Effects of sheep and horse manure and pine bark amendments on metal distribution and chemical properties of contaminated mine soils.

Mine soils usually contain large levels of heavy metals and poor fertility conditions which limit their reclamation and the application of phyto-remediation technologies. Two organic waste materials (pine bark compost and sheep and horse manure compost), with different pHs and varying degrees of humification and nutrient contents, were applied as amendments to assess their effects on copper (Cu) and zinc (Zn) bioavailability and on fertility conditions of mine soils. Soil samples collected from two abandoned mining areas near Madrid (Spain) were mixed with 0, 30 and 60 t ha?1 of the organic amendments. The concentrations of metals among the different mineral and organic fractions of soil were determined by several extraction procedures to study the metal distribution in the solid phase of the soil affected by the organic amendments. The results showed that the manure amendment increased the soil pH and the cation exchange capacity and enhanced the nutrient levels of these soils. The pine bark amendment decreased the soil pH and did not significantly change the nutrient status of soil. Soil pH, organic matter content and its degree of humification, which were altered by the amendments, were the main factors affecting Cu fractionation. Zn fractionation was mainly affected by soil pH. The addition of manure not only improved soil fertility, but also decreased metal bioavailability resulting in a reduction of metal toxicity. Conversely, pine bark amendment increased metal ioavailability. The use of sheep and horse manure could be a cost-effective practice for the restoration of contaminated mine soils.

Effects of forage type on diversity in bacterial pellets isolated from liquid and solid phases of the rumen content in sheep and goats.

Two sheep and two goats, fitted with a ruminal cannula, received two diets composed of 30% concentrate and 70% of either alfalfa hay (AL) or grass hay (GR) as forage in a two-period crossover design. Solid and liquid phases of the rumen were sampled from each animal immediately before feeding and 4 h post-feeding. Pellets containing solid associated bacteria (SAB) and liquid associated bacteria (LAB) were isolated from the corresponding ruminal phase and composited by time to obtain 2 pellets per animal (one SAB and one LAB) before DNA extraction. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal DNA was used to analyze bacterial diversity. A total of 78 and 77 bands were detected in the DGGE gel from sheep and goats samples, respectively. There were 18 bands only found in the pellets from sheep fed AL-fed sheep and 7 found exclusively in samples from sheep fed the GR diet. In goats, 21 bands were found only in animals fed the AL diet and 17 were found exclusively in GR-fed ones. In all animals, feeding AL diet tended (P < 0.10) to promote greater NB and SI in LAB and SAB pellets compared with the GR diet. The dendrogram generated by the cluster analysis showed that in both animal species all samples can be included in two major clusters. The four SAB pellets within each animal species clustered together and the four LAB pellets grouped in a different cluster. Moreover, SAB and LAB clusters contained two clear subclusters according to forage type. Results show that in all animals bacterial diversity was more markedly affected by the ruminal phase (solid vs. liquid) than by the type of forage in the diet.

Technical note: Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) to assess bacterial diversity in the rumen of sheep

The aim of this study was to compare automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques to assess bacterial diversity in the rumen of sheep. Sheep were fed 2 diets with 70% of either alfalfa hay or grass hay, and the solid (SOL) and liquid (LIQ) phases of the rumen were sampled immediately before feeding (0 h) and at 4 and 8 h postfeeding. Both techniques detected similar differences between forages, with alfalfa hay promoting greater (P < 0.05) bacterial diversity than grass hay. In contrast, whereas ARISA analysis showed a decrease (P < 0.05) of bacterial diversity in SOL at 4 h postfeeding compared with 0 and 8 h samplings, no variations (P > 0.05) over the postfeeding period were detected by DGGE. The ARISA technique showed lower (P < 0.05) bacterial diversity in SOL than in LIQ samples at 4 h postfeeding, but no differences (P > 0.05) in bacterial diversity between both rumen phases were detected by DGGE. Under the conditions of this study, the DGGE was not sensitive enough to detect some changes in ruminal bacterial communities, and therefore ARISA was considered more accurate for assessing bacterial diversity of ruminal samples. The results highlight the influence of the fingerprinting technique used to draw conclusions on factors affecting ruminal bacterial diversity.

