Optimizing the dietary calcium (Ca) level is essential to maximize the eggshell quality, egg production and bone formation in poultry. This study aimed to establish the Ca requirements of egg-type duck breeders from 23 to 57 weeks of age on egg production, eggshell, incubation, tibial, plasma and ovary-related indices, as well as the expression of matrix protein-related genes. Totally, 450 Longyan duck breeders aged 21 weeks of age were allotted randomly into five treatments, each with six replicates of 15 individually caged birds. The data collection started from 23 weeks of age and continued over the following 35 weeks. The five groups corresponded to five dietary treatments containing either 2.8%, 3.2%, 3.6%, 4.0% or 4.4% Ca. The tested dietary Ca levels increased (linear, P <0.01) egg production and egg mass, and linearly improved (P <0.01) the feed conversion ratio (FCR). Increasing the dietary Ca levels from 2.8% to 4.4% increased (P <0.01) the eggshell thickness and eggshell content. The tested Ca levels showed a quadratic effect on eggshell thickness and ovarian weight (P <0.01); the highest values were obtained with the Ca levels 4.0% and 3.6%, respectively. Dietary Ca levels affected the small yellow follicles (SYF) number and SYF weight/ovarian weight, and the linear response (P <0.01) was significant vis-à-vis SYF number. In addition, dietary Ca levels increased (P <0.05) the tibial dry weight, breaking strength, mineral density and ash content. Plasma and tibial phosphorus concentration exhibited a quadratic (P <0.01) response to dietary Ca levels. Plasma calcitonin concentration linearly (P <0.01) increased as dietary Ca levels increased. The relative expression of carbonic anhydrase 2 in the uterus rose (P <0.01) with the increment of dietary Ca levels, and the highest value was obtained with 3.2% Ca. In conclusion, Longyan duck breeders fed a diet with 4.0% Ca had superior eggshell and tibial quality, while those fed a diet with 3.6% Ca had the heaviest ovarian weights. The regression model indicated that the dietary Ca levels 3.86%, 3.48% and 4.00% are optimal levels to obtain maximum eggshell thickness, ovarian weight and tibial mineral density, respectively.

Surplus accounting is a method for evaluating trends in how a firm’s productivity factors (intermediate inputs, capital, land, labour) are performing and how the productivity gains are redistributed between agents in the economy. Here the surplus accounting method was applied on a database of 164 Charolais-area suckler cattle farms running from 1980 to 2015. Over this 36-year period – with differences per sub-period – the cumulative productivity surplus (PS) increased at a low rate of +0.17%/year (i.e. cumulative volume of outputs produced increased slightly more than cumulative volume of inputs used). This timid increase in PS is linked to the constant expansion in labour productivity whereas other factor productivities have shrunk. The observable period-wide macrotrends are that commercial farm businesses struggle to protect their revenues, we also observe a slight fall in input prices, land rent and financing costs, and a huge climb in direct support-policy payments. The bulk of the cumulative economic surplus has been captured downstream – 64% downstream of the cattle value chain as a drop in prices, and 22% downstream of other value chains (chiefly cereals). It emerges that the productivity gains in beef cattle farming mostly benefit the downstream value chain (packers–processors, distributors and consumers), whereas it is mainly government money backing this drop in prices of agricultural output. Here we see the principal of the 1992 ‘MacSharry’ reform at work, with a transfer from the taxpayer through direct support-policy payments through to the consumer via lower prices. The simple fact that farmers’ incomes are stagnating is a clear indication that they are net losers in this distribution of productivity gains, despite the improvement in labour factor productivity.

