Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2012 10(1): 88-98 Available online at www.inia.es/sjar ISSN: 1695-971-X doi: http://dx.doi.org/10.5424/sjar/2012101-134-11 eISSN: 2171-9292

Chemical composition and digestibility of some browse species collected from Algerian arid rangelands S. Boufennara1, 2, S. Lopez1, 3, *, H. Bousseboua1, R. Bodas3 and L. Bouazza1, 2 1 Departamento de Producción Animal, Universidad de Léon, E-24007 León, Spain 2 Département de Microbiologie Appliquée, BP 360. Route de Ain El-Bey, Université Mentouri de Constantine, Algeria 3 Instituto de Ganadería de Montaña (CSIC-ULE), E-24346 Grulleros (León), Spain

Abstract 0DQ\ZLOGEURZVHDQGEXVKVSHFLHVDUHXQGHUYDOXHGPDLQO\EHFDXVHRILQVXI¿FLHQWNQRZOHGJHDERXWWKHLUSRWHQWLDO feeding value. The objective was to evaluate some nutritional attributes of various Algerian browse and shub species (Atriplex halimus, Artemisia campestris, Artemisia herba-alba, Astragalus gombiformis, Calobota saharae, Retama rae- tam, Stipagrostis pungens, Lygeum spartum and Stipa tenacissima). Chemical composition, phenols and tannins concentra- tion, in vitro digestibility, in vitro JDVSURGXFWLRQNLQHWLFVDQG in vitro bio-assay for assessment of tannins using buffered UXPHQÀXLGDQG in situ GLVDSSHDUHQFHRIWKHHGLEOHSDUWVRIWKHSODQWV OHDYHVWKLQWZLJVDQGÀRZHUV ZHUHGHWHUPLQHG,Q general, protein content in dicotyledon species was always greater than in grasses, these showing higher QHXWUDODQGDFLGGHWHUJHQW¿EUHDQGORZHUOLJQLQFRQWHQWVWKDQGLFRWV7KHWDQQLQFRQFHQWUDWLRQVYDULHGFRQVLGHUDEO\EHWZHHQ species, but in general the investigated in this study had low tannin contents (except for Artemisia spp. and S. tena- cissima). Monocots showed lower in vitro and in situ digestibilities, fermentation rate, cumulative gas production and extent of degradation than dicot species. The plants were clustered by principal components analysis in two groups: SRRUTXDOLW\JUDVVHVDQGWKHPRVWGLJHVWLEOHGLFRWVSHFLHV&KHPLFDOFRPSRVLWLRQ QHXWUDOGHWHUJHQW¿EUHDQGSURWHLQ DQG GLJHVWLELOLW\ZHUHWKHPDLQLQIOXHQWLDOYDULDEOHVGHWHUPLQLQJWKHUDQNLQJ,QFRQFOXVLRQA. halimus, A. campestris, A. herba-alba and A. gombiformis FDQEHFRQVLGHUHGRIJUHDWHUQXWULWLRQDOYDOXHWKDQWKHKLJKO\¿EURXVDQGORZGLJHVWLEOH grasses (S. pungens, L. spartum and S. tenacissima) that should be considered emergency roughages. Additional key words: chemical composition; forage; gas production; in vitro digestibility; tannins.

Resumen Composición química y digestibilidad de varias especies arbustivas características de pastizales en zonas áridas de Argelia El objetivo de este trabajo fue el de evaluar varias especies arbustivas de Argelia (Atriplex halimus, Artemisia cam- pestris, Artemisia herba-alba, Astragalus gombiformis, Calobota saharae, Retama raetam, Stipagrostis pungens, Lygeum spartum y Stipa tenacissima). Se determinó la composición química, la concentración de fenoles y taninos, la produc- ción de gas y digestibilidad in vitro y la degradabilidad in situ de la parte comestible del pasto arbustivo (hojas, tallos ¿QRV\ÀRUHV /RVFRQWHQLGRVHQSURWHtQD\OLJQLQDIXHURQVXSHULRUHVHQODVGLFRWLOHGyQHDVTXHHQODVPRQRFRWLOHGy- QHDVPLHQWUDVTXHORVFRQWHQLGRVHQ¿EUDIXHURQPiVHOHYDGRVHQODVPRQRFRWLOHGyQHDV/DFRQFHQWUDFLyQHQWDQLQRV fue variable entre especies y, excepto para Artemisia spp. y S. tenacissima, los contenidos de estos compuestos fueron exiguos en la mayoría de las especies. Las monocotiledóneas fueron menos digestibles, con menores valores de ritmo GHIHUPHQWDFLyQSURGXFFLyQGHJDV\GHJUDGDELOLGDGUXPLQDO$SDUWLUGHXQDQiOLVLVGHFRPSRQHQWHVSULQFLSDOHVVH observaron dos agrupaciones de las plantas: en un grupo las monocotiledóneas de baja calidad nutritiva y en otro gru- SRODVGLFRWLOHGyQHDVPiVGLJHVWLEOHV(VWHDJUXSDPLHQWRIXHGHWHUPLQDGRIXQGDPHQWDOPHQWHSRUODFRPSRVLFLyQ TXtPLFD ¿EUD\SURWHtQD \ODGLJHVWLELOLGDG(QFRQFOXVLyQA. halimus, A. campestris, A. herba-alba y A. gombiformis pueden ser consideradas de mejor calidad (considerando su composición y digestibilidad), mientras que S. pungens, L. spartum y S. tenacissima podrían considerarse como recursos de baja calidad que sólo serían utilizados cuando no hay disponibilidad de otros alimentos. Palabras clave adicionales: composición química; digestibilidad in vitro; forrajes; producción de gas; taninos.

