Detection of bovine immunodeficiency virus DNA in the blood and semen of experimentally infected bulls

Five 18- to 24-month-old bulls were inoculated with either a cell suspension containing bovine immunodeficiency virus (BIV-FL112; 3 bulls) or a BIV-free cell suspension (2 bulls). Blood and semen specimens were collected once a week for 14 weeks, and seroconversion was confirmed by indirect immunofluorescent antibody (IFA) testing. The presence of BIV in blood and semen was determined by virus isolation and/or polymerase chain reaction (PCR) assays. Antibodies to BIV were detected in the 3 experimentally infected bulls as early as day post inoculation (DPI) 17, and levels peaked at DPI 37-58. BIV was isolated from the peripheral blood mononuclear cells (MNCs) of the infected bulls at DPI 9 (2 bulls) and DPI 23 (1 bull), and could be isolated from one animal up to DPI 65. PCR analysis of MNC DNA, using BIV pol gene primers, detected virus in all three of the experimentally infected bulls from DPI 9 until the termination of the experiment at DPI 98. Efforts to isolate a significant number of non-spermatozoal cells (NSC) by gradient separation from the semen of the experimentally infected bulls were unsuccessful. Two methods for the extraction of total NSC DNA from up to 2 ml of non-extended semen were employed; however, no BIV pol fragment was amplified from these DNA preparations. Additionally, 30 bulls from artificial insemination (AI) centers were evaluated for BIV infection by PCR. No amplification products were obtained from MNC DNA from the AI submissions using primer sets for both the BIV pol and env genes.     

Seminal shedding of bluetongue virus in experimentally infected mature bulls

Sixteen bulls ranging in age from 2 to 11 years were experimentally inoculated with bluetongue virus to investigate the frequency and duration of seminal shedding of the virus. All bulls developed typical viremia, lasting 21 to 58 days, and they seroconverted 2 to 3 weeks after inoculation. Virus isolation was attempted from a total of 232 ejaculates, 163 (70%) of which were collected during the period of viremia. Bluetongue virus was not isolated from any of the ejaculates collected from 11 of the 16 bulls. Virus was isolated from 9 of 52 ejaculates collected from the other 5 bulls during the period of viremia. In no instance was virus isolated from semen without concurrent isolation from blood.     

Virus isolation from semen of bulls serologically positive for bluetongue virus

Isolation of bluetongue virus was attempted from 85 semen samples taken from 3 long-term seropositive bulls and 9 short-term seropositive bulls in an artificial breeding service unit. Two types of cell cultures susceptible to bluetongue virus were used for virus isolation. Extended sonication, centrifugation of specimens, and treatment of cell cultures with dimethyl sulfoxide and diethylaminoethyl-dextran were used to enhance virus attachment and infection of cell cultures. Virus isolation results were negative on all specimens. These results indicate that at the limits of the methods used, bluetongue virus-seropositive bulls do not have long-term latent bluetongue virus in their semen.     

Noninfectivity of semen from bulls infected with bovine leukosis virus

An opportunity for study of the potential role of semen in the transmission of bovine leukosis virus (BLV) was provided when a Jersey herd was found to be BLV-seronegative. This was a closed herd; new genetic material had been introduced by artificial insemination (AI), using semen collected and processed at 7 AI centers in the United States. Of 66 donor bulls from which semen had been collected for AI use in the herd during the 5 years the herd remained seronegative, 24 were consistently BLV-seropositive and 2 became seropositive for BLV during the study. Semen collected from the BLV-seropositive bulls accounted for 1,019 semen units, representing 48.3% of the semen purchased. The maintenance of BLV seronegativity in this herd for 5 years, when semen from BLV-seropositive bulls was used for AI, provided evidence for the lack of infectivity of BLV in bovine semen.     

