Evaluation of probiotic supplementation to enhance growth, immune response, intestinal function, and survival in extensively raised neonatal alpacas in the Peruvian Central-southern Andes

Highlights

• •Supplementing newborn alpacas with multi-strain probiotics, minerals, and vitamins improves their growth.
• •Multi-strain probiotics are a preventive option for reducing mortality in neonatal alpacas during the first months of life.
• •More studies are needed to understand the effect of probiotics on neonatal alpacas.

Abstract

This study aimed to evaluate the effects of multi-strain probiotics on growth, immune response, intestinal function, and survival in neonatal alpacas in the Peruvian Central-southern Andes. A total of 96 neonatal alpacas were selected, with an average weight of 7.89 ± 0.93 kg, regardless of breed (Huacaya and Suri). The study employed a completely randomized design. The neonates were randomly assigned to three groups (n = 32): control (T1), Probiolyte® WS (T2), and Polimeve Solúve (T3). Probiotics were administered orally on days 1, 7, 14, 21, and 30 of age. Body weight gain and size were recorded on days 1, 15, 30, 60, and 90; immune response was assessed on 30 and 90 days; and intestinal functional status was evaluated on day 90. The neonates supplemented with Polimeve exhibited greater thoracic circumference after 60 days of age, and increased height at the withers and rump at 15 days (p < 0.05). At 90 days, the Polimeve group had a higher proportion of basophils, while the Probiolyte and control groups showed higher proportions of monocytes (p < 0.05). IgG concentrations at 30 and 90 days did not differ significantly among groups. Probiotic supplementation did not affect ileal villus length; however, it influenced the depth of jejunal and cecal crypts (p < 0.05). Morbidity and mortality rates did not differ significantly between groups. In conclusion, probiotics did not significantly influence growth, immune response, or intestinal function; however, Polimeve may improve weight gain in neonates, while Probiolyte can increase IgG concentrations during the first 30 days of life.

1. Introduction

Approximately 87% of the global alpaca population (4491,671 head) is concentrated in Peru, primarily in the departments of Puno (2030,675 alpacas), Cusco (677,810 alpacas), Arequipa (470,840 alpacas), and Ayacucho (295,856 alpacas) (MIDAGRI, 2025). Alpacas are predominantly raised in high-Andean ecosystems under extensive production systems and play a crucial role in the cultural, social, and economic sustainability of rural communities (Mallma et al., 2021). In recent years, alpaca have gained increasing recognition as a valuable zoogenetic resource, and their population has grown significantly (Gómez-Quispe et al., 2022; Zarrin et al., 2020). The species exhibits remarkable adaptations to the harsh environmental conditions found above 3800 m above sea level, including efficient forage utilization, reduced soil degradation due to its footpads, and the presence of unique immunoglobulins with potential therapeutic applications (Mallma et al., 2021; MIDAGRI, 2019). In addition to their ecological adaptability, alpacas provide essential products such as meat, fiber, and leather, as well as by-products like manure (Zarrin et al., 2020).

Despite these advantages, neonatal alpacas are particularly vulnerable during the first weeks of life. Animal between 6 and 12 weeks of age, especially those with low birth weight, are highly susceptible to environmental stressors and colonization by enteric pathogens (Alawneh et al., 2020; Gómez-Quispe et al., 2022; Zarrin et al., 2020). These factors compromise gut health, leading to diarrhea a potentially fatal condition due to excessive loss of electrolytes and water (Rojas et al., 2016). Diarrhea-related nutritional deficiencies contribute significantly to neonatal morbidity (23%) and mortality (7%), generating substantial economic losses for alpaca producers. Estimated losses reach approximately 23 soles per neonatal alpacas and at least 5500 soles annually, disproportionately affecting of small and medium-scale farmers (Caman, 2018; Hancco, 2018).

Several pathogens have been associated with enteric disorders in alpacas. Epidemiological studies have reported a high prevalence of Eimeria spp. (80%), followed by coronavirus (40%), E. coli (34%), rotavirus (32%), Clostridium spp. (22%), and Cryptosporidium spp. (20%) (Rojas et al., 2016). Additional bacterial taxa linked to intestinal disease include Clostridium spiroforme, Blautia, and Bacteroides fragilis (Zapata et al., 2024). In many production systems, antibiotics are frequently used to control diarrheal diseases (Lorenz et al., 2011); however, increasing concerns regarding antimicrobial residues and the emergence of antimicrobial resistance have raised important challenges for animal production and public health (Ali et al., 2024; Kelsey & Colpoys, 2018; Lee et al., 2021). Consequently, alternative strategies to maintain gut health and prevent enteric diseases in livestock are increasingly being explored. As a result, the use of antimicrobials has been banned in many countries (Lee et al., 2021).

Among these alternatives, probiotics have attracted considerable attention due to their capacity to modulate intestinal microbiota and improve host health. Probiotics are defined as live microorganisms, including bacteria and fungi, administered individually or as multi-strain formulations to confer health benefits to the host (Wang et al., 2019a). Their use is well established in humans and monogastric livestock species such as pigs and poultry (Guo et al., 2024; Konieczka et al., 2022; Plaza-Diaz et al., 2019). Meanwhile, their application in ruminants has only recently expanded (Ali et al., 2024; Taboada et al., 2022). The most commonly used probiotic strains in livestock production include Bifidobacterium, Enterococcus, Lactobacillus, Saccharomyces, and Streptococcus (Guo et al., 2022; Konieczka et al., 2022; Piewngam et al., 2018; Wang et al., 2022a).

