Effects of Nutrient Metabolism and Excess Protein Catabolism on
Dairy Cow Fertility
M.L. Rhoads, T.R. Bilby, R.P. Rhoads and L.H. Baumgard
Department of Animal Sciences
The University of Arizona
Corresponding Author: [email protected]
SUMMARY
•
•
•
•
The metabolism of excess dietary protein results in high plasma urea nitrogen (PUN)
concentrations
High PUN concentrations (and ammonia to a lesser extent) are associated with decreased
fertility in lactating dairy cows
High PUN concentrations probably affect fertility via direct toxic effects on the oocyte
and embryo, alterations in the uterine environment and decreased progesterone secretion
The effects on fertility may be further exacerbated by negative energy balance and heat
stress
INTRODUCTION
Successfully managing the health and productivity of dairy cattle during early lactation is
one of the most challenging and important aspects of dairy farming. In order to boost milk
production, early lactation dairy cows are often fed diets that exceed their requirements for
degradable or undegradable protein. The amino acids from the diet are metabolized to ammonia
in the rumen which is then either converted to microbial protein or absorbed into the blood
stream. Circulating ammonia is quickly converted to urea by the liver, after which it can be
either excreted or recycled (Van Soest, 1994; Visek, 1984). When protein is fed in excess of
requirements, the result is elevated blood urea nitrogen levels, which have been linked to
decreased fertility in most (Blanchard et al., 1990; Butler et al., 1996; Canfield et al., 1990;
Ferguson et al., 1993; Jordan and Swanson, 1979a; Kaim et al., 1983), but not all (Barton et al.,
1996; Carroll et al., 1988; Howard et al., 1987) studies dealing with feeding high protein diets to
dairy cattle.
The conception rates of both dairy cows and heifers have been shown to plummet
dramatically in response to diets containing excess protein. During these studies, when plasma
urea nitrogen (PUN) levels exceeded 19 or 20 mg/dL there was approximately a 20 percentage
point decrease in conception rates (Butler et al., 1996; Elrod and Butler, 1993; Ferguson et al.,
1988; Ferguson et al., 1993). Such a significant decrease in conception rates represents a large
economic loss to dairy producers. Therefore, it is of the utmost importance that dairy rations are
carefully formulated and closely monitored; particularly during early lactation when cows are
being synchronized for estrus and inseminated. Extensive research has been conducted in an
effort to determine the mechanisms involved in the detrimental effects of excess protein
metabolism on reproduction and fertility. However, these mechanisms are not yet completely
understood.
Reproductive processes that are likely to be affected by excess protein metabolism
include follicular, luteal and embryonic development. Therefore, previous research has focused
primarily on direct detrimental effects of PUN or ammonia on the gametes (especially the
oocyte), embryonic development and survival and progesterone production and secretion.
Discrepancies in the results of these studies have fostered a debate amongst scientists concerning
the true impact of excess protein intake on fertility. Clearly the high PUN values that are the
consequence of excess protein intake have a dramatic effect on reproduction in most (but not all)
cases. The key to these discrepancies may be a synergistic interaction with the early lactation
cow’s physiological state. In particular, a reproductively detrimental interaction may exist
between excess protein intake and the severity of negative energy balance during early lactation.
RUMINANT DIGESTION OF DIETARY PROTEIN AND NON-PROTEIN NITROGEN
The digestion and metabolism of dietary protein and non-protein nitrogen is a process
dependent on several conditions. Factors such as degradability, rate of fermentation and/or
passage determine whether dietary protein entering the rumen is degraded or escapes to the lower
gastrointestinal tract (Clark et al., 1992; Firkins et al., 1998). The portion of protein that is
degraded by the microbes is hydrolyzed through peptides to amino acids and ammonia, which
may be used in microbial protein synthesis. Ammonia which is not used to meet microbial
requirements leaves the rumen by either diffusing across the rumen wall into the blood stream or
passes with the liquid phase to the lower gastrointestinal tract. The amount of ammonia
absorbed from the rumen is primarily a function of the concentration of unionized ammonia in
the rumen fluid and rumen pH (Leng and Nolan, 1984).
