Protective effects of microencapsulated Lactoferrin in enterotoxigenic Escherichia coli- challenged piglets

Protective effects of microencapsulated Lactoferrin in enterotoxigenic Escherichia coli- challenged piglets | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective effects of microencapsulated Lactoferrin in enterotoxigenic Escherichia coli- challenged piglets Agustina Cots, Nadya Mura, María Carolina Flores Bracamonte, Diego Fernando Acevedo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6889295/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Enterotoxigenic Escherichia coli (ETEC) is a significant cause of diarrhea in neonatal and weanling pigs, leading to considerable morbidity, mortality, and economic losses in swine production. The present study evaluated the protective effects of microencapsulated lactoferrin (Lf) in ETEC-challenged piglets, focusing on growth performance, intestinal health, and oxidative stress parameters, thereby contributing to sustainable practices in animal husbandry. The microencapsulation process, utilizing bio-based polymers, enhances the bioavailability and stability of Lf, which is for its application in veterinary medicine. An in vivo assay was conducted with 40 piglets divided into four groups: CON − (not challenged, not treated), CON + (challenged, no treated), T1 (challenged treated with free Lf) and T2 (challenged, treated with microencapsulated Lf). Key metrics, including growth rates, diarrhea incidence, and blood parameters, were recorded over ten days. Microscopic evaluations of intestinal morphology and oxidative stress markers in liver tissues were also conducted. The results demonstrated that the T2 group exhibited a significant reduction in diarrhea incidence, enhanced survival rates (100% by day 10), and improved average daily weight gain compared to the other groups, aligning with the health and well-being goals of sustainable development. Blood analyses revealed that the CON + group showed signs of anemia, whereas those receiving microencapsulated Lf had normalized levels of red blood cells, hemoglobin, and hematocrit. Additionally, liver MDA levels were significantly lower, while SOD levels were significantly increased in the Lf treatment groups, indicating reduced oxidative stress. This study confirms their microencapsulation of Lf using bio-based polymers can enhance its bioactivity, contributing to better intestinal health and overall performance in piglets challenged with ETEC. This approach not only mitigates the negative impacts of porcine neonatal diarrhea on swine production but also supports sustainable agricultural practices by promoting animal health and reducing dependency on antibiotics. Lactoferrin infectious diseases piglets bio-based polymers neonatal diarrhea Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Neonatal porcine diarrhea is a significant health challenge in swine production, causing substantial economic losses due to rapid dehydration, growth delays and mortality in piglets (Fairbrother & Nadeau, 2019 ). This disease is multifactorial and complex to diagnose; however, the most implicated agent in this pathology is the enterotoxigenic Escherichia coli (ETEC) (Lv et al., 2018 ; Wu et al., 2018 ). Piglets infected with ETEC can experience intense, secretory diarrhea within 1 to 2 hours after birth. They show symptoms such as abundant, non-bloody, watery diarrhea, along with nausea, abdominal cramps, and shivering. As a result, the piglets quickly become dehydrated, undernourished, and weak (Jacobson et al., 2022). Additionally, this pathology can also alter porcine blood parameters (Ider et al., 2023; Chethan et al., 2017 ). The metabolic response to the infection typically generates reactive oxygen species (ROS), which play a critical role in the pathophysiology of the disease and contribute to cellular damage (Zhang et al., 2023 ; Zhou et al., 2023 ; Sun et al., 2021 ). To counteract the harmful effects of ROS, various antioxidant mechanism function synergistically to prevent or mitigate damage to macromolecules (Dalle-Done et al., 2006; Andersen et al., 1997 ). Evidence suggests that a decline in antioxidant activity, coupled with increased ROS production, contributes to intestinal dysfunction and oxidative stress (Zhu et al., 2012 ). Moreover, numerous studies have demonstrated that intestinal dysfunction plays a pivotal role in growth impairment (Cai et al., 2023 ; Kikusato et al., 2021; Upadhaya et al., 2021; Campbell et al., 2013 ). Over the past two decades, extensive research has focused on developing alternatives to antibiotics to maintain the health and performance of pigs, particularly on the light of the public health issue posed by bacterial resistance to antibiotics and its potential transfer to humans (Mounsey et al., 2024 ; Rhouma et al., 2016 ). In this context, the use of a safe agents to promote growth and bolster immunity, while concurrently preventing diseases that affect piglets at early stages, is of paramount importance and urgency. Lactoferrin (Lf), an 80 kDa non-heme iron-binding glycoprotein, is recognized as a critical host defense molecule (González-Chávez et al., 2009 ). When incorporated into early nutritional intervention, it has the potential to enhance digestion and nutrient absorption, thereby optimizing growth. Additionally, Lf influences numerous vital physiological processes, modulates intestinal function, and alleviates neonatal and post-weaning diarrhea in piglets (Hao et al., 2019 ). Recent studies have demonstrated that Lf possesses antimicrobial activity (Yan et al., 2021 ; Duarte et al., 2022 ) and antioxidant capacity both in vivo and in vitro (Sarkar et al., 2023 ; Narmuratova et al., 2022 ; Oussaief et al., 2022 ; Farid et al., 2021 ). Furthermore, Lf is regarded as safe, with no known toxic or adverse side effects, and it does not contribute to drug resistance (Hao et al., 2019 ). However, due tits rapid degradation in gastrointestinal conditions, achieving a successful therapeutic effect often requires a substantial amount or frequent dosing (Abad et al., 2021 ). In recent studies conducted by our research group, we demonstrated that the encapsulating Lf in chitosan-alginate microcapsules preserves its structural integrity, thereby enhancing its antibacterial activity against an ETEC strain (Cots et al., 2025 ). These materials are biodegradable, non-toxic, and have been extensively studied for agricultural and food applications. Their use in microencapsulation not only enhances the stability and bioavailability of Lf but also supports environmental objectives by reducing persistent waste and reliance on synthetic polymers that may pose ecological risks (Lewicka et al., 2024 ; Nahar et al., 2025 ; Nanda et al., 2025 ). The objective of this study is to evaluate the protective effect of the oral administration of microencapsulated lactoferrin in piglets challenged with E. coli, using biodegradable polymer-based microencapsulation techniques. MATERIALS AND METHODS Lf encapsulation To promote environmentally responsible practices, Lf was encapsulated utilizing biodegradable and renewable polymers. Lf loaded Chi/Alg microparticles were produced following Cots et al ( 2025 ) through green methodologies. Initially, Alg microparticles were generated via ionic gelation using calcium chloride in aqueous, mild conditions, avoiding harmful solvents or cross-linking agents. This process resulted in biodegradable microspheres. Subsequently, these Alg microparticles were coated with Chi through an aqueous-phase electrostatic interaction. Bacterial isolation ETEC strain was obtained from a from a neonatal pig with diarrhea in an intensive pig production farm located in Córdoba province, Argentina. Initially, the strain was cultured on MacConkey agar (Britania®, Argentina) at 37 ◦C for 48 h. Then, the identification as ETEC strain was confirmed using molecular techniques. The PCR analysis revealed that the strain was F4 + (K88 + ) and capable of producing of the heat-stable toxin (STb), and heat-labile toxin (LT) (Bessone et al., 2017 ). ETEC strain management ETEC strain, maintained at -20°C in glycerol, was thawed and cultured in trypticase soy broth (TSB) for 18 h at 37°C. Then, streaking was performed on trypticase soy agar (TSA), and it was incubated at 37°C for 24 h. Subsequently, 1 colony was resuspended in 5 mL of TSB, incubating for 18 h at 37°C to obtain the stock inoculum and carry out a bacterial count using the micro drop technique. Serial dilutions (1:10) of the stock inoculum were made up to the 1 × 10 − 12 dilution. Dilutions were plated on TSA plates in triplicate and incubated for 18 h at 37°C. After the incubation time, the CFU/mL count of the stock inoculum was carried out using the following formula: CFU/mL = average number of colonies× 100 × dilution factor Subsequently, a bacterial suspension was prepared with a multiplicity of infection (MOI) of 1:1 bacterium per cell in the media without antibiotics. Animals and housing The experiment was conducted at the INTA Marcos Juárez Agricultural Experimental Station. Two multiparous sows with similar numbers of litters (2–3 births), that had not received the E. coli vaccine and were in good health, were chosen for this study. The sows were moved to the farrowing room on day 108 of gestation, and after giving birth, each sow and her piglets were housed in farrowing crates (2.2 x 0.60 m), with a plastic slatted floor and infrared lamps for the piglets. At the time of birth, the litter size was homogenized to sixteen piglets with a birth weight of 1.51 ± 0.31 kg, and they were allowed to nurse without any handling on the first day of life. Experimental design A total of 16 newborn piglets of the Genetiporc x Choice cross were randomly assigned to 4 groups: CON + (positive control, n = 3), CON − (negative control, n = 3), T1 (Free Lf, n = 5), and T2 (Lf loaded Chi/Alg microparticles, n = 5). The adaptation period lasted for 2 days after bird and the experiment period lasted for 10 days. At the beginning of the experiment, the animals were identified with a plastic ear tag on the right ear. On the third day of life, each piglet received an intramuscular injection of 200 mg of dextran iron (Cali-Dex 200, Calier®) and a dose of 1.5% diclazuril orally for coccidia control (Vetribac Doser, Vetanco®). During the experimental period (10 days), the only source of food for the piglets was maternal milk. The piglets in group T1 were orally administered with a dose of 5 mL of Lf solution, dissolved in cow's milk, divided into two daily doses (10:00 and 17:00) from day 1 to day 7 of the experiment (0.5 g/kg of body weight/day) as described by Hu et al. ( 2019 ). On the other hand, the piglets in group T2 were fed the same dose of Lf loaded in Chi/Alg microparticles, delivered in cow's milk. Finally, the animals in the CON groups (positive and negative) were given the same dose of cow's milk. All piglets (except the CON − group) were orally challenged on day 6 of the experiment with a dose of 1 mL (1x10 8 UFC/mL per pig) of porcine ETEC (Fig. 1 ). Animal clinical evaluation During the 10-day period, the clinical status of all animals was examined considering parameters such as sensorium status, hydration status, presence/absence of diarrhea, and rectal temperature. Lethargic behavior was defined as slow reactions, unstable gait, and resistance to movement (Rossi et al., 2012 ). Piglets were considered dehydrated when the skin folded on the neck took more than 2–4 sec to return to its normal position (Huang et al., 2018 ). The presence or absence of diarrhea was evaluated through daily rectal swabs and direct observation of the hygiene of the perineal area. Rectal temperature was recorded immediately prior to the challenge and the following day in the morning, before any handling of the animal (Rossi et al., 2012 ). Health and well-being assessment Growth performance The live weight of the animals was recorded individually on days 0 (birth weight, BW), 2, 5, 8, and 10, to calculate average daily gain (ADG). The diarrhea score (DS) was assessed each morning at 10:00 am. Diarrhea score and incidence A scoring scale from 0 to 3 was established based on Rossi et al ( 2012 ), where 0 was considered normal (firm stools), 1 was semi-solid (soft, formed stools), 2 was semi-liquid (loose stools, generally yellowish), and 3 was liquid (watery and projectile stools). A fecal consistency score of ≤ 1 (0, 1) was considered normal, while a fecal score > 1 (2, 3) was indicative of diarrhea. The incidence of diarrhea (%) was calculated as the percentage of affected piglets during the experimental period divided by the total number of piglets in each group (Huang et al., 2018 ). Survival rate The survival rate was calculated as: Number of live piglets/Total number of piglets in that group at the end of the experiment * 100. Hematological analyses Prior to euthanasia, blood samples were collected via venipuncture from the cava vein, both with and without anticoagulant (EDTA), and were stored refrigerated at 4°C for hematological analyses. The samples with and without EDTA were centrifuged at 1500 rpm for 15 min to extract the plasma. The samples without anticoagulants were also centrifuged immediately after extraction at 3200 rpm for 10 min for serum separation, which was stored at -20°C until analysis. From whole blood, the following determinations were made red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, plasma proteins, fibrinogen, total white blood cell count (WBC), and leukocyte differential count. Finally, in the serum, total protein, albumin (Alb), globulins (G), and the Alb:G ratio were evaluated. Euthanasia of Animals and Post-Mortem Evaluation