Growing evidence suggests that multi-strain probiotics (MSPs) may provide more consistent and synergistic effects than single-strain formulations (Kumar et al., 2025; Wang et al., 2022b). The reported benefits include improved weight gain and growth, as well as higher milk production of good quality (Lele et al., 2019; Wang et al., 2019b); enhanced stimulation of the immune system through increased production of IgG and IgM (Barba-Vidal et al., 2019; Konieczka et al., 2023); improved gastrointestinal health through the modulation of beneficial microbiota, as well as the prevention and correction of digestive disorders by promoting a protective microflora, and stimulation of goblet cells (Wang et al., 2022b). For optimal functionality, probiotic strains must tolerate gastrointestinal conditions, including acidic pH, digestive enzymes, and bile salts, while also exhibiting antagonistic activity against enteric pathogens (Plaza-Díaz et al., 2018; 2019).

Different probiotic formulations have demonstrated beneficial effects in livestock. Bacillus-based probiotics have been associated with improvements in animal performance and gut health (Piewngam et al., 2018; Varada et al., 2022). Similarly, Lactobacillus and Bifidobacterium species have been shown to enhance immune responses increasing cytokines such as IL-2, IL-4, IL-6, and IL-10 and immunoglobulins IgG, IgE, and reduced tumor necrosis factor-alpha (TNF-α) (Kumar et al., 2022). Saccharomyces cerevisiae-based probiotics contribute to maintaining intestinal barrier integrity (Ali et al., 2024). Nevertheless, the effectiveness of probiotics in vivo may be limited by factors such as environmental complexity, rapid gastrointestinal transit, and reduced intestinal colonization, which can affect their bioavailability and persistence in the host (Wang et al., 2021).

Despite growing evidence supporting the use of probiotic supplementation in various ruminants (Ali et al., 2024; Konieczka et al., 2023; Singh et al., 2021; Varada et al., 2022), there is insufficient information on the effect of probiotics in neonatal alpacas. Given the beneficial properties of probiotics, it would be important to incorporate them into the feeding programs for neonatal alpacas as a preventive strategy to improve health indicators, since in the first days of life they are prone to contracting diseases such as clostridiosis and coccidiosis, which are the leading causes of mortality. Therefore, the objective of the present study was to evaluate the effect of MSPs on growth, survival, immune response, and intestinal functional status in neonatal alpacas raised under extensive systems in the Andean region of Central-southern Peru.

2. Materials y methods

2.1. Animal ethics statement

This experiment was approved by the Research Ethics Committee of the Vice-Rectorate for Research at the Universidad Nacional de San Cristóbal de Huamanga, Ayacucho, Peru (No. 003-2025/CEI-VRI-UNSCH).

2.2. Location

The study was led in the Chuschi district, Ayacucho, Peru (Fig. 1), between March and July 2024. The area is characterized by a cold, dry climate, with a distinct rainy season (December to March) and dry season (May to October). Average temperatures range from 0.1 °C in July to 28.7 °C in November, with monthly precipitation between 5.5 and 154.7 mm (SENAMHI, 2018). The ecosystem features gentle slopes, small wetland areas, and extensive grasslands dominated by native vegetation including Phoaceae, Ciperaceae, Rosaceae, Asteraceae, and Fabaceae (Trillo et al., 2021).

Fig. 1.

Geographic location of the Chuschi district, Ayacucho department, Peru.

2.3. Experimental design

The study employed a completely randomized design. The Ninety-six (96) experimental units were randomly assigned to one of three groups of n = 32 neonates each. The groups consisted of two probiotic supplementation treatments and a control. No treatment was administered to the control group (T1), while the supplemented groups received either Probiolyte® WS (Agrovet Market Animal Health, Peru) (T2) (Table 1) or Polimeve Solúve® (Imeve Laboratory, Brazil) (T3) (Table 2), both composed of quantified colony-forming units (CFUs). The activation of the product's additives was verified in accordance with the manufacturer's instructions.

Table 1.

Composition per 1 g of PMC Probiolyte® WS ¹.

Components	Quantity	Unit
Bacterial strains
Lactubacillus plantarum	5.916×10^3	CFU/g
Lactobacillus acidophilus	11.834×10^3	CFU/g
Lactobacillus rhamnosus	5.917×10^3	CFU/g
Bacillus subtilis	473.373×10^3	CFU/g
Bacillus licheniformis	355.030×10^3	CFU/g
Bifidobacterium longun	3.55×10^3	CFU/g
Bifidobacterium bifidum	3.55×10^-3	CFU/g
Streptococcus thermophilus	3.55×10^3	CFU/g
Saccharomyces cerevisiae	47.337×10^3	CFU/g
Enterococcus faecium	3.55×10^3	CFU/g
Aspergillus oryzae	1.183×10^3	CFU/g
Bacillus coagulans	85.207×10^3	CFU/g
Total	1× 10^6	CFU/g
Vitamins
Retinol Palmitate (Vitamin A)	10,000.00	IU
Cholecalciferol (Vitamin D3)	2500.00	IU
DL-Alfa Tocopherol Acetate (Vitamin E)	5.00	mg
Menadione (as Menadione sodium bisulfite (Vitamin K3)	4.50	mg
Thiamine (as Thiamine hydrochloride) (Vitamin B1)	1.10	mg
Riboflavin (as Riboflavin 5-phosphate sodium dyhydrate)	1.50	mg
(Vitamin B2)
Nicotinamide (Vitamin B3)	10.00	mg
Calcium Pantothenato (Vitamin B5)	5.50	mg
Pyridoxine (as Pyridoxine hydrochloride) (Vitamin B6)	0.75	mg
Biotin (Vitamin B7)	0.40	mg
Folid Acid (Vitamin B9)	0.20	mg
Cyanocobalamin (Vitamin B12)	0.01	mg
Sodium Pangamate (Vitamin B15)	0.20	mg
Inositol (Vitamin BH)	0.50	mg
Ascorbic Acid (as Sodium Ascorbate) (Vitamin C)	1.00	mg
Amino Acids
Aspartic Acid (L-Aspartic Acid)	1.91	mg
Glutamic Acid (as Monosodium Glutamate)	3.30	mg
Alanine (L-Alanine)	0.84	mg
Arginine (as L-Arginine Hydrochloride)	0.47	mg
Cysteine (as L-Cysteine Hydrochloride Monohydrate)	0.43	mg
Glycine	4.13	mg
Histidine (as L-Histidine Hydrochloride Monohydrate)	0.42	mg
Isoleucine (L-Isoleucine)	1.12	mg
Leucine (L-Leucine)	1.78	mg
Lysine (L-Lysine hydrochloride)	15.00	mg
Methionine (DL-Methionine)	10.00	mg
Proline (L-Proline)	1.01	mg
Serine (L-Serine)	3.30	mg
Tyrosine (L-Tyrosine)	0.40	mg
Threonine (L-Threonine)	1.26	mg
Tryptophan (L- Tryptophan)	0.32	mg
Valine (L-Valine)	1.07	mg
Minerals and Electrolytes
Anhydrous Citric Acid	50.00	mg
Sodium Chloride	50.00	mg
Potassium Chloride	87.50	mg
Cobalt (as Cobalt Gluconate)	0.33	mg
Copper (as Copper-Edetate)	1.88	mg
Chromium (as Chromium-Methionine)	0.00	mg
Ferrun (as Ferrum-Edetate)	0.65	mg
Manganese (as Manganese-Edetate)	1.63	mg
Sodium Selenite (as Sodium Selenite Pentahydrate)	0.50	mg
Magnesium Sulfate Anhydrous	12.50	mg
Potassium Iodide	2.50	mg
Zinc (as Zinc-Edetate)	1.88	mg
Other
Inulin	5.00	mg

