Effect of Concentrate: Roughage Ratio and the Addition of Kefir on the Production Characteristics of Ruminant in vitro

1. Introduction

Feed additives are extremely important in livestock ration due to the increase in nutrient utilization, change in rumen fermentation, and optimization of efficiency in livestock production processes. Due to the prohibition of synthetic hormones and antibiotics in livestock feed additives in many countries, the use of probiotics, feed enzymes, herbs, and other “natural" supplements is becoming increasingly popular and considerable. In addition to increasing the efficacy, these natural food additives minimize the transmission risk of human infections, reduce antibiotic use and risk of developing antibiotic resistance, and limit the removal of contaminants. Recently, many additives have been examined to replace or reduce the use of antibiotics, such as probiotics ( 1 ). Probiotic bacteria are live bacterial feed additives that increase the microbial balance of the host animal. It has been shown that probiotics have multiple functions, including the prevention of young animals from enteropathy diseases, enhancement of feed quality, animal growth, and immunity system ( 2 - 4 ).

Kefir is saline, thick, lightly carbonated, and fermented milk mixture that is often cultured with bacteria and yeasts. Kefir is made from the kefir grains through the inoculation of cow, sheep, or goat milk ( 5 ). It includes proteins, polysaccharides, ethyl alcohol, lactic acid, salt, minerals, and vitamins ( 5 ). Kefir grains are comprised of lactic acid bacteria, acetic acid bacteria (e.g., species Lactobacillus sp., Lactobacillus acidophilus, Leuconostoc sp., Acetobactersp. and Streptococcus sp.), yeasts (e.g., Saccharomycessp., Torula sp.),and other microorganisms.

The health effects of kefir on mice, rats, fowl, and goat kids have been investigated widely ( 6 , 7 ). However, there has been very little research regarding the usage of kefir in calves.

Manipulation of the rumen microbial environment and the creation of optimal conditions have always been important for animal nutritionists. Yeast in ruminants helped to keep the rumen pH stable and allowed low pH-sensitive cellulolytic bacteria to grow. Rumen yeast assisted in the protection of required anaerobes from feed ingested in the rumen as well as feed consumption. In sheep, total VFA production, acetate to propionate ratio, and dry matter digestibility increased in vitro ( 8 ). Accordingly, the use of feed additives "probiotics and enzymes" have been considered due to the lack of long-term consequences. The current study aimed to investigate the efficiency of kefir in vitro fermentation and kinetics of various concentrate and roughage ratios combinations as a probiotic source using cattle rumen liquor.

2. Materials and Methods

Experimental diets were designed to evaluate in vitro digestion of six concentrate to forage ratios (100: 0, 80:20, 60:40, 40:60, 20:80 and 0: 100, respectively). Kefir was used in diet in the amount of 0, 0.8, 1.6, 2.4, and 3.2 ml. The concentrate feed consisted of corn (61.00%), soybean (27.00%), wheat bran (8.50%), and minerals mixture (3.5%, including calcareous [0.79%], sodium bicarbonate (0.99%), di-calcium phosphates (0.59%), trace premix (0.40%), and salt (0.79%). Table 1 presentsthe chemical composition data for the feed ingredients and the tested ratios.

2.1. Gas Production

The quantity of the in vitro gas production was estimated according to Menke, Raab ( 9 ). Fluids from rumen were gathered from two bulls slaughtered at the slaughterhouse of the Basrah Governorate. The rumen fluid was poured into a sealed container that was pre-warmed and filtered using sterile gauze. Rumen fluid was washed and fixed well mixed with CO2 stream. For each experiment, 200 mg of samples from each diet were collected into 125 ml glass bottles. A buffer solution was prepared as described by Tilley and Terry ( 10 ). During sample inoculation, CO2 was constantly pumped at 39°C. The syringes were immediately filled with buffered rumen fluid (30 ml) and placed in a shaking water bath set at 39°C. Cumulative sample volume measurements from the three replicates were read manually at 0, 4, 6, 8, 12, 24, 48, 72, and 96 h of incubation. Fermentation syringes without samples (blanks) were included to control group gas generated directly from rumen fluid. The data were fitted to the exponential model used by Ørskov and McDonald ( 11 ) and the equation (Y= A+B(1-exp-ct)) following the subtraction of gas output from blanks, where 'Y' is the cumulative gas produced over time in ml; 't' is time in hours; 'A' and 'B' parameters are used to explain the fermentation potential ('A+B' represents the maximum fermentation potential), and 'c' is the constant gas output rate used to explain fermentation speed. Gas production was calculated as followed after 24 h:

GPDM= total gas production (ml)/ substrate DM (g)

GPOM= total gas production (ml)/ substrate OM (g)

GPNDF= total gas production (ml)/ substrate NDF (g)

GPADF= total gas production (ml)/ substrate ADF (g)

Metabolizable energy was estimated using the method adopted by Menke, Raab (9): ME in MJ= 2.20 + 0.136 GP + 0.057 CP. The short chain fatty acid (SCFA) output (mM) was calculated as SCFA= 0.0239 GP-0.0601,using the equation described by Getachew, Makkar ( 12 ).According to Menke, Raab (9), invitro digestibility of organic matter (ivOMD, g/kg OM) was calculated as ivOMD= 14.88 + 0.889 GP+ 4.5 CP (%) + 0.0651 ash (%), where GP is a net GP in ml after 24 h of incubation from 200 mg of dry sample.

2.2. in vitro Degraded Dry Matter (ivTDDM)

Using methods described by Anele, Südekum ( 13 ),in vitro degraded dry matter (ivTDDM) was determined and was calculated as ivTDDM= feed (DM) incubated − residue (DM) recovered in the crucibles/feed (DM) incubated ( 14 ). Partitioning factor (PF, a measure of fermentation efficiency) was calculated as: PF= ivTDDM (mg) /GP₂₄ (mL). Following the method of Blümmel, Makkar ( 15 ), the in vitro microbial mass production can be calculated as Microbial mass (mg)= ivTDDM (mg) - (GP₂₄ (mL)×2.2),where: 2.2: stoichiometric Ally factor, according to the amounts (mg) of C, H, and O required for the production of 1 mM of SCFA and associated 1 mL gas.

2.3. Statistical Analysis

Data on in vitro degradability and fermentation parameters have been statistically analyzed using SPSS software (version 20) ( 16 ) through a two-factor general linear model (six levels of concentrate: roughage and five kefir levels). The separation of means was achieved using the Bonferroni test within the same statistical program. Regression analysis of various parameters was also carried out. A p-value less than 0.05 (P<0.05) was considered statistically significant.

3. Results and Discussion

The ratio of concentrate and roughage (C: R) and the addition of kefir on the gas output parameters, including soluble gas production (a), insoluble gas (b), and constant gas production rate (P<0.05) were evaluated in this study. Insoluble gas production (c), and the potential degree of gas production (a plus b) are tabulated in tables 2 and 3. The total gas production produced in 96 h was presented in figures 1, 2, and 3. In the C: R ratio, accumulated gas output was highest at 96 h, at 100:0, and 80:20 (Figure 1). There was no interaction effect between the ratios of C: R and kefir (P>0.05) on gas kinetics (Table 4).

Figure 1. Gas production (mL/ 200 mg) for different ratios of concentrates: roughages(Concentrate=100% concentrate, 80CR=80% concentrate, 60CR=60% concentrate, 40CR=40% concentrate, 20CR=20% concentrate, and Roughage=0% concentrate: 100%alfalfa hay).

