Fear responses and postmortem muscle characteristics of turkeys of two genetic lines 1

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1 Fear responses and postmortem muscle characteristics of turkeys of two genetic lines 1 M. A. Erasmus, H. C. Lee, I. Kang,, and J. C. Swanson,2 Department of Animal Science, Michigan State University, East Lansing, MI 48824; and Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI ABSTRACT Commercial turkey production has increased greatly in recent decades. Along with increased production, problems with turkey meat quality have also increased. Research with other species has demonstrated that differences in meat quality exist among pigs and cattle differing in characteristics such as fearfulness. However, associations between fear responses and postmortem (PM) muscle characteristics related to the meat quality of turkeys have not been examined. This study evaluated the test retest repeatability of responses of male commercial (COMM) and randombred (RB) turkeys in an open field (OF) test, which is used to assess fear and activity levels of poultry. Another objective of this study was to evaluate the relationship between behavioral OF responses and PM breast muscle characteristics (ph and R-value) that are related to meat quality. Thirdly, this study evaluated differences in ph and R-value between the turkey lines. Male COMM and RB turkeys were each housed in groups in 4 pens. Turkeys were individually tested in an OF ( m, divided into 81 squares) at 1, 4, and 11 wk (COMM N = 27; RB N = 33). Turkeys were then grouped into clusters based on a cluster analysis of OF behavior. Turkeys were processed and meat quality characteristics were evaluated at wk for COMM and wk for RB turkeys. Results were analyzed using a mixed model (SAS 9.4). Breast muscle ph and R-value did not differ between genetic lines, and there were no differences in ph and R-value among clusters within genetic lines. These findings suggest that OF responses measured during rearing are not related to PM breast muscle ph and R-value, which ultimately affect meat quality. Further research is needed to assess whether other types of fear responses are associated with meat quality and whether differences in R-value between genetic lines are associated with differences in other meat quality characteristics. Key words: turkey, fear responses, meat quality, ph, R-value 2015 Poultry Science 94: INTRODUCTION Annual turkey production in the United States has increased from 90 million in 1962 to over 250 million in 2012 (USDA National Agricultural Statistics Service, 2014). Increased turkey production was facilitated by changes in production, such as intensive housing and selective breeding methods, which reduced costs associated with raising turkeys (Martin, 2009). However, concurrent with improved production efficacy, problems with meat quality have increased (Petracci and Cavani, 2012). Previous research demonstrated that meat from C 2015 Poultry Science Association Inc. Received January 27, Accepted June 10, A preliminary report of this research was presented in a proceedings paper (Erasmus, M., H. C. Lee, I. Kang, and J. Swanson Fear in turkeys: Implications for productivity and well-being. Proc. Midwest Poult. Fed. Conv. Accessed Jun amazonaws.com/item files/b3ba/attachments/35641/original/ erasmus marisa - fear in turkeys - implications for productivity and well-being.pdf). 2 Corresponding author: swansoj@anr.msu.edu modern faster-growing chicken and turkey lines is lower in some quality parameters compared to meat from slower growing lines (reviewed in Petracci and Cavani, 2012). Meat quality refers to the attributes of meat that influence consumer acceptance, including appearance, texture, juiciness, flavor, and functionality (Fletcher, 2000). Appearance and texture are considered to be the most important attributes determining meat quality (Fletcher, 2000). Appearance is affected by many factors, including the ph and dryness of the muscle (Le Bihan-Duval, 2004; reviewed in Hamoen et al., 2013; Jeong et al., 2011a). Specifically, meat color is affected by ph such that meat with a high ph is darker in color, whereas a lower ph is associated with a lighter color (Fletcher, 2000). Texture, on the other hand, is influenced by the contractile state of the myofibrillar proteins, which in turn is affected by the rate at which rigor mortis develops (Fletcher, 2000). Rigor mortis development also affects the ultimate ph and color (Castigliego et al., 2012). Rigor mortis development is indirectly measured using the R-value, which is an 2018

