Effect of Sperm Cryopreservation on the European Eel Sperm Viability and Spermatozoa Morphology

The main objective of the present work was to study the effect of cryopreservation of European eel sperm both on the sperm viability and the spermatozoa head morphology. Spermatozoa morphology was evaluated with computer-assisted morphology analysis after collection in fresh samples, after adding the freezing medium containing dimethyl sulfoxide as cryoprotectant and, finally, after the cryopreservation process and thawing. Cell viability was assessed, in both fresh and thawed samples, by Hoechst 33258 staining. Computer-assisted sperm analysis (CASA) was used to determine the percentage of motile cells and to measure motility parameters in sperm samples. A significant decrease of head perimeter (12.56%) and area (17.90%) was detected from spermatozoa in fresh to thawed samples, indicating that cells do not recover the original size after the cryopreservation process. CASA was used to measure the percentage of motile cells (51.9%) and spermatozoa motility parameters such as curvilinear, straight line and angular path velocities, as well as beating cross frequency. This technique was employed in the fresh sperm samples but proteins present at the freezing medium (L-alpha-phosphatidylcholine) made impossible to use this last technique in thawed samples. When sperm viability was assessed by Hoechst staining, a significant decrease of approximately 15% (73.10 vs 58.26%) of alive spermatozoa was registered from fresh to thawed samples. The percentage of motile cells measured by CASA in fresh samples (51.9%) was lower than the percentage of alive cells determined by Hoechst stainning, suggesting the existence of different batches of spermatozoa in different stages of development, even during the eight to tenth weeks of treatment, when the highest sperm quality was found.     

Single Kernel Near‐Infrared Analysis of Tetraploid (Durum) Wheat for Classification of the Waxy Condition

Plant breeding programs are active worldwide in the development of waxy hexaploid (Triticum aestivum L.) and tetraploid (T. turgidum L. var. durum) wheats. Conventional breeding practices will produce waxy cultivars adapted to their intended geographical region that confer unique end use characteristics. Essential to waxy wheat development, a means to rapidly and, ideally, nondestructively identify the waxy condition is needed for point‐of‐sale use. The study described herein evaluated the effectiveness of near‐infrared (NIR) reflectance single‐kernel spectroscopy for classification of durum wheat into its four possible waxy alleles: wild type, waxy, and the two intermediate states in which a null allele occurs at either of the two homologous genes (Wx‐1A and Wx‐1B) that encodes for the production of the enzyme granule bound starch synthase (GBSS) that controls amylose synthesis. Two years of breeders' samples (2003 and 2004), corresponding to 47 unique lines subdivided about equally into the four GBSS genotypes, were scanned in reflectance (1,000–1,700 nm) on an individual kernel basis. Linear discriminant analysis models were developed using the best set of four wavelengths, best four wavelength differences, and best four principal components. Each model consistently demonstrated the high ability (typically >95% of the time) to classify the fully waxy genotype. However, correct classification among the three other genotypes (wild type, wx‐A1 null, and wx‐B1 null) was generally not possible.     

Hypoxia and a hypoxia mimetic up-regulate matrix metalloproteinase 2 and 9 in equine laminar keratinocytes

The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulate the activity of matrix metalloproteinase (MMP)-2 and -9 in cultures of equine hoof keratinocytes. These effects were assessed in primary cultures of laminar keratinocytes using gelatin zymography. Incubation of keratinocytes with cobalt chloride significantly increased the levels of active MMP-2 compared to untreated controls. Hypoxia significantly increased the expression of active MMP-2 and -9 in keratinocyte cultures. This up-regulation was observed after 6h and peaked at 24h. The study findings provide novel evidence of a potential link between hypoxia within the hoof and up-regulation of MMPs which may in turn result in damage to the lamellar basement membrane.     

Up‐Regulation of Insulin‐Like Growth Factor I and Uteroglobin in In Vivo‐Developed Parthenogenetic Embryos

Parthenote embryos are being considered as an alternative source of embryonic stem cells. However, as there is still a dearth of knowledge of this kind of embryos, a better understanding of their biology is needed for their application. In this work, we studied the differences and similarities between parthenotes and normal embryos at the blastocyst stage in vivo developed. We analysed the expression of factor OCT‐4, vascular endothelial growth factor (VEGF), insulin‐like growth factor I (IGF‐I) and uteroglobin (UG) by real‐time PCR. To do so, oocytes were recovered and after activation procedure were transferred by ventral middle laparoscopy to receptive does to undergo completely in vivo development. Does were slaughtered 6 days post‐ovulation induction, and parthenote and normal embryos were recovered for mRNA expression analysis. Our results reported that parthenotes and normal embryos showed similar mRNA expression for OCT‐4 and VEGF. However, IGF‐I and UG showed to be over‐expressed in parthenote embryos. Thus, our study highlights that despite the in vivo development of parthenotes, they still seem to have an altered expression and, therefore, to be different to normal embryos. The altered expression pattern of parthenote embryos suggests that these embryos should be studied carefully before future application.     

Lythrum hyssopifolia (lesser loosestrife) poisoning of sheep in Victoria

Objective To document an ovine disease attributed to the consumption of Lythrum hyssopifolia (lesser loosestrife). Procedures Historical and histological review of field and experimental cases. Results 1–20% mortality occurred in sheep flocks grazing paddocks where L. hyssopifolia was the predominant green vegetation. Well‐documented disease outbreaks occurred in summer on nine farms across Victoria between 1974 and 2002. Liver damage occurred in all nine outbreaks, with kidney damage in at least eight. Hepatocyte necrosis was usually zonal to midzonal (zone 2) in the liver samples from four farms and periacinar (zone 3) in those from three farms, but some livers showed only single‐cell necrosis. Multinucleate hepatocytes near necrotic areas were a feature in six cases. Proximal tubular epithelium appeared to be the primary renal target and brown granules were often present in renal tubules. Biochemical and histological evidence of liver and kidney damage was obtained from two sheep experimentally pen‐fed harvested L. hyssopifolia. Conclusion Chemicals in L. hyssopifolia are toxic to ovine hepatocytes and renal tubular epithelial cells.     

Field comparison of the interferon-gamma assay and the intradermal tuberculin test for the diagnosis of bovine tuberculosis

An extensive field comparison of the gamma interferon (IFN-gamma) assay and the single intradermal tuberculin test for the diagnosis of bovine tuberculosis was conducted in Australia. The specificity of the IFN-gamma assay was determined by testing more than 6000 cattle from tuberculosis-free herds and varied from 96.2% to 98.1%, depending on the cut-off point chosen to define a positive reactor. For the sensitivity trial, cattle from herds being de-populated because of bovine tuberculosis were examined with both assays. The sensitivity of the IFN-gamma assay was shown to be significantly higher than the single intradermal tuberculin test and varied from 76.8% to 93.6% depending on the method of interpretation. A maximum overall sensitivity of 95.2% was obtained by testing with the IFN-gamma and the tuberculin test in parallel. The superior sensitivity of the IFN-gamma assay and the ability to adjust the sensitivity of the system depending on the task involved, will provide the Australian Tuberculosis Eradication Campaign with a valuable additional test to enable it to accomplish its goals.     

Frequency of a FAS ligand gene variant associated with inherited feline autoimmune lymphoproliferative syndrome in British shorthair cats in New Zealand

Abstract

AIMS

To determine the frequency of the FAS-ligand gene (FASLG) variant associated with feline autoimmune lymphoproliferative syndrome (FALPS) and the proportion of carriers of the variant in three British shorthair (BSH) breeding catteries in New Zealand.