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Background: The setting and following of phytosanitary standards for weed seeds can lessen the impacts of weeds on agriculture. Standards adopted by seed companies, laboratories and regulators ensure the contamination rates do not exceed some thresholds. Globally sample size standards are set based on the amount needed to obtain a contaminant in a random sample of the seed lot, not detectability. New Zealand requires a 95% confidence that the maximum pest limit of 0.01% of quarantine weed seed contamination is not exceeded in an imported seed lot. We examined 24 samples each containing approximately 150 000 seeds of either perennial ryegrass (12 samples) or white clover seeds (12 samples) that were then spiked with seeds (contaminants) from 12 non-crop species (3-8 seeds of each). We considered factors that may impact detection rates: shape, color, size, and texture relative to the crop, and technician (including a commercial seed laboratory)., Results: A linear mixed model fitted to the data indicated significant observer, crop, and seed color, shape, and size effects on detection. Detectability increased by 20% ± 7.7 (± standard error) when seeds had a distinct shape or color (28% ± 8.1), or were larger (23% ± 8.7) rather than smaller, relative to the crop. Commercial laboratory identifications were usually correct at the level of genus, and species for common weeds, but some misidentifications occurred., Conclusion: Sample sizes for border inspections should be based on detectability of regulated weed seeds in the crop in combination with weed risk for the crop and location. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

A 60% pregnancy success for inseminations is targeted to optimize production efficiency for dairy cows within a seasonal, pasture-grazed system. Routine measures of pregnancy success are widely available but are limited, in practice, to a gestation stage beyond the first 28 d. Although some historical data exist on embryonic mortality before this stage, productivity of dairy systems and genetics of the cows have advanced significantly in recent decades. Accordingly, the aim was to construct an updated estimate of pregnancy success at key developmental stages during the first 70 d after insemination. Blood samples were collected for progesterone concentrations on d 0 and 7. A temporal series of 4 groups spanning fertilization through d 70 were conducted on 4 seasonal, pasture-grazed dairy farms (n = 1,467 cows) during the first 21 d of the seasonal breeding period. Morphological examination was undertaken on embryos collected on d 7 (group E7) and 15 (group E15), and pregnancy was diagnosed via ultrasonography on approximately d 28 and 35 (group E35) as well as d 70 (group E70). Fertilization, embryo, and fetal evaluation for viability established a pregnancy success pattern. Additionally, cow and on-farm risk factor variables associated with pregnancy success were evaluated. We estimated pregnancy success rates of 70.9%, 59.1%, 63.8%, 62.3%, and 56.7% at d 7, 15, 28, 35, and 70, respectively. Fertilization failure (15.8%) and embryonic arrest before the morula stage (10.3%) were the major developmental events contributing to first-week pregnancy failures. Embryo elongation failure of 7% contributed to pregnancy failure during the second week. The risk factors for pregnancy success that were related to the cows included interval between calving and insemination, and d-7 plasma progesterone concentrations, whereas insemination sire was associated with pregnancy outcome. Most pregnancy failure occurs during the first week among seasonal-calving pasture-grazed dairy cows., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

The study aims to determine the timing of application for high efficacy of Metarhizium anisopliae as a biocontrol agent. A field experiment was undertaken with M. anisopliae applied to the soil at five intervals during the peanut crop lifecycle, at seed germination (day 0) through to pod filling period [75 days after sowing (DAS)], and assessed the change of M. anisopliae density by sampling rhizospheric soil, subsequently at regular intervals and testing counts (CFU/g dry soil) through to harvest. The crop was sown into soil with an established white grub population, with larval density determined at harvest when the trial was concluded. Applications at 0, 15 and 30 days in the crop growth cycle, saw M. anisopliae mean propagule counts drop significantly after 15 days before increasing over the following 15-45 days. We observed an elevated mean increase in counts 30-45 days after application at the early flowering stage (30 DAS). Irrespective of application timing, in general, M. anisopliae densities declined to less than the initial 10% in the late stages of peanut development. At harvest, larval densities in all M. anisopliae treatments were significantly less compared to the control, with the highest mortality (72%) in M. anisopliae treatment applied at early flowering (30 DAS). Relationship analysis showed that white grub density was significantly related to peanut yield. A regression of yield on number of damaged pods also supported that treatment at the early flowering caused the highest impact in terms of reducing damage to pods and improving yield. These results suggest that applying M. anisopliae at the early flowering stage optimizes survival of M. anisopliae in the soil profile, meaning greater probability of larvae contacting the pathogen, leading to greater mortality., Competing Interests: Conflict of interestThe authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results., (© King Abdulaziz City for Science and Technology 2020.)

