Reduction in Rumen Tetracycline-Insensitive Bacteria during a Grain Challenge Using the Isoflavone Biochanin A.

The isoflavone biochanin A was previously shown to promote weight gain in growing steers by selectively inhibiting rumen bacteria-like growth-promoting feed antibiotics. The hypothesis that biochanin A inhibited the action of drug efflux pumps was tested by enumerating tetracycline-insensitive bacteria from steers in a subacute rumen acidosis (SARA) challenge. Steers (n = 3/group) treatment groups were forage only, SARA control, SARA with monensin (0.2 g d-1), and SARA with biochanin A (6.0 g d-1). As the steers were stepped up from the forage-only basal diet to 70% cracked corn, the number of rumen bacteria enumerated on two tetracycline-containing media types (nutrient glucose agar and tetracycline, and bile esculin azide and tetracycline) increased (p < 0.05) from as little as 1.7(105) to as great as 6.7(106) cfu mL-1 on the nutrient glucose agar in the SARA and monensin control groups. The biochanin A group maintained the same number of tetracycline-insensitive bacteria as the forage-only controls (p > 0.05). The effects were similar to the more selective media type, but the differences were smaller. These results support the hypothesis that biochanin A inhibits the activity of drug efflux pumps in vivo.

Profiling of cool-season forage arabinoxylans via a validated HPAEC-PAD method.

Cool-season pasture grasses contain arabinoxylans (AX) as their major cell wall hemicellulosic polysaccharide. AX structural differences may influence enzymatic degradability, but this relationship has not been fully explored in the AX from the vegetative tissues of cool-season forages, primarily because only limited AX structural characterization has been performed in pasture grasses. Structural profiling of forage AX is a necessary foundation for future work assessing enzymatic degradability and may also be useful for assessing forage quality and suitability for ruminant feed. The main objective of this study was to optimize and validate a high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) method for the simultaneous quantification of 10 endoxylanase-released xylooligosaccharides (XOS) and arabinoxylan oligosaccharides (AXOS) in cool-season forage cell wall material. The following analytical parameters were determined or optimized: chromatographic separation and retention time (RT), internal standard suitability, working concentration range (CR), limit of detection (LOD), limit of quantification (LOQ), relative response factor (RRF), and quadratic calibration curves. The developed method was used to profile the AX structure of four cool-season grasses commonly grown in pastures (timothy, Phleum pratense L.; perennial ryegrass, Lolium perenne L.; tall fescue, Schedonorus arundinaceus (Schreb.) Dumort.; and Kentucky bluegrass, Poa pratensis L.). In addition, the cell wall monosaccharide and ester-linked hydroxycinnamic acid contents were determined for each grass. The developed method revealed unique structural aspects of the AX structure of these forage grass samples that complemented the results of the cell wall monosaccharide analysis. For example, xylotriose, representing an unsubstituted portion of the AX polysaccharide backbone, was the most abundantly-released oligosaccharide in all the species. Perennial rye samples tended to have greater amounts of released oligosaccharides compared to the other species. This method is ideally suited to monitor structural changes of AX in forages as a result of plant breeding, pasture management, and fermentation of plant material.

Red clover supplementation modifies rumen fermentation and promotes feed efficiency in ram lambs.

