Research Article

Effect of Dietary Supplementation of Mannan Oligosaccharide on Gut Morphology of Pre-Weaning Calves

Muhammad Usman1, Saima Ashraf2, Muhammad Shahbaz Yousaf3, Hafiz Iftikhar Hussain4, Nabeel Ijaz5, Qudrat Ullah6*, Haseeb Khaliq1, Mubasher Rauf4, Sherish Raiz8, Mushtaq Ahmad Gondal7, Hafsa Zaneb2*

1Department of Anatomy and Histology, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100, Pakistan; 2Department of Anatomy and Histology, University of Veterinary and Animal Sciences, Lahore-54000, Pakistan; 3Department of Physiology, University of Veterinary and Animal Sciences, Lahore-54000, Pakistan; 4Department of Pathology, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100, Pakistan; 5Department of Clinical Science, Faculty of Veterinary Sciences, Bahauddin Zakariya University Multan, Pakistan; 6Department of Theriogenology, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100, Pakistan; 7Institute of Continuing Education & Extension, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100, Pakistan; 8Department of Zoology, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100, Pakistan.

Received | July 14, 2025; Accepted | September 5, 2025; Published | February 16, 2026

*Correspondence | Hafsa Zaneb, Department of Anatomy and Histology, University of Veterinary and Animal Sciences, Lahore-54000, Pakistan; Qudrat Ullah, Department of Anatomy and Histology, University of Veterinary and Animal Sciences, Lahore-54000, Pakistan; Email: [email protected]

Citation | Usman, M., S. Ashraf, M.S. Yousaf, H.I. Hussain, N. Ijaz, Q. Ullah, H. Khaliq, M. Rauf, S. Raiz, M.A. Gondal and H. Zaneb. 2026. Effect of dietary supplementation of mannan oligosaccharide on gut morphology of pre-weaning calves. Sarhad Journal of Agriculture, 42(1): 326-338.

DOI | https://dx.doi.org/10.17582/journal.sja/2026/42.1.326.338

Keywords | Pre-weaning calves, Mannan oligosaccharide, Dietary supplementation, Lymphocytes, Goblet cell count, Morphometry

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

The livestock sector plays a crucial role in supporting Pakistan’s economy, contributing 58.5% to the agricultural sector and 11.61% to the national GDP. Pakistan inhabits 42.8 million cattle and 36.6 million of buffalo, and ranks 2nd after India in buffalo meat production (Anonymous, 2016). Calf rearing is labor-intensive and cost-effective sector of animal production. During the first 3-4 weeks of age, a calf undergoes a transitional phase from monogastric to compound stomach. This transitional phase is immunocompromised and leads to higher disease burden and economic losses i.e., treatment cost and death losses. Approximately 84% of total calf mortality occurs during the first month of life, with the highest risk observed in the third week (Tiwari et al., 2007; Muner et al., 2025). However, supply of milk to pre-weaning calves in Pakistan is limited as farmers prefer to use calves only for milk let-down, and later on sale the milk. It results in poor growth and a high mortality rate (Abbas et al., 2017). Sudden alteration in the diet results in imbalanced enteric flora, whereas diversity of gut microbiota is important for normal growth of animals. In ruminants, different supplementation strategies are utilized during this transitional phase to support health and production of the calves (Kaufhold et al., 2000; Quigley et al., 2006)

Antibiotics are being utilized as animal feed additives for optimal health and increased milk and meat yield (Lei et al., 2017). In milk industry antibiotics have been extensively utilized as milk replacer for calves (Heinrich et al., 2003). Currently their use as feed additive is being discouraged because:

• Sub-therapeutic application of antibiotics results in antibiotic resistance development in pathogenic bacteria of animal origin.
• Transfer of antibiotic residues occurs to the human consumer from animals’ products with subsequent development of antibiotic resistance in pathogenic bacteria of human origin.
• There is increased risk of epidemics and economic losses due to resultant antibiotic inefficacy.
• Decreased population of intestinal beneficial microbiota due to antibiotics use.

