Determining the protocol requirements of in-home cat food digestibility testing.

In-home cat food digestibility testing has the potential to yield data that are highly representative of the pet population for which the food is intended. However, no standardized and validated in-home digestibility test protocols are currently available. Such protocols for in-home testing should address key factors that explain variation in cat food digestibility values and here we investigated the required period of adaptation, fecal collection and sample sizes. Thirty privately-owned indoor housed cats of various breeds (20♀ 10♂, 5.9 ± 3.9 yr, 4.5 ± 1.3 kg) received a relatively low and high digestible complete dry extruded food with the marker titanium (Ti) dioxide. Foods were given in a cross-over design of 2 periods of 8 consecutive days each. Owners collected feces daily for the determination of daily fecal Ti concentrations and digestibility of dry matter, crude protein, crude fat, and gross energy. Data originating from 26 cats were analyzed as mixed models and broken line regressions to investigate the required adaptation and fecal collection period. Bootstrap sampling was used to assess the impact of increasing the number of fecal collection days and sample size on the precision of the digestibility estimates. Feces were collected on 347 out of 416 study days (16 days/cat; 26 cats), implying the necessity for multiple collection days to account for cats not defecating every day. Cats showed stable fecal marker concentrations from day 2 onwards when fed the low digestible food and from 3 onwards when fed the high digestible food. Digestibility values were stable from day 1, 2 or 3 onwards, depending on the test food and nutrient. Increasing the number of fecal collection days from 1 to 6 days did not result in more precise digestibility estimates, whereas increasing the number of animals from 5 to 25 cats did. For future in-home digestibility tests of cat food, the findings support a minimum of 2 adaptation days and 3 fecal collection days. Appropriate sample sizes depend on the test food, the nutrient of interest, and the acceptable margin of error. The findings of this study support the protocol development for future in-home digestibility testing of cat foods.

Performance comparison of fecal preservative and stock solutions for gut microbiome storage at room temperature.

The gut microbiome, which is symbiotic within the human body, assists in human digestion. It plays significant roles in identifying intestinal disease as well as in maintaining a healthy body with functional immune and metabolic activities. To confirm the consistency of fecal intestinal microbial research, it is necessary to study the changes in intestinal microbial flora according to the fecal collection solution and storage period. We collected fecal samples from three healthy Korean adults. To examine the efficacy of fecal collection solution, we used NBgene-Gut, OMNIgene-Gut, 70% ethanol (Ethanol-70%), and RNAlater. The samples were stored for up to two months at room temperature using three different methods, and we observed changes in microbial communities over time. We analyzed clusters of changes in the microbial flora by observing fecal stock solutions and metagenome sequencing performed over time. In particular, we confirmed the profiling of alpha and beta diversity and microbial classification according to the differences in intestinal environment among individuals. We also confirmed that the microbial profile remained stable for two months and that the microbial profile did not change significantly over time. In addition, our results suggest the possibility of verifying microbial profiling even for long-term storage of a single sample. In conclusion, collecting fecal samples using a stock solution rather than freezing feces seems to be relatively reproducible and stable for GUT metagenome analysis. Therefore, stock solution tubes in intestinal microbial research can be used without problems.

Effects of Collection Durations on the Determination of Energy Values and Nutrient Digestibility of High-Fiber Diets in Growing Pigs by Total Fecal Collection Method.

This study was conducted to evaluate the effect of collection durations on the energy values and nutrient digestibility of high-fiber diets in growing pigs with a time-based total fecal collection method. A total of 24 barrows (body weight (BW): 31.1 ± 1.5 kg) were allotted to a completely randomized design with three diets. Diets included a corn-soybean meal (CSM) basal diet and two additional diets containing 20% sugar beet pulp (SBP) or defatted rice bran (DFRB) by replacing corn, soybean meal, and soybean oil in the CSM diet, respectively. Each diet was fed to eight barrows for a 7-day adaptation period followed by a 7-day total feces and urine collection period. The 7-day collection duration was divided into three collection phases, namely, phase 1 (days 8 to 11), phase 2 (days 11 to 13), and phase 3 (days 13 to 15). Then, similar portions of feces and urine from the different collection phases were composited into three additional samples (days 8 to 11, days 8 to 13, and days 8 to 15, respectively). The results showed that the digestible energy (DE), metabolizable energy (ME), and apparent total tract digestibility (ATTD) of gross energy (GE) and nutrient in experimental diets decreased linearly as the collection durations increased from a 3-day to a 7-day collection (p < 0.05). However, there were no differences in the energy values, GE, and nutrient digestibility of diets and of high-fiber ingredients between the 5-day and 7-day collection durations. In conclusion, this study suggests that a 5-day collection duration is adequate to determine the energy values and nutrient digestibility of high-fiber diets containing SBP or DFRB in growing pigs by the time-based total fecal collection method.