Some observations on the semen of bulls persistently infected with bovine virus diarrhoea virus

As a result of screening procedures employed for animals entering the AI service, two bulls were identified as being persistently infected with bovine virus diarrhoea virus by isolation of the virus from blood. Semen was collected on two occasions from these bulls; its quality as measured by density and motility was poor. Gross abnormalities of the sperm head, termed 'collapsed' heads, were seen in 28 to 45 per cent of sperm from one bull and in 1 per cent of sperm from the other. The collapsed heads were small and the whole head or its anterior part had the appearance of a dried pea. Electron microscopy showed the defect to consist of convoluted nuclear material with membrane-bound vacuoles and invaginations containing membranous debris and lamellar structures. In the 'high incidence' bull there was a corresponding increase in enlarged sperm heads. The 'low incidence' bull had sperm with heads of similar mean size to sperm from control bulls but with an increased variance. The semen was diluted in a lactose diluent, frozen and stored in liquid nitrogen. The distribution of viral antigen was determined and virus was isolated from several fractions of the semen, both before and after processing and cryopreservation. In one animal raw semen failed to yield virus but virus was recovered after processing, suggesting that raw semen may not be suitable for the efficient detection of the virus.     

Libido, hormone concentrations in blood plasma and semen characteristics in Holstein bulls

Relationships among bull libido, serum hormone concentrations and semen characteristics were studied using 18 Holstein bulls that were 4 to 5 yr old. The hormones studied included testosterone, estradiol (E2), prolactin (PRL), LH and cortisol. Two ejaculates were collected three times per week from each bull during a 5-wk trial. During the last week of the trial, on a day semen was not collected, blood was collected from indwelling catheters every 15 min for 6 h to determine the hormonal profiles of each bull. On the following day, blood was sampled every 10 min before and after the time of semen collection. Libido factors were quantified, and semen volumes and sperm concentrations were recorded. The libido factors included reaction time to first service, latency time between the first and second semen collections, and duration of time the bull mounted the teaser prior to the first (TM1) and second (TM2) semen collection. Average reaction and latency times were correlated (r = .524; P = .026), as were TM1 and TM2 (r = .597; P = .015). Latency times were correlated with average TM2 (r = .669; P = .003). Average PRL concentrations were correlated with average latency times (r = .467; P = .05). Low libido bulls tended to have higher E2:testosterone ratios than did high libido bulls. Both PRL and cortisol concentrations increased at semen collection.     

Distribution of Mycobacterium avium subsp. paratuberculosis in organs of naturally infected bull-calves and breeding bulls

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, has particular importance in cattle due to the resulting chronic diarrhoea, weight loss, decreased production, infertility and eventual death. While faecal oral route of infection is generally recognised, reports about semen-derived infection are rare. The objective of this work was to assess whether M.a. paratuberculosis may disseminate from the gastrointestinal tract to reproductive organs, and compare this event between naturally infected bull-calves and breeding bulls. Ten bull-calves, aged 6-28 weeks and four breeding bulls were tested by serology, faecal and tissue culture, IS900 PCR and RFLP. In seven bull-calves M.a. paratuberculosis was isolated predominantly from mesenteric lymph nodes (75%); isolates from mucosa of the intestine constituted 25%. In three breeding bulls, M.a. paratuberculosis was isolated both from intestinal mucosa and mesenteric lymph nodes. Head and mediastinal lymph nodes, liver, spleen and semen of bull no. 1 (Holstein-Friesian); testes and epididymis of bull no. 2 (Piemonte); testes, epididymides and seminal vesicle of bull No. 3 (Hereford); and seminal vesicle of bull No. 4 (Simmental) tested positive by culture. Hot-start PCR revealed M.a. paratuberculosis in semen, seminal vesicle and intestinal tissue where culture isolation was difficult. Isolates from bull-calves and breeding bulls were of RFLP types B-C9 and B-C1, respectively. Bull-calves born in infected herd can be sources of infection when later used for natural mating or artificial insemination. Sub-clinically infected bulls release M.a. paratuberculosis into semen, consequently infecting the uterine environment of cows.     

Transmission of bovine viral diarrhoea virus through the semen of acutely infected bulls under field conditions

Epidemiological investigations implicated the semen of artificial insemination (ai) bulls as the only plausible source of infection with bovine viral diarrhoea virus (bvdv) in 10 Finnish dairy herds. The infection was traced back to two northern Finncattle bulls that had been transiently infected when their semen had been collected while they were in a gene bank herd containing persistently infected (pi) animals. The isolates of bvdv from the animals in the gene bank herd, from the semen of the two bulls and from a pi calf born in one of the herds using the semen belonged to a rare genetic type in Finland and, on the basis of the nucleotide sequences in the 5' untranslated region, were identical. Cross-contamination of batches of semen at the ai station and an external source of bvdv were ruled out for the recipient herds. It was concluded that bvdv infection can be transmitted through the semen of transiently infected bulls under field conditions.     