Red clover produces isoflavones, including biochanin A, which have been shown to have microbiological effects on the rumen while also promoting growth in beef cattle. The objective was to determine if supplementation of biochanin A via red clover hay would produce similar effects on the rumen microbiota and improve growth performance of lambs. Twenty-four individually-housed Polypay ram lambs (initial age: 114 ± 1 d; initial weight: 38.1 ± 0.59 kg) were randomly assigned to one of three experimental diets (85:15 concentrate:roughage ratio; N = 8 rams/treatment): CON-control diet in which the roughage component (15.0%, w/w, of the total diet) consisted of orchardgrass hay; 7.5-RC-red clover hay substituted for half (7.5%, w/w, of the total diet) of the roughage component; and 15-RC-the entire roughage component (15.0%, w/w, of the total diet) consisted of red clover hay. Feed intake and weight gain were measured at 14-d intervals for the duration of the 56-d trial, and rumen microbiological measures were assessed on days 0, 28, and 56. Red clover supplementation impacted growth performance of ram lambs. Average daily gains (ADG) were greater in ram lambs supplemented with red clover hay (7.5-RC and 15-RC) than for those fed the CON diet (P < 0.05). Conversely, dry matter intake (DMI) was lower in 7.5-RC and 15-RC than for CON lambs (P = 0.03). Differences in ADG and DMI resulted in greater feed efficiency in ram lambs supplemented with red clover hay (both 7.5-RC and 15-RC) compared to CON (P < 0.01). Rumen microbiota were also altered by red clover supplementation. The total viable number of hyper-ammonia-producing bacteria in 7.5-RC and 15-RC decreased over the course of the experiment and were lower than CON by day 28 (P ≤ 0.04). Amylolytic bacteria were also lower in 15-RC than in CON (P = 0.03), with a trend for lower amylolytic bacteria in 7.5-RC (P = 0.08). In contrast, there was tendency for greater cellulolytic bacteria in red clover supplemented lambs than in CON (P = 0.06). Red clover supplementation also increased fiber utilization, with greater ex vivo dry matter digestibility of hay for both 7.5-RC and 15-RC compared to CON by day 28 (P < 0.03). Results of this study indicate that low levels of red clover hay can elicit production benefits in high-concentrate lamb finishing systems through alteration of the rumen microbiota.

Red clover is rich in the bioactive isoflavone, biochanin A. The goal was to evaluate the impacts of biochanin A supplementation via red clover hay on growth performance of ram lambs as well as the rumen microbiota and fermentation. Low levels of red clover hay inclusion (7.5% and 15.0%, w/w, of the total diet) in high-concentrate finishing diets improved feed efficiency of ram lambs, promoting weight gain while decreasing feed intake. Red clover hay supplementation suppressed ruminal protein-wasting, peptide- and amino-acid degrading and starch-utilizing bacteria compared to control diets without isoflavones. Red clover hay also promoted fiber degrading bacteria and fiber utilization. Lamb growth and microbiological effects of red clover were consistent regardless of supplementation level in the diet. Results of this study indicate that low levels of red clover hay can produce production benefits in lamb finishing systems and demonstrated the efficacy of red clover as a functional feed, or feed with biological activities, in the context of its traditional use as a forage feedstuff.

Calibrating temperature reconstructions from fish otolith oxygen isotope analysis for California's critically endangered Delta Smelt.

RATIONALE: Oxygen isotope ratios (δ18 O values) of fish otoliths (ear bones) are valuable geochemical tracers of water conditions and thermal life history. Delta Smelt (Hypomesus transpacificus) are osmerid forage fish endemic to the San Francisco Estuary, California, USA, that are on the verge of extinction. These fish exhibit a complex life history that allows them to survive in a dynamic estuarine environment; however, a rapidly warming climate threatens this thermally sensitive species. Here we quantify the accuracy and precision of using δ18 O values in otoliths to reconstruct the thermal life histories of Delta Smelt. METHODS: Delta Smelt were reared for 360 days using three different water sources with different ambient δ18 Owater values (-8.75‰, -5.28‰, and -4.06‰) and different water temperatures (16.4°C, 16.7°C, 18.7°C, and 20.5°C). Samples were collected after 170 days (n = 28) and 360 days (n = 14) post-hatch. In situ δ18 O values were measured from the core of the otolith to the dorsal edge using secondary ion mass spectrometry (SIMS) to reconstruct temporally resolved thermal life histories. RESULTS: The δ18 Ootolith values for Delta Smelt varied as a linear inverse function of water temperature: 1000 ln α = 18.39 (±0.43, 1SE)(103 TK-1 ) - 34.56 (±1.49, 1SE) and δ18 Ootolith(VPDB) - δ18 Owater (VPDB) = 31.34(±0.09, 1SE) - 0.19(±0.01, 1SE) × T ° C. When the ambient δ18 Owater value is known, this species-specific temperature-dependent oxygen isotope fractionation model facilitated the accurate (0.25°C) and precise (±0.37°C, 2σ) reconstruction of the water temperature experienced by the fish. In contrast, the use of existing general fractionation equations resulted in inaccurate temperature reconstructions. CONCLUSIONS: The species-specific δ18 Ootolith fractionation equation allowed for accurate and precise reconstructions of water temperatures experienced by Delta Smelt. Characterization of ambient δ18 Owater values remains a critical next step for reconstructing thermal life histories of wild Delta Smelt. This tool will provide new insights into habitat utilization, potential thermal refugia, and resilience to future warming for this critically endangered fish.

