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J. Anim Sci. 2008. 86:738-747. doi:10.2527/jas.2007-0189
© 2008 American Society of Animal Science

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ANIMAL PRODUCTION

Learned appetites for calcium, phosphorus, and sodium in sheep1,2

J. J. Villalba*,3, F. D. Provenza* and J. O. Hall{dagger}

* Department of Forest, Range and Wildlife Sciences; and and {dagger} Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan 84322-5230


    Abstract
 Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
If supplemental minerals are needed to promote optimal animal performance, what is the best way of providing them: free choice or in the diet? We hypothesized that herbivores discriminate among feeds containing Na, P, and Ca and modify their choices as a function of need. One group of lambs was fed a basal diet low in P and high in Ca (low P-high Ca), whereas another group was fed a basal diet high in P and low in Ca (high P-low Ca). After 73 d of exposure to the unbalanced diets, the lambs were conditioned by offering flavored grape pomace containing NaCl, CaCO3, or NaH2PO4. Preference for pomace + minerals was determined when all lambs were fed a basal diet of alfalfa pellets and barley grain (initial preference) and during 4 phases. Phases 1 and 2 occurred after 40 and 67 d of feeding the unbalanced basal diets, phase 3 occurred after conditioning with NaCl, CaCO3, or NaH2PO4, and phase 4 occurred 22 d after the groups were moved to 2 new (separate) locations so the animals in the different groups could not eat dirt, urine, or feces from the other pen. Preference for pomace did not differ between the groups during the initial preference tests (P = 0.62); both groups preferred NaCl > CaCO3 = NaH2PO4 (P < 0.001). As the study progressed, and lambs fed low P-high Ca had lower P and greater Ca concentrations in serum than lambs fed high P-low Ca (P < 0.001), the preference between groups diverged. In phase 2, lambs in high P-low Ca continued to prefer NaCl (P < 0.001), but lambs in low P-high Ca preferred NaH2PO4 (P < 0.05). After conditioning, both groups preferred NaCl = NaH2PO4 > CaCO3 (P < 0.01 to 0.11). After the groups were moved to different locations, lambs fed low P-high Ca showed the lowest concentration (3.7 mg/dL) of inorganic P in serum for all phases (P < 0.001), and they preferred NaH2PO4 > NaCl = CaCO3 (P < 0.001). In contrast, lambs in high P-low Ca avoided NaH2PO4 (P < 0.05). Lambs offered high P-low Ca showed a greater preference for CaCO3 (P = 0.12) and NaCl (P < 0.05) and a lower preference for NaH2PO4 compared with lambs fed low P-high Ca (P < 0.001). In summary, lambs discriminated among different flavored feeds containing NaCl, CaCO3, and NaH2PO4 and displayed preferences as a function of the mineral imbalance in their basal diets. Thus, it may be possible to feed Ca and P supplements free choice, such that individual animals within a group can manifest preferences based on their specific needs.

Key Words: calcium • learning • phosphorus • preference • sheep • sodium


    INTRODUCTION
 Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Calcium and P play vital roles in most body tissues with structural roles in cell membranes, bones, and teeth (McDowell and Arthington, 2005Go). They are involved in cellular processes including membrane potentials, DNA synthesis, intracellular communication, cell membrane fluidity, and metabolic pathways (Underwood and Suttle, 1999Go). The need for cells to recognize and relate their needs (Ca and P hunger) could be an important behavioral adaptation to maintain homeostasis. State-dependent appetites for Ca and P have been questioned because cattle and sheep did not select Ca and P in amounts researchers thought would meet their needs (Coppock, 1970Go). Thus, some suggest offering minerals in a blended feed so that livestock receive all nutrients in proper proportion with each mouthful (Muller et al., 1977Go). In contrast, other studies support the notion of Ca and P appetites in mammals (Denton et al., 1986Go; Tordoff, 2002Go).

Sheep discriminated between the postingestive effects of NaH2PO4, NaCl, and vehicle (water); they showed a strong avoidance to NaH2PO4 when the need for P was low, but they manifested a modest preference for P when P deprivation was high (Villalba et al., 2006aGo). In that study, sheep received intraruminal infusions of salts and thus they could not regulate mineral intake, which likely caused aversions in animals that received too-high doses (Provenza, 1996Go; Villalba et al., 2006aGo). Aversions could have been minimized by offering supplements containing minerals so that each animal could regulate its intake on a daily basis and thus avoid over-ingestion.

The objectives of the current study were to determine whether lambs displayed state-dependent preferences for supplements containing Na, Ca, and P such that preference increased with P and Ca deprivation, and decreased with high levels of P and Ca in the basal diet, and if lambs discriminated supplements containing salts of P, Ca, and Na when presented at the same molar concentrations in different supplements.


