The influence of food processing methods on serum parameters, apparent total-tract macronutrient digestibility, fecal microbiota and SCFA content in adult beagles.

Food processing methods may influence the health of dogs. However, previous studies have mostly been based on a comparison of several commercial dog foods with different ingredients. In this study, eighteen adult beagles of the same age and health status (assessed by routine blood tests) were used in the experiments. This study analyzed the effects of the following different processing methods: raw, pasteurized, and high temperature sterilization (HTS) made with the same ingredients and nutrients (based on dry matter) on serum parameters, apparent total-tract macronutrient digestibility, fecal microbiota and short-chain fatty acid (SCFA) content in beagle dogs. The data showed, after a test lasting 56-days, the apparent digestibility (ATTD) of protein and fat in HTS food was 91.9%, which was significantly higher (P< 0.05) than that in dry food (89.2%, P < 0.05). The serum content of triglyceride increased in beagles fed HTS food (P < 0.05), and the number of neutrophils in beagles fed raw food and pasteurized food increased significantly (P < 0.05), and the platelet count in beagles fed raw food showed an increasing trend compared with the beagles fed HTS food. Different processing methods had an impact on the intestinal microbiota and SCFA of beagles; at least 14 genera were significantly affected by the food produced using different processing methods. In particular, the abundance of Allprevotella, Escherichia-Shigella and Turicibacter, and the total acid content were lower in beagles fed the raw diet, whereas Streptococcus, Collinsella, Bacteroides and Ruminococcus gnavus were more abundant following the HTS diet, and Lactococcus showed the highest abundance in beagles fed the pasteurized diet. This study showed that dog food produced by different processing methods affected the health of adult beagles.

1. Introduction

Over the years, dog food has changed dramatically. It is recognized that dogs are derived from wolves [1]; thus, original dog food would have been raw meat. However, due to close integration with humans over 10,000 years, dogs now accept human carbohydrate-based cooked food [2]. The industrial revolution has changed the form of dog food. Since the invention of puffed dog food, this type of food has rapidly grown. In recent years, some people think that we should return dogs to “natural food”, so more and more owners feed their dogs wet food, especially, raw food [3].

There is controversy regarding which form of food is better for pet health. Algya et al. [4] analyzed four different processing methods (extruded, high moisture roasted refrigerated, high-moisture grain-free roasted refrigerated, and raw) for producing dog food, and found that “the lightly cooked and raw diets were highly palatable, highly digestible, reduced blood triglycerides, maintained fecal quality and serum parameters, and modified the fecal microbial community of healthy adult dogs.” However, Algya et al. [4] used different formula food in their study; therefore, it is difficult to judge whether the impact was caused by the processing method itself or due to the change in raw materials or nutrients. The study by Schmidt et al. had similar limitations, as different formula food was adopted in the study [5]. Therefore, to eliminate the interference of raw materials and produce diets with the same raw materials and nutrients, it is necessary to conduct an analysis of wet dog food produced using different processing methods.

We hypothesized that the sterilization method at different temperatures had an impact on the characteristics of the food itself, thus affecting the health of dogs. Therefore, this study aimed to analyze the effects of the following different processing methods: raw, pasteurized, and high temperature sterilization (HTS) on serum parameters, apparent total-tract macronutrient digestibility, fecal characteristics and microbiota in adult beagle dogs, in order to provide a basis for improving the processing of dog food and promoting dog health.

2. Methods and materials

2.1. Diets

The dog food was prepared using rice, chicken breast, corn, sugar beet meal, meat and bone meal, chicken fat and palatability enhancer (Table 1). Rice, corn, sugar beet meal, meat and bone meal were all commercially available finished powders, and the chicken fat and flavoring agents were pasty. The food was prepared in a clean environment. First, chicken breast meat was made into a meat emulsion, mixed with other raw materials, water was added and thoroughly stirred, and the raw food was directly packaged. The sterilized food was sterilized at 80°C for 20 min and then packaged under aseptic conditions. The HTS food was autoclaved at 121°C for 30 min and packaged after cooling to room temperature. All foods were divided and stored at -20°C. The raw, pasteurized and HTS dog foods used in this experiment were all processed by Shanghai Weita Pet Products Co., Ltd., Shanghai, China.

Table 1

Ingredients in the experimental diets.

Ingredients	Amount (%)
Broken rice	14.5
Chicken meat	32.4
Corn	3.4
Poultry fat	3.1
Meat and bone meal	1.8
Palatability enhancer 1	1.5
Beet pulp	1.0
Water	42.3

1 Palatability enhancer was mainly made up of chicken liver extract.

The dry mass, crude protein, fat (ether extract), ash, crude fiber, total Ca and P contents of the excreta and diet were determined according to the AOAC (1990) 930.15, 984.13, 954.02, 942.05, 962.09, 927.02 and 965.05 methods, respectively (Table 2).

Table 2

Chemical composition of the experimental diets.

Nutrients	Raw	Pasteurized	HTS2	SEM	P-value
Dry mass (DM)	34.64	34.03	34.89	8.01	0.94
Crude protein (CP)	30.59	30.27	30.42	0.15	0.92
Fat (EE)	9.44	9.35	9.51	0.22	0.83
Ash	7.54	7.63	7.40	0.35	0.74
Crude fiber (CF)	1.05	0.98	1.01	0.03	0.88
Calcium	1.42	1.39	1.40	0.10	0.78
Phosphorous	1.34	1.31	1.33	0.02	0.94

All data are measured values. All foods were tested at least 3 times (n = 3).

1 Data expressed as %DM basis except DM; DM expressed as-fed basis.

2 HTS: High temperature sterilization.

2.2. Animals and treatment

The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The experimental procedures were approved by the Ethics Committee for Research using Laboratory Animals of Shanghai Vocational College of Agriculture and Forestry.

