S. Meier

Modulation of the maternal immune system by the pre-implantation embryo

BackgroundA large proportion of pregnancy losses occur during the pre-implantation period, when the developing embryo is elongating rapidly and signalling its presence to the maternal system. The molecular mechanisms that prevent luteolysis and support embryo survival within the maternal environment are not well understood. To gain a more complete picture of these molecular events, genome-wide transcriptional profiles of reproductive day 17 endometrial tissue were determined in pregnant and cyclic Holstein-Friesian dairy cattle.ResultsMicroarray analyses revealed 1,839 and 1,189 differentially expressed transcripts between pregnant and cyclic animals (with ≥ 1.5 fold change in expression; P-value < 0.05, MTC Benjamini-Hochberg) in caruncular and intercaruncular endometrium respectively. Gene ontology and biological pathway analysis of differentially expressed genes revealed enrichment for genes involved in interferon signalling and modulation of the immune response in pregnant animals.ConclusionThe maternal immune system actively surveys the uterine environment during early pregnancy. The embryo modulates this response inducing the expression of endometrial molecules that suppress the immune response and promote maternal tolerance to the embryo. During this period of local immune suppression, genes of the innate immune response (in particular, antimicrobial genes) may function to protect the uterus against infection.

Evaluation of real-time PCR endogenous control genes for analysis of gene expression in bovine endometrium

BackgroundQuantitative real-time PCR gene expression results are generally normalised using endogenous control genes. These reference genes should be expressed at a constant level across all sample groups in a study, and should not be influenced by study treatments or conditions. There has been no systematic investigation of endogenous control genes for bovine endometrium to date. The suitability of both commonly used and novel endogenous control genes was evaluated in this study, with the latter being selected from stably expressed transcripts identified through microarray analysis of bovine endometrium. Fifteen candidate endogenous control genes were assessed across different tissue subtypes in pregnant and cycling Holstein-Friesian dairy cows from two divergent genetic backgrounds.ResultsThe expression profiles of five commonly used endogenous control genes (GAPDH, PPIA, RPS9, RPS15A, and UXT) and 10 experimentally derived candidate endogenous control genes (SUZ12, C2ORF29, ZNF131, ACTR1A, HDAC1, SLC30A6, CNOT7, DNAJC17, BBS2, and RANBP10) were analysed across 44 samples to determine the most stably expressed gene. Gene stability was assessed using the statistical algorithms GeNorm and Normfinder. All genes presented with low overall variability (0.87 to 1.48% CV of Cq). However, when used to normalise a differentially expressed gene (oxytocin receptor - OXTR) in the samples, the reported relative gene expression levels were significantly affected by the control gene chosen. Based on the results of this analysis, SUZ12 is proposed as the most appropriate control gene for use in bovine endometrium during early pregnancy or the oestrus cycle.ConclusionThis study establishes the suitability of novel endogenous control genes for comparing expression levels in endometrial tissues of pregnant and cycling bovines, and demonstrates the utility of microarray analysis as a method for identifying endogenous control gene candidates.

Relationships between cytology, bacteriology and vaginal discharge scores and reproductive performance in dairy cattle

The objective was to compare three diagnostic approaches for intrauterine infection and inflammation: scoring of vaginal contents; quantification of percentage of nucleated cells that were polymorphonuclear leukocytes (PMN) following endometrial cytology; and intra-uterine bacteriology. Dairy cows (n = 303) were examined twice, Days 28 (D28) and 42 (D42), where Day 0 = day of calving. Associations between gross vaginal inflammation scores, uterine cytology, and bacteriology, and subsequent reproductive performance were examined using multivariable models. There was fair agreement at D28 (Kappa = 0.29), but only slight agreement at D42 (Kappa < 0.15), between PMN% and gross vaginal inflammation score. Cows were categorized as having PMN% in the highest quartile (H), or not (L), at both D28 and D42; therefore, cows were categorized as PMNLL, PMNLH, PMNHL, or PMNHH. Cows in the highest PMN% quartile at both time periods were slower to conceive (P < 0.001) than those in all other quartiles (mean ± SEM 32.2 ± 2.3, 37.0 ± 5.3, 40.8 ± 4.1, and 55.3 ± 7.3 d from start of breeding to conception for PMNLL, PMNLH, PMNHL, and PMNHH PMN% cows, respectively). Milk yield was greater (P = 0.001) in cows in the lower quartiles for PMN% at D28 and D42 (i.e., PMNLL) than those in the PMNHH and PMNHL categories, with PMNLH intermediate (P = 0.001). We concluded that PMN% was a better predictor of reproductive performance than either intra-uterine bacteriology or gross vaginal inflammation score. Cows in the highest quartile for PMN% at both D28 and D42 had lower pregnancy rates, took longer to conceive, and had a lower milk yield than those in the lower PMN% categories.