    MATERIALS AND METHODS
 Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The study was conducted at the Green Canyon Ecology Center, located at Utah State University in Logan according to procedures approved by the Utah State University Institutional Animal Care and Use Committee.

During the study, 20 commercial crossbred lambs (4 mo of age) with BW of 31 ± 0.6 kg were individually penned outdoors, under a protective roof in individual adjacent pens measuring 2.4 x 3.6 m. Lambs had free access to fresh water and trace mineral salt blocks (Table 1Go). Before exposures to the experimental feeds and diets, lambs were given an adjustment period of 2 wk, during which they received 350 g of rolled barley grain/d and free access to alfalfa pellets.


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Table 1. Mineral concentrations (% as-fed basis) in test feeds, traced mineral blocks, and basal diets
 
We hypothesized that sheep would discriminate among feeds containing Na, P, and Ca and modify their feed choices as a function of the benefit supplied by each element relative to the minerals present in the basal diet. On that basis, we predicted that if ingestion of a specific supplement was followed by recovery from a mineral deficiency, such that the concentration of that mineral in blood increased, then animals would prefer that supplement at the onset of the occurrence of the same mineral deficiency. Thus, we determined whether sheep learned to prefer flavored supplements containing NaCl, CaCO3, or NaH2PO4 as a function of the deficiency/unbalance in their basal diet.

General Protocol
To assess preferences for minerals in ruminants, we developed a protocol in which 2 groups of lambs first received a diet balanced for Ca and P and subsequently 2 diets that supplied contrasting amounts of P and Ca (low P-high Ca; high P-low Ca). Animals then experienced 3 flavored supplements that supplied different amounts of P, Ca, and Na. Before and after this experience, the 2 groups of lambs were offered choices of the supplements to determine whether their preference was a function of the basal diets offered. Thus, this study compared supplement intake by lambs fed contrasting diets, such that each group served as a control (or reference) for the other group. Lambs within each group also acted as their own controls, because they were initially fed a balanced diet, followed by exposure to the unbalanced diets (see Table 2Go for a summary of the methods).


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Table 2. Conditioning and preference tests during the study
 
Adaptation to Flavored Grape Pomace + Minerals
Lambs were familiarized with a low-quality feed, grape pomace, which contained different flavors known to be easily discriminated by sheep (Villalba and Provenza, 1997a,b,cGo), and minerals (Table 1Go). In a previous study, no interaction between flavors and minerals was found (Villalba et al., 2006aGo).The pomace was mixed (40 g of flavor/kg of grape pomace) with ground onion powder (Pacific Foods, Kent, WA), coconut, and maple flavors (Lucta USA, Northbrook, IL). A different salt was added to each flavored feed, as follows: NaCl to coconut-flavored pomace, NaH2PO4 to maple-flavored pomace, and CaCO3 to onion-flavored pomace (Table 1Go). Flavors were assigned at random to the supplements. Salts were added to flavored pomace at the same molar concentrations (0.32 mol/kg of flavored pomace) to control for the molarity of the salts and the osmotic loads. During adaptation, lambs were offered flavored grape pomace at 0900, refusals were collected and weighed at 1200, and all animals then received 1 kg of alfalfa pellets and 350 g of rolled barley grain. No other feed was offered until the next day. Lambs were exposed to each flavor during 3 consecutive periods of 3 d each (from July 20 to July 28, 2005).

The sequence of flavor/mineral exposure in flavored grape pomace, which was chosen at random, was 1) NaCl + coconut, 2) NaH2PO4 + maple, and 3) CaCO3 + onion. Lambs consumed 121 ± 10 g, 117 ± 11 g, and 153 ± 13 g of NaCl + coconut-, NaH2PO4 + maple-, and CaCO3 + onion-flavored grape pomace, respectively during the 3 periods. At the end of this period (July 28, 2005), jugular blood samples were taken from each lamb to determine the initial serum concentrations of inorganic-P (Pi) and Ca, before the lambs were fed the basal diets.

Initial Preference Test
After the last day of exposure to flavored grape pomace, lambs received NaCl + coconut-, NaH2PO4 + maple-, and CaCO3 + onion-flavored grape pomace simultaneously for 15 min at 1000, and intake of each feed was determined (Table 2Go). Percentage preference for each feed was calculated as: (intake of a flavored feed/total flavor intake) x 100. From 1030 to 1300, all lambs were offered 1 kg of alfalfa pellets and 350 g of rolled barley grain. No other feed was offered until the next day.

