Balancing consumer and societal requirements for sheep meat production: an Australasian perspective.

Although there has been a decline in sheep numbers in Australia and New Zealand, both countries remain significant producers and exporters of sheep meat. The ongoing demand for more sustainable and ethical animal farming systems and practices requires sheep production industries to be both vigilant and responsive to consumer and the broader societal needs. Demonstration of continuous improvement in animal welfare is paramount and the welfare risks and challenges confronting Australasian sheep industries now and into the future are discussed.

Key features of 'environmental fit' that promote good animal welfare in different husbandry systems.

In animal production there are two core dimensions to environmental fit, one that centres on the capacity of the environment to meet an animal's needs and the other concerns the capacity of the animal to match or 'fit' the environment. Efforts to increase capacity in both of these dimensions can contribute substantially to the continuous improvement of animal welfare within different livestock production systems. Achieving this will require an integrated approach that combines genetic, environmental and management strategies.

Motivation to obtain a food reward of pregnant ewes in negative energy balance: behavioural, metabolic and endocrine considerations.

Low food availability often coincides with pregnancy in grazing animals. This study investigated how chronic reductions in food intake affected feeding motivation, and metabolic and endocrine parameters in pregnant sheep, which might be indicative of compromised welfare. Ewes with an initial Body Condition Score of 2.7±0.3 (BCS; 0 indicates emaciation and 5 obesity) were fed to attain low (LBC 2.0±0.0,), medium (MBC 2.9±0.1) or high BCS (HBC 3.7±0.1) in the first trimester of pregnancy. A feeding motivation test in which sheep were required to walk a set distance for a palatable food reward was conducted in the second trimester. LBC and MBC ewes consumed more rewards (P=0.001) and displayed a higher expenditure (P=0.02) than HBC ewes, LBC ewes also tended to consume more rewards than MBC ewes (P=0.09). Plasma leptin and glucose concentrations were inversely correlated to expenditure (both P<0.05) and appear to be associated with hunger in sheep. LBC ewes were in negative energy balance, with lower muscle dimensions, plasma glucose, leptin, insulin, cortisol, and insulin-like growth factor-1 concentrations and higher free fatty acids concentrations compared to HBC ewes; metabolic and endocrine parameters of the MBC ewes were intermediate. The high feeding motivation and negative energy balance of low BCS ewes suggested an increased risk of compromised welfare. Imposing even a small cost on a food reward reduced motivation substantially in high BCS ewes (despite high intake when food was freely available). Assessment of a willingness to work for rewards, combined with measures of key metabolic and endocrine parameters, may provide sensitive barometers of welfare in energetically-taxed animals.

Temperament and hypothalamic-pituitary-adrenal axis function are related and combine to affect growth, efficiency, carcass, and meat quality traits in Brahman steers.

Associations between temperament, stress physiology, and productivity were studied in yearling Brahman steers (n = 81). Steers differed in calpain system gene marker status; 41 were implanted with a hormonal growth promotant at feedlot entry. Temperament was assessed with repeated measurements of flight speed (FS) and crush score (CS) during 6 mo of backgrounding at pasture and 117 d of grain finishing. Adrenal responsiveness was assessed with ACTH challenge, with plasma samples collected immediately before and 60 min after challenge. Steers with higher FS and CS had higher prechallenge plasma cortisol, glucose, lactate, and nonesterified fatty acid concentrations. The ACTH-induced cortisol response was unrelated to FS or CS, but glucose remained higher after challenge in flightier steers. The hormonal growth promotant reduced adrenal responsiveness; tenderness genotype had no effect. When temperament assessments and cortisol concentrations before and after challenge were combined in a principal components analysis, four vectors accounting for 38%, 25%, 18%, and 9% of the variation were identified. The first vector had significant loadings on temperament and prechallenge cortisol; increasing scores were associated with increased plasma glucose, lactate, and nonesterified fatty acid and with reductions in BW and feedlot growth rates, carcass fatness, and muscle pH. The second vector loaded only on ACTH-induced cortisol response; increased scores related to increased residual feed intake, number of daily feed sessions, and meat marbling score. The third and fourth vectors had different loadings on FS and CS and appeared to identify different aspects of temperament measured by FS or CS. Fewer associations were found between the third or fourth vectors and productivity traits, possibly because of lower variance accounted for by these vectors. In conclusion, temperament was related to prechallenge cortisol but not to ACTH-induced cortisol response. Principal components analysis separated these traits into separate components, which in turn had different relations with productivity traits. The largest component of temperament was described similarly by FS and CS, but there were smaller components that these described differently. There were some temperament-related differences in the metabolic status of the steers which were not related to the variation in cortisol, suggesting involvement of the sympatho-adrenal-medullary axis in these temperament-related effects.