In this study, 10 samples of rapeseed meal (RSM) from 10 different oil plants in Germany were examined. In situ rumen degradation of CP was determined by incubation over 1, 2, 4, 8, 16, 32 and 72 h in duplicate per time point using three rumen fistulated dry cows. Degradation kinetics were estimated by an exponential model and effective CP degradation was calculated. Degradation was corrected for small particle loss as the difference between washing loss and water-soluble fraction. Amino acid analysis was carried out in the samples and in the residues after 8 and 16 h of incubation in situ and degradation of individual amino acids was calculated for these incubation times. In vitro pepsin–pancreatin digestibility of CP (IPD) was determined in the samples as well as in the 8 and 16 h residues. Effective CP degradation for a rumen outflow rate of 8%/h (ED8) averaged 54.3% with a considerable variation among samples ranging from 44.3% to 62.7%. A multiple regression equation containing acid detergent insoluble N, total glucosinolates and petroleum ether extract as independent variables predicted ED8 with satisfying accuracy (R2 = 0.74; RSD = 6.4%). Degradation of amino acids was different from that of CP for most amino acids studied, especially after 8 h of incubation. Compared with CP, degradation of essential amino acids was predominantly lower while degradation of non-essential amino acids was higher in most cases. However, for lysine and methionine no distinct difference with CP degradation was found. Degradation of individual amino acids was predicted from CP degradation with high accuracy using linear regression equations. Average IPD of RSM was 79.8 ± 2.6%. IPD was lower in the incubation residues and decreased with longer incubation time and increasing rumen degradation, respectively.

In two experiments over 2 years, 57 North Country Cheviot and 82 South Country Cheviot hill ewes were differentially group-fed indoors over a 2-month period to achieve either good or moderate body con- dition. Over 5 weeks prior to mating, ewes in good condition were brought down in condition by restricted feeding and ewes in mod- erate condition were raised in condition by a high level of feeding. The ewes were thus in moderately-good condition at mating. After mating, ewes were maintained in this condition until killed either on return to service or at 29 ± 8 days for counts of corpora lutea and viable embryos.

Ovulation rate in each breed was positively related to the level of pre-mating food intake at the condition level studied. Embryo mortality, as ova loss, was not influenced overall by the level of pre-mating food intake but loss of multiple-shed ova was greater than that of single-shed ova in ewes which had been on restricted feeding before mating. Although a greater proportion of ewes in the North Country Cheviot breed were not pregnant at slaughter, this could not be identified as a breed difference since the breeds were studied in different years.

In many equestrian pursuits such as dressage and show-jumping, it is important that the horse exhibits the same level of balance when ridden to the left as when ridden to the right in canter – that is, to show no motor bias. It is a long-held belief within such disciplines that to reduce bias that exists in horses and thus to enhance symmetry of performance to the left and right, the horse needs to be worked equally in both directions, although there is a lack of scientific evidence of this influencing bias. There also is little compelling evidence for either the existence or absence of motor bias in unridden (and therefore younger) or ridden (and therefore older) horses. In this study, we tested whether there was a difference in motor bias between unridden (n = 15) and ridden (n = 15) horses when their balance was challenged by cantering them in circles both to the left and to the right on the lunge. As indicators of a difference in balance between the left and right and thus as indicators of motor bias, we conducted three lunging tests – time spent in canter, whether the horse cantered on the correct lead and whether it became disunited. A grazing stance test, where the extended foreleg during grazing was recorded as the preferred forelimb, was also used to compare responses in a test where balance was not actively challenged, to the three lunging tests where balance was actively challenged. No bias was found in either the unridden or ridden groups when their balance was challenged, but ridden horses exhibited a motor bias in grazing stance – when their balance was not challenged. There was also a correlation between the responses in all three lunging tests, but none between the grazing stance test and any of the three lunging tests. We therefore conclude that neither ridden nor unridden horses are biased when their balance is challenged; thus it cannot be concluded that ambidextrous training affects an inherent bias, and that estimation of motor bias in horses is affected by the test conditions. Finally, if ridden horses are truly unbiased, strong human motor bias might be responsible for the common perception amongst riders that horses are biased.

Forty-eight pigs were used to compare the feeding value of barley which had been stored dry, and then hammer-milled, with that of barley stored moist, either anaerobically or treated with 1·3% propionic acid, and then rolled. The pigs were fed from 30 kg live weight for a period of 11 weeks and during this period all consumed the same allowance of dry matter. The growth rate and feed conversion ratio of the pigs given the rolled moist barley were significantly poorer than those of others given dry milled or acid treated rolled barley. There were no treatment differences in carcass attributes.

