INTRODUCTION
Pets positively affect people’s physical health and emotional stability [1]. These effects improve their quality of life and increase people’s preference for pet ownership [2]. Pets are raised in about 66%, 69%, and 60% of households in the United States, Australia, and the United Kingdom, respectively [3-5]. Interest in pets has increased as the majority of the population is raising them, which raises nutritional and health anxiety about their diets [6]. Because dogs are normally provided nutrients from complete and balanced diets, the nutrient content of diets and nutrient digestibility are important [7]. Pet food companies routinely perform digestibility testing to provide important information on the nutrient content of their diets [8]. Several nations have recognized the importance of the nutrient digestibility of dogs and offered related information [9–11].
In the Republic of Korea, pets have become a fundamental component of daily life, and the number of households with dogs has increased dramatically in recent years [12]. According to Joo et al. [13], dogs represent 77.4% of the total household pets. However, research on domestic dog diets is insufficient in the Republic of Korea compared to the increasing number of dogs being raised. Most domestic dog diets developed in the Republic of Korea consult overseas nutritional requirements, such as NRC [9] and AAFCO [10]. Few nutritional studies have been conducted on dog diets, so it is necessary to investigate and establish nutrient digestibility standards.
Both in vitro and in vivo methods are used to evaluate the nutrient digestibility of diets [14]. Among them, in vitro methods have positive features, such as being cheaper, ethical, and more time-saving, and can be utilized as an alternative to in vivo methods [15]. Numerous studies have used two-step in vitro methods to simulate digestion in the stomach and small intestines of dogs [16,17]. Most in vitro studies have compared feedstuff digestibility to in vivo studies and generated predictive equations for their relationships [18]. However, few studies based in the Republic of Korea have used dog diets to study in vitro digestibility and compared them with in vivo digestibility. Therefore, this study was conducted to evaluate in vitro prediction of digestibility at each age (puppy, adult, and senior) of dry matter (DM), organic matter (OM), crude protein (CP), gross energy (GE), crude fiber (CF), and ether extract (EE) using dog diets.
MATERIALS AND METHODS
The experimental diet using in vitro and in vivo methods based on hydrolyzed chicken powder, soy protein, and brown rice was manufactured in extruded form. The diet was formulated to meet or exceed the nutrient requirements according to the AAFCO guideline (Table 1).
Vitamin and mineral premix supplied per kg of diets: 3,500 IU vitamin A; 250 IU vitamin D3; 25 mg vitamin E; 0.052 mg vitamin K; 2.8 mg vitamin B1 (thiamine); 2.6 mg vitamin B2 (riboflavin); 2 mg vitamin B6 (pyridoxine); 0.014 mg vitamin B12; 6 mg Cal-d-pantothenate; 30 mg niacin; 0.4 mg folic acid; 0.036 mg biotin; 1,000 mg taurine; 44 mg FeSO4; 3.8 mg MnSO4; 50 mg ZnSO4; 7.5 mg CuSO4; 0.18 mg Na2SeO3; 0.9 mg Ca (IO3)2.
The in vitro method described by Hervera et al. [19] method was conducted in two steps with 6 replicates of dog diet.
Step 1: The samples were prepared in finely ground (< 1.0 mm) form. In stomach simulation, weigh (1.000 ± 0.001 g) of each sample in 250 mL Erlenmeyer flasks, then add 25 mL of phosphate buffer (0.1 M, pH 6.0) and 10 mL of HCl solution (0.2 M, pH 0.7) to each flask. The pH was adjusted to 2.0 using 1 M HCl and 1 M NaOH solution, and 1 mL pepsin solution (10 mg/mL; ≥ 250 units/mg solid, P7000, pepsin from porcine gastric mucosa; Sigma-Aldrich, St. Louis, MO, USA) was added to the flask to simulate stomach digestion in the dog. In addition, 1 mL of chloramphenicol solution (C0378, chloramphenicol; Sigma-Aldrich with 5 g/L ethanol) was also added to avoid bacterial fermentation. The flasks were closed with a Parafilm M® film and incubated in a shaking incubator (SWB-35, Hanyang Science Lab, Seoul, Korea) at 39°C for 2 h.
