Effect of sodium chloride, sodium sulfate or sodium nitrite in drinking water on intake, digestion, growth rate, carcass traits and meat quality of Barbarine lamb

Accepted Manuscript Title: Effect of sodium chloride, sodium sulfate or sodium nitrite in drinking water on intake, digestion, growth rate, carcass traits and meat quality of Barbarine lamb Author: Ibrahim Yousfi Hichem Ben Salem Dorra Aouadi Sourour Abidi PII: DOI: Reference:

S0921-4488(16)30207-3 http://dx.doi.org/doi:10.1016/j.smallrumres.2016.08.013 RUMIN 5278

To appear in:

Small Ruminant Research

Received date: Revised date: Accepted date:

28-6-2016 18-8-2016 20-8-2016

Please cite this article as: Yousfi, Ibrahim, Salem, Hichem Ben, Aouadi, Dorra, Abidi, Sourour, Effect of sodium chloride, sodium sulfate or sodium nitrite in drinking water on intake, digestion, growth rate, carcass traits and meat quality of Barbarine lamb.Small Ruminant Research http://dx.doi.org/10.1016/j.smallrumres.2016.08.013 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.

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Effect of sodium chloride, sodium sulfate or sodium nitrite in drinking water on intake, digestion, growth rate, carcass traits and meat quality of Barbarine lamb

Ibrahim Yousfia,b, Hichem Ben Salema, Dorra Aouadia, Sourour Abidia

a

Institut National de la Recherche Agronomique de Tunisie (INRAT), Université de Carthage,

Laboratoire des Productions Animales et Fourragères, Rue Hédi Karray, 2049 Ariana, Tunisia b

Institut National Agronomique de Tunisie (INAT), Université de Carthage, 43 Avenue

Charles Nicolle, 1082 Tunis, Tunisia

Corresponding author: [email protected]

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Highlights

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Response of sheep to NaCl, Na2SO4 or NaNO2 added to drinking water was investigated There was no effect on intake, diet digestibility and nitrogen metabolism Carcass traits and the proportions of SFA, MUFA and PUFA in meat were similar among treatments The three salts reduced substantially protozoa concentration in the rumen Blood profile changed with the addition of these salts to water

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Abstract The 79 day-experiment reported herein was conducted to determine through a completely randomized design the effects of the enrichment of drinking water for lamb with sodium chloride (NaCl), sodium sulfate (Na2SO4), or sodium nitrite (NaNO2) on water and feed intakes, apparent diet digestibility, nitrogen balance, microbial nitrogen supply, rumen fermentation (pH, ammonia nitrogen and protozoa concentrations), blood metabolites, carcass traits and meat quality (i.e. ultimate pH, cooking loss, and fatty acids (FA) profile). Four groups of eight Barbarine lamb each (initial BW 21.12.4 kg, 5–6-month old) were housed in individual boxes and received oat hay ad libitum and 400 g concentrate. Each group was assigned randomly to one of the following treatments: ad libitum access to fresh water (control, group 1) or to fresh water enriched with either 7 g NaCl /L (group 2), or 2 g Na2SO4/L (group 3) or 40 mg NaNO2/L (group 4). Lambs having access to salt-enriched water consumed more water than control lambs. Such effect was important (P<0.05) in group 2 (4.6 vs 2.48 L/day). Compared to control group, the three sources of salt administrated in water had no effect (P>0.05) on diet intake and apparent digestibility, N retention and microbial N supply. Rumen pH, ammonia-N and protozoa remained unchanged (P>0.05) with the consumption of saline water. The lack of variation of diet intake and digestibility and rumen fermentation could justify the absence of significant variation of the growth rate of lambs (44-63 g/day). Serum creatinine increased (P<0.05) with the administration of the three salts in drinking water. Serum glucose was higher (P< 0.001) in lamb assigned to Na2SO4 and NaNO2 treatments. Nitrite treatment (40 mg NaNO2/L) reduced (P = 0.034) the concentration of triglycerides in the blood. The enrichment of water with the three sources of salts had no adverse effect on carcass yield and the FA profile of lamb meat, except for iso-heptadecanoic acid (C17:0 iso) which was higher (P = 0.024) in the meat of lambs in group 3 than in the meat of control group. The proportion of 10-heptadecenoic acid (C17:1(n-7)) in the meat of lambs drinking NaNO2-enriched water was 25 to 30% lower (P = 0.041) than that of the three other groups. The ratio Omega 6 to Omega 3 was similar among the four lamb groups (4.685.01). It is concluded that the doses and types of salts intentionally used in the present study to reflect water quality in some Tunisian zones home to Barbarine sheep had no effect on lamb’s growth, carcass traits and meat quality. However, the increased creatinine and reduced triglycerides in the blood justify the need to emphasize further investigations on the effect of these salts on lamb’s health.

Keywords: Drinking water, salts, digestion, growth, carcass traits, meat quality, lamb.

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1. Introduction The dry areas worldwide share many features including reliance on livestock breeding as main source of farmers’income, arid to semiarid climate, limited water resources compromising agriculture development and high economic and social value of water. Annual rainfall is in major parts of the dry areas like North Africa low (e.g. below 200 mm in southern Tunisia) meaning that surface water is proportionally low among the available water sources. Under such circumstances farmers are intensively using groundwater (i.e. deep and phreatic aquifers) for food-feed cropping and livestock watering. The overexploitation of groundwater rendered water a scarce public good and is, at the same time, causing soil and water salinization. Currently, major proportion of saline water available in southern Tunisia falls in the range of 3000-7000 ppm of salt. Higher salt levels (> 7000 ppm) in water could be encountered in some zones where farming is difficult to practice. The presence of some other water pollutants, mainly sulfates, nitrates and fluoride leads to further deterioration of water quality (Rahaingomanana, 1998; Hannachi et al., 2014) in Tunisia. According to Zaara (2008) tap water salinity in the major part of southern Tunisia ranges between 1500 and 3580 ppm in which around 700 to 1500 ppm are sulfates. The sulfate ion is available in most natural waters and it originates from the dissolution of soil and rocks minerals. Under anaerobic conditions, bacteria in water could reduce sulfate to sulfur resulting in the release of hydrogen sulfur that makes water taste and smell unpleasant. According to (ANZECC, 2000; MAF, 2004; CCME, 2008), 1000 mg sulfate/Liter of water represent a maximum threshold above which the sulfate in drinking water could have serious negative effects on the animal (e.g. diarrhea). Nitrates and nitrites are oxidized forms of nitrogen. Groundwater could contain high levels of nitrates originating mainly from the contamination of water by certain pollutants (e.g. nitrogen fertilizers, manure, industry residues, etc.). The content of tap water could exceed in some parts of Tunisia the threshold (50 mg/L) fixed for human safety (Zghibi et al. 2013).

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Concentrations of nitrite in water above 30 mg/L could be dangerous for livestock health (ANZEEC, 2000; CCME, 2008). Salinization of groundwater is expanding in the arid zones; therefore, the development of biosaline agriculture has become a prerequisite for the improvement of farmers’ livelihood (Gaaloul and Zouari, 2004). The introduction of salttolerant crops for food (e.g. vegetables and cereals) and fodder (e.g. halophytes) productions and building the adaptation of livestock to salty diets and or to saline drinking water have become key elements in the strategies targeting agriculture development in these zones. However, when it comes to human nutrition and health, the effects of saline water containing the above mentioned contaminants on the quality of crop and animal products should be taken into consideration when developing strategies for saline agriculture. Barbarine sheep, a fattailed breed, is the main breed raised in almost all livestock-based production systems in Tunisia from humid to arid climates. This sheep breed withstands the harsh conditions prevailing in southern Tunisia. The merits of this breed have been discussed by Ben Salem et al. (2011), and include adaptation to heat stress and feed restriction and high feed efficiency, but its adaptation to drinking saline water and its response to the main water contaminants (i.e. sulfates and nitrates or nitrites) is, to our knowledge, not yet investigated. In general, ruminants having access to salt-rich diets consume more water to help the kidney removing excessive salt from their body. However, when the drinking water is excessively high in salts, the animal could face digestive and metabolic disorders which may affect productive performance (Masters et al., 2007) and could provoke intoxication (Mckenzie et al., 2004). Research studies on the later topic had been focusing on the response of ruminants, mainly sheep, to NaCl and occasionally potassium in the diet or in drinking water. Sulfate or other sulfur-containing compounds may induce anorexia mediated through the formation of sulfide in the rumen. Nitrate in the diet and or in drinking water is transformed to ammonia and nitrite. Therefore, the level of nitrite in the rumen is also important to control (Olkowski, 2009). To the best of our knowledge the response of sheep to sulfate and nitrite in water is not

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well documented. Also, information on the effect of water pollutants (e.g. sodium chloride, sulfate and nitrite) on meat quality is scarce. Having data on the response of Barbarine sheep to these salts would help developing watering strategies to increase productive performance of this breed. Therefore, the current study was designed to determine the effects of sodium chloride, sodium sulfate and sodium nitrite in drinking water at amounts close to those in the groundwater available in southern Tunisia on feed and water intakes, apparent diet digestibility, nitrogen balance, rumen fermentation parameters, blood profile, growth rate, carcass traits and meat quality in Barbarine lamb.

