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Lactation performance and diet digestibility of dairy cows in response to the supplementation of Bacillus subtilis spores
Livestock Science 200 (2017) 35–39
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Lactation performance and diet digestibility of dairy cows in response to the supplementation of Bacillus subtilis spores
MARK
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V.L. Souzaa, , N.M. Lopesb, O.F. Zacaronib, V.A. Silveirab, R.A.N. Pereirac, J.A. Freitasd, ⁎ R. Almeidaa, G.G.S. Salvatib, M.N. Pereirab, a
Department of Animal Science, University of Paraná, Curitiba, PR 80035-050, Brazil Department of Animal Science, University of Lavras, Lavras, MG 37200-000, Brazil c Minas Gerais State Ag Research Enterprise, EPAMIG, Lavras, MG 37200-000, Brazil d Department of Animal Science, University of Paraná, Palotina, PR 85950-000, Brazil b
A R T I C L E I N F O
A B S T R A C T
Keywords: Bacillus subtilis Dairy cattle Milk composition Milk yield Probiotic
Bacillus subtilis is a transitory microorganism of the digestive tract, non-pathogenic to animals, and capable of forming spores that are resistant to heat and cold. As an animal feed probiotic the microorganism is supposedly capable of increasing diet digestibility and immunity. Two experiments were conducted to evaluate the effect of Bacillus subtilis spores on milk yield and composition and diet digestibility. In both experiments lactating dairy cows were fed in tie stalls and treatments were force-fed once per day. In experiment 1, 18 Holsteins in late lactation (246 ± 75 days in milk) received a sequence of the treatments Bacillus subtilis strain C-3102 (3.0×109 colony-forming units of spores per day) or Placebo in a crossover design with 39-day periods, a 10-day wash-out between periods, and response evaluated after the 28th day of the periods. The supplementation of Bacillus subtilis spores did not elicit detectable changes in intake (18.3 kg/d, P=0.91), milk (25.3 kg/d, P=0.66) and solids yield and concentration, total tract nutrient digestibility, and chewing activity. In experiment 2, 30 cows (161 ± 72 days in milk) with high milk somatic cell count (725,000 cells/mL) received the same treatments for 16 weeks, in a covariate adjusted randomized block design with repeated measures over time. Bacillus subtilis spores increased the yields of milk (25.3 vs. 23.6 kg/d, P=0.02), protein (0.816 vs. 0.763 kg/d, P=0.01), total solids (2.718 vs. 2.566 kg/d, P=0.05), and energy (60.7 versus 56.5 MJ/d, P=0.02) and milk urea-N tended to be reduced (19.3 vs. 20.8 mg/dL, P=0.06). Milk somatic cell count did not differ between treatments. The positive lactation response to Bacillus subtilis spores supplementation occurred when the probiotic was fed for 16 weeks and there was no evidence to suggest that increased diet digestibility was a mediator of the response.
1. Introduction Human food production from animals supplemented with live microorganisms as a replacement to chemical feed additives is in line with the natural trend of the consumer market. The addition of probiotics to the diet may have beneficial effect on gut physiology and immune function (Reid, 2008). Spore forming bacteria, such as Bacillus subtilis, have been used as probiotic supplements for humans and animals (Cutting, 2011). Bacillus subtilis is a transitory microorganism of the digestive tract, non-pathogenic to animals, and capable of forming spores resistant to heat and cold, having high stability in the diet (Sanders et al., 2003; Carlin, 2011; Cutting, 2011). The spores germinate at the intestinal lumen, which is required for its action as an animal feed probiotic. The bacterial population of Bacillus subtilis in the digestive tract is reduced after the cessation of the supplementation
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(Sanders et al., 2003). For this reason, the dietary supplementation of Bacillus subtilis should be performed daily. Probiotics based on bacterial spores can survive passage through the stomach (Hoa et al., 2000) with the potential to be more resistant to the low gastric pH compared with probiotic supplementation in the form of vegetative cells. The supplementation of Bacillus subtilis has improved the performance of non-ruminants (Fritts et al., 2000; Hooge, 2008; Zhang et al., 2012, 2013; Lee et al., 2014) and calves (Sun et al., 2010). Bacillus subtilis can increase anaerobiosis in the digestive tract, which favors native proliferation of Lactobacilli capable of producing lactic acid and inhibiting pathogenic bacteria growth (Maruta et al., 1996; Sanders et al., 2003) and can improve immune function (Sun et al., 2010). Although increased diet digestibility is frequently proposed as a plausible response to spore forming bacterial supplements in nonruminants, currently no study in lactating dairy cows have demon-
Corresponding authors. E-mail addresses: [email protected] (V.L. Souza), [email protected]fla.br (M.N. Pereira).
http://dx.doi.org/10.1016/j.livsci.2017.03.023 Received 15 June 2016; Received in revised form 29 March 2017; Accepted 30 March 2017 1871-1413/ © 2017 Elsevier B.V. All rights reserved.
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were 0.3 g/d of viable spores of Bacillus subtilis on calcium carbonate or Placebo (calcium carbonate). The active agent in the probiotic consisted of viable endospores of a single strain of Bacillus subtilis originally isolated from soil in Japan and dried and pasteurized to kill vegetative cells. The final product supplied Bacillus subtilis strain C-3102 (Calpis Co. Ltd., Tokyo, Japan) to provide a minimum daily intake of 3.0×109 cfu of viable spores. Gelatin capsules were filled with the treatments for daily oral dosing of each cow on return to their tie stalls after the morning milking.
strated this mechanism of action. This research addresses the hypothesis that lactating dairy cows fed Bacillus subtilis can improve the lactational performance with positive changes in milk composition. Thus, two independent experiments were performed to investigate the response of dairy cows in lactation performance, milk composition, and diet digestibility to the supplementation with Bacillus subtilis spores. 2. Material and methods The experimental procedures were in agreement with the ethical principles in animal experimentation of the Committee of Ethics in Animal Experimentation of the University of Paraná, Brazil (protocol number 050/2010).
