Ovine rumen papillae biopsy via oral endoscopy; a rapid and repeatable method for serial sampling

AIMS: To explore and validate the utility of rumen endoscopy for collection of rumen papillae for gene expression measurement.

METHODS: Four adult Coopworth ewes were fasted for either 4 or 24 hours. Animals were sedated, placed in a dorsally recumbent position at 45 degrees with the head upright, and an endoscope inserted via a tube inserted into the mouth. Biopsies of rumen papillae were taken from the ventral surface of the rumen atrium under visual guidance. Two biopsies were collected from one of the animals that had been fasted for 4 hours, and three from one of the animals that had been fasted for 24 hours. Video of the rumen atrium and reticulum was also collected. The animals recovered uneventfully. Biopsies were subsequently used for extraction and sequencing of mRNA.

RESULTS: The ventral surface of the rumen atrium was accessible after 4 hours off pasture, but a larger region was accessible after 24 hours of fasting. Sedation allowed access for endoscope use for around 5 to 10 minutes after which increased saliva flow was noted. Rumen papillae biopsies were easily collected, with samples from a variety of sites collected in the ～10 minute time window. High quality RNA was obtained for stranded mRNA sequencing. Of the resulting reads, 69–70% mapped uniquely to version 3.1 of the ovine genome, and 48–49% to a known gene. The rumen mRNA profiles were consistent with a previously reported study.

CONCLUSIONS: This method for obtaining rumenal tissue was found to be rapid and resulted in no apparent short or long term effects on the animal. High quality RNA was successfully extracted and amplified from the rumen papillae biopsies, indicating that this technique could be used for future gene expression studies. The use of rumen endoscopy could be extended to collection of a variety of rumen and reticulum anatomical measurements and deposition and retrieval of small sensors from the rumen. Rumen endoscopy offers an attractive and cost effective approach to repeated rumen biopsies compared with serial slaughter or use of cannulated animals.     

Distribution of thiobacillus ferrooxidans and leptospirillum ferrooxidans: implications for generation of acid mine drainage

Although Thiobacillus ferrooxidans and Leptospirillum ferrooxidans are widely considered to be the microorganisms that control the rate of generation of acid mine drainage, little is known about their natural distribution and abundance. Fluorescence in situ hybridization studies showed that at Iron Mountain, California, T. ferrooxidans occurs in peripheral slime-based communities (at pH over 1.3 and temperature under 30 degreesC) but not in important subsurface acid-forming environments (pH 0.3 to 0.7, temperature 30 degrees to 50 degreesC). Leptospirillum ferrooxidans is abundant in slimes and as a planktonic organism in environments with lower pH. Thiobacillus ferrooxidans affects the precipitation of ferric iron solids but plays a limited role in acid generation, and neither species controls direct catalysis at low pH at this site.     

Proteins Associated With the Early Intrauterine Equine Conceptus

A critical period of early gestation in the mare involves the immobilization (fixation) of the encapsulated conceptus at around days 16–17. We compared the major proteins in the normal equine embryonic capsule and endometrial secretions around the period of fixation with those from pregnancies in the process of termination induced by administration of an analogue of prostaglandin F_2α (PGF_2α). Uterocalin and β₂-microglobulin (β₂M) associated with the embryonic capsule were proteolytically converted to smaller forms during the fixation period. These conversions were similar in conceptuses from control and treated mares. A 17 kDa cationic protein identified as a secretory phospholipase A2 (sPLA2) type IIA was detected bound to normal capsules but increased substantially in response to PGF_2α. Two forms of uteroglobin were distinguished by partial amino acid sequences of ∼6 kDa bands in flush fluids from normal pregnant uteri. After administration of PGF_2α one immunoreactive form of uteroglobin was preferentially increased. These studies demonstrate that failure of pregnancy in this model is associated with an increase in secretory phospholipase in the capsule and a change in the forms of uteroglobin in the uterine secretions.     

Efficiency of inspection procedures for the detection of tuberculous lesions in cattle

The sensitivity of the abattoir inspection procedure introduced for Australian export beef in 1976 was compared to a detailed necropsy procedure for the detection of tuberculous lesions in cattle. In a sample of cattle that were reactors to the tuberculin test, abattoir inspection failed to detect an estimated 47% of cattle with lesions. The detailed necropsy examination of cattle with lesions of tuberculosis identified 21 sites of infection compared with 13 to 18 in cattle examined by routine meat inspection procedures. Of the lesions detected during detailed necropsy, 15.9% did not involve the thoracic cavity or the medial retropharyngeal lymph nodes. The failure to detect lesions during abattoir inspection has its greatest significance in an animal with a single lesion. If the 245 cattle found with single lesions during detailed necropsy had been examined by abattoir inspection using the 1976 or the 1986 procedures, 0.8 and 8.9%, respectively, of these animals would not have been detected because the diseased tissues would not have been examined. If meat inspection is to provide an effective means of monitoring the level of bovine tuberculosis during the final stages of eradication, a procedure no less sensitive than that introduced in 1976 should be used.     