2. Materials and methods A 79-day experiment was carried out from April to July 2012 at the National Institute of Agricultural Research of Tunisia (INRAT) experimental unit of Bourbia station (Zaghouan governorate).The average temperature during this period is 30°C and 10 to 12 sunshine hours. 2.1. Animals, diets and experimental design Thirty-two Barbarine male lambs (5–6-month old) with an initial body weight (BW) of 21.1±2.4 kg were treated with an enterotoxemia vaccine then with Ivermectyl (1 mL/50 kg BW, 1 g Ivermectine/100 ml; Médivet Santé Animale, Soliman—Tunisia) to free them from external and internal parasites. Animals were housed in individual pens, each equipped with feed and water troughs. All of them received oaten hay ad libitum (0.2 kg in excess of the previous day’s intake) and 400 g concentrate (Table 2). The concentrate was composed of ground barley (570 g/kg), faba bean (400 g/kg) and commercial mineral and vitamin supplement (MVS, 30 g/kg). The declared composition of MVS (g/kg) was 600 g calcium carbonate, 300 g sodium chloride, 50 g trace minerals and 50 g vitamins. The concentrate was offered once daily at 0900 while oaten hay was offered twice a day at 0900 and 1600. Lambs

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were used in a completely randomized design, with four treatments and eight replicates: Group 1 (Control) had free access to water taken from general running water available on tap in the Experimental Station of INRAT at Bourbia where this experiment was conducted and provided by the National Water Distribution Utility through the drinking water network (i.e. without salt addition). The three remaining groups received each ad libitum tap water enriched with NaCl (7 g/L, Group 2), Na2SO4 (2 g/L, Group 3) or NaNO2 (40 mg/L, Group 4). The amount of salt was setup to simulate the quality of drinking water for livestock available in areas, which are prone to water salinization. Experimental drinking water was prepared daily in 40 L plastic pail by totally dissolving the respective doses of these salts in tap water initially containing 760 mg/L TDS. The physico-chemical composition of the tap water is reported in Table 1. Doses of these compounds were close to those available in the groundwater in some zones of Tunisia where sheep production is the main source of smallholders’ income. Animals were adapted to housing conditions and diets for 10 days before starting a 50-day growth trial. The first adjustment 5-day was allowed to adapt to water by gradually increasing the compound concentration of the drinking water until it reached the required concentration for the concerned groups, the second 5-day period was for the animal’s adaptation to the experimental watering regimes. At the end of the growth trial, animals were transferred into individual metabolic cages. They were acclimated for 4 days to new housing conditions before starting a 7-day total collection period for diet digestibility determination. One week later all lambs assigned to the same treatments were slaughtered.

2.2. Measurements and sampling Lambs were weighed at the beginning, the end and at weekly intervals throughout the feeding trial to determine their growth rates. Daily feed and water intakes were calculated on the basis of the amounts of distributed and refused feed and water.

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Along with the total collection period, feed intake was determined daily by weighing the amounts of feeds distributed and corresponding individual refusals. Feces and urine were collected daily between 0800 and 0900, weighed, and a 10% aliquot (by weight) was collected from each sample, and then stored in a cooling chamber. Fecal and urine aliquots were composited by lamb within the 7-day collection period, and a subsample of feces and urine were frozen for later analyses. Amounts of feed and water offered and refused were weighed daily and subsamples of feeds and refusals were taken. At the end of the collection period, refusal subsamples were composited by sheep, and stored until analyzed. Samples of feeds offered to lambs were daily stored until analyzed. Samples of feeds offered, individual refusals (feeding and digestibility trials) and feces (digestibility trial) were taken for dry matter determination (80°C for 48 h). Stored subsamples of feeds offered, refusals and feces were dried at 50°C then ground to pass through a 1mm screen and stored until analyzed. Urine excreted daily by each animal was collected in a plastic bucket containing 100 ml of aqueous sulfuric acid solution (1.8 moles/l) to keep urine pH below 3 (Chen and Gomes, 1992). Individual urine samples collected daily were stored at −15°C until analyzed for the predominant purine derivatives (i.e. allantoin) and for nitrogen. Blood samples were taken from the jugular vein of each animal the day following the end of the collection period just before the distribution of the morning meal. After 3–4-min coagulation, samples were centrifuged (3000×g at 4°C) and serum was collected and stored at −20°C until analyzed. An additional blood sample was taken from each animal. It was then transported to the laboratory in an icebox for automated complete blood counts (CBC) which was performed in the same day. In the day following the end of collection period, about 20 ml of rumen fluid were collected before morning feeding (0 h) then 2 and 6 h post-feeding using a flexible stomach tube and a 100 ml syringe for pumping. The pH of rumen fluid was measured on a digital pH-meter.

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Five-ml subsample of unfiltered rumen fluid from each lamb was mixed with 5 ml of a solution composed of 10 ml formol (350–400 ml/l), 90 ml distilled water and 60 mg of bromocresol for protozoa counting using Malassez chamber (depth 0.2 mm, Superior, Germany). The remaining rumen fluid was filtered through two layers of cheesecloth, then 5 ml of filtrate were added three drops of sulfuric acid (18 moles/l) and stored in the freezer until analyzed for ammonia nitrogen (NH3–N). Lambs were slaughtered one week after the total collection period. Animals were weighed just before slaughtering (BWS). After slaughtering, the hot carcass (HCW) and tail were weighed separately. The full and the empty digestive tracts were weighed. Carcasses were hung by Achilles tendon and were chilled at 4°C in complete darkness. After 24 h, each carcass was weighed again and referred to as chilled carcass weight (CCW), and then the muscle Longissimus dorsi (from the 1st to the 13th thoracic rib) was excised from the carcass and weighed. Part of this muscle (from the 1st to the 10th rib) was vacuum-packed and stored at −25°C until analyzed. The remaining part was immediately subjected to pH measurement using an Orion 9106 penetrating probe and for the determination of the cooking loss. The hot carcass yield (HCY), chilled carcass yield (CCY) and shrink after chilling (SC) were calculated as follows: HCY = (HCW/BWS) x 100 CCY = (CCW/BWS) x 100 SC = ((HCW – CCW)/HCW) x 100

2.3. Laboratory analyses Ground samples of offered feeds, refusals and feces were analyzed for dry matter (DM, #930.15), ash (#924.05) and N (Kjeldahl-N, #984.13) according to AOAC (1990). The

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ANKOM220 system was used to determine fibre fractions of feeds, refusals and feces. The ash-free neutral detergent fibre (NDF) contents of feeds, refusals and feces were determined following the procedure described by Van Soest et al. (1991). Only for concentrate the heat stable α-amylase was used for the analysis of NDF. Sodium sulphite was not used. Ash-free acid detergent fibre (ADF) of feeds was determined and expressed exclusive of residual ash (AOAC, 1990; #973.18). Lignin (sa) of feeds was analyzed by solubilisation of cellulose with 720 g/kg sulphuric acid (Robertson and Van Soest, 1981). Urine was analyzed for Kjeldahl-N (#984.13; AOAC, 1990) and allantoin (Chen and Gomes, 1992). Rumen fluid sub-samples were centrifuged (3000×g for 15 min) and supernatant fractions of ruminal contents were analyzed for NH3–N (Weatherburn, 1967). Serum metabolites were analyzed using Biomaghreb kits (Soukra, Tunisia). Urea in serum was analyzed using an enzymatic colorimetric technique based on the Berthelot reaction. Urea in the presence of urease and water forms ammonium carbonate. Ammonium ions react with phenol and hypochlorite of reagents resulting in colored complexes for which the intensity, measured on spectrophotometer UV–vis (Spectronic 601) at 590 nm, is proportional to urea concentration. Serum glucose and triglycerides were analyzed by a colorimetric enzymatic method after enzymatic reaction and enzymatic oxidation for glucose or the enzymatic hydrolysis to glycerol for triglycerides, quinoneimine was formed giving pink color which is measured photometrically at 505 nm to indicate glucose or triglycerides concentration. Total proteins were analyzed using the Biuret colorimetric test. Serumal proteins formed in alkaline medium a colored complex; the intensity of which is proportional to proteins in the sample (Biuret reaction) and measured at 546 nm. Albumin was analyzed using the green of bromocresol (BCG) method. Albumin, in a buffered solution, reacts with the BCG to form a red-color complex; the intensity of which is measured at 628 mm. Gamma-glutamyltransferase (Gamma-GT) activity was measured by Kinetic Colorimetric method at 405 nm.