Calf housing often only meets the basic needs of calves, but there is a growing interest in providing enrichments. This study described the behaviour of calves when they were given the opportunity to interact with two commonly available enrichment items. Female and male calves (approximately 11 days old) were pair-housed in 8 identical pens fitted with an automated brush and a hanging rope. Frequency and duration of behaviours were recorded on 3 separate days (from 12:00 until 08:00 the following day. Calves spent equal time using the brush and rope (27.1 min/day), but there was less variation in the use of the brush as opposed to the rope (coefficient of variation, CV: 23 vs. 78%, respectively). Calves had more frequent (94 bouts, CV: 24%) and shorter (17.8 s/bout, CV: 24%) brush use bouts compared to fewer (38 bouts, CV: 43%) and longer (38.3 s/bout, CV: 53%) rope use bouts. There was a diurnal pattern of use for both items. Frequency of play was similar to rope use, but total time playing was 8% of rope and brush use. Variability among calves suggested that individual preference existed; however, the social dynamics of the pair-housed environment were not measured and therefore could have influenced brush and rope use. Multiple enrichment items should be considered when designing improvements to calf housing., Competing Interests: The authors declare no conflict of interest.

The aim of this study was to investigate heterogeneity in tissue morphology, milk protein and immune-related gene expression, and apoptosis of epithelial cells in the lactating and involuting mammary glands of the dairy cow. Mammary tissue from different regions of the gland (alveolar, cisternal, and peripheral) was collected postmortem from nonpregnant, pasture-fed, Holstein-Friesian primiparous cows in mid-lactation that were killed at different time points postmilking: 0, 6, 12, 18, 24, 36, and 72 h (n = 6 per time point). The CSN1NS1 and LALBA mRNA was decreased in alveolar, cisternal, and peripheral tissue by 12 to 36 h postmilking. In contrast, lactoferrin (LF) and mammary serum amyloid A3 (M-SAA3) mRNA was increased in these regions by 36 to 72 h. During lactation, more variability was present in gene expression in alveolar tissue between cows and between quarters within a cow, than within quarters. Histological analysis indicated the alveolar tissue from lactating cows was mostly uniform in structure; however, in situ hybridization indicated that although most of the alveolar tissue expressed milk proteins, the level of expression varied within and between alveoli. This heterogeneity became more pronounced with involution and with increasing regions of alveoli expressing lactoferrin, indicating that alveoli enter involution asynchronously. The peripheral and cisternal tissue had more variability in gene expression between cows compared with the alveolar tissue. The M-SAA3 signal was more intense in the cisternal tissue and less intense than the peripheral compartment compared with LF particularly in the earlier time points. In addition, between cows within the later time points, differences were observed in tissue morphology, the levels of milk protein and immune-related gene expression, and phosphorylated signal transducer and activator of transcription (STAT) 5-P and STAT3-P proteins, and degree of apoptosis, indicating that involution of the mammary gland occurs at different rates between cows. Understanding the mechanisms initiating the process of involution of the mammary gland provides an opportunity for enhancing milk production of the dairy cow., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In dairy cows, mammary gland involution, and