Kathrin Herzog

Luteal blood flow is a more appropriate indicator for luteal function during the bovine estrous cycle than luteal size

The objective of this study was to assess the reliability of luteal blood flow (LBF) as recorded by color Doppler sonography to monitor luteal function during the estrous cycle of dairy cows and to compare the results with that for the established criterion luteal size (LS) as determined by B-mode sonography. In total, 14 consecutive sonographic examinations were carried out in 10 synchronized lactating Holstein-Friesian cows (Bos taurus) on Days 4, 5, 6, 7, 8, 10, 12, 14, 16, -5, -4, -3, -2, -1 of the estrous cycle (Day 1=ovulation). Plasma progesterone concentrations in venous blood (P(4)) were quantified by enzyme immunoassay. Luteal size was determined by sonographic measurement of the maximal cross-sectional area of the corpus luteum (CL). Luteal blood supply was estimated by calculating the maximum colored area of the CL from power Doppler sonographic images. Luteal size doubled during the luteal growth phase (until Day 7) and remained at this level during the luteal static phase (Day 8 to 16) before decreasing rather slowly during luteal regression (Days -5 to -1). Luteal blood flow doubled during the growth phase, doubled furthermore during the static phase, and decreased rapidly during luteal regression. Thus, LBF values represented highly reliable predictors of luteal status. Luteal blood flow predicted reliably a P(4)>1.0 ng/mL by reaching only 35% of the maximal values, whereas LS had to exceed 60% of the maximal values to indicate reliably a functional CL. It is concluded that LBF reflected luteal function better than LS specifically during luteal regression.

Plasma progesterone concentrations in the mid-luteal phase are dependent on luteal size, but independent of luteal blood flow and gene expression in lactating dairy cows

The objective of the present study was to investigate if plasma progesterone (pP(4)) concentrations are dependent on luteal size, blood flow, or gene expression in luteal tissue. To induce cycles with high and low pP(4) concentrations, respectively, 20 lactating dairy cows received either a single treatment with 25 mg prostaglandin F(2α) (PGF(2α)) on Day 4 Hour 12 (PG1; n=8), or two treatments (25 mg PGF(2α) each) on Day 4 Hours 0 and 12 (PG2; n=12) of the estrous cycle (Day 1, Hour 0=ovulation). In four cows, ovulation occurred between 4 and 6d after the second PGF(2α) treatment; these cows and one lame cow were excluded from the study. In the 15 remaining cows with physiological interovulatory intervals, pP(4), area (LTA) and volume (LTV) of luteal tissue, as well as absolute (LBF) and relative (rLBF) luteal blood flow were determined on Day 9, and relative luteal P(4) (rLP(4)) as well as luteal mRNA expression of important receptors, angiogenic, vasoactive, and steroidogenic factors were quantified on Day 11 (±1) during two successive estrous cycles. Furthermore, rLP(4) was multiplied by LTV to produce a semiquantitative assessment of absolute luteal P(4) (LP(4)). There was no effect (P>0.05) of treatment (one or two PGF(2α) treatments), neither on pP(4) concentrations nor on any other parameter in the present study. Nevertheless, there was a lower LP(4) (P=0.01), LTA (P=0.03), and LTV (P=0.02), as well as tendencies of lower pP(4) (P=0.06) and LBF (P=0.09) at first compared with second diestrus. Plasma P(4) was related with LP(4) (r=0.43, P=0.04), LTA (r=0.65, P=0.0001), and LTV (r=0.43, P=0.02), but not with rLBF (r=-0.18, P=0.34). Furthermore, there was no significant correlation between gene expression of important steroidogenic factors and P(4) concentrations in luteal tissue. Results indicate that plasma P(4) concentrations in the mid-luteal phase were dependent on luteal size, but independent of blood flow and gene expression per luteal tissue unit.

Transrectal Doppler sonography of uterine blood flow during the first 12 weeks after parturition in healthy dairy cows.

The aim of this study was to determine if transrectal colour Doppler sonography of uterine arteries is a useful method to quantify uterine changes during the first 12 weeks after parturition in cows. Examinations were carried out on days 1, 7, 14, 28, 56 and 86 after parturition (day 0) in 42 Holstein-Friesian multiparous dairy cows (mean lactation number: 2.63+/-0.73). Findings obtained by transrectal manual palpation were quantified using scores for uterine size (SUS) graded from 1 to 6. The mean diameter of intrauterine fluid accumulation within the uterine body and the presence of a dominant follicle and/or a corpus luteum were examined using B-mode sonography. Blood flow was measured by determining blood flow volume (BFV) and pulsatility index (PI) of the uterine arteries. Clinical uterine involution was complete on day 28 as demonstrated by transrectal palpation and B-mode sonography. BFV declined (P<0.05) steeply between days 1 and 7 from 4312 ml/min to 1443 ml/min and moderately (P<0.05) to 230 ml/min on day 28. From this time on there were no significant changes (P>0.05) of BFV until the end of the study. The pulsatility index rose from 1.54 on day 1 reaching a peak value of 5.56 on day 28 and decreased (P<0.05) linearly to 3.13 on day 86. The results show that transrectal colour Doppler sonography is an additional tool for examining uterine changes during the first 12 weeks after parturition in cows. While uterine blood flow changes were only demonstrated during the first 4 weeks of the puerperium, the pulsatility index was also suitable to investigate alterations in uterine perfusion during the next 8 weeks after parturition.