¹Composition of the Product commercial Probiolyte® WS of Agrovet Market Animal Health, Peru. ProBiolyte is a nutritional supplement that includes 15 vitamins, 18 amino acids, chelated organic minerals, and a blend of probiotics. Registration SENASA Peru A.16.07.N.015. CFU= colony-forming units; IU= international units.

Table 2.

Composition per 1 g of PMC Polimeve Solúve®¹.

Components	Concentration	Unit
Bacterial strains Bacillus cereus var. Toyoi	4 × 10^8	CFU/g
Bacillus sutilis	4 × 10^8	CFU/g
Bifidubacteriom bifidum	3.5 × 10^8	CFU/g
Enterococcus faisum	3.5 × 10^8	CFU/g
Lactobacillus acidophilus	3.5 × 10^8	CFU/g
Folic acid (mg)	1.00	mg
Calcium pantothenate (min)	10.00	mg
Niacin (min)	19.00	mg
Methionine	0.21	g
Lysine (min)	0.10	g
Chloride (min)	0.81	mg
Potassium (min)	0.80	mg
Sodium (min)	0.06	mg
Magnesium (min)	0.07	mg
Vitamin B1 (min)	0.01	mg
Vitamin B2 (min)	0.02	mg
Vitamin B6 (min)	0.02	mg
Vitamin B12 (min)	0.09	µg
Vitamin C (min)	0.01	mg
BHA	0.001	mg

¹Composition of the Product commercial Polimeve Solúve® of Imeve Laboratory, Brazil. Polimeve Solúve® is a supplement with probiotics, vitamins, amino acids, and electrolytes. CFU= colony-forming units.

2.4. Animals and supplementation

Neonatal alpacas with an average live weight 7.89 ± 0.92 kg were included, both males and females, regardless of coat color or breed (Huacaya and Suri). With between 1 and 3 births per day in the herds, the 96 experimental units were completed within one month. All neonates were confirmed to have consumed colostrum before being randomly allocated to one of the three groups and monitored for 90 days.

Probiotic products were administered orally at 1, 7, 14, 21, and 30 days of age: 10 g (1 × 10⁷ CFU/g) of Probiolyte® WS and 5 g (1.85*10⁹ CFU/g) of Polimeve Solúve®, according to the assigned treatment. The dose was determined based on the manufacturer’s instructions and previous studies by Hancco (2018); Wang et al. (2022) y Konieczka et al. (2023). The product was dissolved in 15 mL of boiled non-potable water cooled to 38–40 °C and administered orally using a 30 mL dosing syringe equipped with a cannula. Doses were given between 6:00 and 8:00 a.m., while the herds were still in their resting pen. Neonates were either allowed to graze freely with the herd or kept in fenced native pastures with their dams for approximately 10 days before being released to join the herd. The alpacas access to water has been thru local springs and rivers. It is worth noting that during the study, two neonates from the Polimeve group were preyed upon by predators (foxes), reducing the final number in that group to 30 animals.

2.5. Sampling and laboratory analysis

2.5.1. Body weight and growth

At 1, 14, 30, 60, and 90 days of age, five zoometric variables were recorded (Table 3). The variables evaluated withers height (WH), rump height (RH), body length (BL), thoracic girth (TG), and body weight gain (BW), to assess body size. BW was measured using a portable scale, and zoometric measurements were taken using a millimetric measuring tape and a wooden Vernier-type zoometric stick (Ablondi et al., 2023; Espinoza, 2018). Based on the collected data, final body weight (BW; kg), average daily gain (ADG; kg/d), and percentage weight gain (PWG) were calculated (Lee et al., 2024; Wang et al., 2022a).

Table 3.

Description of zoometric variables based on anatomical foundations in alpacas.

