Bacterial chondronecrosis with osteomyelitis in broilers: Influence of sires and straight-run versus sex-separate rearing

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1 Bacterial chondronecrosis with osteomyelitis in broilers: Influence of sires and straight-run versus sex-separate rearing R. F. Wideman Jr.,* 1 A. Al-Rubaye, D. Reynolds,* D. Yoho,* H. Lester,* C. Spencer,* J. D. Hughes Jr., and I. Y. Pevzner * Division of Agriculture, and Department of Biological Sciences, University of Arkansas, Fayetteville 72701; and Cobb-Vantress Inc., Siloam Springs, AR ABSTRACT Two experiments (E1, E2) were conducted to compare the influence of sires (sire A on dam C vs. sire B on dam C) and straight-run versus sex-separate rearing on the incidence of bacterial chondronecrosis with osteomyelitis (BCO) in broilers. Fertile eggs from commercial breeder flocks were incubated and hatched at the University of Arkansas Poultry Research Hatchery. Male and female chicks were reared together (straight-run) or separately (sex-separate) in 3 3 m pens on litter or flat wire flooring with 65 (E1) or 60 (E2) birds per pen. Necropsies revealed lesions that are pathognomonic for BCO in 98% of the birds that became lame. The SigmaStat Z-test was used to compare cumulative BCO incidences through 8 wk of age. For birds reared on litter, the incidences of BCO were low regardless of cross or sex (range: 1.7 to 5.1%; P 0.6). Within a cross and sex, rearing the broilers straight-run versus sex-separate on wire flooring did not significantly affect the incidence of BCO. Significant incidences INTRODUCTION Bacterial chondronecrosis with osteomyelitis (BCO) is a prevalent cause of lameness in commercial broiler flocks, with most cases developing in large birds after 35 d of age (Nairn and Watson, 1972; McNamee et al., 1998; Butterworth, 1999; McNamee and Smyth, 2000; Bradshaw et al., 2002). Lesions associated with BCO occur most commonly in the proximal tibiotarsus (tibia) and proximal femur where the wide, thick growth plates are susceptible to mechanical damage (osteochondrosis) that predisposes the cartilage to secondary bacterial infection (Nairn and Watson, 1972; Howlett, 1980; Mutalib et al., 1983a,b; Thorp and Duff, 1988; of BCO did not develop until after d 40. Males from the sire A cross developed a higher incidence of BCO than males from the sire B cross in E1 (27 vs. 17%, respectively; P = 0.009) but not in E2 (28.5 vs. 22.6%, respectively; P = 0.141). In both experiments, males from the sire A cross developed higher incidences of BCO than females from the sire B cross (27 vs. 11.9%, in E1; 28.5 vs. 14.8%, in E2). With the sexes pooled, broilers from the sire A cross consistently developed higher incidences of BCO than broilers from the sire B cross (21.4 vs. 14.9%, P = in E1; 26.5 vs. 18.7%, P = in E2). High susceptibilities to both femoral head (all femoral head necrosis = 66 to 85% incidences) and tibial head (all tibial head necrosis = 81 to 96% incidences) BCO lesions were demonstrated in lame birds from both sexes and crosses. This study supports a sire influence on the susceptibility of broilers to BCO. Sire lines can be chosen to reduce BCO susceptibility when broilers are grown beyond 6 wk of age. Key words: broiler lameness, bacterial chondronecrosis with osteomyelitis, wire floor model, sire line 2014 Poultry Science 93 : Poultry Science Association Inc. Received January 16, Accepted February 25, Corresponding author: rwideman@uark.edu McNamee and Smyth, 2000; Wideman and Prisby, 2013). Rearing broilers on flat or sloping wire flooring triggers high incidences of BCO without purposefully exposing the birds to pathogenic bacteria (Wideman et al., 2012, 2013; Gilley et al., 2014). The unstable footing created by wire flooring apparently accelerates the formation of osteochondrotic micro-fractures and clefts in the proximal epiphyseal-physeal growth plates of the femora and tibiae. Wire flooring (or the lack of access to litter) also creates physiological stress that can lead to immunosuppression and enhanced bacterial translocation from the gastrointestinal tract (El-Lethey et al., 2003; Quinteiro-Filho et al., 2012a,b; Wideman and Pevzner, 2012; Wideman and Prisby, 2013). Opportunistic bacteria pass through the intestinal epithelium and spread hematogenously to micro-fractures in the proximal femoral and tibial growth plates. Bacterial proliferation and the ensuing immunological response generate the necrotic abscesses and voids that 1675

