Increasing dietary levels of citral oil on nutrient total tract digestibility, ruminal fermentation, and milk composition in Saanen goats

Accepted Manuscript Title: Increasing dietary levels of citral oil on nutrient total tract digestibility, ruminal fermentation, and milk composition in Saanen goats Authors: Ta´ıssa S. Canaes, Filipe Zanferari, Bruna L. Maganhe, Caio S. Takiya, Thiago H. Silva, Tiago A. Del Valle, Francisco P. Renn´o PII: DOI: Reference:

S0377-8401(17)30043-3 http://dx.doi.org/doi:10.1016/j.anifeedsci.2017.05.002 ANIFEE 13780

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7-1-2017 28-4-2017 3-5-2017

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Please cite this article as: Canaes, Ta´ıssa S., Zanferari, Filipe, Maganhe, Bruna L., Takiya, Caio S., Silva, Thiago H., Del Valle, Tiago A., Renn´o, Francisco P., Increasing dietary levels of citral oil on nutrient total tract digestibility, ruminal fermentation, and milk composition in Saanen goats.Animal Feed Science and Technology http://dx.doi.org/10.1016/j.anifeedsci.2017.05.002 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.

Increasing dietary levels of citral oil on nutrient total tract digestibility, ruminal fermentation, and milk composition in Saanen goats Taíssa S. Canaes, Filipe Zanferari, Bruna L. Maganhe, Caio S. Takiya, Thiago H. Silva, Tiago A. Del Valle, Francisco P. Rennó* Department of Animal Nutrition and Production, University of Sao Paulo, Pirassununga, Brazil. 13635-900. *

Corresponding author: Prof. Francisco Palma Rennó, mailing address: Department of Animal Nutrition and

Production, University of Sao Paulo, Av. Duque de Caxias Norte, 225 – campus da USP, CEP 13635-900, Pirassununga, SP-Brazil. e-mail: [email protected], phone number: +55 19 3565-4248, fax number: +55 19 3565-4300.

Highlights     

Four incrementing doses of high purity citral oil (CO) were evaluated. CO consumption by goats linearly decreased neutral detergent fiber digestibility. CO showed a quadratic positive effect on rumen propionate concentration. CO had neither effect on milk yield nor composition of goats. CO had no effects on milk fatty acid profile.

Abstract This study was undertaken to evaluate the effects of increasing dietary doses of high purity citral oil on nutrient total tract digestibility, ruminal fermentation, blood metabolites, milk yield and composition, and N utilization in dairy goats. Twenty-four Saanen goats [62±1.4 kg of body weight (BW), 75±20 days in milk, and 3.0±0.27 kg/d of milk yield, at the start of experiment], being eight of them rumen-cannulated, were used in a 4×4 Latin square design experiment with 21-d periods in which the first 14 d were allowed to treatment adaptation. Animals were assigned to the following treatments: control, with no citral supply; and dietary addition of 0.08, 0.16 or 0.24 mL of citral oil per kg of BW. Increasing doses of citral oil did not affect dry matter (DM) and nutrient intake, but it linearly decreased neutral detergent fiber total tract digestion in dairy goats. Treatments neither affected ruminal pH nor NH3-N, but citral oil linearly increased butyrate proportion in ruminal fluid of goats. Citral oil consumption had a positive quadratic effect on 1

ADF, acid detergent fiber; BW, body weight; CP, crude protein; DM, dry matter; EE, ether extract; EO, essential oil; FA, fatty acid; FCM, fat-corrected milk; iADF, indigestible detergent fiber; N, nitrogen; NDF, neutral detergent fiber; NH3-N, ammonia nitrogen; td, truly digestible; TDN, total digestible nutrient; VFA, volatile fatty acid.

ruminal propionate (mmol/dL and mmol/100 mmol) and butyrate (mmol/dL) in goats. Treatments had a negative quadratic effect on ruminal acetate to propionate ratio and acetate proportion. Citral consumption by dairy goats linearly decreased blood urea concentration. Although treatments did not affect milk and fat-corrected milk yield, citral oil linearly decreased milk fat production in goats. Citral had no effect on milk fatty acid profile and N utilization in goats. Citral consumption had no effect on DM and nutrient intake as well as on fat-corrected milk yield, but it may increase ruminal propionate concentration in dairy goats. Keywords: antimicrobial; ; ; ; , 3,7-dimethyl-2,6-octadienal, essential oil, lemongrass, monoterpene aldehyde.

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Introduction Public concern regarding the use of antibiotics as growth promoters in livestock production

has increased since their use have been limited by the European Union, because of the possible relationship with antibiotic resistant bacteria emergence (Casewell et al., 2003). Consequently, animal scientists, microbiologists and ruminant nutritionists have reduced the use of antibiotics and started seeking for alternative additives that promote feed efficiency and productivity of cattle. Essential oils (EO) are naturally occurring volatile components from plant extracts, which compounds - such as citral, carvacrol, tymol, limonene and cineole - have shown inhibitory effects upon gram-negative and gram-positive bacteria, due to its terpenoid and phenolic compounds (Benchaar et al., 2008). Essential oils have a broad range of effects on rumen fermentation and biohydrogenation, protein metabolism and animal performance (Benchaar et al., 2008). For example, lactating dairy cows fed a most-concentrate diet and supplemented a blend of EO exhibited higher ruminal pH and acid detergent fiber (ADF) digestibility, and a trend to higher milk conjugated linoleic acid concentration than control group (Benchaar et al., 2006). In an in vitro experiment, Chaves et al. (2008) evaluated several EO (anethol, cinnamon oil, garlic oil, juniper berry, and p-cymene) on ruminal fermentation and bacterial deaminative activity. These authors reported that all EO maintained pH at greater values than control and most of the EO increased the deaminative activity of ruminal bacteria. Finally, EO provision to dairy ewes increased the milk production through an entire lactation, decreased milk urea content, and had no effect on serum parameters (Giannenas et al., 2011). Although literature have reported several effects of EO, both in in vitro or in vivo studies, it is important to highlight that the active components in plant extracts may vary widely depending