Sensitivities of an endemic, endangered California smelt and two non-native fishes to serial increases in temperature and salinity: implications for shifting community structure with climate change.

In many aquatic systems, native fishes are in decline and the factors responsible are often elusive. In the San Francisco Estuary (SFE) in California, interactions among native and non-native species are key factors contributing to the decline in abundance of endemic, endangered Delta Smelt (Hypomesus transpacificus). Climate change and drought-related stressors are further exacerbating declines. To assess how multiple environmental changes affect the physiology of native Delta Smelt and non-native Mississippi Silverside (Menidia beryllina) and Largemouth Bass (Micropterus salmoides), fishes were exposed to serial exposures of a single stressor (elevated temperature or salinity) followed by two stressors (elevated temperature and salinity) to determine how a single stressor affects the capacity to cope with the addition of a second stressor. Critical thermal maximum (CTMax; a measure of upper temperature tolerance) was determined after 0, 2, 4 and 7 days following single and multiple stressors of elevated temperature (16°C vs. 20°C) and salinity (2.4 vs. 8-12 ppt, depending on species). Under control conditions, non-native fishes had significantly higher CTMax than the native Delta Smelt. An initial temperature or salinity stressor did not negatively affect the ability of any species to tolerate a subsequent multiple stressor. While elevated salinity had little effect on CTMax, a 4°C increase in temperature increased CTMax. Bass experienced an additive effect of increased temperature and salinity on CTMax, such that CTMax further increased under multiple stressors. In addition, Bass demonstrated physiological sensitivity to multiple stressors demonstrated by changes in hematocrit and plasma osmolality, whereas the physiology of Silversides remained unaffected. Non-native Bass and Mississippi Silversides showed consistently higher thermal tolerance limits than the native Delta Smelt, supporting their abundance in warmer SFE habitats. Continued increases in SFE water temperatures predicted with climate change may further impact endangered Delta Smelt populations directly if habitat temperatures exceed thermal limits.

Combined effects of warming and hypoxia on early life stage Chinook salmon physiology and development.

Early life stages of salmonids are particularly vulnerable to warming and hypoxia, which are common stressors in hyporheic, gravel bed, rearing habitat (i.e. a 'redd'). With the progression of global climate change, high temperatures and hypoxia may co-occur more frequently within redds, particularly for salmonid species at their southern range limit. Warming and hypoxia have competing effects on energy supply and demand, which can be detrimental to energy-limited early life stages. We examined how elevated temperature and hypoxia as individual and combined stressors affected the survival, physiological performance, growth, and development of Chinook salmon (Oncorhynchus tshawytscha). We reared late fall-run Chinook salmon from fertilization to the fry stage in a fully factorial design of two temperatures [10°C (ambient) and 14°C (warm)] and two oxygen levels [normoxia (100% air saturation, 10 mg O2/l) and hypoxia (50% saturation, 5.5 mg O2/l)]. Rearing in hypoxia significantly reduced hatching success, especially in combination with warming. Both warming and hypoxia improved acute thermal tolerance. While rearing in hypoxia improved tolerance to acute hypoxia stress, warming reduced hypoxia tolerance. Hypoxia-reared fish were smaller at hatch, but were able to reach similar sizes to the normoxia-reared fish by the fry stage. High temperature and normoxia resulted in the fastest rate of development while low temperature and hypoxia resulted in the slowest rate of development. Despite improved physiological tolerance to acute heat and hypoxia stress, hypoxia-reared embryos had reduced survival and growth, which could have larger population-level effects. These results suggest that both warming and hypoxia are important factors to address in conservation strategies for Chinook salmon.

