Disruption of bovine oocytes and preimplantation embryos by urea and acidic pH.
ABSTRACT Feeding cattle diets high in degradable crude protein (CP) or in excess of requirements can reduce fertility and lower uterine pH. Objectives were to determine direct effects of urea and acidic pH during oocyte maturation and embryonic development. For experiment 1, oocytes were matured in medium containing 0, 5, 7.5, or 10 mM urea (0, 14, 21, or 28 mg/dl urea nitrogen, respectively). Cleavage rate was not reduced by any concentration of urea. However, the proportion of oocytes developing to the blastocyst stage at d 8 after insemination was reduced by 7.5 mM urea. In addition, the proportion of cleaved oocytes becoming blastocysts was decreased by 5 and 7.5 mM urea. For experiment 2, putative zygotes were collected -9 h after insemination and cultured in modified Potassium Simplex Optimized Medium (KSOM). Urea did not reduce the proportion of oocytes developing to the blastocyst stage, although 10 mM urea reduced cleavage rate slightly. For experiment 3, dimethadione (DMD), a weak nonmetabolizable acid, was used to decrease culture medium pH. Putative zygotes were cultured in modified KSOM containing 0, 10, 15, or 20 mM DMD for 8 d. DMD reduced cleavage rate at 15 and 20 mM and development to the blastocyst stage at all concentrations. Results support the idea that feeding diets rich in highly degradable CP compromises fertility through direct actions of urea on the oocyte and through diet-induced alterations in uterine pH.
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ABSTRACT: During early postpartum, high-producing dairy cows undergo a period of extensive tissue catabolism because of negative nutrient balance. Homeorrhetic controls assure that nutrients are partitioned to favor lactation at the same time that homeostasis secures survival. However, unrestrained metabolic disturbances often lead to diseases which, in turn, dramatically decrease both productive and reproductive performance. Negative nutrient balance has been associated with compromised immune and reproductive functions in dairy cows. Low circulating concentrations of glucose and insulin associated with elevated concentrations of non-esterified fatty acids and ketone bodies postpartum have disruptive and detrimental effects on the oocyte, granulosa and immune cells. Negative nutrient balance is associated with changes in the pattern of ovarian follicle growth which can indirectly affect oocyte quality. Some of this disruption seems to be the result of endocrine and biochemical changes that alter the micro-environment of the growing and maturing oocyte. In addition, cows under negative nutrient balance have extended periods of anovulation. Postpartum anestrus, as well as infertility, is magnified by losses of body condition during the early postpartum period. The underlying mechanism for resumption of ovulatory cycles seems to be associated with metabolic signals and regulatory hormones primarily insulin and insulin-like growth factor (IGF)-1, which link nutritional status with gonadotropin secretion, recoupling of the growth hormone-IGF system, and follicle maturation and ovulation. Feeding diets that promote increases in plasma glucose and insulin may improve the metabolic and endocrine status of cows in early lactation. Furthermore, fertility in postpartum cows is also determined by uterine health. Reductions in circulating concentrations of Ca and antioxidant vitamins around parturition are also linked with impaired immune competence and result in greater risk of uterine diseases that impair reproduction. Specific nutrients and dietary ingredients have been implicated to affect reproduction in cattle. Excess intake of dietary protein has been suggested as detrimental to fertility, although feeding excess of dietary protein can no longer be justified. Addition of moderate amounts of supplemental fat to the diet improves caloric intake, modulates prostaglandin F2 secretion by the uterus, affects ovarian dynamics, enhances luteal function and embryo quality, and has moderate positive effects on fertility. More specifically, some fatty acids might impact fertilization rate and embryo quality in dairy cows. On the contrary, some dietary ingredients, such as gossypol, when ingested in large quantities decrease fertility of dairy cows because of its negative effects on embryo quality and pregnancy maintenance.Animal Reproduction. 10/2012; 9(3):260-272.