2 1676 Wideman et al. are pathognomonic for BCO (Nairn and Watson, 1972; McNamee et al., 1999; Wideman et al., 2012; Wideman and Prisby, 2013). The wire flooring model imposes a rigorous, sustained challenge that consistently reveals innate susceptibility to BCO in experimental flocks. Genotype and selection for growth performance have been implicated in the susceptibility of broilers and turkeys to leg disorders and impaired walking ability (Leach and Nesheim, 1972; Riddell, 1973, 1976; Kestin et al., 1992, 1999, 2001; Sorensen, 1992; Hester, 1994; Kuhlers and McDaniel, 1996; Nestor and Anderson, 1998; Julian, 2005). In a previous study, the wire flooring model was used to compare the potential impact of genotype on the susceptibility of broiler crosses to BCO (Wideman et al., 2013). Four commercially available crosses were evaluated in 2 experiments, including 2 standard crosses that grow rapidly at an early age and 2 highyield crosses that initially tend to grow more slowly. Males and females were reared together (straight-run) in the first experiment and sex-separate in the second experiment. Within each cross females reared straightrun developed only modestly lower incidences of BCO than their male hatch mates, whereas females reared sex-separate had markedly lower BCO incidences than males. These observations tentatively suggested that female broilers might experience less competitive stress when reared separately from males, thereby potentially attenuating a source of chronic stress that otherwise might aggravate the pathogenesis of BCO. In both experiments, the standard broiler crosses developed higher incidences of lameness attributable to BCO than the high-yield crosses (Wideman et al., 2013), results that match the experience of broiler integrators who have used these crosses. As a caveat, integrators also suggested that, regardless of initial growth performance, broilers derived from crossing sire A on dam C are more likely to develop higher incidences of BCO than broilers derived from crossing sire B on dam C (R. F. Wideman, personal communications). Accordingly, 2 experiments (E1, E2) were conducted to evaluate the influence of the sire line on the susceptibility of broiler crosses to BCO, to quantify the lesion locations and categories associated with BCO in both sire crosses, and to compare the BCO incidences of males and females reared straight-run versus sex-separate. MATERIALS AND METHODS Experimental Setup Animal procedures were approved by the University of Arkansas Institutional Animal Care and Use Committee (Protocols #08036 and #11002). Two experiments (E1 and E2) were conducted using 24 pens in building A364 East at the University of Arkansas Poultry Research Farm. The pens were 3.05 m 3.05 m with flooring consisting of clean wood shavings litter (pens 1, 6, 12, 13, 18, and 24) or flat wire flooring wire panels (all remaining pens). The wire flooring panels were constructed from 5 cm 5 cm lumber and were 3 m long and 1.5 m wide, with 5 cm 5 cm cross members added for support. Hardware cloth (1.3 cm 2.54 cm mesh = 0.5 inch 1 inch, gauge, galvanized welded wire cloth; Direct Metals, Kennesaw, GA) was fastened to the top of the frame and cross-members. Two panels per pen were elevated on 30 cm high masonry blocks to permit manure to pass through and accumulate underneath the wire surface. Tube feeders were positioned at the front, and nipple waterers were positioned at the rear of each pen, thereby forcing the chicks to traverse the length of the floor to eat and then drink (Wideman et al., 2012, 2013). Between experiments the pens were disassembled, the manure was removed, and the floor panels were cleaned using a pressure washer and detergent. Fertile eggs for the sire A dam C cross and the sire B dam C cross were obtained from commercial breeder flocks and were incubated at the University of Arkansas Poultry Research Farm Hatchery. The chicks hatched and were vent-sexed on 1/25/2013 for E1, and on 9/27/2013 for E2. Three litter pens and 9 wire flooring pens were assigned per sire cross. The chicks were placed unvaccinated in pens according to sire cross (A, B) and sex: sex-separate females (1 litter and 3 wire pens per sire); sex-separate males (1 litter and 3 wire pens per sire); and, straight-run females with males (1 litter and 3 wire pens per sire). Eighty chicks initially were placed per pen. On d 14 the