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on the geographic location which the plant was cropped, methods of extraction, purity degree, and whether they were provided as a blend or as a pure extract (Marino et al., 2001; Burt, 2004). Thus, it is necessary to evaluate the effects of high purity and commercially available substances in in vivo experiments to generate consistent results. Citral is a major component (~80%) of volatile oil obtained from lemongrass (Cymbopogon citratus), Litsea cubeba, and lemon myrtle (Backhousia citriodora) (Nhu-Trang et al. 2006) and it is comprised by two isomeric acyclic monoterpene aldehydes - geranial and neral - responsible for its broad spectrum antibacterial and antifungal activities (Khunkitti, 2010; Liakos et al., 2016). In addition, lemongrass oil has been associated with anti-inflammatory (Katsukawa et al., 2010), anticarcinogenic (Puatanachokchai et al., 2002), and antioxidant (Rabbani et al., 2005) effects. Nanon et al. (2014) evaluated lemongrass oil on in vitro ruminal fermentation using a substrate with 50:50 of forage to concentrate ratio and reported that lemongrass oil increased microbial attachment to feed particles, especially neutral detergent fiber (NDF) from forage, and consequently improved DM digestibility. Despite citral has been used in cosmetics, food, and pharmaceutical industries (Enjalbert et al., 1983; Carnat et al., 1998), little is known regarding its effects on ruminant nutrition. The objective of this study was to determine the effects of increasing dietary levels of high purity citral oil on total tract digestibility of nutrients, ruminal fermentation, blood metabolites, milk yield and composition, and N utilization in dairy goats. Our hypothesis was that citral oil consumption would improve ruminal fermentation and nutrient digestibility, increasing milk production of dairy goats in a dose-dependent manner.

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Material and Methods This experiment was conducted at the Small Ruminant Sector from University of Sao

Paulo, Pirassununga, Brazil. All experimental procedures were carried out under the approval from the School of Veterinary Medicine and Animal Science’s Ethics Committee, University of São Paulo, São Paulo, Brazil.

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Animals and Design Twenty-four Saanen goats [62±1.4 kg of body weight (BW), 75±20 days in milk and

3.0±0.27 kg/d milk yield, at the start of experiment], being eight of them rumen cannulated and 16 non-cannulated, were used in a 4×4 Latin square design experiment. Periods lasted 21 days whereas 14 days were allowed to treatment adaptation (Machado et al., 2016) and seven days used for sampling. Animals were blocked within square according to DIM, BW and milk production, then goats were randomly assigned to one sequence containing the following treatments: control, with no citral oil; and dietary addition of 0.08, 0.16 or 0.24 ml of citral oil per kg of BW. Treatment sequences were balanced to minimize carryover effects (Williams, 1949). Citral oil was acquired from Sigma Aldrich (CAS number 5392-40-5; St. Louis, MO) and had the following specifications according to its manufacturer: cis and trans, >96% purity, 0.88 g/mL density at 25ºC. Citral doses were chosen based on Canaes (2011) who studied the partial replacement of corn silage by lemongrass in diets of dairy goats and reported improvements on milk production and milk fatty acid (FA) profile when feeding lemongrass at 354 g/kg diet DM. Citral oil was added to 100 g of concentrate and then, the concentrate containing citral was top dressed onto the TMR to guarantee that animal consume all citral supplied. Cannulated and non-cannulated goats were treated similarly. Citral oil was stored according to manufacturer’s 5

recommendation and it was supplied twice daily in equal amounts. The basal diet was formulated according to NRC (2007), consisted of 50:50 forage to concentrate ratio, and was provided at 6h00 and 13h00 in equal amounts to keep the daily orts between 5 and 10% of feed offered on as fedbasis (Table 1). Throughout the experiment, animals were housed in individual pens (1.8 m 2 of area), containing feed bunks, free access to water and slatted floor. 2.2

Nutrient Intake Ration and refusals were daily weighed to determine nutrient intake. Over the sampling

periods, dietary ingredients were collected during the concentrate preparation (4 samples); corn silage and orts samples of each animal were taken daily during the sampling periods and composited into samples per animal and period. All samples were dried in a forced air oven at 55ºC for 72 h and ground 1-mm or 2-mm screen Wiley mill (MA340, Marconi, Piracicaba, Brazil). All samples were analyzed for DM (950.15), total N (984.13) and ether extract (920.39) according to AOAC (2000) methods. Neutral detergent fiber, ADF, and lignin content in samples were assessed according to Van Soest et al. (1991) using a fiber analyzer (TE-149, Tecnal Equipment for Laboratory Inc., Piracicaba, Brazil). 2.3