Step 2: 5 mL of NaOH solution (0.6 M) and 10 mL of phosphate buffer (0.2 M, pH 6.8) were added to the flask after cooling at room temperature. The pH was adjusted to 6.8 using 1 M HCl and 1 M NaOH solution, and 1 mL of pancreatin solution (100 mg/mL; 4 × USP, P1750, pancreatin from the porcine pancreas; Sigma-Aldrich) was added in the flask to simulate digestion conditions in the small intestine of the dog. Then, the flasks were closed with a Parafilm M® film and incubated in a shaking incubator (SWB-35; Hanyang Science Lab) at 39°C for 4 h.
Then, the collected undigested samples were filtered through pre-dried and pre-weighed glass filter crucibles (Gooch Type Filter Crucibles, PYREX®, Sunderland, UK). During filtering, the flasks were rinsed three times with distilled water. Additionally, 10 mL of 95% ethanol and 10 mL of 99.5% acetone were added twice to the glass filter crucibles.
At the end of the in vitro procedure, the filter crucibles containing undigested residues were dried at 70°C for 24 h to calculate DM. Then, they were burned at 550°C for 4 h to calculate OM. After being dried and combusted, it was cooled to room temperature and then weighed. The methods utilized for the determination of DM (method 930.15), OM (method 942.05), CF (method 978.10) and EE (method 920.39) were conducted with the methods of AOAC [20]. The CP and GE content were analyzed by using the dumas (Rapid MAX N-Exceed, Elementar, Langenselbold, Germany) and bomb calorimeter (Parr 6400 Bomb Calorimeter, Parr Instrument, Moline, IL, USA), respectively.
Calculating the in vitro digestibility of DM using the following formula:
Calculating the in vitro digestibility of OM, CP, GE, CF and EE used the following formula:
Nr = nutrient concentration in residues (DM %), Nd = nutrient concentration in diet (DM %), and IDDM =in vitro digestibility (DM %)
This experiment was examined and approved (approval # 202310A-CNU-179) by the Institutional Animal Care and Use Committee of Chungnam National University, Daejeon, Korea. In experiment, dogs were collected and managed by the procedures.
A total of 18 mixed-sex beagles were used in this experiment. Beagles were divided into 3 groups according to their age: six puppies (under 1 year old), six adult dogs (2 to 7 years old), and six senior dogs (over 8 years old). Total experimental period was 17 days which included 7 days adaptation period. Each dog was managed in individual cage (0.9 m × 0.9 m × 0.9 m), and the temperature was maintained at 23°C. The maintenance energy requirements (MER) for each growth stage were calculated using metabolic body weight (mBW).
Calculating the MER used the following formula:
Daily feed requirements were calculated in accordance with MER applied to each dog and fed twice a day at 9:00 and 17:00.
At the bottom of each kennel, dense mesh was attached to separate urine and feces for collecting pure fecal samples. Pee pads absorbed urine through the mesh, and the fecal samples remained on the mesh. Fecal samples for calculating digestibility by the total fecal collection method were collected during 8 days of experimental periods. Fresh fecal and feed samples were stored in a freezer at -20°C after collection immediately. The stored fecal samples were dried at 103°C for 12 h and then finely ground (< 1 mm) for chemical analysis at the end of the experiment. The total fecal collection digestibility of DM, OM, CP, GE, CF and EE were analyzed using samples. The methods utilized for the determination of DM (method 930.15), OM (method 942.05), and EE (method 920.39) were conducted with the methods of AOAC [20]. The CP and GE content were analyzed by using the dumas (Rapid MAX N-Exceed, Elementar, Langenselbold, Germany) and bomb calorimeter (Parr 6400 Bomb Calorimeter, Parr Instrument), respectively. The equation for the total fecal collection method described by Donadelli and Aldrich [21].
Total fecal collection digestibility was determined by the following formula:
Dog means served as the experimental unit. The means of the treatments were also compared by using orthogonal contrasts: in vitro digestibility vs. other treatments. Variability in the data was expressed as the SEM. The relationship between in vitro and in vivo digestibility measured in dogs was determined by regression analyses using a general linear model (GLM) in a JMP (JMP® Pro version 16.0.0, SAS Institute, Cary, NC, USA). The model was y = ax + b, where y = in vivo digestibility, a = slope, x = in vitro digestibility and b = intercept. Statistical differences were determined to be significant at p < 0.05.