The availability of carbohydrates to the rumen microbes plays an important role in
determining the fate of the amino acids formed from the degradation of dietary protein.
Carbohydrate availability promotes the use of ammonia in microbial amino acid synthesis and
growth (Clark et al., 1992; Nocek and Russell, 1988). Therefore, the more quickly
carbohydrates are fermented, the greater the capacity the microbes have to incorporate and utilize
ammonia. Because of this interaction, the balance between the amount of available
carbohydrates and protein in the diet is extremely important to the efficiency of nitrogen
utilization in the dairy cow. If protein is degraded well in advance of the peak in carbohydrate
fermentation, ammonia absorption across the rumen wall into the blood stream is favored (Van
Soest, 1994), thereby further increasing circulating ammonia and urea concentrations.
Ammonia is a toxic compound, and capable of causing death if large amounts are
allowed to accumulate in the body. This rarely occurs, however, because ammonia in the blood
stream is taken up by the liver and rapidly detoxified as it is converted to urea (Visek, 1984).
Urea is then either excreted in the urine via the kidney or recycled; mostly for use in the rumen
(Van Soest, 1994). Urea is known to equilibrate with body water (DePeters and Ferguson,
1992), and is therefore capable of diffusing into all tissues and organs of the body, including
those essential to reproductive success. This characteristic makes urea a strong candidate for
involvement in the interaction between metabolism and fertility in lactating dairy cattle.
HIGH LEVELS OF DIETARY PROTEIN INTAKE AND FERTILITY
Over the past three decades, numerous studies have been conducted in an attempt to
define the interaction between high levels of protein intake and fertility in dairy cows. Many
researchers have presented clear evidence of the damaging effects of excess dietary protein on
pregnancy rates and other reproductive indices. Almost as convincing, however, are experiments
concluding that high levels of protein intake have no effect on the reproduction of dairy cows.
Subsequent reviews have examined these studies and found discrepancies which may explain the
variation in results (Ferguson and Chalupa, 1989; Westwood et al., 1998) including age, lactation
number and uterine health of the animals enrolled in the experiments, energy content of the diet,
and the proportion of dietary protein that is rumen degradable or undegradable.
During one of the early studies conducted by Jordan and Swanson (1979a), lactating
dairy cows were fed diets containing 12.7, 16.3 or 19.3% crude protein. They found that the
cows consuming the 19.3% crude protein diet had fewer days to their first observed estrus, but
that the two groups consuming lower protein levels had fewer services per conception. The
group consuming the lowest crude protein diet also had fewer days open than the cows on the
higher protein diets (69 vs. 96 and 106 days, respectively). The results of a similar study by
Kaim et al. (1983) showed no difference in the number of days to the first observed estrus or day
of insemination between cows fed low protein or high protein diets. There was, however, a
significant difference in conception rate between the two groups (56 vs. 43% for low and high
protein diets, respectively). These results illustrate an interesting paradox because cows that
reach their first observed postpartum estrus sooner (cows consuming the high protein diets in
some studies) are perceived to have a reproductive advantage. Therefore, we would expect them
to conceive sooner. However, as these results show, the animals consuming the high protein
diets had lower conception rates, more services per conception, and more days open than cows
consuming lower protein diets.
More recent studies that have measured PUN levels in addition to evaluating
reproduction have found a consistent decrease in fertility when PUN is around or above 19
mg/dL. Canfield et al. (1990) found that the first service conception rate of lactating cows
dropped from 48 to 31% in animals with high PUN concentrations. The cows that conceived to
first service had a plateau PUN of 15.7 mg/dL, while those that did not conceive had a plateau
PUN of 18.6 mg/dL. Results of a study by Ferguson et al. (1993) suggested that the conception
rate of lactating cows significantly decreases when serum urea nitrogen is greater that 20 mg/dL.
The results of another large study reaffirmed these findings with 18 and 21 percentage point
decreases in conception rates when PUN and milk urea nitrogen (MUN) rose above 19 mg/dL
(Butler et al., 1996). These results demonstrate the importance of regularly monitoring
circulating urea nitrogen concentrations on commercial dairy farms, which is easily
accomplished by evaluating MUN values. On farms where conception rates are lower than
expected, MUN concentrations near or above 19 mg/dL suggest that excess dietary protein may
be the culprit responsible for decreased fertility.
