Effects of small milking stalls on stress responses in dairy cows during milking in group milking parlors.

Milking stall dimensions have not been adapted to the increase in cow body size caused by selection for better milking performance over the past decades. Improper milking stall dimensions might limit cow comfort, could lead to stress responses during milking and thus could negatively affect cow welfare. A crossover study was conducted in an experimental milking parlor that was converted from a herringbone (HB) to a side-by-side (SBS) parlor. The milking stall dimensions were modified in length and width and for HB also in depth (perpendicular distance between rump rail and breast rail). The stall dimensions applied during the experiments ranged from much smaller than common in European dairy farming to much larger. Treatments were applied for 2 wk per milking parlor type. In each milking parlor type, a total of 30 cows, kept in 2 groups were observed during milking for behavioral and physiological stress responses and for milking performance. In addition, milk cortisol levels and somatic cell counts were measured at the end of the 2-wk period. Outcome variables were selected based on a principal component analysis and analyzed using mixed effects models reflecting the experimental design. The results showed that the first cow per milking batch required more time (on average >40 s) to enter very small HB stalls than to enter small, large and very large stalls (<30 s). Also, cows yielded more milk per milking in very small and very large HB stalls than in the small and large milking stall dimensions [very small: 15.8 kg; 95% confidence interval (CI), 14.2-17.4 kg); small: 14.3 kg (95% CI, 12.8-15.9 kg); large: 14.6 kg (95% CI, 13.1-16.1 kg); very large: 16.1 kg (95% CI, 14.6-17.6 kg)]. The other behavioral, physiological and milk flow parameters as well as udder health were not affected by stall dimensions. For the SBS parlor, effects of milking stall dimensions were not detectable in any of the parameters. Despite the strong avoidance behavior to enter the milking parlor (measured as latency), no acute stress responses were found during milking. However, the study cannot exclude long-term effects of narrow stall dimensions on stress levels and possibly udder health, which should be investigated in future studies.

Simultaneous quantification of progestogens in plasma and serum by UHPLC-HRMS employing multiplexed targeted single ion monitoring.

Progesterone is the predominant gestagen in most mammals studied so far. It plays a substantial role in the regulation of the female reproductive cycle and in providing support for pregnancy maintenance. Despite its known functions, gaps in knowledge are present regarding its reduced metabolites that potentially exert biological activity. Therefore, a new UHPLC-HRMS method based on a Q Exactive™ mass spectrometer was developed to detect and quantify simultaneously progesterone, its hormone precursor pregnenolone and 10 reduced progestogens (20α-DHP, 20ß-DHP, 3α,5α-THP, 3α,5ß-THP, 3ß,5α-THP, 3ß,5ß-THP, 3α-DHP, 3ß-DHP, 5α-DHP and 5ß-DHP) in plasma and serum samples. Purification was achieved by an optimized solid phase extraction (SPE) and the analysis was conducted in positive electrospray ionization (ESI) mode with the application of multiplexed selected ion monitoring (msx-t-SIM). The method validation included the study of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery and matrix effects. Despite the poor ionization properties of underivatized steroids, a high sensitivity in the range of pg/mL was achieved.

The ACTH challenge and its repeatability in fattening bulls-influences of physiological state, challenge time standardization, and horn status.

Adrenocorticotropic hormone (ACTH) challenges are frequently performed in repetition when evaluating stress or welfare in animals. To our knowledge, the repeatability of ACTH challenges in cattle, although fundamental to further studies of this type, has not yet been the subject of research. Therefore, the objective of this study was to evaluate the repeatability of ACTH challenges in fattening bulls of different horn status. Eight-one bulls were subjected to 3 consecutive ACTH challenges. The first challenge (C1) was performed in calves aged 1.5 mo. Subsequently, animals were characterized as high or low cortisol responders and either disbudded or left with horns. They were then assigned to 1 of 3 rearing groups: a horned group (H+), a disbudded group (H-), and a mixed group (M; 50% horned and 50% disbudded), with each group containing an equal number of high and low responders. The second ACTH challenge (C2) was performed at the age of 11 mo. Time of day (TOD) of challenge was either fixed (ST = same TOD) or alternated (AT = alternate TOD) between C1 and C2. The third ACTH challenge (C3) was performed 7 d after and at the same TOD as C2. Saliva samples were taken 60 and 30 min before and 30, 60, 90, 120, and 150 min after each intravenous ACTH injection. The area under the curve (AUC) was calculated with respect to both ground (AUCG) and to increase (AUCI). The AUCI increased markedly between C1 and C2 (P < 0.05) in ST bulls, and no effects were observed for AUCG between C1 and C2 in ST or AT bulls, nor for any AUC between C2 and C3 (P > 0.1). The overall repeatability of AUCG and AUCI between C1 and C2, reflecting the repeatability between 2 different physiological states, was poor and moderate, respectively, for ST bulls (AUCG: r = 0.24, P > 0.1, intraclass correlation coefficient (ICC) = 0.21; AUCI: r = 0.48, P < 0.01, ICC = 0.41) and lacked in AT bulls (AUCG: r = 0.07, P > 0.1; ICC = 0.03; AUCI: r = 0.08, P > 0.1, ICC = 0.06). The repeatability of AUCG and AUCI between C2 and C3, reflecting the repeatability within the same physiological state, was moderate (AUCG: r = 0.59, P < 0.001; ICC = 0.53; AUCI: r = 0.58, P < 0.001, ICC = 0.52). Assignment to high and low responder groups based on peak cortisol concentration in C1 did not persist over time. H+ bulls showed higher AUCI in C2 and C3 (P < 0.1 and P < 0.05, respectively) than H- bulls. The M group differed from the H- group only in C3 (P < 0.05). Thus, the effect of horn status on ACTH challenges needs further investigation. In conclusion, our results report poor repeatability of the cortisol response to ACTH challenges for challenges performed in different physiological states and moderate repeatability for challenges performed within the same physiological state. Moreover, they point out the importance of standardization of TOD when performing repeated ACTH challenge.

