Effects of Providing Enrichment to Broilers in an Animal Welfare Environment on Productivity, Litter Moisture, Gas Concentration (CO₂ and NH₃), Animal Welfare Indicators, and Stress Level Concentration

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for the considerable effort invested in completing this study. It is a well-written and systematically written article. Yet, the similar idea was easily found as a published article [e.g. https://www.animbiosci.org/journal/view.php?viewtype=pubreader&number=25157] that investigated closely similar object with similar parameters.  After a comprehensive peer review, it has been determined that significant improvement are necessary for this study to align with the standards of the Agriculture Journal by MDPI. Detailed comments are provided in the attached document. Good luck for the revision.

Sincerely,

Comments for author File: Comments.pdf

Author Response

Reviewer #1:

 

Comments:

 

Effects of providing enrichment to broilers in an animal welfare environment on productivity, litter moisture, gas emission, animal welfare indicators and stress level concentration

 

 

Dear authors,

Thank you for the considerable effort invested in completing this study. It is a well-written and systematically written article. Yet, the similar idea was easily found as a published article [e.g.

https://www.animbiosci.org/journal/view.php?viewtype=pubreader&number=25157] that investigated closely similar object with similar parameters.  After a comprehensive peer review, it has been determined that significant improvement are necessary for this study to align with the standards of the Agriculture Journal by MDPI. Detailed comments are provided in the attached document. Good luck for the revision.

 

Sincerely,

Response: We are extremely thankful to the reviewer for considering our manuscript toward revision. We are grateful for all the queries and suggestions that helped us to improve the original version of the manuscript. We hope that all queries, comments, and suggestions were adequately justified in the revised version of the manuscript.

 

Abstract section :

 

Please kindly provide detailed sample condition, e.g. what strain of broiler, age, replications at each treatment, total sample number, etc. Thus, improve the understanding of experimental conditions.

Response: Thank you for the reviewer's comments. In the abstract of the revised manuscript, information on the age and breed of the broilers used was provided, and the experimental design, such as the creation of an identical environment and provision of grain blocks, was written in more detail.

 

The revised contents are as follows :

 

“As animal welfare becomes more active in livestock industry, research is being conducted on ways to improve poor housing environments, reduce stress, and meet welfare standards. Among these, environmental enrichment methods are effective in reducing stress and creating a welfare-friendly rearing environment, but there are few cases of actual application to farms. Therefore, we aimed to investigate the effect of providing pecking materials (grain blocks), known as one of the environmental enrichment methods, to commercial broiler farms. This study used two facilities that can accommodate 32,000 1-day-old broilers (Arbor acres) per building, and two groups (control and treatment groups) were designed after creating two identical areas within each building (total 2 treatments, 2 replicates, 16,000 birds per replicate). Two identical zones within the house were created by installing a partition in the center, one side provided with grain blocks (One grain block per 1,000 birds) and the other side did not. Analysis items included productivity (body weight, uniformity), environmental variables (litter and air), welfare indicators (leg, gait score, feather cleanliness score) and serum corticosterone levels. Analysis of all items was conducted twice, on the 19th and 27th, taking into account the farm's feed change date and slaughter schedule. Other environmental conditions (density, lighting, ventilation, temperature, humidity, feed, and water) were the same. As a result, no difference in productivity was observed according to enrichment, and the quality of litter and air was similar. Also, there was no significant difference in welfare indicators. Interestingly, however, provision of the environment enrichment lowered serum corticosterone levels (p<0.05). The implications of our study are that grain blocks as a pecking material are an effective way to reduce stress without adversely affecting broiler performance and rearing environment. However, it is still necessary to explore optimal enrichment materials that can help not only the welfare level but also the broiler performance.” (Pg. 1, Line 12-33)

 

Keywords: Please kindly revise in alphabetic order

Response: In the revised manuscript, keywords are arranged in alphabetical order.

 

Keywords : Animal welfare; Broiler; Pecking block; Physiological indicator; Welfare indicator (Pg. 1, Line 34)

 

The abstract was written in a great fashion. Apart from presenting the strength and weakness of study at a glimpse of reading, it was written in a clear and systematic way, thus only minor amendment at this section needs to be addressed.

Response: Thank you for your valuable comments. In the revised manuscript, we have reviewed and revised the content to improve the delivery of information and understanding to readers.

 

“As animal welfare becomes more active in livestock industry, research is being conducted on ways to improve poor housing environments, reduce stress, and meet welfare standards. Among these, environmental enrichment methods are effective in reducing stress and creating a welfare-friendly rearing environment, but there are few cases of actual application to farms. Therefore, we aimed to investigate the effect of providing pecking materials (grain blocks), known as one of the environmental enrichment methods, to commercial broiler farms. This study used two facilities that can accommodate 32,000 1-day-old broilers (Arbor acres) per building, and two groups (control and treatment groups) were designed after creating two identical areas within each building (total 2 treatments, 2 replicates, 16,000 birds per replicate). Two identical zones within the house were created by installing a partition in the center, one side provided with grain blocks (One grain block per 1,000 birds) and the other side did not. Analysis items included productivity (body weight, uniformity), environmental variables (litter and air), welfare indicators (leg, gait score, feather cleanliness score) and serum corticosterone levels. Analysis of all items was conducted twice, on the 19th and 27th, taking into account the farm's feed change date and slaughter schedule. Other environmental conditions (density, lighting, ventilation, temperature, humidity, feed, and water) were the same. As a result, no difference in productivity was observed according to enrichment, and the quality of litter and air was similar. Also, there was no significant difference in welfare indicators. Interestingly, however, provision of the environment enrichment lowered serum corticosterone levels (p<0.05). The implications of our study are that grain blocks as a pecking material are an effective way to reduce stress without adversely affecting broiler performance and rearing environment. However, it is still necessary to explore optimal enrichment materials that can help not only the welfare level but also the broiler performance.” (Pg. 1, Line 12-33)