smaller chicks were culled to reduce bird numbers to 65 per pen in E1 or 60 per pen in E2. The early culling protocol was instituted after necropsies of runts and culls during the first 2 wk revealed macroscopic evidence of systemic bacterial infection including osteomyelitis (Wideman et al., 2012). The photoperiod was set for 23 h light per day. Thermoneutral temperatures were maintained throughout with target temperatures set at 32 C for d 1 to 3, 30 C for d 4 to 6, 28 C for d 7 to 10, 26 C for d 11 to 14, and 24 C thereafter. A heat lamp was positioned in one corner of each pen to provide a source of supplemental heat through d 28. Feed and water were provided ad libitum. The starter diet was a commercial corn and soybean-meal based chick starter (crumbles), and after d 35 all birds were switched to a pelleted commercial corn and soybean-meal-based finisher diet. Feed was formulated without meat or animal byproducts to meet or exceed minimum NRC (1994) standards for all ingredients. Beginning on d 15, the birds were observed daily to detect the onset of lameness. Affected broilers had difficulty standing, sat down quickly, exhibited an obvious limping gait while dipping one or both wing tips, and if not removed, became completely immobilized within 3 d. As soon as the onset of lameness was noticed, affected birds were humanely euthanized and necropsied within 20 min postmortem. Lame birds with BCO can die quickly because they have difficulty accessing food and water, and they are picked on and trampled by their flock mates. Therefore birds found dead also

3 SIRE CROSS AND BACTERIAL CHONDRONECROSIS WITH OSTEOMYELITIS 1677 Table 1. Necropsy categories for broilers in experiment 1 that died or became lame through 56 d of age: incidences within each diagnostic category 1 for females (F) and males (M) from the A C cross or the B C cross 1 Sire (A or B) and sex (M or F) PHS SDS KB TW were necropsied to ascertain the cause of death and assess leg lesions. Each bird s sex was confirmed by necropsy. Broilers that died spontaneously or that developed lameness were assigned to one of the following diagnostic categories: cull (runts and moribund individuals that failed to thrive); UNK (unknown cause of death); SDS (sudden death syndrome, flipover, heart attacks); PHS (pulmonary hypertension syndrome, ascites); KB (kinky back or spondylolisthesis: diagnosed based on the characteristic posterior paraparesis and hock-resting posture, and the absence of other macroscopic BCO lesions; Wise, 1971); TW (twisted leg or slipped tendon, perosis, chondrodystrophy); TD (tibial dyschondroplasia); lame-unk (lame for unknown or undetermined reasons); normal F (no macroscopic abnormalities of the proximal femur); FHS (proximal femoral head separation or epiphyseolysis); FHT (proximal femoral head transitional degeneration); FHN (proximal femoral head necrosis); normal T (no macroscopic abnormalities of the proximal tibia); THN (mild proximal tibial head necrosis, a subcategory of BCO in the tibiotarsus); THNs (severe THN in which the growth plate was imminently to threatened or damaged); and THNc (caseous THN in which caseous exudates or bacterial sequestrae were macroscopically evident). Previously published photographs illustrate typical BCO lesions of the proximal femora TD Lame- UNK and tibiae (Wideman et al., 2012; Wideman and Prisby, 2013). Proximal femoral head lesions (FHS, FHT, FHN) and tibial head lesions (THN, THNs, THNc) were categorized separately to emphasize the progressive development of BCO in the proximal ends of both long bones of the legs (Mutalib et al., 1983a; Thorp and Waddington, 1997; McNamee et al., 1998, 1999; Butterworth, 1999; McNamee and Smyth, 2000; Dinev, 2009; Durairaj et al., 2009; Wideman et al., 2012, 2013; Wideman and Pevzner, 2012). Proximal femora and tibiae that appeared to be normal macroscopically were not routinely evaluated microscopically. The birds were not weighed during these experiments to avoid the nonuniform stress associated with catching, handling, and weighing. Stress promotes bacterial translocation and BCO (Quinteiro-Filho et al., 2012a,b; Wideman and Pevzner, 2012). Previous studies have demonstrated that lameness is minimal through wk 6 when fast growing broilers reared on wire flooring are culled heavily on d 14 and are not weighed during the experiment (R. F. Wideman, personal observations). Statistical Analysis All BCO lame Total lame A, F 0.8 (3/390) 2.8 (11/390) (53/390) bc 13.6 (53/390) bc A, M 2.3 (9/390) 5.4 (21/390) (2/390) (80/390) a 21.0 (82/390) a A, F+M 1.5 (12/780) 4.1 (32/780) (2/780) (133/780) ab 17.3 (135/780) ab B, F 2.3 (9/390) 2.1 (8/390) (38/390) c 9.7 (38/390) c B, M 2.1 (8/390) 2.8 (11/390) (2/390) (57/390) b 15.1 (59/390) b B, F+M 2.2 (17/780) 2.4 (19/780) (2/780) (95/780) bc 12.4 (97/780) bc a c Values with different superscripts within a category differed significantly at P 0.05; z-tests were used to compare proportions. 