* Corresponding author: [email protected] Received: 18-03-11. Accepted: 18-01-12 Chemical composition and digestibility of Algerian browse plants 89

Introduction annual herbaceous vegetation as fodder from these species is available at different times of the year, con- The problem of animal feed supply and quality is sidering their phenology and growth season (Delillis aggravated in arid, semi-arid and tropical regions with & Fontanella, 1992). Although these resources gain scarce and erratic rainfall that limits the growth of LQFUHDVLQJVLJQL¿FDQFHDVWKHQXWULWLRQDOYDOXHRIJUDVV herbaceous species and biomass yield in rangelands. drops, they never reach a prominent place in the diet, 7KXVOLYHVWRFNLQVXFKUHJLRQVKDYHWRVXUYLYHRQUH- EHFDXVHWKHLUORZ&3FRQWHQWDQGKLJK¿EUHFRQWHQWV FXUUHQWVKRUWDJHRIIHHGUHVRXUFHVRILQVXI¿FLHQWQX- and low digestibility (Wilson, 1977; Cabiddu et al., tritional value for most part of the year (Robles et al., 2000). Moreover, some of these plants contain anti- 2008). As an example, the crude protein (CP) content nutritional secondary compounds (phenolics, tannins) of herbaceous rangeland vegetation declines drasti- with potential adverse effects such as inhibition of cally during the dry season in semi arid regions, lead- rumen microbial fermentation, as well as decreased LQJWRSURORQJHGSHULRGVRIXQGHUQXWULWLRQRIOLYHVWRFN feed digestibility and animal performance (Min et al., raised under such adverse environmental conditions 2003; Waghorn & McNabb, 2003; Mueller-Harvey, (Yayneshet et al., 2009). In addition, uncontrolled and 2006). In spite of their limited nutritional value, these excessive use of increasingly scarce communal grazing forage resources are indispensable as feeds for herbiv- areas on dry rangelands has contributed to their degra- ores when production systems are based on grazing GDWLRQUHGXFLQJIXUWKHUWKHDYDLODELOLW\RIOLYHVWRFN rangelands (Papanastasis et al., 2008). feed resources. Considering all these aspects related 7KHREMHFWLYHRIWKLVZRUNZDVWRHYDOXDWHYDULRXV WROLYHVWRFNIHHGLQJLQGU\DUHDVWKHUHLVDQLQFUHDVLQJ browse and shrub species collected from an arid zone in LQWHUHVWLQWKHUDWLRQDOXWLOL]DWLRQRISRWHQWLDOOLYHVWRFN Algeria, based on the determination of their chemical feed resources such as browse species that are adapted composition, in vitro digestibility, in situ disappearance to these environments (Robles et al., 2008). Indige- and tannin concentration in the edible part of the plants, nous browse species are useful sources of animal considered as useful indicators for the preliminary evalu- feeds, as these plants remain green during the dry ation of previously uninvestigated feeding resources. season and provide vegetation with better nutritive value than other annual grass and herbaceous species that become withered (Aregawi et al., 2008). Indig- Material and methods enous shrub and bush species have also a potential to prevent desertification, mitigating the effects of Description of the study site droughts, allowing soil fixation and enhancing the restoration of the vegetation and the recuperation of The study was conducted in Bou Saâda district, north rangelands (Robles et al., 2008). central Algeria (35° 15.768’ N, 04° 13.885’ E), in the Rangelands in Algeria represent two-thirds of the Saharan Atlas region, at the northern edge of the Sa- total land widespread, mainly in the arid regions, where hara Desert between the Atlas Mountains and the el- browse species can be decisive for grazing ruminants +RGQDGHSUHVVLRQDQGVDOWODNH7KHDUHDLVDQDULGKLJK in periods of feed scarcity. However, in spite of their SODWHDX PDOWLWXGH ZLWKVWHSSHOLNHSODLQV abundance, many wild browse and bush species have and extensive barren soils. According to the Köppen been generally undervalued mainly because of insuf- FODVVL¿FDWLRQWKHFOLPDWHRIWKLVUHJLRQLVBWh (dry ¿FLHQWNQRZOHGJHDERXWWKHLUSRWHQWLDOIHHGLQJYDOXH desert climate), characterized by high temperatures Some of the ligneous species studied herein are con- ranging between 24 and 41°C, and scarce and erratic sumed to a certain degree by small ruminants grazing annual precipitations for a total of 350-700 mm. Under in Mediterranean rangelands (Kababya et al., 1998). these environmental conditions, the plant species stud- These forage species may be complementary to the ied show a slow vegetative growth and phenological

