Hilden, Germany). The DNA concentration was measured with a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, USA). Bacteria suchAnaerovibrio lipolytica(Gudla et al., 2012)Ruminococcus albus(Wang et al., 2004),Fibrobacter succinogenes(Denman and McSweeny, 2006),Butyrivibrio fibrisolvens(Li et al., 2009),Ruminococcus flavevaciens (Koike and Kobayashi, 2001), and total bacteria (Maeda et al., 2003) were quantified using the standard primers. The specificity of primers was confirmed using the primer-BLAST program in the GenBank Database. The quantification of each bacterial species and total bacteria was performed with a QuantStudio 12 Flex PCR system (Life Technologies, Grand Island, NY, USA). Methanogens were quantified using the fluorescencein situhybridization (FISH) technique as described bySzczechowiak et al. (2016).

2.3.6. Analysis of chemical composition of milk

Composite milk samples were analyzed for basic milk constituents using an infrared analyzer (Milko-Scan 255 A/S N; Foss Electric, Hillerød, Denmark). Urea concentration in milk was determined in an accredited milk quality laboratory by means of infrared spectrometry using a CombiFoss 6000 analyzer (Foss Electric, Hillerød, Denmark).

2.4. Feed cost calculation

The FeedExpert (Rovecom, Hoogeveen, The Netherlands) software was used to calculate dairy cows feeding costs. The cost was calculated on the basis of current prices during thein vivoexperiment.

2.5. Statistical analysis

All data were analyzed using SAS statistical software (Univ. Edition, version 9.4). In Experiment 1, all data were analyzed using one-way ANOVA model with PROC GLM procedure, where levels of the LSM used in the diet were considered as a fixed factor and each run was considered as a random factor. The different levels of LSM included in the diet were then tested with orthogonal contrasts (linear and quadratic responses) followed by Tukey’s post hoc test. Significant differences among groups were determined at P < 0.05. In Experiment 2, results were tested with independentt-test where means of both groups were compared through PROC TTEST procedure. Means between the controlversusLSM group were considered as significantly different at P < 0.05 or tended to be significant at 0.05 <p≤ 0.10. All values are shown as the means with pooled standard errors of means.

3. Results

L. angustifoliusTango seeds contained high protein (345 g/kg DM) and moderate NDF (202 g/kg DM) contents (Table 1). For the FA composition, LSM contained higher proportion of UFA, especially oleic and linoleic acids (approximately 26.5 g/100 g FA and 31.7 g/100 g FA). For carbohydrate composition, the LSM had high proportion of NSP (321 g/kg DM), as well as galactose and glucose (103 and 104 g/kg DM, respectively).

3.1. In vitro experiments

In Experiment 1, changes in ruminal fermentation parameters were LSM dose dependent (Table 2). Ruminal ammonia con- centrations of 40 and 80 g/kg of LSM groups were lower compared with the control group (P = 0.01). The ruminal ammonia concentration results indicated a quadratic response due to the level of LSM supplementation (P = 0.02) with the lowest con- centration at 80 g/kg of LSM. Total gas production was lower only in 20 g/kg of LSM group compared with the control group.

Compared with the control, LSM supplementation linearly decreased methane production (up to about 17 %; P < 0.01). The methane production expressed per gram DM orin vitrodigested dry matter orin vitrodigested organic matter decreased linearly (P < 0.01).

Total VFA as well as the individual VFA concentrations (acetate, butyrate, isovalerate and valerate) and acetate to propionate ratio linearly decreased (P < 0.01) due to LSM supplementation. Butyrate, isovalerate and acetate to propionate ratio showed quadratic response (P < 0.01). Propionate and isobutyrate concentration increased with a linear and a quadratic response (P < 0.01).

Total bacterial populations of 20 and 40 g/kg of LSM groups were higher than the control and other LSM groups (Table 2). In 8 % and 100 g/kg LSM groups, lower bacterial population was detected compared with the control group. Archaea populations in 20, 40, 80 and 100 g/kg of LSM groups were lower than the control, and showed a linear response (P = 0.02). Total protozoa, entodiniomorphids, and holotrichs protozoa decreased linearly (P < 0.05). The decreased protozoa population was noted at 40 g/kg of LSM compared with the control, which was resulted from decreased entodiniomorphids, while the holotrich protozoa decreased at 100 g/kg of LSM diet.

