Effect of Dietary Rumen-Protected Choline in Lactating Dairy Cows1 R. A. ERDMAN and B. K. SHARMA2 Animal ScIence Department University of Maryland College Park 20742
ABSTRACT
(Key words: choline, dairy cows, lactation, romen-protected)
Two experiments were conducted to test the effects of graded amounts of romen-protected choline on milk yield and composition in lactating dairy cows fed 40% com silage and 60% concentrate diets (OM basis). In Experiment 1, 48 Holstein cows were fed 0, .078, .156, and .234% rumen-protected choline (choline chloride basis) from wk 5 to 21 postpartum. Increasing choline had no effect on OM! and tended to increase milk yield only from 1 to 2.2 kg/d. Milk fat percentage was reduced in the .078% choline treatment and increased to control levels thereafter with .156 and .234% choline. In Experiment 2, 16 Holstein cows in midlaetation were assigned randomly to either 13.0 or 16.5% dietary CP (OM basis). Within CP concentration, cows were fed 0, .08, .16, and .24% rumenprotected choline in a replicated 4 x 4 Latin square design. Dietary protein had no effect on milk yield, although milk protein percentage and yield were increased .25 percentage units and 63 gld, respectively, by increased dietary CPo Increasing dietary choline to .24% linearly increased milk yield 2.6 kg/d, although it had no consistent effects on milk fat or protein percentage. There was only a slight tendency for greater responses in milk yield to dietary choline with lower dietary CP. Data from these experiments confirm earlier results with postruminal choline infusions, suggesting that choline may be a limiting nutrient for milk pr0duction.
Abbreviation key: PP :::: postpartum, RPC :::: romeo-protected choline. INTRODUCTION
Received Iune 27, 1990. ~ted November 21, 1990. IScientific Article Number A 6014. CODlribution Number 8174 of the Malyland Agricultural E.xpmment Station. 2ADimal Scieoces Department, Michigan State Univ~ lily, East LaDsiDg, MI 48824.
1991 I Dairy Sci 74:1641-1647
Choline is a required nutrient for many animal species, including rats (14), poultry (12, 15, 18, 26), swine (16, 24), preruminant sheep (1), and calves (11). However, few research studies have been conducted feeding choline to rominants and, in particular, to lactating dairy cows. Our earlier work (23) has shown that postrominal supplementation of choline results in increased milk production and milk fat percentage. The average response in three experiments to postruminal choline infusions ranging from 30 to 90 gld per cow was 1.7 kg milk, .25 percentage units of fat, and 2.4 kg FCM (23). A study with posttuminal infusion of choline and methionine, with or without an inhibitor of methyl transfer for choline synthesis, suggested that a portion of the dairy cow's methionine requirement is as a methyl donor for choline synthesis (21). This suggests a possible interaction of choline and dietary protein (e.g., postruminal methionine availability) as shown in other species (8, 18). Dietary choline is degraded rapidly in the rumen (3, 6, 17). In vivo trials with dairy cows in which choline intake was increased up to 303 gld over controls resulted in only a 1.3 gld increase in choline flow to the duodenum (22). Data from these experiments indicated that dietary supplementation of choline chloride is not a practical method of meeting the dairy cow's choline requirement. Recently, a rumen-protected choline product (Showa Denko, K.K., Tokyo, Japan) that could potentially be used to supplement the dairy cow's choline needs has become available. In vitro studies in our laboratoIy showed that at least 87% of the choline was protected from rumen degradation, but choline was not detected in the feces of animals fed this product.
1641
1642
ERDMAN AND SHARMA
TABLE 1. Ingredient composition (percentage as-fed basis) of control grain mixes and chemical composition of total mixed diets (conttols) used in Experiments 1 and 2. Experiment 2 Experiment 1
13.0% CP
16.5% CP
Ingredient
Ground com1,2 Soybean meal (48% CP)
Limestone Dicalcium phosphate
Trace-mineralized saI~
Potassium magnesium sulfate Potassium chloride Vitamin A, D, and E 4 Chemical compositionS Choline,6 mg/g CP, % OM, % NDF, % ADF, % Ca, %
74.75 20.76 1.72 1.31
.73 .43 .26 .02 1.02 14.8 94.4 30.4 13.9 .91
82.98 11.80 2.36 .99 .83
68.94 26.74 2.46
55 .02
.63 .83 .23 .15 .02
1.03 13.0 93.6 30.8 12.1 .93
1.11 16.5 93.8 30.2 12.3 .90 ~
.47
~%
~
~
Mg, %
22
20
20
~%
~
~
~
126 26 155 35 7
1.17 29 228 41 7
1.19 .29 205 44 8
K, % Na, % Fe, mgIkg Zn, mgIkg CD, mgIkg -mgIkg
~
~
~
lRumen-protected choline containing 30% choline chloride was substituted for com at 0, .39•.78, and 1.17% of the grain mix (as-fed basis). 2Rumen-protected choline containing 30% choline chloride was substituted for 0, .41, .83, and 1.24% of the grain mix (as-fed basis). 3Contains : NaCl >94%; Fe, .10%; _ 20%; Zn, .008%; Mg, .10%; S, .05% CD, .025%; Co, .010%; and I, .007%. 4Contains: 2,063,630 IU/kg vitamin A; 454,545 IU/kg vitamin D; and 2227 IU/kg vitamin E. >rotal mixed diet, DM basis. ~pressed as choline chloride equivalents.