Considering all these consequences, the EU Member States prohibited the use of antibiotics in feed for farm animals in 2006 (Castanon, 2007). Therefore, replacing antibiotic-laden growth promoters in livestock production system is now essential. Researchers are now actively pursuing establishment of different classes of feed additives as replacer of in-feed antibiotics (Gibson and Roberfroid, 1995). Probiotics, prebiotics, phytogenic, organic acid, microelements have been reported as the alternative growth enhancers (Qamar et al., 2015).

Prebiotics are indigestible carbohydrates in feed that help host by promoting the growth and performance of beneficial bacteria. MOS, a mannan–based oligosaccharide prebiotic, has been proposed as one such alternative to antibiotics. These complex sugars are of interest because certain microbes (bifidobacteria and lactobacilli) can utilize them for energy. The basic components of MOS ae Glucan, mannan and chitin. MOS is a cell-wall component of Saccharomyces cerevisiae (Spring et al., 2000) and contains protein and phosphate-radicals as well as mannose. MOS is also known to augment host’s resistance to disease and stress (Grieshop et al., 2004).

Among gut pathogens, gram-negative bacteria like E. coli and Salmonella bind to the wall of intestine through mannose-specific (type I) fimbriae. The MOS functions through occupying attachment sites of these pathogenic microbes thus facilitating the flushing out of bacteria due to unavailability of the binding sites (Fairchild et al., 2001).

Prebiotics improve growth performance, fecal parameter and digestion in calves. Particularly, MOS enhances growth in dairy calves during preweaning stage (Brown et al., 2005) and improves feed intake, weight gain, and decreases severity of fecal score in Holstein calves (Ghosh et al., 2012). Prebiotics lead to increase in population of Bifidobacterial species and Lactobacilli species in intestine (Smiricky et al., 2003) and enhancement of mucosal immune system in pigs (White et al., 2002; Lei et al., 2019). Intestinal lumen diameter also improves with the inclusion of prebiotics (Hosono et al., 2003). Disaccharide lactulose amplify the gut microflora by activating the growth of intestinal probiotic bacteria (Fleige et el., 2009; Ren et al., 2024). The changes in gut microflora are known to alter the organism’s immune system (Choct, 2009; Khan et al., 2024). Xylooligosaccharide and fructooligosaccharide are reported to be beneficial to gastrointestinal health in the rats (Hsu et al., 2004). As constituent of synbiotics i.e., dextran and L. casei, prebiotics improve milk letdown in Holstein cow (Yasuda et al., 2007; Huang et al., 2023)

Dietary supplementation considerably improves the intestinal morphology. The epithelium of intestine functions as natural barricade against harmful microorganisms and toxic matters. Nutrient absorption is enhanced by morphological adaptations in the intestine, such as improved villus height, decreased crypt depth, and more villus height-to-crypt depth ratio. These structural modifications either expand the absorptive surface area or optimize cellular turnover, thereby improving overall digestive efficiency (Awad et al., 2008).

Optimal production performance in cattle is primarily determined by the status of gut health. This in turn is a function of enhanced nutrient absorption and integrity of micro-architecture. Keeping in view the immunogenic potential of MOS and evidence of its effect on maintenance of gut micro-architecture, this study investigates the impact of MOS-supplementation on intestinal morphology in calves.

Materials and Methods

Experimental animals

For this experimental study twenty Holstein calves aged 4-5 days were purchased from local dairy farm and were divided into 2 groups having 10 animals each (Detailed are described in section 2.5).

Housing of experiments animals

The trial was performed at the Pattoki Campus, University of Veterinary and Animal Sciences (UVAS), Lahore under standard husbandry conditions for 56 days. Before the start of trial, the barn was thoroughly cleaned. Calves were kept in individual calf cages bedded with straw.