Juvenile rockfish show resilience to CO2-acidification and hypoxia across multiple biological scales.

California's coastal ecosystems are forecasted to undergo shifting ocean conditions due to climate change, some of which may negatively impact recreational and commercial fish populations. To understand if fish populations have the capacity to respond to multiple stressors, it is critical to examine interactive effects across multiple biological scales, from cellular metabolism to species interactions. This study examined the effects of CO2-acidification and hypoxia on two naturally co-occurring species, juvenile rockfish (genus Sebastes) and a known predator, cabezon (Scorpaenichthys marmoratus). Fishes were exposed to two PCO2 levels at two dissolved oxygen (DO) levels: ~600 (ambient) and ~1600 (high) µatm PCO2 and 8.0 (normoxic) and 4.5 mg l-1 DO (hypoxic) and assessments of cellular metabolism, prey behavior and predation mortality rates were quantified after 1 and 3 weeks. Physiologically, rockfish showed acute alterations in cellular metabolic enzyme activity after 1 week of acclimation to elevated PCO2 and hypoxia that were not evident in cabezon. Alterations in rockfish energy metabolism were driven by increases in anaerobic LDH activity, and adjustments in enzyme activity ratios of cytochrome c oxidase and citrate synthase and LDH:CS. Correlated changes in rockfish behavior were also apparent after 1 week of acclimation to elevated PCO2 and hypoxia. Exploration behavior increased in rockfish exposed to elevated PCO2 and spatial analysis of activity indicated short-term interference with anti-predator responses. Predation rate after 1 week increased with elevated PCO2; however, no mortality was observed under the multiple-stressor treatment suggesting negative effects on cabezon predators. Most noteworthy, metabolic and behavioral changes were moderately compensated after 3 weeks of acclimation, and predation mortality rates also decreased suggesting that these rockfish may be resilient to changes in environmental stressors predicted by climate models. Linking physiological and behavioral responses to multiple stressors is vital to understand impacts on populations and community dynamics.

Antarctic emerald rockcod have the capacity to compensate for warming when uncoupled from CO2 -acidification.

Increases in atmospheric CO2 levels and associated ocean changes are expected to have dramatic impacts on marine ecosystems. Although the Southern Ocean is experiencing some of the fastest rates of change, few studies have explored how Antarctic fishes may be affected by co-occurring ocean changes, and even fewer have examined early life stages. To date, no studies have characterized potential trade-offs in physiology and behavior in response to projected multiple climate change stressors (ocean acidification and warming) on Antarctic fishes. We exposed juvenile emerald rockcod Trematomus bernacchii to three PCO2 treatments (~450, ~850, and ~1,200 µatm PCO2 ) at two temperatures (-1 or 2°C). After 2, 7, 14, and 28 days, metrics of physiological performance including cardiorespiratory function (heart rate [fH ] and ventilation rate [fV ]), metabolic rate (M˙O2), and cellular enzyme activity were measured. Behavioral responses, including scototaxis, activity, exploration, and escape response were assessed after 7 and 14 days. Elevated PCO2 independently had little impact on either physiology or behavior in juvenile rockcod, whereas warming resulted in significant changes across acclimation time. After 14 days, fH , fV and M˙O2 significantly increased with warming, but not with elevated PCO2 . Increased physiological costs were accompanied by behavioral alterations including increased dark zone preference up to 14%, reduced activity by 12%, as well as reduced escape time suggesting potential trade-offs in energetics. After 28 days, juvenile rockcod demonstrated a degree of temperature compensation as fV , M˙O2, and cellular metabolism significantly decreased following the peak at 14 days; however, temperature compensation was only evident in the absence of elevated PCO2 . Sustained increases in fV and M˙O2 after 28 days exposure to elevated PCO2 indicate additive (fV ) and synergistic (M˙O2) interactions occurred in combination with warming. Stressor-induced energetic trade-offs in physiology and behavior may be an important mechanism leading to vulnerability of Antarctic fishes to future ocean change.