1 Data (% of n starting on d 14) are grouped by sire and sex independent of floor treatment (litter and wire flooring pens pooled). PHS: pulmonary hypertension syndrome (ascites); SDS: sudden death syndrome; KB: kinky back or spondylolisthesis; TW: twisted leg, perosis; TD: tibial dyschondroplasia; lame-unk: cause of lameness could not be determined = unknown; all BCO lame = lameness was attributed to proximal femoral or tibial heads with bacterial chondronecrosis with osteomyelitis lesions; total lame = KB + TW + TD + lame-unk + all BCO. The total incidence of femoral BCO lesions was calculated as all FH = FHS + FHT + FHN. The total incidence of tibial BCO lesions was calculated as all Table 2. Necropsy categories for broilers in experiment 2 that died or became lame through 56 d of age: incidences within each diagnostic category for females (F) and males (M) from the A C cross or the B C cross 1 Sire (A or B) and sex (M or F) PHS SDS KB TW TD Lame- UNK All BCO lame Total lame A, F (2/360) 0.3 (1/360) (1/360) (68/360) ab 19.4 (70/360) ab A, M 0.3 (1/360) 1.1 (4/360) 0.3 (1/360) 0.3 (1/360) 0.3 (1/360) (78/360) a 22.5 (81/360) a A, F+M 0.1 (1/720) 0.8 (6/720) 0.3 (2/720) 0.1 (1/720) 0.3 (2/720) (146/720) a 21.0 (151/720) a B, F 1.3 (4/360) 1.1 (4/360) (1/360) 11.7 (42/360) b 11.9 (43/360) b B, M 0.6 (2/360) 1.9 (7/360) (66/360) ab 18.3 (66/360) ab B, F+M 0.8 (6/720) 1.5 (11/720) (1/720) 15.0 (108/720) b 15.1 (109/720) b a,b Values with different superscripts within a category differed significantly at P 0.05; z-tests were used to compare proportions. 1 Data (% of n starting on d 14) are grouped by sire and sex independent of floor treatment (litter and wire flooring pens pooled). PHS: pulmonary hypertension syndrome (ascites); SDS: sudden death syndrome; KB: kinky back or spondylolisthesis; TW: twisted leg, perosis; TD: tibial dyschondroplasia; lame-unk: cause of lameness could not be determined = unknown; all BCO lame = lameness was attributed to proximal femoral or tibial heads with bacterial chondronecrosis with osteomyelitis lesions; total lame = KB + TW + TD + lame-unk + all BCO.

4 1678 Wideman et al. Table 3. Incidence of bacterial chondronecrosis with osteomyelitis lameness in experiment 1 by floor treatment, sex, and cross for females (F) and males (M) from the A C cross or the B C cross that were reared sex-separate or straight-run (% of n starting on d 14) Floor Sex Sire A Sire B P-value (sire A vs. B) Litter F (all) 6.2 (6/98) 4.1 (4/98) Litter M (all) 4.1 (4/98) 6.2 (6/98) Litter F+M (all) 5.1 (10/196) 5.1 (10/196) Wire F (all) 15.7 (46/293) b 11.9 (35/293) y Wire M (all) 27.0 (79/293) a 17.7 (52/293) x Wire F+M (all) 21.4 (125/586) ab 14.9 (87/586) xy Wire F (straight-run) 16.4 (16/98) 11.3 (11/98) Wire F (sex-separate) 15.4 (30/196) 12.3 (24/196) Wire M (straight-run) 32.8 (32/98) 18.5 (18/98) Wire M (sex-separate) 24.1 (47/196) 17.4 (34/196) a,b Values with different superscripts within a floor category and sire differed significantly at P x,y Values with different superscripts within a category differed significantly at P > 0.05, THN = THN + THNs + THNc. The total incidence of lameness was calculated as: total lame = KB + TW + TD + lame UNK + all BCO. For comparisons of lesion and lameness incidences, the individual bird was used as the experimental unit, and the SigmaStat Z-test procedure was used to compare proportions (Jandel Scientific, 1994, San Rafael, CA). RESULTS Chick mortality and culling averaged less than 1.5% per cross through d 14 in both experiments. Between d 14 and 56, 5 birds died from unknown causes or were culled for poor performance in both experiments, and the remaining nonlame mortality was attributable to PHS and SDS (Tables 1 and 2). Within each experiment the incidences of PHS and SDS did not differ by sire or sex. The incidences of KB (spondylolisthesis), TW, TD, and lame UNK were 0.5% in both experiments. Several birds that initially appeared