Microbial diversity and metabolic function in duodenum, jejunum and ileum of emu (Dromaius novaehollandiae).

Emus (Dromaius novaehollandiae), a large flightless omnivorous ratite, are farmed for their fat and meat. Emu fat can be rendered into oil for therapeutic and cosmetic use. They are capable of gaining a significant portion of its daily energy requirement from the digestion of plant fibre. Despite of its large body size and low metabolic rate, emus have a relatively simple gastroinstetinal (GI) tract with a short mean digesta retention time. However, little is known about the GI microbial diversity of emus. The objective of this study was to characterize the intraluminal intestinal bacterial community in the different segments of small intestine (duodenum, jejunum, and ileum) using pyrotag sequencing and compare that with the ceca. Gut content samples were collected from each of four adult emus (2 males, 2 females; 5-6 years old) that were free ranged but supplemented with a barley-alfalfa-canola based diet. We amplified the V3-V5 region of 16S rRNA gene to identify the bacterial community using Roche 454 Junior system. After quality trimming, a total of 165,585 sequence reads were obtained from different segments of the small intestine (SI). A total of 701 operational taxonomic units (OTUs) were identified in the different segments of small intestine. Firmicutes (14-99%) and Proteobacteria (0.5-76%) were the most predominant bacterial phyla in the small intestine. Based on species richness estimation (Chao1 index), the average number of estimated OTUs in the small intestinal compartments were 148 in Duodenum, 167 in Jejunum, and 85 in Ileum, respectively. Low number of core OTUs identified in each compartment of small intestine across individual birds (Duodenum: 13 OTUs, Jejunum: 2 OTUs, Ileum: 14 OTUs) indicated unique bacterial community in each bird. Moreover, only 2 OTUs (Escherichia and Sinobacteraceae) were identified as core bacteria along the whole small intestine. PICRUSt analysis has indicated that the detoxification of plant material and environmental chemicals seem to be performed by SI microbiota, especially those in the jejunum. The emu cecal microbiome has more genes than SI segments involving in protective or immune response to enteric pathogens. Microbial digestion and fermentation is mostly in the jejunum and ceca. This is the first study to characterize the microbiota of different compartments of the emu intestines via gut samples and not fecal samples. Results from this study allow us to further investigate the influence of the seasonal and physiological changes of intestinal microbiota on the nutrition of emus and indirectly influence the fatty acid composition of emu fat.

Seasonal and sex-dependent gene expression in emu (Dromaius novaehollandiae) fat tissues.

Emu (Dromaius novaehollandiae) farming has been gaining wide interest for fat production. Oil rendered from this large flightless bird's fat is valued for its anti-inflammatory and antioxidant properties for uses in therapeutics and cosmetics. We analyzed the seasonal and sex-dependent differentially expressed (DE) genes involved in fat metabolism in emus. Samples were taken from back and abdominal fat tissues of a single set of four male and four female emus in April, June, and November for RNA-sequencing. We found 100 DE genes (47 seasonally in males; 34 seasonally in females; 19 between sexes). Seasonally DE genes with significant difference between the sexes in gene ontology terms suggested integrin beta chain-2 (ITGB2) influences fat changes, in concordance with earlier studies. Six seasonally DE genes functioned in more than two enriched pathways (two female: angiopoietin-like 4 (ANGPTL4) and lipoprotein lipase (LPL); four male: lumican (LUM), osteoglycin (OGN), aldolase B (ALDOB), and solute carrier family 37 member 2 (SLC37A2)). Two sexually DE genes, follicle stimulating hormone receptor (FSHR) and perilipin 2 (PLIN2), had functional investigations supporting their influence on fat gain and loss. The results suggested these nine genes influence fat metabolism and deposition in emus.

Seasonal and sexual variation in mRNA expression of selected adipokine genes affecting fat deposition and metabolism of the emu (Dromaius novaehollandiae).

