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Reduced Fertility Associated with Low Progesterone Postbreeding and Increased Milk Urea Nitrogen in Lactating Cows
Reduced Fertility Associated with Low Progesterone Postbreeding and Increased Milk Urea Nitrogen in Lactating Cows S. F. LARSON, W. R. BUTLER, and W. B. CURRIE1 Department of Animal Science, Cornell University, Ithaca, NY 14853
ABSTRACT The primary objectives of this study were to determine whether a delay in the onset of the luteal phase, or high milk urea nitrogen at breeding, or both were associated with failure of pregnancy early in gestation. Milk samples were collected twice daily from cows in a single herd during the week following breeding; single samples were collected on d 14 and 21 postbreeding. Progesterone was measured in all samples, and a total of 156 sample sets was used. The progesterone data combined with results from pregnancy examinations were used to distribute the cows into three groups: 1 ) pregnant, 2 ) nonpregnant with a low concentration (<2 ng/ml) of progesterone on d 21, and 3 ) nonpregnant with a high concentration ( ≥2 ng/ml) of progesterone on d 21. The interestrous interval for cows in group 3 was longer than that for cows in group 2. Beginning 4.5 d after breeding, pregnant cows had higher concentrations of progesterone than did cows in group 3. Pregnant cows also had higher concentrations of progesterone than did all open cows on d 14 and 21. The onset of the luteal phase was earlier in pregnant cows than it was in cows in group 3. Milk urea nitrogen at breeding was similar in pregnant cows and in cows in group 3, but was higher in cows in group 2. Increased milk urea nitrogen was also statistically associated with decreased fertility. We propose that the cows in group 3 likely had embryos that initiated pregnancy recognition and prolonged luteal function, but these embryos were compromised by suboptimal exposure to progesterone early in development. ( Key words: progesterone, pregnancy, milk urea nitrogen) Abbreviation key: MUN = milk urea nitrogen, NPH = nonpregnant and high circulating concentrations of progesterone ( ≥2 ng/ml) on d 21 postbreeding, NPL =
Received May 13, 1996. Accepted November 18, 1996. 1Corresponding author. 1997 J Dairy Sci 80:1288–1295
nonpregnant and low circulating concentrations of progesterone (<2 ng/ml) on d 21 postbreeding. INTRODUCTION As the size of dairy herds increases, more intensive management systems are needed to maximize milk production, and overall herd fertility often decreases. In New York dairies, conception rates after first service have declined from 66% in 1951 to 40% in 1995, and milk production has doubled during the same period (4, 5). Most pregnancy losses occur during the first 30 d postbreeding and are due to a variety of causes [reviews (1, 31)]. The role of progesterone in pregnancy maintenance is undisputed; however, the possible role of progesterone around the time of maternal recognition of pregnancy is not as apparent (23). Some studies have indicated that peripheral concentrations of progesterone are higher in pregnant cows than in nonpregnant cows as early as d 4 ( 3 ) or d 6 (9, 22) postbreeding or that low concentrations of progesterone during the 1st wk after breeding are associated with abnormal embryonic development (17). Other researchers have reported no differences in peripheral concentrations of progesterone that are related to pregnancy status until d 10 ( 1 6 ) or d 13 ( 2 ) . Many of these studies, however, either used limited numbers of cattle or failed to follow individual animals throughout the onset of the luteal phase. We hypothesized that variation among cattle in the establishment of luteal function might be appreciable and that individuals with delayed onset of luteal function would be less likely to become pregnant or to maintain pregnancy. Another factor that has been recently implicated in the subfertility of dairy cows is excessive circulating concentrations of urea; values for urea nitrogen measured in plasma and milk are comparable ( 3 ) . High circulating concentrations of urea nitrogen are usually caused by dietary protein, especially highly degradable protein, in excess of the nutritional requirements of the animal. Concentrations of blood urea nitrogen >19 or >20 mg/dl are associated with decreased pregnancy rates of lactating cows and heif-
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ers (3, 10, 11). Results of other studies (7, 8 ) have indicated that increased plasma urea nitrogen is associated with lower conception rates and decreased uterine pH for several days after estrus. The objectives of this study were 1 ) to determine whether peripheral concentrations of progesterone differed between pregnant and nonpregnant cows during the 1st wk after breeding, 2 ) to investigate further the contribution of increased milk urea nitrogen ( MUN) to subfertility, and 3 ) to determine factors other than circulating progesterone concentrations that might influence pregnancy outcome or bias the statistical calculations. MATERIALS AND METHODS Cows All cows were grade Holsteins in a dairy herd that had a rolling herd average milk production of 11,000 kg of milk/yr per cow. The cows were housed at the Teaching and Research Center, Department of Animal Science, Cornell University (Ithaca, NY). No aspect of herd management was changed for the purpose of the study; most cows were housed in free-stall pens, and all were fed a TMR that was based on corn silage. All lactating cows that were presented for breeding during the first 3 d of each week (to take advantage of the estrous synchronization schedule) from January to July were used in the study; the only exclusions were cows that were in use in other experiments that might have compromised fertility. Information was obtained for a total of 228 breedings of 162 cows; 34 cows were sampled twice, and 14 cows were sampled three times. When the cows were assigned to the study, the following information was recorded: body condition score at breeding, lactation number, number of times bred during the current lactation, DIM, mean daily milk production, number of times milked per day, time of day bred (a.m. or p.m.), whether estrus had been synchronized by use of PGF2a, and whether the cow was being bred at a fixed time (80 h after PGF2a administration) without observation of estrus. This information would be potentially useful to account for part of the random variation in the planned analyses of variance. Study Design For 7 d, starting on the day of insemination, milk samples were collected twice daily from all cows that were assigned to the study. One sample was collected on d 14 and 21 ( ±1 d ) postbreeding to verify luteal function and to evaluate accurately cows that had
returned to estrus. At approximately 7 wk postbreeding, if the cow had not returned to estrus, she was palpated rectally by the herd veterinarian to determine pregnancy status. After all analyses were completed, the data file was sorted to remove sample sets that exhibited any of the following characteristics: 1 ) progesterone concentrations >1 ng/ml on day of breeding (regarded as misbred, n = 33), 2 ) no increase in progesterone noted throughout the sampling period (cystic or anestrous, n = 11), 3 ) missing >6 samples ( n = 6), 4 ) grossly erratic patterns of progesterone concentrations (likely caused by a sampling error, n = 15), 5 ) culled before pregnancy evaluation ( n = 1), or 6 ) rebred <21 d after entering the study (based on the assumption that the second breeding might have interfered with a conceptus that was present from the initial breeding, n = 6). A total of 156 sample sets were retained and used in the final analysis. Sample Collection and Processing Milkers collected 5- to 10-ml premilking strip samples from one quarter of each cow. Samples were refrigerated after collection, and milk fat was allowed to separate. Within 2 d of collection, cream was aspirated from the sample. Samples were centrifuged for 10 min at 100 × g, reaspirated to eliminate milk fat, and stored at –20°C. Samples were sorted into complete sets from each breeding so that each sample set was analyzed for progesterone concentration in the same radioimmunoassay. All samples of skim milk were analyzed for progesterone content, and samples from the day of breeding were also analyzed for MUN. Radioimmunoassay A double-antibody radioimmunoassay adapted from the methodology described by Nara and First ( 1 9 ) was used to measure concentrations of progesterone in skim milk. A 100-ml aliquot of sample or standard was diluted with 400 ml of PBS containing 0.1% (wt/vol) gelatin. Progesterone labeled with 125I (ICN Biomedicals Inc., Costa Mesa, CA) was also diluted in PBS containing 0.1% (wt/vol) gelatin, and 20,000 cpm were added. Standards were assembled in 0.5% BSA in PBS at the following concentrations: 0.5, 1, 2, 4, 8, and 16 ng/ml. Progesterone antiserum [rabbit number 8; (24)] was used at a final dilution of 1:80,000 (vol/vol) in 1:400 (vol/vol) normal rabbit serum in PBS containing 50 mM EDTA. Tubes were incubated at 37°C for 1 h and then at 4°C overnight. Ovine anti-rabbit gamma globulin (produced in-house) was added to all tubes to a final Journal of Dairy Science Vol. 80, No. 7, 1997
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dilution of 1:125 (vol/vol). The tubes were then incubated at 4°C for an additional 48 h. Antibody-bound and unbound radioactivity was separated by the addition of 4 ml of PBS at 4°C, centrifugation for 30 min at 1000 × g, decanting, and draining. Antibody-bound radioactivity was quantified in a gamma spectrometer. Under the conditions described, 25 to 35% of 125I-labeled progesterone was bound by antibody in the absence of standard or unknown ligand ( B 0) . Standard concentrations of progesterone (0.5 to 16 ng/ml) generated linear logit-log standard curves ( r 2 = 97%) with 75% (0.5 ng/ml) to 22% (16 ng/ml) binding, using B0 as the reference. Either 0.5% BSA in PBS or solvent-extracted skim milk could be used as the diluent. Nonspecific binding of radioactivity (no antiprogesterone) in the presence of skim milk rarely exceeded twice the background of the gamma spectrometer. Data were compared from matched plasma and milk samples taken from cows in estrus and from cows in the early and midluteal phases to verify that progesterone in skim milk accurately reflected the circulating concentrations of progesterone. Plasma samples were extracted and analyzed ( 1 9 ) and were compared with measurements in skim milk samples as described previously: plasma progesterone = 0.2 + 0.96 milk progesterone; r2 = 76%. Pools of milk with low and high progesterone contents were analyzed in all assays in the present study; coefficients of variation within and between assays were 17 and 13%, respectively. Recovery was measured by the addition of 100 or 200 pg of progesterone to aliquots of the pooled milk with low progesterone content, which resulted in 103% recovery of the added progesterone.
