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SYMPOSIUM: OPTIMIZING PROTEIN NUTRITION
FOR REPRODUCTION AND LACTATION
Review: Effect of Protein Nutrition on Ovarian
and Uterine Physiology in Dairy Cattle
W. R. BUTLER
Department of Animal Science,
Cornell University,
Ithaca, NY 14853-4801
Received June 22, 1997.
Accepted December 8, 1997.
ABSTRACT
Milk production and dry matter intake of dairy
cows are stimulated in response to increased intake of
dietary protein, but, unfortunately, decreased fertility
is often associated with this nutritional strategy. Ruminally
degradable protein or ruminally undegradable
protein in excess of requirement can contribute to
reduced fertility in lactating cows. Dietary protein
nutrition or utilization and the associated effects on
ovarian or uterine physiology have been monitored
with urea nitrogen in plasma or milk; concentrations
above 19 mg/dl have been associated with altered
uterine pH and reduced fertility in dairy cows. The
uterine pH changed dynamically and inversely with
plasma urea nitrogen, signaling possible changes in
the uterine milieu. Mechanisms for reduced fertility
include exacerbation of negative energy balance and
reduced plasma progesterone concentrations when
cows were fed rations that were high in ruminally
degradable intake protein. Alternatively, changes in
uterine secretions that are associated with high protein
intake and elevated plasma urea nitrogen might
be detrimental to embryos. Bovine endometrial cells
in culture respond directly to increasing urea concentrations
with alteration in pH gradient but respond
most notably with increased secretion of prostaglandin
F 2a (PGF 2a ). Increased uterine luminal PGF 2a
interferes with embryo development and survival in
cows, thus providing a plausible link between
elevated plasma urea nitrogen concentrations and
decreased fertility. Poor fertility in high producing
dairy cows reflects the combined effects of a uterine
environment that is dependent on progesterone and
rendered suboptimum by the antecedent effects of
negative energy balance or postpartum health
problems and that is further compromised by the
effects of urea resulting from intake of high dietary
protein.
( Key words: protein, uterus, urea, progesterone)
Abbreviation key: BUN = blood urea nitrogen,
MUN = milk urea nitrogen, PUN = plasma urea
nitrogen, SUN = serum urea nitrogen.
INTRODUCTION
Increased genetic potential for milk production has
been associated with a decline in fertility of lactating
dairy cows [(6); Figure 1]. Strategies for meeting the
nutritional requirements of high producing cows has
necessarily changed in conjunction with genetic gains.
Diets that are high in protein (17 to 19% CP) both
support and stimulate high milk production in early
lactation (20, 26, 32). Currently, the NRC (30)
recommends that diets contain 18 to 19% CP for high
producing cows. Although high dietary protein stimulates
milk production, increased protein has been
found to be detrimental to reproductive performance
in most, but not all, studies (15, 38). Concerns about
high dietary protein and impaired fertility are not
new (23, 25, 35) but have become investigated more
intensively in the last decade.
Previous reviews (15, 38) on protein intake and
reproduction in dairy cows have been useful in identifying
several mechanisms that underlie the discrepancies
in effects on reproductive performance observed
among various studies:
1. Dietary protein fractions (RDP and RUP) fed at
optimum ratios relative to requirements must be
considered rather than simply using CP percentage.
2. Any detrimental effects of high dietary protein
may be confounded with or exacerbate reproductive
health problems (1, 10).
3. Excess RDP may exacerbate negative energy
balance during early lactation and thereby
reduce fertility.
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BUTLER
Figure 1. The inverse relationship between conception rate
(CR) and annual milk production of Holstein dairy cows in New
York.
Conception and the establishment of pregnancy are
an ordered progression of interrelated events involving
all of the various tissues of the reproductive tract:
follicular development resulting in ovulation, fertilization
of the oocyte, embryo transport and development,
maternal recognition, and implantation.
Hypothetically, ammonia, urea, or some other toxic
product of protein metabolism may intercede at one or
more of these steps to impair reproductive efficiency.
This review focuses on the detrimental effects of high
dietary protein that may be exerted on the physiological
mechanisms of the ovary and uterus.
MONITORING PROTEIN METABOLISM
The biological value of protein substrates for the
lactating dairy cow is directly related to the energy
status of the cow and the balance of absorbed amino
acids relative to their requirements. In lactating
cows, dietary protein comprises RDP and RUP fractions.