It was our objective to evaluate this product in early lactation dairy cows fed com silage-based diets and to determine the interaction between choline feeding and dietary CP concentration. MATERIALS AND METHODS Animals and Sampling Procedures
Holstein cows (n = 48) in early lactation were used in Experiment 1. They were fed a total mixed diet consisting of 40% com silage and 60% concentrate (DM basis, Table 1) during wk 1 to 21 postpartum (PP). During wk 1 to 4 PP, cows were fed a control diet containing no supplemental choline. Cows were asJournal of Dairy Science Vol. 74, No.5, 1991
signed in blocks of 4 by date of calving and parity (36 multiparous and 12 primiparous) and at wk 5 PP were assigned randomly within block to one of four levels of added dietary rumen-protected choline chloride (RPC): 0, .078, .156, and .234% (DM basis, substituted for ground com) to be fed through wk 21 PP. These amounts, assuming an average DMI of 20 kgId per cow, should have resulted in intakes of 0, 15, 30, and 45 g choline chloride daily. Amounts of choline in treatment diets encompassed optimal levels suggested in an experiment by Sharma and Erdman (23) to give a dose response to added choline. Cows were fed individually the total mixed diets twice daily and were housed in tie stalls
1643
RUMEN-PROTECTED CHOLINE FOR DAIRY COWS
fitted with rubber mats and bedded with wood shavings. Feed refusals were recorded once daily. Milk production was measured twice daily. Milk samples, composited from the a.m. and p.m. milkings once a week. were submitted to the Maryland DillC laboratory for fat, protein, and lactose by infrared analysis (Milk-OScan, Foss Technology, Denmark) and SCC (Fossomatic Somatic Cell Counter, Foss Technology, Denmark). Cows were allowed 3 h of exercise daily and were weighed every other week prior to the p.m. milking. Blood plasma samples at 4 h after feeding were taken during wk 4, 6, and 10 PP for triglyceride (Wako Kit Number 997-69801, Wako Biochemical, Osaka, Japan) and free fatty acids (Wako Kit Number 990-75401, Wako Biochemical) analysis by enzymatic procedures and for phosphatidylcholine by the procedure of Takayama et al. (25). Health and reproductive data were recorded during the trial, including incidence of mastitis and off-feed problems. Reproductive data included days open, days to first estrus, number of breedings, pregnancy rates, and services per conception. In Experiment 2, the potential effect of dietary CP on responses to dietary choline was tested. Sixteen Holstein cows in midlactation (151 d PP, SD = 12) were assigned randomly to either 13.0 or 16.5% CP diets in a 12-wk experiment (Table 1). Cows were fed individually a 40% com silage and 60% concentrate diet as a total mixed diet once daily, with feed refusals collected once daily. Within CP concentration, cows were fed diets containing 0, .08, .16, and .24% RPC (OM basis) in two replicated 4 x 4 Latin squares with 3-wk experimental periods. Cows were milked twice daily and allowed exercise on a concrete lot for 3 h daily as in Experiment 1. Milk samples from consecutive a.m. and p.m. milkings were collected 2 d weekly and analyzed for fat, protein, lactose, and SCC as in Experiment 1.
pIes were stored at -10·C and later composited on a bimonthly basis in Experiment 1 and by experimental period in Experiment 2. Composite samples of com silage were oven-dried at 6S·C for 48 h and ground using a 2-mm screen. Composite silage and concentrate were analyzed for DM, ash, CP (2), NDF (19), and ADF (10). Samples were submitted to the New York DID laboratory for analysis of Ca, P, Mg, S, K, Na, Fe, Zn, Cu, and Mn content. Feed choline content of feed was detennined after initial acid hydrolysis using 15% nitric acid (15% voVvol) (4), followed by choline oxidase enzymatic procedures (25) as described by Atkins et al. (3). In Experiment 1, means for intake, milk production, and milk composition by individual cows were analyzed statistically using the general linear models procedure of the SAS (9). Data collected during lactation wk 3 and 4 were used as the covariate, using the model: Y = covariate + block + treatment + residual error. In Experiment 2, to minimize carry-over effects of previous choline treatment, data from the 3rd wk only of each period were used in the analysis. Statistical analysis was by ANOVA, using the general linear models procedure of SAS, using a split-plot model where: Y protein + cow within protein + period + choline + choline by protein + residual error. A similar model using individual cow and period within square effects showed no effect (P > .2) of square assignment Protein effects were tested using cow within protein as the test term, whereas choline and choline by protein interactions were tested using residual error. Linear and quadratic effects of RPC were analyzed by orthogonal polynomial comparisons in both experiments. Health and reproductive categorical data in Experiment 1 were analyzed by chisquare analysis. Probabilities less than .05 were regarded as significant, and data were interpreted accordingly.
=
RESULTS Chemical and Statistical Analysis
Concentrate mixtures and com silage in both experiments were sampled weekly for DM analysis. Dry matter content of concentrate mixtures was determined by oven drying overnight at l00·C (2), and DM content of com silage was determined by toluene distillation (7). Weekly com silage and concentrate sam-
Experiment 1
Control diet composition is shown in Table 1. Diets should have been nutritionally adequate except for CP and ADF (13). The relatively low ADF concentration can be attributed to the high percentage of concentrate fed and to the relatively low content of ADF in the com Journal of Dairy Science Vol. 74, No.5, 1991
1644
ERDMAN AND SHARMA
TABLE 2. Production responses1 of early lactation cows to increasing amounts of rumen-protected (RP) choline supplementation (Bxperiment 1). Choline chloride % Item
o
.078
.156
234
BW,kg RP Choline intake,2 g/da..
598
592 36.1
DMI, kg/d
21.8 3.66 36.7 3.69 3.30 4.97 372 345
599 16.9 21.7 3.62 37.7 3.18 3.15 4.87 35.0 147 1
588 51.1 21.9 3.70 37.4 3.71 3.23 4.91 38.9 166 0
171
160
Percentage of BW Milk, kgf.d Milk fa1,3 % Milk proteiD,3 %
Milk lactose % 3.5% FCM,4 ~d Milk sec, 1~/ml CliDica1 mastitis, n Plasma, mg/dl Phosphatidylcholine Free fatty acids Triglycerides
o
o
158 4.0 29
23.2
3.92 38.9 3.56 3.10 5.00 39.6 131 1
29
9.1 .5 .7 .11 1.0 .14
.OS .07 1.01 91
154 4.8 28
4.0 29
5.5
SEM
9 .5 2
1Least squares means.
2calculatcd assuming 30% choline chloride in product
3Quadratic effect of choline (P < .05).
4unear
effect of choline (P
< .OS).
=
silage used (X 23.4%). By design, dietary CP was lower than NRC (13) recommendations because of the interaction between CP and choline (8, 18, 21). Dietary choline had no effect on average BW or DMI (Table 2). Intake of RPC expressed as choline chloride equivalents increased significantly to 51 g/d in the .234% dietary RPC treatment. There was no effect of RPC on DMI, although it tended to be higher in the .156 and .234% treatments. There was no significant effect on milk yield, although there was a tendency for increased milk yield with choline treatments. Figure 1 shows actual milk production by week PP. Milk production for animals fed choline was higher than for controls throughout the study. However, milk production during the preliminary period (wk 1 to 4 PP) also was higher. Thus, differences in covariance-adjusted means for milk yield were much smaller (Table 2). Milk fat percentage changed in a quadratic fashion with increasing choline, with reduced fat percentage at .078% choline, and with fat percentages near control as choline increased to .234%. Milk protein percentage also responded quadratically to increasing choline, although the Journal of Daily Science Vol. 74, No.5, 1991
magnitude of the response was much smaller than for fat percentage. Milk lactose and sec were not affected by choline administration. Added choline resulted in a linear increase in 3.5% FCM: .156 and .234% choline increased FCM 2.4 and 1.7 kg/d over controls, respectively. Milk SCC tended to be lower for each of the choline treatments than for the control group. Choline supplementation had no effect
45
Q
a
40
~ C
...I
W
>= =:.c:: ...I
:E 25'
I
1
I
4
I
I
I
I
7 10 13 16 19 WEEK POSTPARTUM
Figure 1. Effect of 0 (0), .078 (0), .153 (e), and 234% (11) rumen-Pl'Otected choline chloride on milk yield by week poslpaItum.