 

 

 

Introduction section :

 

The introduction ideally provides all the substantial information that guides readers to the author’s proposed idea. In addition, it should also accommodate the written title, particularly mentioning all the information that is presented in the title. While the written title was “Effects of providing enrichment to broilers in an animal welfare environment on productivity, litter moisture, gas emission, animal welfare indicators and stress level concentration,” reviewer sees that this manuscript would be well benefited from additional paragraphs, mainly provision of previously conducted study on pecking block addition (hay, grain based, mineral based) on productivity, litter moisture, gas emission, animal welfare indicators and stress level. Additionally, please provide a concise but information on the mechanism of environment on animal welfare indicators and stress status of broilers, and how would this study benefit broilers animal welfare status.

Response: Thank you for the reviewer's comments. The revised manuscript includes the following additions to provide more information on the impact of environmental enrichment on various aspects of poultry:

 

“The provision of environmental enrichment can have a number of positive effects on poultry in a variety of ways. In broilers that were not provided with environmental enrichment, they were affected by poor locomotion, leg lameness, increased time spent lying down, increased litter contact, which led to breast blister and contact dermatitis [7]. In addition, leg disorders and skin diseases caused by lack of activity can affect the quality of the air by generating ammonia in the litter [7]. These atmospheric problems can be a welfare problem because they cause chemical burns and pain in the poultry [7]. Spieß et al. [8] reported that environmental enrichment can make poultry more active for 80% of the time except for eating and drinking time, which induces physical activity and reduces lameness and dermatitis [7]. In addition, increased activity can reduce contact with litter, improve litter quality [9], and ultimately affect air quality, which can affect welfare improvement. In terms of productivity, it can have a positive impact by reducing mortality in poultry and increasing body weight more uniformly [10].” (Pg. 2, Line 50-62)

 

Also, the proposed pecking block materials is not comprehensively mentioned. Only slightly mentioning the grain-based pecking block but no further information is accessible.

Response: Thank you for the reviewer's comments. Unfortunately, there are very few studies that provide environmental enrichment in the form of grain-based blocks. However, we have supplemented the content as follows to provide readers with an understanding of the characteristics of various blocks and their effects on livestock. In the revised manuscript, we also explain the rationale for our use of grain-based blocks, which we believe will help readers understand the background of this study.

 

“Meanwhile, in order to maximize the effect of providing environmental enrichment materials, it is important to explore materials that have low risk factors, are interesting to livestock, and can be used for a long time. Several studies have shown that environmental enrichment materials in the form of blocks or stones (hard and durable) or feed (nutritious feed such as hay, carrots, mealworms, etc.) can have different effects on livestock welfare and environmental improvement depending on the material provided. Although hay has been reported to be effective in reducing pecking behavior (e.g., cannibalism), stimulating foraging behavior, and lowering stress hormones [11], it may be a factor in aggravating plantar dermatitis and increasing the risk of bacterial infections [17]. Insects such as mealworms have the advantage of being nutritious and popular with chicks, but the effects vary depending on the insect species and can be problematic for broiler health, especially if they are consumed contaminated [18, 19]. Enrichment materials such as carrots, silage, and corn straw have been reported to reduce negative poultry behaviors but have potential nutritional imbalances and safety concerns associated with spoilage on farms [20]. Therefore, the environmental enrichment materials provided to poultry must be raw materials that are guaranteed to be safe when consumed, and one such material can be grains used in feed [21].

The provision of commercially available and durable pecking blocks can provide enrichment to the poultry environment [22]. And heavy structured environmental enrichment can be used by livestock for a long time, improve welfare indicators such as stress, and eliminate risk factors such as beak trimming [11, 22, 23]. Although the study by Beaudoin et al. [23] targeted pigs, it was confirmed that among various environmental enrichment materials, wood blocks were accessed and used for the longest period of time. The porous concrete blocks used in the study by Holcman et al. [24] were also of great interest to poultry due to their complex and destructible properties. Considering this, it was hypothesized that a somewhat rigid but breakable and edible material would enrich the behavior of poultry, reducing stress and improving welfare. However, there are few cases in which grain-based blocks are provided as environmental enrichment that are safe for broilers, provide nutrients, and are breakable by pecking behavior.

Against this backdrop, we have specially manufactured grain blocks based on broiler feed raw materials. In addition, most studies on environmental enrichment through pecking material provision have been conducted at the laboratory scale, so they need to be verified at the farm level. Therefore, this study aimed to investigate the effect of providing grain-based pecking blocks using two facilities in an animal welfare-certified broiler farm. We investigated welfare indicators, stress levels, productivity, and housing environment (air conditions, litter quality), and these results can confirm the practical effect of providing pecking material (grain-based blocks) on farms.” (Pg. 2-3, Line 73-109)

 

 

 

Materials and methods section :

 

Delete " on L71

This section would be helpful to include more details about the machines, chemicals, and materials used (e.g., model, manufacturer name, province, country) to ease reproducibility

Response: Thank you for the reviewer's comments. We have removed " from the revised manuscript. And in the revised manuscript, we reviewed manuscript again to provide more information for readers to reproducibly research.

 

Could the author please provide detailed sample conditions, e.g. what strain of broiler, age, replications at each treatment, total sample number, etc. Thus, improve the understanding of experimental condition.

Response: Thank you for the reviewer's comments. We have supplemented the content to make it easier for readers to understand the information about the facilities used in our study, the research design, and the management methods, and rearranged the order of the text to make the flow of the context smooth.