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Creatinine was analyzed using kinetic test without deproteinization. Creatinine in a basic picrate solution forms a colored complex. The ∆ extinction at predetermined times during conversion is proportional to the concentration of creatinine in the sample. The complex color intensity is measured at 492 nm. Complete blood counts were performed within 3 hours after whole blood sample's collection using a Coulter MaxM automated hematology analyzers (Beckman Coulter, Inc). Intramuscular fat was extracted, from 4 g of ground meat sample, according to Folch et al. (1957) as modified by French et al. (2000). The FA methylesters (FAME) derivatives were prepared by reacting with methanolic BF3 as described by Thurnhofer and Vetter (2005). The gas chromatograph used was interfaced to a flame ionization detector (F.I.D.). Separation was done by a capillary column Omegawax 250 (material: fused silica; L × I.D. 30 m × 0.25 mm, df 0.25 μm; Sigma-Aldrich Co. LLC). The injector and detector were set to 220°C. Column temperature was programmed at 160°C and to reach 205°C. Fatty acid proportions were reported as mg/100 mg of the total FA detected. The sum of short FA (SFA), monounsaturated FA (MUFA), and polyunsaturated FA (PUFA) was calculated. We also determined the ratios PUFA to SFA and n-6 FA to n-3 FA. The cooking loss was determined by weighing meat samples in plastic bags (Wi) and immersing them in water bath until the internal temperature of the meat reached 75°C (Boccard et al., 1981). The bags were then placed under cold running water for 30 min, then the cooked meat was patted dry with paper towels and reweighed (Wf). Cooking loss was calculated as (Wi - Wf)/Wi. 2.4. Calculation and statistical analyses The average daily gain was calculated for each lamb from regression analysis of body weight against time from day 1 to day 50.

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The metabolisable energy (ME) content for diet was estimated using the following equation (AFRC, 1993): ME (MJ/kg DM) = 0.0157 × D-value (g/kg DM) Where D-value (g/kg DM) is calculated as: [OM intake (g) − faecal OM (g)]/DM intake (kg) Urinary excretion of allantoin (Y, mmol/day) was used to calculate microbial purines absorbed (X, mmol/day) from the equation: Y = 0.84X + (0.150 ×W0.75 × e−0.25X) Where: W is the animal body weight (kg). Microbial N supply (g/day) was calculated from using the equation relationship: 70X/(0.83×0.116×1000) , where 70 is the N content of purines (mg N/mmol), X is as defined above, 0.83 is the assumed digestibility of microbial purines, 0.116 is the ratio of purine N/total N in mixed rumen microbes and 1000 converts mg to g (Chen and Gomes, 1992). Data on the intake, nutrient digestibility, N balance, purine derivatives and microbial N supply, blood metabolites, average daily gain, carcass traits and FA profile were analyzed using the SAS (1991) MIXED procedure according to the model: Y = µ + Ai + Wj + eij, where Y = dependent variable, µ = overall mean, Ai = effect of animal (i = 1-32), Wj = effect of water quality (j=1-4), and eij = residual error. Rumen fermentation parameters (pH, NH3-N and protozoa) were analyzed as repeated measures over time by sing the SAS (1991) MIXED procedure according to the following model: Y = µ + Ai + Wj + Dk + Tl + (WD)jk + (WT)jl + (WDT)jkl + eijkl, where Y = dependent variable, µ = overall mean, Ai = effect of animal (i = 1-32), Wj = effect of water quality (j=14), Dk = effect of sampling day (k = 1-2), Tl = effect of sampling time (i.e. hours post-feeding, l = 1-3), (WD)jk = effect of the interaction between water quality and sampling day, (WT)jl =

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effect of the interaction between water quality and sampling time, (WDT)jkl = effect of the interaction between water quality, sampling day and sampling time, and eij = residual error. In both statistical models Animal (Ai) was considered as random effect and the remaining independent variables served as fixed effects. Means of all dependent variables were obtained by using the LSMEANS command. The effects were considered significant when P<0.05.

3. Results 3.1. Water and feed intakes and growth rate Compared to control group, the enrichment of drinking water with 7 g NaCl/L increased (P< 0.001) by almost two-folds the volume of water consumed by sheep in group 2. However, the two other salts in drinking water did not affect water intake (Table 3). Total nutrient intakes (DM, OM, CP and NDF) and the average daily gains were similar (P 0.05) among the four groups (Table 3).

3.2. Diet digestibility, N balance and microbial N supply The administration of the three salts separately in drinking water (7 g NaCl/L, 2g Na2SO4/L or 40 mg NaNO2/L) didn't affect the apparent digestibility of the diet DM, OM, CP and NDF (Table 4). The experimental watering regimens had no effect (P>0.05) on N intake and N losses, thus on N retention (Table 4). Urinary excretion of allantoin increased (P = 0.03) with the administration of sodium nitrate in drinking water but the efficiency of microbial N supply was similar (P > 0.05) among all groups (Table 4).

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3.3. Rumen fluid pH, ammonia nitrogen and protozoa concentrations Rumen fermentation parameters are reported in Table 5. Average pH was not affected (P>0.05) by each of the three salts added to drinking water. However, it dropped at 3 h postfeeding then remained unchanged until 6 h post-feeding. The average concentration of NH3-N ranged from 9.61 and 10.32 mg/dl. It was not affected by the administration of the three salts in water. It picked at 2 h post-feeding (11.3 mg/dl) then slightly decreased at 6 h (10.8 mg/dl). Irrespective of sampling times, the three salts, mainly Na2SO4, added to water reduced substantially (P=0.009) the concentration of protozoa in the rumen. The defaunating effect of salt-enriched water was important after the distribution of the morning meal (P<0.001). The interaction water quality x sampling time was not significant (P>0.05) for the three fermentation parameters.

3.4. Blood metabolites and complete blood count The concentrations of serum metabolites are presented in Table 6. Albumin, total protein, and urea concentrations and the Gamma-Glutamyl-transferase (Gamma-GT) activity were similar (P>0.05) among the four groups. Nitrite treatment (Group 4, 40 mg NaNO2/L) reduced (P = 0.034) the concentration of triglycerides when compared to control. Creatinine concentrations was similar (P > 0.05) among the three groups receiving salts-enriched water but was 29 to 38% lower (P = 0.003) in control group. Serum glucose concentration was higher (P< 0.001) in groups 3 and 4 than in control group. Table 6 reports data on the complete blood count. Platelets count (PLT) and plateletcrit (PCT) values were lower (P=0.04 and P=0.049, respectively) in lambs consuming drinking water enriched with 40 mg/L sodium nitrite than in lambs assigned to the three other watering regimens.

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3.5. Carcass traits and meat quality Data on carcass traits and meat quality are reported in Table 7. The three sources of salts dissolved in the drinking water had no effect (P>0.05) on carcass traits. Neither meat pH at 24h nor cooking loss was affected (P>0.05) by watering regimens. The proportions of SFA, MUFA, and PUFA and the ratio of PUFA to SFA in meat were not affected (P> 0.05) by the addition of one of the three salts in water. The FAs profile was similar among all groups except for iso-heptadecanoic acid (C17:0 iso) which was higher (P=0.024) in the meat of lamb having access to sodium sulfate (2 g Na2SO4/L) containing water than in the meat of control group and group 2. The proportion of 10-heptadecenoic acid (C17:1(n-7)) detected in meat of sheep in group 4 (40 mg NaNO2/L) was 25 to 30% lower (P = 0.041) than that of the three other groups.

4. Discussion

4.1. Effects on feed and water intakes and daily gain Feed and diet intakes were not affected by the incorporation of any of the three salts added to drinking water. There was a substantial increase of water consumption by lambs receiving NaCl-enriched water (7 g/L) and a numerical increase of water intake resulting from the addition of Na2SO4 (2 g/L) or NaNO2 (40 mg/L) to water as compared to control treatment. The increase in the consumption of drinking water is the most common result obtained in different studies on the response of ruminants to saline water. This increase is proportional to the level of salinity in the drinking water if the animals are slowly acclimated to. In general, the ruminant tries to adapt to salty diets and or saline water by increasing water consumption to dilute the excessive amount of salt in the body. According to Potter (1961), increased