2.1.2. Sample collection and analysis Between days 28 and 39, samples of feed ingredients and orts from each cow were sampled daily and frozen. Composite samples per period were formed on an as fed basis for further analysis. The contents of dry matter (DM; method 934.01), organic matter (OM; ash method 924.05), crude protein (CP; method 984.13) and ether extract (EE; method 920.39) were analyzed according to the Association of Official Analytical Chemists (AOAC, 1990). Ash free neutral detergent fiber (aNDFom) was analyzed with porous crucibles following the recommendations of Van Soest et al. (1991) with heat-stable amylase and sodium sulfite. Acid detergent fiber was analyzed non-sequentially. Samples from nine consecutive milkings were taken on days 29, 30, and 31 and a daily composite was formed in proportion to the volume secreted on each milking. The concentrations of protein, fat, lactose, and total solids were measured by infrared spectrophotometry using a Bentley 2000 analyzer (Bentley Instruments, Chaska, USA). The SCC was determined in a Somacount 150 equipment (Bentley Instruments, Chaska, USA) based on the principle of laser-based flow cytometry. Milk urea-N (MUN) was analyzed with a ChemSpec 150 (Bentley Instruments, Chaska, USA) that utilizes a modified Berthelot reaction. The daily secretion of milk energy (MJ/day) was calculated (NRC, 2001): {[(0.0929×% fat)+(0.0547×% protein)+(0.0395×% lactose)]×kg of milk}×4.184. The SCC was transformed to a linear scale from 0 to 9 (SCC score) in which scores represented the following values of SCC (×1000 cells/mL): 12.5 for SCC score 0; 25 for SCC score 1; 50 for SCC score 2; 100 for SCC score 3; 200 for SCC score 4; 400 for SCC score 5; 800 for SCC score 6; 1600 for SCC score 7; 3200 for SCC score 8; and 6400 for SCC score 9. The SCC score was calculated from the natural logarithm of the measured SCC (1000 cells/mL) and the above mentioned SCC value of each score: SCC score=−3.6438+1.4427× Ln(SCC). Negative values were rounded to zero. Body weight (BW) and body condition score (BCS) were determined on day 35 to describe experimental units. The BCS was scored using a 1–5 scale, thin to fat (Wildman et al., 1982). The same three independent evaluators scored each cow. Cows were weighed immediately after the morning milking. The total tract apparent digestibility coefficient of DM, OM, aNDF, and Non-aNDF OM was determined on days 33–35 by total fecal collection. Feces were collected concurrent to defecation during three 8-h sampling periods and weighed. The second and third sampling periods were each delayed by 8 h to avoid a major disturbance to the animals, while still representing a 24-h collection period. Fecal aliquots (equal fresh weight basis) were immediately frozen along the collection period and a composite sample was formed. Daily digestible OM intake (DOMI, kg/day) was calculated. Feed efficiency was determined as the ratio of milk yield to DM intake. The efficiency of energy use was defined by the ratio between milk energy (MJ/day) and DOMI. On day 28, chewing activity was assessed by visual observation of the buccal activity of each cow, at 5-min intervals, continuously for 24 h. Chewing time (min/ day) was defined as the sum of ingestion and rumination times. Chewing, ingestion, and rumination per unit of DM intake were calculated using the DM intake of the day in which chewing activity was evaluated.
2.1. Experiment 1 2.1.1. Cows, design, and treatments Eighteen Holstein cows, with 246 ± 75 days in milk (DIM), 29.7 ± 5.8 kg/d of milk yield, and with somatic cell count (SCC) below 100,000 cells/mL at the start of the experiment, were individually fed in sand bedded tie stalls at the Better Nature Research Center (www. holandesflamma.com.br) and were millked three times per day in a adjacent herringbone parlor. Cows were paired blocked based on parity (primiparous and multiparous) and milk yield and were assigned to a sequence of two treatments in a crossover design, with 39-day periods. Between the two experimental periods, a 10-day washout was adopted, in which the animals received the same basal diet with no treatment addition. Response variables were evaluated on the last 12 days of each period. Cows were fed on a Total Mixed Ration (TMR) mixed and offered twice daily starting at 0500 and 1300 h. The TMR was offered in amount sufficient to provide at least 10% of the offered as daily refusal. The composition of the TMR is reported in Table 1. Diet offered and orts from each cow were measured daily during the experiment. Treatments
Table 1 Composition of the offered total mixed ration in ingredients and of the consumed in nutrients (g/kg of dry matter) on treatments Bacillus subtilis spores or Placebo in Experiment 1 and of the basal diet in Experiment 2. Experiment 1 Bacillus subtilis Corn silage Tifton hay Soybean meal Citrus pulp High moisture corn Finely ground mature corn Calcium salt of fatty acids Urea Magnesium oxide Limestone NaCl Sodium bicarbonate Minerals and vitaminsa Crude protein Neutral detergent fiber (aNDFom) aNDFom from forage Acid detergent fiber Ether extract Ash Non-fiber carbohydratesb
Experiment 2
Placebo
500 41 202 102 112
424 218 158 156 13 4
13 4 3 8 3 8 4 172 337 268 238 48 93 350
171 335 267 238 48 93 353
8 4 9 6 177 301 221 215 52 88 385
a Composition per kg: 185 g of Ca; 150 g of P; 30 g of Mg; 30 g of S; 240 mg of Co; 3000 mg of Cu; 8000 mg of Mn; 12,000 mg of Zn; 90 mg of Se; 180 mg of I; 1000 KIU Vitamin A; 250 KIU Vitamin D3; 6250 IU Vitamin E. b NFC (g/kg of dry matter)=1000 – (crude protein+aNDFom+ether extract+ash).
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2.2. Experiment 2
Table 2 Intake, lactation performance, feed efficiency, milk somatic cell count (SCC) and urea-N (MUN) concentration, and body weight (BW) and condition score (BCS) on treatments Bacillus subtilis spores or Placebo (Experiment 1).
2.2.1. Cows, design, and treatments Experiment 2 was performed in a commercial dairy farm located 40 km from the University of Lavras. The 50 cow herd was housed in a sand bedded tie stall and was milked twice per day in a herringbone parlor. The herd was chosen to represent a high SCC scenario in which the effect of Bacillus subtilis supplementation on SCC and lactation performance could be evaluated. Thirty Holsteins (seven primiparous) with 161 ± 72 DIM at the beginning of the experiment were paired blocked based on SCC and milk yield and were randomly assigned to the same two treatments of experiment 1 for 16 weeks. The mean SCC at blocking was 716,000 cells/mL for treatment Bacillus subtilis and 734,000 cells/mL for Placebo, and milk yield was 29.6 and 29.9 kg/d, respectively. Data used for blocking was used as covariate in the statistical model. The daily dosage of each treatment per cow was mixed to a cup of ground corn and top-dressed to the feed offered in the morning. A technician was hired to perform the daily allocation of treatments to each cow and to assist with data collection and feed management during the experiment. Samples of feed ingredients and the daily refusal were collected and frozen for further analysis as previously described. Individual feed intake was not recorded to avoid disturbance on herd routine and because of constraint in skilled personal and equipment continuously at the farm. The ingredient and nutrient composition of the basal diet is presented in Table 1. Milk samples were obtained at 7day intervals along the 16-week comparison period for analysis as previously described. The BW and BCS were determined at 4-week intervals.
Variables
Bacillus subtilis
Placebo
s.e.m.a
P-value
18.3 3.07 11.99 25.4 0.700 0.787 1.094 2.871 29.0 32.8 44.1 114.9 66.1 1.39 5.65 2.48 17.5 673 3.8
18.3 3.08 11.94 25.2 0.683 0.790 1.079 2.845 29.0 33.3 43.9 114.7 65.3 1.37 5.48 2.55 17.0 668 3.8
0.16 0.078 0.263 0.38 0.0137 0.0160 0.0211 0.0467 0.060 0.023 0.038 0.100 1.05 0.023 0.126 0.171 0.40 7.3 0.05
0.91 0.89 0.90 0.66 0.39 0.89 0.61 0.69 0.94 0.17 0.73 0.89 0.60 0.53 0.43 0.76 0.39 0.65 0.95
Dry matter intake (kg/day) Dry matter intake (% of BW) DOMIb (kg/day) Milk yield (kg/day) Fat yield (kg/day) Protein yield (kg/day) Lactose yield (kg/day) Solids yield (kg/day) Fat (g/kg) Protein (g/kg) Lactose (g/kg) Solids (g/kg) Milk energy (MJ/day) Milk/Dry matter intake Milk energy/DOMI (MJ/kg) SCC scorec (0–9) MUN (mg/dL) BW (kg) BCS (1–5) a b c
Standard error of the means. Digestible organic matter intake. Equivalency of the linear scores. 2.48=70,000 cells/mL. 2.55=73,000 cells/mL.