 

2. Materials and Methods

We received review and approval (approval No. NIAS-2021-534) from the Animal Experiment Ethics Committee at the National Institute of Animal Science, Rural Development Administration, Republic of Korea.

 

Farm Selection and experimental design

    The experiment was conducted on a farm certified for animal welfare. The farm was situated in Boseong-gun, Jeollanam-do, and consisted of two houses, each measuring 120 meters in length and 16 meters in width, covering an area of 1,920 m². Each house housed approximately 32,000 birds, resulting in a stocking density of 16.7 birds/m². The bedding material provided in each house was fresh rice hulls. The houses were windowless and equipped with tunnel-type forced ventilation. Each house had 2-meter-long wooden perches, with one perch for every 1,000 birds.

Other management specifications followed the guidelines recommended by the integrated company. Briefly, the lighting schedule started at 23 hours of lightness (L) and 1 hour of darkness (D) on day 1, with the day length gradually decreasing until reaching 18L:6D by day 5. After that, lighting schedule was then maintained continuously until the end of the experiment. The house temperature was maintained at 30℃ during the first week of the experiment and gradually decreased to 24℃ at the end of the experiment. The humidity of the broiler house was maintained at 50 to 70% during the experiment (Table 1).

 

Table 1. Characteristics of commercial experimental farms.

	Farm
Farm	Animal welfare certified farms
Region	Boseong-gun, Jeollanam-do, South Korea
Strain	Arbor acres
Housing type	Windowless
Ventilation type	Forced exhaust
Flock size, number of size	32,000
House size, m, m2	120 × 16, 1,920
Stock density, birds/m2	16.7
Litter type	Rice hulls
Perches installed	Wood type, 2 m length, 1,000 birds/m
Lighting schedule1	Gradually from 23L:1D to 18L:6D in the first 5 days, and continued at 18:6D

¹ The lighting schedule is represented at the number of hours of lightness(L): darkness(D).

 

The broiler strain used 1-day-old Arbor Acres (body weight 42.54±1.34g) supplied by the integrated company, and was allocated to a total of 2 treatments, 2 replicates, and 16,000 birds per replicate. A wall was built in the center of the house to prevent the chickens from mixing, and one side was provided with enrichment (Enrichment group) and the other was not provided (Control). The enrichment was a grain-based block, with one grain block per 1,000 birds (16 pecking blocks per replicate). The locations of the pecking blocks were alternated across different sections of the house. The housing facilities and experimental design used in this study are summarized in Figure 1.

 

“Water and feed were available ad libitum, and commercial diets were provided. In addition, considering the feed supply program (Stater: 1d - 21d; Finisher: 22d – before slaughter) and slaughter date (30d) operated by the farm, the analysis dates for each index in this study were conducted at 19 and 27 days of age. This experiment lasted 27 days.”

 

We have also tried to improve the understanding by creating a more detailed schematic diagram within the account.

 

 

Figure 1. Schematic diagram of the sampling locations and placement of pecking blocks in house. The symbol ‘□’ represents the placement of enrichment (grain-based pecking block), while the symbol ‘●’ represent specific sampling locations. The ‘●’ symbols indicate where productivity, blood sampling, litter sampling, litter ammonia, carbon dioxide, footpad dermatitis, hock burn and feather dirtiness were determined.

 

Water and feed were available ad libitum, and commercial diets were provided. In addition, considering the feed supply program (Stater: 1d - 21d; Finisher: 22d – before slaughter) and slaughter date (30d) operated by the farm, the analysis dates for each index in this study were conducted at 19 and 27 days of age. Therefore, this experiment lasted 27 days. (Pg. 3-4, Line 110-154)

 

Also, more importantly, provides the average temperature and relative humidity during experimental conduction.

Response: In the revised manuscript, the following was added regarding the environment (temperature, humidity) set during the broiler rearing period.

 

“The house temperature was maintained at 30℃ during the first week of the experiment and gradually decreased to 24℃ at the end of the experiment. The humidity of the broiler house was maintained at 50 to 70% during the experiment (Table 1).” (Pg. 3, Line 127-130)

 

Many of the parameters and variables detailed in the materials and methods section lack accompanying reference protocols. To enhance the reproducibility, reliability, and relevance of this work, it is essential to provide pertinent references for these elements.

Response: Thank you for the reviewer's comments. In the revised manuscript, the following content has been supplemented and referenced protocols and machine information have been added to provide information to readers and to ensure sufficient study reproducibility.

 

Productivity

In terms of productivity, we investigated body weight and uniformity. The body weight measurement was conducted twice in total, at 19 and 27 days of age, and an electronic automatic measuring scale (GI-1000, G tech Co. Ltd., Gyeonggi-do, Korea) was used to measure the body weight of each individual. For each measurement, 90 broilers were measured per treatment group, and for uniformity, they were randomly selected and surveyed at the points indicated in Figure 1 (7 to 8 birds were measured at each point). Afterwards, to investigate the uniformity of body weight (expressed as coefficient of variation (CV)) for each measurement day, the average value of the measurement location was used and the value was calculated and determined.

 

Litter moisture and gas emissions

Litter samples were randomly collected from six designated locations (Figure 2) within each barn at 19 and 27 days of age, with moisture content analyzed according to AOAC method 934.01 [25]. Briefly, the moisture content in the litter was analyzed by collecting 100 g of litter from each location on the day of measurement. The moisture content was measured by drying in dry oven at 105℃ for 24 hours, and the moisture content was expressed as a percentage (%) of the difference in weight after drying from the weight before drying.

