Ultrasonographic Screening of Dairy Cows with Normal Uterine Involution or Developing Postpartum Uterine Disease Using B-Mode, Color, and Spectral Doppler

Uterine involution, ovarian activity, and incidence of postpartum uterine disease (PUD) were assessed in forty-eight dairy cows from calving until the 10th postpartum week. Postpartum follow-up included evaluation of uterine involution and ovarian structures by B-mode, Doppler color, and Doppler spectral ultrasound of the right uterine artery in cows with no calving or postpartum uterine problems (healthy cows). Data from cows that developed PUD (PUD cows) were compared with healthy cows matched by herd and days in milk (DIM). Data were analyzed by descriptive statistics, simple regression, one-way ANOVA, or repeated ANOVA measures, and in data analysis of healthy cows, uterine horn diameter assessed by B-mode ultrasound ranged from 22.9 ± 2.4 to 19.4 ± 1.4 mm and 19.9 ± 2.2 to 20.5 ± 2.3 mm from the fourth to the seventh postpartum week in the left and right uterine horns, respectively (P > 0.05). During the study, 15 and 7 cows had corpus luteum in the left and right ovaries, respectively. The mean time for the first postpartum CL was 30.1 ± 3.2 DIM (min 8, max 67 DIM). In data analysis of PUD cows, uterine blood flow assessed by color Doppler ranged from 7.4 ± 4.0 to 43.75 ± 10.3% in cows that developed PUD compared to 16.7 ± 11.0% in healthy cows (P > 0.05). No statistically significant changes were found in resistance index, pulsatility index, time-averaged maximum velocity, time-averaged mean velocity, or diastole/systole ratio (D/S) in cows that developed PUD compared to healthy cows (P > 0.05). Finally, no correlation was found between Doppler spectral parameters and uterine involution (P > 0.05). Our data suggest that cows receiving transition diets and exhibiting normal calving undergo a rapid macroscopic uterine involution and ovarian follicular dynamics resumption. Complete ultrasound evaluation provides valuable data for assessing uterine involution in postpartum dairy cows.

B-mode ultrasound exam allows a direct assessment of the postpartum uterus, improving Met and CE diagnosis [19].Besides, successful evaluation of uterine circulation by color Doppler and spectral Doppler was reported [20].Furthermore, uterine blood fow changes have been reported during the estrus cycle's follicular and luteal phases [21], superovulation, and early pregnancy [22,23].In addition, the blood fow of specifc vessels can be semi-quantitatively evaluated through Doppler indices, which allow measurement of the resistance of peripheral vessels to blood fow (reviewed in [19]).In Doppler ultrasound values, S represents the maximum systolic fow and M represents the enddiastolic or minimum-diastolic fow.Doppler indices are calculated from two consecutive blood fow waves captured by the probe and set up according to the equipment software (see Figure 4 in [19]).
Krueger et al. [24] evaluated uterine blood fow and the pulsatility index (PI) of uterine arteries from calving to the twelfth postpartum week in dairy cows.Also, Heppelmann et al. [25] found changes in uterine artery diameter, timeaveraged maximum velocity (TMAX), and PI within the frst two postpartum weeks of Simmental cows.However, to our knowledge, no studies on Doppler parameters of uterine arteries in postpartum cows afected by PUD have been reported.
Our working hypothesis proposed that the uterine artery blood fow parameters of cows developing PUD difered from those of healthy postpartum cows.Te objectives of this study were to evaluate uterine involution, ovarian structures, serum calcium, milk beta-hydroxybutyrate concentration, uterine and ovarian blood fow by B-mode ultrasound and color Doppler, and uterine artery Doppler spectral values in postpartum dairy cows that received transitional dietary management, had normal calving, and did not sufer from PUD.We evaluated uterine involution by ultrasonography and compared the color and spectral Doppler parameters between dairy cows that develop PUD or remain healthy between the frst and the seventh postpartum week.

Approval. Te University of Antioquia Institutional
Board for Using Animal Subjects in Experimental Designs supplied ethical support for the project (approved by Act # 111, June 8, 2017).Also, all farmers signed informed consent.