Through normal ruminal fermentation, RDP
provides a source of ammonia for microbial protein
synthesis. Some of the ammonia that is produced
escapes incorporation by the microorganisms, diffuses
out of the rumen into the portal blood, and is detoxified
in the liver by conversion to urea. The quantity of
ammonia that is produced and the amount that escapes
for conversion to urea directly reflects both
dietary RDP and the availability of fermentable carbohydrates
to support microbial growth and protein
synthesis.
A second source of urea produced by the liver is
from the deamination and metabolism of amino acids.
Circulating amino acids originate from RUP,
microbial protein, and body stores. Amino acids such
as glutamine and arginine are important interorgan
transporters of ammonia nitrogen in a nontoxic form.
These and other available amino acids that are not
taken up for utilization in milk protein synthesis or
deposited elsewhere are deaminated by the liver to
yield energy substrates and urea. As the urea that is
produced by the liver circulates through the blood, it
equilibrates into all tissues, is recycled through the
rumen, and is excreted in urine. Although the production
of ammonia and urea can be minimized by
balancing RDP and RUP, high dietary intake to support
milk production and variation in rumen
microbial protein yield make accurate prediction of
the availability of amino acids very difficult. Consequently,
most high producing cows consume protein
in excess of the requirements, and urea concentrations
are increased in accordance with intake (16).
Urea is the metabolic end product of protein
catabolism in the body and is easily measured by the
nitrogen content (i.e., urea nitrogen concentration).
Urea nitrogen concentrations circulating in the
bloodstream are measured in either plasma or serum
fractions ( PUN or SUN, respectively) and are often
referred to generically as blood urea nitrogen ( BUN).
Typically, BUN peaks about 4 to 6 h after meals
because of RDP catabolism, and the metabolism of
RUP contributes to BUN continuously throughout the
day (13). The fluctuations in BUN during the day are
usually smaller (2 to 3 mg/100 dl) in cows fed a TMR
than in cows fed concentrates and forages separately
(11, 13, 21). Urea is a small, water-soluble molecule
that permeates all cells and tissues in the body and
passes easily between the blood and milk within the
mammary gland (21). With a lag time of less than 1
h for equilibration with blood concentrations, milk
urea nitrogen ( MUN) provides a rapid, noninvasive,
and inexpensive means of assessing the dynamics of
BUN (21, 31) and of monitoring overall protein
metabolism in lactating cows (33). Also, MUN provides
an integrated measure of metabolism and utilization
of RDP and RUP, reflecting both intake and
energy availability (8, 9, 31, 33).
Measurements of BUN or MUN have provided a
useful index for studying the association between
metabolism of dietary protein and reproductive efficiency.
Conception rate decreased when SUN concentrations
exceeded 20 mg/dl on the day of insemination,
suggesting that degradation of excessive
amounts of dietary protein in the rumen contributed
to infertility (14). A more detailed field trial (16)
with nine commercial dairy herds confirmed the inverse
relationship of high SUN on conception rate and
emphasized the detrimental effects of fertility when
SUN exceeded 20 mg/dl. More recently, both PUN
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SYMPOSIUM: OPTIMIZING PROTEIN NUTRITION FOR REPRODUCTION AND LACTATION 2535
TABLE 1. High dietary CP and postpartum ovarian activity in
dairy cows.
Effect on days
CP
to first estrus
in diet or ovulation Reference
(%)
20 1 Delay of 13 d (38)
20 2 Delay of 4 d ( 1 )
19 NS (7)
19.3 Reduced 9 d (23)
19.4 NS (25)
20 NS (10)
1Excessive RDP (72.5% of CP).
2Effect of reproductive health status included.
and MUN concentrations were used to monitor pregnancy
rate, and the range of urea nitrogen concentrations
in high producing cows and the inverse relationship
between PUN (>19 mg/dl) and fertility were
both confirmed (5). Most reports (38) have found
that elevated BUN concentrations are associated with
some deficiency in the reproductive performance of
cows.
OVARIAN CYCLES AND CIRCULATING
PROGESTERONE CONCENTRATIONS
The intake of high CP diets by lactating cows
following parturition has had inconsistent effects on
the reinitiation of ovarian activity (Table 1). Diets
containing 20% CP prolonged the interval of days to
first ovulation when RDP was particularly in excess
[72.5% of CP; (38)] or when reproductive health status
was included in the statistical assessment (1).