Total Tract Digestibility of Nutrients Fecal samples were collected at 6h00, 10h00, 14h00, 18h00 and 22h00 on days 18, 19 and

20 directly from the rectum of goats after digital stimulation. Samples were pooled into one sample per animal per period and then, dried in a forced air oven at 55ºC during 72 h. Dried samples of feed ingredients, orts and feces were ground in a 2-mm screen Wiley mill (MA340, Marconi). Total DM fecal excretion was determined based on indigestible ADF (iADF) intake and iADF concentration in feces (Lippke et al., 1986). For analysis of iADF content, dried and ground samples (25 mg DM/cm2) of feed, orts and feces were placed in bags of non-woven fabric tissue 6

(pore size 50 µm, 100 g/m2) with dimensions of 5 × 5 cm. Then, samples were incubated in the rumen of two cannulated dry cows receiving a similar diet (ad libitum) of the current experiment. After 288-h incubation, samples were washed in running tap water, and analyzed for ADF, as previously described. Nutrient digestibility was estimated based on total intake and total excretion of each nutrient. 2.4

Ruminal Fermentation Ruminal fluid samples were collected at 0, 2, 4, 6, 8, 10 and 12 h relative to the morning

feeding on day 21 of each experimental period. Ruminal digesta, collected from different sites (anterior dorsal, anterior ventral, medium ventral, posterior dorsal, and posterior ventral) was pooled for each animal and squeezed through four layer cheesecloth. Ruminal fluid pH was measured using a pHmeter (MB-10, Marte, Santa Rita Sapucai, Brazil). Aliquots of 1600 µL of ruminal fluid samples were mixed with 400 µL methanoic acid (98–100% H2CO2), being centrifuged at 7,000 × g for 15 min at 4ºC, and the supernatant of each sample was stored at -20 ºC for volatile fatty acids (VFA) analysis. Also, ruminal fluid aliquots of 2 mL were mixed with 1 mL of sulfuric acid (0.5 Mol/L H2SO4) and frozen for subsequent analysis of NH3-N by the colorimetric phenol-hypochlorite method (Broderick and Kang, 1980). Volatile fatty acids were measured using a gas chromatograph (GC-2014, Shimadzu, Tokyo, Japan) equipped with a capillary column (Stabilwax, Restek, Bellefonte, USA). The gases used were helium as the carrier gas (8.01 mL/min flow), hydrogen as the fuel gas (pressure of 60 kPa), and synthetic air as the oxidizer gas (pressure of 40 kPa). Steamer temperature was set at 220 °C; ionization detector flames at 250 °C; and separation column at 145 °C for 3 min, which was then raised to 10 °C/ min until it reached 200 °C.

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Blood metabolites On day 16 of each period, blood samples were collected from all goats by puncture of

jugular vessels into sterile vacutainers without clot activator (BD Vacutainer systems, Franklin Lakes, NJ, USA) prior to the morning feeding. After clotting, samples were centrifuged for 15 min at 2,000 × g and 4 °C; the supernatant serum was transferred into labeled plastic tubes and stored at -20 °C. Blood serum was analyzed for glucose, urea, triacylglycerides, high density lipoprotein, urea, albumin, creatinine kinase, aspartate-aminotransferase, gamma-glutamyltransferase and alanine transaminase using colorimetric commercial kits (Bioclin®, Belo Horizonte, Brazil) and absorbances measured in a semi-automatic biochemistry analyzer (SBA-200, CELM®, Sao Caetano do Sul, Brazil). 2.6

Milk Yield and Composition Goats were mechanically milked (WestfaliaSurge, GEA, Campinas, Brazil) once daily

(8h00) and milk samples were collected from each goat on days 15, 16 and 17 of each experimental period. Immediately after collection, milk samples were analyzed for fat, protein and lactose contents by infrared analysis (Lactoscan®, Entelbra, Londrina, Brazil). Sub-samples were stored at -20 °C for milk FA profile analysis. Fat-corrected milk (FCM) was calculated according to Mavrogenis and Parachristoforou (1988), as follows: FCM 4% = milk yield (kg/d) × (0.411 + 0.147× milk fat percentage). Milk lipid extraction was performed according to Feng et al. (2004) and methylated according to Kramer et al. (1997). Fatty acids were quantified by gas chromatography (GC Shimatzu 2010 with automatic injection, Shimadzu Corporation, Kyoto, Japan) equipped with a SP-2560 capillary column (100 m × 0.25 mm i.d. with 0.02 µm film thickness; Supelco SigmaAldrich Group, Bellefonte, PA). The oven temperature was held on 70 °C for 4 min, increased by 8

13 °C/min until 175 ºC, and then held at this temperature for 27 min. Finally, temperature was increased by 4 °C/min until it reaches 215 °C, and then was kept at this temperature for 31 min. Hydrogen was used as the carrier gas flowing at a rate of 40 cm 3/s. Four standards were used for FA identification: standard C4-C24 (TM 37; Supelco Sigma-Aldrich Group), C18:1 trans-11 (V038- 1G; Supelco Sigma-Aldrich Group), C18:2 trans-10, cis-12 (UC-61M 100 mg; NUCHEKPREP, Inc. Elysian, MN), and C18:2 cis-9, trans-11 (UC-60M 100 mg; NU-CHEKPREP, Inc. Elysian). 2.7

