Harold V. Henderson

Human absorption and metabolism of oleuropein and hydroxytyrosol ingested as olive (Olea europaea L.) leaf extract.

Phenolic compounds derived from the olive plant (Olea europaea L.), particularly hydroxytyrosol and oleuropein, have many beneficial effects in vitro. Olive leaves are the richest source of olive phenolic compounds, and olive leaf extract (OLE) is now a popular nutraceutical taken either as liquid or capsules. To quantify the bioavailability and metabolism of oleuropein and hydroxytyrosol when taken as OLE, nine volunteers (five males) aged 42.8 ± 7.4 years were randomized to receive either capsulated or liquid OLE as a single lower (51.1 mg oleuropein, 9.7 mg hydroxytyrosol) or higher (76.6 mg oleuropein, 14.5 mg hydroxytyrosol) dose, and then the opposite strength (but same formulation) a week later. Plasma and urine samples were collected at fixed intervals for 24 h post-ingestion. Phenolic content was analyzed by LC-ESI-MS/MS. Conjugated metabolites of hydroxytyrosol were the primary metabolites recovered in plasma and urine after OLE ingestion. Peak oleuropein concentrations in plasma were greater following ingestion of liquid than capsule preparations (0.47 versus 2.74 ng/mL; p = 0.004), but no such effect was observed for peak concentrations of conjugated (sulfated and glucuronidated) hydroxytyrosol (p = 0.94). However, the latter peak was reached earlier with liquid preparation (93 versus 64 min; p = 0.031). There was a gender effect on the bioavailability of phenolic compounds, with males displaying greater plasma area under the curve for conjugated hydroxytyrosol (11,600 versus 2550 ng/mL; p = 0.048). All conjugated hydroxytyrosol metabolites were recovered in the urine within 8 h. There was wide inter-individual variation. OLE effectively delivers oleuropein and hydroxytrosol metabolites to plasma in humans.

Changes in electrical conductivity and somatic cell count between milk fractions from quarters subclinically infected with particular mastitis pathogens

Cows with subclinical intramammary infections were identified by milk bacteriology. The mastitis pathogens included Staphylococcus aureus (n = 9), Streptococcus uberis (n = 10) and coagulase-negative staphylococci (n = 10). Samples of first fore milk, main flow milk and strippings milk fractions were collected from each quarter and laboratory measurements were made of electrical conductivity, milk fat concentration and somatic cell count. Conductivity measurements were corrected for milk fat concentration and within-cow inter-quarter conductivity ratios calculated. Repeatability estimates of all measurements between days were calculated. In the case of infected quarters, all conductivity values decreased markedly (P < 0.05) from first fore milk to main flow milk fractions. Conductivity differences between quarters of infected cows were substantially lower during the main milk flow phase. For quarters infected with Staph. aureus an increase in conductivity was observed (P < 0.05) from main flow to strippings fractions. For uninfected quarters, conductivity declined as milk fat concentration increased with successive milk fractions. Variation, both within and between milk fractions, was greater for somatic cell count than for conductivity. Differences in conductivity between milk fractions from individual infected quarters were not accounted for by changes in fat concentration and may result from the mixing of milk from infected and uninfected regions of the gland. Localized infection may produce a decrease in conductivity between fore milk and mid-flow fractions while differential drainage from an infection site in the secretory tissue may additionally produce an increase in conductivity from mid-flow to strippings fractions. Such changes may thus provide information on the location and magnitude of an infection. The results clearly demonstrate the importance of the milk fraction when using conductivity as a diagnostic of intramammary infection, the highest diagnostic sensitivity being achieved by using first fore milk samples.

Response of Jersey and Friesian cows to once a day milking and prediction of response based on udder characteristics and milk composition

The response of dairy cows to once a day (OAD) milking was investigated in four trials. Two trials involved Friesians (F) and Jersey cows selected for high (HP) or low (LP) milk protein concentration. Trial 1 compared 74 HP, LP and F cows milked either OAD or twice daily (TAD) for 2 weeks in both mid and late lactation. Trial 2 compared 98 HP, LP and F cows milked OAD or TAD for 12 weeks in early lactation. Trial 3 investigated the repeatability of production loss by milking identical twin cows either OAD or TAD during three 1 week periods. Trial 4 compared the time course of milk production in 32 cows milked every 12 or 24 h for 72 h. Cows milked OAD produced 10–28% less milk and milk solids in early to mid lactation and 9–13% less in late lactation than cows milked TAD. Losses in fat and protein yield were slightly lower than those of milk. HP, LP and F cows did not differ in percentage loss in milk and milk solids in Trial 1, except for loss in protein yield which was lower for HP than LP and F cows. In Trial 2 the losses in milk and milk solids were lower for HP cows than for F cows but the other differences were not significant. Milk storage ability of the udder (hours worth of secretion) was higher and productivity of the udder was lower for HP cows than F cows. Levels of residual milk did not differ among groups. The loss in udder capacity during lactation was greater for cows milked OAD than for those milked TAD. The correlations for repeated estimates of percentage losses in yields of milk and milk solids between successive OAD periods ranged from 0·29 to 0·49. Secretion rate of cows milked OAD was lower during the second 12 h than the first 12 h during each 24 h milking interval. Prediction of OAD performance based on milk composition and udder characteristics was poor. A high level of residual milk was the factor most consistently associated with high loss on OAD milking.