Type of Study.
We performed a descriptive analysis including forty-seven cows from four dairy farms of the northern region of Antioquia State (Colombia), including Donmatias, Entrerrios, and Santa Rosa de Osos municipalities, settled within 2400 and 2600 meters over sea level, and temperatures ranging from 6 to 16 °C all along the year.
At the beginning of the study, all cows preselected in each farm that exhibited normal calving were followed up for their postpartum outcome.All cows included in the study were within the frst and seventh lactation.Te calves' birth weight record was not considered for analysis because of the absence of dystocia.

Inclusion Criteria and Clinical Examination.
Each dairy farm was visited to select the prepartum cows included in the study.Once cows with normal calving and no clinical signs of metabolic diseases were selected, farms were visited from the frst postpartum week and then weekly until the tenth postpartum week.At each visit, cows were evaluated by clinical exam and reproductive assessment by rectal palpation and ultrasound exam (Figure 1).

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Clinical Exam.
Cows were evaluated for their general condition to assess clinical signs indicative of metabolic or other prevalent diseases.Each cow was sampled at the time of inclusion and at each time point of evaluation, including blood and milk samples.According to the results at each time point of evaluation, cows were classifed as healthy or developing PUD (Figure 1).[30,31].Te cutof point was the presence of more than 5% PMNs in a 200-cell count.No information about uterine cervicitis was recorded in the study (Figure 1).

Blood and Milk
Sampling.Blood samples were taken from the coccygeal vein using a vacutainer with no anticoagulant.Blood samples were transported to the laboratory under ice and centrifuged to obtain serum.Serum was stored at −4 °C until processing.Calcium concentration was measured using an automated serum analyzer (Mindray autoanalyzer IN-B2400 Plus) with Randox calcium kits (Randox Laboratories Canada Ltd.) in a certifed diagnostic laboratory in Rionegro (Antioquia, Colombia).Milk samples were assessed for BHB (mM) using infrared spectroscopy (Mil-koScan FT2, Foss Analytics) at the Raw Milk Quality and Safety Laboratory of the University of Antioquia (Medellin, Colombia).

Postpartum Uterine Involution and Ovarian Status
3.1.Uterine Involution.Cows meeting eligible criteria and selected for the study started at 3 ± 2 days postpartum were evaluated by ultrasound using a Mindray Z5 portable ultrasound equipped with a 5.0 to 7.5 MHz gradable probe (Mindray Medical Colombia, Marquetingnet S.A.S., Bogotá, Colombia).Each cow was assessed by a transrectal exam, including an exhaustive evaluation of the uterine horns with cross-sectional and longitudinal images captured by B-mode ultrasound.In addition, cross section uterine horn images taken at the greater uterine curvature were recorded.Te cursor was set up at the uterine curvature to measure the distance between the dorsal, ventral, and lateral spaces corresponding to the uterine mucosa's 3 and 9-o'clock sides (Figure 2).Te same veterinarian (MHG) performed all measurements.Te average measurements were considered as the diameter of each uterine horn.Also, the presence of uterine content indicated by a hyperechogenic line or variable amounts of hyperechogenic content was deemed abnormal and indicative of CE if corroborated by positive endometrial cytology.
According to the clinical progress of the reproductive tract of the cows, two datasets were obtained.Dataset one depicts fndings of healthy cows that remained nonafected by general disease or PUD through the study period.Tables 1  and 2 show overall (n � 27) and specifc farm data, respectively.Dataset 2 depicts fndings of cows with normal calving that developed Met, CE, or SE during the follow-up period (n � 15).See Tables 1 and 2 for overall and specifc farm data, respectively.In addition, PUD cows were retrospectively matched to healthy cows by DIM and farm for data comparison purposes (Figure 1).

Ovarian Follicles and Corpus Luteum Assessment.
Te ovaries were analyzed for the presence of follicles and corpus luteum by rectal palpation followed by B-mode, color Doppler, and spectral Doppler ultrasound.Findings were recorded for each cow.Only the greatest follicle was measured in each ovary, and any corpus luteum detected was also recorded, although the corpus luteum diameter was not recorded (Figure 3).

Doppler Color Assessment of Uterine Horns, the Greatest Dominant Follicle, and Corpus Luteum.
To collect the color Doppler data, a box (green line in Figures 2-4) was drawn over the image of the uterine horn in B-mode ultrasound.Next, a cross (yellow lines in Figure 3) was drawn to defne the four quadrants where blood fow was evaluated in color Doppler mode.Blood fow was then recorded in the resulting quadrants.Next, the data were taken in pixels and transformed into fow percentages in each quadrant by the equipment software.Finally, the fnal fow values for each quadrant were averaged, and the values were evaluated as mean ± standard error of the mean.