Nitrogen Utilization Nitrogen excreted in milk was calculated based on the following equation: milk N (g/d) =

milk CP concentration (g/kg) × milk yield (kg/d) ÷ 6.38. Nitrogen excreted in feces was calculated based on the equation: fecal N (g/d) = CP in feces (g/kg) ×DM fecal excretion (kg/d) ÷ 6.25. Urine samples were collected on days 17 and 18 of each period, 4 h after the morning feeding. After being collected, urine samples were filtered and stored at -20 °C, for further assessment of N and creatinine concentrations. Total urine volume was estimated according to Fonseca et al. (2006), and urine N was determined according to AOAC (method 984.13, AOAC, 2000); hence, urinary N excretion was calculated by multiplying urine N by urine volume. Creatinine and uric acid concentrations were determined using commercial kits (Bioclin®) by colorimetric method and absorbance was measured by a semi-automatic biochemistry analyzer (SBA-200, CELM®). Daily creatinine urinary excretion was estimated assuming the daily rate of creatinine excretion as a ratio to BW fixed at 26.05 mg/kg BW (Fonseca et al., 2006). Body weights were measured using an electronic livestock scale for small ruminants, after milking and before the morning feeding on days 7 and 21 of each experimental period. 2.8 Statistical Analysis 9

Data from all animals (n = 24) were utilized in the statistical analysis of nutrient intake and digestibility, blood metabolites, milk yield and composition, and N utilization. The ruminal fermentation parameters were analyzed from data of cannulated goats (n = 8). Data, except for ruminal fermentation parameters, were submitted analyses of variance using the PROC MIXED SAS 9.4 (Statistical Analysis Software – SAS Institute Inc., Cary, NC) according to the following model: Yijkl = μ + Si + gj:i + Tk + Pl + eijkl with 𝑔j:i  𝑁 (0, 𝑔2 ) and 𝑒𝑖𝑗𝑘𝑙  𝑁 (0, 2𝑒 ) where: Yijkl is the value of the dependent variable, µ is the overall mean, Si is the fixed effect of the ith Latin square (i = 1 to 6), gj:i is the random effect of the jth goat within the ith Latin square (j = 1 to 24), Tk is the fixed effect of the kth treatment (k = 1 to 4), Pl is the fixed effect of the lth experimental period (k = 1 to 4), eijkl is the random residual error, 𝑔2 is the variance due to cows, and 2𝑒 is the residual variance. Data of ruminal fermentation were analyzed as a Latin square design, with repeated measurements (0, 2, 4, 6, 8, 10, 12 h relative to the morning feeding). The resulting model was: Yijkl = μ + Si + gj:i + Tk + Pl + ωijkl + Im + TIkm + eijklm with 𝑔j:i  𝑁 (0, 𝑔2 ), 𝜔𝑖𝑗𝑘𝑙  𝑁 (0, 2𝜔 ) and 𝑒𝑖𝑗𝑘𝑙𝑚  𝑀𝑉𝑁 (0, 𝑅) where: Yijkl is the value of the dependent variable, µ is the overall mean, Si is the fixed effect of the ith Latin square (i = 1 to 2), gj:i is the random effect of the jth goat within the ith Latin square (j = 1 to 8), Tk is the fixed effect of the kth treatment (k = 1 to 4), Pl is the fixed effect of the lth experimental period (k = 1 to 4), ωijkl is the random effect of the jth goat within the lth 10

experimental period, Im is the fixed effect of the mth evaluation time (k = 1 to 7), TIkm is the interaction term, eijklm is the random residual error, 𝑔2 is the variance due to goat, 2𝜔 is the variance

due to goat within in experimental period (parcel). MVN stands multivariate normal, and R is the variance-covariance matrix of residuals due to the repeated measurements on parcel. Various structures of the error covariance matrix R were fitted: compound symmetry (CS), autoregressive [AR(1)], Huynh-Felt (HF), Toeplitz (TOEP) and unstructured (UN) and was chosen based on the Akaike Criterion (BIC). Means were adjusted by LSMEANS procedure, and degrees of freedom were calculated by Kenward and Rogers (1997) method. Linear and quadratic contrasts were performed to evaluate the dose-response of citral. Significance level was set at P ≤ 0.05. Tendency was considered at 0.10 ≥ P > 0.05.

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Results Despite of increasing doses of citral oil had no effect (P ≥ 0.11) on DM and nutrient intake

of goats, they linearly decreased (P ≤ 0.04) NDF digestibility in dairy goats (Table 2). No effects were detected (P≥ 0.24) of citral oil on ruminal pH or NH3-N in goats (Table 3). Citral oil provision linearly increased (P < 0.01) ruminal butyrate proportion in goats. Further, citral oil had a positive quadratic effect (P ≤ 0.03) on ruminal propionate (concentration and proportion) and butyrate (concentration), in which intermediary levels (0.08 and 0.16 mL/kg BW) showed higher values than control and higher level (0.24 mL/kg BW) treatments. Additionally, citral oil had a negative quadratic effect (P < 0.01) on ruminal acetate to propionate ratio and acetate proportion in ruminal fluid of goats. No treatment by time interaction effect were observed (P ≥ 0.15) on ruminal parameters.