On day 10 of the experiment, all animals (n = 16) were euthanized. This was carried out through exsanguination in accordance with the American Veterinary Medical Association Euthanasia Guidelines (Leary et al. 2020 ). During the necropsy, macroscopic damage was assessed, particularly in the digestive system, and data were collected, including the coloration of the digestive mucosa, appearance and consistency of the intestinal contents, and pH value of the contents. Histological analysis For sampling, the small intestine was divided into two segments: duodenum (5–8 cm from the pylorus) and jejunum-ileum: jejunum (middle portion) and ileum (distal portion, approximately 5 cm in length proximal to the ileocecal junction). Samples were fixed in 4% paraformaldehyde, dehydrated in ethanol, then embedded in paraffin and sectioned into 5 µm thick slices, mounted on glass slides. Subsequently, the samples were deparaffinized, rehydrated, and stained with hematoxylin-eosin (Du et al., 2019). For microscopic evaluation, a Carl Zeiss trinocular optical microscope (AxioStar PLUS) coupled with a digital camera (Moticam 3.0) was used. The length of at least 10 randomly oriented well-aligned villi (Woliński et al., 2020 ) and the depth of the crypts were measured. Images were captured using Motic Image Plus 3.0 software and parameters were measured using Image J software. Study of Oxidative Parameters in Liver Tissue Determination of MDA concentration Lipid peroxidation levels were assessed using the thiobarbituric acid reactive substances (TBARS) metho, which quantifies MDA concentrations in liver tissue. This measurement was based on the protocol established by Marcincák et al (2003). Briefly, a 1 mL sample of 30% liver homogenate was mixed with butylated hydroxytoluene (5 mM) and 15% (v/v) trichloroacetic acid, followed by incubation at 90°C for 30 min. After allowing the mixture to cool, it was centrifuged to obtain the supernatant. Subsequently, 1 mL of the supernatant was extracted and reacted with hydrochloric acid (0.25 N) and thiobarbituric acid 0.375% (v/v), followed by another incubation. The colored layer was measured at 535 nm, using 1,1,3,3-tetramethoxypropane (Sigma) as a standard. MDA concentration was calculated as nmol/g of tissue, according to the formula: log y (DO) = log A + log B x (MDA concentration) x = log⁻¹ (log y – log A) / B Where A corresponds to the colorimetric readings and B to known concentrations of MDA. Determination of SOD activity SOD activity was determined by its ability to inhibit the auto-oxidation of epinephrine at alkaline pH. Liver tissue was homogenized in a buffer, and an aliquot was taken to measure protein content using the Bradford method, using bovine serum albumin as a standard. The remaining homogenate was centrifuged. Next, 50 µL of epinephrine and 2.85 mL of glycine buffer (50 mM, pH 10–11) were added, and the appearance of epinechrome was measured at 480 nm. The slope of the graph of epinephrine oxidation (a) and the slope of the graph of epinephrine oxidation in the presence of the sample (b) were obtained and subsequently, log b/a was plotted as a function of µL of sample. SOD activity was calculated as U SOD/mg of protein = 1000/µL (sample corresponding to 50% inhibition). One unit of SOD corresponds to the µL of sample that inhibits epinechrome formation by 50%. Statistical Analysis All experimental data were analyzed using Infostat software version 2020. Data were expressed as mean ± standard deviation (SD). Statistical differences were considered significant when the p-value was less than 0.05. RESULTS Health and well-being indicators Growth performance Daily weight gain (DWG) is a key parameter for assessing growth performance in animals. In this study, all piglets started at the same age (1 day old). The birth weights (BW) of each piglet were recorded, and then on days 2, 5, 8, and 10 of the experiment to determine DWG. As shown in Table 1 , statistically significant differences were found between the CON + and T1 groups compared to the T2 group on days 2, 5, 8, and 10. Table 1 Daily weight gain in piglets challenged with ETEC CON- CON + T1 T2 p-value Day o (BW) 1.53 ± 0.11 1.50 ± 0.05 1.51 ± 0.13 1.52 ± 0.09 - Day 2 0.26 ± 0.10 ab 0.08 ± 0.03 a 0.18 ± 0.13 a 0.64 ± 0.11 b 0.0078 Day 5 0.79 ± 0.20 ab 0.22 ± 0.03 a 0.42 ± 0.39 a 0.96 ± 0.11 b 0.0212 Day 8 1.34 ± 0.06 ab 0.28 ± 0.25 a 0.53 ± 0.73 a 1.50 ± 0.11 b 0.0108 Day 10 1.56 ± 0.12 ab 0.45 ± 0.40 a 0.63 ± 0.86 a 2.02 ± 0.13 b 0.0096 The results are expressed as mean ± SD. Different letters indicate a significant difference (p < 0.05). Infostat, 2020. BW: birth weight. Diarrhea score and incidence Regarding the diarrhea score and incidence (%), piglets in the CON - group did not show any episodes compatible with intestinal disease during the evaluated period (Fig. 2 A). In the case of the CON + group, two piglets exhibited diarrhea (scores 2 and 3, incidence: 66.67% on day 1 post-challenge), both with dehydration levels of 6% and lethargy (Fig. 2 B). In the T1 group, two animals also presented diarrhea episodes (scores 2 and 3, incidence: 40%), showing signs of severe dehydration (> 6%), lethargy, and sensory depression (Fig. 2 C). Finally, in the T2 group, only one piglet was observed with mild and transient diarrhea lasting one day (score 2, incidence: 10% on day 0) (Fig. 2 D). According to these data, T1 reduces the incidence of diarrhea compared to the positive control group (CON +). However, the values of the evaluated parameters clearly improved when using Lf administered in Chi-Alg microcapsules. Survival rate The survival rates (SR%) of the days following the challenge in piglets from the four experimental groups are shown in Fig. 3 . The results indicated that the piglets in the T2 group recovered more quickly after the ETEC infection, with 0% mortality, due to a lower incidence of diarrhea and lower diarrhea scores, similar to the CON - group, where none of the piglets’ exhibited symptoms of diarrhea. Both groups had a 100% SR by day 10 of the trial (day 4 post-inoculation). In contrast, the CON + and T1 groups showed lower SR%, at 66.67% (2/3* 100) and 40% ( 2/5 * 100) respectively, as one animal from the CON + group and three animals from the T1 group died before day 10. Hematological parameters The results of the hematological profile of the piglets evaluated on day 10 are shown in Table 2 . Table 2 Hematological parameters Complete blood count Parameter CON - CON + T1 T2 p-value GR (mill/µl) 4.28 ± 0.19 b 2.61 ± 0.55 a 3.59 ± 0.69 ab 3.76 ± 0.37 ab 0.0100 Hb (g/dl) 7.80 ± 0.20 b 3.60 ± 0.14 a 6.85 ± 0.49 ab 7.40 ± 0.50 ab 0.0355 Hto (%) 24.4 ± 1.1 b 16.3 ± 0.4 a 22.3 ± 0.4 ab 22.6 ± 1.6 ab 0.0340 VCM (fl) 56.0 ± 3.0 a 70.0 ± 2.8 b 72.5 ± 3.5 b 62.0 ± 1.6 ab 0.0005 HCM (pg) 18 ± 3 19 ± 1 16 ± 4 19 ± 2 0.5925 CHCM (%) 32 ± 1 28 ± 2 21 ± 1 31 ± 2 0.0523 Platelets (mil/µL) 675.53 ± 38.8 903 ± 118.8 930 ± 84.9 732.2 ± 176.5 0.1577 Plasma prot. (g/dL) 5.30 ± 0.20 b 5.40 ± 0.28 b 4.90 ± 0.28 ab 4.80 ± 0.16ª 0.0260 Fibrinogen (mg/dL) 100 ± 50ª 675 ± 35 b 635 ± 21 b 525 ± 86 ab 0.0228 Leukogram Leukocytes (n°/µL) 11600 ± 0.10 9150 ± 0.64 12150 ± 1.77 11350 ± 2.83 0.4802 Segmented Neutrophils 5337 ± 432 3555 ± 140 5764 ± 3218 5659 ± 2223 0.6251 Lymphocytes 5724 ± 723 5071 ± 159 5873 ± 3064 4848 ± 817 0.7598 Monocytes 579.3 ± 230.5 91.5 ± 6.4 565.5 ± 337.3 652.3 ± 683.5 0.2048 Eosinophils 193.3 ± 67 - 297.5 ± 41.7 190 ± 182 0.1367 Basophils - - - - - Results are expressed as mean ± SD. Different letters indicate significant differences (p < 0.05). Infostat, 2020. CON -: Negative control (n = 3). CON +: Positive control (n = 2, 1 dead). T1: Free Lf, n = 2 (3 dead). T2: Encapsulated Lf, n = 4 (1 coagulated). Means with a common letter are not significantly different (p > 0.05). Abbreviations: GR: Red blood cells, Hb: Hemoglobin, Hto: Hematocrit, VCM: Mean corpuscular volume, HCM: Mean corpuscular hemoglobin, CHCM: Mean corpuscular hemoglobin concentration. Significant statistical differences were found in red blood cell parameters (GR, Hb, and Hto) between the CON + and CON − groups (p < 0.05). The CON + group showed the lowest values for GR, Hb, and Hto. No significant differences were observed in leukogram. Additionally, there was a significant increase in the MCV in the T1 and CON + groups compared to CON − . Fibrinogen levels were found to be altered, with statistical analysis showing a difference between the CON + and T1 groups compared to the CON − group. The serum protein levels measured on day 10 of the experiment are summarized in Table 3 . Table 3 Serum protein levels Serum Parameters CON - CON + T1 T2 p-value TP 5.16 ± 0.10 b 4.68 ± 0.35 ab 4.54 ± 0.35ª 4.85 ± 0.13 ab 0.0333 Alb 2.97 ± 0.02 ab 2.73 ± 0.36 ab 3.11 ± 0.14 b 2.61 ± 0.30ª 0.0318 G 2.22 ± 0.02 ab 1.83 ± 0.53ª 1.43 ± 0.03ª 2.31 ± 0.02 b 0.0007 Alb/G 1.34 ± 0.02 ab 1.53 ± 0.25 ab 2.17 ± 0.06 b 1.13 ± 0.13ª 0.0010 Results expressed as mean ± SD. Different letters represent significant differences (p < 0.05). Infostat, 2020. TP: Total Proteins (g/dL). A: Albumins (g/dL). G: Globulins (g/dL). Alb/G: Albumin/Globulins Ratio. CON − : Negative control (n = 3), CON + : Positive control (n = 2, 1 dead), T1: Treatment 1 (n = 2, 3 dead), T2: Treatment 2 (n = 4, 1 coagulated). Although the total protein values (g/dL) fall within the reference range, the results show a statistically significant increase for the CON − group compared to the T1 group. Additionally, the T2 group exhibited lower levels of Alb, higher G levels, and consequently a decreased in Alb/G ratio compared to the T1 group (p < 0.05). Postmortem evaluation The macroscopic evaluation of the digestive system in piglets revealed stomach and intestinal dilation in animals from the CON + group and in 3 of the 5 animals from the T1 group. Additionally, marked congestion throughout the intestinal tract was observed in the T1 group, and some animals had watery fecal matter inside (data not shown). In contrast, the T2 group exhibited milder dilation, particularly in the large intestine (Fig. 4 ). These findings align with Luppi et al. (2023), whose study focused on the diagnostic approach to enteric disorders in pigs caused by common pathogens, including E. coli . Histopathological parameters of the intestine In this study, the morphology of two intestinal sections (duodenum and jejunum-ileum) of piglets challenged with ETEC was evaluated at the end of the experiment (day 10). As seen in the histological images, animals in the CON + and T1 groups exhibited villi with varying degrees of atrophy, areas of marked hyperemia, and shedding epithelial tissue in both the duodenum and jejunum-ileum (Figs. 5 and 6 ). Table 4 presents the results obtained from the measurements of average villus height (VH), crypt depth (CD), and the ratio between the two (VH/CD). Piglets of CON + group displayed altered intestinal architecture, characterized by a reduction in average of VH and the VH/CD ratio in both duodenum, and jejunum-ileum sections, along with an increase in CD. These changes were statistically significant when compared to the values obtained in the CON − and T2 groups (p < 0.05). The values found in the piglets of the T1 group showed no significant differences compared to the CON + group. Table 4 Effect of oral administration of encapsulated lactoferrin on histological parameters of intestine in piglets challenged with ETEC Intestine section Parameter CON - CON + T1 T2 p-value Duodenum VH (µm) 755 ± 74ª 314 ± 15 b 305 ± 27 b 647 ± 60ª < 0.01 CD (µm) 237 ± 19 ab 277 ± 21 c 276 ± 74 bc 205 ± 22ª < 0.01 VH/CD 3.2 ± 0.4ª 1.1 ± 0.1 b 1.2 ± 0.2 b 3.2 ± 0.5ª < 0.01 Jejunum-Ileum VH (µm) 598 ± 82ª 153 ± 24 b 216 ± 32 b 475 ± 49ª < 0.01 CD (µm) 143 ± 30ª 95 ± 12 b 97 ± 14 b 161 ± 23 a < 0.01 VH/CD 4.4 ± 1.2 c 1.6 ± 0.3ª 2.3 ± 0.5 ab 3.0 ± 0.5 bc < 0.01 Results expressed as mean ± SD. Different superscript letters indicate significant differences (p < 0.01). VH: Villus height. CD: Crypt depth Oxidative parameters in hepatic tissue MDA concentration As shown in Fig. 7 , statistical analyses of MDA values reveal significant differences between the CON – group (147.59 ± 7.13) and T2 group (152.19 ± 14.82) compared to the T1 group (206.12 ± 15.21) and CON + group (212.87 ± 9.34) (p < 0.05). SOD concentration The levels of SOD were significantly higher (p < 0.05) in the T2 group (18.91 ± 2.35) and the CON − group (19.78 ± 2.50) compared to the CON + group (12.23 ± 1.12) and the T1 group (11.78 ± 1.13) (Fig. 8 ). DISCUSSION Lf exhibits a broad spectrum of biological activity, making it a valuable alternative for biomedical applications. In a previous study, we demonstrated that the encapsulation of Lf in Chi/Alg microcapsules is a strategy that allows maintaining its structural and functional integrity in vitro (Cots et al., 2025 ). In the present study, we aim to evaluate the protective effect of the oral administration of microencapsulated lactoferrin in piglets challenged with ETEC, using biodegradable polymer-based microencapsulation techniques. The study of health and well-being indicators revealed a statistically significant increase on daily weight gain in T2 piglets on days 2, 5, 8, and 10 comparing to CON + and T1 groups. Similar results were found by Ma et al. ( 2023 ) in a study that evaluated the effects of dietary Lf supplementation on growth performance, immune function, and intestinal health in weaned piglets. In their study, four groups were used with different diets: negative control (basic diet); positive control (basic diet + antibiotics); lactoferrin A (bLF-A) (basic diet + 1 g/kg of Lf); and lactoferrin B (bLF-B) (basic diet + 3 g/kg of Lf). The results demonstrate that dietary supplementation of Lf