To measure gas concentrations, a gas sampling pump (Gastec Corp., Ayase-city, Japan) was used along with specific detector tubes—No. 3M and 3La for ammonia, and No. 4LL and 4LK for carbon dioxide.

 

Gait score

To assess animal welfare, gait scoring was conducted using a 3-point scale according to AssureWel Meat Chicken Assessment Protocol [26], chosen for its effectiveness and practicality in a farm setting. This analysis examined 180 broilers randomly selected per treatment (15 birds per location × 6 locations × 2 houses) on each measurement day. Broilers exhibiting abnormal gait while walking across 20% of the house area were identified, and the flock’s movement was recorded using a GoPro Hero 12 camera (CPST1, GoPro, Inc., CA., USA) along their walking path. In terms of scoring, a score of 3 indicated an abnormal gait and impaired mobility, a score of 4 reflected a severe gait issue with only a few steps possible, and a score of 5 signified the inability to walk at all [15, 21].

 

Footpad dermatitis, hock burn, and feather cleanliness

Footpad dermatitis, hock burn, and feather cleanliness were assessed following the Welfare Quality Assessment protocol for poultry [27]. On days 19 and 27, a total of 180 birds were randomly selected from each house (15 birds per location × 6 locations × 2 houses) and inspected. Both footpad dermatitis and hock burn were scored on a 3-point scale (1 to 3), where a score of 1 indicated no signs of the condition, 2 indicated mild symptoms, and 3 indicated visible symptoms. Feather cleanliness was also rated using a 3-point scale (1 to 3), with 1 representing clean feathers, 2 representing slightly dirty feathers, and 3 representing significantly dirty feathers. Figure 3 shows examples of the score criteria used in our study to measure foot pad dermatitis and feather cleanliness.

                                                                           

(a) Footpad dermatitis
Score 1; minor (light)	Score 2; mild (medium)	Score 3; severe (heavy)
(b) Feather cleanliness
	<Front>
	<Back>
Score 1; minor (light)	Score 2; mild (medium)	Score 3; severe (heavy)

Figure 3. Footpad dermatitis of broilers showing how the degree of damage was scored (a) and feather condition and cleanliness for the scores of 1~3 on the body of each broiler (b).

 

Stress hormones (corticosterone) in the blood

    To assess the stress levels associated with pecking block provision, the stress hormone corticosterone was analyzed from blood samples. Blood samples were collected from the wing vein of 12 birds randomly selected from twelve locations per treatment group (six locations per replicate) on days 19 and 27, using EDTA-coated BD Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, USA). After collection, the serum was separated and stored at -70℃ until further analysis. The analysis was performed using a chicken stress hormone enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Fine Biotech Co., Ltd., Hubei, China) according to the protocol provide in the manual. Absorbance was measured with a spectrophotometer (Epoch 2; BioTek Instrument, Inc., Winooski, VT, USA) following the analysis (Pg. 5-7, Line 171-232)

 

Please also include software vendor details. (type, origin, country)

Response: Thank you for the reviewer's comments. In the revised manuscript, we added the following information about the statistical software we used, and supplemented the normality verification and outlier removal before statistical analysis to help readers understand.

 

In this study, we attempted to remove outliers and missing values by checking the homogeneity of variance and normality of data. If the residuals were more than three times the standard error of the parameter, they were considered outliers to be excluded from the data set before statistical analysis. Subsequently, all data were analyzed using analysis of variance (ANOVA) in a completely randomized design in statistical analysis system (SAS) software (version 9.4; Statistical Analysis System Institute Inc., Cary, NC, USA) through the Proc Mixed procedure. Outliers were checked with the UNIVARIATE procedure in SAS; however, none were identified. Differences among least-squares means were assessed using the PDIFF option with a t-test. Animal welfare indicators were analyzed using a chi-square test, with Fisher’s Exact Test applied when the expected frequency was less than 5. A macro program was utilized to categorize output values into letter groups. Statistical significance was defined at p < .05, with trends considered for .05 ≤ p ≤ .10. (Pg. 7, Line 234-247)

 

Could the author please present a detailed composition on the utilized grain-based pecking blocks?

Response: Thank you for the reviewer's comments. The composition of the pecking block material used in this study has already been presented in the Materials and Methods section. However, we have supplemented the content as follows to provide readers with more information about our study.

 

In this study, to provide environmental enrichment for broilers, grain based pecking blocks have been developed by Sinaebio Co. Ltd (Sungnam-si, Korea). These blocks were designed as cubic structures with dimensions of 25 × 25 × 25 cm (Figure 2) and consisted mainly of 50~60% by-products such as wheat bran, 10~20% grains and 10~15% limestone, supplemented with additional components such as moisture and glycerine. The grain blocks were changed regularly every 14 days for a total of 27 days of experiment. (Pg. 4, Line 158-163)

 

Why did the 19 and 27 days of age selected in measuring most parameters in this study?

Response: Thank you for the reviewer's comments. Feeding programs for broilers are generally divided into early and late stages. (Stater: 1d - 21d; Finisher: 22d - before slaughter)

In the farm we used, starter diet was provided until 22 days before and then changed to finisher diet from day 22. Also, the reason why the analysis was performed at 27 days of age is that most broiler farms slaughter around 30 days of age, so sampling and analysis were conducted at 27 days of age to minimize the impact. Therefore, additional data on 42-day-old broilers could not be obtained because they had already been slaughtered.

 

“In addition, considering the feed supply program (Stater: 1d - 21d; Finisher: 22d – before slaughter) and slaughter date (30d) operated by the farm, the analysis dates for each index in this study were conducted at 19 and 27 days of age. Therefore, this experiment lasted 27 days.” (Pg. 4, Line 151-154)

 

Results and Discussions section :

 

The result is sufficiently written, where the author successfully presented all captures phenomenon according to the data.