$EEUHYLDWLRQVXVHG$ DV\PSWRWLFJDVSURGXFWLRQ $') DFLGGHWHUJHQW¿EUHH[SUHVVHGZLWKUHVLGXDODVK $'/ DFLGGHWHUJHQW lignin); c (fractional rate of gas production); CP (crude protein); DM (dry matter); D144 (dry matter disappearance after 144 h of incubation); ED (extent of degradation); G24 (cumulative gas production at 24 h); IVD-TT (in vitro digestibility of Tilley & Terry); ivDMloss (in vitro dry matter loss); L ODJWLPH 1') QHXWUDOGHWHUJHQW¿EUHH[SUHVVHGZLWKUHVLGXDODVK 3&$ SULQFLSDOFRPSRQHQWV analysis); PEG (polyethylene glycol); TIVD (true in vitro digestibility); TCT (total condensed tannins); TEP (total extractable phenols); TET (total extractable tannins). 90 S. Boufennara et al. / Span J Agric Res (2012) 10(1): 88-98 development throughout most of the year, often lagged Ciolateau reagent and tannic acid as standard. Total in response to the infrequent major rainfalls. extractable tannins (TET) were estimated indirectly after adsorption of TEP to insoluble polyvinylpyrro- lidone, and measuring the remaining total phenols (or Forage and roughage material QRQSUHFLSLWDEOHSKHQROV LQWKHVXSHUQDWDQW 0DNNDU et al., 1993). Concentration of TET was calculated Nine browse plant species were used in this study: WKURXJKVXEWUDFWLRQDVIROORZV7(7 7(3íQRQ six dicotyledon plants namely Atriplex halimus L., Ar- precipitable phenols. Free condensed tannins were temisia campestris L., Artemisia herba-alba Asso, As- measured in the extract using the butanol-HCl assay tragalus gombiformis Pomel, Calobota saharae (Coss. (Porter et al ZLWKWKHPRGL¿FDWLRQVRI0DNNDU 'XULHX %RDWZU %(YDQ:\N IRUPHUO\Genista  DQGXVLQJSXUL¿HGTXHEUDFKRWDQQLQDVVWDQGDUG saharae or Spartidium saharae), and Retama raetam The bound condensed tannins were measured in the solid )RUVVN :HEE %HUWKHODQGWKUHHPRQRFRW\OHGRQ residue remaining after extraction of phenolic com- plants namely Stipagrostis pungens (Desf.) De Winter pounds. Concentration of total condensed tannins (TCT) (formerly pungens), Lygeum spartum/RHÀH[ was calculated as follows: TCT = free condensed tan- L. and Stipa tenacissima L. Selection of the species was nins + bound condensed tannins. Concentration of phe- based on the available information on their consumption nols and tannins were expressed in g tannic acid equiv- by grazing small ruminants, and on their relative abun- DOHQWNJí DM, whereas the concentration of condensed dance in the area of study. Samples were collected in WDQQLQVZDVH[SUHVVHGLQJTXHEUDFKRHTXLYDOHQWNJí -XQHZKHQSODQWVZHUHDWDÀRZHULQJ A. halimus, DM. All chemical analyses were performed in triplicate. A. gombiformis, R. raetam and L. spartum) or at a mature stage (the rest of species). Sampling was during the dry season, because this is the time of the year when these $QLPDOVDQGUXPHQÀXLGH[WUDFWLRQIRU plants may be more important for grazing. Between six in vitro and in situ studies and ten specimens of each plant species were sampled to obtain a representative aliquot of the edible biomass. )RXUPDWXUH0HULQRVKHHS ERG\ZHLJKW“NJ  /HDYHVWKLQWZLJV \RXQJVWHPV DQGVRPHÀRZHUV ZKHQ ¿WWHGZLWKDSHUPDQHQWUXPLQDOFDQQXOD PPGLDP- existing) were clipped with scissors from the aerial part HWHU ZHUHXVHGIRUWKHH[WUDFWLRQRIUXPHQÀXLGRUIRU RIWKHSODQWVDQGWDNHQLQPHGLDWHO\WRWKHODERUDWRU\ in situ incubation of nylon bags. Animals were fed with where the samples from the different specimens were lucerne hay ad libitum (167 g CP, 502 g NDF, 355 g SRROHGRYHQGULHGDWƒ& 0DNNDU DQGVXEVH- $')DQGJ$'/NJí DM) and had free access to quently ground to pass a 1 mm screen. ZDWHUDQGPLQHUDOYLWDPLQEORFN6DPSOHVRIUXPHQ contents were withdrawn prior to morning feeding, WUDQVIHUUHGLQWRWKHUPRVÀDVNVDQGWDNHQLPPHGLDWHO\ Chemical analysis to the laboratory, where rumen fluid was strained WKURXJKYDULRXVOD\HUVRIFKHHVHFORWKDQGNHSWDWƒ&