The purpose of the initial preference test was to balance each treatment group based on individual lamb biases for flavored pomace. Preference tests were performed on 2 consecutive days, and the mean of the 2 d was used to assign lambs to 2 groups (low P-high Ca and high P-low Ca; n = 10 lambs/group). Lambs were stratified according to initial preference for one feed (e.g., NaCl + coconut-flavored grape pomace), and pairs of lambs were randomly assigned to the 2 groups. Thus, differences between groups due to initial feed preferences were balanced. Lambs belonging to the 2 groups were housed randomly in adjacent pens.

Test Diets
The day after the initial preference tests, we stopped feeding alfalfa pellets and rolled barley, and low P-high Ca lambs were fed a ground basal diet (1- to 2-mm particle size) low in P and high in Ca consisting of (as-fed basis) 97% beet pulp, 2.0% urea, and 1% calcium carbonate (Table 1Go). In contrast, high P-low Ca lambs were fed a basal diet low in Ca and high in P consisting of wheat bran (1- to 2-mm particle size) ad libitum and 400 g of alfalfa pellets. These diets were chosen due to their contrasting concentrations of Ca and P. Given the low concentrations of Ca in wheat bran (Table 1 and NRC, 1985Go), we added a source of Ca (alfalfa pellets) to the basal diet to avoid severe hypocalcemia.

Lambs were offered the low P-high Ca or the high Plow Ca diets from 0900 to 1300. At 1300 refusals were collected and weighed. No other feed was offered until the next day. We began feeding the unbalanced diets on July 31, 2005 (d 1) and ended on November 18, 2005 (d 111; Table 2Go).

To determine the status of P and Ca in the 2 groups of lambs, jugular blood samples were taken from each animal on September 8 (d 40), October 5 (d 67), October 19 (d 81), and November 17 (d 110). Concentrations of Pi and Ca in the blood are good indicators of phosphate and calcium deficiency in ruminants (Denton et al., 1986Go; Miller et al., 1987Go; Underwood and Suttle, 1999Go).

Conditioning
After 73 d of exposure to the low P-high Ca or the high P-low Ca diets, lambs were conditioned with flavored grape pomace containing NaCl, CaCO3, or NaH2PO4 during 3 consecutive 2-d periods between October 12 and 17, 2005 (Table 2Go). Lambs received the NaCl + coconut treatment in the first conditioning period, such that they would experience the "salty taste" of NaCl before they received Ca or P supplements. Treatment sequences for conditioning Periods 2 and 3 were chosen at random and were CaCO3 + onion-, and NaH2PO4 + maple-flavored grape pomace, respectively. Each day from 0700 to 0900, flavored pomaces + minerals were offered, and subsequent refusals were collected and weighed. Lambs were offered 150 g of flavored grape pomace with added NaCl (period 1), CaCO3 (period 2), or NaH2PO4 (period 3), which supplied approximately 95, 42, and 67% of the lambs’ Na, Ca, and P daily requirements, respectively (NRC, 1985Go). After conditioning, the BW of the lambs was 34 ± 0.7 kg.

Group Separation
Lambs belonging to the 2 groups were randomly distributed in individual adjacent pens, and thus lambs fed the low P-high Ca diet had neighbors fed the high P-low Ca diet and vice versa. Individual pens were built using panels of steel rod mesh (20.5 x 14.5 cm), and we noted that the lambs licked and chewed the dirt not only in their own pens but also in their neighbors’ pens. We also saw lambs eating feces and licking urine while their neighbors were urinating in the adjacent pens. As a result, concentrations of P in serum were not declining as fast and steadily as in a previous study (Villalba et al., 2006aGo), when the lambs were exposed to a diet low in P and high in Ca, similar to the basal diet used in the present study. Thus, after 87 d (October 25, 2005) of feeding the basal diets in individual pens, lambs in the 2 groups were moved to 2 new and separate locations and housed in pens (Table 2Go) where urine or feces had not been deposited on the ground before the experiment, and lambs belonging to one group could not eat soil, feces, or urine from the other pen. Group pens were under protective roofs and were separated by 20 m.

Lambs remained in the separated groups from d 88 to 111. On d 107, animals were again moved to individual and adjacent pens to conduct preference tests during d 110 and 111, but this time the 2 groups remained at separate locations. Throughout the group separation, lambs continued to receive their respective diets: low P-high Ca and high P-low Ca. The amount of feed offered during group feeding represented an average of the amount of feed consumed by lambs in the individual pens during d 81 to 87, with low P-high Ca lambs offered 900 g/lamb daily and high P-low Ca lambs offered 730 g/lamb daily.