thus a decline in milk production, occurs following peak lactation. To examine the cell signaling pathways regulating involution of the mammary gland, signal transducer and activator of transcription factors (STAT5 and 3), suppressors of cytokine signaling (SOCS1-3 and CIS), insulin-like growth factors (IGF1 and 2), and protein kinase B (Akt) were examined. Mammary involution was induced by termination of milking, and alveolar tissue was collected from 52 nonpregnant, primiparous, mid-lactation Holstein-Friesian cows killed at 0, 6, 12, 18, 24, 36, 72, and 192h postmilking. Qualitative immunohistochemistry showed that activated (phosphorylated) STAT5-P was localized in nuclei of mammary epithelial cells at the early time points, with detection levels decreasing by 24h postmilking. In contrast, STAT3-P was barely detectable at the early time points, with detection levels increasing following longer postmilking periods. This was supported by Western analysis, which showed a decline in STAT5 and STAT5-P protein levels by 24h postmilking, no change in STAT3 levels, and an increase in STAT3-P protein (barely detectable at the early time points) by 72h postmilking. Quantitative real-time reverse transcription PCR analysis showed SOCS1 and SOCS3 mRNA increased by 72h postmilking compared with 6h postmilking. The SOCS2 mRNA remained unchanged across the time series, whereas CIS decreased by 18h postmilking and remained lower compared with that at 6h postmilking until 72h postmilking. The IGF1 mRNA increased by 192h postmilking, whereas IGF2 mRNA decreased by 18h postmilking compared with 6h postmilking. The IGFBP5 mRNA and protein levels of Akt and Akt-P remained unchanged over the time series. These results show that reciprocal activation of STAT5 and STAT3 occurs at the onset of mammary gland involution in the bovine, albeit at a slower rate than in rodents. Mathematical modeling of the pathways indicated that activated STAT3 could block the STAT5 pathway by upregulating SOCS3. The regulation of IGF1-Akt signaling suggests that by 192h postmilking in dairy cows, the involution process is still in the reversible phase, with quiescent mammary epithelial cells not yet in the senescent phase., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Aims: We aimed to evaluate different formulations for their ability to adhere Trichoderma atroviridae spores to wheat seeds, and promote survival during storage at a range of temperatures and relative humidities (RH)., Methods and Results: We tested a range of formulations for their ability to adhere T. atroviridae spores to wheat seeds. Treated seeds were stored for 6 months at a range of temperatures and RH, and spore viability among formulation was compared over time. Spore survival within formulations interacted significantly with environmental conditions. Notably, under optimum conditions (low temperatures and RH) best spore survival was recorded with a xanthan-gum-based formulation. Conversely under suboptimum conditions (high temperatures and RH), survival of spores was best in a waxy-starch formulation, but very poor in the xanthan-gum formulation., Conclusions: These results indicate that T. atroviridae spores can be effectively delivered on to seeds and that a xanthan-gum formulation is promising when optimal storage conditions can be maintained., Significance and Impact of the Study: Most published formulation papers/patents only report survival of organisms over time at a single or limited number of temperatures and RH. For the first time, this study shows how different formulations are better suited to certain temperature and RH combinations., (© 2015 The Society for Applied Microbiology.)

