Protein Composition of Seminal Plasma in Fractionated Stallion Ejaculates

Seminal plasma (SP) contains several types of compounds derived from the epididymides and accessory glands. The aim of this study was to examine the protein composition of different ejaculate fractions. Trial I: fractionated ejaculates were collected from two normal and two subfertile stallions. Samples containing pre‐sperm fluid and the first sperm‐rich jets (HIGH‐1), the main sperm‐rich portion (HIGH‐2), the jets with low sperm concentrations (LOW), and a combined whole‐ejaculate (WE) sample was centrifuged, and the SP was filtered and frozen. A part of each SP sample was stored (5°C, 24 h) with spermatozoa from HIGH‐2 and skim milk extender. Sperm motility was evaluated after storage in extender mixed with the stallion’s own SP or SP from one of the other stallions (sperm from a normal stallion stored in SP from a subfertile stallion and vice versa). Protein composition was analysed using reverse‐phase liquid chromatography (RP‐HPLC), N‐terminal sequencing and mass spectrometry. The area‐under‐the‐curve (AUC) was used for quantitative comparison of proteins within fractions. Trial II: semen samples were collected from seven stallions. Fractions with the highest (HIGH) and lowest (LOW) sperm concentrations and WE samples were examined using SDS‐PAGE and densitometry. No significant differences emerged between fractions in the AUC‐values of the Horse Seminal Protein‐1 (HSP‐1) and HSP‐2 peaks, or the peak containing HSP‐3 and HSP‐4 (HSP‐3/4). Levels of HSP‐1, HSP‐2 and HSP‐3/4 were not significantly correlated with total sperm motility, progressive sperm motility or average path velocity after storage. Significant differences between ejaculate fractions in the amount of different protein groups present in SP were not found in Trial I; but in Trial II, the proteins in the 60–70 kDa range were more abundant in LOW than in HIGH and WE, indicating that this band contained proteins derived mainly from the seminal vesicles, which produce most of the SP in LOW.     

Unilateral scrotal pyocele in ram caused by Staphylococcus capitis

A massive unilateral scrotal pyocele caused by Staphylococcus capitis in a 6‐year‐old ram is reported. Ultrasound examination of the right hemiscrotum showed an irregular hyperechoic mass in an anechoic fluid. A dense exudate was collected from the scrotum for microbiological analysis. Grossly, there was an extensive greenish purulent exudate and a completely atrophied right testis. Coagulase‐negative S. capitis was isolated in pure culture. To the authors' knowledge, this is the first report of genital infection by S. capitis in rams. This microorganism should be included in the differential diagnosis of ovine genital infections.     

Microbial origin of the abdominal swelling affecting farmed larvae of gilt-head seabream, Sparus aurata L.

The aetiological agents of the abdominal swelling affecting farmed larvae of gilt-head seabream, Sparus aurata L., were studied. Four Vibrio strains were isolated from larvae of S. aurata affected by this disease, and all strains reproduced the disease in healthy larvae under controlled infection experiments, producing a significant increase of the mortality rates compared to the control (non-inoculated larvae). Several enzymatic properties, which can act as .virulence factors, were demonstrated both in the extracellular products (ECPs) and In live cells of the strains tested. Histopathological examinations of the infected fish larvae revealed important changes of the anterior intestine and liver characterized by a marked hyperthrophy of the intestinal epithelium and hepatocytes, and by a separation of the mucosal and submucosal layers in the digestive tube. These histological alterations were associated with the constant presence of cocobacillar bacteria in the anterior intestine and in the liver. However, the precise pathogenic mechanisms of the strains tested have not been completely elucidated yet.     

Thermodynamics of calcite growth: baseline for understanding biomineral formation

The complexity of biomineralized structures suggests the potential of organic constituents for controlling energetic factors during crystal synthesis. Atomic force microscopy was used to investigate the thermodynamic controls on carbonate growth and to measure the dependence of step speed on step length and the dependence of critical step length on supersaturation in precisely controlled solutions. These data were used to test the classic Gibbs-Thomson relationship and provided the step edge free energies and free energy barriers to one-dimension nucleation for calcite. Addition of aspartic acid, a common component in biomineralizing systems, dramatically affected growth morphology and altered the magnitude of the surface energy.     

Percentage of Ubiquitinated Spermatozoa does not Correlate with Fertilizing Capacity of Thawed Bovine Semen

In the spermatozoa of some species, the ubiquitin–proteasome system detects altered proteins and tags them for elimination by the proteasome. In some species' ejaculates, a high proportion of ubiquitinated spermatozoa (i.e. those having ubiquitin bound to the altered or damaged membrane proteins) has been related to infertility. The aim of this study was to assess whether the percentage of ubiquitinated spermatozoa relates to fertility of dairy bulls and whether ubiquitination increases during protein remodelling that occurs during in vitro spermatic capacitation. Thirty‐two frozen semen straws from four high‐fertility (ReproMax^®) and four normal‐fertility (Normal) Holstein‐Friesian sires were evaluated. Ubiquitinated and capacitated spermatozoa were quantified by sperm ubiquitin tag immunoassay and chlortetracycline stain, respectively. Fertilizing capacity of sires was assessed by in vitro fertilization. No differences were found between Normal and ReproMax^® sires with regard to the observed percentage of ubiquitinated spermatozoa (42.97 ± 3.69% and 49.68 ± 9.27%, respectively; p > 0.05). Additionally, no differences were found in the percentage of ubiquitinated spermatozoa as a consequence of spermatic capacitation in either Normal (42.97 ± 3.69% before capacitation vs 44.67 ± 7.5% after; p > 0.05) or ReproMax^® sires (49.68 ± 9.27% before vs 45.05 ± 7.51% after; p > 0.05). The percentage of ubiquitinated spermatozoa in a thawed sperm samples did not correlate with its in vitro fertilizing capacity; thus, this assay does not prove useful to detect in vivo fertility differences between sires. Additionally, protein degradation occurring during remodelling of the spermatozoon plasma membrane during the capacitation process does not seem to involve the ubiquitin–proteasome system.     

