Measurement of precipitin reactions in the millimicrogram protein-nitrogen range

Exploitation of newer instrumentation and a dye-binding method for protein measurement made possible reduction in volume down to 1/1000 that commonly used, with no greater error. The procedure was tested at two levels, 1- and 10-microl volumes, with human gamma globulin and rabbit antiserum. Of the dyes tested, bromsulfalein proved best for the protein estimation.     

Intracytoplasmic Glutathione Levels in Heifer Oocytes Cultured in different Maturation Media and its Effect on Embryo Development

The present study was carried out to study the effect of different maturation media on embryo development of heifer oocytes and on their glutathione (GSH) synthesis during in vitro maturation (IVM). Immature heifer oocytes were matured in parallel in one of four maturation media: (i) Tissue Culture Medium (TCM)-199 supplemented with 10 ng/ml of epidermal growth factor (EGF); (i) TCM-199 supplemented with 10 ng/ml of EGF plus 1 microg/ml of FSH; (iii) TCM-199 supplemented with 10% of foetal bovine serum (FBS) and (iv) TCM-199 supplemented with 10% of FBS plus 1 microg/ml of FSH. Cow oocytes were used as control and were matured in TCM-199 supplemented with 10 ng/ml of EGF. No differences were observed in blastocyst rate among the different heifer oocyte groups (8.8, 7.5. 8.4 and 6.8%, respectively) however, the percentage of blastocysts obtained from cow oocytes was significantly higher (30%; p < 0.01) than those obtained from heifer oocytes. De novo GSH synthesis during oocyte maturation of heifer and cow oocytes was detected. No significant differences in intracytoplasmic GSH levels were observed among the experimental heifer oocyte groups or between heifer and cow oocytes both before and after IVM. In conclusion, the blastocyst yield obtained from heifer oocytes was lower than that from cow oocytes and this fact could not be explained by significant differences in intracytoplasmic GSH contents of oocytes before or after IVM.     

THE PLACE OF A SPECIES IN THE FOREST, WITH SPECIAL REFERENCE TO WESTERN NORTH AMERICAN SPECIES OF CONIFER USED IN BRITAIN

The nature of the differences between the climates of the QueenCharlotte Islands and of England and Wales are discussed briefly.The author then examines the site tolerance of the principalspecies within their own natural range, comparing particularlythe place of Sitka spruce (Picea sitcbensis Carr.) in the foreston Graham Island with its place around Terrace and describingthe Douglas fir forests of the drier eastern side of VancouverIsland and the place that Sitka spruce has in them. Factorsaffecting the growth of Douglas fir (Pseudotsuga taxifolia Brit.)are discussed. It is suggested that care is needed before itis assumed that a markedly dry summer period such as occursin much of the Douglas fir region entails a ‘dry’period as regards supply to the tree. Illustrations are givenfrom eastern North America of changes in type of site occupiedby a species with change in climatic or edaphic environment.Observation of the relative position of a species in the forestas climate changes makes it possible to suggest the place ofSitka spruce and Douglas fir in Great Britain.     

Blood Gas Values During Intermittent Positive Pressure Ventilation and Spontaneous Ventilation in 160 Anesthetized Horses Positioned in Lateral or Dorsal Recumbency

