Interaction of forage provision (alfalfa hay) and sodium butyrate supplementation on performance, structural growth, blood metabolites and rumen fermentation characteristics of lambs during pre-weaning period

Accepted Manuscript Title: Interaction of forage provision (alfalfa hay) and sodium butyrate supplementation on performance, structural growth, blood metabolites and rumen fermentation characteristics of lambs during pre-weaning period Authors: M. Soltani, M. Kazemi-Bonchenari, A.H. Khaltabadi-Farahani, O. Afsarian PII: DOI: Reference:

S0377-8401(16)30652-6 http://dx.doi.org/doi:10.1016/j.anifeedsci.2017.06.002 ANIFEE 13801

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Received date: Revised date: Accepted date:

12-9-2016 5-5-2017 2-6-2017

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Please cite this article as: Soltani, M., Kazemi-Bonchenari, M., Khaltabadi-Farahani, A.H., Afsarian, O., Interaction of forage provision (alfalfa hay) and sodium butyrate supplementation on performance, structural growth, blood metabolites and rumen fermentation characteristics of lambs during pre-weaning period.Animal Feed Science and Technology http://dx.doi.org/10.1016/j.anifeedsci.2017.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Interaction of forage provision (alfalfa hay) and sodium butyrate supplementation on performance, structural growth, blood metabolites and rumen fermentation characteristics of lambs during pre-weaning period

Running head; interaction of forage and sodium butyrate in pre-weaning lambs

M. Soltani1, M. Kazemi-Bonchenari1*, A.H. Khaltabadi-Farahani1, O. Afsarian2 1

Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University,

38156-8-8349, Arak, Iran 2

Department of Animal Science, University of Zanjan, 45371-38791, Zanjan, Iran

*Corresponding author: Tel: 0098 912 558 7005 E-mail address: [email protected] and [email protected] Mail Address; Department of Animal Science, Faculty of Agriculture and Natural Resources, Arak University, 38156-8-8349, Arak, Iran

Highlights •

This study was the first to investigate the interaction effects of forage and sodium butyrate in preweaning period in lambs

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Forage improved faecal score as well as rumen pH however intake, gain and feed efficiency did not influenced

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Sodium butyrate improved intake and gain and increased SCFA in to more efficient profile Forage inclusion in pre-weaned lambs was not efficient but simultaneous using with sodium butyrate could be recommendable

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Abstract The present study evaluated the interaction of forage provision (F) and sodium butyrate (SB) supplementation in pre-weaning lambs on performance, structural growth, blood metabolites and rumen fermentation characteristics. Twenty eight 3-days old individually housed Chaal breed lambs averaging BW of 6.02 ± 0.57 kg were randomly assigned to 4 treatments (n = 7 lambs/treatment: 4 males and 3 females). Experimental treatments were; 1) starter diet provided neither with F nor with SB (NF-NSB), 2) starter diet supplemented only with SB (NF-SB), 3) starter diet provided only with forage (F-NSB), and 4) starter diet simultaneously provided by forage and SB (F-SB). The F provision was 10% chopped alfalfa hay included in whole concentrate starter based diet (DM basis) and the SB supplementation was 3 g of SB/kg of starter DM. The manual milk feeding was performed for all lambs. The lambs were weaned on d 59 of age but the study lasted until d 73 of age. Performance statistical analysis was carried out for preweaning (8 weeks), post-weaning (2 weeks) and entire experimental periods (10 weeks). The results show that starter intake influenced with SB (P = 0.03) and weaning weight was greater in lambs supplemented with SB (P < 0.01). Neither intake nor gain was influenced with forage provision (P > 0.05). The interaction was observed between SB and F for gain in overall period of experiment (P = 0.03). The interaction of SB and F caused the greatest wither height in F-SB treatment (P = 0.03). Body barrel was greater in forage fed lambs (P = 0.01). No blood parameter was influenced with separate or simultaneous provision of F and SB in the current study. The short chain fatty acid (SCFA) concentration (P = 0.02) as well as butyrate concentration (P < 0.01) was increased by SB supplementation. Forage inclusion improved ruminal fluid pH (P = 0.02) and increased acetate (P < 0.01); however both butyrate (P = 0.02) and propionate (P = 0.01) concentrations were 2

reduced. In conclusion we found that F provision was not efficient as maximum as whole concentrate starter diet but simultaneous providing of SB could alleviate the negative effects of provided forage in pre-weaning lambs. Future studies warranted to evaluate microscopic development of rumen papillae in simultaneous provision of forage and butyrate in pre-weaning lambs. Keywords; pre-weaning lamb, rumen development, forage, sodium butyrate 1. Introduction There are controversial literatures with respect the introducing forage during the milk feeding period in ruminants. Different beneficial points of view for forage inclusion in pre-weaned ruminants had been stated in literatures. Some of these benefits were stimulating effect of forage for the muscular layer of the rumen (Tamate et al., 1962), promote rumination (Phillips, 2004), maintain the integrity and healthiness of the rumen wall (Suarez et al., 2007), and reduce behavioral problems (Phillips, 2004). Moreover absence of forage in starter diets may decrease rumen motility and cause hyperkeratinization and clumping of ruminal papillae (Bull et al., 1965); thus, decreasing the rumen mucosa ability to absorb nutrients (Hinders and Owen, 1965). From the other side of view some of literatures discourage forage inclusion in pre-weaned ruminants. They believe that forage may displace concentrate intake and shift rumen fermentation in favor of acetate rather than butyrate production and, thus, delay rumen papillae development (Tamate et al., 1962). Moreover it has been stated that provision of forage to young ruminants has not been recommended because it has been shown to reduce starter feed intake (Phillips, 2004), impair rumen papillae development (Nocek and Kesler, 1980), and decrease body weight (BW) and dry matter (DM) digestibility (Leibholz, 1975). The literatures frequently (Coverdale et al., 2004;

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Suarez et al., 2007; Hill et al., 2008, 2010; Beiranvand et al., 2014) evaluated the effect of forage inclusion in starter diet of dairy calves. Although some of literatures (Papi et al., 2011; AlvarezRodríguez et al., 2012) were evaluated the forage levels in growing or finishing lamb production, limited literatures had been evaluated the forage provision in lambs in pre-weaning period.