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Citral oil had small effects on blood metabolites, that included a linear decrease (P = 0.02) on urea blood concentration and a trend to linear decrease (P = 0.07) blood glucose concentration in goats (Table 4). Citral oil consumption linearly reduced (P = 0.04) milk fat yield and tended to decrease (P = 0.08) FCM production of goats (Table 5). In addition, citral oil linearly decreased (P = 0.02) BW of goats. No effects (P ≥ 0.19) of citral oil consumption were detected on milk FA profile of goats (Table 6). Citral tended to show a positive quadratic effect (P =0.06) on fecal N of goats (Table 7).

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Discussion Lemongrass derived products seems not alter DM intake in ruminants, even being

supplemented as ground leaves at 100 g/d to dairy steers (Hosoda et al., 2006), as a dry powder (Wanapat et al., 2008), or as lemongrass meal up to 300 g/d to beef cattle (Wanapat et al., 2013). Although no statistical differences were observed on nutrient intake, we need to highlight that the highest dose of citral oil showed the lowest value of NDF intake in goats which is likely related to the linear decrease of NDF digestibility and ruminal fermentation changes. Few studies have evaluated lemongrass products effects on nutrient digestibility in ruminants, but decreases on nutrient digestibility have been reported in literature. Wanapat et al. (2008) noticed a decrease up to 45 g/kg on NDF digestibility after supplying lemongrass powder to beef cattle. In an in vitro ruminal fermentation experiment, Pawar et al. (2014) incubated a substrate containing wheat straw and grain mixture at 1:1 ratio with increasing doses of lemongrass oil and reported a linear decrease on total organic matter digestibility. In addition, an in vitro study using a substrate with 51:49 of forage to concentrate ratio incubated with high purity (>98%) EO (eugenol, carvacrol, and thymol) demonstrated a decrease on in vitro NDF digestibility in relation to control (Benchaar et al., 2007). Thus, the lower NDF

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digestibility when feeding citral oil reported in the current experiment and the results of previous studies suggest that EO negatively affect fibrolytic bacteria. There are some evidences that lemongrass derivative products may alter rumen microorganism population and jeopardize the fiber digestion. Wanapat et al. (2008) reported that amylolytic bacteria and cellulolytic bacteria counts increased when lemongrass powder was supplied at low doses (100 g/d), whereas they decreased when lemongrass powder was fed at high doses (200 and 300 g/d). Lin et al. (2013) supplied a mixture of EO containing citral to sheep and found a decrease on the percentage of F. succinogenes and B. fibrisolvens, which are major degraders of cellulose and hemicellulose in ruminants (Cheng et al., 1991). The mechanisms by which lemongrass derivative products may manipulate rumen microbiota include loss of cellular chemiosmotic control due to changes in electron transport, failure on protein translocation, and enzyme-dependent reactions (Ultee et al., 1999). Previous in vitro study showed that high purity EO had positive effects on ruminal fluid pH with a substrate consisted of 50:50 forage to concentrate ratio, without altering the proportion of VFA (Benchaar et al., 2007). We hypothesized that high purity citral oil could exert similar effect on ruminal pH of goats fed a diet with relative high concentrate content in comparison with diets provided in extensive systems, but citral consumption had no effect on ruminal pH. Although citral had no effects on ruminal pH and ammonia concentration, citral had a quadratic effect on ruminal propionate and butyrate production. In addition, treatments had a negative quadratic effect on ruminal acetate to propionate ratio. Effects of any feed additive on ruminal fermentation are considered positive either when is reported an increase on total VFA and propionate production or when is found a decrease on acetate to propionate ratio (Castillejos et al., 2008).

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If in one hand EO often present antimicrobial activity against Gram-positive bacteria in rumen, including acetate-producing bacteria (Patra and Yu, 2012); on the other hand, EO indirectly favor the growth of Gram-negative bacteria (due to low competitiveness for space and substrate) whose fermentation pathway produce propionate in rumen (Russel, 1996). Further, bacteria species that produce propionate are more resistant to EO activity (Holley and Patel, 2005). In the current experiment, the highest values of ruminal propionate and total VFA concentration were found when feeding 0.16 mL/ kg of BW which is around 9.6 mL of citral oil per day. Further, the highest dose of citral showed the lowest values of ruminal acetate, propionate and butyrate concentrations, suggesting an inhibition of microbial fermentation. Agreeing with the current study, Pawar et al. (2004) found an increase on in vitro rumen propionate proportion of substrates incubated with lemongrass oil. In the current experiment, besides the decrease on acetate proportion we observed an increase on molar proportion of ruminal butyrate in goats fed citral. Although the reasons for these effects are unclear, in vitro studies have reported similar effects of EO on ruminal acetate and butyrate molar proportions (Busquet et al., 2005; Busquet et al., 2006). The results of this study clearly demonstrate that high purity citral oil modulate ruminal fermentation, but more studies are necessary to recommend a dosage and moment that citral oil should be provided. Essential oils effects on VFA profile are dependent on pH (Cardozo et al., 2005), thus other diets likely promote different outcomes. In the present study, we found a time effect on all evaluated ruminal measurements. Before the morning feeding, goats had the lowest values of VFA concentration and highest values of ruminal pH. After the morning feeding, protein degradation rapidly increased NH3-N concentration, resulting in the highest NH3-N concentration two hours after feeding, agreeing with reported in literature (Galina et al., 2004). Further, the second feeding (13h00)

