DOI: 10.1111/j.1439-0396.2009.00966.
ORIGINAL ARTICLE
Effect of rumen-protected choline and methionine
on physiological and metabolic disorders and reproductive
indices of dairy cows
M. Ardalan, K. Rezayazdi and M. Dehghan-Banadaky
Department of Animal Science, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
Keywords
dairy cow, physiological disorder, rumen-
protected methionine, rumen-protected
choline
Correspondence
M. Ardalan, Department of Animal Science,
University College of Agriculture and Natural
Resources, University of Tehran, PO Box
31587-77871, Karaj, Iran. Tel: +98 261
2248082; Fax: +98 261 2246752; E-mail:
m.ardalan87@gmail.com
Received: 17 March 2009;
accepted: 18 July 2009
Introduction
Management and nutrition during the transition
period influence milk production, the incidence of
peripartal metabolic disorders, and reproductive per-
formance. Therefore, management of cows during
this critical stage is crucial for the productivity of
dairy cattle (Overton and Waldron, 2004). To meet
their energy requirements during the periparturient
period and early lactation, dairy cows mobilize large
amounts of fatty acids from adipose tissue, resulting
in increased circulating concentrations of non-
esterified fatty acids (NEFA) in the bloodstream.
Although NEFA can be used by other tissues for
energy and for milk fat, the liver typically extracts
Journal of Animal Physiology and Animal Nutrition 94 (2010) e259–e265 ª 2010 Blackwell Verlag GmbH
Summary
The objective of this study was to investigate the effect of feeding differ-
ent levels of ruminally protected methionine and choline on the
incidence of physiological and metabolic disorders, production, and
some of the reproductive indices of Holstein dairy cows. Forty Holstein
dairy cows in their first and second lactation were used from 4-week
pre-partum through 20-week post-partum and randomly assigned to
receive one of the following treatments: 18 g/day of rumen-protected
methionine (RPM), 60 g/day of rumen-protected choline (RPC), 18 g/
day of RPM + 60 g/day of RPC, and neither supplement (control). The
treatments significantly affected services per conception and open days
of lactating dairy cows (p < 0.05), but did not affect significantly on days
to first oestrus and number of pregnant cows. RPM + RPC-fed cows had
the lowest open days, days to first oestrus and services per conception
compared with other groups. The effect of treatments was significant on
the incidence of metabolic and physiological problems except for foot/
leg problems. Cows fed RPM+RPC had the lowest health problems com-
pared with other groups (p < 0.05). Results indicate that the supplemen-
tation of RPM and RPC can improve reproductive performance and
health status of dairy cows.
NEFA in proportion to its supply (Emery et al.,
1992). Hepatic capacities for fatty acid oxidation
and export as very low density lipoproteins (VLDL)
are low in ruminants (Grummer, 1993); therefore,
excessive uptake of NEFA by the liver can lead to
the development of hepatic lipidosis (Overton et al.,
1998).
Choline is sometimes classified as a B vitamin,
even though it does not fulfil the standard vitamin
definition, and is a small water-soluble molecule that
has been found in all mammalian cells. It is a qua-
ternary amine that features three methyl groups,
which enable it to function as a methyl donor when
choline is converted to betaine. Choline is a nutrient
involved with the transport of fat from the liver, and
e259
Rumen-protected methionine and choline for Holsteins
required for the synthesis of phosphatidylcholine, a
phospholipid found in the membranes of VLDL
(National Research Council, 2001). Hence, choline
deficiency in transition dairy cattle may be associ-
ated with hepatic lipidosis. There is an estimated
requirement for gram quantities of choline for
normal tissue metabolism and milk production in
lactating dairy cows (Erdman and Sharma, 1991).
Research has suggested that early lactation dairy
cattle can produce more milk when they receive
supplemental choline (Erdman and Sharma, 1991).