Furthermore high non-structural carbohydrate included in lamb’s diet revealed to induce parakeratosis (Steel et al., 2012) and because forage inclusion may prevent that (Suarez et al., 2007) forage provision in starter diet of pre-weaning lambs need to conduct more research. Butyrate has been reported to be the short chain fatty acid (SCFA) that stimulates the growth of rumen mucosa papillae (Warner, 1991; Mentschel et al., 2001). Supplementation of sodium butyrate (SB) in milk replacer during the first 4 weeks of life improved rumen papillae development, production performance and health status in calves (Guilloteau et al., 2004; Górka et al., 2009). In addition, calves fed milk replacer supplemented with SB improved the digestibility of most nutrients of the diet (Guilloteau et al., 2010). Guilloteauet al. (2009) observed that when SB was administered during the first month of life of calves, positive effects on growth occurred. The addition of SCFA salts were evaluated in lamb nutrition as well. Lee-Rangel et al. (2012) and Mendoza-Martínez et al. (2014) had been used calcium propionate in fattening lambs. The above mentioned studies evaluated the propionate salt in fattening period. Furnish higher SCFA concentrations or alter the rumen fermentation pattern may be used as a tool for reducing the milk feeding period in pre-weaned ruminants (Ferreira and Bittar, 2010). Recently Cavini et al. (2015) has been found positive effects of SB in pre-weaned lambs on rumen development of pre-weaned lambs. The forage did not provided in starter diet in their study. Moreover lambs were sucked their

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dams throughout that study and hence they did not report milk values fed by lambs which could affected the results of performance. To the best knowledge of the authors there is no data regarding the interaction of butyrate supplementation with forage provision in starter diet of pre-weaning lambs. We postulated that simultaneous use of butyrate and forage may be more effective than either offered separately. The objective of current study was to evaluate the effects of, and interactions between, different levels of forage (alfalfa hay) and sodium butyrate on performance, structural growth, blood metabolites, and ruminal fermentation characteristics in pre-weaning lambs. 2. Materials and Methods 2.1. Animals, treatments and management The experiment was carried out at Gole-Reyhan industrial sheep farm (Khomeyn, Arak, Iran). A total of twenty eight 3-day-old Chaal breed lambs (16 males and 12 females) with a starting BW of 6.02 ± 0.57 kg were randomly assigned in a 2 × 2 factorial arrangement with the factors of supplementation or non-supplementation of sodium butyrate (NSB vs. SB) and dietary forage level (NF vs. F). Lambs were separated from their dams immediately after birth, weighed, and moved to individual pens. Colostrum feeding was continued for the first 2 days of life. The lambs were fed 0.3 L of colostrum at each of the first 2 feedings (i.e., within 2 h of life and at 12 h after the first feeding). Colostrum feeding was performed for the first 2 d of life. Day 3 of age was considered as start of experiment. The study lasted 10 weeks which the first eight weeks was preweaning and the last two weeks was as post-weaning period. All the lambs were fed the milk manually throughout the experiment. The ewe’s milk was collected two times daily and stored in 4 C° for later feeding of lambs. Lambs received 0.3 L/d whole milk in galvanized tin buckets twice a day at 0730 and 1600 h from d 3 to 15, thereafter they received 0.7 L/d from d 16 to 50 of the 5

study and then 0.3 L/d from d 51 to d 56 of the study. All the lambs were weaned on d 59 of age (i.e. d 56 of the study) but the experimental diets were continued for two weeks thereafter (until d 73 of age). Experimental treatments were; 1) starter diet provided neither with F nor with SB (NFNSB), 2) starter diet supplemented only with SB (NF-SB), 3) starter diet provided only with forage (F-NSB), and 4) starter diet simultaneously provided by forage and SB (F-SB). Ingredients and chemical composition of experimental diets are presented in Table 1. Experimental diets were formulated to meet NRC 2007 small ruminant requirements for lamb nutrients (NRC, 2007). The F was 10% alfalfa hay inclusion in starter diet (DM basis). The SB was incorporated in to the diet at the dose of 3 g/kg of starter DM. Sodium butyrate salt was product of Adimix® Butyrate c, Inve Nutri-Ad, Karterlee, Belgium which was provided by commercial company (Mina-Tajhiz Aria, Tehran, Iran). Add libitum starter intake was achieved by offering an amount that resulted in residue of 5-10% after 24 h. Feed offered and refused was weighed daily and total daily intake calculated to determine individual daily feed intakes throughout the experiment. The vaccination schedule and rearing system protocols were as farm conventional protocol. The lambs had ad libitum access to water. 2.2. Sample collection and chemical analysis Intake of starter was measured daily, and individual BW was recorded using an electronic balance every 7 d throughout the experiment (eight records in pre-weaning and two records in postweaning periods). The amounts of feed offered and refused were recorded daily for each individual lamb. Pre- and post-weaning, and overall means of daily gain (DG) and feed efficiency (kg of BW gain/kg of total intake) were also calculated. Samples from feeds and orts were dried at 60°C using a convection oven for 48 h. Subsamples of feeds and refusals were mixed thoroughly, dried, and ground to pass a 1 mm screen in a mill (Ogaw Seiki CO., Ltd., Tokyo, Japan) before chemical 6