Relationships between endometritis and metabolic state during the transition period in pasture-grazed dairy cows

The primary objective of this study was to identify relationships between endometritis and metabolic state during the calving transition and early lactation periods. A subset of mixed age and breed dairy cows (n=78) from a seasonal, pasture-grazed herd of 389 cows was examined. The selected cows were grouped as having endometritis at d 42 postpartum or being unaffected by endometritis. Endometritis was defined as >6% (upper quartile) of uterine nucleated cells being polymorphonuclear cells (H-PMN; n=38); unaffected by endometritis was defined as ≤1% of nucleated cells being polymorphonuclear (L-PMN; n=40). Milk yield was determined at each milking, and milk composition (fat and protein) was determined at 2-wk intervals. Blood samples collected on d -14, 0 (d of calving), 4, 7, 14, 28, and 42 were analyzed for indicators of energy status (nonesterified fatty acids, glucose, and urea), liver function (albumin, globulin, glutamate dehydrogenase, and aspartate aminotransferase), inflammation (haptoglobin), and mineral status (Ca and Mg). Samples collected weekly from d 21 to 63 or 70 were analyzed for progesterone content. The postpartum anovulatory interval was defined to end on the first day postpartum that plasma progesterone concentration was ≥1 ng/mL. A greater percentage of H-PMN cows failed to ovulate before d 63 or 70 (34%) compared with L-PMN cows (10%), although the proportions of cows ovulating within either polymorphonuclear group was similar through d 56 postpartum. Plasma concentrations of albumin and the albumin:globulin ratio were consistently lower in H-PMN cows. Plasma Mg was lower, whereas glutamate dehydrogenase and aspartate aminotransferase were higher, in H-PMN cows during early lactation compared with L-PMN cows. Circulating metabolites indicative of energy status (nonesterified fatty acids, glucose, and urea) were not different between polymorphonuclear groups. Among 3- to 5-yr-old cows, daily milk yield for the first 42 d after calving was lower for H-PMN cows than for L-PMN cows. Among cows >5 yr old, protein percentage was lower in H-PMN cows compared with L-PMN cows. In summary, endometritis at 42 d postpartum in the herd studied was associated with an increased likelihood of remaining anovulatory. These cows had lower albumin concentrations throughout the calving transition period, perhaps indicating impaired liver function, with lower plasma Mg and evidence of hepatocellular damage in early lactation. Similar profiles of nonesterified fatty acids and glucose indicated that energy status was not a risk factor for endometritis.

Body condition score at calving affects systemic and hepatic transcriptome indicators of inflammation and nutrient metabolism in grazing dairy cows.

Calving body condition score (BCS) is an important determinant of early-lactation dry matter intake, milk yield, and disease incidence. The current study investigated the metabolic and molecular changes induced by the change in BCS. A group of cows of mixed age and breed were managed from the second half of the previous lactation to achieve mean group BCS (10-point scale) that were high (HBCS, 5.5; n=20), medium (MBCS, 4.5; n=18), or low (LBCS, 3.5; n=19). Blood was sampled at wk -4, -3, -2, 1, 3, 5, and 6 relative to parturition to measure biomarkers of energy balance, inflammation, and liver function. Liver was biopsied on wk 1, 3, and 5 relative to parturition, and 10 cows per BCS group were used for transcript profiling via quantitative PCR. Cows in HBCS and MBCS produced more milk and had greater concentrations of nonesterified fatty acids and β-hydroxybutyrate postpartum than LBCS. Peak concentrations of nonesterified fatty acids and β-hydroxybutyrate and greater hepatic triacylglycerol concentrations were recorded in HBCS at wk 3. Consistent with blood biomarkers, HBCS and MBCS had greater expression of genes associated with fatty acid oxidation (CPT1A, ACOX1), ketogenesis (HMGCS2), and hepatokines (FGF21, ANGPTL4), whereas HBCS had the lowest expression of APOB (lipoprotein transport). Greater expression during early lactation of BBOX1 in MBCS and LBCS suggested greater de novo carnitine synthesis. The greater BCS was associated with lower expression of growth hormone/insulin-like growth factor-1 signaling axis genes (GHR1A, IGF1, and IGFALS) and greater expression of gluconeogenic genes. These likely contributed to the higher milk production and greater gluconeogenesis. Despite greater serum haptoglobin around calving, cows in HBCS and MBCS had greater blood albumin. Cows in MBCS, however, had a higher albumin:globulin ratio, probably indicating a less pronounced inflammatory status and better liver function. The marked decrease in expression of NFKB1, STAT3, HP, and SAA3 coupled with the increase in ALB on wk 3 in MBCS cows were consistent with blood measures. Overall, results suggest that the greater milk production of cows with higher calving BCS is associated with a proinflammatory response without negatively affecting expression of genes related to metabolism and the growth hormone/insulin-like growth factor-1 axis. Results highlight the sensitivity of indicators of metabolic health and inflammatory state to subtle changes in calving BCS and, collectively, indicate a suboptimal health status in cows calving at either BCS 3.5 or 5.5 relative to BCS 4.5.

