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Effect of BioFresh™ Boluses on Milk Yield, Milk Composition, and In Situ Digestibility in Lactating Holstein Dairy Cattle1 February 2003 K. E. Griswold, D. L. Hastings, B. N. Jacobson, D. Devore Department of Animal Science, Food & Nutrition Southern Illinois University, Carbondale This research was graciously funded by Agrarian Marketing Corporation, Middlebury, IN and Select Sires, Inc., Plain City, OH. The authors would like to thank Select Sires, Inc. and Agrarian Marketing Corporation for supplying the BioFresh™ Boluses and placebo boluses used in this study. 1 Griswold et al. 2003 Introduction The current economic climate for dairy farmers dictates the need for keeping feed costs low while optimizing milk production. Factors that affect milk quality have direct economic bearing on farm income. In a survey of over 100,000 cows, Wilson et al. (1997) estimated dollar losses per lactation ranging from $52 to $312 for cows infected with mastitis pathogens. Further, infected cows had a higher linear somatic cell count (LSCC) than non-infected cows, 4.4 vs. 3.0, respectively. Infection by mastitis-causing pathogens is the major factor affecting quarter, cow and bulk tank somatic cell count (SCC) (Harmon, 1994). However, regardless of SCC, milk production is negatively affected by increasing SCC (Kehrli and Shuster, 1994). Efforts to reduce SCC focus mainly on prevention of infection by improving hygiene (Kehrli and Shuster, 1994) and boosting the immune response of the animal (Hogan et al., 1993). Once elevated by infection, SCC can vary greatly and may not decline substantially even if the infection-causing pathogen has been cleared from the quarter (Harmon, 1994). Antibiotic therapy at the time of infection or at dry-off is the most prevalent method for reducing elevated SCC (Kehrli and Shuster, 1994). However, the current concerns over the use of antibiotics in animal agriculture and the development of antibiotic resistance warrant the development of antibiotic alternatives to reduce SCC. Direct feeding of yeast culture (YC) or supplemental enzymes to increase milk yield has produced varied results. Yeast culture has been shown to increase (Piva et al., 1993) or have no effect (Dann et al., 2000) on milk yield and milk components. Dann et al. (2000) reported no changes in SCC when YC was fed to primiparous and multiparous Jersey for 21 day prior to parturition. Vicini et al. (2003) reported no effect on milk production when fibrolytic enzymes were fed to cows in two separate trials, and reported that responses in other studies ranged from 0 to 6.3 kg/d. Direct-fed enzymes (DFE) have been shown to increase milk fat and milk protein percentages (Beauchemin et al., 1999; Beauchemin et al., 2000; Bowman et al., 2002), but an equal number of studies have reported no changes in milk composition with DFE (Yang et al., 2000; Kung et al., 2002; Vicini et al., 2003) Vicini et al. (2003) was the only study to report LSCC and found no effect of feeding DFE on LSCC in two separate studies. However, Vicini et al. (2003) pointed out that responses to DFE are dependent on a number of factors including method of delivery and specific enzyme complexes. Therefore, our objective was to test shortterm feeding of a combination of YC, DFE, and other ingredients, including L-form Griswold et al. 2003 lactobacillus, (BioFresh™) in a bolus form as an effective method for reducing SCC in lactating Holstein cows. The effects of BioFresh™ Boluses on milk yield, milk composition and in situ dietary digestibility were also evaluated. Materials and Methods Cows and Treatments: Forty-two primiparous and multiparous Holstein cows averaging 179 days in lactation were used in the study at the SIU Dairy Center. Cows were paired together by lactation number, days in milk, projected 305-day milk yield, and LSCC from the two