Dry matter (DM, method ID 934.01), ash (method XQGHUDFRQVWDQWÀRZRI&22. ID 942.05) and CP (method ID 954.01) contents were determined following the methods of AOAC (2000). Neutral and acid detergent fibre (NDF and ADF, In vitro digestibility respectively) and sulphuric acid detergent lignin (ADL) ZHUHGHWHUPLQHGZLWKWKH$1.20¿EUHDQDO\VHUDV In vitro DM digestibility was determined using the described by Van Soest et al. (1991). Sodium sulphite, ANKOM-DAISY procedure (Ammar et al., 1999) fol- EXWQRWĮDP\ODVHZDVDGGHGWRWKHVROXWLRQIRUWKH lowing two different approaches, the proposed by Tilley 1')GHWHUPLQDWLRQ%RWK¿EUHIUDFWLRQVZHUHH[SUHVVHG & Terry (1963), and the one described by Van Soest including residual ash. et al. (1966). Both techniques were carried out sepa- Phenolic compounds were extracted following the rately in different incubations. SURFHGXUHVGHVFULEHGE\0DNNDU  7RWDOH[WUDFW- A culture medium containing macro- and micro- able phenols (TEP) were determined according to the mineral solutions, a bicarbonate buffer solution and PHWKRGRI-XONXQHQ7LLWWR  XVLQJWKH)ROLQ UHVD]XULQZDVSUHSDUHGDVGHVFULEHGE\0HQNH 6WH- Chemical composition and digestibility of Algerian browse plants 91 ingass (1988). The medium was maintained at 39°C WKHDEVHQFHRIVXEVWUDWH2QFH¿OOHGXSDOOWKHERWWOHV and saturated with CO2. Oxygen in the medium was were closed with rubber stoppers, crimped with alu- removed by the addition of a reducing solution contain- PLQLXPVHDOVVKDNHQDQGSODFHGLQDQLQFXEDWRUWR ing cysteine-HCl and sodium sulphide, as described by controlled temperature 39°C. Volume of gas produced Van Soest et al  5XPHQÀXLGZDVWKHQDGGHG was recorded at several incubation times (3, 6, 9, 12, 16, to the medium in the proportion 1:4 (v/v). Samples of 21, 26, 31, 36, 48, 72, 96, 120 and 144 h after inoculation SODQWPDWHULDO PJ ZHUHZHLJKHGLQWRDUWL¿FLDO time) using a pressure transducer (Delta Ohm DTP704- ¿EUHEDJV VL]HFPîFPSRUHVL]H—P ZKLFK 2BGI, Herter Instruments SL, Barcelona). At the end of were heat-sealed and placed in incubation jars (5 L the incubation (after 144 h), the contents of each serum glass recipients with a plastic lid provided with a sin- ERWWOHZHUH¿OWHUHGXVLQJVLQWHUHGJODVVFUXFLEOHV FRDUVH gle-way valve which avoids the accumulation of fer- SRURVLW\QRSRUHVL]H—P XQGHUYDFXXP PHQWDWLRQJDVHV (DFKLQFXEDWLRQMDUZDV¿OOHGZLWK Then the residue was washed out with a neutral detergent /RIWKHEXIIHUHGUXPHQÀXLGWUDQVIHUUHGDQDHUREL- solution at 100°C during 1 h and oven-dried at 100°C cally, and closed with the lid, mixing the contents for 48 h to estimate the potential DM disappearance (g thoroughly. The jars were then placed in a revolving gí DM). Incubations were performed using three dif- LQFXEDWRU $QNRP'DLV\,,GLJHVWLRQV\VWHP$1.20 IHUHQWLQRFXOD UXPHQÀXLGIURPWKUHHVKHHSXVHGVHSD- Technology Corp., Fairport, NY, USA) at 39°C, with UDWHO\ ZLWKWZRERWWOHVSHUUXPHQÀXLGLQRFXOXP IRUD continuous rotation to ensure the effective immersion total of six observations —three replicates— per sam- RIWKHEDJVLQWKHUXPHQÀXLG$IWHUKRILQFXEDWLRQ SOH ,QRUGHUWRHVWLPDWHWKHIHUPHQWDWLRQNLQHWLFSD- LQEXIIHUHGUXPHQÀXLGVDPSOHVZHUHGULHGWRHVWLPDWH UDPHWHUVJDVSURGXFWLRQGDWDZHUH¿WWHGXVLQJWKHH[- in vitro DM loss after 48 h incubation. Then, bags used ponential model proposed by France et al. (2000): to measure in vitro digestibility following the original ⎡ −−ct( L) ⎤ method of Tilley & Terry (1963) were subjected to a GA=−⎣1e ⎦ for t •L, 48 h acid pepsin-HCl digestion, and the dry residue remaining in the bag was considered as the apparently where G (mL gí) denotes the cumulative gas production indigestible DM to estimate in vitro digestibility (IVD- at time t; A (mL gí) is the asymptotic gas production; TT). On the other hand, the other batch of bags were c (hí) is the fractional rate of substrate fermentation and gently rinsed in cold water followed by an extraction L (h) is the lag time. Volume of gas (mL gí DM) produced with a neutral detergent solution at 100°C during 1 h after 24 h of incubation was used as an index of digest- as described by Van Soest et al. (1966). According to LELOLW\DQGHQHUJ\IHHGYDOXHDVVXJJHVWHGE\0HQNH Van Soest (1994), the extraction with the neutral de- & Steingass (1988). According to France et al. (2000), tergent removes bacterial cell walls and other endog- the extent of degradation in the rumen (ED, g gíDM) enous products, and therefore can be considered as a for a given rate of passage (k, hí) was estimated as: determination of the true in vitro digestibility. With cD× 144 each procedure, each browse sample was incubated in ED = e−kL , tetraplicate, with one bag per sample incubated in each ck+ MDUDQGUXPHQÀXLGIURPHDFKRIWKHIRXUVKHHSEHLQJ where D144 is the DM disappearance after 144 hours incubated separately in each of the four jars. of incubation. To calculate ED, a rate of passage of 0.03 hí (characteristic for sheep fed with forage diet at maintenance level) was used. In vitro gas production kinetics

*DVSURGXFWLRQSUR¿OHVZHUHREWDLQHGXVLQJDQDG- Polyethilenglycol (PEG) bio-assay for the aptation of the technique described by Theodorou assessment of tannins et al. (1994). Ground samples (500 mg) were incu- EDWHGLQP/RIGLOXWHGUXPHQÀXLG P/PL[HG The gas production technique described above was

UXPHQÀXLGP/PHGLXPSUHSDUHGXQGHUD&22 also used for this biological assay. Incubations were FRQVWDQWÀRZ LQP/VHUXPERWWOHV6L[ERWWOHV carried out in serum bottles with or without the addition FRQWDLQLQJRQO\GLOXWHGUXPHQÀXLGZHUHLQFXEDWHGDV of 500 mg PEG. Ground samples (300 mg) were EODQNVDQGXVHGWRFRPSHQVDWHIRUJDVSURGXFWLRQLQ ZHLJKHGRXWLQWRVHUXPERWWOHVNHSWDWDSSUR[LPDWHO\ 92 S. Boufennara et al. / Span J Agric Res (2012) 10(1): 88-98