In dairy cows, extended periods of nonmilking results in reduced milk secretion, modifications in milk composition, and eventually involution of the mammary glands. The aim of this study was to determine the effect of extended nonmilking periods on the recovery of milk yield and composition, and levels of prolactin and insulin-like growth factor-I in pasture-fed cows after resuming milking. Pasture-fed, primiparous, nonpregnant, Friesian dairy cows at mid lactation (mean ± standard deviation, 97 ± 2d in milk, 14.0 ± 2.5 L/d) were divided into 3 groups (n=6 per group). The cows were subjected to nonmilking periods of 7, 14, or 28d. Twice-daily milking was resumed for 7d following the nonmilking periods. Milk yield recoveries at the end of the 7-d remilking period were 91, 51, and 29% for the 7, 14, and 28-d nonmilked groups, respectively. The somatic cell count declined to less than 400,000 cells/mL by d 3 and 6 of remilking for the 7- and 14-d-nonmilked groups, respectively, but remained greater than 800,000 cells/mL in the 28-d-nonmilked group through the 7-d remilking period. By d 7 of remilking, the somatic cell count for the 7-d-nonmilked group was not different from pretrial values. Upon remilking, the milk fat content returned to pretrial values for the 7- and 14-d-nonmilked groups, although it remained lower than pretrial for the 28-d-nonmilked group. All 3 nonmilked groups had a higher milk protein content following 7d of remilking, compared with pretrial values. The lactose content returned to pretrial values for the 7-d-nonmilked group but remained lower for the 14- and 28-d-nonmilked groups. Circulating prolactin concentrations increased once remilking was resumed, compared with the pretrial and nonmilking periods. Prolactin concentrations did not majorly differ between the groups, with the levels upon 7d of remilking remaining higher than the pretrial concentrations and the nonmilked periods. Plasma concentrations of insulin-like growth factor-I increased during the nonmilking period and were greater in all 3 nonmilked groups on d 1 of remilking than pretrial values and returned to pretrial concentrations following remilking for the 7-d-nonmilked group, whereas the 14- and 28-d-nonmilked groups remained higher than the pretrial values. These data indicate that the process of involution is fully reversed after remilking following 7d of milk stasis but more extended periods of nonmilking prevent the complete recovery of lactation. However, even after 28d of milk stasis, the milk synthesis capacity of the mammary gland could still be partially recovered., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (© 2015 Stichting International Foundation for Animal Genetics.)

Study Question: Are childhood measures of phenotype associated with peri-conception parental, IVF treatment and/or embryonic characteristics of IVF children?, Summary Answer: Birthweight, childhood body mass index (BMI) and height of pre-pubertal IVF children were strongly associated with peri-conception factors, including follicular and embryonic characteristics., What Is Known Already: A growing number of studies have identified a range of phenotypic differences between IVF and naturally conceived pre-pubertal children; for example, birthweights are lower following a fresh compared with a thawed embryo transfer., Study Design, Size, Duration: This retrospective cohort study included IVF children (n = 96) born at term (>37 weeks) after a singleton pregnancy from the transfer of either fresh or thawed embryos in New Zealand. Between March 2004 and November 2008, these children were subjected to clinical assessment before puberty., Participants/materials, Setting, Methods: Clinical assessment provided anthropometric measures of children aged 3.5-11 years old. Peri-conception factors (n = 36) derived retrospectively from parental, treatment, laboratory and embryonic variables (n = 69) were analysed using multiple stepwise regression with respect to standard deviation scores (SDSs) of the birthweight, mid-parental corrected BMI and height of the IVF children. Data from children conceived from fresh (n = 60) or thawed (n = 36) embryos, that met inclusion criteria and had high-quality data with >90% completeness, were analysed., Main Results and the Role of Chance: Embryo treatment at transfer was identified as a predictor of birthweight with thawed embryos resulting in heavier birthweights than fresh embryos [P = 0.02, 95% confidence interval (CI) fresh minus thawed: -1.047 to -0.006]. Birthweight SDS was positively associated with mid-parental corrected BMI SDS (P = 0.003, slope 0.339 ± 0.100). Four factors were related (P < 0.05) to mid-parental corrected height SDS. In particular, child height was inversely associated with the diameter of lead follicles at oocyte retrieval (P < 0.0001, slope -0.144 ± 0.040) and with the quality score of embryos at transfer (P = 0.0008, slope -0.425 ± 0.157), and directly associated with the number of follicles retrieved (P = 0.05, slope 1.011 ± 0.497). Child height was also positively associated with the transfer of a fresh as opposed to thawed embryo (P < 0.001, 95% CI 0.275-0.750)., Limitations, Reasons for Caution: More than one embryo was transferred in most cycles so mean development and quality data were used. The large number of variables measured was on a relatively small sample size. Large cohorts from multiple clinics using a variety of treatment protocols and embryology methods are needed to confirm the associations identified and ultimately to test these factors as possible predictors of phenotype., Wider Implications of the Findings: This is the first study to directly associate peri-conception measures of IVF treatment with a pre-pubertal child's phenotype. Demonstration that peri-conception measures relate to a pre-pubertal child's phenotype extends the range of factors that may influence growth and development. These findings, if corroborated by larger studies, would provide invaluable information for practitioners, who may want to consider the impact of ovarian stimulation protocols as well as the quality of the embryo transferred on a child's growth and development, in addition to their impact on pregnancy rate., Study Funding/competing Interests: This work was supported by grants from the National Research Centre of Growth and Development New Zealand (grant 3682065) and the Australasian Paediatric Endocrine Group (APEG; grant 3621994), as well as a fellowship from Fertility Associates New Zealand awarded to M.P.G. In terms of competing interest, J.C.P is a shareholder of Fertility Associates. M.P.G. currently holds the position of Merck Serono Lecturer in Reproductive Biology. W.S.C. and P.L.H. have also received grants and non-financial support from Novo Nordisk, as well as personal fees from Pfizer that are unrelated to the current study. The other authors have no conflict of interest to declare., (© The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