Cattle temperament: persistence of assessments and associations with productivity, efficiency, carcass and meat quality traits.

Relationships between temperament and a range of performance, carcass, and meat quality traits in young cattle were studied in 2 experiments conducted in New South Wales (NSW) and Western Australia (WA), Australia. In both experiments, growth rates of cattle were assessed during backgrounding on pasture and grain finishing in a feedlot. Carcass and objective meat quality characteristics were measured after slaughter. Feed intake and efficiency during grain finishing were also determined in NSW. Brahman (n = 82 steers and 82 heifers) and Angus (n = 25 steers and 24 heifers) cattle were used in the NSW experiment. In NSW, temperament was assessed by measuring flight speed [FS, m/s on exit from the chute (crush)] on 14 occasions, and by assessing agitation score during confinement in the crush (CS; 1 = calm to 5 = highly agitated) on 17 occasions over the course of the experiment. Brahman (n = 173) and Angus (n = 20) steers were used in the WA experiment. In WA, temperament was assessed by measuring FS on 2 occasions during backgrounding and on 2 occasions during grain feeding. At both sites, a hormonal growth promotant (Revalor-H, Virbac, Milperra, New South Wales, Australia) was applied to one-half of the cattle at feedlot entry, and the Brahman cattle were polymorphic for 2 calpain-system markers for beef tenderness. Temperament was not related (most P > 0.05) to tenderness gene marker status in Brahman cattle and was not (all P > 0.26) modified by the growth promotant treatment in either breed. The Brahman cattle had greater individual variation in, and greater correlations within and between, repeated assessments of FS and CS than did the Angus cattle. Correlations for repeated measures of FS were greater than for repeated assessments of CS, and the strength of correlations for both declined over time. Average FS or CS for each experiment and location (NSW or WA × backgrounding or finishing) were more highly correlated than individual measurements, indicating that the average values were a more reliable assessment of cattle temperament than any single measure. In Brahman cattle, increased average FS and CS were associated with significant (P < 0.05) reductions in backgrounding and feedlot growth rates, feed intake and time spent eating, carcass weight, and objective measures of meat quality. In Angus cattle, the associations between temperament and growth rates, feed intake, and carcass traits were weaker than in Brahmans, although the strength of relationships with meat quality were similar.

Genetic and environmental effects on meat quality.

In order for livestock industries to consistently produce high quality meat, there must be an understanding of the factors that cause quality to vary, as well as the contribution of genetics. A brief overview of meat tenderness is presented to understand how genotype and environment may interact to influence this trait. Essentially, meat tenderness is determined from the contribution of connective tissue, sarcomere length determined pre-rigor and rate of proteolysis during ageing, as well as contributions from intramuscular fat and post-mortem energy metabolism. The influence of mutations in myostatin, the callipyge gene, the Carwell or rib eye muscle gene as well as the calpain system on meat tenderness is presented. Specific examples of interactions between the production or processing environment and genetics are presented for both sheep and cattle. The day-to-day variation in tenderness is evident across experiments and this variation needs to be controlled in order to consistently produce tender meat.

The effects of 12, 30, or 48 hours of road transport on the physiological and behavioral responses of sheep.

To identify long-distance transport durations compatible with acceptable animal welfare, the aim of this study was to determine the responses of healthy sheep to road transport under good conditions for 12, 30, or 48 h. Merino ewes (n = 120; 46.9 +/- 0.39 kg) were allocated to road transport treatments of 12, 30, or 48 h, with 2 replicates per treatment. Blood and urine samples and BW were taken pretransport and at 0, 24, 48, and 72 h posttransport. Lying time was measured using data loggers. Increasing transport durations resulted in reduced (P < 0.001) BW and increased (P < 0.05) hemoconcentration, but these effects did not exceed clinically normal ranges for any transport duration, and sheep generally recovered to pretransport values within 72 h posttransport. Sheep transported for 30 and 48 h had less BW on arrival than sheep transported for 12 h (P < 0.001). There were no differences (P > 0.05) between the 12- and 30-h treatments in sheep BW at 24, 48, or 72 h after arrival. Sheep transported for 30 and 48 h had greater total plasma protein concentrations on arrival than sheep transported for 12 h (P < 0.001). Although the white cell count and neutrophil:lymphocyte ratio increased with transport, there were no consistent effects of transport duration. There were also no effects (P = 0.10) of transport duration on plasma cortisol concentrations. There were no treatment differences (P > 0.05) in lying times during the first 18 h after arrival. Sheep transported for 30 or 48 h lay down less (P < 0.05) than sheep transported for 12 h between 18 and 24 h after arrival, but there were no other differences over 72 h. These findings indicate that healthy adult sheep, transported under good conditions, can tolerate transport durations of up to 48 h without undue compromise to their welfare.