Emus are farmed for fat production. Oil rendered from their back and abdominal fat pads has good anti-oxidant and anti-inflammatory properties and has ingredients that promote cell growth. Our objective is to examine the mRNA expression of 7 emu adipokine genes (eFABP4, eSCD1, eAdipoQ, eAdipoR1, eAdipoR2, eLEP and eLepR) to identify gene markers that may help improve emu fat production. Back and abdominal fat tissues from 11 adult emus were biopsied at four time points (April, June, August and November). Total RNA was isolated and cDNA was synthesized. Gene specific primers were designed for partial cloning fragments to amplify the open reading frame of the 7 genes. eLEP was not expressed in emu fat tissue. Nucleotides and amino acids sequences of the 6 expressed gene were compared with homologs from other species and phylogenetic relationships established. Seasonal mRNA expression of each gene was assessed by quantitative RT-PCR and differential expression analysed by the 2-ΔΔCT method. The 6 expressed genes showed seasonal variation in expression and showed association of expression level with back fat adiposity. More whole-genome scanning studies are needed to develop novel molecular markers that can be applied to improve fat production in emus.

Bioaccumulation and effects of selenium from surface coal mining in an aquatic songbird.

Open-pit mining operations are hailed for safe working conditions for miners as well as economically and logistically favourable outcomes for mining companies. However, ecological impacts of these operations may persist for decades. Expansions of open-pit coal mining in British Columbia of Western Canada are planned. Governmental regulation of background contaminants leached from these mines into nearby water systems were established to mitigate environmental impacts associated with these operations. We analyzed water, periphyton, invertebrate, and American dipper (Cinclus mexicanus) blood, egg, and feather samples for selenium and mercury exposure related to mining operations in the Elk Valley. We also quantified effects associated with exposure on clutch size and hatching success. Selenium concentrations in water, periphyton, and invertebrates were significantly higher downstream of mines compared to reference sites within and outside of the Elk River watershed. Selenium concentrations in water from exposed sites exceeded current regulatory levels established to protect wildlife by up to 20 times. Mercury concentrations were below toxic levels for birds in all media and did not factor into determination of selenium exposure or effects. Egg selenium concentrations were on average 0.9 times the regulatory threshold. Our stable isotope analyses showed that diets of nesting females included a negligible proportion of higher order consumers. We did not detect a significant effect of selenium on the rate of hatching success or clutch size of dippers nesting in exposed sites. We conclude that at the time of the investigation, selenium exposure was not impairing hatching of American dippers at the sampled locations.

River otters (Lontra canadensis) "trapped" in a coastal environment contaminated with persistent organic pollutants: Demographic and physiological consequences.

Productive coastal and estuarine habitats can be degraded by contaminants including persistent organic pollutants (POPs) such as PCBs, dioxins, and organochlorine insecticides to the extent of official designation as contaminated sites. Top-predatory wildlife may continue to use such sites as the habitat often appears suitable, and thus bioaccumulate POPs and other contaminants with potential consequences on their health and fitness. Victoria and Esquimalt harbours are located on southern Vancouver Island, British Columbia (BC) and are federally designated contaminated sites due mainly to past heavy industrial activities, such as from shipyards and sawmills. We collected scat samples from river otters (Lontra canadensis) throughout an annual cycle, and combined chemical analysis with DNA genotyping to examine whether the harbour areas constituted a contaminant-induced ecological trap for otters. We confirmed spatial habitat use by radio telemetry of a subsample of otters. Fifteen percent of otter scat contained PCB concentrations exceeding levels considered to have adverse effects on the reproduction of mink (Neovison vison), and there were significant positive correlations between concentrations of PCBs and of thyroid (T3) and sex (progesterone) hormones in fecal samples. Radio telemetry data revealed that otters did not show directional movement away from the harbours, indicating their inability to recognize the contaminated site as a degraded habitat. However, analysis and modeling of the DNA genotyping data provided no evidence that the harbour otters formed a sink population and therefore were in an ecological trap. Despite the highly POP-contaminated habitat, river otters did not appear to be adversely impacted at the population level. Our study demonstrates the value of combining chemical and biological technologies with ecological theory to investigate practical conservation problems.

Interaction of genotype and diet on small intestine microbiota of Japanese quail fed a cholesterol enriched diet.