Response: Thank you for your detailed review of our study.

 

The reviewer’s suggestion is to revise the table format in a similar manner to body text.

Response: Thank you for the reviewer's comments. In the revised manuscript, we attempted to supplement the results section of the original manuscript so that the tables and text would be consistent.

 

Not separating mean and SD at different columns, but in the same column 1.78 ± 0.341, representing mean and SD.

Response: Thank you for the reviewer's comments. In the revised manuscript, the overall result expression method (SD values) was revised.

 

At table and figure caption, please provide detailed treatment for control, enrichment group, superscript [if available].

Response: In the revised manuscript, information was provided by adding descriptions of treatment groups to the footnotes of tables and figure titles.

 

Control, environmental enrichment (grain based pecking blocks) not provided; Enrichment, environmental enrichment (grain based pecking blocks) provided (one grain block per 1,000 birds). (Each tables and figures)

 

We welcome the detailed and constructive comments from the reviewer, and we have attempted our best to justify those in the revised manuscript addressing most of the concerns. However, we welcome any further query and constructive suggestions posed by the reviewer. 

 

Thank you for your consideration. I look forward to hearing from you. 

 

Yours Sincerely,

 

Chan-Ho Kim, Ph. D.

Animal Welfare Research Team, National Institute of Animal Science, Rural Development Administration, Wanju, 55365 Korea

E-mail: kch8059@korea.kr

Tel: +82-63-238-7053

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The present study investigated the effects of enrichment on many parameters of broiler chickens. The results showed that providing the grain blocks could reduce the stress of broiler chickens under intensive production. Following are some comments on the manuscript.

 

L89: How long was the experimental period? From hatching day?

 

L90: The authors indicated that the aim of grain blocks was to stimulate the pecking behavior in broiler chickens. However, no data related to this behavior was provided. This sentence should be revised.

 

Figure 1: Was the grain block renewed regularly?

 

L108: Why d19 and d27 were chosen to measure the body weight? The time interval was a bit short. What about d42? Similar with all measurements, why d19 and d27?

 

L119: Could the authors provide photos regarding the characteristic of different scores of footpad dermatitis, hock burn, and feather cleanliness? Photos could provide better understanding for the readers.

 

Figure 2: The authors indicated that the blocks were given to only half chickens. However, this figure showed that all chickens had access to the blocks. The separating method should be clearly pointed out in the figure.

 

L136: Photos of different gait score are suggested to be provided.

 

L148: The authors indicated that 10 blood samples were collected for the measurement of corticosterone. However, in Table 4, the replicate was 12. Which is right?

 

L157: Did the authors calculate the normal distribution of the data? Normal distribution is the prerequisite of ANOVA.

 

Table 4: The value difference between two groups was very low, whereas the SD was indeed very high (almost 15-19% of the average value). The p value should be significant under this circumstance. The authors should provide the original data of corticosterone levels.

 

Discussion: Environmental enrichment is required for promoting the occurrence of natural behavior in captive animals. The main objective is to satisfy relevant behavior in animals. The authors were supposed to analyze the effect of enrichment on the pecking behavior in broiler chickens as they have pointed out the aim of this study was to stimulate the instinctive behavior. Unfortunately, no behavior data was provided. The authors are suggested to discuss it in the manuscript.

Author Response

Dated: 27/12/2024

Response to Reviewer's Comments

Animal Welfare Research Team, National Institute of Animal Science, Rural Development Administration, Wanju, 55365 Korea

Dear Editor,

Many greetings, please find enclosed the revised manuscript entitled "Effects of providing enrichment to broilers in an animal welfare environment on productivity, litter moisture, gas emission, animal welfare indicators and stress level concentration".

 

Answers to the reviewer's comments

 

NOTE: All the changes made in the revised manuscript are highlighted in red color.

 

Reviewer #2:

 

Comments:

 

The present study investigated the effects of enrichment on many parameters of broiler chickens. The results showed that providing the grain blocks could reduce the stress of broiler chickens under intensive production. Following are some comments on the manuscript.

 Response: We are extremely thankful to the reviewer for considering our manuscript toward revision. We are grateful for all the queries and suggestions that helped us to improve the original version of the manuscript. We hope that all queries, comments, and suggestions were adequately justified in the revised version of the manuscript.

 

L89: How long was the experimental period? From hatching day?

Response: Thank you for the reviewer's comments. Our study was conducted for a total of 27 days, from the time the broilers first entered the farm (1 day of age) to the 27th day considering the slaughter date. We have supplemented the abstract and material & methods sections to make it easier for readers to obtain information.

 

In abstracts :

 

“This study used two facilities that can accommodate 32,000 1-day-old broilers (Arbor acres) per building, and two groups (control and treatment groups) were designed after creating two identical areas within each building (total 2 treatments, 2 replicates, 16,000 birds per replicate). Two identical zones within the house were created by installing a partition in the center, one side provided with grain blocks (One grain block per 1,000 birds) and the other side did not. Analysis items included productivity (body weight, uniformity), environmental variables (litter and air), welfare indicators (leg, gait score, feather cleanliness score) and serum corticosterone levels. Analysis of all items was conducted twice, on the 19th and 27th, taking into account the farm's feed change date and slaughter schedule.” (Pg. 1, Line 17-25)

 

In material & methods :

 

“The broiler strain used 1-day-old Arbor Acres (body weight 42.54±1.34g) supplied by the integrated company, and was allocated to a total of 2 treatments, 2 replicates, and 16,000 birds per replicate.” (Pg. 3, Line 134-136)

 

L90: The authors indicated that the aim of grain blocks was to stimulate the pecking behavior in broiler chickens. However, no data related to this behavior was provided. This sentence should be revised.

