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. 2021 Oct 17;99(11):skab291. doi: 10.1093/jas/skab291

Grazing diverse combinations of tanniferous and nontanniferous legumes: implications for foraging behavior, performance, and hair cortisol in beef cattle

Sebastian Lagrange 1,2,✉, Jennifer W MacAdam 3, Bryan Stegelmeier 4, Juan J Villalba 1
PMCID: PMC8575690  PMID: 34657159

Abstract

A diversity of forages with different types and concentrations of nutrients and plant secondary compounds may lead to complementary relationships that enhance cattle performance and welfare. We determined whether grazing combinations of tanniferous legumes (Lotus corniculatus, birdsfoot trefoil [BFT], Onobrychis viciifolia, sainfoin [SF]), and alfalfa [ALF] (Medicago sativa) influence foraging behavior, performance, and hair cortisol concentration in beef cattle compared with grazing the same legumes as monocultures. Twenty-one pairs of heifers grazed three spatial replications of seven treatments: monocultures of BFT, SF, or ALF, and all possible two- and three-way choices among strips of these legumes: SF-BFT, ALF-BFT, ALF-SF, and ALF-SF-BFT in two periods of 25 d each (adaptation phase + experimental period) during two consecutive years. The lowest incidence of grazing events occurred in the BFT treatment (42.0% of the total scans recorded; P < 0.10), with the rest of the treatments ranging between 47.8% (SF-BFT) and 52.6% (ALF-SF) of the total scans recorded. Heifers selected a varied diet, preferring SF over BFT or ALF in a 46:27:27 ratio for the three-way choice, and in a 70:30 ratio for both two-way choices. Heifers preferred BFT over ALF (62:38 ratio) in a two-way choice. All treatments followed similar daily grazing patterns (P > 0.10), with two major grazing events (1 h after sunrise and 3 h before dark). No differences among treatments were observed for the number of steps taken by heifers on a daily basis, motion index, or the percentage of time heifers spent standing (1,599, 5,356, and 45.3%, respectively; P > 0.10), suggesting that heifers on choice treatments did not invest extra time in walking, searching, or patch switching activities relative to heifers grazing monocultures. Heifers grazing the three-way choice gained more body weight (1.27 kg/d) than the average gains observed for animals grazing in all legume monocultures (1.00 kg/d; P = 0.014) or two-way choices (0.97 kg/d; P = 0.007), suggesting a synergism among pasture species for the treatment with the highest diversity. No differences in hair cortisol concentration were observed among treatments, with values ranging between 1.4 (BFT) and 2.12 ng/g (three-way choice; P > 0.10). Thus, forage diversity has the potential to enhance animal performance without affecting grazing efficiency, likely explained by the spatial arrangement of the forage species presented in the study.

Keywords: condensed tannins, choice, feeding pattern, forage diversity, one welfare, preference

Introduction

Legume-based finishing systems take advantage of the unique ability of ruminants to utilize significant amounts of plant fiber for energy and the high nutritional quality of legumes relative to grasses (Van Soest et al., 2018). Nevertheless, monocultures of legumes like alfalfa (ALF; Medicago sativa L.) impose limitations to production in part caused by the risk of bloat and by the inefficient use of nitrogen due to imbalances in the ratio of nitrogen to energy commonly observed in these species (Wang et al., 2012). One solution to this problem involves offering a diversity of forages with different types and concentrations of biochemicals (e.g., protein, nonfibrous carbohydrates, and plant secondary compounds such as condensed tannins; CT) that promote complementary relationships among multiple feed resources that improve animal fitness (Provenza et al., 2003) and reduce carbon and nitrogen (N) footprints (Patra and Saxena, 2010). For instance, the use of ALF in association with tanniferous legumes like birdsfoot trefoil (BFT; Lotus corniculatus), or sainfoin (SF; Onobrychis viciifolia) overcomes the problem of excessive ruminal protein degradability, which reduces urinary N excretions and improves N retention in sheep and cattle (Lagrange and Villalba, 2019; Lagrange et al., 2020a).

In addition to the aforementioned benefits, forage diversity provides animals with varied sensorial and postingestive stimuli that increase the motivation to eat (Villalba et al., 2011). Herbivores grazing monocultures of single species satiate on the orosensorial characteristics of single feeds due to transient food aversions caused by flavors, nutrients and toxins ingested too frequently or in excess, and satiety can be stressful (Provenza, 1996). Consistent with this notion, a monotonous diet offered to sheep in confinement increase plasma cortisol levels relative to animals fed a diversity of feed items (Villalba et al., 2012). However, if diverse options are available, animals may continue responding to other orosensorial or postingestive stimuli, achieving an adequate state of nutrition based on their individual and changing needs (Provenza et al., 2003). Thus, forage diversity contributes to enhanced animal welfare because generalist herbivores are less likely to experience stressful situations, like frustration due to lack of food alternatives available to build a balanced diet, or satiety due to repeated or excessive exposure to the same single feeds (Catanese et al., 2013).

Finally, the level of spatial aggregation of forage species in diverse systems, ranging from uniform mixes to separated swards may influence ingestive behavior and performance in ruminants (Chapman et al., 2007). In a finely intermingled mix pasture, animals may have to search for and handle the preferred plant species; these time-consuming activities may reduce intake rate relative to grazing monocultures (Prache et al., 1998). On the other hand, spatial segregation of plant species into patches may reduce the time animals need to select and handle desired amounts of specific forages, while overcoming many agronomic difficulties inherent in establishing and maintaining mixed swards (Chapman et al., 2007). There is a gap in knowledge regarding the potential complementarity among patches of legumes of different chemistries and their potential associative effects on beef production systems. Therefore, the aim of this study was to evaluate the synergistic effect of offering increasingly diverse combinations of tanniferous (BFT; SF) and nontanniferous (ALF) legumes on foraging behavior, animal performance, and welfare in grazing cattle during the finishing process during a study where animal environmental impacts were previously assessed (Lagrange et al., 2020a). Preliminary results have been published in an abstract form (Lagrange et al., 2020b).

Materials and Methods

The study was conducted at the Utah State University irrigated pasture research facility in Lewiston, UT (41 56′ N 111 52′W, 1382 m altitude) from June 21 to September 2 in 2016 and from June 5 to August 23 in 2017. All procedures herein described were previously approved by the Utah State University Institutional Animal Care and Use Committee (approval number 2566).