ƒ&DQGÀXVKHGZLWK&22 before use. Two bottles tion time. Two bags per sample were washed following were used for each substrate with each inoculum source the same procedure without being previously incubated UXPHQÀXLGIURPWKUHHVKHHSZDVXVHGVHSDUDWHO\DV in the rumen to estimate DM disappearance at 0 h (esti- three different inocula giving three replicates per treat- mate of DM solubility and particle loss from the bag). ment), one for each treatment (with or without PEG). Bottles were tightly closed and placed in the incubator DWƒ&EHLQJVKDNHQDWUHJXODUWLPHV7KHYROXPHRI Statistical analysis gas produced in each bottle was recorded at 6, 12, 24 and 48 h after inoculation time, using a pressure trans- One way analysis of variance (Steel & Torrie, 1980) ducer. Gas production was corrected by subtracting the was performed on in vitro digestibility, gas production YROXPHRIJDVSURGXFHGIURPEODQNFXOWXUHV7DQQLQ IHUPHQWDWLRQNLQHWLFVDQGin situ degradability data, with activity was calculated as the ratio between cumulative EURZVHVSHFLHVDVWKHRQO\VRXUFHRIYDULDWLRQ ¿[HGHI- gas measured in the PEG bottle and that recorded in IHFW DQGVRXUFHRILQRFXOXP UDQGRPHIIHFW DVDEORFN- the control (no PEG) bottle, for each sample and in- LQJIDFWRU7XNH\¶VPXOWLSOHFRPSDULVRQWHVWZDVXVHGWR oculum. For each sample, values from the three repli- determine which means differed from the rest (p < 0.05). cates (inoculum sources) were averaged. 3HDUVRQOLQHDUFRUUHODWLRQFRHI¿FLHQWVZHUHGHWHUPLQHG pair-wise between the variables studied. Clustering of browse species from a multivariate analysis of all data In situ degradability recorded (chemical composition, tannin content and bio- logical activity, in vitro digestibility, gas production The procedure to measure in situ disappearance has IHUPHQWDWLRQNLQHWLFVDQGin situ degradability data), and been described in detail by López et al. (1991, 1999). In DVVHVVPHQWRIWKHUHODWLYHLQÀXHQFHRIHDFKYDULDEOHRQ situ DM degradability in the rumen of each browse spe- that clustering were established by principal components cies was determined as the DM disappearance when analysis (PCA). Analysis of variance, correlation and VDPSOHV J'0 ZHLJKHGLQQ\ORQEDJV —PSRUH PCA were performed using the procedures MIXED, VL]HDQGîFPVL]H ZHUHLQFXEDWHGIRUDQG &255DQG)$&725RIWKH6$6VRIWZDUHSDFNDJH 6$6 KLQWKHUXPHQRIWKUHH¿VWXODWHG0HULQRVKHHSIHG Institute, 2008), respectively. with alfalfa hay (3 bags per sample and incubation time, one in each sheep). At the end of incubation, bags were removed from the rumen, rinsed with cold tap water and Results washed in a washing machine with cold water for 3 cycles of 3 min each. The washed bags were dried in Chemical composition of the plant material collected a forced draft oven at 100°C for 48 h, and the residual from the different species is shown in Table 1. The CP DM used to calculate DM disappearance at each incuba- content of the plant species samples varied widely, being

Table 1.&KHPLFDOFRPSRVLWLRQ JNJ–1 dry matter) of Algerian forages Neutral Acid Acid Organic Crude Plant family Plant species detergent detergent detergent matter protein ¿EUH ¿EUH lignin Dicotyledons Chenopodiaceae Atriplex halimus 805 153.6 360 181 59.9 Asteraceae Artemisia campestris 898 115.0 330 212 97.5 Artemisia herba-alba 920 123.9 378 258 101.1 Fabaceae - Leguminosae Astragalus gombiformis 871 223.4 340 218 46.7 Calobota saharae 955 109.8 574 427 135.2 Retama raetam 956 108.7 623 445 199.5 - Gramineae Stipagrostis pungens 945 95.2 771 425 58.3 Lygeum spartum 936 72.7 801 535 62.5 Stipa tenacissima 964 74.6 793 476 73.2 Chemical composition and digestibility of Algerian browse plants 93 particularly high for A. gombiformis JNJí DM) In vitro digestibility and in situ DM disappearance and low for the grasses L. spartum and S. tenacissima were variable (p < 0.05) across the examined forages DQGJNJí DM, respectively). Protein content (Table 3). The lowest in vitro and in situ DM digest- in dicotyledon species ranged widely from 109 to ibilities were observed in monocotyledons (being JNJí DM and was always greater than in mono- particularly low for S. tenacissima), whereas dicots cotyledon grasses. In general, monocots had higher had significantly higher values. Similar trends were NDF and ADF and lower lignin contents than dicots, observed for the in vitroIHUPHQWDWLRQNLQHWLFVHVWL- although material collected from C. saharae and mated form the gas production curves (Table 4). Al- R. raetam had high NDF and lignin contents, whereas though the monocotyledons showed higher asymp- that from A. halimus and A. gombiformis showed low totic gas (parameter A) than dicots (p < 0.05), their lignin contents. fermentation rate (parameter c) was significantly Tannin composition of the plant species is presented lower (p < 0.05), resulting in lower gas production (at in Table 2. The highest contents of TEP and TET were 24 h incubation) and ED for grasses than for dicot observed in the Asteraceae family (Artemisia spp.), species. whereas grasses, A. halimus and leguminous plants The PCA based on data recorded on chemical and showed lower concentrations. TCT varied widely tannin composition, in vitro and in situ DM digestibil- among species, being highest in S. tenacissima and LW\DQGIHUPHQWDWLRQNLQHWLFVJURXSHGWKHVWXGLHG lowest for A. halimus. Based on the results observed species in two clusters (Fig. 1). One of them included with the PEG bioassay, the species with highest tannin the grasses and the other one some of the dicots biological activity on gas production would be A. camp- (A. gombiformis and Artemisia spp), with the legu- estris and, to a lesser extent, S. tenacissima (p < 0.001), minosae C. saharae and R. raetam in an intermediate negligible for S. pungens, and it did not exist for the position. The same analysis was used to examine the other species. Tannin values observed with the different most influential variables on the observed clustering WHFKQLTXHVZHUHVLJQL¿FDQWO\FRUUHODWHGDQG7(7ZHUH (Fig. 2). All variables, with the exception of lignin, positively correlated with tannin biological activity were highly influential. Chemical composition (NDF (r = 0.75, p = 0.020 at 48 h incubation). There were no and CP) and DM digestibility were the main variables incubation time effect (p  RUDVLJQL¿FDQWLQ- GHWHUPLQLQJWKHUDQNLQJRIVSHFLHVRQIDFWRU  teraction (p = 0.070) between incubation time and plant of variance accounted for), whereas the second factor species, thus effects of PEG on gas production (in- RIYDULDQFHDFFRXQWHGIRU ZRXOGEHH[SODLQHG dicative of the presence of tannins) were similar at all mainly by the content and activity (PEG bioassay) of incubation times. tannins.

