Johannes Lüttgenau

LPS-mediated effects and spatio-temporal expression of TLR2 and TLR4 in the bovine corpus luteum

When given intravenously (iv), lipopolysaccharide (LPS) transiently suppresses the structure and function of the bovine corpus luteum (CL). This is associated with increased release of prostaglandin (PG) F2α metabolite. The underlying regulatory mechanisms of this process remain, however, obscure. Therefore, the aims of this study were: i) to investigate the expression of the LPS receptor toll-like receptor 4 (TLR4) and 2 (TLR2) in the bovine CL during early, mid- and late luteal phases; and ii) to further dissect the mechanisms of LPS-mediated suppression of luteal function. As revealed by semi-quantitative qPCR and immunohistochemistry, both receptors were detectable throughout the luteal lifespan. Their mRNA levels increased from the early toward the mid-luteal phase; no further changes were observed thereafter. The TLR4 protein seemed more highly represented than TLR2. The cellular localization of TLRs was in blood vessels; weaker signals were observed in luteal cells. Additionally, cows were treated either with LPS (iv, 0.5 μg/kg BW) or with saline on Day 10 after ovulation. Samples were collected 1200 h after treatment and on Day 10 of the respective subsequent (untreated) cycle. The mRNA expression of several possible regulatory factors was investigated, revealing the suppression of PGF2α receptor (PTGFR), STAR protein and 3β-hydroxysteroid dehydrogenase, compared with controls and subsequent cycles. The expression of TLR2 and TLR4, interleukin 1α (IL1A) and 1β (IL1B) and of PGF2α and PGE2 synthases (HSD20A and mPTGES respectively) was increased. The results demonstrate the presence of TLR2 and TLR4 in the bovine CL, and implicate their possible involvement in the deleterious effects of LPS on its function.

Repeated intrauterine infusions of lipopolysaccharide alter gene expression and lifespan of the bovine corpus luteum

Inflammation of the uterus is associated with disturbed ovarian function and reduced reproductive performance in dairy cows. To investigate the influence of endometritis on the bovine corpus luteum, 8 heifers received intrauterine infusions with either phosphate-buffered saline (PBS; 9mL) or Escherichia coli lipopolysaccharide (LPS; 3µg/kg of body weight diluted in 9mL of PBS) at 6-h intervals from 12h before and until 9d after ovulation during 2 cycles in a random order (ovulation=d 1). An untreated cycle was examined before and after PBS and LPS cycles, and the mean values from both untreated cycles were used as control. In all cycles, blood sampling and ultrasonography of the ovaries were performed on d 0, 1, 2, 4, 6, 8, 9, 10, 12, 15, 18, and then every 2d until ovulation. Endometrial cells were collected for cytology and quantitative real-time reverse transcriptase PCR on d 0, 6, and 9, and on d 0 and 6, respectively, and luteal tissue was collected for quantitative real-time reverse transcriptase PCR on d 6 and 9. Both, PBS and LPS infusions induced subclinical endometritis, which was accompanied by increased endometrial mRNA abundance of proinflammatory cytokines IL1β, IL8, and tumor necrosis factor α. Additionally, LPS challenge induced premature luteolysis, which was characterized by increased plasma concentrations of PGF2α metabolite, decreased plasma progesterone concentrations, and reduced luteal size and blood flow compared with the control. The luteal mRNA expression of the LPS receptor TLR4, PGE synthase, and the apoptosis-related factor CASP3 were higher, and those of steroidogenic factors STAR and HSD3B, the PGF receptor, and the angiogenic factor VEGFA121 were lower after LPS challenge compared with the control. In conclusion, repeated intrauterine LPS infusions during the first 9d of the estrous cycle alter gene expression and shorten the lifespan of the bovine corpus luteum.

Ultrasonographic Doppler Use for Female Reproduction Management

Transrectal color Doppler ultrasonography is a useful technique to get new information about physiologic and pathophysiologic alterations of the uterus and ovaries in female cattle. During all reproductive stages characteristic changes in uterine blood flow are observed. Cows with puerperal disturbances show delayed decrease in uterine blood flow in the first few weeks postparturition compared with healthy cows. Measurement of follicular blood flow is used to identify normally developing follicles and predict superovulatory response. Determination of luteal blood is more reliable than B-mode sonography to distinguish between functional and nonfunctional corpora lutea. Color Doppler ultrasonography is a promising tool to improve reproductive management in female cattle.

Different chronological patterns of appearance of blood derived milk components during mastitis indicate different mechanisms of transfer from blood into milk.

