Effect of single or mixed strain probiotics on milk yield of dairy cows

Brendah Kembabazi, James O Ondiek and Perminus K Migwi

Egerton University, Department of Animal Science, P O Box 536 - 20115, Egerton, Kenya
brendabakesigaki@gmail.com

Abstract

An experiment was conducted to determine the effect of single and mixed strain probiotics on milk yield of dairy cows. Treatments were CTL: basal diet, LP40: basal diet + 40g of Lactobacillus plantarum, SC40: basal diet + 40g of Saccharomyces cerevisiae, SC20LP20: basal diet + 20g of Lactobacillus plantarum + 20g of Saccharomyces cerevisiae and SC40LP40: basal diet + 40g of Lactobacillus plantarum + 40g of Saccharomyces cerevisiae. The basal diet consisted of 70% Rhodes grass hay and 30% dairy meal. Fifteen dairy cows in their early and mid-lactation stage had 14 days of adaptation and 21 days of data collection for milk yield and composition.

The combination of Lactobacilli plantarum and Saccharomyces cereviciae and greater DM intakes by the cows.

Key-words: in-vitro digestibility, Lactobacillus plantarum, Saccharomyces cerevisiae

Introduction

Growing public concern towards the use of antibiotics in animal feeding paved the way for probiotics (Suiryanrayna et al 2015). Probiotics are being used in diets of ruminants to modulate rumen metabolism (Bodas et al 2012) and ultimately enhance nutrient utilisation and animal performance (Mutsvangwa et al 2010). The live microorganisms which are the basis of probiotics are considered to benefit the host animal by improving the microbial balance of its gastrointestinal tract (Chen et al 2013; Vierra et al 2014; Murad et al 2019).

Probiotic benefits are strain-specific and not species-specific or genus-specific according to Anadón et al (2016). The mechanism by which probiotics produce their beneficial effects vary widely and include; maintaining a beneficial microbial population in the gastrointestinal tract, altering bacterial metabolism by increasing digestive enzymes and decreasing bacterial enzyme activity (Musa et al 2009).

Many commercial products are available; however, the benefits of using more than one strain or species in a single product have not been established according to FAO (2016).

Lactobacillus spp, Bifidobacterium spp, Enterococcus spp, and Saccharomyces spp are the most widely used probiotic genera in animals (Ripamonti et al 2011). Saccharomyces cerevisiae in ruminants may stabilize the pH of the rumen and therefore favor the growth of cellulolytic bacteria sensitive to low pH. The oxygen scavenging nature of Saccharomyces cerevisiae in the rumen is thought to help to mop up any traces of the gas and therefore protecting the obligate anaerobes from the air ingested in the rumen along with feed (Sheikh et al 2017). Saccharomyces cerevisiae may exert a positive effect on the digestibility especially the fibre fraction probably by stimulating cellulolytic microbial populations in the rumen (Patra 2012). It has been introduced to ruminants feeding on fibrous roughages because it can utilize part of the free sugar in the rumen and create a fermentation shift due to rapid degradation of fibrous material. Furthermore, it can secrete some metabolites that are useful for other rumen microorganisms.

Saccharomyces cerevisiae contains B-complex vitamins, amino acids, and organic acids, particularly malate, which may stimulate the growth of other rumen bacteria that digest cellulose (Kashongwe et al 2017). They also have a positive effects on milk production and some milk quality characteristics (fat, protein and lactose yield) in lactating cows under field conditions (Yalçın et al 2011)

Probiotics from lactic acid-producing bacteria (LAB) have been administered to improve rumen fermentation and therefore enhance feed efficiency by stimulating microbial fermentation. (Ridwan et al 2018). One specie of LAB that has the potential to serve as a probiotic is Lactobacillus plantarum which produces lactic acid from its metabolism (O^’Brien, 2013). The effect of L. plantarum on in-vitro rumen fermentation was influenced by dosage and the bacterial strain used (Ellis et al 2016). The cause of improved animal performance by the addition of LAB is not completely clear and the response in ruminants is inconsistent. Strain differences affect the probiotic ability to improve rumen fermentation (Wulansih et al 2018). Information on the effect of L. plantarum on rumen fermentation is lacking and shows several variations consistent with the strains of probiotic used (Wulansih et al 2018).