to have KB instead had severe bilateral BCO lesions of the proximal femora. Accordingly, total lameness in both experiments (floor treatments pooled) was overwhelmingly attributable to BCO (Tables 1 and 2). The incidence of BCO on litter flooring was low and did not differ between the sire crosses or sexes in either experiment (Tables 3 and 4). Figure 1 illustrates the time course of cumulative BCO lameness for broilers reared in pens with wire flooring. Significant levels of BCO did not develop until after d 40. On d 50 in E1 and d 49 in E2 males from the sire A cross had higher cumulative BCO incidences than females from the sire B cross. By d 56 the BCO incidence for males from the sire A cross was higher than the incidence for males from the sire B cross in E1 but not in E2. In both experiments, males from the sire A cross had higher incidences of BCO than females from the sire B cross on d 56 (Figures 1 and 2). With the sexes pooled, broilers from the sire A cross consistently developed higher incidences of BCO on wire flooring than broilers from the sire B cross (Figure 2, Tables 3 and 4). Within each cross and sex, rearing the broilers straight-run versus sex-separate on wire flooring did not affect the incidence of BCO (Figure 3, Tables 3 and 4). Figures 4 and 5 illustrate the BCO lesion incidences within the proximal femoral head and proximal tibial head diagnostic categories for broilers that became lame on wire flooring in E1 and E2, respectively. The Table 4. Incidence of bacterial chondronecrosis with osteomyelitis lameness in experiment 2 by floor treatment, sex, and cross for females (F) and males (M) from the A C cross or the B C cross that were reared sex-separate or straight-run (% of n starting on d 14) Floor Sex Sire A Sire B P-value (sire A vs. B) Litter F (all) 2.2 (2/90) 2.2 (2/90) Litter M (all) 1.1 (1/90) 5.6 (5/90) Litter F+M (all) 1.7 (3/180) 3.9 (7/180) Wire F (all) 24.4 (66/270) 14.8 (40/270) b Wire M (all) 28.5 (77/270) 22.6 (61/270) a Wire F+M (all) 26.5 (143/540) 18.7 (101/540) ab Wire F (straight-run) 21.1 (19/90) 15.6 (14/90) Wire F (sex-separate) 26.1 (47/180) 14.4 (26/180) Wire M (straight-run) 22.2 (20/90) 25.6 (23/90) Wire M (sex-separate) 31.7 (57/180) 21.1 (38/180) a,b Values with different superscripts within a floor category and sire differed significantly at P 0.05; z-tests were used to compare proportions.

5 SIRE CROSS AND BACTERIAL CHONDRONECROSIS WITH OSTEOMYELITIS 1679 Figure 1. Time course of cumulative bacterial chondronecrosis with osteomyelitis (BCO) lameness for female (F) and male (M) broilers from the sire A cross or sire B cross that were reared in pens with wire flooring in experiment 1 (upper panel) or experiment 2 (lower panel). Values are calculated as the percentage of the total number of birds on wire flooring per cross on d 14 (n = 293 or 270 per sex and sire in experiments 1 and 2, respectively). Values with different letters (a c) differed on d 50 (experiment 1), d 49 (experiment 2), or d 56 (P 0.05; SigmaStat Z-test). values for these incidences also are provided in Tables 5 and 6. Lameness clearly was associated with comparably high susceptibilities to both femoral head (all FH = 66 to 85% incidences) and tibial head (all THN = 81 to 96% incidences) lesions in lame birds from both sexes and crosses. Males from the sire B cross tended to have low incidences of FHT and THNc lesions (Figure 4), whereas females from the sire A cross tended to have the high incidences of FHT and THNc lesions (Figure 5). Necropsies of large 7- to 8-wk-old broilers exhibiting the earliest symptoms of lameness nevertheless occasionally revealed the presence of very serious THNc

6 1680 Wideman et al. Figure 2. Cumulative bacterial chondronecrosis with osteomyelitis (BCO) lameness on d 56 for all female (F) or all male (M) broilers, and for F and M combined, from the sire A cross or sire B cross that were reared in pens with wire flooring in experiment 1 (upper panel) or experiment 2 (lower panel). Values are calculated as the percentage of the total number of birds per sire cross and sex on d 14. Values with different letters (a c) differed among crosses and sexes (n = 293 or 270 per sex and sire in experiments 1 and 2, respectively; P 0.05; SigmaStat Z-test). Values with different letters (x,y) differed among sire crosses with the sexes combined (n = 586 or 540 per sex and sire in experiments 1 and 2, respectively; P 0.05; SigmaStat Z-test).