Zoometric variables	Anatomical points	References
Live Weight (LW)	Live weight of the animal in kg	(Ablondi et al., 2023)
Withers Height (WH)	Distance in cm from the ground to the top of the withers	(Ablondi et al., 2023)
Rump Height (RH)	Distance in cm from the ground to the top of the rump	(Ablondi et al., 2023)
Body Length (BL)	Distance in cm from the scapulohumeral joint to the tip of the rump	(Ablondi et al., 2023)
Thoracic Girth (TG)	Measurement in cm of the thoracic circumference, taken around the sternum and the seventh thoracic vertebra.	(Ablondi et al., 2023)

2.5.2. Immune response

Blood samples were collected from 7 randomly selected neonates per treatment group at 30 and 90 days of age. Blood was drawn from the jugular vein into 10 mL tubes without additives and 6 mL tubes with EDTA, during the morning before the animals went out to graze. Samples were refrigerated for transport to the laboratory. The additive-free tubes were centrifuged at 3500 × g for 15 min (Wang et al., 2022a), and the serum was aliquoted into 1.5 mL Eppendorf tubes and stored at –20 °C until analysis. IgG concentration was determined using a radial immunodiffusion kit (Triple J Farms, USA) following the manufacturer’s instructions. For leukocyte counts, EDTA blood samples were used. A 0.7 µL aliquot of blood was used to prepare two smears on a slide with the aid of another slide at a 30° angle. After air drying for 5 min, the slides were stained with Wright stain for 1 min, treated with buffer (distilled water) for 10 min, rinsed gently with tap water, and air-dried (Wang et al., 2021). The two stained smears were examined under a microscope (AmScope, China) with immersion oil at 100 × magnification, where a total of 100 leukocyte cells (neutrophils, basophils, eosinophils, lymphocytes, and monocytes) were quantified and calculated in percentage values (Salgado-Schüler, 2017).

2.5.3. Functional state of the intestine

Sample collection followed the guidelines of Páucar et al. (2017). At 90 days of age, following an 8-hour fast, two neonates per treatment group were sedated and euthanized (Parasuraman and Christapher, 2022). It should be noted that social and cultural factors among producers have influenced the limited availability of samples for sacrifice. Four intestinal segments (duodenum, jejunum, ileum, and cecum) were carefully dissected and 4 cm sections from each were collected. Using forceps, the samples were rinsed 5–10 times in saline solution to remove mucosal impurities that could accelerate villi autolysis. Clean samples were preserved in 10% formalin and stored at 4 °C until analysis. Histological sections were prepared using an automatic microtome (YIDI model YD-355AT, China). Ileal villi height, crypt depth, muscle layer thickness, and mucosal health status were assessed under a light microscope (Leica ICC50W, Germany) with 4 × magnification and a digital camera (Leica DM500, Germany) (Fig. 2).

Fig. 2.

Morphometric evaluation of intestinal structure. The intestinal mucosal layer (a), submucosal layer (b), muscular layer (c), serosal layer (d), intestinal villi (e), and intestinal crypts (f) are shown. Villi length was measured only in the ileum, as it was the only section that preserved its histological structure for proper evaluation. H-E staining, 4 × magnification.

2.5.4. Survival rate

The presence of diarrhea was recorded from the beginning of the experiment (first probiotic dose at day 1 of age) until 90 days of age to determine morbidity rates. Mortality due to digestive tract infections was also recorded to determine the mortality rate.

2.6. Statistical análisis

For the analysis of the information, descriptive and inferential statistics were used. Normal distribution and homogeneity of variances were verified. The least squares means (Mlmeans) and standard error (SE) of body weight, body size, immune response, and intestinal function were calculated. The effect of the treatments was analyzed via analysis of variance (ANOVA) with a univariate linear model. Mean comparisons were performed using Tukey’s multiple range test. Data that did not meet normality or homogeneity assumptions were analyzed using the non-parametric Kruskal-Wallis test, and means were compared with the Wilcoxon rank-sum test. Morbidity and mortality rates were determined using frequency tables, and associations with treatments were tested using the Chi-square test. Statistical significance was set at P < 0.05 or P < 0.001. Data were processed using R-statistical software version 4.4.0. The statistical model used was:

$$\mathrm{Y}ijk=\mu +\tau \mathrm{i}+\epsilon ijk$$

Where: Yijk = k observations in treatment i at age j, μ = mean, τi= effect of treatment or type of probiotic i (i = 1, 2, 3), εijk = residual.

3. Results

3.1. Growth performance

PMC supplementation had no significant effect on weight gain (p > 0.05). The initial live weight of neonatal alpaca was 7.90±1.03, 7.69±0.69, and 8.11±1.01 kg for the control, Probiolyte, and Polimeve groups, respectively (Table 4). The Polimeve-treated group tended to increase live weight (LW) and average daily gain (ADG) at 60 and 90 days compared to the other groups; similarly, they showed improvements in percentage weight gain (PWG) and specific growth rate (SGR), although these differences were not statistically significant (p > 0.05). The PMC effect on body size is shown in Table 5 and Fig. 3. A positive response was observed in the Polimeve-supplemented groups, which showed higher body size values. Polimeve-treated groups showed a significantly greater increase in thoracic girth after 15 days of age, as well as withers and rump height at 15 days, compared to the other groups (p < 0.05). However, body length did not differ significantly between groups (p > 0.05).

Table 4.

Effect of PMC supplementation on body weight gain in alpaca neonatal up to 90 days of age.

Variable	Control	Probiolyte	Polimeve	SE	p-value
LW (kg)
1 d	7.90	7.69	8.11	0.09	0.190
15 d	10.39	9.84	10.34	0.14	0.193
30 d	11.84	11.70	11.99	0.17	0.800
60 d	15.89	15.66	16.10	0.24	0.766
90 d	19.15	18.73	19.80	0.29	0.151
ADG (kg/day)
1–15 d	0.17	0.14	0.15	0.01	0.211
16–30 d	0.13	0.13	0.13	0.00	0.801
31–60 d	0.13	0.13	0.14	0.00	0.736
61–90 d	0.10	0.10	0.13	0.00	0.056
PWG (%)	1.38	1.44	1.49	0.03	0.374
SGR	0.12	0.12	0.13	0.00	0.196

LW: Live weight, ADG: Average daily gain, PWG: Percentage of weight gain, SGR: Specific growth rate, SE: Standard error of the mean.