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ABSTRACT: Lactating dairy cows (n=177) feed with grass and corn silage ad libitum kept in pasture, were randomly assigned to evaluate how urea nitrogen in plasma and milk can be related to their pregnancy rate. Blood and milk samples were collected on the artificial insemination (AI) day to evaluate plasma urea nitrogen (PUN) and milk urea nitrogen (MUN) as well as progesterone levels, excluding cows with progesterone higher than 0.5 ng/ml. Cows were considered pregnant if six weeks after artificial insemination, they did not return to estrus. Concentrations of PUN or MUN greater than the average (16 mg/dl) were associated with decreased pregnancy rates (13% and 14%, respectively) (p< 0.05) as compared to the cows with urea levels less than this value on the insemination day. As PUN and MUN increased to greater than 16 mg/dl, the likelihood ratio for pregnancy decreased. There was a high correlation between PUN and MUN concentrations (r2= 0.97, p≤ 0.001). The results of this study indicate that an increase in PUN or MUN can exert direct or indirect effects in reproduction, impairing the conception of grazing dairy cows.J Phys Pharm Adv. 12/2011; 1(1):9-14.
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ABSTRACT: Large amounts of protein intake are associated with elevated ammonia and urea concentrations in both plasma and uterine fluid in dairy cows. These increased concentrations affect successful embryo development and subsequent pregnancy establishment. The objective of the present study was to examine the effects of ammonia and urea on the expression of some candidate genes in the endometrium of mid-luteal phase of the estrous cycle of dairy cows. Endometrial explants were cultured and treated with 0, 75, 150, 300, 600μM of ammonium chloride or 0, 4, 8, 12, 16mM of urea. After the RNA extraction and reverse transcription, real time PCR was performed to assess the treatment effects on relative amounts of mRNA of candidate genes. BCL2 mRNA was greater in explants treated with 150μM of ammonium chloride compared to explants treated with 0, 75 and 300μM. Relative amounts of IGFBP1 mRNA were less in explants treated with 600μM of ammonium chloride when compared with other concentrations. Relative FGF2 gene expression was less in explants treated with a greater concentration (600μM) of ammonium chloride or urea (16mM) when compared with lesser concentrations. Expression of HSPA1A, IGFBP3 and SERPINA14 genes was greater in explants exposed to lesser concentrations (150μM) of ammonium chloride or urea (4mM). Relative amounts of IGF1 and BAX mRNA were not affected by any of the ammonium chloride or urea concentrations tested. In conclusion, greater concentrations of ammonia and urea have negative effects on some endometrial gene expression, while moderate concentrations have positive effects.Animal reproduction science 07/2013; · 1.56 Impact Factor
J. Dairy Sci. 86:1194–1200
American Dairy Science Association, 2003.
Disruption of Bovine Oocytes and Preimplantation Embryos
by Urea and Acidic pH1
O. M. Ocon2and P. J. Hansen
Department of Animal Sciences, University of Florida,
Feeding cattle diets high in degradable crude pro-
direct effects of urea and acidic pH during oocyte mat-
uration and embryonic development. For experiment
1, oocytes were matured in medium containing 0, 5,
respectively). Cleavage rate was not reduced by any
concentration of urea. However, the proportion of oo-
cytes developing to the blastocyst stage at d 8 after
insemination was reduced by 7.5 mM urea. In addi-
tion, the proportion of cleaved oocytes becoming blas-
tocysts was decreased by 5 and 7.5 mM urea. For ex-
periment 2, putative zygotes were collected ∼9 h after
plex Optimized Medium (KSOM). Urea did not reduce
the proportion of oocytes developing to the blastocyst
stage, although 10 mM urea reduced cleavage rate
slightly. For experiment 3, dimethadione (DMD), a
weak nonmetabolizableacid, was used todecrease cul-
ture medium pH. Putative zygotes were cultured in
modified KSOM containing 0, 10, 15, or 20 mM DMD
for 8 d. DMD reduced cleavage rate at 15 and 20 mM
and development to the blastocyst stage at all concen-
trations. Results support the idea that feeding diets
rich in highly degradable CP compromises fertility
through direct actions of urea on the oocyte and
through diet-induced alterations in uterine pH.