Piepenbrink and Overton (2003) reported that feed-
ing a rumen-protected form (RPC) tended to
increase yields of milk fat, 3.5% fat-corrected milk,
and total solids. The microbial populations in the
rumen quickly degrade dietary choline; therefore,
the only practical means of increasing choline to the
dairy cows is to feed it in RPC (Atkins et al., 1988).
If synthesis of choline and related compounds during
the periparturient period is insufficient for maximal
hepatic metabolism of NEFA, the severity of hepatic
lipid infiltration may be exacerbated by providing
additional choline through the diet.
Methionine is a sulphur-containing amino acid
(AA) that is involved in many pathways including
the synthesis of phospholipids, carnitine, creatine
and the polyamines (Bequette et al., 1998). In addi-
tion to being used for protein synthesis, methionine
can be used to provide methyl groups for a variety
of reactions and to provide sulfur groups from the
synthesis of cysteine. One approach that has been
used to supply additional methionine to the cow has
been to protect it from ruminal degradation for sub-
sequent absorption in the small intestine (rumen-
protected methionine, RPM). Methionine is the
source of the methyl donor S-adenosylmethionine,
the metabolite that provides methyl groups for the
de novo synthesis of choline. Choline and methionine
metabolism are closely associated, and as much as
28% of absorbed methionine is used for choline syn-
thesis (Emmanuel and Kennelly, 1984). Methionine
plays a direct role in VLDL synthesis in bovine and
acts to reduce plasma ketones during early lactation
(Durand et al., 1992). Increasing the availability of
AA may increase glucose production, increase
hepatic AA oxidation, or directly promote protein
synthesis. The objective of this research was to study
the effect of feeding different levels of ruminally
protected methionine and choline from 4-week
pre-partum through 20-week post-partum on the
incidence of physiological and metabolic disorders,
production and some of the reproductive indices of
Holstein dairy cows in Iran.
e260
M. Ardalan, K. Rezayazdi and M. Dehghan-Banadaky
Materials and methods
Cows, treatments, and data collection
Forty Holstein dairy cows in their first and second
lactation were used in a lactation study from 4-week
pre-partum (body condition score, BCS = 3.6 ± 0.04;
mean ± SE) through 20-week post-partum. Selection
of cows for initializing this experiment was based on
parity, milk yield of previous lactation (milk yield of
dams for the cows in their first lactation), and body
condition score. Therefore, the similarity of produc-
tion and BCS was used as selection criterion of dairy
cows for starting the study. Cows (six cows in first
lactation and four cows in second lactation per treat-
ment) were randomly assigned to receive one of the
following treatments: 18 g/day of RPM, 60 g/day of
RPC, 18 g/day of RPM + 60 g/day of RPC, and nei-
ther supplement (control). The RPM product [Smar-
tamineM (Adisseo, Antony, France)] was a white
solid granular powder coated with a polymer (2-vinyl
pyridine-Co Styrene) sensitive to acidic pH in aboma-
sum, and contained 75% dl-methionine. The RPC
product [COL 24 (Soda Feed Ingredients, Monaco,
France)] is a rumen-protected source of choline chlo-
ride. COL 24 is produced by encapsulating choline
chloride with a coating matrix able to resist rumen
breakdown and release choline in the intestine. The
control cows received the same diets but without
RPM and RPC. The DM-based forage to concentrate
ratio was 57:43 for dry period diet and 44:56 for lac-
tation diet. The RPM and RPC were top dressed onto
the TMR. Cows were fed for ad libitum intake the total
mixed diets and feed refusals were recorded once
daily, allowing 5% orts at each feed delivery. Pre-
partum and post-partum diets (Tables 1 and 2) were
formulated using the CNCPS program. The CNCPS
program is a model designed to evaluate diets and ani-
mal performance for all classes of cattle in unique pro-
duction situations, using science-based principles of
rumen function, microbial growth, feed digestion and
passage, and physiological state (Fox et al., 2004).
Reproductive data were recorded, including days
open, days to first oestrus, and services per concep-
tion. Also, health problems were recorded during the
trial, including incidence of dystocia, mastitis,
retained placenta, displaced abomasum, ketosis, milk
fever, foot/leg problems, and uterine problems.