Preference Tests
Preference tests were conducted during 4 consecutive 2-d phases (Table 2Go). During the tests, lambs were offered NaCl + coconut-, NaH2PO4 + maple-, and CaCO3 + onion-flavored grape pomace simultaneously for 15 min at 1000, and intake of each feed was measured. From 1030 to 1300, all lambs were offered their respective basal diets. Tests for phases 1 and 2 were conducted 40 (d 40 to 41) and 67 (d 67 to 68) d after we began feeding the unbalanced basal diets. Tests for phase 3 were conducted the day after conditioning ended (d 81 to 82), whereas tests for phase 4 were conducted after the groups were separated and placed into the different locations (d 110 to 111). During the preference tests, both groups continued to receive their respective diets every day, as described previously (Table 2Go). During d 1 of each phase and 1 h before the preference tests, jugular blood was obtained from each lamb to determine serum Pi and Ca.

Chemical Analyses
Feed Mineral Determinations.
Representative samples of trace mineral blocks, grape pomace, flavored grape pomace, and low P basal diets were analyzed for mineral content. Briefly, 0.5 g of each diet material was digested in 9.5 mL of trace mineral grade, nitric acid (Fisher Scientific, Pittsburgh, PA) in 50-mL, Oak Ridge, Teflon digestion tubes (Nalge Nunc International, Rochester, NY). Samples were digested for 2 h at 90 C. After digestion, samples were brought to a final volume of 10 mL with trace mineral grade, nitric acid. Then, 0.5 mL of the digest was added to 9.5 mL of 18.3 mohm water in 15-mL polypropylene, trace-metal–free tubes (Elkay, Mansfield, MA). This provided a 5% nitric acid matrix for the analysis, which was matrix-matched for all standard curve and quality control samples. Mineral content analysis was performed using an ELAN 6000 inductively coupled plasma mass spectrometer (ICP-MS, Perkin Elmer, Shelton, CT). Five-point standard curves from 0.01 to 0.50 mg/L were used to quantify the minerals. Sequential 1:10 (vol/vol) dilutions were made using 5% nitric acid for minerals exceeding the standard curve. Standard curves and quality control samples were analyzed for every 5 samples. National Institute of Standards and Technology standards were analyzed to verify accuracy of the analytical results. Assay sensitivities for mineral determinations were 0.00001% for Fe; 0.0001% for Ca, P, Na, and K; and 0.001% for Mg. The CV for accuracy and repeatability of the assays was less than 5%.

Blood Determinations.
Blood Ca and Pi values were assessed throughout the study to monitor their evolution as a function of the diets fed (balanced and unbalanced rations) and conditions during feeding (animals from different groups in adjacent pens vs. group separation). Blood samples collected by jugular venipuncture were put in 10-mL vacuum tubes (Fisher Scientific). After allowing the blood to clot for 1 h, the samples were centrifuged for 20 min at 1,100 x g for final separation, and serum was pipetted into trace mineral-free, 15-mL centrifuge tubes. Inorganic P and Ca analyses were performed at the local hospital (Logan Regional Hospital) by the dry slide techniques of Vitros Products Chemistry (Ortho-Clinial Diagnostics, Rochester, NY). Calcium and P analyses had an analytical sensitivity of 0.1 mg/dL, and the CV for within and between days was less than 5%.

Statistical Analyses
Feed intake during conditioning and intake and preference during preference tests were analyzed as split-plot designs with lambs nested within groups. Group (low P-high Ca; high P-low Ca) was the between-animal factor; and feed offered during conditioning [1) NaCl + coconut-, 2) CaCO3 + onion-, 3) NaH2PO4 + maple-flavored grape pomace], day (1–2), and phase (initial preference tests, phases 1–4) were the within-animal factors in the analyses. Day (1 to 2) was the repeated measure. Intake of the basal diets and serum Pi and Ca values were analyzed as a split-plot design. Group (low P-high Ca; high P-low Ca) was the whole-plot factor, assigned randomly to lambs. Day (basal diets) or phase (serum determinations) was the sub-plot.

Analyses were computed using the MIXED procedure (SAS Inst. Inc., Cary, NC). Means were compared using the LSD test. Intake during preference tests was subjected to square root [sqrt (intake)] transformations to better meet the assumptions of normality and homogeneity of variance.


    RESULTS
 Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Test Diets
Averaged across days, lambs consumed 733 ± 44 g (as fed) of the low P-high Ca diet and 1,065 ± 44 g (as fed) of the high P-low Ca diet (group effect; P < 0.001). Intake fluctuated for both groups across days, which caused a day effect and a group x day interaction (P < 0.001; Figure 1Go).


Figure 1
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Figure 1. Consumption of a low P-high Ca (97% beet pulp, 2.0% urea, and 1% calcium carbonate; as-fed basis) and high P-low Ca basal (wheat bran + 400 g of alfalfa pellets) diets by 2 groups of lambs. After 73 d of exposure to the diets, lambs were conditioned by offering flavored grape pomace containing NaCl (d 74 to 75), CaCO3 (d 76 to 77), or NaH2PO4 (d 78 to 79). Values are means for 10 animals/group; SE are represented by the vertical bars.