Despite the convincing results of the previously mentioned studies, several experiments
have shown no effect of high protein diets on reproduction in dairy cows under similar
conditions. During some studies PUN concentrations in the high protein groups reached levels
of 20 to 24 mg/dL while the PUN values of the control groups remained between 8 and 16
mg/dL. Despite the extreme differences in PUN concentrations, there were no differences in the
number of days to first observed estrus, number of days open, services per conception or
percentage of cows eventually diagnosed pregnant between experimental groups (Barton et al.,
1996; Carroll et al., 1988; Howard et al., 1987). However, the study by Carroll et al. (1988) did
find more days to first estimated ovulation for the cows fed the high protein diet, suggesting that
those cows were exhibiting estrus without ovulating during early lactation. Barton and coworkers (1996) also found an interaction between poor health status and the high protein diet,
resulting in increased number of days open. This interaction indicates that the high protein diet
may have exacerbated the effects of postpartum health conditions.
One compelling hypothesis that would explain discrepancies in results across studies is
that an interaction between high dietary protein and negative energy balance exists and results in
the most dramatic effects on reproduction. Due to the sudden demands of their physiological
state, dairy cows in early lactation experience a period of negative energy balance. However,
this period of negative energy balance can vary in severity and duration depending on individual
cow physiology and management during early lactation. In addition to the production demands
of early lactation, cows consuming excess levels of dietary protein have the added expense of
detoxifying the extra ammonia in their blood system. It is estimated that this conversion costs
the animal 12 kcal/g of nitrogen (Van Soest, 1994). Staples and co-workers (1993) suggested
that this exacerbation of negative energy balance may play a role in the observed decreases in
reproductive performance associated with high levels of dietary protein. More recently, the
effects of prepartum and postpartum urea concentrations were evaluated separately for
primiparous and multiparous cows. The effects of urea concentrations on some reproductive
indices varied between the two experimental groups who also exhibited divergent postpartum
metabolic and endocrine profiles. For example, elevated urea concentrations at 7 weeks
postpartum increased the calving to conception intervals in both parity groups but in primiparous
animals the interval was also extended in response to high prepartum urea concentrations
(Wathes et al., 2007). An additional complication of negative energy balance is its effects on
liver composition and function. The extensive mobilization of body reserves that occurs during
negative energy balance results in the accumulation of non-esterified long chain fatty acids
within the liver. This accumulation impairs the ability of the liver to detoxify circulating
ammonia through conversion to urea (Tamminga, 2006), thereby intensifying toxic effects on the
reproductive system.
Interestingly, another deterrent to dairy cow fertility, heat stress, appears to increase
circulating PUN concentrations. In terms of effects on fertility, most research has focused on the
urea produced as a result of protein metabolism within the rumen. However, elevated urea
concentrations are also a consequence of increased skeletal muscle breakdown. A better
indicator of muscle catabolism is 3-methyl-histidine, which is reported to increase in heatstressed poultry, and is independent of reduced DMI (Yunianto et al., 1997). In addition, body
composition data indicate reduced body protein retention in heat-stressed vs. pair-fed chickens
(Geraert et al., 1996). Direct effects of heat stress on muscle breakdown (3-methyl-histidine or
creatine) have been reported in exercising men (Febbraio, 2001), rabbits (Marder et al., 1990)
and lactating cows (Kamiya et al., 2006; Schneider et al., 1988). The end result of these
physiological changes that occur during heat stress are elevated PUN concentrations in heat-
stressed cows compared to pair-fed cows in thermal-neutral conditions (Wheelock et al.,
unpublished). Therefore, elevated PUN concentrations may be exacerbating the decrease in
fertility that is frequently observed during periods of heat stress.