most recent DHIA test dates. Pairs of animals were then randomly split between the two treatments (n = 21). The treatments were either BioFresh™ Boluses or placebo boluses containing the carrier used in manufacturing BioFresh™. The cows were bolused for each of 3 consecutive days at 0900 h one wk after the initiation of the study. Diet and Management: All cows received the same diet throughout the study. The diet was formulated to meet the nutrient requirements of a lactating Holstein cow producing 85 lbs. of 3.8% fat milk (NRC, 2001), and consisted of corn silage, milage, wet brewers grain, alfalfa hay, ground corn, soybean meal 48%, extruded soybeans, cottonseed, and mineral-vitamin premix. The ingredient composition and nutrient analyses of the diet are provided in Table 1. Cows were fed the diet at ad libitum intake plus 5% as a TMR once per day at 0800 h. Cows had continual access to clean water and were housed in a curtained freestall barn oriented east to west with the prevailing wind from the west. Cows had daily access to a dirt lot for exercise. Freestalls were bedded with sand, which was cleaned 2x per day when cows were gathered for milking. Clean sand was added every 2 wk using a sandshooter system attached to a skidsteer. Freestall alleys and the feedlot were cleaned on a daily basis using a skidsteer with a rubber tire scraper. Cows were milked 2x per day at 0400 and 1600 h in a double-four herringbone parlor, using Surge milking equipment. All cows received bST throughout the study. In situ Digestibility: Two ruminally-cannulated cows were used to determine in situ digestibility of the diet as affected by BioFresh™ Boluses. The in situ procedures were performed twice, once prior to the administration of the BioFresh™ Boluses, and then during and after administration of the 3 Griswold et al. 2003 BioFresh™ Boluses. Samples of the diet were dried (24 h, 55ºC), ground through a 2-mm screen in a Wiley mill (Thomas Scientific, Philadelphia, PA), and weighed (2.5 g) into Dacron bags (pore size 50 ± 10 μ, Ankom Co., Fairport, NY). Bags were sealed and placed in the rumen after a 15 min. preincubation in water at 0800 h. Once bags were placed in the rumen, they were sequentially removed to achieve the following incubation times: 0, 1, 3, 6, 9, 12, 24, 36, 48, 60, 72, and 96 h. Quadruplicate bags were prepared for each cow for each time point, and blank bags were included at each time point to correct for bacterial contamination. Upon removal, bags were thoroughly rinsed in cold water until no coloration of the water occurred and dried (24 h, 55ºC). The rate of disappearance of potentially digestible components was determined by the log-linear procedure to provide initial estimates for the nonlinear procedure of SAS (SAS Inst. Inc., Cary, NC), as described by Callison et al. (2001). Sampling and analyses: Dietary TMR samples and orts were collected on a daily basis, stored at -20ºC, and composited on a weekly basis. Composite samples were analyzed for DM, OM, ash and N (AOAC, 1990), NDF, ADF, and ADIN (Ankom Technology, Fairport, NY), and lipid content (Hara and Radin, Milk weights were recorded on a daily basis. Milk samples were collected on the following days of the study: -7 through 14, 16, 19, 22, 25, 27, 49, and 78. Milk samples from each milking were composited on a daily basis, and analyzed for fat, true protein, lactose, solids, and somatic cell count. In situ samples were analyzed for DM, NDF, ADF, and N. Experimental Design and Statistical Analyses: The experimental design was a split plot with treatment as the main plot and time as the subplot. Cow was the experimental unit. Data were statistically analyzed using the MIXED procedures of SAS (SAS Inst. Inc., Cary, NC) with repeated measures. The model included the effects of cow, day, initial LSCC, treatment, and all interactions. Cow and day were used as covariates in