Deworming and vaccination

Deworming was done during the 3rd week using prescribed anthelmintic. Upon arrival, FMD vaccine were inoculated to the calves the calves with Aftovaxpur (Merial, France). They were then vaccinated at one-week intervals against Clostridium perfringens C and D types (Marush Pvt. Ltd., Pakistan), hemorrhagic septicemia (Niab Pvt. Ltd., Pakistan), and bovine respiratory disease (Boehringer Ingelheim GmbH, Germany). Each booster vaccination takes place 21 days after the initial vaccination.

Experimental diets

From the first to the sixth week, the calves were offered milk twice daily in an amount equal to 10% of their body weight. During the week 7 and 8, milk allowance was reduced to 5.0% and 2.5% of the body weight, respectively. The animals were provided with corn-based feed and water ad libitum. The calf starter was formulated according to NRC (2001) recordation with approximate chemical composition as described previously. The calves were divided into two groups based on presence or absence of dietary MOS supplementation as follows:

Group-1: Control –Calf starter without dietary MOS supplementation

Group-2: Treated – Calf starter with dietary MOS supplementation at a rate of 04g/day/calf.

Sample collection and measurement of organ

Upon completion of d-56, five animals were randomly selected from each group. After an overnight fasting, the animals were weighed at the time of sampling. The animals were slaughtered by severing the carotid arteries to allow exsanguination. Samples of SI (ileum, duodenum, jejunum) were then taken for examination. The intestines length (ileum, duodenum, jejunum) was measured through measuring tape. The filled and empty weights of SI were measured with electrical balance.

Tissue sampling

Immediately after slaughter, fixed and mesenteric parts of SI were identified and isolated. SI was identified as the segment beyond the pylorus up to the termination of mesenteric part, clearly divisible into fixed and mesenteric part. The fixed portion of the SI is referred to as the duodenum, whereas the mesenteric portion is conventionally divided into the jejunum and ileum (Grossman and Getty 1975). Five cm long segments of each part of SI were removed, cleaned with saline and preserved in 10% neutral-buffered-formalin (NBF). One liter of NBF was prepared as; 10 % formalin, Sodium dihydrogen phosphate monohydrate 4g and Disodium dihydrogen phosphate anhydrous 6.5 g (Bancroft, 2013).

Tissue processing

For light microscopy, tissues processing was done by using the technique of paraffin embedding (Bancroft et al., 2013). Light-microscopy requires thin tissue slices (5-7um) that were attained with embedding technique through following steps:

Washing and dehydration

Following fixation in 10% NBF for 24-h, the sectioned were rinsed under constant stream of tap-water for 2 hours. Dehydration was then carried out by passing the samples through a graded ethanol series of 60%, 70%, 90%, and 100%. The tissue cassettes were immersed for 2 hours each in 60% and 70% ethanol, followed by 4 hours in 90% ethanol. Final dehydration was completed in two changes of absolute (100%) ethanol, with the tissues kept for 2 hours in each (Bancroft et al., 2013).

Clearing and infiltration

For the clearing step, the tissue cassettes were placed in two changes of xylene, each for 2 hours. Infiltration was performed using molten paraffin wax, with tissues kept in the wax for 4–6 hours at 58 °C (Liu et al., 2018). Embedding was carried out by transferring the tissues into molds filled with paraffin to form blocks, which were then cooled and solidified to facilitate proper sectioning.

Tissue mounting / sectioning

Sectioning was performed using a semi-automatic microtome (AMOS-Scientific AEM-50). The paraffin blocks were mounted on the microtome, and rough trimming was carried out at 20 µm to expose the tissue, followed by sectioning at 5 µm thickness. Ribbon sections were floated in a water-bath maintained at 45°C. To enhance adhesion, gelatin was added to the water bath, and the slices were mounted onto glass slides. These slides were now desiccated in a hot air-oven at 90°C for 10-15 minutes. For each tissue, three sections were made. These tissue sections were then stained using Mayer’s H&E technique (Bancroft, 2013). For differentiation of goblet cell count, slides were stained using PAS stain.

