Collection of tissue and culture samples from the canine reproductive tract

Definitive diagnosis of reproductive tract infection or other disease often requires sampling of tissue, either for culture or histopathology. Indications, sample collection technique, possible side-effects and interpretation of results are reviewed. Pertinent facts include: (1) collection of uterine biopsy specimens via laparotomy was associated with higher yield of diagnostic samples and fewer side-effects than other less invasive techniques; (2) vaginal culture samples should be collected from the anterior vagina to minimize number of contaminants in the sample; (3) collection of culture samples from the anterior vagina during proestrus or estrus, in the presence of discharge originating in the uterus, was a non-invasive technique for assessment for uterine infection; (4) samples for bacterial culture from mucosal surfaces, including the vagina and penis, must be quantitated to allow interpretation, with moderate to heavy growth of any single aerobic bacterial organism relevant; (5) mycoplasma and ureaplasma are part of the normal flora of the genitourinary tract in dogs and bitches and, because most laboratories cannot give reliable quantitative results, interpretation of positive results often is difficult; (6) collection of prostatic tissue samples for cytology or culture was more likely to yield a correct diagnosis than submission of ejaculated prostatic fluid.     

Anaerobic seminal fluid micro-flora in chronic prostatitis/chronic pelvic pain syndrome patients

We investigated the seminal micro-flora of 116 men. Eighty-four men had chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), and 34 of them were also leukocytospermic. Thirty-two asymptomatic men formed the control group. Micro-organisms were found in all of the 116 seminal fluid specimens. More than 20 different micro-organisms were found in both groups. Neisseria gonorrhoeae and Chlamydia trachomatis were not found. A high frequency of anaerobic bacteria was found in all groups (68-79%), and in most of the specimens, anaerobic micro-organisms were equal to or outnumbered the aerobic strains. We found 1-8 different micro-organisms in each semen sample, the total count of micro-organisms ranged from 10(2) to 10(7)/mL of semen. Both parameters were significantly higher in leukocytospermic CP/CPPS (NIH IIIA category) patients (median=5 different micro-organisms; total median count 5 x 10(4)) than in the control group (median=3 different micro-organisms; total median count 10(3)). In the CP/CPPS patients, the prevalence and/or count of some opportunistic bacteria was higher than in the control group. To show that the micro-organisms do not originate from the urethra, first voided urine was also investigated in 17 prostatitis patients and 15 controls. One patient had significantly fewer micro-organisms (median 1 vs. 4) and a lower total count of micro-organisms (median 10(2) vs. 10(4)/mL) in the first-catch urine than in the seminal fluid. We found only one third of the micro-organisms to be similar in urine and semen while anaerobic bacteria and some aerobic opportunists were infrequent in urine. Semen is a suitable specimen for the diagnosis of prostatitis.     

Characterization of fecal bacterial populations in canines: effects of age,breed and dietary fiber

The effects of age, breed, and diet on fecal chemistry, enzyme activity, and bacterial populations of dogs were studied. Eighteen dogs from two age groups (young: 2.5 +/- 0.5 years, old: 10.9 +/-0.7 years) and three different breeds (German shepherds, miniature schnauzers, and English setters) were rotated through a Latin Square design such that every dog was fed each of the diets. The test diets included a low-fiber (control) diet and a 10% fiber diet which contained 5% soybean hulls and 5% beet pulp. Inclusion of 10% fiber in the diet decreased the fecal concentration of ammonia, sulfide, and indole. Fiber inclusion significantly increased acetic, propionic, and butyric acid concentrations, while fecal pH decreased by 0.4 units. Fresh fecal samples were plated on selected aerobic and anaerobic culture media and DNA extracted for denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S ribosomal DNA fragments. Plate counts showed significant effects of breed (p < or = 0.05) and age (p < or = 0.01) on selected aerobic and anaerobic bacterial counts, while no significant effect of diet was found. Analysis of PCR-DGGE banding patterns showed there was a tendency for individual dogs to cluster together according to age (young or old dogs) and also for size (large or small dogs). However, the outstanding conclusion obtained from the DGGE analysis of fecal bacterial profiles was that individual dogs had their own characteristic banding pattern which was unique and stable. The relative stability and individuality of the patterns indicates that each individual harbored a characteristic fecal bacterial community which was independent of diet.     

Herapin-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.     