 
Conditioning
Intake of flavored grape pomace with added minerals did not differ (P > 0.27) between groups or among feeds. Lambs in low P-high Ca consumed 110 ± 10, 106 ± 10, and 120 ± 10 g, whereas lambs in high P-low Ca ate 124 ± 10, 112 ± 10, and 115 ± 10 g of NaCl + coconut-, CaCO3 + onion-, and NaH2PO4 + maple-flavored grape pomace, respectively (group x feed interaction; P = 0.26). Thus, intake of pomace, and consequently amount of exposure to the flavors and minerals in the test feeds, was similar between groups and across different conditioning periods.

Preference Tests
Lambs in both groups did not differ (P > 0.62) in initial intake or preference for flavored pomace containing NaCl, CaCO3, or NaH2PO4 (Figure 2Go). However, as testing progressed, lambs in both groups differed in their preference for flavored grape pomace with added minerals (group x feed x phase interaction; intake: P = 0.10; preference: P = 0.02; Figure 2Go). During phase 1, lambs in high P-low Ca ate NaCl > CaCO3 = NaH2PO4 (P < 0.001), and both groups preferred NaCl > CaCO3 = NaH2PO4 (P < 0.001; Figure 2Go). During phase 2, lambs offered the high P-low Ca basal diet continued to prefer flavored pomace with NaCl over the alternative feeds (P < 0.001), but lambs offered the low P-high Ca basal diet had the greatest preference for NaH2PO4 (P < 0.05; Figure 2Go). After conditioning during phase 3, when all animals ate each flavored feed with added salts, both groups preferred NaCl = NaH2PO4 > CaCO3 (P < 0.01 to 0.11; Figure 2Go). After groups were moved to separate locations in phase 4, lambs fed the low P-high Ca basal diet had the lowest concentration of Pi in serum for all phases and groups (see below) and preferred NaH2PO4 > NaCl = CaCO3 (P < 0.001; Figure 2Go). In contrast, lambs fed the high P-low Ca basal diet avoided NaH2PO4 (P < 0.05; Figure 2Go); they tended to prefer CaCO3 (P = 0.12), and they had a greater preference for NaCl (P < 0.05) than did lambs fed the low P-high Ca basal diet.


Figure 2
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Figure 2. Intake and preference for flavored grape pomace containing CaCO3 (Ca), NaCl (Na), or NaH2PO4 (P), and changes in serum concentrations of inorganic-P (Pi) and Ca, of 2 groups of lambs. Preference tests were conducted when all lambs were fed a basal diet of alfalfa pellets and rolled barley grain (initial preference) and during 4 phases, where the lambs in group 1 had a basal diet low in P and high in Ca (low P-high Ca; 97% beet pulp, 2.0% urea, and 1% calcium carbonate; as-fed basis), and lambs in group 2 had a basal diet high in P and low in Ca (high P-low Ca; wheat bran + 400 g of alfalfa pellets). Before phase 3, the lambs were conditioned by offering flavored grape pomace containing NaCl, CaCO3, and NaH2PO4. Twenty-two days before phase 4, the groups were moved to separate locations. Intake and preference for flavored pomace did not differ during initial preference tests, but as the study progressed preference for flavored pomace with NaCl, Values are means for 10 lambs/group; SE are represented by the vertical bars.

 
Serum Determinations
Serum Pi.
Lambs in low P-high Ca had lower Pi concentrations than did lambs in high P-low Ca (group effect; P < 0.001; Figure 2Go). However, Pi concentrations did not differ (P = 0.92) between groups initially (i.e., during initial preference test), before groups started to receive their respective basal diets.

Compared with the basal diet of alfalfa and barley grain, lambs in the low P-high Ca group had lower serum Pi concentrations in phases 1 and 2 (d 40 and 67; P < 0.05; Figure 2Go). In contrast, serum Pi values increased from phase 2 to phase 3 after conditioning with flavored pomace + minerals (P < 0.05). After the 2 groups were separated (phase 4) so they could not eat dirt, urine, and feces from neighbors belonging to a different group, lambs in low P-high Ca showed the lowest (3.7 mg/dL) concentration of Pi in serum for all phases (P < 0.001). This sharp decline occurred 29 d after lambs had marginally deficient serum values (6.3 mg/dL, phase 3; Figure 2Go). For ruminants, marginally deficient values range from 3.9 to 5.4 mg/dL (Underwood and Suttle, 1999Go).

Serum Pi increased for lambs in high P-low Ca during phase 1 relative to when the basal diet was alfalfa and barley grain (P < 0.001; Figure 2Go). After conditioning, serum Pi values increased relative to all other phases (P < 0.001; phase 3).