Pastures and experimental design

Three contiguous fields (blocks) containing seven pasture treatments were established on irrigated land in September 2015. Treatments included monocultures of two tanniferous legume species: (1) SF (Onobrychis viciifolia “Shoshone”; SF) and (2) BFT (Lotus corniculatus “Langille”; BFT), (3) the nontanniferous legume ALF (Medicago sativa “Vernal”; ALF), and all two- and three-way choices among these legumes presented in strips: (4) ALF-SF, (5) ALF-BFT, (6) SF-BFT, and (7) ALF-SF-BFT. All treatment plots had an area of 0.5 ha each, and were randomly distributed within each block. For monocultures, the entire 0.5 ha was planted to a single species; for choice treatments, there were either two 0.25-ha strips ~30 m wide × 82 m long, or three 0.165-ha strips of 20 m wide × 82 m long; strips within each block were randomly assigned to ALF, SF, or BFT, depending on treatment. Thus, in each two- and three-way choice plot, cattle could freely graze on any of the two or three species on offer. The perimeters of the experimental plots were defined by electric fence.

Animals and grazing protocol

Pastures were grazed during two periods (P1 and P2) in two consecutive years, 2016 and 2017. During each year, a different set of 42 Angus heifers were sorted by body weight (BW), and distributed among seven groups of six animals with similar total weight per group. Groups were randomly assigned to the seven treatments. Heifers within treatments were grouped in pairs (n = 3) with similar individual weight and each pair was randomly assigned to one of three treatment replications (blocks). The heifers’ initial and final mean BW was 394 ± 54 kg, and 436 ± 55 kg, respectively, for 2016, and 352 ± 40 and 421 ± 42 kg, respectively, for 2017.

Prior to the experimental periods, animals were adjusted to their respective diet treatments in a 10-d adaptation phase. Experimental period 1 of 2016 (18 d) occurred from June 30 to July 18, and P2 from August 18 to September 2 (15 d). During 2017, P1 occurred from June 15 to June 28 (14 d), and P2 from August 10 to August 23 (14 d). Samples were collected during five consecutive days at the end of each experimental period (collection period).

During 2016, the first growth cycle for all pastures had some forbs and weeds. In order to work with pure stands, BFT, ALF, and SF pastures were mowed on May 19. Subsequently, at the beginning of P1 (June 30), BFT and ALF swards were in the early bloom stage of their second growth cycle, whereas SF swards were in the full bloom stage. On August 18 (P2), ALF and BFT’s third growth cycle was in late bud to early bloom stage, whereas SF (with a slower regrowth) was in the late vegetative to early bud stage. During 2017, the first growth cycle of all legumes was not contaminated with weeds, and thus the first growth was not mowed. Birdsfoot trefoil and ALF were grazed at full bloom stage and SF at the early seed pod stage, beginning June 15. On August 10 (P2), ALF and BFT regrowth was in full bloom but SF was at the late bud and early flowering stage. All pastures were irrigated once in 2016 and twice in 2017 in-between grazing periods (July 19 to August 7; and June 29 to July 30 for 2016 and 2017, respectively), using hand-line sprinklers in 12-h sets that applied ~105 mm of water every 2 wk. Considering rainfall and irrigation, all pastures received 523 and 777 mm of water during the growing season of 2016 and 2017, respectively.

Heifers strip-grazed their respective plots behind electric fences that were moved approximately every 3 d to give access to fresh forage, and back-fenced to prevent access to previously grazed forage and allow legumes to re-grow. The area of each new paddock offered to all animals during the 3-d period was ~550 m2 during both years of the study. In P2, heifers grazed legumes that had regrown for ~45 d. Heifers were moved to a new section of the same treatment once they grazed 20% to 30% of the initial available biomass for monocultures, or when any of the legume strips was grazed to that extent in two- or three-way choice treatments. This procedure ensured ad libitum forage availability for all the species present in each treatment.

Throughout the adaptation and sample collection phases, animals had free access to water and trace-mineral salt blocks (mineral composition: minimum 960 g/kg NaCl, 320 mg/kg Zn, 380 mg/kg Cu, 2,400 mg/kg Mn, 2,400 mg/kg Fe, 70 mg/kg I, and 40 mg/kg Co). Animals on all treatments had access to bloat protectant blocks with Poloxalene 6.6% (Sweetlix Pressed Bloat Guard, Ridley USA Inc., Mankato, MN) for 2 d before entering the adaptation phase in order to reduce the likelihood of frothy bloat in animals that were assigned to ALF. Heifers did not have access to any supplemental bloat protectants during the adaptation phase or experimental periods.

Herbage biomass

Herbage biomass in each plot was assessed as described by Lagrange et al. (2020a). Forage biomass was determined before (pregrazing herbage biomass) and after (postgrazing herbage biomass) animals entered their respective paddocks. Measurements involved taking 60 readings per forage species from each paddock using a rising plate pasture meter (Electronic Plate Meter Jenquip EC-10, Agriworks Ltd, NZ) while walking in a “lazy” W pattern. Calibration curves for each species were built from pre- and postgrazing herbage readings by collecting all forage inside a 0.10-m2 quadrant frame, the same area covered by the plate meter, and by drying the forage collected at 60 °C to constant weight. Linear relationships for each experimental period and each species were estimated from calibration curves of dry matter (DM) herbage biomass on plate meter readings.

Forage sampling

Representative samples of the herbage ingested by heifers were collected on day 3 of each experimental period from each forage and replication of each treatment (one sample per pasture strip = 12 samples per block = 36 samples per period). Herbage samples were collected between 1000 and 1300 hours by walking a transect across a pasture section and hand-plucking the top 15 to 20 cm of the sward every few steps, mimicking the plant parts grazed by heifer. This was achieved by relying on previous multiple observations of the animals’ foraging behavior while grazing the experimental pastures. Samples were placed in plastic bags, covered with dry ice, and frozen at –20 °C until they were freeze-dried (Free Zone 18 Liters, Labconco Corporation, Kansas City, MO), and ground to pass the 1-mm screen of a Wiley mill (model 4; Thomas Scientific Swedesboro, NJ) for chemical analyses.

Scan sampling

The foraging behavior of the pair of heifers in each treatment plot was recorded using game cameras (PC800 HyperFire Professional IR, Reconyx Inc., Holmen, WI), and the incidence of feeding on each of the forage species in the choice treatments was then determined. During the experimental period, seven cameras were distributed among the seven treatment plots in a spatial replication (block), allocating a camera at one side of each paddock. Cameras were placed immediately after heifers had access to fresh strips of pasture, and they were kept in the same plot for 48 h in order to capture images of the heifer’s locations and behaviors (see below) with a time-lapse of 5 min intervals. Cameras were active from 0500 (dawn) until 2200 (last light) hours, a period of 17 h. Subsequently, cameras were moved to a different block for the first 48 h on fresh pasture breaks, and then to the third replication for the same amount of time. These rotations continued until the three replicates for each treatment were recorded twice in each one of the experimental periods in 2016 and 2017. Pictures were then visualized individually using Preview version 10.1 (Apple Inc.).