Doppler Spectral Assessment of the Uterine Artery.
Te equipment was adjusted for Doppler spectral parameters to evaluate the uterine arteries.Te right uterine artery was assessed in each cow within a 5-minute average duration.Te lineal transductor was put in a dorsal-oriented cross-sectional orientation on the uterine artery, according to Bollwein et al. [19].In brief, the aorta artery was found with the probe, which was moved caudally until the origin of the external iliac artery, which follows ventrally aside from the iliopectineal line.Ten, the internal iliac artery was set caudally (Figure 4), followed by the common trunk, before branching the umbilical and uterine arteries.Otherwise, the middle uterine artery is palpated as a mobile vessel in the mesometrium.Following its origin in the umbilical artery, we visualized and evaluated the uterine artery.Doppler spectral parameters were recorded with the US probe at a 60% angle for more accurate measurement [19,32].Doppler parameters recorded were resistance index (RI), pulsatility index (PI), time-averaged maximum velocity (TMAX), time-averaged mean velocity (TMEAN), and systole-to-diastole ratio (S/D) (Figure 4).

Endometrial Cytology.
Endometrial samples were taken by cytobrush, smeared on glass slides, and stained by Wright staining.Te percentage of PMN after counting two hundred cells in 40x magnifcation was calculated as previously described, considering positive values greater than 5% PMN [30].

Statistical Analysis.
For data analysis, data from fortythree cows in which three or four consecutive evaluations were performed were fnally included for analysis.Data from Doppler measurements-RI, PI, TMAX, TMEAN, and S/D, the left and right uterine horn diameter, and serum calcium concentration were assessed for normal distribution and homogeneity of variance using Levine's test.Tese variables ft a normal distribution.Comparisons of Doppler values according to ovarian fndings, e.g., between cows exhibiting corpus luteum, only follicles, or no ovarian structures, were compared by one-way ANOVA, including Tukey HSD.A corpus luteum presence was compared between cows suffering PUD and healthy paired cows by chi-square test with Yates's correction.Comparison of nonparametric data from color Doppler quadrants of the left and right ovaries and uterine horns between cows sufering PUD and healthy paired cows were analyzed by T-Test Calculator for two independent means.It was also applied to compare uterine horn diameter and Doppler spectral values between cows exhibiting corpus luteum, follicles, or no structures to assess if ovarian structures had a statistically signifcant efect on these variables.One-way ANOVA analyzed the comparison between PUD incidences farm by farm.Finally, Doppler parameter values of healthy cows between postpartum weeks 1 and 7 were evaluated by repeated ANOVA measures.Tis analysis did not apply to data from PUD cows due to few case numbers (Table 2).

Results
Te mean body condition score (BCS) of cows included in the analysis was 3.0 (1.0 to 5.0 scale) (data not shown).Te average parity of healthy cows was 2.65 (mean ± SEM) (Table 1).Twenty-seven cows (62.79%) did not develop PUD and were in the group of healthy postpartum cows.In contrast, sixteen episodes of PUD were found (37.2%), including clinical metritis (n � 4), clinical endometritis (n � 7), and subclinical endometritis (n � 5) (Tables 1 and 2).No cases of puerperal metritis were diagnosed.No statistically signifcant diferences were found when comparing parity and DIM data from healthy cows to cows that developed PUD according to farm (Table 2).Interestingly, no cases of PUD were found on farm one.Due to sample size, no statistically signifcant diferences were assessed for PUD between farms (Table 2).

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Dataset One: Uterine Involution in Cows Presenting
Normal Calving with No PUD.Data were grouped into weekly intervals for comparison even though data were recorded since the frst postpartum week; only since the fourth week postpartum were we able to record uterine diameter data from healthy cows not developing PUD (Table 3).Veterinary Medicine International difer during the study period nor between the right and the left horns (P > 0.05).Te uterine horn diameter ranged from 19.4 to 22.9 mm (Table 3).Tese values remained without statistically signifcant diferences up to the study's endpoint at the seventh postpartum week (Supplementary Figures 1A  and 1B).No cases of delayed uterine involution were found in these healthy postpartum cows.

Ovarian Follicles and First Postpartum Ovulation.
Ovarian follicles were frst detected in the second and third postpartum weeks onward.Te average (± standard deviation) follicle diameter was 10.6 ± 5.9 and 11.