Our previous study has shown that genetic selection for susceptibility/resistance to diet-induced atherosclerosis has affected the Japanese quail's cecal environment to accommodate distinctly different cecal microbiota. In this study, we fed the Atherosclerosis-resistant (RES) and -susceptable (SUS) quail a regular and a cholesterol enriched diet to examine the interaction of host genotype and diet on the diversity, composition, and metabolic functions of the duodenal and ileal microbiota with relations to atherosclerosis development. In the duodenal content, 9 OTUs (operational taxonomic units) were identified whose abundance had significant positive correlations with plasma total cholesterol, LDL level and/or LDL/HDL ratio. In the ileal content, 7 OTUs have significant correlation with plasma HDL. Cholesterol fed RES hosted significantly less Escherichia and unclassified Enterobacteriaceae (possibly pathogenic) in their duodenum than SUS fed the same diet. Dietary cholesterol significantly decreased the duodenal microbiome of SUS's biosynthesis of Ubiquinone and other terpenoid-quinone. Cholesterol fed RES had significantly more microbiome genes for Vitamin B6, selenocompound, taurine and hypotaurine, and Linoleic acid metabolism; Bisphenol degradation; primary bile acid, and butirosin and neomycin biosynthesis than SUS on the same diet. Microbiome in the ileum and ceca of RES contributed significantly towards the resistance to diet induced atherosclerosis.

Increased rodenticide exposure rate and risk of toxicosis in barn owls (Tyto alba) from southwestern Canada and linkage with demographic but not genetic factors.

Among many anthropogenic drivers of population decline, continual rapid urbanization and industrialization pose major challenges for the survival of wildlife species. Barn owls (Tyto alba) in southwestern British Columbia (BC) face a multitude of threats ranging from habitat fragmentation to vehicle strikes. They are also at risk from secondary poisoning of second-generation anticoagulant rodenticides (SGARs), a suite of toxic compounds which at high doses results in a depletion of blood clotting factors leading to internal bleeding and death. Here, using long-term data (N = 119) for the hepatic residue levels of SGAR, we assessed the risk of toxicosis from SGAR for the BC barn owl population over the past two decades. We also investigated whether sensitivity to SGAR is associated with genetic factors, namely Single Nucleotide Polymorphisms (SNPs) found in the CYP2C45 gene of barn owls. We found that residue concentration for total SGAR was significantly higher in 2006-2013 (141 ng/g) relative to 1992-2003 (57 ng/g). The proportion of owls exposed to multiple SGAR types was also significantly higher in 2006-2013. Those measures accordingly translate directly into an increase in toxicosis risk level. We also detected demographic differences, where adult females showed on average lower concentration of total SGAR (64 ng/g) when compared to adult males (106 ng/g). Juveniles were overall more likely to show signs of toxicosis than adults (33.3 and 6.9 %, respectively), and those symptoms were positively predicted by SGAR concentrations. We found no evidence that SNPs in the CYP2C45 gene of barn owls were associated with intraspecific variation in SGAR sensitivity. We recommend several preventative measures be taken to minimize wildlife exposure to SGAR.

The effect of diet and host genotype on ceca microbiota of Japanese quail fed a cholesterol enriched diet.

Two Japanese quail strains, respectively atherosclerosis-susceptible (SUS) and -resistant (RES), have been shown to be good models to study cholesterol metabolism and transportation associated with atherosclerosis. Our objective was to examine possible difference in cecal microbiota between these strains when fed a control diet and a cholesterol enriched diet, to determine how host genotype and diet could affect the cecal microbiome that may play a part in cholesterol metabolism. A factorial study with both strains and two diets (control, cholesterol) was carried out. Cecal content was collected from 12 week old quail that have been on their respective diets for 6 weeks. DNA was extracted from the samples and the variable region 3-5 of the bacterial 16S rRNA gene was amplified. The amplicon libraries were subjected to pyrosequencing. Principal Component Analysis (PCA) of ß-diversity showed four distinct microbiota communities that can be assigned to the 4 treatment groups (RES/control, RES/cholesterol, SUS/control, SUS/cholesterol). At the Phylum level, the 4 treatment groups has distinct Firmicutes community characteristics but no significant difference in Bacteroidetes. Eubacterium dolichum was rare in RES/control but became overabundant in RES/cholesterol. An unclassified species of Lactobacillaceae was found in abundance in SUS/control but the same species was rare in RES/cholesterol. On the other hand, two Lactobacillus species were only found in RES/control and an unclassified Lachnospiraceae species was abundant in RES/cholesterol but rare in SUS/control. The abundance of four species of Lachnospiraceae, three species of Ruminococcaceae and one species of Coprobacillaceae was positively correlated with plasma Total Cholesterol, plasma LDL, and LDL/HDL ratio. Our study of cecal microbiota in these quail has demonstrated that selection for susceptibility/resistance to diet induced atherosclerosis has also affected the quail's cecal environment to host distinctly different cecal microbiome.