Effect of glycogen concentration and form on the response to electrical stimulation and rate of post-mortem glycolysis in ovine muscle.

The associations between the muscle glycogen concentration and form and the rate of post-mortem glycolysis in ovine muscle were investigated. Twenty-two merino wethers (18-24 months) were allocated to either roughage or concentrate pelleted diets for 34 days prior to slaughter. An exercise depletion/repletion model was applied four days prior to slaughter to generate differences in muscle glycogen levels at slaughter. Muscle biopsies were taken from the m. semimembranosus (SM) and m. semitendinosus (ST) prior to and immediately after exercise for muscle glycogen determination. At slaughter, one side was electrically stimulated and both sides were conventionally chilled for 24h. The pH response to electrical stimulation (ΔpH) and the rate of pH decline adjusted to a constant temperature of 38°C over the initial 6h post-mortem period was determined in three muscles (m. longissimus thoracis et lumborum LTL, SM and ST). In addition, the concentrations of glycogen, proglycogen (PG), macroglycogen (MG) and lactate in the three muscles immediately after slaughter were determined. The glycogen loss due to exercise was influenced by diet (P<0.01; concentrate 63% and roughage 73%) but did not differ between muscles. The rates of repletion significantly varied between muscles (SM>ST) and diet (concentrate>roughage). The available glycogen (glycogen(A)) and MG concentrations at slaughter varied significantly depending on the diet (P<0.01) and muscle (P<0.001). The percentage of MG relative to MG+PG varied between muscles (46%, 50% and 57% for the ST, LTL and SM). The concentration and form of available glycogen at slaughter did not influence the response to electrical stimulation after adjusting for pre-stimulation pH (P<0.01). The ΔpH varied significantly between muscles (0.39±0.03, 0.26±0.02 and 0.20±0.03 for the ST, LTL and SM) after adjusting for pre-stimulation pH. Differences in the temperature adjusted rate of pH decline were observed between the muscles (LTL>SM>ST). Importantly, a positive linear association (P=0.05) was found between muscle glycogen(A) concentration at slaughter and the rate of pH decline (temperature adjusted).

Have we underestimated the impact of pre-slaughter stress on meat quality in ruminants?

Stress is the inevitable consequence of the process of transferring animals from farm to slaughter. The effects of chronic stress on muscle glycogen depletion and the consequent dark cutting condition have been well documented. However, there has been little examination of the consequences of acute stress immediately pre-slaughter on ruminant meat quality. New evidence is emerging to show that non pH-mediated effects on meat quality can occur through pre-slaughter stress in cattle and sheep. This paper reviews the general aspects of pre-slaughter stress in the pre-slaughter context. It then examines the impacts of pre-slaughter stressors on ruminant carcass and meat quality and considers remedial strategies for remediating and preventing pre-slaughter stress. Further quantification of the biological costs of pre-slaughter stress and the consequences to meat quality is required.

Epistasis between calpain 1 and its inhibitor calpastatin within breeds of cattle.

The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactions of causative mutations for quantitative trait loci (QTL) at calpain 1 (CAPN1), located on chromosome 29, with causative mutations for QTL variation at calpastatin (CAST), located on chromosome 7, in cattle. First we identified potential causative mutations at CAST and then genotyped these along with putative causative mutations at CAPN1 in >1500 cattle of seven breeds. The maximum allele substitution effect on the phenotype of the CAPN1:c.947G>C single nucleotide polymorphism (SNP) was 0.14 sigma(p) (P = 0.0003) and of the CAST:c.155C>T SNP was also 0.14 sigma(p) (P = 0.0011) when measured across breeds. We found significant epistasis between SNPs at CAPN1 and CAST in both taurine and zebu derived breeds. There were more additive x dominance components of epistasis than additive x additive and dominance x dominance components combined. A minority of breed comparisons did not show epistasis, suggesting that genetic variation at other genes may influence the degree of epistasis found in this system.

Comparison of 16S rRNA sequencing with conventional and commercial phenotypic techniques for identification of enterococci from the marine environment.