Figure 2. Gas production (mL/ 200 mg) for different levels of kefir and 100% concentrates (conck0=100 %concentrat+0 kefir, conck 0.8= 100% concentrate+ 0.8 ml kefir, = 100%concentrate+ 1.6 ml kefir, = 100% concentrate+ 3.2ml kefir, and conck 4.0= 100% concentrate+ 2.4 ml kefir)

Figure 3. Gas production (mL/200 mg) for different levels of kefir and 100% Alfalfa hay (alfalfak0=100 %alfalfa+0 kefir, alfalfak0.8= 100% alfalfa+ 0.8 ml kefir, alfalfak1.6 = 100% alfalfa + 1.6 ml kefir, alfalfak3.2= 100% alfalfa + 3.2 ml kefir and alfalfk4.0= 100%alfalfa+2.4 ml kefir)

The presence of a high concentrate ration improved the rate and duration of fermentation ( 17 ). Similarly, Kang, Wanapat ( 18 ) observed that total gas production improved with the increase in the percentage of concentrate in the diet. The addition of kefir increased gas dynamic and aggregation which may be attributed to the fact that kefir might activate the rumen microbes and increase the incubated substratum digestibility, leading to progress in the kinetics of gas output.

Tang, Tayo ( 19 ) reported that accumulated gas production was improved by supplementation of a yeast culture. These findings agreed with those obtained by Wang, He ( 20 ), indicating that probiotic supplementation improved overall gas output following the incubation of various types of diets. In addition to improving the overall production of gas, the incorporation of yeast can also lead to qualitative improvements in the gases ( 21 ).

Table 5 demonstrated the influence of the C: R ratio and the incorporation of kefir on in vitro digestibility rumen pH, and NH3-N. Total gas production at 24 h of incubation decreased significantly (P<0.05) with the rise of roughage percentage in the ratio, from 67.82 to 52.97 ml for 100% concentrate to 0% alfalfa hay, respectively. Both IVDMD and IVOMD improved with the increase of concentrate proportions (P<0.05). Consistently, the results of the studies conducted by Phesatcha, Phesatcha ( 22 ) and Kang, Wanapat ( 18 ) revealed that digestibility can be optimized by the elevated percentage of concentrated feeds in the ration. This may be attributed to the resultant induction of microorganisms’ growth, which led to increased digestibility. The inclusion of kefir dosage improved the analysis of IVDMD and IVOMD as well. The maximal IVDMD and IVOMD were obtained at 24 h of incubation in the R: C ratio of 80:20 withthe addition of Kefir, which was higher than those in the group of control by 4.76% and 3.15%.Wang, He ( 20 ), observed that the increase in IVDMD and in vitro NDF (IVNDFD) occurred due to the inclusion of Kefir. Tang, Tayo ( 19 ) showed that the addition of yeast increased digestibility in vitro with low-quality roughage. The inclusion of Saccharomyces cerevisiae (S. cerevisiae) improved the NDF and digestibility of both extracts in bovine animals at 2.5 g/ d ( 23 ).Cagle, Fonseca ( 24 ) reported that the addition of dry yeast at 10 g/d improved the digestibility of DM and NDF in finishing beef cattle ( 24 ). The fact that the digestibility of nutrients has been increased with the addition of kefir may be attributed to the enhancement of the rumen microorganisms. In addition, S.cerevisiae was proposed to be able to scavenge the available oxygen on the tops of freshly consumed feed to sustain metabolism activity which in turn reduced the ability of rumen redox ( 25 - 27 ).

The beneficial effect of kefir and its mode of action can be summed up in the changes occurring in the rumen fermentation rates and forms. Certain probiotics are successful in increasing and stabilizing the pH by stimulating and inducing the protozoa to rapidly consume the starch and thereby with the bacteria that digest the starch and generate lactate ( 17 ). The reduction of rumen acidity increased the growth and activity of fibrolytic bacteria ( 28 ). Prado, Blandón ( 29 ) demonstrated that kefir was made through the fermentation of milk with kefir starting grain that contain a symbiotic consortium of microbes impacted by grain origin and growth conditions. However, there are variations in the total number of microorganisms in grains ( 29 ), including lactic acid bacteria (LAB) (Lactobacillus spp.), yeast (Saccharomyces spp., Kluyveromyces spp., Kazachstania spp., and Lachancea spp.), and acetic acid bacteria (Acetobacter spp.) ( 30 ). Proteins, lipids, and lactose, as well as ethanol and lactic acid produced kefir. In the diet, kefir could be a rich source of calcium, essential amino acids, and vitamins. Some microbial strains of kefir community have been used as probiotics or antibacterial substance producers in the past ( 31 ). Although there has been few studies on the use of milk kefir in livestock or companion animals ( 32 ), it has been suggested that kefir can be a protein-rich livestock feed ( 33 ) or probiotics in both ruminant and non-ruminant herbivores. Several studies have described the anti-inflammatory, anti-allergic ( 34 ), and probiotic effects of kefir on the digestive systems ( 5 ). Moreover, kefir has been shown to interact with the gut microbiota and reduce pathogenic diseases, such as Clostridium difficile ( 35 ).