Uterine blood flow during the first 3 weeks of pregnancy in dairy cows

Transrectal color Doppler sonography was used to compare changes in uterine blood flow between cyclic and early pregnant lactating dairy cows. Examinations were carried out in 53 multiparous lactating cows on days 3, 6, 9, 11, 13, 15, 18 and 21 (day 0=estrus). Fourteen cows were examined during the estrous cycle and thirty-nine cows after insemination with frozen/thawed sperm. Uterine blood flow was reflected by the time-averaged maximum velocity (TAMV) and the pulsatility index (PI) in the uterine artery ipsilateral to the corpus luteum. Twenty-one cows that were not pregnant on day 25 were excluded from the study. There was high inter-individual variability in PI- (CV: 22-62%) and TAMV-values (CV: 22-42%) at all examinations. In cyclic cows, TAMV values increased between days 13 and 18 and PI values decreased between days 15 and 21 (P<0.05). In pregnant cows (n=18), TAMV values increased from days 9 to 11 and then decreased to minimal values by day 18 (P<0.05). The PI values decreased between days 3 and 11 and then increased to maximum levels on day 18 (P<0.05). On day 21, both variables reached (P<0.05) values that did not differ (P>0.05) from those on day 11. The changes in TAMV were correlated with estrogen and progesterone concentrations (r=0.69 and -0.70, respectively; P<0.05) in cycling cows, but not in pregnant cows (P>0.05). The PI values did not correlate with steroid hormone levels (P>0.05). Differences in uterine blood flow between cycling and early pregnant cows were observed only on day 18 (P<0.05). The results show that in pregnant cows changes in uterine blood supply can be detected already in the second week after insemination; these changes do not occur in the second week of the cycle.

Luteal blood flow increases during the first three weeks of pregnancy in lactating dairy cows.

The objectives of this experiment were to characterize luteal blood flow in pregnant and non-pregnant cows and to determine its value for early pregnancy diagnosis. Lactating dairy cows (n = 54), 5.2 ± 0.2 y old (mean ± SEM), average parity 2.4 ± 0.2, and ≥ 6 wk postpartum at the start of the study, were used. The corpus luteum (CL) was examined with transrectal color Doppler ultrasonography (10.0-MHz linear-array transducer) on Days 3, 6, 9, 11, 13, 15, 18, and 21 of the estrus cycle (estrus = Day 0). Artificially inseminated cows (n = 40) were retrospectively classified as pregnant (embryonic heartbeat on Day 25; n = 18), nonpregnant (interestrus interval 15 to 21 d, n = 18), or having an apparent early embryonic loss (interestrus interval >25 d, n = 4). There was a group by time interaction (P < 0.001) for luteal blood flow from Days 3 to 18; it was approximately 1.10 ± 0.08 cm(2) (mean ± SEM) on Day 3, and increased to approximately 2.00 ± 0.08 cm(2) on Day 13 (similar among groups). Thereafter, luteal blood flow was numerically (albeit not significantly) greater in pregnant cows, remained constant in those with apparent embryonic loss, and declined (not significantly) between Days 15 and 18 in nonpregnant cows. Luteal blood flow was greater in pregnant than in nonpregnant (P < 0.05) and nonbred cows (P < 0.05, n = 14) on Day 15 (2.50 ± 0.16, 2.01 ± 0.16, and 2.00 ± 0.18 cm(2), respectively) and on Day 18 (2.40 ± 0.19, 1.45 ± 0.19, and 0.95 ± 0.21 cm(2)). In cows with apparent early embryonic loss, luteal blood flow was 2.00 ± 0.34 and 2.05 ± 0.39 cm(2) on Days 15 and 18, which was less (not significantly) than in pregnant cows, but greater (P < 0.05) than in nonbred cows on Day 18. Although mean luteal blood flow was significantly greater in pregnant than nonpregnant (and nonbred) cows on Days 15 and 18, due to substantial variation among cows, it was not an appropriate diagnostic tool for pregnancy status.