2 FEAR RESPONSES AND MEAT QUALITY OF TWO TURKEY LINES 2019 indicator of the level of adenosine-triphosphate (ATP) breakdown in muscle (Calkins et al., 1982). Higher R- values are associated with lower levels of ATP in the muscle (Alvarado and Sams, 2000). If ATP is depleted, toughening occurs at rigor mortis, which affects meat tenderness (Hamoen et al., 2013). Both the ph and R-value are measures of postmortem (PM) muscle metabolism and are affected by the amount of glycogen present in the muscle at the time of slaughter (Wariss, 2000). Two conditions associated with different levels of muscle glycogen are PSE meat and dark, firm, dry (DFD) meat. In pigs, PSE meat results when pigs experience stress before slaughter, which results in rapid glycolysis, increased lactic acid production, a decrease in muscle ph (reviewed in Broom and Johnson, 1993), and accelerated rigor mortis (Sosnicki et al., 1998). PSE-like meat has also been documented in chickens and turkeys (Fletcher, 2000) and has increased over the years in turkeys (Sosnicki et al., 1998) as turkeys have been selectively bred for higher growth rates and breast muscle yield (Strasburg and Chaing, 2009). In contrast to PSE meat, DFD meat results when there is little or no glycogen in the muscle before slaughter, resulting in low lactic acid production and high ph (Broom and Johnson, 1993). High levels of physical activity before slaughter result in DFD meat because glycogen reserves are depleted (Terlouw, 2005). DFD meat has been extensively studied in cattle, but DFD-like meat has also been reported in chickens (Fletcher, 2000). The amount of glycogen in the muscle depends on several factors, including the nutritional background of the animal during rearing and the amount of preslaughter stress the animal experiences (McGilchrist et al., 2012; Coombes et al., 2014). It is widely accepted that individual animals differ in characteristics such as stress reactivity and fearfulness and that these characteristics are consistent over time (Koolhaas et al., 1999; Cockrem, 2007, 2013). Fear and stress are closely linked because the physiological stress-related pathways are activated when an animal experiences fear (Cockrem, 2007; Armario et al., 2012). Consequently, stimuli that elicit fear have been described as emotional stressors (Armario et al., 2012). The slaughter process is associated with a number of events and conditions that may induce stress. Antemortem stressors, such as transportation, heat stress, and struggling of birds before slaughter, promote faster PM glycolysis, which is associated with the development of PSE meat (McCurdy et al., 1996; McKee and Sams, 1997). Therefore, it can be hypothesized that if individual animals are consistent in their stress and fear responses and the slaughter process is associated with events that elicit stress and fear, then animals that differ in fearfulness should differ in meat quality characteristics, regardless of whether fearfulness is assessed immediately prior to slaughter or weeks or months before slaughter. If fearfulness is associated with meat quality, then it would be expected that more fearful animals would experience a greater level of glycogen breakdown, with the result that meat from more fearful animals would have a lower initial PM muscle ph. Research with pigs has demonstrated that pigs displaying less fear of humans when tested a few months before slaughter were less reactive to slaughter procedures, and pigs displaying less fear of novel objects had higher muscle ph levels (Terlouw and Rybarczyk, 2008). Similarly, steers characterized as flighty (indicated by high flight speed, defined as the time taken to move a specified distance after being released from a weighing crate) were more susceptible to stress before slaughter and had lower live weights, poorer feed conversion efficiency, lower carcass dressing percentages, and lower initial muscle ph levels (Petherick et al., 2002; reviewed in Norris et al., 2014). In contrast to studies with cattle and pigs, there is scant research examining the relationship between temperament or fearfulness and meat quality characteristics of poultry. Fearfulness is perhaps the most widely studied temperament characteristic of poultry. Research with broiler chickens found no relationship between fear (measured using tonic immobility) and meat quality measures (Debut et al., 2003), whereas Remignon et al. (1998) reported that meat quality parameters differed between quail selected for high and low fear (increased and decreased tonic immobility duration, respectively) when exposed to acute stress before slaughter. The open field (OF) test and tonic immobility test are two of the most widely used tests for assessing fear responses of poultry. The OF test for poultry is intended to measure fear of novelty and subjects animals to social isolation and is also used to assess social reinstatement motivation (Forkman et al., 2007). It is believed that birds that are inactive and silent experience greater fear than birds that move around the arena and vocalize (Forkman et al., 2007). When tests such as the OF test are used to evaluate the fearfulness of animals, it is important that these tests be repeatable over time. It was recently demonstrated that commercial (COMM) male turkeys behavioral responses in an OF test were repeatable between weeks (Erasmus and Swanson, 2014). However, the test retest reliability of responses of a slower-growing, randombred (RB) turkey line has not been examined. Before the association between fear responses and meat quality measures can be assessed, it is necessary to evaluate the repeatability of fear responses. The objectives of this study were to examine the repeatability of OF responses of two genetic lines of turkeys: a faster-growing COMM line and a slowergrowing RB line and to determine whether differences in OF responses are associated with differences in PM breast muscle ph and R-values. MATERIALS AND METHODS All procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University.

