Characteristics of seminal plasma proteins and their correlation with canine semen analysis

The objectives were to separate canine seminal plasma proteins (with SDS-PAGE) and to determine the correlation between specific proteins and semen characteristics. Three ejaculates from 20 mixed-breed dogs, of unknown fertility, were collected by digital manipulation. Ejaculate volume and color, sperm motility, sperm vigor, percentage of morphologically normal spermatozoa, and membrane integrity (hypoosmotic swelling test and fluorescent staining) were assessed. For each dog, seminal plasma was pooled from all three ejaculates and proteins were separated with SDS-PAGE, using polyacrylamide concentrations of 13% and 22% in the separation gels. After staining, gel images were digitized to estimate molecular weights (MW) and integrated optical density (IOD) of each lane and of individual bands. Total seminal plasma protein concentration was 2.19 +/- 1.56 g/dL (mean +/- SD; range 1.12-5.19 g/dL). A total of 37 protein bands were identified (although no dog had all 37 bands). In the 13% gel, molecular weights ranged from 100.6 to 17.1 kDa, with four bands (49.7, 33.2, 26.4, and 19.5 kDa) present in samples from all dogs. In the 22% gel, molecular weights ranged from 15.6 to 3.6 kDa, with nine bands (15.6, 13.5, 12.7, 11.7, 10.5, 8.7, 7.8, 5.6, and 4.9 kDa) present in samples from all dogs. Combined for both gels, the majority of bands (85%) had molecular weights < 17 kDa, with B20 (15.6 kDa) in high concentrations in samples from all dogs. There were positive correlations (P <= 0.01) between two bands, 134 (67 kDa) and B5 (58.6 kDa), and sperm motility (r = 0.66 and r = 0.46), sperm vigor (r = 0.56 and r = 0.66), percentage of morphologically normal spermatozoa (r = 0.55 and r = 0.59), the hypoosmotic swelling test (r = 0.76 and r 0.68), and fluorescent staining (r = 0.56 and r = 0.59), respectively. In conclusion, 37 proteins were identified in seminal plasma; two were significantly correlated with semen characteristics. (c) 2007 Elsevier B.V. All rights reserved.

Canine corpus luteum regression: Apoptosis and caspase-3 activity

The present study evaluated the occurrence of apoptosis and caspase-3 activity in the canine corpus luteum during the period of luteal regression in eight pregnant and nine nonpregnant diestrus bitches. Intact luteal cells were obtained from corpora lutea in both peripartum pregnant bitches and nonpregnant diestrus bitches at approximately 65 d (range 63-68) after estrus, but not at days 75 and 85 in nonpregnant bitches. In all bitches, apoptotic cells were rarely detected and when present, those cells were more easily detected using the hematoxylin and eosin technique than using the critical electrolyte concentration technique. The luteal structures at 75 and 85 d of diestrus had histological characteristics similar to a corpus albicans. Caspase-3 activity was detected in morphologically normal corpora lutea from both pregnant and diestrus bitches around day 65, and also in the later structures considered corpus albicans tissue. These results suggested that apoptosis may not be the major mechanism involved in canine functional luteal regression, and that caspase-3 participated in both functional and morphological luteolysis and in the tissue reorganization involved in corpus albicans formation. (c) 2006 Published by Elsevier B.V.

Vasectomy effect on canine seminal plasma biochemical components and their correlation with seminal parameters