Eighteen healthy beagle dogs (9 male and 9 female) of the same age (3 years) and similar weight were selected from the Training Base of Shanghai Vocational Technical College of Agriculture and Forestry. The test dogs were randomly divided into three groups with 6 dogs in each group according to the principle of half male and female, and fed raw, pasteurized, and HTS food, respectively. The experiment was conducted over 8 weeks, and the dogs were adaptively fed in the kennel for 1 week before the experiment. Each dog was individually raised in a room 2 m × 2 m × 3 m in size. The rooms were separated by cement walls, and each room was equipped with a food bowl, automatic drinking fountain and cotton pads. The temperature in the rooms was maintained at (25.0 ± 3.0°C during the test period. All beagles were fed once a day at 09:00 am, and were fed 750 g each time. Drinking water was always available. The rooms were cleaned at 09:00 am every day.

2.3. Blood sample collection and analyses

On day 0 and 56, blood was collected from the beagles for routine analyses of blood and serum metabolite concentrations. About 5 ml of blood from each beagle was collected via the forelimb vein in two appropriate vacutainer tubes: one with K2EDTA added for routine blood analyses, and the other for serum separation. The tubes with K2EDTA added were analyzed by an automated blood cell counter (BC-2800Vet, Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China) immediately. The blood samples without anticoagulant were kept at 4°C overnight and then centrifuged at 3,000 × g at room temperature for 10 min to obtain serum, and the serum was then transported to the laboratory for analysis using a clinical chemistry analyzer.

2.4. Fecal collection

Fresh fecal samples were collected after 8 weeks of treatment (54–56 d). Total feces excreted during the collection phase were collected from the bottom of the kennel immediately after observing the beagle’s bowel behavior. All fecal samples were divided into two, and frozen at -20°C for further analysis.

2.5. Apparent total tract digestibility study

One fecal sample from each beagle was thawed and dried in an air-dry oven at 60°C for 48 h. The dry mass, crude protein and fat (ether extract) were analyzed as described above. Acid-insoluble ash was used as an indigestible marker, and was analyzed using the method of Cai et al. [6].

2.6. DNA extraction and 16S rRNA gene processing

The fecal samples used for sequencing analysis were carefully wiped off the surface of the ultra-clean workbench, and about 0.1 g of the sample not exposed to the external environment was selected for DNA extraction. Microbial genomic DNA was extracted from the feces using a QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentrations and integrity of genomic DNA were verified with a Nanodrop 2000 spectrophotometer and 1.5% agarose gel electrophoresis. The variable region of 16S rRNA V4 was amplified using the universal primer sequence, 343F: 5′-TACGGRAGGCAGCAG-3′ and 798R: 5′-AGGGTATCTAATCCT-3′. Library construction was performed on barcoded V4 PCR amplicons and sequenced on the Illumina MiSeq PE250 platform (San Diego, CA, USA).

2.7. Data processing

Raw sequences were first filtered, and reads with adapter contamination at the ends of the reads, reads <50 bp, and reads with low quality (quality score <20) were removed with the Trimmomatic program [7]. Subsequently, the qualified double-ended raw data were spliced to obtain paired end sequences with a maximum overlap of 200 bp using Flash [8]. The clean tag sequence was then obtained using the split libraries software in QIIME [9] to remove sequences containing N bases in the paired end sequences, single base repeat sequences greater than six, and sequences with a length less than 200 bp. Finally, UCHIME [10] software was used to remove the chimerism in clean tags, and valid tags were obtained for subsequent operational taxonomic unit (OTU) partition. Sequence clustering was subsequently performed with the Vsearch algorithm [11] and clustered into OTUs. The most abundant sequence in each OTU was selected as a representative.

The taxonomy of each OTU was assigned by blasting the representative sequence against the Green genes reference database (Release 13.8, http://greengenes.secondgenome.com/) using the RDP classifier Naive Bayesian classification algorithm [12]. Unknown archaeal or eukaryotic sequences were filtered and removed. Diversity index data were analyzed statistically with analysis of variance, and significant differences between group means were determined with the least significant difference test. These sequence data have been submitted to the GenBank databases under accession number PRJNA733866.

2.8. Short-chain fatty acid detection

For SCFA detection, 1 g of fecal sample was diluted with distilled water, homogenized, and centrifuged at 12,000 × g for 10 min. Metaphosphoric acid (0.2 ml, 25% w/v) containing crotonic acid solution was added into 1 ml of supernatant. After storage overnight at -20°C, the samples were centrifuged for 10 min at 12,000 ×g. The supernatant was filtered through a 0.22 μm filter, and 0.5 μL of filtrate was injected into a gas chromatograph (7890B, Agilent Technologies, CA, USA) equipped with a flame ionization detector and a capillary column (30 m × 0.32 mm × 0.25 μm film thickness). To measure SCFAs, we used crotonic acid as an internal standard (1.077 mg/L). The column, injector, and detector temperatures were 130, 180, and 180°C, respectively. Hydrogen gas, produced by a gas generator (Parker Chrom Gas, Parker Hannifin Corporation, MN, USA), was used as the carrier gas at a flow rate of 40 mL/min. A standard SCFA mixture containing acetate, propionate, butyrate, isovaleric, and valeric acid was used for calculation.

2.9. Statistical analysis

Data on the growth performance (feed intake, body weight), ATTD, routine blood parameters, serum biochemical parameters, and SCFA content were expressed as means ± standard error of the mean (mean ± SEM). All the data mentioned above were validated by a Kolmogorov–Smirnov test and the results showed that all data followed normal distribution. These data were then analyzed by one-way ANOVA followed by post-hoc multiple comparison tests using SPSS v.21.0 statistical software.

The data on 16S RNA processing are described in the “Data processing” section. The statistical differences in the final data were also expressed as mean ± SEM except the relative abundance of the top 10 bacteria at the phylum level (Fig 1A). Fig 1A also describes the relative abundance in each dog. The data on 16S RNA processing was also analyzed by one-way ANOVA followed by post-hoc multiple comparison tests using SPSS v.21.0 statistical software.

Fig 1

Distribution of gut microbiome composition and relative change in beagles fed different processed food at the phylum level.

(a) Relative abundance. (b) Relative change in the top 10 bacteria at the phylum level. HTS: High temperature sterilization.

A P value < 0.05 was considered significant, whereas 0.05 < P value < 0.10 was considered a tendency.

