Heat-induced redistribution of disulfide bonds in milk proteins. 1. Bovine beta-lactoglobulin

Changes in the structure and chemistry of beta-lactoglobulin (beta-LG) play an important role in the processing and functionality of milk products. In model beta-LG systems, there is evidence that the aggregates of heated beta-LG are held together by a mixture of intermolecular non-covalent association and heat-induced non-native disulfide bonds. Although a number of non-native disulfide bonds have been identified, little is known about the initial inter- and intramolecular disulfide bond rearrangements that occur as a result of heating. These interchange reactions were explored by examining the products of heat treatment to determine the novel disulfide bonds that form in the heated beta-LG aggregates. The native protein and heat-induced aggregates were hydrolyzed by trypsin, and the resulting peptides, before and after reduction with dithiothreitol, were separated by high-performance liquid chromatography and their identities confirmed by electrospray ionization mass spectrometry. Comparisons of these peptide patterns showed that some of the Cys160 was in the reduced form in heated beta-LG aggregates, indicating that the Cys160-Cys66 disulfide bond had been broken during heating. This finding suggests that disulfide bond interchange reactions between beta-LG non-native monomers, or polymers, and other proteins could occur largely via Cys160.     

Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre‐exposure of oocytes to Ricinus communis (RCA‐1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm–egg binding and fertilization. In vitro‐matured bovine oocytes were incubated (39°C, 5% CO₂ in air) for 2 h in the following treatments: (i) 500 μl of fertilization medium (FM); (ii) 250 μl of FM with 0.25 ml of non‐luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 μl of FM with 250 μl of NLAUTF and 4 μl of RCA‐1 lectin; (iv) 250 μl of FM with 250 μl of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA‐1 lectin; (v) 500 μl of FM and RCA‐1 lectin. Following incubation, oocytes were washed, placed in FM with 2 μg heparin, and incubated with 1 × 10⁵ frozen–thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate‐orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean ± SEM) when oocytes were incubated in treatment 3 (59.0 ± 5.5) than in treatments 2 (46.4 ± 5.6), 4 (18.1 ± 5.4), 5 (33.4 ± 5.6) or 1 (32.5 ± 5.6). More oocytes were fertilized when incubated in treatment 3 (91% ± 3.0) than in 2 (84% ± 3.0), 4 (40% ± 3.0), 5 (77% ± 3.0) or 1 (76% ± 3.0). As in previous studies, this study suggests that RCA‐1 lectin enhances binding of UTF‐derived OPN to bovine oocytes, resulting in increased sperm–egg binding and fertilization in vitro and a possible role in fertilization.     

Different Culture Media Requirements of IVF and Nuclear Transfer Bovine Embryos

Important differences exist between in vitro fertilized (IVF) and nuclear transfer (NT) bovine embryos. Studies have shown that although in vitro development is comparable, post-implantation survival is greatly reduced in NT embryos. In this study, we compare serum and bovine serum albumin (BSA) supplementation during oocyte maturation and embryo culture of IVF and NT embryos. In experiment 1, oocytes and embryos were randomly distributed into different treatment groups consisting of synthetic oviductal fluid (SOF) medium supplemented with either serum, fatty acid-free BSA (FAF) or fraction V BSA during maturation and/or culture to assess IVF embryo development. In experiment 2, oocytes were matured in SOF + serum or SOF + FAF and reconstructed embryos were cultured in SOF + FAF to assess NT embryo development. Among the IVF treatment groups, a greater number of blastocysts were observed in the steer serum (SER) group (IVM and IVC in SOF + serum) on day 6; however, no significant differences were seen in blastocyst development from day 8 onwards. Hatching frequencies on days 8 and 9 were significantly greater in groups with serum, with the exception of FAF (IVM and IVC in SOF + FAF) on day 9. For the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes and increased blastocyst development and hatching rates were compared with supplementation of FAF. These results indicate that both serum and FAF provide comparable embryo development for IVF but not for NT bovine embryos.     

A Satellite RNA of Ophiostoma novo-ulmi Mitovirus 3a in Hypovirulent Isolates of Sclerotinia homoeocarpa

ABSTRACT Two genetically distinct double-stranded RNA (dsRNA) elements were identified in hypovirulent isolates of Sclerotinia homoeocarpa, the causal agent of dollar spot of turfgrass. The large dsRNA (L-dsRNA) was consistently present in all hypovirulent isolates, whereas the small dsRNA (S-dsRNA) was found only in some hypovirulent isolates. Virulence comparisons revealed that there was no significant difference between isolates containing one or both dsRNAs. Therefore, the L-dsRNA appears to be the genetic determinant of hypovirulence, while the S-dsRNA is not essential for hypovirulence in S. homoeocarpa. The L-dsRNA in hypovirulent isolate Sh12B of S. homoeocarpa was previously characterized as a fungal mitochondrial virus and designated Ophiostoma novo-ulmi mitovirus 3a-Sh12B (OnuMV3a-Sh12B) because it was conspecific with O. novo-ulmi mitovirus 3a-Ld from O. novo-ulmi, the causal agent of Dutch elm disease. In the present study, the nucleotide sequences of the S-dsRNAs (738 to 767 nucleotides) in hypovirulent isolates Sh12B, Sh279B, and Sh286B were determined. Nucleotide sequence analysis indicated that the S-dsRNA was not derived from the OnuMV3a dsRNA and it could not encode an RNA-dependent RNA polymerase. These results are consistent with biological data that the S-dsRNA was always associated with the L-dsRNA and was never found independently. Therefore, the S-dsRNA can be regarded as a satellite RNA of OnuMV3a in S. homoeocarpa. Northern blotting analysis indicated that nucleic acid extracts from isolate Sh12B of S. homoeocarpa contained more single (+) stranded RNA than dsRNA for this satellite RNA. The 5'- and 3'-terminal sequences of the positive strand of the S-dsRNA each could be folded into a stem-loop structure and the terminal 21 nucleotides were complementary to each other, potentially forming a panhandle structure.     