Table 5.

Effect of PMC supplementation on growth variables of alpaca neonatal up to 90 days of age.

Variable	Control	Probiolyte	Polimeve	SE	p-value
TG (cm)
1 d	45.21	44.30	44.53	0.31	0.449
15 d	48.82ab	48.30b	50.00a	0.28	0.010 *
30 d	51.97	51.09	52.38	0.30	0.200
60 d	59.16ab	57.41b	59.54a	0.32	0.014 *
90 d	61.75b	61.55b	63.96a	0.41	0.036 *
WH (cm)
1 d	53.36	53.15	53.20	0.24	0.932
15 d	56.88a	55.85b	57.67a	0.24	0.016 *
30 d	58.64	58.06	59.24	0.26	0.192
60 d	62.91	61.55	63.00	0.32	0.114
90 d	64.81	64.79	66.30	0.31	0.081
RH (cm)
1 d	55.18	55.48	55.10	0.27	0.833
15 d	58.97a	57.88b	59.47a	0.25	0.029 *
30 d	60.82	60.00	61.10	0.22	0.114
60 d	64.72	64.08	64.75	0.27	0.525
90 d	66.81	66.98	68.30	0.30	0.099
BL (cm)
1 d	39.21	38.30	38.53	0.22	0.183
15 d	43.00	42.48	43.47	0.22	0.202
30 d	45.42	45.27	45.83	0.22	0.576
60 d	49.97	50.25	51.14	0.36	0.227
90 d	51.48	51.68	52.56	0.27	0.162

TG: Thoracic girth, WH: Withers height, RH: Rump height, BL: Body length, SE: Standard error of the mean. Superscript letters ^(a,b) in rows indicate significant differences at p ≤ 0.05 (*).

Fig. 3.

Effect of PMC supplementation on body size performance up to 90 days of age. a) Thoracic Girth (TG), b) Withers Height (WH), c) Rump Height (RH), d) Body length (BL).

3.2. Immune response

At 90 days of age, the Polimeve group (9.00%) had a higher proportion of basophils (BA) compared to the other groups, while the Probiolyte (9.00%) and control (9.00%) groups had higher proportions of monocytes (MO) relative to Polimeve (p ≤ 0.05). PMC supplementation had no significant effect on the proportion of neutrophils, eosinophils, or lymphocytes (p > 0.05). At 30 days of age, all three groups had a higher proportion of band neutrophils (BNE) than segmented neutrophils (SNE), and this ratio reversed at 90 days. IgG concentrations at both 30 and 90 days of age showed no significant differences among groups (p > 0.05) (Table 6).

Table 6.

Effect of PMC supplementation on immune response in alpaca neonatal up to 90 days of age.

Variable	Control	Probiolyte	Polimeve	SE	p-value
BNE (%)
30 d	0.16	0.14	0.16	0.01	0.628
90 d	0.09	0.12	0.10	0.01	0.472
SNE (%)
30 d	0.20	0.17	0.14	0.01	0.712
90 d	0.33	0.34	0.32	0.01	0.765
EO (%)
30 d	0.06	0.10	0.08	0.01	0.149
90 d	0.03	0.03	0.05	0.00	0.223
BA (%)
30 d	0.15	0.14	0.16	0.01	0.063
90 d	0.06b	0.06b	0.09a	0.01	0.050*
LI (%)
30 d	0.35	0.37	0.39	0.01	0.600
90 d	0.39	0.36	0.37	0.01	0.659
MO (%)
30 d	0.08	0.08	0.08	0.01	0.459
90 d	0.09a	0.09a	0.06b	0.01	0.023*
IgG (mg/dL)
30 d	883.83	1162.10	689.04	124.68	0.313
90 d	2066.63	1479.78	1609.04	123.56	0.125

BNE: Band neutrophils, SNE: Segmented neutrophils, EO: Eosinophils, BA: Basophils, LI: Lymphocytes, MO: Monocytes, IgG: Immunoglobulin G, SE: Standard error of the mean. Superscript letters ^(a,b) in rows indicate significant differences at p ≤ 0.05 (*).

3.3. Intestinal functional status

Groups supplemented with PMC had shorter ileal villi compared to the control group (Table 7; p < 0.05). PMC supplementation significantly affected crypt depth in the jejunum and cecum (p < 0.05). Histological signs of inflammatory processes were observed across all treatment groups, with the intestinal mucosa of neonates showing structural damage, including a discontinuous epithelial layer and autolytic changes (Fig. 4). Especially in the ileum portion of the neonates showed mild to moderate enteritis to severe acute diffuse catarrhal enteritis associated with Coccidia (Fig. 5, Fig. 6).

Table 7.

Effect of PMC supplementation on intestinal morphometry in alpaca neonatal up to 90 days of age.

Variable	Control	Probiolyte	Polimeve	SE	p-value
VL (µm)
Ileum	367.70a	311.50ab	268.65b	14.34	0.013 *
CD (µm)
Duodenum	211.70	194.55	184.90	5.56	0.136
Jejunum	139.85a	208.15b	163.10a	7.66	<0.001***
Ileum	219.00	196.20	194.45	6.01	0.180
Cecum	253.35b	358.60a	347.45a	11.15	<0.001***
CM (µm)
Duodenum	237.00	145.00	182.00	23.83	0.350
Jejunum	224.50	170.50	287.00	30.38	0.363
Ileum	144.00	187.00	171.00	20.74	0.789
Cecum	151.00	314.50	277.50	34.34	0.069
LM (µm)
Duodenum	100.50	92.50	80.50	4.74	0.252
Jejunum	109.50	110.50	133.00	10.13	0.676
Ileum	107.00	132.50	79.00	16.43	0.519
Cecum	41.00	137.00	80.50	19.11	0.058

VL: villus length, CD: crypt depth, CM: circular muscle, LM: longitudinal muscle, SE: Standard error of the mean. Superscript letters ^(a,b) in rows indicate significant differences at p ≤ 0.001 (***), p ≤ 0.05 (*).

