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Total mixed ration pellets for light fattening lambs: effects on animal health
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C. Blanco1, F. J. Giráldez1, N. Prieto1,a, J. Benavides1, S. Wattegedera2, L. Morán1,b,
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S. Andrés1, R. Bodas1,c
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1 Instituto
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Grulleros, León, Spain.
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2 Moredun
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de Ganadería de Montaña (CSIC-Universidad de León). E-24346
Research Institute, Bush Loan, Penicuik EH26 0PZ, Midlothian, Scotland,
UK.
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a
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University of Alberta, 4-10 Agriculture/Forestry Centre, Edmonton, Alberta, T6G 2P5,
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Canada. Lacombe Research Centre, Agriculture and Agri-Food Canada, 6000 C&E
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Trail, Lacombe, Alberta, T4L 1W1, Canada.
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b
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c Present
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119. E-47071 Valladolid, Spain.
Present address: Department of Agricultural, Food and Nutritional Science,
Present address: Ashtown Food Research Centre, Teagasc, Dublin 15, Ireland
address: Instituto Tecnológico Agrario de Castilla y León. Ctra. Burgos, km
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Corresponding author: Raúl Bodas. Email address: bodrodra@itacyl.es
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Short title: Health effects of TMR for light fattening lambs
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Abstract
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Fifty male Merino lambs (6–8 wk, 14.1 kg; n=10 per group) were used to study the
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effect of feeding system: barley straw in long form and concentrate pellets in
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separate troughs (Control), ad libitum alfalfa supplemented with concentrate in
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separate troughs (Alfalfa) or including various levels of ground barley straw in
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concentrate pellets (B05, B15 and B25 for 50, 150 and 250 g barley straw/kg), on
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rumen characteristics, acid-base status, blood cell counts and lymphocyte
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stimulation. Alfalfa lambs had the heaviest digestive tract contents, highest rumen pH
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values, lowest VFA concentration, highest papillae counts and best mucosa colour
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and the greatest blood pCO2 values, lowest sodium and chloride and highest
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potassium concentrations (P<0.05). Including ground barley straw in the concentrate
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pellet or providing straw in long form separately from the concentrate reduces rumen
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pH and darkens ruminal mucosa as compared to alfalfa-fed lambs, thus affecting
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acid-base status.
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Keywords: rumen; hematology; concentrate; acidosis; sheep
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Implications
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Using concentrate-based pellets which include up to 25% of ground barley straw,
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instead of giving concentrate and straw separately, is a suitable way of feeding
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lambs, thus allowing automatic feed delivery and reducing labor and storage costs.
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These systems are still liable to cause ruminal acidosis if compared to alfalfa-based
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diets, although none of them seem to cause serious problems to animal health on top
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of all of this in young fattening lambs.
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Introduction
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Traditional fattening systems for lambs in Mediterranean countries are based on an
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ad libitum supply of concentrates generally supplemented with cereal straw in
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separate feeding troughs (Joy et al., 2008; Rodríguez et al., 2008). However,
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concentrate pellets are being used more often nowadays in order to make feedlot
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feeding management easier. The traditional system, which allows the animals to
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maximize their growth potential, is not devoid of problems, subacute acidosis being
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the most common. Concentrate diets lead to subacute ruminal acidosis by increasing
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volatile fatty acid (VFA) production in the rumen and the proportion of propionate and
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lactate, thus decreasing rumen pH (Enemark et al., 2002; Bodas et al., 2007;
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Enemark, 2008). These circumstances may entail a drop in blood pH and deplete
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blood base excess (Brossard et al., 2003). In this sense, if animal health is
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threatened by a disease (as in the case of rumen acidosis), the risk of associated
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disorders increases (e.g. parakeratosis-rumenitis, liver abscesses, laminitis) (Penner
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et al., 2011) and blood cell counts, especially leukocyte counts, and their response to
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stimuli (LPS, endotoxins) can be modified, thereby compromising the animal’s
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immune response (Penner et al., 2011; Ceroni et al., 2012).
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Forage intake could be increased by including barley straw in the concentrate pellet
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(thus forming a total mixed ration, TMR), which could help animals to raise rumen pH
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to some extent without compromising animal performance. However, the effect of
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forage on rumen pH depends, to a great extent, on forage particle size, this being
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particularly small in concentrate pellet-based diets, where the barley straw is almost
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ground. As a result, using pelleted diets may have no positive effects on rumen
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physiology, the consequences for animal health being even more detrimental than
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those from the traditional feeding system due to a decrease in physically effective
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fibre contents (Zhao et al., 2011). So far, the best strategy to promote optimal rumen
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fermentation is the use of forage-based rations, although it is known that the feed-to-
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gain ratio and average daily gain may be worsened as compared to concentrate-
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based rations (Carrasco et al., 2009b; Tufarelli et al., 2011). In a previous work,
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Blanco et al. (2014) showed that it is possible to fatten light lambs on a TMR pellet
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including ground barley straw and enhance animal performance without modifying
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meat characteristics compared to the traditional feeding system for these animals in
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Mediterranean countries, based on concentrate and long form barley supplied
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separately. However, despite the improvements achieved when some of the above-
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mentioned strategies are applied to concentrate-fed lambs, there is a scarcity of data
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showing their impact on rumen physiology and health when compared to animals
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receiving forage-based diets (Álvarez-Rodríguez et al., 2010 and 2012).
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Therefore, the objective of this study was to evaluate the effect of feeding system:
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barley straw in long form and concentrate pellets in separate troughs (Control), ad
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libitum alfalfa supplemented with concentrate in separate troughs (Alfalfa) or
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including various levels of ground barley straw in concentrate pellets (B05, B15 and
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B25 for 50, 150 and 250 g barley straw/kg), on rumen characteristics and
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fermentation, acid-base status, blood cell counts and cytokine production in fattening
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lambs; feed intake, growth rate and weight of digestive tract and contents are also
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considered.