Progestogen profiling in plasma during the estrous cycle in cattle using an LC-MS based approach.

In many mammalian species, corpus luteum derived progesterone (P4) is the main functional gestagen during the estrous cycle and pregnancy. P4 can be metabolized into various metabolites, of which some are biologically active. While some metabolites target the classical nuclear progesterone receptor (PR), neurosteroids bind the receptors of type A γ-aminobutyric acid (GABAA-r) in the brain. According to the position of reduction within the molecule, metabolites of P4 can be characterized into C20-reduced progestogens (20α-dihydroprogesterone (20α-DHP) and 20ß-dihydroprogesterone (20ß-DHP)), C3-reduced progestogens (3α-dihydroprogesterone (3α-DHP) and 3ß-dihydroprogesterone (3ß-DHP)), 5α-reduced progestogens (5α-dihydroprogesterone (5α-DHP), allopregnanolone and isopregnanolone) and 5ß-reduced progestogens (5ß-dihydroprogesterone (5ß-DHP), pregnanolone and epipregnanolone). We questioned whether the reduced progestogens are present in bovine plasma during the estrous cycle and whether their profiles differed from the profile of the common precursor P4 around the time of luteolysis. The analytes were monitored in plasma samples using liquid chromatography mass spectrometry (LC-MS). While progestogens lagged behind the drop of P4 at luteolysis, they followed the profile of P4 during the estrous cycle. The abundance of P4 was predominant followed by allopregnanolone, pregnanolone, epipregnanolone and 20ß-DHP. Further studies will need to focus particularly on the period around luteolysis.

Interferon-τ induced gene expression in bovine hepatocytes during early pregnancy.

Interferon-tau (IFNτ) is the conceptus derived specific early pregnancy signal in bovidae. Locally, IFNτ induces an IFNτ specific gene expression (ISG) in endometrial cells and by this it averts luteolysis of the corpus luteum (CL) by suppressing prostaglandin production. Moreover, it was shown that IFNτ also induces ISG in the liver in pregnant sheep and in liver biopsies from Holstein Friesian heifers on Day 18 of pregnancy. The objective of the present study was to confirm increased hepatic ISG in vivo on Day 18 of pregnancy and to prove if hepatocytes and not non-parenchymal cells react to IFNτ by using immunohistochemistry and primary bovine hepatocytes stimulated in vitro with recombinant bovine IFNτ. For the animal experiment, Angus heifers (n = 12) were cycle synchronized and the Day of ovulation (Day 0) was defined by ovarian ultrasonography and verified by progesterone < 0.1 ng/ml. Heifers were artificially inseminated either with sperm (n = 9) or with seminal plasma (mock control, n = 3). Early pregnancy was defined and detected by progesterone and pregnancy associated glycoprotein (PAG) concentration in blood before and after induced luteolysis by a PGF-injection on Day 21 in n = 3 inseminated heifers. A liver biopsy was taken on Day 18 for the analysis of gene (ISG 15, MX 1, MX 2 and OAS 1) and protein (OAS1) expression using qPCR and immunohistochemistry, respectively. Primary bovine hepatocytes were collected from bull liver using a two-step collagenase perfusion, cultured short-term in a monolayer and stimulated with IFNτ. Thereafter, gene expression was measured by qPCR. In liver biopsies obtained from pregnant heifers ISG was numerically higher expressed compared to biopsies from non-pregnant heifers. Furthermore, the OAS 1 protein expression was localized in hepatocytes on Day 18 of pregnancy. In vitro, primary bovine hepatocytes showed an increased mRNA expression of ISG after IFNτ stimulation. In conclusion, the findings confirm that IFNτ induces ISG in the parenchymal part of the liver in early pregnancy of cattle.

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 IL1ß, 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.