Fig. 4.

Micrograph of the ileum of a young alpaca showing histological signs of acute inflammation. Catarrh and mucosal desquamation (green arrow), immature microgamete (black arrow), and mature schizont (red arrow). H-E stain, 4 × magnification.

Fig. 5.

Micrographs of the ileum in alpaca neonatal showing histological signs of mild, moderate, and severe inflammation according to treatment group. A) Ileum with moderate to severe catarrhal enteritis; B) Ileum with mild to moderate catarrhal enteritis; C) Ileum with moderate to severe catarrhal enteritis; D) Ileum with severe acute catarrhal enteritis.

Fig. 6.

Micrographs of intestinal health status in alpaca neonatal supplemented with PMC. The top panel shows images of alpacas from the control group. The middle panel shows images of alpacas supplemented with the probiotic Probiolyte. The bottom panel shows images of alpacas supplemented with the probiotic Polimeve. H&E staining, 4 × magnification.

3.4. Morbidity and mortality indices

No neonatal mortality was recorded during the experiment, and morbidity did not show a significant association among groups (p > 0.05), although the Polimeve and Probiolyte groups had a proportional increase of diarrhea cases (Table 8). It is worth noting that the diarrheas observed in neonates were physiological in nature, resulting from probiotic use an expected response due to microbial shifts in the gastrointestinal tract. Additionally, this is a pilot study that demonstrates an alternative supplement to improve survival and resilience in neonates.

Table 8.

Effect of PMC supplementation on survival in alpaca neonatal up to 90 days of age.

Variables	Treatments	%	X2	p-value
Morbidity	Control	3.03 (1/32)	1.047	0.592
	Probiolyte	9.09 (3/32)
	Polimeve	6.67 (2/30)
Mortality	Control	-	-	-
	Probiolyte	-
	Polimeve	-

4. Discussion

4.1. Growth performance

In recent years, the use of PMC-based additives have significantly raised expectations among livestock producers' (Cholewińska et al., 2020). It is widely used to promote the development of a protective microflora at the intestinal barrier level, especially in neonates (Wang et al., 2022b). Studies have shown that PMCs can improve milk quality, increase weight gain, and feed efficiency (Adjei-Fremah et al., 2018; Hasunuma et al., 2011; Liu et al., 2021). In the early stages of life, animals go through critical periods for their survival, as their immune systems are not yet fully developed, making them entirely dependent on the maternal immunity they receive through colostrum. These physiological factors, combined with environmental ones, can seriously impair the normal growth of newborns (Liu et al., 2014).

In this study, PMC supplementation had no significant effect on daily weight gain (DWG) or final body weight in neonatal alpacas. No previous research was found on the effect of PMC supplementation on the growth of neonatal alpaca. In other species, such as guinea pigs (Valdizán et al., 2019), lambs (Esfiokhi et al., 2024), and calves (Wang et al., 2022a), no significant effect on weight gain was reported. In contrast, favorable effects have been observed in suckling piglets (Konieczka et al., 2023). Differences between studies may be related to factors such as animal species, physiological and immunological status, age, probiotic concentration and dose, rearing conditions, diet composition, and route of administration (Hoseinifar et al., 2018; Lambo et al., 2021). Esfiokhi et al. (2024) confirm that the viability and efficacy of probiotics are related to the specific strains used, the composition of the gut microbiota, and the host’s diet.

The use of PMC in this study had no significant effect on growth. No comparable studies were found in alpacas; however, Wang et al. (2022a) reported a negative effect on withers and hip height in neonatal calves, whereas Noori et al. (2016) found a positive response in piglets. Probiotics are capable of synthesizing amylase, cellulase, protease, improving digestive function and support growth in newborn calves (Cangiano et al., 2020). The bioavailability of minerals such as calcium, phosphorus, and magnesium contained in the supplement would also be influencing growth (Noori et al., 2016). In other studies, it was demonstrated that probiotics based on Bacillus subtilis and Bacillus amyloliquefaciens can enhance the production of GH/IGF-1 because these bacteria stimulate the gene expression responsible for that hormone, which has a direct relationship with body growth (Du et al., 2018; Salehizadeh et al., 2019). Furthermore, variations in the animals’ physical development are a result of the environmental conditions in which they are raised and the production model adopted (Heinrichs et al., 2007)

4.2. Immune response in neonates

The immune system in neonatal animals is underdeveloped. Studies on the use of PMC as a supplement in ruminants (Ali et al., 2024; Konieczka et al., 2023; Varada et al., 2022; Wang et al., 2022a) and pseudo-ruminants (Hancco, 2018) have shown improved innate immunity and enhanced preparation of adaptive (both humoral and cellular) immune responses (Ali et al., 2024; Varada et al., 2022). Immunity can be subdivided into innate and adaptive components. Innate immunity comprises neutrophils, monocytes, basophils, eosinophils, and natural killer cells (NKCs), and acts as the first line of defense against pathogenic microbial colonization (Rasmussen et al., 2009). Adaptive immunity is represented by T and B lymphocytes (Schenten and Medzhitov, 2011), with B cells being responsible for producing IgG, which function as antigen receptors (Ramos-Medina et al., 2012).