Calving body condition score affects indicators of health in grazing dairy cows

The objectives of this study were to determine the effect of calving body condition score (BCS) on cow health during the transition period in a pasture-based dairying system. Feed inputs were managed during the second half of the previous lactation so that BCS differed at drying off (BCS 5.0, 4.0, and 3.0 for high, medium, and low treatments, respectively: a 10-point scale); feed allowance was managed after cows were dried off, such that the BCS differences established during lactation remained at the subsequent calving (BCS 5.5, 4.5, and 3.5; n=20, 18, and 19, for high, medium, and low treatments, respectively). After calving, cows were allocated pasture and pasture silage to ensure grazing residuals >1,600 kg of DM/ha. Milk production was measured weekly; blood was sampled regularly pre- and postpartum to measure indicators of health, and udder and uterine health were evaluated during the 6 wk after calving. Milk weight, fat, protein, and lactose yields, and fat content increased with calving BCS during the first 6 wk of lactation. The effect of calving BCS on the metabolic profile was nonlinear. Before calving, cows in the low group had lower mean plasma β-hydroxybutyrate and serum Mg concentrations and greater mean serum urea than cows in the medium and high BCS groups, which did not differ from each other. During the 6 wk after calving, cows in the low group had lower serum albumin and fructosamine concentrations than cows in the other 2 treatment groups, whereas cows in the low- and medium-BCS groups had proportionately more polymorphonucleated cells in their uterine secretions at 3 and 5 wk postpartum than high-BCS cows. In comparison, plasma β-hydroxybutyrate and nonesterified fatty acid concentrations increased linearly in early lactation with calving BCS, consistent with a greater negative energy balance in these cows. Many of the parameters measured did not vary with BCS. The results highlight that calving BCS and, therefore, BCS through early lactation are not effective indicators of functional welfare, with the analyses presented indicating that both low and high BCS at calving will increase the risk of disease: cows in the low group were more prone to reproductive compromise and fatter cows had an increased risk of metabolic diseases. These results are important in defining the welfare consequences of cow BCS.

Endometrial gene expression during early pregnancy differs between fertile and subfertile dairy cow strains

A receptive uterine environment is a key component in determining a successful reproductive outcome. We tested the hypothesis that endometrial gene expression patterns differ in fertile and subfertile dairy cow strains. Twelve lactating dairy cattle of strains characterized as having fertile (n = 6) and subfertile (n = 6) phenotypes underwent embryo transfer on day 7 of the reproductive cycle. Caruncular and intercaruncular endometrial tissue was obtained at day 17 of pregnancy, and microarrays used to characterize transcriptional profiles. Statistical analysis of microarray data at day 17 of pregnancy revealed 482 and 1,021 differentially expressed transcripts (P value < 0.05) between fertile and subfertile dairy cow strains in intercaruncular and caruncular tissue, respectively. Functional analysis revealed enrichment for several pathways involved in key reproductive processes, including the immune response to pregnancy, luteolysis, and support of embryo growth and development, and in particular, regulation of histotroph composition. Genes implicated in the process of immune tolerance to the embryo were downregulated in subfertile cows, as were genes involved in preventing luteolysis and genes that promote embryo growth and development. This study provides strong evidence that the endometrial gene expression profile may contribute to the inferior reproductive performance of the subfertile dairy cow strain.

Effects of an acute feed restriction at the onset of the seasonal breeding period on reproductive performance and milk production in pasture-grazed dairy cows.

Reproduction and milk production responses were compared between dairy cows offered a high (HPA; n=453 in 4 replicates) or low (LPA; n=317 in 3 replicates) pasture allowance for the first 14 d after the planned start of mating (d 0 of experiment) in 2 seasonal, pasture-based dairy herds. Estimated pasture dry matter intake (DMI) between d -1 and 13 were 14.3+/-2.8 and 8.0+/-1.7kg of DM/cow per day for HPA and LPA cows, respectively. The LPA cows produced 22% less energy-corrected milk during the 14-d feeding treatment period, and milk yield remained less in LPA cows at d 53 despite all cows receiving pasture allowances to support DMI of 16kg of dry matter/cow per day from d 14 onwards. The 3-wk submission rate (percentage of cows inseminated at least once from d 0 to 20) and the 3- and 6-wk pregnancy rates of LPA cows (88, 45, and 71%, respectively) were lower than those of HPA cows (94, 53, and 78%, respectively), but conception rates to first (50%) or second (47%) service and final pregnancy rates (93%) did not differ between treatments. These results quantify the immediate and subsequent responses in reproductive performance and milk production to a severe restriction in DMI at the onset of a seasonal breeding period in pasture-grazed dairy cows.