Grazing behavior was recorded using scan sampling (Altmann, 1974) at 5 min intervals during the 17 h observation period. If the heifer was performing any other behavior than grazing, this was scored as “nongrazing.” The incidence of feeding in each treatment was evaluated as the percentage of the total number of scans in which the pair of heifers was feeding during each experimental period relative to the total number of scans recorded (grazing and nongrazing events). In two- or three-way choices, frequency of feeding on each legume (preference) was calculated as a percentage of the number of grazing scans recorded for the pair of heifers on each of the legume species within each treatment relative to the total number of grazing scans recorded for each experimental period.

Behavioral levels of activity

One animal from each of the 21 pairs of heifers used in the study was used for activity measurements and fitted with a pedometer (Icetag3D, IceRobotics, Roslin, UK) on their left rear leg from the beginning of each experimental period, which was removed during the last day of the period. Activity levels and posture (number of steps taken, motion index, lying and standing bouts) were measured with the use of these pedometers which took second-to-second readings throughout the period. The motion index provides a broader measure of the animal’s activity level and complements the step count, considering the magnitude of the 3D acceleration, and as such it is related to the total amount of energy used by the animal over a given period. The calculation is performed per second, and then summed to provide the total activity per minute in G’s/10 (Ice Robotics, 2020). Data were downloaded with the provided IceRobotics software (version 2012) in a format of 1 summary record per day. The percentage of time heifers spent standing and lying were calculated by summing the time in seconds during the day animals spent standing and lying, respectively, and dividing by the total time pedometers were recording data each day. Activity and posture data for each heifer was then averaged over dates within each experimental period.

Average daily gain calculations

Heifers were weighed individually using a load cell scale (Rice Lake weighing systems, Rice Lake, WI) located under the squeeze chute at the beginning and end of each experimental period to estimate average daily gains (ADG). Feed and water were withheld from 1800 hours until the following morning, when animals were weighed at 0900 hours before transfer to pastures.

Hair sampling

Hair samples were taken from one animal of each pair of heifers. Heifers were shaved the first day of the adaptation phase and hair samples were collected on the last day of the experimental period, a hair growth period of 18 and 25 d in 2016 and 21 and 19 d in 2017, for P1 and P2, respectively. The hair samples contained only new black hair grown during each period, and was taken from a 100 cm2 square area on the forehead, as described by Tallo-Parra et al. (2015). Each hair sample was placed into prelabeled zip-lock plastic bags and stored in the freezer until cortisol extraction.

Chemical analyses

Forage samples were analyzed for DM, total N concentration, acid detergent fiber (ADF), acid detergent lignin (ADL), and CT. DM was determined by drying the samples at 105 °C for 3 h in a forced-air drying oven (AOAC, 1990; method 930.04). Total N concentration was analyzed using a Leco FP-528 N combustion analyzer (AOAC, 2000; method 990.03) with crude protein (CP) concentration calculated as N concentration × 6.25. Concentration of ADF was determined according to (AOAC, 2000; method 973.18), modified by using Whatman 934-AH glass micro-fiber filters with 1.5 μm particle retention and a Buchner funnel in place of a fritted glass crucible. Determinations of ADL were modified by Robertson and Van Soest (1981). Analyses of total CT in legume samples were conducted in triplicate, according to the butanol–HCl–acetone spectrophotometric assay of the study by Grabber et al. (2013), using CT isolated from SF and birdsfoot trefoil as the standards.

Cortisol extraction and determinations were determined according to protocols described by Tallo-Parra et al. (2015).

Statistical analyses

Average daily gain was analyzed using a two-way factorial treatment structure (year × period) in a randomized complete block design using a generalized linear mixed model with a normal distribution. Treatment (7; single forage species, two- and three-way combinations), Period (2), Year (2) and all interactions were the fixed factors. Block, Block × Treatment, and Block × Treatment × Year were included in the model as random factors. Hair cortisol concentration was analyzed with the same model but using a Lognormal distribution, and back-transformed LSmeans and SE were reported.

Number of steps per day, daily motion index, and percentage of standing time per day were analyzed using a normal distribution with Period nested within Year, because experimental periods were not performed at the same time in both years and there was a photoperiod shift that may affect the response. Thus, the fixed factors were Treatment, Year and Period (Year), and random factors were Block, Block × Treatment, and Block × Treatment × Period (Year). Similarly, percentage of total grazing scans was analyzed with Period nested within Year but using a binomial distribution with grazing events/total events (grazing and not grazing) as the response variable.

Percentage of grazing scans and standing time at each hour of the day (grazing patterns) were analyzed separately for each experimental period of each year of the study, due to differences in daylight hours at each experimental period affecting these variables. Thus, the generalized lineal mixed model included Treatment and time of the day and their interaction as fixed factors and Block and Block × Treatment were included in the model as random factors. Percentage of standing time per time interval was analyzed using a Logit distribution and back-transformed LSmeans and SE were reported. Percentage of grazing scans per time interval also included Block × Time as random factors and used a binomial distribution which better fitted the nature of the scans data. In order to address overdispersion of the data for the binomial distribution, the residual variance Block × Treatment × Time was also included in this model as a random factor.

Finally, CP, ADF, ADL concentrations in legume species as well as DM forage biomass were analyzed separately for each period and year of the study using a generalized linear mixed model and normal distribution, with Species rather than Treatment as a fixed factor (ALF, SF, or birdsfoot trefoil). Measurements of forage biomass and nutritional quality for each species were taken from four different locations within each block (monoculture, two 2-way choice, and three-way choice treatments), resulting in 12 replicates for each period-year combination. Block and Block × Species were included in the model as random factors. Condensed tannins concentrations for each species were analyzed using a Lognormal distribution and back-transformed LSmeans and SE were reported.

All analyses were computed using PROC GLIMMIX in SAS/STAT 14.2 (SAS Inst., Inc. Cary, NC; Version 9.4 for Windows). Assumptions of homoscedasticity of variance and normality were tested using studentized residuals when analysis used a normal distribution. Least squares means (LSmeans) were compared pairwise using the least significant difference test when the overall test for Treatment effect was significant (P ≤ 0.10). Means were reported along with their standard errors (SEM). Treatment differences were considered a tendency when 0.10 < P ≤ 0.15. Additionally, preplanned contrasts were performed to compare the three-way choice LSmean vs. the average LSmean for the three monocultures treatments or the average LSmean for all two-way choices, using the LSMESTIMATE statement in PROC GLIMMIX. Contrasts were specified as the arithmetic difference between ALF-SF-BFT and (0.33ALF + 0.33SF + 0.33BFT) or (0.33ALF-SF + 0.33ALF-BFT + 0.33SF-BFT), respectively. Contrasts between the average of two-way choices and the average of monoculture treatments were also performed. A difference was considered significant when P-values were of <0.10 and considered a tendency when 0.10 < P ≤ 0.15, given the relatively low sample size/treatment, typical of grazing studies, and the high number of treatments assayed.