Comparison of four digestibility markers to estimate fecal output of dogs.

Twelve adult beagle dogs (10.6 ± 1.4 kg) were fed extruded dog diets in which the starch sources were whole sorghum, sorghum flour, sorghum mill-feed, or an equal combination of rice, corn, and wheat. The experiment was conducted as a replicated Latin square design digestibility study. Estimates of fecal organic matter (OM), crude protein (CP), crude fat (CF), and gross energy (GE) outputs were determined by four methods: total fecal collection (TFC), chromic oxide (Cr2O3), titanium dioxide (TiO2), and acid insoluble ash (AIA). The correlation among the fecal output estimates by the four methods by partial correlation coefficients from the Error SSCP Matrix (Pearson) were considered significant at P < 0.05. The external markers, Cr2O3 and TiO2, had a higher (P < 0.05) OM fecal output Pearson correlation to TFC than the intrinsic marker AIA (R = 0.931 for Cr2O3 vs. TiO2; R = 0.559 for TFC vs. Cr2O3; R = 0.592 for TFC vs. TiO2; R = 0.291 for AIA vs. TFC). Interestingly, TiO2 highly correlated (P < 0.05) to Cr2O3 (R = 0.93 for OM), and was also correlated highly to TFC and AIA. The study suggests that TiO2 may be a preferred marker to estimate fecal output in dogs vs. Cr2O3. The use of AIA represents a potential option for determining digestibility for diets in which external markers are impractical.

Reliability of a participant-friendly fecal collection method for microbiome analyses: a step towards large sample size investigation.

BACKGROUND: The effects of gut microbiota on human traits are expected to be small to moderate and adding the complexity of the human diseases, microbiome research demands big sample sizes. Fecal samples for such studies are mostly self-collected by participants at home. This imposes an extra level of complexity as sample collection and storage can be challenging. Effective, low-burden collection and storage methods allowing fecal samples to be transported properly and ensuring optimal quality and quantity of bacterial DNA for upstream analyses are necessary. Moreover, accurate assessment of the microbiome composition also depends on bacterial DNA extraction method. The aim of this study was to evaluate the reliability and efficiency of the OMNIgene•GUT kit as a participant-fecal friendly collection method (storage at room temperature for 24 h (O24h) or 7 days (O7d)) in comparison to the standard collection method (Fresh, storage at 4 °C for less than 24 h) in terms of amount of variability and information content accounting for two common DNA extraction methods. RESULTS: Fourteen fecal samples were collected from healthy individuals (7 males, 7 females). Collection and storage methods did not differ significantly in terms of DNA concentration and Shannon diversity index. Phylum relative abundance showed significant differences for Bacteroidetes, Actinobacteria and Cyanobacteria. The differences were observed between control (Fresh) and O24h methods, but not between Fresh and O7d. These differences were not seen when performing bacterial DNA quantification based on three bacterial groups: Bacteroides spp., Bifidobacterium spp. and Clostridium cluster IV, which represent three major phyla: Bacteroidetes, Actinobacteria and Firmicutes respectively. The two DNA extraction methods differ in terms of DNA quantity, quality, bacterial diversity and bacterial relative abundance. Furthermore, principal component analysis revealed differences in microbial structure, which are driven by the DNA extraction methods more than the collection/storage methods. CONCLUSION: Our results have highlighted the potential of using the OMNIgene•GUT kit for collection and storage at ambient temperature, which is convenient for studies aiming to collect large samples by giving participants the possibility to send samples by post. Importantly, we revealed that the choice of DNA extraction method have an impact on the microbiome profiling.

Improving the standards for gut microbiome analysis of fecal samples: insights from the field biology of Japanese macaques on Yakushima Island.