Response: Thank you for the reviewer's advice. In the revised manuscript, the following sentences were revised to fit the purpose of providing grain blocks.

 

“In this study, to provide environmental enrichment for broilers, grain based pecking blocks have been developed by Sinaebio Co. Ltd (Sungnam-si, Korea).” (Pg. 4, Line 158-159)

 

Figure 1: Was the grain block renewed regularly?

Response: Thank you for the reviewer's comments. The grain blocks provided in this study were provided at 1 day of age and replaced after 14 days, so that there was no problem in analyzing the effect of providing environmental enrichment.

The following content was supplemented to provide information on the replacement date.

 

In material & methods :

 

“The grain blocks were changed regularly every 14 days for a total of 27 days of experiment.” (Pg. 4, Line 162-163)

 

L108: Why d19 and d27 were chosen to measure the body weight? The time interval was a bit short. What about d42? Similar with all measurements, why d19 and d27?

Response: Thank you for the reviewer's comments. Feeding programs for broilers are generally divided into early and late stages. (Stater: 1d - 21d; Finisher: 22d - before slaughter)

In the farm we used, starter diet was provided until 22 days before and then changed to finisher diet from day 22. Also, the reason why the analysis was performed at 27 days of age is that most broiler farms slaughter around 30 days of age, so sampling and analysis were conducted at 27 days of age to minimize the impact. Therefore, additional data on 42-day-old broilers could not be obtained because they had already been slaughtered.

 

In material & methods :

 

“In addition, considering the feed supply program (Stater: 1d - 21d; Finisher: 22d – before slaughter) and slaughter date (30d) operated by the farm, the analysis dates for each index in this study were conducted at 19 and 27 days of age. Therefore, this experiment lasted 27 days.” (Pg. 4, Line 151-154)

 

L119: Could the authors provide photos regarding the characteristic of different scores of footpad dermatitis, hock burn, and feather cleanliness? Photos could provide better understanding for the readers.

Response: Thank you for the reviewer's comments. We have provided images of the criteria for the scores of feather cleanliness and toe burn in the revised manuscript. However, in relation to the hock burn score, no score higher than 1 was observed in our study, so there is no picture for reference. However, to help readers understand our research method, we have referred to the literature that used the same method and presented the protocol.

                                                                           

(a) Footpad dermatitis
Score 1; minor (light)	Score 2; mild (medium)	Score 3; severe (heavy)
(b) Feather cleanliness
	<Front>
	<Back>
Score 1; minor (light)	Score 2; mild (medium)	Score 3; severe (heavy)

Figure 3. Footpad dermatitis of broilers showing how the degree of damage was scored (a) and feather condition and cleanliness for the scores of 1~3 on the body of each broiler (b).

(Pg. 5, Line 216-220)

 

Figure 2: The authors indicated that the blocks were given to only half chickens. However, this figure showed that all chickens had access to the blocks. The separating method should be clearly pointed out in the figure.

Response: Thank you for the reviewer's comments. In the revised manuscript, we have revised the figures to provide readers with a more accurate information of our research facility and the placement of grain blocks, and we have included in the text the addition of a wall between the houses used to prevent mixing of broilers.

 

Materials and methods section :

 

“The broiler strain used 1-day-old Arbor Acres (body weight 42.54±1.34g) supplied by the integrated company, and was allocated to a total of 2 treatments, 2 replicates, and 16,000 birds per replicate. A wall was built in the center of the house to prevent the chickens from mixing, and one side was provided with enrichment (Enrichment group) and the other was not provided (Control). The enrichment was a grain-based block, with one grain block per 1,000 birds (16 pecking blocks per replicate). The locations of the pecking blocks were alternated across different sections of the house. The housing facilities and experimental design used in this study are summarized in Figure 1.”

 

 

Figure 1. Schematic diagram of the sampling locations and placement of pecking blocks in house. The symbol ‘□’ represents the placement of enrichment (grain-based pecking block), while the symbol ‘●’ represent specific sampling locations. The ‘●’ symbols indicate where productivity, blood sampling, litter sampling, litter ammonia, carbon dioxide, footpad dermatitis, hock burn and feather dirtiness were determined.

(Pg. 4, Line 142-149)

 

L136: Photos of different gait score are suggested to be provided.

Response: Thank you for the reviewer's comments. Gait score is an index that analyzes behavior by observing it, and there is no data on images. However, we have provided information on the gait score evaluation method and presented several research cases that have been analyzed so that readers can easily obtain information. The supplemented content is as follows.

 

 

Gait score

To assess animal welfare, gait scoring was conducted using a 3-point scale according to AssureWel Meat Chicken Assessment Protocol [26], chosen for its effectiveness and practicality in a farm setting. This analysis examined 180 broilers randomly selected per treatment (15 birds per location × 6 locations × 2 houses) on each measurement day. Broilers exhibiting abnormal gait while walking across 20% of the house area were identified, and the flock’s movement was recorded using a GoPro Hero 12 camera (CPST1, GoPro, Inc., CA., USA) along their walking path. In terms of scoring, a score of 3 indicated an abnormal gait and impaired mobility, a score of 4 reflected a severe gait issue with only a few steps possible, and a score of 5 signified the inability to walk at all [15, 21]. (Pg. 5, Line 194-203)

 

Reference:

 

• Meat Chicken Assessment Protocol: Instructions. Available online: http://assurewel.org/broilers.html (accessed on 5 January 2023)
• Kwon, B.Y.; Lee, H.G.; Jeon, Y.S.; Song, J.Y.; Park, J.; Kim, S.H.; Kim D.W.; Lee, K. W. Effects of grain-based pecking blocks on productivity and welfare indicators in commercial broiler chickens. Anim. Biosci. 2023, 37, 536. https://doi.org/10.5713/ab.23.0384
• Kwon, B.Y.; Lee, H.G.; Jeon, Y.S.; Song, J.Y.; Park, J.; Kim, S.H.; Kim, D.W.; Lee, K.W.; Effects of grain-based pecking blocks on productivity and welfare indicators in commercial broiler chickens. Anim. Biosci. 2024, 37, 536-546. https://doi.org/10.5713/ab.23.0384

 

L148: The authors indicated that 10 blood samples were collected for the measurement of corticosterone. However, in Table 4, the replicate was 12. Which is right?