This study aimed to describe chronological patterns of changes of various candidate blood components in milk during the acute phase of a mammary immune response in detail. Eight dairy cows were challenged with Escherichia coli lipopolysaccharide in one udder quarter. Milk from challenged and control quarters and blood samples were taken before, and 1 and 2 h after challenge and then every 15 min until 5 h after challenge. The SCC, serum albumin, immunoglobulin (Ig)G1, IgG2, lactate dehydrogenase (LDH), and L-lactate in milk and blood, and α-lactalbumin in blood were analysed. All selected parameters in milk increased in challenged quarters but did not increase in control quarters. Milk IgG1, IgG2, serum albumin, and LDH were already significantly increased at 2 h after challenge whereas a significant increase of SCC was detectable at 2.75 h and L-lactate was increased at 2.25 h after challenge. In blood L-lactate was increased at 3.75 h after challenge, however, other factors in blood did not change significantly within the 5 h of experiment. In conclusion, the increase of blood components in milk during inflammation follows two different patterns: There is a rapid increase for IgG1, IgG2, or LDH, before the increase of SCC, and their concentrations reach a plateau within 3 h. On the other hand, SCC and L-lactate show a slower but consistent increase not reaching a plateau within 5 h after LPS challenge. L-lactate increases to higher concentrations in milk than in blood. This clearly shows that the increase of blood components follows different patterns and is therefore a controlled and compound-specific process and not exclusively an unspecific type of leakage.

Ultrasonographic examination reduces the percentage of unsuccessful inseminations in dairy cows

The objective of this study was to investigate whether confirmation of suitability for service by traditional methods, observation and transrectal palpation, is improved in cattle by additional ultrasonographic examination of the uterus and ovaries. The data from 600 lactating cows from Southern Germany submitted for the first service and alternately assigned to manual (group M, n = 301) and manual and sonographic examination (group S, n = 299) were evaluated. Cows of group M with a large, soft follicle with an estimated diameter of 12.0 to 22.5 mm, without a palpable CL (>10 mm), and with a contractile uterus without palpable content were considered suitable for service and inseminated. Cows of group S with a soft follicle measuring 12.0 to 22.5 mm, without a CL (>10 mm), and with a contractile uterus and no echoic content were inseminated. Pregnancy was determined by ultrasonography at 28 to 35 days after the first service. In group S, 10.9% fewer cows (P = 0.003) were considered suitable for service than in group M. The main reason (29.6%) for exclusion from service in group S was the presence of a CL greater than 10 mm in addition to a preovulatory follicle (12.0-22.5 mm), which was diagnosed less frequently in group M (4.5%; P < 0.0001). The first-service conception rate tended to be higher in group S than in group M (62.7% vs. 54.9%; P = 0.10), and the percentage of open cows, based on all cows submitted for service, was higher in group M than in group S (35.2% vs. 25.1%; P = 0.007). We concluded that sonographic examination of cows submitted for service facilitates the exclusion from service of proestrous and diestrous cows and reduces the number of pointless services.

Body condition loss and increased serum levels of nonesterified fatty acids enhance progesterone levels at estrus and reduce estrous activity and insemination rates in postpartum dairy cows

Data from 96 Holstein Friesian cows on a commercial dairy farm were used to investigate whether body condition and serum levels of nonesterified fatty acids (NEFAs) postpartum (pp) affect progesterone (P4) levels, estrous activity, and fertility in dairy cows. The examination period started 14 days before the expected calving date and ended either when a cow was inseminated or at a maximum of 90 days pp. Body condition score (BCS; 1-5 scale) and backfat thickness (BFT) were determined every 2 weeks. Blood for analysis of NEFA and P4 concentrations was sampled weekly during the first 35 days pp and then every 48 hours until an ovulation was observed. Transrectal ultrasonography of the ovaries started at 21 days pp and was performed after blood sampling. If cows were not inseminated because of silent ovulation, sampling and ultrasonography continued on Days 7, 14, and 18 after ovulation and again every 48 hours until the next ovulation. Estrous activity was continuously measured with the Heatime estrus detection system. Pregnancy controls were performed ultrasonographically 28 and 42 days after AI. Cows with increased NEFA levels at 28 days pp had an increased risk of maintaining minimum P4 levels above 0.4 ng/mL at first recognized estrus (P = 0.03). Higher NEFA levels at Day 7 were associated with lower probability for a cow to have elevated P4 levels (≥2 ng/mL) by Day 35 pp, indicating delayed commencement of luteal activity (C-LA). Estrous activity was not influenced (P > 0.10) by minimum P4 concentrations at estrus, but more animals with C-LA until Day 35 pp showed estrous activity compared to cows without C-LA throughout this period (P = 0.006). Estrous activity was lower in cows with a low BCS 14 days pp (P = 0.02) and with a low BFT 42 days pp (P = 0.03). Moreover, the probability to exhibit estrus was reduced with higher NEFA levels at 21 days pp (P = 0.01). Eighty-five cows were inseminated and 37 (44%) got pregnant after insemination. Higher NEFA levels, especially 14, 28, and 42 days pp, were associated with lower probability to inseminate a cow. Regarding the cows that were inseminated, no influence of BCS, BFT, or NEFA levels on pregnancy per artificial insemination was found. In conclusion, cows with higher loss of body condition and elevated NEFA concentrations had higher P4 concentrations during estrus, a reduced estrous activity and insemination rate, but no depression in pregnancy per artificial insemination.