Changes in rumen microbes in sheep in response to dietary forage quality and relationships with fermentation characteristics

An in vitro experiment was carried out using the Hohenheim gas production technique to evaluate 24-h gas production, apparently and truly degraded dry matter (DM), partitioning factor (PF), short chain fatty acids, crude protein (CP) and carbohydrate (CHO) fractionation of grass and multipurpose tree species (MPTS) foliage diets. Four grasses and three MPTS were used to formulate 12 diets of equal mixtures (0.5:0.5 on DM basis) of each grass with each MPTS. In vitro gas production was terminated after 24 h for each diet. True DM degradability was measured from incubated samples and combined with gas volume to estimate PF. Diets had greater (P<0.001) CP (102–183 g/kg DM) content than sole grasses (66–131 g/kg DM) and lower (P<0.001) concentrations of fibre fractions. Contrary to in vitro apparently degraded DM, in vitro truly degraded DM coefficient was greater (P<0.001) in diets (0.63–0.77) than in sole grasses (0.48–0.68). The PF was on average higher in diets than in sole grasses. The proportion of potentially degradable CP fractions (A1, B1, B2 and B3, based on the Cornell Net Carbohydrate and Protein System) in the diets ranged from 971 to 989 g/kg CP. Crude protein fractions, A and B2 were greater in diets but B1 and B3 fractions were less in diets than in sole grasses. A similar trend was also observed in the CHO fractions. Results showed that the nutritive value of the four grasses was improved when MPTS leaves were incorporated into the diet and this could ensure higher productivity of the animals.

Influence of castration on growth performance and carcass and meat quality of heavy female pigs

The main objetive of this Doctoral Thesis was to study the influence of female castration and pig sex on growth performance and carcass and meat quality of white pigs slaughtered at different final weights. Three experiments (Exp.) were conducted. In Exp. 1, a total of 200 (Landrace * Large White dam x Pietrain * Large White sire) gilts of 50 ± 3 days of age (23.3 ± 1.47 kg BW) was used to investigate the effects of castration (intact females, IF vs. castrated feamles, CF) and slaughter weight (106 vs. 122 kg BW) on productive performance and carcass and meat quality. There were four experimental treatments arranged as a 2 x 2 factorial and 5 replicates of 10 pigs each per treatment. Half of the gilts were ovariectomized at 58 d of age (8 days after the beginning of the trial; 29.8 ± 1.64 kg BW) whereas the other half remained intact. Meat samples were taken at m. Longissimus thoracis at the level of the last rib and subcutaneous fat samples were taken at the tail insertion. For the entire experiment period, CF had higher BW gain (P<0.05) and backfat and m. Gluteus medius (GM) fat thickness (P<0.001) than IF. However, IF had higher loin and trimmed primal cut yields (P<0.05) than CF. Meat quality was similar for IF and CF but the proportion of linoleic acid in subcutaneous fat was higher (P<0.001) for IF. Pigs slaughtered at 122 kg BW had higher (P<0.001) feed intake and poorer feed efficiency than pigs slaughtered at 106 kg BW. An increase in slaughter weight (SW) improved (P<0.001) carcass yield but decreased (P<0.05) trimmed primal cut yield. Meat from females slaughtered at the heavier BW was redder (a*; P<0.001) and had more (P<0.01) intramuscular fat and less thawing (P<0.05) and cooking (P<0.10) loss than meat from females slaughtered at the lighter BW. Also, females slaughtered at 122 kg BW had less (P<0.01) linoleic acid content in the subcutaneous fat than pigs slaughtered at 106 kg BW. Castration of gilts and slaughtering at heavier BW might be useful practices for the production of heavy pigs destined to the dry cured industry in which a certain amount of fat in the carcass is required. In contrast, when the carcasses are destined to fresh meat production, IF slaughtered at 106 kg BW are a more efficient alternative. In Exp. 2, crossbred pigs (n=240) from Pietrain*Large White sires mated to Landrace*Large White dams with an average of 100 d of age (60.5 ± 2.3 kg) were used to investigate the effects of gender and slaughter weight (SW) on growth performance and carcass and meat quality characteristics. There were 6 treatments arranged factorially with 3 genders (IF vs. CF vs.castrated males, CM) and 2 SW (114 vs. 122 kg BW). Each of the 6 combinations of treatments was replicated 4 times and the experimental unit was a pen with 10 pigs. Castrated males and CF ate more feed, grew faster and had more carcass backfat depth and fat thickness at the GM muscle, but lower loin yield than IF (P<0.05). In addition, CF and CM had more intramuscular fat (P<0.05) and less linoleic acid content in the subcutaneous fat (P<0.01) than IF. Pigs slaughtered at 122 kg BW had lower ADG (P<0.05), poor gain-to-feed ratio (P<0.05), and more GM fat than pigs slaughtered at 114 kg BW (P < 0.05). It is concluded that CF and CM had similar productive performance and meat quality characteristics when slaughtered at the same age, and that the castration of females improved daily gains and increased weight and fat content of primal cuts with respect to IF. Therefore, castration of females is recommended in pigs destined to the dry-cured industry because of the beneficial effects on the quality of the primal cuts. In Exp. 3, the effects of gender and castration of females (IF vs. CF vs. CM) on performance and carcass and meat quality were studied in crossbred pigs (Landrace x Large White dams x Duroc sires) slaughtered at 119.2 (trial 1) or 131.6 (trial 2) kg BW. Intact females had better feed conversion and less carcass fat than CF and CM. Trimmed shoulder yield was higher for CM than for CF with IF being intermediate. Primal cut yield and meat quality, however were similar for all treatments. Proportion of linoleic acid in backfat was lower for CF than for IF or CM, and the differences were significant in pigs slaughtered witn 131.6 kg BW. The higher fat content and the fatty acid profile favour the use of CF and CM over IF for the production of heavy pigs destined to the dry-cured industry.