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glomerular filtration rate following saline water consumption is one of the main adaptation ways exhibited by sheep to excrete salt load excess. Apparently, when salt content of the drinking water is less than 20 g /L and the taste of this water is not repulsive for sheep, the increase in water consumption is ordered by a mechanism reactive to the total quantity of salt ingested. The regulation of the salt balance and water is controlled by the renin-angiotensin system (RAAS) (Cowley et al., 1986). Nevertheless, this would logically happen at the expense of diet intake, which decreases. Meyer et al. (1955) concluded that the incorporation of increasing levels of NaCl (6.6, 48, 94, and 128 g/kg of the ration) in the diet of fattening sheep had no detrimental effects on the efficiency of feed utilization and growth performance. Peirce (1957, 1959, 1960, 1962, 1963) conducted a set of experiments on Merino wethers (BW 40 to 60 kg) and came up with the conclusion that water intake increased with the increase of NaCl concentration, and that the reduction of appetite started with the level of 13 g/L of soluble salts in water. The effect of high salt loads on ruminant production is variable. It can reduce animal production mainly through the reduction of ingestion (Wilson 1966; Wilson and Dudzinski, 1973) or, in the contrary, can improve the supply of protein in the small intestine of ruminants through for example the increase of the dilution rate of ruminal bacteria (Potter et al., 1972). Peirce (1957) and Wilson (1966) reported that the consumption of salt-rich water (> 13 g NaCl / L) causes a decrease in the weight gain resulting from decreased diet intake. Drinking water, which has low to moderate salinity (current study), does not seem to affect the animal body weight; while, some other water contaminants, like manure, could induce important decrease of the growth rate (e.g. Willms et al., 2002; Lardner et al., 2005). Excessive consumption of Na2SO4 by sheep resulted in reduced DM intake (Morrow et al., 2013). In the present study, control lamb grew at a rate of 60 g/day. In previous studies on the same breed (initial body weight ranging from 22 to 32 kg) reared indoor, receiving similar diet (i.e. oat hay ad libitum and 200-300 g barley-based concentrate) and having free access to fresh water, the average daily gain was 46 g (Abidi et al., 2009), 71

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g (Atti and Ben Salem, 2008) and 77 g (Majdoub-Mathlouthi et al., 2013). The administration of lower levels of this salt (2000 ppm) did not affect the growth rate of lamb (current experiment), beef cattle (Grout et al., 2006) and yearling steers (Sexson et al., 2010). Lewis (1951a) postulated that sodium nitrite can be toxic for sheep when administrated directly in the rumen at a rate of 170 mg/kg BW or intravenously at a rate of 34 mg/kg BW. In the present experiment and from the amount of water consumed by animals in group 4, sodium nitrite intake amounted about 120 mg/day or 5-7 mg/kg BW per day which is considerably lower than the toxic threshold reported by the later author.

4.2. Effects on diet digestibility, N balance and microbial N supply The lack of effect of the salts we added to drinking water on apparent diet digestibility and N balance could be related to the lower doses of salts used in the current study as compared to those reported in the literature. In contrast to our results and referring to the significant decrease of N metabolites (total protein, albumin and globulin) and N retention observed in his experiment on Barki rams, Attia-Ismail (2003) concluded that salt water (12494 ppm TDS diluted seawater) affected N metabolism probably as consequence of the alteration of osmotic pressure within the rumen, thus, affecting rumen ecosystem. The TDS concentrations in the water used in the current experiment (ranging from 760 to 8045 ppm TDS) were lower than that used by this author. The three salts in drinking water did not affect significantly N intake and retention and microbial N supply. This means that N metabolism in Barbarine lamb was not constrained by the doses of the salts added to drinking water. These findings are in line with the results obtained on goat (Deng et al., 2013) and lamb (Morrow et al., 2013) receiving Na2SO4-enriched diets (5 and 8.3 g/kg, respectively). Further to unaffected N metabolism, the unchanged ME intake across all watering treatments supports the lack of significant variation of the efficiency of microbial N synthesis.

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4.3. Effects on rumen fermentation Our results show that rumen fermentation in Barbarine lamb was not adversely affected by drinking salt-containing water; evidently considering the amounts of the respective salts we used. Ruminal pH recorded before the distribution of the morning meal was in the range (6– 6.5) of values reported in numerous studies (e.g. Mould and Ørskov, 1983; Leng, 1990; Nasri et al., 2011; Baluch-Gharaei et al., 2015). Irrespective of treatments and times post-feeding, the concentrations of NH3-N in the rumen were all above the recommended concentration (5 mg/dl) for an optimal microbial activity (Satter and Slyter, 1974). Ammonia-N picked at 2 hpost-feeding then decreased slightly at 6 h-post-feeding. Protozoa concentration in the rumen is in the range of numbers reported by several authors (Mould and Ørskov, 1983; Rouissi and Guesmi, 2004). Our results show clearly the defaunating effect, although partially, of the three salts added to water for lamb. The absence of effect of the amounts of salts added to drinking water on diet N intake and crude protein digestibility justifies the stability of NH3-N concentration among the four groups. Lewis (1951a, b) conducted in vivo and in vitro experiments. He postulated that nitrite is an intermediary in the reduction of nitrate to ammonia by the rumen micro-organisms and concluded that when nitrate was administrated (10 and 25 g) in the rumen of sheep the nitrite and ammonia concentrations rose steadily for many hours. The discrepancy of these findings with our results is mainly due to difference in the doses of nitrite used by this other as compared to that we used in the current experiment. In our study, there was significant drop of the concentration of protozoa in the rumen of sheep drinking salinized water, mainly with the provision of NaCl and Na2SO4. This could have originated from increased osmotic pressure in the rumen as shown by Stacy and Warner (1966) and Potter et al. (1972). The concentrations of electrolytes in the rumen of sheep drinking salt-water might have changed,

19

particularly the concentration of sodium and potassium, consequently the rumen osmotic pressure might have increased. The substantial increase of water consumption by sheep drinking NaCl might have increased the dilution rate resulting in reduced concentration of protozoa in the rumen. Irrespective of water quality, the progressive decrease of the concentration of protozoa ciliate after feeding could be ascribed to the sequestration of Entodiniomorphida and to the dilution effect of saliva influx and passage rate (Dehority, 2003).

4.4. Effects on blood metabolites and hematology

The concentrations of serum metabolites and complete blood count measured in the current study were in the range of those reported in the literature (Assad et al., 1997; Jaber et al., 2004; Casamassima et al., 2008; Deghnouche et al., 2011). Albumin, triglycerides, GammaGT activity and total protein levels in the blood are indicators of liver function and the nutritional status of the animal (Barnouin et al., 1981; Braun, 1983). In the experiment reported herein only NaNO2 added to water reduced substantially the concentration of serum triglycerides. We are not aware of any study reported in the literature addressing the effect of NaNO2 in drinking water for ruminants on blood metabolites, particularly triglycerides. Stokes et al. (2009) revealed that nitrite addition to drinking water (50 mg/L sodium nitrite) substantially reduced the level of triglycerides in mice subjected to a high-cholesterol diet. They claimed that this indicates a novel pathway by which exogenous inorganic nitrite may be affecting fat metabolism or energy utilization. In a recent in vitro study, Zhang et al. (2015) demonstrated that sodium nitrite enhanced the autophagic flux and decomposition of triglycerides in steatotic L-02 cells. Serum glucose concentration was similar in lamb assigned to control and NaCl-enriched water treatments; this is evidenced by the unchanged OM and ME intakes and diet digestibility. In line with our findings, Godwin and William (1986)

20

showed that plasma glucose in sheep was not affected by the intra-ruminal administration of increasing doses of NaCl. Also, Assad and El-Sherif (2002) reported no change of glucose concentration in the plasma of Barki ewes drinking freely fresh water (280 ppm TDS) or diluted seawater containing 7650 and 13535 ppm TDS. However, in the current study this metabolite was significantly higher in the serum of sheep assigned to Na2SO4 or NaNO2enriched water than in control group. This happened while energy value of the diets was not affected by any of the watering treatments. To our knowledge, there is no literature information on the effect of Na2SO4 in water or diet on the concentration of blood glucose. Hyperglycemia has been reported (Pankow and Ponsold, 1973) in rats following the administration of certain doses of nitrite but, so far, there is no similar study conducted on ruminants.

The variation of serum creatinine and urea is an indicator of kidney health. In the current study, there was no modification in urea level among the four groups. However, creatinine concentration was 29 to 37% lower in groups having access to salt-enriched water as compared to control. The change of the concentration of the later metabolite could be ascribed to the physiological status (Deghnouche et al., 2011) and to the nutritional conditions of the animals. An increase in blood creatinine is often considered as a sign of a decreased renal clearance of endogenous creatinine, indicating a reduction in glomerular filtration rate. Reciprocally, the decrease in blood creatinine level could be attributed to improved glomerular filtration and thus increased renal clearance of endogenous creatinine accompanying the enhancement of urine production as observed in the current study on animals drinking salt-enriched water. Urine production in the kidneys increases as part of the body's homeostatic maintenance of fluid balance (Diuresis). Complete blood count didn't reflect any variation among the four groups except for platelets count (PLT) and plateletcrit (PCT) which were lowest in NaNO2-group. Assad et al. (1997)

21

did not detect any change in WBC, RBC, Hb, MCH and MCHC in sheep consuming saline water (7.7 or 13.5g TDS/L diluted sea water). However, they observed a reduction of plasma volume suggesting a haemo-concentration induced by the high salt level. The effect of nitrite as regulator of platelets function was investigated by many scientists (Webb et al., 2008; Srihim et al., 2012; Park et al., 2013; Apostoli et al., 2014; Dautov et al., 2014; Park et al., 2014; Gambaryan and Tsikas, 2015). Inorganic nitrate/nitrite is increasingly recognized as a source of bioactive nitric oxide (NO) (Apostoli et al., 2014; Gambaryan and Tsikas, 2015). Nitric oxide is one of the potent inhibitors of platelet function (Park et al., 2013). It could be generated from L-arginine by endogenous nitric oxide synthase (NOS) and from exogenous sources such as diets rich in nitrite and nitrate by two-step reduction pathway in which nitrate is converted first into nitrite and then into nitric oxide via certain mechanisms (enzymatic and non-enzymatic pathways) (Park et al., 2014).