Characterization of a new bioactive protein from bovine seminal fluid

A new acidic seminal fluid protein (aSFP) was purified from bovine seminal fluid, using anion exchange chromatography and FPLC on MonoQ. The purified aSFP displays a pI of 4.8 and an apparent molecular weight of 14 kDa. Homogeneity of aSFP was demonstrated by FPLC and SDS-polyacrylamide gel electrophoresis. Monospecific anti-aSFP IgGs were employed to characterize aSFP in bovine seminal plasma and seminal vesicle secretion by immuno blot analysis. Proteinchemical characterization of aSFP included amino acid analysis as well as determination of 23 amino acid residues of the N-terminal sequence of aSFP. According to this sequence, aSFP appears to represent a hitherto unknown protein. aSFP stimulated cell division and progesterone secretion of bovine granulosa cells in vitro in a potent and dose dependent manner. aSFP appears to be a potent growth factor with effects on ovarian granulosa cells.     

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 < or = 0.01) between two bands, B4 (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.     

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

Three semen samples were collected at 48 h 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.     

The seminal fluid proteome of the honeybee Apis mellifera

Ejaculates contain sperm but also seminal fluid, which is increasingly recognized to be of central importance for reproductive success. However, a detailed biochemical composition and physiological understanding of seminal fluid is still elusive. We have used MS to identify the 57 most abundant proteins within the ejaculated seminal fluid of the honeybee Apis mellifera. Their amino acid sequences revealed the presence of diverse functional categories of enzymes, regulators and structural proteins. A number have known or predicted roles in maintaining sperm viability, protecting sperm from microbial infections or interacting with the physiology of the female. A range of putative glycoproteins or glycosylation enzymes were detected among the 57, subsequent fluorescent staining of glycolysation revealed several prominant glycoproteins in seminal fluid, while no glycoproteins were detected in sperm samples. Many of the abundant proteins that accumulate in the seminal fluid did not contain predictable tags for secretion for the cell. Comparison of the honeybee seminal fluid proteins with Drosophila seminal fluid proteins (including secreted accessory gland proteins known as ACPs), and with the human seminal fluid proteome revealed the bee protein set contains a range of newly identified seminal fluid proteins and we noted more similarity of the bee protein set with the current human seminal fluid protein set than with the known Drosophila seminal fluid proteins. The honeybee seminal fluid proteome thus represents an important addition to available data for comparative studies of seminal fluid proteomes in insects.     

Immunoreactive vasopressin in bull seminal fluid

Immunoreactive arginine vasopressin (irAVP) was measured in seminal fluid with and without extraction using a specific radioimmunoassay (RIA). A large fraction of irAVP was removed after extraction on octadecasilylsilica cartridges. The measured amount of irAVP corresponded to the levels found in blood plasma. Dilutions of seminal plasma extracts were parallel with the RIA standard curve. On reversed phase HPLC the extracted material coeluted with synthetic AVP. These findings suggest an identity of this immunoreactive material with intact AVP. During incubations of synthetic AVP and its analogue 8-D-arginine vasopressing (8-DAVP) in seminal plasma, immunoreactivity decreased considerably with the former peptide, while the concentration of 8-DAVP was not significantly altered.     

Seminal vesicle production and secretion of growth hormone into seminal fluid.

Production of foreign proteins in the tissues of transgenic animals represents an efficient and economical method of producing therapeutic and pharmaceutical proteins. In this study, we demonstrate that the mouse P12 gene promoter specific to the male accessory sex gland can be used to generate transgenic mice that express human growth hormone (hGH) in their seminal vesicle epithelium. The hGH is secreted into the ejaculated seminal fluids with the seminal vesicle lumen contents containing concentrations of up to 0.5 mg/ml. As semen is a body fluid that can be collected easily on a continuous basis, the production of transgenic animals expressing pharmaceutical proteins into their seminal fluid could prove to be a viable alternative to use of the mammary gland as a bioreactor.     

gamma-Glutamyl transpeptidase of human seminal fluid.

γ-Glutamyl transpeptidase, which is present in high levels in human seminal fluid plasma, was purified about 870-fold from this source. The enzyme is present in seminal fluid plasma in particulate form. Purification by a procedure involving treatment with bromelain gave a protein (apparent molecular weight, about 70,000), which exhibited catalytic properties characteristic of γ-glutamyl transpeptidase preparations isolated from rat kidney and other mammalian tissues. The physiological significance of seminal fluid γ-glutamyl transpeptidase and its potential clinical value are considered.     