1645
RUMEN-PROTECTED CHOLINE FOR DAIRY COWS TABLE 3. Effects of rumen-protected choline on reproductive performance and health (Experiment 1). Choline chloride, %
.156
.234
2 10
2 10
3 9
1.2 92 9 8 1.1 93 3
1.5 70 10 9 1.4 90 3
1.9 96 9 5 2.0
1.4 99 10 8
1.5 70 10 10 1.4 98 3
Item
0
.078
Cows not inseminated Cows available for breeding
2 10
SEM
o-21wt~
Services per cowl Days to first breeding Number bred Number pregnant Services per cooception1 Days open Cystic ovaries, n D-44 wk postpartum Services per cow Days to first breeding Number bred Number pregnant Services per conceptionl Days openl Cystic ovaries, n
1.1
93 3
3
1.8 88 8 6 1.8 100 5
1.9 101 10 7 2.0 159 3
1.7 95 9 9 1.7 129 5
lOS
.3 12 .32 14
.3 12 .3 19
lLinear effect of choline (P < .10).
on plasma phosphatidylcholine, FFA, or triglycerides. Reproductive traits are sununarized in Table 3. Two to three cows in each treatment were intentionally not bred because of low milk production or poor conformation. Data were subdivided to include cows bred or pregnant prior to the end of the experiment at wk 21 PP or all cows up to wk 44 PP after the completion of this experiment. Although not significantly different, the number of cows pregnant during the experiment tended to be lower as choline intake increased over .078%. Services per cow and per conception tended to be higher (P < .1) for choline-treated cows bred or pregnant prior to wk 21 also using data up to wk 44 PP. Days open also were greater (P < .1) for the .156 and .234% choline treatments using data up to wk 44 PP. We suspect that, because of the higher actual milk yield (Figure I), reproductive responses were more related to differences in milk yield than to the effect of RPC. When data up to 44 wk PP were summarized, there was no apparent trend due to RPC on number pregnant, services, or days to first breeding. Experiment 2
Data from Experiment 2 are presented in Table 4. One cow in the 16.5% CP group had
abnormally high SCC (>3 x 1()6 cells/ml) during the last three experimental periods. Although no clinical signs of mastitis were 0bserved, data for this cow during these periods were deleted because of these high counts. By random treatment assignment, cows on the 16.5% CP diet: tended to be larger (718 vs. 659 kg) than those on the 13.0% CP diet (Table 3). However, OM! expressed in kilograms per day or as percentage of BW was not affected by diet CP or RPC. Milk yield increased linearly with increasing choline intake. There was no significant choline by protein interaction although the magnitudes of the maximum responses were 2.1 and 3.1 kg/d in the 16.5 and 13.0% CP diets, respectively, and the maximum response occurred in the .16% choline 16.5% CP diet. Neither dietaty CP nor choline affected milk fat percentage or milk fat yield (Table:: 4). Milk protein percentage and yield were increased .23 percentage units and 63 gld, respectively, by increasing diet CP to 16.5%. Choline had no effect on milk protein percentage but increased protein yield because of its effects on total milk yield Because of variation in milk fat percentage, FCM yield was not affected by either diet CP or RPC (Table 4). Although the effect was not explainable, milk lactose responded in a quadJournal of Dairy Science Vol. 74, No.5, 1991
1646
ERDMAN AND SHARMA
TABLE 4. Effect of dietaIy protein and rumen-protected (RP) choline on least squares means for intake, milk yield, and composition (Experiment 2). 16.5% CP Diet
13.0% CP Diet Item
.01
.08
8 Observations, n 653 BW,kg RP Choline intake,4,5 g/d o
.16
.24
8
8
660
660
18.5
37.2
22.1 3.36 31.7
222 3.35 33.4
8 663 56.9
.0
.08
8 723 0
7 719 19.6
DMI. kg/d Percentage BW Milk,5 kg/d Milk fat, Percentage g/d 3.5% FCM, ~d Milk protein, Percentage g/d Milk lactose, 7 % Milk SCC, 103/ml
.16 7 716 40.4
.24 7 716 57.9
sm-Z 25 1.4
SEC3
4 .9
23.2 3.29 33.8
12 .18 1.4
.3 .04 .9
3.98 3.89 4.45 3.45 1271 1243 1498 1207 34.3 33.9 38.8 34.6
3.50 3.21 3.33 3.84 1017 1108 1160 1209 32.6 33.4 33.4 30.1
.17 77 1.7
.15 63 1.3
3.44 3.42 3.33 3.39 1088 1079 1107 1178 4.98 4.92 4.87 4.93 85 231 87 106
3.74 3.61 3.55 3.61 1205 1124 1226 1147 4.97 5.01 4.87 4.92 114 255 198 207
.10 37 .08 46
21.8 3.37 31.7
22.7 3.44 34.8
23.7 3.30 31.8
23.4 3.27 31.6
23.9 3.34 33.9
.05 32 .04 44
1Percentage choline chloride. 2S tandard error of mean for protein effects. 3S tandard error of mean for choline effects.
