Abstract

Temperature (TEM) and O2 concentrations during the plateau stage of oxygen consumption are known to affect yolk utilization, tissue development, and thyroid metabolism in turkey embryos. Three experiments were conducted to evaluate these incubation effects on long bone development. Fertile eggs of Nicholas turkeys were used. In each trial, standard incubation conditions were used to 24 d, when the eggs containing viable embryos were randomly divided into 4 groups. Four experimental cabinets provided 4 TEM (36, 37, 38, or 39°C) or 4 O2 concentrations (17, 19, 21, or 23% O2). In the third experiment, 2 temperatures (36 and 39°C) and 2 O2 concentrations (17 and 23%) were evaluated in a 2 × 2 factorial design. Body and residual yolk weights were obtained. Both legs were dissected, and shanks, femur, and tibia weights, length, and thickness were recorded. Relative asymmetry of each leg section was calculated. Chondrocyte density was evaluated in slides stained with hematoxylin and eosin. Immunofluorescence was used to evaluate the presence of collagen type X and transforming growth factor β. Hot TEM caused reduction of tibia weights and increase of shank weight when compared with cool TEM. The lengths of femur, tibia, and shanks were reduced by 39°C. The relative asymmetry of leg weights were increased at 38 and 39°C. Poult body and part weights were not affected by O2 concentrations, but poults on 23% O2 had bigger shanks and heavier tibias than the ones on 17% O2. High TEM depressed the fluorescence of collagen type X and transforming growth factor β. The O2 concentrations did not consistently affect the immunofluorescence of these proteins. The chondrocyte density was affected by TEM and O2 in resting and hypertrophic zones. In the third experiment, high TEM depressed BW, leg muscle weights, and shank length. Low O2 reduced tibia and shanks as a proportion of the whole body. We concluded that incubation conditions affect long bone development in turkeys.

INTRODUCTION

Skeletal disorders are one of the most prevalent and expensive diseases in poultry meat production (Mench, 2004). Recently, Simsa and Monsonego-Ornan (2007) described the early development of turkey bones. They observed clear signs of ossification, such as collagen type X, alkaline phosphatase, and expression of metalloproteinases at 18 d of incubation in turkey embryos. These findings highlight the importance of the rapid growth phase that occurs in bones during incubation. Poultry long bones have almost linear growth rates from 2 d pre-hatch until 11 d of age (Oviedo-Rondón et al., 2006). Yalçin and Siegel (2003) observed that changes in incubator temperature (TEM) during early incubation (E0-E8) could affect the length and relative asymmetry (RA) of long bones in chickens during embryo development. However, the rates of growth converged and resulted in developmental stability before hatching, when incubations conditions were maintained normal during the rest of the incubation. Relative asymmetry between limbs and other bilateral phenotypic traits reflects the ability of individuals to cope with stressful conditions during ontogeny. The RA of long bones has been correlated with tibial dyschondroplasia (TD), worsen of gait scores and tonic immobility in chickens (Møller et al., 1999).

Incubation TEM has an important impact on the thyroid-IGFI-GH hormonal axis that controls bone development (Robson et al., 2002; Van der Eerden et al., 2003; Christensen et al., 2005; Oviedo-Rondón et al., 2006). Thyroid hormones have a critical role in growth plate chondrocyte differentiation (Ballock and O’Keefe, 2003; Shao et al., 2006). Chondrocyte proliferation and differentiation is affected by heat stress (Yalçin et al., 2007). Degradation of the differentiation-dependent aggrecan proteoglycan in the extracellular matrix by chicken chondrocytes is also TEM dependent (Alonso et al., 1996).

Chondrocytes in the growth plate differentiate from the proliferative zone and undergo hypertrophy before suffering apoptosis and ossification. This process of chondrocyte maturation is regulated by several hormones and cytokines (Ballock and O’Keefe, 2003). Some isoforms of the transforming growth factor β (TGF-β) are the cytokines that regulates production of fibronectins and collagen type II and X in avian growth plates (Schmid and Linsenmayer, 1985; Ling et al., 2000; Janssens et al., 2005). Collagen type II is characteristic of chondrocytes in the proliferative zone, whereas hypertrophic chondrocytes are the unique producers of collagen type X (ColX) necessary for ossification. Failure in the production of these proteins is characteristic of tibial dyschondroplasia lesions (Leach and Monsonego-Ornan, 2007).

Christensen et al. (2007) recently demonstrated that incubator TEM and O2 concentrations during the plateau stage of incubation affect turkey muscle growth. During the plateau stage of incubation, or last 4 d of embryo development, the embryo is obliged to take up oxygen under hypoxic conditions against increasing metabolism with development and encounters a relative respiratory acidosis (Tazawa, 1980). While chondrocytes are very well adapted to low oxygen tension (Rajpurohit et al., 1996), avian osteoclasts are susceptible to acidotic conditions (Carano et al. 1993). The TEM and O2 concentration conditions during the plateau stage of incubation are known to affect yolk utilization, thyroid metabolism (Christensen et al., 1999; 2004a, 2005), heart (Christensen et al., 2004b), gut (Christensen et al., 2004a), and muscle development in turkeys (Christensen et al., 2007).