. Resistance Index (RI)
. RI values showed a high variability during the evaluation period (Supplementary Figure 2).RI values ranged from 0.98 to 0.9 between the frst and seventh weeks postpartum (Table 3), with no statistically signifcant diferences (P > 0.05).RI values showed a slight increase from the frst to the second postpartum week, followed by a decline up to the ffth, increased until the eighth week, and then declined again in the ninth week, with a fnal increase up to the tenth postpartum week (Supplementary Figure 2A), ftting a sixdegree polynomial trend (y � 0.0001 × 6 − 0.0037 × 5 + 0.0323 × 4 − 0.0922 × 3 − 0.0598 × 2+ 0.4186x + 0.691; R 2 � 0.855).

Dataset Two: Postpartum Uterine and Ovarian Parameters in Cows Tat Developed PUD Compared to
Healthy Cows.In this study, the postpartum day at which normal calving cows presented any episode of PUD was recorded, and data from their uterine and ovarian follow-up were retrospectively compared to data from normal calving cows with a healthy postpartum condition.Data from cows that develop PUD were matched to data from healthy cows by DIM and farm.Four, seven, and fve cows developed Met, CE, and SE (Table 1), with no statistically signifcant diferences for DIM (P > 0.05) when compared to healthy postpartum cows.However, serum calcium concentration was signifcantly lower (P < 0.05) in the dataset of cows that developed SE (7.88 ± 0.3 mg/dL) compared to the dataset from healthy postpartum cows (9.17 ± 0.4 mg/dL) (Figure 5(b)).Besides, several cases of PUD occurred in the same cows during the follow-up period: three cows developed Met and then CE and two cows presented two or three episodes of SE.
Te diameter of the right uterine horn (Supplementary Figure 1C) and left uterine horn (Supplementary Figure 1D) did not signifcantly difer from the corresponding values of healthy postpartum cows (P > 0.05).Te ultrasonography exam shows that all cows evaluated presented a complete macroscopic uterine involution at the fourth postpartum week.Uterine horn diameters ranged around 23.57 ± 3.8 and 18.10 ± 1.3 mm and 19.4 ± 2.4 and 17.25 ± 1.4 mm for the right and the left uterine horns in cows that developed Met and CE, respectively.

Ovarian Findings.
In cows that developed Met, CE, and SE, the follicle diameter ranged from 3.8 to 15 mm, 7.8 to 21.2 mm, and 9.8 to 15.7 mm, respectively.In cows with normal calving that developed PUD, we found the frst CL at 25.8 ± 6.2 (min 9, max 35 DIM).One of the cows that developed Met had corpus luteum, whereas two and three cows that developed CE and SE showed corpus luteum.In addition, one healthy cow and two healthy cows matched to CE  2A), PI (Supplementary Figure 2B), TMAX (Supplementary Figure 3A), TMEAN (Supplementary Figure 3B), and D/S (Supplementary Figure 4B) values did not signifcantly difer between cows that developed PUD and healthy cows (P > 0.05).Doppler spectral values were compared between farms and ovarian status, including ovaries with no structures, only follicles, and ovaries with corpus luteum.No statistically signifcant diferences were found (data not shown).Only RI values were signifcantly higher in cows having a corpus luteum (P < 0.05) compared to cows with no structures or only follicles in their ovaries (Supplementary Figure 5).