Three semen samples were collected at 48 It intervals from 20 mature research dogs previously conditioned to manual semen collection. Vasectomy was performed in all dogs, and 15 days after surgery, another three ejaculates were similarly collected. The semen was evaluated, and centrifuged to obtain seminal plasma for measurement of pH, and concentrations of total proteins (TP), total chlorides (Cl), calcium (Ca), potassium (K), and sodium (Na). The seminal plasma protein profile was evaluated by SDS-PAGE; molecular weights and the integrated optical density (IOD) of each band were estimated. There was a negative correlation between K concentration and progressive motility (r = -0.49, P = 0.027), sperm vigor (r = -0.60, P = 0.0053), and plasma integrity, evaluated by both the hypo-osmotic swelling test (r = -0.50, P = 0.026) and a fluorescent stain (r = -0.45, P = 0.046). Positive correlations between Na and K pre- and post-vasectomy (r = 0.88, P < 0.001; r = 0.56, P < 0.01, respectively) were verified. There were a total of 37 bands pre-vasectomy and 35 post-vasectomy (range, 100.6-3.6 kDa). Bands B9 and B13 (42.6 and 29.2 kDa) were not present post-vasectomy. The IOD of band B3 (73.5 kDa) was higher (P 0.03) pre-vasectomy, compared to post-vasectomy; conversely, the IODs of bands B29 and B37 (7.8 and 3.6 kDa) increased (P 0.026 and 0.047). Pre-vasectomy, there was a positive correlation (r = 0.49, P = 0.029) between band B37 band (3.6 kDa) and the Na:K ratio. In conclusion, K appeared to be involved in sperm motility in dogs and could be a tool to evaluate sperm function. The prostate contributed several elements to canine seminal plasma. Vasectomy changed Ca concentrations and the protein profile of the seminal plasma. Further studies must be performed to clarify the function of these elements on the in vivo fertility of dogs. (c) 2006 Elsevier B.V. All rights reserved.

Prognostic value of canine frozen-thawed semen parameters on in vitro sperm-oocyte interactions

Combining the data from conventional semen analysis with oocyte penetration assays should improve the assessment of the fertilizing ability of a semen sample. Thus, the objective of the present study was to evaluate the prognostic value of various semen parameters on the in vitro interactions between frozen-thawed canine sperm and homologous oocytes. Ten ejaculates from five stud dogs (two ejaculates/dog) were collected by digital manipulation. Semen samples were evaluated, extended in Tris-egg yolk-glycerol, frozen and stored in liquid nitrogen, and thawed several weeks later. Samples were evaluated for motility and sperm populations by computer-aided semen analysis (CASA), plasma membrane integrity (carboxy-fluorescein diacetate and propidium iodide), and sperm morphology (Bengal Rose). Thawed spermatozoa were also incubated with homologous oocytes for 18 h in an atmosphere of 5% CO2 and 95% air at 38 degrees C and sperm-oocyte interactions were evaluated. Simple linear regression models were calculated, with sperm parameters as independent variables and sperm-oocyte interactions as the dependent variable. There were significant associations between: percentage of oocytes bound to spermatozoa and beat cross frequency (BCF; R-2 = 63%); percentage of oocytes that interacted with spermatozoa and BCF (R-2 = 73%); and number of penetrated spermatozoa and velocity average pathway (VAP; R-2 = 64%) and velocity straight line (VSL; R-2 = 64%). Although plasma membrane integrity and sperm morphology had little prognostic value for in vitro interactions between canine frozen-thawed sperm and homologous oocytes, some motility patterns (evaluated by CASA) were predictive of in vitro sperm-oocyte interactions. (c) 2005 Elsevier B.V. All rights reserved.

