Guinea Pigs in Balance: Impact of Diet on pH, Microbiota and Intestinal Performance

1. Introduction

The relationship between diet and animal health, as well as the interaction between gastrointestinal microbiota and metabolism, has been studied for decades, and its importance continues to be relevant in the field of animal production [1]. In this context, guinea pigs (Cavia porcellus) emerge as an invaluable model due to their herbivorous nature and their widespread use in meat production in many regions of the world [2]. Diet plays a crucial role in performance and significantly influences various physiological and metabolic aspects of animals. However, the relationship between diet and parameters such as gastrointestinal pH, intestinal microbiota composition, intestinal morphometry, and productive performance has not yet been fully elucidated in guinea pigs.

Diet can alter the functional metabolism of the intestinal microbiome [3] and, along with genetic factors and certain additives, influence the predominance of some microorganisms over others [4]. Moreover, the microbiota is associated with body growth, immune development, and nutrition [5,6,7], while intestinal morphometry provides invaluable information on nutrient absorption capacity and gastrointestinal tract health. Similarly, gastrointestinal pH can either promote or hinder digestion and nutrient absorption [8]. For example, in rabbits, exposure to excessive amounts of easily fermentable substrates (proteins and starch) leads to hyperfermentation when incompletely ingested before reaching the cecum, causing changes in cecal pH and inhibiting normal microbiota [9]. In non-herbivorous monogastrics such as chickens, protein-rich diets cause an alkaline pH at the cecum, promoting intestinal health and productive parameters [10].

Despite existing evidence on the influence of diet on these physiological aspects, a deeper understanding is still needed of how different dietary components can modulate gastrointestinal pH, which in turn may affect probiotic mechanisms. This is especially relevant considering the discrepancies in the results reported in various studies using Lactobacillus to improve the productive performance of guinea pigs.

The present study analyzed the impact of different diets on gastrointestinal pH, intestinal microbiota composition, digestive tract morphometry, and guinea pig performance. It is expected that the findings of this research will contribute to the design of more effective nutritional strategies to improve the health and performance of these animals in production systems.

2. Materials and Methods

2.1. Study Area

The trial was conducted in the Azuay province, Cuenca canton, Ecuador, at an average altitude of 2,500 meters above sea level, with an annual average temperature of 14.6ºC and an average precipitation of 900 mm per year [11]. A total of 160 improved genotype guinea pigs were used, weaned at 21 days of age, with an average weight of 351 g for males and 307 g for females. The animals were randomly assigned to three treatments: T1 (Alfalfa - Medicago sativa), T2 (Alfalfa + Concentrate), and T3 (Concentrate), with four subgroups per treatment and 10 animals per subgroup (9 females and 1 male). After three weeks, 2 females were removed from each subgroup for euthanasia, leaving groups of 7 females and 1 male, as recommended by Cruz [12].

2.2. Zootechnical Management of Guinea Pigs

The identification of the animals was carried out by placing a numbered metal tag on the left ear pavilion. The environmental variables within the pen (temperature, humidity, and light), density, and health program were similar for all experimental units. The nutritional program was adjusted according to the nutritional requirements based on the physiological stage of the guinea pigs.

2.3. Procedure

The study was divided into two phases:

2.3.1. First Phase

The influence of diet on the gastrointestinal pH of the guinea pigs was analyzed. A 7-day adaptation period to the diet was implemented. After 21 days, 8 female guinea pigs per treatment (2 per group) were selected for euthanasia using Sodium Pentobarbital (25-40 mg/kg) administered intravenously, following the recommendations for the euthanasia of experimental animals issued by Close et al. [13,14]. Subsequently, the gastrointestinal tract segments (stomach, duodenum, jejunum, ileum, and cecum) were exposed, and the pH of these segments was measured using pH strips.

2.3.2. Second Phase

The study investigated whether the variability in pH recorded with the three diets influenced the mechanism of action of the enhanced probiotic mixture (Probiolyte® WS), incorporated at 0.5% in T2 and 1% in T3, with T1 serving as the control treatment. To identify the gut microbiota of the lactating guinea pigs and those in T2 and T3, samples were first plated on specific culture media using streaking techniques to facilitate the isolation of individual colonies. The isolated colonies were then purified on fresh culture media to ensure pure cultures. Once pure colonies were obtained, MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry) technology was used for identification due to its high precision and speed in microbial species characterization [15].

Additionally, intestinal morphometric parameters (width and length of intestinal villi and Lieberkühn crypt depth) were analyzed before and after 60 days of including the enhanced probiotic mixture. For this procedure, samples (segments of the duodenum, jejunum, and ileum) were fixed in 10% formalin and analyzed using Hematoxylin-Eosin (H-E) staining, with morphometric changes evaluated through a digital trinocular microscope with 10X magnification. Finally, the productive parameters of the guinea pigs were recorded and analyzed.