Hematoxylin & Eosin staining technique

H&E stains were prepared with following components (Bancroft, 2013). 2.5 g Hematoxylin, 25 ml anhydrous ethanol, 50 g alum, 500ml dH2O, 1.25 g mercuric oxide, 20 ml glacial acetic acid, and 1 % Eosin solution was formulated by through-mixing 1 g eosin in 100 ml dH2O.

Staining protocol

The slides were first deparaffinized by immersing them in xylene twice for 2 minutes each. To remove the residual xylene, they were then passed through the descending grades of alcohol (100%, 90%, 80%, and 70%) for 2 minutes each, followed by rehydration in the distilled water for a duration of 5 minutes. The tissue slices were immersed in hematoxylin for duration of 15 minutes and washed under the running tap water to eliminate extra stain. To differentiate H&E stain, slides were dipped in 1% acid alcohol for 5 seconds, subsequently washed in running water for duration of 5 minutes. Counterstaining was executed with 1% eosin for 10 minutes, residual stain was eliminated by rinsing under running water for 1–5 minutes. The sections were then dehydrated by passing them through an ascending series of various ethanol grades (70%, 80%, 90%, and 100%) for duration of 2 minutes each (Xiao et al., 2019). At last, the tissue-slices were cleared from paraffin by immersing xylene (2 x 2 min), and subsequently DPX were used to mount the cover slips.

Periodic acid schiff staining technique

This staining technique requires the following reagents. Periodic Acid (PA) Solution includes PA (0.5g) and distilled H2O (100ml). For Schiff Reagent dissolved 1g Fuchsin and 1.9g Potassium Metabisulfite in 100ml of distilled water and 2ml of HCL. The solution was intermittently mixed until it turned from yellow to light-brown. Subsequently, 500 mg of activated charcoal was added and the mixture was agitated for 1–2 minutes. Filtration was carried out by Whatman No. 1 filter paper, resulting in a clear, colorless mixture. The prepared solution was kept at 4 °C until required (Bancroft, 2013).

Staining protocol

Following deparaffinization, the slides were oxidized in 0.5% PA solution for duration of 5 minutes and then washed for approximately 5-min in distilled H2O. The slices were subsequently dyed with Schiff’s mixture for 20 minutes, followed by washing in distilled H2O for 5 minutes. Counterstaining was performed with hematoxylin for 2 minutes, and the sections were then rinsed under tap water for 2–5 minutes. Dehydration was achieved by passing the sections through an ascending series of alcohol (70%, 80%, 90%, and 100%) for 2 minutes each. Finally, clearing is done twice (2 min each) in xylene, and cover slips were mounted using DPX (Bancroft, 2013).

Histomorphometry of small intestine

Slides were observed using LABOMED®USA microscope having a digital camera and software to connect with laptop. Morphometric analysis was performed by a commercial software program (Prog Res® 2.1.1 Capture Prog Camera Control Software). Parameters assessed in the SI villi included height, width, surface area, depth of crypt, thickness of epithelium, lamina propria, tunica mucosa, and tunica externa. Height of villus (µm) was counted from the tip to the crypt junction, while crypt depth (µm) was calculated from the base of crypt to the muscularis mucosae (Awad et al., 2009). The height-to-crypt depth ratio of villus was measured as described by Castillo et al. (2008). Villus surface area (mm²) was estimated by the formula: (2π) × (VW/2) × VL, where VW represents villus width and VL represents villus length. For goblet cell identification, sections were stained using a combined Alcian Blue–PAS method (Bancroft and Gamble, 2008).

 

Table 1: Comparison of weights and lengths of small intestine between control and MOS supplemented groups (Mean ± SEM)

Parameters	Control group	MOS group	P- value
Weight of filled intestine (kg)	5.90±0.58	5.28±0.48	0.43
Weight of empty intestine (kg)	2.95±0.18	2.68±0.17	0.31
Length of intestine (inches)	1087.00±34.51	1063.20±12.07	0.53

 

Goblet cell count

The Alcian Blue PAS-stained slides were examined under a bright-field microscope (Labomed USA) at 10X and 40X magnification to count goblet cells. Differential goblet cell counting was performed. The blue-stained goblet cells were acidic mucin goblet cells. Mixed goblet cells, which contained both acidic and neutral mucin, stained purple (Bancroft, 2013).