Table 3 Lactation performance, milk somatic cell count (SCC) and urea-N (MUN) concentration, and body weight (BW) and condition score (BCS) on treatments Bacillus subtilis spores or Placebo (Experiment 2).
2.3. Statistical analysis
P-value Variables
2.3.1. Experiment 1 Data were analyzed using the MIXED procedure of SAS (SAS Institute, 2008) with a model containing the random effect of cow (1–18) and the fixed effects of period (1, 2) and treatment (Bacillus subtilis, Placebo).
Milk yield (kg/day) Fat yield (kg/day) Protein yield (kg/day) Lactose yield (kg/day) Solids yield (kg/day) Fat (g/kg) Protein (g/kg) Lactose (g/kg) Solids (g/kg) Milk energy (MJ/day) SCC scorec (0–9) MUN (mg/dL) BW (kg) BCS (1–5)
2.3.2. Experiment 2 Data were analyzed as repeated measures using the MIXED procedure of SAS (SAS Institute, 2008) with a model containing the continuous effect of covariate (measure of the same variable before treatments allocation), the random effect of block (1–15), and the fixed effects of treatment (Bacillus subtilis, Placebo), week (1–16), and the interaction between treatment and week. The best covariance structure was defined by the Akaike's information criterion among first-order autoregressive, unstructured, and compound symmetry. The mean square for the effect of cow nested within treatment was used as the error term to test the treatment effect. Probability values below 0.05 were considered significant and below 0.10 were considered trend.
Bacillus subtilis
Placebo
s.e.m.a
Treat
Treat×Weekb
25.3 0.605 0.816 1.074 2.718 24.1 32.9 42.6 108.0 60.7 5.34 19.3 635 3.64
23.6 0.572 0.763 1.050 2.566 25.2 33.1 43.5 109.5 56.5 4.81 20.8 623 3.65
0.42 0.0228 0.0127 0.0238 0.0476 0.070 0.047 0.023 0.135 1.05 0.339 0.46 7.9 0.061
0.02 0.35 0.01 0.52 0.05 0.30 0.83 0.02 0.48 0.02 0.31 0.06 0.33 0.91
0.90 0.97 0.98 0.69 0.88 0.74 0.20 0.55 0.44 0.94 0.86 0.34 0.97 0.08
a
Standard error of the means. Interaction between treatment and week. P < 0.01 for the effect of week for all variables. c Equivalency of the linear scores. 5.34=540,000 cells/mL. 4.81=350,000 cells/mL. b
3. Results In experiment 1, Bacillus subtilis supplementation had no detectable effect on intake, milk yield and its components, and feed and energy usage efficiencies (Table 2). On the other hand, in experiment 2 the supplementation of dairy cows with Bacillus subtilis spores increased the yields of milk, protein, and total solids (P < 0.05; Table 3). The positive milk yield response to the supplementation of Bacillus subtilis was detected after 5 weeks of supplementation and was consistent from week 7–16 (Fig. 1). The probiotic increased milk energy secretion (P=0.02) in experiment 2 (Table 3), but had no effect on fat and lactose yields (P > 0.35) and tended to reduce MUN concentration (P=0.06). The treatment had no detectable effect on milk SCC in both experiments (Tables 2 and 3) and on BW and BCS over time (Table 3). As shown in Table 4, there was no difference in chewing activity per day or per kg of
DM intake in experiment 1. Also, no difference was observed in total tract apparent digestibility of nutrients (Table 5).
4. Discussion Although the response in lactation performance in experiments 1 and 2 lacked consistency (Tables 2 and 3), the +1.7 kg/day increase in milk yield for cows fed Bacillus spores in experiment 2 is of large biological significance and would result in favourable ratio of extra income to probiotic cost. In experiment 1, the change in milk yield elicited by the spores approached only a non-statistical increase of 0.2 kg/d compared to Placebo. Some factors can be proposed for the 37
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tic, although the mechanism was not elucidated. However, the supplementation of Bacillus subtilis natto during 7 weeks increased milk and protein yield of lactating cows in late lactation (Liang-ce et al., 2012), suggesting that cows in early and late lactation can respond to probiotic supplementation. Further studies are needed to evaluate the effect of Bacillus subtilis C-3102 on cows differing in stage of lactation. The duration of the supplementation also differed between experiments. In experiment 2, milk yield of cows receiving Bacillus subtilis was greater than Placebo on week 5 of supplementation, but consistent increase was observed from weeks 7–16 (Fig. 1). Long term supplementation may increase the chance of obtaining a positive lactation response to the probiotic, although our experimental design did not evaluate this hypothesis. Diet composition could be another factor on the difference in lactation response to the spores between experiments. The diet of experiment 1 had 12% more forage DM than the diet of experiment 2, although feed ingredients and nutrient concentration were similar (Table 1). In both experiments, milk fat concentration was low, probably the result of adding calcium salts of soybean fatty acids (377 g of linoleic acid/kg of fatty acids) to the diet based on short particle size corn silage (Onetti and Grummer, 2004). Although diets were not identical in both experiments, we cannot ascertain that diet composition was a factor on the discrepancy of results. In both experiments the objective was to guarantee the consumption of a known dosage of the treatments, either orally in gelatine capsules or by top dressing treatments to the feed offered in the morning. Both methodologies pulse dosed the treatments into the rumen. This methodology was also chosen (in substitution to adding treatments to the TMR) to guarantee that the same TMR batch was offered to all animals twice per day, desirable to avoid difference in batches of the same diet, especially in experiment 2, performed in a commercial dairy farm. Method of treatment allocation was probably not a factor on the difference in the response to treatments between experiments. Sampling during data collection and laboratory procedures were also performed using the same methodology and by the same personal in experiments 1 and 2. Understanding the interaction of feed additives with diet and management practices is useful to define when and how a product should be used in commercial production systems with the greatest chance of inducing a viable positive response. We evaluated the same dosage of the treatments under two independent experimental conditions. Any explanation for the difference in the response to treatments between experiments would be purely speculative and could be explored in future experimentation on Bacillus subtilis spores supplementation. In addition to the significant milk yield response to Bacillus spores, milk protein secretion was the major drive for the increase in milk solids yield and energy secretion in experiment 2. The yields of lactose and fat did not respond to the treatment. A trend for a reduction in MUN when cows were fed Bacillus subtilis spores, coupled with the milk protein response, suggests that the spores affected protein metabolism in the rumen or systemically. Qiao et al. (2010) detected an increase in ruminal ammonia 1 and 6 h post-feeding in cows fed Bacillus subtilis. Sun et al. (2013) also reported that Bacillus subtilis natto supplementation resulted in higher ruminal ammonia concentration. Peng et al. (2012) observed no effect of a Bacillus subtilis supplement on blood urea-N of lactating cows, but detected a trend for increased concentration of ruminal ammonia. The available literature suggests that ruminal ammonia accumulation may be increase by Bacillus subtilis supplementation, the opposite to the trend of reduced MUN in experiment 2. Bacillus subtilis supplementation may be capable of affecting rumen fermentation and microbial profile. Sun et al. (2013) showed that Bacillus subtilis natto supplementation increased amylolitic, proteolitic, and total bacteria, and reduced total protozoa in rumen fluid. Moreover, that study reported that rumen fluid of cows fed Bacillus subtilis natto had increased total volatile fatty acids (VFA), propionate, and valerate concentrations, and reduced acetate concentration and pH.