PROTEIN METABOLITE CONCENTRATIONS IN FOLLICULAR AND UTERINE
FLUID
The concentrations of both ammonia and urea equilibrate within reproductive fluids and
increase proportionally with increasing concentrations of PUN. Follicular fluid ammonia
(Hammon et al., 2005) and urea concentrations (Hammon et al., 2005; Leroy et al., 2004) are
highly correlated with PUN concentrations during early lactation (r2 values between 0.61 and
0.98) suggesting that the oocyte within the developing follicle is susceptible to damage by high
PUN concentrations. Indeed, a recent study found that follicular fluid urea nitrogen
concentrations (and presumably PUN concentrations) were a predictor of the developmental
competence of bovine oocytes. Following fertilization, the embryos from the higher urea
nitrogen oocytes had lower cleavage and blastulation rates (Iwata et al., 2006). Thus, the oocytes
that encounter elevated ammonia or urea concentrations within the follicular fluid are less likely
to develop into competent, viable embryos. This data is particularly disconcerting because it
takes over 30 days for ovarian follicles to progress from initial antrum formation to recruitment
(Mihm and Bleach, 2003), presenting a significant window of time during which the oocyte is
susceptible to damage.
Likewise, the concentrations of ammonia and urea are higher in the uterine fluid of cows
with greater PUN concentrations. Urea concentrations within the uterine fluid were higher both
at estrus and during the luteal phase. However, uterine ammonia concentrations were only
elevated during the luteal phase (Hammon et al., 2005). The significance of the fluctuation in
ammonia concentrations from estrous to the luteal phase is not fully understood. But since both
toxic compounds (ammonia and urea) are elevated during the luteal phase within the uterus, it is
likely that they act directly on the embryo and decrease development and viability. The direct
effect of urea on embryonic development has been demonstrated in vitro. Embryos that were
cultured in media containing 21 mg of urea/dL were more likely to degenerate before they
reached the blastocyst stage than those cultured in control media (Ocon and Hansen, 2003). The
results of these experiments indicate that the uterine environment of dairy cattle with high PUN
concentrations is suboptimal for embryonic development and may be one of the primary factors
contributing to the observed decrease in fertility.
HIGH LEVELS OF DIETARY PROTEIN INTAKE AND THE EMBRYO IN THE
UTERINE ENVIRONMENT
A likely mechanism by which circulating urea concentrations affect reproduction in dairy
cattle is by decreasing embryonic development and survival. Effects of urea on embryos may be
manifested through direct actions on the embryo within the oviduct or uterus or by altering the
uterine environment (especially secretory activity) and thereby decreasing the likelihood of
embryonic survival. Many of the changes that have been observed in the uterine environment in
response to high PUN concentrations are occurring at a time during the estrous cycle when a
functional corpus luteum is present and the embryo has migrated to the uterus (Elrod and Butler,
1993; Elrod et al., 1993). Therefore, these changes have the potential to significantly affect the
fertility of cows consuming excess protein.
Even seemingly minor alterations in the uterine milieu can be catastrophic for developing
embryos. Previous research has shown that the uterine environment of dairy cows is indeed
affected by their level of protein intake, particularly during the luteal phase. For example, ion
concentrations (P, Mg, K and Zn) differed within the uterine fluid between cows fed diets
containing relatively high or low protein concentrations (Jordan et al., 1983). In another study,
similar changes in ion concentrations within uterine flushings were associated with the
production of abnormal embryos that would be less likely to continue developing and survive
(Wiebold, 1988).
The uterine luminal pH of cows fed high protein diets is also affected by circulating PUN
concentrations. Both cows and heifers fed high protein diets had lower uterine pH values 7 days
after estrus (luteal phase) than the animals fed the control diets (Elrod and Butler, 1993; Elrod et
al., 1993). Interestingly, there were no differences in uterine pH at estrus regardless of diet.
Based on these results, Rhoads and co-workers (2004) directly infused urea or saline (control)
into the circulation during the luteal phase. Mean PUN concentrations increased from 16.6
mg/dL to 22.6 mg/dL during urea infusion. Uterine pH remained relatively constant during
saline infusion but decreased during urea infusion (Figure 1; from 7.08 at 6 hours to 6.88 at 18
hours of infusion). During embryo culture, lowering the pH of the culture media to similar levels
reduced cleavage rates and almost completely inhibited the development of embryos to the
blastocyst stage (Ocon and Hansen, 2003). Thus, even the minor changes in uterine luminal pH
that are observed in association with high PUN concentrations are capable of decreasing embryo
viability.