Therefore, it was hypothesized that bone development may be affected by environmental conditions in incubators during the plateau stage of O2 consumption in embryo development. Increasing the understanding of changes in early bone development may aid to develop strategies to reduce turkey leg problems grown under commercial conditions.

MATERIALS AND METHODS

Approximately 500 fertilized turkey eggs (Nicholas, Lewisburg, WV) were obtained from a commercial flock (Prestage Farms, Clinton, NC) for each of 3 experiments. The eggs were incubated (Natureform I40, Jacksonville, FL) for 24 d using standard industry procedures. The setter machine operated at a set point of 37.5°C dry bulb temperature and a RH of 53%. Eggs containing viable embryos on the 24th day of development were randomly divided into 4 groups immediately before the plateau stage in O2 consumption. Four experimental cabinets accommodating approximately 100 eggs were used for the hatching process. Each cabinet contained one incubator tray and was regulated by thermistors connected to microprocessors with temperature sensitivity of ± 0.05°C. Humidity was controlled by a similar system using relative humidity sensors. Digital thermometers (Cox, Lexington, NC) were used with each cabinet to verify set point temperatures. Atmospheric gases were measured by using an infrared detector for CO2 (Engelhard Ventostat 2001V, Iselin, NJ) and electrochemical cells for O2 (Teledyne, Los Angeles CA), each with a sensitivity of ±0.1%. Manually operated valves infused gases into each of the cabinets, and the flow rate was adjusted based on the sensors to create the desired gas concentrations. Three experiments were conducted using the experimental cabinets. Experiment 1 and 2 were preliminary experiments to determine the effects of TEM or O2 concentrations on bone development. Experiment 3 was conducted to examine the interactive effects on long bone development using the most extreme treatments affecting bone weights determined in Experiments 1 and 2.

Temperature

The eggs were moved to the treatment cabinet at the beginning of the 24th day of development. The 24th day for turkey embryos is the beginning of the plateau stage in oxygen consumption (Rahn, 1981). All infertile eggs and nonviable embryos were removed before transfer to one of 4 experimental cabinets. At the beginning of the 24th day the eggs were incubated at one of the treatments TEM (36, 37, 38, or 39°C) to expose the embryos to different TEM during the plateau stage.

Tissue Sampling

Ten poults were selected randomly from each incubator at hatch. Poult body weights (BW) were recorded (nearest 0.1 g) with and without yolk and and legs were dissected and frozen in liquid nitrogen as quickly as possible and stored at −20°C for further analyses. Each hatchling was sexed by visual inspection following dissection. Legs were thawed, divided in drums, thighs and shanks and parts weighed (to the nearest 0.0001 g). Muscles were removed and bones were cleaned, weighed (to the nearest 0.0001 g) and length measured in mm (to the nearest 0.01 mm) with electronic calipers (ProMax Fred V. Fowler Co. Inc., Newton, MA). The RA of bilateral traits was defined as (|R –L|/[(R + L)]/2) × 100 (Møller et al., 1999).

Ten tibia bones per treatment were fixed overnight with buffered neutral paraformaldehyde. The following day the tissues were dehydrated, washed, and embedded in paraffin. Five-micron-thick cross sections were cut on a microtome and adhered to glass slides. Tissues were dewaxed and stained using standard hematoxylin and eosin procedures or subjected to Immunoflurescence analyses. Samples were stained with primary monoclonal antibodies against ColX (X-AC9 Developmental Studies Hybridoma Bank; Schmid and Linsenmayer, 1985) and transforming growth factor (TGF) β. The anti-TGF-β1, -β2, -β3 monoclonal Ab was purified from the supernatants of the hybridoma cell line 1D11 and purified using T-gel thiophilic adsorption (Pierce). Protein concentrations of purified 1D11 mAb were determined using BCA assay (Bio Rad). A Leica DMR (Leica Microsystems, Bannockburn, IL) microscope was used to observe the tissue sections. A Spot-RT CCD (Diagnostic Instruments Inc., Sterling Heights, MI) camera was used to capture images of each of the cross sections. The cell chondrocyte numbers in selected section of each growth plate area were measured using UTHSCSA ImageTools Software (Wilcox et al., 2002). Five randomly selected areas on each growth plate region were selected to count chondrocyte cells, and cell density (CellD) per region (cells/mm2) was calculated.