3. Results

3.1. Effects of food processing methods on growth performance and apparent total tract digestibility in adult beagles

Coat color and liveliness in beagle dogs were not changed according to observations by the breeder and other participants in the experiment. As shown in Fig 2A, no differences in food intake were found between the treatments (P > 0.1); but all dogs had a significant increase in body weight during the test period (P<0.05). Analysis of the growth rate in terms of body weight (Fig 2B), showed that food intake of the different foods did not change, but the dogs fed pasteurized food and HTS food gained weight faster than the dogs fed raw food (0.05 < P <0.1). These results showed that the crude protein digestibility of HTS food was significantly higher than that of raw and pasteurized food (P<0.05, Fig 2C).

Fig 2

Effects of food processing methods on food intake, body weight increase (between 0 d and 56 d), and apparent total tract digestibility (ATTD) in adult beagles.

a) Effects of food processing methods on food intake; b) Effects of food processing methods on body weight increase; c) Effects of food processing methods on ATTD. Data are presented as mean ± SEM (n = 6). In each graph, different letters indicated a significant difference using one-way ANOVA (P < 0.05). HTS: High temperature sterilization.

3.2. Food processing methods affected serum parameters in adult beagles

No significant differences were found in beagles for both routine blood analysis and serum biochemical parameters at the beginning (0 d, data are shown in S1 & S2 Tables). However, as shown in Table 3, after treatment, the number of neutrophils (Gran) in beagles fed raw food and pasteurized food increased significantly (P < 0.05), and the platelet count in beagles fed raw food showed an increasing trend compared with the beagles fed HTS food. The number of white blood cells (WBC), mononuclear cells (Mon) and lymphocytes (Lymph) did not change (P > 0.1). The serum biochemical parameters (Table 4) in all dogs after the test were not significantly changed except serum triglyceride (TG). The content of TG increased in beagles fed HTS food compared with dogs fed raw or pasteurized food (P < 0.05). Routine blood analysis in all beagles showed normal levels both before and after the test.

Table 3

The effect of processing methods on the blood routine of adult beagles.

Item	Reference	Raw	Pasteurized	HTS2	P-value
WBC 1 (109·L-1)	6.0–17.0	12.42 ± 0.21	12.35 ± 0.82	11.73 ± 0.35	0.608
Lymph (109·L-1)	0.8–5.1	3.37 ± 0.33	2.85 ± 0.26	3.10 ± 0.18	0.413
Mon (109·L-1)	0.0–1.8	0.92 ± 0.08	0.83 ± 0.07	0.75 ± 0.03	0.207
Gran (109·L-1)	4.0–12.6	10.48 ± 0.49a	10.55 ± 0.68a	8.38 ± 0.58b	0.032
RBC (1012·L-1)	5.5–8.5	7.31 ± 0.34	7.79 ± 0.44	7.80 ± 0.27	0.549
HGB (g·L-1)	110–190	161.83 ± 7.18	159.33 ± 11.93	160.83 ± 5.79	0.979
MCV (fL)	62–72	66.35 ± 1.06	67.77 ± 1.16	66.88 ± 0.72	0.609
PLT (109·L-1)	117–460	349.00 ± 17.77A	342.00 ± 13.23AB	303.33 ± 9.65B	0.074

Data represent the mean ± standard error of the mean of 6 beagles per treatment (n = 6). Blood from each beagle was collected via the forelimb vein in one vacutainer tube containing K2EDTA and instantly analyzed by an automated blood cell counter.

1WBC, white blood cell count; Lymph, lymph cell count; Mon, monocyte cell count; Gran, neutrophilic granulocyte count; RBC, red blood cell count; HGB, hemoglobin concentration; MCV, mean corpuscular volume; PLT, platelet count.

2HTS: High temperature sterilization.

a-b Significant (P < 0.05) differences in the same row following analysis with Duncan’s test are indicated by different superscript letters. A-B A tendency (P < 0.1) for differences in the same row following analysis with Duncan’s test are indicated by different superscript letters.

Table 4

The effect of processing methods on serum biochemical parameters in adult beagles.

Item	Raw	Pasteurized	HTS2	P-value
TP1 (g·L-1)	66.42 ± 1.97	64.63 ± 1.54	64.25 ± 1.72	0.654
GLB (g·L-1)	37.75 ± 1.08	35.55 ± 0.76	35.27 ± 1.68	0.325
ALB (g·L-1)	28.67 ± 1.67	29.08 ± 1.32	28.98 ± 1.35	0.978
TC (mM·L-1)	3.60 ± 0.23	4.17 ± 0.45	3.75 ± 0.31	0.497
TG (mM·L-1)	0.84 ± 0.02b	0.87±0.03b	0.99± 0.04a	0.019
ALT (U·L-1)	39.73 ± 1.20	41.68 ± 1.10	41.12 ± 1.64	0.579
AST (U·L-1)	31.12 ± 1.50	31.02 ± 0.81	32.62 ± 1.74	0.673
Ca (mM·L-1)	2.23 ± 0.14	2.44 ± 0.22	2.23 ± 0.26	0.739
P (mM·L-1)	1.10 ± 0.05	1.25 ± 0.06	1.16 ± 0.04	0.151

Data represent the mean ± standard error of the mean of 6 beagles per treatment (n = 6). Blood from each beagle was collected via the forelimb vein in one vacutainer tube and the serum analyzed using a clinical chemistry analyzer.

1TP, total protein; GLB, globulin; ALB, albumin; TC, total cholesterol; TG, triglyceride; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Ca, calcium content; P, phosphorus content.

2HTS: High temperature sterilization.

a-b Significant (P < 0.05) differences in the same row following analysis with Duncan’s test are indicated by different superscript letters.

3.3. Food processing methods affected fecal microbiota composition of adult beagles

In this study, an average of 39, 938 clean tags were obtained for each group, the average length of the sequences was 416 bp, and a mean of 592 observed species was obtained for each group (S3 Table). Rarefaction analysis of 16S rRNA gene sequences indicated adequate sequencing depth (S1 Fig).