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., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Streptococcus uberis is a prevalent causative organism of mastitis and resides naturally in the environment of the dairy cow making prevention of the disease difficult. A bovine cDNA microarray comprising approximately 22,000 expressed sequence tags was used to evaluate the transcriptional changes that occur in the mammary gland after the onset of clinical Strep. uberis mastitis. Five lactating Friesian heifers were intramammary infused in an uninfected quarter with approximately 1,000 to 1,500 cfu of a wild-type strain of Strep. uberis. Microarray results showed that Strep. uberis mastitis led to the differential expression of more than 2,200 genes by greater than 1.5-fold compared with noninfected control quarters. The most highly upregulated genes were associated with the immune response, programmed cell death, and oxidative stress. Quantitative real-time reverse transcription PCR analysis confirmed the increase in mRNA expression of immune-related genes complement component 3, clusterin, IL-8, calgranulin C, IFN-gamma , IL-10, IL-1beta, IL-6, toll-like receptor-2, tumor necrosis factor-alpha, serum amyloid A3, lactoferrin, LPS-bonding protein, and oxidative stress-related genes metallothionein 1A and superoxide dimutase 2. In contrast, a decrease of mRNA levels was observed for the major milk protein genes. Bovine mammary epithelial cells in culture challenged with the same Strep. uberis strain used to induce clinical mastitis in the in vivo animal experiment did not cause a change in the mRNA levels of the immune-related genes. This suggests that the expression of immune-related genes by mammary epithelial cells may be initiated by host factors and not Strep. uberis. However, challenging epithelial cells with different Strep. uberis strains and Staphylococcus aureus resulted in an increase in the mRNA expression of a subset of the immune-related genes measured. In comparison, an Escherichia coli challenge caused an increase in the majority of immune-related genes measured. Results demonstrate the complexity of the bovine mammary gland immune response to an infecting pathogen and indicate that a coordinated response exists between the resident, recruited, and inducible immune factors.