Mercury in Forage Fish from Mexico and Central America: Implications for Fish-Eating Birds.

Mercury (Hg) is a global contaminant of aquatic food chains. Aquatic birds, such as the osprey (Pandion haliaetus), with migratory populations breeding in Canada and the northern United States and wintering in the Central and South America, can be exposed to mercury on both the breeding and wintering ranges. We examined Hg levels in 14 fish taxa from 24 osprey wintering sites identified from satellite telemetry. Our main goal was to determine whether fish species that feature in the diet of overwintering and resident fish-eating birds reached toxicity thresholds for Hg. Mean Hg levels in fish whole carcasses ranged from a high of 0.18 µg g(-1) (wet weight) in Scomberomorus sierra to a low of 0.009 µg g(-1) in Catostomidae. Average Hg levels were within published toxicity threshold values in forage fish for only two sites in Mexico (Puerto Vallarta and San Blas Estuary), and all were marine species, such as mackerel (Scomberomorus sierra), sea catfish (Ariopus spp.), and sardinas species (Centropomus spp.). Except for one sample from Nicaragua, sea catfish from Puerto Morazan, none of the fish from sites in Central America had Hg levels which exceeded the thresholds. Nonmetric multidimensional scaling revealed geographical differences in Hg levels with significant pairwise differences between sites along the Pacific Ocean (Mexico) versus the Bay of Campeche, partly due to differences in species composition of sampled fish (and species distributions). Hg increased with trophic level, as assessed by nitrogen stable isotope ratios (δ(15)N but not δ(13)C), in freshwater and marine, but not estuarine, environments. Hg concentrations in forage fish do not account for the elevated Hg reported for many osprey populations on the breeding grounds, thus primary sources of contamination appear to be in the north.

Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation.

Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51×faster), ostrich oil (1.46×faster), and rhea oil (1.64×faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35×slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions.

Assessment of toxicity and coagulopathy of brodifacoum in Japanese quail and testing in wild owls.

Based on detection of hepatic residues, scavenging and predatory non-target raptors are widely exposed to second generation anticoagulant rodenticides (SGARs). A small proportion, generally <10%, of tested birds are diagnosed as acutely poisoned. Little is known, however, of sub-lethal effects of SGARs, such as interaction of clotting capacity with traumatic injury. Assessment of coagulation function of birds submitted live to wildlife rehabilitators or veterinarians may provide a means of establishing the proportion of animals suffering sub-lethal coagulopathies, as well as identifying individuals requiring treatment. As a first step in exploring the potential of this approach, we dosed Japanese quail (Coturnix japonica) with the SGAR, brodifacoum, at 0, 0.8, 1.4, 1.9, and 2.5 mg/kg and sampled birds at 1, 3, 5 and 7 days post-dosing. Prothrombin time (PT), which measures the extrinsic coagulation pathway, was significantly prolonged in 98% of brodifacoum-exposed quail in a dose- and time-dependent manner. 50-fold prolongation of PT occurred at higher brodifacoum dosages and correlated to hemorrhage found at necropsy. Activated clotting time (ACT), a measure of the intrinsic pathway also increased with dose and time. Hemoglobin (Hb) and hematocrit (Hct) decreased dose- and time-dependently at doses ≥1.4 mg/kg with no significant change at 0.8 mg/kg. Reference intervals for PT (10.0-16.2 s), ACT (30-180 s), Hb (9.6-18.4 g/dl), and Hct (34-55%) were established in Japanese quail. Species-specific reference intervals are required as barn owl PT (17-29 s) and quail PT were different. The proportion of brodifacoum-exposed quail with hemorrhage was not correlated with liver residues, but was correlated with PT, suggesting that this assay is a useful indicator of avian anticoagulant rodenticide exposure. PTs measured in free-living barn owls sampled between April 2009 and August 2010 in the lower Fraser Valley of BC do not suggest significant exposure to SGARs.

Recovery of fertility from adult ovarian tissue transplanted into week-old Japanese quail chicks.