In this study, no significant effects were observed on the mean proportions of neutrophils, eosinophils, and lymphocytes, but there were effects on basophils and monocytes. Hancco (2018) reported that probiotics had no effect on neutrophils, but positively influenced monocytes (20.14%) and negatively affected lymphocytes (21.33%). This positive effect on monocytes would explain the content of the bacterial strains in the supplement, as Hancco (2018) used Lactobacillus bulgaricus and Estreptococos thermophilus compared to this study.

In apparently healthy 90-day-old neonatal alpaca, Velásquez (2023) reported neutrophil proportions ranging from 50.83 to 60.80%, lymphocytes from 38.00 to 43.50%, monocytes from 3.20 to 5.00%, eosinophils from 1.50 to 2.00%, and basophils from 0.17 to 0.33%. These values were higher than the proportion of neutrophils and lower than the proportion of monocytes, eosinophils, and basophils observed in the control group of this study. Sierra et al. (2010) found that Lactobacillus salivarius-based probiotics increased monocyte percentages, although this strain was not included in any of the PMC used in this study. In another study, Gutiérrez-Castro and Corredor-Matus (2017) found no effect of PMC on leukocyte parameters.

The effects of PMC supplementation on leukocyte concentrations in alpacas have not been well studied. Leukocytes are understood to be the first line of defense against pathogenic microorganisms (Rasmussen et al., 2009); however, they are influenced by environmental conditions, age, sex, nutritional status, and the animal’s health status (Foster et al., 2009; Udeh et al., 2021). Lymphocyte levels tend to be higher in neonatal alpacas than in adult alpacas whereas the opposite trend is observed for eosinophils and neutrophils (Hengrave et al., 2005; Wagener et al., 2024). Similar patterns have been reported in calves (Mohri et al., 2007). Likewise, elevated lymphocyte and neutrophil levels during the first month of life may be attributed to increased cortisol concentrations, which peak during late gestation and parturition (Martín-Barrasa et al., 2023).

PMC supplementation had no significant effect on IgG concentration; however, at 30 days of age, the Probiolyte group showed a numerically higher IgG level (1162.099 mg/dL) compared to the other groups. By contrast, at 90 days, the control group exhibited a higher IgG concentration (2066.63 mg/dL), followed by the Polimeve group (1609.04mg/dL). Hancco (2018) reported lower IgG values (646.40 mg/dL and 664.70 mg/dL) using different probiotic doses. The higher IgG concentrations observed in the present study may be attributed to the specific doses used (Wang et al., 2022a), strain concentrations, strain diversity, and the presence of micronutrients and amino acids in the PMC formulations. Nevertheless, Velásquez (2023) demonstrated that amino acid supplementation had no effect on IgG levels in alpaca crias.

Other studies have shown that probiotics containing Enterococcus, Lactobacillus, and Bifidobacterium can activate the production of IgA, IgE, and IgG (Ali et al., 2024; Letnická et al., 2017; Sierra et al., 2010). To activate IgA, probiotics first activate dendritic cells, through interaction with Toll-like receptors, this process differentiate promotes the differentiation of B lymphocytes into IgA+B cells, thereby increase IgA production (Ali et al., 2024; Dogi et al., 2016). Parada et al. (2019) mentioned that probiotics allow the increase in the gene expression of proteins responsible for the synthesis of immune cells.

In alpacas, due to the epitheliochorial type of placentation, immunoglobulins cannot cross the placenta; for this reason, the immunity of newborns depends on the absorption of immunoglobulins and lymphocytes from the mother’s colostrum (Pachari, 2008; Weaver et al., 2000). It has been demonstrated that by day 79, neonatal alpaca begins to independently produce adequate levels of IgG (Quispe, 2019), suggesting that before this age, their immunity depends entirely on maternal immunoglobulin transfer. Though, Maximiliano et al. (2018) found no association between serum IgG concentrations and mortality in calves due to enterotoxemia.

4.3. Functional status of the intestine

Epithelial cells play a crucial role in maintaining the intestinal ecosystem and integrity to preserve intestinal health (Ding et al., 2021). They also function as a selectively permeable barrier with the capacity to absorb nutrients and provide protection against potential pathogens and allergens (Chen et al., 2015; Groschwitz and Hogan, 2009; Prudencio, 2005). Probiotics have been shown to exert a trophic effect on the barrier function of epithelial cells by increasing Peyer’s patches (Raabis et al., 2019; Rhayat et al., 2019; Shini et al., 2020).

In this study, neonates supplemented with PMC showed shorter ileal villi compared to the control group. No similar reports were found in alpaca neonates; however, in guinea pigs (Puente et al., 2019), pigs (Konieczka et al., 2023), and chickens (Šefcová et al., 2023), probiotics have been shown to increase ileal villus height. The ileum is primarily involved in the reabsorption of electrolytes and water (Ross and Pawlina, 2013). Nutrient absorption is more efficient with longer villi due to increased surface area, whereas shorter villi are associated with poor intestinal health (Awad et al., 2009). The efficacy of probiotics on villus length is influenced by factors such as animal species, product dosage, animal age, probiotic strains, and their viability, persistence in the intestinal tract (Serrano, 2015), and external factors such as the prevalence of protozoa (Centro Experimental Canaán, INIA), since Sharma et al. (2015) have confirmed that infection with Eimeria spp. it causes atrophy and fusion of the villi.

In the Probiolyte and Polimeve groups, the crypts of the jejunum and cecum were deeper than in the control group. Similar studies in guinea pigs (Puente et al., 2019) and pigs (Konieczka et al., 2023) reported no effect of probiotics on crypt depth. It is suggested that shallower crypts are more efficient in nutrient absorption, whereas deeper crypts contain mostly immature cells due to active regeneration of the villi, thus reducing absorption efficiency (Wang et al., 2019a; Bogucka et al., 2019). The results could be attributed to environmental conditions (Torres-Rodríguez et al., 2007), since this study revealed the initial stage of Eimeria spp. infection, which would explain the active regeneration of the crypts. It is also important to note that these results may be influenced by the number of animals evaluated, since at the end of the study, only two animals per group underwent histological analysis.