3 2020 ERASMUS ET AL. Animals and Housing A total of 80 male COMM turkeys (Hybrid Converter line, Hybrid Turkeys, Kitchener, ON, Canada) and 62 male RB turkeys (RBC2 line established in 1966; Nestor et al., 1969) were used in this study. Owing to low availability, only 62 RB poults were obtained. The RB poults were obtained from the Ohio Agricultural Research and Development Center (OARDC) of the Ohio State University, Wooster, OH. COMM poults were placed 1 d prior to placement of RB poults. Turkeys of both genetic lines were beak-trimmed and COMM turkeys were toe-trimmed in accordance with standard commercial practice. Turkeys were brooded in groups of the same genetic line in 4 littered (wood shavings) pens (2 pens per line) measuring m in the same barn from 1 d to 2 wk of age. At 2 wk, birds were distributed throughout the barn and randomly assigned to one of 16 littered pens ( m) in the same barn and housed in groups of the same genetic line (15 16 birds/pen (RB turkeys) and 20 birds/pen (COMM turkeys); 4 pens per line) until 7 wk. At 7 wk, some COMM turkeys (2 4/pen) were removed from their pens for use in another study, and the number of COMM turkeys in each pen was reduced to and maintained at 16. Therefore, group sizes were similar across lines from 7 wk onward (in 2 RB pens there were 15 birds/pen; all other RB and all COMM pens had 16 birds/pen). Stocking density in pens varied slightly as the study progressed owing to head pecking, which was beyond the control of the researchers. Injurious pecking is known to be a problem among COMM male turkeys (Dalton et al., 2013). All birds that were injured because of head pecking were promptly moved to a hospital pen to facilitate recovery or humanely euthanized in severe cases. Between 7 and 11 wk, 2 RB turkeys from the same pen were moved and 1 from another pen was euthanized owing to head pecking. Between 3 and 11 wk, 2 COMM turkeys from the same pen were euthanized because of injuries. Two other turkeys from another pen were euthanized because of lameness and unthriftiness. Temperature and diet were provided as described in Erasmus and Swanson (2014). A photoperiod of 24 light (L): 0 dark (D) was applied for the first 7 d. Thereafter, the light period was reduced by 1 h/d for 7 d until a photoperiod of 16L: 8D was reached (light period: 6:00 a.m. to 10:00 p.m.) and applied from 14 d to 14 wk. Light intensity was maintained at 20 l. To individually identify turkeys, colored (green, black, purple, and orange), nontoxic livestock marker (Prima Tech Marking Stick, Neogen Corp., Lansing, MI, USA) was applied to different parts of the body and in different color combinations for each bird in the pen (e.g., black /or green on shoulders, left or right wing) at 1 wk. Thereafter, the marker was reapplied biweekly to ensure that markings remained visible on overhead cameras. COMM and RB turkeys were weighed on the same d, 4 d before the third open field (OF) test at 11 wk. Open Field (OF) Testing Birds were tested in the OF test at 1 wk (Test 1), 4 wk (Test 2), and 11 wk (Test 3) to examine whether OF behavior was repeatable over time. The OF test arena and testing procedures were conducted as described in Erasmus and Swanson (2014). Briefly, each bird was placed in the center of the OF test arena for 10 min, and behavior was recorded in real time (60i fields/s) from 2 overhead high-definition camcorders (VIXIA HF M41, Canon USA, Inc., Melville, NY, USA). Behavioral data, including the latency to ambulate, number of steps taken, latency to vocalize, number of vocalizations, and number of defecations, were collected from video recordings. Open Field Responses, and ph and R-Value Measurements To examine the relationship between ph, R-value, and OF responses, the results of all 3 OF tests were averaged to obtain an average value for each OF response (latency to ambulate, number of steps taken, latency to vocalize, number of vocalizations, and number of defecations). Only turkeys for which data from all 3 OF tests and PM ph and R-values were available were used in the analysis (COMM: N = 27; RB: N = 33). Cluster analysis was used to group birds with similar OF responses into distinct clusters. Breast muscle ph and R-values were then compared between clusters to determine whether ph and R-values differed among birds that differed in OF responses. Processing COMM turkeys were reared to ± 0.29 kg (average live weight) to mimic current turkey production and slaughtered between 15 and 17 wk. Similar to the study by Chiang et al. (2008), RB turkeys were slaughtered between 20 and 21 wk (11.04 ± 0.10 kg average live weight) because of their slower growth rate relative to COMM turkeys. Owing to the limited number of birds that could be processed daily, turkeys from each genetic line (5 turkeys/pen) were randomly assigned to 1 of 3 slaughter groups (20 turkeys/group). Groups were slaughtered 3 7 d apart. After feed withdrawal for 12 h, turkeys were individually tagged in the wing and transported for approximately min from the Michigan State University Poultry Farm to the Michigan State University Meat Laboratory. The outside temperature at the time of transportation ranged between 5 and 14 C. Upon arrival, the turkeys were shackled and electrically stunned for 3 s (80 ma, 60 Hz, 110 V), bled for 90 s by severing