Pasteurella multocida contains multiple immunogenic haemin- and haemoglobin-binding proteins

Iron-dependent outer membrane proteins (IROMPs) play an important role in bacterial pathogenesis and present several attributes of potential vaccine candidates. TBLASTN analysis of the Pasteurella multocida Pm70 genome using the same molecules of other bacterial pathogens as a query identified eight putative haemin and haemoglobin receptors for this organism. Quantitative binding assays have demonstrated that the proteins PM0040, PM0236, PM0741, PM1081, PM1428, PM0592 and HgbA bind both haemin and haemoglobin, whereas PM0576 and PM1282 ORFs only bind either haemoglobin or haemin, respectively. Furthermore, Western blot analysis showed that P. multocida-infected mice generate specific antibodies against PM0040, PM0236, PM0741, PM1081, PM1428, PM0592 and HgbA proteins. Nevertheless, inoculation of mice with any single one of these receptors alone did not protect against P. multocida infection.     

Assessment of Suitable Porcine Semen for Freezing,According to the Ejaculate Characteristics in the Iberico × Landrace Breed

A total of 35 ejaculates were studied in order to assess the suitability of porcine semen for freezing according to the ejaculate characteristics. The effects of the freezing procedure were identified; a decrease in motility and acrosome quality was found after thawing. The best results on motility were linked to the ejaculates with a volume of less than 100 ml of the sperm‐rich fraction, a concentration lower than 450 × 10⁶ spermatozoa/ml and an agglutination score below 2. However, the best normal apical ridge (NAR) was found when the volume of the sperm‐rich fraction was greater than 150 ml. For this reason, an intermediate volume of the sperm‐rich fraction of the ejaculate for the best motility and the best NAR, a concentration lower than 450 × 10⁶ spermatozoa/ml and a rate of agglutination below 2 should provide the best quality after freezing. This study also attempted to determine whether a positive effect of ejaculate selection on the overall freezing performance might be expected.     

Stabilizing Lead-Salt Diode Lasers: Understanding and Controlling Chaotic Frequency Emission

Lead-salt tunable diode lasers (TDLs) are the only devices currently available that can generate tunable monochromatic radiation at arbitrary wavelengths between 3 and 30 micrometers and are particularly useful for high-resolution spectroscopy over a wide range of spectral regimes. Detailed observations of TDLs show that the observed instrumental linewidth is actually a temporal average of many narrow (less than 0.5 megahertz) emission "modes." The time scale characteristic of these "modes," which appear to be of relatively constant intensity, is of the order of a microsecond. The laser's behavior is highly suggestive of a chaotic process, that is, seemingly random excursions of a dynamic variable (frequency) within a bounded range. This report shows experimentally that TDL emissions are indeed chaotic. Furthermore, in a simple and robust fashion, this chaotic behavior has been successfully controlled with the use of recent techniques that take advantage of chaos to produce a narrow band laser output.     

Mucopolysaccharidosis type VI in a Miniature Poodle-type dog caused by a deletion in the arylsulphatase B gene

Abstract

AIM: To investigate and characterise an inborn error of metabolism in a dog with skeletal and ocular abnormalities.

METHODS: A 2.5-year-old small male Miniature Poodle-like dog was presented with gross joint laxity and bilateral corneal opacities. Clinical examination was augmented by routine haematology, serum chemistry, radiographs, pathology, enzymology and molecular genetic studies. Euthanasia was requested when the dog was 3 years of age because of progressively decreasing quality of life.

RESULTS: Radiology revealed generalised epiphyseal dysplasia, malformed vertebral bodies, luxation/subluxation of appendicular and lumbosacral joints with hypoplasia of the odontoid process and hyoid apparatus. These clinical and radiographic findings, together with a positive urinary Berry spot test for mucopolysaccharides, and metachromatic granules in leucocytes, were indicative of a mucopolysaccharidosis (MPS), a lysosomal storage disease. Histological lesions included vacuolation of stromal cells of the cornea, fibroblasts, chondrocytes, macrophages and renal cells. The brain was essentially normal except for moderate secondary Wallerian-type degeneration in motor and sensory tracts of the hind brain. Dermatan sulphate-uria was present and enzymology revealed negligible activity of N-acetylgalactosamine-4-sulphatase, also known as arylsulphatase B, in cultured fibroblasts and liver tissue. A novel homozygous 22 base pair (bp) deletion in exon 1 of this enzyme's gene was identified (c.103_124del), which caused aframe-shift and subsequent premature stop codon. The “Wisdom pure breed-mixed breed” test reported the dog as a cross between a Miniature and Toy Poodle.

CONCLUSIONS: The clinicopathological features are similar to those of MPS type VI as previously described in dogs, cats and other species, and this clinical diagnosis was confirmed by enzymology and molecular genetic studies. This is an autosomal recessively inherited lysosomal storage disease.

CLINICAL RELEVANCE: The prevalence of MPS VI in Miniature or Toy Poodles in New Zealand and elsewhere is currently unknown. Due to the congenital nature of the disorder, malformed pups may be subject to euthanasia without investigation and the potential genetic problem in the breed may not be fully recognised. The establishment of a molecular genetic test now permits screening for this mutation as a basis to an informed breeding policy.