Fertility of cryopreserved ovarian tissue from immature chickens and Japanese quail has been recovered by transplantation. This is of special importance for non-mammalian vertebrates in which cryopreservation and in vitro maturation of oocytes are challenging because their oogenesis is characterised by vitellogenesis. This study tested whether fertility of adult quail ovarian tissue could be recovered by transplantation. Ovaries were isolated from mature Japanese quail hens, trimmed, cut into 3- to 4-mm2 pieces and transplanted into ovariectomised, week-old chicks. Recipients were administered an immunosuppressant for two weeks. Ten of 12 recipients survived until sexual maturity and seven laid eggs, but all stopped laying by 17 weeks of age. The age at first egg of recipients laying eggs (75.7±4.2 days) was greater than that of untreated hens (51.8±1.7 days) and egg production of recipients during the laying period (21.7±5.7) was less than that of untreated hens (60.8±3.5). Recipients were paired with males from the WB line for test mating. Only two hens laid eggs during the test period but both produced 100% donor-derived offspring. This research demonstrated that the reproductive potential of ovarian tissue from adult quail hens can be restored by transplantation.

Carcass yields and meat quality characteristics of adult emus (Dromaius novaehollandiae) transported for 6h before slaughter.

The meat quality characteristics of adult emus transported for 6h before slaughter were determined. Forty-two emus were used in two trials, undertaken under warm and cool weather conditions, respectively. Male emus had significantly higher fat yields than females (12.43kg vs 9.5kg, P=0.002). About 38.1% of the emus had no wounds or bruises, 40.5% had bruises, while 21.4% had small wounds after transport. Meat from injured emus had significantly higher pH45. In warm weather, emus experienced significantly higher loss in body weight than that under cool weather. Drip loss in meat after 24h of storage was higher in emus which had greater live weight loss after transport (r=0.66, P<0.0001), confirming the adverse effects of transport stress on meat quality. Nutrient supplementation did not significantly affect processing yield or meat quality characteristics. This study points to the need for optimizing transport conditions of emus to maintain meat quality.

Transportation stress and the incidence of exertional rhabdomyolysis in emus (Dromaius novaehollandiae).

Many emu farms are located in areas lacking processing facilities that can handle these birds. Thus, long-distance shipping of birds to an abattoir is necessary. Two experiments were conducted, wherein emus were transported in a modified horse trailer for 6 h to an abattoir. Changes in the indices of stress and metabolic homeostasis (hematology, serum biochemistry, enzymes, and body temperature and weight) were used to evaluate the physiological response to transport. The activities of enzymes alanine aminotransferase, aspartate aminotransferase, and creatine kinase increased significantly (P < 0.001) from pretransport to slaughter, indicating muscle cell wall damages. The body temperature of emus was significantly (P < 0.001) increased from 37.0 to 39.6°C after transport in experiment 1 and from 37.2 to 38.9°C in experiment 2. Transport resulted in significant weight loss in both experiments (P < 0.001; 2.1 ± 0.2 kg vs. 0.6 ± 0.2 kg) and posttransport resting at lairage led to slight regaining (P < 0.01) of BW. Oral administration of supplements before and after transport was effective in protecting against muscle damage and faster recovery of BW losses during lairage. The clinical findings were suggestive of the incidence of exertional rhabdomyolysis and thus underlined the need for careful handling and improved transport conditions of emus.

Effect of 3',5'-cyclic diguanylic acid in a broiler Clostridium perfringens infection model.