Response: First, we apologize for any confusion regarding the number of samples in the original manuscript. In the revised manuscript, we have reconfirmed the number of samples analyzed and corrected the incorrect figures. (It is correct that the corticosterone analysis was conducted on a total of 12 broilers.)

 

L157: Did the authors calculate the normal distribution of the data? Normal distribution is the prerequisite of ANOVA.

Response: Thank you for the reviewer's comments. To ensure the reliability of the study results, we designed the experiment and analyzed the results as follows to explain the differences between the treatment groups.

 

First, our study was conducted using broilers, with two replicates per treatment group, and 8,000 broilers were assigned to each replicate, and the study was conducted in compliance with animal welfare standards.

 

In addition, the number of measurement objects and the number of samples were determined so that the effect depending on the provision of environmental enrichment could be sufficiently analyzed for each analysis item, and the number of samples used for each analysis item is as follows.

 

-              Growth performance: 90 birds (12 sites(replicate); 7 to 8 chickens/site)/treatment

-              Litter quality: 12 sites/treatment

-              Air quality: 12 sites/treatment

-              Gait score: 180 birds(15 birds/site × 6 sites × 2 houses)

-              FPD, Hock burn score, feather cleanliness score: 180 birds(15 birds/site × 6 sites × 2 houses)

-              Corticosterone: 16 birds/treatment

 

This experimental design is considered to be of an appropriate scale of analysis while not causing any harm to the operation of the farm and there is no problem in comparing significant differences between treatment groups in the animal welfare environment of broilers (Provide at least 12 replicates (birds) per treatment).

 

In order to secure the homogeneity of variance and the normality of data in the observed research data, outliers and missing values were removed. The criteria for removing outliers were performed by referring to the references below, and in this study, outliers were considered to be excluded from the data set if they were more than three times the standard error.

 

After going through this process, we tested the significance of each analysis indicator between groups using a statistical program (SAS).

 

In the revised manuscript, we have described in detail the process from data acquisition to statistical analysis to help readers understand our research.

 

“In this study, we attempted to remove outliers and missing values by checking the homogeneity of variance and normality of data. If the residuals were more than three times the standard error of the parameter, they were considered outliers to be excluded from the data set before statistical analysis. Subsequently, all data were analyzed using analysis of variance (ANOVA) in a completely randomized design in SAS (SAS Institute, Inc., Cary, NC, USA) through the Proc Mixed procedure. Outliers were checked with the UNIVARIATE procedure in SAS; however, none were identified. Differences among least-squares means were assessed using the PDIFF option with a t-test. Animal welfare indicators were analyzed using a chi-square test, with Fisher’s Exact Test applied when the expected frequency was less than 5. A macro program was utilized to categorize out-put values into letter groups. Statistical significance was defined at p < .05, with trends considered for .05 ≤ p ≤ .10.”

 

Reference:

 

• Jespersen, J. C., Richert, S., de Paula Dorigam, J. C., Oelschlager, M. L., & Dilger, R. N. (2021). Effects of lysine biomass supplementation on growth performance and clinical indicators in broiler chickens. Poultry science, 100(3), 100971.
• Santos, R. R., & van Eerden, E. (2021). Impaired performance of broiler chickens fed diets naturally contaminated with moderate levels of deoxynivalenol. Toxins, 13(2), 170.
• Dalólio, F. S., Albino, L. F. T., da Silva, J. N., Fireman, A. K. A. T., Burin, Á. M., Busanello, M., & Ribeiro, V. (2021). Dietary chromium-methionine supplementation and broiler (22–43 days) responses during heat stress. 1. Growth performance and carcass yield, metabolisable energy and serum biochemistry. Animal production science, 61(6), 586-595.

 

Table 4: The value difference between two groups was very low, whereas the SD was indeed very high (almost 15-19% of the average value). The p value should be significant under this circumstance. The authors should provide the original data of corticosterone levels.

Response: Thank you for the reviewer's comments. The raw data files investigating the concentration of corticosterone in this study were submitted along with the revised paper.

No.	19 day		27 day
	Control	Enrichment	Control	enrichment
1	1.711	1.835	1.844	1.969
2	1.687	1.830	1.727	2.157
3	1.716	1.512	1.502	1.633
4	1.835	1.582	1.544	1.566
5	1.698	1.759	1.877	2.448
6	1.989	1.590	1.702	2.448
7	1.652	2.032	1.867	1.528
8	1.698	1.913	1.791	1.479
9	1.512	1.605	1.575	1.477
10	1.985	1.544	1.617	1.544
11	1.999	1.581	2.009	1.575
12	1.757	1.549	1.991	1.636
13	1.625	1.558	2.438	1.429
14	1.725	1.538	2.248	1.789
15	1.789	1.494	1.620	1.982
16	1.898	1.489	1.685	2.169
Mean	1.767	1.651	1.818	1.780
SD	0.140	0.167	0.185	0.182

 

 

Discussion: Environmental enrichment is required for promoting the occurrence of natural behavior in captive animals. The main objective is to satisfy relevant behavior in animals. The authors were supposed to analyze the effect of enrichment on the pecking behavior in broiler chickens as they have pointed out the aim of this study was to stimulate the instinctive behavior. Unfortunately, no behavior data was provided. The authors are suggested to discuss it in the manuscript.