Oxygen

Four O2 concentrations were evaluated in the second trial. All procedures to the 24th day of development were the same as in experiment 1. The O2 concentrations within the cabinets were 17, 19, 21, or 23% of the atmosphere. The fractional concentration at sea level (Raleigh, NC) corresponded to O2 partial pressures of 129, 144, 160, and 175 mmHg, respectively. Concentrations lower than ambient O2 concentrations (20.9%) were maintained by infusing nitrogen gas into the cabinet at a rate that resulted in the desired concentration of 17 or 19% O2. Concentrations were measured with an O2 meter and flow rates from O2 or nitrogen storage tanks were adjusted to maintain the desired O2 level. The TEM was maintained at 37°C in all 4 cabinets. Poult samples were collected and analyzed as in experiment 1.

Temperature and Oxygen

The most and least effective TEM (36 and 39°C) and O2 treatments (17 and 23%) to modify bone weights and cause relative asymmetry in the preliminary experiments were combined in a factorial arrangement for the third experiment. The TEM and O2 concentrations were maintained as described previously. Fertilized eggs were again incubated 24 d when viable embryos were assigned randomly to 1 of the 4 cabinets. The conditions were TEM of 36°C with 17 or 23% O2 and 39°C with 17 or 23% O2 in a factorial arrangement. Birds were sampled identically to the previous experiments.

Statistical Analysis

Data for all 3 experiments were analyzed using the general linear models procedure (SAS Institute, 1998). Experiments 1 and 2 were arranged as a 4 levels of TEM or O2 treatments. In experiment 3, the data were arranged as 2 × 2 factorial arrangement of treatments with 2 TEM and 2 O2 concentrations as main effects. In each experiment we had 10 replicates per treatment. Histological analyses were conducted with a minimum of 7 samples per treatment. Means differing significantly were separated by the t-test or Tukey’s test procedures. Means in tables are least square means. All possible interaction and main effects were tested. All probabilities were based on P < 0.05 unless otherwise noted.

RESULTS

Temperature Trial

The BW and yolk utilization of poults were not affected (P > 0.05) by incubation temperatures, but absolute and relative weights of tibia and shanks were affected by TEM (Tables 1 and 2). The 39°C TEM caused reduced tibia weight and increased shank weight when compared with 36°C. Lengths of femur and tibia were reduced at 39°C compared with lengths of these bones at 36°C. Shank length was reduced at 39°C compared with 37 and 38°C, but was similar to the shank length of poults at 36°C (Table 2). The RA of shank weights increased at 38 and 39°C compared with 36 and 37°C (Table 1).

Oxygen Trial

The BW and yolk utilization of poults were not affected (P > 0.05) by O2 concentrations (Figure 1A). However, absolute and relative weights of femur and tibia were depressed in the 19% O2 compared with 23 and 17%, but these weights were similar to weights observed in chickens at 19% O2 (Figure 1B). The RA of shank length was also increased by the 19% O2 compared with 23 and 17% O2 (Table 3).

Histology Results

High TEM depressed the fluorescence of ColX and TGFβ (Figures 2 and 3). The O2 concentrations did not affect consistently the fluorescence of these proteins. The CellD in the resting zone was increased (P < 0.05) by the high TEM in the incubator, while the CellD in the hypertrophic zone was reduced by the same treatment (P < 0.001). The high O2 concentrations (23%) decreased (P < 0.05) CellD in the resting and hyperthrophic zones (Table 4).

Temperature with Oxygen

In this trial BW and yolk utilization were affected by TEM. The poults at 36°C TEM were heavier (Table 5) than those at 39°C. There was a TEM × O2 interaction (P < 0.001) effect on leg weight. Poults at 36°C and 23% O2 had the heaviest legs compared with the ones at 39°C. The leg weights of poults at 36°C and 17% O2 were not different from all other treatments. The TEM and O2 had independent effects on absolute and relative weights of bones (Tables 6, 7, 8, and 9). The relative weights of tibia and femur were lower at 36°C (Table 6). The weights of tibia and shanks were lower (P < 0.05) for poults incubated at 17% O2 independently of the TEM (Tables 7 and 9). No significant effects of treatments were observed in femur parameters (Table 8). Shanks of poults incubated at 39°C or 17% O2 were lighter (P < 0.05) and thinner (P < 0.01) than the ones incubated at 36°C and 23% O2. Poults in the high TEM incubator also had shorter (P < 0.05) shanks (Table 9).

DISCUSSION

Incubator TEM and O2 concentrations may affect chick embryo development (Christensen et al., 1999, 2004a,b, 2005, 2007). Embryos are sensitive to incubation conditions at any age of development but especially during the plateau stage of O2 consumption (Christensen et al., 2004a,b, 2005, 2007) when most of the organs, including the skeletal system, are under rapid maturation processes. We proposed the hypothesis that incubator TEM and O2 concentrations during the plateau stage in O2 consumption may interact to affect long bone physiology and growth in developing chick embryos. We examined femur, tibia, and shanks based on their growth, maturation and RA immediately after hatching. In the first 2 experiments both factors were evaluated independently. Higher TEM and lower O2 concentrations than optimum may affect relative bone weights, length, and may cause asymmetry between the 2 limbs. Higher O2 concentrations seem to increase bone development and reduce average RA. The histological analyses of the proximal tibia growth plates showed the effects of extreme environmental TEM and O2 conditions during incubation on growth plate maturation. Based on changes in chondrocyte density and lower immunofluorescence responses to ColX and TGFβ, we suggest that growth plate differentiation may be affected by high TEM and low O2 concentration conditions. These tissue specific responses may have implications on TD incidence since both proteins have been related to TD lesions (Ling et al., 2000; Leach and Monsonego-Ornan, 2007; Yalçin et al., 2007).