AIMS: To compare accuracy of genus and species level identification of presumptive enterococci isolates from the marine environment using conventional biochemical testing, four commercial identification systems and 16S rRNA sequence analysis. METHODS AND RESULTS: Ninety-seven environmental bacterial isolates identified as presumptive enterococci on mEI media were tested using conventional and Enterococcus genus screen biochemical tests, four commercial testing systems and 16S rRNA sequencing. Conventional and Enterococcus genus screen biochemical testing, 16S rRNA sequencing and two commercial test systems achieved an accuracy of > or = 94% for Enterococcus genus confirmation. Conventional biochemical testing and 16S rRNA sequencing achieved an accuracy of > or = 90% for species level identification. CONCLUSIONS: For confirmation of Enterococcus genus from mEI media, conventional or genus screen biochemical testing, 16S rRNA sequencing and the four commercial systems were correct 79-100% of the time. For speciation to an accuracy of 90% or better, either conventional biochemical testing or 16S rRNA sequencing is required. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of presumptive environmental Enterococcus isolates to genus and species level is an integral part of laboratory quality assurance and further characterization of Enterococcus species from pollution incidents. This investigation determines the ability of six different methods to correctly identify environmental isolates.

Enumeration and speciation of enterococci found in marine and intertidal sediments and coastal water in southern California.

AIMS: To determine the levels and species distribution of enterococci in intertidal and marine sediments and coastal waters at two beaches frequently in violation of bacterial water standards. METHODS AND RESULTS: Faecal indicator bacteria were extracted from sediment and enumerated using membrane filtration. High levels of enterococci were detected in intertidal sediments in a seasonal river and near a storm drain outlet. Low levels were found in marine sediments at 10 m depths and in surf zone sand. Bacterial isolates presumptively identified as Enterococcus on mEI media were speciated. The predominant species found in both water and sediment included Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus casseliflavus and Enterococcus mundtii. A number of isolates (11-26%) from regulatory water samples presumptively identified as enterococci on mEI media were subsequently identified as species other than Enterococcus. At both study sites, the distribution of species present in water was comparable with those in sediments and the distribution of species was similar in water samples passing and exceeding bacterial indicator standards. CONCLUSIONS: High levels of Enterococcus in intertidal sediments indicate retention and possible regrowth in this environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Resuspension of enterococci that are persistent in sediments may cause beach water quality failures and calls into question the specificity of this indicator for determining recent faecal contamination.

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed.

AIMS: The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. METHODS AND RESULTS: Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. CONCLUSIONS: None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds.

Quantitation of Cryptosporidium parvum infection in cell culture using a colorimetric in situ hybridization assay.

A quantitative colorimetric in situ hybridization assay was developed for detecting Cryptosporidium parvum infection in cell cultures using a digoxigenin-labeled probe targeting 18S rRNA. Intra-cellular developmental stages of C. parvum such as trophozoites and meronts were clearly discerned by light microscopy as localized areas of dark purple/black precipitate against a colorless background. Infections developed focally and the term infectious focus was applied to each cluster of developmental stages. There were no significant differences in the number of infectious foci following 24 h or 48 h incubation. However, 24 h and 48 h dose response curves were significantly different when infectivity was measured as the number of developmental stages per monolayer, with an average of 5.3-fold more stages following 48 h incubation. When infectivity was expressed as the number of infectious foci per inoculum oocyst converted to a percentage, it was demonstrated that the rate of infection decreased with increasing oocyst age. Oocysts of the Iowa isolate that were 7-10 days old demonstrated 7.8+/-2.4% infectivity (mean +/- standard deviation) compared to 4.2+/-0.8% for 21-28 day-old oocysts and 1.4+/-1.3% for 42-70 day-old oocysts. The assay also detected infection with other genotype 2 oocysts and a genoptye 1 isolate. This assay provides a direct quantitative approach for measuring C. parvum infectivity in cell culture.

Transformation of a kappa-opioid receptor antagonist to a kappa-agonist by transfer of a guanidinium group from the 5'- to 6'-position of naltrindole.

The importance of the indole scaffold of GNTI 3 in directing its address (5'-guanidinium group) to associate with the Glu297 residue of the kappa-opioid receptor was investigated by the synthesis and biological evaluation of its 4'- (4a), 6'- (4b), and 7'- (4c) regioisomers. The finding that only the 5'-regioisomer (GNTI) possessed potent kappa-opioid antagonist activity and high affinity at kappa-receptors illustrates the importance of the 5'-position in orienting the guanidinium group to the proper recognition locus (Glu 297) for potent kappa-antagonist activity. The discovery that the 6'-regioisomer of GNTI was a potent kappa-agonist, together with the results of site-directed mutagenesis studies that are consistent with association between the 6'-guanidinium group and Glu297, suggest that the transition from an inactive to an active state of the kappa-receptor involves a conformational change of TM6. We propose that association of the 6'-guanidinium group of 4b with Glu297 promotes axial rotational motion of transmembrane helix VI which leads to receptor activation via a conformational change of inner loop 3.