Occurrence of fungal pathogens of carrots on wooden boxes used for storage

Infested wooden boxes, previously used for carrot storage, were sampled in four commercial carrot production farms in Bradford Marsh, Ontario, and screened for fungal occurrence. At least 128 and 465 fungal isolates were recovered from these boxes in 2001 and 2002, respectively, and were classified into 10 taxonomic groups, including Alternaria spp., Aspergillus spp., Botrytis cinerea , Fusarium spp., Mucor spp., Penicillium spp., Rhizoctonia carotae , Rhizopus spp., Sclerotinia sclerotiorum and Trichoderma spp. A subsample of 27 putative pathogenic isolates was further tested for the ability to cause disease on carrots and to colonize wood surfaces under growth room and cold storage conditions. Approximately 60% of the taxa growing on wood caused lesions upon contact with intact carrots in cold storage. Isolates of S. sclerotiorum , B. cinerea and R. carotae caused the most severe diseases, developed most extensively on wooden surfaces in cold storage, and represented 12% of the recovered fungi. Isolates of Alternaria spp., Aspergillus spp., Fusarium spp., Mucor spp., Penicillium spp., Rhizopus spp. and Trichoderma spp. caused negligible or no disease on carrots and represented 88% of recovered fungi. Several of these fungi, however, showed potential to colonize wooden surfaces and cause disease on sliced carrots. This study suggests that pathogenic inocula occurring on used wooden boxes can initiate disease upon contact with healthy carrots and reusing infested boxes can affect carrots in storage.     

Demographic profile of the veterinary profession in New Zealand, 1985

The demographic characteristics of the veterinary profession in New Zealand are examined using information supplied by registered veterinarians to the Veterinary Surgeons Board in their applications for annual practising certificates in 1985. Comparisons are made with the veterinary professions in other similar countries, and with the medical and paramedical professions in New Zealand. In 1985 1308 registered veterinarians were working in New Zealand and 304 were overseas, whereas in 1976 the equivalent figures were 748 and 182. This represents an increase of 73% in the number of registered veterinarians in the last 10 years. Eighteen percent of veterinarians were female. The mean age of veterinarians was 38 years, but females in the profession were on average much younger (32 years) than males (42 years). Clinical practice provides employment for 70% of veterinarians: of these 59% work principally with farm animals and 41% with non-farm animals. The remaining veterinarians are employed by Government (22%), University (5%) and Industry (3%). The current demographic structure of the profession has been markedly influenced by the opening of New Zealand's only veterinary school at Massey University in 1963. Differences from other medical professional populations in New Zealand and overseas principally reflect the marked change in the number and sex ratio of graduates entering the veterinary profession since that time. It will take about another twenty years before the population reaches a stable age and sex structure, assuming that current graduation patterns persist throughout that period. Because the structure of the population is changing, considerable caution is needed in predicting future employment trends from data for a single year.     

The effects of autumn closing date on sward leaf area index and herbage mass during the winter period

The provision of grass for early spring grazing in Ireland is critical for spring calving grass‐based milk production systems. This experiment investigated the effect of a range of autumn closing dates (CD), on herbage mass (kg DM ha^?1), leaf area index (LAI) and tiller density (m^?2) during winter and early spring. Thirty‐six grazing paddocks, closed from 23 September to 1 December 2007, were grouped to create five mean CD treatments – 29 September, 13 October, 27 October, 10 November, 24 November. Herbage mass, tiller density and LAI were measured every 3 weeks from 28 November 2007 to 20 February 2008; additionally, herbage mass was measured prior to initial spring grazing and tiller density was measured intermittently until September 2008. Delaying CD until November significantly (P < 0·05) reduced herbage mass (by approximately 500 kg DM ha^?1) and LAI (by approximately 0·86 units) in mid‐February. On average, 35% of herbage mass present on swards on 20 February was grown between 28 November and 30 January. LAI was positively correlated with herbage mass (R² = 0·78). Herbage mass increased by approximately 1000 kg DM ha^?1 as spring grazing was delayed from February to April. Tiller density increased from November to February, although it did fluctuate, and it was greatest in April (9930 m^?2). This experiment concludes that in the south of Ireland adequate herbage mass for grazing in early spring can be achieved by delaying closing to early mid‐October; swards required for grazing after mid‐March can be closed during November.