Table 2.3KHQROLFFRPSRXQGV JNJ–1 DM, standard equivalent) and tannin biological activitya of Algerian forages

a Total Total Free Total Tannin biological activity Plant family Plant species H[WUDFWDEOH H[WUDFWDEOH condensed condensed at incubation times: phenols tannins tannins tannins 6 h 12 h 24 h 48 h Dicotyledons Chenopodiaceae Atriplex halimus 16.1 8.4 42.1 69.1 0.85 0.93 0.97 0.97 Asteraceae Artemisia campestris 84.3 57.1 62.7 114.3 1.40 1.36 1.27 1.23 Artemisia herba-alba 63.7 36.4 80.6 118.8 0.84 0.92 0.97 0.99 Fabaceae - Leguminosae Astragalus gombiformis 13.5 3.0 51.6 78.3 0.93 0.95 0.94 0.95 Calobota saharae 29.8 9.6 76.4 109.7 0.90 0.94 0.95 0.96 Retama raetam 8.5 2.0 40.5 77.9 0.92 0.99 1.01 1.01 Monocotyledons Poaceae - Gramineae Stipagrostis pungens 10.2 4.8 46.5 78.7 1.09 1.08 1.05 1.03 Lygeum spartum 35.6 11.1 77.2 102.4 1.00 1.00 1.01 1.01 Stipa tenacissima 12.2 3.8 165.5 213.9 1.16 1.13 1.07 1.04 a Tannin biological activity as the ratio between gas production measured at different incubation times adding PEG vs. control (i.e., gas PEG / gas control). 94 S. Boufennara et al. / Span J Agric Res (2012) 10(1): 88-98

Table 3. In vitro dry matter (g g–1 DM) digestibility and in situ dry matter disappearance (g g–1 DM) at different incubation times of Algerian forages In situ DM disappearance Plant family Plant species ivDMloss1 IVD-TT2 TIVD3 after incubation times: 0 h 24 h 96 h Dicotyledons Chenopodiaceae Atriplex halimus 0.542ab 0.756a 0.755a 0.404b 0.716a 0.804a Asteraceae Artemisia campestris 0.550a 0.741a 0.731a 0.366c 0.633b 0.789a Artemisia herba-alba 0.539ab 0.660b 0.686a 0.331d 0.505c 0.634b Fabaceae - Leguminosae Astragalus gombiformis 0.519b 0.742a 0.755a 0.495a 0.675ab 0.849a Calobota saharae 0.444c 0.560c 0.550b 0.265e 0.479c 0.523cd Retama raetam 0.517b 0.580c 0.595b 0.195f 0.369d 0.442d Monocotyledons Poaceae - Gramineae Stipagrostis pungens 0.349d 0.458d 0.455c 0.135g 0.305d 0.528c Lygeum spartum 0.359d 0.449d 0.451c 0.265e 0.506c 0.614b Stipa tenacissima 0.252e 0.269e 0.341d 0.117g 0.198e 0.328e SEM 4 0.0152 0.0153 0.0154 0.0063 0.0142 0.0170

1 ivDMloss: in vitro dry matter loss; 2 IVD-TT: in vitro digestibility of Tilley & Terry; 3 TIVD: true in vitro digestibility; 4 Standard error of the mean a, b, c, d, e, f, g0HDQVLQDFROXPQZLWKGLIIHUHQWVXSHUVFULSWVDUHVLJQL¿FDQWO\GLIIHUHQW p<0.05).

Discussion feedstuffs with a CP content lower than 70 mg gí DM require a supplementation of nitrogen to improve their In these arid areas small domestic ruminants have ingestion and digestion by the ruminants. Our CP val- to resort more and more to natural standing shrubs, ues are similar to those reported for other Mediterra- forbs and ligneous grasses as the only forage resourc- nean shrubs (Cabiddu et al., 2000; Frutos et al., 2002; es available during the dry season. The CP content of Ammar et al., 2004a,b; Arhab et al., 2009). The low the browse species studied herein is higher than the CP contents in L. spartum and S. tenacissima can be PLQLPXPOHYHORI'0UHTXLUHGIRURSWLPXP probably due to high proportions of mature leaves and UXPHQIXQFWLRQDQGIHHGLQWDNHLQUXPLQDQWOLYHVWRFN twigs in the samples. Protein was lower in these grass- (Van Soest, 1994). According to Paterson et al. (1996), es than in the dicots. Leguminous forages and trees