Fecal DNA-based 16S ribosomal RNA (rRNA) gene sequencing using next-generation sequencers allows us to understand the dynamic gut microbiome adaptation of animals to their specific habitats. Conventional techniques of fecal microbiome analysis have been developed within the broad contexts defined by human biology; hence, many of these techniques are not immediately applicable to wild nonhuman primates. In order to establish a standard experimental protocol for the analysis of the gut microbiomes of wild animals, we selected the Japanese macaques (Macaca fuscata yakui) on Yakushima Island. We tested different protocols for each stage of fecal sample processing: storage, DNA extraction, and choice of the sequencing region in the bacterial 16S rRNA gene. We also analyzed the gut microbiome of captive Japanese macaques as the control. The comparison of samples obtained from identical macaques but subjected to different protocols showed that the tested storage methods (RNAlater and lysis buffer) produced effectively the same composition of bacterial operational taxonomic units (OTUs) as the standard frozen storage method, although the relative abundance of each OTU was quantitatively affected. Taxonomic assignment of the detected bacterial groups was also significantly affected by the region being sequenced, indicating that sequencing regions and the corresponding polymerase chain reaction (PCR) primer pairs for the 16S rRNA gene should be carefully selected. This study improves the current standard methods for microbiome analysis in wild nonhuman primates. Japanese macaques were shown to be a suitable model for understanding microbiome adaptation to various environments.

A Non-invasive Method to Collect Fecal Samples from Wild Birds for Microbiome Studies.

Over the past few decades, studies have demonstrated that the gut microbiota strongly influences the physiology, behavior, and fitness of its host. Such studies have been conducted primarily in humans and model organisms under controlled laboratory conditions. More recently, researchers have realized the importance of placing host-associated microbiota studies into a more ecological context; however, few non-destructive methods have been established to collect fecal samples from wild birds. Here, we present an inexpensive and easy-to-use kit for the non-invasive collection of feces from small birds. The portability of the collection kit makes this method amenable to field studies, especially those in remote areas. The main components of the collection kit include a flat-bottomed paper bag, a large modified weigh boat (tray), vinyl-coated hardware cloth fencing (grate), a clothespin, and a 10% bleach solution (to sterilize the tray and grate). In the paper bag, a sterile tray is placed under a small grate, which prevents the birds from contacting the feces and reduces the risk of contamination. After capture, the bird is placed in the bag for 3-5 min until it defecates. After the bird is removed from the bag, the tray is extracted and the fecal sample is moved to a collection tube and frozen or preserved. We believe that our method is an affordable and easy option for researchers studying the gut microbiota of wild birds.

Dietary maifanite supplementation did not affect the apparent total tract digestibility of calcium and phosphorus in growing pigs.

OBJECTIVE: This study was conducted to determine the effects of dietary maifanite supplementation and fecal collection method on the apparent total tract digestibility (ATTD) of calcium (Ca) and phosphorus (P) and blood parameters in growing pigs. METHODS: Thirty-six growing barrows (Duroc×Landrace×Yorkshire; 27.0±2.6 kg) were allotted to six dietary treatments with 6 pigs per treatment according to body weight in a completely randomized design. The experimental treatments were: i) Low Ca+cornstarch (2.25%), ii) Low Ca+maifanite (2.25%), iii) Medium Ca+cornstarch (1.42%), iv) Medium Ca+maifanite (1.42%), v) High Ca+cornstarch (0.64%), and vi) High Ca+maifanite (0.64%). Feces were collected by the total collection (TC) and indicator method (IM). At the beginning and the end of the experiment, blood samples were collected from each pig. RESULTS: For the TC method, there were no difference in Ca intake, fecal Ca output, Ca retention and the ATTD of Ca between cornstarch and maifanite diets at the same dietary Ca level. However, urinary Ca excretion was lower (p = 0.01) in pigs fed low Ca diets without maifanite supplementation compared with other dietary treatments. Dietary maifanite supplementation had no effect on the P metabolism in growing pigs. For the IM method, there was no difference in Ca digestibility between cornstarch and maifanite diets at the same dietary Ca level. The ATTD of P was greater (p<0.01) in pigs fed the high Ca diet with maifanite supplementation compared with the high Ca diet with cornstarch treatment. Dietary inclusion of maifanite had no effect on blood parameters in growing pigs. CONCLUSION: Dietary maifanite supplementation had no effect on the ATTD of Ca and P and serum parameters in growing pigs. The IM resulted in lower digestibility values than the TC method.