Discussion
Tis study evaluated uterine involution in dairy cows presenting normal calving in high-altitude tropical dairies using B-mode, Doppler color, and spectral ultrasound.Besides, in a retrospective analysis, we evaluated uterine involution in a group of cows that developed PUD.We compared it with data from healthy postpartum cows matched to PUD by farm and DIM.All cows enrolled in the study showed complete macroscopic uterine involution at the fourth week postpartum based on uterine horn diameter assessed by Bmode ultrasound.Serum calcium concentration did not signifcantly difer in healthy postpartum cows during the three postpartum weeks.Still, cows with SE but not Met or CE had signifcantly lower calcium concentrations than their matched healthy cows (P < 0.05).
In this study, cows that developed Met or CE showed rapid macroscopic uterine involution, as evidenced by uterine horn diameters in both uterine horns from the fourth week postpartum, as reported previously [33].Tis fnding could result from the objective inclusion criteria for the study, highlighting the importance of providing the cows with a transition diet for improving postpartum immune response and metabolic status [34,35].Besides, PUD is a risk factor for culling lactating dairy cows due to its negative impact on pregnancy rates [36][37][38].Interestingly, we performed a B-mode ultrasound evaluation of uterine involution, which provided a confdent result for the absence of abnormal uterine contents and accurate measurements of the uterine horn's diameter.Furthermore, even though cows that developed Met were diagnosed on average at 12 days postpartum, their macroscopic uterine involution was not signifcantly afected compared to healthy matched cows (Supplementary Figure 1) in agreement with Kawashima et al. [8].
Te curve for uterine diameter presented a polynomial trend for the left and right uterine horns (data not shown).Tis fnding agrees with Okano and Tomizuka [39], who found a polynomial trend in uterine horn diameter, uterine body, and cervix diameter in normal calving Holstein dairy cows evaluated from the third to the 48 th DIM.Tese authors reported uterine diameters around 3.2 cm in the previously pregnant and nonpregnant uterine horns at 21 st DIM.Our study's diameter was around 2.2 cm on the same postpartum days (Supplementary Figure 1).Our results on uterine horn diameter agree with the report by Scully et al. [40], who evaluated uterine involution in lactating and nonlactating primiparous Holstein dairy cows, reporting 25 mm diameter at 21 postpartum days in the previously gravid uterine horn [40], and fndings correspond to cows having normal calving as performed in our study (Figures 2(a) and 2(b)).
Cows were evaluated from the frst postpartum week onward, and we were primarily focused on uterine involution, although data collection from ovaries could be achieved from a few cows.However, recording data from all cows was possible since the frst postpartum day.In addition to great uterine content in cows with Met, we also found a hyperechogenic content in the uterine lumen in cows that showed no signs of CE at frst, a fnding further accompanied by positive endometrial cytology, suggesting that some cows that developed CE did not necessarily exhibit purulent vaginal discharge (PVD).Tis fnding highlights the importance of using ultrasound in experimental studies on postpartum uterine diseases.As much as possible, its use must be encouraged to be progressively incorporated as a diagnostic tool by the bovine practitioner.
In our study, 35% of cows presented subclinical hypocalcemia with no cases of milk fever.Tree studies reported a relationship between subclinical hypocalcemia and Met [41,42].Only 10% of cows in our study developed Met due to the inclusion criteria that granted normal calving cows entering the study.However, our research did not evaluate a relationship between low calcium concentration and Met.
We found an early resumption of ovarian follicular function in both ovaries according to the presence of dominant follicles and early postpartum ovulation in at least a third of cows.
Te indices included in the present study comprise the time-averaged peak velocity (TAMEAN or TMEAN), the time-averaged maximum velocity (TMAX), the pulsatility index (PI), the resistance index (RI), and the S/D ratio [43,44].PI and RI represent the semi-quantitative assessment of blood fow resistance.While S/D and RI show a parabolic relationship with increased vascular resistance, PI shows a linear correlation with vascular resistance [19].Resistance refers to factors that impede or reduce blood fow, e.g., resistance faced by blood fow as it passes through a blood vessel.High resistance fows are more pulsatile and correspond to extremities arteries and sites not requiring constant blood fow, exhibiting low diastolic values and a reverse fow area in diastole.Terefore, this fact could Veterinary Medicine International apply to the uterine artery during uterine involution in the postpartum dairy cow.Doppler spectral parameters found in healthy postpartum cows were like Herzog and Bollwein's report [45], which found reduced blood fow during the frst postpartum week in healthy cows.Uterine blood fow appears to vary in response to phases of the estrus cycle and the reproductive status of the cow, probably because of the predominant hormonal milieu in the uterus, with a positive relationship found between progesterone concentration and RI values [46], and it was found to be related to heat stress [47,48] but not in our study.RI and PI are the most common parameters evaluated for studying uterine blood fow.If RI values are low, it shows increased uterine blood fow [19].Nevertheless, we found no statistically signifcant diferences between cows sufering PUD and healthy postpartum cows.
Kaya et al. [49] found no correlation between Doppler parameters, follicular dynamics, and ovulation in cows exhibiting a PGF2a-induced estrus.Contrary to the fnding by Moonmanee et al. [50], who reported a high relationship between ovarian blood fow dynamics and reproductive parameters in beef cows, even though we did not evaluate the progesterone or estrogen concentration in our study, we found that cows with corpus luteum showed signifcantly higher values of RI (P < 0.05) than cows with no ovarian structures or follicles.Tis parameter is also afected by gestational age and hormonal status [51,52], environmental temperatures [53], and other not evaluated factors in the present study.Te RI found in healthy cows presents an oscillatory pattern of three weeks, representing blood fow fuctuations related to the increased ovarian follicular activity and the elevation of circulating estrogens.Estrogen receptors in the tunica media of uterine arteries respond to calcium concentration [54], infuencing vessel contractibility and blood fow.In the report by Debertolis et al. [20], the authors reported a negative relationship between TMAX and PI, with increased TMAX, indicating a high blood fux in cows with acute endometritis.Te authors also found that TMAX signifcantly decreased in the fourth and ffth postpartum weeks and then increased in the seventh week, agreeing with our TMAX results (Supplementary Figure 3A), which reached a lower value in the ffth and sixth postpartum week and then increased in the seventh postpartum week.
Interestingly, TMEAN values showed a similar pattern (Supplementary Figure 3B), representing the uterine involution period until ovarian follicular dynamics resumption.Besides, our study's average DIM at the frst postpartum corpus luteum was 39.6 ± 4.6 days.Progesterone and estrogen serum concentrations play a critical role in uterine blood fow by their efect on uterine arteries; estrogens cause vasoconstriction of the arterial smooth muscle, and progesterone causes vasodilation [19,20].Although no hormonal measurements were performed in this study to evaluate if the presence of ovarian structures could afect Doppler spectral parameters (as indirect evidence of the steroid hormonal status), we found no statistically signifcant diferences in Doppler spectral data between cows with no ovarian structures, cows with dominant follicles, or cows with corpus luteum (data not shown).Tese data support the concept that the ovarian status of the cows did not signifcantly afect Doppler spectral parameters in our sample.Only the resistance index was signifcantly increased (P < 0.05) in healthy cows, showing corpus luteum compared to cows with no ovarian structures and cows having only follicles (Supplementary Figure 5).No comparisons could be evaluated with cows sufering PUD due to the low sample size in these groups.
Blood fux volume was signifcantly reduced proportionally to DIM due to progressive uterine size and weight reduction resulting from uterine involution.In contrast, IP values decreased to 30 DIM due to caruncular vascular supply reduction.In this context, PUD may alter the pattern of uterine blood fow [19,25].According to our results, cows reached a complete macroscopic uterine involution within the fourth postpartum week.However, values of RI increased between the ffth and seventh postpartum week, a fnding that could refect decreased blood fow, probably because several cows exhibited a corpus luteum early postpartum.RI increased due to a high circulating progesterone concentration and reduced blood fow.On the contrary, when luteolysis occurs, RI values decrease with progesterone concentration.Accordingly, Hassan et al. [46] reported a proportional relationship between RI and progesterone.
In cows with CE, the Doppler parameters were more variable, where the PI was higher in CE cows than in healthy matched cows, while the RI was similar in both groups (P > 0.05).Te results on blood fow are like other studies where authors found increased PI and RI in severe and moderate cases of CE.In this study, the higher PI value in CE cows compared to healthy cows suggests PI could be increased in cases of CE, in agreement with the report by Sharma et al. [55] and with the fnding of Doppler color in CE.
Te present study provides evidence of rapid uterine involution in cows with normal calving, some presenting a single or several repeated Met and CE episodes.No statistically signifcant diferences were found for the average diameter of both uterine horns, ranging from 25 to 20 mm from the fourth to the seventh postpartum week.Tis suggests a rapid macroscopic involution almost completed after the fourth postpartum week.No diferences in the diameter of uterine horns were found between cows that developed PUD compared to matched healthy cows.At least at the beginning of the third postpartum week, we found dominant follicles in both ovaries, accompanied by rapid detection of the frst postpartum corpus luteum, in 36.9% of cows not developing PUD on average at 30 postpartum days, compared to 16% of cows sufering Met or CE on average at 46 postpartum days.An intriguing result was the fnding of rapid macroscopic involution of uterine horns in cows that developed PUD.Further studies must include comparing uterine ultrasound parameters in cows with normal calving or dystocia and comparing the frequency of PUD according to calving status.
In conclusion, this study provides evidence of evaluating postpartum uterine involution by B-mode ultrasound evaluation of uterine horns and Doppler ultrasound and Doppler spectral of the uterine artery.Our results evidenced Veterinary Medicine International a rapid involution of the uterus in cows calved with no complications, where almost 100% of cows presented a complete macroscopic uterine involution at the fourth postpartum week.Also, Postpartum involution fnished earlier than one month even in cows presenting PUD, a fnding suggesting the time elapsed between the diagnosis of PUD and its remission was lower than one week.One concluding remark of our study is using ultrasonography for a more accurate diagnosis of uterine involution, ovarian follicular dynamics and ovulation, and PUD diagnosis.