Forage preference (percentage of grazing scans recorded for any single species relative to the total number of grazing scans recorded in a choice treatment) was assessed separately for each of the two- and three-way choice treatments. Data were analyzed using a generalized linear mixed model for a two-way factorial treatment structure (Year and Period) in an randomized complete block design with a binomial distribution (y/n: number of grazing scans of any species/number of total grazing scans in the choice). The residual Block × Period × Year was included as random factor in order to address overdispersion. Due to lack of independence of scans data within each treatment, the overall mean percentage of each species in a specific choice treatment was estimated as the average over the 4 yr × period combinations, and reported along with their 90% confidence intervals. A legume species was considered “preferred” or “not preferred” in a specific two- or three-way choice treatment, when the overall mean percentage selected for the legume was higher or lower than 50% or 33%, respectively, and the confidence interval for the mean did not include 50% or 33%.

Results

Nutritional composition of the forages

The average nutritional composition of the legumes used in the study for both years 2016 and 2017 is reported in Table 1. All forage legumes contained high concentrations of CP (20% to 30%; DM basis), low levels of ADF (<25%) and intermediate levels of ADL (3.5% to 5.5%). The nutritional composition of birdsfoot trefoil and ALF was similar in both years of the study, and both legumes showed declines in their concentrations of CP and incremental increases in their concentrations of ADF and ADL from P1 (e.g., late June to early July) to P2 (e.g., middle to late August). In contrast, SF contained the lowest concentrations of CP and the greatest concentrations of ADF and ADL during P1 in both years, although the concentration of ADF was less in P2 regrowth, and contents of CP and ADL became similar to the rest of the legumes assayed.

Table 1.

Nutritional composition (g kg−1 dry matter) of legumes during both periods (P1 and P2) and years (2016 and 2017) of study

Species Period 1 Period 2
Alfalfa Birdsfoot trefoil Sainfoin SEM P-value Alfalfa Birdsfoot trefoil Sainfoin SEM P-value [DF]
2016
CP 289.8aA 264.8bA 217.8c 5.40 0.002 246.7aB 215.2bB 219.9b 6.19 0.044
ADF 176.7b 169.2b 224.5aA 15.05 0.078 175.3a 157.1b 172.3aB 4.39 0.082
ADL 35.6bB 45.0a 50.0a 2.64 0.018 39.9cA 46.2b 52.2a 1.42 0.009
CT 1.5cA ± 0.13 15.0b ± 1.31 47.8aB ± 4.24 <0.001 1.0cB ± 0.10 14.3b ± 1.48 90.5aA ± 9.39 <0.001
2017
CP 275.7aA 287.1aA 218.0b 7.27 0.005 235.4B 248.6B 224.8 11.10 0.302
ADF 183.6bB 146.4cB 256.6aA 9.04 0.002 227.4aA 175.5bA 237.5aB 10.15 0.020
ADL 37.9bB 42.1bB 53.5a 2.07 0.013 51.5A 50.0A 55.0 2.97 0.363
CT 1.5cB ± 0.09 20.3bA ± 1.23 40.8aB ± 2.47 <0.001 1.8cA ± 0.20 17.5bB ± 1.99 61.7aA ± 7.02 < 0.001
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a–cLSmeans in a row with different lower-case superscripts differ within periods (P < 0.10).

A,BLSmeans in a row with different upper-case superscripts differ between periods (P < 0.10). Values are means for three spatial replications (blocks).

Condensed tannin concentration in SF was three- to sixfold (2016), and two- to fourfold (2017) the concentration observed in birdsfoot trefoil for P1 and P2, respectively. Alfalfa is a nontanniferous legume, confirmed by the low levels of CT revealed in the assay (Table 1).

Herbage biomass

Herbage biomass across periods and years ranged from 4 to 8 Mg/ha (Table 2; Lagrange et al., 2020a), with greater values during P1 than during P2 of 2017 for ALF, SF, and birdsfoot trefoil. Forage biomass in ALF and birdsfoot trefoil was greater in 2017 than in 2016 (P = 0.008 and P = 0.022; respectively).

Table 2.

Average of pre- and postgrazing DM herbage biomass (Mg/ha) (LSmeans) for alfalfa, sainfoin, and birdsfoot trefoil across treatments during two periods (P1 and P2) and years (2016 and 2017), and the overall mean across treatments, periods, and years

Species Alfalfa, Mg DM/ha Birdsfoot trefoil, Mg DM/ha Sainfoin, Mg DM/ha
P1 P2 SEM P-value P1 P2 SEM P-value P1 P2 SEM P-value
2016 pregrazing 4.6bA 4.2bB 0.14 0.065 5.4b 5.5b 0.12 0.873 4.3 4.1 0.41 0.775
2017 pregrazing 8.0aA 6.1aB 0.24 0.021 7.3aA 6.1aB 0.17 0.035 5.9A 3.8B 0.29 0.035
SEM 0.23 0.13 0.16 0.17 0.47 0.17
Year effect, P-value 0.009 0.009 0.014 0.046 0.132 0.362
Average Average Average SEM P-value
2016 pregrazing 4.4bB 5.5bA 4.2B 0.15 0.008
2017 pregrazing 7.0aA 6.7aA 4.9B 0.24 0.007
SEM 0.17 0.13 0.23
Year effect, P-value 0.008 0.022 0.178
Overall
Species Alfalfa (Mg DM/ha) Birdsfoot trefoil (Mg DM/ha) Sainfoin (Mg DM/ha)
Pregrazing 5.6 6.1 4.5
Postgrazing 4.5 5.0 3.3
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a,bLSmeans in a column with different lower-case superscripts differ (P < 0.10).

A,BAverage LSmeans in a row with different upper-case superscripts differ (P < 0.10). Values are means for three spatial replications (blocks), and four treatments within each species (n = 12). Values at the bottom half of the table are means for three blocks, four treatments within each species, 2 yr and two periods within each year of the study (n = 48).

Pregrazing biomass was greater for birdsfoot trefoil than for ALF or SF during 2016, whereas pregrazing biomass of both birdsfoot trefoil and ALF was greater than pregrazing biomass of SF during 2017 (Table 2). The proportion of herbage disappearance after grazing averaged across periods and years was 0.27 (SF), 0.20 (ALF), and 0.18 (birdsfoot trefoil) of pregrazing measurements.