Table 4. In vitro fermentation NLQHWLFV HVWLPDWHGIURPJDVSURGXFWLRQFXUYHV RI$OJHULDQIRUDJHV A 1 c 2 G24 3 D144 4 ED 5 Plant family Plant species (mL g–1 DM) (h–1) (mL g–1 DM) (g g–1 DM) (g g–1 DM) Dicotyledons Chenopodiaceae Atriplex halimus 174c 0.0412b 103.6c 0.835bc 0.452b Asteraceae Artemisia campestris 226abc 0.0784a 188.2a 0.894a 0.623a Artemisia herba-alba 208bc 0.0818a 175.4a 0.822c 0.578a Fabaceae - Leguminosae Astragalus gombiformis 206bc 0.0760a 171.6a 0.874ab 0.620a Calobota saharae 207bc 0.0472b 139.1b 0.666de 0.401b Retama raetam 226abc 0.0391b 132.9b 0.707d 0.385b Monocotyledons Poaceae - Gramineae Stipagrostis pungens 295a 0.0169c 76.5d 0.634e 0.190c Lygeum spartum 277ab 0.0154c 74.7d 0.550f 0.172c Stipa tenacissima 253abc 0.0118c 56.1d 0.469g 0.126c SEM6 16.8 0.00247 5.24 0.0087 0.0135

1 A: asymptotic gas production, 2 c: fractional rate of fermentation; 3 G24: gas production at 24 h of incubation; 4 D144: DM disap- pearance after 144 h of incubation; 5 ED: extent of degradation for a fractional passage rate of 0.03 h–1; 6 Standard error of the mean; a, b, c, d, e, f, g0HDQVLQDFROXPQZLWKGLIIHUHQWVXSHUVFULSWVDUHVLJQL¿FDQWO\GLIIHUHQW p<0.05). Chemical composition and digestibility of Algerian browse plants 95

4 have been used as a basic feed animal supply in many 3 regions of the world, mainly because of their high Agom protein contents (Norton, 1994; Tolera et al., 1997; 2 Ahal Ammar et al., 2004b) throughout the year. 1 Spun Based on their chemical composition, these feed- Csah Lspa 0 Rrae VWXIIVFRXOGEHFODVVL¿HGDVKLJKO\¿EURXVDVDOOIRU- Aher Sten –1 DJHVVKRZHGKLJK¿EUH 1')DQG$') DQGOLJQLQ contents, particularly the grasses. The high level of

Factor 2: 19.39% 2: Factor –2 Acam ¿EUHFRQWHQWLQVRPHRIWKHIRUDJHVSHFLHVFRXOGEH –3 explained partly by the environmental conditions pre- –4 vailing in the area of Bou Saâda, as high temperatures –5 and low precipitations tend to increase the cell wall –5–4–3–2–10123456 fraction and to decrease the soluble contents of the Factor 1: 58.18% plants (Pascual et al., 2000). Our values are similar to Figure 1. Plant species discrimination on the basis of principal those reported for other browse forages (Larbi et al., component analysis performed on data recorded on chemical 1998; Ammar et al.*DVPL%RXEDNHUet al., and tannin composition, in vitro and in situ digestibility and 2005; Salem et al., 2006), with some differences among IHUPHQWDWLRQNLQHWLFV $KDO Atriplex halimus, Acam = Ar- all studies, probably because of the different propor- temisia campestris, Aher = Artemisia herba-alba, Agom = As- tions of foliage and twigs in the samples and the dif- tragalus gombiformis, Csah = Calobota saharae, Rrae = Retama raetam, Spun = Stipagrostis pungens, Lspa = Lygeum spartum ferent phenological stage of the plants at sampling (our and Sten = Stipa tenacissima). plants were collected at a mature stage). There was a considerable variation between species in the tannin concentrations in the material collected. 1.0 7KHDQDO\VLVRIVSHFL¿FWDQQLQVJLYHVDQLQGLFDWLRQRI the presence of some anti-nutritive factors. Except for some few species (Artemisia spp. and S. tenacissima), CP the plants investigated in this study had low tannin con- 0.5 tents (in particular R. raetam, A. halimus and S. pun- DIS96 gens ZKLFKZRXOGEHRIOLWWOHVLJQL¿FDQFHLQWKHLU TIVD effects on digestion of nutrients by ruminants. With NDF 0.0 ED KLJKSURWHLQFRQWHQWDQGORZ¿EUHDQGWDQQLQFRQWHQWV rate some of the leguminous species could be regarded as