4 FEAR RESPONSES AND MEAT QUALITY OF TWO TURKEY LINES 2021 both the carotid artery and jugular vein on one side of the neck, and scalded for 120 s at 59 o C. Breast Muscle ph and R-value The ph and R-value of the breast muscle (Pectoralis major) were measured at 15 min PM as measures of PM muscle metabolism. The ph was measured according to the procedures used by Jeong et al. (2011b). Briefly, 5 g muscle tissue was taken from the cranial area of the breast fillet and homogenized in 25 ml of distilled/deionized water. The ph was then measured using a ph electrode (Model , Fisher Scientific Inc., Houston, TX) that was attached to a ph meter (Accumet AR15, Fisher Scientific Inc., Pittsburgh, PA). The R-value was measured following the procedures of Thompson et al. (1987). Statistical Analysis All statistical analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC). Similar to Erasmus and Swanson (2014), the test retest reliability of OF responses was assessed using the Spearman rank correlation coefficient to compare test 1 with test 2, test 1 with test 3, and test 2 with test 3. Based on Martin and Bateson (1993), correlation coefficients between 0.2 and 0.4 were regarded as low, correlation coefficients between 0.4 and 0.7 were regarded as moderate, correlation coefficients between 0.7 and 0.9 were regarded as high, whereas correlation coefficients between 0.9 and 1.0 were considered very high. Similar to a study examining individual differences in the behavior of goats (Miranda-de la Lama et al., 2011), hierarchical cluster analysis (PROC CLUSTER) was used to assign turkeys to a particular cluster. Hierarchical cluster analysis produces a unique set of nested categories or clusters by sequentially pairing variables (Bridges, 1966). Prior to cluster analysis, variables (latency to ambulate, number of steps taken, latency to vocalize, number of vocalizations, and number of defecations) were standardized (mean = 0, SD = 1). Ward s method of clustering was specified. The purpose of cluster analysis is to group individuals into clusters, where individuals in the same cluster are similar to one another, but different clusters are different (SAS, 2012). Therefore, the goal of cluster analysis was to identify groups of animals that differed significantly in OF responses. To verify that turkeys that were part of a particular cluster differed in OF responses from turkeys in other clusters, the nonparametric Kruskall Wallis test (PROC NPAR1WAY) was used. The post hoc Bonferroni adjustment was used to evaluate multiple comparisons between clusters. Live weight at 11 wk, ph, and R-value were compared between genetic lines and among clusters within genetic lines using a mixed model with Tukey s adjustment for multiple comparisons. Pen was included as a random effect. Slaughter group was additionally included as a random effect in models evaluating differences in ph and R-value. The Spearman rank correlation coefficient was used to assess whether individual OF test measures were associated with ph and R-value. Statistical significance was declared at P < RESULTS AND DISCUSSION Test Retest Reliability of OF Responses Spearman rank correlation coefficients for correlations between OF tests are presented in Table 1. For COMM turkeys, a significant, moderate correlation was found for the number of steps taken in test 1 vs. test 2, whereas for RB turkeys there was a significant, low correlation for the number of defecations produced in test 1 vs. test 2. No significant correlations were found between tests 1 and 3 for any OF responses of RB turkeys. In contrast, there were significant, low correlations between tests 1 and 3 for the latency to ambulate and number of steps taken for COMM turkeys. Significant, moderate correlations were found between tests 2 and 3 for all OF responses for COMM turkeys, with the number of vocalizations having the highest correlation (r S = 0.68). For RB turkeys, significant, low correlations were found between tests 1 and 3 for the number of vocalizations and defecations produced, whereas significant, moderate correlations were found for the latency to ambulate and number of steps taken. There were no significant correlations between tests 1 and 3 for the latency of RB turkeys to vocalize. Previously, we evaluated the test retest reliability of OF responses of COMM male turkeys between 4 6 and 8 10 wk (Erasmus and Swanson, 2014), but early OF responses (1 wk of age) had not been examined. The Table 1. Spearman rank correlation (r S ) of open field (OF) behavior among tests for male commercial (COMM) and randombred (RB) turkeys. OF response Test 1 vs. 2 Test 1 vs. 3 Test 2 vs. 3 COMM turkeys N=36 N=30 N=47 Latency to ambulate Latency to vocalize Step number Vocalization number Defecation number RB turkeys N=36 N=35 N=47 Latency to ambulate Latency to vocalize Step number Vocalization number Defecation number P < P < P < P < P <