tus. The model for the analysis of variance for repeated measures was Yijkm = m + ai + bj + tk( i) + ( ab) ij + em( ijk) , where Yijkm = progesterone concentration; m = overall mean, ai = fixed effects of pregnancy status i, bj = fixed effects of sampling day j, tk( i) = random effects of cow k nested within pregnancy status i, ( ab) ij = effects of interaction of fixed pregnancy status i and sampling day j, and em( ijk) = random error terms. As expected, the interaction of sampling day and pregnancy status was highly significant, and Fisher’s protected least significant difference post hoc test was used to assess differences between means for pregnancy status groups on individual days. On the basis of all available information, cows were categorized into three groups: 1 ) pregnant (confirmed by palpation), 2 ) nonpregnant and with <2 ng/ml of milk progesterone at d 21 postbreeding ( NPL) (some, but not all, of these cows were observed in estrus around d 21), and 3 ) nonpregnant and with ≥2 ng/ml of progesterone on d 21 postbreeding ( NPH) . Interestrous intervals for the NPL and NPH groups were calculated based on the dates that cows were rebred after entering the study initially; means were compared using a t test. Time of onset of the luteal phase was calculated for individual cows. Mean periestrus concentrations of progesterone and the associated standard deviation were calculated for the first three samples collected from each cow. The onset of the luteal phase was defined as the time that the concentration of progesterone persistently exceeded two standard deviations above the periestrus concentrations (Figure 1). Mean days until the onset of the luteal phase and MUN concentration at breeding were calculated for each pregnancy status
MUN Analysis Analysis of MUN was performed as described by Butler et al. ( 3 ) . Aliquots of the milk samples taken on the day of breeding were centrifuged at 100 × g to remove gross particulate matter and then were analyzed by an automated diacetylmonoxamine method (Technicon industrial method 339-01; Technicon Industries, Tarrytown, NY). A few samples ( n = 7 ) were not analyzed because the volume was insufficient to conduct both progesterone and MUN analyses. Statistical Analysis Analysis of variance for repeated measures was used to assess differences in concentrations of progesterone over time as related to pregnancy staJournal of Dairy Science Vol. 80, No. 7, 1997
Figure 1. Calculated day of onset of the luteal phase for two cows ( ⁄ = cow 5391; ÿ = cow 5276). Dashed lines indicate mean ( ±SD) periestrus concentrations of progesterone and the samples from which they were calculated. * Day of onset of the luteal phase.
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group and assessed by analysis of variance and Fisher’s least significant difference post hoc test for differences between means. Likelihood ratios were calculated to determine the likelihood that open cows would return to estrus 21 d after breeding (NPL) or would have extended cycles (NPH). Ranges of MUN were selected to yield a relatively balanced distribution of cows in five ranges. Within each MUN range, the number of cows in the NPH group was divided by the total number of NPH cows; the same calculations were performed for the NPL group (Table 1). To determine the likelihood that cows would have extended cycles in each MUN category, the proportion of NPH cows was divided by the proportion of NPL cows (Table 1); the reverse calculation was performed to determine the likelihood that cows would be in estrus on d 21 (Table 1). Management factors (e.g., body condition score and lactation number), including MUN, were examined as potential, very simple predictors of pregnancy outcome using logistic regression analysis, including a forward stepwise procedure to select variables for the outcomes of pregnant or nonpregnant. Logistic regression analysis was selected rather than multiple linear regression because of the binomial nature of the dependent variable (pregnant vs. nonpregnant); variable selection was determined based on maximum likelihood estimates. A second logistic procedure as-
TABLE 1. Likelihood that open lactating dairy cows would have an extended cycle (NPH 1) or would return to estrus (NPL) as categorized by milk urea nitrogen (MUN) concentrations on the day of breeding. Proportions2 MUN
Cows
NPH
(mg/dl) ≤19 19.1–21 21.1–23 23.1–25 >25
(no.) 14 16 14 12 14
23.8 28.6 14.3 14.3 19
Likelihood
NPL
NPH3
NPL4
14.3 14.3 28.6 21.4 21.4
1.67 2 0.5 0.67 0.89
0.6 0.5 2 1.5 1.13
(%)
1NPH = Nonpregnant cows with circulating concentrations of progesterone ≥2 ng/ml on d 21 postbreeding; NPL = nonpregnant cows with circulating concentrations of progesterone <2 ng/ml on d 21 postbreeding. 2Number of NPH or NPL cows divided by the total number of NPH or NPL cows, respectively. 3Proportion of NPH cows divided by the proportion of NPL cows. 4Proportion of NPL cows divided by the proportion of NPH cows.
sessed the factors that could predict whether the nonpregnant cows would fall into the NPL or NPH categories. The basic linear logistic models used were Prob(Y j = 0 ) = 1/[1 + exp(–(b0 + b1X1j + b2X2j + . . . + bpXpj) ) ] and Prob ( Y j = 1 ) = 1 – Prob(Y j = 0). Number Cruncher Statistical Systems ( 2 0 ) was used for statistical calculations. RESULTS
Figure 2. Mean concentrations of progesterone in milk after artificial insemination of dairy cows by pregnancy status group: pregnant ( ÿ; n = 82; pooled SEM = 0.1), nonpregnant with progesterone concentrations <2 ng/ml on d 21 postbreeding ( o; n = 32; pooled SEM = 0.16), and nonpregnant with progesterone concentrations >2 ng/ml on d 21 postbreeding ( ⁄; NPH; n = 42; pooled SEM = 0.14). Asterisks indicate mean values for NPH cows were different ( P < 0.05) from those for pregnant cows. Differences ( P < 0.05) in concentrations of progesterone within days between pregnancy status groups are indicated by letters (a, b, c).
Of the 156 breedings that satisfied all criteria for retention in the analyses, 82 (52.6%) cows were determined to be pregnant by the 7th wk. After examination of the progesterone data obtained at d 21 ( ± 1 d ) from cows that failed to become pregnant, open cows could be assigned to either of two subcategories. One group of cows (32 breedings, 20.5%) had low progesterone concentrations on d 21 and was assumed to be close to estrus (NPL); the remaining 42 breedings resulted in elevated concentrations of progesterone on d 21 ( ≥2 ng/ml), but the pregnancy was terminated before the examination for pregnancy status at 40 to 50 d (NPH); some, but not all, of the NPH cows were observed in estrus. Figure 2 shows changes in concentrations of milk progesterone after breeding for the three groups of cows. Because of the substantial data file, the error mean square from the analysis of variance (0.8627; 2295 df) estimated a standard error for the analysis of just 0.06. Nonpregnant cows were partitioned post hoc as explained previously; tests of differences between means were adjusted appropriately. Progesterone concentrations were distributed normally around Journal of Dairy Science Vol. 80, No. 7, 1997
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LARSON ET AL. TABLE 2. Least squares means of interestrus interval, day of onset of the luteal phase, and milk urea nitrogen (MUN) concentrations among pregnancy status groups.1 Interestrus interval
Day of onset of luteal phase
MUN
(d) Pregnant NPL NPH
(mg/dl) SEM
(no.)
X
SEM
(no.)
X
SEM
(no.)