can improve growth performance and reduce diarrhea, but in a dose-dependent manner. Additionally, it enhanced immunity and the intestinal architecture in the mucosa of the small intestine. Regarding diarrhea score and incidence, the results revealed that T1 reduces the incidence of diarrhea compared to the CON + group. However, the values of the evaluated parameters clearly improved when the Lf was administered encapsulated in Chi/Alg microcapsules. Hu et al. ( 2019 ) worked with two groups of animals: a Control group consisting of 30 nursing animals and another group of 30 animals that received an oral dose of 0.5 g/kg/day of a Lf solution. They found that Lf was able to reduce the incidence of diarrhea, increase villus height in the jejunum, and improve growth performance. Therefore, early-life intervention with Lf is beneficial for intestinal health. In a study by Comstock et al. ( 2014 ), the effect of oral supplementation with free bovine Lf on the survival of piglets deprived of colostrum was evaluated, using increasing doses: Control group: 130 mg, Lf1: 367 mg, and Lf2: 1300 mg/kg/day. The authors found that none of the doses significantly increased survival, though the highest dose showed a trend toward improved survival compared to the control diet. The survival rates (SR%) indicated that the piglets in the T2 group recovered more quickly after the ETEC infection, with 0% mortality, due to a lower incidence of diarrhea and lower diarrhea scores, similar to the CON − group, where none of the piglets’ exhibited symptoms of diarrhea. Both groups had a 100% SR by day 10 of the trial. Considering these findings, it could be inferred that the T1 group, treated with free Lf, likely did not receive a sufficient dose to enhance piglet survival. Alternatively, Lf may require protection to reach the small intestine intact, preserving its bioactive properties to exert its beneficial effects. While there are currently few or no reports on in vivo studies conducted with encapsulated Lf in Chi/Alg microcapsules in piglets, many studies demonstrate that encapsulation helps protect this protein during its passage through the stomach (acidic pH and digestive enzymes) and allows it to reach its site of action in its bioactive form (Bokkhim et al. 2016 ). On the contrary, a study of Resouzakov et al (2018) reported that piglets fed bLf mounted a more effective immune response and exhibited lower bacterial abundance providing biological underpinnings to the clinical benefits of bLf during S. aureus infection. There were significant differences in red blood cell parameters between the CON + and CON − groups (p < 0.05). The CON + group showed the lowest values for GR, Hb and Hto, while no significant differences were noted in the leukogram. Additionally, there was a significant increase in the MCV in the T1 and CON + groups compared to CON − . According to Chen et al. ( 2019 ), these indicators suggest that the CON + group animals experienced anemia, characterized by low GR, Hb, and Hto levels. The elevated MCV may indicate a regenerative type of anemia, as described by Newcomer et al. ( 2020 ). The wide variability in Hb and Hto levels is influenced by factors such as diet, management systems, and genetics (Cooper et al., 2014 ). Although the values in the T1 group were not indicative of clinical anemia, the Hb levels suggest the presence of subclinical anemia (Fjelkner et al., 2024 ). Piglets are born with limited iron, since the only food available in the first two weeks is maternal milk, which is low in iron, and piglets in intensive farming systems lack access to soil (often raised on concrete or plastic floors), the high incidence of anemia in swine operations is understandable. Iron deficiency is one of the most common nutritional deficiencies in mammals, with iron-deficiency anemia being the most severe consequence (Chaud et al., 2019; Parsons & Tummaruk, 2025 ). Additionally, in the first 12 h of life, there is a significant increase in plasma volume without a corresponding increase in red blood cell count, leading to physiological anemia due to rapid blood volume expansion (Quiles & Hevía, 2004). The altered fibrinogen levels were observed, with statistical analysis indicating a difference between the CON + and T1 groups compared to the CON − group. Increased fibrinogen levels in the blood are a clear indicator of acute inflammation. Kang et al. (2020) investigated acute phase proteins (APP), including fibrinogen, in a study on acid-base and electrolyte imbalances in calves with enteropathogenic diarrhea. They found no statistically significant differences in APP levels between diarrheic and healthy calves suggesting that changes in APP levels do not always correlate with the severity of the disease. According to our results and considering that one of the main functions of Lf is to enhance iron absorption (Baker & Baker, 2012 ), the improvement observed in the blood parameters of the T2 group may reflect a positive response to treatment. Unprotected Lf did not have the same effect as microencapsulated Lf. Previous studies indicate that Lf can be absorbed by binding to its receptor located on enterocytes and intestinal crypts, influencing the differentiation and proliferation of intestinal epithelial cells (Ma et al., 2023 ). This process helps regulate iron absorption and protects newborn piglets from certain gastrointestinal infections. Xu et al. ( 2005 ) demonstrated that Lf supplementation is an effective way to alleviate symptoms of iron deficiency by improving its bioavailability in intestinal cells and increasing the expression of the Lf receptor gene. In pig production, iron supplementation for piglets is a routine and mandatory practice involving various iron supplements administered through different routes, dosages, and schedules. Consequently, it is not surprising that red blood cell parameters vary significantly among piglets depending on the iron treatment protocols used, making it challenging to compare results across different studies. Rieznik et al (2018) reported that piglets supplemented with bovine Lf had lower NRBC populations after infection. In another study, Hu et al., ( 2019 ) demonstrated that oral Lf supplementation increased serum Hb concentrations in suckling piglets. Serum globulins include immunoglobulins and proteins involved in blood coagulation and nutrient transport. The increase in serum globulin levels may indicate enhanced immune function and growth potential (Husain & Arif, 2019; Garas et al., 2016 ). However, it is important to note that active immunity begins to strengthen around the fourth week of life. Therefore, the functional immaturity of the immune system means that piglets can only generate a limited response when confronted with pathogens (Ogawa et al., 2016 ). Lf plays several key roles in gut health, including immune regulation, intestinal barrier protection, and modulation of gut microbiota (Kanwar et al., 2015 ; Godinez et al., 2017, Hao et al., 2021 ). The microscopic evaluation of intestinal architecture is a common indicator of intestinal morphological development. Increases in VH, VH/CD, and villus surface area are associated with enhanced intestinal absorption capacity, while a shortening of the villi will reduce the surface area available for nutrient absorption (Walton et al., 2016 ). The crypt is the area where stem cells divide to facilitate villus renewal; therefore, a deeper crypt indicates a rapid turnover of tissue in response to increased demand (Yang & Liao, 2019 ). Numerous studies indicate that ETEC infection damages the morphology of the intestinal mucosa (Zhang et al., 2023 ; Sun et al., 2021 ; Lv et al., 2018 ; Wu et al., 2018 ). In this study, histological images revealed that animals in the CON + and T1 groups exhibited villi with varying degrees of atrophy, areas of marked hyperemia, and shedding epithelial tissue in both the duodenum and jejunum-ileum. Similar results were found by Hu et al. ( 2019 ) in a study on the impact of Lf on growth performance, intestinal function, and gastrointestinal microbiota in suckling piglets. These authors reported that early-life intervention with Lf is beneficial for intestinal health, as it was able to promote the development of intestinal function and improve the composition of the gut microbiota. On the other hand, Huang et al. ( 2018 ) obtained similar data in a study on how lysozyme, an antimicrobial peptide present in milk, impacts intestinal performance in neonatal piglets challenged with E. coli . They found that lysozyme facilitated recovery from E. coli infection and reduced mortality and morbidity following exposure to the disease, whether through oral inoculation of the bacteria or infection acquired after contact with infected animals. They observed an increase in VH in all three evaluated intestinal sections (duodenum, jejunum, and ileum) and an increase in the VH/CD ratio in the jejunum and ileum. Therefore, it was demonstrated that this protein is beneficial for the intestinal performance of neonatal piglets. Finally, Zhang et al. ( 2023 ) studied the effect of dietary supplementation with a natural tannin on intestinal lesions and oxidative stress in 7-day-old piglets challenged with ETEC. Their results show that the infected untreated animals exhibited damage to intestinal morphology, experiencing a reduction in VH and an increase in CD, while the group of animals treated with tannin showed a significant improvement in intestinal morphology and relief from oxidative damage. Oxidative stress is one of the main factors that compromise the integrity of the gastrointestinal barrier and increase intestinal permeability (Kaplan et al., 2007). In our study we measured SOD levels and MDA levels to investigate the antioxidant activity of encapsulated Lf. Previous studies have reported that experimental infection with ETEC in neonatal piglets can induce oxidative stress through a reduction in SOD levels and an increase in MDA values (Zhang et al., 2023 ). MDA values reveal significant differences between CON – and T2 groups compared to T1 and CON + groups (p < 0.05). According to Zeng et al. ( 2023 ), higher concentrations of MDA indicate greater damage to the organism. On the other hand, the levels of SOD were significantly higher (p < 0.05) in the T2 and CON − groups compared to CON + and T1 groups. These results align with a recent study that reported that Lf reduces ROS levels by activating the expression of SOD, GSH-Px, and CAT (Pan et al., 2021 ). Similar results were obtained by Hu et al. ( 2021 ), who studied the effect of early intervention with Lf on hepatic metabolism in suckling piglets. They found that piglets receiving the Lf supplement showed significantly higher levels of SOD and GR compared to the control group, which only received saline solution. Conversely, MDA levels were significantly lower in the Lf group compared to the CON + group. Therefore, they concluded that treatment with Lf was able to improve the antioxidant capacity of the liver. In another study conducted by Zhang et al. (2022), they investigated the effect of maternal supplementation with Lf on iron content and antioxidant capacity in neonatal pigs. They used sixty sows and divided them into five groups: Control (basal diet without supplements), Lf1, Lf2, and Lf3 supplemented with 100, 200, and 300 mg/kg of Lf in the basal diet, respectively, and finally the Fe-Gly group supplemented with 100 mg/kg of ferrous glycine. They analyzed the levels of the enzymes SOD, GSH-Px, total antioxidant capacity (T-AOC), and MDA in the blood and tissues. Their results showed, similarly to our study, that both LF and Fe-Gly improved the activities of GSH-Px, T-AOC, and SOD while reducing MDA levels in both the mother sows and the piglets. The mechanism by which Lf exerts its antioxidant activity may be related, on one hand, to its structural characteristics, as it is capable of binding to ferric ions, thereby reducing the formation of free radicals. On the other hand, it may also be linked to its ability to stimulate the activation of antioxidant enzymes (Zhang et al., 2022). CONCLUSION Employing biodegradable polymers like chitosan and alginate in the encapsulation of lactoferrin not only improves piglet health outcomes but also advances the goal of sustainable animal production. The results of this study substantiate the protective role of encapsulated Lf in enhancing the health and performance of piglets challenged with ETEC. The T2 group demonstrated not only a 100% survival rate but also significantly improved average daily weight gain and a marked reduction in diarrhea incidence compared to unprotected counterparts. Blood analysis revealed a recovery from anemia and reduced oxidative stress indicators, indicating enhanced overall health due to Lf supplementation. These findings highlight the efficacy of encapsulating Lf in chitosan-alginate microparticles in preserving its bioactive properties, thereby improving gastrointestinal resilience in neonatal piglets. Given the increasing prevalence of porcine neonatal diarrhea, integrating encapsulated Lf into swine diets could offer a practical approach to mitigate its detrimental effects, ultimately promoting sustainable livestock production. Overall, this study underscores the potential for encapsulated Lf as a valuable dietary supplement in swine production, fostering both immediate health benefits and long-term productivity in livestock. Declarations Consent for publication All authors gave their consent for research publication. Competing interests The authors confirm that there are no conflicts of interest with any financial organization regarding the material discussed in the manuscript. Ethics approval All activities conducted in this trial were approved by the Institutional Committee for the Care and Use of Experimental Animals (CICUAE, EEA INTA Marcos Juárez No. E04-21). Funding This work was supported by the grants from PICT 3575/19. Author Contribution C. A. Methodology, Wrote the original draftM. N. Methodology, review & editing.F. B. M. C. Methodology, review & editing.A. D. Formal análisis, Methodology, review & editing.A. F. Formal análisis, Methodology, review & editing.P. L. Formal análisis, Methodology, review & editing.B. F. Formal análisis, Methodology, review & editing.B. A. Supervision, Formal análisis, Methodology, review & editing.B. R. Supervision, Formal análisis, Methodology, review & editing. Funding acquisition and project administration Acknowledgements Cots A., Mura N., Flores Bracamonte M.C and Pedraza M.L. have Fellowship from CONICET. Dr. D. Acevedo and Dra. R. Bellingeri are Members of the Research Career of CONICET. Thanks to the Pig Production Group of the EEA INTA Marcos Juarez: German Cottura, Dario Panichelli, Mariano Lattanzi, Raul Franco, and Jorge Brunori for providing animals and feed. Data availability The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials. References Abad I, Conesa C, Sánchez L (2021) Development of encapsulation strategies and composite edible films to maintain Lactoferrin bioactivity: A review. Materials 14:7358. https://doi.org/10.3390/ma14237358 Andersen HR, Nielsen JB, Nielsen F, Grandjean P (1997) Antioxidative enzyme activities in human erythrocytes. 