Fig. 1. Milk yield on treatments Bacillus subtilis spores or Placebo (Experiment 2). P=0.02 for treatment, P < 0.01 for week, P=0.90 for the interaction between treatment and week. * P < 0.05 – Slice option. Table 4 Chewing activity on treatments Bacillus subtilis spores or Placebo (Experiment 1). Variables
Bacillus subtilis
Placebo
s.e.m.a
P-value
260 432 691 14.2 23.8 38.0
258 421 679 14.0 23.1 37.1
8.2 12.3 14.9 0.40 0.62 0.68
0.92 0.54 0.58 0.74 0.42 0.35
Ingestion (min/day) Rumination (min/day) Chewingb (min/day) Ingestion (min/kg DMIc) Rumination (min/kg DMIc) Chewingb (min/kg DMIc) a b c
Standard error of the means. Ingestion+Rumination. DMI=Dry matter intake.
Table 5 Total tract apparent digestibility of nutrients on treatments Bacillus subtilis spores or Placebo (Experiment 1). Variables b
DM (g/kg of DM) OMc (g/kg of OM) NDFd (g/kg of NDF) Non-NDF OMe (g/kg of Non-NDF OM) a b c d e
Bacillus subtilis
Placebo
s.e.m.a
P-value
694 721 526 845
710 726 558 847
0.9 1.7 4.4 1.3
0.20 0.69 0.28 0.81
Standard error of the means. Dry matter. Organic matter. Neutral detergent fiber. Non-NDF organic matter.
absence of detectable treatment effect on lactation performance in experiment 1 and for the detection in experiment 2. Stage of lactation is a plausible factor in the response of dairy cows to nutritional manipulations (Oba and Allen, 1999). Cows in experiment 1 were in a more advanced stage of lactation than cows in experiment 2 (246 vs. 161 DIM), although daily milk yield was similar. Cows of experiment 1 also had slightly higher BCS than cows of experiment 2 (3.80 vs. 3.64), suggestive of increased tissue deposition with advance in lactation (+84 DIM). The cows in experiment 1 may have been less sensitive to the probiotic action. Experimentally, the detection of a positive response in lactation performance in later lactation cows would be evidence of the efficacy of the product as a supplement for the majority of the lactating herd, and not only for a specific group of cows in early lactation. When cows in early lactation (29 ± 6 DIM) were supplemented with a fermentation product from Bacillus subtilis the plasma concentration of non-esterified fatty acids was reduced (Peng et al., 2012), suggesting that lipid metabolism was responsive to the probio38
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of Higher Education Personnel (CAPES) in the form of the masters degree scholarship granted to the first author. Appreciation is extended to the members of Grupo do Leite of the University of Lavras for the help in data collection. Thanks to Uniquimica for supplying Bacillus subtilis spores and to Iury Rocha Rios (in memorian).
Peng et al. (2012) also observed an increase in propionate and a reduction in acetate proportions in total VFA, suggesting increased energetic efficiency of the ruminal fermentation when Bacillus products were fed. However, Qiao et al. (2010) did not observe a response in rumen fermentation profile to the supplement. The ratio of milk energy secretion to DOMI did not respond to treatments in experiment 1, suggesting that if a change in rumen fermentation profile occurred in response to the treatment, it was not of sufficient magnitude to alter the proportion of the digestible energy intake captured as metabolizable energy, assuming that the efficiency of converting metabolizable into net energy was similar across treatments. Unfortunately this experiment did not evaluate rumen microbial yield and fermentation profile to verify if they would explain the change in milk and protein secretions in experiment 2. Bacterial spores supplementation may have the capacity to act on the immune function of animals (Cutting, 2011; Sun et al., 2010). The cows subjected to increased mastitis pathogen challenge in experiment 2 may have had increased chance of showing a positive performance response to Bacillus supplementation. Sun et al. (2013) observed that Bacillus subtilis natto supplementation reduced SCC, but the specific mode of action was unclear and this observation was not supported by our data. An effect of treatment on SCC was not detected in both experiments. Based on experiment 1, Bacillus subtilis spores had no effect on DM intake and total tract nutrient digestibility. The similarity in chewing behavior also supports these findings. Qiao et al. (2010) observed no response to Bacillus subtilis on intake of lactating cows over a 10-week period and on ruminal digestibility and microbial yield evaluated in cows with rumen and duodenal cannulas. Our data did not support the hypothesis that Bacillus subtilis spores would increase total tract nutrient digestibility in dairy cows. The mechanism by which the spores increased lactation performance in experiment 2 was unclear, but may deserve future consideration, since the response was of large magnitude. The supplementation of yeast probiotics, for example, is expected to increase milk yield of a 650 kg cow by +0.78 kg/day based on the meta-analysis of Desnoyer et al. (2009), approaching +1.2 to +1.3 kg/day response in some studies (Bruno et al., 2009; Salvati et al., 2015), smaller than the +1.7 kg/day response to the spores detected in experiment 2. There is not enough published literature on Bacillus subtilis supplementation to subsidiate the prediction of a lactation response to the supplement.
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Peng, H., Wang, J.Q., Kang, H.Y., Dong, S.H., Sun, P., Bu, D.P., Zhou, L.Y., 2012. Effect of feeding Bacillus subtilis natto fermentation product on milk production and composition, blood metabolites and rumen fermentation in early lactation dairy cows. Anim. Physiol. Anim. Nutr. 96, 506–512. Qiao, G.H., Shan, A.S., Ma, N., Ma, Q.Q., Sun, Z.W., 2010. Effect of supplemental Bacillus cultures on rumen fermentation and milk yield in Chinese Holstein cows. Anim. Physiol. Anim. Nutr. 94, 429–436. Reid, G., 2008. Review, probiotics and prebiotics – progress and challenges. Int. Dairy J. 18, 969–975. Salvati, G.G.S., Morais Junior, N.N., Melo, A.C.S., Vilela, R.R., Cardoso, F.F., Aronovich, M., Pereira, R.A.N., Pereira, M.N., 2015. Response of lactating cows to live yeast supplementation during summer. J. Dairy Sci. 98, 4062–4073. Sanders, M.E., Morelli, L., Tompkins, T.A., 2003. Sporeforms as human probiotics, Bacillus, Sporolactobacillus, and Brevibacillus. Compr. Rev. Food Sci. F 2, 101–110. SAS Institute, 2008. SAS/STAT Software Version 9.1. SAS Institute Inc., Cary, NC, USA. Sun, P., Wang, J.Q., Zhang, H.T., 2010. Effects of Bacillus subtilis natto on performance and immune function of preweaning calves. J. Dairy Sci. 93, 5851–5855. Sun, P., Wang, J.Q., Deng, L.F., 2013. Effects of Bacillus subtilis natto on milk production, rumen fermentation and ruminal microbiome of dairy cows. Animal 7, 216–222. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fibre and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Wildman, E.E., Jones, G.M., Wagner, P.E., Boman, R.L., Troutt Jr., H.F., Lesch, T.N.A., 1982. A dairy cow body condition scoring system and its relationship to selected production characteristics. J. Dairy Sci. 65, 495–501. Zhang, Z.F., Cho, J.H., Kim, I.H., 2013. Short communication: Effects of Bacillus subtilis UBT-MO2 on growth performance, relative immune organ weight, gas concentration in excreta, and intestinal microbial shedding in broiler chickens. Livest. Sci. 155, 343–347. Zhang, Z.F., Zhou, T.X., Ao, X., Kim, I.H., 2012. Effects of β-glucan and Bacillus subtilis on growth performance, blood profiles, relative organ weight and meat quality in broilers fed maize-soybean meal based diets. Livest. Sci. 150, 419–424.
