Mitochondrial function and sperm viability were quantified in samples of cryopreserved bovine spermatozoa from 12 bulls using fluorometric techniques. The active mitochondria of the spermatozoa were fluorescently stained using three different fluorophores: rhodamine 123 (R123), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyan ine iodide (JC-1) or MitoTracker Green FM (MITO). The stained spermatozoa, and companion aliquots that had been stained with SYBR-14 (a living-cell nucleic acid stain) and propidium iodide to assess viability, were quantified using flow cytometry. The resulting fluorescent measurements of mitochondrial function were compared with microscopic assessments of progressive sperm motility immediately after thawing, with motility after 3-h incubation at 37 degrees C, and with the fluorescent assessment of sperm viability. Staining with either R123 or MITO resulted in a single green population. In contrast, the JC-1 staining of mitochondria produced both green and red-orange populations of spermatozoa and sometimes a progressive gradient between the two populations. The ability of JC-1 to discriminate between mitochondria exhibiting high membrane potential from those having low to medium membrane potential provided a more rigorous estimate of metabolic function than the other two fluorescent stains. Overall, the three fluorometric measurements of mitochondrial function were highly correlated with each other, with the SYBR-14 assessment of viability, and with the microscopic estimates of motility.

In mammals, red/yellow and brown/black colorations are determined by the distribution of two pigments, phaeomelanin and eumelanin, respectively, the relative amounts of which are controlled primarily by two loci, extension and agouti. Dominant alleles at the extension locus increase brown/black pigmentation, while recessive alleles block eumelanin synthesis, thereby extending red/ yellow pigmentation within the hair follicle melanocyte. Robbins and associates (1993) have shown that the pigmentation phenotypes in mice controlled by the extension locus result from point mutations altering the function of the melanocyte-stimulating hormone receptor (MSHR). Johannson and colleagues (1994) demonstrated cosegregation of the chestnut (red) coat color in horses and polymorphisms at the MSHR locus. The black and red coat colors, respectively, in cattle are also controlled at the extension locus, the red color being due to a recessive allele (Searle 1968). The Holstein breed is stratified in red and black subpopulations. Gene flow from the black and white to the red and white subpopulation is through rare black carriers of the recessive red allele.To develop a direct test for determining the presence of the recessive allele causing red coat color in black Holstein cattle, we attempted to PCR amplify the bovine MSHR gene, assuming that the molecular basis of the bovine coat color phenotypes determined at the extension locus was similar to the one in horses and mice. The human (Chhajlani and Wikberg 1992) and mouse (Mountjoy et al. 1992) MSHR sequences were aligned to identify highly homologous regions suitable for designing primers based on the human sequences. In a first attempt we tried to amplify bovine DNA corresponding to a 544-bp segment of the human sequence using primer 1 and primer 2 (see Table 1). PCR was carried out in a reaction volume of 25 Ixl containing 100 ng of bovine or human genomic DNA, 10 mM Tris-HC1 pH 8.9, 50 mM KC1, 200 ~M of each deoxynucleotide, 0.4 ~M of each primer, and 2.5 U of Taq DNA polymerase (Boehringer Mannheim, Germany) in a thermal cycles (Hybaid Teddington, UK). MgC12 concentrations ranging from 1.5 to 3.0 mM with 0.5-mM increments were tested to optimize the PCR for specific amplification. After initial denaturation for 5 min at 95~ the PCR profile consisted of a denaturation step at 94~ for 30 s, an annealing step at 62~ for 30 s, and an elongation step at 72~ for 30 s for a total of 30 cycles, followed by a final extension of 7 min at 72~ The PCR product obtained when using bovine template DNA of a black Holstein animal consisted of two major bands between 500 and 600 bp, that is in the range of the human band at 544 bp. The two bovine bands were recovered from the agarose gel according to Heery and coworkers (1990) and subjected individually to another round of *Present address:

Summary. Background: Large animal models that accurately mimic human hemophilia A (HA) are in great demand for developing and testing novel therapies to treat HA. Objectives: To re-establish a line of sheep exhibiting a spontaneous bleeding disorder closely mimicking severe human HA, fully characterize their clinical presentation, and define the molecular basis for disease. Patients/methods: Sequential reproductive manipulations were performed with cryopreserved semen from a deceased affected ram. The resultant animals were examined for hematologic parameters, clinical symptoms, and responsiveness to human FVIII (hFVIII). The full coding region of sheep FVIII mRNA was sequenced to identify the genetic lesion. Results and conclusions: The combined reproductive technologies yielded 36 carriers and 8 affected animals. The latter had almost non-existent levels of FVIII:C and extremely prolonged aPTT, with otherwise normal hematologic parameters. These animals exhibited bleeding from the umbilical cord, prolonged tail and nail cuticle bleeding time, and multiple episodes of severe spontaneous bleeding, including hemarthroses, muscle hematomas and hematuria, all of which responded to hFVIII. Inhibitors of hFVIII were detected in four treated animals, further establishing the preclinical value of this model. Sequencing identified a premature stop codon and frame-shift in exon 14, providing a molecular explanation for HA. Given the decades of experience using sheep to study both normal physiology and a wide array of diseases and the high homology between human and sheep FVIII, this new model will enable a better understanding of HA and facilitate the development and testing of novel treatments that can directly translate to HA patients.