Strengths and Weaknesses of the Study
One strength of the study is the assessment of uterine involution using an objective measurement with B-mode ultrasound, which calls for an exact diagnosis.Similarly, ultrasound allowed us to detect the cases of CE otherwise not seen because there was not enough endometrial content to be observed by clinical inspection.In addition, fnding early postpartum corpus luteum in some cows allows for a search for those cows that could be programmed for insemination early in the voluntary waiting period, once the microscopic involution is reached, from 40 to 45 days onward.Tis fnding resembles cases where cows that show second estrus before 60 days postpartum receive a fertile insemination whose successful pregnancy results in shorter calving intervals.Tis fact must encourage increasing B-mode ultrasound examinations to assess dairy cattle's postpartum uterine diagnosis.
Tis study performed no hormone measurements to corroborate the reestablishment of follicular dynamics, which were only evaluated by ultrasound examination.Hormonal measures could provide evidence of interactions between hormones and Doppler spectral values as reported for cycling dairy cows.However, because Doppler spectral and Doppler color fndings were inconclusive, these measurements must be evaluated in a cohort including cows with dystocia compared to cows with normal calving.
Further studies should evaluate the behavior of uterine involution variables and ultrasonographic evaluation in cows that have not received the transition period diet and compare the clinical outcome at calving and during the frst 45 days postpartum to determine if there are diferences between cows that present calving problems such as dystocia and retained placenta and cows that do not ofer these conditions.In addition, further studies will allow us to evaluate whether there is a relationship between the degree of loss of postpartum body condition score and the incidence of metabolic problems and their impact on ultrasonographic parameters such as those evaluated in this study.