7 SIRE CROSS AND BACTERIAL CHONDRONECROSIS WITH OSTEOMYELITIS 1681 Figure 3. Cumulative bacterial chondronecrosis with osteomyelitis (BCO) lameness on d 56 for female (F) or male (M) broilers from the sire A cross or sire B cross in pens with wire flooring that were reared sex-separate (F or M) or straight-run (F with M, or M with F) in experiment 1 (upper panel) or experiment 2 (lower panel). Values with different letters (a,b) differed among crosses and sexes (n = 98 or 90 per sex and sire for straight-run, and n = 196 or 180 per sex and sire for sex separate in experiments 1 and 2, respectively; P 0.05; SigmaStat Z-test). lesions (Figure 6). These lesions either were extremely aggressive in their rate of development, or perhaps subclinically affected birds were capable of behaviorally masking the presence of progressive tibial damage over the course of days or weeks. DISCUSSION In both experiments, the chicks were incubated and hatched together at the University of Arkansas Poultry Research Farm. Transportation stress was avoided

8 1682 Wideman et al. Figure 4. Incidences within proximal femoral head diagnostic categories for female (F) and male (M) broilers from the sire A cross or sire B cross that became lame through d 56 while being reared on wire flooring. The proximal femoral heads of both legs were evaluated and were diagnosed as being macroscopically normal (normal femur; no apparent abnormalities) or they exhibited FHS (femoral head separation), FHT (femoral head transitional degeneration), or FHN (femoral head necrosis). Total femoral head lesions (all FH = FHS+FHT+FHN). Values reflect the percentages of all legs evaluated; right and left legs pooled. Values with different letters (a,b) differed between sires and sexes within a diagnostic category (P 0.05; SigmaStat Z-test). ns Values did not differ within a category (P 0.05; SigmaStat Z-test). by transferring the vent-sexed chicks directly to their pens on the same farm. Additional attempts to minimize stress included not wing banding, vaccinating, or weighing the birds. All pens were culled rigorously on d 14 to ensure that only the fastest growing, clinically healthiest chicks would contribute to the final outcome. If these precautions are effective, then lameness attributable to BCO is assumed to reflect the innate

9 SIRE CROSS AND BACTERIAL CHONDRONECROSIS WITH OSTEOMYELITIS 1683 Figure 5. Incidences within proximal tibial head diagnostic categories for female (F) and male (M) broilers from the sire A cross or sire B cross that became lame through d 56 while being reared on wire flooring. The proximal tibial heads of both legs were evaluated and were diagnosed as being macroscopically normal (no apparent abnormalities) or they exhibited THN (mild proximal tibial head necrosis, a subcategory of bacterial chondronecrosis with osteomyelitis in the tibiotarsus), THNs (severe THN in which the growth plate was imminently threatened or damaged), THNc (caseous THN in which caseous exudates or bacterial sequestrae were macroscopically evident), or TD (tibial dyschondroplasia). Total tibial head lesions (all THN = THN + THNs + THNc + TD). Values reflect the percentages of all legs evaluated; right and left legs pooled. Values with different letters (a,b) differed between sires and sexes within a diagnostic category (P 0.05; SigmaStat Z-test). ns Values did not differ within a category (P 0.05; SigmaStat Z-test). characteristics of the broilers being evaluated, with significant incidences occurring predominately after the fifth week posthatch (Wideman et al., 2012, 2013). Indeed, in the present study lameness attributable to BCO remained negligible until approximately d 40, after which the cumulative incidences accelerated dramatically. The sires clearly influenced the susceptibility of their progeny to BCO, with broilers derived from

10 1684 Wideman et al. Table 5. Proximal femoral and tibial necropsy incidences within each category in experiment 1 for lame females (F) and males (M) from the A C cross or the B C cross (% of all legs evaluated) 1 Sire (A or B) and sex (M or F) (n legs) Normal femora FHS FHT FHN FHS+FHT +FHN Normal tibiae THN THNs THNc THN+THNs +THNc A, F (132) ab ab 39.1 a 85.9 A, M (154) ab b 28.5 ab 81.0 B, F (80) a ab 25.7 ab 88.6 B, M (122) b a 19.2 b 87.5 a,b Values with different superscripts within a category differed significantly at P 0.05; z-tests were used to compare proportions for all legs evaluated. 