Escherichia coli lipopolysaccharide administration transiently suppresses luteal structure and function in diestrous cows

The objective was to characterize the effects of Escherichia coli lipopolysaccharide (LPS) endotoxin (given i.v.) on luteal structure and function. Seven nonlactating German Holstein cows, 5.1 ± 0.8 years old (mean ± s.e.m.), were given 10  ml saline on day 10 (ovulation=day 1) of a control estrous cycle. On day 10 of a subsequent cycle, they were given 0.5 μg/kg LPS. Luteal size decreased (from 5.2 to 3.8 cm², P≤0.05) within 24 h after LPS treatment and remained smaller throughout the remainder of the cycle. Luteal blood flow decreased by 34% (P≤0.05) within 3 h after LPS and remained lower for 72 h. Plasma progesterone (P₄) concentrations increased (P≤0.05) within the first 3 h after LPS but subsequently declined. Following LPS treatment, plasma prostaglandin (PG) F metabolites concentrations were approximately tenfold higher in LPS-treated compared with control cows (9.2 vs 0.8 ng/ml, P≤0.05) within 30 min, whereas plasma PGE concentrations were nearly double (P≤0.05) at 1 h after LPS. At 12 h after treatment, levels of mRNA encoding Caspase-3 in biopsies of the corpus luteum (CL) were increased (P≤0.05), whereas those encoding StAR were decreased (P≤0.05) in cattle given LPS vs saline. The CASP3 protein was localized in the cytoplasm and/or nuclei of luteal cells, whereas StAR was detected in the cytosol of luteal cells. In the estrous cycle following treatment with either saline or LPS, there were no significant differences between groups on luteal size, plasma P₄ concentrations, or gene expression. In conclusion, LPS treatment of diestrus cows transiently suppressed both the structure and function of the CL.

Noninvasive detection of hepatic lipidosis in dairy cows with calibrated ultrasonographic image analysis.

The aim was to test the accuracy of calibrated digital analysis of ultrasonographic hepatic images for diagnosing fatty liver in dairy cows. Digital analysis was performed by means of a novel method, computer-aided ultrasound diagnosis (CAUS), previously published by the authors. This method implies a set of pre- and postprocessing steps to normalize and correct the transcutaneous ultrasonographic images. Transcutaneous hepatic ultrasonography was performed before surgical correction on 151 German Holstein dairy cows (mean +/- standard error of the means; body weight: 571+/-7 kg; age: 4.9+/-0.2 yr; DIM: 35+/-5) with left-sided abomasal displacement. Concentration of triacylglycerol (TAG) was biochemically determined in liver samples collected via biopsy and values were considered the gold standard to which ultrasound estimates were compared. According to histopathologic examination of biopsies, none of the cows suffered from hepatic disorders other than hepatic lipidosis. Hepatic TAG concentrations ranged from 4.6 to 292.4 mg/g of liver fresh weight (FW). High correlations were found between the hepatic TAG and mean echo level (r=0.59) and residual attenuation (ResAtt; r=0.80) obtained in ultrasonographic imaging. High correlation existed between ResAtt and mean echo level (r=0.76). The 151 studied cows were split randomly into a training set of 76 cows and a test set of 75 cows. Based on the data from the training set, ResAtt was statistically selected by means of stepwise multiple regression analysis for hepatic TAG prediction (R(2)=0.69). Then, using the predicted TAG data of the test set, receiver operating characteristic analysis was performed to summarize the accuracy and predictive potential of the differentiation between various measured hepatic TAG values, based on TAG predicted from the regression formula. The area under the curve values of the receiver operating characteristic based on the regression equation were 0.94 (<50 vs. >or=50mg of TAG/g of FW), 0.83 (<100 vs. >or=100mg of TAG/g of FW), and 0.97 (<50 vs. >or=100mg of TAG/g of FW). The CAUS methodology and software for digitally analyzing liver ultrasonographic images is considered feasible for noninvasive screening of fatty liver in dairy herd health programs. Using the single parameter linear regression equation might be ideal for practical applications.

Bovine luteal blood flow: basic mechanism and clinical relevance

The introduction of transrectal colour Doppler sonography (CDS) has allowed the evaluation of luteal blood flow (LBF) in cows. Because appropriate angiogenesis plays a decisive role in the functioning of the corpus luteum (CL), studies on LBF may provide valuable information about the physiology and pathophysiology of the CL. Studies on cyclic cows have shown that progesterone concentrations in blood plasma can be more reliably predicted by LBF than by luteal size (LS), especially during the regression phase of the CL. In contrast with non-pregnant cows, a significant increase in LBF is seen in pregnant cows during the third week after insemination. However, because there are high interindividual variations in LBF between animals, LBF is not useful for the early diagnosis of pregnancy. Determination of LBF is more sensitive than LS for detecting the effects of acute systemic inflammation and exogenous hormones on the CL. Cows with low progesterone levels have smaller CL during the mid-luteal phase, but LBF related to LS did not differ between cows with low and high progesterone levels. In conclusion, LBF determined by CDS provides additional information about luteal function compared with LS and plasma progesterone concentrations, but its role concerning fertility in the cow is yet to be clarified.