Many FA concentrations in the ruminal fluid were altered due to LSM supplementation (Table 3). The C12:0 proportions in 20, 40 and 80 g/kg of LSM groups were higher compared to the control group following the quadratic response (P = 0.04), while C14:0 and C16:0 proportions were lowered i quadratic manners, especially when supplemented with the maximum level of LSM in the diet (P < 0.01). The proportions of UFA in the LSM groups were higher than in the control group (P < 0.01) with a quadratic response.

The improved UFA proportions of LSM groups were contributed by increased monounsaturated fatty acids (MUFA) and poly- unsaturated fatty acids (PUFA) with quadratic responses (P < 0.01). Proportions of C18:1trans-9and C18:1trans-10 was higher when LSM was supplemented (P < 0.01). The proportions of C18:1cis-9 and C18:1cis-11 in LSM groups were higher than the control group with quadratic responses (P < 0.01). For other PUFA proportion, C18:3cis-9cis-12cis-15 and C18:2cis-9cis-12 proportion were dose dependent (P < 0.01, respectively) showed by their linear responses.

3.2. In vivo experiments

In Experiment 2, the pH in the LSM groups was higher than in the control group (P = 0.05;Table 4). The methane concentration was estimated to be lower in LSM groups than in the control (P < 0.03). Supplementation of LSM significantly decreased total VFA concentrations (P = 0.03) and acetic acid proportion (P = 0.05), whereas isobutyrate, isovalerate and valerate proportions were increased by LSM. Total bacteria, archaea,Methanobacteriales, andMethanomicrobialespopulations were lowered due to the LSM supplementation in the diet (P < 0.05;Table 4). The abundances ofA. lipolytica,F. succinogenes, andR. albusin the LSM groups were significantly lower compared with the control (Fig. 1).

Many changes in FA proportions in ruminal fluid were noted due to LSM included in the diet (Table 5). Ruminal C10:0, C12:0, C14:0, C18:1trans-9 and C18:1trans-11 FA proportions were lower in LSM groups than in the control group (P < 0.05). In contrast, Table 2

The effect ofLupinus angustifoliusTango seed meal supplementation on ruminal fermentation characteristics and microbial populationsin vitroafter 24 h incubation (Experiment 1).

Parameters Control LSM, g/kg diet SEM P value L Q

20 40 60 80 100

pH 6.35^ab 6.40^a 6.37^ab 6.37^ab 6.35^b 6.33^b 0.005 < 0.01 0.01 0.01

NH3,mM 11.8^a 11.2^ab 10.4^b 10.9^ab 10.1^b 10.9^ab 0.15 0.01 0.01 0.02

IVDMD 0.61 0.59 0.59 0.60 0.60 0.63 0.05 0.38 0.34 0.06

IVOMD 0.65 0.62 0.61 0.63 0.64 0.66 0.06 0.16 0.20 0.03

CH4, mmol/bottle 0.87^a 0.80^b 0.81^b 0.79^b 0.73^c 0.72^c 0.01 < 0.01 < 0.01 0.82

H2, mmol/bottle 0.08^a 0.06^b 0.06^ab 0.07^ab 0.08^ab 0.07^ab 0.0015 < 0.01 0.22 0.14

Total gas, mL/g DM 300^a 293^b 295^ab 297^ab 297^ab 296^ab 0.65 0.02 0.60 0.08

CH4, mmol/g DM 2.17^a 2.01^b 2.03^b 1.98^b 1.83^c 1.80^c 0.02 < 0.01 < 0.01 0.83

CH4/total gas, mmol/L 7.21^a 6.86^b 6.74^b 6.66^b 6.20^c 6.13^c 0.05 < 0.01 < 0.01 0.47

CH4/IVDMD, mmol/g 3.53^a 3.39^a 3.43^a 3.27^ab 3.04^bc 2.87^c 0.06 < 0.01 < 0.01 0.17

CH4/IVOMD, mmol/g 3.34^a 3.25^ab 3.32^a 3.10^bc 2.87^c 2.72^c 0.06 < 0.01 < 0.01 0.04

Total VFA, mM 47.2^a 45.0^b 43.5^bc 42.1^bc 41.3^c 42.7^bc 0.39 < 0.01 < 0.01 0.05