Factor 2: 19.39% 2: Factor G24 with a potentially high nutritive value. It is pertinent Lignin TCT to mention that a high proportion of TCT was recovered –0.5 as free condensed tannins that may be responsible for the possible detrimental effects of condensed tannins Tannins on microbial fermentation of nutrients in the rumen. PEG48 –1.0 There was also a considerable variation between the –1.0 –0.5 0.0 0.5 1.0 GLIIHUHQWPHWKRGVRIDQDO\VLVLQWKHUDQNLQJRISODQW Factor 1: 58.18% species according to their tannin concentrations. This Figure 2.'LVFULPLQDWLRQRIWKHPRVWLQÀXHQWLDOYDULDEOHVRQWKH variation is expected since the chemical properties that observed clustering on the basis of principal component analy- are involved in the activity of polyphenols determined sis performed on data recorded on chemical and tannin compo- by the different methods are widely different. The sition, in vitro and in situGLJHVWLELOLW\DQGIHUPHQWDWLRQNLQHWLFV colorimetric methods should be used with caution as a (CP = crude protein; DIS 96 = in situ DM disappearance after quantitative assay. Nevertheless, the strong correlations 96 h incubation; ED = extent of degradation; G24 = cumulative between the different methods of analysis and the val- gas production at 24 h; Lignin = acid detergent lignin; ues observed with the PEG bioassay support the idea 1') QHXWUDOGHWHUJHQW¿EUH3(* WDQQLQELRORJLFDODFWLY- ity at 48 h incubation; rate = fractional rate of gas production; that in vitro gas production technique coupled with the TIVD = true in vitro digestibility; TCT = total condensed tan- use of PEG is a useful tool for screening the potential nins; Tannins = total extractable tannins). effects of tannins in shrub species due to its simplicity 96 S. Boufennara et al. / Span J Agric Res (2012) 10(1): 88-98 and accuracy. The percentage increase in gas produc- low CP content and digestibility. On the contrary, WLRQDVUHVXOWRIWKHEORFNLQJHIIHFWRI3(*RQWDQQLQV A. gombiformis and Artemisia spp. were the most di- would be an indicator of the biological activity of tan- gestible species and with highest CP content, showing nins on rumen microbial fermentation. Indeed, a sig- an interesting potential as fodder resources for small QL¿FDQWLQFUHDVHLQin vitro gas production when mate- ruminants during this time of the year. The leguminosae rial was incubated in the presence of PEG has been species C. saharae and R. raetam were intermediate reported for different tanniferous substrates (Barber between both clusters, with higher NDF and lower et al., 1990; Canbolat et al., 2005; Rubanza et al., 2005; digestibility than the dicot species included in the high- Singh et al., 2005). Based on this analysis, the species quality group. The shrub A. halimus was close to the with a highest tannin anti-nutritional activity would be group of most digestible dicots, but differed slightly A. campestris followed by S. tenacissima. The addition probably due to the lower gas production volumes of PEG was not always accompanied by an increase in observed with this plant species. With high digestibil- gas volume. The extent of positive or negative effect LW\DQGORZJDVSURGXFWLRQIHUPHQWDWLRQHI¿FLHQF\ PJ of tannin in the shrubs would vary depending not only digestible DM mLí gas) would be greater with this on the level of tannins in plants but also on their type shrub species than with any other species. Therefore, and their biological activity (Hagerman et al., 1992; A. halimus should be considered of high potential for Barry & McNabb, 1999). range ruminants browsing shrubs and trees when graz- Digestibility of the forages browse samples was LQJKHUEDFHRXVPDWHULDOEHFRPHVLQVXI¿FLHQWWRPDWFK determined by two conventional and extensively used their nutritional requirements. According to the position in vitro techniques (Tilley & Terry, 1963; Van Soest et of the variables in the factors derived from the PCA, al., 1966), and also assessed from the in situ DM disap- ZKLFKH[SODLQHGDWRWDORIRIWKHYDULDQFH pearance when samples were incubated in the rumen. chemical composition (NDF and CP) and digestibility The ED and degradation rate were estimated from gas ZHUHWKHPDLQYDULDEOHVGHWHUPLQLQJWKHUDQNLQJRQ SURGXFWLRQSUR¿OHVGHULYHGIURPPHDVXUHPHQWRIIHU- species on factor 1. The position of the variables on mentation gas produced when the material was incu- RQHHGJHRIWKLVD[LVFRQ¿UPVWKDWDOOGLJHVWLELOLW\ bated in vitro (Theodorou et al., 1994). With some variables were strongly and positively correlated to differences among techniques in the values estimated, each other and also to CP, with high and negative cor- DOOWHFKQLTXHVUHVXOWHGLQVLPLODUUDQNLQJDQGJURXSLQJ relations of all these variables with NDF (positioned of forages. With all the digestibility measures it was on the opposite end of the axis). The second factor observed a large variability among species, with a clear ZRXOGEHGH¿QHGPDLQO\E\WKHFRQWHQWDQGDFWLYLW\ differentiation between the most digestible species (PEG bioassay) in tannins, having a considerable im- (A. halimus, A. campestris, and A. gombiformis) and SDFWRQWKHFODVVL¿FDWLRQRIEURZVHVSHFLHVDFFRUGLQJ those showing the lowest digestibility coefficients to their nutritional quality. (grass species). These differences among browse spe- $V¿QDOFRQFOXVLRQVDOOWKHFKHPLFDOin vitro and cies in digestibility may be partly attributed to the in situ measurements are useful tools in initial screen- variations in chemical composition (mainly cell wall LQJVWXGLHVWRUDQNWKHIRUDJHVDFFRUGLQJWRWKHLUQXWUL- content and composition). In vitro DM loss showed WLYHTXDOLW\$VLJQL¿FDQWYDULDWLRQLQin vitro, in situ VLJQL¿FDQWQHJDWLYHFRUUHODWLRQVZLWK1') r = – 0.92; GLJHVWLELOLW\DQGIHUPHQWDWLRQVNLQHWLFVZHUHREVHUYHG p < 0.001) and ADF (r = – 0.81; p=0.008), whereas among the samples. Part of this variation was associ- free condensed tannins was negatively correlated with ated with crude protein and cell wall contents. The IVD-TT (r = – 0.71; p=0.033). The rate of degradation PRGHUDWHWRKLJK&3DQGORZ¿EUHFRQWHQWDORQJZLWK c (hí) was correlated with NDF (r = – 0.91; p<0.001) high in vitro digestibility and in situ DM disappearance and ADF (r = – 0.79; p=0.012). found in A. halimus, A. campestris, A. herba-alba and The PCA clustered the studied species in two groups A. gombiformis suggest that these plants could have considering jointly all the measures of chemical com- a greater nutritional value than the highly fibrous position and digestibility: one including the poor- and low digestible grasses (S. pungens, L. spartum and quality grasses and another one including the most S. tenacissima) that should be considered emergency digestible dicot species (A. gombiformis and Artemisia roughages of poor quality, to be used by the ruminants spp.). Grasses examined in the present study were, at only when drought decreases grazing herbaceous bio- WKHWLPHSODQWVZHUHVDPSOHGKLJKO\¿EURXVDQGZLWK mass yield. Chemical composition and digestibility of Algerian browse plants 97

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