4.5. Effects on carcass traits and meat quality The average hot carcass yield (HCY) of control lambs (0.41) is similar to that (0.42) reported by Abidi et al. (2009) on Barbarine lamb receiving oat hay ad libitum and 300 g concentrate. Studies on the effect of saline water on carcass traits and meat quality are scarce. Lambs assigned to the four watering treatments had similar carcass characteristics. Neither meat ultimate pH nor cooking loss was affected by the addition of the three types of salts in drinking water. These two parameters are in the range of those reported in other studies carried out on Barbarine lamb receiving diets composed of oat hay and concentrate and having continuous access to fresh water (Abidi et al., 2009; Nasri et al., 2011; MajdoubMathlouthi et al., 2013). Overall, the addition of the three salts in water did not produce major effects on the intramuscular FA composition of Barbarine lamb meat. Only two long chain FAs were

22

affected. The proportion of 10-Heptadecenoic acid (17:1(n-7)) decreased with the incorporation of NaNO2 in drinking water. Also, the proportion of C17:0 iso increased substantially in the meat of lambs drinking Na2SO4-enriched water as compared to control treatment. Omega-6 and Omega-3 FAs have different metabolic and function traits, and often have important opposing physiological functions (Simopoulos, 2009). In human nutrition, low omega-6 to omega-3 ratio is recommended to reduce the risk of contracting a wide range of chronic diseases (e.g. coronary heart disease, Simopoulos, 2009). The optimal n-6 to n-3 ratio varies between 1 and 4 depending on the disease (Simopoulos, 2002). In the current experiment n-6/n-3 ratio (4.7-5.0) surpassed the recommended value. In conformity with previous studies conducted in Tunisia (Nasri et al., 2011; Majdoub-Mathlouthi et al., 2013) on the same breed fed on oat hay and concentrate, n-6/n-3 ratio was above 6. In the current experiment, the experimental diets were extremely low in α-linolenic acid (ALA; 18:3ω3); which is the precursor of the endogenous synthesis of omega-3and their long-chain derivatives. This is in line with the low proportions of n-3 FAs in the meat analyzed in the present study. N-heptadecanoic acid (margaric acid) is present as a trace component in sheep fat (Hansen et al., 1957). Total SFA, MUFA and PUFA were not influenced by the addition of the three types of salt in drinking water.

5. Conclusion Our results show that the enrichment of drinking water with either NaCl (7 g/L), Na2SO4 (2 g/L) or NaNO2 (40 mg/L) increased water consumption but did not affect diet intake, apparent nutrient digestibility, N balance and microbial N supply. This is, à priori, the reason behind the absence of a significant effect on the growth rate of

Barbarine lamb. This is also

supported by the unchanged fermentation pattern in the rumen due to salt addition to water. The variation of some blood metabolites would suggest that salt provision through drinking

23

water could have affected sheep health. For example, the decreased concentration of serum creatinine in sheep drinking water high in TDS reflects a possible alteration of kidney function. Sodium nitrite in drinking water provoked hyperglycemia and caused a decrease in serum triglyceride, platelets count and plateletcrit. The above mentioned salt sources and their respective amounts added to water did not affect carcass traits and meat quality, mainly FAs profile. In brief, the Barbarine lamb copes with the quality of drinking water tested in this experiment without detrimental effect on its production performance, however, the impact of these salts on sheep health warrants further investigation.

Conflict of interest

There is no conflict of interests associated with this publication.

Acknowledgements

This study was funded by the Ministry of High Education and Scientific Research as part of the research program of the laboratory of Animal and Forage Productions of INRA-Tunisia. The authors are grateful to Prof. Mohamed Gharbi for facilitating the haematological analysis in his laboratory at the National School of Veterinary Medicine of Sidi Thabet (Tunisia).

24

References Abidi, S., Ben Salem, H., Vasta, V., Priolo, A., 2009. Supplementation with barley or spineless cactus (Opuntia ficus indica f. inermis) cladodes on digestion, growth and intramuscular fatty acid composition in sheep and goats receiving oaten hay. Small Rumin. Res. 87, 9–16. Agricultural Food Research Council (AFRC), 1993. Energy and protein requirements of ruminants. Technical Committee on Responses to Nutrients. CAB International Publication, Wallingford, UK. ANZECC (Australian and New Zealand Environment and Conservation Council), 2000. Primary Industries — Rationale and Background Information (Irrigation and general water uses, stock drinking water, aquaculture and human consumers of aquatic foods) Water Quality Guidelines. Volume 3. AOAC, 1990. Association of Official Analytical Chemists, Official Methods of Analysis, 15th ed. AOAC, Arlington, VA, USA. Apostoli, G. L., Solomon, A., Smallwood, M. J., Winyard, P. G., Emerson, M., 2014. Role of inorganic nitrate and nitrite in driving nitric oxide–cGMP-mediated inhibition of platelet aggregation in vitro and in vivo. J. Thromb. Haemostasis. 12, 1880–1889. Assad, F., Bayoumi, M. T., Khamis, H. S., 1997. Impact of long-term administration of saline water and protein shortage on the haemograms of camels and sheep. J. Arid Environ. 37, 71–81. Assad, F., El-Sherif, M.M.A., 2002. Effect of drinking saline water and feed shortage on adaptive responses of sheep and camels. Small Rumin. Res. 45, 279–290. Atti, N., Ben Salem, H., 2008. Compensatory growth and carcass composition of Barbarine lambs receiving different levels of feeding with partial replacement of the concentrate with feed blocks. Anim. Feed Sci. Technol. 147, 265–277.

25

Attia-Ismail, S. A., 2003. Metabolism and nitrogen balance in the rumen of sheep under the influence of drinking salt water and flavomycin. J. Nutr. Feeds 6, 1143-1152. Baluch-Gharaeia, H. Rouzbehana,Y., Fazaelib, H., Rezaeia, J., 2015. Effect of deep-stacking broiler litter on pathogenic bacteria, intake, digestibility, microbial protein supply and rumen parameters in sheep. Anim. Feed Sci. Technol. 199, 73–83. Barnouin, J., Mialot, M., Levieux, D., 1981. Evaluation de la pathologie hépatique des bovins sur un prélèvement de sang. Relations avec l'histopathologie. Ann. Rech. Vét. 12, 363– 369. Ben Salem, H., Lassoued, N., Rekik, M., 2011. Merits of the fat-tailed Barbarine sheep raised in different production systems in Tunisia: digestive, productive and reproductive characteristics. Trop. Anim. Health. Prod. 43, 1357–1370. Bird, P.R., 1972. Sulphur metabolism and excretion studies in ruminants. X. Sulphide toxicity in sheep. Aust. J. Biol. Sci. 25, 1087–1098. Boccard, R.L., Buchter, E., Casteels, E., Cosentino, E., Dransfield, D., Hood, E., Joseph, R.L., MacDougall, D.B., Rhodes, D.N., Schon, I., Tinbergen, B.J., Touraille, C., 1981. Procedures for measuring meat quality characteristics in beef production experiments. Report of a working group in the Commission of the European Communities (CEC) beef production research programme. Livest. Prod. Sci. 8, 385–397. Braun, J.P., Bénard, P., Burgat, V., Rico, A.G., 1983. Gamma Glutamyl Transferase in domestic animals. Vet. Res. Commun. 6, 77-90. Casamassima, D., Pizzo, R., Palazzo, M., D’Alessandro, A. G., Martemucci, G., 2008. Effect of water restriction on productive performance and blood parameters in Comisana sheep reared under intensive condition. Small Rumin. Res. 78, 169–175. CCME (Canadian Council of Resource and Environment Ministers), 2008. Canadian Water Quality Guidelines1987-1997. Environment Canada, Ontario.)