Effect of gestation length on the levels of five innate defence proteins in human milk

BACKGROUND Human milk contains a range of host defence proteins that appear to contribute to health and wellbeing, but their variability in abundance among individuals has not been very well characterised. Milk from mothers of premature infants has altered composition, but the effect of gestation length on the host-defence properties of milk is not known. A study was therefore undertaken to determine the variability and effect of gestation length on the abundance of five host-defence proteins in milk; lactoferrin, secretory IgA, IgG, secretory component, and complement C3. METHODS Milk was obtained from 30 mothers at their second and fifth week of lactation. These were from three groups of ten mothers having had very premature (V; 28-32 weeks gestation), premature (P; 33-36 weeks) or full term deliveries (T; 37-41 weeks). The concentration of each of the five proteins was measured in each milk sample by either ELISA or quantitative western blotting. RESULTS The concentration of IgG, and complement C3 ranged 22- and 17-fold respectively between mothers, while lactoferrin, secretory IgA, and secretory component ranged 7-, 9-, and 4-fold, respectively. The V group had significantly lower concentrations of four of the five proteins, the exception being IgG. Levels of these four proteins also decreased between weeks 2 and 5 of lactation in the P and T groups. Significant correlation was found between the concentrations of the host defence proteins within individual mothers, indicating some degree of co-ordinate regulation. CONCLUSIONS Mothers vary widely in the levels of host defence proteins in milk. Very short gestation length results in decreased abundance of host-defence proteins in milk. This may have functional implications for very premature infants.

Genome-wide DNA methylation patterns and transcription analysis in sheep muscle.

DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS). While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep Longissimus dorsi muscles. RRBS analysis of ∼1% of the genome from Longissimus dorsi muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep Longissimus dorsi muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species.

Signal Inhibition Reveals JAK/STAT3 Pathway As Critical for Bovine Inner Cell Mass Development

ABSTRACT The inner cell mass (ICM) of mammalian blastocysts consists of pluripotent epiblast and hypoblast lineages, which develop into embryonic and extraembryonic tissues, respectively. We conducted a chemical screen for regulators of epiblast identity in bovine Day 8 blastocysts. From the morula stage onward, in vitro fertilized embryos were cultured in the presence of cell-permeable small molecules targeting nine principal signaling pathway components, including TGFbeta1, BMP, EGF, VEGF, PDGF, FGF, cAMP, PI3K, and JAK signals. Using 1) blastocyst quality (by morphological grading), 2) cell numbers (by differential stain), and 3) epiblast (FGF4, NANOG) and hypoblast (PDGFRa, SOX17) marker gene expression (by quantitative PCR), we identified positive and negative regulators of ICM development and pluripotency. TGFbeta1, BMP, and cAMP and combined VEGF/PDGF/FGF signals did not affect blastocyst development while PI3K was important for ICM growth but did not alter lineage-specific gene expression. Stimulating cAMP specifically increased NANOG expression, while combined VEGF/PDGF/FGF inhibition up-regulated epiblast and hypoblast markers. The strongest effects were observed by suppressing JAK1/2 signaling with AZD1480. This treatment interfered with ICM formation, but trophectoderm cell numbers and markers (CDX2, KTR8) were not altered. JAK inhibition repressed both epiblast and hypoblast transcripts as well as naive pluripotency-related genes (KLF4, TFCP2L1) and the JAK substrate STAT3. We found that tyrosine (Y) 705-phosphorylated STAT3 (pSTAT3Y705) was restricted to ICM nuclei, where it colocalized with SOX2 and NANOG. JAK inhibition abolished this ICM-exclusive pSTAT3Y705 signal and strongly reduced the number of SOX2-positive nuclei. In conclusion, JAK/STAT3 activation is required for bovine ICM formation and acquisition of naive pluripotency markers.