细菌性阴道病的优势菌群半定量培养分析

目的探讨临床适用的菌群培养方法,用于检测分析细菌性阴道病患者阴道优势菌群,以更好地指导临床诊治。方法采集32例细菌性阴道病（BV）患者的阴道分泌物标本,在不同气体环境中用非选择性、半定量方法做细菌培养,比较培养结果,分析患者阴道优势菌群。结果在厌氧环境中培养时菌落数量最多,检出的优势菌共15种,每份标本的优势细菌种类多数为2种（20/32）,少数为3种（3/32）;而在微需氧及需氧环境中检出的主要菌分别为8种及5种。结论非选择性半定量、厌氧培养的方法可有效、简便地了解BV阴道优势菌群的构成,可用于临床对BV菌群的检测分析研究。     

Mutagenicity testing of seminal fluid: seminal fluid increases the mutagenicity of the precursor mutagen benzo[a]pyrene in the presence of S9 mix

Small amounts of seminal fluid strongly enhanced the mutagenicity of the precursor mutagen benzo[a]pyrene (BP) in the Salmonella/microsome test. This previously unreported effect was found only in the presence of S9 mix for metabolic activation. The increase far exceeded the additive effect expected from experiments where seminal fluid and BP were tested separately with S9 mix. Testing of the direct-acting mutagen 4-nitro-o-phenylene-diamine (NPD) together with seminal fluid resulted in a lower mutagenic activity than that of NPD alone. Seminal fluid had a bactericidal effect on the Salmonella bacteria, thus only volumes up to 40 microliter could be used per plate. The mutagenic effect of only seminal fluid and S9 mix was slightly increased over controls in a standard Ames test, but was equal to the spontaneous mutation rate with a preincubation test modified according to Kado and coworkers. There were no significant differences between seminal plasma from smokers and non-smokers in any experimental series. Seminal fluid concentrated 20-fold by extraction with the mutagen-removing adsorbant Mutasorb did not have any enhancing effect on the mutagenicity of BP, nor did it exhibit any mutagenic activity in itself with or without S9 mix.     

Identification and function of proteolysis regulators in seminal fluid

Proteins in the seminal fluid of animals with internal fertilization effect numerous responses in mated females that impact both male and female fertility. Among these proteins is the highly represented class of proteolysis regulators (proteases and their inhibitors). Though proteolysis regulators have now been identified in the seminal fluid of all animals in which proteomic studies of the seminal fluid have been conducted (as well as several other species in which they have not), a unified understanding of the importance of proteolysis to male fertilization success and other reproductive processes has not yet been achieved. In this review, we provide an overview of the identification of proteolysis regulators in the seminal fluid of humans and Drosophila melanogaster, the two species with the most comprehensively known seminal fluid proteomes. We also highlight reports demonstrating the functional significance of specific proteolysis regulators in reproductive and post‐mating processes. Finally, we make broad suggestions for the direction of future research into the roles of both active seminal fluid proteolysis regulators and their inactive homologs, another significant class of seminal fluid proteins. We hope that this review aids researchers in pursuing a coordinated study of the functional significance of proteolysis regulators in semen. Mol. Reprod. Dev. © 2012 Wiley Periodicals, Inc.     

Detection of prostatic tissue and seminal plasma peptides reacting with human seminal fluid acid phosphatase antibodies

IgG1 monoclonal antibody to purified seminal fluid phosphatase was raised by fusion of spleen cells from immunized mice with cell line Sp2/O-Ag 14 using simple method of screening for antiphosphatase antibody secreting clones. All molecular forms of catalytically active seminal fluid phosphatase and prostatic tissue phosphatase, resolved by chromatofocusing in pH gradient, react with this monoclonal antibody and with rabbit antiserum to purified seminal fluid phosphatase. Peptides of Mr 25,000 to 76,000 and of Mr 13,000 to 76,000 were adsorbed from the prostatic tissue extract and from seminal plasma on the monoclonal antibody-Sepharose column.     

Comparative evaluation of seminal,vaginal, and rectal bacterial flora in the cheetah and domestic cat

