The study was carried out at Uludag University Applied Research Center for Veterinary Faculty Unit in Bursa located within the North West Turkey. This study was conducted under an approved protocol by Animal Care and Use Committee of University of Uludag.

Twenty healthy calves (5 days old) of ten each male and female were selected by clinical examination (body temperature, respiration, and pulsation rates), general appearance and occurrence of diarrhoea. Calves were randomly divided into two equal groups (control [CG] and experimental [EG]) of five male and five female.

Calves were fed colostrum within 2 h of birth and housed in individual pens until the end of the study at 56 days of age. Calves received 4 L/d of the whole milk from day 2 to 49 and 2 L/d of the whole milk during last 7 days of the study. Milk was offered twice daily. Pelleted calf starter and water were given ad libitum from day 5 (the beginning of the study). Pelleted calf starter was containing 19.58% of crude protein, 5.7% of crude fat, 22.46% of neutral detergent fiber, 9.99% of acid detergent fiber, 3.55% of acid detergent lignin, 43.23% of non-fiber carbohydrate and 9.03% of ash on a dry matter basis. Calves were weaned at 56 days of age.

Each calf in EG was supplemented with 7 g/d of a MOS product from 5 days to 56 days of age. MOS supplement was mixed with the whole milk once in the morning and administered to the calves in EG via nipple bottle at the amount recommended by the manufacturer (Celmanax, Vi-COR, Mason City, IA, USA). The prebiotic product obtained from cell wall fragments of S. cerevisiae contained 51% of glucan and 25.5% of mannan according to the certificate of analysis reported by Vi-COR.

Individual pelleted calf starter intake was measured daily by collecting residual feed after every feeding during the study. Calves were weighed before feeding at day 5 and 56. ADG, ADFI, and feed efficiency (ADG/ADFI) were calculated for each calf.

Faecal samples were collected from each calf by retrieval from the rectum on day 5, 7, 14, 21, 28, 35, 42, 49, and 56. Faecal samples were scored in respect to consistency by the same researcher on all collection days according to the following system: 1 = watery, diarrhoea; 2 = soft, unformed; 3 = soft, formed; 4 = hard, formed; and 5 = hard, dry pellets. On day 5 and 56, each faecal sample was diluted 10 folds with distilled water for pH measurement. The mixture of faecal sample and distilled water was homogenized and faecal pH was immediately measured using an electronic pH meter (PT-10, Sartorius AG, Goettingen, Germany) fitted with a glass electrode. Faecal samples could be collected from 8 of 10 and 9 of 10 calves in CG and EG, respectively, on day 56 by retrieval from rectum using sterile gloves. Faecal samples were placed in sterile sampling bags and immediately transported to the laboratory for bacterial enumeration. One g of each faecal sample was homogenized with 9 mL of saline peptone water in a stomacher laboratory blender (Stomacher 80 Biomaster, Seward Inc., Port Saint Lucie, FL, USA). Subsequently, serial 10-fold dilutions were made in saline peptone water and plated onto relevant selective media. Lactobacillus was grown on Man Rogosa Sharpe agar (MRS agar, Hypet Media, Diatek, Istanbul, Turkey). Each plate was incubated at 37°C for 48 h in an anaerobic jar (Oxoid AN0035A, Basingstoke, Hampshire, UK) with gas generating sachet (Oxoid CN0020C, Basingstoke). Non–spore-former rods, gram-positive and catalase-negative isolates were regarded as Lactobacillus. Bifidobacterium was grown on Bifidobacterium Selective Medium agar (BSM agar, Hypet Media, Diatek). The plates were incubated at 37°C for 48 to 72 h in anaerobic jar with gas generating sachet. The selected bacterial colonies were investigated with regard to cell morphology by Gram staining. The colonies with gram-positive rods and characteristic bifurcated “Y” and “V” shapes were recorded as Bifidobacterium. C. perfringens was grown on 4-methylumbelliferyl phosphate-supplemented (MUP, Merck 1.00888, Darmstadt, Germany) Tryptose Sulfite Cycloserine agar (TSC agar, Hypet Media, Diatek) containing egg yolk emulsion and selective supplement. Each plate was incubated at 37°C for 24 h in an anaerobic jar with gas generating sachet. Each presumptive black colony was added to 10 mL of Thioglycolate Broth (Merck 1.08190) and then incubated at 37°C for 16 to 18 h. Activated cultures were prepared for identifying by characteristic colony morphology, Gram staining and biochemical tests. The colonies with gram-positive and nonmotile rods, lactose positive, nitrate reduction positive, gelatine positive and motility negative were considered to be C. perfringens. Total coliform was grown on Violet Red Bile (VRB, Oxoid CM0107, Basingstoke) agar. After 24 to 48 h of incubation at 37°C, the red colonies with halos were typical for E. coli. The colonies typical for E. coli were enumerated and inoculated into Lactose Broth (LB, Oxoid CM0137, Basingstoke) with Durham tube. After 24 to 48 h of incubation at 44°C, gas and lactic acid production positive tubes were streaked onto Eosin Methylen Blue (EMB, Oxoid CM0069, Basingstoke) agar and confirmed to be E. coli by biochemical tests, such as Indol (SIM, Merck 1.05470), Methyl red and Voges-Proskauer (MR-VP, Merck 1.05712,) and Citrate (Oxoid CM0155, Basingstoke). Indol positive, MR positive, VP negative and citrate negative indicated the presence of E. coli type-1. The bacterial counts were expressed as log10 CFU (colony forming unit) per gram of faecal samples.

Body temperatures were taken per rectum using a digital thermometer at the same times and recorded on day 5, 7, 14, 21, 28, 35, 42, 49, and 56. Health status of calves was monitored daily during the study. Faecal score of one was considered to be diarrhoea. In addition, tail and/or hind limbs stained with faeces were evaluated as a finding of diarrhoea. Calves with diarrhoea were treated by the farm veterinarian according to protocols established by the Department of Internal Medicine, Faculty of Veterinary Medicine. In the case of calves with diarrhoea, medication used (antibiotics, anti-inflammatory, immun stimulant, and electrolyte products) and dosage and the duration of treatments applied for diarrhoea were recorded throughout the study. Costs associated with diarrhoea treatments were calculated based on current costs for each product and daily dosage for each medication for individual calves.

All statistical analyses were conducted by using SPSS ver. 5.0 (SPSS Inc., Chicago, IL, USA) (SPSS, 2004). Data for body weight, ADFI, ADG, and feed efficiency were tested to determine normal distribution by Kolmogorov-Smirnov test. Body weight, ADFI, ADG, and feed efficiency were tested for homogeneity of variance and analysed by independent sample T-test. Data for faecal score and faecal pH were tested to determine normal distribution by Kolmogorov-Smirnov test and F-test, respectively. Faecal score, faecal pH, faecal bacterial populations and body temperature were analysed by independent sample T-test. The incidence of diarrhoea was analysed by chi-square test and Fisher exact test. The statistical analyses for the duration of treatment applied for diarrhoea and the costs associated with diarrhoea treatments were performed by Mann-Whitney test. Differences between groups were considered significant at p≤0.05.