5. Conclusions The supplementation of dairy cows for 16 weeks with Bacillus subtilis spores increased milk and protein yield. Data from a short-term crossover experiment did not support that increased total tract diet digestibility would be a plausible mediator of the positive lactation response. Competing interests The authors declare that they have no competing interests. Authors' contributions V.L. Souza, M.N. Pereira and R.A.N. Pereira performed the project, conducted the trials in the farms, and wrote the draft. N.M. Lopes, O.F. Zacaroni, V.A. Silveira and G.G.S. Salvati conducted the trials at dairy farms and helped in laboratory analysis. J.A. Freitas and R. Almeida helped finalize manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by the Coordination for the Improvement 39
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Lactation performance and diet digestibility of dairy cows in response to the supplementation of Bacillus subtilis spores
MARK
⁎
V.L. Souzaa, , N.M. Lopesb, O.F. Zacaronib, V.A. Silveirab, R.A.N. Pereirac, J.A. Freitasd, ⁎ R. Almeidaa, G.G.S. Salvatib, M.N. Pereirab, a
Department of Animal Science, University of Paraná, Curitiba, PR 80035-050, Brazil Department of Animal Science, University of Lavras, Lavras, MG 37200-000, Brazil c Minas Gerais State Ag Research Enterprise, EPAMIG, Lavras, MG 37200-000, Brazil d Department of Animal Science, University of Paraná, Palotina, PR 85950-000, Brazil b
A R T I C L E I N F O
A B S T R A C T
Keywords: Bacillus subtilis Dairy cattle Milk composition Milk yield Probiotic
Bacillus subtilis is a transitory microorganism of the digestive tract, non-pathogenic to animals, and capable of forming spores that are resistant to heat and cold. As an animal feed probiotic the microorganism is supposedly capable of increasing diet digestibility and immunity. Two experiments were conducted to evaluate the effect of Bacillus subtilis spores on milk yield and composition and diet digestibility. In both experiments lactating dairy cows were fed in tie stalls and treatments were force-fed once per day. In experiment 1, 18 Holsteins in late lactation (246 ± 75 days in milk) received a sequence of the treatments Bacillus subtilis strain C-3102 (3.0×109 colony-forming units of spores per day) or Placebo in a crossover design with 39-day periods, a 10-day wash-out between periods, and response evaluated after the 28th day of the periods. The supplementation of Bacillus subtilis spores did not elicit detectable changes in intake (18.3 kg/d, P=0.91), milk (25.3 kg/d, P=0.66) and solids yield and concentration, total tract nutrient digestibility, and chewing activity. In experiment 2, 30 cows (161 ± 72 days in milk) with high milk somatic cell count (725,000 cells/mL) received the same treatments for 16 weeks, in a covariate adjusted randomized block design with repeated measures over time. Bacillus subtilis spores increased the yields of milk (25.3 vs. 23.6 kg/d, P=0.02), protein (0.816 vs. 0.763 kg/d, P=0.01), total solids (2.718 vs. 2.566 kg/d, P=0.05), and energy (60.7 versus 56.5 MJ/d, P=0.02) and milk urea-N tended to be reduced (19.3 vs. 20.8 mg/dL, P=0.06). Milk somatic cell count did not differ between treatments. The positive lactation response to Bacillus subtilis spores supplementation occurred when the probiotic was fed for 16 weeks and there was no evidence to suggest that increased diet digestibility was a mediator of the response.
1. Introduction Human food production from animals supplemented with live microorganisms as a replacement to chemical feed additives is in line with the natural trend of the consumer market. The addition of probiotics to the diet may have beneficial effect on gut physiology and immune function (Reid, 2008). Spore forming bacteria, such as Bacillus subtilis, have been used as probiotic supplements for humans and animals (Cutting, 2011). Bacillus subtilis is a transitory microorganism of the digestive tract, non-pathogenic to animals, and capable of forming spores resistant to heat and cold, having high stability in the diet (Sanders et al., 2003; Carlin, 2011; Cutting, 2011). The spores germinate at the intestinal lumen, which is required for its action as an animal feed probiotic. The bacterial population of Bacillus subtilis in the digestive tract is reduced after the cessation of the supplementation
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(Sanders et al., 2003). For this reason, the dietary supplementation of Bacillus subtilis should be performed daily. Probiotics based on bacterial spores can survive passage through the stomach (Hoa et al., 2000) with the potential to be more resistant to the low gastric pH compared with probiotic supplementation in the form of vegetative cells. The supplementation of Bacillus subtilis has improved the performance of non-ruminants (Fritts et al., 2000; Hooge, 2008; Zhang et al., 2012, 2013; Lee et al., 2014) and calves (Sun et al., 2010). Bacillus subtilis can increase anaerobiosis in the digestive tract, which favors native proliferation of Lactobacilli capable of producing lactic acid and inhibiting pathogenic bacteria growth (Maruta et al., 1996; Sanders et al., 2003) and can improve immune function (Sun et al., 2010). Although increased diet digestibility is frequently proposed as a plausible response to spore forming bacterial supplements in nonruminants, currently no study in lactating dairy cows have demon-
Corresponding authors. E-mail addresses: [email protected] (V.L. Souza), [email protected]fla.br (M.N. Pereira).
http://dx.doi.org/10.1016/j.livsci.2017.03.023 Received 15 June 2016; Received in revised form 29 March 2017; Accepted 30 March 2017 1871-1413/ © 2017 Elsevier B.V. All rights reserved.
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were 0.3 g/d of viable spores of Bacillus subtilis on calcium carbonate or Placebo (calcium carbonate). The active agent in the probiotic consisted of viable endospores of a single strain of Bacillus subtilis originally isolated from soil in Japan and dried and pasteurized to kill vegetative cells. The final product supplied Bacillus subtilis strain C-3102 (Calpis Co. Ltd., Tokyo, Japan) to provide a minimum daily intake of 3.0×109 cfu of viable spores. Gelatin capsules were filled with the treatments for daily oral dosing of each cow on return to their tie stalls after the morning milking.
strated this mechanism of action. This research addresses the hypothesis that lactating dairy cows fed Bacillus subtilis can improve the lactational performance with positive changes in milk composition. Thus, two independent experiments were performed to investigate the response of dairy cows in lactation performance, milk composition, and diet digestibility to the supplementation with Bacillus subtilis spores. 2. Material and methods The experimental procedures were in agreement with the ethical principles in animal experimentation of the Committee of Ethics in Animal Experimentation of the University of Paraná, Brazil (protocol number 050/2010).