Scan sampling

Preference

Figure 1 shows the percentage of grazing scans in each species relative to the total number of grazing events recorded for animals grazing a choice of legumes for each year and period. Heifers offered three-way choices were observed more times grazing SF (46% of the total grazing scans recorded) than birdsfoot trefoil or ALF (27% each) (Figure 1a). During P1 of both years of the study, heifers spent approximately half of their grazing activity during daily 17-h sessions grazing SF (47% and 49% of the grazing events recorded for 2016 and 2017, respectively, Figure 1a). However, preference for this legume declined during P2 to 43% and 44% of the total grazing events recorded as a consequence of an increment in grazing activity on birdsfoot trefoil. However, the confidence intervals of these means included 33%, indicating indifference or no selection preference.

Figure 1.

Figure 1.

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Percentage of grazing scans where heifers recorded a preference for a legume species in three- or two-way choices among alfalfa, sainfoin, and birdsfoot trefoil, during two grazing periods (P1 and P2) in 2016 and 2017. Values are means for three spatial replications. Bars represent upper and lower values of 90% confidence intervals. Dashed lines indicate indifference or no preference (33% and 50% for three- and two-way choices, respectively) for any species. A legume species was considered “preferred” or “not preferred” when the confidence interval for the mean did not include the indifference threshold.

When animals were offered two-way choices containing SF, they preferred this legume over ALF or birdsfoot trefoil, particularly during P1 of 2016, with 80% of the total grazing events recorded on SF strips (Figure 1b and c, respectively). Percentage of grazing scans recorded on birdsfoot trefoil and ALF slightly increased throughout periods and years, but they were always the least preferred species in a choice with SF. On average across years and periods, heifers preferred SF over ALF or birdsfoot trefoil in a 69:31 and 71:29 ratio, respectively (Figure 1b and c).

When heifers were exposed to two-way choices between ALF and BFT, they preferred birdsfoot trefoil over ALF in three out of the four grazing periods of the study and only in P1 of 2017, heifers showed no preference among species (Figure 1d). Averaged across years and periods, heifers preferred birdsfoot trefoil over ALF in a 62:38 ratio.

Total grazing scans

The average percentage of total grazing events recorded across years and periods was the lowest for the BFT treatment (P < 0.10; Table 3), and no treatment × period (P = 0.679) or treatment × year (P = 0.255) interactions were detected. Consistent with the overall pattern, the BFT treatment showed the lowest percentages of grazing scans in P1 of both years (P < 0.10; data not shown). No differences among treatments were observed during P2 in both years of the study (2016; P = 0.332 and 2017; P = 0.496).

Table 3.

Percentage of grazing scans, behavioral levels of activity, hair cortisol concentration, and average daily gains [LS means (SEM)] by heifers grazing single legumes or two- and three-way choices

Treatment ALF BFT SF ALF-SF ALF-BFT SF-BFT ALF-SF-BFT
Grazing scans1, % 48.2a (2.0) 42.4b (1.9) 50.4a (2.0) 52.6a (2.0) 49.3a (2.0) 47.8a (2.0) 48.7a(2.1)
Standing time2, % 44.2 (1.4) 44.3 (1.7) 46.2 (1.4) 47.6 (1.4) 45.2 (1.4) 44.1 (1.4) 45.7 (1.6)
Steps3, number/d 1,511 (148) 1,447 (172) 1,634 (145) 1,731 (145) 1,566 (148) 1,648 (145) 1,653 (162)
Motion index4 5,033 (494) 4,550 (576) 5,180 (483) 5,726 (483) 5,652 (494) 5,132 (483) 6,222 (538)
Hair cortisol5, ng/g 2.00 (0.34) 1.44 (0.24) 1.86 (0.31) 1.81 (0.31) 1.90 (0.32) 2.36 (0.40) 2.12 (0.40)
ADG6, kg/d 0.93c (0.08) 1.14ab (0.08) 0.93c (0.08) 0.89c (0.08) 0.97bc (0.08) 1.03bc (0.08) 1.27a (0.08)
P-values
Parameter Grazing scans Standing time Number of steps Motion index Hair cortisol ADG
Treatment 0.063 0.534 0.877 0.423 0.584 0.054
Period 0.001 0.192 0.208 0.312 0.001 0.009
Year 0.093 0.341 0.083 0.051 0.024 0.768
Treatment × period 0.679 0.604 0.544 0.515 0.761 0.198
Treatment × year 0.255 0.593 0.982 0.999 0.793 0.139
Two-way choice vs. monocultures7 0.080 0.548 0.359 0.172 0.337 0.618
Three-way choice vs. monocultures 0.478 0.682 0.521 0.052 0.391 0.014
Three-way vs. two-way choices 0.588 0.994 0.979 0.250 0.814 0.007
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1Grazing scans were recorded for both heifers of the pair. Treatment values are the average of two heifers in each combination treatment × block.

2–4Variables were measured on one animal of the pair of heifers in each combination treatment × block.

5Hair cortisol was measured on one animal of the pair of heifers in each combination treatment × block. Reported values are back-transformed LSmeans.

6Average daily gain values are the average of two heifers in each combination treatment × block.

7Preplanned contrasts between two-, three-way choices, and monoculture treatments.

a–cLSmeans in a row with different letters differ (P < 0.10). Values are means for three spatial replications (blocks), 2 yr, and two periods within each year of study.

Preplanned contrasts showed that the average of the grazing events recorded for 2two-way choices was greater than the average value for all single species (49.9% vs. 47.0%, SEM = 2.0%, respectively; P = 0.080; Table 3), with no additional differences observed for the rest of the contrasts performed (Table 3). No differences in grazing scans were observed between P1 and P2 during 2016 (50.4% vs. 48.1%, SEM = 1.1%), but the percentage of grazing scans was greater in P1 than in P2 during 2017 (49.9% vs. 45.6%, SEM = 1.1%, respectively).

Feeding patterns

When grazing events were analyzed across daily 17-h sessions in 1-h time intervals (from 0500 to 2200 hours), no treatment × time intervals interactions were detected for both periods of 2016 (P = 0.133 and 0.707; for P1 and P2, respectively; Figure 2a and b) and during P1 of 2017 (P = 0.274; Figure 2c), showing that all treatments followed similar grazing patterns throughout the day. All animals in P1 (mid-June to mid-July) during both years started to graze at the same time between 0500 and 0600 hours (dawn time: 0530 and 0518 hours for 2016 and 2017, respectively), and by the next hour (0600 to 0700 h) 69.2% (2016) and 64.9% (2017) of the scans recorded represented grazing events (Figure 2a–c). After this interval, and from mid-day to afternoon, heifers showed grazing events that alternated between 40% and 50% of the total scans recorded, ending at dusk with the greatest percentages of grazing events (70% to 90%) between 1900 and 2200 hours (dusk time: 2135 and 2139 hours for 2016 and 2017, respectively).

Figure 2.

Figure 2.