5 2022 ERASMUS ET AL. results reported here for correlations between test 2 (4 wk) and test 3 (11 wk) are generally in accordance with previous results that indicated that moderate, significant correlations were found for all OF responses when OF behavior tested at 4 6 wk was compared to OF behavior tested 4 wk later at 8 10 wk (Erasmus and Swanson, 2014). In contrast to previous results, a moderate, significant correlation was found between 4 and 11 wk for the latency to vocalize, whereas the latency to vocalize was not significantly correlated between 4 6 wk and 8 10 wk (Erasmus and Swanson, 2014). Tests 2 and 3 were conducted 7 wk apart, whereas in the previous study tests were conducted 4 wk apart (Erasmus and Swanson, 2014), which may account for differences in correlation coefficients between studies. The test retest reliability of OF responses of RB turkeys has not previously been examined. However, based on the present results, the behavior of RB turkeys related to ambulation (latency to ambulate and number of steps taken) was repeatable between 4 and 11 wk, whereas behavior related to vocalization (latency to vocalize and number of vocalizations produced) and defecation had low correlations. Further research is needed to understand whether turkeys of different genetic lines differ in fear responses and whether there are quantitative trait loci (QTL) for OF responses of turkeys as there are for chickens and quail (reviewed in Forkman et al., 2007; Jensen 2014). Assessing whether individual animals are consistent in their responses to OF testing necessitates that the same animals be tested repeatedly in the same test. Therefore, animals may become habituated to testing conditions over time such that the OF test arena can no longer be considered a novel environment by the second and third tests. In addition to habituation, age-related changes in behavior may also influence test retest reliability. For example, early research with chickens suggested that behavior associated with predator avoidance, which is related to fearfulness, increases as birds age (Suarez and Gallup, 1983). Therefore, OF and other fear-related behavior may be more consistent at later ages owing to the development of fear-related behavior over time. Age and habituation due to repeated testing may influence OF test responses, but it is not possible to separate the effects of age from the effects of habituation (Erasmus and Swanson, 2014). The percentages of COMM turkeys that ambulated during OF testing decreased with each successive test (test 1: 67%, test 2: 63%, test 3: 43%). In contrast, the percentages of RB turkeys that ambulated increased from test 1 to test 2 but decreased from test 2 to test 3 (test 1: 33%, test 2: 70%, test 3: 57%). Ambulation is influenced by fear (Forkman et al., 2007). If repeated testing resulted in habituation, turkeys would be less fearful as a result of habitation, and the percentage of turkeys that ambulated during OF testing would be expected to increase with repeated testing. However, the percentages of COMM turkeys that ambulated decreased with each successive test, whereas the percentages of RB turkeys that ambulated increased from test 1 to test 3. Turkeys are much larger than other poultry species, which may limit at what ages turkeys can be tested in the OF test. The reduced percentages of COMM turkeys that ambulated with each successive test may be due to changes in physical ability and BW. No turkeys were tested that displayed observable signs of lameness or difficulty in walking in the home pen. However, as turkeys aged and their body conformation changed and BW increased, it may have become increasingly difficult for them to move around the OF test arena. Therefore, some turkeys that ambulated in OF test 1 and OF test 2 may have been less likely to ambulate during OF test 3 because of greater BW and not because of fearfulness. However, 43% of turkeys ambulated during testing at 11 wk and results were moderately correlated between 4 wk (when turkeys would have been more agile) and 11 wk. Further research examining whether physical ability changes over time, in addition to examining physiological stress responses to OF testing, may provide important insights into whether turkeys that ambulate less in the OF test do so because of difficulty walking or because of fear. A limitation of this study is that toe trimming was performed for COMM turkeys but not for RB turkeys because COMM turkeys were obtained from a COMM hatchery where they were subjected to standard hatchery procedures. Toe trimming in combination with heavier BWs may have affected the performance of COMM turkeys in OF tests, possibly resulting in