Variability of uterine blood flow in lactating cows during the second half of gestation

The main goal of the present study was to measure uterine blood flow volume (BFV) in the second half of gestation in lactating German Holstein cows. Furthermore, it was investigated, if there are individual variations in uterine blood flow and correlations between uterine blood flow and maternal weight and the birth weight of the calf. Forty-four cows were examined via color Doppler sonography in gestation weeks (GW) 21, 25, 29, 33, 37 and 39. The cows were allocated in groups based on the following variables: body weight (light ≤ 575 kg, heavy > 575 kg) and birth weight of the calf (light ≤ 42 kg, heavy > 42 kg). The BFV was measured via transrectal Doppler sonography of both uterine arteries. There was a linear increase in uterine BFV throughout the study period from 3053 ± 1143 ml/min to 16912 ± 5793 ml/min. Variation coefficients for inter-individual variations ranged from 34 to 37%. There was a moderate correlation between uterine BFV and birth weight of the calf in weeks 21 to 37 (0.30 ≤ r < 0.49; P < 0.05) and a good correlation in week 39 (r = 0.60; P < 0.0001). Uterine BFV in week 21 was significantly (P < 0.01) higher in heavy cows (3394 ± 1119 ml/min) than in light cows (2658 ± 1064 ml/min). Compared with light cows, the increase in uterine BFV was 32% higher in heavy cows during the study period. In week 21, there was no difference (P > 0.05) in uterine BFV between cows carrying a heavy calf (3351 ± 1130 ml/min) and those carrying a light calf (2796 ± 1115 ml/min). Thereafter, the increase of BFV was 43% higher in cows with a heavy calf than in those carrying a light calf. Cows with different body weights, but same birth weight of calf showed no differences (P > 0.05) in the increase of BFV, while in cows with the same body weight the rise in BFV was higher (P < 0.05) in those cows producing a heavy calf compared to cows carrying calves with light birth weights. In conclusion, there was a linear increase in uterine BFV in lactating Holstein cows during the second half of pregnancy with marked individual variations. Differences in the rise of BFV were more caused by the fetus than by body weight of cows.

Relationships between ovarian blood flow and ovarian response to eCG-treatment of dairy cows

The goal of the present study was to investigate ovarian blood flow and ovarian response in cows undergoing a gonadotropin treatment to induce a superovulatory response, using transrectal colour Doppler sonography. Forty-two cows including 19 cross-bred, 14 German Holstein and 9 German Black Pied cows were examined sonographically before hormonal stimulation on Day 10 of the oestrous cycle, three days after administration of eCG (Day 13) and seven days after artificial insemination (Day 7(p.i.)). After each Doppler examination, blood was collected for determination of total oestrogens (E) and progesterone (P4) in peripheral plasma. The blood flow volume (BFV) and pulsatility index (PI), which is a measure for blood flow resistance, were determined in the ovarian artery, and B-mode sonography was used to count dominant follicles and corpora lutea. Important criteria to assess the ovarian response following the hormonal treatment were the number of follicles >5mm in diameter on Day 13 and the number of corpora lutea on Day 7(p.i.) per cow. The number of follicles ranged from 2 to 61 (mean+/-S.E.M.: 17.5+/-1.7) and corpora lutea from 0 to 50 (mean+/-S.E.M.: 17.0+/-1.6). The BFV increased from 28.4 to 45.0 ml/min between Days 10 and 13 and reached a maximum of 108.5 ml/min on Day 7(p.i.) The PI decreased from 6.25 on Day 10 to 4.70 on Day 13 and to 2.10 on Day 7(p.i.) The BFV and PI on Day 13 did not correlate with the number of follicles (P>0.05). However, on Day 7(p.i.) the number of corpora lutea correlated positively with the BFV (r=0.64; P<0.0001), and an inverse relationship was found for the PI (r=-0.51; P=0.0005). There were no correlations (P>0.05) between the BFV and PI on Day 10 and the number of follicles on Day 13 or the number of corpora lutea on Day 7(p.i.) Results of the present study show that in cows, a hormonal treatment to induce a superovulatory response yielded a marked increase in BFV and a marked decrease in PI in the ovarian artery. However, there was no correlation between BFV and PI in the ovarian arteries before hormonal stimulation and the number of follicles and corpora lutea that developed after stimulation. Thus BFV and PI measured in the ovarian arteries have limited diagnostic value to predict the outcome of a gonadotropin treatment.