In an experiment where early lactating dairy cows were fed on a diet based on 60:40 concetrate and alfalfa hay ratio supplemented with Lactobacillus plantarum, Lactobacillus plantarum had no effect on milk yield and composition. The 40 % dietary alfalfa hay may have improved ruminal environment and nullified the impact of probiotics on ruminal fermentation and microbial growth, suggesting that further studies are needed to be planned with different source and level of roughage in the diet with probiotic supplementation (Boğa 2007).

The advantages of administering mixed-strain/species as probiotics may include the enhanced capability of colonizing the gastrointestinal tract and to combine the different mechanisms of action of each strain in a synergistic way (Agazzi et al 2011; Ripamonti et al 2011). The mixed-strain probiotics have a broad-spectrum effect from the different strains against infections and could increase the beneficial effects of the combined probiotics due to their synergistic adhesion effect (Adjei-Fremah et al 2018).

Using Lactobacillus plantarum and Saccharomyces cerevisiae as a mixed culture could create a low steady concentration of lactate in the rumen, thus providing a low pH medium for the activity of Saccharomyces cerevisiae which frequently increases bacterial numbers in the rumen, competing with starch utilizing bacteria. This prevents lactate build up because of their pH regulation and oxygen scavenging actions creating better conditions for cellulolytic activity by bacteria leading to increased forage utilization (Thomas 2017).

On this basis, it was considered appropriate to compare the effects of Lactobacillus plantarum and Saccharomyces cerevisiae both as single and mixed strains on feed intake and milk production of lactating dairy cows.

Materials and methods

Study site

The experiment was conducted at Tatton Agriculture Park TAP, Egerton University, Njoro. Tatton Agriculture Park TAP is within longitude 36º 36’E and latitude 0º 22’S and at an elevation of 2238 metres above sea level. The site receives annual rainfall ranging from 1000 to 1200 mm with a bimodial distribution pattern. The temperature at the locality varies between 19 and 22^oC (Egerton University Weather Station, 2018 unpublished data).

Animals and experimental design

Fifteen lactating dairy cows (8 Friesian and 7 Guernsey) in their early and mid-lactation stage with average milk production of 5 L per day and an average liveweight of 440 kg were allocated to five treatments;

CTL: basal diet of dairy meal and Rhodes grass hay;

LP40: basal diet + 40g of Lactobacillus plantarum;

SC40: basal diet + 40g of Saccharomyces cerevisiae;
SC20LP20: basal diet + 20g of Saccharomyces cerevisiae + 20g of Lactobacillus plantarum

SC40LP40: basal diet + 40g of Saccharomyces cerevisiae + 40g of Lactobacillus plantarum. The probiotics were mixed with the dairy meal on a daily basis

The cows were sprayed with DuoDip 55% E.C at the start of the experiment to control external parasites. They were housed in a zero-grazing unit each cow with individual cubicles.

Feed preparation

Culturing Saccharomyces cerivisea

The starter culture of Saccharomyces cerevisiae was purchased from a local retailer Four hundred grams (400g) of the dairy meal were placed in an anaerobic jar and with 400 ml of water and mixed to make a slurry which was autoclaved at 121^oC. The slurry was left to cool and the pH adjusted to 4.0 using citric acid. Saccharomyces cerevisiae (5g) was mixed with the slurry and incubated for seven days at 32^oC, after which a sample of the slurry with grown yeast cells was diluted with peptone water (3.75 g of peptone in 250 ml of distilled water) and put on 18 plates each with 20 ml of the diluted slurry and incubated at 32 ^oC for seven days. The plates were then removed for colony counting.