Microbiological counts made throughout the experiment showed that there was no significant fungal or bacterial development on the acid treated barley. Similar numbers of bacteria were present on the dry barley as on the moist, but the fungal count for the moist grain was always at least 500 times higher than that of the corresponding dry sample. The dominant organisms were identified.

In a further experiment the apparent digestibility of dry matter and nitrogen of diets containing dry, moist or propionic acid treated barley, processed either by milling or rolling, were determined. The highest values were obtained for dry barley. Acid treated moist barley had lower values than moist barley. The method of processing did not affect the apparent digestibility of the dry matter, but that of the N was significantly lower for the rolled barleys.

Forty-eight Suffolk cross lambs were reared artificially from 2 days of age. Sixteen entire males, 16 castrates (castrated by rubber ring at 2 days) and 16 female lambs were allocated at random to a set of 2×2×2 factorial treatments in which feeding was ad libitum or restricted for each of three growth periods—from 2 days old to 15 kg, from 15 to 25 kg and from 25 to 40 kg live weight.

The results showed no apparent interaction of sex and feeding treatment and differences due to sex were apparent only in the period after 25 kg live weight. Entire males grew faster and were more efficient at this stage although there was little difference in voluntary feed intake. Carcasses of the males were lower in fat and energy content and higher in protein content than those of females and castrates.

The feeding treatments in the main had similar effects to those shown in other studies in the series. Restricted milk feeding was compensated for by increased solid food consumption during the milk-feeding stage but in this experiment there was no tendency for restricted lambs to eat more and grow faster when fed ad libitum in the following period. Restricted feeding in the last period, particularly if preceded by restricted feeding, gave higher killing-out percentage, higher fat content and lower protein content in the carcass than feeding ad libitum. This finding was partly explained by the higher weight of the carcasses of the restricted lambs.

The incidence, heritability and effects on lactation performance of twinning in 185 000 calvings in Israeli-Friesian dairy herds are presented. The incidence of twinning in heifer and cow calvings (parities 2 to 6 inclusive) was 0·7 and 5·4% respectively in the large Kibbutz herds and 0·7 and 3·5% in smallholders' herds. The heritability estimate for 144 sires each with over 200 calvings of daughters of parity 2 to 6 was 0·024 ± 0·002. The range of incidence of twinning in these parities among sire progeny groups each with more than 1000 calvings was 2·8 to 7·8 %, apart from one sire, whose daughters had 12·2% twins. His progeny excelled in five characters and as an explanation it is suggested that a rare gene is segregating in the breed with a major effect on the endocrine system and associated pleiotropic effects.

Three dominant morphological fractions (i.e. leaf blade (LB), leaf sheath (LS) and stem) were analysed for chemical composition and ruminal degradability in three rice straw varieties. In one variety treated with alkali, cell wall features were also characterized using Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscopy. The highest concentrations of cell wall carbohydrates (hemicellulose and cellulose) were observed in LS, whereas the highest concentrations of non-fibre (silica, phenolic compounds and CP) and lignin were recorded for LB. The stem had the lowest silica and hemicellulose contents but intermediate levels of other components. In terms of ruminal degradability, stem ranked higher than LB, which was followed by LS. Hemicellulose was found to be less degradable than either dry matter or cellulose in all the three fractions investigated. FTIR results indicated that the highest levels of hydrogen bonding, esterification and crystallinity within the cell wall components belonged to LS. In the alkaline treatment, these indices decreased to a larger extent for leaf fractions and a greater improvement was achieved in the degradability of LB and LS compared with that of stem. In the 24-h ruminal incubation, the silicified layer of epidermis and the underlying cell walls showed a rigid structure in the control fractions, whereas the treatment with NaOH resulted in crimping of the silicified cuticle layer and the loss of integrity in cell structure. Despite the highest silica and lignin contents observed in LB, LS showed the lowest degradability, which might be due to its high level of hydrogen bonding, crystallinity and esterification within its cell wall components as well as its high hemicellulose content.