analysis for crude protein (method 988.05; AOAC, 1990) and lipid (method 920.39; AOAC, 1990). Neutral detergent fibre (NDF) was analysed without using sodium sulfite and with the inclusion of α-amylase (Van Soest et al., 1991). Faecal scores were recorded by a day interval based on Khan et al. (2007) and body temperature was recorded weekly. Body measurements, including heart girth (circumference of the chest), body length (distance between the points of shoulder and rump), body barrel (circumference of the belly before feeding), wither height (distance from the base of the front feet to wither) and hip height (distance from the base of the rear feet to the hook bones) were taken at the start of the experiment (d 3), at weaning (d 59) and at the end of the study (d 73) according to the method described by Khan et al. (2007) for dairy calves. Blood samples were collected 4 h after morning feeding from the jugular vein into 10 mL tubes on d 35 (pre-weaning sample) and 70 (post-weaning sample) of the study. Blood samples were heparinized and stored at 2°C for about 6 h; samples were centrifuged (3,000 × g 4°C, 20 min) and the harvested plasma was stored at –20°C. Plasma then was analysed to determine the concentration of glucose and blood urea nitrogen (BUN) using Pars Azmoon kits and associated procedures (Pars Azmoon Co., Tehran, Iran). Other kit was used to measure Beta-Hydroxybutyrate (BHBA) concentration (Abbott Diabetes Care Ltd. Rang Road. Witney, Oxin, OX29 OYL., UK). Rumen fluid (80 ml) was collected on d 36 of the experiment using a stomach tube fitted to a vacuum pump 3-4 h after morning feeding; the first 50 ml was discarded because of possible saliva contamination and rumen pH was measured immediately (HI 8314 membrane pH meter, Hanna Instruments, Villafranca, Italy). The rumen samples were squeezed through 4-layers of cheesecloth. A 10 ml aliquot was preserved with 2 ml of 25% meta-phosphoric acid and frozen at −20°C until analysis for SCFA. After thawing at room temperature, the rumen samples were

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analysed for SCFA using gas chromatography (model CP-9002,Chrompack, Delft, the Netherlands) as described in our previous work (Kazemi-Bonchenari et al., 2016). Ruminal fluid samples were thawed at room temperature and clarified by centrifuging at 15,000 x g for 20 minutes. The clarified supernatant were then decanted and analysed for ruminal ammonia-N using a modified phenol-hypochlorite method adapted from Broderick and Kang (1980). 2.3. Statistical analysis Statistical analyses were conducted for 3 periods: pre-weaning (d 3 to 59), post-weaning (d 59 to 73) and the entire period (d 3 to 73). Statistical analyses were performed using PROC MIXED of SAS (version 9.1; SAS Institute, Cary, NC) with the individual lamb as experimental unit. Starter intake, ADG, and feed efficiency were statistically analysed as repeated measures with week as repeated variable using the following model: Yijk = μ + SBi + Fj + Wk + (SB×W)ik + (F×W)jk + (SB×F)ij + (SB×F×W)ijk + β(Xi- X̅ ) + εijk where Yijk is the dependent variable; µ is the overall mean; SBi is the effect of sodium butyrate supplementation, Fj is the effect of forage provision, Wk is the effect of week, (SB×W)ij is the effect of the interaction between sodium butyrate and week; (F×W)ik is the effect of the interaction between forage and week; (SB×F)jk is the interaction between sodium butyrate and forage; (SB×F×W)ijk is the tripartite effect of sodium butyrate, forage and week; β(Xi-X̅ ) is the covariate variable (used only for BW with initial BW as covariate) and εijk is the overall error term. Because lamb sex was not significant, it was removed from the final model. An autoregressive (order 1) covariance structure was chosen based on the Akaike and Baysian information criteria. The variables for rumen fermentation, growth and blood were analysed using a similar model but without the time (week) effect. For growth variables, the initial measurements were included as a covariate for the statistical analysis of body measurements. The differences among treatment 8

means were determined using Tukey’s multiple range tests. Effects were considered to be significant when P < 0.05, and a tendency was considered when 0.05 < P < 0.10. All reported values are least square means. 3. Results 3.1. Lamb performance and rumen fermentation pattern The results of dietary treatments on feed intake, BW, ADG and feed efficiency are presented in Table 2. Sodium butyrate supplementation caused to greater feed intake in pre-weaned lambs (P = 0.03) and entire period of experiment (P = 0.01). No interaction was found for starter intake neither pre- nor post-weaning period between forage provision and SB supplementation. Milk intake was statistically similar among treatments (P > 0.05). Average daily gain was positively affected in pre-weaning period with SB supplementation (P < 0.01) and the interaction was significantly meaningful for overall study period (P = 0.03). The results clear that the best ADG was acquired for F-SB treatment. Sodium butyrate supplementation caused to the greater weights in weaning time compared to non-supplemented treatments (P < 0.01), but forage provision did not effect on gain. The interaction of SB and F was tended be significant with respect to the feed efficiency in pre-weaning period (P = 0.08). Forage dietary provided lambs had better faecal scores compared to forage un-supplemented lambs. Body temperatures were similar among all treatments. The ruminal SCFA concentration, individual SCFAs and ruminal ammonia-N concentrations were presented in Table 3. Forage provision improved rumen pH (P = 0.02). The SCFA concentration in was affected by SB supplementation (P = 0.02). With the respect to individual SCFAs, molar proportion of acetate was negatively affected by SB supplementation (P < 0.01) but positively with F inclusion (P = 0.04) in diets. The molar proportion of propionate was negatively affected by F 9

provision as well (P = 0.02). The results showed that proportional rate of butyrate positively affected by SB supplementation (P < 0.01) however negatively with F provision (P < 0.01). The acetate: propionate ratio (C2:C3) increased (P < 0.01) with F inclusion with the greatest value observed for N-NSB treatment. The proportional concentrations of valerate and iso-valerate were similar among treatments (P > 0.05). The concentration for ruminal ammonia-N was not differed among treatments (P > 0.05). No interaction was found in the present study for SB and F for rumen fermentation profile. 3.2. Skeletal growth Body measurements data are presented in Table 4. Heart girth and body length were similar among treatments (P > 0.05). However wither height was affected by interaction of SB and F in diets in pre-weaning period (P = 0.03). Body barrel was greater in lambs provided by forage both in preand post-weaning times. The interaction of SB and F was tended to be significant for hip height in weaning time (P = 0.08). 3.3. Blood metabolites The blood metabolite results are given in Table 5. No interactions were detected between SB and F with respect to the blood concentrations on either day 35 or 70 of study. The week effect was significant for glucose and BHBA (P < 0.05). 4. Discussion 4.1. Lamb performance and rumen fermentation pattern There was no interaction between SB supplementation and forage provision on feed intake in the current study. However, supplementation of starter with SB resulted in an increase in intake in preweaning period and in whole trail. Consistent with our findings Górka et al., (2011) reported 10