~pressed as choline chloride (grams per day). Sr.inear effect of choline (P < .05). 6t>rotein effect (P < .05). 7Quadratic effect of choline (P < .05).
ratic fashion to dietary choline when lactose was lower, in the .08 and .16% treatment, than the controls. As in Experiment 1, dietary choline had no effect on milk: sec. DISCUSSION
The range in supplemental choline intakes used in these experiments was somewhat smaller than used in previous postmminal infusion studies in which 0 to 90 g of choline or an equivalent 0 to 120 g/d choline chloride were used (23). One previous study suggested 30 g/d choline (23) or 40 g/d choline chloride as a possible requirement. Results of Experiment 1 showed highest milk yield was associated with the .156% choline, which equals 36 g/d choline. In Experiment 2, no maximwn for milk. yield was observed with up to 59 g/d RPC. Future experiments need to examine higher concentrations of dietary choline before feeding recommendations can be made. In contrast with previous infusion studies (21, 23), choline did not have a positive effect Journal of Dairy Science Vol. 74, No.5, 1991
on milk. fat percentage. Responses in milk. fat were inconsistent in that low concentrations of dietary choline reduced its percentage in Experiment 1 and had variable effects in Experiment 2. In a previous postruminal infusion experiment (21) comparing choline and methionine, milk fat percentage was increased by postruminal choline both with and without an inhibitor of methyl transfer for choline synthesis. Milk protein also was greater during choline infusion than during methionine infusions when an inhibitor of methyl transfer was included (21). Dietary methionine hydroxy analog was shown to increase milk fat percentage in low protein diets in a multi-location study (5). This suggested that methionine and, indirectly, protein requirements might be functions of methyl donor needs rather than specifically amino acid needs (20) for milk protein synthesis. There was little evidence from Experiment 2 to suggest a choline by protein interaction. The magnitude of milk yield response to choline in the 13.0% CP diet was greater than that observed in Experiment 1 or in
RUMEN-PROTECTED CHOLINE FOR DAIRY COWS
the 16.5% CP diet in Experiment 2. The interaction of choline and methionine will need further exploration before choline could be substituted for methionine in a rumen-protected form. In summary, data from these experiments confirm earlier postmminal infusion studies in which increasing postruminal choline increased milk yield up to 3.1 kg/d. However, no positive effects on milk fat percentage were observed in these studies in contrast with our earlier postruminal infusion experiments (21, 23). ACKNOWLEDGMENTS
The authors wish to acknowledge financial support provided by Showa Denko, K.K. for these experiments. We also wish to thank Charles Anderson, Tom Junis, Tom Moreland, and Laure Giles for conducting the experiments and data analysis. REFERENCES
1 AI-Ali, S. I., N. M. Malouf, and D. M. Walker. 1985. Choline requirement of the preruminant lamb during the ftrst two or three weeks of life. Aust I. Agric. Res. 36:829. 2 Association of Official Analytical Chemists. 1975. Official methods of analysis. 12th ed. AOAC, Washington, DC. 3 Atkins, K. B., R A. Erdman, and I. H. Vandersall. 1988. Dietary choline effects on milk and duodenal choline flow in dairy cattle. I. Daily Sci. 71:109. 4Atwal, A. S., N.A.M. Eskin, and M. Vaisey-Genser. 1980. Note on the estimation of choline in plant protein sources. Cereal Chem. 57:368. 5 Chandler, P. T., C. A. Brown, R P. Iohnston, Ir., G. K. Macleod, R D. McCarthy, B. R Moss, A. H. Rakes, and L. D. Satter. 1976. Protein and methionine hydroxy analog for dairy cows. I. Dairy Sci. 59:1897. 6 Dawson, RM.C., D. W. Grime, and D. B. Lindsey. 1981. On the insensitivity of sheep to the almost complete microbial destruction of dietary choline before alimentary tract absorption. Biochem. I. 196:499. 7 Dewar, W. A., and P. McDonald. 1961. Determination of dry matter in silage by distillation with toluene. I. Sci. Food Agric. 12:790. 8 Emanual, B., and I. I. Kennelly. 1984. Kinetics of methionine and choline and their incorporation into plasma lipids and milk components in lactating goats. I. Dairy Sci. 67:1912. 9 Freund, R I., and R C. Littell. 1981. SASQll for linear models. SAS Inst., Inc., Cary, NC. 10 Goering, H. K., and P. I. Van Soest. 1970. Forage fiber
1647
analyses (apparatus, reagents, procedures, and some applications). Agric. Handbook No. 379. ARS-USDA, Washington, DC. 11 Iohnson, B. C., H. H. Mitchell, 1. A. Pinkos, and C. C. Morrill 1951. Choline deficiency in the calf. 1. NUll. 43:37. 12 Molitoris, B. A., and D. H. Baker. 1976. The choline requirement of broiler chicks during the seventh week of life. Poult. Sci. 55:220. 13 National Research Council. 1988. Nutrient requiremmts of dairy cattle. Nat!. Acad. Sci., Washington, DC. 14 National Research CounciL 1978. Nutrient requirements of laboratory animals. Natl. Acad. Sci., Washington, DC. 15 National Research Council. 1984. Nutrient requirements of poultry. Natl. Acad. Sci., Washington, DC. 16 National Research Council. 1988. Nutrient requirements of swine. Nail. Acad. Sci., Washington, DC. 17 Neill, A. R. D. W. Grime, A. M. Snoswell, A. I. Northrop, D. B. Lindsay, and RM.C. Dawson. 1979. The low availability of dietary choline for the nutrition of the sheep. Biochem. 1. 180:559. 18 Pesti, G. M., A. E. Harper, and M. L. Sunde. 1980. Choline/methionine nutrition of starting broiler chicks. Three models for estimating the choline requirements with economic considerations. Poult. Sci. 59: 1973. 19 Robertson, I. B .• and P. 1. Van Soest. 1977. The detergmt system of analysis and its application to human foods. Page 125 in The analysis of dietary fiber in food. W.P.T. lames and O. Theander, ed. Marcel Dekker, New York, NY. 20 Schwab, C. G., and L. D. Satter. 1974. Effect of abomasal infusion of amino acids on lactating dairy cows. I. Dairy Sci. 57:632. 21 Sharma, B. K., and R. A. Erdman. 1988. Abomasal infusion of choline and methionine with or without 2-amino-2-methy1-1-propano1 for lactating dairy cows. I. Dairy Sci. 71:2406. 22 Sharma, B. K., and R. A. Erdman. 1988. Effect of high amounts of dietary choline supplementation on duodenal choline flow and production responses of dairy cows. I. Daily Sci. 71:2670. 23 Sharma, B. K., and R. A. Erdman. 1989. Effect of dietary and abomasally infused choline on milk production responses of lactating dairy cows. I. Nutr. 119: 248. 24 Stocldand, W. L.• and L. G. Blaylock. 1974. Choline requirement of pregnant sows and gilts under restricted feeding conditions. I. Anim. Sci. 39:1113. 25 Takayama, M., S. !tob, T. Nagasaki. and I. Tanimiju. 1977. A new enzymatic method for determination of serum choline-containing phospholipids. Clin. Chim. Acta 79:93. 26 Tsiagbe, V. K., C. W. Kang, and M L. Sunde. 1982. Effect of choline supplementation in growing pullet and laying hen diets. Poult. Sci. 61:2060.