To determine the status and potential impact of microorganisms on reproductive health, bacterial cultures were evaluated from cheetah seminal, vaginal, and rectal swabs and the results compared to those from clinically healthy, domestic cats. Aerobic bacteria were isolated in the semen from 26 of the 40 (65.0%) cheetahs and 25 of the 27 (92.6%) domestic cats. Gram-negative organisms predominated in the electroejaculates of both species, accounting for >70% of the total bacterial isolates. The most common seminal organism in both species was hemolytic Escherichia coli. Bacteria were isolated from vaginal samples obtained from 49 of the 67 (73.1%) cheetahs and 46 of the 49 (93.9%) domestic cats. Gram-negative organisms dominated, representing >63% of the vaginal bacteria, and again hemolytic E. coli was the most prevalent isolate in both species. None of the cheetah or domestic cat vaginal cultures contained Mycoplasma spp. or Ureaplasma spp. Numerous gram-negative and gram-positive bacteria were identified in rectal cultures of 73 cheetahs and 60 domestic cats, but hemolytic E. coli clearly was the most common isolate. Within each species, a comparison between electroejaculates that were positive vs. negative for hemolytic E. coli growth revealed no differences in sperm concentration, sperm motility ratings, or the proportion of structurally abnormal spermatozoa. Neutrophils were not detected in any of the 67 felid ejaculates, and the presence of seminal hemolytic E. coli was unrelated to fertility, on the basis of past ability to sire young or fertilize oocytes in vitro. Vaginal cytologic evaluations in both the cheetah and domestic cat indicated that hemolytic E. coli was not associated with a pathologic inflammatory response. Overall fecundity and proven ability to produce young were similar between females producing positive or negative vaginal cultures for E. coli. These findings indicate that commensal bacteria exist in the reproductive tract of the cheetah and domestic cat, and these organisms constitute normal, apparently innocuous bacterial microflora in the semen and vagina. © 1993 Wiley-Liss, Inc.     

A simple and sensitive method for detecting bacterial elastase production

A sensitive method for detecting bacterial elastase production in growing cultures is described. A variety of commonly isolated clinically relevant aerobic and anaerobic bacteria have been shown to produce the enzyme.     

Creatine kinase and ATPase in human seminal fluid and prostatic fluid

A creatine kinase assay based on estimation of creatine liberated from creatine phosphate was accurate and reproducible for use with seminal or prostatic fluid, after allowance was made for acid phosphatase interference. Comparison of this method with one which relies on enzymic coupling of ATP formation to NADP+ oxidation shows that the latter under-estimates creatine kinase activity by a factor of about 3. This discrepancy could be due to the high ATPase activity found in prostatic and seminal fluid. Uncritical use of the NADP+ assay might account for different seminal creatine kinase values reported in the literature. Interrelationships between ATPase, creatine kinase and zinc suggest that seminal ATPase is a prostatic secretory product while creatine kinase may be multiglandular in origin.     

Male mating rate is constrained by seminal fluid availability in bedbugs, Cimex lectularius

Sexual selection, differences in reproductive success between individuals, continues beyond acquiring a mating partner and affects ejaculate size and composition (sperm competition). Sperm and seminal fluid have very different roles in sperm competition but both components encompass production costs for the male. Theoretical models predict that males should spend ejaculate components prudently and differently for sperm and seminal fluid but empirical evidence for independent variation of sperm number and seminal fluid volume is scarce. It is also largely unknown how sperm and seminal fluid variation affect future mating rate. In bedbugs we developed a protocol to examine the role of seminal fluids in ejaculate allocation and its effect on future male mating rate. Using age-related changes in sperm and seminal fluid volume we estimated the lowest capacity at which mating activity started. We then showed that sexually active males allocate 12% of their sperm and 19% of their seminal fluid volume per mating and predicted that males would be depleted of seminal fluid but not of sperm. We tested (and confirmed) this prediction empirically. Finally, the slightly faster replenishment of seminal fluid compared to sperm did not outweigh the faster decrease during mating. Our results suggest that male mating rate can be constrained by the availability of seminal fluids. Our protocol might be applicable to a range of other organisms. We discuss the idea that economic considerations in sexual conflict research might benefit from distinguishing between costs and benefits that are ejaculate dose-dependent and those that are frequency-dependent on the mating rate per se.     

An ectopic expression screen reveals the protective and toxic effects of Drosophila seminal fluid proteins

In Drosophila melanogaster, seminal fluid regulates the reproductive and immune responses of mated females. Some seminal fluid proteins may provide protective functions to mated females, such as antimicrobial activity and/or stimulation of antimicrobial gene expression levels, while others appear to have negative effects, contributing to a "cost of mating." To identify seminal proteins that could participate in these phenomena, we used a systemic ectopic expression screen to test the effects on unmated females of proteins normally produced by the male accessory gland (Acps). Of the 21 ectopically expressed Acps that we tested for ability to assist in the clearance of a bacterial infection with Serratia marcescens, 3 Acps significantly reduced the bacterial counts of infected females, suggesting a protective role. Of the 23 Acps that we tested for toxicity, 3 were toxic, including one that has been implicated in the cost of mating in another study. We also tested ectopic expression females for other Acp-induced effects, but found no additional Acps that affected egg laying or receptivity upon ectopic expression.