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stimulated feeding and ruminal fermentation of goats. Ruminal fermentation, specially of carbohydrates, increases VFA concentration while decreases ruminal pH (Giger-Reverdin et al., 2014). In the current study, the highest VFA concentration and lowest ruminal pH were observed at 12h after the morning feeding. Citral consumption by dairy goats linearly decreased urea concentration in blood and tended to linearly decrease blood glucose concentration. Decreased blood urea concentration was not expected since CP digestibility and ruminal NH3-N concentration were not affected by citral dietary addition. Hayes and Markovic (2002) demonstrated that citral has cytotoxic effects on human liver derived cells. Impairment of liver function would reduce urea cycle as well as gluconeogenesis, that could partially explain the decreased blood urea concentration and the trend to decrease on blood glucose concentration of goats fed citral. In addition, blood urea concentration may be affected by its transfer to the lumen of the gastrointestinal tract; the rate of blood urea transfer to gut can be positively influenced by factors that affect capillary blood flow (Huntington and Archibeque, 1999). Citral has demonstrated positive effects on vasodilatation and blood flow in mice (Scolnik et al., 1994). However, no effects were observed either on ruminal ammonia concentration or N fecal excretion by goats fed citral in the current study. In addition, Hosoda et al. (2006) did not find differences on blood concentration of high density lipoprotein, and triglycerides, but reported no effect on glucose and a tendency to increase blood urea nitrogen of Holstein steers fed chopped lemongrass. Lemongrass oil (which the major constituent is citral) has been reported to reduce cholesterol in mice (Santhosh Kumar et al., 2011), but citral oil had no effect on goats in this experiment. It is important to highlight that lemongrass products may present pharmacological activity affecting liver function (Viana et al., 2000), and no differences were found in enzymes

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related to liver damage (aspartate amino-transferase and gamma-glutamyl transferase) in the current experiment. Although treatments did not affect milk yield, they linearly decreased FCM and fat production in goats. The decrease on NDF digestibility is partially responsible for lower ruminal acetate proportion and milk fat production of goats fed citral in comparison to control. Mammary gland in ruminants utilizes acetate from ruminal fermentation to synthesize about one-half of the total milk FA (Bauman and Griinari, 2003). Sutton et al. (1988) estimated up to 80% of changes on milk fat proportion are explained by the VFA proportion variation in the rumen; and either acetate deficiency or high propionate concentration in rumen can limit the fat synthesis in the mammary gland (Bauman and Griinari, 2001). Citral consumption tended to decrease blood glucose concentration which is critical for mammary gland metabolism and synthesis of lactose. Lactose is an osmotic component of milk being directly associated with water secretion and milk volume (Miglior et al., 2006). Although the outcomes of milk yield were not statistically significant, the highest dose of citral promoted a decrease of 7.8%, 9.2%, and 9.8% on milk, FCM, and protein productions, respectively. Goats fed the highest dose of citral also exhibited the lowest values of DM intake, NDF digestibility, propionate and VFA production. These results suggest that citral provision at high doses negatively affects ruminal fermentation, microbial growth, and consequently milk and components yield. Inhibition of rumen bacteria by lemongrass powder at relative high doses was demonstrated by Wanapat et al. (2008). According to Bowden (1971), blood concentration of glucose is one of the important indicator of body energy status and this could be related with lower BW of animals fed citral oil. Further, studies have demonstrated the potential of citral in reducing the BW gain and abdominal fat in mass in rats fed high-energy ration in a dose-dependent manner, being recognized as a

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naturally occurring antiadipogenic molecule (Modak and Mukhopadhaya, 2011). Citral had no effect on milk FA profile. Goats have a more stable milk composition due to Δ9-desaturatase (enzyme of de novo synthesis) has lower sensitivity to dietary and ruminal environment changes than presented in other ruminant species (Toral et al., 2015). Furthermore, the absence of citral effect on milk FA profile in this study was probably due to citral did not affect conjugated linoleic acid content in the rumen. Besides of that, Gunal et al. (2013) did not find effects of citronelal, citronelol, geraniole, geranial, and d-limonene on ruminal cultures concentrations of conjugated linoleic acids.

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Conclusion Intermediary dietary levels (0.16 mL/ kg BW) of citral oil shifted ruminal fermentation

towards a more energetically efficient pathway, decreasing the acetate to propionate ratio in lactating goats. Despite citral positively affected ruminal fermentation, it decreased NDF digestibility and milk fat production of goats. Additionally, citral decreased blood urea concentration and had no effect on milk FA profile and N utilization in goats. Conflict of interests The authors declare no conflict of interests in the current manuscript. Acknowledgments The authors acknowledge the Dairy Cattle Research Laboratory of University of Sao Paulo, for providing the infrastructure and staff necessary for this study. Authors acknowledge Sao Paulo Research Foundation (FAPESP) for providing the scholarship of the first author and financial support for this experiment (grant # 12/01863-0 and grant # 11/06260-9).