In the third experiment, we evaluated the interactions between these 2 incubation factors. Both TEM and O2 concentrations can act together but almost always independently on bone weights and lengths during the plateau stage of incubation, taking into consideration the lack of consistent interaction effects.

Yalçin and Siegel (2003) observed increased asymmetries in skeletal traits when eggs were cooled to 36.9°C 6 h/d between E0 and E8, during embryo development but not at hatch. In this initial experiment, Yalçin and Siegel (2003) concluded that although bilateral skeletal traits developed at different rates during incubation due to cold early incubation (E0-E8), they converge for a developmental stability before hatch if TEM is maintained under normal conditions (37.8°C) thereafter. However, Yalçin et al. (2007) using similar incubation conditions observed that the incidence of TD at 49 d of age was higher (14.4 and 12.8% vs. 5.0%) for chicken embryos exposed to cool (36.9°C) or hot (39.6°C) TEM between E0 and E8. In this second experiment, tibia weight at hatch and 49 d of age was reduced by heating eggs (39.6°C) for 6 h/d during E10 to E18. Therefore, the authors proposed that growth plate differentiation and tibia growth do not share the same critical stage. Based on results of the experiments presented herein, we suggest that appropriate TEM and O2 concentrations during the plateau stage of incubation are critical for both bone growth and growth plate differentiation.

The RA of several traits of long bones was increased by hot temperatures during incubation. The symmetry on limb dimensions, weight, or stage of development may have implications on development of normal gait patterns, adult gait scores, and TD incidence (Møller et al., 1999; Yalçin and Siegel, 2003).

Taking in consideration the results of these experiments, we conclude that elevated incubator temperatures and low oxygen concentrations during the plateau stage of O2 consumption can affect bone development by changing CellD, expression of ColX, TGFβ in the tibia growth plate, and altering long bone weight, length, and thickness. Temperatures greater than 37°C and oxygen concentrations less than 21% O2 should be avoided to ensure optimal bone development at hatching. It is important to evaluate the long-term effects of incubator temperature and oxygen concentrations at market age.

Table 1

Body, body without yolk, and leg weights (g) of poults at hatch exposed to different temperatures (TEM) during the plateau stage of incubation

   Leg bones  
     Shanks  
TEM (°C) BW BW without yolk Femur Tibia Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R–L|/[(R+L)]/2) × 100. 
36 56.2 50.98 0.28 0.48a 1.54b 1.57b 3.65b 
37 62.2 52.58 0.24 0.44ab 1.63ab 1.64ab 3.68b 
38 59.7 51.05 0.26 0.43ab 1.58b 1.69ab 6.87a 
39 60.6 50.26 0.26 0.40b 1.77a 1.82a 6.98a 
SEM 1.66 1.27 0.01 0.01 0.04 0.05 0.89 
CV% 8.29 7.42 8.25 9.50 6.01 6.03 — 
 ——— P-value ——— 
TEM 0.1650 0.6276 0.5091 0.0167 0.0039 0.0184 0.0186 
   Leg bones  
     Shanks  
TEM (°C) BW BW without yolk Femur Tibia Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R–L|/[(R+L)]/2) × 100. 
36 56.2 50.98 0.28 0.48a 1.54b 1.57b 3.65b 
37 62.2 52.58 0.24 0.44ab 1.63ab 1.64ab 3.68b 
38 59.7 51.05 0.26 0.43ab 1.58b 1.69ab 6.87a 
39 60.6 50.26 0.26 0.40b 1.77a 1.82a 6.98a 
SEM 1.66 1.27 0.01 0.01 0.04 0.05 0.89 
CV% 8.29 7.42 8.25 9.50 6.01 6.03 — 
 ——— P-value ——— 
TEM 0.1650 0.6276 0.5091 0.0167 0.0039 0.0184 0.0186 
Table 2

Leg bones as proportion (%) of whole body without yolk and length (mm) of bones of poults at hatch exposed to different temperatures (TEM) during the plateau stage of incubation