that starter intake had been shown to be increased by more than 30%, when butyrate was mixed with the starter diet in dairy calves. Cavini et al. (2015) showed that butyrate increases feed palatability. Using the double choice test, sheep were observed to prefer wheat hay treated with aqueous solution of butyric acid compared with non-treated wheat hay (Gherardi and Black, 1991). The SB supplementation improved daily gain in pre-weaning period that it was caused to greater BW both in pre-weaned and at the end of the experiment. Considering the rumen fermentation profile in the present study (Table 3) clear that butyrate proportion in rumen fluid was positively affected by SB supplementation. This make it clear in part, that why SB supplementation improved gain on weaning time. Butyrate has long been considered as chemical stimulator in rumen development that may be associated with improved animal performance as well as the rumen development (Sander et al., 1959; Mentschel et al., 2001). Moreover Orskov and Ryle (1990) stated that propionate and butyrate had greater energy retention in comparison with acetate for animal from the stoichiometry point of view. Acetate was lower and butyrate was higher in ruminal fluid of SB supplemented lambs which this may cause greater gain in these animals. The effect of SB supplementation on elevation of SCFA concentration in rumen fluid may be attributed to greater starter intake in lambs. The starter intake was about 40 g/d more for lambs supplemented with SB in comparison to un-supplemented lambs (Table 2). Greater starter intake offer more organic matter in to rumen microbes and hence enhanced ruminal fermentation which could cause greater SCFA production (Lesmeister, 2003). Our results suggest that supplementation of SB either in whole concentrate or forage provided concentrate diets could increase butyrate concentration which may due to the effect of greater starter intake or direct effect of SB supplementation.

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Previous studies which has been used SB in starter diet for lambs (Cavini et al., 2015) or calves (Ferreira and Bittar, 2010) did not measure ruminal SCFA concentration. However consistent with our results supplementation of sodium propionate in pre-weaned lambs (Majdoub et al., 2003) or in pre-weaned calves (Beiranvand et al., 2014) showed to increase propionate concentration in rumen fluid. It seems that more works need to evaluate the direct or indirect mechanisms contributed in influence of individual SCFA on rumen fermentation in pre-weaned ruminants. In addition to the favorable modifications of SB supplementation on rumen fermentation profile, improved gain and efficiency observed in SB supplemented lambs in the current study may be attributed to some other mechanisms which have been stated in previous works such as nutrients digestibility mostly due to the altered pancreatic juice volume and trypsin secretion (Guilloteau et al., 2009), improvement in immune function (Millard et al., 2002) and modification the gut microbes (Biagi et al., 2007). In the present study inclusion of forage in starter diet of lambs did not affect intake. Contradict with our results Norouzian et al., (2011) cleared that feeding starter diet provided by 7.5 or 15% alfalfa hay for Balouchi lambs reduced intake. However they reported similar feed efficiency in comparison with control treatment (whole concentrate starter fed lamb). Inclusion of forage in preweaned ruminants showed to increased (Yang et al., 2015) or decreased starter intake (Phillips, 2004; Hill et al., 2008, 2010). Castells et al. (2012) stated that the performance responses of preweaned ruminants in to forage provision in starter diet are related to ratio of forage inclusion, the forage sources provided, and the acid detergent fibre content of the basal diet. The researchers evaluated frequently the forage sources/levels in dairy calves in pre-weaning period (Suarez et al., 2007; Hill et al., 2008, 2010; Beiranvand et al., 2014; Mirzaei et al., 2015) however limited data is available for forage inclusion in starter diet of milk feeding lambs in indoor system. Intake was 12

not influenced in the presents study by this level of forage inclusion probably because of having no negative effect on rumen fill; therefore different levels of forage inclusion need to be evaluated in suckling lambs diet. In contrast with SB supplementation, forage provision in starter diet reduced gain of lambs in preweaning period in the current research. Lower propionate and higher acetate concentrations (Table 3) were observed in rumen fluid of forage provided lambs that probably would cause the lower energy retention in these animals as described by Orskov and Ryle (1990). In line with our results Suarez et al., (2007) and van Ackerena et al., (2009) reported lower propionate concentration in rumen fluid of dairy calves provided forage in their starter diet. Forage inclusion in pre-weaned ruminants has been reported to have controversial effects on ADG, as some studies reported a decrease in ADG (Hill et al., 2008, 2010) whereas others reported an increase of gain (Khan et al., 2011; Castells et al., 2012; Beiranvand et al., 2014; Yang et al., 2015). Based on what we observed on lambs in the present study inclusion the forage had negative effect on gain during pre-weaning period, however supplementation with SB in forage provided diet could alleviate its negative effect. In the present study, faecal score improved with feeding forage compared with

forage un-fed lambs. In contrast to these results, Coverdale et al. (2004) reported that faecal score was not affected by feeding bromegrass hay at the different inclusion levels with restricted or ad libitum starter feed provision to calves. However Mirzaei et al., (2015) reported increased faecal score when calves fed alfalfa forage. In the present study, lambs supplemented with forage presented greater rumen pH compared with un-supplemented one. Similar to our results, an increase in rumen pH was reported by Castells et al. (2013) in young calves when forage (chopped alfalfa hay or chopped oat hay) was included in their diets during the pre-weaning period. Laarman and Oba (2011) cleared the forage benefits on rumen fermentation which subsequently affect 13