INTRACRANIAL SUBARACHNOID HEMORRHAGE FOLLOWING LUMBAR MYELOGRAPHY IN TWO DOGS

Intracranial subarachnoid hemorrhage is a rare but serious complication of lumbar puncture in humans. Possible sequelae include increased intracranial pressure, cerebral vasospasm, or mass effect, which can result in dysfunction or brain herniation. We describe two dogs that developed intracranial subarachnoid hemorrhage following lumbar myelography. In both dogs, myelography was performed by lumbar injection of iohexol (Omnipaque). Both the dogs underwent uneventful ventral decompressive surgery for disk herniation; however, the dogs failed to recover consciousness or spontaneous respiration following anesthesia. Neurologic assessment in both dogs postoperatively suggested loss of brain stem function, and the dogs were euthanized. There was diffuse subarachnoid hemorrhage and leptomeningeal hemorrhage throughout the entire length of the spinal cord, brain stem, and ventrum of brain. No evidence of infectious or inflammatory etiology was identified. The diagnosis for cause of brain death was acute subarachnoid hemorrhage. Our findings suggest that fatal subarachnoid hemorrhage is a potential complication of lumbar myelography in dogs. The cause of subarachnoid hemorrhage is not known, but may be due to traumatic lumbar tap or idiosyncratic response to contrast medium. Subsequent brain death may be a result of mass effect and increased intracranial pressure, cerebral vasospasm, or interaction between subarachnoid hemorrhage and contrast medium.     

Cytologic and Histopathologic Evaluation of Extruded Canine Degenerate Disks

Objective— To describe the cytologic and histopathologic appearance of degenerate disk material in dogs with Hansen type I intervertebral disk disease (IVDD).
Study Design— Case series.
Animals— Dogs (n=45) that had surgical intervention for Hansen type I IVDD (January–November 2007).
Methods— Impression smears and histopathologic sections were prepared from surgically removed degenerate disk material. All slides were evaluated for overall cellularity, quantity and attributes of extracellular matrix, types of cells present, and their cytomorphology. Histopathologic sections were also examined for presence of neovascularization and hemorrhage.
Results— Cytologically, 11 of 45 samples consisted of only extracellular matrix, 30 had evidence of inflammation, and 20 contained dysplastic spindloid cells. Histologically, hyaline cartilage predominated in 35 of 45 samples, fibrocartilage in 4, and spindloid cells in 6; 37 of 45 were inflamed, 37 were hemorrhagic, and 13 had neovascularization.
Conclusions— The cytologic and histopathologic appearance of extruded degenerate disk material in dogs is variable and can include dysplastic spindloid cells.
Clinical Relevance— The variability in cytologic findings and frequent presence of dysplastic spindloid cells suggest that cytology alone may not be a reliable tool to differentiate degenerate canine disk material from a mesenchymal neoplasm.     

Systemic Concentrations of Endogenous and Exogenous FSH in Anoestrous Ewes Superstimulated with Folltropin®-V

The synchronization of follicular waves with medroxyprogesterone acetate (MAP) and oestradiol‐17β (E₂‐17β) prior to ovarian superstimilation in anoestrous ewes reduces the variability in superovulatory responses by an unknown mechanism. Follicle stimulating hormone (FSH) is a primary promoter of antral follicular development, but the relevance of circulating FSH concentrations to the superovulation performance in ewes has not been examined. Eighteen anoestrous Rideau Arcott ewes (May–June) were superovulated with Folltropin^®‐V (porcine FSH), with (n = 8; treated ewes) or without (n = 10; control ewes) a single i.m. dose of 350 μg of E₂‐17β, given on the sixth day of a 14‐day treatment with MAP‐releasing intravaginal sponges (60 mg). The superovulatory treatment, begun 6 days after E₂‐17β injection, consisted of six i.m. applications of Folltropin^®‐V given twice daily (at 08:00 and 16:00 h), followed by an i.m. injection of GnRH (50 μg). Blood samples collected every 8 h throughout the 3‐day treatment, were analysed by radioimmunoassays for concentrations of ovine and porcine FSH, using species‐specific standards and primary antibodies. Serum concentrations of oFSH were greater (p < 0.05) in the controls compared to treated ewes at 40, 64 and 72 h and the variability in mean oFSH concentrations was greater (p < 0.05) in control ewes at 40, 48, 64 and 72 h after the 1^st Folltropin^®‐V injection. There were no differences (p > 0.05) between the two groups in serum concentrations of pFSH. Significant correlations were recorded between the number of corpora lutea (CL) and oFSH concentrations at 8 h (r = 0.72, p < 0.05), 16 h (r=0.63, p < 0.05) and 64 h (r = 0.84, p < 0.01) after the 1^st Folltropin^®‐V injection. The total number of recovered embryos was positively correlated to oFSH concentrations at 56 h (r = 0.69, p < 0.05). We concluded that changes in endogenous FSH concentrations during ovarian superstimulation with pFSH might contribute to the variability in superovulatory responses in ewes.