Body condition scoring is a common tool to assess the subcutaneous fat reserves of dairy cows. Because of its subjectivity, which causes limits in repeatability, it is often discussed controversially. Aim of the current study was to evaluate the impact of considering the cows overall appearance on the scoring process and on the validity of the results. Therefore, two different methods to reveal body condition scores (BCS), ‘independent BCS’ (iBCS) and ‘dependent BCS’ (dBCS), were used to assess 1111 Swiss Brown Cattle. The iBCS and the dBCS systems were both working with the same flowchart with a decision tree structure for visual and palpatory assessment using a scale from 2 to 5 with increment units of 0.25. The iBCS was created strictly complying with the defined frames of the decision tree structure. The system was chosen due to its formularized approach to reduce the influence of subjective impressions. By contrast, the dBCS system, which was in line with common practice, had a more open approach, where – besides the decision tree – the overall impression of the cow’s physical appearance was taken into account for generating the final score. Ultrasound measurement of the back fat thickness (BFT) was applied as a validation method. The dBCS turned out to be the better predictor of BFT, explaining 67.3% of the variance. The iBCS was only able to explain 47.3% of the BFT variance. Within the whole data set, only 31.3% of the animals received identical dBCS and iBCS. The pin bone region caused the most deviations between dBCS and iBCS, but also assessing the pelvis line, the hook bones and the ligaments led to divergences in around 20% of the scored animals. The study showed that during the assessment of body condition a strict adherence to a decision tree is a possible source of inexact classifications. Some body regions, especially the pin bones, proved to be particularly challenging for scoring due to difficulties in assessing them. All the more, the inclusion of the overall appearance of the cow into the assessment process counteracted these errors and led to a fair predictability of BFT with the flowchart-based BCS. This might be particularly important, if different cattle types and breeds are assessed.

The objective of the present retrospective cohort study was to evaluate potential associations between environmental factors and dam characteristics, including level of milk production during gestation, and insulin traits in newborn Holstein calves. Birth weight and gestational age of the calves at delivery were determined. On the next day, heart girth, wither height and diagonal length of both the calves and their dams were measured. Parity, body condition score and age at calving were recorded for all dams. For the cows, days open before last gestation, lactation length (LL), length of dry period (DP) and calving interval were also calculated. The magnitude and shape of the lactation curve both quantified using the MilkBot model based on monthly milk weights, were used to calculate the amount of milk produced during gestation. Using the same procedure, cumulative milk production from conception to drying off (MGEST) was calculated. A blood sample was collected from all calves (n=481; 169 born to heifers and 312 born to cows) at least 5 h after a milk meal on day 3 of life to measure basal glucose and insulin levels. In addition, an intravenous glucose-stimulated insulin secretion test was performed in a subset of the calves (n=316). After descriptive analysis, generalized linear mixed models were used to identify factors that were significantly associated with the major insulin traits (Insb, basal insulin level; QUICKI, quantitative insulin sensitivity check index; AIR, acute insulin response; DI, disposition index) of the newborn calves. The overall average birth weight of the calves was 42.7±5.92 kg. The insulin traits were significantly associated with gender and season of birth when data of all calves were analyzed. In addition, the insulin traits in calves born to cows were significantly associated with MGEST, DP and LL. The Insb was estimated to be higher in calves born to the cows having passed a higher MGEST (P=0.076) and longer DP (P=0.034). The QUICKI was estimated to be lower in calves born to the cows having passed a higher MGEST (P=0.030) and longer DP (P=0.058). Moreover, the AIR (P=0.009) and DI (P=0.049) were estimated to be lower in male compared with female calves. Furthermore, the AIR (P=0.036) and DI (P=0.039) were estimated to be lower in calves born to cows having passed a longer LL. The decisive effects of MGEST, DP and LL in cows on the insulin traits of their calves may provide a basis for developing managerial interventions to improve metabolic health of the offspring.