Intraepithelial lymphocytes count

The H&E-stained slides were studied under a high magnification microscope (Labomed, USA) at 40× magnification to quantify intraepithelial lymphocytes (IELs). From each intestinal cross-section, five clear and intact villi were randomly chosen based on occurrence of a preserved lamina propria. IELs were identified as globular cells with centrally or marginally eccentrically located nuclei with minimal cytoplasm, resulting in a high nucleus-to-cytoplasm ratio (Ashraf et al., 2013).

 

Table 2: Histomorphometric parameters of duodenum in control and MOS supplemented groups (Mean ± SEM)

Parameters	Control group	MOS group	P- value
Villus height (µm)	314.23±37.18	328.13±47.34	0.82
Villus width (µm)	67.70±9.35	72.35±7.30	0.70
Villus surface area (mm2)	68.17±12.30	73.44±11.54	0.76
Lamina propria thickness (µm)	36.41±3.43	36.85±4.54	0.94
Crypt depth (µm)	92.59±17.18	94.60±13.13	0.92
Muscularis mucosa thickness (µm)	24.79±1.91a	12.23±0.58b	0.00
Tunica muscularis thickness (µm)	256.22±83.18	145.08±18.94	0.22

 

Statistical analysis

Independent sample t-test was used (SPSS v14.0) to analyze and compare data. Data were illustrated as mean ± standard error. Significant of differences were considered at p < 0.05.

Results

Weight and length of small intestine

The results for the length as well as filled and empty weights of SI are presented in the Table 1. There were non-significant differences of filled or empty weights and length of SI between control and MOS-supplemented groups.

Duodenum

The results of histomorphometric parameters of Duodenum are demonstrated in Table 2. Comparison between the control group and the MOS-supplemented group showed non-significant variations (p > 0.05) in villus morphology or related tissue parameters, including villus height, villus width, villus surface area, lamina propria thickness, crypt depth, and tunica muscularis thickness. However, the muscularis mucosae was significantly (p < 0.05) thick in the control group as compared to the MOS-supplemented group. Morphometric measurements of the duodenum, including villus length and width, surface area, depth of crypt, epithelial thickness, mucosal connective tissue, muscularis mucosae, and external layer of smooth muscle (Figure 1).

Jejunum

The results of histomorphometric parameters of Jejunum are illustrated in Table 3. Non-significant

 

 

Table 3: Histomorphometric parameters of jejunum in control and MOS supplemented groups (Mean ± SEM)

Parameters	Control group	MOS group	P- value
Villus height (µm)	284.62±21.36b	434.56±29.95a	0.004
Villus width (µm)	68.27±5.56	67.07±7.71	0.90
Villus surface area (mm2)	61.57±7.96	93.67±15.94	0.10
Lamina propria thickness (µm)	36.39±2.82	37.01±3.43	0.89
Crypt depth (µm)	139..68±21.19a	86.75±7.24b	0.04
Muscularis mucosa thickness (µm)	24.95±0.64	20.91±1.89	0.07
Tunica muscularis thickness (µm)	151.58±26.27	98.95±5.84	0.08

 

variations (p > 0.05) were detected in width, surface area, thickness of lamina propria, and muscularis layer thickness across the control and MOS-supplemented treatment. Morphometric measurements of the jejunum, including villus length and width, villus surface area, crypt depth, epithelial thickness, lamina propria, muscularis mucosae, and muscularis externa, are presented in Figure 2 (4X magnification). The MOS-supplemented group revealed significantly higher villus height and lower crypt depth (p < 0.05) compared to the control group.