Intramammary lipopolysaccharide infusion alters gene expression but does not induce lysis of the bovine corpus luteum.

Data from various studies indicate that the ovarian function in dairy cows can be compromised during intramammary infections. Therefore, in this study, we investigated if an experimentally induced mastitis has an effect on corpus luteum (CL) function in 14 lactating cows. On d 9 of the estrous cycle (d 1=ovulation), cows received a single dose of 200 μg of Escherichia coli lipopolysaccharide (LPS; dissolved in 10 mL of NaCL; n=8) or 10 mL of saline (control; n=6) into one quarter of the mammary gland. Measurements included plasma cortisol, haptoglobin, and progesterone (P4) concentrations, as well as luteal size (LTA) and relative luteal blood flow (rLBF). Sampling was performed on d 1, 4, and 8. On d 9, the main examination day, sampling was performed immediately before (0 h), every 1h (or at 3-h intervals for LTA and rLBF) until 9 h, as well as 12 and 24 h after treatment. Thereafter, measurements were taken on d 12, 15, 18, and then every 2 d until ovulation. Luteal tissue was collected for biopsy 24 h before and 6 h after treatment. Quantitative real-time PCR was applied to assess mRNA expression of steroidogenic factors (STAR, HSD3B), caspase 3, toll-like receptors (TLR2, -4), tumor necrosis factor α (TNFA), and prostaglandin-related factors (PGES, PGFS, PTGFR). Intramammary LPS infusion caused considerable inflammatory responses in the treated udder quarters. No decrease in plasma P4 concentrations was noted after LPS-challenge, and P4 levels did not differ between LPS-treated and control cows. Furthermore, LTA and rLBF values were not decreased after LPS challenge compared with the values obtained immediately before treatment. However, LPS infusion increased plasma levels of cortisol and haptoglobin compared with the control group. In the CL, mRNA abundance of TLR2 and TNFA was increased in cows after LPS-challenge (but not in control cows), whereas TLR4, steroidogenic, and prostaglandin-related factors remained similar to the mRNA abundance before treatment. In conclusion, intramammary LPS challenge induces systemic inflammatory reactions which alter the luteal mRNA abundance of TLR2 and TNFA but does not induce lysis of the CL.

Effects of GnRH or PGF2α in week 5 postpartum on the incidence of cystic ovarian follicles and persistent corpora lutea and on fertility parameters in dairy cows.

Resumption of ovulatory activity and the timely lysis of the first CL postpartum (pp) are important determinants for the reproductive performance of dairy cows. Cystic ovarian follicles (COFs) and persistent CLs preclude normal ovarian cyclicity and increase the calving interval. The objective of this study was to investigate the effect of GnRH on the incidence of COFs and the effect of PGF2α on the incidence of a prolonged luteal phase (PLP) and on fertility parameters in dairy cows. A total of 476 cows were examined ultrasonographically for the presence of a dominant follicle (12-25 mm, without CL >10 mm; n = 237) or a functional CL (≥20 mm; n = 239) between 28 and 35 days pp and were allocated to one of four groups. Cows with a dominant follicle received 10-μg GnRH (buserelin; group F-T; n = 118) or saline (group F-C; n = 119), and cows with a functional CL received 0.5 mg of a PGF2α analogue (cloprostenol; group CL-T; n = 119) or saline (group CL-C; n = 120) on the day of initial examination, defined as Day 0. Cows were reexamined 7 and 21 days (F-T and F-C) and 3 and 24 days (CL-T and CL-C) later, and COFs were treated immediately after diagnosis in all cows. On the basis of the ovarian findings on Days 21 and 24, cows were treated according to a protocol aimed at timely breeding. The incidence of COFs by Days 7 (F-T vs. F-C; 7.6% vs. 16.8%) and 21 (11.0% vs. 21.8%) decreased (P ≤ 0.03) with GnRH; however, this did not lead to a substantial improvement of calving-to-conception interval (means ± standard error of the mean; 107.91 ± 5.70 vs. 117.94 ± 6.63 days), first-service conception rate (42.3% vs. 41.3%), and number of services per conception (2.06 ± 0.12 vs. 2.31 ± 0.15). Treatment with PGF2α decreased (P < 0.0001) the incidence of PLP by Day 24 (CL-T vs. CL-C; 1.7% vs. 17.5%), decreased calving-to-conception interval(91.28 ± 4.77 vs. 101.75 ± 5.03 days), increased first-service conception rate (63.3% vs. 38.7%), and reduced the number of services per conception (1.65 ± 0.10 vs. 2.08 ± 0.12; each P ≤ 0.01). The results indicate that strategic treatment with GnRH or PGF2α in week 5 pp to induce early ovulation and luteolysis reduces the incidence of COFs and PLP, respectively. Initial treatment with PGF2α also enhanced reproductive performance when used in conjunction with a standardized treatment protocol for all cows in week 8 pp (aimed at timely breeding). In contrast, GnRH did not improve fertility parameters of cystic cows in herds where all cows with COFs were treated as expeditiously as possible.