Cows
Forty-seven primiparous and pluriparous lactating Holstein-Friesian cows from a research farm of the University of Veterinary Medicine Hannover, Germany, were used in the study. Cows were milked twice a day. Cows were 38 ± 12 [median ± MAD] months old, weighed 616 ± 66 kg and had a 305-day milk yield of 9085 ± 1657 kg. The cows were fed a total mixed ration, and concentrate was supplemented according to production. The study was approved by the independent ethics committee of the Lower Saxony Federal State Office for Consumer Protection and Food Safety, Oldenburg, Germany (research permit number 33.9 – 42502 – 04-09/1782).
Group allocation
Based on the results of the clinical examination of the reproductive tract, the cows were classified as healthy (group H) or as having metritis (group M; Figure 1). Uterine size was assessed subjectively according to a defined score system [26]. Transrectal palpation was carried out on days 4, 8 and 11 (±1 day) post partum and then three times a week until day 21. On day 21 post partum, cows with a uterus which could be gathered up with the hand, and horns of the uterus were the thickness of three or four fingers [26] or larger, were assumed to have a delayed uterine involution. When uterine discharge was released from the vulva during transrectal examination, it was noted, and its odor assessed. Furthermore, vaginoscopy was carried out using a tube speculum on days 4, 8 and 11, and the odor of the lochia was assessed. All examinations were carried out by the same person.
Based on Sheldon et al. [6], cows with a uterine enlargement and discharge having an offensive odor at any point of the examination were assigned to group M, and all other cows to group H. Metritis-cows which had fever (body temperature > 39.5°C) were treated daily for 3 days with ceftiofur (2.2 mg/kg, im, Excenel RTU®, Pfizer Animal Health, Germany) and on the day of diagnosis with a single dose of flunixin meglumine (2.2 mg/kg, iv, Finadyne®, MSD Intervet, Germany).
Observation of cyclicity
Cows were monitored by ultrasonography three times a week (Monday, Wednesday, Friday) to detect the first ovulation starting on day 11 post partum. Ovulation was diagnosed when a dominant follicle, which had been seen at the previous examination, was no longer visible. A CL with a diameter of more than 7 mm was deemed to be the result of ovulation two days previously (day of ovulation = day 1), and the cycle stage was designated as day 3. When the dominant follicle was no longer present, and a CL was not visible or was smaller than 7 mm, the previous day was considered the day of ovulation. On days 5 ± 1 and 11 ± 2 of cycle, the presence of a CL was verified sonographically. The following three ovulations were diagnosed accordingly. To determine the occurrence of the second ovulation, monitoring (three times per week) started on day 14, and for detection of the third and fourth postpartum ovulations, monitoring (three times per week) started on day 18 of the previous cycle.
Examinations of cyclic corpora lutea
Luteal size was measured sonographically once between days 9 and 13 of the first, second and fourth cycles, and blood samples were collected at the same time for measuring the serum P4 (Figure 1). In the first, second and fourth cycles, the CL of 23 (group H, n = 11; group M, n = 12), 41 (group H, n = 23; group M, n = 18) and 18 cows (group H, n = 11; group M, n = 7) was measured, respectively. At the beginning of the study, cyclic CLs of the second and fourth postpartum cycles were analyzed to examine the long-term effect of metritis on luteal activity. An additional number of cows was investigated during the first and second postpartum estrous cycles to evaluate the short-term effect of metritis on CL. All cows were grouped together to simplify the presentation of results. For that reason, the number of individuals in the second cycle included most of the animals. During the second and fourth cycles, 16 cows (group H, n = 9; group M, n = 7) underwent transvaginal biopsy of the CL once between days 9 and 13. Luteal biopsies were taken as described by Onnen-Lübben et al. [27] and Herzog et al. [28]. Cows which did not ovulate within the first 42 days post partum were excluded from analysis regarding cyclic CLs.