rumen pH. Krause et al. (2002) reported that mean ruminal pH was positively correlated with the time spent ruminating and chewing which it has been affect with forage inclusion in diet. Although Mgbeahuruike (2007) cleared that the greater fibre content in diet caused to increased faecal consistency and improved faecal score in early lactating dairy cows, but the mechanism of increasing faecal score with forage inclusion in pre-weaned ruminants remains unclear. In the current study SCFA concentration did not influence by forage provision in diet. Castells et al. (2013) found a decrease in ruminal total SCFA concentrations in forage supplemented calves (alfalfa hay or oat hay). In contrast, Suárez et al. (2007) did not observe significant differences in total SCFA concentration in the ruminal fluid between corn silage fed calves and control calves. The discrepancy between studies might be attributed to the source and level of forage supplementation. Results reported herein for ruminal acetate proportions are in accordance with previous report (Terré et al., 2013). They reported that greater acetate concentration in rumen usually was observed in forage included. The acetate-to-propionate ratio observed in the current research was greater in forage provided lambs compared with un-provided lambs was in line with previous works (Castells et al., 2013; Mirzaei et al., 2015). Our results clear that both butyrate and propionate proportions negatively influenced with provision of forage in starter diet. In agreement with our findings propionate was decreased in forage provided dairy calves in pre-weaning period (Suarez et al., 2007; van Ackerena et al., 2009) or in pre-weaned lambs (Yang et al., 2015). Butyrate and propionate has long been considered as chemical stimuli in rumen development (Sander et al., 1959). However forage fermentation does not provide sufficient concentrations of these short chain fatty acids (Zitnan et al., 2005). Greater concentrate would be favorable for starch-digesting bacteria in the rumen that shift rumen fermentation toward proportionally more propionate (Dijkstra, 1994) that is what we observed in non-forage provided diets. The results 14

show that whole concentrate starter diet compared with forage included one alter rumen fermentation pattern toward more efficient energy metabolism which subsequently cause to obtain more gain in pre-weaned lambs. Ruminal ammonia-N was not influenced with SB supplementation or forage provision or their interaction. Yang et al. (2015) clarified the freechoice feeding of alfalfa hay in lambs fed pelleted starter based diet reduced ruminal ammonia-N. It seems that more works need to be evaluated the interaction of physical starter form and level of forage provided for lambs with respect to nitrogen metabolism in rumen. 4.2. Skeletal growth No differences were found between forage-provided lambs in comparison to un-provided forage as well as SB supplemented with un-supplemented ones with respect to heart girth, body length, wither height and hip height. Beiranvand et al., (2014) reported no differences in body length for calves fed 10% provided forage in starter diet compared with whole concentrate starter feeding. Considering the main effects of the present study (forage and SB), among the structural growth indicators only body barrel size was influenced by forage inclusion in starter diet in pre-weaned lambs. To the knowledge of the authors there are scare documents for measurements of forage effects on structural growth of lambs before weaning. Working on dairy calves Mirzaei et al., (2015) reported the greater body barrel in animal fed corn silage in comparison with non-forage fed animal. They cleared that rumen fill could be a confounding factor when determining forage (alfalfa hay) effects on body measurement of dairy calves. The interaction of SB and forage caused the greatest wither height in F-SB treatment. Moreover this interaction also caused a tendency for increase in hip height of lambs. Both of these interactions were observed in weaning time and not in final measurement of experiment. The results of the preset study show that the best gain was observed in lambs provided with forage and 15

supplemented with SB. There seem to be a synergic effect of these two factors (i.e. forage and SB) to obtain optimum rumen fermentation profile and animal health which consequently resulted in better daily gain and growth. Looking in to daily gains reported for different treatments in the presents study, the greatest ADG in pre-weaning period was observed in lambs provided simultaneously with forage and SB (F-SB treatment; 232 g/d). The greatest value for daily gain in F-SB treatment in pre-weaning period consequently caused the greatest weaning weight in this treatments (18.96 kg). Because main body development measures have high correlation with live weight (Heinrichs et al., 2007), therefore expectedly greater wither height was observed in F-SB treatment where the greatest body live weight was obtained. 4.3. Blood metabolites Glucose and BHBA concentrations as well as urea nitrogen concentration in blood were not affected neither with forage inclusion, nor with SB supplementation. No interaction between F and SB was observed for above mentioned items as well. The concentrations of blood metabolites in the present study were in range of previous works (Norouzian et al., 2011; Vosooghi-poostindoz et al., 2014). Consistent with our results providing 7.5 or 15% of starter with alfalfa forage Norouzian et al., (2011) did not find any differences for glucose and BHBA in comparison with control (whole concentrate) diet. Although in the current study forage inclusion decreased proportional ratio of propionate in ruminal fluid of experimental lambs (Table 3), it has been expected to observe lower glucose concentration in forage-fed lambs. This is because propionate is the main glucose precursor in the liver (Bergman, 1990). However we found that the lower propionate did not negatively affect glucose concentration in forage fed provided lambs. This is in part may attributed in to statistically similar intake among treatments. Furthermore based on Ferreira and Bittar, (2011) findings no elevation in glucose concentration was found when they 16

supplemented dairy calves with calcium propionate, it seems that the greater glucose concentration in response to greater ruminal propionate concentration would be seen only in animals with the rumen already developed. Form the other hand as Lane and Jesse (1997) found the greater propionate may not increase gluconeogenesis perhaps because the greater propionate could increase in circulating insulin (DeJong, 1982) which subsequently insulin could inhibit the gluconeogenesis. Because we found the proportional rate of individual SCFA altered in rumen fluid of experimental animals, however no difference for glucose and BHBA were observed, more research are granted to make clear the effects of SCFA on blood metabolite concentrations in under-developing ruminants. Independent of the forage provision or SB supplementation in the present study, the glucose and BHBA concentrations did differ with aging the lambs. Glucose concentration turned lower and BHBA turned greater in post-weaning period in comparison with pre-weaning period. In the present study the first blood sampling was on d 35 and the second one was in d 70 of experiment. Ferreira and Bittar, (2011) observed a reduction of the glucose concentration as calves aged which has been stated that is a response correlated to the rumen development and the transition of animal from pre-ruminant to functional ruminant. Other authors have also found a similar pattern for glucose plasma concentrations according to animal age (Quigley et al., 1991). As it has been clear in calves evaluation of BHBA reveals an increase in plasma concentrations with advancing age (Quigley et al., 1992). This mainly because of the increased solid feed intake as animals mature (Ferreira1 and Bittar, 2011). Blood urea nitrogen was not affected with treatments. Because the concentration of ruminal ammonia-N was not affected by treatments (Table 3), the urea nitrogen concentration in blood was not affected as well. This is partly because of relatively similar protein content of the diets. 5. Conclusion 17