1 Proximal femoral heads of both legs were evaluated and were diagnosed as being macroscopically normal (normal femora; no apparent abnormalities) or they exhibited FHS (femoral head separation), FHT (femoral head transitional degeneration), or FHN (femoral head necrosis). Total femoral head bacterial chondronecrosis with osteomyelitis (BCO) lesions = FHS+FHT+FHN. Proximal tibial heads were diagnosed as being macroscopically normal (normal tibiae; no apparent abnormalities) or they exhibited THN (mild proximal tibial head necrosis, a subcategory of BCO in the tibiotarsus), THNs (severe THN in which the growth plate was imminently threatened or damaged), or THNc (caseous THN in which caseous exudates or bacterial sequestrae were macroscopically evident). Total tibial head BCO lesions = THN + THNs + THNc. sire A being more likely to develop higher incidences than broilers derived from sire B (Figure 2, sexes combined). The male progeny from sire A consistently developed significantly higher incidences of BCO than the female progeny from sire B. The biological basis for this sire contribution remains to be determined. In previous studies, the susceptibility of poultry to leg disorders and lameness has been associated with growth performance (see Introduction). For example, standard broiler crosses that grow rapidly at an early age were shown to develop higher incidences of BCO than highyield crosses that initially tend to grow more slowly. However, by d 56 the standard broiler crosses did not have higher BW when compared with their companion yield crosses within a sex (Wideman et al., 2013). Body weights were not recorded during the present study, but prior experience has shown that, regardless of sire, the females have a modestly slower growth rate and a lower final BW than their male hatch mates, whereas within a sex the final BW differs minimally between the progeny of sires A and B mated on dam C. Additional research will be required to further clarify the innate factors responsible for triggering BCO, but at the present time we are not convinced that patterns of early growth or final BW made major contributions to the different cumulative incidences of BCO between the progeny of sires A and B within a sex. It is clear that sires can be chosen to reduce the incidence of BCO when broilers are grown beyond 6 wk of age to heavy yield weights. As summarized previously, BCO lesions develop predominately in the proximal ends of the femora and tibiae, which likely reflects the enhanced susceptibility to osteochondrosis within the elongated columns of unmineralized hypertrophic chondrocytes in the faster growing proximal growth plates when compared with the more slowly growing distal growth plates of the leg bones (Church and Johnson, 1964; Thorp and Duff, 1988; Kirkwood et al., 1989a,b; Wideman and Prisby, 2013). By quantifying the locations (e.g., proximal tibial vs. proximal femoral) and relative severity of BCO lesions (e.g., FHS vs. FHN, or THN vs. THNc), it was hoped that unique lesion patterns might help differentiate biological traits associated with susceptibility, such as leg conformation, stance, gait, or overall body morphometrics. Previous studies demonstrated that proximal femoral and tibial lesion incidences in lame broilers reared on wire flooring differed only minimally for left versus right leg comparisons (Wideman et al., 2012, 2013), as was true in the present study (not shown). Table 6. Proximal femoral and tibial necropsy incidences within each category in experiment 2 for lame females (F) and males (M) from the A C cross or the B C cross (% of all legs evaluated) 1 Sire (A or B) and sex (M or F) (n legs) Normal femora FHS FHT FHN FHS+FHT +FHN Normal tibiae THN THNs THNc THN+THNs +THNc A, F (132) 15.2 y a y a 93.9 A, M (154) 18.8 xy ab xy b 90.3 B, F (80) 20.0 xy ab xy ab 96.2 B, M (122) 25.4 x b y ab 93.4 a,b Values with different superscripts within a category differed significantly at P x,y Values with different superscripts within a category differed significantly at P > 0.05, Proximal femoral heads of both legs were evaluated and were diagnosed as being macroscopically normal (normal femora; no apparent abnormalities) or they exhibited FHS (femoral head separation), FHT (femoral head transitional degeneration), or FHN (femoral head necrosis). Total femoral head bacterial chondronecrosis with osteomyelitis (BCO) lesions = FHS+FHT+FHN. Proximal tibial heads were diagnosed as being macroscopically normal (normal tibiae; no apparent abnormalities) or they exhibited THN (mild proximal tibial head necrosis, a subcategory of BCO in the tibiotarsus), THNs (severe THN in which the growth plate was imminently threatened or damaged), or THNc (caseous THN in which caseous exudates or bacterial sequestrae were macroscopically evident). Total tibial head BCO lesions = THN + THNs + THNc.