(Key words: urea, pH, preimplantation embryo,
Abbreviation key: COC = cumulus-oocyte com-
plexes, DMD = dimethadione, KSOM = Potassium
Simplex Optimized Medium.
Received April 5, 2002.
Accepted August 6, 2002.
Corresponding author: P. J. Hansen; e-mail: Hansen@animal.
Experiment Station. Research was supported in part by USDA-
CSREES grant # 2001-52101-11318.
vania State University, University Park, 16802-3503.
Feeding dairy cows large amounts of degradable
protein in excess of requirements can reduce preg-
nancy rates per insemination (Canfield et al., 1990;
Elrod and Butler, 1993). Diets high in protein content
elevate urea nitrogen concentrations in plasma and
uterine secretions (Jordan et al., 1983; Canfield et al.,
1990; Elrod and Butler, 1993; Roseler et al., 1993),
and elevations in blood or milk urea nitrogen concen-
trations have also been associated with low fertility
(Butler et al., 1996; Larson et al., 1996; Rajala-Schultz
et al., 2001).
The mechanism by which feeding large amounts of
protein affects fertility is not completely known. One
possibility is that high concentrations of urea associ-
ated with excess feeding of CP could disrupt oocyte
growth or maturation, fertilization, or development.
Feeding a diet generating large amounts of urea and
ammonia increased growth of the second-wave domi-
nant follicle, decreased capacity of oocytes from small
(1 to 4 mm) and medium (>4 to 8 mm) follicles to cleave
when fertilized in culture, and decreased ability of
cleaved embryos formed from oocytes obtained from
tocyst stage (Sinclair et al., 2000). Moreover, exposure
of oocytes to 6 mM of urea during maturation in vitro
impaired meiosis and fertilization rate (De Wit et
Feeding large amounts of protein can also alter the
uterine environment by reducing concentrations of
magnesium, potassium, and phosphorus in uterine se-
cretions (Jordan et al., 1983) and by reducing uterine
pH (Elrod et al., 1993; Elrod and Butler, 1993). Effects
ofprotein diet on potassium,phosphorus, andpH were
only observed during the luteal phase. Although con-
onic function are not known, a reduction in pH from
mise embryonic development in mice (Edwards et al.,
1998). In contrast, development of hamster preim-
plantation embryos was unaffected by a range of pH
from 6.5 to 7.4 (Bavister et al., 1983; Carney and
EFFECTS OF UREA AND ACIDIC pH ON BOVINE EMBRYOS
Objectives of the present study were to 1) determine
direct effects of urea on oocyte maturation and embry-
onicdevelopmentand 2)evaluatewhetherlow pHdur-
ing the period of embryonic growth disrupts develop-
ment. To test effect of pH, embryos were cultured in
varying concentrations of dimethadione (DMD), a
nonmetabolizable weak acid that lowers pH of the cul-
ture medium and has been used to study effects of
pH on development of hamster and mouse embryos
(Carney and Bavister, 1987; Edwards et al., 1998).
MATERIALS AND METHODS
Materials for in vitro maturation, fertilization, and
embryo culture were obtained as described previously
(Rivera and Hansen, 2001). Urea was purchased from
Research Organics (Cleveland, OH), and hyaluroni-
dase and dimethadione (5,5-dimethyl-2, 4-oxazol-idi-
nedione) were from Sigma (St. Louis, MO). Frozen
semen from bulls of several breeds (Angus, Holstein,
and Brangus) was donated by Select Sires Inc. (Rocky
Mount, VA) or was purchased from Southeastern Ser-
In Vitro Production of Embryos
In vitro production of embryos was performed as
described by Rivera and Hansen (2001) using oocytes
recovered from ovaries obtained at a local abattoir.