Cows were milked three times daily in a milking
parlour at 06.00, 14.00, and 22.00, with no provision
of water or concentrate while milking. Milk yield was
recorded weekly for all cows. The milk samples from
individual cows were sent to the laboratory weekly
for the analysis of milk percentages of fat and protein
Journal of Animal Physiology and Animal Nutrition. ª 2010 Blackwell Verlag GmbH
M. Ardalan, K. Rezayazdi and M. Dehghan-Banadaky Rumen-protected methionine and choline for Holsteins

Table 1 Ingredient composition of pre- and post-partum diets (% of (Proc GLM) of SAS (SAS Institute, 2002) and the
DM) statistical model included the effects of treatment,
Ingredient Pre-partum TMR Post-partum TMR parity, and treatment · parity. Also, because the
incidences of health problems were coded as a
Alfalfa hay 36.00 17.81 dichotomous variable (0 health; 1 disease), a logistic
Corn silage 20.89 25.84
regression model was used to analyze the effect of
Barley grain 10.44 17.39
Corn grain 5.22 7.61
treatments on the incidence of health problems,
Soybean meal 10.68 7.79 using the maximum likelihood method of the
Wheat bran 10.56 6.60 LOGISTIC procedure of sas (SAS, 2002).
Whole cottonseed – 6.82 The repeated measurements of milk yield and
Canola meal – 6.82 composition were analyzed as a linear mixed model
Beet pulp 3.60 – (Proc Mixed) with the best fitted covariance struc-
Minerals and vitamins supplement* 0.78 0.61
ture of sas (SAS, 2002). The least square means
Salt 0.16 0.49
Calcium carbonate – 0.37
were estimated by restricted maximum likelihood
Sodium bicarbonate – 0.62 method. The statistical model included the effects of
Fat supplement – 1.23 treatment, parity, time (week of lactation), and
Ammonium chloride 0.27 – treatment · time. The covariance structure used to
Calcium chloride 1.16 – analyze the repeated measures of milk yield and
Magnesium sulphate 0.23 – components was first-order heterogeneous autore-
*Contained 180 g/kg Ca, 70 g/kg P, 30 g/kg Mg, 4 g/kg Fe, 0.5 g/kg gressive structure, as it produced the least Akaike’s
Cu, 5 g/kg Mn, 4 g/kg Zn, 0.1 g/kg Co, 0.1 g/kg I, 0.03 g/kg Se, 0.4 g/ information criterion (AIC). Week of lactation were
kg antioxidant, 5 · 105 IU/kg of vitamin A, 105 IU/kg of vitamin D, and used in the REPEATED statement with cow within
2 · 103 IU/kg of vitamin E. treatment as the error term, where significant, treat-
by Milk-O-Scan (Foss Electric, Hillerod, Denmark). ment means were separated using PDIFF option of
4% fat corrected milk (FCM) of each cow was calcu- sas (SAS, 2002). Acceptable significant levels were
lated as follows: FCM = [(0.4 · kg milk) + (0.15 · kg declared at p < 0.05.
milk · fat%)]. The yield of energy corrected milk
(ECM) was calculated by the following formula) Results and discussion
(DeFrain et al., 2006): ECM = [(0.327 · kg milk) +
Reproductive indices and health problems
(12.95 · kg fat) + (7.2 · kg protein)].
The treatments significantly affected services per
conception, and open days of lactating dairy cows
Statistical analyses (p < 0.05), but did not affect significantly on days to
Statistical analysis of reproductive data was per- first oestrus and number of pregnant cows (Table 3).
formed using the general linear models procedure Higher-producing cows are usually may experience
longer periods of negative energy balance than
Table 2 Chemical composition of pre- and post-partum diets lower-producing cows. As a result of a more nega-
tive energy balance, the higher producing cows may
Item Pre-partum TMR Post-partum TMR
be at greater risks of reproductive/conception failure
DM, % 66.0 62.0 than lower-producing cows (Ravagnolo and Misztal,
CP, % 16.5 18.0 2002). Therefore, providing RPM was expected to
Ash, % 8.9 7.1 enable the higher-producing cows to maintain pro-
Total fat, % 2.4 5.1 ductivity but at reduced expenses of reproductive
NDF, % 39.7 34.7
malfunction.