In an effort to explore strategies to control Clostridium perfringens, we investigated the synergistic effect of a ubiquitous bacterial second messenger 3',5'-cyclic diguanylic acid (c-di-GMP) with penicillin G in a broiler challenge model. All chicks were inoculated in the crop by gavage on d 14, 15, and 16 with a mixture of 4 C. perfringens strains. Birds were treated with saline (control group) or 20 nmol of c-di-GMP by gavage or intramuscularly (IM) on d 24, all in conjunction with penicillin G in water for 5 d. Weekly samplings of ceca and ileum were performed on d 21 to 35 for C. perfringens and Lactobacillus enumeration. On d 35 of age, the IM treatment significantly (P < 0.05) reduced C. perfringens in the ceca, suggesting possible synergistic activity between penicillin G and c-di-GMP against C. perfringens in broiler ceca. Moreover, analysis of ceca DNA for the presence of a series of C. perfringens virulence genes showed a prevalence of 30% for the Clostridium perfringens alpha-toxin gene (cpa) from d 21 to 35 in the IM-treated group, whereas the occurrence of the cpa gene increased from 10 to 60% in the other 2 groups (control and gavage) from d 21 to 35. Detection of ß-lactamase genes (blaCMY-2, blaSHV, and blaTEM) indicative of gram-negative bacteria in the same samples from d 21 to 35 did not show significant treatment effects. Amplified fragment-length polymorphism showed a predominant 92% similarity between the ceca of 21-d-old control birds and the 35-d-old IM-treated c-di-GMP group. This suggests that c-di-GMP IM treatment might be effective at restoring the normal microflora of the host on d 35 after being challenged by C. perfringens. Our results suggest that c-di-GMP can reduce the colonization of C. perfringens in the gut without increasing the selection pressure for some ß-lactamase genes or altering the commensal bacterial population.

A model for cryobanking female germplasm in Japanese quail (Coturnix japonica).

Cryopreservation and transplantation of ovarian tissue can be used for cryobanking female germplasm of avian species. Here we report a model of cryobanking ovarian tissue of Japanese quail. Ovaries of 1-wk-old quail chicks from the recessive white-breasted (WB) line were cryopreserved using a vitrification method. The cryopreserved tissue was warmed and transplanted orthotopically into ovariectomized, 1-wk-old female quail chicks from the homozygous wild-type QO line. At sexual maturation, recipients were mated with WB males and the plumage coloration was used as a marker to determine the origin of their ovaries. Seven of the 15 recipients laid eggs and produced donor-derived offspring, and 5 produced 100% donor-derived offspring. No difference was seen in egg production between recipients and untreated birds. The high efficiency of the vitrification and transplantation procedures in preserving and recovering quail ovarian tissue shows that this model can be used for maintaining commercial and experimental quail strains and may benefit other poultry species and endangered avian species.

Characterization of cecal microbiota of the emu (Dromaius novaehollandiae).

Emus (Dromaius novaehollandiae), large flightless ratites native to Australia, are farmed for their fat and meat. They are omnivorous and feed on a wide variety of plants and insects. Despite having a relatively simple gastrointestinal tract and a short digesta retention time, emus are able to digest a significant portion of the ingested dietary neutral detergent fibre. However, nothing is known about the microbial diversity in their gastrointestinal tract. In this study, we evaluated the phylogenetic diversity of the cecal microbiota of four emus (2 males, 2 females) that were fed a barley-alfalfa-canola based diet, using 454 pyrosequencing after amplification for V3-V5 region of bacterial 16S rRNA gene. Emus were slaughtered in early November, just prior to the onset of their breeding season, but after the seasonal decline in their feed intake had begun. A total of 822 operational taxonomic units (OTUs) (335.3 ± 70.5 OTUs/sample) belonging to 9 bacterial phyla were identified. The most predominant bacterial phyla were Bacteroidetes (≈ 57% of total classified diversity), Proteobacteria (≈ 24%), Fusobacteria (≈ 11.3%), and Firmicutes (≈ 7%). Our results indicate that the emus' ceca may have a higher microbial richness (Chao1: 624 ± 170 OTUs, and ACE: 586 ± 161 OTUs) than other species of birds, but they have a lower microbial diversity (Shannon diversity index: 3.4 ± 0.2, Simpson index: 0.79 ± 0.02), possibly reflecting their decrease feed intake. This is the first study to characterize the microbial community of the gastrointestinal tract of a ratite using pyrosequencing, providing a baseline for further study.

Production of live offspring from testicular tissue cryopreserved by vitrification procedures in Japanese quail (Coturnix japonica).