One hundred sixty horses were anesthetized with xylazine, guaifenesin, thiamylal, and halothane for elective soft tissue and orthopedic procedures. Horses were randomly assigned to one of four groups. Group 1 (n = 40): Horses positioned in lateral (LR_G1,; n = 20) or dorsal (DR_G1,; n = 20) recumbency breathed spontaneously throughout anesthesia. Group 2 (n = 40): Intermittent positive pressure ventilation (IPPV) was instituted throughout anesthesia in horses positioned in lateral (LR_G2; n = 20) or dorsal (DR_G2; n = 20) recumbency. Group 3 (n = 40): Horses positioned in lateral (LR_G3; n = 20) or dorsal (DR_G3; n = 20) recumbency breathed spontaneously for the first half of anesthesia and intermittent positive pressure ventilation was instituted for the second half of anesthesia. Group 4 (n = 40): Intermittent positive pressure ventilation was instituted for the first half of anesthesia in horses positioned in lateral (LR_G4; n = 20) or dorsal (DR_G4; n = 20) recumbency. Spontaneous ventilation (SV) occured for the second half of anesthesia. The mean time of anesthesia was not significantly different within or between groups. The mean time of SV and IPPV was not significantly different in groups 3 and 4. Variables analyzed included pH, PaCO₂, PaO₂, and P(A-a)O₂ (calculated). Spontaneous ventilation resulted in significantly higher PaCO₂ and P(A-a)O₂ values and significantly lower PaO₂ values in LR_G1, and DR_G1, horses compared with LR_G2 and DR_G2 horses. Intermittent positive pressure ventilation resulted in normocarbia and significantly lower P(A-a)O₂ values in LR_G2 and DR_G2 horses. In LR_G2 the Pao₂ values significantly increased from 20 minutes after induction to the end of anesthesia. The PaO₂ and P(A-a)O₂ values were not significantly different from the beginning of anesthesia after IPPV in DR_G2 or DR_G3. The PaO₂ values significantly decreased and the P(A-a)O₂ values significantly increased after return to SV in horses in LR_G4, and DR_G4. The PaO₂ values were lowest and the P(A-a)O₂ values were highest in all horses positioned in dorsal recumbency compared with lateral recumbency and in SV horses compared with IPPV horses. The pH changes paralleled the changes in PaCO₂. Blood gas values during right versus left lateral recumbency in all groups were also evaluated. The PaO₂ values were significantly lower and the P(A-a)O₂ values were significantly higher during SV in horses positioned in left lateral (LR_LG1) compared with right lateral (LR_RG1) recumbency. No other significant changes were found comparing left and right lateral recumbency. Arterial hypoxemia (PaO₂ < 60 mm Hg) developed in 35% of DR_G1 horses and 20% of DR_G2 horses at the end of anesthesia. Arterial hypercarbia (PaCO₂= 50–60 mm Hg) developed in DRoi horses. Arterial hypoxemia that developed in 20% of DR_G3 horses was not improved with IPPV. Arterial hypoxemia developed in 55% of DR_G4 horses after return to SV. Some DR_G4 horses with hypoxemia also developed hypercarbia, whereas some had PaCO₂ values within normal limits. Arterial hypoxemia developed in one LR_G1, and two LR_G4, horses. Hypercarbia developed in onlv one LR_G4 horse.     

Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction

OBJECTIVE: To develop rapid (< 8 hour) tests using polymerase chain reaction (PCR) for the diagnosis of equine herpesvirus 3 (EHV3; equine coital exanthema virus), equine gammaherpesviruses 2 (EHV2) and EHV5, equine adenovirus 1 (EAdV1), EAdV2, equine arteritis virus (EAV), equine rhinitis A virus (ERAV; formerly equine rhinovirus 1) DESIGN: Either single round or second round (seminested) PCRs were developed and validated. METHODS: Oligonucleotide primers were designed that were specific for each virus, PCR conditions were defined and the specificity and sensitivity of the assays were determined. The application of the tests was validated using a number of independent virus isolates for most of the viruses studied. The PCRs were applied directly to clinical samples where samples were available. RESULTS: We developed a single round PCR for the diagnosis of EHV3, a seminested PCR for EHV2 and single round PCRs for EHV5, EAdV1, EAdV2 and RT-PCRs for EAV and ERAV. The PCR primer sets for each virus were designed and shown to be highly specific (did not amplify any recognised non-target template) and sensitive (detection of minimal amounts of virus) and, where multiple virus isolates were available all isolates were detected. CONCLUSION: The development and validation of a comprehensive panel of PCR diagnostic tests, predominantly for viruses causing equine respiratory disease, that can be completed within 8 hours from receipt of clinical samples, provides a major advance in the rapid diagnosis or exclusion diagnosis of these endemic equine virus diseases in Australia.     