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26

Table 1 Ingredients and chemical composition of basal diet (g/kg DM, otherwise stated). Item Ingredient

Diet

Corn silage

500

Ground corn

240

Soybean meal

220

Limestone

10.0

Mineral mixa

30.0

Chemical

a

Dry matter (kg/kg fresh matter)

455

Organic matter

916

Non-fiber carbohydrateb

405

Neutral detergent fiber

317

Acid detergent fiber

225

Crude protein

161

Ether extract

32.0

Lignin

22.3

Net energy of lactationc (Mcal/kg DM)

1.54

Contained per kg of product: 125 mg Co, 5625 mg Cu, 9 mg S, 312 mg I, 5000 mg Fe, 18125 mg

Mn, 144 mg Se, 23,750 mg Zn, 2,000 IU vitamin A, 500 IU vitamin D and 12,500 IU vitamin E. b

Non-fiber carbohydrate = 1000 – (crude protein + neutral detergent fiber + ether extract + ash),

with values expressed in g/kg DM, from Hall (2000). c

Net energy of lactation (Mcal/kg DM) = 0.0245 × total digestible nutrient (%) - 0.12, using 1 as

processing factor, from Weiss et al. (1992).

27

Table 2 Influence of increasing dietary levels of citral oil on intake and total tract digestibility of nutrients in dairy goats. Treatmenta

Item 0

8

16

P-valueb 24

SEM

L

Q

Intake (g/d) Dry matter 1914 1943 1917 1796 93.36 0.37 0.42 Organic matter 1738 1766 1742 1630 62.17 0.36 0.42 Non-fiber carbohydrate 845 869 859 830 26.38 0.76 0.51 Neutral detergent fiber 494 496 481 428 18.88 0.11 0.34 Crude protein 332 336 335 316 10.71 0.54 0.49 Ether extract 62.6 68.3 65.2 61.2 2.09 0.65 0.18 Intake (% body weight) Dry matter 3.26 3.40 3.36 3.12 0.09 0.52 0.25 Neutral detergent fiber 0.84 0.87 0.84 0.75 0.29 0.23 0.25 Total tract digestion (g/kg) Dry matter 729 730 736 731 0.53 0.82 0.79 Organic matter 747 747 755 740 0.52 0.75 0.76 Crude protein 708 745 758 749 0.97 0.12 0.21 Neutral detergent fiber 433 412 393 349 1.34 0.04 0.67 Ether extract 806 815 819 819 0.68 0.49 0.72 a Dietary inclusion of 0, 0.08, 0.16 or 0.24 mL of citral oil per kg BW; or an approximately citral oil consumption of 0, 4.8, 9.6 and 14.0 mL/d for treatments 0,8,16 and 24, respectively. b

Linear (L) and quadratic (Q) contrasts.

28

Table 3 Influence of increasing dietary levels of citral oil on ruminal variables in dairy goats. Treatmenta P-valueb Item

SEM

0

8

16

24

Time

pH

6.17

6.14

6.11

6.11

NH3-N (mg/dL)

24.5

24.6

23.6

Acetate

62.3

58.3

Propionate

18.9

Butyrate

INT

L

Q

0.02 <0.01 0.95

0.24

0.84

26.1

0.56 <0.01 0.98

0.59

0.48

64.1

59.6

0.94 <0.01 0.96

0.74

0.88

20.5

21.7

18.0

0.49 <0.01 0.94

0.67 <0.01

9.59

10.1

11.9

10.9

0.22 <0.01 0.97 <0.01 0.03

Volatile fatty acids

92.3

90.9

97.2

90.3

1.41 <0.01 0.77

0.99

Acetate to propionate ratio

3.55

3.02

3.14

3.43

0.05 <0.01 0.15

0.42 <0.01

Acetate

0.69

0.65

0.65

0.67

0.01 <0.01 0.52

0.01 <0.01

Propionate

0.21

0.22

0.21

0.20

0.01 <0.01 0.66

0.11 <0.01

Butyrate

0.11

0.11

0.12

0.12

0.01

Concentration (mmol/dL)

0.25

Proportion (mmol/100 mmol)

0.01

0.33 <0.01 0.39

a

Dietary inclusion of 0, 0.08, 0.16 or 0.24 mL of citral oil per kg BW; or an approximately citral oil consumption of 0, 4.8, 9.6 and 14.0 mL/d for treatments 0,8,16 and 24, respectively. b INT: treatment by time interaction effect; Linear (L) and quadratic (Q) contrasts.

29

Table 4 Influence of increasing dietary levels of citral oil on blood metabolites in dairy goats. Treatment1 Item

P-value2

0

8

16

24

Glucose (mg/dL)

61.2

62.0

59.2

58.7

Triacylglycerides (mg/dL)

12.4

12.5

12.6

Cholesterol (mg/dL)

86.9

89.4

High density lipoprotein (mg/dL)

53.7

Urea (mg/dL)

SEM

L

Q

1.02

0.07

0.58

13.3

0.58

0.48

0.71

86.1

87.6

1.65

0.89

0.81

54.4

53.8

54.5

1.07

0.75

0.98

26.9

27.7

25.7

25.4

0.62

0.02

0.38

Total Protein (g/dL)

7.66

7.52

7.50

7.47

0.07

0.11

0.45

Albumin (g/dL)

3.22

3.23

3.22

3.27

0.03

0.61

0.70

Creatinine kinase (U/L)

75.1

78.6

77.0

72.9

1.79

0.45

0.19

Aspartate amino-transferase (U/L)

58.2

59.9

56.7

60.3

1.46

0.55

0.61

Gamma-glutamyl transferase (U/L)

54.7

56.4

53.8

55.3

1.17

0.84

0.96

Alanine transaminase (U/L)

13.6

13.2

13.3

13.4

0.37

0.97

0.68

1

Dietary inclusion of 0, 0.08, 0.16 or 0.24 ml of citral oil per kg BW; or an approximately citral oil consumption of 0, 4.8, 9.6 and 14.0 mL/d for treatments 0,8,16 and 24, respectively. 2 Linear (L) and quadratic (Q) contrasts.