The calculation of the relationship between the produced gases, the in vitro organic matter digestibility coefficient, and the percentage of the concentrated diet showed a positive linear relationship with an accuracy of 0.9862 for gas production and 0.9140 for the digestibility coefficient of organic matter (Figure 4). The increase in the percentage of concentrated feed by one unit increased the percentage of gas by an average of 0.153 ml/200 mg and the digestibility coefficient of organic matter by 0.0978%.Moreover, ivTDDM correlated positively with less accuracy than the previous parameters (0.8730). The mean increase in ivTDDM was 0.0008 mg after increasing the concentrated feed by one unit (Figure 5). On the other hand, PF had a linear and negative correlation with concentrated feed with high accuracy (0.9648) and the mean of reduction in PF was 0.0016 ml/mg after the increase of concentrate by one unit.

Figure 4. Gas production (GP) and in vitro organic matter digestibility (IVOMD %) associated with percentages of dietary concentration.

Figure 5. Association between partitioning factor (PF) and in vitro total dry matter digestibility (ivTDDM) with concentration percentages in the diet.

Animal efficiency is the most direct indicator for measuring the quality of nutrition. Performance data, on the other hand, is hard to determine and describe the rumen’s potential interactions. Only a few studies exanimated cause-effect interactions of roughage combination in terms of energy or protein substrate involvement, rumen-degrading chemicals, and potentially associated effects that contribute to favorable or poor animal performance results. The need to evaluate VFAs in in vivo digestibility tests was crucial to determine the concentration, difference, and uptake of fermented VFAs. However, VFA levels were only determined at the end of the 48-hdigestion cycle in the phase of in vitro gas digestibility, providing useful information when concentrations were comparable in a variety of feed forms. As the most abundant source of energy which accounts for at least half of all digestible energy, VFAs were formed by fermentation of the rumen substrate and consumed subsequently ( 36 ).

In conclusion, the high ratio of concentrate to roughage and the addition of 1.6 ml of kefir to the overall dietary substrate could improve the fermentation of the rumen and boost the digestibility of the feed with no changes in the counts of microorganisms.

Authors' Contribution

Study concept and design: H. A. J. A. and M. S. A.

Acquisition of data: H. A. J. A. and M. S. A.

Analysis and interpretation of data: H. A. J. A. and M. S. A.

Drafting of the manuscript: H. A. J. A. and M. S. A.

Critical revision of the manuscript for important intellectual content: H. A. J. A. and M. S. A.

Statistical analysis: H. A. J. A. and M. S. A.

Administrative, technical, and material support: H. A. J. A. and M. S. A.

Ethics

This study was ethically approved by the Institutional Animal Care and Use Committee at the University of Basrah, Basrah, Iraq.

Conflict of Interest

The authors declare that they have no conflict of interest.