 

Table 4: Histomorphometric parameters of ileum in control and MOS supplemented groups (Mean ± SEM)

Parameters	Control group	MOS group	P- value
Villus height (µm)	336.22±24.12	335.71±11.95	0.98
Villus width (µm)	80.55±9.66	65.12±5.23	0.19
Villus surface area (mm2)	86.43±14.09	69.04±5.06	0.27
Lamina propria thickness (µm)	34.71±5.07	35.91±5.24	0.87
Crypt depth (µm)	126.74±13.92	96.27±13.42	0.15
Muscularis mucosa thickness (µm)	24.98±1.38	24.35±1.29	0.74
Tunica muscularis thickness (µm)	117.74±14.52b	196.58±18.45a	0.01

 

 

Ileum

The outcomes of histomorphometric parameters of ileum are presented in Table 4. There were non-significant (p > 0.05) alterations seen in the villus structure and associated layers (height, width, surface area, crypt depth, and muscularis layer size) between the control and the MOS treated groups. Tunica muscularis thickness was greater (p < 0.05) significantly in the MOS-group relative to non-MOS-group. Morphometric measurement of ileum for villus length, area, width, thickness, depth, and muscularis-externa with 40X showing is illustrated in Figure 3.

 

Table 5: Intraepithelial lymphocytes count in the segments of small intestine in control and MOS supplemented group (Mean ± SEM)

Intestinal segment	Control group	MOS group	P-value
Duodenum	18.26±1.63	21.80±3.87	0.17
Jejunum	20.40±4.68	22.13±4.08	0.55
Ileum	18.60±1.33	23.00±2.58	0.18

 

 

Table 6: Acidic goblet cell counts in the segments of small intestine in control and MOS supplemented group (Mean ± SEM)

Segment	Control group	MOS group	P-value
Duodenum	43.06±3.71	40.40±4.75	0.67
Jejunum	32.40±7.99b	43.80±7.13a	0.04
Ileum	41.53±1.22	45.66±2.27	0.16

 

 

Intraepithelial lymphocyte count

The results of IELs count are shown in Table 5. Non-significant difference (p > 0.05) in the IELs count when comparing control and MOS-supplemented groups, though their number was consistently higher in the MOS group. Intraepithelial lymphocytes in duodenum (H& E) with 40X showing in Figure 4.

Goblet cell count in small intestine (Duodenum, Jejunum and Ileum)

Data on goblet cell amount in small-gut are shown in Table 6. Only acidic mucins containing goblet cells were found in the three intestinal sections. In duodenum and ileum there was non-significant difference (p > 0.05) of cell count of goblet cell across the control and the MOS-supplemented group. The cell count was elevated significantly (p < 0.05) in jejunum of MOS-group of treatment as compared to control. Acidic type of goblet cells in duodenum (PAS) with 40X shown in Figure 5.

Discussion

This study was executed to determine the outcome of supplementation of MOS on mucosal morphology of SI (duodenum-jejunum-ileum). Effects of prebiotics and probiotics on the morphology of gut have been thoroughly reported in the poultry and pigs. However, current knowledge about the significance of MOS-prebiotic to modulate gut morphology of calves is limited. MOS and fructo-oligosaccharide are prebiotics which are present in the yeast’s cellular wall such as Saccharomyces cerevisiae (Jiang et al., 2025; Khan et al., 2023). MOS are extracted from the surface layer of the yeast cell wall and contain mannan, glucan, protein in the ratio of 30%, 30% and 12.5% respectively, as well as a small amount of phosphate radicals. The MOS has positive effect on gut morphology and serves as an alternative binding site in the intestine for gram -iv morbific organism (Hady et al., 2012; Ghaffar et al., 2018).