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Vet Res 54:9. https://doi.org/10.1186/s13567-023-01139-z Zhu LH, Zhao KL, Chen XL, Xu JX (2012) Impact of weaning and an antioxidant blend on intestinal barrier function and antioxidant status in pigs. J Anim Sci 90(8):2581–2589 https://doi.org/10.2527/jas.2012-4444 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6889295","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":473453159,"identity":"e839fc83-ece5-45b8-8bdd-d2e943ffb334","order_by":0,"name":"Agustina Cots","email":"","orcid":"","institution":"Consejo Nacional de Investigaciones Científicas y Técnicas (INCIVET, CONICET), Universidad Nacional de Río Cuarto (UNRC)","correspondingAuthor":false,"prefix":"","firstName":"Agustina","middleName":"","lastName":"Cots","suffix":""},{"id":473453160,"identity":"8cb0b1d8-341d-42d1-80b8-92d34ff14171","order_by":1,"name":"Nadya Mura","email":"","orcid":"","institution":"Consejo Nacional de Investigaciones Científicas y Técnicas (IITEMA, CONICET), Universidad Nacional de Río Cuarto (UNRC)","correspondingAuthor":false,"prefix":"","firstName":"Nadya","middleName":"","lastName":"Mura","suffix":""},{"id":473453161,"identity":"078a9827-0044-4fe4-a84b-786658749ee8","order_by":2,"name":"María Carolina Flores Bracamonte","email":"","orcid":"","institution":"Consejo Nacional de Investigaciones Científicas y Técnicas (IITEMA, CONICET), Universidad Nacional de Río Cuarto (UNRC)","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Carolina Flores","lastName":"Bracamonte","suffix":""},{"id":473453162,"identity":"14e2b85b-6f3e-4383-9841-02fd6f6bd3fa","order_by":3,"name":"Diego Fernando Acevedo","email":"","orcid":"","institution":"Consejo Nacional de Investigaciones Científicas y Técnicas (IITEMA, CONICET), Universidad Nacional de Río Cuarto (UNRC)","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"Fernando","lastName":"Acevedo","suffix":""},{"id":473453163,"identity":"d4a7f98a-0a9a-4e2f-9362-d82fd28d0934","order_by":4,"name":"Fabrisio Alustiza","email":"","orcid":"","institution":"Instituto Nacional de Tecnología Agropecuaria (INTA)","correspondingAuthor":false,"prefix":"","firstName":"Fabrisio","middleName":"","lastName":"Alustiza","suffix":""},{"id":473453164,"identity":"bfe2f716-0b68-4b2d-9088-5060e41a3556","order_by":5,"name":"Luján Pedraza","email":"","orcid":"","institution":"Instituto Nacional de Tecnología Agropecuaria (INTA)","correspondingAuthor":false,"prefix":"","firstName":"Luján","middleName":"","lastName":"Pedraza","suffix":""},{"id":473453165,"identity":"5c8d6fb0-bc19-49e9-8116-a532dc42b36c","order_by":6,"name":"Fernando Bessone","email":"","orcid":"","institution":"Instituto Nacional de Tecnología Agropecuaria (INTA)","correspondingAuthor":false,"prefix":"","firstName":"Fernando","middleName":"","lastName":"Bessone","suffix":""},{"id":473453166,"identity":"3ad20841-8fd4-4547-8e68-5281c55f5554","order_by":7,"name":"Andrea Bozzo","email":"","orcid":"","institution":"Consejo Nacional de Investigaciones Científicas y Técnicas (INCIVET, CONICET), Universidad Nacional de Río Cuarto (UNRC)","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Bozzo","suffix":""},{"id":473453167,"identity":"ec1b64f7-88f6-4617-94ec-74cee15052cb","order_by":8,"name":"Romina Bellingeri","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABGElEQVRIie3QMUvDQBTA8XcE0iU165NEP8NBB4eW5qtcKFyWgrpIB4dI4CZrZ/HLBA7iEp0jZkiWTg5xFFrxkqg4JFU3wftDyCXkx7sLgE73F0MgcUHrFbkovrw3dhGIWUsi+gvSLk38ETm6ieKYneTTlSPF4kVIoI/LsoLF2A8Hd0UXcfOEqY2tZ9dXvnhY1iS/HSGkgR9aAe0iiHOqiJzRlIhsKDjQjJtAhPRD4N1HweOqIZ4ip9tP8qqIve4h8/qPySm1iDCGYvJOQkWwZ4qbNBtjmJLIce8n1n7GDWRJMBLYM8WJyqLaSM++HJTPT2d4uJdxUlXn44OV3T3lI3VYFTHBah6ZusydAMBrb5tvPtPpdLp/2Rts7mFxJ4ZKTQAAAABJRU5ErkJggg==","orcid":"","institution":"Consejo Nacional de Investigaciones Científicas y Técnicas (IITEMA, CONICET), Universidad Nacional de Río Cuarto (UNRC)","correspondingAuthor":true,"prefix":"","firstName":"Romina","middleName":"","lastName":"Bellingeri","suffix":""}],"badges":[],"createdAt":"2025-06-13 15:08:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6889295/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6889295/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85818777,"identity":"9203e4cc-d460-4a66-b663-4c132f7afa4b","added_by":"auto","created_at":"2025-07-02 06:10:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1038636,"visible":true,"origin":"","legend":"\u003cp\u003eAnimal experimentation design.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6889295/v1/fe3035f82e0979d81b447988.png"},{"id":85817633,"identity":"3d2151c2-a08c-4743-98f1-cc36f13d7e4b","added_by":"auto","created_at":"2025-07-02 06:02:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":868902,"visible":true,"origin":"","legend":"\u003cp\u003eDiarrhea score (scale 0-3) in relation to days post-challenge with ETEC\u003cem\u003e.\u003c/em\u003e Asterisks indicate the death of those piglets. Day 0 represents the day of inoculation. A: Negative Control Group. B: Positive Control Group. C: Treatment Group 1 (non-encapsulated Lf). D: Treatment Group 2 (Lf loaded Chi/Alg microcapsules). The reference numbers in the upper right margin indicate the identification number of each piglet.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6889295/v1/c6f4bafbad36ce3e41287ecf.png"},{"id":85817631,"identity":"f0e9f6cc-d550-4307-8b69-e795d3add9f4","added_by":"auto","created_at":"2025-07-02 06:02:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":208202,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival rate (%) during the days post-challenge with E. coli in the different experimental groups. CON -: Negative control. T2: Treatment 2. CON +: Positive control. T1: Treatment 1.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6889295/v1/dc00be9bc0cbf1506c643575.png"},{"id":85817634,"identity":"fa656042-32c4-4cff-ade0-81db9aea42e1","added_by":"auto","created_at":"2025-07-02 06:02:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6477579,"visible":true,"origin":"","legend":"\u003cp\u003eNecropsy images of piglets. A: CON\u003csup\u003e-\u003c/sup\u003e group, B: CON\u003csup\u003e+\u003c/sup\u003e group, C: T1 group, D: T2 group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6889295/v1/38bf71292ce6cdf91bb303da.png"},{"id":85817637,"identity":"05423278-5edd-4d0e-98e2-72aba444102d","added_by":"auto","created_at":"2025-07-02 06:02:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2984779,"visible":true,"origin":"","legend":"\u003cp\u003eHistological images of two intestinal sections (duodenum and jejunum-ileum) from 10-day-old suckling piglets. Staining with H\u0026amp;E, 5x objective. Scale: 200 μm. Asterisks: lymphoid accumulations (Peyer’s patches). Arrows: areas of congestion.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6889295/v1/9ac4a286f1918ffe43dfb8fd.png"},{"id":85817636,"identity":"e0564ea4-0ed5-4a16-ba66-e3660ba2e164","added_by":"auto","created_at":"2025-07-02 06:02:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":954095,"visible":true,"origin":"","legend":"\u003cp\u003eHistological images with greater detail of jejunum-ileum samples from the CON + and T1 groups. Marked hyperemia (rectangles) and detachment of epithelial tissue (arrows) can be observed. H\u0026amp;E staining. 5x objective. Scale: 200 μm.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6889295/v1/a637d6b6848fcc57ffff25c3.png"},{"id":85817635,"identity":"87e1c86a-db1a-4688-8d7e-5b8837ceb141","added_by":"auto","created_at":"2025-07-02 06:02:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":157721,"visible":true,"origin":"","legend":"\u003cp\u003eHepatic MDA levels in 10-day-old suckling piglets. Results are presented as mean ± standard deviation. Different letters represent significant differences (p \u0026lt; 0.05). Infostat, 2020.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6889295/v1/3d090f4fea29802031a00679.png"},{"id":85817638,"identity":"8323cabd-4f85-435b-99bb-0311d48587af","added_by":"auto","created_at":"2025-07-02 06:02:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":156102,"visible":true,"origin":"","legend":"\u003cp\u003eHepatic SOD levels in 10-day-old suckling piglets. Results are expressed as mean ± SD. Different letters represent significant differences (p \u0026lt; 0.05). Infostat, 2020.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6889295/v1/dbe3a52638687e5b6ebd5125.png"},{"id":86719139,"identity":"999fc41b-e730-4710-b5b6-6b86487cba7c","added_by":"auto","created_at":"2025-07-14 23:01:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12738047,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6889295/v1/e8eb337c-b602-498a-92f9-41a644c942e3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Protective effects of microencapsulated Lactoferrin in enterotoxigenic Escherichia coli- challenged piglets","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNeonatal porcine diarrhea is a significant health challenge in swine production, causing substantial economic losses due to rapid dehydration, growth delays and mortality in piglets (Fairbrother \u0026amp; Nadeau, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This disease is multifactorial and complex to diagnose; however, the most implicated agent in this pathology is the enterotoxigenic \u003cem\u003eEscherichia coli\u003c/em\u003e (ETEC) (Lv et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Piglets infected with ETEC can experience intense, secretory diarrhea within 1 to 2 hours after birth. They show symptoms such as abundant, non-bloody, watery diarrhea, along with nausea, abdominal cramps, and shivering. As a result, the piglets quickly become dehydrated, undernourished, and weak (Jacobson et al., 2022). Additionally, this pathology can also alter porcine blood parameters (Ider et al., 2023; Chethan et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The metabolic response to the infection typically generates reactive oxygen species (ROS), which play a critical role in the pathophysiology of the disease and contribute to cellular damage (Zhang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To counteract the harmful effects of ROS, various antioxidant mechanism function synergistically to prevent or mitigate damage to macromolecules (Dalle-Done et al., 2006; Andersen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Evidence suggests that a decline in antioxidant activity, coupled with increased ROS production, contributes to intestinal dysfunction and oxidative stress (Zhu et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Moreover, numerous studies have demonstrated that intestinal dysfunction plays a pivotal role in growth impairment (Cai et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kikusato et al., 2021; Upadhaya et al., 2021; Campbell et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOver the past two decades, extensive research has focused on developing alternatives to antibiotics to maintain the health and performance of pigs, particularly on the light of the public health issue posed by bacterial resistance to antibiotics and its potential transfer to humans (Mounsey et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Rhouma et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this context, the use of a safe agents to promote growth and bolster immunity, while concurrently preventing diseases that affect piglets at early stages, is of paramount importance and urgency. Lactoferrin (Lf), an 80 kDa non-heme iron-binding glycoprotein, is recognized as a critical host defense molecule (Gonz\u0026aacute;lez-Ch\u0026aacute;vez et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). When incorporated into early nutritional intervention, it has the potential to enhance digestion and nutrient absorption, thereby optimizing growth. Additionally, Lf influences numerous vital physiological processes, modulates intestinal function, and alleviates neonatal and post-weaning diarrhea in piglets (Hao et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies have demonstrated that Lf possesses antimicrobial activity (Yan et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Duarte et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and antioxidant capacity both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e (Sarkar et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Narmuratova et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Oussaief et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Farid et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, Lf is regarded as safe, with no known toxic or adverse side effects, and it does not contribute to drug resistance (Hao et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, due tits rapid degradation in gastrointestinal conditions, achieving a successful therapeutic effect often requires a substantial amount or frequent dosing (Abad et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In recent studies conducted by our research group, we demonstrated that the encapsulating Lf in chitosan-alginate microcapsules preserves its structural integrity, thereby enhancing its antibacterial activity against an ETEC strain (Cots et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These materials are biodegradable, non-toxic, and have been extensively studied for agricultural and food applications. Their use in microencapsulation not only enhances the stability and bioavailability of Lf but also supports environmental objectives by reducing persistent waste and reliance on synthetic polymers that may pose ecological risks (Lewicka et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Nahar et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Nanda et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe objective of this study is to evaluate the protective effect of the oral administration of microencapsulated lactoferrin in piglets challenged with E. coli, using biodegradable polymer-based microencapsulation techniques.