Journal of Dairy Science Vol. 74, No.5. 1991
ABSTRACT
(Key words: choline, dairy cows, lactation, romen-protected)
Two experiments were conducted to test the effects of graded amounts of romen-protected choline on milk yield and composition in lactating dairy cows fed 40% com silage and 60% concentrate diets (OM basis). In Experiment 1, 48 Holstein cows were fed 0, .078, .156, and .234% rumen-protected choline (choline chloride basis) from wk 5 to 21 postpartum. Increasing choline had no effect on OM! and tended to increase milk yield only from 1 to 2.2 kg/d. Milk fat percentage was reduced in the .078% choline treatment and increased to control levels thereafter with .156 and .234% choline. In Experiment 2, 16 Holstein cows in midlaetation were assigned randomly to either 13.0 or 16.5% dietary CP (OM basis). Within CP concentration, cows were fed 0, .08, .16, and .24% rumenprotected choline in a replicated 4 x 4 Latin square design. Dietary protein had no effect on milk yield, although milk protein percentage and yield were increased .25 percentage units and 63 gld, respectively, by increased dietary CPo Increasing dietary choline to .24% linearly increased milk yield 2.6 kg/d, although it had no consistent effects on milk fat or protein percentage. There was only a slight tendency for greater responses in milk yield to dietary choline with lower dietary CP. Data from these experiments confirm earlier results with postruminal choline infusions, suggesting that choline may be a limiting nutrient for milk pr0duction.
Abbreviation key: PP :::: postpartum, RPC :::: romeo-protected choline. INTRODUCTION
Received Iune 27, 1990. ~ted November 21, 1990. IScientific Article Number A 6014. CODlribution Number 8174 of the Malyland Agricultural E.xpmment Station. 2ADimal Scieoces Department, Michigan State Univ~ lily, East LaDsiDg, MI 48824.
1991 I Dairy Sci 74:1641-1647
Choline is a required nutrient for many animal species, including rats (14), poultry (12, 15, 18, 26), swine (16, 24), preruminant sheep (1), and calves (11). However, few research studies have been conducted feeding choline to rominants and, in particular, to lactating dairy cows. Our earlier work (23) has shown that postrominal supplementation of choline results in increased milk production and milk fat percentage. The average response in three experiments to postruminal choline infusions ranging from 30 to 90 gld per cow was 1.7 kg milk, .25 percentage units of fat, and 2.4 kg FCM (23). A study with posttuminal infusion of choline and methionine, with or without an inhibitor of methyl transfer for choline synthesis, suggested that a portion of the dairy cow's methionine requirement is as a methyl donor for choline synthesis (21). This suggests a possible interaction of choline and dietary protein (e.g., postruminal methionine availability) as shown in other species (8, 18). Dietary choline is degraded rapidly in the rumen (3, 6, 17). In vivo trials with dairy cows in which choline intake was increased up to 303 gld over controls resulted in only a 1.3 gld increase in choline flow to the duodenum (22). Data from these experiments indicated that dietary supplementation of choline chloride is not a practical method of meeting the dairy cow's choline requirement. Recently, a rumen-protected choline product (Showa Denko, K.K., Tokyo, Japan) that could potentially be used to supplement the dairy cow's choline needs has become available. In vitro studies in our laboratoIy showed that at least 87% of the choline was protected from rumen degradation, but choline was not detected in the feces of animals fed this product.
1641
1642
ERDMAN AND SHARMA
TABLE 1. Ingredient composition (percentage as-fed basis) of control grain mixes and chemical composition of total mixed diets (conttols) used in Experiments 1 and 2. Experiment 2 Experiment 1
13.0% CP
16.5% CP
Ingredient
Ground com1,2 Soybean meal (48% CP)
Limestone Dicalcium phosphate
Trace-mineralized saI~
Potassium magnesium sulfate Potassium chloride Vitamin A, D, and E 4 Chemical compositionS Choline,6 mg/g CP, % OM, % NDF, % ADF, % Ca, %
74.75 20.76 1.72 1.31
.73 .43 .26 .02 1.02 14.8 94.4 30.4 13.9 .91
82.98 11.80 2.36 .99 .83
68.94 26.74 2.46
55 .02
.63 .83 .23 .15 .02
1.03 13.0 93.6 30.8 12.1 .93
1.11 16.5 93.8 30.2 12.3 .90 ~
.47
~%
~
~
Mg, %
22
20
20
~%
~
~
~
126 26 155 35 7
1.17 29 228 41 7
1.19 .29 205 44 8
K, % Na, % Fe, mgIkg Zn, mgIkg CD, mgIkg -mgIkg
~
~
~
lRumen-protected choline containing 30% choline chloride was substituted for com at 0, .39•.78, and 1.17% of the grain mix (as-fed basis). 2Rumen-protected choline containing 30% choline chloride was substituted for 0, .41, .83, and 1.24% of the grain mix (as-fed basis). 3Contains : NaCl >94%; Fe, .10%; _ 20%; Zn, .008%; Mg, .10%; S, .05% CD, .025%; Co, .010%; and I, .007%. 4Contains: 2,063,630 IU/kg vitamin A; 454,545 IU/kg vitamin D; and 2227 IU/kg vitamin E. >rotal mixed diet, DM basis. ~pressed as choline chloride equivalents.