Others (7, 10, 23, 25), however, have reported that
diets with 19 to 21% CP did not delay the postpartum
interval to first ovulation or estrus. Follicular development
in nonlactating cows was not perturbed by
high CP diets (18), but, thus far, no effects during
the development of ovarian follicles in postpartum
lactating cows has been reported. Overall, high CP in
the diet does not appear to have a strong impact on
the reinitiation of ovarian activity in the postpartum
period.
Jordan and Swanson (24) were the first to report
that cows fed low CP (12.7%) during the breeding
period had higher serum progesterone concentrations
than did cows fed 16.3 or 19.3% CP. In other studies,
progesterone concentrations in circulation were lower
(36, 38) or were not affected (1, 18) in dairy cows fed
high protein diets (Table 2). One explanation for
these conflicting responses is the difference in the
lactational status of the cows in the various studies.
High dietary CP reduced plasma progesterone concentrations
in lactating cows in three of four studies (4,
24, 36, 38), but did not reduce concentrations in
nonlactating cows (4, 18) or heifers (12). Plasma
progesterone concentrations progressively increase
over the first three ovarian cycles during early lactation,
and the rate of increase is reduced or moderated
by the effects of negative energy balance (37, 39).
When cows are fed excess RDP during early lactation,
the negative energy balance is exacerbated because of
the energy cost of detoxifying ammonia escaping from
the rumen (38), but changes in metabolic clearance
rate of progesterone should also be considered. Therefore,
the deleterious effects of a negative energy
balance that is more extreme might explain the lower
plasma progesterone concentrations that were observed
in studies with postpartum cows fed high CP
(24, 36, 38). Reduced concentrations of plasma
progesterone during the early breeding period appears
to be a likely component of the reduction in
fertility associated with feeding high dietary protein.
EMBRYO DEVELOPMENT
The effects of high protein intake on early embryo
development have been studied in dairy cows. Early
degeneration and poor development of embryos occurred
in lactating cows that were fed excess RDP
(3), but a similar, well-controlled study of nonlactating
cows found that excess intake of CP failed to
affect the health and number of embryos (18). Because
the energy status of the cows preceding each of
these studies was markedly different, depending on
whether or not they were lactating, those researchers
(18) suggested that the consequence of increasing
TABLE 2. High dietary CP and plasma progesterone concentrations
(PPC) during the estrous cycle of lactating and nonlactating dairy
cows.
CP
Effects
in diet on PPC Lactating Reference
(%)
19.3 Reduced 25% 1 Yes (24)
20 Reduced 30% 1 Yes (36)
20 2 Reduced 50% Yes (38)
20 NS 3 Yes ( 1 )
27.4 NS No (18)
25 NS No (4)
21.8 4 NS No (12)
1During more than one estrous cycle.
2Excessive RDP (72.5% of CP).
3Nonsignificant ( P > 0.05).
4Heifers >14 mo of age fed diet containing 70% ME required.
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BUTLER
dietary CP content or degradability in lactating cows
might be to exacerbate one or more factors affecting
nutrition, metabolism, or energy balance that would
result in lower reproductive performance (i.e., impaired
embryo development). Indeed, increased embryo
losses were reported for heifers that were fed an
energy-restricted diet containing high amounts of
degradable protein (12). Furthermore, negative
energy balance during the early postpartum period
might exert residual effects during the 40 to 60 d
required for follicular development that might impair
the health of preovulatory follicles later during the
breeding period (27). Therefore, the combined effects
of excess RDP and energy status might explain why
the embryo quality of lactating cows was compromised
(3).
A recent study with sheep supports the detrimental
effects of high RDP on embryo development. Bishonga
et al. ( 2 ) suggested that elevated circulating concentrations
of urea and ammonia resulting from feeding
high RDP exerted an adverse effect on early embryo
development (d 4 to 11). However, because their
study of dietary treatments followed by insemination
were conducted within 30 d after termination of lactation,
the effects attributed to high RDP might be
confounded with the possible residual effects of antecedent
energy status of the ewes during lactation
(e.g., negative energy balance).