X
. . . 16 14
21.9f 34.6e
. . . 0.05 0.5
82 32 42
3.27d 3.71cd 3.99c
0.16 0.25 0.21
79 29 41
21.5b 23.3a 21.7b
0.7 0.4 0.6
a,bMeans
within a column with different superscript letters differ ( P < 0.1). within a column with different superscript letters differ ( P < 0.05). e,fMeans within a column with different superscript letters differ ( P < 0.0001). 1NPL = Nonpregnant cows with circulating concentrations of progesterone <2 ng/ml on d 21 postbreeding; NPH = nonpregnant cows with circulating concentrations of progesterone ≥2 ng/ml on d 21 postbreeding. c,dMeans
the time that statistically significant differences among the groups became apparent; no data transformation was necessary before analysis. Differences among groups were not significant during the first 3 d after insemination. Starting at 4.5 d after breeding and continuing thereafter, pregnant cows had higher ( P < 0.05) concentrations of milk progesterone than did NPH cows. Milk progesterone concentrations were different ( P < 0.05) among all three pregnancy status groups on d 14 and 21; pregnant cows had the highest concentrations of progesterone, NPH cows had intermediate concentrations, and NPL cows had the lowest concentrations of progesterone. A summary of the mean values for interestrus interval, day of onset of the luteal phase, and MUN is presented in Table 2. The interestrus interval, when it could be determined, was significantly longer for NPH cows than for NPL cows. The time of onset of the luteal function, calculated as described previously, was delayed ( P < 0.05) in NPH cows versus pregnant cows (3.99 vs. 3.27 d; Table 2). Time of onset (3.71 d ) was intermediate for NPL cows and was not significantly different from that of the other groups. The NPL cows had higher ( P = 0.09) MUN concentrations than did both the pregnant and NPH cows. The MUN concentrations of pregnant and NPH cows did not differ. Likelihood ratios for open cows that had extended cycles or that had returned to estrus on d 21 according to the ranges of MUN are shown in Table 1. Open cows were more likely to have had extended cycles if MUN concentrations at breeding were <21 mg/dl, but cows with MUN concentrations >21 mg/dl were more likely to return to estrus on d 21. Variables that were identified by logistic regression analyses as the best predictors of pregnancy status Journal of Dairy Science Vol. 80, No. 7, 1997
are shown in Table 3. The first analysis indicated that, as lactation number and MUN values increased, cows were marginally less likely to become pregnant. For breedings that did not result in pregnancy, the second analysis found that higher MUN concentrations were associated with NPL cows, which is consistent with the ratios presented in Table 1. Some association was found between higher milk production or cows bred in the p.m. and the NPH cows (Table 3). No other management variables were identified by these analyses to be useful predictors of breeding outcome. DISCUSSION Data from this study support the hypothesis that the timing of the onset of the luteal phase can affect pregnancy outcome in lactating dairy cows. Cows that were confirmed to be pregnant had the earliest onset
TABLE 3. Logistic regression analyses of a selection of basic management variables as predictors of pregnancy status in lactating dairy cows. Prediction factors
b Estimate
Pregnancy Lactation number –0.233 –0.071 MUN2 Incidence of extended cycles in open cows Bred in p.m. 1.805 MUN –0.132 Milk production 0.02
SE
pb=0
Last r2 1
0.14 0.05
0.09 0.12
0.02 0.02
0.69 0.07 0.01
0.01 0.06 0.15
0.09 0.05 0.03
1Contribution to the overall model r2 when that variable is added to the linear logistic regression model. 2Milk urea nitrogen.
PROGESTERONE IN EARLY PREGNANCY
of the luteal phase and had consistently higher peripheral concentrations of progesterone than did NPH cows by 4.5 d. These findings are consistent with those from other studies of this period of pregnancy (3, 22), including one study ( 1 7 ) in which normal embryonic development was associated with higher progesterone. Compared with cows that failed to maintain pregnancy, pregnant cows had higher concentrations of progesterone, which might have been because their corpora lutea produced progesterone earlier, thus promoting embryonic development. Alternatively, the presence of healthy embryos might have had luteotrophic effects, causing the earlier increase in progesterone of the pregnant cows. Other studies (2, 16) have failed to expose differences in peripheral progesterone as early after breeding as was shown here, but those studies used fewer cows with less frequent sampling, or they failed to follow individual cows throughout the complete sampling period. Significant differences in mean concentrations of milk progesterone among the pregnancy outcome groups were apparent during the 1st wk after breeding in the present study, which was partially reflecting the power of the statistical model ( r 2 = 73%) to reduce residual variance. Measurements of progesterone in individual cows around d 6 were, however, of little use in predicting pregnancy; the distribution of pregnancy rates within quartiles of the progesterone concentration data for given samplings failed to show consistent trends (data not shown). The population of NPH cows was interesting in both physiological and economic terms. The mean concentration of progesterone on d 21 (4.5 ng/ml) was more likely to have been due to the concurrent or earlier presence of an embryo than to the failure of conception and an estrous cycle that was coincidentally longer than normal. This hypothesis was further supported by the much longer interestrus interval of NPH cows than that of NPL cows. Concentrations of MUN were similar for both the pregnant and NPH groups; both were lower ( P = 0.09) than the MUN concentrations of NPL cows. Cows in the NPH group exhibited a statistically significant delay in the onset of the luteal phase and lower concentrations of progesterone starting 4.5 d after insemination than did the pregnant cows. This result suggested that the suboptimal concentrations of progesterone of the NPH cows may have compromised the early development of the embryo; thus, even though maternal recognition of pregnancy occurred and luteal function was prolonged, the pregnancy could not be maintained. The NPH group represented 26.9% of the cows in the present study; assuming this represents embryonic
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loss between ∼d 16 and d 35, the rate of loss during this period is higher than that suggested by early studies that investigated the timing of embryonic loss in dairy cows [reviews (1, 31)]. In the present study, embryonic loss could be a reflection of the metabolic demands of high milk production, based on the demonstrated association of higher milk production of cows in the NPH group. Onset of the luteal phase, which was derived according to statistical comparison with periestrus concentrations of progesterone, occurred when milk progesterone concentrations were approximately 1 ng/ ml or 3.2 nM. The dissociation constant of the bovine progesterone receptor has been reported to range from 1.2 nM ( 1 8 ) to 1.7 nM (26). Because the concentration of circulating progesterone at the derived onset time exceeded the dissociation constant of the receptors, the onset times derived using this method were probably physiologically meaningful. The mean time for pregnant cows to initiate luteal function (3.27 d after breeding) approximates the time of transport of the embryo from the oviduct to the uterus (25), an event that is linked to an early influence of progesterone on the musculature of the isthmus ( 6 ) . Whether the delay in onset of the luteal phase to 3.99 d results in similar delay in embryo transport and whether this process has deleterious consequences for timely embryonic development remain to be determined. The need for synchrony between the uterus and early embryo, particularly for embryo transfer, is well documented in several species (21, 30), and experiments that have demonstrated the ability of progesterone to advance the stage of development of the uterus (13, 14) confirm the role of progesterone in affecting uterine and embryo synchrony. The effect of asynchronous embryo transfers on embryonic development has been well documented for sheep and cattle. Delayed development occurs when the uterus is less advanced than the embryo (15), and accelerated development occurs when the uterus is more advanced than the embryo (12, 15, 28, 29). We postulate that an embryo, delayed in its development but otherwise normal, might be capable of signaling its presence to the dam, thereby prolonging luteal function, but, if the embryo fails to signal with sufficient vigor, maintenance of the pregnancy might not be ensured. Nonpregnant cows that have extended cycles have a longer interval before rebreeding, which is an economic concern. Consequently, the number of days open in the lactation is increased, which would likely lead to a longer than desired calving interval, thereby decreasing the profitability of the milking operation. Journal of Dairy Science Vol. 80, No. 7, 1997