Briefly, oocytes were collected by slashing the ovaries
and washing in oocyte collection medium (Tissue Cul-
ture Medium 199 with Hank’s Salts without phenol
red, supplemented with 2% [vol/vol] bovine steer se-
rum [containing 2 U/ml heparin], 1 mM glutamine,
100 U/ml penicillin-G, and 0.1 mg/ml streptomycin).
Groups of 10 cumulus-oocyte complexes (COC) were
ration medium (Tissue Culture Medium 199 with
Earle’s Salts supplemented with 10% [vol/vol] bovine
steer serum, 50 µg/ml gentamicin, 20 µg/ml FSH, 2
µg/ml estradiol-17β, 22 µg/ml sodium pyruvate, and 1
mM glutamine) for 21 to 24 h at 38.5°C and 5% (vol/
were washed and groups of 30 oocytes were placed in
four well plates of 600 µl of IVF-TALP (Parrish et al.,
1986) supplemented with 25 µl of a mixture of 0.5
mM penicillamine, 0.25 mM hypotaurine, and 25 µM
epinephrine in 0.9% (wt/vol) NaCl. The COC were fer-
tilized with ∼1 × 106Percoll-purified sperm/well (Par-
rish et al., 1986). For each replicate, sperm were pre-
pool of bulls was sometimes but not always used for
Journal of Dairy Science Vol. 86, No. 4, 2003
each replicate. Following incubation for 8 to 10 h at
38.5°C and 5% (vol/vol) CO2, putative zygotes were
denuded of cumulus cells by vortexing and, if neces-
sary, by incubation in 300 µg/ml of hyaluronidase in
Hepes-TALP. Embryos were then cultured in Pot-
assium Simplex Optimized Medium (KSOM), modi-
fied by adding 3 mg/ml bovine serum albumin (essen-
tially fatty-acid free), 50 µg/ml gentamicin, Basal Me-
dium Eagle essential AA solution, and Minimum
Essential Medium nonessential AA solution. For em-
bryo culture, putative zygotes were placed in drops
of modified KSOM at 38.5°C and 5% CO2(vol/vol) in
humidified air until d 8 after insemination. Cultures
were performed in 25-µl drops, and each contained 10
to 20 embryos per drop. Within a replicate, numbers
of embryos per drop were constant for all treatments.
For each replicate, one or more drops (typically two)
of embryos were prepared for each treatment.
Effect of urea during oocyte maturation on
cleavage and subsequent development. Groups of
∼10 COC were transferred to drops of oocyte matura-
concentrations are equivalent to 0, 14, 21, and 28 mg/
dl urea nitrogen, respectively. Oocytes were matured
for 21 to 24 h, washed in Hepes-TALP medium, and
then subjected to fertilization in wells without the
presence of urea. After fertilization, putative embryos
wereculturedin 25-µldropsofmodified KSOM.Cleav-
age rate was recorded on d 3 and the percentage of
oocytes reaching the blastocyst stage recorded at d 8.
The experiment was replicated seven times with a
total of 204 to 229 oocytes per treatment group.
Effect of urea on early embryonic development.
Putative zygoteswere producedby invitro maturation
and fertilization. After fertilization, groups of 10 to 20
putative zygotes were transferred to 25-µl drops of
modified KSOM containing 0, 5, 7.5, or 10 mM urea
for the duration of culture. Embryos were examined
on d 3, 5, 7, and 8 for development. The experiment
was replicated five times with a total of 167 to 180
putative zygotes per treatment group.
Effect of DMD on early embryonic development.
A preliminary experiment was performed one time in
which pH was measured in medium containing 0, 10,
15, or 20mMDMD atvarioustimepoints afterincuba-
tion at 38.5°C and 5% CO2(vol/vol) in humidified air.
After fertilization, groups of 10 to 20 putative zy-
gotesproduced byinvitromaturation andfertilization
were transferred to 25-µl drops of modified KSOM
containing 0, 10, 15, or 20 mM DMD. Embryos re-
mained in these drops for the duration of culture.