Grazing dairy cows had decreased interferon-γ, tumor necrosis factor, and interleukin-17, and increased expression of interleukin-10 during the first week after calving

Peripartum, and especially during the transition period, dairy cows undergo dramatic physiological changes. These coincide with an increased risk of disease during the first 2 wk after calving and have been linked to dairy cows failing to achieve production as well as reproductive targets. Previous evidence suggests that these physiological changes affect the immune system and that transition dairy cows experience some form of reduced immunocompetence. However, almost all of these studies were undertaken in high-production, housed dairy cows. Grazing cows have much lower levels of production and this study aimed to provide clarity whether or not the dysfunctional attributes of the peripartum immune system reported in high production housed cows are evident in these animals. Therefore, cell culture techniques, flow cytometry, and quantitative PCR were applied to analyze the cellular composition of peripheral blood mononuclear cells from transition dairy cows as well as the performance of these cells in an in vitro assay. First, a combination of in vitro stimulation and quantitative PCR for cytokines was validated as a quantifiable immunocompetence assay in 29 cattle and a correlation of quantitative PCR and ELISA demonstrated. Second, the relative number of T helper cells, cytotoxic T cells, B cells, γδ T cells, natural killer cells, and monocytes in peripheral blood was measured, of which B cells and natural killer cells increased in number postcalving (n=29) compared with precalving. Third, following in vitro stimulation cytokine profiles indicated decreased expression of IFNγ, tumor necrosis factor, and IL-17 and increased expression of IL-10 wk 1 after calving, which later all returned to precalving values (n=39). Additionally, treatment of transition cows with a nonsteroidal anti-inflammatory drug (i.e., carprofen) administered on d 1, 3, and 5 postcalving (n=19; untreated control n=20) did not affect the cytokine expression at any time point. In conclusion, an immunocompetence assay has been developed that highlights a characteristic expression pattern for IFNγ, tumor necrosis factor, IL-17, and IL-10 that reflects a state of reduced immunocompetence in moderate-yielding pasture-based transition cows after calving, which is similar to that described for higher-yielding housed cows.

Production and reproduction of Fleckvieh, Brown Swiss, and 2 strains of Holstein-Friesian cows in a pasture-based, seasonal-calving dairy system

The first objective of this study was to compare the productive and reproductive performance of Holstein-Friesian (CH HF), Fleckvieh (CH FV), and Brown Swiss (CH BS) cows of Swiss origin with New Zealand Holstein-Friesian (NZ HF) cows in pasture-based compact-calving systems; NZ HF cows were chosen as the reference population for such grazing systems. The second objective was to analyze the relationships within and between breeds regarding reproductive performance, milk yield, and body condition score (BCS) dynamics. On 15 commercial Swiss farms, NZ HF cows were paired with Swiss cows over 3 yr. Overall, the study involved 259 complete lactations from 134 cows: 131 from 58 NZ HF, 40 from 24 CH HF, 43 from 27 CH FV, and 45 from 25 CH BS cows. All production parameters were affected by cow breed. Milk and energy-corrected milk yield over 270 d of lactation differed by 1,000 kg between the 2 extreme groups; CH HF having the highest yield and CH BS the lowest. The NZ HF cows had the greatest milk fat and protein concentrations over the lactation and exhibited the highest lactation persistency. Body weight differed by 90 kg between extreme groups; NZ HF and CH BS being the lightest and CH HF and CH FV the heaviest. As a result, the 2 HF strains achieved the highest milk production efficiency (270-d energy-corrected milk/body weight(0.75)). Although less efficient at milk production, CH FV had a high 21-d submission rate (86%) and a high conception rate within 2 inseminations (89%), achieving high pregnancy rates within the first 3 and 6 wk of the breeding period (65 and 81%, respectively). Conversely, poorer reproductive performance was recorded for CH HF cows, with NZ HF and CH BS being intermediate. Both BCS at nadir and at 100 d postpartum had a positive effect on the 6-wk pregnancy rate, even when breed was included in the model. The BCS at 100 d of lactation also positively affected first service conception rate. In conclusion, despite their high milk production efficiency, even in low-input systems, CH HF were not suited to pasture-based seasonal-calving production systems due to poor reproductive performance. On the contrary, CH FV fulfilled the compact-calving reproduction objectives and deserve further consideration in seasonal calving systems, despite their lower milk production potential.