2.1.2. Sample collection and analysis Between days 28 and 39, samples of feed ingredients and orts from each cow were sampled daily and frozen. Composite samples per period were formed on an as fed basis for further analysis. The contents of dry matter (DM; method 934.01), organic matter (OM; ash method 924.05), crude protein (CP; method 984.13) and ether extract (EE; method 920.39) were analyzed according to the Association of Official Analytical Chemists (AOAC, 1990). Ash free neutral detergent fiber (aNDFom) was analyzed with porous crucibles following the recommendations of Van Soest et al. (1991) with heat-stable amylase and sodium sulfite. Acid detergent fiber was analyzed non-sequentially. Samples from nine consecutive milkings were taken on days 29, 30, and 31 and a daily composite was formed in proportion to the volume secreted on each milking. The concentrations of protein, fat, lactose, and total solids were measured by infrared spectrophotometry using a Bentley 2000 analyzer (Bentley Instruments, Chaska, USA). The SCC was determined in a Somacount 150 equipment (Bentley Instruments, Chaska, USA) based on the principle of laser-based flow cytometry. Milk urea-N (MUN) was analyzed with a ChemSpec 150 (Bentley Instruments, Chaska, USA) that utilizes a modified Berthelot reaction. The daily secretion of milk energy (MJ/day) was calculated (NRC, 2001): {[(0.0929×% fat)+(0.0547×% protein)+(0.0395×% lactose)]×kg of milk}×4.184. The SCC was transformed to a linear scale from 0 to 9 (SCC score) in which scores represented the following values of SCC (×1000 cells/mL): 12.5 for SCC score 0; 25 for SCC score 1; 50 for SCC score 2; 100 for SCC score 3; 200 for SCC score 4; 400 for SCC score 5; 800 for SCC score 6; 1600 for SCC score 7; 3200 for SCC score 8; and 6400 for SCC score 9. The SCC score was calculated from the natural logarithm of the measured SCC (1000 cells/mL) and the above mentioned SCC value of each score: SCC score=−3.6438+1.4427× Ln(SCC). Negative values were rounded to zero. Body weight (BW) and body condition score (BCS) were determined on day 35 to describe experimental units. The BCS was scored using a 1–5 scale, thin to fat (Wildman et al., 1982). The same three independent evaluators scored each cow. Cows were weighed immediately after the morning milking. The total tract apparent digestibility coefficient of DM, OM, aNDF, and Non-aNDF OM was determined on days 33–35 by total fecal collection. Feces were collected concurrent to defecation during three 8-h sampling periods and weighed. The second and third sampling periods were each delayed by 8 h to avoid a major disturbance to the animals, while still representing a 24-h collection period. Fecal aliquots (equal fresh weight basis) were immediately frozen along the collection period and a composite sample was formed. Daily digestible OM intake (DOMI, kg/day) was calculated. Feed efficiency was determined as the ratio of milk yield to DM intake. The efficiency of energy use was defined by the ratio between milk energy (MJ/day) and DOMI. On day 28, chewing activity was assessed by visual observation of the buccal activity of each cow, at 5-min intervals, continuously for 24 h. Chewing time (min/ day) was defined as the sum of ingestion and rumination times. Chewing, ingestion, and rumination per unit of DM intake were calculated using the DM intake of the day in which chewing activity was evaluated.
2.1. Experiment 1 2.1.1. Cows, design, and treatments Eighteen Holstein cows, with 246 ± 75 days in milk (DIM), 29.7 ± 5.8 kg/d of milk yield, and with somatic cell count (SCC) below 100,000 cells/mL at the start of the experiment, were individually fed in sand bedded tie stalls at the Better Nature Research Center (www. holandesflamma.com.br) and were millked three times per day in a adjacent herringbone parlor. Cows were paired blocked based on parity (primiparous and multiparous) and milk yield and were assigned to a sequence of two treatments in a crossover design, with 39-day periods. Between the two experimental periods, a 10-day washout was adopted, in which the animals received the same basal diet with no treatment addition. Response variables were evaluated on the last 12 days of each period. Cows were fed on a Total Mixed Ration (TMR) mixed and offered twice daily starting at 0500 and 1300 h. The TMR was offered in amount sufficient to provide at least 10% of the offered as daily refusal. The composition of the TMR is reported in Table 1. Diet offered and orts from each cow were measured daily during the experiment. Treatments
Table 1 Composition of the offered total mixed ration in ingredients and of the consumed in nutrients (g/kg of dry matter) on treatments Bacillus subtilis spores or Placebo in Experiment 1 and of the basal diet in Experiment 2. Experiment 1 Bacillus subtilis Corn silage Tifton hay Soybean meal Citrus pulp High moisture corn Finely ground mature corn Calcium salt of fatty acids Urea Magnesium oxide Limestone NaCl Sodium bicarbonate Minerals and vitaminsa Crude protein Neutral detergent fiber (aNDFom) aNDFom from forage Acid detergent fiber Ether extract Ash Non-fiber carbohydratesb
Experiment 2
Placebo
500 41 202 102 112
424 218 158 156 13 4
13 4 3 8 3 8 4 172 337 268 238 48 93 350
171 335 267 238 48 93 353
8 4 9 6 177 301 221 215 52 88 385
a Composition per kg: 185 g of Ca; 150 g of P; 30 g of Mg; 30 g of S; 240 mg of Co; 3000 mg of Cu; 8000 mg of Mn; 12,000 mg of Zn; 90 mg of Se; 180 mg of I; 1000 KIU Vitamin A; 250 KIU Vitamin D3; 6250 IU Vitamin E. b NFC (g/kg of dry matter)=1000 – (crude protein+aNDFom+ether extract+ash).
36
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2.2. Experiment 2
Table 2 Intake, lactation performance, feed efficiency, milk somatic cell count (SCC) and urea-N (MUN) concentration, and body weight (BW) and condition score (BCS) on treatments Bacillus subtilis spores or Placebo (Experiment 1).
2.2.1. Cows, design, and treatments Experiment 2 was performed in a commercial dairy farm located 40 km from the University of Lavras. The 50 cow herd was housed in a sand bedded tie stall and was milked twice per day in a herringbone parlor. The herd was chosen to represent a high SCC scenario in which the effect of Bacillus subtilis supplementation on SCC and lactation performance could be evaluated. Thirty Holsteins (seven primiparous) with 161 ± 72 DIM at the beginning of the experiment were paired blocked based on SCC and milk yield and were randomly assigned to the same two treatments of experiment 1 for 16 weeks. The mean SCC at blocking was 716,000 cells/mL for treatment Bacillus subtilis and 734,000 cells/mL for Placebo, and milk yield was 29.6 and 29.9 kg/d, respectively. Data used for blocking was used as covariate in the statistical model. The daily dosage of each treatment per cow was mixed to a cup of ground corn and top-dressed to the feed offered in the morning. A technician was hired to perform the daily allocation of treatments to each cow and to assist with data collection and feed management during the experiment. Samples of feed ingredients and the daily refusal were collected and frozen for further analysis as previously described. Individual feed intake was not recorded to avoid disturbance on herd routine and because of constraint in skilled personal and equipment continuously at the farm. The ingredient and nutrient composition of the basal diet is presented in Table 1. Milk samples were obtained at 7day intervals along the 16-week comparison period for analysis as previously described. The BW and BCS were determined at 4-week intervals.