OCON AND HANSEN
Cleavage rate was recorded on d 3, and the percentage
of oocytes reaching the blastocyst stage recorded at d
8. The experiment was replicated three times with a
total of 102 to 124 putative zygotes per treatment
The proportion of oocytes that cleaved and the pro-
portion of oocytes and cleaved embryos that became
blastocysts was calculated for each replicate (i.e., for
embryos from all drops treated alike in a given repli-
cate). Data were analyzed by least squares ANOVA
using the general linear models procedure of the Sta-
tistical Analysis System (SAS, 1989). The mathemati-
cal model included effects of treatment and replicate;
the error term represents treatment × replicate. When
the main effect of treatment was significant, means
separation procedures were carried out using least-
mine concentrations of urea that differed from un-
treated controls. Data were analyzed two ways—as
untransformed data and after performing arcsin
transformation. Probability values were similar for
from the analysis of transformed data, whereas re-
ported least-squares means and standard errors are
derived from analysis of untransformed data.
Effect of Urea During Maturation
Cleavage rates were not reduced by urea at any
concentration tested (Figure 1A). As compared with
control oocytes, the proportion of oocytes that became
blastocysts at d 8 after insemination was reduced (P
< 0.001) by maturation in 7.5 mM urea but not by
maturation in 5 or 10 mM urea (Figure 1B). The pro-
portion of cleaved embryos developing to blastocysts
was decreased by 5 (P < 0.05) and 7.5 mM urea (P <
0.001) but not by 10 mM urea (Figure 1C).
Effect of Urea During Embryonic Development
The highest urea concentration tested (10 mM) re-
duced (P < 0.05) cleavage rate slightly (Figure 2A).
Nonetheless, in contrast to effects during maturation,
urea did not reduce the proportion of oocytes (Figure
2B) or cleaved embryos (Figure 2C) developing to the
Effect of DMD During Embryonic Development
A preliminary study was conducted to describe the
changes in the pH over time in media containing vari-
Journal of Dairy Science Vol. 86, No. 4, 2003
Figure 1. Development of bovine embryos treated with various
concentrations of urea during in vitro maturation. The experiment
was replicated seven times with a total of 204 to 229 oocytes per
treatment group. Results are least-squares means ± SEM. Means
significantly different from 0 mM urea are indicated by asterisks (*P
< 0.05; ***P < 0.001).
ous concentrations of DMD (Figure 3). The initial pH
values for modified KSOM with 0, 10, 15, and 20 mM
DMD were 7.2, 6.6, 6.4, and 6.3, respectively. After
24 h in 5% CO2, the respective pH values were 7.4,
7.1, 7.0, and 6.8.
As compared with controls, cleavage rate was not
affected by 10 mM DMD but was reduced by 15 (P =
EFFECTS OF UREA AND ACIDIC pH ON BOVINE EMBRYOS
Figure 2. Development of bovine embryos treated with various
concentrations ofurea duringculture. Theexperiment wasreplicated
five times with a total of 167 to 180 putative zygotes per treatment
group. Results are least-squares means ± SEM. Means significantly
different from 0 mM of urea are indicated by an asterisk (*P < 0.05).
0.05) and 20 mM (P < 0.01) DMD (Figure 4A). Develop-
ment to the blastocyst stage was significantly reduced
by all concentrations of DMD regardless of whether
data were expressed as percentage of oocytes devel-
oping to blastocysts (Figure 4B) or percentage of
The inhibition to development caused by DMD was
nearly total; only 1.0% of oocytes cultured with 5 mM
Journal of Dairy Science Vol. 86, No. 4, 2003
Figure 3. Effects of various concentrations of DMD on culture
medium pH at 38.5°C and 5% CO2. Values are plotted for modified
KSOM, modified KSOM containing vehicle for DMD (0 mM), and
modified KSOM containing 10 to 20 mM DMD.
DMD became blastocysts, and no oocytes cultured in
15 or 20 mM DMD reached the blastocyst stage.
In this study, exposure of oocytes to physiologically
relevant concentrations of urea during the process of
maturation interfered with ability of the embryos
formed after fertilization to develop to the blastocyst
stage. Exposure of the embryo to urea after fertiliza-
tion had no effects on development, indicating that
the embryoitselfwas resistanttodirecteffectsofurea.