Given the results of studies of dairy cows involving
embryo collection and evaluation, no evidence exists
that high dietary protein has an impact on ovarian
follicular development, ovulation, or fertilization of
oocytes (3, 18). However, there is an interaction between
the effects of CP and energy status during
lactation that may impair embryo development. The
relative significance of the separate effects and their
interactions are not yet fully resolved.
UTERINE PHYSIOLOGY
Successful development of the embryo during early
pregnancy depends upon the nature of the uterine
luminal environment (29). The luminal environment
is dynamic and exhibits marked differences between
the stages of the estrous cycle, and further evolution
occurs during early pregnancy in support of the developing
embryo. The cyclic nature of the microenvironment
of the lumen is a consequence of ovarian
steroidal regulation of endometrial secretion (17, 29).
The local signaling effects of secretions from the
blastocyst further modify the milieu and induce the
secretion of specific proteins by the uterine
epithelium (29). Early embryonic mortality (by d 7
of pregnancy) in lactating dairy cows has been found
(40) to be associated with concentrations of ions and
protein in the uterine environment that were significantly
( P < 0.05) different from those in cows with
normal embryos.
Jordan et al. (22) examined the effects of dietary
CP on the constituents of uterine secretions at various
stages of the estrous cycle in high producing dairy
cows. Blood ammonia and urea concentrations and
urea in uterine secretion were all higher for cows fed
23% CP than for cows fed 12% CP. Intake by cows of
23% CP altered the concentrations of magnesium,
potassium, and phosphorus in uterine secretions, but
only during the luteal phase and not at estrus.
Differential effects during the estrous cycle of high CP
on the uterine environment were also observed for
uterine luminal pH (12, 13). Uterine pH normally
increases from about 6.8 at estrus to 7.1 on d 7 of the
estrous cycle, but this increase failed to occur in both
heifers and lactating cows fed excess RDP or RUP
(12, 13). Uterine pH was inversely related to PUN,
regardless of diet (13). The combined results from
those studies suggest that high dietary CP may
reduce fertility by interfering with the normal induc-
TABLE 3. Changes in plasma urea nitrogen (PUN) and ammonia concentrations following feeding in
dairy cows.
0h 4h 8h
Cow Ammonia PUN Ammonia PUN Ammonia PUN
( mg/dl) (mg/dl) ( mg/dl) (mg/dl) ( mg/dl) (mg/dl)
4783 42 24 33 25 12 25
4788 76 20.5 82 22 24 21
4890 76 20 115 22.5 39 23
5292 21 24 21 26 1 26
5311 15 28 9 28.5 1 28
5331 36 23 24 25.5 1 22
5339 145 23 27 25.5 1 23
5345 12 21.5 1 21.5 1 20
5359 70 26 49 28 3 27
X 55.0 23.3 40.0 24.9 9.2 23.9
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SYMPOSIUM: OPTIMIZING PROTEIN NUTRITION FOR REPRODUCTION AND LACTATION 2537
Figure 2. The concurrent measurements and correlation of
plasma urea nitrogen (PUN) and uterine luminal pH in lactating
dairy cows (n = 8) sampled during 36 h. For the regression
equation, R 2 = 0.22.
tive effects of progesterone on the microenvironment
of the uterus, thereby providing suboptimal conditions
for the support of embryo development.
LINKAGES BETWEEN PROTEIN METABOLISM
AND THE UTERINE ENVIRONMENT
The intake of high dietary CP results in elevated
blood concentrations of ammonia and urea. Conceivably,
one or both of these metabolites is responsible for
altering the luminal microenvironment of the uterus.
When diets with 22 to 27% CP were fed, the ammonia
in blood was increased by 25 to 50% (18, 22) or not at
all (12). In the latter study, blood ammonia was low
even though the animals were fed an energyrestricted
diet. From recent measurements in cows
fed a 19% CP TMR, plasma ammonia concentrations
were low (Table 3) and unrelated to elevated PUN
concentrations, which peaked 4 to 8 h after feeding
(11, 13). Because accurate measurements of blood
ammonia require special procedures and rapid analysis
(12), differences in methodology may account for
the discrepancies in ammonia values among the
studies. It remains uncertain whether blood ammonia
concentrations in lactating cows are sufficient to affect
fertility. Furthermore, ammonia would be
directly involved only when RDP, not RUP, was in
excess.