VFA proportion, mol/100 mol

Acetic, (A) 57.2^ab 58.2^a 58.1^a 56.2^bc 54.9^c 52.9^c 0.55 < 0.01 < 0.01 0.37

Propionic, (P) 26.4^d 27.1^c 27.3^c 29.5^b 30.6^b 32.3^a 0.22 < 0.01 < 0.01 < 0.01

Isobutyric 0.45^c 0.38^d 0.37^d 0.38^d 1.14^b 1.38^a 0.13 < 0.01 < 0.01 < 0.01

Butyric 12.4^a 10.9^b 11.0^b 10.9^b 10.4^b 10.5^b 0.09 < 0.01 < 0.01 < 0.01

Isovaleric 1.19^a 1.02^c 1.10^b 1.08^b 1.04^c 1.05^c 0.05 < 0.01 < 0.01 0.01

Valeric 2.36^a 2.42^a 2.11^b 1.92^c 1.87^c 1.83 0.07 < 0.01 < 0.01 0.90

A/P ratio 2.16^a 2.15^a 2.13^a 1.90^b 1.79^bc 1.64^c 0.03 < 0.01 < 0.01 0.01

Total bacteria, × 10⁷/mL 8.66^b 11.7^a 12.4^a 9.63^b 6.85^c 5.57^c 0.29 < 0.01 < 0.01 < 0.01

Archaea, × 10⁶/mL 6.01^a 4.10^bc 3.97^bc 5.30^ab 3.61^c 4.31^bc 0.21 < 0.01 0.02 0.06

Total protozoa, × 10⁴/mL 4.20^a 3.58^ab 2.89^b 3.69^ab 3.34^ab 3.23^ab 0.105 0.03 0.04 0.12

Holotrichs,×10³/mL 0.94^ab 1.03^a 0.69^bc 0.81^ab 0.73^bc 0.44^c 0.034 < 0.01 < 0.01 0.20

Entodiniomorphids, ×10⁴/mL 4.11^a 3.48^ab 2.82^b 3.60^ab 3.27^ab 3.19^ab 0.104 0.03 0.05 0.11

LSM: lupine seed meal; IVDMD:in vitrodry matter digestibility coefficient; IVOMD:in vitroorganic matter digestibility coefficient; CH4: methane;

H2: hydrogen; DM: dry matter; VFA; volatile fatty acids; NH3: ammonia; SEM: standard error of means; L: Linear; Q: Quadratic; Different superscript letters in the same row are considered as significantly different (P < 0.05).

Table 3

The effect ofLupinus angustifoliusTango seed meal supplementation on ruminal fatty acid compositionin vitroafter 24 h incubation (Experiment 1).