We have used cDNA microarray analysis to identify genes that play a role in bovine mammary involution. Involution was induced by termination of milking, and alveolar tissue was collected from 48 nonpregnant Friesian cows in mid lactation sacrificed at 0, 6, 12, 18, 24, 36, 72, and 192 h (n = 6/group) postmilking. The most highly upregulated genes were those associated with oxidative stress. Quantitative real-time reverse-transcription PCR analysis confirmed that mRNA expression of spermidine/spermine N(1)-acetyltransferase was increased by 24 h, superoxide dismutase 2 and metallothionein 1A by 36 h, and glutathione peroxidase by 72 h postmilking. The mRNA expression of the host defense proteins lactoferrin and lingual antimicrobial peptide were increased by 192 h postmilking. A dramatic increase in the protein expression of lactoferrin by 192 h postmilking was also detected by Western analysis. Decreased mRNA expression of the milk protein genes alpha(S1)-, beta-, and kappa-casein, and alpha-lactalbumin were early events in the process of involution occurring within 24 to 36 h postmilking, whereas beta-lactoglobulin mRNA was decreased by 192 h postmilking. Decreases in alpha-lactalbumin and beta-lactoglobulin protein levels in alveolar tissue occurred by 24 and 192 h postmilking, respectively, and the cell survival factors beta1-integrin and focal adhesion kinase were decreased by 72 and 192 h postmilking, respectively. The results demonstrate that in the bovine mammary gland, decreased milk protein gene expression and cell survival signaling are associated with multiple protective responses to oxidative stress that occur before the induction of immune responses and mammary epithelial cell apoptosis during involution.

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.

Aim: To obtain information on serum and liver vitamin B12 and urinary methylmalonic acid concentrations as diagnostic tests to predict a weight gain response to supplementation with vitamin B12 in young dairy cattle when grazing pasture of low cobalt content. Methodology. Forty dairy cattle (12 Friesian, 14 Friesian x Jersey and 14 Jersey) were allocated to two equal sized groups, treated and untreated, based on liveweight. At monthly intervals for 14 months, all animals were weighed, their serum and urine sampled, their liver biopsied and the pasture sampled from the paddocks they were grazing and going to graze. Serum and liver were assayed for vitamin B12 concentrations. For the first 5 months of the trial, urine was assayed for methylmalonic acid concentrations. Both washed and unwashed pasture samples were assayed for cobalt concentrations., Results: No weight gain response occurred vitamin B12 supplementation in young growing cattle grazing pasture with a cobalt concentration of 0.04-0.06 mg/kg DM. For 5 months of the trial, liver vitamin B12 concentrations from untreated calves were in the range 75-220 nmol/kg and serum vitamin B12 concentrations were as low as 72 pmol/1. There was no associated growth response to supplementation., Conclusion: Further trials involving young cattle grazing pastures with cobalt concentrations less than 0.04 mg/kg DM are required to reliably determine liver and serum vitamin B12 concentrations at which growth responses to vitamin B12 or cobalt supplementation are likely under New Zealand pastoral grazing conditions.

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.

We investigated the distribution of radiolabelled IGF-1 in the late gestation ovine fetus by exclusion gel chromatography following intravenous injection of 125I rh (recombinant human) met-IGF-1 into the chronically instrumented fetal lamb (120-130 days, n = 7). One minute after injection of 125I rh met-IGF-1 into the fetal femoral vein, 20.9 +/- 3.1% of the counts circulated in the 150K binding protein region, 55.0 +/- 3.7% in the 50K binding protein region and 18.7 +/- 0.6% in the free or 7K region. The chromatographic profiles obtained in the fetus were in general similar to those previously seen in the adult sheep. After an initial equilibration phase the half life of IGF-1 associated with the 150K binding fractions were 412.1 +/- 103.6 min. Two phases of clearance were observed for IGF-1 in association with the 50K binding fractions, an initial phase with a half life of 30.6 +/- 4.5 min followed by a second phase with a half life of 202.3 +/- 10.3 min. The 7K or 'free' form of IGF-1 had an initial half life of 12.6 +/- 5.1 min. Chromatography of samples of fetal tracheal fluid, fetal urine, amniotic fluid, maternal uterine venous plasma and maternal systemic plasma showed no movement of intact IGF-1 out of the fetal circulation into the fetal fluids or into the maternal circulation. However, when simultaneous samples were obtained from the fetal femoral artery and umbilical vein, higher radioactivity was consistently observed in the fetal femoral artery raising the possibility of placental uptake of IGF-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Three cows received injections of thyroxine (T4; 20 mg/day), four cows GH (40 mg/day) and three cows saline (control; 10 ml/day) on days 5-8 of a 16-day experimental period during peak lactation. Milk yield increased 13% in cows given GH (from 14.6 to 16.5 kg/day) and 15% in cows given T4 (from 15.8 to 18.2 kg/day) but did not change in control cows. Injection of T4 increased milkfat and lactose content but reduced milk protein content. Injection of GH was without effect on milk composition during the injection period but milk protein rose after injections ceased. Injection of T4 increased plasma concentrations of T4 and tri-iodothyronine six- to sevenfold, with maxima occurring on day 9. Injection of GH increased the plasma concentration of GH five- to tenfold 5 h after injection. The plasma concentration of insulin-like growth factor I (IGF-I) was increased in cows given GH in both morning (08.30 h) and afternoon (14.30 h) blood samples, the difference being greatest in afternoon samples in which plasma IGF-I content increased from 3.3 to 6.8 nmol/l. Injection of T4 reduced the plasma concentration of IGF-I in morning samples but the concentration in afternoon samples remained relatively constant throughout the 16-day experimental period. The plasma concentration of IGF-II rose in morning samples in all treatment groups to reach a maximum of 200-250 nmol/l by day 9. The galactopoietic response to injection of GH but not T4 was associated with an increase in plasma concentration of IGF-I. Changes in plasma concentration of IGF-II were not associated with changes in milk yield.