greater correlations between OF test responses than correlations found for RB turkeys. Another limitation of this study is that the same numbers of COMM and RB turkeys could not be obtained at hatch, resulting in different group sizes until 7 wk, when 2 4 COMM turkeys were removed from each pen to be used in another study. Thus, group sizes were similar for COMM and RB turkeys from 7 wk onward. Removal of COMM turkeys may have influenced social and other behavior of COMM turkeys in the home pen. In addition to environmental effects during rearing, conditions before hatch may affect behavior subsequently. It is unknown how the environmental and rearing conditions of the flocks from which these turkeys were produced may have affected the behavior of turkeys in this study. Owing to the limitations of this study, including differences in prehatch conditions, toe trimming, and group sizes before 7 wk, OF behavior was not compared between COMM and RB lines. Open Field Responses and Meat Quality Measures To examine the relationship between OF responses and PM muscle ph and R-values, a cluster analysis was conducted using the average OF responses of all 3 OF tests to identify distinct behavioral groups of turkeys. Three distinct clusters were identified for

6 FEAR RESPONSES AND MEAT QUALITY OF TWO TURKEY LINES 2023 Figure 1. Dendogram (visual representation) of clusters of commercial (COMM) male turkeys (N = 27) based on responses in an open field test (latency to ambulate, latency to vocalize, number of steps taken, number of vocalizations produced, and number of defecations produced). Open field responses were calculated as the average response across 3 tests (test 1: 1 wk, test 2: 4 wk, test 3: 11 wk). Each turkey is represented by a number. COMM turkeys (Figure 1), and 2 distinct clusters were identified for RB turkeys (Figure 2). Statistical analyses (Kruskall Wallis tests) were conducted to verify that clusters of each genetic line differed significantly (Table 2). Initially, 3 clusters were tested for RB birds, but data from 2 of the clusters were merged into a single cluster, cluster 2 (Figure 2), because the OF responses of birds in these 2 clusters did not differ significantly from one another. The OF test measures fear responses but can also be used to assess activity levels. Birds that are less fearful (or more active) vocalize more and ambulate more and take less time to vocalize and ambulate compared to more fearful (less active) birds (reviewed in Forkman et al., 2007). Based on OF behavior, COMM turkeys in cluster 1 were more active and displayed behavior consistent with being less fearful compared to turkeys in clusters 2 and 3. Specifically, the latency to ambulate and latency to vocalize were lower for COMM turkeys in cluster 1. Turkeys in cluster 1 also took more steps and vocalized and defecated more compared to turkeys in other clusters. Behavioral responses of COMM turkeys in cluster 3 were intermediate, whereas COMM turkeys in cluster 2 were the least active and displayed behavior consistent with being more fearful. RB turkeys in cluster 1 were more active and displayed behavior indicative of less fearfulness compared to RB turkeys in cluster 2. Cluster differences in live weight at 11 wk, ph, and R-values are presented in Table 3 for COMM and RB turkeys. Within genetic lines, clusters did not differ sig- Figure 2. Dendogram (visual representation) of clusters of randombred (RB) male turkeys (N = 33) based on responses in an open field test (latency to ambulate, latency to vocalize, number of steps taken, number of vocalizations produced, and number of defecations produced). Open field responses were calculated as the average response across 3 tests (test 1: 1 wk, test 2: 4 wk, test 3: 11 wk). Each turkey is represented by a number. nificantly in live weight, ph, or R-values. Spearman rank correlation coefficients of ph and R-values and OF responses are presented in Table 4. No significant correlations were found between any OF responses and ph or R-values. Therefore, results do not support the hypothesis that turkeys that are more fearful in an OF test produce meat with a lower ph and higher R-value indicative of a faster rate of rigor mortis development. Within genetic lines, turkeys were not slaughtered on the same day. It is possible that PM ph and R-values may have been affected by the different slaughter times, reducing the ability to detect differences in ph and R-values between clusters. To account for possible slaughter-day effects, turkeys were randomly assigned to a particular slaughter day, and slaughter day was included as a random effect in statistical models. Therefore, it is unlikely that the differences in slaughter times significantly affected the variability between the ph and R-values of turkeys in different clusters. Few studies have been conducted to examine the relationship between fear responses and PM muscle characteristics or meat quality of poultry. However, our results are in agreement with those of Debut et al. (2003), who examined the association between meat quality and fear responses, measured using the tonic immobility test, of female broiler chickens. Tonic immobility responses were assessed 1 wk before slaughter (Debut et al., 2003). No association was found between tonic immobility and meat ph or color (Debut et al., 2003). In another study using quail from genetic lines selected for long (increased fear) and short (decreased fear)