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLf encapsulation\u003c/h2\u003e \u003cp\u003eTo promote environmentally responsible practices, Lf was encapsulated utilizing biodegradable and renewable polymers. Lf loaded Chi/Alg microparticles were produced following Cots et al (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) through green methodologies. Initially, Alg microparticles were generated via ionic gelation using calcium chloride in aqueous, mild conditions, avoiding harmful solvents or cross-linking agents. This process resulted in biodegradable microspheres. Subsequently, these Alg microparticles were coated with Chi through an aqueous-phase electrostatic interaction.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBacterial isolation\u003c/h3\u003e\n\u003cp\u003eETEC strain was obtained from a from a neonatal pig with diarrhea in an intensive pig production farm located in C\u0026oacute;rdoba province, Argentina. Initially, the strain was cultured on MacConkey agar (Britania\u0026reg;, Argentina) at 37 ◦C for 48 h. Then, the identification as ETEC strain was confirmed using molecular techniques. The PCR analysis revealed that the strain was F4\u003csup\u003e+\u003c/sup\u003e (K88\u003csup\u003e+\u003c/sup\u003e) and capable of producing of the heat-stable toxin (STb), and heat-labile toxin (LT) (Bessone et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eETEC strain management\u003c/h3\u003e\n\u003cp\u003eETEC strain, maintained at -20\u0026deg;C in glycerol, was thawed and cultured in trypticase soy broth (TSB) for 18 h at 37\u0026deg;C. Then, streaking was performed on trypticase soy agar (TSA), and it was incubated at 37\u0026deg;C for 24 h. Subsequently, 1 colony was resuspended in 5 mL of TSB, incubating for 18 h at 37\u0026deg;C to obtain the stock inoculum and carry out a bacterial count using the micro drop technique. Serial dilutions (1:10) of the stock inoculum were made up to the 1 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;12\u003c/sup\u003e dilution. Dilutions were plated on TSA plates in triplicate and incubated for 18 h at 37\u0026deg;C. After the incubation time, the CFU/mL count of the stock inoculum was carried out using the following formula:\u003c/p\u003e \u003cp\u003eCFU/mL\u0026thinsp;=\u0026thinsp;average number of colonies\u0026times; 100 \u0026times; dilution factor\u003c/p\u003e \u003cp\u003eSubsequently, a bacterial suspension was prepared with a multiplicity of infection (MOI) of 1:1 bacterium per cell in the media without antibiotics.\u003c/p\u003e\n\u003ch3\u003eAnimals and housing\u003c/h3\u003e\n\u003cp\u003eThe experiment was conducted at the INTA Marcos Ju\u0026aacute;rez Agricultural Experimental Station. Two multiparous sows with similar numbers of litters (2\u0026ndash;3 births), that had not received the \u003cem\u003eE. coli\u003c/em\u003e vaccine and were in good health, were chosen for this study. The sows were moved to the farrowing room on day 108 of gestation, and after giving birth, each sow and her piglets were housed in farrowing crates (2.2 x 0.60 m), with a plastic slatted floor and infrared lamps for the piglets. At the time of birth, the litter size was homogenized to sixteen piglets with a birth weight of 1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 kg, and they were allowed to nurse without any handling on the first day of life.\u003c/p\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eA total of 16 newborn piglets of the Genetiporc x Choice cross were randomly assigned to 4 groups: CON\u003csup\u003e+\u003c/sup\u003e (positive control, n\u0026thinsp;=\u0026thinsp;3), CON\u003csup\u003e\u0026minus;\u003c/sup\u003e (negative control, n\u0026thinsp;=\u0026thinsp;3), T1 (Free Lf, n\u0026thinsp;=\u0026thinsp;5), and T2 (Lf loaded Chi/Alg microparticles, n\u0026thinsp;=\u0026thinsp;5). The adaptation period lasted for 2 days after bird and the experiment period lasted for 10 days. At the beginning of the experiment, the animals were identified with a plastic ear tag on the right ear. On the third day of life, each piglet received an intramuscular injection of 200 mg of dextran iron (Cali-Dex 200, Calier\u0026reg;) and a dose of 1.5% diclazuril orally for coccidia control (Vetribac Doser, Vetanco\u0026reg;). During the experimental period (10 days), the only source of food for the piglets was maternal milk.\u003c/p\u003e \u003cp\u003eThe piglets in group T1 were orally administered with a dose of 5 mL of Lf solution, dissolved in cow's milk, divided into two daily doses (10:00 and 17:00) from day 1 to day 7 of the experiment (0.5 g/kg of body weight/day) as described by Hu et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). On the other hand, the piglets in group T2 were fed the same dose of Lf loaded in Chi/Alg microparticles, delivered in cow's milk. Finally, the animals in the CON groups (positive and negative) were given the same dose of cow's milk. All piglets (except the CON\u003csup\u003e\u0026minus;\u003c/sup\u003e group) were orally challenged on day 6 of the experiment with a dose of 1 mL (1x10\u003csup\u003e8\u003c/sup\u003e UFC/mL per pig) of porcine ETEC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnimal clinical evaluation\u003c/h2\u003e \u003cp\u003eDuring the 10-day period, the clinical status of all animals was examined considering parameters such as sensorium status, hydration status, presence/absence of diarrhea, and rectal temperature. Lethargic behavior was defined as slow reactions, unstable gait, and resistance to movement (Rossi et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Piglets were considered dehydrated when the skin folded on the neck took more than 2\u0026ndash;4 sec to return to its normal position (Huang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The presence or absence of diarrhea was evaluated through daily rectal swabs and direct observation of the hygiene of the perineal area. Rectal temperature was recorded immediately prior to the challenge and the following day in the morning, before any handling of the animal (Rossi et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHealth and well-being assessment\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGrowth performance\u003c/h2\u003e \u003cp\u003eThe live weight of the animals was recorded individually on days 0 (birth weight, BW), 2, 5, 8, and 10, to calculate average daily gain (ADG). The diarrhea score (DS) was assessed each morning at 10:00 am.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDiarrhea score and incidence\u003c/h2\u003e \u003cp\u003eA scoring scale from 0 to 3 was established based on Rossi et al (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), where 0 was considered normal (firm stools), 1 was semi-solid (soft, formed stools), 2 was semi-liquid (loose stools, generally yellowish), and 3 was liquid (watery and projectile stools). A fecal consistency score of \u0026le;\u0026thinsp;1 (0, 1) was considered normal, while a fecal score\u0026thinsp;\u0026gt;\u0026thinsp;1 (2, 3) was indicative of diarrhea. The incidence of diarrhea (%) was calculated as the percentage of affected piglets during the experimental period divided by the total number of piglets in each group (Huang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSurvival rate\u003c/h2\u003e \u003cp\u003eThe survival rate was calculated as: Number of live piglets/Total number of piglets in that group at the end of the experiment * 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHematological analyses\u003c/h2\u003e \u003cp\u003ePrior to euthanasia, blood samples were collected via venipuncture from the cava vein, both with and without anticoagulant (EDTA), and were stored refrigerated at 4\u0026deg;C for hematological analyses. The samples with and without EDTA were centrifuged at 1500 rpm for 15 min to extract the plasma. The samples without anticoagulants were also centrifuged immediately after extraction at 3200 rpm for 10 min for serum separation, which was stored at -20\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eFrom whole blood, the following determinations were made red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, plasma proteins, fibrinogen, total white blood cell count (WBC), and leukocyte differential count. Finally, in the serum, total protein, albumin (Alb), globulins (G), and the Alb:G ratio were evaluated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEuthanasia of Animals and Post-Mortem Evaluation\u003c/h2\u003e \u003cp\u003eOn day 10 of the experiment, all animals (n\u0026thinsp;=\u0026thinsp;16) were euthanized. This was carried out through exsanguination in accordance with the American Veterinary Medical Association Euthanasia Guidelines (Leary et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). During the necropsy, macroscopic damage was assessed, particularly in the digestive system, and data were collected, including the coloration of the digestive mucosa, appearance and consistency of the intestinal contents, and pH value of the contents.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eFor sampling, the small intestine was divided into two segments: duodenum (5\u0026ndash;8 cm from the pylorus) and jejunum-ileum: jejunum (middle portion) and ileum (distal portion, approximately 5 cm in length proximal to the ileocecal junction). Samples were fixed in 4% paraformaldehyde, dehydrated in ethanol, then embedded in paraffin and sectioned into 5 \u0026micro;m thick slices, mounted on glass slides. Subsequently, the samples were deparaffinized, rehydrated, and stained with hematoxylin-eosin (Du et al., 2019). For microscopic evaluation, a Carl Zeiss trinocular optical microscope (AxioStar PLUS) coupled with a digital camera (Moticam 3.0) was used. The length of at least 10 randomly oriented well-aligned villi (Woliński et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and the depth of the crypts were measured. Images were captured using Motic Image Plus 3.0 software and parameters were measured using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStudy of Oxidative Parameters in Liver Tissue\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of MDA concentration\u003c/h2\u003e \u003cp\u003eLipid peroxidation levels were assessed using the thiobarbituric acid reactive substances (TBARS) metho, which quantifies MDA concentrations in liver tissue. This measurement was based on the protocol established by Marcinc\u0026aacute;k et al (2003). Briefly, a 1 mL sample of 30% liver homogenate was mixed with butylated hydroxytoluene (5 mM) and 15% (v/v) trichloroacetic acid, followed by incubation at 90\u0026deg;C for 30 min. After allowing the mixture to cool, it was centrifuged to obtain the supernatant. Subsequently, 1 mL of the supernatant was extracted and reacted with hydrochloric acid (0.25 N) and thiobarbituric acid 0.375% (v/v), followed by another incubation. The colored layer was measured at 535 nm, using 1,1,3,3-tetramethoxypropane (Sigma) as a standard. MDA concentration was calculated as nmol/g of tissue, according to the formula:\u003c/p\u003e \u003cp\u003elog y (DO)\u0026thinsp;=\u0026thinsp;log A\u0026thinsp;+\u0026thinsp;log B x (MDA concentration)\u003c/p\u003e \u003cp\u003ex\u0026thinsp;=\u0026thinsp;log⁻\u0026sup1; (log y \u0026ndash; log A) / B\u003c/p\u003e \u003cp\u003eWhere A corresponds to the colorimetric readings and B to known concentrations of MDA.