Livestock Research for Rural Development 22 (10) 2010 Notes to Authors LRRD Newsletter

Citation of this paper

Influence of feeding complete diet supplemented with thermotolerant probiotic yeast (Saccharomyces cerevisiae) on humoral immune response in Nellore ram lambs

Ch Harikrishna, M Mahender, Y Ramana Reddy, M G Prakash, D B V Ramana*, A Sarat Chandra, Ch Venkatasesaiah and M Venkateshwarlu

Department of Livestock Production and Management, College of Veterinary Science, Sri Venkateswara Veterinary University, Rajendranagar, Hyderabad – 500030, Andhra Pradesh State, India
* Senior Scientist, Central Research Institute for Dryland Agriculture, Hyderabad, India


Eighteen Nellore ram lambs (87 1.8 days of age and 12.33 0.08 kg body weight) were used to evaluate the effect of supplementing yeast (Saccharomyces cerevisiae) on humoral immunity using heat killed Brucella abortus plain immunogen. Lambs were fed complete diets and assigned randomly to one of three dietary treatments. Treatment diets were without yeast (CON; n = 6), with mesophilic yeast-MTCC-1813 (MPY; n = 6) and with thermotolerant yeast-OBV-9 (TPY; n = 6). Blood samples (10 ml) were collected from the jugular vein of all the experimental lambs on 7, 14, 21 and 28 days of post sensitization into a clean, dry, sterilized test tube without adding anti-coagulant prior to the morning feeding to monitor antibody titers by standard tube agglutination test (STAT).


Antibody titers in lambs fed CON, MPY and TPY diets varied from 4.49 to 6.52, 4.99 to 9.16 and 5.99 to 9.16, respectively. TPY group had significantly (P<0.01) higher antibody titers on 7th and 14th day post sensitization compared to MPY and CON groups of lambs, whereas both MPY and TPY groups had significantly (P<0.01) higher antibody titers on 21st and 28th day post sensitization, compared to CON group of lambs. Peak titers were achieved by 21st day post sensitization in lambs fed CON diet, while it was by 14th day for lambs fed both MPY and TPY diets. By 28th day post sensitization, STAT titers were reduced in lambs fed CON diet, whereas further improved in lambs fed both MPY and TPY diets.


It is concluded that, complete diet with theromotolerant yeast appears to be beneficial when compared to mesophilic yeast in achieving higher serum antibody titres from STAT revealing higher humoral immune response in ram lambs.

Keywords: antibody titres, Brucella abortus antigen, standard tube agglutination test


Probiotics are used as feed additives to enhance the animal performance by improving the balance of microbial flora in the gastrointestinal tract and nutrient utilization (Wallace and NewBold 1992). One of the most commonly used probiotic in livestock enterprise is yeast (Saccharomyces cerevisiae). But the yeast used so far was mesophilic in nature and may not exert more beneficial action due to the harsh environmental (temperature, variation in pH, and bile concentration) conditions under which they have to survive in the gastrointestinal tract of the animal (Lankaputra and Shah 1995). Identification of a thermo, acid, osmo and bile tolerant strain of Saccharomyces cerevisiae will have more pronounced beneficial effect on meat and milk producing animals. Two strains of thermotolerant Saccharomyces cerevisiae have been identified (Bhima et al 2008). Some researchers like Soltan (1998), Coppola and Turnes (2004), Dhole (2004), Haghighi et al (2005), Al Homidan and Fahmy (2007), Kumprechtova and Illek (2007) and Nayebpor et al (2007) conducted studies on probiotics and reported that yeast cells have ability to influence animal health by increasing humoral immune response. Fuller (1989) defined probiotics as the live microbial supplements which benefits the host organism. Similarly Netherwood et al (1999), Fooks and Gibson (2002) and Patterson and Burkholder (2003) defined probiotics as “A live microbial feed that is beneficial to health”.