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Material and methods
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Animals and diets
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Fifty male Merino lambs (6–8 wk old and mean BW 14.1 ± 0.17 kg at the beginning of
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the experiment) were used in this study and managed as described elsewhere
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(Blanco et al., 2014). Each lamb was housed in an sawdust bedded individual pen
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during the whole experimental period and, after randomization on the basis of BW,
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assigned to one of five experimental diets (n = 10): Control (conventional system:
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barley straw in long form (>25 mm) and concentrate pellet in separate feeding
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troughs), B05 (concentrate-based TMR pellet including 50 g ground barley straw/kg),
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B15 (concentrate-based TMR pellet including 150 g ground barley straw /kg), B25
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(concentrate-based TMR pellet including 250 g ground barley straw /kg) and Alfalfa
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(restricted concentrate pellets plus ad libitum alfalfa hay). Alfalfa lambs received 17
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g/kg LBW of concentrate pellet and alfalfa hay ad libitum to keep a minimum supply
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of long form forage of 50% (this group was included to obtain data from animals fed
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on a forage-based diet). The rest of the lambs were fed the corresponding
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experimental diets ad libitum (TMR pellets), fresh drinking water being always
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available. The ingredients and chemical composition of the feeds are shown in Table
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1. The amount of feed offered was adjusted daily on the basis of the previous day’s
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intake, allowing refusals of ca. 200 g/kg feed offered. Samples of the feeds offered
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and orts were collected daily and pooled in weekly composites for each animal
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analyzed for DM content.
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All handling practices followed the recommendations of Directive 2010/63/EU of the
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European Parliament and of the Council on the Protection of Animals Used for
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Scientific Purposes, and all of the animals used were able to see and hear the other
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lambs.
[Table 1]
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Blood sampling
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Blood samples from all the animals were obtained from the jugular vein early in the
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morning, before feeding time (0800 h) on day 15 of the experimental period (approx.
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18 kg LW), and again at the same time on the day each animal was slaughtered (27
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kg LW). Blood was collected into 5 ml vacutainers containing lithium heparin tubes
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(for blood gas analyses, peripheral blood mononuclear cell separation and cytokine
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assay) and EDTA (for blood cell count analysis).
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Peripheral blood mononuclear cell (PBMC) separation and cytokine assay
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Blood was mixed with 30 ml of PBS and then PBMC were obtained by centrifuging
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the cells over Lymphoprep (Axis-Shield). The PBMC recovered were washed three
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times in Hank´s balanced salt solution (Sigma-Aldrich) and resuspended in complete
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medium constituted of RPMI 1640 (Lonza) with 10% FCS (Lonza) and 1% antibiotic–
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antimycotic solution (Santa Cruz Biotechnology). PBMC were counted with an
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automated cell counter (BioRad) and 1×106 cells/ml were added to each well of a 24
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well plate. Cells were stimulated in duplicate with Concanavalin A (ConA) at 10
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g/ml, or with 25 l of complete medium as control (PBS). Cultures were incubated at
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37 ºC in 5 % CO2 for 72 h. Then, supernatants were collected, centrifuged and stored
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at -20 ºC until cytokine quantification.
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Interferon-gamma (IFN-γ) and interleukin 4 (IL-4) productions were measured in
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culture supernatants by sandwich ELISAs. For IFN-γ, a commercially available kit
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was used (AbD Serotec) and for IL-4 an in-house ELISA using commercially
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available monoclonal antibodies (mAbs) raised against bovine IL-4 (mAb CC313 and
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CC314 mAb clones, AbD Serotec). The IL-4 mAbs have been shown to be species
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cross-reactive for sheep and the ELISA protocol followed has been in described in
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full by Hope et al (2005). The results for each animal were expressed as the optical
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density ratio between stimulation with ConA or PBS and with complete medium only.
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Blood gases, acid-base and haematological variables
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Blood samples for acid-base variables were assayed an hour after being sampled in
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a Stat Profile pHOx Plus blood analyser (Nova Biomedical, USA) for pH, bicarbonate
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(HCO3-), CO2 pressure (pCO2), anion Gap, total CO2 (tCO2), and Na, K and Cl
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concentrations.
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Blood samples for red blood cell (RBC) and white blood cell (WBC) counts
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(leukocytes, lymphocytes, monocytes and granulocytes), packed cell volume and
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haemoglobin were assayed in an electronic cell counter (Cellanalyzer CA530,
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Bromma, Sweden).
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Rumen samples and digestive tract characteristics
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Body weight was recorded twice a week, before the morning feeding, until the lambs
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reached the intended slaughter BW (27 kg BW). Once each animal was slaughtered
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(27 kg BW) and the white offal was obtained, full and empty weight of the reticulum-
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rumen-omasum and abomasum-intestine portions was recorded (Blanco et al.,
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2014). Afterwards, rumen fluid samples from each animal were collected, strained
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through 2 layers of cheesecloth, and pH was determined immediately. Then, two
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square samples of 3×3 cm from two locations on the ruminal wall (posterior part of
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dorsal sac and anterior part of ventral sac) were placed into histological cassettes,
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washed under tap water and then fixed by immersion in 10% buffered formalin for
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one week. Gross digital pictures of the ruminal mucosa were taken to measure the
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colour of the ruminal epithelium as an indicator of the degree of keratinisation
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(Benavides et al., 2013). Pictures of all the samples were taken with the same
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camera (Nikon D100, Tokyo, Japan), where focus, lens aperture and exposure time
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were set to manual, adjusted on the first sample and then kept unchanged until all
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the pictures were taken. During the whole process, the camera was attached to a
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stand with tungsten incandescent bulbs for illumination; the position of the camera
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and the bulbs remained unchanged for all the pictures.
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Once downloaded into the computer, a 1×1 cm representative area of the region of
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interest located over a representative area of each sample of ruminal epithelium was
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selected and the ruminal papillae within this area were counted. The picture was then
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converted to grey scale and the mean gray value, ranging from 0 to 256, where 0 is
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black and 256 is white, of the selected region of interest was measured. A mean
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value was obtained for the two areas studied from each rumen. All these operations
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were performed with ImageJ 1.43 software (Rasband, W.S., ImageJ, U. S. National
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Institutes of Health, Bethesda, Maryland, USA).