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This result would be of particular significance for the herd used in this study because 60% of the open cows were in the NPH category. Data from the MUN analysis were included in a review by Butler et al. ( 3 ) , which indicated that MUN at a concentration >19 mg/dl was associated with decreased pregnancy rates, in agreement with results from other studies (10, 11). Making a distinction between NPL and NPH cows based on progesterone concentrations on d 21 in this study yielded information (not previously reported) about MUN on the day of breeding. Based on the logistic regression analyses, higher MUN was associated with decreased fertility, and open cows with higher MUN were more often identified in the NPL category. The observations regarding open cows were consistent with the likelihood ratios presented in Table 1; cows that failed to become pregnant and had MUN concentrations >21 mg/dl were much more likely to be included in the NPL category. The NPL cows also had higher ( P = 0.09) mean MUN concentrations than did the pregnant or NPH cows. Elrod et al. ( 8 ) reported that increased blood urea nitrogen decreased uterine pH several days after breeding, which could make the uterine environment more hostile to the early embryo. Wiebold ( 2 7 ) reported that uterine flushings from cows with abnormal embryos had higher concentrations of glucose, total protein, and several minerals than did flushings from cows with normal embryos. The similar mean MUN concentrations for pregnant and NPH cows supported the suggestion that the NPH cows did have an embryo present at some stage that was able to prolong the luteal function because lower MUN should be an indication that the uterine environment was more conducive to early embryonic development. Therefore, high MUN concentrations at breeding may cause fertilization failure or very early embryonic losses prior to maternal recognition of pregnancy. The only management factors that were identified as predictors of overall fertility were higher MUN concentration and lactation number; both were negatively related to fertility. The r2 values indicated that these factors were not very strong predictors, but most did corroborate observations from other studies. The apparent association between p.m. breeding and cows that had extended cycles was unexpected. These results were not likely to have been caused by the fixed breeding time of cows or by the p.m. breeding of most cows that had been synchronized with PGF2a because neither timed breeding nor PGF2a synchronization was selected as a predictor in either analysis. Additionally, pregnancy rates were nearly identical Journal of Dairy Science Vol. 80, No. 7, 1997
for the cows that were bred at a fixed time and for cows that were bred based on behavior indicating estrus. The association of p.m. breeding with the NPH group may be explained by slightly mistimed breeding. In mistimed breeding, either the oocyte or sperm become aged to the point that fertilization and early embryonic development can occur, but pregnancy is not maintained successfully. The timing of breeding in this study was probably suboptimal, because nearly 80% of the cows were bred in the p.m. Cows that produced more milk were more likely to have been in the NPH group, which could again have a negative economic impact on the dairy operation. CONCLUSIONS This study clearly supports the hypothesis that a delay in the onset of the luteal phase could be associated with decreased fertility of lactating dairy cows. The delay was most prevalent in cows with apparently extended cycles (NPH); these cows were presumed to have initiated pregnancy but subsequently failed to maintain the embryo. A delay in the onset of the luteal phase could be a consequence of high milk production, which was associated with NPH cows, and these cows had the longest time until the onset of the luteal phase. The use of progesterone data to establish the three pregnancy status groups also elicited new information regarding the role of increased circulating urea nitrogen and its association with suboptimal fertility in dairy cows; MUN concentrations >21 mg/dl were associated with a recycling pattern in which cows were detected in estrus at 21 d following breeding. This pattern might have occurred because of a disruption in the oviductal or uterine environment, rendering it inhospitable to the early embryo. The prolongation of luteal function in NPH cows might have been the result of a naturally occurring uterine and embryo asynchrony caused by a delay in the onset of the luteal phase. ACKNOWLEDGMENTS The authors thank Susanne Pelton for assistance with the radioimmunoassays, Jordana Calaman for MUN analysis, and the many employees at the Teaching and Research Center who helped coordinate the study and collected samples. REFERENCES 1 Ayalon, N. 1978. A review of embryonic mortality in cattle. J. Reprod. Fertil. 54:483. 2 Bulman, D. C., and G. E. Lamming. 1978. Milk progesterone levels in relation to conception, repeat breeding and factors influencing acyclicity in dairy cows. J. Reprod. Fertil. 54:447.
PROGESTERONE IN EARLY PREGNANCY 3 Butler, W. R., J. J. Calaman, and S. W. Beam. 1996. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J. Anim. Sci. 74:858. 4 Butler, W. R., D.J.R. Cherney, and C. C. Elrod. 1995. Milk urea nitrogen (MUN) analysis: field trial results on conception rates and dietary inputs. Page 89 in Proc. Cornell Nutr. Conf., Rochester, NY. Cornell Univ., Ithaca, NY. 5 Butler, W. R., and R. D. Smith. 1989. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci. 72:767. 6 Chang, M. C. 1966. Effects of oral administration of medroxyprogesterone acetate and ethinyl estradiol on the transportation and development of rabbit eggs. Endocrinology 79:939. 7 Elrod, C. C., and W. R. Butler. 1993. Reduction of fertility and alteration of uterine pH in heifers fed excess ruminally degradable protein. J. Anim. Sci. 71:694. 8 Elrod, C. C., M. Van Amburgh, and W. R. Butler. 1993. Alterations of pH in response to increased dietary protein in cattle are unique to the uterus. J. Anim. Sci. 71:702. 9 Erb, R. E., H. A. Garverick, R. D. Randel, B. L. Brown, and C. J. Callahan. 1976. Profiles of reproductive hormones associated with fertile and nonfertile inseminations of dairy cows. Theriogenology 5:227. 10 Ferguson, J. D., T. Blanchard, D. T. Galligan, D. C. Hoshall, and W. Chalupa. 1988. Infertility in dairy cattle fed a high percentage of protein degradable in the rumen. JAVMA 192: 659. 11 Ferguson, J. D., D. T. Galligan, T. Blanchard, and M. Reeves. 1993. Serum urea nitrogen and conception rate: the usefulness of test information. J. Dairy Sci. 76:3742. 12 Garrett, J. E., R. D. Geisert, M. T. Zavy, and G. L. Morgan. 1988. Evidence for maternal regulation of early conceptus growth and development in beef cattle. J. Reprod. Fertil. 84: 437. 13 Geisert, R. D., T. C. Fox, G. L. Morgan, M. E. Wells, R. P. Wetteman, and M. T. Zavy. 1991. Survival of bovine embryos transferred to progesterone-treated asynchronous recipients. J. Reprod. Fertil. 92:475. 14 Lawson, R.A.S., and L. P. Cahill. 1983. Modification of the embryo-maternal relationship in ewes by progesterone treatment early in the oestrous cycle. J. Reprod. Fertil. 67:473. 15 Lawson, R.A.S., R. A. Parr, and L. P. Cahill. 1983. Evidence for maternal control of blastocyst growth after asynchronous transfer of embryos to the uterus of the ewe. J. Reprod. Fertil. 67: 477.
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16 Lukaszewska, J., and W. Hansel. 1980. Corpus luteum maintenance during early pregnancy in the cow. J. Reprod. Fertil. 59:485. 17 Maurer, R. R., and S. E. Echternkamp. 1982. Hormonal asynchrony and embryonic development. Theriogenology 17:11. 18 Moffatt, R. J., W. G. Zollers, W. V. Welshons, K. R. Kieborz, H. A. Garverick, and M. F. Smith. 1993. Basis of norgestomet action as a progestogen in cattle. Domest. Anim. Endocrinol. 10: 21. 19 Nara, B. S., and N. L. First. 1981. Effect of indomethacin and prostaglandin F2a on parturition in swine. J. Anim. Sci. 52: 1360. 20 Number Cruncher Statistical Systems. 1990. Users Guide, Version 5.03. NCSS, Kaysville, UT. 21 Pope, W. F. 1988. Uterine asynchrony: a cause of embryonic loss. Biol. Reprod. 39:999. 22 Shelton, K., M. F. Gayerie De Abreu, M. G. Hunter, T. J. Parkinson, and G. E. Lamming. 1990. Luteal inadequacy during the early luteal phase of subfertile cows. J. Reprod. Fertil. 90:1. 23 Sreenan, J. M., and M. G. Diskin. 1983. Early embryonic mortality in the cow: its relationship with progesterone concentration. Vet. Rec. 112:517. 24 Staigmiller, R. B., R. E. Short, R. A. Bellows, and J. B. Carr. 1979. Effect of nutrition on response to exogenous FSH in beef cattle. J. Anim. Sci. 48:1182. 25 Thibault, C. 1972. Physiology and physiopathology of the fallopian tube. Int. J. Fertil. 17:1. 26 Vesanan, M., V. Isomaa, M. Alanko, and R. Vihko. 1988. Cytosol estrogen and progesterone receptors in bovine endometrium after uterine involution postpartum and in the estrous cycle. Anim. Reprod. Sci. 17:9. 27 Wiebold, J. L. 1988. Embryonic mortality and the uterine environment in first-service lactating dairy cows. J. Reprod. Fertil. 84:393. 28 Wilmut, I., C. J. Ashworth, A. J. Springbett, and D. I. Sales. 1988. Effect of variation in embryo stage on the establishment of pregnancy, and embryo survival and growth in ewes with two embryos. J. Reprod. Fertil. 83:233. 29 Wilmut, I., and D. I. Sales. 1981. Effect of asynchronous environment on embryonic development in sheep. J. Reprod. Fertil. 61:179. 30 Wilmut, I., D. I. Sales, and C. J. Ashworth. 1985. The influence of variation in embryo stage and maternal hormone profiles on embryo survival in farm animals. Theriogenology 23:107. 31 Wilmut, I., D. I. Sales, and C. J. Ashworth. 1986. Maternal and embryonic factors associated with prenatal loss in mammals. J. Reprod. Fertil. 76:851.
