Probiotic Attributes of Lactobacillus fermentum NKN51 Isolated from Yak Cottage Cheese and the Impact of Its Feeding on Growth, Immunity, Caecal Microbiology and Jejunal Histology in the Starter Phase of Broiler Birds

Augmentation in the perception of society towards the scientific acquaintance has led the people to turn out to be more vigilant concerning their health aspects. Therefore, the demand for animal protein has also increased in recent years. Poultry meat, being the most widely accepted world wide with no religious taboos, is well known for its specific taste, flavor and low-fat virtues. After the discovery of antibiotics, the poultry industry flourished more with their use as growth promoters in poultry production. Since decades, the antibiotics have been an imperative part of poultry production as a growth promoter (AGPs) but the extravagant use of these has resulted in amplified trouble of antibiotic resistance (Furtula et al., 2010; Forgetta et al., 2012), which eventually influences living beings in terms of their health issues (Diarra et al., 2010). Thus, with the ultimate aim of reducing resistance development in bacterial pathogens, there is an urgent need to look for such alternatives, which can be utilized as growth-promoters in broilers in addition to the ability to control pathogenic infections in them.
 
Therefore, the exploration of the probiotic bacteria in the current scenario is the need of the hour for their further expedition as an alternative to antibiotics in the poultry industry. Among the probiotics, genus Lactobacillus is the most common group of probiotic bacteria to be used widely in the industry today. Although the probiotic properties of genus Lactobacillus, the natural gut inhabitants with GRAS status (Generally Recognized as Safe) are well known to researchers but even after being used extensively in the food industry, various inconsistent and adverse reports regarding the use of probiotic at some point of time are not uncommon, which may be as a result of lack of the information at genetic level itself (Sharma et al., 2018). Thus, at present, it becomes elemental to have a detailed study regarding the use of the respective probiotic bacteria with some known genetic properties before introducing into an animal model. L. fermentum, a promising probiotic bacterium, has undergone various research studies and is found to be bestowed with the various functional attributes which can be utilized for further application from a scientific point of view. Lactobacillus fermentum NKN51, a novel probiotic, isolated from Yak cottage cheese by IIT, Roorkee, India is reported to have a novel phytase, PhyLf of class PTP that can enhance the bioaccessibility of mineral ions. Its resistance to oxidative environment and gastric milieu along with the capability of releasing micronutrients are distinctive attributes of PhyLf which depicts a valid point for its further use in ameliorating the nutritional value of cereals and animal feed (Sharma et al., 2018). The present study was thus conducted to explore the effects of Lactobacillus fermentum NKN51 on performance parameters of slow-growing broilers including growth, immunity and gut physiology in their starter phase so as to address the wellbeing of the poultry birds with improved gut physiological conditions leading to better utilization of nutrients for maximum gains without the use of antibiotics.

The present study was conducted at the division of Post-Harvest Technology, ICAR-Central Avian Research Institute, Izatnagar in the year 2019 with due consent from the Institute’s Animal Ethics Committee (IAEC). A total of one hundred and twenty-eight day-old broiler chicks (n=128) of CARIBRO-VISHAL were procured from broiler farm, ICAR-Central Avian Research Institute, India, which were allotted randomly in four experimental groups in a completely randomized design (CRD). Each treatment group included 4 replicates with 8 chicks in each making a total of 32 chicks per treatment group. The broiler birds were raised under the standardized conditions of a deep litter system using paddy husk as a litter material with a floor space provision of 0.5-1 square ft per bird. Initially, the birds were provided with 23 hrs light with the light intensity of 3-4 foot-candle which was then reduced up to 16-18 hours with the light intensity of 0.5-1 foot-candle. On the first day, the brooding temperature was maintained around 95 °F which was then reduced 3-5 °F weekly till it reached 70 °F. The birds were reared for a period of 28 days to assess the effect of feeding of L. fermentum NKN51 in slow-growing broilers in the early phase of life (pre-starter and starter period). The culture of Lactobacillus fermentum NKN51 was isolated from Yak cottage cheese samples of Himachal Pradesh by the Indian Institute of Technology, Roorkee, India and was revitalized with the use of MRS broth and agar. Each of the four treatment groups were fed different levels of Lactobacillus fermentum along with the basal feed i.e. T1 (basal feed + L. fermentum @ 10⁷ cfu/gm of feed), T₂ (basal feed + L. fermentum @ 10⁶ cfu/gm of feed), T₃ (basal feed + L. fermentum @ 10⁵ cfu/gm of feed) and T₄ (basal feed only). The T₄ was served as the control group. The components of the basal diet have been summarized in Table 1 (Mandal et al., 2013). During the experimental period of 28 days, growth performance parameters including weekly body weight, feed intake and feed conversion ratio (FCR) were evaluated. The in-vivo cell-mediated immunity response was evaluated as per the methodology of Cheng and Lamont (1988) on 25^th  day post-hatch (n=8) as a response to phyto-haemagglutinin type P (PHA-P), whereas the humoral immunity was evaluated by using the method of Siegel and Gross (1980) with slight modification by injecting 1 ml of 1% sheep RBC suspension intravenously at 22^nd  day post-hatch in broilers (n=8). On the 28^th day (5 days post-immunization), 2 ml of blood was collected from the jugular vein, after which, immune serum was harvested and determination of antibody titer was done by haemagglutination test (HA) in which, the reciprocal of the highest dilution showing clear agglutination was taken as the end titer which was expressed as log₂. Further, the gut microbiology was conducted as per the methodology of Tatini et al., (1984), where samples (n=8) of the caecal contents were serially diluted in 0.85% sterile saline solution to evaluate the Lactobacillus, total plate and E. coli counts using lactobacilli MRS agar, plate count agar and EMB agar, respectively.  The colony counts were noted from the countable dilutions and were expressed as log₁₀ value of colony forming units per gram of intestinal contents (log₁₀ cfu/g). In addition to these parameters, jejunal histomorphometry was also done for each of the treatment groups (n=8) to measure villus height, villus width, crypt depth, crypt width, villus height-crypt depth ratio and intestinal absorptive surface area. The formula used for calculating the mean absorptive surface area as follows (Kisielinski et al., 2002).
   