Canada,

Ontario

(In

land

Water

Directorate,

26

Chen, X.B., Gomes, M.J., 1992. Estimation of microbial protein supply to sheep and cattle based on urinary excretion of purine derivatives. An overview of the technical details. Occasional Publication. International Feed Resources Unit, Rowett Research Institute, Aberdeen. UK, p. 19. Cowley, A. W., Skelton, M. M., Merrill, D. C., 1986. Osmoregulation during high salt intake: relative importance of drinking and vasopressin secretion. Am. J. Physiol.-REG I, 251(5), R878-R886. Dautov R. F., Stafford, I., Liu, S., Cullen, H., Chirkov, Y. Y., Horowitz, J D., 2014. Hypoxic potentiation of nitrite effects in human vessels and platelets. Nitric Oxide. 40, 36–44. Deghnouche, K., Tlidjane, M., Meziane, T., Touabti, A., 2011. Influence du stade physiologique sur divers paramètres biochimiques sanguins chez la brebis Ouled Djellal des zones arides du Sud-Est algérien. Revue Méd. Vét. 162, 1, 3-7. Dehority, B. A., 2003. Rumen Microbiology. Nottingham Univ. Press, Nottingham, UK. Deng, H. W., Cong, Y. Y., Feng, Y. L., Chen, J., 2013. Effects of dietary supplementation with different sulfate sources on nitrogen and sulfur metabolism and rumen fermentation in cashmere goats. Livest. Res. Rural Develop. 25, Article #41. Dryden, G. M., 2008. Animal nutrition science. CABI Press, Wallingford, United Kingdom. p 115–129. Emery, R.S., Liesman, J.S., Herdt, T.H., 1992. Metabolism of long chain fatty acids by ruminant liver. J. Nutr. 122, 832-837. Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509. French, P., Stanton, C., Lawless, F., O’Riodan, E.G., Monahan, F.J., Caffrey, P.J., Moloney A.P., 2000. Fatty acid composition, including conjugated linoleic acid, of intramuscular fat from steers offered grass, grass silage, or concentrate-based diets. J. Anim. Sci. 78, 2849-2855.

27

Gaaloul, N., Zouari, K., 2004. Harnessing salty water to enhance sustainable livelihoods of the rural poor in Tunisia. Report. International Center for Biosaline Agriculture. 32 p.

Gambaryan, S., Tsikas, D., 2015. A review and discussion of platelet nitric oxide and nitric oxide synthase: do blood platelets produce nitric oxide from l‑arginine or nitrite? Amino Acids47, 1779-1793. Godwin, I.R., Williams, V.J., 1986. Effects of intraruminal sodium chloride infusion on rumen and renal nitrogen and electrolyte dynamics in sheep. Br. J. Nutr. 56, 379-394. Grout, A. S., Veira, D. M., Weary, D. M., von Keyserlingk, M. A. G., Fraser, D., 2006. Differential effects of sodium and magnesium sulfate on water consumption by beef cattle consumption by beef cattle. J. Anim. Sci. 84, 1252-1258. Hannachi, Ch., Guesmi, F., Missaoui, K., Hamrouni, B., 2014. Application of adsorption models for fluoride, nitrate, and sulfate ion removal by AMX membrane. Int. J. Technol. 1, 60-69.

Hansen, R. P., Shorland, F. B., June Cooke, N., 1957. "Occurrence in Butterfat of nHeptadecanoic Acid (Margaric Acid)". Nature. 179, 98. Jaber, L.S., Habre, A., Rawda, N., Abi Said, M., Barbour, E. K., Hamadeh, S., 2004. The effect of water restriction on certain physiological parameters in Awassi sheep. Small Rumin. Res. 54, 115–120. Keenan, D. M., Allardyce, C. J., 1986. Changes in plasma creatinine levels of sheep during submaintenance feeding. Aust. Vet. J. 63, 29–30. Lardner, H. A., Kirychuk, B. D., Braul, L., Willms, W. D., Yarotski, J., 2005. The effect of water quality on cattle performance on pasture. Crop Pasture Sci., 56, 97-104. Leng, R. A., 1990. Factors affecting the utilization of poor quality forages by ruminants particularly under tropical conditions. Nutr. Res. Rev. 3, 277-303. Lewis, D., 1951a. The metabolism of nitrate and nitrite in the sheep. 1. The reduction of nitrate in the rumen of the sheep. Biochem. J. 48, 175–180.

28

Lewis, D., 1951b. The metabolism of nitrate and nitrite in the sheep. 2. Hydrogen donators in nitrate reduction by rumen micro-organisms in vitro. Biochem. J. 49, 149–153. MAF (Ministry of Agriculture and Fisheries), 2004. Livestock production gains from improved drinking water: Literature review. New Zealand. MAF Technical Paper No 2004/07. Prepared for MAF Policy by: Abacus Biotech Ltd. ISBN No: 0-478-07826-9. 42 p. Majdoub-Mathlouthi, L., Saïd, B., Say, A., Kraiem, K., 2013. Effect of concentrate level and slaughter body weight on growth performances, carcass traits and meat quality of Barbarine lambs fed oat hay based diet. Meat Sci. 93, 557–563. Masters, D. G., Benes, S. E., Norman, H. C., 2007. Biosaline agriculture for forage and livestock production. Agric. Ecosyst. Environ. 119, 234–248. Masters, D.G., Rintoul A.J., Dynes, R.A., Pearce, K.L., Norman, H.C., 2005. Feed intake and production in sheep fed diets high in sodium and potassium. Aust. J. Agric. Res. 56, 427–434. McKenzie, R. A., Rayner, A. C., Thompson, G.K., Pidgeon, G. F., Burren, B. R., 2004. Nitrate-nitrite toxicity in cattle and sheep grazing Dactyloctenium radulans (button grass) in stockyards. Aust. Vet. J. 82, 630-634. Meyer, J.H., Weir, W.C., Ittner, N.R., Smith, J.D., 1955. The influence of high sodium chloride intakes by fattening sheep and cattle. J. Anim. Sci. 14, 412–418. Morrow, L. A., Felix, T. L., Fluharty, F. L., Daniels, K. M., Loerch, S. C., 2013. Effects of sulfur and acidity on performance and digestibility in feedlot lambs fed dried distillers grains with solubles. J. Anim. Sci. 91, 2211–2218. Mould, F.L., Ørskov, E.R., 1983. Manipulation of rumen fluid pH and its influence on cellulolysis in sacco, dry matter degradation and the rumen microflora of sheep offered either hay or concentrate. Anim. Feed Sci. Technol. 10, 1-14.

29

Nasri, S., Ben Salem, H., Vasta, V., Abidi, S., Makkar, H.P.S., Priolo, A., 2011. Effect of increasing levels of Quillaja saponaria on digestion, growth and meat quality of Barbarine lamb. Anim. Feed Sci. Technol. 199, 73–83. Olkowski, A. A., 2009. La qualité de l'eau d'abreuvement du bétail : Guide de terrain relatif aux bovins, aux chevaux, à la volaille et aux porcs. ISBN 978-1-100-12443-8. Pankow, D., Ponsold, W., 1973. Blood glucose level after sodium nitrite injection and combined administration of sodium nitrite and carbon monoxide in rats. Int. Archiv für Arbeitsmedizin, 31, 237-241. Park, J. W., Piknova, B., Nghiem, K., Lozier, J. N., Schechter, A. N., 2014. Inhibitory effect of nitrite on coagulation processes demonstrated by thrombelastography. Nitric Oxide. 40, 45–51. Park, J.W., Piknova, B., Huang, P. L., Noguchi, C. T., Schechter, A. N., 2013. Effect of Blood Nitrite and Nitrate Levels on Murine Platelet Function. PLoS ONE. 8(2), e55699. Peirce, A.W., 1957. Studies on salt tolerance of sheep. I. The tolerance of sheep for sodium chloride in the drinking water. Aust. J. Agric. Res. 8, 711-722. Peirce, A.W., 1959. Studies on salt tolerance of sheep II. The tolerance of sheep for mixture of sodium chloride and magnesium chloride in the drinking water. Aust. J. Agric. Res. 10, 725-735. Peirce, A.W., 1960. Studies on salt tolerance of sheep. III. The tolerance of sheep for mixtures of sodium chloride and sodium sulphate in the drinking water. Aust. J. Agric. Res. 11, 548-556. Peirce, A.W., 1962. Studies on salt tolerance of sheep. IV. The tolerance of sheep for mixtures of sodium chloride and calcium chloride in the drinking water. Aust. J. Agric. Res. 13, 479–486.

30

Peirce, A.W., 1963. Studies on salt tolerance of sheep. V. The tolerance of sheep for mixtures of sodium chloride, sodium carbonate, and sodium bicarbonate in the drinking water. Aust. J. Agric. Res. 14, 815–823. Potter, B. J., 1961. The renal response of sheep to prolonged ingestion of sodium chloride. Crop Pasture Sci. 12, 440-445. Potter, B.J., Walker, D.J., Forrest, W.W., 1972. Changes in intraruminal function of sheep when drinking saline water. Brit. J. Nutr. 27, 75-83. Rahaingomanana, N., 1998. Water chemistry characteristics in small reservoirs of semiarid Tunisia. Rain water harvesting and management of small reservoirs in arid and semiarid areas. Lund University, Sweden, 29 June - 2 July, 1998. Robertson, J.B., Van Soest, P.J., 1981. The detergent system of analysis and its application to human foods. In: James, W.P.T., Theander, O. (Eds.), The Analysis of Dietary Fiber in Food. Marcel Dekker, NY, USA, pp. 123–158. Rouissi, H., Guesmi, A., 2004. Etude comparée de la population des protozoaires ciliés dans le rumen des ovins et caprins. Option Méditerranéenne, Série A, 57-59. SAS. 1991. SAS user’s guide. SAS Institute, Inc. Cary, N.C. Satter, L. D., Slyter, L. L., 1974. Effect of ammonia concentration on rumen microbial protein production in vitro. Brit. J. Nutr. 32, 199-208. Sexson, J. L., Wagner, J. J., Engle, T. E., Spears, J. W., 2010. Effects of water quality and dietary potassium on performance and carcass characteristics of yearling steers. J. Anim. Sci. 88, 296-305. Simopoulos, A. P., 2002. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomed. Pharmacother. 56, 365–379. Simopoulos, A. P., 2009. The Importance of the Omega-6/Omega-3 Fatty Acid Ratio in Cardiovascular Disease and Other Chronic Diseases. Exp. Biol. Med. 233, 674-688.