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Feed analysis
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Feed samples were assayed to determine dry matter, ash, crude protein (AOAC,
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2003), neutral and acid detergent fibre (Van Soest et al., 1991).
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Calculations and statistical analysis
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Average daily weight gain was estimated as the regression coefficient (slope) of BW
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against time using the REG procedure in the SAS package (SAS Inst. Inc., Cary,
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NC). Feed to gain ratio was estimated by regression as the inverse of the slope of
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BW against cumulative feed intake. All the data obtained were subjected to one way
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analysis of variance with the diet as the fixed effect and the animal as the random
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effect, using the MIXED procedure of SAS. The level of significance was determined
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at P < 0.05 and means were separated using the least significant difference (LSD)
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procedure; a trend towards significance was declared when P < 0.10.
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Results
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Mean values of feed intake, growth rate, feed conversion and weight of digestive
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tract and contents are shown in Table 2. Alfalfa-fed lambs had the highest forage and
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lowest concentrate and total feed intake (P<0.001). Forage intake increased with the
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level of barley straw included in the TMR pellet (P<0.001), whereas concentrate
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intake (proportion of TMR that is not forage) remained constant for Control, B05, B15
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and B25 lambs. Neutral and acid detergent fibre intake increased with the level of
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forage ingested, B25 and alfalfa lambs showing the highest values and Control and
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B05 lambs having the lowest fibre intakes (P<0.001). Alfalfa lambs had the lowest
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growth rate, while B15 and B25 lambs showed higher daily gains than Control and
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B05 animals (P<0.001). Alfalfa-fed lambs showed higher feed to gain ratio than the
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rest of lambs (P<0.001). Animals in the Alfalfa group had the greatest rumen
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contents weight (P<0.001) and the lowest empty rumen weight (P<0.05). The highest
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weight of abomasums+intestine contents was shown by alfalfa and B25-fed lambs,
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while B05 lambs had the lowest values (P<0.05). No differences were observed in
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empty abomasum+intestine weight (P>0.10).
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[TABLE 2 NEAR HERE, PLEASE]
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Table 3 shows the effects of diet on ruminal characteristics. Alfalfa lambs had the
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highest rumen pH values, followed by Control, B25 and B15 lambs; B05 animals
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showed the lowest rumen pH values (P<0.05). There were no differences in
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ammonia concentration. Total VFA concentration was lower in the Alfalfa group
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compared to the rest of the lambs, as were individual fatty acid concentrations
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(except butyrate) (P<0.05). However, rumen samples from Alfalfa lambs tended to
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have the highest proportion of acetate (P<0.10) and the lowest proportion of
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propionate (P<0.05), thus raising the acetate to propionate ratio compared to the
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other groups (P<0.01).
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The highest counts of papillae were observed for Alfalfa lambs, followed by B25,
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Control and B05 lambs, with B15 animals showing the lowest counts (P<0.001).
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Regarding rumen mucosa colour, animals receiving alfalfa were clearly different from
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the others, showing the greatest grey, red, green and blue values (P<0.001).
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[TABLE 3 NEAR HERE, PLEASE]
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Mean values of parameters related to blood gases are shown in Table 4. B15, B25
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and Alfalfa lambs had the highest pH values on day 15, although these differences
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had disappeared by the day of slaughter. Animals in the Alfalfa group tended to have
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higher HCO3- values on day of slaughter (P<0.10) and had significantly (P<0.05)
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higher pCO2 values on day of slaughter than lambs in the other groups, whereas
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anion gap values showed the opposite trend (the lowest values for Alfalfa lambs). Na
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and Cl concentrations were always lower for Alfalfa than for the rest of the groups
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(P<0.001), while K concentration was the highest for Alfalfa lambs (P<0.001).
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[TABLE 4 NEAR HERE, PLEASE]
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The type of diet consumed by the animals did not affect white blood cells counts
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(P>0.10), as can be seen in Table 5. However, red blood cell counts and packed cell
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volume were lower for alfalfa-fed lambs on day 15 of the experiment.
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[TABLE 5 NEAR HERE, PLEASE]
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No effects were observed on IFN-γ or IL-4 production depending on the type of diet
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offered to the animals (P>0.10; Table 6).
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[TABLE 6 NEAR HERE, PLEASE]
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Discussion
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Feed intake and animal performance
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Feed intake increased with the proportion of barley straw in the TMR, with animals
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fed on TMR diets showing values for average daily gain and feed-to-gain ratio similar
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to or better than animals fed the control (traditional, concentrate and forage supplied
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separately) diet. However, alfalfa-fed lambs (which can be considered as a positive
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control for animal health and welfare) had lower growth rates and higher feed-to-gain
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ratios than animals in the other groups. These differences are on account of the
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higher fibre and lower protein and energy intakes in alfalfa-fed lambs as compared to
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concentrate-fed animals, in agreement with Carrasco et al. (2009a and 2009b), Papi
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et al. (2011) and Tufarelli et al. (2011).
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Digestive tract characteristics
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The high weight of rumen contents observed in animals receiving the alfalfa diet is
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mainly due to the composition of the dry matter intake, which was rich in fibre. This
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determines a slow fermentation rate and, therefore, low passage rate (Ndlovu and
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Buchanan-Smith, 1985; Poore et al., 1990; Carro et al., 2000). As a consequence,
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feed requires more time to be digested, staying longer in the digestive tract
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(especially in the rumen) and filling it. In fact, the fermentation pattern is different
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depending upon the diet, as is rumen pH, which is increased in response to forage
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intake (Carro et al., 2000), especially if it is alfalfa (Ha et al., 1983), as observed in
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our study. In the case of alfalfa-fed lambs, its intrinsic buffering capacity (Giger-
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Reverdin et al., 2002), together with its coarse structural fiber that stimulates
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ruminative chewing and salivation, increases rumen buffering capacity (Mirzaei-
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Aghsaghali et al., 2008). Within the other groups, Control and B25 had the highest
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pH values and B05 the lowest. Control group lambs had free access to barley straw
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and, although they had low forage intake (around 5%), it was in the long form, whose
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fibre effect is high (Allen, 1997). Moreover, barley straw intake in Control lambs could
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be associated not with concentrate intake, but with the moments at which pH was
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lowest (between 3 and 6 hours after intake) (Brossard et al., 2003). Therefore, these
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animals could regulate rumen pH by ingesting forage in a long form (Allen, 1997).