ADF, % 27.8 20.8
NFC*, % 32.5 35.1
Polan et al. (1991) reported that supplementation
Ca, % 0.89 0.56 of rumen-protected forms of lysine and methionine
P, % 0.41 0.45 had not significant effects on days to first service,
NEL, Mcal/kg 1.43 1.63 services per conception, and calving interval in dairy
RUP, % of CP 27.1 34.5 cows. Erdman and Sharma (1991) reported that
RDP, % of CP 72.9 65.5 although not significantly different, the number of
Met, % 1.94 1.82
cows pregnant during the experiment tended to be
Lys, % 6.60 6.42
lower as choline intake increased. Services per cow
*NFC = 100 - (CP + Ash + total fat + NDF). and per conception tended to be higher for choline-

Journal of Animal Physiology and Animal Nutrition. ª 2010 Blackwell Verlag GmbH e261
Rumen-protected methionine and choline for Holsteins M. Ardalan, K. Rezayazdi and M. Dehghan-Banadaky

Table 3 The effects of supplemental rumen-protected methionine and choline on reproductive indices of Holstein dairy cows

Treatment p-Value

Item Control RPM RPC RPM + RPC SEM Trt Parity Trt·Parity

Days to first oestrus 61.8 48.8 64 31.6 7.4 0.325 0.08 0.364
Open days 115.4a 88.6ab 106.6ab 73.8b 9.3 0.002 0.663 0.739
Services per conception 3b 2.2ab 2.4ab 2a 0.2 0.034 0.096 0.425
Number of pregnant cows 3 6 6 8 0.6 0.064 0.125 0.534

Means with different superscripts differ significantly (p < 0.05). Trt, treatment; RPM, rumen-protected methionine; RPC, rumen-protected choline,
SEM, standard error of means.

treated cows bred or pregnant prior to week 2. Days limited by VLDL production causing triglycerides to
open also were greater for choline treatments using accumulate in the liver (Zeisel, 1993). During early
data up to week 44 post-partum. Also, there was no lactation, dairy cattle are in a negative energy
apparent trend due to RPC on number pregnant, balance and must mobilize fat stores from adipose
services, or days to first breeding. They were pro- tissue. This increases blood NEFA concentrations
posed that reproductive responses were more related which are taken up by the liver and esterified to
to differences in milk yield and the effect of RPC on triglycerides oxidized to ketone bodies (Van den Top
reproductive performance was minimal. Our results et al., 1995). Erdman (1991) suggested that since
are contrary to the results of Erdman and Sharma clinical ketosis often leads to fatty liver, choline may
(1991), but in the case of number of pregnant cows, be useful in reducing the incidence of clinical keto-
our result is consistent with the result of them. sis. Choline potentially could affect the incidence of
The incidence of health problems is summarized sub-clinical ketosis as well, which is defined by high
in Table 4. The effect of treatments was significant levels of circulating ketone bodies when no clinical
on the incidence of metabolic and physiological symptoms are evident (Andersson, 1988). Economic
problems except for foot/leg problems and the losses from cases of sub-clinical ketosis are due to
overall incidence of health-related disorders was lower milk production and poor reproductive perfor-
numerically lowest for cows fed RPM + RPC mance (Duffield, 2000). Also, there is a higher risk
(p < 0.05). Overton et al. (2000) reported that for developing displaced abomasum or clinical keto-
reported rumen-protected choline significantly sis in cows with sub-clinical ketosis (Duffield, 2000).