Response: Thank you for the reviewer's comments. We also believe that if we had observed behavioral changes in broilers when grain-based blocks were provided as environmental enrichment in this study, the quality of the study would have been improved and readers would have gained more information about environmental enrichment.

We are very sorry that we have not analyzed and presented this data, and we will make sure to provide it in order to investigate changes in behavioral patterns when conducting further research on environmental enrichment in the future.

 

Meanwhile, in the revised manuscript based on the reviewer's comments, an additional paragraph was created on the change in behavioral patterns of poultry through the provision of environmental enrichment to provide information to readers and suggest necessary future research directions.

 

Additional information is provided below, and we hope that providing this discussion will improve the quality of our research paper.

 

“Meanwhile, environmental enrichment methods through providing pecking materi-als can influence the behavioral patterns of poultry, stimulate instinctive activities, and provide a satisfactory rearing environment [43]. In particular, pecking between poultry birds is a bad behavior that causes great injury and stress to the flock, and causes great damage to the farm economy [11, 44, 45]. When poultry raised in harsh environments are provided with pecking materials such as strings, highly valued feed items (e.g., hay, in-sects, mealworms), their pecking behavior decreases and other behavioral patterns emerge (foraging, dustbathing, and locomotion) [31, 43]. In the case of straw bales, they also serve as protection and shelter, but after two weeks, they have been observed to function as rest-ing areas, where they rest or lie down [46]. Environmental enrichment using hay and perches stimulated standing, resting, and moving behaviors [47], while studies providing insects resulted in more wing flapping, stretching, scratching, preening, and ground pecking. Dixon et al. [43] reported that providing environmental enrichment reduced the frequency of pecking behavior as poultry spent more time with the enriched objects. Addi-tionally, several studies have shown that the lack of foraging material on the ground af-fects feather pecking behavior [20]. Although it is known to be difficult to completely re-duce pecking behavior using environmental enrichment materials, continued exploration of environmental enrichment materials that are effective in improving poultry behavior is warranted [20, 43].” (Pg. 12, Line 417-435)

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been improved. It is acceptable for publication now.

Author Response

Line 118- 120  factors such as peak trimming …and then you cite a reference related to pigs. This is not acceptable. Furthermore it  is questionable to transfer pig behaviour data to poultry. So please delete the pig reference from this manuscript and change all sentences related to them if necessary.

(Response) As suggested by the reviewer, we have removed the description of the pig and the reference to it

(Delete ) 23. Beaudoin, J.M.; Bergeron R…………………. Growing pigs’ interest in enrcichment------

 

Line 181 …certified for animal welfare.  Please include here something. We need to know the requirements that have to be fulfilled to be certified for animal welfare (or give a reference where these criteria are described preferably in English)

(Response) As suggested by the reviewer, we have included the main Korean animal welfare certification standards.

South Korea’s broiler animal welfare certified must provided stocking densities 30kg/m², perch, and enrichments.

 

Line 551, 556, 558 …do not use significant and p-value… this is kind of redundant…use p value or significant never both!! Please change throughout the entire manuscript

(Response) As suggested  by the reviewer, we have included delete p-value

 

Line 556-557 please rewrite!! When not significant it is not slightly higher … they are the same, a difference that was not significant is not a difference!!!

(Response) We have changed it to the sentence below as suggested

NH₃ emissions were also not significantly different between treatments on both days 19 and 27 (Table 4).

 

Line 579- 590 and table 3  this needs to be rewritten because you only describe the gas concentration. You did not measure emission because emission would the amount of gas per time or per airflow or complete experiment. Please change gas emission to gas concentration!! ppm is no SI Unit please recalculate to mg per cubic meter or liter

(Response) We have converted to the units from ppm to mg/m³ as suggested by the reviewer

 

Line 657 change corn forage to maize forage, please check if you used corn anywhere else in the text and change it to maize

(Response)

As suggested by the reviewer, we have changed corn forage

 

Line 701  change to Si Units

(Response) We changed from ppm to mg/m³ as suggested.

 

Line 789 –800 your corticosterone data  were taken by single venpuncture. Venepuncture procedures have also been conceived as ‘‘mild pain’’ , ‘‘traumatic’’  or ‘‘potent psychological and physiological stressors’’ and might therefore elicit humoral stress responses themselves. In consequence, stress responses elicited by venepuncture could superimpose the responses elicited by target stressors, and consequently distort research findings. Please indicate shortly that venepuncture itself may induce corticosterone levels to increase, therefore these results have to be discussed with caution. Please give the normal physiological range of corticosterone level in poultry blood. If the values are within the typical physiological range the question arises if this small difference you found is of bioogival relevance even so it is significant . Under stress continous increase would be much higher  I would guess.

(Response) As suggested by the reviewer, we have inserted a note about the normal range od stress hormones in the blood and the interpretation of the data blood taken from a subacromial vein.

Absoulte corticosterone measures in chickens differ heavily among studies [39]. To summarize, basal corticosterone concentrations have been described to range between 0.27 and 2.7 ng/mL [39, 40]. ~ In addition, the venipuncture itself may induce an increase in corticosterone concentrations levels, and therefore the interpretation of the results should be discussed with caution, and future experiments should be repeated.

Author Response File: Author Response.docx