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Grazing patterns (percentage of grazing scans recorded at each hour of the day) by heifers grazing single forages, two- or three-way choices of alfalfa (ALF), birdsfoot trefoil (BFT), and sainfoin (SF) during two grazing periods in two consecutive years. Values represent the average of 6 heifers across 4 d in each period of 2016 and 2017. Time intervals 0800 to 0900 hours were dropped from the analysis in P1 2017 due to missing values. Time intervals 0500 to 0600 hours were dropped from the analysis in P2 2016 and P2 2017 because most of the observed grazing percentages were zero.

Due to differences in photoperiod, animals during P2 (mid-August to early September) started to graze ~1 h later between 0600 and 0700 hours (dawn time: 0617 and 0605 hours for 2016 and 2017, respectively) and showed their first peak of daily grazing events between 0700 and 0800 hours (Figure 2b). Consistent with P1, although 1 h earlier, animals showed a second peak of grazing events at dusk, with percentages ranging between 75% and 87% of all the scans recorded (dusk time: 2040 and 2058 hours for 2016 and 2017, respectively). In contrast to the rest of the periods, a treatment × time interval interaction was observed for P2 in 2017 (Figure 2d), driven by a sharp decline in grazing events for all treatments except for the three-way choice treatment during the 0800 to 0900 hours time interval, and for the high percentage of grazing events (79.2% of all scans) observed at noon for the same treatment.

Behavioral levels of activity

Averaged across periods and years in the study, no differences among treatments were observed for the number of steps taken by heifers on a daily basis (P = 0.877; Table 3). On average across treatments, the number of steps was also similar for both periods in 2016 (P = 0.110) and in 2017 (P = 0.447; data not shown), although 2017 heifers took more steps than 2016 heifers (1,707 vs. 1,490; SEM = 80; P = 0.083).

No differences among treatments were observed for levels of activity measured as a motion index (P = 0.423; Table 3). However, when contrasting the three-way choice against the average of the three-single species in preplanned contrasts, the former showed a motion index 26.4% greater than the average value recorded for single species treatments (6,222 vs. 4,921; P = 0.052). In contrast, no differences in motion index were detected between two-way choices and single-legume species (5,504 vs. 4,921; P = 0.172). Consistent with number of steps, the motion index also showed greater values during 2017 than during 2016 (5,764 vs. 4,949; SEM = 18; P = 0.051).

No differences were detected among treatments in the percentage of time heifers spent standing (P = 0.534; Table 3). The same response applies to the percentage of time animals spent lying down, as both variables are linear combinations (i.e., standing time = total time –time lying down). No interactions were detected between treatments and periods (P = 0.604) or treatments and years (P = 0.593), with similar standing times across grazing periods and years.

Figure 3 shows the percentage of time that heifers spent standing in each treatment at each hour (time interval) for each grazing period in each year. No interactions between treatments and time intervals were observed for both periods of 2016 (P = 0.307 and 0.979; for P1 and P2, respectively) and 2017 (P = 0.164 and 0.107). Consistent with the pattern observed for the percentage of grazing scans, animals during P1 and between 0600 and 0700 hours (73.2% to 79.4% of the time recorded) and 1900 and 2200 hours (70% to 98.6%) spent most of the time standing. During P2, due to differences in photoperiod, peaks shifted to the 0700 to 0800 hours interval in 2016 (89.4%) and to the 0900 to 1000 hours interval in 2017 (90.1%). At dusk, peaks of standing time occurred earlier in P2 than P1, from 1800 to 2100 hours, both during 2016 (67.7% to 96.5%) and 2017 (70.0% to 97.9%).

Figure 3.

Figure 3.

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Percentage of standing time recorded in each hour of the day of heifers grazing single forages, two- or three-way choices of those forages: alfalfa (ALF), birdsfoot trefoil (BFT), and Sainfoin (SF) during two grazing periods in two consecutive years. Values represent the average of 3 heifers across 7 d in each period of 2016 and 9 d in each grazing period of 2017. Time intervals 7 to 8 and 8 to 9 were dropped from the analysis in both periods of 2017 due to missing values.

Hair cortisol

Cortisol concentrations in the hair of the heifers during the study are reported in Table 3. No differences in the levels of cortisol were observed among treatments (treatment effect; P = 0.584), and no treatment × period or treatment × year interactions were detected (P = 0.761 and 0.793, respectively). Similarly, no differences were observed for the preplanned contrasts between the three-way choice (ALF-SF-BFT) and the average cortisol values for monocultures (ALF, SF, and BFT; P = 0.391), or averages between three- and two-way choices (P = 0.814). Averaged across treatments, the levels of cortisol were greater at the end of P1 than at the end of the P2 (2.35 ± 0.21 vs. 1.55 ± 0.14 ng/g; P = 0.001), and they were greater during the first than during the second year of the study (2.25 ± 0.20 vs. 1.62 ± 0.15 ng/g; P = 0.024 for 2016 and 2017, respectively).

Average daily gains

Averaged across periods and years, cattle grazing monoculture BFT gained more BW than cattle grazing monoculture ALF or SF (P = 0.077 and 0.073, respectively); no differences were observed between SF and ALF treatments (P = 0.980; Table 3). When SF or birdsfoot trefoil was offered with ALF in two-way choices (ALF-BFT or ALF-SF), BW gains did not differ from those observed in animals under the ALF treatment (P > 0.10). In contrast, when the three species were offered in the three-way choice (ALF-SF-BFT), heifers gained 27% more BW than the average of all monoculture (ALF, SF, and BFT) treatments (P = 0.014), and 30.0% more than the average of all two-way choice treatments (P = 0.007). In contrast, no differences were observed in ADG between the average of two-way choices and the average of all monoculture treatments (P = 0.618).

Discussion

Foraging behavior by cattle grazing choices of legumes

When heifers were allowed to choose among strips of different legume species, they selected a diverse diet, which was consistent with behaviors typically observed in generalist herbivores (Provenza, 1996; Provenza et al., 2003). The frequent moves to fresh paddocks and the high forage allowances in each paddock prevented restrictions in selectivity, as confirmed by the low levels of legume utilization apparent from high postgrazing pasture DM (Table 2). Despite the high biomass availability for all forages in two- and three-way choices, where heifers could have selected the preferred species, significant amounts of all legumes were incorporated into the diet.

In addition to choosing a diverse diet, forage selection by heifers was not random. Based on scan sampling data, SF was the preferred species in three-way (almost 50% of all grazing events), and two-way (70% of all grazing events) choices. Previous studies have also reported a preference for SF over ALF by cattle grazing strips of these legumes and tall fescue (Villalba et al., 2015). Several explanations have been provided for selection of varied diets by herbivores. Some contend that no single forage species is capable of providing all the nutrients and the appropriate proportions that herbivores need (Westoby, 1978). Others proposed the need to minimize the ingestion of foods with plant toxins (i.e., the toxin dilution hypothesis; Freeland and Janzen, 1974). Finally, the satiety hypothesis states that varied diets are the consequence of transient food aversions caused by flavors, nutrients, and toxins ingested too frequently or in excess (Provenza, 1996). These hypotheses are not mutually exclusive, and they may all have contributed to the heifers’ foraging decisions in this study.