Journal of Dairy Science Vol. 80, No. 7, 1997
ABSTRACT The primary objectives of this study were to determine whether a delay in the onset of the luteal phase, or high milk urea nitrogen at breeding, or both were associated with failure of pregnancy early in gestation. Milk samples were collected twice daily from cows in a single herd during the week following breeding; single samples were collected on d 14 and 21 postbreeding. Progesterone was measured in all samples, and a total of 156 sample sets was used. The progesterone data combined with results from pregnancy examinations were used to distribute the cows into three groups: 1 ) pregnant, 2 ) nonpregnant with a low concentration (<2 ng/ml) of progesterone on d 21, and 3 ) nonpregnant with a high concentration ( ≥2 ng/ml) of progesterone on d 21. The interestrous interval for cows in group 3 was longer than that for cows in group 2. Beginning 4.5 d after breeding, pregnant cows had higher concentrations of progesterone than did cows in group 3. Pregnant cows also had higher concentrations of progesterone than did all open cows on d 14 and 21. The onset of the luteal phase was earlier in pregnant cows than it was in cows in group 3. Milk urea nitrogen at breeding was similar in pregnant cows and in cows in group 3, but was higher in cows in group 2. Increased milk urea nitrogen was also statistically associated with decreased fertility. We propose that the cows in group 3 likely had embryos that initiated pregnancy recognition and prolonged luteal function, but these embryos were compromised by suboptimal exposure to progesterone early in development. ( Key words: progesterone, pregnancy, milk urea nitrogen) Abbreviation key: MUN = milk urea nitrogen, NPH = nonpregnant and high circulating concentrations of progesterone ( ≥2 ng/ml) on d 21 postbreeding, NPL =
Received May 13, 1996. Accepted November 18, 1996. 1Corresponding author. 1997 J Dairy Sci 80:1288–1295
nonpregnant and low circulating concentrations of progesterone (<2 ng/ml) on d 21 postbreeding. INTRODUCTION As the size of dairy herds increases, more intensive management systems are needed to maximize milk production, and overall herd fertility often decreases. In New York dairies, conception rates after first service have declined from 66% in 1951 to 40% in 1995, and milk production has doubled during the same period (4, 5). Most pregnancy losses occur during the first 30 d postbreeding and are due to a variety of causes [reviews (1, 31)]. The role of progesterone in pregnancy maintenance is undisputed; however, the possible role of progesterone around the time of maternal recognition of pregnancy is not as apparent (23). Some studies have indicated that peripheral concentrations of progesterone are higher in pregnant cows than in nonpregnant cows as early as d 4 ( 3 ) or d 6 (9, 22) postbreeding or that low concentrations of progesterone during the 1st wk after breeding are associated with abnormal embryonic development (17). Other researchers have reported no differences in peripheral concentrations of progesterone that are related to pregnancy status until d 10 ( 1 6 ) or d 13 ( 2 ) . Many of these studies, however, either used limited numbers of cattle or failed to follow individual animals throughout the onset of the luteal phase. We hypothesized that variation among cattle in the establishment of luteal function might be appreciable and that individuals with delayed onset of luteal function would be less likely to become pregnant or to maintain pregnancy. Another factor that has been recently implicated in the subfertility of dairy cows is excessive circulating concentrations of urea; values for urea nitrogen measured in plasma and milk are comparable ( 3 ) . High circulating concentrations of urea nitrogen are usually caused by dietary protein, especially highly degradable protein, in excess of the nutritional requirements of the animal. Concentrations of blood urea nitrogen >19 or >20 mg/dl are associated with decreased pregnancy rates of lactating cows and heif-
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ers (3, 10, 11). Results of other studies (7, 8 ) have indicated that increased plasma urea nitrogen is associated with lower conception rates and decreased uterine pH for several days after estrus. The objectives of this study were 1 ) to determine whether peripheral concentrations of progesterone differed between pregnant and nonpregnant cows during the 1st wk after breeding, 2 ) to investigate further the contribution of increased milk urea nitrogen ( MUN) to subfertility, and 3 ) to determine factors other than circulating progesterone concentrations that might influence pregnancy outcome or bias the statistical calculations. MATERIALS AND METHODS Cows All cows were grade Holsteins in a dairy herd that had a rolling herd average milk production of 11,000 kg of milk/yr per cow. The cows were housed at the Teaching and Research Center, Department of Animal Science, Cornell University (Ithaca, NY). No aspect of herd management was changed for the purpose of the study; most cows were housed in free-stall pens, and all were fed a TMR that was based on corn silage. All lactating cows that were presented for breeding during the first 3 d of each week (to take advantage of the estrous synchronization schedule) from January to July were used in the study; the only exclusions were cows that were in use in other experiments that might have compromised fertility. Information was obtained for a total of 228 breedings of 162 cows; 34 cows were sampled twice, and 14 cows were sampled three times. When the cows were assigned to the study, the following information was recorded: body condition score at breeding, lactation number, number of times bred during the current lactation, DIM, mean daily milk production, number of times milked per day, time of day bred (a.m. or p.m.), whether estrus had been synchronized by use of PGF2a, and whether the cow was being bred at a fixed time (80 h after PGF2a administration) without observation of estrus. This information would be potentially useful to account for part of the random variation in the planned analyses of variance. Study Design For 7 d, starting on the day of insemination, milk samples were collected twice daily from all cows that were assigned to the study. One sample was collected on d 14 and 21 ( ±1 d ) postbreeding to verify luteal function and to evaluate accurately cows that had
returned to estrus. At approximately 7 wk postbreeding, if the cow had not returned to estrus, she was palpated rectally by the herd veterinarian to determine pregnancy status. After all analyses were completed, the data file was sorted to remove sample sets that exhibited any of the following characteristics: 1 ) progesterone concentrations >1 ng/ml on day of breeding (regarded as misbred, n = 33), 2 ) no increase in progesterone noted throughout the sampling period (cystic or anestrous, n = 11), 3 ) missing >6 samples ( n = 6), 4 ) grossly erratic patterns of progesterone concentrations (likely caused by a sampling error, n = 15), 5 ) culled before pregnancy evaluation ( n = 1), or 6 ) rebred <21 d after entering the study (based on the assumption that the second breeding might have interfered with a conceptus that was present from the initial breeding, n = 6). A total of 156 sample sets were retained and used in the final analysis. Sample Collection and Processing Milkers collected 5- to 10-ml premilking strip samples from one quarter of each cow. Samples were refrigerated after collection, and milk fat was allowed to separate. Within 2 d of collection, cream was aspirated from the sample. Samples were centrifuged for 10 min at 100 × g, reaspirated to eliminate milk fat, and stored at –20°C. Samples were sorted into complete sets from each breeding so that each sample set was analyzed for progesterone concentration in the same radioimmunoassay. All samples of skim milk were analyzed for progesterone content, and samples from the day of breeding were also analyzed for MUN. Radioimmunoassay A double-antibody radioimmunoassay adapted from the methodology described by Nara and First ( 1 9 ) was used to measure concentrations of progesterone in skim milk. A 100-ml aliquot of sample or standard was diluted with 400 ml of PBS containing 0.1% (wt/vol) gelatin. Progesterone labeled with 125I (ICN Biomedicals Inc., Costa Mesa, CA) was also diluted in PBS containing 0.1% (wt/vol) gelatin, and 20,000 cpm were added. Standards were assembled in 0.5% BSA in PBS at the following concentrations: 0.5, 1, 2, 4, 8, and 16 ng/ml. Progesterone antiserum [rabbit number 8; (24)] was used at a final dilution of 1:80,000 (vol/vol) in 1:400 (vol/vol) normal rabbit serum in PBS containing 50 mM EDTA. Tubes were incubated at 37°C for 1 h and then at 4°C overnight. Ovine anti-rabbit gamma globulin (produced in-house) was added to all tubes to a final Journal of Dairy Science Vol. 80, No. 7, 1997
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dilution of 1:125 (vol/vol). The tubes were then incubated at 4°C for an additional 48 h. Antibody-bound and unbound radioactivity was separated by the addition of 4 ml of PBS at 4°C, centrifugation for 30 min at 1000 × g, decanting, and draining. Antibody-bound radioactivity was quantified in a gamma spectrometer. Under the conditions described, 25 to 35% of 125I-labeled progesterone was bound by antibody in the absence of standard or unknown ligand ( B 0) . Standard concentrations of progesterone (0.5 to 16 ng/ml) generated linear logit-log standard curves ( r 2 = 97%) with 75% (0.5 ng/ml) to 22% (16 ng/ml) binding, using B0 as the reference. Either 0.5% BSA in PBS or solvent-extracted skim milk could be used as the diluent. Nonspecific binding of radioactivity (no antiprogesterone) in the presence of skim milk rarely exceeded twice the background of the gamma spectrometer. Data were compared from matched plasma and milk samples taken from cows in estrus and from cows in the early and midluteal phases to verify that progesterone in skim milk accurately reflected the circulating concentrations of progesterone. Plasma samples were extracted and analyzed ( 1 9 ) and were compared with measurements in skim milk samples as described previously: plasma progesterone = 0.2 + 0.96 milk progesterone; r2 = 76%. Pools of milk with low and high progesterone contents were analyzed in all assays in the present study; coefficients of variation within and between assays were 17 and 13%, respectively. Recovery was measured by the addition of 100 or 200 pg of progesterone to aliquots of the pooled milk with low progesterone content, which resulted in 103% recovery of the added progesterone.