2.4. Statistical Analysis

The collected data were analyzed using the R statistical software. First, a normality test was performed using the Shapiro-Wilk test to verify the distribution of the data. For each studied variable, including gastrointestinal pH, intestinal morphometry, and productive parameters, a one-way analysis of variance (ANOVA) was conducted to compare means among the three treatments (T1, T2, and T3). The composition of the gut microbiota was analyzed through frequency and prevalence analysis. Additionally, Pearson correlation analysis was used to assess the influence of pH on intestinal morphometric variables in each intestinal segment. Finally, a linear regression model was employed to investigate how independent variables (diet, gastrointestinal pH, and microbiota composition) affect dependent variables (productive parameters and intestinal morphometry). A value of p < 0.05 was considered statistically significant in all analyses.

3. Results

3.1. Gastrointestinal pH

The pH of the gastrointestinal tract in guinea pigs fed with different diets showed statistically significant differences (p<0.05) across the various segments analyzed. Guinea pigs fed an alfalfa-based diet had the highest pH in the stomach (5.88). In contrast, those fed a concentrate diet exhibited the highest pH values in the duodenum and jejunum, with readings of 7.13 and 8.38, respectively. No statistically significant differences (p>0.05) in pH were observed in the ileum and cecum based on the type of diet. The data are presented in Table 1.

3.2. Identification of the Intestinal Microbiota

A total of 17 bacteria were identified in the fecal microbiota of suckling guinea pigs, with Staphylococcus vitulinus and Escherichia coli being the most frequent. These bacteria belong to the Firmicutes and Proteobacteria phyla, respectively. In weaned guinea pigs integrated into treatments T2 and T3, a total of ten bacteria were identified, with Escherichia coli being the predominant bacterium. See Table 2 and Table 3.

3.3. Intestinal Morphometry

Statistical differences (p<0.05) were observed between treatments, and it was determined that age also influences the intestinal morphometric variables, with these variables increasing with age. Mixed feeding with the addition of 0.5% of the enhanced probiotic mixture (Probiolyte® WS) significantly increased the length of intestinal villi in the duodenum (537.83 µm) and ileum (293.64 µm) and the density of villi in the duodenum (67 per 4X field). Mixed feeding with the addition of 1% improved the width of intestinal villi in the duodenum (56.90 µm) and the depth of villi in the ileum (48.83 µm). Guinea pigs fed with concentrate increased the length of intestinal villi in the jejunum (433.70 µm) and the width in the ileum (51.87 µm). Concentrate feeding with the addition of 1% reinforced probiotic blend improved the depth of villi in the jejunum (52.93 µm). See Table 4.

3.4. Productive Parameters

The productive parameters (weight gain, feed intake, and feed conversion ratio) showed a significant difference (p < 0.05) between treatments. The guinea pigs fed with a mixed diet plus 0.5% of the reinforced probiotic blend reached the highest weight gain (284.67 g), the highest feed intake (1046.08 g), and the best feed conversion ratio (3.70) by the seventh week of the study, compared to the other treatments. See Table 5.

3.5. Correlation Analysis

The correlation analysis suggests that pH has a variable influence on the morphometric characteristics of intestinal villi depending on the segment of the digestive tract. In the jejunum and ileum, pH shows a stronger association with the length and width of the villi, whereas in the duodenum, the influence of pH is more limited. These findings highlight the importance of considering the specific segment when evaluating the impact of pH on intestinal morphometry. See (Table 6).

4. Discussion

4.1. Gastrointestinal pH

Previous studies on pH in the digestive system of guinea pigs and other species have reported varied results. For example, Merchant et al. [16] recorded a gastric pH of 2.9 in guinea pigs fed ad libitum, a pH in the small intestine ranging from 6.4 to 7.4, and pH values between 6.0 and 6.4 in the cecum and between 6.1 and 6.6 in the colon. Rechkemmer et al. [17] indicated that the pH of the jejunal microclimate in guinea pigs is 7.37, while the luminal pH is 7.27.

García et al. [18] demonstrated that pH influences the activity of amylolytic, cellulolytic, proteolytic, and lipolytic enzymes in the cecum of guinea pigs, observing lower cellulolytic enzyme activity at a pH of 5 and lipolytic activity at a pH of 9, without significantly affecting amylolytic and proteolytic activity. Pinchao et al. [19] reported that lactic acid bacteria (LAB) are more adaptable at intestinal pH levels of 2.9, 5.0, 6.4, and 7.4. On the other hand, Ramón [20] found that the pH in the stomach and cecum of guinea pigs performing cecotrophy is 1.53 and 6.63, respectively.

Vásquez et al. [8] measured pH in alpaca crias during the first 45 days of life, recording a gastric pH ranging from 6.91 to 5.95, and in the duodenum, jejunum, ileum, and cecum, pH values of 6.49 to 6.41, 6.64 to 6.71, 7.18 to 6.63, and 6.49 to 6.68, respectively. Gastrointestinal pH variations may be influenced by fiber levels in the diet, as high-fiber diets can neutralize some acidity by increasing saliva production, which is alkaline. The presence of certain minerals such as calcium and magnesium, as well as the amount and frequency of feeding, may also affect pH. Increased food intake may elevate hydrochloric acid production in the stomach, while long periods without feeding can reduce acidity. According to Miranda et al. [21], the inclusion of fermented bioadditives with lactic acid bacteria and yeasts produces organic acids (lactic and acetic acids) as metabolic byproducts, which can alter pH values along the gastrointestinal tract, Goichochea et al. [22] and thereby reduce bacterial growth, especially at pH equal to or below 4.