Examinations of persistent corpora lutea
Cows with a CL with no sign of luteolysis at the end of a cycle underwent sonographic luteal examination and transvaginal luteal biopsy once between days 29 and 33 of the estrous cycle as well as blood collection. The existence of a persistent CL was confirmed by a serum P4 concentration of ≥ 1.0 ng/mL. After sonographic examination of the persistent CL, the cows were treated with cloprostenol (0.5 mg, im, Estrumate®, Intervet, Unterschleißheim, Germany) to induce luteolysis, and the subsequent ovulation was diagnosed as described for spontaneous ovulations. In the following induced cycle, these cows underwent another luteal biopsy as well as sonographic luteal examination and blood collection once between days 9 and 13 of the induced cycle (Figure 1).
Endometrial biopsy and fertility
To verify whether the clinically based diagnosis of metritis leads to endometritis, transvaginal endometrial biopsies were taken during the second and third postpartum estrus (Figure 1). Biopsies were taken when a dominant follicle (Ø ≥ 10 mm) and only a small CL (Ø < 10 mm) were apparent during transrectal, sonographic examination. Artificial insemination was started at the fourth postpartum estrus. Only cows observed in standing heat were bred. Two pregnancy examinations were carried out using sonography on days 29 ± 1 and 40 ± 3 after breeding. An embryonic heart beat was used to confirm pregnancy. Days to first service and days open as well as insemination (total number of inseminations in all cows per number of pregnant cows) and pregnancy indices (number of inseminations per achieved pregnancy) were calculated for cows in the two groups. Cows not ovulating within the first 42 days post partum were excluded from analysis regarding endometrial biopsies and fertility.
Ultrasonography
A portable ultrasound machine (HS-101 V, Honda Electronics CO., Tokio, Japan) with a 5-MHz linear-array transducer was used to monitor cows for ovulation, and another machine (LOGIQ Book XP, General Electric Medical System, Solingen, Germany), equipped with a 10-MHz linear-array transducer, was used to measure luteal size. According to Lüttgenau et al. [29], a CL was assumed to have the shape of a prolate spheroid. For each detectable CL, maximum longitudinal and cross-sectional images were frozen and recorded three times. The maximum height and width of the cross-sectional area of the CL were measured (PixelFlux Version 1.0, Chameleon Software, Leipzig, Germany) and taken as the major and minor diameters of the spheroid, respectively. The volume of the CL was calculated as follows:
with a = major diameter (rotational axis) and b = minor diameter (transverse axis). If a CL had a cavity, the volume of the cavity was determined accordingly and subtracted from the previously calculated luteal volume. The difference between the total volume of the CL and the volume of its cavity was defined as the volume of luteal tissue. In double ovulations, the volumes were added, based on a previous study showing that the weight of a CL resulting from a single ovulation does not differ from the combined luteal weight resulting from a double ovulation [30].
Measurement of progesterone and prostaglandin E
Blood samples from a jugular vein were collected (serum- and EDTA-tubes, Sarstedt, Nümbrecht, Germany) and placed on ice. Serum and plasma were separated by centrifugation (3 000 × g, 15 min) and frozen at −20°C. Serum P4 concentration was determined using a commercial coat-a-count radioimmunoassay according to the manufacturer’s instructions (Progesterone Coat-a-Count, TKPG1, Siemens Medical Diagnostics, CA, USA). For PGE analysis, cows of both groups were sampled once during the first postpartum luteal phase (between days 9 and 13) and cows with a persistent CL were tested between days 29 and 33 after the first ovulation. As described by Herzog et al. [28], PGE plasma concentration was determined using a commercial PGE2 enzyme immunoassay (Prostaglandin E2 EIA Kit, Biotrend Chemikalien GmbH, Cologne, Germany).
Luteal RNA extraction and cDNA production
Luteal biopsies were used to quantify the levels of mRNA for StAR, cytochrome P450 and 3β-HSD. These substances are important factors for luteal steroidogenesis [22, 24, 25, 31]. As described by Shirasuna et al. [32], total RNA was extracted from luteal biopsy samples, and cDNA was synthesized.