Serum Calcium.
Lambs in high P-low Ca had lower Ca concentrations than lambs in low P-high Ca (group effect; P < 0.0001; Figure 2Go). However, Ca concentrations did not differ (P = 0.22) between groups until they began to receive their respective and contrasting basal diets following the initial preference test. During phase 3, serum Ca declined for both groups of lambs (low P-high Ca: P < 0.05; high P-low Ca: P = 0.16). In phase 4, after groups were moved to 2 separate locations, lambs in low P-high Ca had the greatest (11.7 mg/dL) concentration of Ca in serum for all phases (P < 0.001; Figure 2Go).


    DISCUSSION
 Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Discrimination Among Flavored Feeds with NaCl, CaCO3, or NaH2PO4
Although minerals frequently co-occur in nature, and thus requirements for minerals may be satisfied by searching for a "salty taste" (Schulkin, 1991Go), we hypothesized that sheep discriminate among feeds containing Na, P, and Ca and modify their feed choices in accordance with the benefit supplied by each element relative to mineral deficits in their basal diet. On that basis, we predicted that if ingestion of a specific supplement were followed by recovery from a mineral deficiency such that the concentration of that mineral in blood increased, then animals would prefer that supplement while experiencing the mineral deficiency. We determined whether sheep preferred flavored supplements containing NaCl, CaCO3, or NaH2PO4 as a function of the deficiency/unbalance in their basal diets: low P-high Ca or high P-low Ca. CaCO3, or NaH2PO4 diverged for the 2 groups (Figure 2Go). Lambs discriminated among the 3 feeds, even when they all contained the same filler (grape pomace) and had salts added at the same molar concentrations. Intake of flavored pomace was similar during different conditioning periods, which supports the notion that the amount of exposure to the flavors was similar for both groups of lambs and for the different feeds containing NaCl, CaCO3, or NaH2PO4. Different degrees of exposure to feeds could have influenced preference, because increased exposure tends to enhance preference (Pliner, 1982). Thus, our results suggest that lambs discriminated among specific flavors associated with the postingestive effects of NaCl and NaH2PO4, consistent with previous findings (Villalba et al., 2006aGo). Sheep also discriminated among 3 flavors associated with ruminal infusions of starch, casein, and water (Villalba and Provenza, 1999Go), and among the benefits of ingesting sodium bicarbonate, polyethylene glycol, and sodium bentonite when fed diets high in grain, tannins, and oxalates, respectively (Villalba et al., 2006bGo). Taken together, these findings suggest that sheep make flavor-feedback associations that enable them to discriminate among multiple internal states and select diets according to needs (Provenza, 1995Go; Provenza and Villalba, 2006Go).

Rodents and ruminants respond to mineral deficits. Calcium-deficient rats associate flavor ingestion with the postingestive benefits of consuming Ca (Tordoff, 2002Go) and prefer CaCl2 over MgCl2 or NaCl (Leshem et al., 1999Go). Phosphorus-deficient rats prefer KH2PO4 when offered in a choice with CaCO3, even after only 2 d of dietary P restriction (Sweeny et al., 1998Go). Likewise, P-deficient sheep decrease intake and preference for flavors associated with intraruminal infusions of NaH2PO4 when serum concentrations of Pi are greatest, and increase intake and preference for P as serum concentrations decline (Villalba et al., 2006aGo).

Preference for Flavored Feeds with NaCl, CaCO3, or NaH2PO4
If supplemental minerals are needed to promote optimal animal performance, what is the best way to provide them: free choice or in the diet? The answer to this question is controversial. Mineral supplements offered free choice assumes that animals will consume enough to meet their requirements (Coppock et al., 1972Go).

In a recent study, P-depleted sheep showed a modest preference for flavors associated with intraruminal infusions of NaH2PO4 (Villalba et al., 2006aGo). During conditioning, lambs received fixed intraruminal infusions of NaH2PO4 and thus they could not regulate P intake. The doses of P likely were too high for some animals, which could have created mild to strong food aversions rather than preferences (Provenza, 1996Go). As a critical change in design in the current study, we offered minerals in flavored supplements free choice so that animals could regulate their intake of Na, Ca, and P during conditioning and preference tests, thereby making aversions to excessive doses of minerals less likely (Table 2Go).

Animals were familiarized with the mineral supplements and their postingestive effects during adaptation (adaptation to flavored grape pomace + minerals). We offered choices of supplements before conditioning, and 40 and 67 d after we started feeding the unbalanced basal diets (phases 1 and 2) to determine whether animal preferences for the flavored mineral supplements changed based on their short experiences with the supplements during adaptation. Preference tests during phase 2 (d 67 to 68) support this hypothesis. Lambs offered the high P-low Ca basal diet preferred flavored pomace with NaCl over the alternative feeds, whereas lambs offered the low P-high Ca basal diet showed the greatest preference for NaH2PO4 (Figure 2Go).