Histological evaluation of the newborns treated with PMC revealed structures consistent with coccidia. The histopathological diagnosis of the ileum revealed acute catarrhal enteritis associated with coccidia, with few lymphocytes and epithelial cell desquamation. These results suggest that these probiotics may be effective only against bacterial pathogens, not protozoal ones (Maria, 2021). Furthermore, the occurrence of coccidiosis in alpaca newborns may be related to the duration of PMC supplementation, because in this study, the newborns received probiotics only on five occasions during the first month of life, which reduces the probiotic’s efficacy, since daily administration of probiotics is recommended in the animal industry (Wang et al., 2022a; Konieczka et al., 2023).

Likewise, there seems to be a high prevalence of Eimeria spp. in breeding areas, which may be compromising intestinal health status, where the protozoan would take advantage of the susceptibility of the neonates. Damage to the intestinal mucosa may impair the mode of action of probiotics, as they primarily interact with epithelial cells, dendritic cells, macrophages, and intraepithelial lymphocytes (Kalita et al., 2021; Raabis et al., 2019).

4.4. Morbidity and mortality rates in neonates

In this study, PMC supplementation had no significant effect on morbidity or mortality rates in alpaca neonates. A higher morbidity rate was observed in PMC-treated groups (9.09 to 9.38%) compared to the control group (3.03%). The diarrhea observed in the treated neonates may be due significant changes in the gastrointestinal microbiota resulting from supplementation, which may involve anaerobic shifts and an increase in lactic acid bacteria, leading to dysbiosis in the intestinal tract (Gomez et al., 2022). Llamas and alpacas between 3 and 7 days of age are affected by diseases such as coccidiosis, clostridiosis, and colibacillosis in approximately 23% of cases, which is often associated with septicemia and ultimately leads to high mortality among newborns (Foster et al., 2008; Rojas et al., 2016).

No mortality in neonates was reported in this study, while Hancco (2018), working in Puno, reported a mortality rate of 6.67% in neonates supplemented with probiotics. High mortality rates due to enterotoxemia (Clostridium perfringens) have been reported in alpaca neonates, particularly between 8 and 35 days of age (Wiedner, 2021). In this region, Escherichia coli has been identified as a primary cause of neonatal mortality (Centro Experimental Canaán, INIA). It is crucial that neonates receive colostrum within the first hours after birth and that it is effectively absorbed in the intestinal barrier. The absence of maternal antibodies can lead to death from colibacillosis (E. coli) by 3 or 4 days of age (Tsur et al., 1996).

As final considerations, the implementation of probiotics as a supplement in neonatal alpacas with the aim of improving growth and gastrointestinal health in alpaca neonates is an emerging strategy that warrants greater scientific attention, particularly given the promising results reported in other ruminants such as buffaloes (Varada et al., 2022), cattle (Wang et al., 2022a), and goats (Taboada et al., 2022).

More research is needed to better understand the relationship between probiotic bacterial strains and the functional integrity of the intestinal mucosa in alpacas. Several reports indicate that probiotics release various metabolites (bacteriocins, organic acids, amines, etc.), with bacteriocins being the main metabolite because they act at the level of the bacterial cytoplasmic membrane and alter the driving force of the bacteria (Kumar et al., 2013; Umu et al., 2017). Probiotics also act as a competitive exclusion factor, as they compete for receptor sites in the intestinal tract, leaving no space for pathogenic bacteria (Bermudez-Brito et al., 2012).

5. Conclusion

The use of probiotics in alpaca neonates could serve as an alternative supplementation and therapeutic strategy against digestive disorders. The administration of Polimeve can improve neonatal vigor, promoting faster weight gain and body growth. At 90 days of age, Polimeve was found to improve basophil concentration. No significant effect was observed on the IgG concentration; however, an increase in the average IgG levels was noted during the first 30 days with the use of Probiolyte. The supplemented animals showed shorter ileal villi compared to the control group, as well as greater crypt depth in the jejunum and cecum. Likewise, epithelial cell loss from the villi was observed in all three groups, indicating structural damage to the intestinal mucosa, likely associated with enteritis caused by Coccidia. This gastrointestinal issue in neonates may be related to the grazing area, which appears to be contaminated with protozoa of the Coccidia genus. The use of PMC showed no effect on neonatal survival. Among the limitations are the limited availability of samples and the lack of specialized laboratories nearby to process samples. It is worth noting that further studies are required on the use of probiotics in alpaca neonates, including the identification of potential probiotic microorganisms and the relationship between digestive physiology and the intestinal microbiota.

Funding

This research was funded by the project “Mejoramiento de los Servicios de Investigación y Transferencia de Tecnología en Ganadería Altoandina en 33 Distritos de los Departamentos de Apurímac, Arequipa, Ayacucho, Cusco, Huancavelica, Junín, Moquegua, Pasco, Puno y Tacna”, CUI N° 2491159, of the Instituto Nacional de Innovación Agraria.

CRediT authorship contribution statement

Walter Palomino-Guerrera: Conceptualization, Methodology, Software, Data curation, Writing – original draft, Visualization, Writing – review & editing. Wenfil Daisi Mariño Huaraca: Conceptualization, Methodology. Mijail Contreras Huamani: Conceptualization, Visualization, Supervision, Validation, Writing – review & editing. Pedro Coila: Validation, Writing – review & editing. Natalia Verónica Taboada: Visualization, Validation, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The raw data supporting the conclusions of this article will be made available by the authors on request.