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of SOD activity\u003c/h2\u003e \u003cp\u003eSOD activity was determined by its ability to inhibit the auto-oxidation of epinephrine at alkaline pH. Liver tissue was homogenized in a buffer, and an aliquot was taken to measure protein content using the Bradford method, using bovine serum albumin as a standard. The remaining homogenate was centrifuged. Next, 50 \u0026micro;L of epinephrine and 2.85 mL of glycine buffer (50 mM, pH 10\u0026ndash;11) were added, and the appearance of epinechrome was measured at 480 nm. The slope of the graph of epinephrine oxidation (a) and the slope of the graph of epinephrine oxidation in the presence of the sample (b) were obtained and subsequently, log b/a was plotted as a function of \u0026micro;L of sample. SOD activity was calculated as U SOD/mg of protein\u0026thinsp;=\u0026thinsp;1000/\u0026micro;L (sample corresponding to 50% inhibition). One unit of SOD corresponds to the \u0026micro;L of sample that inhibits epinechrome formation by 50%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were analyzed using Infostat software version 2020. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical differences were considered significant when the p-value was less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eHealth and well-being indicators\u003c/h2\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003eGrowth performance\u003c/h2\u003e \u003cp\u003eDaily weight gain (DWG) is a key parameter for assessing growth performance in animals. In this study, all piglets started at the same age (1 day old). The birth weights (BW) of each piglet were recorded, and then on days 2, 5, 8, and 10 of the experiment to determine DWG. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, statistically significant differences were found between the CON\u0026thinsp;+\u0026thinsp;and T1 groups compared to the T2 group on days 2, 5, 8, and 10.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDaily weight gain in piglets challenged with ETEC\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCON-\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCON +\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay o (BW)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0078\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0212\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0108\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDay 10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0096\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eThe results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Different letters indicate a significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Infostat, 2020. BW: birth weight.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eDiarrhea score and incidence\u003c/h2\u003e \u003cp\u003eRegarding the diarrhea score and incidence (%), piglets in the CON - group did not show any episodes compatible with intestinal disease during the evaluated period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In the case of the CON\u0026thinsp;+\u0026thinsp;group, two piglets exhibited diarrhea (scores 2 and 3, incidence: 66.67% on day 1 post-challenge), both with dehydration levels of 6% and lethargy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In the T1 group, two animals also presented diarrhea episodes (scores 2 and 3, incidence: 40%), showing signs of severe dehydration (\u0026gt;\u0026thinsp;6%), lethargy, and sensory depression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Finally, in the T2 group, only one piglet was observed with mild and transient diarrhea lasting one day (score 2, incidence: 10% on day 0) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to these data, T1 reduces the incidence of diarrhea compared to the positive control group (CON +). However, the values of the evaluated parameters clearly improved when using Lf administered in Chi-Alg microcapsules.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSurvival rate\u003c/h2\u003e \u003cp\u003eThe survival rates (SR%) of the days following the challenge in piglets from the four experimental groups are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The results indicated that the piglets in the T2 group recovered more quickly after the ETEC infection, with 0% mortality, due to a lower incidence of diarrhea and lower diarrhea scores, similar to the CON - group, where none of the piglets\u0026rsquo; exhibited symptoms of diarrhea. Both groups had a 100% SR by day 10 of the trial (day 4 post-inoculation). In contrast, the CON\u0026thinsp;+\u0026thinsp;and T1 groups showed lower SR%, at 66.67% (2/3*\u003cem\u003e100) and 40% (\u003c/em\u003e2/5\u003cem\u003e*\u003c/em\u003e100) respectively, as one animal from the CON\u0026thinsp;+\u0026thinsp;group and three animals from the T1 group died before day 10.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eHematological parameters\u003c/h2\u003e \u003cp\u003eThe results of the hematological profile of the piglets evaluated on day 10 are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHematological parameters\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eComplete blood count\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCON -\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCON +\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGR (mill/\u0026micro;l)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHb (g/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHto (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0340\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVCM (fl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e70.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e72.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHCM (pg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u0026thinsp;\u0026plusmn;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.5925\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCHCM (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlatelets (mil/\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e675.53\u0026thinsp;\u0026plusmn;\u0026thinsp;38.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e903\u0026thinsp;\u0026plusmn;\u0026thinsp;118.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e930\u0026thinsp;\u0026plusmn;\u0026thinsp;84.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e732.2\u0026thinsp;\u0026plusmn;\u0026thinsp;176.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlasma prot. (g/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFibrinogen (mg/dL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;50\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e675\u0026thinsp;\u0026plusmn;\u0026thinsp;35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e635\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e525\u0026thinsp;\u0026plusmn;\u0026thinsp;86\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLeukogram\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeukocytes (n\u0026deg;/\u0026micro;L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11600\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9150\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12150\u0026thinsp;\u0026plusmn;\u0026thinsp;1.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11350\u0026thinsp;\u0026plusmn;\u0026thinsp;2.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4802\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSegmented Neutrophils\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5337\u0026thinsp;\u0026plusmn;\u0026thinsp;432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3555\u0026thinsp;\u0026plusmn;\u0026thinsp;140\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5764\u0026thinsp;\u0026plusmn;\u0026thinsp;3218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5659\u0026thinsp;\u0026plusmn;\u0026thinsp;2223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.6251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5724\u0026thinsp;\u0026plusmn;\u0026thinsp;723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5071\u0026thinsp;\u0026plusmn;\u0026thinsp;159\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5873\u0026thinsp;\u0026plusmn;\u0026thinsp;3064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4848\u0026thinsp;\u0026plusmn;\u0026thinsp;817\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.7598\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonocytes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e579.3\u0026thinsp;\u0026plusmn;\u0026thinsp;230.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e565.5\u0026thinsp;\u0026plusmn;\u0026thinsp;337.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e652.3\u0026thinsp;\u0026plusmn;\u0026thinsp;683.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEosinophils\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e193.3\u0026thinsp;\u0026plusmn;\u0026thinsp;67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e297.5\u0026thinsp;\u0026plusmn;\u0026thinsp;41.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e190\u0026thinsp;\u0026plusmn;\u0026thinsp;182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.1367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasophils\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eResults are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Different letters indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Infostat, 2020. CON -: Negative control (n\u0026thinsp;=\u0026thinsp;3). CON +: Positive control (n\u0026thinsp;=\u0026thinsp;2, 1 dead). T1: Free Lf, n\u0026thinsp;=\u0026thinsp;2 (3 dead). T2: Encapsulated Lf, n\u0026thinsp;=\u0026thinsp;4 (1 coagulated). Means with a common letter are not significantly different (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Abbreviations: GR: Red blood cells, Hb: Hemoglobin, Hto: Hematocrit, VCM: Mean corpuscular volume, HCM: Mean corpuscular hemoglobin, CHCM: Mean corpuscular hemoglobin concentration.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c7\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSignificant statistical differences were found in red blood cell parameters (GR, Hb, and Hto) between the CON\u003csup\u003e+\u003c/sup\u003e and CON\u003csup\u003e\u0026minus;\u003c/sup\u003e groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The CON\u003csup\u003e+\u003c/sup\u003e group showed the lowest values for GR, Hb, and Hto. No significant differences were observed in leukogram. Additionally, there was a significant increase in the MCV in the T1 and CON\u003csup\u003e+\u003c/sup\u003e groups compared to CON\u003csup\u003e\u0026minus;\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFibrinogen levels were found to be altered, with statistical analysis showing a difference between the CON\u003csup\u003e+\u003c/sup\u003e and T1 groups compared to the CON\u003csup\u003e\u0026minus;\u003c/sup\u003e group.\u003c/p\u003e \u003cp\u003eThe serum protein levels measured on day 10 of the experiment are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerum protein levels\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum Parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCON -\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCON +\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTP\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0333\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlb\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0318\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAlb/G\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eResults expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Different letters represent significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Infostat, 2020. TP: Total Proteins (g/dL). A: Albumins (g/dL). G: Globulins (g/dL). Alb/G: Albumin/Globulins Ratio. CON\u003csup\u003e\u0026minus;\u003c/sup\u003e: Negative control (n\u0026thinsp;=\u0026thinsp;3), CON\u003csup\u003e+\u003c/sup\u003e: Positive control (n\u0026thinsp;=\u0026thinsp;2, 1 dead), T1: Treatment 1 (n\u0026thinsp;=\u0026thinsp;2, 3 dead), T2: Treatment 2 (n\u0026thinsp;=\u0026thinsp;4, 1 coagulated).