Where,
M = intestinal absorptive surface area, villus W = mean width of villi, villus L = mean length of villi and crypt W = mean width of crypts.


 
The data obtained for each of the parameters were subjected to test of significance by analysis of variance (one way ANOVA) as per the methodology of Snedecor and Cochran (1994) using IBM-SPSS software package. The statistical significance was expressed as P<0.05 using Duncan’s multiple comparison tests to compare means.

The key indicator for the wellbeing of broilers can certainly be attributed to their growth performance since their performance is straightly associated with their feed utilization efficiency. The novel findings in this experiment revealed that the supplementation of Lactobacillus fermentum NKN51 @ 10⁷ cfu/gm i.e. T₁ in the feed of slow broiler chicks resulted in significant (P<0.05) improvement in growth performance parameters including body weight (Table 2) and feed conversion ratio (Table 3). These findings are in agreement with the previous studies (Walkunde et al., 2011; Bai et al., 2013; Zhang and Kim, 2014; Li et al., 2019).
 

 

 
One of the most imperative attributes of probiotic bacteria is considered to be its effect on the immune response. In the present study, the birds of the treatment groups fed probiotics @ 10⁷ cfu/gm i.e. T₁ had significantly (P<0.05) better cell-mediated and humoral immunity as compared to that of the control group (Table 4). These results are in agreement with the results of Fathi et al., (2017), describing that the dietary supplementation of probiotics imparted the positive response towards the serum immunoglobulin M (IgM) and cell-mediated immunity as compared to that of the control, however, the immunoglobulin A (IgA) and immunoglobulin Y (IgY) were also improved but not significantly. The satisfactory immune response of dietary probiotics in broilers was also supported by other recent studies too (Seidavi et al., 2017). According to earlier studies, there is a close association between the microbiota of the gut and the intestinal immune system (Gabriel et al., 2006; Applegate et al., 2010). In another study conducted by Park et al., (2018), it was stated that the genome of L. fermentum constitutes candidate genes involved in the immune mechanism. The oral supplementation of L. fermentum can augment innate and adaptive immune cell generation, trafficking and function in the host (Westfall et al., 2018).
 

 
With the supplementation of Lactobacillus fermentum NKN51 in broiler feeding, the total plate and E.coli counts were found to be reduced significantly (P<0.05) as compared to that of the control group fed basal diet only, however, the Lactobacillus count was significantly (P<0.05) higher in probiotic fed broiler groups (Table 5). These results are in agreement with the results of Smulikowska et al., (2005) who documented that a probiotic in diets decreased the fecal microflora of broiler chickens. A comparable conclusion was presented by Gunal et al., (2006) explaining that the counts of these microbes were affected (P<0.05) by dietary treatments such as probiotic mixture. The decrease in the microbial count recovered from the faecal matter of chickens fed with Lactobacillus may have been attributable to the lactic acid production or production of antimicrobial substances and these were accredited to the concept of competitive exclusion of pathogens. Further, Yun et al., (2017) also concluded that faecal Lactobacillus count was found significantly higher in broilers fed probiotic as compared to control; however, there was no significant difference in the E. coli counts.  In contrast, Cengiz et al., (2015) proclaimed that the dietary probiotic supplementation in broilers did not affect the population of gut microflora.
 

 
Further, supplementation of L. fermentum in broiler feed significantly (P<0.05) improved the villus height, villus width and villus height-crypt depth ratio thus enhancing the nutrient absorption capacity of the intestine (Table 6), which is in agreement with the earlier research findings (Al-Baadani et al., 2016: Ding et al., 2019). Increased villus height and villus width contributed towards the better intestinal absorptive surface area in the L. fermentum fed groups exhibiting maximum nutrient absorption in groups fed L. fermentum @ 10⁷cfu/gm of basal feed.
 

Thus, the data obtained from the present study revealed that the incorporation of Lactobacillus fermentum NKN51, a novel probiotic in broiler diet @ 10⁷cfu/gm of feed can impart the better weight gain, improved feed efficiency, enriched immune status including both cell-mediated as well as humoral immunity along with the boosted gut health assuring reduced level of microbes and better nutrients absorption capacity of intestine.