Chao1 and observed species indices showed the number of possible taxa, while Shannon and Simpson diversity were used to describe community richness and evenness. These are all alpha diversity indices. Table 5 shows that HTS food decreased both Chao1 and observed species in dogs (P< 0.05), and no differences in all alpha diversity indices (P > 0.1) were found between raw and pasteurized fed dogs.

Table 5

Alpha diversity indices of the bacterial communities.

Item	Raw	Pasteurized	HTS1	P-value
Chao1	1024 ± 28ab	1095 ± 56a	910 ± 31b	0.018
observed species	614 ± 29ab	657 ± 51a	515 ± 20b	0.036
Shannon	5.0 ± 0.21	5.3 ± 0.11	4.8 ± 0.28	0.272
Simpson	0.92 ± 0.01	0.94 ± 0.01	0.91 ± 0.02	0.404

Data represent the mean ± standard error of the mean of 6 beagles per treatment (n = 6). A fresh fecal sample from each beagle was collected after 8 weeks of treatment. DNA extraction was performed on barcoded V4 PCR amplicons and sequenced on the Illumina MiSeq PE250 platform.

1 HTS: High temperature sterilization.

a-b Significant (P < 0.05) differences in the same row are indicated by different superscript letters.

Firmicutes (54.17%, means for all samples, and the same below) was the only dominant phylum in the samples from all groups, Firmicutes, Bacteroidetes (18.79%), Fusobacteria (11.57%), Proteobacteria (9.88%) and Actinobacteria (5.29%) comprised over 99% of the bacterial phyla in the samples from dogs at the beginning of the test (Fig 1A). Fig 1B shows that the abundance of Actinobacteria, Acidobacteria and Gemmatimonadetes in HTS fed dogs decreased compared with those fed pasteurized food (P< 0.05).

Fusobacterium, Streptococcus, Lactococcus, Prevotella, and Alloprevotella were the top 5 abundant genera in dog feces, which consisted of almost 50% (exactly 48.05%) of the abundance on average in all 502 genera detected in this analysis. The principal component analysis (PCA) plot (S2 Fig) shows microbial communities in the feces of dogs fed food prepared by the different processing methods with no obvious distinction. Table 6 shows at least 14 genera were affected by the food prepared using different processing methods (genera with a relative abundance less than 0.1% were ignored in this analysis). Streptococcus was one of the most abundant genera; it increased rapidly in the dogs fed raw food. Lactococcus was also one of the main genera in dogs; data showed that the number of Lactococcus in dogs fed with pasteurized food increased. Alloprevotella was another main genus in dogs; this study showed that the abundance of Alloprevotella in dogs fed with HTS food markedly increased. With regard to other bacteria, the abundance of Collinsella, Bacteroides, Ruminococcus gnavus, Megasphaera, Erysipelatoclostridium and Lachnospiraceae decreased in dogs fed HTS food; the abundance of Escherichia-Shigella and Prevotellaceae decreased in dogs fed raw food, and the abundance of Ruminococcaceae increased in dogs fed raw food; the abundance of Turicibacter and Paeniclostridium increased in dogs fed pasteurized food.

Table 6

Genus-level taxonomic composition of the bacterial communities.

Genera	Raw, %	Pasteurized, %	HTS1, %	P-valueb
Streptococcus	17.04 ± 4.26a	6.19 ± 1.95b	4.11 ± 0.81b	0.010
Lactococcus	4.79 ± 1.14b	15.12 ± 3.37a	7.93 ± 1.89b	0.020
Alloprevotella	6.61 ± 2.06b	8.11 ± 2.35ab	16.42 ± 3.7a	0.054
Collinsella	4.11 ± 0.53a	4.44 ± 0.76a	0.95 ± 0.65b	0.003
Bacteroides	3.02 ± 0.85ab	3.67 ± 1.13a	1.01 ± 0.33b	0.097
Escherichia Shigella	0.45 ± 0.06b	6.17 ± 1.90a	4.66 ± 1.82ab	0.045
Ruminococcus gnavus	1.47 ± 0.31a	1.32 ± 0.16a	0.58 ± 0.09b	0.018
Turicibacter	0.53 ± 0.22b	1.53 ± 0.29a	0.83 ± 0.11b	0.015
Megasphaera	0.34 ± 0.08ab	0.82 ± 0.37a	0.04 ± 0.02b	0.067
Prevotellaceae unclassified	0.19 ± 0.05b	0.87 ± 0.27a	0.68 ± 0.23ab	0.090
Erysipelatoclostridium	0.30 ± 0.11a	0.26 ± 0.03a	ND2b	0.009
Lachnospiraceae	0.26 ± 0.04a	0.21 ± 0.04ab	0.12 ± 0.01b	0.036
Paeniclostridium	0.10 ± 0.04b	0.32 ± 0.10a	0.14 ± 0.03ab	0.056
Ruminococcaceae unclassified	0.19 ± 0.06a	0.09 ± 0.03ab	0.05 ± 0.02b	0.073

Data represent the mean ± standard error of the mean of 6 beagles per treatment (n = 6). A fresh fecal sample from each beagle was collected after 8 weeks of treatment. 502 genera were observed, and only the data of relative abundance more than 0.1% and a P-value in at least one group less than 0.1 are shown here.

1 HTS: High temperature sterilization.

2 ND: The data were less than 0.01, and were defined as “not detected”.

a-b Significant (P < 0.05) differences in the same row are indicated by different superscript letters.

3.4. Food processing methods changed the gut SCFA composition in adult beagles

Table 7 shows that dog food prepared using different processing methods had a certain effect on the SCFA content in beagle feces. The feces of beagles fed raw food showed significantly lower acetic acid, propionic acid, butyric acid and total acid content (P < 0.05); the feces of beagles fed HTS food showed a significantly lower valeric acid content (P < 0.05); and the food processing method did not alter isovaleric acid content in beagles (P > 0.1).

Table 7

SCFA changes in adult beaglesa.