It is likely that heifers reduced their grazing time in ALF when it was offered in a two-way choice with SF or birdsfoot trefoil, despite the fact that ALF showed the greatest concentration of CP and the lowest contents of ADL, in order to reduce the potentially toxic effects of rapid protein breakdown and ammonia accumulation in the rumen and blood (Provenza, 1996). It is also likely that the lower preferences for alfalfa are partially explained by the incidence of subacute frothy bloat caused by the ingestion of this legume (Wang et al., 2012). As an example of negative influences of CP and bloat on preference, sheep develop aversions to forages associated with high levels of ammonia in the rumen (Villalba and Provenza, 1997), and they learn to avoid foods that cause rumen distension and to prefer foods that attenuate this effect (Villalba et al., 2009). In contrast, SF had a lower concentration of CP and a greater concentration of fiber and ADL, and yet it was preferred over ALF or birdsfoot trefoil. The presence of relatively high concentrations of CT in SF likely contributed to this preference, as CT reduce the incidence of bloat and SF is a nonbloating legume (Wang et al., 2012). Thus, incorporation of high levels of SF in the diet did not cause, or could have even alleviated, the discomfort caused by subacute levels of frothy bloat caused by the consumption of ALF (Wang et al., 2006).

The greater ADG observed in heifers grazing BFT and the reduced percentage of grazing scans recorded for this species suggest that less birdsfoot trefoil was more satisfying, probably because the primary nutrients were more concentrated and given that BFT contains CT that reduce the rate of proteolysis in the rumen and thus the accumulation of ammonia in the animal’s tissues (Waghorn, 2008). Consistent with this notion, significantly greater concentrations of blood urea nitrogen (BUN) and urinary urea nitrogen were observed in this study in heifers grazing ALF than in those grazing tanniferous monocultures (Lagrange et al., 2020a).

Preference for SF over birdsfoot trefoil could also be explained by lower rates of proteolysis and ammonia formation with SF consumption (e.g., 4× the concentration of CT in SF relative to birdsfoot trefoil). In addition, greater concentrations of BUN have been observed in animals consuming birdsfoot trefoil than in those consuming SF (Lagrange and Villalba, 2019). Alternatively, the lower CP concentration in SF than in ALF or birdsfoot trefoil may have contributed to dilute total protein ingestion and thus balance the ratio of energy to soluble protein ingested by heifers. Finally, the type of CT present in SF do not appear to cause toxic effects in ruminants; on the contrary, sheep prefer high- to low-tannin-containing SF pellets (Costes-Thiré et al., 2018).

When SF was not present in the choice (i.e., ALF-BFT treatment), heifers preferred birdsfoot trefoil over ALF in a 60:40 ratio. The presence of CT—even when at lower concentrations than in SF—could also explain this pattern as described above. In addition, the concentration of nonstructural carbohydrates in birdsfoot trefoil may be greater than in ALF (Christensen et al., 2015) which could improve the imbalance of high protein/energy ratios typical of legume diets. Finally, differences in sward structure (i.e., that lead to a greater bite sizes for BFT; see below) may also contribute to explain a preference for birdsfoot trefoil over ALF.

Levels of activity by cattle grazing monocultures vs. choice

Total grazing events were similar among treatments, except for BFT which showed lower values across periods and years. Likewise, grazing patterns were not influenced by treatment, suggesting that grazing activity was not constrained by the availability of forage alternatives in choice treatments relative to monocultures. Given that animals in choice treatments selected a diverse diet (see previous section), a reduced number of grazing events in three- and two-way choice treatments could have been expected relative to monocultures due to an increased investment in searching and forage switching activities that reduce foraging efficiency (Bailey et al., 1996). Nevertheless, the spatial distribution of legumes in the present study (i.e., in patches), typically reduce searching activities relative to mixed swards as the manifestation of a preference occurs automatically after the selection of a specific feeding station (Chapman et al., 2007). Searching activities may also be minimized given that cattle manifest spatial memory, which contributes to increased foraging efficiency (Bailey et al., 1996). Heifers in our study were familiar with the distribution of strips in their paddocks, which were fixed, a design that reduces searching time relative to random distribution. Switching activities from one strip to the next may also reduce the number of grazing bouts as animals need to move among feeding stations, but this outcome was likely minimized by the proximity and size of the legume strips relative to the body size of the heifers. Under this context, it is likely that the time invested in switching between strips in choice treatments was similar to the time invested in switching between feeding stations by animals grazing monocultures during the process of moving along the grazing pathway (Bailey et al., 1996).

Grazing efficiency is the ratio between grazing and walking time, which increases with increments in short-term herbage intake rates and in residence time per feeding station (Gregorini et al., 2009). Consistent with grazing scans and patterns, no differences among treatments were observed regarding number of daily steps, suggesting that the spatial distribution of patches in choice treatments led to similar grazing efficiencies to those in animals grazing monocultures, with the added benefit of building a diverse diet, typical of generalist herbivores. These benefits could be summarized as the incorporation of beneficial (i.e., antioxidant, antiparasitic, and nutraceutical) secondary compounds such as CT (Waghorn, 2008), improved ADG and lower levels of excretion of urinary N, which have been also observed in this study (Lagrange et al., 2020a).

Despite all treatments showing similar numbers of daily steps, the motion index for the three-way choices was 26.4% greater than the average observed for monocultures. This suggests that heifers in the treatment with highest diversity moved faster, likely to maintain their foraging efficiency when more legume species were available for selection. Consistent to number of steps taken or grazing scans, no differences among treatments were detected regarding total standing time (Table 3), suggesting similar residence time per feeding station across all treatments, which further supports the idea that grazing efficiency was similar for choice or no-choice treatments.

A possible explanation for the lower number of grazing events by the BFT treatment entails sward structure. Birdsfoot trefoil plants present a more prostrate growth habit relative to ALF or SF, with greater biomass per unit of area (Table 2) and higher bulk density (i.e., herbage weight per unit of canopy volume), which is correlated with a greater leaf area index (Gibb and Orr, 1997). These characteristics might have led to a greater bite mass, a fundamental variable determinant of intake rate which is dependent on sward structure (Laca et al., 1992). Thus, heifers on this treatment likely invested longer times per bite in order to process and swallow a greater bolus, but possibly with greater intake rates that led to lower grazing times. In contrast, heifers in treatments containing SF and/or ALF with a different sward structure, with an erect growth habit, larger stems and lower bulk density in the upper layers likely promoted a lower bite size (Carvalho, 2013). These characteristics might have involved more time invested in handling activities, and consequently greater likelihoods of being captured by scan sampling in a grazing position. In addition, such differences in forage structure and bite size may partially explain the greater proportion of grazing scans recorded for SF in the SF-BFT treatment.