Culturing Lactobacillus plantarum

Lactobacillus plantarum isolates were taken through a resuscitation process to make them viable. MRS broth was used as the nutrient media; 2.08 g of broth were dissolved in 40 ml of distilled water. The solution was autoclaved for 15 minutes then allowed to cool to room temperature. Lactobacillus plantarum cells were then placed into the solution, and kept at 37^oC for 16 hr after which the solution was checked for turbidity as an indicator of live cells. After 24 hr, the cells were ready for culturing.

Potato Dextrose Agar PDA 19.5 g was dispersed in 200 ml of water. The solution was autoclaved for 15 minutes and allowed to cool. 20 ml of solution was poured in each petri dish (n=8) and allowed to settle and solidify in readiness for inoculation. The cells were inoculated by streaking and then placed in an anaerobic jar, and transferred to the oven at 37^oC. After 16 hr the cells were harvested and introduced into the probiotic diet.

Colony count

The test sample was diluted following the serial dilution technique. Each dilution bottle was filled with 9 ml of peptone water and 1ml of diluted sample was added to the solution making it up to 10 ml. Holding the bottle in the right hand, the cap was removed and the neck of the bottle flamed for sterilizing. 1 ml of the sample was injected into the bottle and the cap replaced and the bottle gently shaken for a uniform mixture. Ten dilution bottles were prepared. The 10^th, 11^th and 8 ^th dilution bottles were used to draw samples for inoculating in the petri dishes for colony count. The lid of the petri dish was slightly opened and the sample poured onto the petri dish and the lid replaced. The neck of the bottle was flamed and replaced with a cap. The Petri dishes were gently rotated to mix the culture and the medium to ensure that the medium covered the plate evenly. The Potato Dextrose Agar was allowed to completely gel.

The plates were incubated at 32°C for Saccharomyces cerevisiae for 7 days and 37°C for 48 hr for Lactobacillus plantarum. After 7 days or 48 hours all colonies were counted using a magnifying colony counter. Colony-forming units (cfu) were counted and ranged from 1*10⁷cfu to 1*10¹⁰cfu. These were used to form the probiotic supplements with the autoclaved dairy meal being the carrier.

Formulation of the probiotic supplements

400 g of dairy meal mixed with 400 ml of water were sterilized at 121^oC and 1.5 atm for 30 minutes then left to cool. For Saccharomyces cerevisiae, the pH of the dairy meal was adjusted to 4.0 using citric acid to attain optimum growing conditions. For Lactobacillus plantarum the pH was maintained at 7. After inoculation, 20 g and 40 g of the probiotic supplement were measured and placed into separate tubes to be added in the dairy meal before feeding. Probiotic supplemented diets were given to the treatment groups continuously for 35 days: 14 days of adaptation and 21 days of data collection.

Milk yield and composition

Cows were milked twice daily at 06:00 and 16:00 hr. Samples of milk were stored at 4 °C until analysis in a lactoscan apparatus for: fat, protein, total solids, solids-not fat, lactose and ash.

Chemical Analysis

Samples of feeds were analysed according to procedures of AOAC (1990). Neutral detergent fibre, and acid detergent fibre were determined using the methods prescribed by Van Soest et al (1991).

Data analysis

The data were analysed using the general linear model of the Statistical Analysis Systems computer package (SAS 2009). Sources of variation were treatments and error. Milk yields were corrected by covariance for yields during a 21 day period prior to incorporating the microbial additives in the diets.

Results

Feed intake was increased on those treatment which contained Saccharomyces cerevisiae but this effect was not manifested in cow milk yields (Table 1) nor in milk composition (Table 2, which did not differ among treatments.

Discussion

The reason why the 16% improvement in feed intake was not reflected in increased milk production may be because pf the low nutritive value of the diet, 70% of which was provided by Rhodes grass hay of very low quality

Conclusion

• A combination of Lactobacilli plantarum and Saccharomyces cereviciae appeared to act as a probiotic as evidenced by increased DM intakes in dairy cows.

Acknowledgements

The authors would like to thank the Center of Excellence in Sustainable Agriculture and Agribusiness Management CESAAM of Egerton University for financial support and the Department of Dairy Science, Egerton University for offering us strains of Lactobacillus plantarum used in the experiment.

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