In vitro Capacitation and Acrosome Reaction of Dog Spermatozoa can be Feasibly Attained in a Defined Medium Without Glucose

Incubation of dog spermatozoa in a medium without glucose and in the presence of lactate and pyruvate (l-CCM) for 4 h at 38.5 degrees C in a 5% CO(2) atmosphere induced in vitro capacitation of these cells. This was verified after the combined specific capacitation-like changes in percentages of viability and altered acrosomes, motility characteristics, sperm location of reactivity against Pisum sativum, Arachis hypogaea and Helix pomatia lectins and the tyrosine phosphorylation pattern. Furthermore, a feasible acrosome reaction (AR) was induced when spermatozoa incubated in l-CCM for 4 h were further co-incubated for 1 h with canine oocytes. This was demonstrated by AR-like changes in percentages of viability, altered acrosomes, motility characteristics and sperm location of reactivity against P. sativum, A. hypogaea and H. pomatia lectins. All these results clearly indicate that in vitro capacitation, and subsequent AR, can be feasibly achieved without the presence of sugars. This ability can be related to the specific characteristics of energy-metabolism regulation reported in dog spermatozoa.     

Differential expression of insulin‐like growth factor family members in immature cumulus–oocyte complexes from dairy cows with different genotypes

It has been evident the improvement of in vitro embryo production (IVEP) in dairy cows. Nevertheless, it is known that differences in the number and quality of oocytes between taurine and zebu females impact the efficiency and economic viability of IVEP. As the insulin‐like growth factor (IGF) system is related to follicular and oocyte development, we aimed to quantify mRNA abundance of IGF system members and pregnancy‐associated plasma protein‐A (PAPPA) in the cumulus–oocyte complexes (COCs) of Gir, 1/2 Holstein × 1/2 Gir and Holstein cows. Four pools of 30 immature COCs from Gir, 1/2 Holstein × 1/2 Gir and Holstein cows were obtained by ovum pickup (OPU), and the oocytes and cumulus cells (CC) were mechanically separated and stored at ?80°C. Total RNA was extracted from pools of 30 oocytes and their respective CC. Expression of target genes was assessed by real‐time RT‐PCR. In oocytes, the abundance of IGFR1 mRNA was higher (p < .05) in Gir cows compared with the other breeds. In contrast, in CC, mRNA encoding IGF2 (p < .05), IGFR2 (p < .05) and IGFBP4 (p < .01) was higher in Holstein donors compared with Gir and 1/2 Holstein × 1/2 Gir cows. Additionally, the abundance of PAPPA mRNA was higher in oocytes (p < .001) and CC (p < .01) in Gir and 1/2 Holstein × 1/2 Gir cows compared with the Holstein donors. In conclusion, the higher abundance of PAPPA mRNA in the oocytes and CC from Gir and cross‐breed donors combined with the low expression of IGFBP4 in the CC suggests an enhancement of the bioavailability of IGF‐free when compared with Holstein COCs.     

Evidence for absence of equine arteritis virus in the horse population of New Zealand

AIM: To summarise investigation and laboratory data collected between 2001 and 2011 to provide evidence that equine arteritis virus is not present in the horse population of New Zealand.

METHODS: Analysis was carried out on results from laboratory tests carried out at the Ministry for Primary Industries Animal Health Laboratory (AHL) for equine arteritis virus from horses tested prior to being imported or exported, testing of stallions as part of the New Zealand equine viral arteritis (EVA) control scheme and testing as part of transboundary animal disease (TAD) investigations for exclusion of EVA. Horse breeds were categorised as Thoroughbred, Standardbred or other.

RESULTS: A total of 7,157 EVA serological test records (from import and export testing, EVA control scheme testing and TAD investigations) were available for analysis between 2005 and 2011. For the three breed categories a seroprevalence of ≤1.6% at the 95% confidence level was determined for each category. Between 2001 and 2011, as part of the EVA control scheme, the EVA status of 465 stallions was determined to be negative. During 2005–2011 EVA was excluded from 84 TAD investigations.

CONCLUSIONS: There was no evidence of equine arteritis virus being present in the general horse population outside of carrier stallions managed under the EVA control scheme.

CLINICAL RELEVANCE: Equine arteritis virus is absent from the general horse population of New Zealand.