the analysis. Post-hoc testing was performed using LSD. Significance was determined at P ≤ 0.05 and P-values of 0.05 > P ≤ 0.10 are discussed as trends. Results and Discussion The objective of this study was to determine the effect of short-term feeding of YC and DFE (i.e. BioFresh™) in a bolus form on SCC in lactating Holstein cows. In addition, the effects of BioFresh™ Boluses on milk yield, milk composition and in situ dietary digestibility 4 Griswold et al. 2003 were also determined. In examination of the raw data, it became apparent that the effect of BioFresh™ on SCC may depend on the SCC of the cow prior to administration. Therefore, data were analyzed by grouping cows according to initial LSCC, which resulted in four “treatment” groups. The four groups are designated as follows: 1.) Initial LSCC of ≤ 4 and BioFresh™ (B4), 2.) Initial LSCC ≥ 5 and BioFresh™ (B5), 3.) Initial LSCC ≤ 4 and Placebo (P4), and 4.) Initial LSCC ≥ 5 and Placebo (P5). These groupings contained 13, 9, 13, and 9 cows, respectively. The effects of BioFresh™ Boluses on milk yield and milk composition for the entire trial are provided in Table 2. There were no effects of treatment on yields of actual milk or fat corrected milk (FCM) in the current study. This is not surprising as effects of YC or DFE on milk yield are highly variable (Piva et al., 1993; Dann et al., 2000; Vicini et al., 2003). There was a trend (P = 0.08) for an interaction between initial LSCC and treatment on milk yield (Table 2) with the B4 group having greater yields than all other groups. There are no findings in the literature to support this potential interaction. Further, visual evaluation of daily milk yields (Figure 1) suggest that cows in group B4 had numerically greater milk production at the start of the trial compared to the other groupings, which may have led to the trend described above. More research needs to be conducted with a greater number of cows to confirm this finding. As a result of the trend for the interaction on milk yield, there was a trend (P = 0.09) for an interaction of initial LSCC and treatment to affect milk fat % with B4 having a lower % compared to all other groupings. Further, the greater milk production with B4 caused trends for greater yields of milk protein (P = 0.09) and lactose (P = 0.09) with the B4 grouping compared to all other groupings. Cows with an initial LSCC of ≤ 4 had significantly greater (P = 0.0005) milk lactose % than cows having an initial LSCC of ≥ 5. Ballou et al. (1995) showed that increasing SCC caused a decrease in lactose % of bulk tank milk samples. The main objective of this study was to determine if bolusing BioFresh™ for three consecutive days would affect SCC of lactating cows. There was a significant interaction (P = 0.05) of initial LSCC by treatment by day on LSCC, which meant that cows in group B5, those with initial of LSCC of ≥ 5 and bolused with BioFresh™, had significantly lower LSCC that cows in group P5, those with initial of LSCC of ≥ 5 and bolused with the placebo, on days 22, 25, and 27 (Figure 2, 2A, 2B). The level of decrease averaged approximately one LSCC unit (i.e. 200,000 somatic cells). Dann et al. (2000) reported no effect of feeding YC for 21 days 5 Griswold et al. 2003 prior to parturition on SCC of Jersey cows for the first 140 days of lactation. Vicini et al. (2003) found no effect of DFE on SCC in cows from two separate trials. Delineating a reason for this effect on SCC was beyond the capacity of this study. However, it would appear that the effect of reducing SCC is only evident in cows with a LSCC of ≥ 5, which would suggest that BioFresh™ has an affect on the immune response of cows that are actively trying to clear an intramammary infection (IMI). The response seemed to start occurring approximately 18 to 21 d after initial bolusing of the BioFresh™. The length of the response is difficult to