Two animals with XY gonadal dysgenesis both had a reproductive tract similar in size to that found in sexually immature heifers, but neither had normal testicular or ovarian tissue. All cells examined in both animals contained XY chromosomes and spinal cord neurones were sex chromatin negative. Basal LH concentrations averaged 3.1 ng/ml in Animal 1 and 2.4 ng/ml in Animal 2 but increased within 12 h of injecting oestradiol to peak concentrations of 125 and 11 ng/ml respectively. Animal 1 displayed a distinct pulsatile LH release pattern with a highly repeatable decline phase at each pulse. A GnRH injection produced a rapid rise in plasma LH in both animals, sustained in Animal 1 at greater than 500 ng/ml for more than 2 h. Each animal displayed behavioural symptoms of oestrus within 12 h of being injected with 3 mg oestradiol benzoate and was repeatedly served by a bull. These studies indicated that both animals differed from freemartins and had some hypothalamic and pituitary response patterns resembling those reported for female cattle.

The metabolic clearance of insulin-like growth factor-I (IGF-I) has been examined in sheep using a radioiodinated hormone preparation (131I-labelled IGF-I). Following i.v. administration, 131I-labelled IGF-I was distributed in a volume equivalent to plasma (60 ml whole blood/kg liveweight) and demonstrated a triphasic pattern of clearance with apparent half-lives (t 1/2) of 4.0 +/- 0.4 (S.E.M.), 52.4 +/- 3.4 and 792 +/- 26.5 min (n = 10). No significant differences in the t1/2 of the three phases were identified in fed compared with starved animals (fed, n = 4, phase 1 = 3.1 +/- 0.64, phase 2 = 46 +/- 5.9 and phase 3 = 756 +/- 27 min; starved, n = 6, phase 1 = 4.6 +/- 0.58, phase 2 = 57 +/- 3.2 and phase 3 = 816 +/- 38.5 min). Similarly, no significant differences in the distribution volume (fed, n = 4, 44 +/- 4 ml/kg live-weight; starved, n = 6, 39 +/- 2 ml/kg liveweight) or metabolic clearance rate (fed, n = 4, 2.9 +/- 0.15 ml/min; starved, n = 6, 3.2 +/- 0.5 ml/min) of the IGF-I were found in fed compared with starved animals. High-performance gel filtration chromatography of sequential plasma samples following injection of 131I-labelled IGF-I revealed three clear peaks of radioactivity which demonstrated markedly different patterns of clearance. These correspond to hormone complexed to binding proteins of 150,000 and 50,000 daltons and to 'free' hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Critical leaf concentrations for deficiency of nitrogen, potassium, phosphorus, sulphur, and magnesium were estimated for perennial ryegrass (Lolium perenne L.) grown in sand culture. The values associated with a 10% reduction in dry matter yield were as follows (all results expressed as g kg -1 DM except for the nitrate-nitrogen value which appears as μg -1 DM): Kjeldahl-nitrogen 32; nitrate-nitrogen 500; potassium 28; phosphorus 2.1; sulphur 1.8; and magnesium 0.7. A major difference between the critical leaf concentrations estimated in this study and the tentative values published elsewhere was for potassium. Concentrations required in the shoot for near maximum growth were higher than previously reported. Differences in the nitrogen status of the plants probably account for this result. The depressing effect of potassium on the absorption of magnesium and calcium is discussed in relation to plant and animal nutrition. Sulphur was found to be inefficiently absorbed by phosphorus deficient plants despite non-limiting amounts of sulphur applied in the nutrient solution. A possible explanation for this effect may be linked to the observation that the transport of sulphate into the chloroplast was coupled with that of phosphate. The concentration of macroelements required in the nutrient solution to produce maximum shoot growth of perennial ryegrass was very much greater than that generally applied to plants grown in sand culture.