Intestinal villi have important part in absorption and digestion of feed. As the length of villi increases it provides increased upper zone for captivation of nutrients in intestine. Increased absorption of nutrients not only increases the body weight but also acts as precursor of active cell mitosis and enterocytes turnover (Yamauchi et al., 2010; Haroon et al., 2022; Khaliq et al., 2025). The current trial showed that supplementation of MOS at 4g significantly increased the villus height in the jejunum. A similar outcome on the height of villi in Jejunum in chicken has been observed previously (Pourabedin et al., 2014) with MOS-supplementation. Similar observations were reported by Yang et al. (2008) in broilers fed with MOS. Ashraf et al. (2013) reported that height of villus improved in duodenum and jejunum of broiler chicken with MOS-supply. The MOS is also reported to decline the depth of crypt in jejunum of nursery pigs (Poeikhampha and Bunchasak 2011). It is well established that greater villus-height with reduced crypt depth are certainly associated with enhanced gastrointestinal and absorptive roles in the gut, as they provide a larger absorptive surface area and lower tissue-turnover-rate. (Munyaka et al., 2012). The current study found that crypt depth reduced significantly in Jejunum of MOS supplemented group. Though comparable studies could not be traced for calves but reduction of crypt-depth in jejunum of chicks has been previously studied with use of MOS (Sohail et al., 2010). A comparable decrease in depths of duodenum and jejunum crypt in broilers resulting from MOS supplementation has been testified by (Ashraf et al., 2013) and Padihari et al. (2014). Fructose-oligosacsharide, another prebiotic, when supplemented at the rate of 0.4% lead to decline in depth of jejunum and ileum crypts in the broiler chicks (Xu et al., 2003). Peker et al. (2014) studied that Saccharomyces cerevisia decreased the crypt depth in jejunum of rabbit. Crypt depth is associated with enterocytes replacement rate. An increase in enterocytes replacement rate requires additional energy which may deprive other tissues and organs of growth and development. So, the decreased crypt depth may be associated with a significant growth rate. The findings of our study indicated that MOS-supplementation did not significantly affect area of villi in the duodenum-ileum; however, an increase was observed in the area of jejunum. Although scientific literature on the actions of MOS to this parameter in calves is inadequate, previous studies have reported a surge in surfer area of villus across different intestinal segments in both healthy and heat-stressed broilers (Ashraf et al., 2013; Jahania et al., 2015). It may therefore be assumed that the mechanisms underlying villus surface area enlargement in broilers could also be functional in calves. In the present experiment, a lack of effect of MOS on the thickness of lamina propria was observed. Similar findings have been published for turkey poults (De Los Santos et al., 2007) and weanling pigs (Davis et al., 2004). Lamina propria thickness may serve as a gauge of gut-health, as it comprises dendritic-cells playing a significant part in immune function. By monitoring the luminal contents, the lamina propria contributes to protection against infection through stimulation of the adaptive-immune reaction, enhancement of gut-motility, and regulation of mucin production. In the current trail, a lessening in the thickness of the duodenal muscularis mucosa was observed Padihari et al. (2014) observed that width of muscularis-mucosa reduced in all segments of SI in broiler chicken. Yang et al. (2008) stated attenuation of muscularis-mucosa only in jejunum of the broilers supplemented with mannan oligosaccharide. Red yeast supplementation is also known to reduce muscular layer thickness in the broiler chicken (Tapingkae et al., 2016). The attenuation of muscularis-mucosa stimulates the rate of absorption of nutrients (Eurell and Frappier 2013).

The current study indicated that thickness of muscularis externa increased significantly in Ileum in MOS supplemented group, and remained unaltered in Duodenum and Jejunum. A similar observation was made by Shang et al. (2015) with FOS supplementation and was attributed to a possible increase in the bacterial fermentation and absorption area of intestine in broilers. Comparative influence of MOS-prebiotic and zinc on intestinal morphology of broiler and found that zinc supplemented group did not show significant alterations in thickness of muscularis externa. Increase in the muscularis externa thickness may be co-related with inflammation which leads to bacterial infections. Subsequent to aggregation of morbific germs lymphocytes accrue to eradicate the pathogens. It provokes the inflammatory process which may result in increased muscularis externa thickness. In such conditions, antibiotics application diminishes the pathogenic bacteria and their toxin production (Gunal et al., 2006; Sammad et al., 2022)