4.2. Identification of the Intestinal Microbiota

Different data from those found in this study have been reported by Frias et al. [23], who identified four phyla: Bacteroidota, Firmicutes, Spirochaetota, and Synergistota in the cecal microbiome of three guinea pig breeds: Andina, Inti, and Perú. The authors concluded that genetics could influence the structure and composition of the guinea pig cecal microbiome by finding unique genera for each breed.

On the other hand, Wu et al. [24] point to Ruminococcus_albus as the predominant bacterial community in the guinea pig intestinal microbiota. Meanwhile, Murga et al. [25] highlight Bifidobacterium longum (Phylum Actinomycetota), Fibrobacter succinogenes (Fibrobacterota), and Faecalibacterium prausnitzii (Bacillota), attributing that the composition or alteration of the microbiome may be related to the development of certain diseases or genetics. Zhu et al. [26] indicate that the addition of non-nutritive sweeteners (rebaudioside) to the diet of guinea pigs significantly alters the relative abundance of Lactobacillus.

Hildebrand et al. [27] demonstrated that the guinea pig intestinal microbiota is dominated by two phyla: Bacteroidetes and Firmicutes. Pinchao et al. [19] isolated 29 strains of lactic acid bacteria obtained from the intestines, cecum, and colon of adult and young guinea pigs, with Ligilactobacillus salivarius being the most prevalent strain; however, Alayande et al. [28] state that the origin of the intestinal microbiota is not well established.

These discrepancies between studies could be due to several reasons, including differences in methodologies used for bacterial identification, variations in diets and living conditions of the guinea pigs, and genetic diversity among different populations and breeds of guinea pigs. Additionally, it is important to consider the physiological state of the animals (infants, juveniles, or adults) and the impact of the immediate environment on the configuration of the microbiome.

4.3. Intestinal Morphometry

The results of the study differ from those reported by Carcelén et al., and Puente et al. [29,30], who added a probiotic mixture (Enterococcus hirae, Lactobacillus reuteri, L. frumenti, L. johnsonii, Streptococcus thoraltensis, and Bacillus pumilus) to a mixed diet (wheat bran plus forage) for guinea pigs, showing only benefits in the ratio of villi length to depth in the ileum and duodenum, respectively. Saccharomyces cerevisiae, added as a probiotic to the mixed diet of guinea pigs, also has a positive effect on villi width and the length of the crypt-villi axis.

Studies conducted on broiler chickens demonstrate the benefits of probiotics on intestinal morphometry; Roa et al. [31] report that the individual or mixed inclusion of probiotics (Saccharomyces cerevisiae, Lactobacillus sp., and Bacillus sp.) has a positive effect on the intestinal morphometry of the birds.

4.4. Productive Parameters

Several authors have studied the effect of additives such as probiotics, prebiotics, symbiotics, and enzymes on the diet of guinea pigs. Miranda et al. [32] assessed the impact of probiotics derived from a substrate of agro-industrial residues fermented with lactic acid bacteria and/or yeasts (Lactobacillus acidophilus, L. bulgaricus, Saccharomyces cerevisiae, and Kluyveromyces fragilis), with a molasses-vinasse substrate, and observed improvements in weight gain in guinea pigs. Andía & Lazo [33] report that the inclusion of a probiotic mixture of Lactobacillus acidophilus, L. casei, and Saccharomyces cerevisiae in the guinea pig diet has a significant effect on food consumption and carcass yield. Cuenca et al. [34] added garlic powder at 1%/kg of concentrated feed as a prebiotic and observed improvements in the productive parameters of guinea pigs starting from the third week of the experiment.

Contrarily, [29,35,36] report that the addition of probiotics, prebiotics, or symbiotics to guinea pig feed does not have a significant or consistent effect on the productive parameters of this species. Bazay et al. [37] indicate that mannan-oligosaccharides (MOS) included in the diet of guinea pigs during the fattening phase do not improve production parameters. Criollo et al. [38] report that the addition of Saccharomyces cerevisiae to the guinea pig diet does not have favorable effects on productive parameters.

5. Conclusions

The diet significantly influences gastrointestinal pH, intestinal microbiota, intestinal morphometry, and productive parameters in guinea pigs. These findings highlight the importance of diet composition and supplementation in the nutritional management of guinea pigs, with direct implications for optimizing their digestive health and productive performance. An optimal pH in the gastrointestinal tract promotes a balanced intestinal microbiota, which in turn positively impacts productive parameters and strengthens the immune system, emphasizing the interdependence of these factors in the health and performance of the animals.