Interactions between different media and follicle‐stimulating hormone supplementation on in vitro culture of preantral follicles enclosed in ovarian tissue derived from collared peccaries (Pecari tajacu Linneaus, 1758)

The aim was to verify the effect of follicle‐stimulating hormone (FSH) supplementation to α‐MEM+ or TCM199+ media on the in vitro development of ovarian preantral follicles (PFs) derived from collared peccaries. Ovaries (n = 5 pairs) were collected and divided into fragments destined to control group (non‐cultured) or treatments that were cultured for 7 days. The PFs morphology, growth and activation were evaluated by classical histology. The immunohistochemistry markers Ag‐NOR and PCNA were used for nuclear proliferation analysis, and the picrosirius red labelling was used for ovarian extracellular matrix (ECM) evaluation. After 7‐day culture, only the TCM199+ treatment maintained the proportion of intact PFs similar to day 1 (63.2%), but no differences were found among treatments (p > .05). In addition, a significant increase in the growing follicles proportion was verified for all the treatments, indicating follicular activation (p > .05). By the Ag‐NOR analysis, only the TCM199+/FSH maintained the nuclear proliferation similar to the first day (p > .05). The picrosirius red staining revealed that the ECM remained intact in all the treatments (p > .05). We suggest the use of TCM199+ medium supplemented of FSH for the in vitro development of peccaries PFs under 7‐day culturing conditions.     

Development and evaluation of a highly sensitive and specific blocking enzyme-linked immunosorbent assay and polymerase chain reaction assay for diagnosis of bovine leukemia virus infection in cattle

OBJECTIVE: To develop a blocking ELISA for detection of bovine leukemia virus (BLV) antibodies that is comparable to a radioimmunoprecipitation (RIP) assay, to evaluate use of this ELISA for identification of BLV-infected herds, and to develop a polymerase chain reaction (PCR) assay for direct diagnosis of infection with BLV. SAMPLE POPULATION: Serum samples and pooled bulk-tank milk samples from cattle. PROCEDURE: The blocking ELISA was developed, using BLV gp51 as antigen, captured by a selected bovine polyclonal serum. A nested PCR was conducted with primers specific for a segment of the pol region of the BLV genome. RESULTS: Sensitivity and specificity of the ELISA were comparable to those of the RIP assay. Use of the ELISA on pooled milk samples allowed identification of herds in which prevalence of BLV infection among lactating cows was as low as 2.5%. Pooled milk samples from BLV-free herds did not react in the ELISA. All cattle that had positive results for the nested PCR had BLV antibodies, but cattle with consistantly low antibody titers required examination of sequential DNA samples to detect viral sequences. None of the 63 antibody-negative cattle had positive results for the PCR. CONCLUSIONS AND CLINICAL RELEVANCE: This ELISA is a highly specific and sensitive assay for the detection of BLV antibodies in serum and milk samples of cattle. Examination of pooled milk samples with the ELISA provides a reliable, practical, and economic procedure for identification of BLV-infected herds. The nested PCR also constitutes a specific procedure for direct diagnosis of infection with BLV.     

Deep‐water cirripedes colonizing dead shells of the cephalopod Nautilus macromphalus from New Caledonian waters

Fossil cephalopods are frequently encrusted by epibionts; however, determining whether encrustation occurred prior to or post‐mortem to the host, and whether the final environment of deposition corresponds to the habitat of encrustation is complex. The present paper describes cirripede epibionts, their calcareous bases and their attachment scars on 6 post‐mortem shells of Nautilus macromphalus, collected from deep water off New Caledonia. The cirripedes have left both cemented calcareous bases of Hexelasma and scars associated with bioerosion and discoloration produced by verrucomorph barnacles. Live cirripedes included a Metaverruca recta, with articulated opercular plates and organic tissue (on a shell that had been exposed on the sea floor for at least 150 years), and specimens of Hexelasma velutinum, one of which was partly attached to an internal surface of a shell. The disposition of verrucomorphs indicates that most Nautilus shells were colonized post‐mortem rather than during a floating stage. However, as cirripedes are known to have colonized living Nautilus, some Hexelasma, preserved only as calcareous eroded bases, may represent specimens that settled on a living Nautilus. The degree of bioerosion and discoloration induced by verrucomorph barnacles varies according to the surface preservation of Nautilus shells, with deeper and discolored traces preserved on old and degraded shells. Traces made by verrucomorphs described here are ellipsoidal and a new ichnotaxon, Anellusichnus ellipticus, is proposed to accommodate them. Importantly, verrucomorphs and other cirripede taxa with membranous bases that were attached to pristine shells may not leave any substantial scars, and, thus, will be difficult to detect in the fossil record.     

A comparison of two intramuscular doses of xylazine–ketamine combination and tolazoline reversal in llamas

The anesthetic and cardiorespiratory effects of a low dose (LD, 0.4 mg kg^?1 xylazine and 4 mg kg^?1 ketamine) and a high dose (HD, 0.8 mg kg^?1 xylazine and 8 mg kg^?1 ketamine) of IM xylazine–ketamine combination were compared in a randomized cross‐over study using six castrated male llamas. Three llamas in each dosage group (LDT, HDT) were assigned to receive IM tolazoline (2 mg kg^?1) after 30 minutes of recumbency. All IM injections were given in the semitendinosus or semimembranosus muscles. Pulse, respiratory rate, and indirect arterial blood pressure were recorded every 10 minutes, and hemoglobin oxygen saturation was recorded every 5 minutes during lateral recumbency. Samples for arterial blood gas analysis were collected 5 minutes following recumbency and every 30 minutes thereafter. Base‐to‐apex ECG was monitored continuously. Analgesia was evaluated every 5 minutes by both a 30 minutes skin pinch and a needle prick of the toe. Most llamas breathed room air throughout anesthesia. Two llamas that developed severe hypoxemia (SpO₂ < 75%) received 5 minutes of nasal oxygen insufflation, but were maintained on room air for the rest of the anesthetic period. anova for repeated measures and Tukey's test were used to analyze cardiorespiratory data. Fischer's exact test was used to compare the ability of each to provide >30 minutes of lateral recumbency and analgesia. A p‐value < 0.05 was considered significant. Both dosages provided reasonably rapid induction following injection (LD: 10.8 ± 6.3 minutes; HD: 5.0 ± 1.1 minutes; p = 0.07). Duration of lateral recumbency and analgesia were 34.7 ± 6.7 and 27.3 ± 4.6 minutes, respectively, in the LDT llamas. None of the three remaining LD llamas remained in lateral recumbency for longer than 12 minutes. Duration of lateral recumbency and analgesia were 87.3 ± 18.5 and 67.7 ± 16.0 minutes, respectively, for the HD llamas that did not receive tolazoline. The HDT llamas were recumbent for a significantly shorter time (43.3 ± 0.6 minutes; p = 0.05). The ability to provide >30 minutes of recumbency and analgesia was better in the HD group (6/6) than in the LD group (2/6) (p = 0.03). No differences between dosages were seen in pulse rate, respiratory rate, or arterial pressures. No ECG abnormalities were seen. Transient hypoxemia was seen in the first 10 minutes of lateral recumbency in the HD group by both hemoglobin oxygen saturation (84 ± 9.5%) and by blood gas PaO₂ (44.5 ± 5.8 mm Hg). It was concluded that the HD provided more consistent results than the LD, but induced transient hypoxemia. Tolazoline shortened the recovery time in llamas receiving the HD.