However, developmentwas impaired byembryonic ex-
posure to acidic pH similar to those of uterine secre-
tions from cows fed excess CP (Elrod et al., 1993; Elrod
and Butler, 1993). Taken together, the results impli-
cate changes in urea concentrations and uterine pH
as a cause of infertility associated with feeding diets
containing excess CP.
As in the present study, De Wit et al. (2001) also
observed an effect of urea exposure during maturation
on the proportion of oocytes becoming blastocysts.
However, the cause for the decline in development to
the blastocyst stage was different than was the case
here. In particular, De Wit et al. (2001) observed that
the addition of 6 mM urea to maturation medium has-
tened completion of metaphase I, inhibited completion
of metaphase II, reduced fertilization rate, and de-
creased the proportion of oocytes that became blasto-
cysts. There was no effect of urea exposure during
maturation on development of cleaved embryos to the
blastocyst stage. Thus, urea reduced the proportion of
OCON AND HANSEN
Figure 4. Effect of DMD during embryonic development. The ex-
Means significantly different from 0 mM DMD are indicated by sym-
bols (†P = 0.05; *P < 0.05; **P < 0.01).
oocytes becoming blastocysts because it reduced fertil-
ization and cleavage rate, not because the embryos
that were formed after fertilization were compro-
mised. In the present study, in contrast, addition of
to become blastocysts. Thus, the overall reduction in
the proportion of oocytes becoming blastocysts is at-
tributed to decreased developmental competence of
embryos formed from oocytes exposed to urea.
Journal of Dairy Science Vol. 86, No. 4, 2003
Differences in concentrations of urea between the
two studies cannot account for the discrepancy be-
tween the current results and those of De Wit et al.
(2001) because the reduction in development of
cleaved embryos was observed in the present study at
5 and 7.5 mM urea. Perhaps differences in embryo
culture media between the two studies are responsible
for the discrepancy in effectsof urea on embryo compe-
tence. The culture medium used in the study of De
Wit et al. (2001) was complex and included serum,
epidermal growth factor, insulin, and other constit-
uents not present in the modified KSOM used in the
present study. Future studies to determine whether
specific nutrients or regulatory factors can overcome
damage to the embryo caused by exposure of oocytes to
embryonic survival under adverse conditions.
One surprising result of the current study was the
observation that, in contrast to effects of 5 and 7.5
mM urea, exposure of oocytes to 10 mM urea during
maturation did not affect cleavage or subsequent de-
velopment. Conceivably, high concentrations of urea
trigger some compensatory mechanism in the oocyte
that increases resistance to urea toxicity. The nature
of this phenomenon is unknown but is unlikely to in-
volve transcription, because the oocyte is transcrip-
tionally inactive during maturation and cannot re-
spond to a cellular stress by increasing transcription
of genes involved in cytoprotection. Heat shock, for
example, can induce heat shock protein 70 synthesis
at the two-cell stage but not in oocytes evaluated be-
fore or after completion of maturation (Edwards and
Hansen, 1996). One possibility is that high concentra-
tions (>7.5 mM) of urea trigger changes in membrane
transport of urea to either inhibit movement of urea
inside the cell or remove urea from the cytoplasm.
Carrier-mediated exchange of urea occurs in other
cells(Verkman etal.,1985;Hisanagaetal.,1991; Kato
and Sands, 1998), but the system of urea transport in
the oocyte and its regulation by urea concentration is
The impairment of events during oocyte maturation
by urea occurred at concentrations that are probably
trations affecting maturation (5 and 7.5 mM; equiva-
lent to plasma urea nitrogen concentrations of 14 and
21 mg/dl) closely match the concentrations of urea in
the plasma or milk of cows experiencing infertility due
to excess feeding of CP (Canfield et al., 1990; Elrod
and Butler, 1993; Butler et al., 1996; Larson et al.,
1996; Rajala-Schultz et al., 2001).It is likely that feed-
ing excessamountsof CPalsoelevatesureaconcentra-
tions in the follicle, the site of oocyte maturation, al-