SCFA	RAW	Pasteurized	HTS2	P-value
Acetic acid (mg·g-1)	3.54 ± 0.37b	5.22 ± 0.28a	4.91 ± 0.25a	0.003
Propionic acid (mg·g-1)	3.59 ± 0.26b	4.38 ± 0.29a	4.60 ± 0.17a	0.027
Butyric acid (mg·g-1)	0.79 ± 0.10b	1.35 ± 0.20a	1.53 ± 0.16a	0.011
Isovaleric acid (mg·g-1)	0.22 ± 0.04	0.25 ± 0.03	0.31 ± 0.02	0.165
Valeric acid (mg·g-1)	0.27 ± 0.09A	0.28 ± 0.08A	0.04 ± 0.02B	0.063
Total acid (mg·g-1)	8.39 ± 0.71b	11.47 ± 0.69a	11.38 ± 0.46a	0.005

Data represent the mean ± standard error of the mean of 6 beagles per treatment (n = 6). A fresh fecal sample from each beagle was collected after 8 weeks of treatment. The SCFA in samples were analyzed by gas chromatography.

1 SCFA, short-chain fatty acids.

2 HTS: High temperature sterilization.

a-b Significant (P < 0.05) differences in the same row following analysis with Duncan’s test are indicated by different superscript letters. A-B A tendency (P < 0.1) for differences in the same row following analysis with Duncan’s test are indicated by different superscript letters.

4. Discussion

People’s expectations of dog and cat food are very different to those of other animals’ food. People no longer care about “production performance indicators” such as daily weight gain and feed-to-meat ratio for dogs and cats, but instead use “health indicators”, such as coat color, routine blood analysis, serum parameters, gut health and liveliness. However, the standard for dog and cat health and the food processing method conducive to the health of dogs and cats are controversial.

Weight, coat color, and liveliness are the easiest indicators for dog owners to observe, and they are also the indicators that owners are most concerned about. However, it is difficult to quantitatively describe the coat color and liveliness, and most rely on the subjective feelings of the observer. In this study, the different food processing methods examined had no effect on the coat color and liveliness of beagles. However, the weight of all dogs in the experiment increased significantly (P< 0.05), and the weight of beagles fed pasteurized food and HTS food increased more. On the one hand, this change in body weight may be related to higher energy in the food in this study than the food originally consumed by the beagles, and on the other hand, the change may also be related to the difference in digestibility of the foods prepared using different processing methods, although this was not statistically significant (P> 0.1). This study demonstrated that the digestibility of protein processing at high temperature was higher.

Routine blood and serum biochemical parameters are commonly used to evaluate animal health. This study also tested routine blood and some routine serum biochemical parameters in beagles. However, it was found that only the number of neutrophils in beagles fed raw and pasteurized food increased, but there was no corresponding significant change in other parameters except platelet count, and it was difficult to determine the cause of this change. The study by Algya et al. suggested that serum triglyceride content in beagles fed extruded food was significantly higher than that in dogs fed wet food. Our results also showed that HTS food caused a rise in serum triglyceride content in beagles compared with raw or mildly processed (pasteurized) foods.

The intestinal microbiota plays a key role in the efficient absorption and utilization of nutrients, the maintenance of normal intestinal functions, regulation of immune responses, and protection from pathogenic bacteria [13, 14]. Species in the intestinal microbiota may vary greatly. This study showed that Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria and Actinobacteria were the main genera in dogs, which was the same as that reported by Paßlack et al. [15]. There have been many studies on the influence of food processing methods on animal intestinal microbiota [4, 16, 17].

As mentioned above, the intestinal microbiota plays a key role in the nutrient metabolism efficiency of the host. At the genus level, the abundance of over 14 genera showed significant differences in beagles fed different diets. Some results in this study were consistent with previous studies. Alloprevotella was positively associated with weight, fat mass, and energy metabolism [18]. This may be one of the factors related to the increase in body weight of beagles fed HTS food. Collinsella species are usually considered pathobionts as they can affect metabolism by altering intestinal cholesterol absorption, decrease glycogenesis in the liver and increase triglyceride synthesis [19]. This study also showed that the relative abundance of Collinsella was positively associated with triglycerides in dogs (P < 0.05, by Pearson’s analysis). Bermingham et al. [20] reported large reductions in the relative abundance of fecal Prevotella in dogs consuming raw-meat diets, and this study also showed that two genera of Prevotellaceae decreased in beagles fed raw diets compared to those fed HTS diets. However, some results are not completely consistent: the results of Algya et al. [4] showed that beagles fed raw diets had higher fecal Lactobacillus, Pediococcus and Sutterella compared with those fed pasteurized diets. This study showed no difference in Lactobacillus count between beagles fed raw diets and pasteurized diets, while Lactococcus count was lower in beagles fed raw diets compared with those fed pasteurized diets. In addition, no significant differences in Pediococcus and Sutterella in beagles fed different diets were observed. Interestingly, Streptococcus is a Lactobacillales, and this study showed that it increased in beagles fed pasteurized diets. Due to the huge difference in processing conditions between different diets, it is difficult to judge whether this effect is the result of the processing method itself or other reasons (such as the storage of raw materials, sanitary conditions of the processing environment, etc.)

Some non-starch polysaccharides in food will be fermented by microorganisms to produce SCFAs. In recent years, it has become apparent that SCFAs may play a key role in the prevention and treatment of the metabolic syndrome and bowel disorders [21]. Many intestinal microbiota are related to SCFA in the intestine, such as Bacteroides, Turicibacter, Prevotellaceae etc. Studies have shown that Turicibacter was correlated with butyric acid and dietary fiber metabolism [22], and may have an adverse effect on intestinal health [23]. The data from our study showed that the relative abundance of Turicibacter in dogs fed pasteurized food significantly increased. Studies have shown that the food processing method could also affect the content of SCFAs [4, 24]. Research by Algya et al. showed that the feces of beagles fed raw food had a higher SCFA content [4], which was confirmed by Sandri et al. [25]. Although this study found that the valeric acid content in the feces of dogs fed raw food increased, the total SCFA content showed the opposite trend. This may be due to the difference in food composition which affected the composition of the intestinal microbiota in beagles, and may in turn have affected the SCFA content in feces. It also shows that there are limits to comparing the effects of two processing methods on animals without guaranteed food ingredients and composition.