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAlthough the total protein values (g/dL) fall within the reference range, the results show a statistically significant increase for the CON\u003csup\u003e\u0026minus;\u003c/sup\u003e group compared to the T1 group. Additionally, the T2 group exhibited lower levels of Alb, higher G levels, and consequently a decreased in Alb/G ratio compared to the T1 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003ePostmortem evaluation\u003c/h2\u003e \u003cp\u003eThe macroscopic evaluation of the digestive system in piglets revealed stomach and intestinal dilation in animals from the CON\u003csup\u003e+\u003c/sup\u003e group and in 3 of the 5 animals from the T1 group. Additionally, marked congestion throughout the intestinal tract was observed in the T1 group, and some animals had watery fecal matter inside (data not shown). In contrast, the T2 group exhibited milder dilation, particularly in the large intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings align with Luppi et al. (2023), whose study focused on the diagnostic approach to enteric disorders in pigs caused by common pathogens, including \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eHistopathological parameters of the intestine\u003c/h2\u003e \u003cp\u003eIn this study, the morphology of two intestinal sections (duodenum and jejunum-ileum) of piglets challenged with ETEC was evaluated at the end of the experiment (day 10). As seen in the histological images, animals in the CON\u003csup\u003e+\u003c/sup\u003e and T1 groups exhibited villi with varying degrees of atrophy, areas of marked hyperemia, and shedding epithelial tissue in both the duodenum and jejunum-ileum (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents the results obtained from the measurements of average villus height (VH), crypt depth (CD), and the ratio between the two (VH/CD). Piglets of CON\u003csup\u003e+\u003c/sup\u003e group displayed altered intestinal architecture, characterized by a reduction in average of VH and the VH/CD ratio in both duodenum, and jejunum-ileum sections, along with an increase in CD. These changes were statistically significant when compared to the values obtained in the CON\u003csup\u003e\u0026minus;\u003c/sup\u003e and T2 groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The values found in the piglets of the T1 group showed no significant differences compared to the CON\u003csup\u003e+\u003c/sup\u003e group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of oral administration of encapsulated lactoferrin on histological parameters of intestine in piglets challenged with ETEC\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntestine section\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCON\u003csup\u003e-\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCON\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVH (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e755\u0026thinsp;\u0026plusmn;\u0026thinsp;74\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e314\u0026thinsp;\u0026plusmn;\u0026thinsp;15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e305\u0026thinsp;\u0026plusmn;\u0026thinsp;27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e647\u0026thinsp;\u0026plusmn;\u0026thinsp;60\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e237\u0026thinsp;\u0026plusmn;\u0026thinsp;19\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e277\u0026thinsp;\u0026plusmn;\u0026thinsp;21\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e276\u0026thinsp;\u0026plusmn;\u0026thinsp;74\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e205\u0026thinsp;\u0026plusmn;\u0026thinsp;22\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVH/CD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eJejunum-Ileum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVH (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e598\u0026thinsp;\u0026plusmn;\u0026thinsp;82\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e153\u0026thinsp;\u0026plusmn;\u0026thinsp;24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e216\u0026thinsp;\u0026plusmn;\u0026thinsp;32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e475\u0026thinsp;\u0026plusmn;\u0026thinsp;49\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD (\u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e143\u0026thinsp;\u0026plusmn;\u0026thinsp;30\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95\u0026thinsp;\u0026plusmn;\u0026thinsp;12\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97\u0026thinsp;\u0026plusmn;\u0026thinsp;14\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e161\u0026thinsp;\u0026plusmn;\u0026thinsp;23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVH/CD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u0026ordf;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e \u003cp\u003eResults expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Different superscript letters indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). VH: Villus height. CD: Crypt depth\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eOxidative parameters in hepatic tissue\u003c/h2\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003eMDA concentration\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, statistical analyses of MDA values reveal significant differences between the CON\u003csup\u003e\u0026ndash;\u003c/sup\u003e group (147.59\u0026thinsp;\u0026plusmn;\u0026thinsp;7.13) and T2 group (152.19\u0026thinsp;\u0026plusmn;\u0026thinsp;14.82) compared to the T1 group (206.12\u0026thinsp;\u0026plusmn;\u0026thinsp;15.21) and CON\u003csup\u003e+\u003c/sup\u003e group (212.87\u0026thinsp;\u0026plusmn;\u0026thinsp;9.34) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eSOD concentration\u003c/h3\u003e\n\u003cp\u003eThe levels of SOD were significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the T2 group (18.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35) and the CON\u003csup\u003e\u0026minus;\u003c/sup\u003e group (19.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50) compared to the CON\u003csup\u003e+\u003c/sup\u003e group (12.23\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12) and the T1 group (11.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eLf exhibits a broad spectrum of biological activity, making it a valuable alternative for biomedical applications. In a previous study, we demonstrated that the encapsulation of Lf in Chi/Alg microcapsules is a strategy that allows maintaining its structural and functional integrity \u003cem\u003ein vitro\u003c/em\u003e (Cots et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In the present study, we aim to evaluate the protective effect of the oral administration of microencapsulated lactoferrin in piglets challenged with ETEC, using biodegradable polymer-based microencapsulation techniques.\u003c/p\u003e \u003cp\u003eThe study of health and well-being indicators revealed a statistically significant increase on daily weight gain in T2 piglets on days 2, 5, 8, and 10 comparing to CON\u003csup\u003e+\u003c/sup\u003e and T1 groups. Similar results were found by Ma et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) in a study that evaluated the effects of dietary Lf supplementation on growth performance, immune function, and intestinal health in weaned piglets. In their study, four groups were used with different diets: negative control (basic diet); positive control (basic diet\u0026thinsp;+\u0026thinsp;antibiotics); lactoferrin A (bLF-A) (basic diet\u0026thinsp;+\u0026thinsp;1 g/kg of Lf); and lactoferrin B (bLF-B) (basic diet\u0026thinsp;+\u0026thinsp;3 g/kg of Lf). The results demonstrate that dietary supplementation of Lf can improve growth performance and reduce diarrhea, but in a dose-dependent manner. Additionally, it enhanced immunity and the intestinal architecture in the mucosa of the small intestine.\u003c/p\u003e \u003cp\u003eRegarding diarrhea score and incidence, the results revealed that T1 reduces the incidence of diarrhea compared to the CON\u003csup\u003e+\u003c/sup\u003e group. However, the values of the evaluated parameters clearly improved when the Lf was administered encapsulated in Chi/Alg microcapsules. Hu et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) worked with two groups of animals: a Control group consisting of 30 nursing animals and another group of 30 animals that received an oral dose of 0.5 g/kg/day of a Lf solution. They found that Lf was able to reduce the incidence of diarrhea, increase villus height in the jejunum, and improve growth performance. Therefore, early-life intervention with Lf is beneficial for intestinal health.\u003c/p\u003e \u003cp\u003eIn a study by Comstock et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), the effect of oral supplementation with free bovine Lf on the survival of piglets deprived of colostrum was evaluated, using increasing doses: Control group: 130 mg, Lf1: 367 mg, and Lf2: 1300 mg/kg/day. The authors found that none of the doses significantly increased survival, though the highest dose showed a trend toward improved survival compared to the control diet.\u003c/p\u003e \u003cp\u003eThe survival rates (SR%) indicated that the piglets in the T2 group recovered more quickly after the ETEC infection, with 0% mortality, due to a lower incidence of diarrhea and lower diarrhea scores, similar to the CON\u003csup\u003e\u0026minus;\u003c/sup\u003e group, where none of the piglets\u0026rsquo; exhibited symptoms of diarrhea. Both groups had a 100% SR by day 10 of the trial. Considering these findings, it could be inferred that the T1 group, treated with free Lf, likely did not receive a sufficient dose to enhance piglet survival. Alternatively, Lf may require protection to reach the small intestine intact, preserving its bioactive properties to exert its beneficial effects. While there are currently few or no reports on \u003cem\u003ein vivo\u003c/em\u003e studies conducted with encapsulated Lf in Chi/Alg microcapsules in piglets, many studies demonstrate that encapsulation helps protect this protein during its passage through the stomach (acidic pH and digestive enzymes) and allows it to reach its site of action in its bioactive form (Bokkhim et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). On the contrary, a study of Resouzakov et al (2018) reported that piglets fed bLf mounted a more effective immune response and exhibited lower bacterial abundance providing biological underpinnings to the clinical benefits of bLf during \u003cem\u003eS. aureus\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eThere were significant differences in red blood cell parameters between the CON\u003csup\u003e+\u003c/sup\u003e and CON\u003csup\u003e\u0026minus;\u003c/sup\u003e groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The CON\u003csup\u003e+\u003c/sup\u003e group showed the lowest values for GR, Hb and Hto, while no significant differences were noted in the leukogram. Additionally, there was a significant increase in the MCV in the T1 and CON\u003csup\u003e+\u003c/sup\u003e groups compared to CON\u003csup\u003e\u0026minus;\u003c/sup\u003e. According to Chen et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), these indicators suggest that the CON\u003csup\u003e+\u003c/sup\u003e group animals experienced anemia, characterized by low GR, Hb, and Hto levels. The elevated MCV may indicate a regenerative type of anemia, as described by Newcomer et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The wide variability in Hb and Hto levels is influenced by factors such as diet, management systems, and genetics (Cooper et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although the values in the T1 group were not indicative of clinical anemia, the Hb levels suggest the presence of subclinical anemia (Fjelkner et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Piglets are born with limited iron, since the only food available in the first two weeks is maternal milk, which is low in iron, and piglets in intensive farming systems lack access to soil (often raised on concrete or plastic floors), the high incidence of anemia in swine operations is understandable. Iron deficiency is one of the most common nutritional deficiencies in mammals, with iron-deficiency anemia being the most severe consequence (Chaud et al., 2019; Parsons \u0026amp; Tummaruk, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Additionally, in the first 12 h of life, there is a significant increase in plasma volume without a corresponding increase in red blood cell count, leading to physiological anemia due to rapid blood volume expansion (Quiles \u0026amp; Hev\u0026iacute;a, 2004).\u003c/p\u003e \u003cp\u003eThe altered fibrinogen levels were observed, with statistical analysis indicating a difference between the CON\u003csup\u003e+\u003c/sup\u003e and T1 groups compared to the CON\u003csup\u003e\u0026minus;\u003c/sup\u003e group. Increased fibrinogen levels in the blood are a clear indicator of acute inflammation. Kang et al. (2020) investigated acute phase proteins (APP), including fibrinogen, in a study on acid-base and electrolyte imbalances in calves with enteropathogenic diarrhea. They found no statistically significant differences in APP levels between diarrheic and healthy calves suggesting that changes in APP levels do not always correlate with the severity of the disease.\u003c/p\u003e \u003cp\u003eAccording to our results and considering that one of the main functions of Lf is to enhance iron absorption (Baker \u0026amp; Baker, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), the improvement observed in the blood parameters of the T2 group may reflect a positive response to treatment. Unprotected Lf did not have the same effect as microencapsulated Lf. Previous studies indicate that Lf can be absorbed by binding to its receptor located on enterocytes and intestinal crypts, influencing the differentiation and proliferation of intestinal epithelial cells (Ma et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This process helps regulate iron absorption and protects newborn piglets from certain gastrointestinal infections. Xu et al. (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) demonstrated that Lf supplementation is an effective way to alleviate symptoms of iron deficiency by improving its bioavailability in intestinal cells and increasing the expression of the Lf receptor gene.\u003c/p\u003e \u003cp\u003eIn pig production, iron supplementation for piglets is a routine and mandatory practice involving various iron supplements administered through different routes, dosages, and schedules. Consequently, it is not surprising that red blood cell parameters vary significantly among piglets depending on the iron treatment protocols used, making it challenging to compare results across different studies. Rieznik et al (2018) reported that piglets supplemented with bovine Lf had lower NRBC populations after infection. In another study, Hu et al., (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) demonstrated that oral Lf supplementation increased serum Hb concentrations in suckling piglets.