    Length (mm)  
 Bone percentage (%)   Shanks  
TEM (°C) Femur Tibia Shanks Femur Tibia Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R –L|/[(R + L)]/2) × 100. 
36 0.55 0.94a 3.06b 23.71a 35.74a 28.29ab 28.34ab 0.52 
37 0.47 0.81b 3.21b 23.13ab 36.21a 28.23a 28.46a 1.71 
38 0.51 0.87ab 3.18b 23.30ab 36.74a 29.16a 29.56a 1.92 
39 0.52 0.80b 3.47a 22.37b 34.04b 27.02b 27.08b 1.55 
SEM 0.03 0.02 0.06 0.27 0.33 0.32 0.38 0.56 
CV% 7.53 4.06 2.64 3.22 2.65 3.24 3.74 — 
 ——— P-value ——— 
TEM 0.2653 0.0055 < 0.0001 0.0269 < 0.0001 0.0004 0.0004 0.4418 
    Length (mm)  
 Bone percentage (%)   Shanks  
TEM (°C) Femur Tibia Shanks Femur Tibia Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R –L|/[(R + L)]/2) × 100. 
36 0.55 0.94a 3.06b 23.71a 35.74a 28.29ab 28.34ab 0.52 
37 0.47 0.81b 3.21b 23.13ab 36.21a 28.23a 28.46a 1.71 
38 0.51 0.87ab 3.18b 23.30ab 36.74a 29.16a 29.56a 1.92 
39 0.52 0.80b 3.47a 22.37b 34.04b 27.02b 27.08b 1.55 
SEM 0.03 0.02 0.06 0.27 0.33 0.32 0.38 0.56 
CV% 7.53 4.06 2.64 3.22 2.65 3.24 3.74 — 
 ——— P-value ——— 
TEM 0.2653 0.0055 < 0.0001 0.0269 < 0.0001 0.0004 0.0004 0.4418 
Table 3

Leg bones as proportion (%) of whole body without yolk and length (mm) of bones of poults at hatch exposed to different oxygen concentrations (%) during the plateau stage of incubation

   Length (mm)  
 Relative bone weight (%)   Shanks  
O2 (%) Femur Tibia Shanks Femur Tibia Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R –L|/[(R+L)]/2) × 100. 
17 0.67a 0.98a 3.13 23.76 35.95 28.29 28.31 1.80b 
19 0.46b 0.80b 3.25 23.41 36.14 27.95 29.01 3.72a 
21 0.63ab 0.92ab 3.06 24.23 36.78 28.66 29.52 2.20ab 
23 0.61ab 0.97a 3.27 23.86 36.01 29.25 29.25 1.16b 
SEM 0.04 0.05 0.06 0.32 0.39 0.31 0.30 0.46 
CV% 8.93 6.23 2.58 3.45 2.76 3.29 2.97 — 
 ——— P-value ——— 
Oxygen 0.0094 0.0232 0.1055 0.4341 0.5009 0.0631 0.0502 0.0024 
   Length (mm)  
 Relative bone weight (%)   Shanks  
O2 (%) Femur Tibia Shanks Femur Tibia Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R –L|/[(R+L)]/2) × 100. 
17 0.67a 0.98a 3.13 23.76 35.95 28.29 28.31 1.80b 
19 0.46b 0.80b 3.25 23.41 36.14 27.95 29.01 3.72a 
21 0.63ab 0.92ab 3.06 24.23 36.78 28.66 29.52 2.20ab 
23 0.61ab 0.97a 3.27 23.86 36.01 29.25 29.25 1.16b 
SEM 0.04 0.05 0.06 0.32 0.39 0.31 0.30 0.46 
CV% 8.93 6.23 2.58 3.45 2.76 3.29 2.97 — 
 ——— P-value ——— 
Oxygen 0.0094 0.0232 0.1055 0.4341 0.5009 0.0631 0.0502 0.0024 
Table 4

Chondrocyte density (cells/mm2) in proximal tibia growth plates of poults at hatch exposed to 2 temperatures (36 or 19°C) or 2 oxygen concentrations (17 and 23%) during the plateau stage of oxygen consumption

  Growth plate area 
Treatment  Resting Proliferation Hypertrophic 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
Temperature, °C 36 4,039b 1,923 1,281a 
 39 4,423a 1,904 1,028b 
Pooled SEM  142 82 42 
P-value  0.0591 0.8700 < 0.0001 
Oxygen, % 17 4,296a 2,192 1,388a 
 23 3,772b 1,984 1,100b 
Pooled SEM  38 30 21 
P-value  0.0187 0.2300 0.0214 
  Growth plate area 
Treatment  Resting Proliferation Hypertrophic 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
Temperature, °C 36 4,039b 1,923 1,281a 
 39 4,423a 1,904 1,028b 
Pooled SEM  142 82 42 
P-value  0.0591 0.8700 < 0.0001 
Oxygen, % 17 4,296a 2,192 1,388a 
 23 3,772b 1,984 1,100b 
Pooled SEM  38 30 21 
P-value  0.0187 0.2300 0.0214 
Table 5

Body, body without yolk, and leg weights (g) of poults at hatch exposed to different temperatures (TEM) and oxygen concentrations during the plateau stage of incubation