Growth performance, apparent total tract digestibility, routine blood analysis, serum parameters, intestinal microbiota structure, and fecal SCFA content often have complex internal links. Thus, it is meaningful to explore more food processing methods for dogs and cats.

This study showed that dog food produced by different processing methods could affect the health of adult beagle dogs. The apparent digestibility of protein and fat in HTS food was higher than that in raw food. The content of TG increased in beagles fed HTS food compared with those fed raw or pasteurized food (P < 0.05). The number of neutrophils (Gran) in beagles fed raw food and pasteurized food increased significantly (P < 0.05), and the platelet count in beagles fed raw food showed an increasing trend compared with the beagles fed HTS food. Different processing methods had a huge impact on the intestinal microbiota and SCFA in beagles: at least 14 genera were significantly affected by the food produced using different processing methods, but it is difficult to define whether these changes are good or bad. This research provided data on the use of the same raw materials and the same nutritional ingredients to prepare dog food using different processing methods. However, it is limited by the scarcity of experimental animals, sampling conditions, and the lack of mechanism analysis in this study. Experiments with various designs and larger sample sizes are necessary to examine the internal mechanism of dog food processing methods and their effect on dog health.

OTUs detected in this study.

The curve tends to be flat, indicating adequate sampling.

(DOCX)

Click here for additional data file.

Principal component analysis (PCA) plot showing clustering of microbial communities from feces of dogs fed food after different processing methods were not different.

(DOCX)

Click here for additional data file.

Routine blood analysis in adult beagles at the beginning (0 d).

(DOCX)

Click here for additional data file.

The effect of processing methods on serum biochemical parameters in adult beagles (0 d).

(DOCX)

Click here for additional data file.

Quality control of 16S rRNA sequencing.

(DOCX)

Click here for additional data file.

Raw data on growth performance and apparent total tract digestibility.

(DOCX)

Click here for additional data file.

Raw data on routine blood and serum biochemical parameters.

(DOCX)

Click here for additional data file.

Raw data on SCFAs.

(DOCX)

Click here for additional data file.

21 Jul 2021

PONE-D-21-20446

The influence of food processing methods on serum parameters, apparent total-tract macronutrient digestibility, fecal microbiota and SCFA content in adult beagles

PLOS ONE

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18 Aug 2021

Revision checklist

1. Manuscript is not formatted for Plos One.

Re: Thank you for your careful check. We have modified the format of this manuscript according to the instructions provided in the website. Although we have tried our best to modify the format of the manuscript, we believe that there are still some format problems in the manuscript. Please point it out in the next revision, and we will correct it seriously.

2. Provide specific number of each of the AOAC methods.

Re: Thank you for your careful check. This has been corrected (line 86-88).

3. Statistical analysis is poor described. Authors must breakdown the statistical analysis for the animal experiment and amplicon sequencing.

Re: Thank you for your careful check. We have rewritten the "statistical analysis" and I don't know if it meets the requirements of the magazine. The data processing in the part of "amplicon sequencing" is described in a section in "Materials and Methods", so the description was not distinguished.

4. For the animal experiment, what were the random and fixed effected used in the model? What was the experimental unit? Were repeated measure analysis used? What is the power for the number of animals used? Why Duncan?

Re: Thank you for your advice. I'm sorry I think we may not fully understand what you mean. I guess your suggestion is for the use of SAS as statistical software for analysis. We are not familiar with SAS software. In this study (except for PCA analysis of sequencing data), all comparisons between groups are based on SPSS software. We did not mention the concept of "random and fixed effected used in the model" in our previous analysis. Correspondingly, we guessed and answered based on the literal meaning. In this study, the health status of the dogs, the raw materials and ingredients of the feed are fixed, and the only variable is the processing method of the feed.

In this study, one dog is a repeat. Therefore, the number of repetitions in this study is six. The supplementary description was added to line 101 and table notes to allow readers to more clearly define the experimental design.

As for why Duncan analysis is used. Well, it seems that there is no absolute standard for which hypothesis testing method to choose. We are based on the results of a Chinese degree thesis (Evalluation of multiple comparison methods with quantitative data): "When the first aim is to explore the difference between the neans of each groups, the LSD and Duncan methods are first selected..." At the same time, the SPPS software expresses the results of Duncan analysis more clearly. Many hypothesis tests in the literature with similar experimental design also use Duncan analysis (Liu et al., 2014. doi: 10.1371/journal.pone.0106412; Song et al., doi: 2017. 10.3382/ps/pex163), so we use Duncan analysis for comparison.

5. Tables 3 to 5: please provide the P-values Type 3 Tests of Fixed Effects. Defined HTS: Tables must stand alone so any abbreviation must be defined regardless if previously defined.

Re: Thank you for your careful check. These have been corrected (Tables 2 to 7, Fig 1 & Fig 2).

6. LSMEANS of Erysipelatoclostridium zero? It should be either not detected or increase the decimals.

Re: Thank you for your careful check. These have been corrected (Line 296).

7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Re: Thank you for your careful check. These have been corrected.

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Re: Thank you for your careful check. We have modified the format of this manuscript according to the instructions provided in the website. Although we have tried our best to modify the format of the manuscript, we believe that there are still some format problems in the manuscript. Please point it out in the next revision, and we will correct it seriously.

2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1)housing condition, (2) methods of sacrifice, (3) amount of blood taken from each animal (4) where the blood was taken and (5) methods of anaesthesia if used for blood collection.

Re: Thank you for your careful check. (1) Housing condition was described in Line 105-108; (2) No animal sacrificed in this study; (3) Amount of blood taken from each animal was described in Line 113; (4) The place blood was taken described in Line 114; (5) Anaesthesia was not used in this study.

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

Re: Thank you for your advice. Raw sequences reads for this study can be found in the NCBI sequence read archive BioProject ID: PRJNA733866. Other relevant data are within the paper and its Supporting Information files.

Submitted filename: Response to Reviewers0819.docx

Click here for additional data file.