Supplementation of lamb’s diet with SB in pre-weaning period improved intake and gain. Providing the lamb starter diet with alfalfa forage increased ruminal pH but failed to improve performance. In conclusion supplementation SB could alleviate forage negative effects on rumen fermentation and performance in pre-weaning period in lambs. More research are warranted to evaluate interaction of other SCFA sources with different forage sources/levels in pre-weaned lambs in indoor feeding system. Acknowledgments The data in this study was developed as a part of the first author thesis. Special thanks to Arak University for covering the financial supports of the present study. Appreciations to management board and staff of Gole-Reyhan sheep farm to help in collecting the data throughout the study. Great thanks for useful technical comments of Mr. Sajedi (Animal Nutrition Laboratory, University of Tehran) in measuring ruminal parameters. Literature cited Alvarez-Rodríguez, J., Monleón, E., Sanz, A., Badiola, J.J., Joy, M., 2012. Rumen fermentation and histology in light lambs as affected by forage supply and lactation length. Res. Vet. Sci. 92, 247-253. Anderson, K.L., Nagaraja, T. G., Morrill, J.L., Avery, T.B., Galitzer, S.J., Boyer, J.E., 1987. Ruminal microbial development in conventionally or early-weaned calves. J. Anim. Sci. 64, 1215–1226. AOAC. 1990. Official Methods of Analysis. 15th ed. Association of Official Analytical Chemists, Arlington, VA.

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Beiranvand, H., Ghorbani, G.R., Khorvash, M., Nabipour, A., Dehghan-Banadaky, M., Homayouni, A., Kargar, S., 2014. Interactions of alfalfa hay and sodium propionate on dairy calf performance and rumen development. J. Dairy Sci. 97, 2270–2280. Bergman, E.N., 1990. Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol. Rev. 70, 567–590. Biagi, G., Piva, A., Moschini, M., Vezzali, E., Roth, F.X., 2007. Performance, intestinal microflora, and wall morphology of weanling pigs fed sodium butyrate. J. Anim. Sci. 85, 1184– 1191. Broderick, G.A., Kang, J.H., 1980. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J. Dairy Sci. 63, 64-75. Bull, L.S., Bush, L.J., Friend, J.D., Harris Jr, B., Jones, E.W., 1965. Incidence of ruminal parakeratosis in calves fed different rations and its relation to volatile fatty acid absorption. J. Dairy Sci. 48, 1459–1466. Castells, L., Bach, A., Arís, A., Terré, M., 2013. Effects of forage provision to young calves on rumen fermentation and development of the gastrointestinal tract. J. Dairy Sci. 96, 5226– 5236. Castells, L., Bach, A., Araujo, G., Montoro, C., Terre, M., 2012. Effect of different forage sources on performance and feeding behavior of Holstein calves. J. Dairy Sci. 95, 286–293. Cavini, S., Iraira, S., Siurana, A., Foskolos, A., Ferret, A., Calsamiglia, S., 2015. Effect of sodium butyrate administered in the concentrate on rumen development and productive performance of lambs in intensive production system during the suckling and the fattening periods. Small Rum. Res. 123, 212–217.

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Khan, M.A., Weary, D.M., von Keyserlingk, M.A.G., 2011. Hay intake improves performance and rumen development of calves fed higher quantities of milk. J. Dairy Sci. 94, 3547–3553. Khan, M.A., Lee, H.J., Lee, W.S., Kim, H.S., Ki, K.S., Hur, T.Y., Suh, G.H., Kang, S.J., Choi, Y.J., 2007. Structural growth, rumen development, and metabolic and immune responses of Holstein male calves fed milk through step-down and conventional methods. J. Dairy Sci. 90, 3376–3387. Krause, K.M., Combs, D.K., Beauchemin, K.A., 2002. Effects of forage particle size and grain fermentability in midlactation cows. II. Ruminal pH and chewing activity. J. Dairy Sci. 85, 1947–1957. Laarman, A.H., Oba, M., 2011. Short communication: Effect of calf starter on rumen pH of Holstein dairy calves at weaning. J. Dairy Sci. 94, 5661–5664. Lane, M.A., Jesse B.W., 1997. Effect of volatile fatty acid infusion on development of the rumen epithelium in neonatal sheep. J Dairy Sci. 80, 740–746. Lee-Rangel, H.A., Mendoza, G.D., Gonzalez, S.S., 2012. Effect of calcium propionate and sorghum level on lamb performance. Anim. Feed Sci. Technol. 177, 237–241. Leibholz, J., 1975. Ground roughage in the diet of the early-weaned calf. Anim. Prod. 20, 93– 100. Lesmeister, K.E., 2003. Dietary alterations and their influence on rumen development in neonatal dairy calves. PhD Thesis, The Penn State University, State College city, PA. Majdoub, I., Vermorel, M., Ortiques-Marty, I., 2003. Intraruminal propionate supplementation modifies indlibm energy metabolism without changing the splanchnic release of glucose in growing lambs. Brit. J. Ntur. 89, 39-50. Mendoza-Martínez, G.D., Pinos-Rodríguez, J.M., Lee-Rangel, H.A., Hernández-García, P.A., 22