Cryopreservation of testicular tissue can be used for ex situ conservation of male germplasm of avian species. The possibility of using vitrification and transplantation of testicular tissue for fertility preservation and recovery was tested in Japanese quail. Testes were removed from 1-wk-old Japanese quail; transfixed on acupuncture needles; equilibrated with dimethyl sulphoxide, ethylene glycol, and sucrose; plunged into liquid nitrogen; and stored in 2-ml straws. Cryopreserved tissue was warmed in sucrose solution at room temperature or at 40°C. Fresh and cryopreserved tissue were transplanted subcutaneously into castrated, 1-wk-old recipients. Twenty of 21 recipients survived the surgery, and 18 had viable transplants at maturity, with no difference in transplantation success between fresh and cryopreserved tissue. Fluid extrusion from 11 of the transplants was collected and inseminated surgically into the magnum of 22 quail hens, and 10 inseminations included foam from the proctodeal gland of the same recipients. Egg production in the 2 wk after insemination was reduced, and none of the hens inseminated with foam produced fertile eggs. Five hens inseminated without foam produced a total of eight live offspring; four of these hens had been inseminated with fluid extrusion from cryopreserved tissue. Histological examination showed spermatogenesis in the transplants, and the tubules, lumens, and epithelium of the seminiferous tubules were of comparable size to those of testicular tissue from intact males. These results demonstrate that testicular tissue of Japanese quail can be preserved using vitrification procedures and recovered through transplantation.

Expressed sequence tag analysis of the emu (Dromaius novaehollandiae) pituitary by 454 GS Junior pyrosequencing.

Emus (Dromaius novaehollandiae) are farmed for their oil for pharmaceutical and cosmetic uses. This emu pituitary expressed sequence tag study was undertaken to identify novel transcripts in the emu pituitary to propel their identification and functional studies. By mapping reads derived from the Roche 454 GS Junior pyrosequencer to 8 reference species (human, mouse, chicken, zebra finch, fruit fly, turkey, round worm, and Carolina anole lizard) from the UniGene database, a total of 81,788 reads (53,312 mapped reads) were obtained and assembled with Reference Sequence (RefSeq). We annotated 6,676 potential emu genes by referencing 7 species (excluding lizard) and identified 1,232 potential genes common among 3 species (human, mouse, and chicken) with complete available reference genomes. Gene Ontology analysis revealed 376 Gene Ontology terms showing, with the highest counts, their involvements in biological processes, metabolism, and cellular components. These potential genes were detected to associate with 20 pathways including mitogen-activated protein kinase, insulin, neurotrophin signaling pathways, and carbohydrate digestion and absorption pathway. We also revealed a panel of tissue-specific genes including regulator of G-protein signaling protein (RGS), glucagon-like peptide receptor (GLPR), and growth hormone-inducible transmembrane protein (GHITM). Additionally, fatty acid binding protein (FABP), fatty acid desaturase (FAS), and stearoyl-coenzyme A desaturase (SCD), key enzyme genes in fat metabolism, were found to be also expressed in emu pituitary. This expressed sequence tag study represents the first step in functional characterization of emu pituitary gene expression and SNP identification for the improvement of fat production in the emu.

Novel needle-in-straw vitrification can effectively preserve the follicle morphology, viability, and vascularization of ovarian tissue in Japanese quail (Coturnix japonica).

Cryopreservation of ovarian tissue has been the only effective way of ex situ conservation of female germplasm in avian species. A novel needle-in-straw (NIS) vitrification method was developed to store tissue in straws instead of cryovials. Fragments of ovarian tissue from one-week old Japanese quail were transfixed on an acupuncture needle. They were immersed in equilibration and vitrification solutions containing dimethyl sulphoxide, ethylene glycol and sucrose. A layer of tin foil was rolled over the tissue fragments and the tin foil package was plunged into liquid nitrogen and inserted into a pre-cooled, 0.5-ml straw which was stored in liquid nitrogen. Tissue was also preserved using a needle immersed vitrification (NIV) method, in which tissue fragments transfixed on needles without tin foil and were stored in cryovials filled with liquid nitrogen. Cryopreserved tissue was warmed at room temperature (RT) or 37°C and the ratio of normal follicles to total visible follicles was determined by histological methods. In addition, cryopreserved and warmed tissue was cultured on the chorioallantoic membranes of fertilized chicken eggs for 5-6 days. The viability and vascularization of the grafts were evaluated. The tissue cryopreserved by NIS and warmed at RT showed comparable follicle morphology to fresh tissue and to that preserved by NIV and warmed at RT. No significant impairment on the viability or vascularization of the grafted tissue was observed. The NIS method allows tissue to be stored and transported safely and efficiently and can be used instead of cryovials in tissue cryobanking.