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Regarding B25 lambs, although they had a relatively high fibre intake, similar to
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Alfalfa lambs, the small particle size, together with their high concentrate (rapidly
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fermentable) intake, reduced saliva production and buffering capacity, thus promoting
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lower pH than the alfalfa diet. The animals with the highest concentrate and lowest
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long forage intake, B05 lambs, had the lowest rumen pH.
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According to the pH profile found in the rumen, fermentation pattern and VFA
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concentration were different for animals receiving concentrate than for alfalfa-fed
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lambs. Forage based diets are associated with slow fermentation rates, low VFA
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production, high proportions of acetate and valerate and high acetate to propionate
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ratios in the rumen (Carro et al., 2000), as observed in this experiment. At the
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opposite end of the scale, animals receiving concentrate-based diets with up to 25%
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ground barley straw have a high concentration of VFA, which is related to faster
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fermentation rate, high acid (mainly propionic) production and total acid load and,
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therefore low rumen pH (Bodas et al., 2007). Likewise, the type and amount of feed
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fermented in the rumen and the fermentation rate profile are some of the factors that
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influence the development of the rumen wall in the early stages. Thus, increased
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VFA concentration would determine further development of rumen mucosa, which
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could have led to a heavier empty rumen for concentrate-fed animals as compared to
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those receiving alfalfa (Baldwin, 1999; Baldwin et al., 2007).
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On the other hand, the heavy rumens observed in concentrate-fed animals
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(regardless of the amount of straw included in the TMR) could also be caused by the
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stimulant effect that feeding concentrate has on ruminal development (Odongo et al.,
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2006). However, it must be borne in mind that the rumen wall undergoes structural
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changes in response to the inflammation process (rumenitis and parakeratosis)
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associated with high acid load and low pH (Krehbiel et al., 1995; Steele et al., 2009
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and 2011).
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The changes occurring in rumen fermentation and dynamics eventually reflect in the
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state of the mucosa. Thus, in response to acidosis, the rate of metabolism and
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proliferation of stratified squamous epithelium increase dramatically, resulting in
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premature transition of cells into the keratinous layer, a condition known as
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parakeratosis (Kleen et al., 2003; Steele et al., 2011; Plaizier et al., 2012). All
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animals that received any type of concentrate had a darker colour than those fed on
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alfalfa. In the table, high values are positive indicators of lightness, whereas lower
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values indicate darker colours. Parakeratosis processes are associated with a
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darkening and hardening of the rumen mucosa, due to the low pH and the high
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concentrations of AGV. In contrast, alfalfa induces lighter brown epithelium colour
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than concentrate feed, which turned darker and grey (Álvarez-Rodríguez et al.,
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2012).
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Regarding papillae counts, this parameter was significantly higher in the animals
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receiving alfalfa, with no differences between the other groups. Although earlier work
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has shown that concentrate diets with high grain content markedly increase the
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number and size of ruminal papillae in lambs (Odongo et al., 2006), the underlying
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mechanisms of the dietary energy-dependent physiologic, biochemical, and
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histological alterations in the rumen epithelium are not known. Conversely, recent
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work carried out with lambs fed hay or concentrate diets suggests that forage supply
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did not affect papillae height or width but reduced the surface area of the papillae
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(Álvarez-Rodríguez et al., 2012). In any case, the parakeratosis process together
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with the increased rumen osmolality can result in rapid influx of water from the blood
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circulation into rumen epithelial cells and into the rumen, causing swelling and
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rupturing of rumen papilla (Owens et al., 1998; Plaizier et al., 2012), thus reducing
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their numbers.
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Consequently, although including up to 25% barley straw in the TMR for fattening
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lambs improves feed intake and animal performance (Blanco et al., 2014), it does not
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seem to promote a rumen as healthy as including 50% alfalfa hay in the diet.
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Acid-base blood parameters
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Alfalfa is rich in potassium, and its administration in large amounts has been
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associated with increases in K and reduced levels of Na and Cl blood concentrations
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(Kume et al., 2004). In our experiment, animals receiving the alfalfa diet experienced
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an increase in K and a decrease in Na and Cl, changes that became more noticeable
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by the end of the fattening period, when the lambs had the highest alfalfa intakes.
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Although the modifications in the concentrations of these ions accounted for the
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decrease in anion gap observed for alfalfa-fed lambs, it must be highlighted that
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substantial increases in this parameter usually indicate the presence of a metabolic
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acidosis (Emmett and Narins, 1977; Odongo et al., 2006).
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Changes in rumen pH may cause changes in blood base excess and blood pH.
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Albeit all values observed in this experiment were within the normal range, Alfalfa
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lambs had significantly higher blood pH values than the rest of the animals by mid-
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fattening period, but these differences were not maintained until the end of the
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period. This perhaps could be related to increased keratinization of ruminal mucosa
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or its development in the final stages of the fattening period, which may reduce acid
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absorption from the rumen (Krehbiel et al., 1995; Kleen et al., 2003). Therefore, the
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development of parakeratosis as a result of a chemical injury, such as that caused by
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acidosis, may protect the animals against a continuous drop in blood pH. This does
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not mean that nutrient (VFA) absorption from the rumen is impaired, but only reduced
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as compared to the initial stages, when the mucosa is probably not as yet thickened
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(as in animals receiving alfalfa). As a matter of fact, the lower HCO3- and pCO2
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values observed in concentrate-fed compared to alfalfa-fed lambs indicate that
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excess base (reserve) in the blood that helps to maintain blood pH is also delivered
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to the rumen, thus leading to an exhaustion of the reserves (Brossard et al., 2003;
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Ceroni et al., 2012). Moreover, the long-term effect of an acidogenic diet on the
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alkaline reserves of the blood requires a longer recovery period than that required for
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rumen parameters (Brossard et al., 2003). It is important to say that no clinical signs
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of acidosis were observed, which suggests the lambs likely did not experience
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discomfort attributable to the diets (Commun et al., 2009). It is noteworthy, however,
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that the B15 group showed the best HCO3- values after the alfalfa animals by the end
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of the fattening period, thus indicating a better state of alkaline reserves.