5 Oct 2021

PONE-D-21-20446R1The influence of food processing methods on serum parameters, apparent total-tract macronutrient digestibility, fecal microbiota and SCFA content in adult beaglesPLOS ONE

Dear Dr. Cai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Alex V Chaves, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Discussion section has not considered the mechanism for the observed changes. Please fix this on the revised version.

Reviewers' comments:

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Reviewer #2: Partly

**********

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Reviewer #1: Additional comments:

PLOS one review: The influence of food processing methods on serum parameters, apparent total tract macronutrient digestibility, fecal microbiota, and SCFA content in adult beagles

The authors in this study examined food processing methods on the health of beagle dogs. Three types of processing methods were assessed to see their resulting effects on the following parameters: apparent total tract digestibility, serum parameters, SCFA content, and fecal microbiota. Overall, a very interesting read with great results. Authors have fixed many of the corrections from the first round of reviews. However, the paper needs to be professionally edited to correct for grammatical, spelling, and punctuation errors. In addition to this, I have included a few minor corrections to improve the quality of the paper.

Major correction:

1. This paper requires a review by a professional editor in order to correct the grammatical, spelling, and punctuation errors. I began to fix some of the mistakes, but they were too numerous to continue. Some of these errors make it difficult to understand the sentences, especially in the materials and methods, and results sections. Therefore, I strongly recommend a professional editor.

Minor corrections:

2. L179: Please provide a brief description validating the use of one-way ANOVA. Ex.: does the data follow parametric assumptions, validated by a KS test (normality) and Levene’s test (homogeneity of variance)?

3. Table 4: The significance letters are not labeled consistently. Specifically, refer to item “TG” under HTS

4. Table 6: In addition to the p-values, please add significance letters to keep it consistent with the other tables.

5. L407-414: Written exactly as in the abstract. Please re-word this.

6. Figure 1c: It would be better to start the y-axis numbering from 0% instead of 80%, and then include a break in the bar graphs instead.

Reviewer #2: The authors have set about testing the question whether there is any nutritional difference between feeding a diet that has been processed in 3 different ways: raw, pasteurised or high temperature sterilization. The premise underlying this project is the statement in the first line of the manuscript: “Food processing methods have a huge influence on the health of dogs.” The experimental design and the methods of analysis are all appropriate for determining any biological effects of feeding to a group of beagle dogs, the same diet processed in 3 different ways. A wide range of assays were undertaken, and statistically different mean values were found in protein digestibility, serum triglycerides, platelet and neutrophil populations in serum and the species range and population proportion of faecal microorganisms of dogs fed on these differently processed diets.

The experimental approach and its technical execution were entirely appropriate to test the hypothesis that the 3 methods of food preparation would show differences in various biological features of the dogs being studied. However, how do the authors justify the findings in terms of the opening sentence of their manuscript: “Food processing methods have a huge influence on the health of dogs.”? What is the evidence that there was any difference between the processing methods and the health of the dogs being studied? Certainly, statistical differences were found in some of the responses being measured, but these differences were so small that it is difficult to see how they would have had any impact on the “health of the dogs”. It is not surprising that heat treatment would affect digestibility of protein. It probably also would have affected starch digestibility in the rice component of the food formulation. Hence the composition of the digestion residue being presented to the microbiome in the large intestine would be different between the 3 processing methods. This would be the likely explanation for the findings of this study. This conclusion should have been pointed out in the Discussion section of the paper, along with an assessment that all of the significantly different changes, observed between the 3 processing methods, would have had negligible effects on the “health” of the dogs. The study was worth doing, but the results are entirely unsurprising.

**********

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Reviewer #1: No

Reviewer #2: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

25 Oct 2021

Revision checklist

1. This paper requires a review by a professional editor in order to correct the grammatical, spelling, and punctuation errors. I began to fix some of the mistakes, but they were too numerous to continue. Some of these errors make it difficult to understand the sentences, especially in the materials and methods, and results sections. Therefore, I strongly recommend a professional editor.

Re: Thank you for your careful check. We have carefully checked the manuscript and the language of this manuscript has been edited by a company named “International Science Editing” (https://www.internationalscienceediting.com/).

2. L179: Please provide a brief description validating the use of one-way ANOVA. Ex.: does the data follow parametric assumptions, validated by a KS test (normality) and Levene’s test (homogeneity of variance)?

Re: Thank you for your advice. This has been corrected (Line 183-186).

3. Table 4: The significance letters are not labeled consistently. Specifically, refer to item “TG” under HTS

Re: Thank you for your careful check. It has been corrected (Table 4).

4. Table 6: In addition to the p-values, please add significance letters to keep it consistent with the other tables.

Re: Thank you for your careful check. It has been corrected (Table 6, Line 303).

5. L407-414: Written exactly as in the abstract. Please re-word this.

Re: Thank you for your careful check. It has been corrected (Line 412-414)..

6. Figure 1c: It would be better to start the y-axis numbering from 0% instead of 80%, and then include a break in the bar graphs instead.

Re: Thank you for your advice. These have been corrected.

7. The authors have set about testing the question whether there is any nutritional difference between feeding a diet that has been processed in 3 different ways: raw, pasteurised or high temperature sterilization. The premise underlying this project is the statement in the first line of the manuscript: “Food processing methods have a huge influence on the health of dogs.” The experimental design and the methods of analysis are all appropriate for determining any biological effects of feeding to a group of beagle dogs, the same diet processed in 3 different ways. A wide range of assays were undertaken, and statistically different mean values were found in protein digestibility, serum triglycerides, platelet and neutrophil populations in serum and the species range and population proportion of faecal microorganisms of dogs fed on these differently processed diets.

The experimental approach and its technical execution were entirely appropriate to test the hypothesis that the 3 methods of food preparation would show differences in various biological features of the dogs being studied. However, how do the authors justify the findings in terms of the opening sentence of their manuscript: “Food processing methods have a huge influence on the health of dogs.”? What is the evidence that there was any difference between the processing methods and the health of the dogs being studied? Certainly, statistical differences were found in some of the responses being measured, but these differences were so small that it is difficult to see how they would have had any impact on the “health of the dogs”. It is not surprising that heat treatment would affect digestibility of protein. It probably also would have affected starch digestibility in the rice component of the food formulation. Hence the composition of the digestion residue being presented to the microbiome in the large intestine would be different between the 3 processing methods. This would be the likely explanation for the findings of this study. This conclusion should have been pointed out in the Discussion section of the paper, along with an assessment that all of the significantly different changes, observed between the 3 processing methods, would have had negligible effects on the “health” of the dogs. The study was worth doing, but the results are entirely unsurprising.