    Leg weight 
TEM (°C) O2 (%) BW BW without yolk Right Left 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
  ——— g ——— 
36 17 67.4 58.4 6.32ab 6.25ab 
 23 67.9 60.6 7.07a 7.11a 
TEM mean  67.6a 59.5a 6.70 6.68 
39 17 63.1 50.2 5.84b 5.82b 
 23 61.7 51.5 5.75b 5.69b 
TEM Mean  62.4b 50.9b 5.79 5.76 
O2 Mean 17 65.3 54.3 6.08 6.03 
 23 64.8 56.0 6.41 6.40 
SEM  1.2 0.8 0.15 0.16 
CV%  8.05 8.17 7.34 8.27 
Source of variation  ——— P-value ——— 
    TEM  0.0002 < 0.0001 0.0004 0.0010 
    O2  0.7182 0.1382 0.1210 0.1307 
    TEM × O2  0.4358 0.6947 0.0587 0.0491 
    Leg weight 
TEM (°C) O2 (%) BW BW without yolk Right Left 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
  ——— g ——— 
36 17 67.4 58.4 6.32ab 6.25ab 
 23 67.9 60.6 7.07a 7.11a 
TEM mean  67.6a 59.5a 6.70 6.68 
39 17 63.1 50.2 5.84b 5.82b 
 23 61.7 51.5 5.75b 5.69b 
TEM Mean  62.4b 50.9b 5.79 5.76 
O2 Mean 17 65.3 54.3 6.08 6.03 
 23 64.8 56.0 6.41 6.40 
SEM  1.2 0.8 0.15 0.16 
CV%  8.05 8.17 7.34 8.27 
Source of variation  ——— P-value ——— 
    TEM  0.0002 < 0.0001 0.0004 0.0010 
    O2  0.7182 0.1382 0.1210 0.1307 
    TEM × O2  0.4358 0.6947 0.0587 0.0491 
Table 6

Bones as proportion (%) of body weights of poults without yolk at hatch exposed to two temperatures (TEM) and oxygen concentrations during the plateau stage of incubation

  Tibia Femur Shanks 
TEM (°C) O2 (%) 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
  ——— % ——— 
36 17 0.88 0.91 0.54 0.57 3.05 3.03 
 23 0.92 0.90 0.51 0.54 3.14 3.13 
TEM mean  0.90b 0.90b 0.53b 0.55 3.10 3.08 
39 17 0.99 0.95 0.59 0.59 3.05 3.08 
 23 1.09 1.04 0.63 0.62 3.17 3.23 
TEM mean  1.04a 0.99a 0.61a 0.60 3.11 3.15 
O2 Mean 17 0.93 0.93 0.57 0.58 3.05 3.05b 
 23 1.00 0.97 0.57 0.58 3.16 3.18a 
SEM  0.03 0.03 0.02 0.02 0.04 0.04 
CV%  4.47 4.38 5.94 5.08 7.21 6.94 
Source of variation  ——— P-value ——— 
    TEM  0.0028 0.0303 0.0026 0.0879 0.8671 0.1773 
    O2  0.0840 0.2800 0.8019 0.8700 0.0649 0.0308 
    TEM × O2  0.5097 0.2097 0.0869 0.3038 0.7394 0.6959 
  Tibia Femur Shanks 
TEM (°C) O2 (%) 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
  ——— % ——— 
36 17 0.88 0.91 0.54 0.57 3.05 3.03 
 23 0.92 0.90 0.51 0.54 3.14 3.13 
TEM mean  0.90b 0.90b 0.53b 0.55 3.10 3.08 
39 17 0.99 0.95 0.59 0.59 3.05 3.08 
 23 1.09 1.04 0.63 0.62 3.17 3.23 
TEM mean  1.04a 0.99a 0.61a 0.60 3.11 3.15 
O2 Mean 17 0.93 0.93 0.57 0.58 3.05 3.05b 
 23 1.00 0.97 0.57 0.58 3.16 3.18a 
SEM  0.03 0.03 0.02 0.02 0.04 0.04 
CV%  4.47 4.38 5.94 5.08 7.21 6.94 
Source of variation  ——— P-value ——— 
    TEM  0.0028 0.0303 0.0026 0.0879 0.8671 0.1773 
    O2  0.0840 0.2800 0.8019 0.8700 0.0649 0.0308 
    TEM × O2  0.5097 0.2097 0.0869 0.3038 0.7394 0.6959 
Table 7

Left and right tibia weights (g) and lengths and relative asymmetry of poults at hatch exposed to different temperatures (TEM) and oxygen concentrations during the plateau stage of incubation