Variables
Bacillus subtilis
Placebo
s.e.m.a
P-value
18.3 3.07 11.99 25.4 0.700 0.787 1.094 2.871 29.0 32.8 44.1 114.9 66.1 1.39 5.65 2.48 17.5 673 3.8
18.3 3.08 11.94 25.2 0.683 0.790 1.079 2.845 29.0 33.3 43.9 114.7 65.3 1.37 5.48 2.55 17.0 668 3.8
0.16 0.078 0.263 0.38 0.0137 0.0160 0.0211 0.0467 0.060 0.023 0.038 0.100 1.05 0.023 0.126 0.171 0.40 7.3 0.05
0.91 0.89 0.90 0.66 0.39 0.89 0.61 0.69 0.94 0.17 0.73 0.89 0.60 0.53 0.43 0.76 0.39 0.65 0.95
Dry matter intake (kg/day) Dry matter intake (% of BW) DOMIb (kg/day) Milk yield (kg/day) Fat yield (kg/day) Protein yield (kg/day) Lactose yield (kg/day) Solids yield (kg/day) Fat (g/kg) Protein (g/kg) Lactose (g/kg) Solids (g/kg) Milk energy (MJ/day) Milk/Dry matter intake Milk energy/DOMI (MJ/kg) SCC scorec (0–9) MUN (mg/dL) BW (kg) BCS (1–5) a b c
Standard error of the means. Digestible organic matter intake. Equivalency of the linear scores. 2.48=70,000 cells/mL. 2.55=73,000 cells/mL.
Table 3 Lactation performance, milk somatic cell count (SCC) and urea-N (MUN) concentration, and body weight (BW) and condition score (BCS) on treatments Bacillus subtilis spores or Placebo (Experiment 2).
2.3. Statistical analysis
P-value Variables
2.3.1. Experiment 1 Data were analyzed using the MIXED procedure of SAS (SAS Institute, 2008) with a model containing the random effect of cow (1–18) and the fixed effects of period (1, 2) and treatment (Bacillus subtilis, Placebo).
Milk yield (kg/day) Fat yield (kg/day) Protein yield (kg/day) Lactose yield (kg/day) Solids yield (kg/day) Fat (g/kg) Protein (g/kg) Lactose (g/kg) Solids (g/kg) Milk energy (MJ/day) SCC scorec (0–9) MUN (mg/dL) BW (kg) BCS (1–5)
2.3.2. Experiment 2 Data were analyzed as repeated measures using the MIXED procedure of SAS (SAS Institute, 2008) with a model containing the continuous effect of covariate (measure of the same variable before treatments allocation), the random effect of block (1–15), and the fixed effects of treatment (Bacillus subtilis, Placebo), week (1–16), and the interaction between treatment and week. The best covariance structure was defined by the Akaike's information criterion among first-order autoregressive, unstructured, and compound symmetry. The mean square for the effect of cow nested within treatment was used as the error term to test the treatment effect. Probability values below 0.05 were considered significant and below 0.10 were considered trend.
Bacillus subtilis
Placebo
s.e.m.a
Treat
Treat×Weekb
25.3 0.605 0.816 1.074 2.718 24.1 32.9 42.6 108.0 60.7 5.34 19.3 635 3.64
23.6 0.572 0.763 1.050 2.566 25.2 33.1 43.5 109.5 56.5 4.81 20.8 623 3.65
0.42 0.0228 0.0127 0.0238 0.0476 0.070 0.047 0.023 0.135 1.05 0.339 0.46 7.9 0.061
0.02 0.35 0.01 0.52 0.05 0.30 0.83 0.02 0.48 0.02 0.31 0.06 0.33 0.91
0.90 0.97 0.98 0.69 0.88 0.74 0.20 0.55 0.44 0.94 0.86 0.34 0.97 0.08
a
Standard error of the means. Interaction between treatment and week. P < 0.01 for the effect of week for all variables. c Equivalency of the linear scores. 5.34=540,000 cells/mL. 4.81=350,000 cells/mL. b
3. Results In experiment 1, Bacillus subtilis supplementation had no detectable effect on intake, milk yield and its components, and feed and energy usage efficiencies (Table 2). On the other hand, in experiment 2 the supplementation of dairy cows with Bacillus subtilis spores increased the yields of milk, protein, and total solids (P < 0.05; Table 3). The positive milk yield response to the supplementation of Bacillus subtilis was detected after 5 weeks of supplementation and was consistent from week 7–16 (Fig. 1). The probiotic increased milk energy secretion (P=0.02) in experiment 2 (Table 3), but had no effect on fat and lactose yields (P > 0.35) and tended to reduce MUN concentration (P=0.06). The treatment had no detectable effect on milk SCC in both experiments (Tables 2 and 3) and on BW and BCS over time (Table 3). As shown in Table 4, there was no difference in chewing activity per day or per kg of
DM intake in experiment 1. Also, no difference was observed in total tract apparent digestibility of nutrients (Table 5).
4. Discussion Although the response in lactation performance in experiments 1 and 2 lacked consistency (Tables 2 and 3), the +1.7 kg/day increase in milk yield for cows fed Bacillus spores in experiment 2 is of large biological significance and would result in favourable ratio of extra income to probiotic cost. In experiment 1, the change in milk yield elicited by the spores approached only a non-statistical increase of 0.2 kg/d compared to Placebo. Some factors can be proposed for the 37
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tic, although the mechanism was not elucidated. However, the supplementation of Bacillus subtilis natto during 7 weeks increased milk and protein yield of lactating cows in late lactation (Liang-ce et al., 2012), suggesting that cows in early and late lactation can respond to probiotic supplementation. Further studies are needed to evaluate the effect of Bacillus subtilis C-3102 on cows differing in stage of lactation. The duration of the supplementation also differed between experiments. In experiment 2, milk yield of cows receiving Bacillus subtilis was greater than Placebo on week 5 of supplementation, but consistent increase was observed from weeks 7–16 (Fig. 1). Long term supplementation may increase the chance of obtaining a positive lactation response to the probiotic, although our experimental design did not evaluate this hypothesis. Diet composition could be another factor on the difference in lactation response to the spores between experiments. The diet of experiment 1 had 12% more forage DM than the diet of experiment 2, although feed ingredients and nutrient concentration were similar (Table 1). In both experiments, milk fat concentration was low, probably the result of adding calcium salts of soybean fatty acids (377 g of linoleic acid/kg of fatty acids) to the diet based on short particle size corn silage (Onetti and Grummer, 2004). Although diets were not identical in both experiments, we cannot ascertain that diet composition was a factor on the discrepancy of results. In both experiments the objective was to guarantee the consumption of a known dosage of the treatments, either orally in gelatine capsules or by top dressing treatments to the feed offered in the morning. Both methodologies pulse dosed the treatments into the rumen. This methodology was also chosen (in substitution to adding treatments to the TMR) to guarantee that the same TMR batch was offered to all animals twice per day, desirable to avoid difference in batches of the same diet, especially in experiment 2, performed in a commercial dairy farm. Method of treatment allocation was probably not a factor on the difference in the response to treatments between experiments. Sampling during data collection and laboratory procedures were also performed using the same methodology and by the same personal in experiments 1 and 2. Understanding the interaction of feed additives with diet and management practices is useful to define when and how a product should be used in commercial production systems with the greatest chance of inducing a viable positive response. We evaluated the same dosage of the treatments under two independent experimental conditions. Any explanation for the difference in the response to treatments between experiments would be purely speculative and could be explored in future experimentation on Bacillus subtilis spores supplementation. In addition to the significant milk yield response to Bacillus spores, milk protein secretion was the major drive for the increase in milk solids yield and energy secretion in experiment 2. The yields of lactose and fat did not respond to the treatment. A trend for a reduction in MUN when cows were fed Bacillus subtilis spores, coupled with the milk protein response, suggests that the spores affected protein metabolism in the rumen or systemically. Qiao et al. (2010) detected an increase in ruminal ammonia 1 and 6 h post-feeding in cows fed Bacillus subtilis. Sun et al. (2013) also reported that Bacillus subtilis natto supplementation resulted in higher ruminal ammonia concentration. Peng et al. (2012) observed no effect of a Bacillus subtilis supplement on blood urea-N of lactating cows, but detected a trend for increased concentration of ruminal ammonia. The available literature suggests that ruminal ammonia accumulation may be increase by Bacillus subtilis supplementation, the opposite to the trend of reduced MUN in experiment 2. Bacillus subtilis supplementation may be capable of affecting rumen fermentation and microbial profile. Sun et al. (2013) showed that Bacillus subtilis natto supplementation increased amylolitic, proteolitic, and total bacteria, and reduced total protozoa in rumen fluid. Moreover, that study reported that rumen fluid of cows fed Bacillus subtilis natto had increased total volatile fatty acids (VFA), propionate, and valerate concentrations, and reduced acetate concentration and pH.