The daily grazing pattern followed by heifers on different diets was analyzed by grazing period due to the observed differences in photoperiod, which affects the time that animals spend eating, ruminating, and resting (Gregorini et al., 2006). The grazing and standing activities of cattle appear to be synchronized for all treatments (Figures 2a–d and 3a–d). Internal motivations for synchrony induced by daylight may be stronger than external factors such as feeding time in dairy cows (Flury and Gygax, 2016). The proximity of animals from different treatments using contiguous plots separated by an electrical fence might have also induced heifers to mimic behaviors of cattle allocated to other treatments, thus leading to synchrony (Stoye et al., 2012). Heifers showed a typical grazing pattern with two major grazing events during the day, as reported in previous research (Gregorini et al., 2006), one early in the morning 1 h after sunrise and one in the evening, with greater numbers of grazing events toward the last 3 h before dark. Shorter photoperiod by period 2 shifted the peaks of grazing activity to 1 h later in the morning and 1 h earlier in the afternoon than in period 1, “compressing” the grazing activity within those limits. In-between peaks of grazing activity, heifers were less synchronous than during dawn or dusk grazing events, reflecting what other authors have reported in previous studies (Stoye et al., 2012). It is likely that factors imposed by different treatments, such as motivation to consume diverse diets vs. reduction in feeding bouts due to monotony, influenced feeding during those in-between periods, like the sharp decline in grazing events during the 0800 to 0900 hours time interval for all treatments except for the three-way choice treatment, and for the high percentage of grazing events observed at noon for the same treatment of greatest diversity. Nevertheless, such pattern only occurred for period 2 in 2017, and thus they were not consistent for all periods or years.

Performance and cortisol levels

Heifers grazing BFT performed better than animals grazing ALF or SF, gaining an average of 22.5% more BW across periods and years of study. This effect may be attributed in part to the particular molecular weight and chemical structure of CT in BFT and their high affinity to bind with proteins in the rumen, which reduces proteolysis and subsequently provides a better profile of dietary amino acids to the small intestine (Waghorn, 2008). Reduced ammonia formation also prevents the extra energy cost needed for ammonia detoxification by the animal (Lobley and Milano, 1997). Alternatively, a greater proportion of nonstructural carbohydrates in birdsfoot trefoil that reduces the N/energy imbalance typically observed in legumes may also explain the greater BW gains by heifers in the BFT treatment (Christensen et al., 2015), as well as potential greater bite sizes in BFT swards as discussed above. In contrast, the effects of subclinical bloat and excess protein may explain the lower ADG in the ALF treatment.

When both tanniferous legumes were consumed together along with ALF in the three-way choice, ADG was greater than for ALF or SF. This may be explained by increments in DM intake when heifers were exposed to a greater degree of forage diversity, which is consistent with previous studies where sheep were exposed to a diversity of flavors and feeds (Villalba et al., 2011). In addition, by consuming a mixed diet, animals obtain a more balanced mixture of nutrients allowing for greater growth rates than grazing a monoculture (Provenza et al., 2003). Thus, chemical complementarities induced by the incorporation of forages such as birdsfoot trefoil and SF with high concentrations of soluble carbohydrates (Christensen et al., 2015) and moderate levels of CT may have allowed for an improved utilization of the high contents of rumen-degradable protein in ALF and therefore, greater animal performance. These benefits translate into reduced enteric methane and nitrogen emissions to the atmosphere (Lagrange et al., 2020a), which in addition to the diversity of forages provided to livestock are consistent with the concept of One Welfare (Pinillos et al., 2016).

Frequent or excessive exposure to the same orosensorial or postingestive stimuli, like those experienced when ruminants consume monotonous diets or forages can be stressful (Provenza, 1996). Consistent with this notion, a diversity of food items offered to sheep in confinement reduces plasma cortisol levels relative to animals fed monotonous rations (Villalba et al., 2012), and reduces lymphocyte counts (Catanese et al., 2013) and stress-induced hyperthermia in open field tests (Villalba et al., 2012). Nevertheless, no differences in hair cortisol levels were observed in this study for animals exposed to choice or no-choice treatments. It is likely that monotony is experienced differently by animals at pasture than at confinement. In fact, dairy cows in confinement are willing to perform work to gain access to pasture (von Keyserlingk et al., 2017). Locomotor activities may promote increased levels of hair cortisol in cattle (Comin et al., 2011). Results from our study show no differences in number of steps among treatments, consistent with the lack of differences observed in hair cortisol.

In conclusion, heifers offered a choice among tanniferous and nontanniferous legumes preferred the former (particularly SF) over alfalfa, although they selected significant amounts of all three species, building a diverse diet that led to improvements in BW gains. We did not observe any effect of forage diversity on grazing events or other types of activities such as standing, walking, moving, or resting, or hair cortisol concentrations relative to monocultures. Thus, spatial segregation of forage species into patches has the potential to enhance animal performance without influencing foraging behavior. The incorporation of a diverse array of chemicals into the diet, such as the ingestion of different types and concentrations of CT or soluble carbohydrates may promote synergisms that improve animal nutrition and health, with environmental benefits (i.e., enhanced biodiversity, reduced methane, and nitrogen emissions to the atmosphere) that are in line with the One Welfare approach.

Acknowledgments

The authors thank Justin Tylor, Gavin Johnson, Raul Guevara, Marcello Pira, Peter Armstrong, Matt Endicott, Elizabeth Stewart, and Edward Knoppel for technical support. We also thank Dr. Susan Durham for his expertise and advising with the statistical analysis. This research was supported by grants from the National Institute of Food and Agriculture (NIFA), USDA (award no. 2016-67019-25086), the Utah Agricultural Experiment Station, Irrigated Pasture Project (UTA1321), and a fellowship to S.L. (INTA, Instituto Nacional de Tecnología Agropecuaria, Buenos Aires, Argentina). The paper is published with the approval of the Director, Utah Agricultural Experiment Station, and Utah State University, as journal paper number 9337.

Glossary

Abbreviations

ADF

acid detergent fiber

ADG

average daily gain

ADL

acid detergent lignin

BFT

birdsfoot trefoil

BUN

blood urea nitrogen

BW

body weight

CP

crude protein

CT

condensed tannins

DM

dry matter

N

nitrogen

SF

sainfoin

Conflict of Interest Statement

The authors declare no real or perceived conflicts of interest.

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