  Weight (g) Length (mm) 
TEM (°C) O2 (%) Right Left RA1 Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R − L|/[(R + L)]/2) × 100. 
36 17 0.50 0.52 3.69 37.5 37.7 0.85 
 23 0.58 0.57 3.03 38.8 38.3 1.58 
TEM mean  0.54 0.54a 3.34 38.1 37.9 1.21 
39 17 0.50 0.48 8.46 37.8 38.1 1.85 
 23 0.54 0.52 6.75 38.1 37.6 1.71 
TEM mean  0.52 0.50b 7.61 37.9 37.9 1.77 
O2 mean 17 0.50b 0.50b 6.08 37.7 37.9 1.34 
 23 0.56a 0.54a 4.89 38.4 38.0 1.64 
SEM  0.26 0.14 1.70 10 0.4 0.59 
CV%  9.72 8.66 — 10.58 2.99 — 
Source of variation  ——— P-value ——— 
    TEM  0.4131 0.0444 0.0970 0.4337 0.7388 0.5145 
    O2  0.0146 0.0407 0.6291 0.3152 0.1506 0.7221 
    TEM × O2  0.3541 0.6002 0.8314 0.8801 0.3147 0.6146 
  Weight (g) Length (mm) 
TEM (°C) O2 (%) Right Left RA1 Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R − L|/[(R + L)]/2) × 100. 
36 17 0.50 0.52 3.69 37.5 37.7 0.85 
 23 0.58 0.57 3.03 38.8 38.3 1.58 
TEM mean  0.54 0.54a 3.34 38.1 37.9 1.21 
39 17 0.50 0.48 8.46 37.8 38.1 1.85 
 23 0.54 0.52 6.75 38.1 37.6 1.71 
TEM mean  0.52 0.50b 7.61 37.9 37.9 1.77 
O2 mean 17 0.50b 0.50b 6.08 37.7 37.9 1.34 
 23 0.56a 0.54a 4.89 38.4 38.0 1.64 
SEM  0.26 0.14 1.70 10 0.4 0.59 
CV%  9.72 8.66 — 10.58 2.99 — 
Source of variation  ——— P-value ——— 
    TEM  0.4131 0.0444 0.0970 0.4337 0.7388 0.5145 
    O2  0.0146 0.0407 0.6291 0.3152 0.1506 0.7221 
    TEM × O2  0.3541 0.6002 0.8314 0.8801 0.3147 0.6146 
Table 8

Left and right femur weights (g) and lengths and relative asymmetry of poults at hatch exposed to two temperatures (TEM) and oxygen concentrations during the plateau stage of incubation

  Weight (mg) Length (mm) 
TEM (°C) O2 (%) Right Left RA1 Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R − L|/[(R + L)]/2) × 100. 
36 17 312 323 7.99 24.3 24.7 2.03 
 23 325 348 7.74 25.1 25.0 0.98 
TEM mean  319 335 7.86a 24.7 24.9 1.51 
39 17 298 298 1.90 24.5 24.6 0.90 
 23 316 311 3.08 24.8 24.7 1.61 
TEM mean  307 304 2.49b 24.7 24.7 1.25 
O2 mean 17 305 311 4.94 24.4 24.6 1.46 
 23 321 329 5.41 24.9 24.9 1.30 
SEM  10 14 1.9 0.3 0.3 0.4 
CV%  10.58 13.60 — 3.38 3.39 — 
Source of variation  ——— P-value ——— 
    TEM  0.4337 0.1311 0.0594 0.9495 0.5836 0.7188 
    O2  0.3152 0.3482 0.8682 0.1746 0.4791 0.8144 
    TEM × O2  0.8801 0.7446 0.7993 0.4958 0.7974 0.2173 
  Weight (mg) Length (mm) 
TEM (°C) O2 (%) Right Left RA1 Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R − L|/[(R + L)]/2) × 100. 
36 17 312 323 7.99 24.3 24.7 2.03 
 23 325 348 7.74 25.1 25.0 0.98 
TEM mean  319 335 7.86a 24.7 24.9 1.51 
39 17 298 298 1.90 24.5 24.6 0.90 
 23 316 311 3.08 24.8 24.7 1.61 
TEM mean  307 304 2.49b 24.7 24.7 1.25 
O2 mean 17 305 311 4.94 24.4 24.6 1.46 
 23 321 329 5.41 24.9 24.9 1.30 
SEM  10 14 1.9 0.3 0.3 0.4 
CV%  10.58 13.60 — 3.38 3.39 — 
Source of variation  ——— P-value ——— 
    TEM  0.4337 0.1311 0.0594 0.9495 0.5836 0.7188 
    O2  0.3152 0.3482 0.8682 0.1746 0.4791 0.8144 
    TEM × O2  0.8801 0.7446 0.7993 0.4958 0.7974 0.2173 
Table 9

Left and right shank weights (g), thickness (mm), lengths (mm), and relative asymmetry of poults at hatch exposed to two temperatures (TEM) and oxygen concentrations during the plateau stage of incubation