elucidate, as milk sampling did not continue on a regular basis past day 27 of the study. By day 49, the LSCC of groups B5 and P5 were similar (Figure 2, 2A, 2B), suggesting that the response lasted less than 28 days. Future research should focus on determining the length and scope of the SCC response. Examination of in situ digestibility with two ruminally-cannulated cows showed mixed response to BioFresh™ Boluses (Table 3, Figure 3). Cow A responded positively to the BioFresh™ with an increase in the rate and extent of digestion of the diet, whereas, Cow B showed a decrease in the rate and extent of diet digestibility. Future research should include a greater number of cows with a crossover experimental design to more clearly assess potential effects on digestibility. However, any effects on digestibility would likely be transient given the method of administering the BioFresh™. Summary Administering BioFresh™ Boluses for three consecutive days to cows with an initial LSCC of ≥ 5 caused LSCC to decrease roughly one LSCC unit (i.e. 200,000 SCC) starting approximately 18 to 21 days after initial administration of the boluses. The decrease in LSCC lasted < 28 days. Other effects from BioFresh™ Boluses were limited, and more research is needed to more clearly delineate potential effects on milk yield, milk composition, and diet digestibility. 6 Griswold et al. 2003 Literature Cited Association of Official Analytical Chemists, 1990. Official Methods of Analysis, vol. I, 15th ed. AOAC, Arlington, VA. Ballou, L. U., M. Pasquini, and R. D. Bremel. 1995. Factors affecting herd milk composition and milk plasmin at four levels of somatic cell counts. J. Dairy Sci. 78:2186-2195. Beauchemin, K. A., W. Z. Yang, and L. M. Rode. 1999. Effects of grain source and enzyme additive on site and extent of nutrient digestion in dairy cows. J. Dairy Sci. 82:378-390. Beauchemin, K. A., L. M. Rode, M. Maekawa, D. P. Morgavi, and R. Kampen. 2000. Evaluation of a nonstarch polysaccharidase feed enzyme in dairy cow diets. J. Dairy Sci. 83:543-553. Bowman, G. R., K. A. Beauchemin, and J. A. Shelford. 2002. The proportion of the diet to which fibrolytic enzymes are added affects nutrient digestion by lactating dairy cows. J. Dairy Sci. 85:3420-3429. Callison, S. L., J. L. Firkins, M. L. Eastridge, and B. L. Hull. 2001. Site of nutrient digestion by dairy cows fed corn of different particle sizes or steam-rolled. J. Dairy Sci. 84:1485-1467. Dann, H. M., J. K. Drackley, G. C. McCoy, M. F. Hutjens, and J. E. Garrett. 2000. Effects of yeast culture (Saccharomyces cerevisiae) on prepartum intake and postpartum intake and milk production of Jersey cows. J. Dairy Sci. 83:123-127. Hara, A., Radin, N. S., 1978. Lipid extraction of tissues with a low toxicity solvent. Anal. Biochem. 90, 420-426. Harmon, R. J. 1994. Physiology of mastitis and factors affecting somatic cell counts. J. Dairy Sci. 77:2103-2112. Hogan, J. S., W. P. Weiss, and K. L. Smith. 1993. Role of vitamin E and selenium in host defense against mastitis. J. Dairy Sci. 76:2795-2803. Kehrli, Jr., M. E., and D. E. Shuster. 1994. Factors affecting milk somatic cells and their role in health of the bovine mammary gland. J. Dairy Sci. 77:619-627. Kung, Jr., L. M. A. Cohen, L. M. Rode, and R. J. Teather. 2002. The effect of fibrolytic enzymes sprayed onto forages and fed in a total mixed ration to lactating dairy cows. J. Dairy Sci. 85:2396-2402. NRC. 2001. Nutritional requirements of dairy cattle, 7th rev. ed. National Academy Press. Washington, D.C. Piva, G., S. Belladonna, G. Fusconi, and F. Sicbaldi. 1993. Effects of yeast on dairy cow performance, ruminal fermentation, blood components, and milk manufacturing properties. J. Dairy Sci. 76:2717-2722. 