Therefore, the present experiment was conducted with an objective to study the influence of feeding complete diet supplemented with thermotolerant yeast in comparison with mesophilic yeast and control on humoral immune response of Nellore ram lambs by challenging with Brucella abortus plain antigen.


Materials and methods 

Experimental design, animals and diets


Experimentation was conducted at the Livestock Experimental Station, Livestock Research Institute (LRI) and Department of Veterinary Microbiology and Immunology, College of Veterinary Science of Sri Venkateswara Veterinary University (SVVU), Rajendranagar-500 030, Hyderabad, India and the study area was classified as Deccan plateau in southern part of India, at latitude 17O20/ N, longitude 78O30/ E and elevation of 536 m above sea level. Eighteen Nellore ram lambs on average 87 1.8 days of age and 12.33 0.08 kg body weight were purchased from livestock farmers of Hyderabad District born in the rainy season of 2008 and assigned randomly to one of three treatment diets (6 ram lambs/treatment diet). These diets (Table 1) were complete diets processed into mash without yeast (CON; n = 6), with 0.1% mesophilic yeast (MPY; n = 6), and with 0.1% thermotolerant yeast (TPY; n = 6).

Table 1.  Ingredients and chemical composition of experimental complete diets


Complete dietsa




Ingredients, g/kg DM

Sorghum straw








Groundnut cake




Sunflower cake








Mineral and vitaminsb




Common salt




Mesophilic yeast culture




Thermotolerant yeast culture




     Feed cost/tones, Rs.




Nutrient, g/kg DM




Dry matter




Organic matter




Crude protein




Neutral detergent fibre




Acid detergent fibre




aComplete diets were (1) without yeast (CON; n = 6) (2) with 0.1% mesophilic yeast     
 (MPY; n = 6) (3) with 0.1% thermotolerant yeast (TPY; n = 6).

bVitamin supplement was added @ 10 g/100 kg diet and composition per 1 kg contained 

(vitamin A, 4,50,000 IU; vitamin D3, 1,100,0000 IU; vitamin E, 3.18g; Mn,10.9 g; I,1.09g
Zn, 22.73 g; Fe, 22.73 g; Cu, 2.73 g; Co, 0.635; Mg, 100 g; Se, 0.1 g).

The lyophilized thermotolerant yeast (Saccharomyces cerevisiae, OBV-9), and mesophilic yeast (Saccharomyces cerevisiae, MTCC-1813) obtained from DBT project on “Development and Application of Thermotolerant Probiotic Yeast for Enhanced Animal Productivity”, Department of Animal  Nutrition, College of Veterinary Science, Rajendranagar, Hyderabad, India were used in the present study. The complete diets were formulated to have 12% CP (DM basis). Feeding of animals lasted for 180 days.


All the experimental animal groups were kept separately under hygienic conditions in well ventilated pens (4 m x 3 m) and were not allowed for grazing. Deworming was done to all animals at the beginning of the experiment and once again in the middle of the experiment i.e. after 90 days. Healthy surroundings and proper cleanliness were maintained in the experimental sheds. The treatment diets (CON, MPY and TPY) were offered to the animals twice a day (two equal meals at 09:00 and 15:00 h) with free access to clean, fresh and wholesome drinking water for the duration of the study.


Preparation of Brucella abortus plain antigen


Heat killed Brucella abortus plain antigen was procured from Institute of Veterinary Preventive Medicine, Ranipet-632 403, Tamilnadu State, India. Heat killed phenolised suspension of Brucella abortus plain antigen was used for the sensitization of lambs. The suspension was aseptically drawn into a centrifuge tubes and centrifuged at 10,000 x g for 15 min. The supernatant was discarded and sediment was re-suspended in normal saline. The procedure was repeated for three times to make the antigen suspension in normal saline and turbidity of the cell suspension was adjusted between Mc Farland tube number three and four (i.e. between 9 x 108 cells/ml and 1 x 109 cells/ml) by adding required quantity of sterile normal saline and was used as T independent antigen for inoculation.