Heparin-binding proteins of canine seminal plasma

Heparin-binding proteins (HBP) from seminal plasma have been expected to participate in modulation of the acrosomal reaction, and have been correlated with fertility in some species. However, they have not been described in the dog. The aim of this study was to document the HBPs of canine seminal plasma. Six pooled samples of seminal plasma from three crossbred dogs were used. The HBPs were isolated by heparin affinity chromatography and the fractions recovered were pooled. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 12 and 18% vertical minigels. The stained gels were scanned and the molecular weight (kDa) values for each band within a lane were calculated by image analysis software. The electrophoresis analysis of the pooled eluded fractions identified 19 bands, with molecular weights varying from 61.5 to 5.2 kDa. Previous studies, using one-dimensional SDS-PAGE, identified two bands (67 and 58.6 kDa), which were positively correlated with some semen parameters (sperm motility, sperm vigor, percentage of morphologically normal sperm and plasma membrane integrity). The 61.5 kDa band detected in the present study apparently corresponded to the 58.6 kDa band identified previously. Canine seminal plasma contained HBP; since HBP modulate the acrosome reaction in other species, they may have the same function in the dog. Further studies are necessary to better characterize this protein and determine if it is associated with fertility in the dog. (c) 2006 Elsevier B.V. All rights reserved.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Acetabular deepening in the treatment of severe canine hip dysplasia

Uma técnica para restituir a anatomia acetabular com aprofundamento da cavidade acetabular e reconstrução do ligamento da cabeça do fêmur e da cápsula articular, foi testada em nove cães, de raças de grande porte, portadores de displasia coxofemoral grave com subluxação acentuada ou luxação. O procedimento cirúrgico foi constituído de duas fases. Inicialmente, foi realizada a pectineotomia bilateral em todos os cães. A segunda intervenção nos mesmos cães incluiu abordagem e aprofundamento do acetábulo, reconstrução do ligamento da cabeça do fêmur e da cápsula. em geral, 30 dias após a cirurgia, os cães apoiavam o membro operado para se locomover. Com exceção de dois cães, todos os outros recuperaram a função locomotora do membro pélvico dentro de 60-90 dias. É concluído que a técnica de acetabuloplastia é uma boa alternativa para o tratamento da displasia coxofemoral grave.

Post-thaw viability of in vivo-produced canine blastocysts cryopreserved by slow freezing

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Canine rabies epidemiology in Araçatuba and neighborhood, Northwestern São Paulo State - Brazil

São apresentadas algumas características epidemiológicas da raiva canina na região de Araçatuba, no período de 1993 a 1997. Dentre 1.984 cães submetidos ao diagnóstico de raiva, 351 foram positivos sendo que 89% dos casos (312/351) ocorreram na zona urbana e destes, 85% (266/312) eram de animais com proprietário. As informações sobre sexo estavam presentes em 83% das fichas e destas 61% eram de machos. A média de idade dos cães positivos foi de 34 meses. A agressividade foi um dos sintomas mais comumente observado (77%), seguido por incoordenação motora e/ou paralisia (42%) e 48% dos cães positivos haviam agredido pessoas ou outros animais. Das 182 fichas epidemiológicas que possuíam dados de vacinação, 51% (92/182) dos cães não eram vacinados. Aponta-se como possíveis razões para o aparecimento da epidemia no município de Araçatuba a baixa cobertura vacinal demonstrada pelo índice de cães não vacinados que adquiriram a doença aliado à elevada relação cão/homem (1:3,5) revelada em estudo anterior.

Avaliação da leishmaniose visceral canina em Poxoréo, Estado do Mato Grosso, Brazil

O cão doméstico desempenha importante papel como reservatório na transmissão da leishmaniose visceral ao homem, zoonose de grande importância em saúde pública. Realizou-se avaliação epidemiológica da leishmaniose visceral em 1.112 cães domiciliados no município de Poxoréo, estado do Mato Grosso e observou-se prevalência de 7,8%. Observou-se ainda associação estatisticamente significativa entre a prevalência de leishmaniose visceral canina e as variáveis faixa etária, presença de sinais clínicos e presença de outra espécie animal co-habitando com os cães avaliados, tendo sido as galinhas mais freqüentemente observadas entre os animais soropositivos. O sexo, a coleta de lixo domiciliar bem como a renda familiar não apresentaram associação estatisticamente significativa com a prevalência da leishmaniose visceral canina. A análise dos resultados sugere que cães com idade superior a sete anos e a , presença de outra espécie animal co-habitando com os cães podem ser fatores de risco para a leishmaniose visceral canina.