Bovine mastitis, the inflammation of the udder, is a major problem for the dairy industry and for the welfare of the animals. To better understand this disease, and to implement two special techniques for studying mammary gland immunity in vitro, we measured the innate immune response of primary bovine mammary epithelial cells (pbMEC) from six Brown Swiss cows after stimulation with the heat-inactivated mastitis pathogens, Escherichia coli 1303 and Staphylococcus aureus 1027. The cells were extracted and cultivated from milk instead of udder tissue, which is usually done. The advantages of this technique are non-invasiveness and less contamination by fibroblasts. For the first time, pbMEC gene expression (GE) was measured with a microfluidic high-throughput real-time reverse transcription-quantitative PCR platform, the BioMark HD™ system from Fluidigm. In addition to the physiological analysis, the precision and suitability of this method was evaluated in a large data set. The mean coefficient of variance (± s.e.) between repeated chips was 4.3 ± 0.4% for highly expressed and 3.3 ± 0.4% for lowly expressed genes. Quantitative PCR (qPCR) replicate deviations were smaller than the cell culture replicate deviations, indicating that biological and cell culture differences could be distinguished from the background noise. Twenty-two genes (complement system, chemokines, inflammatory cytokines, antimicrobial peptides, acute phase response and toll-like receptor signalling) were differentially expressed (P < 0.05) with E. coli. The most upregulated gene was the acute phase protein serum amyloid A3 with 618-time fold. S. aureus slightly induced CCL5, IL10, TLR4 and S100A12 expression and failed to elicit a distinct overall innate immune response. We showed that, with this milk-derived pbMEC culture and the high-throughput qPCR technique, it is possible to obtain similar results in pbMEC expression as with conventional PCR and with satisfactory precision so that it can be applied in future GE studies in pbMEC.

In stall-feeding conditions, food diversity has been shown to increase an animal's motivation to eat and its level of food intake. This effect has also been suggested, but not demonstrated, for free-grazing animals. In free-grazing conditions, however, animals may have to search for the preferred plant species and this may reduce their intake rate. Moreover, the management of diverse swards faces a risk of overgrazing the preferred species and undergrazing the less preferred species, leading to resource degradation. We investigated the effect of food diversity on foraging behaviour and intake in grazing sheep, in interaction with the type of diversity (conterminal monocultures v. mixtures) and management (two monocultures offered simultaneously v. successively over time). Five treatments were compared with five groups of five dry INRA 401 ewes for five periods, using a Latin-square design: LOL=grazing a monoculture of Lolium perenne cv. Herbie (L); FES=grazing a monoculture of Festuca arundinacea cv. Florine (F); STRIPS=grazing conterminal monocultures of L and F, 0·5:0·5 by ground area, with animals having a free choice between both species; TIME=grazing L from 16:00 to 09:00 h and F from 09:00 to 16:00 h; MIX=grazing an intermeshed mixture of L and F. Sward height was maintained at 9 cm. Each period comprised a 5-day adaptation subperiod followed by a 5-day measurement subperiod. Dietary choices and intake were measured using n-alkanes, and grazing time was assessed using the Ethosys® recording system. Intake rate was calculated as the ratio of intake to grazing time. Intake in the monoculture treatments were higher than predictions made for housed animals given similar forages ad libitum, demonstrating that sward conditions were favourable for the expression of voluntary intake in all treatments. Intake was higher in LOL than in FES, this result being consistent with predictions for stall-feeding conditions. There was no difference in diet composition, foraging behaviour and intake between the bispecific treatments (STRIPS, TIME and MIX). Intake was higher in the bispecific treatments than in LOL (1666 v. 1492 g digestible organic matter per day, P<0·001). Grazing time was higher in STRIPS and MIX than in LOL (420 and 430 min respectively v. 366 min, P<0·01). Dry ewes increase their intake when offered a choice of herbage species at pasture, this being mediated via an increase in grazing time rather than an increase in intake rate. There was no interaction with type of diversity or type of management.