tus. The model for the analysis of variance for repeated measures was Yijkm = m + ai + bj + tk( i) + ( ab) ij + em( ijk) , where Yijkm = progesterone concentration; m = overall mean, ai = fixed effects of pregnancy status i, bj = fixed effects of sampling day j, tk( i) = random effects of cow k nested within pregnancy status i, ( ab) ij = effects of interaction of fixed pregnancy status i and sampling day j, and em( ijk) = random error terms. As expected, the interaction of sampling day and pregnancy status was highly significant, and Fisher’s protected least significant difference post hoc test was used to assess differences between means for pregnancy status groups on individual days. On the basis of all available information, cows were categorized into three groups: 1 ) pregnant (confirmed by palpation), 2 ) nonpregnant and with <2 ng/ml of milk progesterone at d 21 postbreeding ( NPL) (some, but not all, of these cows were observed in estrus around d 21), and 3 ) nonpregnant and with ≥2 ng/ml of progesterone on d 21 postbreeding ( NPH) . Interestrous intervals for the NPL and NPH groups were calculated based on the dates that cows were rebred after entering the study initially; means were compared using a t test. Time of onset of the luteal phase was calculated for individual cows. Mean periestrus concentrations of progesterone and the associated standard deviation were calculated for the first three samples collected from each cow. The onset of the luteal phase was defined as the time that the concentration of progesterone persistently exceeded two standard deviations above the periestrus concentrations (Figure 1). Mean days until the onset of the luteal phase and MUN concentration at breeding were calculated for each pregnancy status
MUN Analysis Analysis of MUN was performed as described by Butler et al. ( 3 ) . Aliquots of the milk samples taken on the day of breeding were centrifuged at 100 × g to remove gross particulate matter and then were analyzed by an automated diacetylmonoxamine method (Technicon industrial method 339-01; Technicon Industries, Tarrytown, NY). A few samples ( n = 7 ) were not analyzed because the volume was insufficient to conduct both progesterone and MUN analyses. Statistical Analysis Analysis of variance for repeated measures was used to assess differences in concentrations of progesterone over time as related to pregnancy staJournal of Dairy Science Vol. 80, No. 7, 1997
Figure 1. Calculated day of onset of the luteal phase for two cows ( ⁄ = cow 5391; ÿ = cow 5276). Dashed lines indicate mean ( ±SD) periestrus concentrations of progesterone and the samples from which they were calculated. * Day of onset of the luteal phase.
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group and assessed by analysis of variance and Fisher’s least significant difference post hoc test for differences between means. Likelihood ratios were calculated to determine the likelihood that open cows would return to estrus 21 d after breeding (NPL) or would have extended cycles (NPH). Ranges of MUN were selected to yield a relatively balanced distribution of cows in five ranges. Within each MUN range, the number of cows in the NPH group was divided by the total number of NPH cows; the same calculations were performed for the NPL group (Table 1). To determine the likelihood that cows would have extended cycles in each MUN category, the proportion of NPH cows was divided by the proportion of NPL cows (Table 1); the reverse calculation was performed to determine the likelihood that cows would be in estrus on d 21 (Table 1). Management factors (e.g., body condition score and lactation number), including MUN, were examined as potential, very simple predictors of pregnancy outcome using logistic regression analysis, including a forward stepwise procedure to select variables for the outcomes of pregnant or nonpregnant. Logistic regression analysis was selected rather than multiple linear regression because of the binomial nature of the dependent variable (pregnant vs. nonpregnant); variable selection was determined based on maximum likelihood estimates. A second logistic procedure as-
TABLE 1. Likelihood that open lactating dairy cows would have an extended cycle (NPH 1) or would return to estrus (NPL) as categorized by milk urea nitrogen (MUN) concentrations on the day of breeding. Proportions2 MUN
Cows
NPH
(mg/dl) ≤19 19.1–21 21.1–23 23.1–25 >25
(no.) 14 16 14 12 14
23.8 28.6 14.3 14.3 19
Likelihood
NPL
NPH3
NPL4
14.3 14.3 28.6 21.4 21.4
1.67 2 0.5 0.67 0.89
0.6 0.5 2 1.5 1.13
(%)
1NPH = Nonpregnant cows with circulating concentrations of progesterone ≥2 ng/ml on d 21 postbreeding; NPL = nonpregnant cows with circulating concentrations of progesterone <2 ng/ml on d 21 postbreeding. 2Number of NPH or NPL cows divided by the total number of NPH or NPL cows, respectively. 3Proportion of NPH cows divided by the proportion of NPL cows. 4Proportion of NPL cows divided by the proportion of NPH cows.
sessed the factors that could predict whether the nonpregnant cows would fall into the NPL or NPH categories. The basic linear logistic models used were Prob(Y j = 0 ) = 1/[1 + exp(–(b0 + b1X1j + b2X2j + . . . + bpXpj) ) ] and Prob ( Y j = 1 ) = 1 – Prob(Y j = 0). Number Cruncher Statistical Systems ( 2 0 ) was used for statistical calculations. RESULTS
Figure 2. Mean concentrations of progesterone in milk after artificial insemination of dairy cows by pregnancy status group: pregnant ( ÿ; n = 82; pooled SEM = 0.1), nonpregnant with progesterone concentrations <2 ng/ml on d 21 postbreeding ( o; n = 32; pooled SEM = 0.16), and nonpregnant with progesterone concentrations >2 ng/ml on d 21 postbreeding ( ⁄; NPH; n = 42; pooled SEM = 0.14). Asterisks indicate mean values for NPH cows were different ( P < 0.05) from those for pregnant cows. Differences ( P < 0.05) in concentrations of progesterone within days between pregnancy status groups are indicated by letters (a, b, c).
Of the 156 breedings that satisfied all criteria for retention in the analyses, 82 (52.6%) cows were determined to be pregnant by the 7th wk. After examination of the progesterone data obtained at d 21 ( ± 1 d ) from cows that failed to become pregnant, open cows could be assigned to either of two subcategories. One group of cows (32 breedings, 20.5%) had low progesterone concentrations on d 21 and was assumed to be close to estrus (NPL); the remaining 42 breedings resulted in elevated concentrations of progesterone on d 21 ( ≥2 ng/ml), but the pregnancy was terminated before the examination for pregnancy status at 40 to 50 d (NPH); some, but not all, of the NPH cows were observed in estrus. Figure 2 shows changes in concentrations of milk progesterone after breeding for the three groups of cows. Because of the substantial data file, the error mean square from the analysis of variance (0.8627; 2295 df) estimated a standard error for the analysis of just 0.06. Nonpregnant cows were partitioned post hoc as explained previously; tests of differences between means were adjusted appropriately. Progesterone concentrations were distributed normally around Journal of Dairy Science Vol. 80, No. 7, 1997
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LARSON ET AL. TABLE 2. Least squares means of interestrus interval, day of onset of the luteal phase, and milk urea nitrogen (MUN) concentrations among pregnancy status groups.1 Interestrus interval
Day of onset of luteal phase
MUN
(d) Pregnant NPL NPH
(mg/dl) SEM
(no.)
X
SEM
(no.)
X
SEM
(no.)