Preference tests after groups were separated (phase 4) suggest that the learned preference was specific for Ca and P. Lambs fed the low P-high Ca basal diet clearly preferred NaH2PO4 > NaCl = CaCO3. In contrast, lambs fed the high P-low Ca basal diet avoided NaH2PO4 (Figure 2Go). Lambs offered the low Ca basal diet showed a greater preference for CaCO3 and NaCl than lambs fed the low P basal diet. Moreover, when lambs fed the low P basal diet had serum levels above the marginal values recommended for ruminants, their preference for NaH2PO4 did not differ from preference for NaCl, a pattern that did not differ from lambs fed the high P-low Ca basal diet (Figure 2Go). The preference for NaH2PO4 by lambs fed the low P-high Ca diet may also represent avoidance of CaCO3 due to the excess Ca fed in the basal diet. However, if sheep avoided only CaCO3 they should have manifest similar preferences for NaH2PO4 and NaCl, but their preference for NaH2PO4 was significantly greater than that for NaCl. This is consistent with the finding that dietary Ca is extensively excreted in feces at relatively little cost to the animal (Underwood and Suttle, 1999Go), such that acquiring preferences for Ca during deficits may be more important than avoiding excesses. Likewise, lambs fed the high P diet likely excreted greater concentrations of P than lambs fed the low P diet, because excess intake of P increases P excretion in urine (Underwood and Suttle, 1999Go).

Lambs fed the high P-low Ca basal diet had a greater preference for CaCO3 and NaCl than lambs fed the low P-high Ca basal diet, but their preference values were low (Figure 2Go), which may reflect a mild Ca deficiency. Their preference for Na likely was due to changes in various physiological processes. Rats deprived of Ca and then given a choice between the chloride salts of Na, K, and Ca, initially prefer Na (Schulkin, 2001Go). Small increases in plasma Na concentrations release bound Ca into the ionized pool, which temporarily reduces the severity of Ca deficiency and likely reinforces a preference for Na (Tordoff, 2001Go). This pattern may explain similar intakes and preferences for NaCl and CaCO3 in lambs fed the low Ca basal diet. Moreover, Ca-deficient rats avoid P (Schulkin, 1991Go), and thus in addition to the high levels of P ingested, the pattern of preference by lambs fed the low Ca-high P diet may also represent an avoidance of NaH2PO4. In contrast, preference for NaCl in rats is unaltered by P deprivation (Tordoff, 1992Go). Preferences for flavors associated with NaCl did not increase and in some phases they declined in P-deprived lambs (Villalba et al., 2006aGo). Because lambs had free access to mineral blocks with NaCl throughout the study, they did not need to consume supplemental NaCl from the test feeds. When the need for NaCl is met, lambs (Villalba and Provenza, 1996Go) and rats (Revusky et al., 1971Go) avoid flavors associated with infusions of NaCl.

Generalist herbivores manifest partial preferences such that when given a choice they select a variety of food items (Provenza, 1996Go). In our experiment lambs always consumed some of each of the foods available during a choice, even though they manifested clear preferences for specific minerals (Figure 2Go). Moreover, preference tests were conservative, because all supplements were formulated with the same base feed (grape pomace), and animals could have generalized preferences/aversions based on similar flavors in the feeds (Launchbaugh and Provenza, 1993Go). However, lambs discriminated among supplements and preferred the ones that rectified their imbalances; they did not select randomly.

Serum Minerals and Consumption of Nonfood Items
Experience with the supplements during adaptation occurred while animals were fed a mineral-balanced diet (alfalfa-barley) and while they had serum P and Ca concentrations within normal ranges. Although serum concentrations of P and Ca changed subsequently during phases 1 and 2, when lambs were in adjacent pens, they were within near-normal ranges, even though lambs in low P-high Ca had somewhat lower serum Pi and greater Ca concentrations than lambs in high Plow Ca (Figure 2Go). High concentrations of dietary Ca reduce plasma Pi because P retention by the skeleton increases in response to Ca supplementation (Underwood and Suttle, 1999Go). However, serum P for lambs in low P-high Ca declined to only 5.7 (phase 1) and 5.4 mg/dL (phase 2; Figure 2Go), values marginal for ruminants (3.9 to 5.4 mg/dL; Underwood and Suttle, 1999Go); serum Ca for lambs in high P-low Ca declined to only 9.3 (phase 1) and 9.2 mg/dL (phase 2), values in the normal range for Ca (8.8 to 11.6 mg/dL; Underwood and Suttle, 1999Go).