Figure 1 :
Figure1: Study design and data collection schedule.All cows were evaluated from the frst to the tenth postpartum week (descriptive study, dataset 1).Cows that developed PUD were matched to healthy cows for comparison purposes (retrospective analysis, dataset 2).

Figure 2 :
Figure 2: B-mode and Doppler color ultrasound evaluation.(a, b) Cross section and longitudinal measurements of uterine diameter (a dashed line between asterisks) in the uterine horn and its corresponding Doppler color image.(c) B-mode ultrasound image of a uterine lumen having a nonechogenic fuid (asterisks) and foating debris (arrows) corresponding to caruncular involution.Te uterine mucosa is shown in the bracket.(d) B-mode and color Doppler ultrasound of the uterine horn in a longitudinal section showing intraluminal hyperechogenic material (asterisks).

Figure 5 :
Figure 5: Serum calcium concentration in postpartum weeks 1 to 3 in dairy cows with normal calving that have not developed the postpartum uterine disease, PUD (a), or cows that developed PUD (b).Values are expressed as mean ± SEM.

Table 1 :
Te study included epidemiological data of cows with normal calving and healthy postpartum from high-altitude tropical dairy herds.

Table 2 :
Days in milk and parity values of cows of the study comparing healthy cows with cows developing PUD.

Table 3 :
Ultrasound and Doppler spectral values of cows with normal calving and healthy postpartum from high-altitude tropical dairy herds.: days in milk.RUH: right uterine horn diameter.LUH: left uterine horn diameter.FRO: follicle diameter in right ovary.FLO: follicle diameter in left ovary.RI: resistance index.PI: pulsatility index.TMAX: maximal blood fow.TMEAN: mean blood fow.D/S: diastole-to-systole ratio.Values are expressed as mean ± SEM. * Tere were no statistically signifcant diferences within weeks (P > 0.05).n.r.: not recorded. DIM

Table 4 :
Mean percentage of blood fow in four quadrants of Doppler color ultrasound images from uterine horns and ovaries in cows sufering PUD compared to matched healthy cows (data records from the fourth to seventh postpartum weeks).