Sensitization of lambs


At the end of the experimental feeding, all ram lambs were sensitized with heat killed Brucella abortus plain antigen administered intramuscularly as per the body weight and a booster dose was inoculated after 15 days. Prior to sensitization, all ram lambs were screened for Brucellosis with the help of Rose Bengal Plate Test (RBPT). The RBPT was performed according to the method described by Alton et al (1975).


Blood collection and monitoring of humoral immune response


Blood samples (10 ml) were collected from the jugular vein of all the experimental animals into a clean, dry, sterilized test tube without adding anti-coagulant prior to the morning feeding at weekly intervals i.e. 7, 14, 21 and 28 days of post sensitization. Serum was separated by centrifugation at 2000 x g for 15 min. and stored at –20C till use. Clean glassware and analytical grade chemicals were used for the study. The antibody titres against Brucella abortus plain antigen in serum samples were measured by standard tube agglutination test (STAT) and titers were expressed as log2/0.05 ml. STAT was performed according to Alton et al (1975).


Statistical methods


Data were analyzed according to the procedures suggested by Snedecor and Cochran (1994) and the difference between treatment means was tested for significance by Duncan’s multiple range and F Test (Duncan 1955).


Results and discussion 

The Nellore ram lambs fed TPY diet had significantly (P<0.01) higher antibody titers against Brucella abortus plain antigen compared to lambs fed other two test diets (MPY and CON) on 7th and 14th day post sensitization, whereas both probiotic yeasts had significantly (P<0.01) higher antibody titers compared to lambs fed CON diet on 21st and 28th day post sensitization (Table 2).

Table 2.  Least square means of STAT titers (log2) against Brucella abortus plain antigen in  Nellore ram lambs fed complete diets containing yeast cultures

Days post sensitization

Complete diets1 (STAT titers (log2/0.05 ml)





7 days





14 days





21 days





28 days





1Complete diets were (1) without yeast (CON; n = 6) (2) with 0.1% mesophilic yeast                     (MPY; n = 6) (3) with 0.1% thermotolerant yeast (TPY; n = 6)

 a, b, c:Means with different superscripts row wise differ significantly (P<0.01); *(P<0.05)

The results of current study were in agreement with the findings of Yorong et al (2005) and Milewski (2009) in lambs fed yeast based diets. Several workers like, Soltan (1998) in cattle, Nayebpor et al (2007) and Al Homidan and Fahmy (2007) in broiler chickens and Kumprechtova and Illek (2007) in calves also reported, dietary supplementation of Saccharomyces cerevisiae and DFM probiotics enhanced humoral immune response and thus, the findings of present study corroborate with their results.


Peak antibody titer was achieved by 21st day post sensitization in lambs fed basal diet (CON), while for lambs fed MPY and TPY diets, the peak titer was attained by 14th day. By 28th day post inoculation, the STAT titer was reduced drastically in lambs fed CON diet, whereas the titer was further improved in probiotic yeasts (MPY and TPY) fed lambs. The reasons for higher antibody titers against Brucella abortus plain antigen might be attributed to thermotolerant and mesophilic probiotic yeasts, which had positive effects on the balance and the role of the intestinal flora, which reinforced the immune defense, as reported by Coppola and Turnes (2004). The results of the present study were also in agreement with the findings of Dhole (2004) and Haghighi et al (2005), who reported probiotics are known to enhance the immune response along with restoration of mucosal barrier function and oral administration of probiotics increasing the natural antibodies against several different antigens in both the gut and the serum, respectively.




The authors are extremely thankful to the financial assistance from DBT Project, Department of Animal Nutrition and appreciation is expressed to Dr. K. Dhanalaxmi and Dr. Y. N. Reddy, Professors, Department of Veterinary Microbiology and Immunology, College of Veterinary Science, Hyderabad, India for conducting laboratory analyses.



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Received 10 August 2010; Accepted 15 August 2010; Published 1 October 2010

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