11 SIRE CROSS AND BACTERIAL CHONDRONECROSIS WITH OSTEOMYELITIS 1685 Both the present and previous studies revealed consistent tendencies for females to develop moderately lower incidences of BCO than males from the same broiler cross or sire, yet lesion incidences and distributions did not differ markedly between the sexes in either study (Wideman et al., 2013; Tables 5 and 6; Figures 4 and 5). Finally, susceptibility to BCO clearly differed between broiler crosses in a previous study (Wideman et al., 2013) and between the progeny of different sires in the present study (Figure 2); nevertheless, the proximal femoral and tibial lesion incidences and distributions failed to identify key differences among crosses or sires. These observations do not support a consistent correlation between susceptibility to BCO and the location or severity of the lesions considered to be broadly pathognomonic for BCO (Wideman et al., 2013; Figures 4 and 5). Instead, susceptibility to BCO may be related to innate differences associated with lesion initiation and persistence, such as susceptibility to physeal osteochondrosis, chronic ischemia, and bacterial translocation, or immunological competence and responsiveness to perceived stress. Figure 6. Examples of tibiae having THNc lesions (tibial head necrosis in which caseous exudates or bacterial sequestrae were macroscopically evident) in 54-d-old broilers that developed bacterial chondronecrosis with osteomyelitis (BCO) lameness in experiment 1. Caseous abscesses and sequestrae are indicated by black arrowheads in the secondary center of ossification (panel A, sire B cross); the growth plate or physis (panel B, sire B cross); the subphyseal metaphysis (panel C, sire A cross); and extending from the metaphysis through the growth plate and into the secondary center of ossification (panel D, sire A cross). The growth plate thickens (*) when underlying infection impairs the metaphyseal vasculature and delays chondrocyte calcification (panels A, C). White arrowheads designate necrotic voids in the metaphysis (panels A and D), physis (panel A), and secondary center of ossification (panel C). White arrows designate lytic channels (panel A) and subphyseal fracture lines (panels B, C, D) that are commonly observed in affected tibiae from broilers with BCO.

12 1686 Wideman et al. Attempts to identify key patterns of lesion development may be confounded by euthanizing and necropsying broilers as soon as possible after the earliest symptoms of lameness become apparent. This protocol is followed to minimize the birds distress, but by doing so the apparent severity of lesion progression clearly may be affected. Experience has shown that broilers tend to exhibit relatively mild BCO lesions (FHS, THN) when they are euthanized at the earliest onset of clinical symptoms (hesitancy to stand, eagerness to sit, slight wing-tip dipping), whereas birds permitted to persist until they become fully immobilized exhibit much more severe lesions (FHN, THNc). Lesion severity clearly increases dramatically if lame birds are permitted to remain alive for several days after exhibiting the earliest symptoms of discomfort. It also is apparent from necropsying survivors of a wire flooring experiment that severe lesions may occasionally be present in very large, apparently robust individuals that exhibit no signs of lameness or leg weakness on d 56 (Wideman et al., 2012). We speculate that broilers may purposefully avoid exhibiting overt symptoms of lameness to avoid being victimized by the predatory behavior of their flock mates. The examples of severe caseous proximal tibial lesions shown in Figure 6 are from 54-dold broilers that on the day before necropsy exhibited no signs of lameness. We speculate that advanced lesions such as these may have been present for days if not weeks until the heavy BW of the bird combined with the severity of the lesion to overwhelm the bird s ability to mask its distress. Accordingly, patterns of lesion development are likely to be confounded by several factors, including the stage of clinical lameness at the time of necropsy as well as the bird s tolerance for or ability to mask perceived distress. Theoretically a bird that is less tolerant or more perceptive of discomfort might have less severe lesions but still exhibit clear early symptoms of lameness when compared with a bird that is more tolerant or less perceptive of joint discomfort. It is important to note that the majority of robust clinically healthy survivors of a wire flooring challenge tend to have proximal femora and tibiae that appear macroscopically normal or that have only mild early macroscopic BCO lesions (FHS, THN). Female broilers reared straight-run on wire flooring typically exhibit only moderately lower incidences of BCO compared with their male pen mates. However in one previous experiment broilers were reared sexseparate and the females developed uniformly low incidences of BCO that did not differ despite significantly different early growth rates for their respective crosses. The possibility was suggested that the females might experience less social/psychological or physical/competitive stress when reared separately from their male hatch mates, thereby attenuating a source of sustained stress that otherwise could aggravate the pathogenesis of BCO (Wideman et al., 2013). That possibility was not confirmed in the present study. Regardless of sire and sex, rearing the broilers straight-run versus sexseparate on wire flooring did not consistently affect their susceptibility to BCO. In summary, rearing broilers on wire flooring triggered lameness attributable overwhelmingly to BCO of the proximal femora and tibiae. Precautions taken to avoid stressing the chicks and to ensure that only clinically healthy chicks remained in the experiment after d 14 produced a pattern of cumulative lameness that remained negligible until approximately d 40, after which the incidence of BCO accelerated dramatically. Sires did influence the susceptibility of their progeny to BCO, with broilers derived from sire A being more likely to develop higher incidences than broilers derived from sire B. The biological basis for this sire contribution remains unknown, but at the present time we are not convinced that patterns of early growth or final BW were major contributing factors. Regardless of sex or sire, lame broilers had remarkably consistent patterns of proximal femoral and tibial lesion incidences and distributions. Lameness clearly was associated with comparably high susceptibilities to both femoral head and tibial head lesions in birds from both sexes and sire crosses. 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