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Blood cell counts
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Recent works have suggested that some blood parameters such as white blood cell
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counts could serve as early indicators of subacute ruminal acidosis in dairy cows
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without the need to sample rumen fluid (Ceroni et al., 2012). It has been reported that
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RBC could increase in animals under acidosis challenge as a compensational
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response against the slow reduction in haematocrit (Ceroni et al., 2012). However, in
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opposition to these findings, the increase in RBC in concentrate-fed animals on day
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15 of the experiment was accompanied by an increase in packed cell volume,
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probably as a consequence of the increase in osmolarity.
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Several studies have shown that inducing acidosis by conducting a nutritional
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challenge based on feeding excessively high grain diets increases lipopolysaccharide
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endotoxin (LPS) in the rumen of cattle, and this LPS, after digestive tract epithelial
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barrier failure, may be translocated into the blood stream or the lymphatic system,
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where it can interact with mononuclear cells, endothelial and smooth muscle cells,
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polymor-phonuclear granulocytes and thrombocytes and stimulate the production of
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pro-inflammatory mediators such as cytokines (Plaizier et al., 2012). In addition, low
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extracellular pH and certain organic acids have been shown to activate cellular and
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humoral components of the immune system and exert pro-inflammatory effects
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(Kellum et al., 2004; Danscher et al., 2011), and as such these factors could have
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contributed to the inflammatory response. In a study carried out involving healthy
376
humans, higher serum anion gap and lower bicarbonate levels were associated with
377
increased WBC and concentration of the major acute phase protein C-reactive
378
protein (Farwell and Taylor, 2010). Different organic acids have been suggested to
379
exert different effects on cytokine expression (Kellum et al., 2004; Danscher et al.,
380
2011). Nevertheless, in the current study, neither WBC nor cytokines were either
381
raised in response to concentrate feeding or decreased in response to forage
382
feeding. Although LPS was not measured here, the concentrations of LPS in rumens
383
from animals under acidosis challenge, as well as pH, vary considerably among
384
studies (see extensive review by Plaizier et al., 2012).
385
It can be concluded that traditional feeding system (based on supplying the
386
concentrate and barley straw separately) or including up to 25% ground barley straw
387
in the concentrate-based TMR for fattening lambs imply a notable rumen pH
388
depression and evident rumen wall damage when compared to alfalfa-fed animals.
389
These changes, which affect to some extent the blood’s acid-base status, seem to be
390
insufficiently severe to threaten animal health and compromise their immune
391
response.
392
393
Conflict of interest
16
394
The authors declare no conflict of interests.
395
396
Acknowledgements
397
Financial support received from the Spanish Ministry of Science and Innovation,
398
Project AGL2010-19094. Carolina Blanco is supported by a contract for young
399
researchers (Council of Castile and León and European Social Fund). Raúl Bodas
400
and Nuria Prieto had a JAE-Doc contract under the programme ‘Junta para la
401
Ampliación de Estudios’ (CSIC-European Social Fund). Sean Wattegedera is funded
402
by The Scottish Government Rural & Environment Science & Analytical Services
403
(RESAS) division and through ‘The route to identification of immunological correlates
404
of protection in ruminants’, Industrial Partnership Award funded by BBSRC (grant
405
numbers BB/I019863/1; BB/I020519/1) with the support of AbD Serotec, a Bio-Rad
406
Company.
407
408
References
409
Allen MS 1997. Relationship between fermentation acid production in the rumen and the
410
requirement for physically effective fiber. Journal of Dairy Science 80, 1447–1462.
411
Álvarez-Rodríguez J, Monleón E, Sanz A, Badiola JJ and Joy M 2012. Rumen fermentation
412
and histology in light lambs as affected by forage supply and lactation length. Reseach
413
Veterinary Science 92, 247–253.
414
Álvarez-Rodríguez J, Sanz A, Ripoll-Bosch R and Joy M 2010. Do alfalfa grazing and
415
lactation length affect the digestive tract fill of light lambs? Small Ruminant Research
416
94, 109–116.
417
418
Association of Official Analytical Chemists 2003. Official methods of analysis, 17th edition.
AOAC, Gaithersburg, MD, USA.
17
419
420
421
Baldwin RL 1999. Sheep gastrointestinal development in response to different dietary
treatments. Small Ruminant Research 35, 39–47.
Baldwin RL, El-Kadi SW, McLeod KR, Connor EE and Bequette BJ 2007. Intestinal and
422
ruminal epithelial and hepatic metabolism regulatory gene expression as affected by
423
forage to concentrate ratio in bulls, in: Ortigues-Marty, I, Miraux, N., Brand-Williams, W.
424
(Eds.), Energy and protein metabolism and nutrition, EAAP Publication nº 124. pp.
425
293–294. Wageningen Academic Publishers, Wageningen, The Netherlands.
426
Benavides J, Martínez-Valladares M, Tejido ML, Giráldez FJ, Bodas R, Prieto N, Pérez V
427
and Andrés S 2013. Quercitin and flaxseed included in the diet of fattening lambs:
428
Effects on immune response, stress during road transport and ruminal acidosis.
429
Livestock Science, 158 (1-3): 84-90.
430
Blanco C, Bodas R, Prieto N, Andrés S, López S and Giráldez FJ 2014. Concentrate plus
431
ground barley straw pellets can replace conventional feeding systems for light fattening
432
lambs. Small Ruminant Research 116, 137-143.