Comparison of methods for determination of nutrient digestibility of a dry kibble diet for ocelots / Comparação de métodos para estimar coeficientes de digestibilidade de uma ração comercial de gatos-domésticos para jaguatiricas

In this experiment, methods of total fecal collection (TFC) and internal markers (acid-insoluble ash - AIA, crude fiber - CF, and acid-detergent fiber - ADF) were compared for determination of the coefficients of apparent digestibility (CAD) for dry matter (DM), crude protein (CP), ether extract (EE), nitrogen-free extracts (NFE), and gross energy (GE) of commercial feline dry kibble for ocelots (Leopardus pardalis). Six adult animals, weighing 12.45±1.37 kg, gradually received experimental kibble in their usual diet until the beginning of the experiment and were submitted to an adaptation period ten days prior to the collection period. CAD obtained by TFC, AIA, CF, and ADF were, respectively, 73.7, 76.83, 62.01, and 46.03% for dry matter; 81.9, 84.8, 75.8, and 63.8% for crude protein; 85, 86.7, 78.5, and 69.1% for ether extract; 78.52, 79.55, 69.11, and 53.04% for nitrogen-free extracts; and 80.5, 82.2, 71.4, and 58.4% for gross energy. The AIA method showed to be efficient in determining coefficients of apparent digestibility and may contribute to investigations on the digestibility of diets for wild felines. In comparison to the items of ocelot's usual diet, the kibble used in this paper provided an adequate nutritional supply with reduced daily costs per animal.

Neste experimento foram comparados os métodos de coleta total de fezes (CT) e de indicadores internos (cinza insolúvel em ácido - CIA, fibra bruta - FB e fibra em detergente ácido - FDA) na determinação dos coeficientes de digestibilidade aparente (CDA) da matéria seca (MS), proteína bruta (PB), extrato etéreo (EE), extrativo não nitrogenado (ENN) e energia bruta (EB) de uma ração comercial de gatos-domésticos para jaguatiricas (Leopardus pardalis). Seis animais adultos com peso de 12,45 ± 1,37kg receberam gradativamente a ração experimental na dieta habitual até o início do experimento e foram submetidos a um período de adaptação de 10 dias anteriores ao período de coleta. Os CDA obtidos pela CT, CIA, FB e FDA foram, respectivamente de 73,70; 76,83; 62,01 e 46,03% para matéria seca, 81,9; 84,8; 75,8 e 63,8% para proteína bruta, 85,0; 86,7; 78,5 e 69,1% para extrato etéreo, 78,52; 79,55; 69,11 e 53,04% para extrativo não nitrogenado e de 80,5; 82,2; 71,4 e 58,4% para energia bruta. O método de CIA mostrou-se eficiente na determinação dos coeficientes de digestibilidade aparente e pode contribuir com as investigações sobre a digestibilidade em dietas com felídeos selvagens. A ração utilizada permitiu aos animais um adequado aporte nutricional e apresentou menores custos/animal/dia em comparação aos itens que compunham a dieta habitual.

Uso da LIPE® como indicador externo na determinação da digestibilidade da proteína e matéria seca de alimentos em frangos de corte / Use of LIPE® as an external indicator in the determination of protein and dry matter digestibility of broiler chicken food

Realizou-se um ensaio metabólico para avaliar a lignina purificada de eucalipto (LIPE®), como indicador na determinação da digestibilidade da proteína bruta e matéria seca de alimentos para frangos de corte. Os tratamentos corresponderam a uma dieta basal e outras oito dietas contendo os alimentos-teste. Os alimentos foram: milho, milho expandido, farelo de soja, farelo de soja expandido, glúten de milho 22 por cento, glúten de milho 60 por cento, farinha de carne e ossos e farinha de penas e vísceras. Os alimentos protéicos e os com menor teor de proteína substituíram 25 e 40 por cento da dieta basal, respectivamente. Foram utilizados 270 pintos de corte, linhagem Ross, de ambos os sexos, dos 21 aos 31 dias de idade. Utilizou-se um delineamento experimental inteiramente ao acaso em esquema fatorial 9×3 (rações × métodos), com três repetições. Os coeficientes de digestibilidade (CD) da proteína e da matéria seca e os valores da proteína bruta digestível e da matéria seca digestível de sete das nove dietas, calculados pelos métodos da coleta total de excretas e dos indicadores óxido crômico e LIPE®, foram semelhantes. Com os alimentos-teste, farelo de soja expandido e farinha de penas e vísceras, o uso de óxido crômico resultou em CD e valores de proteína e matéria seca digestível mais altos, quando comparado com o uso da LIPE® e da coleta total de excretas que foram semelhantes entre si. Pode-se considerar que a proteína bruta digestível e matéria seca digestível dos alimentos, calculada pelos três métodos, foram equivalentes. A LIPE® pode ser validada como indicador de digestibilidade da proteína e matéria seca de alimentos para frangos de corte.