Animal performance and meat characteristics in steers reared in intensive conditions fed with different vegetable oils

Enhancing the quality of beef meat is an important goal in terms of improving both the nutritional value for the consumer and the commercial value for producers. The aim of this work was to study the effects of different vegetable oil supplements on growth performance, carcass quality and meat quality in beef steers reared under intensive conditions. A total of 240 Blonde D? Aquitaine steers (average BW = 293.7 ± 38.88 kg) were grouped into 24 batches (10 steers/batch) and were randomly assigned to one of the three dietary treatments (eight batches per treatment), each supplemented with either 4% hydrogenated palm oil (PALM) or fatty acids (FAs) from olive oil (OLI) or soybean oil (SOY). No differences in growth performance or carcass quality were observed. For the meat quality analysis, a steer was randomly selected from each batch and the 6th rib on the left half of the carcass was dissected. PALM meat had the highest percentage of 16:0 ( P< 0.05) and the lowest n-6/n-3 polyunsaturated fatty acids (PUFA) ratio ( P< 0.05), OLI had the highest content of t 11-18:1 ( P< 0.01) and c 9,t 11-18:2 ( P< 0.05) and SOY showed the lowest value of monounsaturated fatty acids (MUFA) ( P< 0.001), the highest percentage of PUFA ( P< 0.01) and a lower index of atherogenicity ( P = 0.07) than PALM. No significant differences in the sensory characteristics of the meat were noted. However, the results of the principal component analysis of meat characteristics enabled meat from those steers that consumed fatty acids from olive oil to be differentiated from that of steers that consumed soybean oil.

Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and batch cultures of rumen microorganisms