It was our objective to evaluate this product in early lactation dairy cows fed com silage-based diets and to determine the interaction between choline feeding and dietary CP concentration. MATERIALS AND METHODS Animals and Sampling Procedures
Holstein cows (n = 48) in early lactation were used in Experiment 1. They were fed a total mixed diet consisting of 40% com silage and 60% concentrate (DM basis, Table 1) during wk 1 to 21 postpartum (PP). During wk 1 to 4 PP, cows were fed a control diet containing no supplemental choline. Cows were asJournal of Dairy Science Vol. 74, No.5, 1991
signed in blocks of 4 by date of calving and parity (36 multiparous and 12 primiparous) and at wk 5 PP were assigned randomly within block to one of four levels of added dietary rumen-protected choline chloride (RPC): 0, .078, .156, and .234% (DM basis, substituted for ground com) to be fed through wk 21 PP. These amounts, assuming an average DMI of 20 kgId per cow, should have resulted in intakes of 0, 15, 30, and 45 g choline chloride daily. Amounts of choline in treatment diets encompassed optimal levels suggested in an experiment by Sharma and Erdman (23) to give a dose response to added choline. Cows were fed individually the total mixed diets twice daily and were housed in tie stalls
1643
RUMEN-PROTECTED CHOLINE FOR DAIRY COWS
fitted with rubber mats and bedded with wood shavings. Feed refusals were recorded once daily. Milk production was measured twice daily. Milk samples, composited from the a.m. and p.m. milkings once a week. were submitted to the Maryland DillC laboratory for fat, protein, and lactose by infrared analysis (Milk-OScan, Foss Technology, Denmark) and SCC (Fossomatic Somatic Cell Counter, Foss Technology, Denmark). Cows were allowed 3 h of exercise daily and were weighed every other week prior to the p.m. milking. Blood plasma samples at 4 h after feeding were taken during wk 4, 6, and 10 PP for triglyceride (Wako Kit Number 997-69801, Wako Biochemical, Osaka, Japan) and free fatty acids (Wako Kit Number 990-75401, Wako Biochemical) analysis by enzymatic procedures and for phosphatidylcholine by the procedure of Takayama et al. (25). Health and reproductive data were recorded during the trial, including incidence of mastitis and off-feed problems. Reproductive data included days open, days to first estrus, number of breedings, pregnancy rates, and services per conception. In Experiment 2, the potential effect of dietary CP on responses to dietary choline was tested. Sixteen Holstein cows in midlactation (151 d PP, SD = 12) were assigned randomly to either 13.0 or 16.5% CP diets in a 12-wk experiment (Table 1). Cows were fed individually a 40% com silage and 60% concentrate diet as a total mixed diet once daily, with feed refusals collected once daily. Within CP concentration, cows were fed diets containing 0, .08, .16, and .24% RPC (OM basis) in two replicated 4 x 4 Latin squares with 3-wk experimental periods. Cows were milked twice daily and allowed exercise on a concrete lot for 3 h daily as in Experiment 1. Milk samples from consecutive a.m. and p.m. milkings were collected 2 d weekly and analyzed for fat, protein, lactose, and SCC as in Experiment 1.
pIes were stored at -10·C and later composited on a bimonthly basis in Experiment 1 and by experimental period in Experiment 2. Composite samples of com silage were oven-dried at 6S·C for 48 h and ground using a 2-mm screen. Composite silage and concentrate were analyzed for DM, ash, CP (2), NDF (19), and ADF (10). Samples were submitted to the New York DID laboratory for analysis of Ca, P, Mg, S, K, Na, Fe, Zn, Cu, and Mn content. Feed choline content of feed was detennined after initial acid hydrolysis using 15% nitric acid (15% voVvol) (4), followed by choline oxidase enzymatic procedures (25) as described by Atkins et al. (3). In Experiment 1, means for intake, milk production, and milk composition by individual cows were analyzed statistically using the general linear models procedure of the SAS (9). Data collected during lactation wk 3 and 4 were used as the covariate, using the model: Y = covariate + block + treatment + residual error. In Experiment 2, to minimize carry-over effects of previous choline treatment, data from the 3rd wk only of each period were used in the analysis. Statistical analysis was by ANOVA, using the general linear models procedure of SAS, using a split-plot model where: Y protein + cow within protein + period + choline + choline by protein + residual error. A similar model using individual cow and period within square effects showed no effect (P > .2) of square assignment Protein effects were tested using cow within protein as the test term, whereas choline and choline by protein interactions were tested using residual error. Linear and quadratic effects of RPC were analyzed by orthogonal polynomial comparisons in both experiments. Health and reproductive categorical data in Experiment 1 were analyzed by chisquare analysis. Probabilities less than .05 were regarded as significant, and data were interpreted accordingly.
=
RESULTS Chemical and Statistical Analysis
Concentrate mixtures and com silage in both experiments were sampled weekly for DM analysis. Dry matter content of concentrate mixtures was determined by oven drying overnight at l00·C (2), and DM content of com silage was determined by toluene distillation (7). Weekly com silage and concentrate sam-
Experiment 1
Control diet composition is shown in Table 1. Diets should have been nutritionally adequate except for CP and ADF (13). The relatively low ADF concentration can be attributed to the high percentage of concentrate fed and to the relatively low content of ADF in the com Journal of Dairy Science Vol. 74, No.5, 1991
1644
ERDMAN AND SHARMA
TABLE 2. Production responses1 of early lactation cows to increasing amounts of rumen-protected (RP) choline supplementation (Bxperiment 1). Choline chloride % Item
o
.078
.156
234
BW,kg RP Choline intake,2 g/da..
598
592 36.1
DMI, kg/d
21.8 3.66 36.7 3.69 3.30 4.97 372 345
599 16.9 21.7 3.62 37.7 3.18 3.15 4.87 35.0 147 1
588 51.1 21.9 3.70 37.4 3.71 3.23 4.91 38.9 166 0
171
160
Percentage of BW Milk, kgf.d Milk fa1,3 % Milk proteiD,3 %
Milk lactose % 3.5% FCM,4 ~d Milk sec, 1~/ml CliDica1 mastitis, n Plasma, mg/dl Phosphatidylcholine Free fatty acids Triglycerides
o
o
158 4.0 29
23.2
3.92 38.9 3.56 3.10 5.00 39.6 131 1
29
9.1 .5 .7 .11 1.0 .14
.OS .07 1.01 91
154 4.8 28
4.0 29
5.5
SEM
9 .5 2
1Least squares means.
2calculatcd assuming 30% choline chloride in product
3Quadratic effect of choline (P < .05).
4unear
effect of choline (P
< .OS).
=
silage used (X 23.4%). By design, dietary CP was lower than NRC (13) recommendations because of the interaction between CP and choline (8, 18, 21). Dietary choline had no effect on average BW or DMI (Table 2). Intake of RPC expressed as choline chloride equivalents increased significantly to 51 g/d in the .234% dietary RPC treatment. There was no effect of RPC on DMI, although it tended to be higher in the .156 and .234% treatments. There was no significant effect on milk yield, although there was a tendency for increased milk yield with choline treatments. Figure 1 shows actual milk production by week PP. Milk production for animals fed choline was higher than for controls throughout the study. However, milk production during the preliminary period (wk 1 to 4 PP) also was higher. Thus, differences in covariance-adjusted means for milk yield were much smaller (Table 2). Milk fat percentage changed in a quadratic fashion with increasing choline, with reduced fat percentage at .078% choline, and with fat percentages near control as choline increased to .234%. Milk protein percentage also responded quadratically to increasing choline, although the Journal of Daily Science Vol. 74, No.5, 1991
magnitude of the response was much smaller than for fat percentage. Milk lactose and sec were not affected by choline administration. Added choline resulted in a linear increase in 3.5% FCM: .156 and .234% choline increased FCM 2.4 and 1.7 kg/d over controls, respectively. Milk SCC tended to be lower for each of the choline treatments than for the control group. Choline supplementation had no effect
45
Q
a
40
~ C
...I
W
>= =:.c:: ...I
:E 25'
I
1
I
4
I
I
I
I
7 10 13 16 19 WEEK POSTPARTUM
Figure 1. Effect of 0 (0), .078 (0), .153 (e), and 234% (11) rumen-Pl'Otected choline chloride on milk yield by week poslpaItum.