Fatty acid, g/100 g FA Control LSM, g/kg diet SEM P-value L Q

20 40 60 80 100

Saturated fatty acids

C8:0 0.08 0.10 0.10 0.10 0.11 0.10 0.01 0.79 0.18 0.53

C10:0 0.09^ab 0.07^ab 0.10^a 0.08^ab 0.05^b 0.05^b 0.01 0.01 0.01 0.37

C12:0 0.98^c 1.58^ab 1.68^a 1.17^bc 1.48^ab 0.99^c 0.05 < 0.01 0.34 < 0.01

C13:0 9.76^a 7.05^bc 7.34^b 6.79^bc 6.95^bc 5.62^c 0.21 < 0.01 < 0.01 0.04

C14:0 3.20^a 3.31^a 3.25^a 3.08^ab 2.90^bc 2.73^c 0.03 < 0.01 < 0.01 < 0.01

C15:0 2.20^a 2.01^ab 1.99^b 1.95^b 1.89^b 1.94^b 0.02 < 0.01 < 0.01 0.02

C16:0 26.1^a 24.4^b 24.1^b 24.4^b 24.5^b 24.5^b 0.13 < 0.01 < 0.01 < 0.01

C17:0 0.93 0.88 0.89 0.90 0.86 0.88 0.01 0.48 0.14 0.46

C18:0 32.9 32.5 31.6 31.0 31.9 32.4 0.19 0.06 0.19 0.01

C20:0 0.08 0.06 0.08 0.07 0.05 0.05 0.004 0.11 0.03 0.50

C21:0 0.28^a 0.08^b 0.10^b 0.12^ab 0.10^b 0.11^b 0.02 < 0.01 0.01 0.01

C22:0 0.20^a 0.07^b 0.08^b 0.06^b 0.05^b 0.06^b 0.01 < 0.01 < 0.01 < 0.01

C23:0 0.13 0.12 0.11 0.11 0.12 0.13 0.004 0.72 0.81 0.12

C24:0 0.45^a 0.43^abc 0.45^ab 0.43^abc 0.34^c 0.36^ab 0.01 < 0.01 < 0.01 0.26

Monounsaturatedfatty acids

C14:1 0.73^ab 0.72^ab 0.74^a 0.70^ab 0.65^b 0.67^ab 0.01 0.04 0.01 0.44

C15:1 0.62^b 0.66^ab 0.71^ab 0.70^ab 0.66^ab 0.78^a 0.01 0.02 < 0.01 0.98

C16:1 0.65^a 0.35^b 0.37^b 0.32^b 0.32^b 0.31^b 0.03 < 0.01 < 0.01 0.01

C17:1 0.07^b 0.10^b 0.28^a 0.13^b 0.12^b 0.14^b 0.01 < 0.01 0.09 < 0.01

C18:1trans-6−8 0.69 0.55 0.55 0.63 0.67 0.58 0.02 0.02 0.69 0.16

C18:1trans-9 0.58^b 0.53^b 0.56^b 0.86^a 0.66^ab 0.70^ab 0.02 < 0.01 < 0.01 0.34

C18:1trans-10 1.23^c 1.48^bc 1.53^bc 1.72^b 2.12^a 2.16^a 0.05 < 0.01 < 0.01 0.68

C18:1trans-11 3.53 4.31 4.21 4.14 3.86 3.72 0.08 0.04 0.88 < 0.01

C18:1cis-9 5.38^b 7.48^a 7.81^a 8.12^a 7.81^a 8.26^a 0.20 < 0.01 < 0.01 < 0.01

C18:1cis-11 1.23^d 1.91^a 1.88^a 1.82^ab 1.60^bc 1.55^c 0.04 < 0.01 0.11 < 0.01

C18:1cis-12 0.34 0.36 0.40 0.38 0.36 0.36 0.01 0.10 0.79 0.01

C18:1cis-13 0.20^ab 0.18^ab 0.21^a 0.19^ab 0.14^b 0.15^ab 0.01 0.02 0.01 0.26

C18:1cis-14 0.42^b 0.49^ab 0.50^a 0.49^ab 0.49^ab 0.51^a 0.01 0.01 0.01 0.07

C20:1trans 0.55 0.53 0.54 0.57 0.56 0.56 0.01 0.69 0.25 0.94

C22:1n9 0.11^a 0.04^b 0.04^b 0.05^b 0.08^ab 0.10^ab 0.01 < 0.01 0.71 < 0.01

C24:1 0.06^ab 0.08^ab 0.10^ab 0.10^ab 0.05^b 0.11^a 0.01 0.03 0.14 0.76

Polyunsaturated fatty acids

C18:2cis-9cis-12 2.94^b 3.88^ab 4.08^ab 4.10^ab 4.07^ab 4.66^a 0.13 < 0.01 < 0.01 0.31

C18:3cis-9cis-12cis-15 0.07 0.09 0.08 0.09 0.11 0.11 0.01 0.07 < 0.01 0.70

c18:2cis-9trans-11 0.33^b 0.40^ab 0.55^a 0.47^ab 0.49^ab 0.46^ab 0.02 0.01 0.02 0.02

C18:2trans-10cis-12 0.15^b 0.22^ab 0.27^a 0.25^a 0.21^ab 0.23^ab 0.01 < 0.01 0.02 < 0.01