reduced NEFA conversion to stored triglyceride and Van den Top et al. (1995) suggested that the rumi-
increased glycogen in livers of dairy cows at calving nant liver is unable to respond to increased NEFA
and in early lactation. These metabolic changes can with increased VLDL production in cases of fatty
reduce the risk of clinical ketosis and improve health liver because hepatic synthesis of either apolipopro-
status. In choline deficiency, fatty liver occurs tein B or phospholipids, especially phosphatidylcho-
because the export of triglycerides from the liver is line, may be limiting. Choline supplementation
routinely increases VLDL secretion from the liver in
Table 4 Incidence of health problems in Holstein cows fed supple- rats (Zeisel, 1993), and methionine supplementation
mental rumen-protected methionine and choline from 4-week pre- has been shown to increase VLDL synthesis in the
partum through 20-week post-partum liver of calves (Auboiron et al., 1995). Overall, iden-
tifying the required amount of choline and methio-
Treatment
nine and determining how to supply adequate
Item Control RPM RPC RPM + RPC amounts to the liver should reduce the incidence of
fatty liver and ketosis and improve the health status
Retained placenta 5a 2b 3b 0c
Mastitis 3a 1b 1b 0b
in early lactation dairy cattle.
Displaced abomasum 4a 1b 0b 0b Janovick Guretzky et al. (2006) reported the inci-
Uterine problem 3a 0b 0b 0b dence of twinning tended to be greater for cows in
Milk fever 4a 2b 2b 0c the RPC group compared with the control group
Dystocia 5a 2b 2b 0c which likely contributed to the tendency for more
Ketosis 3a 1b 1b 0b cows in the RPC group to have a retained placenta.
Foot/leg problems 1 0 0 0
The tendencies for increased twinning and associated
RPM, rumen-protected methionine; RPC, rumen-protected choline. retained placentas would not be related to supple-
Numbers with different superscripts differ significantly (p < 0.05). mental RPC because number of fetuses was

e262 Journal of Animal Physiology and Animal Nutrition. ª 2010 Blackwell Verlag GmbH
M. Ardalan, K. Rezayazdi and M. Dehghan-Banadaky
established long before initiating RPC supplementa-
tion. Other health problems occurred predominantly
post-partum and incidence was not different
between treatments. Piepenbrink and Overton
(2003) reported a larger number of cows fed higher
amounts of RPC treatment seemed to have more dif-
ficult transitions, having numerically more incidence
of displaced abomasum compared to the cows fed
the other treatments. Xu et al. (1998) reported the
overall incidence of health-related disorders was
numerically lowest for cows fed high amount of
rumen-protected lysine and methionine. Erdman
and Sharma (1991) reported that the supplementa-
tion of RPC to the diet of lactating dairy cows had
no significant effect on the incidence of clinical mas-
titis. Their result was not consistent with the result
of our study. Negative energy balance and elevated
serum beta-hydroxybutyrate probably have been
implicated in reducing leukocyte functionality; thus
increasing the risk of intramammary infection in cat-
tle (Suriyasathaporn et al., 2000). Although the goal
of this study was not the study of the effects of RPM
and RPC on plasma metabolites of dairy cows, but it
is likely that the supplementation of RPM and RPC
in the current study may decrease the serum concen-
trations of beta-hydroxybutyrate and this resulted in
the lower incidence of clinical mastitis in RPM +
RPC-fed cows than other groups.
Milk yield and composition
The treatments significantly affected actual milk
yield, FCM, and ECM across lactation weeks
(p < 0.05; Table 5). The actual milk yield (p < 0.05),
FCM (p < 0.01), and ECM (p < 0.01) were greater
for RPM + RPC-fed cows than other groups. Second-
calf cows had greater actual milk yield, FCM, and
ECM than cows in their first lactation (p < 0.001).
Fat percentage of milk was greater for RPM+RPC-fed
Table 5 The effects of supplemental rumen-protected methionine and choline on productive indices of lactating dairy cows across the lactation
weeks
Treatment
Item Control RPM RPC
Milk, kg/day 30.71c 32.64bc 34.23ab
4% FCM, kg/day 26.55c 28.59bc 30.37ab
ECM, kg/day 29.28c 31.47bc 33.21ab
Fat, % 3.29 3.30 3.28
Protein, % 3.10 3.16 3.09
Means with different superscripts at each row differ significantly (p < 0.05). Trt, treatment; RPM, rumen-protected methionine; RPC, rumen-
protected choline.