Fig. 1. Milk yield on treatments Bacillus subtilis spores or Placebo (Experiment 2). P=0.02 for treatment, P < 0.01 for week, P=0.90 for the interaction between treatment and week. * P < 0.05 – Slice option. Table 4 Chewing activity on treatments Bacillus subtilis spores or Placebo (Experiment 1). Variables
Bacillus subtilis
Placebo
s.e.m.a
P-value
260 432 691 14.2 23.8 38.0
258 421 679 14.0 23.1 37.1
8.2 12.3 14.9 0.40 0.62 0.68
0.92 0.54 0.58 0.74 0.42 0.35
Ingestion (min/day) Rumination (min/day) Chewingb (min/day) Ingestion (min/kg DMIc) Rumination (min/kg DMIc) Chewingb (min/kg DMIc) a b c
Standard error of the means. Ingestion+Rumination. DMI=Dry matter intake.
Table 5 Total tract apparent digestibility of nutrients on treatments Bacillus subtilis spores or Placebo (Experiment 1). Variables b
DM (g/kg of DM) OMc (g/kg of OM) NDFd (g/kg of NDF) Non-NDF OMe (g/kg of Non-NDF OM) a b c d e
Bacillus subtilis
Placebo
s.e.m.a
P-value
694 721 526 845
710 726 558 847
0.9 1.7 4.4 1.3
0.20 0.69 0.28 0.81
Standard error of the means. Dry matter. Organic matter. Neutral detergent fiber. Non-NDF organic matter.
absence of detectable treatment effect on lactation performance in experiment 1 and for the detection in experiment 2. Stage of lactation is a plausible factor in the response of dairy cows to nutritional manipulations (Oba and Allen, 1999). Cows in experiment 1 were in a more advanced stage of lactation than cows in experiment 2 (246 vs. 161 DIM), although daily milk yield was similar. Cows of experiment 1 also had slightly higher BCS than cows of experiment 2 (3.80 vs. 3.64), suggestive of increased tissue deposition with advance in lactation (+84 DIM). The cows in experiment 1 may have been less sensitive to the probiotic action. Experimentally, the detection of a positive response in lactation performance in later lactation cows would be evidence of the efficacy of the product as a supplement for the majority of the lactating herd, and not only for a specific group of cows in early lactation. When cows in early lactation (29 ± 6 DIM) were supplemented with a fermentation product from Bacillus subtilis the plasma concentration of non-esterified fatty acids was reduced (Peng et al., 2012), suggesting that lipid metabolism was responsive to the probio38
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of Higher Education Personnel (CAPES) in the form of the masters degree scholarship granted to the first author. Appreciation is extended to the members of Grupo do Leite of the University of Lavras for the help in data collection. Thanks to Uniquimica for supplying Bacillus subtilis spores and to Iury Rocha Rios (in memorian).
Peng et al. (2012) also observed an increase in propionate and a reduction in acetate proportions in total VFA, suggesting increased energetic efficiency of the ruminal fermentation when Bacillus products were fed. However, Qiao et al. (2010) did not observe a response in rumen fermentation profile to the supplement. The ratio of milk energy secretion to DOMI did not respond to treatments in experiment 1, suggesting that if a change in rumen fermentation profile occurred in response to the treatment, it was not of sufficient magnitude to alter the proportion of the digestible energy intake captured as metabolizable energy, assuming that the efficiency of converting metabolizable into net energy was similar across treatments. Unfortunately this experiment did not evaluate rumen microbial yield and fermentation profile to verify if they would explain the change in milk and protein secretions in experiment 2. Bacterial spores supplementation may have the capacity to act on the immune function of animals (Cutting, 2011; Sun et al., 2010). The cows subjected to increased mastitis pathogen challenge in experiment 2 may have had increased chance of showing a positive performance response to Bacillus supplementation. Sun et al. (2013) observed that Bacillus subtilis natto supplementation reduced SCC, but the specific mode of action was unclear and this observation was not supported by our data. An effect of treatment on SCC was not detected in both experiments. Based on experiment 1, Bacillus subtilis spores had no effect on DM intake and total tract nutrient digestibility. The similarity in chewing behavior also supports these findings. Qiao et al. (2010) observed no response to Bacillus subtilis on intake of lactating cows over a 10-week period and on ruminal digestibility and microbial yield evaluated in cows with rumen and duodenal cannulas. Our data did not support the hypothesis that Bacillus subtilis spores would increase total tract nutrient digestibility in dairy cows. The mechanism by which the spores increased lactation performance in experiment 2 was unclear, but may deserve future consideration, since the response was of large magnitude. The supplementation of yeast probiotics, for example, is expected to increase milk yield of a 650 kg cow by +0.78 kg/day based on the meta-analysis of Desnoyer et al. (2009), approaching +1.2 to +1.3 kg/day response in some studies (Bruno et al., 2009; Salvati et al., 2015), smaller than the +1.7 kg/day response to the spores detected in experiment 2. There is not enough published literature on Bacillus subtilis supplementation to subsidiate the prediction of a lactation response to the supplement.
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5. Conclusions The supplementation of dairy cows for 16 weeks with Bacillus subtilis spores increased milk and protein yield. Data from a short-term crossover experiment did not support that increased total tract diet digestibility would be a plausible mediator of the positive lactation response. Competing interests The authors declare that they have no competing interests. Authors' contributions V.L. Souza, M.N. Pereira and R.A.N. Pereira performed the project, conducted the trials in the farms, and wrote the draft. N.M. Lopes, O.F. Zacaroni, V.A. Silveira and G.G.S. Salvati conducted the trials at dairy farms and helped in laboratory analysis. J.A. Freitas and R. Almeida helped finalize manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by the Coordination for the Improvement 39