The metabolic clearance of ovine insulin-like growth factor-II (IGF-II) was examined in sheep using 131I-labelled IGF-II. Following i.v. administration the tracer was distributed in a volume similar to that of the vascular space (58.5 +/- 3.3 ml/kg; mean +/- S.E.M., n = 5) and demonstrated a triphasic pattern of clearance. Size-exclusion chromatography of a plasma sample collected 1 min after injection revealed peaks of radioactivity corresponding to hormone complexed to binding proteins of 150 and 40-50 kDa (relative abundance 21 and 65% respectively), a high molecular weight binding protein (greater than 200 kDa; 5%) and 'free' tracer (9%). Chromatography of sequential plasma samples revealed different patterns of clearance for these constituents. Half-lives of 131I-labelled IGF-II complexed to the 150 and 40-50 kDa binding proteins, as calculated from rate constants for their decay, were 351 +/- 30 and 9.6 +/- 1.8 min respectively (n = 5). These differ markedly from estimates for the clearance of IGF-I (545 +/- 25 min, n = 8, and 34 +/- 2.3 min, n = 6) associated with carrier proteins of the same apparent molecular weights. This was reflected in calculated metabolic clearance rates for IGF-I (3.9 +/- 0.5 ml/min) and IGF-II (7.8 +/- 1.0 ml/min). Chromatography also revealed that free IGF-II was reduced to negligible levels by 12 min. In contrast, radioactivity eluting in the position expected for the greater than 200 kDa binding protein was cleared from the circulation very slowly.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood samples from 433 periparturient recumbent cows submitted by veterinary practitioners to Ruakura Animal Health Laboratory during 1983 and 1984 were analysed and results related to whether cows recovered, died or were euthanased. Generally cows were sampled only once and the time varied from 15 minutes to 20 days after becoming recumbent. During 1983 serum calcium, magnesium, phosphorus, creatine phosphokinase (CK), aspartate amino transferase (AST), glutamate dehydrogenase (GDH), gamma glutamyl transferase (GGT) were analysed. In 1984 serum urea, creatinine, fibrinogen and haematological examination (haemoglobin, haematocrit, total and differential white cell counts) were added to the panel. Overall 39% of cows recovered, 30% died and 32% were destroyed. Precalving cows had 111% more deaths and 7% less survivors than postcalving recumbent cows (P<0.1). There was little difference (3%) in euthanasia prevalence. Tests that were most useful in predicting a lack of recovery were serum urea and muscle enzymes. Using these tests and duration of recumbency when sampled a model was produced to predict the probability of recovery from 254 cases.