There has been a long history of herd health and production management programmes in many dairy industries around the world, but evidence for the efficacy of such programmes is limited. In response to a perceived decline in fertility of dairy cows, a herd reproductive management programme (InCalf) was introduced in New Zealand in 2007. This programme uses a management cycle approach that includes an assessment of the current herd status, identification of areas for improvement, development of a plan, implementation of this plan and finally a review process. The programme uses facilitators who work with farmers either in a one-to-one manner or in a formalised group setting that involves a series of meetings over a 12-month period (the farmer action group). The hypothesis that involvement in a reproductive management programme would improve herd reproductive performance was tested using a herd-level controlled randomised study (the National Herd Fertility Study) involving herds in four geographic regions of New Zealand over 2 years. Within each region, herds were ranked on the basis of the 6-week in-calf rate (i.e. the proportion of the herd pregnant in the first 6 weeks of the seasonal breeding programme) in the year preceding commencement of the study and then randomly assigned to be involved in a farmer action group or left as untreated controls. The key outcome variable of the study was the 6-week in-calf rate. Pregnancy diagnosis was undertaken at 12 weeks after the start of the seasonal breeding programme, which allowed determination of conception dates and hence calculation of the 6-week in-calf rate. Additional measurements including heifer live weight and body condition score (pre-calving and pre-mating) were undertaken to test whether treatment resulted in measurable changes in some of the key determinants of herd reproductive performance. Involvement in the farmer action group of InCalf resulted in a 2 percentage point increase in the 6-week in-calf rate (P=0.05). The following additional observations were made in herds involved in the farmer action group relative to control herds: heifers had live weight closer to target; the pre-mating body condition score of cows was higher; and oestrous detection rates were higher. It was concluded that involvement in this herd reproductive management programme improved reproductive outcomes in this New Zealand study. However, to achieve substantial improvements in herd reproductive performance at the regional or national level a greater response to the programme and a high uptake of such programmes is required, as well as use of other industry-level tools such as genetic management programmes.

The competitiveness and sustainability of low input cost dairy production systems are generally supported by efficient use of pasture in the diets. Therefore, pasture intake directly affects overall efficiency of these systems. We aimed to assess feeding and grazing management main factors that affect pasture dry matter intake (DMI) in commercial dairy farms during the different seasons of the year. Fortnightly visits to 28 commercial dairies were carried out between June 2016 and May 2017 to record production and price, supplement offered and price, pasture access time (PAT), herbage mass (HM) and allowance (HA). Only farms with the most contrasting estimated pasture DMI per cow (eDMI) were compared as systems with high (HPI; N = 8) or low (LPI; N = 8) pasture DMI. Despite a lower individual milk production in HPI than LPI (19.0 v. 23.3 ± 0.7 l/cow, P < 0.01), daily margin over feeding cost was not different between groups (3.07 v. 2.93 ± 0.15 U$S/cow for HPI and LPI, respectively). During autumn and winter, HPI cows ingested more pasture than LPI cows (8.3 v. 4.6 and 5.9 v. 2.9 ± 0.55 kg DM/cow per day, respectively, P < 0.01) although PAT, HM and HA were similar between groups. Both groups offered high supplementation levels during these seasons, even though greater in LPI than HPI (14.7 v. 9.7 ± 0.7 kg DM supplement/cow per day, respectively, P < 0.01). On the other hand, differences between groups for both pasture and supplement DMI were more contrasting during spring and summer (13.1 v. 7.3 ± 0.5 and 4.0 v. 11.4 ± 0.4 kg DM/cow per day for HPI and LPI, respectively, P < 0.01), with higher PAT in both seasons (P < 0.05) and higher HA during summer in HPI than LPI (P < 0.01). Unlike LPI, during these seasons HPI adjusted offered supplement according to HA, achieving a higher pasture eDMI and making more efficient use of available pastoral resource than LPI. As there was no grazing limiting condition for pasture harvesting in either group, the main factor affecting pasture DMI was a pasture by supplement substitution effect. These results reinforce the importance of an efficient grazing management, and using supplements to nutritionally complement pasture intake rather than as a direct way to increase milk production.