\u003c/p\u003e \u003cp\u003eSerum globulins include immunoglobulins and proteins involved in blood coagulation and nutrient transport. The increase in serum globulin levels may indicate enhanced immune function and growth potential (Husain \u0026amp; Arif, 2019; Garas et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, it is important to note that active immunity begins to strengthen around the fourth week of life. Therefore, the functional immaturity of the immune system means that piglets can only generate a limited response when confronted with pathogens (Ogawa et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLf plays several key roles in gut health, including immune regulation, intestinal barrier protection, and modulation of gut microbiota (Kanwar et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Godinez et al., 2017, Hao et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The microscopic evaluation of intestinal architecture is a common indicator of intestinal morphological development. Increases in VH, VH/CD, and villus surface area are associated with enhanced intestinal absorption capacity, while a shortening of the villi will reduce the surface area available for nutrient absorption (Walton et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The crypt is the area where stem cells divide to facilitate villus renewal; therefore, a deeper crypt indicates a rapid turnover of tissue in response to increased demand (Yang \u0026amp; Liao, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Numerous studies indicate that ETEC infection damages the morphology of the intestinal mucosa (Zhang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lv et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, histological images revealed that animals in the CON\u003csup\u003e+\u003c/sup\u003e and T1 groups exhibited villi with varying degrees of atrophy, areas of marked hyperemia, and shedding epithelial tissue in both the duodenum and jejunum-ileum. Similar results were found by Hu et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) in a study on the impact of Lf on growth performance, intestinal function, and gastrointestinal microbiota in suckling piglets. These authors reported that early-life intervention with Lf is beneficial for intestinal health, as it was able to promote the development of intestinal function and improve the composition of the gut microbiota. On the other hand, Huang et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) obtained similar data in a study on how lysozyme, an antimicrobial peptide present in milk, impacts intestinal performance in neonatal piglets challenged with \u003cem\u003eE. coli\u003c/em\u003e. They found that lysozyme facilitated recovery from \u003cem\u003eE. coli\u003c/em\u003e infection and reduced mortality and morbidity following exposure to the disease, whether through oral inoculation of the bacteria or infection acquired after contact with infected animals. They observed an increase in VH in all three evaluated intestinal sections (duodenum, jejunum, and ileum) and an increase in the VH/CD ratio in the jejunum and ileum. Therefore, it was demonstrated that this protein is beneficial for the intestinal performance of neonatal piglets.\u003c/p\u003e \u003cp\u003eFinally, Zhang et al. (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) studied the effect of dietary supplementation with a natural tannin on intestinal lesions and oxidative stress in 7-day-old piglets challenged with ETEC. Their results show that the infected untreated animals exhibited damage to intestinal morphology, experiencing a reduction in VH and an increase in CD, while the group of animals treated with tannin showed a significant improvement in intestinal morphology and relief from oxidative damage.\u003c/p\u003e \u003cp\u003eOxidative stress is one of the main factors that compromise the integrity of the gastrointestinal barrier and increase intestinal permeability (Kaplan et al., 2007). In our study we measured SOD levels and MDA levels to investigate the antioxidant activity of encapsulated Lf. Previous studies have reported that experimental infection with ETEC in neonatal piglets can induce oxidative stress through a reduction in SOD levels and an increase in MDA values (Zhang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). MDA values reveal significant differences between CON\u003csup\u003e\u0026ndash;\u003c/sup\u003e and T2 groups compared to T1 and CON\u003csup\u003e+\u003c/sup\u003e groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). According to Zeng et al. (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), higher concentrations of MDA indicate greater damage to the organism. On the other hand, the levels of SOD were significantly higher (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the T2 and CON\u003csup\u003e\u0026minus;\u003c/sup\u003e groups compared to CON\u003csup\u003e+\u003c/sup\u003e and T1 groups. These results align with a recent study that reported that Lf reduces ROS levels by activating the expression of SOD, GSH-Px, and CAT (Pan et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similar results were obtained by Hu et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), who studied the effect of early intervention with Lf on hepatic metabolism in suckling piglets. They found that piglets receiving the Lf supplement showed significantly higher levels of SOD and GR compared to the control group, which only received saline solution. Conversely, MDA levels were significantly lower in the Lf group compared to the CON\u003csup\u003e+\u003c/sup\u003e group. Therefore, they concluded that treatment with Lf was able to improve the antioxidant capacity of the liver.\u003c/p\u003e \u003cp\u003eIn another study conducted by Zhang et al. (2022), they investigated the effect of maternal supplementation with Lf on iron content and antioxidant capacity in neonatal pigs. They used sixty sows and divided them into five groups: Control (basal diet without supplements), Lf1, Lf2, and Lf3 supplemented with 100, 200, and 300 mg/kg of Lf in the basal diet, respectively, and finally the Fe-Gly group supplemented with 100 mg/kg of ferrous glycine. They analyzed the levels of the enzymes SOD, GSH-Px, total antioxidant capacity (T-AOC), and MDA in the blood and tissues. Their results showed, similarly to our study, that both LF and Fe-Gly improved the activities of GSH-Px, T-AOC, and SOD while reducing MDA levels in both the mother sows and the piglets.\u003c/p\u003e \u003cp\u003eThe mechanism by which Lf exerts its antioxidant activity may be related, on one hand, to its structural characteristics, as it is capable of binding to ferric ions, thereby reducing the formation of free radicals. On the other hand, it may also be linked to its ability to stimulate the activation of antioxidant enzymes (Zhang et al., 2022).\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eEmploying biodegradable polymers like chitosan and alginate in the encapsulation of lactoferrin not only improves piglet health outcomes but also advances the goal of sustainable animal production. The results of this study substantiate the protective role of encapsulated Lf in enhancing the health and performance of piglets challenged with ETEC. The T2 group demonstrated not only a 100% survival rate but also significantly improved average daily weight gain and a marked reduction in diarrhea incidence compared to unprotected counterparts. Blood analysis revealed a recovery from anemia and reduced oxidative stress indicators, indicating enhanced overall health due to Lf supplementation.\u003c/p\u003e \u003cp\u003eThese findings highlight the efficacy of encapsulating Lf in chitosan-alginate microparticles in preserving its bioactive properties, thereby improving gastrointestinal resilience in neonatal piglets. Given the increasing prevalence of porcine neonatal diarrhea, integrating encapsulated Lf into swine diets could offer a practical approach to mitigate its detrimental effects, ultimately promoting sustainable livestock production. Overall, this study underscores the potential for encapsulated Lf as a valuable dietary supplement in swine production, fostering both immediate health benefits and long-term productivity in livestock.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors gave their consent for research publication.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors confirm that there are no conflicts of interest with any financial organization regarding the material discussed in the manuscript.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003e All activities conducted in this trial were approved by the Institutional Committee for the Care and Use of Experimental Animals (CICUAE, EEA INTA Marcos Ju\u0026aacute;rez No. E04-21).\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the grants from PICT 3575/19.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC. A. Methodology, Wrote the original draftM. N. Methodology, review \u0026amp; editing.F. B. M. C. Methodology, review \u0026amp; editing.A. D. Formal an\u0026aacute;lisis, Methodology, review \u0026amp; editing.A. F. Formal an\u0026aacute;lisis, Methodology, review \u0026amp; editing.P. L. Formal an\u0026aacute;lisis, Methodology, review \u0026amp; editing.B. F. Formal an\u0026aacute;lisis, Methodology, review \u0026amp; editing.B. A. Supervision, Formal an\u0026aacute;lisis, Methodology, review \u0026amp; editing.B. R. Supervision, Formal an\u0026aacute;lisis, Methodology, review \u0026amp; editing. Funding acquisition and project administration\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eCots A., Mura N., Flores Bracamonte M.C and Pedraza M.L. have Fellowship from CONICET. Dr. D. Acevedo and Dra. R. Bellingeri are Members of the Research Career of CONICET. Thanks to the Pig Production Group of the EEA INTA Marcos Juarez: German Cottura, Dario Panichelli, Mariano Lattanzi, Raul Franco, and Jorge Brunori for providing animals and feed.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbad I, Conesa C, S\u0026aacute;nchez L (2021) Development of encapsulation strategies and composite edible films to maintain Lactoferrin bioactivity: A review. Materials 14:7358. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ma14237358\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eAndersen HR, Nielsen JB, Nielsen F, Grandjean P (1997) Antioxidative enzyme activities in human erythrocytes. 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Vet Res 54:9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13567-023-01139-z\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eZhu LH, Zhao KL, Chen XL, Xu JX (2012) Impact of weaning and an antioxidant blend on intestinal barrier function and antioxidant status in pigs. J Anim Sci 90(8):2581\u0026ndash;2589\u0026nbsp;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2527/jas.2012-4444\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lactoferrin, infectious diseases, piglets, bio-based polymers, neonatal diarrhea","lastPublishedDoi":"10.21203/rs.3.rs-6889295/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6889295/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnterotoxigenic \u003cem\u003eEscherichia coli\u003c/em\u003e (ETEC) is a significant cause of diarrhea in neonatal and weanling pigs, leading to considerable morbidity, mortality, and economic losses in swine production. The present study evaluated the protective effects of microencapsulated lactoferrin (Lf) in ETEC-challenged piglets, focusing on growth performance, intestinal health, and oxidative stress parameters, thereby contributing to sustainable practices in animal husbandry. The microencapsulation process, utilizing bio-based polymers, enhances the bioavailability and stability of Lf, which is for its application in veterinary medicine. An \u003cem\u003ein vivo\u003c/em\u003e assay was conducted with 40 piglets divided into four groups: CON\u003csup\u003e\u0026minus;\u003c/sup\u003e (not challenged, not treated), CON\u003csup\u003e+\u003c/sup\u003e (challenged, no treated), T1 (challenged treated with free Lf) and T2 (challenged, treated with microencapsulated Lf). Key metrics, including growth rates, diarrhea incidence, and blood parameters, were recorded over ten days. Microscopic evaluations of intestinal morphology and oxidative stress markers in liver tissues were also conducted. The results demonstrated that the T2 group exhibited a significant reduction in diarrhea incidence, enhanced survival rates (100% by day 10), and improved average daily weight gain compared to the other groups, aligning with the health and well-being goals of sustainable development. Blood analyses revealed that the CON\u0026thinsp;+\u0026thinsp;group showed signs of anemia, whereas those receiving microencapsulated Lf had normalized levels of red blood cells, hemoglobin, and hematocrit. Additionally, liver MDA levels were significantly lower, while SOD levels were significantly increased in the Lf treatment groups, indicating reduced oxidative stress. This study confirms their microencapsulation of Lf using bio-based polymers can enhance its bioactivity, contributing to better intestinal health and overall performance in piglets challenged with ETEC. This approach not only mitigates the negative impacts of porcine neonatal diarrhea on swine production but also supports sustainable agricultural practices by promoting animal health and reducing dependency on antibiotics.\u003c/p\u003e","manuscriptTitle":"Protective effects of microencapsulated Lactoferrin in enterotoxigenic Escherichia coli- challenged piglets","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-02 06:02:06","doi":"10.21203/rs.3.rs-6889295/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"14c991ee-8979-4fd0-93a6-0b9d98cde60d","owner":[],"postedDate":"July 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T22:53:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-02 06:02:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6889295","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6889295","identity":"rs-6889295","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}