Effects of high temperatures on threatened estuarine fishes during periods of extreme drought.

Climate change and associated increases in water temperatures may impact physiological performance in ectotherms and exacerbate endangered species declines. We used an integrative approach to assess the impact of elevated water temperature on two fishes of immediate conservation concern in a large estuary system, the threatened longfin smelt (Spirinchus thaleichthys) and endangered delta smelt (Hypomesus transpacificus). Abundances have reached record lows in California, USA, and these populations are at imminent risk of extirpation. California is currently impacted by a severe drought, resulting in high water temperatures, conditions that will become more common as a result of climate change. We exposed fish to environmentally relevant temperatures (14°C and 20°C) and used RNA sequencing to examine the transcriptome-wide responses to elevated water temperature in both species. Consistent with having a lower temperature tolerance, longfin smelt exhibited a pronounced cellular stress response, with an upregulation of heat shock proteins, after exposure to 20°C that was not observed in delta smelt. We detected an increase in metabolic rate in delta smelt at 20°C and increased expression of genes involved in metabolic processes and protein synthesis, patterns not observed in longfin smelt. Through examination of responses across multiple levels of biological organization, and by linking these responses to habitat distributions in the wild, we demonstrate that longfin smelt may be more susceptible than delta smelt to increases in temperatures, and they have little room to tolerate future warming in California. Understanding the species-specific physiological responses of sensitive species to environmental stressors is crucial for conservation efforts and managing aquatic systems globally.

Juvenile Antarctic rockcod (Trematomus bernacchii) are physiologically robust to CO2-acidified seawater.

To date, numerous studies have shown negative impacts of CO2-acidified seawater (i.e. ocean acidification, OA) on marine organisms, including calcifying invertebrates and fishes; however, limited research has been conducted on the physiological effects of OA on polar fishes and even less on the impact of OA on early developmental stages of polar fishes. We evaluated aspects of aerobic metabolism and cardiorespiratory physiology of juvenile emerald rockcod, ITALIC! Trematomus bernacchii, an abundant fish in the Ross Sea, Antarctica, to elevated partial pressure of carbon dioxide ( ITALIC! PCO2 ) [420 (ambient), 650 (moderate) and 1050 (high) µatm ITALIC! PCO2 ] over a 1 month period. We examined cardiorespiratory physiology, including heart rate, stroke volume, cardiac output and ventilation rate, whole organism metabolism via oxygen consumption rate and sub-organismal aerobic capacity by citrate synthase enzyme activity. Juvenile fish showed an increase in ventilation rate under high ITALIC! PCO2 compared with ambient ITALIC! PCO2 , whereas cardiac performance, oxygen consumption and citrate synthase activity were not significantly affected by elevated ITALIC! PCO2 Acclimation time had a significant effect on ventilation rate, stroke volume, cardiac output and citrate synthase activity, such that all metrics increased over the 4 week exposure period. These results suggest that juvenile emerald rockcod are robust to near-future increases in OA and may have the capacity to adjust for future increases in ITALIC! PCO2  by increasing acid-base compensation through increased ventilation.