31

Srihirun, S., Sriwantana, T., Unchern, S., Kittikool, D., Noulsri, E., Pattanapanyasat, K., Fucharoen, S., Piknova, Barbora., Schechter, A. N., Sibmooh, N., 2012. Platelet Inhibition by Nitrite Is Dependent on Erythrocytes and Deoxygenation. PLoS ONE. 7(1), e30380. Stacy, B.D., Warner, A.C.I., 1966. Balances of water and sodium in the rumen during feeding: osmotic stimulation of sodium absorption in the sheep. Quart. J. exp. Physiol. 51, 79-93. Stokes, K. Y., Dugas, T. R., Tang, Y., Garg, H., Guidry, E., Bryan, N. S., 2009. Dietary nitrite prevents hypercholesterolemic microvascular inflammation and reverses endothelial dysfunction. Am. J. Physiol. Heart. Circ. Physiol. 296, 1281-1288. Thurnhofer, S., Vetter, V., 2005. A gas chromatography/electron ionization-mass spectrometry-selected ion monitoring method for determining the fatty acid pattern in food after formation of fatty acid methyl esters. J. Agric. Food Chem. 53, 8896-8903. Van Soest, P.V., Robertson, J.B.,Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583-3597. Weatherburn, M.W., 1967. Phenol hypochlorite reaction for determi-nation of ammonia. Anal. Chem. 39, 971–974. Webb, A.J., Patel, N., Loukogeorgakis, S., Okorie, M., Aboud, Z., Misra, S.,Rashid, R., Miall, P., Deanfield, J., Benjamin, N., MacAllister, R.,Hobbs, A. J., Ahluwalia, A., 2008. Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite. Hypertension. 51, 784-790. Willms, W. D., Kenzie, O. R., McAllister, T. A., Colwell, D., Veira, D., Wilmshurst, J. F., Olson, M. E., 2002. Effects of water quality on cattle performance. J. Range Manage. 452-460.

32

Wilson, A. D., 1966. The tolerance of sheep to sodium chloride in food or drinking water. Crop Pasture Sci. 17, 503-514. Wilson, A. D., Dudzinski, M. L., 1973. Influence of the concentration and volume of saline water on the food intake of sheep, and on their excretion of sodium and water in urine and feces. Aust. J. Agric. Res. 24, 245–256. Yape Kii, W., Dryden, G. McL., 2005. Effect of drinking saline water on food and water intake, food digestibility, and nitrogen and mineral balances of rusa deer stags (Cervus timorensis russa). Anim. Sci. 81, 99-105. Zaara, M., 2008. Le dessalement en Tunisie pour l’amélioration de la qualité de l’eau potable desservie. ADIRA Workshop, Marrakech 26 Avril 2008. Zghibi, A., Tarhouni, J., Zouhri, L., 2013. Assessment of seawater intrusion and nitrate contamination on the groundwater quality in the Korba coastal plain of Cap-Bon (North-east of Tunisia). Journal of African Earth Sciences, 87, 1-12. Zhang, Y.J., Zheng, N.R., Liu, B., Ji, A.L., Li, Y.Z., Huangfu, C.S., 2015. Sodium nitrite reduces lipid accumulation in steatotic cells by enhancing autophagy. Yao Xue Xue Bao [Acta pharmaceutica sinica]. 50, 1000-7.

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Table 1 Contents and properties of drinking water.

Salts added to the drinking water

TDS (mg/l) pH Sodium (mg/l) Potassium (mg/l) Calcium (mg/l) Magnesium (mg/l) Chlorides (mg/l) Sulfates (mg/l) Nitrates (mg/l) Bicarbonates (mg/l) Sodium adsorption ratio Hardness degree (°fH)

Control

NaCl

Na2SO4

NaNO2

760 7.25 70 4.3 120 19 285 105 55 60 4.5 29.1

8045 7.1 2460 4.6 135 25 4830 90 43 79 69.5 35.5

2645 7.35 725 3.5 110 23 375 1230 48 63 30 37

790 7.92 120 4.2 90 17 265 95 50 77 15 35

34

Table 2 Nutrient contents of feeds (n = 6). Oat hay

Concentrate

DM (g/kg)

917

954

OM (g/kg DM)

920

911

CP (g/kg DM) NDF (g/kg DM) ADF (g/kg DM)

58 682 370

148 242 110

ADL (g/kg DM)

70

35

Fatty acids (g/kg total FA) C12:0 C14:0 C15:0 C16:0 C16:1n-7 C17: 0 C17:1n-7 C18:0 iso C18:0 C18:1n-9 C18:1 C18:2n-6 C18:3n-6 C18:3n-4 C18:3n-3 C18:2 conj C20:0 C20:1n-9 C20:2n-6 C20:3n-6 C20:4n-6 C20:5n-3 C22:0 C21:5n-3 C22:5n-6 C22:5n-3 C24:0

9.8 11.1 180.0 4.1 59.6 94.9 6.9 95.5 1.2 1.6 38.6 16.1 123.0 46.1 5.0 56.7 21.5 10.1 29.8

<0.5 1.7 0.5 169.0 0.7 1.4 0.6 <0.5 21.1 191.0 496.0 50.0 <0.5 8.4 11.0 4.7 1.5 2.4 <0.5 20.9 0.6 1.0 <0.5 2.5

DM, dry matter; OM, organic matter; CP, crude protein; NDF, neutral detergent fibre; ADF, Acid Detergent Fiber; ADL, acid detergent lignin.

35

Table 3 Effect of salts in drinking water on feed and water intakes and growth rate of sheep. Salts added to drinking water

SEM

P-value

601.2 55.4

44.22 4.22

0.354 0.402

925.4 847.2 95.32 427.5 83.6

977.2 894.6 100.6 451.5 90.2

44.22 40.48 4.55 20.43 4.61

0.354 0.324 0.365 0.163 0.470

4600b 433.9b 5046.3b

2820a 254.5a 3050.4a

2940a 269.9a 3093.2a

183 17.19 264.61

<0.001 <0.001 <0.001

21.1 23.6 52.3

21.7 23.7 44.1

21.6 24.0 48.0

0.82 0.98 9.11

0.900 0.993 0.498

Control

NaCl

Na2SO4

NaNO2

484.8 45.0

539 51.0

549.4 49.6

860.8 788.7 88.7 397.7 80.0

915 837.7 94.2 422.7 86.5

Drinking water intake (ml/day) 2480a Drinking water intake (ml/kg BW0.75) 231.5a Drinking water intake (ml/kg DMi) 2888.6a Body weight change Initial BW (kg) Final BW (kg) Average daily gain (g)

Hay intake (g DM/day) Hay intake (g DM/kg BW0.75) Total intake (g/day) DM OM CP NDF Total DM intake (g/kg BW0.75)

21.0 23.8 63.1

DM: dry matter; OM: organic matter; CP: crude protein; NDF: ash-free neutral detergent fibre; BW: body weight. DMi; dry matter intake; SEM: standard error of the mean. a,bMeans

in the same line with different letters are significantly different (P<0.05).

36

Table 4 Effect of salts in drinking water ondiet digestibility, N balance and microbial N supply in sheep. Salts added to drinking water

Apparent diet digestibility (g/g) DM OM CP NDF ME intake (MJ/kg DM) Nitrogen (g/day) Intake Voided in feces Voided in urine Retained

SEM

P-value

0.569 0.590 0.469 0.412

0.0178 0.0170 0.0488 0.0261

0.225 0.211 0.755 0.235

9.22

8.01

0.329

0.054

15.8 7.15 1.56 7.1

16.0 7.91 1.27 6.85

15.7 8.19 1.00 6.53

0.27 0.830 0.253 0.812

0.339 0.838 0.290 0.775

31.8ab 3.61

19.6a 2.29

37.5b 4.07

4.77 0.689

0.034 0.052

Control NaCl

Na2SO4

NaNO2

0.618 0.637 0.492 0.472

0.606 0.625 0.543 0.462

0.617 0.636 0.494 0.488

9.21

9.06

15.3 7.69 1.65 5.95

Urinary allantoin (mg/kg BW0.75) 18.9a Microbial N efficiency (g/kg DOMI) 2.38

DM, dry matter; OM, organic matter; CP, crude protein; NDF, neutral detergent fiber; ME, metabolisable energy; DOMI, digestible organic matter intake; SEM, standard error of the mean. a,bMeans

in the same line with different letters are significantly different (P<0.05).