Ageing grey horses are particularly susceptible to melanoma. Using segregation analysis, six genetic and nongenetic (environmental) models in two grey horse family groups (n = 71) were compared. The polygenic model still fits the data significantly better than an environmental model, indicating a strong genetic impact on the phenomenon. Pmel17/gp100 and TYRP1/gp75, two genes which are specific for melanocytes, and Cdkn2a/p16 a gene coding for an inhibitor of a cell cycle regulator were partially cloned, sequenced and mapped. Using Northern blotting analysis a striking difference in mRNA expression of Pmel17/gp100 and TYRP1/gp75 was found comparing skin samples of solid-coloured (normal level) and grey horses (low level) as well as horse melanoma tumour samples (high). Staining of skin samples with antibodies recognizing the product of Pmel17/gp100, confirmed the results of the corresponding Northern blotting analysis. It seems that Pmel17/gp100 and TYRP1/gp75 are involved in progressive greying of horses. No mutation was found in a partial sequence of equine Cdkn2a/p16 analysed so far. Thus, the relation between equine melanoma susceptibility and Cdkn2a/p16 is subject to further investigation. ZusammenfassungAlternde echte Schimmel sind unabhängig von Geschlecht, Rasse und Population auffällig oft von Hautmelanomen betroffen. Phänotypische Daten zweier Schimmel-Pferdefamilien (n = 71) wurden mittels einer Segregationsanalyse auf sechs genetische und nicht-genetische Modelle ü berprü ft. Die Daten passten sich dem rein polygenen Modell immer noch signifikant besser an als dem Umweltmodell. Dies deutet auf einen starken genetischen Einfluss fü r dieses Merkmal hin. Pmel17/gp100 und TYRP1/gp75, zwei melanozytenspezifische Gene, und Cdkn2a/p16 ein Gen welches einen Inhibitor der Zellzyklusregulation codiert, wurden partiell kloniert, sequenziert und kartiert. Eine 'Northern blot' Analyse zeigte beim Vergleich von Hautproben zwischen farbigen Pferden (normal), Schimmeln (keine) und Tumoren (stark) signifikante Unterschiede in der mRNA-Expression von Pmel17/gp100 und TYRP1/gp75. Färbungen von Hautproben mit einem Antikö rper spezifisch fü r das Produkt von Pmel17/gp100 bestätigten diese Expressionsstudie. Demnach scheint es, dass Pmel17/gp100 und TYRP1/gp75 Einfluss auf die progressive Vergrauung bei Pferden ausü ben. In der partiellen Sequenz von Cdkn2a/p16 wurden bis jetzt keine Mutationen gefunden. Der Zusammenhang zwischen melanomanfälligen grauen Pferden und Cdkn2a/p16 bleibt Gegenstand weiterer Untersuchungen. RésuméLa mélanose concerne plus particulièrement les chevaux gris adultes. Par une analyse de ségrégation, six modèles génétiques et nongénétiques (environnement) ont été comparés dans deux familles de chevaux gris (n = 71). Les données s'ajustent significativement mieux au modèle polygène par rapport au modèle environnemental, ce qui implique un effet génétique important. Deux loci exprimés spécifi-quement dans le mélanocyte -Pmel17/gp100 et TYRP1/gp75 -et un locus codant pour un inhibiteur du cycle cellul...

Supernumerary teats represent a common abnormality of the bovine udder. A genome-wide association study was performed based on the proportion of the occurrence of supernumerary teats in the daughters of 1097 Holstein bulls. The heritability of caudal supernumerary teats without mammary gland in this study was 0.604. The largest proportion of the heritability was attributable to BTA 20. The strongest evidence for association was with five SNPs on chromosome 20, referred to as a QTL. The mode of inheritance at this QTL was dominant. These findings reveal that the occurrence of caudal supernumerary teats without mammary gland in Holstein cattle is influenced by a QTL on chromosome 20 and a polygenic part. The data support the high potential of the SNPs in the QTL region as markers for breeding against caudal supernumerary teats.