Floor space allowance for pigs has substantial effects on pig growth and welfare. Data from 30 papers examining the influence of floor space allowance on the growth of finishing pigs was used in a meta-analysis to develop alternative prediction equations for average daily gain (ADG), average daily feed intake (ADFI) and gain : feed ratio (G : F). Treatment means were compiled in a database that contained 30 papers for ADG and 28 papers for ADFI and G : F. The predictor variables evaluated were floor space (m2/pig), k (floor space/final BW0.67), Initial BW, Final BW, feed space (pigs per feeder hole), water space (pigs per waterer), group size (pigs per pen), gender, floor type and study length (d). Multivariable general linear mixed model regression equations were used. Floor space treatments within each experiment were the observational and experimental unit. The optimum equations to predict ADG, ADFI and G : F were: ADG, g=337.57+(16 468×k)−(237 350×k2)−(3.1209×initial BW (kg))+(2.569×final BW (kg))+(71.6918×k×initial BW (kg)); ADFI, g=833.41+(24 785×k)−(388 998×k2)−(3.0027×initial BW (kg))+(11.246×final BW (kg))+(187.61×k×initial BW (kg)); G : F=predicted ADG/predicted ADFI. Overall, the meta-analysis indicates that BW is an important predictor of ADG and ADFI even after computing the constant coefficient k, which utilizes final BW in its calculation. This suggests including initial and final BW improves the prediction over using k as a predictor alone. In addition, the analysis also indicated that G : F of finishing pigs is influenced by floor space allowance, whereas individual studies have concluded variable results.

Roundup is a glyphosate-based herbicide (GBH) widely used in agriculture and may cause toxic effects in non-target organisms. Model organisms, as zebrafish, and analysis of gene expression by reverse transcription-quantitative PCR (RT-qPCR) could be used to better understand the Roundup toxicity. A prerequisite for RT-qPCR is the availability of appropriate reference genes; however, they have not been described for Roundup-exposed fish. The aim of this study was to evaluate the expression stability of six reference genes (rpl8, β-act, gapdh, b2m, ef1α, hprt1) and one expressed repetitive element (hatn10) in organs of males (brain, gill, testis) and females (ovary) of zebrafish exposed to Roundup WG at three concentrations (0.065, 0.65 and 6.5 mg N-(phosphonomethyl) glycine/l) for 7 days. Genes were ranked by geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder, and their best combinations were determined by geNorm and NormFinder programs. The two most stable ranked genes were specific to each organ: gill (β-act; rpl8); brain (rpl8; β-act); testis (ef1α; gapdh); and ovary (rpl8; hprt1). The cat transcript level was used to evaluate the effect of normalization with these reference genes. These are the first suitable reference genes described for the analysis of gene expression in organs of Roundup-exposed zebrafish, and will allow investigations of the molecular mechanisms of Roundup toxicity.

This study was conducted within the context of the Animal Welfare Indicators (AWIN) project and the underlying scientific motivation for the development of the study was the scarcity of data regarding inter-observer reliability (IOR) of welfare indicators, particularly given the importance of reliability as a further step for developing on-farm welfare assessment protocols. The objective of this study is therefore to evaluate IOR of animal-based indicators (at group and individual-level) of the AWIN welfare assessment protocol (prototype) for dairy goats. In the design of the study, two pairs of observers, one in Portugal and another in Italy, visited 10 farms each and applied the AWIN prototype protocol. Farms in both countries were visited between January and March 2014, and all the observers received the same training before the farm visits were initiated. Data collected during farm visits, and analysed in this study, include group-level and individual-level observations. The results of our study allow us to conclude that most of the group-level indicators presented the highest IOR level (‘substantial’, 0.85 to 0.99) in both field studies, pointing to a usable set of animal-based welfare indicators that were therefore included in the first level of the final AWIN welfare assessment protocol for dairy goats. Inter-observer reliability of individual-level indicators was lower, but the majority of them still reached ‘fair to good’ (0.41 to 0.75) and ‘excellent’ (0.76 to 1) levels. In the paper we explore reasons for the differences found in IOR between the group and individual-level indicators, including how the number of individual-level indicators to be assessed on each animal and the restraining method may have affected the results. Furthermore, we discuss the differences found in the IOR of individual-level indicators in both countries: the Portuguese pair of observers reached a higher level of IOR, when compared with the Italian observers. We argue how the reasons behind these differences may stem from the restraining method applied, or the different background and experience of the observers. Finally, the discussion of the results emphasizes the importance of considering that reliability is not an absolute attribute of an indicator, but derives from an interaction between the indicators, the observers and the situation in which the assessment is taking place. This highlights the importance of further considering the indicators’ reliability while developing welfare assessment protocols.