Re: Thank you very much for your pertinent evaluation. Health is a difficult concept to define, and different researchers may think differently. Because dogs and cats must be sampled non-destructively, the observable indicators are usually the measurement indicators of serum, feces, and urine, which are usually measured by researchers during the research process, such as PMID: 29893876, 30110340, 33480132. But this article began to define it as health is indeed not rigorous enough, so we have revised it accordingly.

The prosperity of the pet industry in recent years has brought many novel processing techniques, but the research on the impact of these processing techniques on the health of dogs and cats is still very limited. There have been some studies in the past, but due to processing difficulties, many researchers use different comparing formulas of dry food and wet food, although there are many interesting conclusions, it is difficult to rule out the interference of different formulas and raw materials. This study is trying to use the same formula and the same raw material to process dog food with different characteristics, and analyze its influence on some physiological and biochemical indicators of beagle dogs, and supplement and verify previous studies. In this sense, we think it can help promote the development of the pet food industry.

However, this study does have some problems. Because of the "schedule" problem of experimental animals, we chose a group test. This resulted in a small number of beagle dogs in each group and large differences within the group, so it is difficult to perform in many analyses significant difference. If we have the opportunity to continue to engage in relevant research, we will be more inclined to use a larger sample size staged trial rather than grouped trials.

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Re: Thank you for your careful check. We have modified the format of this manuscript according to the instructions provided in the website. Although we have tried our best to modify the format of the manuscript, we believe that there are still some format problems in the manuscript. Please point it out in the next revision, and we will correct it seriously.

2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1)housing condition, (2) methods of sacrifice, (3) amount of blood taken from each animal (4) where the blood was taken and (5) methods of anaesthesia if used for blood collection.

Re: Thank you for your careful check. (1) Housing condition was described in Line 106-109; (2) No animal sacrificed in this study; (3) Amount of blood taken from each animal was described in Line 113; (4) The place blood was taken described in Line 114-115; (5) Anaesthesia was not used in this study.

3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

Re: Thank you for your advice. Raw sequences reads for this study can be found in the NCBI sequence read archive BioProject ID: PRJNA733866. Other relevant data are within the paper and its Supporting Information files.

Submitted filename: Response to Reviewers_R2.docx

Click here for additional data file.

25 Nov 2021

PONE-D-21-20446R2The influence of food processing methods on serum parameters, apparent total-tract macronutrient digestibility, fecal microbiota and SCFA content in adult beaglesPLOS ONE

Dear Dr. Cai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Alex V Chaves, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The authors have satisfactorily changed the wording of their manuscript to avoid implying that the state of health of the dogs was affected by the method of food processing. Certainly some stattstically significant differences were found in some of the measurements being made. These differences were small and would not indicate any change in the state of health of the dogs.

There is one minor correction that is needed. In Tables 6 and 7, mean values plus and minus standard errors are given. The units for these values should be stated, as has been done for the data in the other tables.

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Reviewer #1: No

Reviewer #2: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

4 Dec 2021

1. The authors have satisfactorily changed the wording of their manuscript to avoid implying that the state of health of the dogs was affected by the method of food processing. Certainly some stattstically significant differences were found in some of the measurements being made. These differences were small and would not indicate any change in the state of health of the dogs.

There is one minor correction that is needed. In Tables 6 and 7, mean values plus and minus standard errors are given. The units for these values should be stated, as has been done for the data in the other tables.

Re: Thank you for your careful check. We corrected this error in Tables 6 and 7 and added units.

Submitted filename: Response to Reviewers_R3.docx

Click here for additional data file.

16 Dec 2021

PONE-D-21-20446R3The influence of food processing methods on serum parameters, apparent total-tract macronutrient digestibility, fecal microbiota and SCFA content in adult beaglesPLOS ONE

Dear Dr. Cai,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Final remarks from the associate editor:

Did the authors run stats on Table S1? if so please present the P-values.

I just noticed that in Table S2, authors mentioned "Significant (P < 0.05) differences among Duncan analyze in the same row are indicated by different superscript letters." but there are no letters on the LSMEANS. Can you please also add a column with P-values?

S1, S2 & S3 files must be written in English not in Chinese.

Please submit your revised manuscript by Jan 30 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Alex V Chaves, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

19 Dec 2021

1. Did the authors run stats on Table S1? if so please present the P-values.

Re: Thank you for your careful check. We have added a column with P-values.

2. I just noticed that in Table S2, authors mentioned "Significant (P < 0.05) differences among Duncan analyze in the same row are indicated by different superscript letters." but there are no letters on the LSMEANS. Can you please also add a column with P-values?

Re: Thank you for your careful check. The sentence "Significant (P < 0.05) differences among Duncan analyze in the same row are indicated by different superscript letters." Should deleted here for no significant (P < 0.05) difference existed for the data in this table. We have added a column with P-values in Table S2, and also Table S3.

3. S1, S2 & S3 files must be written in English not in Chinese.

Re: Thank you for your careful check. We have revised these.

Submitted filename: Response to Reviewers_R4.docx

Click here for additional data file.

21 Dec 2021

The influence of food processing methods on serum parameters, apparent total-tract macronutrient digestibility, fecal microbiota and SCFA content in adult beagles

PONE-D-21-20446R4

Dear Dr. Cai,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Alex V Chaves, PhD

Academic Editor

PLOS ONE

27 Dec 2021

PONE-D-21-20446R4

The influence of food processing methods on serum parameters, apparent total-tract macronutrient digestibility, fecal microbiota and SCFA content in adult beagles

Dear Dr. Cai:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof Alex V Chaves

Academic Editor

PLOS ONE