RESULTS
The in vitro and in vivo digestibility of DM, OM, CP, GE, CF and EE of puppies, adult dogs, and senior dogs are presented in Table 2. The in vivo digestibility of DM in senior dogs was significantly higher (p = 0.027) than in vitro digestibility. Also, the in vivo digestibility of CP, GE, CF, and EE in all ages was significantly higher (p < 0.001) than in vitro digestibility. However, there was no significant difference in the in vitro digestibility compared to the in vivo digestibility of DM in adults and senior groups and OM in all age groups, respectively.
The statistical relationships between in vitro and in vivo digestibility as linear regression equations are shown in Table 3. There was a strong relationship between DM and GE (r2 = 0.95 and 0.84, respectively) in puppies. In adult dogs, there was a strong relationship between DM and GE (r2 = 0.97 and 0.84, respectively). However, in senior dogs, there was a low relationship between whole contents (DM, r2 = 0.18; OM, r2 = 0.42; CP, r2 = 0.01; GE, r2 = 0.02; CF, r2 = 0.11; EE, r2 = 0.04).
DISCUSSION
This study evaluated the digestibility of a dog diet using in vivo and in vitro methods and generated predictive equations for the relationships between in vivo and in vitro digestibility. Previous studies reported that in vitro digestibility was higher than in vivo digestibility due to endogenous losses in the body [18,21,22]. In this study, the in vitro digestibility of CP, GE, CF, and EE was higher than the in vivo digestibility at all ages. Consistent with our results, Penazzi et al. [23] suggested that in vitro digestibility overestimated in vivo digestibility. Endogenous losses in the body have a significant influence on in vivo digestibility [18]. In the in vitro method, chloramphenicol was added to avoid bacterial fermentation, and the method was conducted under strictly controlled temperature, digestion time, pH, and enzyme content conditions [24], which explains why in vitro digestibility was higher than in vivo digestibility. Le Bon et al. [25] reported that senior dogs had less inflammation and attributed it to gut microbial diversity decreases in aging dogs. Decreases in gut microbial diversity affect gut health, leading to low digestibility [26]. In this study, a significant difference between DM in vivo and in vitro digestibility was seen due to the low digestibility of senior dogs.
The in vitro method can assist in identifying nutritional availability in non-ruminant animals [27]. Prior studies were conducted on the in vitro digestibility of dog diets compared to in vivo digestibility [17,28,29]. This study adopted a modified two-step in vitro procedure for dogs, which involved reducing the doses of exogenous digestive enzymes to account for the shorter gastrointestinal tract and faster digestion rate in dogs compared to pigs [17].
The wide range of nutrient contents in dog diets may affect the accuracy of in vitro equations for predicting nutrient availability [26]. Endogenous losses, enzymatic secretion, and microbial activity were reported to be other influencing factors [30]. In this study, a predictive equation was generated by comparing in vivo and in vitro digestibility in each age group. A strong relationship between DM and GE was found in puppy and adult-aged dogs. Satterlee et al. [31] reported that the analysis of animal protein-based diets resulted in lower accuracy, leading to differences in the digestibility relationship. Burrows et al. [32] suggested that the presence of dietary fiber also affects the digestibility of diets. Consistent with previous studies, Biagi et al. [29] assumed that the low relationship between in vitro and in vivo digestibility could be attributed to the fact that feces include bacteria and other endogenous protein sources, as well as to proteins derived from diets, which causes protein digestibility to be underestimated. In this study, the low relationship between the in vitro and in vivo digestibility of CP, CF, and EE was assumed to be caused by endogenous losses. In senior dogs, a low relationship between in vitro and in vivo digestibility was found for all dietary components analyzed. The low level of adjustment may have been affected by the limited number of samples and the consistent in vivo values recorded across samples [33]. Weber et al. [34] reported that growth affected digestibility by altering the transit time of the digestive system. Consistent with previous studies, our findings were likely due to differences in in vivo digestibility due to age differences, resulting in a low correlation with in vitro digestibility values.
Based on these results, we can use equations to predict age-specific digestibility through in vitro experiments. However, additional research is needed to investigate the relationship between in vitro and in vivo methods in senior dogs.
CONCLUSION
There were strong linear relationships between in vivo and in vitro digestibility (DM and GE) in puppies, (DM and GE) in adult dogs. In vitro, prediction of digestibility (DM and GE) in puppies and adult dogs seem to have significant potential for practical application. However, additional research investigating the in vitro method in senior dogs is needed.