C18:3n6 0.42^c 0.84^b 0.96^ab 1.12^a 0.90^ab 0.96^ab 0.04 < 0.01 < 0.01 < 0.01

C20:2 0.12^a 0.04^b 0.06^ab 0.04^b 0.06^ab 0.04^b 0.01 < 0.01 0.01 0.05

C20:3n6 0.47^b 0.60^b 0.56^b 0.52^b 0.68^ab 0.92^a 0.03 < 0.01 < 0.01 0.06

C20:4n6 0.07^ab 0.09^a 0.05^ab 0.08^ab 0.05^b 0.05^b 0.004 0.01 0.01 0.39

C20:5n3 0.08^ab 0.05^b 0.09^ab 0.05^b 0.09^ab 0.14^a 0.01 < 0.01 < 0.01 < 0.01

C22:2 0.05 0.04 0.05 0.08 0.07 0.07 0.005 0.07 0.01 0.85

C22:5n3 0.18 0.14 0.19 0.16 0.11 0.26 0.02 0.38 0.39 0.23

C22:6n3 1.10^a 1.19^a 0.70^b 1.19^a 1.08^a 1.25^a 0.03 < 0.01 0.03 < 0.01

Sum of SFA 77.6^a 72.6^b 71.9^b 70.8^b 71.8^b 69.9^b 0.43 < 0.01 < 0.01 < 0.01

Sum of UFA 22.45^b 27.4^a 28.1^a 29.2^a 28.2^a 30.1^a 0.43 < 0.01 < 0.01 < 0.01

Sum of MUFA 16.4^b 19.7^a 20.4^a 21.1^a 20.2^a 20.9^a 0.27 < 0.01 < 0.01 < 0.01

Sum of PUFA 6.02^c 7.63^abc 7.33^bc 8.17^ab 7.97^ab 9.15^a 0.20 < 0.01 < 0.01 0.34

Sum of n-6 FA 4.28^b 5.82^a 5.89^a 6.28^a 6.16^a 7.02^a 0.17 < 0.01 < 0.01 0.08

Sum of n-3 FA 1.43^b 1.47^b 1.06^c 1.50^ab 1.42^b 1.77^a 0.04 < 0.01 < 0.01 < 0.01

n6/n3 FA ratio 3.21^b 3.77^b 6.49^a 4.26^b 4.37^b 4.02^b 0.16 < 0.01 0.07 < 0.01

Sum of n-6 PUFA 3.95 5.62 5.73 5.90 5.78 6.66 0.17 < 0.01 < 0.01 0.11

Sum of n-3 PUFA 1.43^b 1.47^b 1.06^c 1.50^b 1.42^b 1.77^a 0.04 < 0.01 < 0.01 < 0.01

PUFA/SFA ratio 0.08^a 0.11^ab 0.10^bc 0.12^ab 0.11^ab 0.13^a 0.003 < 0.01 < 0.01 0.26

LNA/LA ratio 0.03 0.02 0.02 0.03 0.03 0.03 0.001 0.19 0.25 0.18

Sum of C18:1 13.6^b 17.2^a 17.7^a 18.5^a 17.8^a 18.1^a 0.29 < 0.01 < 0.01 < 0.01

Sum oftransC18:1 6.03^b 6.78^ab 6.85^ab 7.49^a 7.36^a 7.18^a 0.11 < 0.01 < 0.01 0.02

Sum ofcisC18:1 7.55^b 10.4^a 10.8^a 11.0^a 10.4^a 10.9^a 0.22 < 0.01 < 0.01 < 0.01

Sum of MCFA 44.4^a 40.1^b 40.2^b 39.3^ab 39.7^ab 37.6^c 0.32 < 0.01 < 0.01 0.01

Sum of LCFA 55.5^c 59.8^b 59.6^b 60.5^ab 60.1^ab 62.2^a 0.32 < 0.01 < 0.01 0.01

LSM: lupine seed meal; SFA: saturated fatty acids; UFA: unsaturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; LNA: α-linolenic acid; LA: linoleic acid; MCFA: medium chain fatty acids; LCFA: long chain fatty acids; SEM: standard error of means; L:

Linear; Q: Quadratic; Different superscript letters in the same row are considered as significantly different (P < 0.05).

ruminal C18:0 proportions in the LSM groups were significantly higher than in the control group (P < 0.01). Increased ruminal C18:0 proportions mainly led to the higher SFA as well as the lower UFA proportion in LSM groups compared to the control (P < 0.01).

Milk yield as well as the fat, protein, urea and energy corrected milk content in the LSM group were similar to the control (Table 6). In contrast, lactose and solid non-fat content in milk tended to be higher in the LSM group than in the control group (P = 0.08). Body condition score (BCS) was significantly higher in the LSM cows (3.13vs2.87) compared with the control cows.