Effect of localised antibiotic infusions applied to the teat-canal and teat sinus at drying-off on mastitis in the dry-period and at calving

An experiment using three New Zealand herds and a total of 632 cows, examined the effect of localised prophylactic treatments with antibiotic at drying-off on the incidence of new intramammary infection during the dry period and at calving. Antibiotic was infused either into the teat canal (0.22 g of dry-cow formulation) or the teat sinus (3.1 g of lactating-cow formulation) of uninfected quarters to eliminate any bacteria present in these locations at the last milking of lactation. These treatments were compared with a negative control (nil treatment) and a positive antibiotic control (infusion of 3.6 g of dry-cow formulation). All antibiotic formulations used the same active ingredient, sodium cloxacillin. No significant reduction in new dry period clinical mastitis was observed for the two localised treatments whereas the positive control treatment achieved 100% reduction in new clinical mastitis compared with untreated control quarters. A 41% reduction (P < 0.05) in new Streptococcus uberis infections at calving was associated with the teat canal antibiotic treatment, compared with an 82% reduction (P < 0.001) for the positive antibiotic control. Both localised treatments showed a reduced incidence of new intramammary infection (P < 0.001) when pooled across periods and pathogens. Teats receiving either the teat canal antibiotic treatment or a full infusion of long acting dry-cow antibiotic had a lower incidence of open teat canals (P < 0.05) at 3 weeks after drying-off.

Genome-wide DNA methylation analysis: no evidence for stable hemimethylation in the sheep muscle genome

The importance of maintaining DNA methylation patterns and faithful transmission of these patterns during cell division to ensure appropriate gene expression has been known for many decades now. It has largely been assumed that the symmetrical nature of CpG motifs, the most common site for DNA methylation in mammals, together with the presence of maintenance methylases able to methylate newly synthesised DNA, ensures that there is concordance of methylation on both strands. However, although this assumption is compelling in theory, little experimental evidence exists that either supports or refutes this assumption. Here, we have undertaken a genome-wide single-nucleotide resolution analysis to determine the frequency with which hemimethylated CpG sites exist in sheep muscle tissue. Analysis of multiple independent samples provides strong evidence that stably maintained hemimethylation is a very rare occurrence, at least in this tissue. Given the rarity of stably maintained hemimethylation, next-generation sequencing data from both DNA strands may be carefully combined to increase the accuracy with which DNA methylation can be measured at single-nucleotide resolution.

Bovine blastocyst development depends on threonine catabolism

Increasing evidence suggests that pluripotency is a metabolically specialised state. In mouse, inner cell mass (ICM) cells and ICM-derived pluripotent stem cells (PSCs) critically depend on catabolising the amino acid threonine, while human PSCs require leucine, lysine, methionine or tryptophan. However, little is known about the specific amino acid requirements of putative pluripotent cells in bovine. We selectively depleted candidate essential amino acids (EAAs) from individually cultured bovine embryos to study their role in blastocyst development. Depleting one (-T, -M), two (-MT, -CM, -CT, -IL, -IK, -KL) or three (-CMT, -IKL) EAAs from chemically defined protein-free culture medium did not affect the morula-to-blastocyst transition from day five (D5) to D8 in vitro. By contrast, removing six (-CIKLMT, -FHRYVW), nine (+CMT, +IKL), eleven EAAs (+T, +M) or all twelve EAAs increasingly impaired blastocyst development. As no clear candidate emerged from this targeted screen, we focussed on threonine dehydrogenase (TDH), which catalyses threonine catabolism. TDH mRNA and protein was present at similar levels in trophectoderm (TE) and ICM but absent from several adult somatic tissues. We then treated morulae with an inhibitor (Qc1) that blocks TDH from catabolising threonine. Continuous exposure to Qc1 reduced total and high-quality blastocyst development from 37% to 26% and 18% to 8%, respectively (P<0.005). This was accompanied by ∼2-fold decrease in ICM, TE and total cell numbers (P<0.005), which was due to increased autophagy (P<0.05). At the same time, ICM-(NANOG) and TE-restricted (KRT8) genes were up-and down-regulated, respectively (P<0.05). In summary, bovine blastocyst viability depended on TDH-mediated threonine catabolism. However, ICM and TE cells did not metabolically differ in this regard, highlighting species-specific connections between metabolism and pluripotency regulation in mouse vs cattle.

Animal physiology and genetic aspects of ryegrass staggers in grazing sheep

Abstract Ryegrass staggers (RGS) is a metabolic disease of herbivores, caused by the ingestion of perennial ryegrass (Lolium perenne L.) containing a fungal endophyte (Neotyphodium lolii) which produces a tremorgenic toxin, lolitrem B. RGS has a major economic impact for agriculture in New Zealand as well as internationally. Management of RGS in grazing sheep can be problematic, and there is an incomplete knowledge of the interaction between the toxin and the grazing animal. This review is focused on recent advances in understanding the molecular physiology of RGS in the affected animal as well as the influence of animal genetics on the degree of susceptibility to RGS. Investigations to date suggest that the primary target for toxin is the large conductance, calcium-activated, potassium (BK) channel, resulting in disruption of neuromuscular junction signalling. Genetic investigation has established the existence of genes influencing resistance to RGS, however their identity has not been confirmed and their impact has not been established. Studies to date suggest that a multi-gene selection approach will be necessary in order to develop an effective selection tool for use in the agricultural industries.