433
Bodas R, Giráldez FJ, López S, Rodríguez AB and Mantecón AR 2007. Inclusion of sugar
434
beet pulp in cereal-based diets for fattening lambs. Small Ruminant Research 71, 250–
435
254.
436
Brossard L, Martin C and Michalet-Doreau B 2003. Ruminal fermentative parameters and
437
blood acido-basic balance changes during the onset and recovery of induced latent
438
acidosis in sheep. Animal Research 52, 513–530.
439
Carrasco S, Panea B, Ripoll G, Sanz A and Joy M 2009a. Influence of feeding systems on
440
cortisol levels, fat colour and instrumental meat quality in light lambs. Meat Science 83,
441
50–56.
442
Carrasco S, Ripoll G, Sanz A, Álvarez-Rodríguez J, Panea B, Revilla R and Joy M 2009b.
443
Effect of feeding system on growth and carcass characteristics of Churra Tensina light
444
lambs. Livestock Science 121, 56–63.
18
445
Carro MD, Valdés C, Ranilla MJ and González JS 2000. Effect of forage to concentrate ratio
446
in the diet on ruminal fermentation and digesta flow kinetics in sheep offered food at a
447
fixed and restricted level of intake. Animal Science 70, 127–134.
448
Ceroni V, Turmalaj L, Lika E and Duro S 2012. Haematological Indicators Affected by the
449
Subacute Ruminal Acidosis in Dairy Cows. Journal of Animal and Veterinary Advances
450
11, 927–930.
451
Commun L, Mialon MM, Martin C, Baumont R and Veissier I 2009. Risk of subacute ruminal
452
acidosis in sheep with separate access to forage and concentrate. Journal of Animal
453
Science 87, 3372–3379.
454
Danscher AM, Thoefner MB, Heegaard PMH, Ekstrøm CT and Jacobsen S 2011. Acute
455
phase protein response during acute ruminal acidosis in cattle. Livestock Science 135,
456
62–69.
457
Emmett M and Narins RG 1977. Clinical use of the anion gap. Medicine 56, 38.
458
Enemark J 2008. The monitoring, prevention and treatment of sub-acute ruminal acidosis
459
460
(SARA): A review. The Veterinary Journal 176, 32–43.
Enemark JMD, Jorgensen RJ and Enemark PS 2002. Rumen acidosis with special emphasis
461
on diagnostic aspects of subclinical rumen acidosis: a review. Veterinarija ir
462
Zootechnika 20, 16–29.
463
Farwell WR and Taylor EN 2010. Serum anion gap, bicarbonate and biomarkers of
464
inflammation in healthy individuals in a national survey. Canadian Medical Association
465
Journal 182, 137–141.
466
De Blas C, Mateos GG and García-Rebollar P 2010. Tablas FEDNA de composición y valor
467
nutritivo de alimentos para la fabricación de piensos compuestos. 3rd Ed. Fundación
468
Española para el Desarrollo de la Nutrición Animal, Madrid, Spain.
469
Giger-Reverdin S, Duvaux-Ponter C, Sauvant D, Martin O, Nunes do Prado I and Müller R
470
2002. Intrinsic buffering capacity of feedstuffs. Animal Feed Science and Technology
471
96, 83–102.
19
472
Ha JK, Emerick RJ and Embry LB 1983. In vitro effect of pH variations on rumen
473
fermentation, and in vivo effects of buffers in lambs before and after adaptation to high
474
concentrate diets. Journal of Animal Science 56, 698.
475
Hope JC, Kwong LS, Thom M, Sopp P, Mwangi W, Brown WC, Palmer GH, Wattegedera S,
476
Entrican G and Howard CJ 2005. Development of detection methods for ruminant
477
interleukin (IL)-4. Journal of Immunology Methods 301, 114-23.
478
Joy M, Ripoll G and Delfa R 2008. Effects of feeding system on carcass and non-carcass
479
composition of Churra Tensina light lambs. Small Ruminant Research 78, 123–133.
480
Kellum JA, Song M and Li J 2004. Science review: extracellular acidosis and the immune
481
482
483
484
response: clinical and physiologic implications. Critical Care-London 8, 331–336.
Kleen JL, Hooijer GA, Rehage J and Noordhuizen JPTM 2003. Subacute Ruminal Acidosis
(SARA): a Review. Journal of Veterinary Medicine Series A 50, 406–414.
Krehbiel CR, Britton RA Harmon DL, Wester TJ and Stock RA 1995. The effects of ruminal
485
acidosis on volatile fatty acid absorption and plasma activities of pancreatic enzymes in
486
lambs. Journal of Animal Science 73, 3111–3121.
487
Kume S, Toharmat T, Ridla M, Nonaka K, Oshita T, Nakamura M, Yamada Y and Ternouth J
488
2004. Effects of High Potassium Intake from Alfalfa Silage on Mineral Status in Sheep
489
and Periparturient Cows. Research Bulletin of the National Agricultural Research
490
Center for Hokkaido Region 181, 1–14.
491
Mirzaei-Aghsaghali A, Maheri-Sis N, Mirza-Aghazadeh A, Safaei AR and Aghajanzadeh-
492
Golshani A 2008. Nutritional Value of Alfalfa Varieties for Ruminants with Emphasis on
493
Different Measuring Methods: A Review. Research Journal of Biological Sciences 3,
494
1227–1241.
495
Ndlovu LR and Buchanan-Smith JG 1985. Utilization of poor quality roughages by sheep:
496
Effects of alfalfa supplementation on ruminal parameters, fiber digestion and rate of
497
passage from the rumen. Canadian Journal of Animal Science 65, 693–703.
20
498
Odongo NE, AlZahal O, Lindinger MI, Duffield TF, Valdes EV, Terrell SP and McBride BW
499
2006. Effects of mild heat stress and grain challenge on acid-base balance and rumen
500
tissue histology in lambs. Journal of Animal Science 84, 447–455.