37

Table 5 Effect of salts in drinking water on average pH, ammonia nitrogen (mg/dl) and protozoa (×105/ml) concentrations in the rumen of sheep

pH NH3-N Protozoa 1

Salts added to drinking water Control NaCl Na2SO4 NaNO2 6.26 6.19 6.20 6.28 9.61 9.66 10.06 10.32 7.16 4.32 3.70 5.03

SEM 0.052 0.778 0.647

Time1 0h 6.52 7.61 8.34

SEM 2h 6h 6.09 6.09 11.37 10.76 3.88 2.94

Sampling times of rumen fluid were 0 h (before morning meal), and 2 h and 6 h postfeeding.

0.033 0.464 0.535

P-values Diet Time 0.598 <0.001 0.903 <0.001 0.009 <0.001

Diet x Time 0.059 0.680 0.546

38

Table 6 Effect of salts in drinking water onblood metabolites and complete blood count in sheep. Salts added to drinking water

SEM

P-value

41.6 8.1b 14.9 0.715c 87.1 0.198b 0.305

1.70 0.81 1.53 0.0323 6.24 0.0451 0.0339

0.068 0.003 0.362 <0.001 0.548 0.034 0.189

9.5 9.4 79.5 28.6 30.5 8.43 278.1 16.7 330.3b 4.81 13.6 0.158b

0.955 0.349 2.630 0.945 0.716 0.143 4.23 0.342 38.11 0.104 0.135 0.0207

0.069 0.508 0.343 0.506 0.594 0.122 0.890 0.447 0.040 0.495 0.383 0.049

Control

NaCl

Na2SO4

NaNO2

Albumin (g/L) Creatinine (mg/L) Gamma GT (U/L) Glucose (g/L) Total proteins (g/L) Triglycerides (g/L) Urea (g/L)

35.2 11.7a 16.1 0.420a 75.3 0.378a 0.341

36.5 8.3b 14.2 0.469a 77.6 0.240ab 0.373

37.1 7.6b 12.2 0.589b 77.2 0.214ab 0.409

WBC (×109/L) RBC (×1012/L) HGB (g/L) HCT (x10-2) MCV (fl) MCH (pg) MCHC (g/L) RDW (x10-2) PLT (×109/L) MPV (fl) PDW (x10-2) PCT (x10-2)

13.2 8.84 77.9 27.9 31.7 8.79 279 17.1 472.0a 4.98 13.9 0.236a

11.8 9.28 78.5 28.3 30.6 8.43 278 16.7 442.6ab 4.86 13.7 0.215ab

10.9 8.78 73.1 26.6 30.5 8.31 274.6 16.3 356.8ab 5.01 13.8 0.176ab

WBC, Total white blood cells; RBC, Total red blood cells; HGB, Hemoglobin; HCT, Hematocrit; MCV,Mean corpuscular volume; MCH, Mean corpuscular hemoglobin; MCHC, Mean corpuscular hemoglobin concentration; RDW, Red blood cell distribution width; PLT, Plateletscount; MPV, Mean platelet volume; PDW, Platelet distribution width; PCT, Plateletcrit. a,bMeans in the same line with different letters are significantly different (P<0.05).

39

Table 7 Effect of salts in drinking water oncarcass characteristics, meat pH, cooking loss, and fatty acids composition in sheep. Salts added to drinking water Control NaCl Na2SO4

NaNO2

Slaughterweight (kg) Hot carcass weight (kg) Chilled carcass weight (kg) Hot carcass yield (kg/100 kg) Chilled carcass yield (kg/100 kg) Shrink after chilling (kg/100 kg) Digestive tract weight (kg) Empty digestive tract weight (kg) Tail weight (g) Liver weight (g) Meat pH 24h Cooking loss

23.4 9.25 8.99 39.5 38.2 3.41 6.44 2.01 664.2 354.7 5.71 0.192

23.1 9.35 9.01 40.3 38.8 3.64 6.34 2.38 701.4 341.1 5.70 0.194

23.6 9.20 8.85 38.9 37.5 3.78 6.38 1.92 775.8 331.4 5.70 0.195

Fatty acids (g/kg total FA) C10:0 C12:0 C14:0 C14:1n-5 C15:0 ante C15:0 iso C15:0 C16:0 iso C16:0 C16:1n-7 C17:0 ante C17:0 iso C17:0 C17:1n-7 C18:0 iso C18:0 C18:1n-9 C18:1 C18:2 C18:2n-6 C18:2 conj C18:2n-4 C18:3n-6 C18:3n-4 C18:3n-3 C20:0 C20:1n-9 C20:2n-6 C20:3n-6 C20:4n-6 C20:5n-3 C22:0 C21:5n-3 C22:5n-6 C22:5n-3 C22:6n-3 C24:0 Omega 3 Omega 6 Omega 6/Omega 3 SFA MUFA PUFA

0.9 2.0 13.1 1.6 0.7 1.5 2.5 0.9 148 13.4 3.0 3.3a 9.1 6.9a 1.5 172.9 407.6 2.7 2.7 74.7 2.0 1.8 1.5 2.0 5.1 3.4 2.9 10.1 4.9 42.1 5.4 3.7 1.3 4.3 14.7 1.3 7.1 27.8 137.7 4.92 371.6 434.5 173.6

1.0 1.7 12.2 1.5 1.1 1.9 4.0 0.9 146.9 14.0 3.2 3.3a 9.2 6.3ab 1.2 177.3 364.4 3.1 1.7 80.7 3.0 1.5 1.7 1.9 5.6 3.0 3.6 11.8 6.0 49.8 6.6 3.5 1.6 5.9 18.0 2.4 5.1 33.3 155.8 4.68 373.4 391.7 196.3

1.5 1.0 13.2 1.2 1.2 1.8 3.2 1.1 164.4 14.2 3.4 4.4b 11.1 6.7ab 1.4 194.8 389.9 1.2 1.6 63.2 2.7 1.4 1.7 1.9 4.7 4.7 3.3 9.8 4.5 36.5 4.7 2.2 1.1 3.6 13.5 1.4 5.6 24.4 118.5 4.84 409.5 414.1 149.1

SEM

P-value

23.3 9.03 8.71 38.6 37.2 3.61 6.96 2.08 576.9 347.1 5.72 0.201

0.74 0.487 0.483 1.08 1.06 0.202 0.380 0.214 90.77 12.41 0.035 0.0141

0.972 0.973 0.967 0.712 0.709 0.636 0.633 0.482 0.488 0.600 0.969 0.973

1.1 2.1 13.6 1.8 0.7 2.0 3.3 1.0 154.8 12.8 3.3 3.9ab 10.3 4.7b 1.8 195.5 375.3 2.0 1.7 70.0 3.5 1.7 1.8 1.6 5.8 4.0 2.7 9.0 5.3 40.9 4.4 6.4 0.7 4.9 14.1 1.9 5.6 26.3 131.8 5.01 405.4 397.6 165.5

0.34 0.75 2.10 0.28 0.28 0.29 0.42 0.11 7.66 0.89 0.19 0.27 0.66 0.55 0.20 9.90 25.22 0.76 0.79 9.28 0.70 0.23 0.32 0.22 0.70 0.64 0.42 1.50 0.85 6.64 0.96 1.27 0.21 1.01 2.39 0.47 1.57 3.74 17.74 0.284 17.55 26.6 20.46

0.633 0.769 0.972 0.680 0.494 0.608 0.105 0.459 0.364 0.685 0.612 0.024 0.121 0.041 0.203 0.262 0.650 0.352 0.753 0.598 0.500 0.559 0.960 0.651 0.680 0.287 0.438 0.585 0.614 0.559 0.383 0.168 0.137 0.465 0.541 0.369 0.836 0.384 0.526 0.963 0.283 0.672 0.443

40 a,bMeans

in the same line with different letters are significantly different (P<0.05); SEM, standard error of the mean; SFA, short chain fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; n6, omega 6 fatty acids; n3, omega 3 fatty acids; SFA = Σ (C10:0, C12:0,C14:0iso, C14:0,C15:0 ante,C15:0 iso, C15:0,C16:0 iso,C17:0 ante,C17:0 iso, C17:0,C18:0 iso, C18:0, C20:0, C22:0, C24:0); MUFA = Σ (C14:1n-5, C16:1n-7, C17:1n-7, C18:1n9); PUFA = Σ (C18:2, C18:2n-4, C18:2n-6, C18:3n-3. C18:3n-4, C18:3n-6, C18:2conj, C20:2n-6, C20:3n-6, C20:4n-6, C20:5n-3, C20:5n-6, C21:5n3, C22:5n-3, C22:6n-3).