A metabolism experiment was carried out to evaluate the use of LIPE® as an external indicator of dry matter and protein digestibility in broilers. The treatments corresponded to a basal diet and eight other different diets. The foods used were: corn grain, expanded corn, soybean meal, expanded soybean meal, gluten meal 22 percent crude protein; gluten meal 60 percent crude protein, meat and bone meal and feather meal. The basal diet was replaced with 40 percent or 25 percent of the food containing more energy or protein, respectively. Two hundred and seventy broiler chickens were used from 21 to 31 days old. A completely randomized design in a 9x3 factorial arrangement (nine forms of rations x three methods) with three replicates was used. The digestible dry matter and protein and metabolizable energy of the food was calculated by the total fecal collection method and by the indicator methods of chromic oxide and LIPE®; the three methods were further compared. Chromic oxide use resulted in higher values of coefficient of digestibility for expanded soybean meal and feathers and viscera meal when compared to the use of LIPE® and total feces collection which were similar. The results validated the LIPE® as an external protein and dry matter digestibility index of the feed of broiler chicks evaluated.

Digestibilidade aparente e verdadeira do fósforo de alimentos de origem animal para suínos / Apparent and true digestibility of phosphorus from animal origin feedstuffs for swines

Determinaram-se os coeficientes de digestibilidade aparente (CDAP) e verdadeira (CDVP) do fósforo de alimentos de origem animal. Foram utilizados 24 suínos, machos castrados, com média de peso de 25,0±3,0kg no período de crescimento e 24 suínos com média de peso de 60,0±5,0kg para o período de terminação. Os tratamentos foram resultantes de um fatorial de duas metodologias (coleta total de fezes e indicador fecal), duas fases (crescimento e terminação) e oito dietas (seis alimentos de origem animal, uma ração referência e uma ração com baixo conteúdo de fósforo total para estimar as perdas de fósforo endógeno), com três repetições e um animal por unidade experimental. Não foram encontradas diferenças entre as metodologias ou entre as fases avaliadas (P>0,05). Os valores médios de CDAP e CDVP encontrados com suínos em crescimento e terminação foram, respectivamente, 61,7 e 62,0 por cento para a farinha de carne e ossos com 35 por cento de proteína bruta (PB); 62,3 e 62,9 por cento para a farinha de carne e ossos com 41 por cento de PB; 49,0 e 52,5 por cento para a farinha de vísceras e penas; 72,3 e 90,8 por cento para a farinha de penas; 85,5 e 88,5 por cento para a farinha de peixe com 55 por cento de PB; e 80,0 e 92,0 por cento para o soro de leite em pó.

The coefficients of apparent (CADP) and true (CTDP) digestibility of the phosphorus from animal origin feedstuffs were determined. Twenty-four barrows in growing phase with initial weight 25.0±3.0kg and the same barrows in finishing phase with initial weight 60.0±5.0kg were used. The treatments were made by a factorial of two methodologies (total collection of feces and fecal marker), two phases (growing and finishing), and eight diets (six animal origin feedstuffs, one reference diet, and one diet with low content of total phosphorus in order to estimate the losses of endogenous phosphorus), with three replicates by treatment. There was no difference between the methodologies or phases evaluated (P>0.05). The average of CADP and CTDP found in growing and finishing phases were, respectively, 61.7 and 62.0 percent for 35 percent crude protein (CP) meat and bone meal; 62.3 and 62.9 percent for 41 percent CP meat and bone meal; 49.0 and 52.5 percent for feather and poultry by-products meal; 72.3 and 90.8 percent for feather meal; 85.5 and 88.5 percent for 55 percent CP fish meal; and 80.0 and 92.0 percent for powder milk whey.