Semen collection from three boars was performed twice a week, and doses (100 ml each) of semen diluted with Beltsville Thaw Solution (BTS) extender were prepared containing 1 × 109, 3 × 109 and 5 × 109 spermatozoa. Diluted semen was then stored at 17°C for a maximum of 120 h. Percentage of motile spermatozoa (PMS) and type of spermatozoa motility (TSM) were determined by microscopic examinations performed at 24-h intervals, from zero time (beginning of storage at 17°C) up to 120 h of storage, and a sperm motility index (SMI) calculated. Sows (no. = 360) were divided into nine experimental groups (40 sows per group) and a combination of number of spermatozoa per dose and storage time was used for artificial insemination in each group. All the animals were inseminated twice at 12 and 36 h after detection of standing oestrus by a teaser boar. Return and farrowing data including litter size were recorded. PMS, TSM and SMI decreased significantly (P < 0·01) from 48 h up to 120 h of storage, this being more marked in semen doses of 5 × 109 spermatozoa. When semen stored for up to 24 h was used, no significant differences (P > 0·05) regarding return rates and farrowing rates were observed among the three groups inseminated with semen containing different numbers of spermatozoa per dose. Inseminations with semen doses of 1 × 109 and 3 × 109 spermatozoa stored for 48 to 72 h have significantly lower return rates (P < 0·05) and significantly higher farrowing rates (P < 0·05) than with doses of 5 × 109 spermatozoa. Inseminations with semen stored for more than 72 h gave relatively higher return rates and lower farrowing rates. Semen storage time and number of spermatozoa per dose appeared to reduce litter size only where semen doses of 5 X109 spermatozoa were inseminated after storage for more than 48 h.

The aim of this paper is to evaluate at a histopathological level the effect of the most commonly used copper (Cu) supplementation (15 mg/kg dry matter (DM)) in the liver of intensively reared beef cattle. This was done by a histochemistry evaluation of (i) the antioxidant capacity in the liver – by the determination of metallothioneins (MT) and superoxide dismutase (SOD) expression – as well as (ii) the possible induction of oxidative damage – by the determination of inducible nitric oxide synthase (iNOS), nitrotyrosine (NITT), malondialdehyde (MDA) and 8-oxoguanine (8-oxo) – that (iii) could increase apoptotic cell death – determined by cytochrome-c (cyto-c), caspase 1 (casp1) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Liver samples from Cu-supplemented (15 mg Cu sulphate/kg DM, n = 5) and non-supplemented calves (n = 5) that form part of other experiments to evaluate Cu status were collected at slaughter and processed for immunohistochemistry and TUNEL. MT expression was diffuse and SOD showed slight changes although without statistical significance. iNOS and NITT positive (+) cells significantly increased, mainly around the central veins in the animals from the Cu-supplemented group, whereas no differences were appreciated for the rest of the oxidative stress and apoptosis markers. Under the conditions of this study, which are the conditions of the cattle raised in intensive systems in NW Spain and also many European countries, routinely Cu supplementation increased the risk of the animals to undergo subclinical Cu toxicity, with no significant changes in the Cu storage capacity and the antioxidant defensive system evaluated by MT and SOD expression, but with a significant and important increase of oxidative damage measured by iNOS and NITT. The results of this study indicated that iNOS and NITT could be used as early markers of initial pathological changes in the liver caused by Cu supplementation in cattle, although more studies in cattle under different levels of Cu supplementation are needed.