Rojo-Rubio, R., Relling, A., 2014. Effects of dietary calcium propionate on growth performance and carcass characteristics of finishing lambs. Anim. Prod. Sci. 56 (7), 11941198. Mentschel, J., Leiser, R., Mulling, C., Pfarrer, C., Claus, R., 2001. Butyric acid stimulates rumen mucosa development in the calf mainly by a reduction of apoptosis. Arch. Tierernahr. 55, 85–102. Mgbeahuruike, A.C., 2007. Fecal characteristics and production of dairy cows in early lactation. MSc Thesis, Swedish University of Agricultural Science, Skara, Sweden. Millard, A.L., Mertes, P.M., Ittelet, D., Villard, F., Jeannesson, P., Bernard, J., 2002. Butyrate affects differentiation, maturation and function of human monocyte-derived dendritic cells and macrophages. Clin. Exp. Immunol. 130, 245–255. Mirzaei, M., Khorvash, M., Ghorbani, G.R., Kazemi-Bonchenari, M., Riasi, A., Nabipour, A., van den Borne, J.J.G.C., 2015. Effects of supplementation level and particle size of alfalfa hay on growth characteristics and rumen development in dairy calves. J. Anim. Physiol. Anim. Nutr. 99, 553–564. National Research Council., 2007. Nutrient requirements of small ruminants. The National Academy Press, Washington, DC, USA. Nocek, J.E., Kesler, E. M., 1980. Growth and rumen characteristics of Holstein steers fed pelleted or conventional diets. J. Dairy Sci. 63, 249–254. Norouzian, M.A., Valizadeh, R., Vahmani, P., 2011. Rumen development and growth of Balouchi lambs offered alfalfa hay pre- and post-weaning. Trop. Anim. Health Prod. 43(6), 1169-74 Orskov, E.R., Ryle, M., 1990. Energy nutrition in ruminants. 1st ed. Elsevier Science Publishers. L.td., Essex. UK. 149pp. 23

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van Ackerena, C., Steinga, H., Hartung, K., Funk, R., Drochner, W., 2009. Effect of roughage level in a total mixed ration on feed intake, ruminal fermentation patterns and chewing activity of earlyweaned calves with ad libitum access to grass hay. Anim. Feed Sci. Technol. 153, 48– 59. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber nonstarch polysaccharide in relation to animal nutrition. J. Dairy Sci. 74, 3583– 3597. Vosooghi-poostindoz, V., Foroughi, A.R., Delkhoroshan, A., Ghaffari, M.H., Vakili, R. Soleimani, A.K., 2014. Effects of different levels of protein with or without probiotics on growth performance and blood metabolite responses during pre- and post-weaning phases in male Kurdi lambs. Small Rum. Res. 117, 1–9. Warner, R.G., 1991. Nutritional factors affecting the development of a functional ruminant-A historical perspective. Proc. Cornell Nutr. Conf., 1-12. Ithaca, NY: Cornell University. Yang, B., He, B., Wang, S.S., Liu, J.X., Wang, J.K., 2015. Early supplementation of starter pellets with alfalfa improves the performance of pre- and postweaning Hu lambs. J Anim. Sci. 93(10), 4984-94. Table 1. Ingredients and chemical composition of experimental diets (% of DM) Experimental diets1

Ingredients

NF

F

Alfalfa hay, chopped

0

10

Corn grain, ground

59

51

Soybean meal Wheat bran

28 7

26 7

25

Vitamin-mineral mix2

3

3

Calcium carbonate

1

1

Sodium bicarbonate

1

1

Dicalcium phosphate

0.5

0.5

Salt

0.5

0.5

ME3, Mcal.kg-1

2.71

2.60

CP

18.78

18.59

NDF

12.9

14.6

NFC4

54.2

51.7

Ether extract

2.80

2.80

Ca

0.52

0.53

P

0.32

0.32

Chemical composition

1

Experimental diets were: NF = whole concentrate starter diet; F = 10% forage (alfalfa hay) on DM basis was included

in starter diet. 2

Contained per kilogram of supplement: 15,000 IU vitamin A, 10 IU vitamin D3, 10 mg/kg vitamin E, 1 g Mn, 30 g

Ca, 1 g Zn, 5 g P, 9 g Mg, 18 g Na, 20 mg Fe, 10 mg S, 14 mg Co, 20 mg Cu, 11 mg I, and 4 mg Se. 3,4

Calculated from NRC (2001) (Non-fibre-carbohydrate was calculated as DM - (NDF + CP + ether extract + ash).

Table 2. Least square means for starter intake, gain, and feed efficiency in lambs fed different alfalfa forage levels (0 vs. 10, % of DM) and sodium butyrate supplementation (0 vs. 3 g/kg starter DM) (n = 7 lambs per treatment). Treatments1

Item

P-value2 SEM

NF NSB

F SB

Starter feed intake, g/day Pre-weaning (d 3-59)

NSB

SB 246.5

F 303.8

275.3 26

SB 299.8

F×SB 12.73

0.46

0.03

0.39

Post-weaning (d 60-73)

913.1

1029.8

984.7

1098.5

46.06

0.26

0.07

0.91

Entire period (d 3-73)

315.1

454.6

410.4

466.6

28.70

0.15

0.01

0.27

Milk DM intake, g/d

96.8

96.5

95.9

95.4

1.0

0.47

0.77

0.96

191.9bc

207.1b

170.8 c

232.2a

11.56

0.87

<0.01

0.06

Post-weaning (d 60-73)

225.0

223.2

235.1

289.3

64.7

0.59

0.69

0.68

Entire period (d 3-73)

197.0b

209.9ab

180.6 c

239.4a

11.83

0.51

<0.01

0.03

6.24

6.18

5.80

5.96

0.20

0.22

0.84

0.68

16.85b

18.04ab

15.35 c

18.96a

0.44

0.64

<0.01

0.06

b

ab

c

22.85

a

0.45

0.89

<0.01

0.02

ADG, g/day Pre-weaning (d 3-59)

BW, kg Initial (d 3) Weaning (d 59) Final (d 73)

20.01

21.29

18.70

Feed efficiency Pre-weaning (d 3-59)

0.519

0.516

0.467

0.593

0.02

0.84

0.15

0.08

Post-weaning (d 60-73)

0.239

0.224

0.258

0.264

0.04

0.64

0.89

0.86

Entire period (d 3-73)

0.366

0.344

0.357

0.401

0.02

0.55

0.78

0.41

Faecal score3

1.52

1.50

1.41

1.33

0.03

0.01

0.28

0.45

Body temperature, C°

39.6

39.6

39.5

39.5

0.05

0.15

0.74

0.94

1

Treatments: 1) starter diet provided neither with F nor with SB (NF-NSB), 2) starter diet supplemented only with SB

(NF-SB), 3) starter diet provided only with forage (F-NSB), and 4) starter diet simultaneously provided by forage and SB (F-SB). 2

Statistical comparisons: F = forage inclusion; SB = sodium butyrate supplementation F×SB= F by SB interaction.