When the groups were moved to separate locations, serum Pi concentration in lambs fed the low P-high Ca diet declined 43%, from 6.5 (phase 3) to 3.7 mg/dL (phase 4) in only 24 d (Figure 2Go). In contrast, when both groups were at the same location serum Pi concentration declined only 31%, from 7.8 (phase 1) to 5.4 mg/dL (phase 3) in 81 d. In a previous study, when lambs were fed a P-deficient diet similar to that fed in the present study, serum Pi concentration declined from 7.5 to 4.4 mg/dL (41% decline) in only 47 d (Villalba et al., 2006aGo). The rapid decline in serum P concentration when groups were separated relative to when they were at the same location in this and previous studies suggests that lambs in the low P-high Ca diet were obtaining P from neighbors under the high P-low Ca diet before group separation took place. Lambs fed the low P-high Ca basal diet also showed the greatest concentration of Ca in serum for all phases (Figure 2Go) likely due to their inability to seek additional sources of P when offered the diet low in P and high in Ca. When both groups were at the same location, we observed P-deficient animals eating dirt, feces, and urine-soaked soil from neighboring pens, which likely diminished the severity of the P deficiency.

Evidence of a specific appetite for substances that contain P in P-deficient livestock ranges from ingesting soil and bones (Blair-West et al., 1992Go) to the unspecific ingestion of nonfood items. Several accounts, dating back to 1785, suggest that cattle eagerly seek and eat bones while experiencing a phosphorus deficiency (see Sweeny et al., 1998Go). Green (1925)Go described the "perverted appetite" or pica when cattle avidly chew on skeletal bones after grazing on phosphate-deficient soils (osteophagia), a behavior that can also take a generalized form known as allotriophagia characterized by animals eating nonfood items (Green, 1925Go; Tordoff, 2001Go). In our study, lambs’ "perverted appetites" led to ingestion of nonfood items from neighbors’ pens such as dirt and feces, which in turn moderated the decline in serum P concentration. Thus, our observations and findings indicate that "perverted appetites" or picas in herbivores are adaptive behaviors, not aberrant behaviors (Provenza and Villalba, 2006Go). Indeed, they provide a mechanism of specific mineral gathering in times of mineral shortage.

Our current results, which suggest that lambs discriminated among the different minerals offered in the choice, differ from earlier studies that did not take into account past experiences or the contingencies necessary for animals to learn specific flavor-feedback associations (Provenza and Villalba, 2006Go). For instance, sulfur-deficient lambs had to discriminate among 4 anions (CO32–, PO42–, SO42–, Cl) that all contained Na (Pamp et al., 1977Go). Animals generalize among substances that share a common flavor (Na), thus making it difficult to discriminate on the basis of similar flavors and excessive feedback from Na. Moreover, previous studies have used calcium phosphate to test preferences for Ca or P in livestock (Coppock et al., 1976Go) probably because calcium phosphate is the most common source of Ca and P for supplementing livestock. However, calcium phosphate is not the best option to test for preferences when animals are fed imbalanced diets with adequate Ca and restricted P or vice versa. Calcium deficiency leads to P avoidance (Schulkin, 1991Go), because blood Pi tends to increase during Ca deficiency (Underwood and Suttle, 1999Go). Thus, to get the element animals need from dicalcium phosphate (e.g., Ca) they have to over-ingest the element they do not need (e.g., P).

Collectively, our results show that lambs discriminated among different flavored feeds containing NaCl, CaCO3, and NaH2PO4 and displayed preference patterns as a function of the mineral imbalance present in their basal diets (Figure 2Go). Differences in preference were more pronounced as lambs fed the low P diet were separated from those fed the high P diet, which caused a sharp decline in the concentrations of serum Pi by lambs fed the low P diet. The ability of sheep to prefer/avoid supplements containing Ca and P implies that it may be possible to offer these supplements free choice so each individual can meet its specific need. Strategic distribution of Ca- and P-containing supplements, in areas where Ca and P are known to be limiting, may also influence grazing pressure and attract herbivores to underutilized areas, such as those remote from water, enabling more uniform use of forage.


    Footnotes
 
1 This research was supported by grants from the Utah Agricultural Experiment Station and the Initiative for the Future of Agriculture and Food Systems, USDA (Agreement No. 2001-52103-11215). This paper is published with the approval of the Director, Utah Agricultural Experiment Station, and Utah State University, as journal paper number 7878. Back

2 We thank T. Lyman for technical support and J. B. Taylor for important suggestions to improve the manuscript. Back

3 Corresponding author: villalba{at}cc.usu.edu

Received for publication March 27, 2007. Accepted for publication December 6, 2007.


    LITERATURE CITED
 Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 


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