30

Table 5 Influence of increasing dietary levels of citral oil on milk yield and composition in dairy goats. Treatmenta P-valueb Item SEM 0 8 16 24 L Q Yield (kg/d) Milk 3.20 3.05 3.06 2.95 0.11 0.28 0.89 Fat-corrected milkc 3.04 2.92 2.91 2.76 0.09 0.08 0.86 Fat (g/d) 116 116 111 105 0.01 0.04 0.47 Protein (g/d) 102 96.0 97.0 92.0 0.01 0.15 0.91 Lactose (g/d) 150 147 146 139 0.01 0.28 0.79 d Efficiency (kg/kg) 1.70 1.61 1.53 1.63 0.04 0.44 0.24 Composition (%) Fat 3.72 3.83 3.80 3.73 0.08 0.99 0.52 Protein 3.18 3.24 3.21 3.18 0.02 0.78 0.11 Lactose 4.77 4.86 4.82 4.76 0.03 0.74 0.13 Body weight change -0.60 -0.53 -0.34 -0.74 0.16 0.88 0.48 (g/d) Body weight (kg) 61.4 60.2 59.9 58.3 0.98 0.02 0.78 a Dietary inclusion of 0, 0.08, 0.16 or 0.24 ml of citral oil per kg BW; or an approximately citral oil consumption of 0, 4.8, 9.6 and 14.0 mL/d for treatments 0,8,16 and 24, respectively. b Linear (L) and quadratic (Q) contrasts. c Fat-corrected milk 4% = milk yield (kg/d) × (0.411 + 0.147× milk fat percentage), from Mavrogenis and Parachristoforou (1988). d Efficiency = milk yield (kg/d) ÷ DM intake (kg/d).

31

Table 6 Influence of increasing dietary levels of citral on milk fatty acid profile in dairy goats (g/100 g fatty acid). Item

Treatmenta 0

8

16

24

4:0

0.80

0.78

0.77

0.78

6:0

1.85

1.83

1.85

8:0

2.12

2.12

10:0

8.34

11:0

SEM

P-valueb L

Q

0.01

0.53

0.47

1.83

0.03

0.96

0.96

2.07

2.07

0.03

0.40

0.95

8.38

8.07

7.94

0.13

0.21

0.75

0.05

0.06

0.03

0.04

0.01

0.25

0.82

12:0

3.96

4.01

3.88

3.76

0.09

0.35

0.64

13:0

0.15

0.15

0.15

0.14

0.01

0.53

0.67

14:0

10.3

10.5

10.41

10.2

0.14

0.68

0.43

14:1

0.14

0.14

0.14

0.14

0.01

0.64

0.99

15:0

0.75

0.76

0.75

0.74

0.02

0.72

0.72

16:0

32.3

33.0

34.1

32.6

0.47

0.59

0.21

16:1

0.67

0.66

0.61

0.67

0.02

0.76

0.19

17:0

0.74

0.73

0.74

0.74

0.01

0.87

0.72

18:0

9.21

9.52

9.35

9.51

0.25

0.64

0.84

18:1 cis-9

24.2

23.3

23.8

25.0

0.41

0.42

0.19

18:1 n9, t

0.09

0.08

0.06

0.08

0.01

0.64

0.38

18:1 trans-11

0.67

0.56

0.65

0.61

0.02

0.57

0.42

18:2 cis-9, cis-12

2.46

2.39

2.39

2.34

0.04

0.34

0.92

18:2 cis-9, trans-11

0.48

0.41

0.46

0.46

0.02

0.99

0.31

18:3 cis-6, cis-9, cis-12

0.09

0.09

0.09

0.09

0.01

0.83

0.75

Otherc

0.22

0.20

0.19

0.21

0.01

0.79

0.22

a

Dietary inclusion of 0, 0.08, 0.16 or 0.24 mL of citral oil per kg BW; or an approximately citral oil consumption of 0, 4.8, 9.6 and 14.0 mL/d for treatments 0,8,16 and 24, respectively. b Linear (L) and quadratic (Q) contrasts. c Unidentified fatty acids or with very low concentration (<0.01 g/100 g fatty acid).

32

Table 7 Influence of increasing dietary levels of citral on N utilization in dairy goats (g/d, otherwise stated). Treatmenta Item

P-valueb

0

8

16

24

N intake

53.1

54.2

53.7

50.6

Fecal N

13.6

14.6

13.7

Urinary N

16.1

15.6

Milk N

15.9

15.3

SEM

L

Q

1.75

0.54

0.47

13.5

0.86

0.71

0.06

15.8

15.1

0.50

0.55

0.92

15.3

14.6

0.51

0.19

0.94

a

Dietary inclusion of 0, 0.08, 0.16 or 0.24 ml of citral oil per kg BW; or an approximately citral oil consumption of 0, 4.8, 9.6 and 14.0 ml/d for treatments 0,8,16 and 24, respectively. b Linear (L) and quadratic (Q) contrasts.

33