X
. . . 16 14
21.9f 34.6e
. . . 0.05 0.5
82 32 42
3.27d 3.71cd 3.99c
0.16 0.25 0.21
79 29 41
21.5b 23.3a 21.7b
0.7 0.4 0.6
a,bMeans
within a column with different superscript letters differ ( P < 0.1). within a column with different superscript letters differ ( P < 0.05). e,fMeans within a column with different superscript letters differ ( P < 0.0001). 1NPL = Nonpregnant cows with circulating concentrations of progesterone <2 ng/ml on d 21 postbreeding; NPH = nonpregnant cows with circulating concentrations of progesterone ≥2 ng/ml on d 21 postbreeding. c,dMeans
the time that statistically significant differences among the groups became apparent; no data transformation was necessary before analysis. Differences among groups were not significant during the first 3 d after insemination. Starting at 4.5 d after breeding and continuing thereafter, pregnant cows had higher ( P < 0.05) concentrations of milk progesterone than did NPH cows. Milk progesterone concentrations were different ( P < 0.05) among all three pregnancy status groups on d 14 and 21; pregnant cows had the highest concentrations of progesterone, NPH cows had intermediate concentrations, and NPL cows had the lowest concentrations of progesterone. A summary of the mean values for interestrus interval, day of onset of the luteal phase, and MUN is presented in Table 2. The interestrus interval, when it could be determined, was significantly longer for NPH cows than for NPL cows. The time of onset of the luteal function, calculated as described previously, was delayed ( P < 0.05) in NPH cows versus pregnant cows (3.99 vs. 3.27 d; Table 2). Time of onset (3.71 d ) was intermediate for NPL cows and was not significantly different from that of the other groups. The NPL cows had higher ( P = 0.09) MUN concentrations than did both the pregnant and NPH cows. The MUN concentrations of pregnant and NPH cows did not differ. Likelihood ratios for open cows that had extended cycles or that had returned to estrus on d 21 according to the ranges of MUN are shown in Table 1. Open cows were more likely to have had extended cycles if MUN concentrations at breeding were <21 mg/dl, but cows with MUN concentrations >21 mg/dl were more likely to return to estrus on d 21. Variables that were identified by logistic regression analyses as the best predictors of pregnancy status Journal of Dairy Science Vol. 80, No. 7, 1997
are shown in Table 3. The first analysis indicated that, as lactation number and MUN values increased, cows were marginally less likely to become pregnant. For breedings that did not result in pregnancy, the second analysis found that higher MUN concentrations were associated with NPL cows, which is consistent with the ratios presented in Table 1. Some association was found between higher milk production or cows bred in the p.m. and the NPH cows (Table 3). No other management variables were identified by these analyses to be useful predictors of breeding outcome. DISCUSSION Data from this study support the hypothesis that the timing of the onset of the luteal phase can affect pregnancy outcome in lactating dairy cows. Cows that were confirmed to be pregnant had the earliest onset
TABLE 3. Logistic regression analyses of a selection of basic management variables as predictors of pregnancy status in lactating dairy cows. Prediction factors
b Estimate
Pregnancy Lactation number –0.233 –0.071 MUN2 Incidence of extended cycles in open cows Bred in p.m. 1.805 MUN –0.132 Milk production 0.02
SE
pb=0
Last r2 1
0.14 0.05
0.09 0.12
0.02 0.02
0.69 0.07 0.01
0.01 0.06 0.15
0.09 0.05 0.03
1Contribution to the overall model r2 when that variable is added to the linear logistic regression model. 2Milk urea nitrogen.
PROGESTERONE IN EARLY PREGNANCY
of the luteal phase and had consistently higher peripheral concentrations of progesterone than did NPH cows by 4.5 d. These findings are consistent with those from other studies of this period of pregnancy (3, 22), including one study ( 1 7 ) in which normal embryonic development was associated with higher progesterone. Compared with cows that failed to maintain pregnancy, pregnant cows had higher concentrations of progesterone, which might have been because their corpora lutea produced progesterone earlier, thus promoting embryonic development. Alternatively, the presence of healthy embryos might have had luteotrophic effects, causing the earlier increase in progesterone of the pregnant cows. Other studies (2, 16) have failed to expose differences in peripheral progesterone as early after breeding as was shown here, but those studies used fewer cows with less frequent sampling, or they failed to follow individual cows throughout the complete sampling period. Significant differences in mean concentrations of milk progesterone among the pregnancy outcome groups were apparent during the 1st wk after breeding in the present study, which was partially reflecting the power of the statistical model ( r 2 = 73%) to reduce residual variance. Measurements of progesterone in individual cows around d 6 were, however, of little use in predicting pregnancy; the distribution of pregnancy rates within quartiles of the progesterone concentration data for given samplings failed to show consistent trends (data not shown). The population of NPH cows was interesting in both physiological and economic terms. The mean concentration of progesterone on d 21 (4.5 ng/ml) was more likely to have been due to the concurrent or earlier presence of an embryo than to the failure of conception and an estrous cycle that was coincidentally longer than normal. This hypothesis was further supported by the much longer interestrus interval of NPH cows than that of NPL cows. Concentrations of MUN were similar for both the pregnant and NPH groups; both were lower ( P = 0.09) than the MUN concentrations of NPL cows. Cows in the NPH group exhibited a statistically significant delay in the onset of the luteal phase and lower concentrations of progesterone starting 4.5 d after insemination than did the pregnant cows. This result suggested that the suboptimal concentrations of progesterone of the NPH cows may have compromised the early development of the embryo; thus, even though maternal recognition of pregnancy occurred and luteal function was prolonged, the pregnancy could not be maintained. The NPH group represented 26.9% of the cows in the present study; assuming this represents embryonic
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loss between ∼d 16 and d 35, the rate of loss during this period is higher than that suggested by early studies that investigated the timing of embryonic loss in dairy cows [reviews (1, 31)]. In the present study, embryonic loss could be a reflection of the metabolic demands of high milk production, based on the demonstrated association of higher milk production of cows in the NPH group. Onset of the luteal phase, which was derived according to statistical comparison with periestrus concentrations of progesterone, occurred when milk progesterone concentrations were approximately 1 ng/ ml or 3.2 nM. The dissociation constant of the bovine progesterone receptor has been reported to range from 1.2 nM ( 1 8 ) to 1.7 nM (26). Because the concentration of circulating progesterone at the derived onset time exceeded the dissociation constant of the receptors, the onset times derived using this method were probably physiologically meaningful. The mean time for pregnant cows to initiate luteal function (3.27 d after breeding) approximates the time of transport of the embryo from the oviduct to the uterus (25), an event that is linked to an early influence of progesterone on the musculature of the isthmus ( 6 ) . Whether the delay in onset of the luteal phase to 3.99 d results in similar delay in embryo transport and whether this process has deleterious consequences for timely embryonic development remain to be determined. The need for synchrony between the uterus and early embryo, particularly for embryo transfer, is well documented in several species (21, 30), and experiments that have demonstrated the ability of progesterone to advance the stage of development of the uterus (13, 14) confirm the role of progesterone in affecting uterine and embryo synchrony. The effect of asynchronous embryo transfers on embryonic development has been well documented for sheep and cattle. Delayed development occurs when the uterus is less advanced than the embryo (15), and accelerated development occurs when the uterus is more advanced than the embryo (12, 15, 28, 29). We postulate that an embryo, delayed in its development but otherwise normal, might be capable of signaling its presence to the dam, thereby prolonging luteal function, but, if the embryo fails to signal with sufficient vigor, maintenance of the pregnancy might not be ensured. Nonpregnant cows that have extended cycles have a longer interval before rebreeding, which is an economic concern. Consequently, the number of days open in the lactation is increased, which would likely lead to a longer than desired calving interval, thereby decreasing the profitability of the milking operation. Journal of Dairy Science Vol. 80, No. 7, 1997