3

Faecal scoring was as follows: 1 = normal, 2 = soft to loose, 3 = loose to watery, 4 = watery, mucous, slightly bloody,

5 = watery, mucous and bloody. Means within a row with different superscript letters are different (P < 0.05).

Table 3. Least square means for rumen fermentation activities in lambs fed different alfalfa forage levels (0 vs. 10, % of DM) and sodium butyrate supplementation (0 vs. 3 g/kg starter DM) (n = 7 lambs per treatment).

Item

Treatments1

27

P-value2

SEM

NF NSB

F SB

NSB

SB

F

SB

F×SB

Rumen pH

5.85

5.84

6.02

5.91

0.07

0.02

0.11

0.15

Total SCFA, mmol/L

76.7 ab

81.8a

72.6 b

80.3 ab

4.23

0.22

0.02

0.54

Acetate

53.0 bc

50.7c

59.9 a

55.9b

2.73

<0.01

0.04

0.54

Propionate

23.1

23.7

20.8

21.5

1.70

0.02

0.45

0.96

Butyrate

17.9 ab

19.7a

13.6 b

16.6 ab

1.17

<0.01

<0.01

0.33

Valerate

3.56

3.59

3.48

3.42

1.40

0.86

0.98

0.94

Isovalerate

2.40

2.13

2.09

2.40

0.64

0.91

0.98

0.40

Acetate/Propionate (C2:C3)

2.30b

2.13c

2.90 a

2.62 ab

0.20

<0.01

0.15

0.69

Ruminal ammonia-N, mg/dl

8.17

7.36

10.44

9.58

3.11

0.19

0.55

0.92

Individual SCFA, mol/100 mol

1

Treatments: 1) starter diet provided neither with F nor with SB (NF-NSB), 2) starter diet supplemented only with SB

(NF-SB), 3) starter diet provided only with forage (F-NSB), and 4) starter diet simultaneously provided by forage and SB (F-SB). Means within a row with different superscript letters are different (P < 0.05).

Table 4. Least square means for structural growth parameters in lambs fed different alfalfa forage levels (0 vs. 10, % of DM) and sodium butyrate supplementation (0 vs. 3 g/kg starter DM) (n = 7 lambs per treatment).

28

Treatments1

Item

P-value2

SEM

NF NSB

F SB

NSB

SB

F

SB

F×SB

Initial (d 3)

52.4

51.7

52.2

52.8

1.34

0.52

0.87

0.42

Weaning (d 59)

58.6

56.7

57.3

56.8

0.80

0.42

0.13

0.40

Final (d 73)

61.8

63.2

62.6

64.5

3.80

0.61

0.20

0.91

Initial (d 3)

40.8

39.5

41.0

40.8

1.84

0.31

0.32

0.41

Weaning (d 59)

49.4

47.4

49.5

49.2

0.60

0.25

0.17

0.32

Final (d 73)

52.2

53.0

53.8

52.9

2.27

0.42

0.92

0.36

Initial (d 3)

51.2

51.5

50.28

51.85

2.28

0.68

0.29

0.46

Weaning (d 59)

56.3ab

54.7 b

55.6b

58.0 a

0.65

0.14

0.65

0.03

Final (d 73)

61.2

60.4

60.5

52.4

2.24

0.55

0.47

0.13

Initial (d 3)

52.4

49.8

51.4

51.5

2.32

0.68

0.18

0.13

Weaning (d 59)

59.5

58.0

58.3

59.9

0.70

0.69

0.91

0.08

Final (d 73)

61.5

60.2

62.3

62.6

3.20

0.25

0.71

0.54

Initial (d 3)

57.5

56.4

58.1

56.5

3.52

0.83

0.29

0.91

Weaning (d 59)

66.3

64.5

67.5

68.6

1.12

0.02

0.75

0.22

Final (d 73)

70.1

71.2

74.1

76.0

3.93

0.01

0.21

0.94

Heart girth

Body length

Wither height

Hip height

Body barrel

1

Treatments: 1) starter diet provided neither with F nor with SB (NF-NSB), 2) starter diet supplemented only with SB

29

(NF-SB), 3) starter diet provided only with forage (F-NSB), and 4) starter diet simultaneously provided by forage and SB (F-SB). 2

Statistical comparisons: F = forage inclusion; SB = sodium butyrate supplementation F×SB= F by SB interaction.

Means within a row with different superscript letters are different (P < 0.05).

Table 5. Least square means for selected blood metabolites in lambs fed different alfalfa forage levels (0 vs. 10, % of DM) and sodium butyrate supplementation (0 vs. 3 g/kg starter DM) (n = 7 lambs per treatment). Treatments1

Item

P-value2

SEM NF NSB

F SB

NSB

SB

F

SB

F×SB

day 35 (Pre-weaning)

4.72

4.39

4.52

4.48

0.41

0.78

0.36

0.48

day 70 (Post-weaning)

2.75

2.82

2.51

2.88

0.32

0.54

0.16

0.34

day 35 (Pre-weaning)

0.49

0.52

0.55

0.47

0.08

0.91

0.78

0.53

day 70 (Post-weaning)

0.83

0.80

0.74

0.75

0.10

0.61

0.92

0.95

day 35 (Pre-weaning)

14.4

13.2

14.8

13.9

4.25

0.83

0.64

0.96

day 70 (Post-weaning)

13.6

11.8

12.2

12.6

2.97

0.82

0.60

0.42

Glucose, mM

BHBA, mM

BUN, mg/dl

1

Treatments: 1) starter diet provided neither with F nor with SB (NF-NSB), 2) starter diet supplemented only with SB

(NF-SB), 3) starter diet provided only with forage (F-NSB), and 4) starter diet simultaneously provided by forage and SB (F-SB). 2

Statistical comparisons: F = forage inclusion; SB = sodium butyrate supplementation F×SB= F by SB interaction.

30