501
502
503
Owens FN, Secrist DS, Hill WJ and Gill DR 1998. Acidosis in cattle: a review. Journal of
Animal Science 76, 275–286.
Papi N, Mostafa-Tehrani A, Amanlou H and Memarian M 2011. Effects of dietary forage-to-
504
concentrate ratios on performance and carcass characteristics of growing fat-tailed
505
lambs. Animal Feed Science and Technology 163, 93–98.
506
Penner GA, Steele, MA, Aschenbach JR and McBride BW 2011. RUMINANT NUTRITION
507
SYMPOSIUM: Molecular adaptation of ruminal epithelia to highly fermentable diets.
508
Journal of Animal Science 89, 1108-1119.
509
Plaizier JC, Khafipour E, Li S, Gozho GN and Krause, DO 2012. Subacute ruminal acidosis
510
(SARA), endotoxins and health consequences. Animal Feed Science and Technology
511
172, 9–21.
512
Poore MH, Moore JA and Swingle RS 1990. Differential passage rates and digestion of
513
neutral detergent fiber from grain and forages in 30, 60 and 90% concentrate diets fed
514
to steers. Journal of Animal Science 68, 2965–2973.
515
Rodríguez AB, Bodas R, Prieto N, Landa R, Mantecón AR and Giráldez FJ 2008. Effect of
516
sex and feeding system on feed intake, growth, and meat and carcass characteristics
517
of fattening Assaf lambs. Livestock Science 116, 118–125.
518
Steele MA, AlZahal O, Hook SE, Croom J and McBride BW 2009. Ruminal acidosis and the
519
rapid onset of ruminal parakeratosis in a mature dairy cow: a case report. Acta
520
Veterinaria Scandinavica 51, 39.
521
Steele MA, Croom J, Kahler M, AlZahal O, Hook SE, Plaizier K and McBride BW 2011.
522
Bovine rumen epithelium undergoes rapid structural adaptations during grain-induced
523
subacute ruminal acidosis. American Journal of Physiology-Regulatory, Integrative and
524
Comparative Physiology 300, R1515–R1523.
21
525
Tufarelli V, Khan RU and Laudadio V 2011. Feeding of wheat middlings in lamb total mixed
526
rations: Effects on growth performance and carcass traits. Animal Feed Science and
527
Technology 170, 130–135.
528
Van Soest PJ, Robertson JB and Lewis BA 1991. Methods for dietary fiber, neutral detergent
529
fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy
530
Science 74, 3583–3597.
531
Zhao XH, Zhan T, Xu M and Yao JH 2011. Effects of physically effective fiber on chewing
532
activity, ruminal fermentation, and digestibility in goats. Journal of Animal Science 89,
533
501-509.
22
534
535
Table 1. Experimental feeds: ingredients and chemical composition.
Concentrate
feed
Ingredients (g/kg)
Barley
530
Corn
230
Soybean meal 44% crude protein
210
Barley straw
-Mineral vitamin mix
30
Chemical composition from analyses (g/kg DM)
DM (g/kg)
900
NDF
166
ADF
58
CP
182
Ash
63
1
Metabolisable energy (MJ/kg DM)
11.5
536
1
B05
B15
B25
490
210
220
50
30
433
150
237
150
30
388
80
252
250
30
900
196
59
181
60
11.2
903
259
94
181
73
10. 5
909
323
138
181
83
9.8
Calculated from feed composition tables (De Blas et al., 2010).
537
23
Barley
Alfalfa straw
917
542
391
137
73
8.0
913
626
413
47
104
5.0
538
Table 2. Effect of feeding concentrate and barley straw separately (Control), a TMR with
539
50, 150 and 250 g barley straw per kg (B05, B15 and B25, respectively) or ad libitum
540
alfalfa supplemented with concentrate (Alfalfa) on feed intake, growth rate and weight of
541
digestive tract and contents.
Intake (g/animal and day)
Concentrate DM1
Forage DM2
Total DM
Neutral detergent fibre
Acid detergent fibre
Average daily gain (g/day)
Feed:gain (g/g)
Weight of digestive
contents (g)
Rumen-reticulumomasum
Abomasum-intestine
Weight of empty digestive
tract (g)
Rumen-reticulumomasum
Abomasum-intestine
Control
B05
B15
B25
Alfalfa
r.s.d.
P-value
790b
27a
817a
148a
57a
299b
2.76a
751b
40b
791a
156a
47a
279b
2.89a
779b
137c
916b
237b
86b
339c
2.74a
792b
264d
1056c
341c
146c
353c
3.03a
422a
465e
887a
322c
206d
194a
4.84b
332
31
73
23
13
43
0.43
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
2634a
1593b
2549a
1529a
2613a
1769bc
2642a
1799c
3646b
1876c
562
260
<0.001
0.023
788ab
1668
903c
1679
868bc
1792
844abc
1784
756a
1646
104
178
0.019
0.219
542
1
543
TMR that is not barley straw (B05, B15 and B25 lambs).
544
b Forage
545
the feeding trough, or calculated from TMR composition (B05, B15 and B25 lambs).
546
r.s.d. = residual standard deviation
547
a, b, c, d, e Means
Concentrate: supplied in the feeding trough (Control and Alfalfa lambs) or the portion of
intake measured as straw (Control lambs) or alfalfa (Alfalfa lambs) ingested from
in the same row with different superscripts differ significantly (P<0.05).