References

1. Frizzo L, Soto L, Zbrun M, Bertozzi E, Sequeira G, Armesto RR, et al. Lactic acid bacteria to improve growth performance in young calves fed milk replacer and spray-dried whey powder. Anim Feed Sci Technol. 2010; 157(3-4):159-67.
2. Novak K, Davis E, Wehnes C, Shields D, Coalson J, Smith A, et al. Effect of supplementation with an electrolyte containing a Bacillus-based direct-fed microbial on immune development in dairy calves. Res Vet Sci. 2012; 92(3):427-34.
3. Sun P, Wang J, Zhang H. Effects of Bacillus subtilis natto on performance and immune function of preweaning calves. J Dairy Sci. 2010; 93(12):5851-5.
4. Timmerman HM, Mulder L, Everts H, Van Espen D, Van Der Wal E, Klaassen G, et al. Health and growth of veal calves fed milk replacers with or without probiotics. J Dairy Sci. 2005; 88(6):2154-65.
5. Leite AMdO, Miguel MAL, Peixoto RS, Rosado AS, Silva JT, Paschoalin VMF. Microbiological, technological and therapeutic properties of kefir: a natural probiotic beverage. Braz J Microbiol. 2013; 44:341-9.
6. Ataþoðlu C, Akbað H, Tölü C, Das G, Savas T, Yurtman I. Effects of kefir as a probiotic source on the performance of goat kids. S Afr J Anim Sci. 2010; 40(4)
7. Toghyani M, kazem Mosavi S, Modaresi M, Landy N. Evaluation of kefir as a potential probiotic on growth performance, serum biochemistry and immune responses in broiler chicks. Anim Nutr. 2015; 1(4):305-9.
8. Ganai A, Sharma T, Dhuria R. Effect of yeast (Saccharomyces cerevisiae) supplementation on ruminal digestion of bajra (Pennisetum glaucum) straw and bajra straw-based complete feed in vitro. Anim Nutr Feed Technol. 2015; 15(1):145-53.
9. Menke K, Raab L, Salewski A, Steingass H, Fritz D, Schneider W. The estimation of the digestibility and metabolizable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor in vitro. J Agric Sci. 1979; 93(1):217-22.
10. Tilley J, Terry dR. A two‐stage technique for the in vitro digestion of forage crops. Grass Forage Sci. 1963; 18(2):104-11.
11. Ørskov E-R, McDonald I. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J Agric Sci. 1979; 92(2):499-503.
12. Getachew G, Makkar H, Becker K. Effect of polyethylene glycol on in vitro degradability ofnitrogen and microbial protein synthesis fromtannin-rich browse and herbaceous legumes. Br J Nutr. 2000; 84(1):73-83.
13. Anele U, Südekum K-H, Hummel J, Arigbede O, Oni A, Olanite J, et al. Chemical characterization, in vitro dry matter and ruminal crude protein degradability and microbial protein synthesis of some cowpea (Vigna unguiculata L. Walp) haulm varieties. Anim Feed Sci Technol 2011; 163(2-4):161-9.
14. Van Soest Pv, Robertson JB, Lewis B. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J dairy Sci. 1991; 74(10):3583-97.
15. Blümmel M, Makkar H, Becker K. In vitro gas production: a technique revisited. J Anim Physiol Anim Nutr. 1997; 77(1‐5):24-34.
16. Spss I. Statistics for windows, version 24. 0 [computer software]. Armonk, NY: IBM Corp. 2016.
17. Polyorach S, Wanapat M, Cherdthong A. Influence of yeast fermented cassava chip protein (YEFECAP) and roughage to concentrate ratio on ruminal fermentation and microorganisms using in vitro gas production technique. Asian-Australas J Anim Sci. 2014; 27(1):36.
18. Kang S, Wanapat M, Phesatcha K, Norrapoke T, Foiklang S, Ampapon T, et al. Using krabok (Irvingia malayana) seed oil and Flemingia macrophylla leaf meal as a rumen enhancer in an in vitro gas production system. Anim Prod Sci. 2016; 57(2):327-33.
19. Tang S, Tayo G, Tan Z, Sun Z, Shen L, Zhou C, et al. Effects of yeast culture and fibrolytic enzyme supplementation on in vitro fermentation characteristics of low-quality cereal straws. J Anim Sci. 2008; 86(5):1164-72.