Real-time reverse transcription-polymerase chain reaction of luteal biopsy
Levels of mRNA for two housekeeping genes, glycerolaldehyde-3-phosphate-dehydrogenase (GAPDH) and β-actin, as well as for StAR, cytochrome P450 and 3β-HSD were quantified by real-time PCR with a LightCycler (Roche Diagnostics Co.) using a commercial kit (LightCycler FastStart DNA Master SYBR Green I: Roche Diagnostics Co.). Based on bovine sequences, primers were designed using Primer-3. Primers used for real-time PCR were as follows: StAR (GenBank: MN174189) forward 5-GTG GAT TTT GCC AAT CAC CT-3 and reverse 5-TTA TTG AAA ACG TGC CAC CA-3; cytochrome P450 (GenBank: K02130) forward 5-CTG CAA ATG GTC CCA CTT CT-3 and reverse 5-CAC CTG GTT GGG TCA AAC TT-3; 3β-HSD (GenBank: X17614) forward 5-TCC ACA CCA GCA CCA TAG AA-3 and reverse 5-AAG GTG CCA CCA TTT TTC AG-3; GAPDH (GenBank: NM001034034) forward 5-CTC TCA AGG GCA TTC TAG GC-3 and reverse 5-TGA CAA AGT GGT CGT TGA GG-3; β-actin (GenBank: K00622) forward 5-CCA AGG CCA ACC GTG AGA AAA T-3 and reverse 5-CCA CAT TCC GTG AGG ATC TTC A-3. The amplification program consisted of 15 min activation at 95°C followed by 40 cycles of PCR steps (15 sec denaturation at 94°C, 30 sec annealing at 58°C and 20 sec extension at 72°C). For the quantification of the target genes, a series of standards were constructed by amplifying a fragment of DNA (150 ~ 250 bp) containing the target sequence for real-time PCR. The PCR products were subjected to electrophoresis, and the target band cut out and purified using a DNA purification kit (SUPRECTM-01, TaKaRa Bio. Inc., Otsu, Japan). The quantification of mRNA expression was performed using Light Cycler Software (Version 3.5, Roche). Primer sets were tested in luteal tissue samples to confirm amplification of single bands. Amplified products were cloned and sequenced to confirm their identity before using primers for analyzing the samples. The values determined for the target genes were normalized against the housekeeping genes GAPDH and β-actin (∆Cq). To avoid negative digits while allowing an estimation of a relative comparison between two genes, data were presented as means ± SD subtracted from the arbitrary value 20 (∆Cq). Thus, a high ∆Cq proportionally resembled high transcript abundance [33].
Procedure of endometrial biopsy
The biopsy instrument, designed by Kevorkian (Fa. Hauptner Herberholz GmbH & Co. KG, Solingen, Germany), was used to collect endometrial tissue samples from both uterine horns approximately 2 cm cranial to the bifurcation. The instrument was introduced into the uterus analogous to an AI pipette. Tissue samples were fixed in 10% neutral formalin, buffered according to Lillie and embedded in paraffin. Sections, 3 to 4 μm thick, were stained with hematoxylin and eosin and examined microscopically for signs of inflammation including the occurrence of lymphocytes, plasma cells, macrophages and neutrophils. Endometritis was defined as an inflammatory cell infiltration of the endometrium which exceeded the normal cellular infiltration associated with repair of the endometrium. Up to 20 neutrophils in the luminal epithelium and 15 mononuclear cells (lymphocytes, plasma cells, macrophages) in the stratum compactum per high-power (x400) field are considered normal.
Statistical analysis
The program SigmaStat 2.03 (Systat Software GmbH, Erkrath, Germany) was used for statistical analysis. Continuous variables were analyzed for normal distribution using the Kolmogorov-Smirnov test. Normally-distributed data are given as mean ± standard deviation. Differences between independent and dependent samples were analyzed using a Student’s t-test and a paired t-test, respectively. Data not normally distributed are presented as the median and mean absolute deviation (MAD). Differences between paired and unpaired samples were analyzed using the Wilcoxon signed ranks test and Mann–Whitney rank sum test, respectively. Most variables had a normal distribution (mean ± SD), and only those which did not were reported as median ± MAD. Days open of groups H and M were plotted by Kaplan Meier curves, and curves were compared by the Mantel-Cox test. Categorical data, such as histopathological findings or occurrence of a persistent CL, were analyzed using a chi-square test or Fisher’s exact test. A p-value ≤ 0.05 was considered significant, and a value 0.05 < p < 0.10 was considered as a trend toward significance.