7.4
7.3
7.2
7.1
7.0
6.9
6.8
6.7
6.6
6.5
Saline Treatment
pH
30
PUN
25
20
15
10
PUN (mg/dL)
Uterine pH
A
5
0
2
4
6
8 10 12 14 16 18 20 22 24
Time (h)
Urea Treatment
7.4
7.3
7.2
7.1
7.0
6.9
6.8
6.7
6.6
6.5
30
pH
PUN
25
†
*
20
15
10
PUN (mg/dL)
Uterine pH
B
5
0
2
4
6
8 10 12 14 16 18 20 22 24
Time (h)
Figure 1. Least-squares means and standard errors for plasma urea nitrogen (PUN)
concentrations and uterine pH during intravenous infusion of saline (panel A; n=8)
and urea (panel B; n=8) in lactating dairy cows. (A) Uterine pH was not significantly
affected by saline infusion. (B) A treatment × time interaction was detected for
uterine pH. *Uterine pH at 18 h differed (P < 0.05) from those at 6 and 12 h.
†
Uterine pH at 24 h tended (P = 0.09) to differ from that at 12 h (Rhoads et al.,
2004).
Likewise, previous investigations have revealed decreased embryonic survival both in
vivo and in vitro when embryos were collected from ewes fed high levels of protein. In addition
to decreased embryo recovery rates and decreased pregnancy rates, embryos that were collected
from donor ewes consuming high levels of urea were less likely to develop to the blastocyst
stage in culture (Bishonga et al., 1994; Bishonga et al., 1996). Embryos from ewes consuming
high urea diets also used more glucose at embryo collection, and after culture, some experienced
up to a 2.8-fold increase in metabolic rate (McEvoy et al., 1997). The availability of nutrients to
the embryo that would be needed to support such an accelerated metabolic rate may be a factor
contributing to the observed decrease in embryo survival. While these studies were conducted
with sheep rather than dairy cows, they clearly demonstrate a dramatic impact on embryo
development and viability.
When bovine embryos were evaluated by Blanchard et al. (1990), no differences were
found in mean number of fertilized, unfertilized, transferable or nontransferable ova from
lactating cows fed either a 73 or 64% rumen degradable intake protein (DIP) diet. However,
more fertilized ova were recovered from cows fed the lower DIP diet. Cows on the lower DIP
diet also tended to have a higher proportion of transferable ova and more of the cows fed the
high DIP diet failed to yield transferable ova.
In another study, bovine embryos from superovulated, non-lactating cows on low and
high protein diets were evaluated. Visual, microscopic and staining techniques were used to
assess the characteristics of embryos from cows with mean PUN levels of 9.8 mg/dL (low
protein diet) and 21.3 mg/dL (high protein diet). No quantitative or qualitative differences were
observed between the two treatment groups (Garcia-Bojalil et al., 1994). However, appearances
can be deceiving. Rhoads and co-workers (2006) collected embryos from lactating dairy cows
consuming diets designed to result in either high (24.4 mg/dL) or moderate (15.5 mg/dL) PUN
concentrations and transferred them to heifers also consuming two levels of dietary protein
(resulting in PUN concentrations of 7.7 and 25.2 mg/dL). In agreement with previous studies,
they also failed to observe any differences in the quantity or quality of embryos collected from
the two experimental groups (Table 1). However, following transfer, differences in embryo
viability became apparent. The transfer of embryos collected from the high PUN donors resulted
in fewer pregnancies than the embryos collected from moderate PUN donors (Table 2; 11 vs.
35% pregnancy rate). Interestingly, there were no differences in pregnancy rate based on the
PUN concentrations of the recipient animals. Since pregnancy rates were only affected by the
PUN concentrations of the donor animals, we can conclude that either the oocyte or embryo was
damaged by high PUN concentrations on or before day 7 of pregnancy (the day that embryos
were collected from donors), resulting in decreased long-term viability.
Table 1. Morphological evaluation and numbers of embryos recovered from donor lactating
cows with moderate or high plasma urea nitrogen (PUN; Rhoads et al., 2006).