20. Wang Z, He Z, Beauchemin KA, Tang S, Zhou C, Han X, et al. Evaluation of different yeast species for improving in vitro fermentation of cereal straws. Asian-Australas J Anim Sci. 2016; 29(2):230.
21. Hristov A, Oh J, Firkins J, Dijkstra J, Kebreab E, Waghorn G, et al. Special topics—Mitigation of methane and nitrous oxide emissions from animal operations: I. A review of enteric methane mitigation options. J Anim Sci. 2013; 91(11):5045-69.
22. Phesatcha K, Phesatcha B, Wanapat M, Cherdthong A. Roughage to Concentrate Ratio and Saccharomyces cerevisiae Inclusion Could Modulate Feed Digestion and In Vitro Ruminal Fermentation. Vet Sci. 2020; 7(4):151.
23. dos Santos Monnerat JPI, Paulino PVR, Detmann E, Valadares Filho SC, Valadares RDF, Duarte MS. Effects of Saccharomyces cerevisiae and monensin on digestion, ruminal parameters, and balance of nitrogenous compounds of beef cattle fed diets with different starch concentrations. Trop Anim Health Prod. 2013; 45(5):1251-7.
24. Cagle CM, Fonseca MA, Callaway TR, Runyan CA, Cravey MD, Tedeschi LO. Evaluation of the effects of live yeast on rumen parameters and in situ digestibility of dry matter and neutral detergent fiber in beef cattle fed growing and finishing diets. Appl Anim Sci. 2020; 36(1):36-47.
25. Campanile G, Zicarelli F, Vecchio D, Pacelli C, Neglia G, Balestrieri A, et al. Effects of Saccharomyces cerevisiae on in vivo organic matter digestibility and milk yield in buffalo cows. Livest Sci. 2008; 114(2-3):358-61.
26. Chaucheyras-Durand F, Walker N, Bach A. Effects of active dry yeasts on the rumen microbial ecosystem: Past, present and future. Anim Feed Sci Technol. 2008; 145(1-4):5-26.
27. Patra AK. The use of live yeast products as microbial feed additives in ruminant nutrition. Asian J Anim Vet Adv. 2012; 7(5):366-75.
28. Chung Y-H, Walker N, McGinn S, Beauchemin K. Differing effects of 2 active dried yeast (Saccharomyces cerevisiae) strains on ruminal acidosis and methane production in nonlactating dairy cows. J Dairy Sci. 2011; 94(5):2431-9.
29. Prado MR, Blandón LM, Vandenberghe LP, Rodrigues C, Castro GR, Thomaz-Soccol V, et al. Milk kefir: composition, microbial cultures, biological activities, and related products. Front Microbiol. 2015; 6:1177.
30. Lopitz-Otsoa F, Rementeria A, Elguezabal N, Garaizar J. Kefir: a symbiotic yeasts-bacteria community with alleged healthy capabilities. Rev Iberoam Micol. 2006; 23(2):67-74.
31. Golowczyc MA, Silva J, Teixeira P, De Antoni GL, Abraham AG. Cellular injuries of spray-dried Lactobacillus spp. isolated from kefir and their impact on probiotic properties. Int J Food Microbiol. 2011; 144(3):556-60.
32. Fouladgar S, Shahraki AF, Ghalamkari G, Khani M, Ahmadi F, Erickson PS. Performance of Holstein calves fed whole milk with or without kefir. J Dairy Sci. 2016; 99(10):8081-9.
33. Plessas S, Trantallidi M, Bekatorou A, Kanellaki M, Nigam P, Koutinas AA. Immobilization of kefir and Lactobacillus casei on brewery spent grains for use in sourdough wheat bread making. Food Chem. 2007; 105(1):187-94.
34. Lee M-Y, Ahn K-S, Kwon O-K, Kim M-J, Kim M-K, Lee I-Y, et al. Anti-inflammatory and anti-allergic effects of kefir in a mouse asthma model. Immunobiology. 2007; 212(8):647-54.
35. Carasi P, Racedo SM, Jacquot C, Romanin DE, Serradell M, Urdaci M. Impact of kefir derived Lactobacillus kefiri on the mucosal immune response and gut microbiota. J Immunol Res. 2015; 2015
36. Sutton J. Digestion and absorption of energy substrates in the lactating cow. Journal of Dairy Science. 1985; 68(12):3376-93.