Journal of Animal Physiology and Animal Nutrition. ª 2010 Blackwell Verlag GmbH
Rumen-protected methionine and choline for Holsteins
cows than other groups, but this effect was not
significant. Also, cows in their first lactation had
greater milk percentage of fat than cows in their sec-
ond lactation (p < 0.01). Although, the treatments
did not significantly affect protein percentage of
milk, RPM-fed cows had greater percentage of milk
protein, which suggested that methionine was the
first limiting AA for milk protein synthesis. In gen-
eral, the treatment · time had not any significant
effect on milk yield and composition. Overton et al.
(1996) reported that the DMI, yields of milk, CP and
percentages of fat and CP in milk were not affected
by RPM. However, yields of fat and 3.5% FCM
increased when RPM was fed. The dietary CP and
fiber have previously been reported to affect the
potential impact of RPM on milk protein (Bateman
et al., 1999). Low dietary NDF may reduce rumen
pH, thereby reducing the efficiency of microbial pro-
tein synthesis. Besides, high intake of CP and rumen
degradable protein may increase both microbial pro-
tein synthesis and rumen NH3, and thereby mask
the potential impact of RPM on milk protein. This is
because the greater microbial protein synthesis can
reduce the demands for essential AA and hence
dilute the potential benefits of RPM. In addition, the
higher NH3 may increase ATP cost for urea biosyn-
thesis and excretion by the liver and kidney. How-
ever, Leonardi et al. (2003) found that RPM can
improve milk protein content to the same extent
regardless of whether it was added to a diet with
14% or a diet with 18% CP. Their finding would
suggest that extra CP elevates nitrogen excretion
(National Research Council, 2001) but may not
essentially neutralize the RPM mechanism of action
(Leonardi et al., 2003). Hence, the positive impact of
RPM on cow productivity appears to be mediated
not exclusively via protein metabolism. Typically,
the increased milk fat by RPM is not as greatly
expected as is the boosted milk protein. To explain,
p-Value
RPM + RPC SEM Trt Parity Time Trt · time
35.14a 0.97 0.014 <0.001 <0.001 0.999
31.40a 1.06 0.003 <0.001 0.016 0.943
34.34a 1.10 0.004 <0.001 0.001 0.983
3.39 0.03 0.904 0.006 <0.001 0.900
3.12 0.02 0.364 0.275 <0.001 0.777
e263
Rumen-protected methionine and choline for Holsteins
both forage and non-forage fibres could adequately
supply milk fat precursors to mammary gland unless
rumen fermentation is truly compromised (National
Research Council, 2001).
The greater ECM requires greater glucose and AA
to uphold. Glucose is essential to provide (i) NADPH
for de novo milk fat synthesis and (ii) ATP for pro-
tein synthesis from AA. The increased ECM suggests
that RPM + RPC can positively impact milk energy
output when early-lactation curve peaks and drops.
Increased yield of 4% FCM resulted from increases
of milk yield, milk fat percentage and DMI. Sharma
and Erdman (1988) speculated that, for experiments
in which supplemental methionine increased milk or
milk fat, choline synthesized from methionine was
likely to have been at least partially responsible.
The results of this study indicate that the supple-
mentation of ruminally protected forms of methio-
nine and choline in the diet can be beneficial and
recommendable for dairy producers, because from
economical perspective, it was expected that the
supplementation of RPM and RPC in the diets of
dairy cows can decrease the costs of health prob-
lems, reproductive disorders and feed because the
incidence of metabolic and reproductive disorders
and negative energy balance would be lower in
these cows due to the possible improvement
in health situation and reproductive performance. In
addition, results indicate that the supplementation of
RPM and RPC can improve the lactation perfor-
mance of lactating dairy cows.
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