Donor Cow Group
Characteristic
Gradea
Stageb
Moderate PUN
1
2
3-5
6
4
5
6
40
1
3
6
30
6
5
High PUN
49
6
6
5
47
5
3
a
Grade 1 = excellent, Grade 2 = good, Grade 3-5 = fair to very poor, Grade 6 = unfertilized
oocyte. Embryos grading ≥ 3 were not transferred to recipient heifers.
b
Stage 4 = compact morula, Stage 5 = early blastocyst, Stage 6 = blastocyst.
Table 2. Pregnancy rates achieved following transfer of embryos from donor cows with high or
moderate plasma urea nitrogen (PUN) concentrations into high or low PUN recipient
heifers. The data is number of pregnancies/number of transfers with pregnancy rate %
in parentheses (Rhoads et al., 2006).
High Recipient
Low Recipient
Total
a, b
High Donor
Moderate Donor
3/18 (17%)
2/27 (7%)
5/45 (11%)a
4/12 (33%)
9/25 (36%)
13/37 (35%)b
Total
7/30 (23%)
11/52 (21%)
Values are significantly different (P<0.02).
HIGH LEVELS OF DIETARY PROTEIN INTAKE AND PLASMA PROGESTERONE
Progesterone is one of the primary hormones responsible for the successful maintenance
of pregnancy. The results of some previous studies indicate that high levels of protein intake
decrease the concentration of plasma progesterone during the luteal phase (days 12 and 14 of the
estrous cycle; Jordan and Swanson, 1979b; Sonderman and Larson, 1989). Decreased plasma
progesterone concentrations during the luteal phase following insemination and fertilization have
profound detrimental effects on the uterus and embryo that are likely to culminate in the loss of
the pregnancy. Thus, decreased progesterone production is yet another plausible explanation for
the observed decrease in fertility during periods of excess protein intake.
The cyclic changes of the bovine uterine environment, which occur in response to
circulating levels of reproductive hormones (including progesterone), are a coordinated
symphony designed to result in the successful establishment of pregnancy. Uterine secretory
activity is a major target of the fluctuating levels of steroid hormones that occur throughout the
estrous cycle. The nature, timing and amount of these secretions are known to be essential to the
preparation of the uterus for pregnancy and subsequent early embryo development. Therefore,
the reported changes in progesterone concentrations and uterine secretions in response to
elevated PUN concentrations are likely catastrophic deterrents to fertility.
Several reports have suggested that secretions from the endometrium influenced by
reproductive hormones are essential for the initiation and regulation of conceptus growth and
development throughout early pregnancy (Barnes, 2000; Garrett et al., 1988; Geisert et al., 1992;
Pope, 1988). Progesterone, in particular, seems to play a very important role in the events
associated with early pregnancy. For example, cows treated with exogenous progesterone during
the early stages of pregnancy produce larger, more advanced embryos (Garrett et al., 1988). A
more recent report suggests that the progesterone produced by the corpus luteum is responsible
for the endometrial secretion of nutrients, growth factors, immunosuppressive agents, enzymes,
ions and steroids. Progesterone also plays an important regulatory role in the production and
secretion of prostaglandin F2α (PGF2α; Geisert et al., 1992). Because of these interactions, a
close synchrony between the uterine environment and the embryo is essential to successful
pregnancy. According to Barnes (2000), asynchronous uterine and embryo conditions can lead
to implantation failure, early embryonic mortality, or altered development and growth, all within
the first week after conception. Fortunately, the embryo is capable of altering its development in
response to the uterine environment, but, if too far out of synchrony, the uterus will not allow the
necessary time for an embryo to modify its growth (Pope, 1988).
CONCLUSIONS
Most previous reports agree that high protein diets, which result in elevated levels of
PUN, are related to decreased fertility in lactating dairy cattle. Evidence suggests that the effects
of excess protein intake (and high PUN concentrations) are enacted on the oocyte within the
developing follicle, the corpus luteum and the early embryo. To complicate matters, the
observed impact on fertility may be exacerbated by synergistic interactions with negative energy
balance and heat stress. The resulting decrease in fertility represents a significant economic loss
to dairy producers. Therefore, in order to optimize reproductive efficiency in dairy cattle close
attention must be paid to dietary formulations and their resulting PUN concentrations.
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