The objective of the current study was to assess how closely batch cultures (BC) of rumen microorganisms can mimic the dietary differences in fermentation characteristics found in the rumen, and to analyse changes in bacterial diversity over the in vitro incubation period. Four ruminally and duodenally cannulated sheep were fed four diets having forage : concentrate ratios (FCR) of 70 : 30 or 30 : 70, with either alfalfa hay or grass hay as forage. Rumen fluid from each sheep was used to inoculate BC containing the same diet fed to the donor sheep, and the main rumen fermentation parameters were determined after 24 h of incubation. There were differences between BC and sheep in the magnitude of most measured parameters, but BC detected differences among diets due to forage type similar to those found in sheep. In contrast, BC did not reproduce the dietary differences due to FCR found in sheep for pH, degradability of neutral detergent fibre and total volatile fatty acid (VFA) concentrations. There were differences between systems in the magnitude of most determined parameters and BC showed higher pH values and NH3–N concentrations, but lower fibre degradability and VFA and lactate concentrations compared with sheep. There were significant relationships between in vivo and in vitro values for molar proportions of acetate, propionate and butyrate, and the acetate : propionate ratio. The automated ribosomal intergenic spacer analysis (ARISA) of 16S ribosomal deoxyribonucleic acid showed that FCR had no effect on bacterial diversity either in the sheep rumen fluid used as inoculum (IN) or in BC samples. In contrast, bacterial diversity was greater with alfalfa hay diets than those with grass hay in the IN, but was unaffected by forage type in the BC. Similarity index between the bacterial communities in the inocula and those in the BC ranged from 67·2 to 74·7%, and was unaffected by diet characteristics. Bacterial diversity was lower in BC than in the inocula with 14 peaks out of a total of 181 detected in the ARISA electropherograms never appearing in BC samples, which suggests that incubation conditions in the BC may have caused a selection of some bacterial strains. However, each BC sample showed the highest similarity index with its corresponding rumen IN, which highlights the importance of using rumen fluid from donors fed a diet similar to that being incubated in BC when conducting in vitro experiments.

Peri-conceptional changes in maternal exposure to sewage sludge chemicals disturbs fetal thyroid gland development in sheep

Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The conceptus regulates tryptophanyl-tRNA synthetase and superoxide dismutase 2 in the sheep caruncular endometrium during early pregnancy

Copyright © 2015 Elsevier Ltd. All rights reserved. This research project was funded by NHS Grampian R&D (project number RG05/019).

Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms

Prion diseases are natural transmissible neurodegenerative disorders in humans and animals. They are characterized by the accumulation of a protease-resistant scrapie-associated prion protein (PrPSc) of the host-encoded cellular prion protein (PrPC) mainly in the central nervous system. Polymorphisms in the PrP gene are linked to differences in susceptibility for prion diseases. The mechanisms underlying these effects are still unknown. Here we describe studies of the influence of sheep PrP polymorphisms on the conversion of PrPC into protease-resistant forms. In a cell-free system, sheep PrPSc induced the conversion of sheep PrPC into protease-resistant PrP (PrP-res) similar or identical to PrPSc. Polymorphisms present in either PrPC or PrPSc had dramatic effects on the cell-free conversion efficiencies. The PrP variant associated with a high susceptibility to scrapie and short survival times of scrapie-affected sheep was efficiently converted into PrP-res. The wild-type PrP variant associated with a neutral effect on susceptibility and intermediate survival times was converted with intermediate efficiency. The PrP variant associated with scrapie resistance and long survival times was poorly converted. Thus the in vitro conversion characteristics of the sheep PrP variants reflect their linkage with scrapie susceptibility and survival times of scrapie-affected sheep. The modulating effect of the polymorphisms in PrPC and PrPSc on the cell-free conversion characteristics suggests that, besides the species barrier, polymorphism barriers play a significant role in the transmissibility of prion diseases.

Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein

Transmissible spongiform encephalopathies, or prion diseases, are fatal degenerative disorders of the central nervous system that affect humans and animals. Prions are nonconventional infectious agents whose replication depends on the host prion protein (PrP). Transmission of prions to cultured cells has proved to be a particularly difficult task, and with a few exceptions, their experimental propagation relies on inoculation to laboratory animals. Here, we report on the development of a permanent cell line supporting propagation of natural sheep scrapie. This model was obtained by stable expression of a tetracycline-regulatable ovine PrP gene in a rabbit epithelial cell line. After exposure to scrapie agent, cultures were repeatedly found to accumulate high levels of abnormal PrP (PrPres). Cell extracts induced a scrapie-like disease in transgenic mice overexpressing ovine PrP. These cultures remained healthy and stably infected upon subpassaging. Such data show that (i) cultivated cells from a nonneuronal origin can efficiently replicate prions; and (ii) species barrier can be crossed ex vivo through the expression of a relevant PrP gene. This approach led to the ex vivo propagation of a natural transmissible spongiform encephalopathy agent (i.e., without previous experimental adaptation to rodents) and might be applied to human or bovine prions.

Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation

Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2/LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for ≈25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.

Direct transformation of rodent fibroblasts by jaagsiekte sheep retrovirus DNA

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary carcinoma, a unique animal model for human bronchioalveolar carcinoma. We previously isolated a JSRV proviral clone and showed that it was both infectious and oncogenic. Thus JSRV is necessary and sufficient for the development of ovine pulmonary carcinoma, but no data are available on the mechanisms of transformation. Inspection of the JSRV genome reveals standard retroviral genes, but no evidence for a viral oncogene. However, an alternate ORF in pol (orf-x) might be a candidate for a transforming gene. We tested whether the JSRV genome might encode a transforming gene by transfecting an expression plasmid for JSRV [pCMVJS21, driven by the cytomegalovirus (CMV) immediate early promoter] into mouse NIH 3T3 cells. Foci of transformed cells appeared in the transfected cultures 2–3 weeks posttransfection; cloned transformants showed anchorage independence for growth, and they expressed JSRV RNA. These results indicate that the JRSV genome contains information with direct transforming potential for NIH 3T3 cells. Transfection of a mutated version of pCMVJS21 in which the orf-x protein was terminated by two stop codons also gave transformed foci. Thus, orf-x was eliminated as the candidate transforming gene. In addition, another derivative of pCMVJS21 (pCMVJS21ΔGP) in which the gag, pol (and orf-x) coding sequences were deleted also gave transformed foci. These results indicate that the envelope gene carries the transforming potential. This is an unusual example of a native retroviral structural protein with transformation potential.

Distribution of endogenous type B and type D sheep retrovirus sequences in ungulates and other mammals.

The jaagsiekte sheep retrovirus (JSRV), which appears to be a type B/D retrovirus chimera, has been incriminated as the cause of ovine pulmonary carcinoma. Recent studies suggest that the sequences related to this virus are found in the genomes of normal sheep and goats. To learn whether there are breeds of sheep that lack the endogenous viral sequences and to study their distribution among other groups of mammals, we surveyed several domestic sheep and goat breeds, other ungulates, and various mammal groups for sequences related to JSRV. Probes prepared from the envelope (SU) region of JSRV and the capsid (CA) region of a Peruvian type D virus related to JSRV were used in Southern blot hybridization with genomic DNA followed by low- and high-stringency washes. Fifteen to 20 CA and SU bands were found in all members of the 13 breeds of domestic sheep and 6 breeds of goats tested. There were similar findings in 6 wild Ovis and Capra genera. Within 22 other genera of Bovidae including domestic cattle, and 7 other families of Artiodactyla including Cervidae, there were usually a few CA or SU bands at low stringency and rare bands at high stringency. Among 16 phylogenetically distant genera, there were generally fewer bands hybridizing with either probe. These results reveal wide-spread phylogenetic distribution of endogenous type B and type D retroviral sequences related to JSRV among mammals and argue for further investigation of their potential role in disease.

Development of a bovine X chromosome linkage group and painting probes to assess cattle, sheep, and goat X chromosome segment homologies.

The X chromosome linkage group is conserved in placental mammals. However, X chromosome morphological differences, due to internal chromosome rearrangements, exist among mammalian species. We have developed bovine chromosome painting probes for Xp and Xq to assess segment homologies between the submetacentric bovine X chromosome and the acrocentric sheep and goat X chromosomes. These painting probes and their corresponding DNA libraries were developed by chromosome micromanipulation, DNA micropurification, microcloning, and PCR amplification. The bovine Xp painting probe identified an interstitially located homologous segment in the sheep and goat Xq region, most probably resulting from chromosome inversion. Ten type II (microsatellite) markers obtained from the bovine Xq library and five other X chromosome assigned, but unlinked, markers were used to generate a linkage map for Xq spanning 89.4 centimorgans. The chromosome painting probes and molecular markers generated in this study would be useful for comparative mapping and tracing of internal X chromosome rearrangements in all ruminant species and would contribute to the understanding of mammalian sex chromosome evolution.