1645
RUMEN-PROTECTED CHOLINE FOR DAIRY COWS TABLE 3. Effects of rumen-protected choline on reproductive performance and health (Experiment 1). Choline chloride, %
.156
.234
2 10
2 10
3 9
1.2 92 9 8 1.1 93 3
1.5 70 10 9 1.4 90 3
1.9 96 9 5 2.0
1.4 99 10 8
1.5 70 10 10 1.4 98 3
Item
0
.078
Cows not inseminated Cows available for breeding
2 10
SEM
o-21wt~
Services per cowl Days to first breeding Number bred Number pregnant Services per cooception1 Days open Cystic ovaries, n D-44 wk postpartum Services per cow Days to first breeding Number bred Number pregnant Services per conceptionl Days openl Cystic ovaries, n
1.1
93 3
3
1.8 88 8 6 1.8 100 5
1.9 101 10 7 2.0 159 3
1.7 95 9 9 1.7 129 5
lOS
.3 12 .32 14
.3 12 .3 19
lLinear effect of choline (P < .10).
on plasma phosphatidylcholine, FFA, or triglycerides. Reproductive traits are sununarized in Table 3. Two to three cows in each treatment were intentionally not bred because of low milk production or poor conformation. Data were subdivided to include cows bred or pregnant prior to the end of the experiment at wk 21 PP or all cows up to wk 44 PP after the completion of this experiment. Although not significantly different, the number of cows pregnant during the experiment tended to be lower as choline intake increased over .078%. Services per cow and per conception tended to be higher (P < .1) for choline-treated cows bred or pregnant prior to wk 21 also using data up to wk 44 PP. Days open also were greater (P < .1) for the .156 and .234% choline treatments using data up to wk 44 PP. We suspect that, because of the higher actual milk yield (Figure I), reproductive responses were more related to differences in milk yield than to the effect of RPC. When data up to 44 wk PP were summarized, there was no apparent trend due to RPC on number pregnant, services, or days to first breeding. Experiment 2
Data from Experiment 2 are presented in Table 4. One cow in the 16.5% CP group had
abnormally high SCC (>3 x 1()6 cells/ml) during the last three experimental periods. Although no clinical signs of mastitis were 0bserved, data for this cow during these periods were deleted because of these high counts. By random treatment assignment, cows on the 16.5% CP diet: tended to be larger (718 vs. 659 kg) than those on the 13.0% CP diet (Table 3). However, OM! expressed in kilograms per day or as percentage of BW was not affected by diet CP or RPC. Milk yield increased linearly with increasing choline intake. There was no significant choline by protein interaction although the magnitudes of the maximum responses were 2.1 and 3.1 kg/d in the 16.5 and 13.0% CP diets, respectively, and the maximum response occurred in the .16% choline 16.5% CP diet. Neither dietaty CP nor choline affected milk fat percentage or milk fat yield (Table:: 4). Milk protein percentage and yield were increased .23 percentage units and 63 gld, respectively, by increasing diet CP to 16.5%. Choline had no effect on milk protein percentage but increased protein yield because of its effects on total milk yield Because of variation in milk fat percentage, FCM yield was not affected by either diet CP or RPC (Table 4). Although the effect was not explainable, milk lactose responded in a quadJournal of Dairy Science Vol. 74, No.5, 1991
1646
ERDMAN AND SHARMA
TABLE 4. Effect of dietaIy protein and rumen-protected (RP) choline on least squares means for intake, milk yield, and composition (Experiment 2). 16.5% CP Diet
13.0% CP Diet Item
.01
.08
8 Observations, n 653 BW,kg RP Choline intake,4,5 g/d o
.16
.24
8
8
660
660
18.5
37.2
22.1 3.36 31.7
222 3.35 33.4
8 663 56.9
.0
.08
8 723 0
7 719 19.6
DMI. kg/d Percentage BW Milk,5 kg/d Milk fat, Percentage g/d 3.5% FCM, ~d Milk protein, Percentage g/d Milk lactose, 7 % Milk SCC, 103/ml
.16 7 716 40.4
.24 7 716 57.9
sm-Z 25 1.4
SEC3
4 .9
23.2 3.29 33.8
12 .18 1.4
.3 .04 .9
3.98 3.89 4.45 3.45 1271 1243 1498 1207 34.3 33.9 38.8 34.6
3.50 3.21 3.33 3.84 1017 1108 1160 1209 32.6 33.4 33.4 30.1
.17 77 1.7
.15 63 1.3
3.44 3.42 3.33 3.39 1088 1079 1107 1178 4.98 4.92 4.87 4.93 85 231 87 106
3.74 3.61 3.55 3.61 1205 1124 1226 1147 4.97 5.01 4.87 4.92 114 255 198 207
.10 37 .08 46
21.8 3.37 31.7
22.7 3.44 34.8
23.7 3.30 31.8
23.4 3.27 31.6
23.9 3.34 33.9
.05 32 .04 44
1Percentage choline chloride. 2S tandard error of mean for protein effects. 3S tandard error of mean for choline effects.