Across the literature, an excess amount of either
RDP or RUP results in lower fertility; consideration of
protein fractions, rather than CP, explained much of
the variation in conception rate that was observed
among the studies (15). Because RDP and RUP are
metabolized and utilized separately and by different
organs in the lactating cow, the common element of
their metabolism when in excess of requirement is the
formation of urea. Excess of either RDP or RUP increased
PUN and altered uterine pH to a similar
degree (13). Those observations further support the
potential for urea as the common mediator of uterine
effects of excess RDP or RUP. In addition, PUN varies
inversely with uterine pH (13) and, thereby, is a
possible mediator of decreased fertility associated
with elevated PUN or MUN (5).
The active involvement of urea in uterine physiology
is being investigated in further studies (W. R.
Butler and R. O. Gilbert, unpublished). Lactating
cows (n = 8) were fitted with intrauterine Foley
catheters (12) to allow the frequent monitoring of
uterine pH in association with PUN concentrations.
Cows were fed a TMR containing 18% CP with RDP
and RUP balanced for requirements (30). Uterine pH
and PUN concentrations across cows during 36 h of
observation were correlated (Figure 2). The sequential
measurement of PUN and uterine pH demonstrated
that uterine pH is quite dynamically attuned
to changes in PUN, given a time lag of several hours
(Figures 3 and 4). These results strongly suggest
that PUN concentrations throughout the range of 12
to 24 mg/dl can exert direct effects on uterine function.
A system of endometrial cell culture has been developed
to study the effects of urea on secretory function
(19). In this three-dimensional system, the endometrial
cells become polarized and establish a pH
Figure 3. The time course of inverse changes in plasma urea
nitrogen (PUN) and uterine luminal pH in a lactating cow (cow
5919). Feeding occurred at the times indicated by arrows during
the 38-h period of study.
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2538
BUTLER
Figure 4. The time course of inverse changes in plasma urea
nitrogen (PUN) and uterine luminal pH in a lactating cow (cow
5776). Feeding occurred at the times indicated by arrows during
the 40-h period of study.
gradient between apical and basal compartments as
soon as the cells reach confluence. The pH gradient is
sensitive to both estradiol and progesterone. The
presence of urea significantly ( P < 0.05) diminished
the effects of progesterone in maintaining a pH
differential between apical and basal compartments.
Large amounts of PGF 2a and PGE 2 are secreted by
the endometrial cultures. Treatment of the cultures
with both estradiol and progesterone suppressed PGF
production, but the presence of urea significantly ( P <
0.01) increased the secretion of PGF 2a and PGE 2 .
The importance of further studies on the effects of
urea on the production of endometrial prostaglandin
stems from the evidence that PGF 2a interferes with
embryo development (28) and viability (34).
CONCLUSIONS
To support and stimulate high milk production,
dairy cows are usually fed diets that are high in
protein (>17% CP), which appears to result in
decreased reproductive performance. Investigations of
this inverse relationship have indicated that excesses
of either RDP or RUP relative to requirements result
in lower fertility and that high amounts of dietary
protein exacerbate the effects of negative energy
balance and reproductive health problems to impair
reproduction further. Because the metabolism of excess
RDP or RUP results in high urea production,
monitoring of BUN or MUN concentrations has
proved to be useful in associating decreased conception
rate with BUN or MUN concentrations above 19
to 20 mg/dl. As part of the progression of events
involved in establishing pregnancy, high dietary RDP
seems not to impact follicle development or ovulation,
but results in reduced concentrations of plasma
progesterone in lactating cows, which appears to be
linked to the effects of exacerbated negative energy
balance. The effects of RDP on energy status may also
impair embryo development. Embryo survival and
growth depend upon the quality of the uterine luminal
environment, and the intake of high dietary protein
alters uterine secretions. Blood urea concentrations,
rather than ammonia, seems to be related to
the detrimental effects on fertility as shown by the
dynamic changes in uterine pH with PUN and in
vitro evidence that urea alters pH and prostaglandin
production in endometrial cell cultures. In conclusion,
the poor fertility of high producing dairy cows reflects
the combined effects of a uterine environment that is
dependent on progesterone, but has been rendered
suboptimal by antecedent effects of negative energy
balance or postpartum health problems and has been
further compromised by the effects of urea resulting
from intake of high dietary protein.
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Journal of Dairy Science Vol. 81, No. 9, 1998