7 2024 ERASMUS ET AL. Table 2. Differences in median (25 75% range) open field (OF) behavior among turkeys of a commercial (COMM) and randombred (RB) line that were grouped into distinct clusters based on open field responses. Median (25 75% range) OF response Latency to ambulate Latency to vocalize Step number Vocalization number Defecation number COMM turkeys (N = 27) Cluster (N = 8) ( ) a ( ) a ( ) a ( ) a ( ) a Cluster (N = 11) ( ) c ( ) c ( ) c ( ) c ( ) c Cluster (N = 8) ( ) b ( ) b ( ) b ( ) b ( ) b RB turkeys (N = 33) Cluster (N = 13) ( ) a ( ) a ( ) a ( ) a ( ) a Cluster (N = 20) ( ) b ( ) b ( ) b ( ) b ( ) b Open field responses were calculated as the average response across 3 tests (test 1: 1 wk, test 2: 4 wk, test 3: 11 wk). a,b,c For each genetic line (COMM or RB), median (25 75% range) values within columns differ (P < 0.05). Table 3. Spearman rank correlation (r S ) of breast muscle meat quality measures (ph and R-values measured at 15 min PM) and open field responses (average responses of open field tests conducted at 1, 4 and 11 wk) of turkeys of a commercial (COMM) and randombred (RB) genetic line. OF response ph R-value COMM turkeys (N = 27) Latency to ambulate Latency to vocalize Step number Vocalization number Defecation number RB turkeys (N = 33) Latency to ambulate Latency to vocalize Step number Vocalization number Defecation number P < Table 4. Breast muscle ph and R-value (measured at 15 min PM) of turkeys of a commercial (COMM) and randombred (RB) line that were grouped into distinct clusters based on open field responses. Cluster Live weight (11 weeks) ph R-value COMM turkeys (N = 27) Cluster 1 (N =8) 8.56± ± ± 0.06 Cluster 2 (N = 11) 8.60 ± ± ± 0.06 Cluster 3 (N =8) 8.75± ± ± 0.06 P-value RB turkeys (N = 33) Cluster 1 (N = 13) 4.45 ± ± ± 0.04 Cluster 2 (N = 20) 4.45 ± ± ± 0.03 P-value Open field responses were calculated as the average response across 3 tests (test 1: 1 wk, test 2: 4 wk, test 3: 11 wk). durations of tonic immobility, respectively, the ph measured at 24 h post-slaughter was significantly higher in quail from the long tonic immobility line when quail were exposed to an acute stressor before slaughter (Remignon et al., 1998). We only measured the initial ph and R-value of the turkey breast meat, and it is possible that other measures of meat quality, such as the ultimate ph, tenderness, or drip loss, may differ between turkeys that differ in fear responses. Another possible explanation for the discrepancies between our results and those of Remignon et al. (1998) and between those of Debut et al. (2003) and Remignon et al. (1998) may be that meat quality differences between birds differing in fear responses are more apparent when birds are exposed to acute stress before slaughter. Although Debut et al. (2003) examined the effects of different preslaughter stressors on meat quality, their study did not evaluate whether chickens that differed in tonic immobility responses differed in their responses to preslaughter stress. Similarly, our study did not assess the effects of preslaughter stress on meat quality, nor did we examine differences in preslaughter stress responses between turkeys in different clusters. Turkeys in our study were likely exposed to acute stress before slaughter, such as handling, crating, and transportation, but the conditions associated with our study may not have elicited large enough stress responses for differences in meat quality between clusters to become apparent. Turkeys were transported a distance of 5.7 km with a duration of 10 to 15 min, and research by Owens and Sams (2000) found that transporting turkeys for as long as 3 h did not adversely affect meat quality. Furthermore, differences between clusters may not have been apparent in turkeys that were processed last because stress levels due to catching, crating, and transportation had returned to lower levels by the time these turkeys were processed. Another explanation for the differences between studies is that the relationship between muscle characteristics related to meat quality and fear responses may depend on the type of fear test used. The OF test was used in the present study because previous research demonstrated that turkeys ambulatory behavior in the