Feeding of LSM decreased (P < 0.05) C10:0, C12:0, C14:0, C16:0, C18:1trans-9, C18:1trans-10, and C18:2 cis-9trans-11 pro- portions in milk compared with the control (Table 7). Higher milk C18:0, C18:2trans-10 cis-11, C18:2 cis-9, and cis-12 proportions in the LSM group were noted (P < 0.01;Table 7). Feeding of LSM resulted in a higher UFA proportions in milk compared with the control, as well as higher total n-6 and total n-3 FA proportions (P < 0.05). Among the desaturase’s indices, LSM diets increased C16:1 desaturase index (P = 0.03;Table 7). Milk thrombogenic and atherogenic indexes were decreased by the LSM supplementation (P = 0.01; P < 0.01). Moreover, the LSM affected the relative transcript abundance of three out of six analyzed genes. Supple- mentation of LSM increased mRNA expressions of theFASNandELOVL5genes whereas theLPLgene was downregulated (Fig. 2).

The daily cost of LSM diet was lower by 4% compared with the control (€4.15vs. €4.31/cow/day respectively). The cost of 1 kg of milk production in LSM-feeding cows was lower than the control cows (€0.128vs. €0.132 per kg, respectively).

4. Discussion

Lupine seeds in animal diets have so far been mainly used due to the high protein content and moderate cost, which allows utilizing lupine seeds as a substitute of soybean meal (Gdala and Buraczewska, 1996;Abraham et al., 2019). It is well known that lupine seeds are rich in alkaloids and thus are unfavorable for animals, except for some lupine varieties with reduced alkaloid content (Zdunczyk et al., 2019). Seeds of the Tango variety of the blue lupine are characterized by a low alkaloid content that amounts up to 0.28 g/kg of the seed mass (Tomczak et al., 2018), and hence it can be a better alternative source of protein in animal nutrition.

Lupine, as mentioned before, is also a rich source of non-starch polysaccharides. Dietary LSM inclusion resulted in 4% cost reduction, which seems important in large scale dairy production.

4.1. Ruminal fermentation, microbial population, methane production and milk yield

The results of the present study validate our hypothesis that the Tango variety ofL. angustifoliushas potential to modulate ruminal fermentation leading to mitigation of enteric methane production and changing the FA proportions in the ruminal fluid and milk of dairy cows. However, thein vivoresults should be considered with caution due to the negative impact of NSP on other rumen parameters (e.g.a decrease in the total VFA). Tango variety LSM contains low level of alkaloids (0.28 g/kg). Intake of 2 kg LSM/cow per day supplying 0.56 g/d alkaloids seems to be to low to influence ruminal metabolism. In Holstein steers, addition of up to 6 g alkaloid extract in the diets did not adversely impact ruminal metabolism including nutient digestibility and VFA concentrations;

rather improved the efficiency of nitrogen utilization decreasing ruminal ammonia concentration and increasing the nonammonia nitrogen flow to the duodenum (Aguilar-Hernández et al., 2016).

To our knowledge, this is the first report documenting the potential of Tango LSM to mitigate methane production in dairy cows Table 4

The effect ofLupinus angustifoliusTango seed meal supplementation on ruminal fermentation and microbial populations in dairy cows (Experiment 2).

Parameters Control LSM SEM P value

pH 5.79 5.89 0.03 0.05

NH3,mM 5.37 8.45 0.30 < 0.01

CH4, mmol/L ruminal fluid¹ 8.83 6.47 0.43 0.01

Total VFA, mM 109.3 93.5 3.69 0.03

VFA proportion, mol/100 mol

Acetate (A) 48.7 47.1 0.42 0.05

Propionate (P) 31.4 31.3 0.49 0.89

Isobutyrate 0.86 1.05 0.03 < 0.01

Butyrate 14.9 15.0 0.19 0.83

Isovalerate 1.65 1.91 0.05 0.01

Valerate 2.31 2.58 0.05 < 0.01

A/P ratio 1.59 1.54 0.04 0.48

Total bacteria, ×10⁹/mL 12.4 3.80 0.97 < 0.01

Archaea, 10⁹/mL 3.60 0.94 0.47 < 0.01

Methanobacteriales,×10⁹/mL 2.26 0.81 0.26 < 0.01

Methanomicrobiales,×10⁹/mL 3.34 0.73 0.46 < 0.01

Total protozoa, ×10⁴/mL 6.80 6.37 0.15 0.14

Holotrichs, ×10³/mL 0.47 0.49 0.03 0.67

Entodiniomorphids, ×10⁴/mL 6.75 6.32 0.14 0.13

LSM: lupine seed meal;¹CH4: methane estimation (Moss et al., 2000); VFA: volatile fatty acids; NH3: ammonia; SEM: standard error of means.

Treatments are considered as significantly different at P < 0.05.