~pressed as choline chloride (grams per day). Sr.inear effect of choline (P < .05). 6t>rotein effect (P < .05). 7Quadratic effect of choline (P < .05).
ratic fashion to dietary choline when lactose was lower, in the .08 and .16% treatment, than the controls. As in Experiment 1, dietary choline had no effect on milk: sec. DISCUSSION
The range in supplemental choline intakes used in these experiments was somewhat smaller than used in previous postmminal infusion studies in which 0 to 90 g of choline or an equivalent 0 to 120 g/d choline chloride were used (23). One previous study suggested 30 g/d choline (23) or 40 g/d choline chloride as a possible requirement. Results of Experiment 1 showed highest milk yield was associated with the .156% choline, which equals 36 g/d choline. In Experiment 2, no maximwn for milk. yield was observed with up to 59 g/d RPC. Future experiments need to examine higher concentrations of dietary choline before feeding recommendations can be made. In contrast with previous infusion studies (21, 23), choline did not have a positive effect Journal of Dairy Science Vol. 74, No.5, 1991
on milk. fat percentage. Responses in milk. fat were inconsistent in that low concentrations of dietary choline reduced its percentage in Experiment 1 and had variable effects in Experiment 2. In a previous postruminal infusion experiment (21) comparing choline and methionine, milk fat percentage was increased by postruminal choline both with and without an inhibitor of methyl transfer for choline synthesis. Milk protein also was greater during choline infusion than during methionine infusions when an inhibitor of methyl transfer was included (21). Dietary methionine hydroxy analog was shown to increase milk fat percentage in low protein diets in a multi-location study (5). This suggested that methionine and, indirectly, protein requirements might be functions of methyl donor needs rather than specifically amino acid needs (20) for milk protein synthesis. There was little evidence from Experiment 2 to suggest a choline by protein interaction. The magnitude of milk yield response to choline in the 13.0% CP diet was greater than that observed in Experiment 1 or in
RUMEN-PROTECTED CHOLINE FOR DAIRY COWS
the 16.5% CP diet in Experiment 2. The interaction of choline and methionine will need further exploration before choline could be substituted for methionine in a rumen-protected form. In summary, data from these experiments confirm earlier postmminal infusion studies in which increasing postruminal choline increased milk yield up to 3.1 kg/d. However, no positive effects on milk fat percentage were observed in these studies in contrast with our earlier postruminal infusion experiments (21, 23). ACKNOWLEDGMENTS
The authors wish to acknowledge financial support provided by Showa Denko, K.K. for these experiments. We also wish to thank Charles Anderson, Tom Junis, Tom Moreland, and Laure Giles for conducting the experiments and data analysis. REFERENCES
1 AI-Ali, S. I., N. M. Malouf, and D. M. Walker. 1985. Choline requirement of the preruminant lamb during the ftrst two or three weeks of life. Aust I. Agric. Res. 36:829. 2 Association of Official Analytical Chemists. 1975. Official methods of analysis. 12th ed. AOAC, Washington, DC. 3 Atkins, K. B., R A. Erdman, and I. H. Vandersall. 1988. Dietary choline effects on milk and duodenal choline flow in dairy cattle. I. Daily Sci. 71:109. 4Atwal, A. S., N.A.M. Eskin, and M. Vaisey-Genser. 1980. Note on the estimation of choline in plant protein sources. Cereal Chem. 57:368. 5 Chandler, P. T., C. A. Brown, R P. Iohnston, Ir., G. K. Macleod, R D. McCarthy, B. R Moss, A. H. Rakes, and L. D. Satter. 1976. Protein and methionine hydroxy analog for dairy cows. I. Dairy Sci. 59:1897. 6 Dawson, RM.C., D. W. Grime, and D. B. Lindsey. 1981. On the insensitivity of sheep to the almost complete microbial destruction of dietary choline before alimentary tract absorption. Biochem. I. 196:499. 7 Dewar, W. A., and P. McDonald. 1961. Determination of dry matter in silage by distillation with toluene. I. Sci. Food Agric. 12:790. 8 Emanual, B., and I. I. Kennelly. 1984. Kinetics of methionine and choline and their incorporation into plasma lipids and milk components in lactating goats. I. Dairy Sci. 67:1912. 9 Freund, R I., and R C. Littell. 1981. SASQll for linear models. SAS Inst., Inc., Cary, NC. 10 Goering, H. K., and P. I. Van Soest. 1970. Forage fiber
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analyses (apparatus, reagents, procedures, and some applications). Agric. Handbook No. 379. ARS-USDA, Washington, DC. 11 Iohnson, B. C., H. H. Mitchell, 1. A. Pinkos, and C. C. Morrill 1951. Choline deficiency in the calf. 1. NUll. 43:37. 12 Molitoris, B. A., and D. H. Baker. 1976. The choline requirement of broiler chicks during the seventh week of life. Poult. Sci. 55:220. 13 National Research Council. 1988. Nutrient requiremmts of dairy cattle. Nat!. Acad. Sci., Washington, DC. 14 National Research CounciL 1978. Nutrient requirements of laboratory animals. Natl. Acad. Sci., Washington, DC. 15 National Research Council. 1984. Nutrient requirements of poultry. Natl. Acad. Sci., Washington, DC. 16 National Research Council. 1988. Nutrient requirements of swine. Nail. Acad. Sci., Washington, DC. 17 Neill, A. R. D. W. Grime, A. M. Snoswell, A. I. Northrop, D. B. Lindsay, and RM.C. Dawson. 1979. The low availability of dietary choline for the nutrition of the sheep. Biochem. 1. 180:559. 18 Pesti, G. M., A. E. Harper, and M. L. Sunde. 1980. Choline/methionine nutrition of starting broiler chicks. Three models for estimating the choline requirements with economic considerations. Poult. Sci. 59: 1973. 19 Robertson, I. B .• and P. 1. Van Soest. 1977. The detergmt system of analysis and its application to human foods. Page 125 in The analysis of dietary fiber in food. W.P.T. lames and O. Theander, ed. Marcel Dekker, New York, NY. 20 Schwab, C. G., and L. D. Satter. 1974. Effect of abomasal infusion of amino acids on lactating dairy cows. I. Dairy Sci. 57:632. 21 Sharma, B. K., and R. A. Erdman. 1988. Abomasal infusion of choline and methionine with or without 2-amino-2-methy1-1-propano1 for lactating dairy cows. I. Dairy Sci. 71:2406. 22 Sharma, B. K., and R. A. Erdman. 1988. Effect of high amounts of dietary choline supplementation on duodenal choline flow and production responses of dairy cows. I. Daily Sci. 71:2670. 23 Sharma, B. K., and R. A. Erdman. 1989. Effect of dietary and abomasally infused choline on milk production responses of lactating dairy cows. I. Nutr. 119: 248. 24 Stocldand, W. L.• and L. G. Blaylock. 1974. Choline requirement of pregnant sows and gilts under restricted feeding conditions. I. Anim. Sci. 39:1113. 25 Takayama, M., S. !tob, T. Nagasaki. and I. Tanimiju. 1977. A new enzymatic method for determination of serum choline-containing phospholipids. Clin. Chim. Acta 79:93. 26 Tsiagbe, V. K., C. W. Kang, and M L. Sunde. 1982. Effect of choline supplementation in growing pullet and laying hen diets. Poult. Sci. 61:2060.
Journal of Dairy Science Vol. 74, No.5. 1991