8 FEAR RESPONSES AND MEAT QUALITY OF TWO TURKEY LINES 2025 OF test was repeatable when measured weeks apart, whereas tonic immobility measures associated with vocalization, but not tonic immobility duration, were repeatable (Erasmus and Swanson, 2014). Vocalizations have not been measured as an indicator of fear per se, whereas tonic immobility duration is generally believed to be an indicator of fear (see Forkman et al., 2007). Moreover, tonic immobility requires that turkeys be placed on their side, which is difficult for large turkeys such as those used in this study, and may risk injury both to the birds and the observer. There may be important species differences in how birds react to fear tests and how this is related to meat quality. Comparison of Muscle ph and R-Value between Genetic Lines Genetic lines differed in live weight at 11 wk (COMM: 8.63 ± 0.10; RB: 4.45 ± 0.09; F 48 = , P < ), which was expected because COMM turkeys have been genetically selected for improved feed efficiency and increased BW. However, ph (COMM: 6.03 ± 0.05; RB: 5.98 ± 0.04, F 48 = 0.56, P = 0.46) and R-value (COMM: 1.18 ± 0.04; RB: 1.23 ± 0.03; F 48 = 1.08; P = 0.30) did not differ between genetic lines. Chiang et al. (2008) reported a higher ph (5.90) at 15 min PM in breast muscle from a COMM turkey line compared to the breast muscle ph (5.64) of the same RB line used here. In our study, the ph of the COMM line (6.03 ± 0.05) was numerically, but not statistically, higher compared to the RB line (5.98 ± 0.04). The ages at which turkeys were processed were similar between our study and that of Chiang et al. (2008), who processed turkeys of the RB line at 22 wk and turkeys from the COMM line at 16 wk. Differences between results reported here and those of Chiang et al. (2008) may be due to the COMM turkey line used. It is not stated whether the COMM line used in the study by Chiang et al. (2008) was the same as the line used here. Another study comparing the same RB line that was used here with a COMM line at 16, 18, and 22 wk found no differences in ph (Updike et al., 2005). However, the ph was measured 2 h post-slaughter (Updike et al., 2005). Therefore, results are not directly comparable to those reported here or those of Chiang et al. (2008). Several studies with other turkey genotypes have reported nodifferencesintherateofphdecline(yostetal., 2002) or initial ph (Werner et al., 2008). Overall, our results are consistent with most other studies showing no difference in initial PM ph from meat of slowergrowing vs. faster-growing turkey lines. There is scant research examining differences in R-values between RB and COMM turkey lines, but the development of early PM rigor mortis greatly affects poultry meat quality (Le Bihan-Duval, 2004). Further research is needed to examine the relationship between turkey lines and meat quality parameters, including R-value. CONCLUSION Our results demonstrated that ambulatory behavior intheoftestofmaleturkeysofarblinewererepeatable between 4 and 11 wk because moderate correlations were found for the latency to ambulate and the number of steps taken. In contrast, all OF responses of male turkeys of a COMM line were repeatable between 4 and 11 wk. Breast muscle ph and R-values did not differ among groups of turkeys that differed in OF behavior, indicating that OF behavior (measured during rearing) is not associated with meat quality characteristics. Regarding initial PM ph, our results are in agreement with most other studies indicating little difference in PM muscle metabolism between turkeys of faster-growing vs. slower-growing lines. Several areas for future research have been identified: 1) examining the effects of acute stress before slaughter on PM muscle characteristics of turkeys; 2) evaluating whether the relationship between fearfulness and meat quality depends on the type of test used; 3) evaluating poultry species differences in fearfulness and meat quality; and 4) examining the relationship between fearfulness and other turkey meat quality characteristics, such as tenderness, color, and water-holding capacity. ACKNOWLEDGMENTS We thank Hybrid Turkeys (Kitchener, ON, Canada) and Cuddy Farms (Strathroy, ON, Canada) for supplying the poults. Many thanks to Darrin Karcher, Angelo Napolitano, and the Michigan State University Poultry Research and Teaching Center staff (Michigan State University, East Lansing, MI) for their assistance. Furthermore, we thank Thanikarn Sansawat, Pranjal Singh, and the student members of the Michigan State University Animal Behavior and Welfare Group (Michigan State University, East Lansing, MI) for assistance with data collection. M.E. was supported by a Natural Sciences and Engineering Research Council of Canada postgraduate scholarship. We further thank the National Research Foundation of Korea (2012R1A6A3A , Seoul, South Korea) and Michigan State University AgBioResearch for providing funding. REFERENCES Alvarado, C. Z., and A. R. Sams Rigor mortis development in turkey breast muscle and the effect of electrical stunning. Poult. Sci. 79: Armario, A., N. Daviu, C. Munoz-Abellan, C. Rabasa, S. Fuentes, X. Belda, H. Gagliano, and R. 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