The current-trial demonstrated that goblet cell-counts across the duodenum-ileum didn’t fluctuate between the control and MOS-supplemented groups; however, a higher goblet cell count was observed in the jejunum of calves receiving MOS. Similar results have been described in other species: Giannenas et al. (2011) noted an increase in jejunal goblet cells in fattening pigs augmented with MOS, while De Los Santos et al. (2007) observed a comparable effect in turkey poults. Likewise, Ashraf et al. (2013), Padharia et al. (2014), and Jahanian et al. (2015) reported that MOS-supplemented chicks showed greater goblet cell amounts in the jejunum, and Baurhoo et al. (2009) documented that MOS increased goblet cell counts throughout the intestinal tract of broilers. An elevated number of goblet cells in the MOS group may enhance mucin production, thereby contributing to intestinal protection under challenging circumstance. The layer of mucus serves as a critical defensive layer by limiting the grip of intestinal germs to epithelial surface (Pourabedin et al., 2014).

Regarding the intraepithelial cells, the lack of effect of MOS on their count may be attributed to a lack of challenge typically presented by enteropathogens. Intraepithelial lymphocytes (IEL) Intraepithelial lymphocytes (IELs) act as local guard cells at the intestinal surface, and their infiltration typically increases in response to pathogen exposure. The precise mechanism underlying MOS-induced immunomodulation remains unclear; however, it is thought to be linked with an upsurge in lactic acid–producing bacteria, which promote competitive exclusion of entero-pathogens like Salmonella and E. coli, thereby reducing their prevalence. This reduction in pathogenic load may explain the absence of significant changes in IEL counts observed in the current experiment. A similar finding was conveyed by Isolauri et al. (2001), who observed non-significant differences in IEL counts across the small intestinal segments of MOS-supplemented broilers.

Conclusions and Recommendations

Dietary supplementation with MOS partially improved gut morphology in pre-weaning calves. Overall performance, particularly weight gain, was significantly enhanced with increased levels of MOS supplementation. However, non-significant differences (p > 0.05) were recorded in the filled or empty weights, SI length, villus morphology (height, width, surface area), thickness of lamina propria, crypt depth, tunica muscularis thickness, or intraepithelial lymphocyte count between control and MOS groups. Notably, the MOS group showed a significantly higher (p < 0.05) count of acidic goblet cells in the jejunum.

Funding Source

This research was not financially supported by any funding agency.

Ethical Approval

The Departmental Committee on Animal Ethics and Welfare, University of Veterinary and Animal Sciences, Lahore, Pakistan approved this study.

Consent to participate and consent to publish

All authors participated equally in this study and agreed to the publication of this work in this journal.

Novelty Statement

This study is among the first to clearly show that dietary MOS can lead to positive, specific changes in the intestinal structure of pre-weaning calves. It provides strong scientific support for using MOS as an alternative to antibiotic growth promoters in calf-rearing, particularly when milk feeding conditions are less than ideal.

Author’s Contribution

Muhammad Usman: Writing – original draft, Methodology, Investigation.

Saima Ashraf: Conceptualization, Validation.

Hafiz Iftikhar Hussain: Validation, Formal analysis.

Qudrat Ullah; Nabeel Ijaz: Formal analysis, Writing – review & editing.

Haseeb Khaliq: Validation, Statistic analysis.

Mubasher Rauf: Methodology, Data curation.

Sherish Raiz; Mushtaq Ahmad Gondal: Formal analysis, Writing.

Hafsa Zaneb: Supervision, Project administration, Final review & editing.

Generative AI or AI assisted technology statement

The authors declare that no generative AI or AI-assisted technologies were used in the writing, analysis, or preparation of this manuscript.

Conflict of interest

The authors have no competing financial interests or personal relationships that could have influenced the results of this study and declare no conflicts of interest with respect to its publication.

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