  Weight (g)  Thickness (mm)  Length (mm)  
TEM (°C) O2 (%) Right Left RA1 Right Left RA1 Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R − L|/[(R + L)]/2) × 100. 
36 17 1,765 1,782 1.39 2.42 2.40 4.16 28.8 29.5 2.68 
 23 1,892 1,903 2.46 2.52 2.49 4.02 29.5 29.7 2.86 
TEM mean  1,829a 1,843a 1.93 2.47a 2.45a 4.09 29.2a 29.6a 2.77 
39 17 1,551 1,531 4.24 2.19 2.14 4.02 28.1 28.0 1.69 
 23 1,658 1,632 1.95 2.35 2.28 3.89 28.6 29.1 2.72 
TEM mean  1,605b 1,582b 3.09 2.27b 2.21b 3.96 28.3b 28.6b 2.21 
O2 mean 17 1,658b 1,657b 2.81 2.31b 2.27b 4.09 28.5 28.8 2.18 
 23 1,775a 1,768a 2.21 2.43a 2.39a 3.96 29.0 29.4 2.79 
SEM  0.031 0.032 0.81 0.03 0.03 0.52 0.2 0.2 0.3 
CV%  10.00 10.25 — 7.05 6.71 — 4.89 4.61 — 
Source of variation  ——— P-value ——— 
    TEM  < 0.001 < 0.001 0.3157 < 0.0001 < 0.0001 0.8558 0.0231 0.0033 0.2213 
    O2  0.0107 0.0175 0.5999 0.0042 0.0054 0.8532 0.1303 0.0903 0.1870 
    TEM × O2  0.8213 0.8135 0.1497 0.5493 0.5712 0.9906 0.7699 0.2382 0.3493 
  Weight (g)  Thickness (mm)  Length (mm)  
TEM (°C) O2 (%) Right Left RA1 Right Left RA1 Right Left RA1 
a,bMeans within a column followed by different superscripts are significantly different (P < 0.05). 
1Relative asymmetry between right and left shank length. RA = (|R − L|/[(R + L)]/2) × 100. 
36 17 1,765 1,782 1.39 2.42 2.40 4.16 28.8 29.5 2.68 
 23 1,892 1,903 2.46 2.52 2.49 4.02 29.5 29.7 2.86 
TEM mean  1,829a 1,843a 1.93 2.47a 2.45a 4.09 29.2a 29.6a 2.77 
39 17 1,551 1,531 4.24 2.19 2.14 4.02 28.1 28.0 1.69 
 23 1,658 1,632 1.95 2.35 2.28 3.89 28.6 29.1 2.72 
TEM mean  1,605b 1,582b 3.09 2.27b 2.21b 3.96 28.3b 28.6b 2.21 
O2 mean 17 1,658b 1,657b 2.81 2.31b 2.27b 4.09 28.5 28.8 2.18 
 23 1,775a 1,768a 2.21 2.43a 2.39a 3.96 29.0 29.4 2.79 
SEM  0.031 0.032 0.81 0.03 0.03 0.52 0.2 0.2 0.3 
CV%  10.00 10.25 — 7.05 6.71 — 4.89 4.61 — 
Source of variation  ——— P-value ——— 
    TEM  < 0.001 < 0.001 0.3157 < 0.0001 < 0.0001 0.8558 0.0231 0.0033 0.2213 
    O2  0.0107 0.0175 0.5999 0.0042 0.0054 0.8532 0.1303 0.0903 0.1870 
    TEM × O2  0.8213 0.8135 0.1497 0.5493 0.5712 0.9906 0.7699 0.2382 0.3493 
Figure 1

Effect of oxygen concentrations during the plateau stage of incubation of turkeys at hatch on (A) body weight, yolk utilization, (B) femur and tibia weights. *P < 0.05.

Figure 1

Effect of oxygen concentrations during the plateau stage of incubation of turkeys at hatch on (A) body weight, yolk utilization, (B) femur and tibia weights. *P < 0.05.

Figure 2

Effect of temperature (36 and 39°C) and oxygen concentrations (17 and 23%) during the plateau stage of incubation of turkeys at hatch on collagen type X expression studied by immunofluorescence in the hypertrophic zone of the tibia growth plate. A) Prehypertrophic zone and B) hypertrophic zone at × 200 magnification.

Figure 2

Effect of temperature (36 and 39°C) and oxygen concentrations (17 and 23%) during the plateau stage of incubation of turkeys at hatch on collagen type X expression studied by immunofluorescence in the hypertrophic zone of the tibia growth plate. A) Prehypertrophic zone and B) hypertrophic zone at × 200 magnification.

Figure 3

Effect of temperature (36 and 39°C) and oxygen concentrations (17 and 23%) during the plateau stage of incubation of turkeys at hatch on transforming growth factor β expression studied by immunofluorescence in the hypertrophic zone of the tibia growth plate. Hypertrophic zone at × 200 magnification.

Figure 3

Effect of temperature (36 and 39°C) and oxygen concentrations (17 and 23%) during the plateau stage of incubation of turkeys at hatch on transforming growth factor β expression studied by immunofluorescence in the hypertrophic zone of the tibia growth plate. Hypertrophic zone at × 200 magnification.

1
The mention of trade names in this publication does not imply endorsement of the products mentioned nor criticism of similar products not mentioned.

We thank US Poultry and Egg Association for funding these studies.

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