7 Griswold et al. 2003 Vicini, J. L., H. G. Bateman, M. K. Bhat, J. H. Clark, R. A. Erdman, R. H. Phipps, M. E. Van Amburgh, G. F. Hartnell, R. L. Hintz, and D. L. Hard. 2003. Effect of feeding supplemental fibrolytic enzymes or soluble sugars with malic acid on milk production. J. Dairy Sci. 86:576-585. Wilson, D. J., R. N. Gonzalez, and H. H. Das. 1997. Bovine mastitis pathogens in New York and Pennsylvania: Prevalence and effects on somatic cell count and milk production. J. Dairy Sci. 80:2592-2598. Yang, W. Z., K. A. Beauchemin, and L. M. Rode. 2000. A comparison of methods of adding fibrolytic enzymes to lactating cow diets. J. Dairy Sci. 2000. 83:2512-2520. 8 Griswold et al. 2003 Table 1. Ingredient composition and nutrient analysis of diet. Ingredient % of diet DM Alfalfa hay 10.0 Corn Silage 24.0 Milage Wet Brewers Grain Cottonseed Ground Corn 8.01 10.0 4.00 28.6 Nutrient DM % 52.1 % of DM OM 93.5 CP 18.5 NDF 45.3 ADF 21.9 Extruded Soybeans 6.01 ADICP 1.9 Soybean meal, 48% 4.00 NFC Minerals and vitamins 5.38 Fat 5.7 Ash 6.5 25.9 Griswold et al. 2003 Table 2. Effect of BioFresh Boluses™ vs. placebo boluses on milk yield and composition of lactating Holstein cows grouped by initial linear somatic cell count (ILSCC). Initial Linear SCC ≤4 ≥5 Treatment2 Placebo BioFresh Placebo BioFresh SEM Probability ILSCC ILSCC Trt x Trt Actual Milk, lb/d 76.8 88.3 81.9 77.1 4.6 NS NS 0.08 FCM, lb/d 72.7 80.7 75.0 72.9 3.9 NS NS NS Fat, % 3.67 3.45 3.48 3.71 0.13 NS NS 0.09 Fat yield, lb/d 2.80 3.03 2.81 2.81 0.15 NS NS NS Protein, % 3.21 3.09 3.15 3.14 0.07 NS NS NS Protein yield, lb/d 2.45 2.70 2.56 2.42 0.12 NS NS 0.09 Lactose, % 5.03 5.04 4.88 4.92 0.04 0.0005 NS NS Lactose yield, lb/d 3.87 4.45 4.01 3.79 0.23 NS NS 0.09 12.83 12.50 12.45 12.69 0.18 NS NS NS 9.83 11.0 10.1 9.71 0.52 NS NS NS <0.0001 NS NS 0.48 <0.0001 NS NS Solids, % Solids yield, lb/d SCC, 1000’s Linear SCC 1 89 1.54 118 1.50 1346 5.22 758 4.88 Cows bolused on three consecutive days at 0800 h. 10 241 Griswold et al. 2003 Table 3. Effect of BioFresh boluses1 on in situ rate and extent of DM, NDF, ADF, and CP disappearance in two ruminally-cannulated cows. Cows A Treatment2 In situ rate of disappearance, h-1 B Pre Post Pre Post Dry matterc 0.038 0.049 0.040 0.043 Neutral detergent fiber 0.033 0.047 0.035 0.034 Acid detergent fiber 0.032 0.050 0.034 0.034 Crude protein 0.032 0.032 0.041 0.038 In situ extent of disappearance, 24 h Dry matterc 69.7 72.7 75.9 67.4 Neutral detergent fiber 44.6 50.6 56.4 42.6 Acid detergent fiber 43.8 48.9 54.7 39.7 Crude protein 67.9 70.9 75.1 68.1 1 Cows bolused on three consecutive days at 0800 h. 2 Pre = In situ digestibility prior to BioFresh bolusing; Post = In situ digestibility during and after BioFresh bolusing c Corrected for bacterial contributions. 11 Griswold et al. 2003 Figure 1. Effect of BioFresh Boluses™ vs. placebo boluses on milk yield by lactating Holstein cows over time grouped by initial linear somatic cell count (ILSCC) 110 Bolus period 105 100 95 Milk, lb. 90 85 80 75 70 65 60 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Day of trial BioFresh Initial LSCC 4 or less Placebo Initial LSCC 4 or less 12 BioFresh Initial LSCC 5 or more Placebo Initial LSCC 5 or more Griswold et al. 2003 Figure 2. Effect of BioFresh Boluses™ vs. placebo boluses on linear somatic cell count (LSCC) of lactating Holstein cows over time grouped by initial LSCC. 8 Bolus period 7 * * * 6 Linear SCC 5 4 ‡ 3 ‡ ‡ 2 1 0 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Day of trial BioFresh Initial LSCC 4 or less BioFresh Initial LSCC 5 or more Placebo Initial LSCC 4 or less Placebo Initial LSCC 5 or more *,‡ Data points within initial LSCC grouping with different superscripts differ significantly, P < 0.05. 13 70 75 80 Griswold et al. 2003 Figure 3. Effect of BioFresh Boluses™ vs. placebo boluses on in situ NDF disappearance over time. 90 Bolus 80 Bolus NDF disappearance, % 70 60 Bolus 50 40 30 Cow A Pre 20 Cow A Post Cow B Pre Cow B Post 10 0 -8 -4 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96 100 Time, h 14