Investigation of the selectivity of oxymorphone- and naltrexone-derived ligands via site-directed mutagenesis of opioid receptors: exploring the "address" recognition locus.

The delta-selective opioid antagonist naltrindole (NTI), as well as the kappa-selective opioid antagonists norbinaltorphimine (norBNI) and 5'-guanidinonaltrindole (GNTI), are derived from naltrexone, a universal opioid antagonist. Previous studies have indicated that extracellular loop III is the key region for discrimination by naltrexone-derived selective ligands between the delta, mu, and kappa opioid receptor types. It has been proposed that selective ligands could bind to all three receptor types if the appropriate portions of the extracellular loops were eliminated. To investigate this possibility, several single-point mutant opioid receptors have been generated with the aim of conferring enhanced affinity of selective ligands for their nonpreferred receptor types. Mutations were made in all three types of opioid receptors with the focus on two positions at the extracellular end of transmembrane regions (TM) VI and VII. It was found that the delta-selective NTI could bind both mu and kappa receptors with significantly enhanced affinity when an aromatic residue in TM VII was replaced with alanine (mu[W318A] and kappa[Y312A]). Similarly, kappa-selective antagonists, norBNI and GNTI, showed enhanced affinity for the mu[W318A] mutant and for both mu and delta receptors when a glutamate residue was incorporated into the extracellular end of TM VI (mu[K303E] and delta[W284E]). These results demonstrate that naltrexone-derived selective ligands achieve their selectivity via a combination of enhanced affinity of the address for a particular subsite along with loss of affinity due to steric interference at nonpreferred types. The results reveal key residues in the "address" recognition locus that contribute to the selectivity of opioid ligands and support the hypothesis that recognition of the naltrexone moiety is essentially the same for all three receptor types.

Exploring the unique pharmacology of a novel opioid receptor, ZFOR1, using molecular modeling and the 'message-address' concept.

Previous studies have probed the structural basis of ligand selectivity in the mu, delta and kappa opioid receptors through the application of molecular modeling techniques in concert with the 'message-address' concept. Here, this approach was used in an attempt to rationalize the unique pharmacological profile of a recently cloned novel opioid receptor, ZFOR1 (ZebraFish Opioid Receptor 1). Specifically, a model of the transmembrane domains of ZFOR1 was constructed and used to explore the binding modes of various prototypical opioid ligands. The results show that the 'message' portion of the binding pocket of ZFOR1 is highly conserved; hence, the binding modes of non-selective opioid ligands are well preserved. In contrast, a small number of variant residues at the extracellular end of the binding pocket, particularly Lys288 (VI:26) and Trp304 (VII:03), are shown to create adverse steric interactions with all delta and kappa selective ligands examined, thereby disrupting their binding modes. These results are consistent with, and serve as an explanation for, the observed pharmacology of this receptor, lending support to both the validity of the 'message-address' concept itself and to the use of molecular modeling approaches in its application.

Synthesis and evaluation of 4-(N,N-diarylamino)piperidines with high selectivity to the delta-opioid receptor: a combined 3D-QSAR and ligand docking study.

A series of 4-(N,N-diarylamino)piperidines are synthesized and evaluated for high affinity binding and selectivity to the delta-opioid receptor using a combination of 3D-QSAR and molecular docking techniques. Based on experimental ligand binding data to both mu- and delta- opioid receptors, CoMFA fields are generated and applied to identify potential ligand modifications to further optimize lead compounds. Molecular docking experiments to the delta-receptor are also reported that explain the CoMFA trends predicted as well as the differential binding and selectivity displayed by various compounds in the series. An analysis of the binding site model proposed indicates the piperidines take advantage of 3 key sites or binding domains within the delta-receptor. These include an aromatic pocket (approximately 1/3 into the receptor cavity), an aspartic acid residue (which serves as a docking point for the piperidinyl cationic amine) and a hydrophobic pocket at the extracellular boundary of the receptor cavity. Links are established between ligand modification and amino acid composition at these sites in mu and delta, providing new insight to the structural basis to binding and selectivity across the series and for related piperazines (i.e. SNC80 and BW373U86). Results are also presented that indicate delta- and mu-selectivity may be determined at alternate sites, suggesting opioid receptors may display multiple binding domains. The model is further supported by comparisons with opiate binding modes and site directed mutagenesis studies and is finally applied to suggest new strategies in ligand design.