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This result would be of particular significance for the herd used in this study because 60% of the open cows were in the NPH category. Data from the MUN analysis were included in a review by Butler et al. ( 3 ) , which indicated that MUN at a concentration >19 mg/dl was associated with decreased pregnancy rates, in agreement with results from other studies (10, 11). Making a distinction between NPL and NPH cows based on progesterone concentrations on d 21 in this study yielded information (not previously reported) about MUN on the day of breeding. Based on the logistic regression analyses, higher MUN was associated with decreased fertility, and open cows with higher MUN were more often identified in the NPL category. The observations regarding open cows were consistent with the likelihood ratios presented in Table 1; cows that failed to become pregnant and had MUN concentrations >21 mg/dl were much more likely to be included in the NPL category. The NPL cows also had higher ( P = 0.09) mean MUN concentrations than did the pregnant or NPH cows. Elrod et al. ( 8 ) reported that increased blood urea nitrogen decreased uterine pH several days after breeding, which could make the uterine environment more hostile to the early embryo. Wiebold ( 2 7 ) reported that uterine flushings from cows with abnormal embryos had higher concentrations of glucose, total protein, and several minerals than did flushings from cows with normal embryos. The similar mean MUN concentrations for pregnant and NPH cows supported the suggestion that the NPH cows did have an embryo present at some stage that was able to prolong the luteal function because lower MUN should be an indication that the uterine environment was more conducive to early embryonic development. Therefore, high MUN concentrations at breeding may cause fertilization failure or very early embryonic losses prior to maternal recognition of pregnancy. The only management factors that were identified as predictors of overall fertility were higher MUN concentration and lactation number; both were negatively related to fertility. The r2 values indicated that these factors were not very strong predictors, but most did corroborate observations from other studies. The apparent association between p.m. breeding and cows that had extended cycles was unexpected. These results were not likely to have been caused by the fixed breeding time of cows or by the p.m. breeding of most cows that had been synchronized with PGF2a because neither timed breeding nor PGF2a synchronization was selected as a predictor in either analysis. Additionally, pregnancy rates were nearly identical Journal of Dairy Science Vol. 80, No. 7, 1997
for the cows that were bred at a fixed time and for cows that were bred based on behavior indicating estrus. The association of p.m. breeding with the NPH group may be explained by slightly mistimed breeding. In mistimed breeding, either the oocyte or sperm become aged to the point that fertilization and early embryonic development can occur, but pregnancy is not maintained successfully. The timing of breeding in this study was probably suboptimal, because nearly 80% of the cows were bred in the p.m. Cows that produced more milk were more likely to have been in the NPH group, which could again have a negative economic impact on the dairy operation. CONCLUSIONS This study clearly supports the hypothesis that a delay in the onset of the luteal phase could be associated with decreased fertility of lactating dairy cows. The delay was most prevalent in cows with apparently extended cycles (NPH); these cows were presumed to have initiated pregnancy but subsequently failed to maintain the embryo. A delay in the onset of the luteal phase could be a consequence of high milk production, which was associated with NPH cows, and these cows had the longest time until the onset of the luteal phase. The use of progesterone data to establish the three pregnancy status groups also elicited new information regarding the role of increased circulating urea nitrogen and its association with suboptimal fertility in dairy cows; MUN concentrations >21 mg/dl were associated with a recycling pattern in which cows were detected in estrus at 21 d following breeding. This pattern might have occurred because of a disruption in the oviductal or uterine environment, rendering it inhospitable to the early embryo. The prolongation of luteal function in NPH cows might have been the result of a naturally occurring uterine and embryo asynchrony caused by a delay in the onset of the luteal phase. ACKNOWLEDGMENTS The authors thank Susanne Pelton for assistance with the radioimmunoassays, Jordana Calaman for MUN analysis, and the many employees at the Teaching and Research Center who helped coordinate the study and collected samples. REFERENCES 1 Ayalon, N. 1978. A review of embryonic mortality in cattle. J. Reprod. Fertil. 54:483. 2 Bulman, D. C., and G. E. Lamming. 1978. Milk progesterone levels in relation to conception, repeat breeding and factors influencing acyclicity in dairy cows. J. Reprod. Fertil. 54:447.
PROGESTERONE IN EARLY PREGNANCY 3 Butler, W. R., J. J. Calaman, and S. W. Beam. 1996. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J. Anim. Sci. 74:858. 4 Butler, W. R., D.J.R. Cherney, and C. C. Elrod. 1995. Milk urea nitrogen (MUN) analysis: field trial results on conception rates and dietary inputs. Page 89 in Proc. Cornell Nutr. Conf., Rochester, NY. Cornell Univ., Ithaca, NY. 5 Butler, W. R., and R. D. Smith. 1989. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci. 72:767. 6 Chang, M. C. 1966. Effects of oral administration of medroxyprogesterone acetate and ethinyl estradiol on the transportation and development of rabbit eggs. Endocrinology 79:939. 7 Elrod, C. C., and W. R. Butler. 1993. Reduction of fertility and alteration of uterine pH in heifers fed excess ruminally degradable protein. J. Anim. Sci. 71:694. 8 Elrod, C. C., M. Van Amburgh, and W. R. Butler. 1993. Alterations of pH in response to increased dietary protein in cattle are unique to the uterus. J. Anim. Sci. 71:702. 9 Erb, R. E., H. A. Garverick, R. D. Randel, B. L. Brown, and C. J. Callahan. 1976. Profiles of reproductive hormones associated with fertile and nonfertile inseminations of dairy cows. Theriogenology 5:227. 10 Ferguson, J. D., T. Blanchard, D. T. Galligan, D. C. Hoshall, and W. Chalupa. 1988. Infertility in dairy cattle fed a high percentage of protein degradable in the rumen. JAVMA 192: 659. 11 Ferguson, J. D., D. T. Galligan, T. Blanchard, and M. Reeves. 1993. Serum urea nitrogen and conception rate: the usefulness of test information. J. Dairy Sci. 76:3742. 12 Garrett, J. E., R. D. Geisert, M. T. Zavy, and G. L. Morgan. 1988. Evidence for maternal regulation of early conceptus growth and development in beef cattle. J. Reprod. Fertil. 84: 437. 13 Geisert, R. D., T. C. Fox, G. L. Morgan, M. E. Wells, R. P. Wetteman, and M. T. Zavy. 1991. Survival of bovine embryos transferred to progesterone-treated asynchronous recipients. J. Reprod. Fertil. 92:475. 14 Lawson, R.A.S., and L. P. Cahill. 1983. Modification of the embryo-maternal relationship in ewes by progesterone treatment early in the oestrous cycle. J. Reprod. Fertil. 67:473. 15 Lawson, R.A.S., R. A. Parr, and L. P. Cahill. 1983. Evidence for maternal control of blastocyst growth after asynchronous transfer of embryos to the uterus of the ewe. J. Reprod. Fertil. 67: 477.
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16 Lukaszewska, J., and W. Hansel. 1980. Corpus luteum maintenance during early pregnancy in the cow. J. Reprod. Fertil. 59:485. 17 Maurer, R. R., and S. E. Echternkamp. 1982. Hormonal asynchrony and embryonic development. Theriogenology 17:11. 18 Moffatt, R. J., W. G. Zollers, W. V. Welshons, K. R. Kieborz, H. A. Garverick, and M. F. Smith. 1993. Basis of norgestomet action as a progestogen in cattle. Domest. Anim. Endocrinol. 10: 21. 19 Nara, B. S., and N. L. First. 1981. Effect of indomethacin and prostaglandin F2a on parturition in swine. J. Anim. Sci. 52: 1360. 20 Number Cruncher Statistical Systems. 1990. Users Guide, Version 5.03. NCSS, Kaysville, UT. 21 Pope, W. F. 1988. Uterine asynchrony: a cause of embryonic loss. Biol. Reprod. 39:999. 22 Shelton, K., M. F. Gayerie De Abreu, M. G. Hunter, T. J. Parkinson, and G. E. Lamming. 1990. Luteal inadequacy during the early luteal phase of subfertile cows. J. Reprod. Fertil. 90:1. 23 Sreenan, J. M., and M. G. Diskin. 1983. Early embryonic mortality in the cow: its relationship with progesterone concentration. Vet. Rec. 112:517. 24 Staigmiller, R. B., R. E. Short, R. A. Bellows, and J. B. Carr. 1979. Effect of nutrition on response to exogenous FSH in beef cattle. J. Anim. Sci. 48:1182. 25 Thibault, C. 1972. Physiology and physiopathology of the fallopian tube. Int. J. Fertil. 17:1. 26 Vesanan, M., V. Isomaa, M. Alanko, and R. Vihko. 1988. Cytosol estrogen and progesterone receptors in bovine endometrium after uterine involution postpartum and in the estrous cycle. Anim. Reprod. Sci. 17:9. 27 Wiebold, J. L. 1988. Embryonic mortality and the uterine environment in first-service lactating dairy cows. J. Reprod. Fertil. 84:393. 28 Wilmut, I., C. J. Ashworth, A. J. Springbett, and D. I. Sales. 1988. Effect of variation in embryo stage on the establishment of pregnancy, and embryo survival and growth in ewes with two embryos. J. Reprod. Fertil. 83:233. 29 Wilmut, I., and D. I. Sales. 1981. Effect of asynchronous environment on embryonic development in sheep. J. Reprod. Fertil. 61:179. 30 Wilmut, I., D. I. Sales, and C. J. Ashworth. 1985. The influence of variation in embryo stage and maternal hormone profiles on embryo survival in farm animals. Theriogenology 23:107. 31 Wilmut, I., D. I. Sales, and C. J. Ashworth. 1986. Maternal and embryonic factors associated with prenatal loss in mammals. J. Reprod. Fertil. 76:851.
Journal of Dairy Science Vol. 80, No. 7, 1997