548
549
24
550
Table 3. Effect of feeding concentrate and barley straw separately (Control), a TMR with
551
50, 150 and 250 g barley straw per kg (B05, B15 and B25, respectively) or ad libitum
552
alfalfa supplemented with concentrate (Alfalfa) on ruminal characteristics.
pH
Ammonia (mg/L)
VFA concentration (mmol/L)
Acetate
Propionate
Butyrate
VFA proportion (mol/100
mol)
Acetate
Propionate
Butyrate
Acetate/Propionate
Rumen mucosa colour
R
G
B
Grey
Papillae/cm2
Control
5.77b
5.77
B05
5.24c
4.64
Groups
B15
5.56bc
5.73
46.6a
28.3a
6.0
49.2a
30.0a
11.2
46.5a
26.0a
7.1
53.4a
36.0a
11.8
31.9b
10.4b
4.8
13.6
13.2
6.6
0.027
0.004
0.127
51.7
29.8a
6.4
1.99b
51.8
30.4a
11.2
1.82b
51.3
27.6ab
7.8
1.90b
49.5
32.5a
11.5
1.65b
60.8
20.3b
9.6
3.32a
8.7
8.0
6.2
0.81
0.076
0.030
0.373
0.001
105b
80b
61b
85b
49.6bc
104b
81b
63b
86b
47.8bc
107b
82b
62b
87b
42.4c
111b
85b
62b
89b
51.9b
149a
117a
76a
121a
74.2a
12
11
7
11
13.7
<0.001
<0.001
<0.001
<0.001
<0.001
553
r.s.d. = residual standard deviation
554
a, b, c Means
B25
5.69b
5.39
Alfalfa
6.94a
6.32
r.s.d.
0.39
2.66
P-value
<0.001
0.750
in the same row with different superscripts differ significantly (P<0.05).
555
25
556
Table 4. Effect of feeding concentrate and barley straw separately (Control), a TMR with
557
50, 150 and 250 g barley straw per kg (B05, B15 and B25, respectively) or ad libitum
558
alfalfa supplemented with concentrate (Alfalfa) on blood gases and ions on day 15 and at
559
the end of fattening period (final, approx 27 kg LBW).
Control
B05
Groups
B15
B25
Day 15
Slaughter day
7.38b
7.43
7.37b
7.41
7.43a
7.44
7.41a
7.44
7.42a
7.42
0.04
0.03
0.003
0.184
HCO3- (mmol/l)
Day 15
Slaughter day
28.1
26.1
28.1
26.5
29.5
27.7
28.2
26.5
28.2
28.2
2.2
1.8
0.576
0.085
pCO2 (mm Hg)
Day 15
Slaughter day
51.3
42.8b
52.5
45.2ab
48.5
44.1b
47.3
42.5b
48.5
47.2a
5.2
3.1
0.178
0.020
Anion Gap (mmol/l)
Day 15
Slaughter day
12.9a
13.4
12.3a
13.4
11.0b
12.9
12.1ab
13.0
11.4b
11.4
1.4
1.5
0.044
0.051
tCO2 (mmol/l)
Day 15
Slaughter day
29.6
27.7
29.8
27.9
31.0
29.1
29.6
27.8
29.7
29.6
2.3
1.8
0.637
0.104
Na (mmol/l)
Day 15
Slaughter day
148a
146a
147a
146a
147a
146a
147a
146a
144b
144b
2
1
0.006
<0.001
K (mmol/l)
Day 15
Slaughter day
5.31
4.34b
5.08
4.53b
4.89
4.42b
4.82
4.47b
5.02
5.04a
0.42
0.38
0.121
0.003
Cl (mmol/l)
Day 15
Slaughter day
112a
110bc
112a
112a
111ab
110bc
111a
112ab
109b
109c
2
2
0.031
0.033
Alfalfa r.s.d. P-value
pH
560
r.s.d. = residual standard deviation.
561
a, b, c Means
in the same row with different superscripts differ significantly (P<0.05).
562
26
563
Table 5. Effect of feeding concentrate and barley straw separately (Control), a TMR with
564
50, 150 and 250 g barley straw per kg (B05, B15 and B25, respectively) or ad libitum
565
alfalfa supplemented with concentrate (Alfalfa) on blood cells counts on day 15 and at the
566
end of fattening period (final, approx 27 kg LBW).
Groups
B15
Control
B05
B25
Alfalfa r.s.d. P-value
Red blood cells (10 /µl)
Day 15
Slaughter day
10.17ab
9.62
10.29a
9.20
9.76bc
9.41
10.72a
9.90
9.19c
9.94
0.92
1.92
0.035
0.929
Packed cell volume (%)
Day 15
Slaughter day
36.2ab
33.7
37.0ab
32.8
36.0b
33.9
39.3a
35.3
33.6c
34.7
3.2
6.5
0.028
0.959
White blood cells (103/µl)
Day 15
Slaughter day
10.3
10.3
9.9
11.4
10.5
12.0
10.6
13.0
11.4
9.8
2.9
2.8
0.867
0.197
Lymphocytes (%)
Day 15
Slaughter day
36.4
48.7
36.5
53.5
40.3
50.7
41.5
56.4
33.7
47.7
10.0
9.1
0.427
0.376
Granulocytes (%)
Day 15
Slaughter day
54.4
43.1
52.7
39.2
49.7
40.8
48.3
35.9
55.7
44.7
8.9
7.5
0.356
0.220
Monocytes (%)
Day 15
Slaughter day
9.4
7.8
10.7
7.4
10.0
8.9
10.1
7.5
10.7
7.6
2.0
2.4
0.662
0.672
6
567
r.s.d. = residual standard deviation.
568
a, b, c Means
in the same row with different superscripts differ significantly (P<0.05).
569
27
570
Table 6. Effect of feeding concentrate and barley straw separately (Control), a TMR with
571
50, 150 and 250 g barley straw per kg (B05, B15 and B25, respectively) or ad libitum
572
alfalfa supplemented with concentrate (Alfalfa) on ratios of interferon-gamma and
573
interleukin-4 production (pg/ml) by peripheral blood mononuclear cells stimulated either
574
with PBS (IFN-γpbs and IL-4pbs) or concanavalin A (IFN-γcon and IL-4con).
IFN-γpbs
IFN-γconA
IL-4pbs
IL-4conA
575
Control
0.00
242
1.52
221
B05
0.00
110
0.93
343
Groups
B15
0.00
0.00
1.71
186
B25
0.02
21
2.18
369
Alfalfa
0.05
327
0.00
267
r.s.d. = residual standard deviation.
28
r.s.d.
0.08
386
3.57
335
P-value
0.623
0.370
0.780
0.823