Detection and isolation of Toxoplasma gondii from fresh semen of naturally infected dogs in Southern Brazil

The aim of this study was to isolate Toxoplasma gondii and determine the viability of the parasite in fresh semen samples of clinically healthy adult dogs naturally infected. Eleven seropositive dogs with T. gondii IgG antibodies from southern Brazil were selected to confirm the presence and viability of T. gondii in fresh semen samples using in vitro isolation in Vero cell culture, polymerase chain reaction (PCR) and sequencing analysis. The presence of viable T. gondii was confirmed by in vitro isolation and PCR in five semen samples. The ITS1 region of the isolated protozoa (TG S4) was amplified and sequenced. The nucleotide sequence obtained was 99% compatible with the T. gondii DNA sequences stored in the GenBank. It has been shown that T. gondii tachyzoites may be isolated in vitro from fresh semen samples of clinically healthy dogs seropositive for T. gondii.     

Demonstration of Tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls.

Portions of penis and prepuce were collected from 24 bulls with current or recent Tritrichomonas foetus infection. Epididymides were collected from seven of the bulls, and seminal vesicles and prostate were collected from four. Following immunohistochemical staining with two monoclonal antibodies (34.7C4.4 and TF1.15) prepared against T. foetus surface antigens, trichomonads were identified in sections from 15 of the bulls. Organisms were most often located in penile crypts in the midshaft and caudal regions and less often in preputial crypts. Trichomonads were not observed in sections from other genitalia or in subepithelial tissue. T. foetus antigen, however, was present in the cytoplasm of some epithelial cells and the cytoplasm of some mononuclear cells in subepithelial lymphoid aggregates and follicles. Preputial smegma was collected from 16 T. foetus-infected bulls and from 16 control bulls with negative T. foetus cultures. Preputial antibody levels to TF1.17, a surface antigen of T. foetus, were determined by an enzyme-linked immunosorbent assay. Preputial secretions from infected bulls contained specific antibody of each isotype and subisotype tested. IgG1 responses were the greatest, IgM and IgA responses were approximately equal, and IgG2 responses were low. Each isotype and subisotype response in infected bulls was significantly greater than that in the controls. These results confirm previous speculation concerning anatomical sites of infection and suggest that parasite antigen can be taken up and processed locally, resulting in deposition of specific IgG1, IgG2, IgA, and IgM antibodies in the preputial cavity.     

Effect of organic and inorganic selenium supplementation on semen quality and blood enzymes in buffalo bulls

The present study aimed to evaluate the effect of organic and inorganic selenium (Se) supplementation on semen quality and blood serum profiles of buffalo bulls. Nine mature buffalo bulls were divided into three groups: control (non‐supplemented); organic Se (10 mg Sel‐Plex®/head twice weekly) and inorganic Se (10 mg sodium selenite/head twice weekly). Semen was collected twice a week for 3 months during Se supplementation. Semen properties were evaluated from fresh ejaculate. Moreover, fructose concentration, aspartate and alanine transaminase (AST and ALT) activities, total protein and total cholesterol were assayed in seminal plasma. Additionally AST, ALT, testosterone and Se levels were determined in the blood serum. Results showed that Se supplementation significantly (P < 0.05) influences the semen parameters during 3 months of treatment. Organic Se significantly (P < 0.05) increased the percentage of viable sperms compared to inorganic Se and the control group. Fructose concentration was significantly higher (P < 0.05) in the seminal plasma of organic Se‐treated bulls. Serum testosterone and Se concentrations were significantly (P < 0.05) increased in the Se supplemented groups than the control group. In conclusion, Se supplementation improved the parameters of buffalo bull semen and more precisely, organic Se was more effective for the improvement of semen quality and some blood components than inorganic Se.     

Detection of Neospora caninum in semen of bulls

In cattle, transplacental infection is the main route of Neospora caninum transmission, but postnatal transmission by the oral uptake of sporozoite-containing oocysts shed by dogs may also be possible. Other routes of horizontal transmission, such as the venereal route, have not been investigated. In this study, we evaluated the presence of N. caninum DNA by a nested-PCR in fresh non-extended semen and frozen extended semen straws of five Holstein-Friesian bulls with naturally-acquired neosporosis. The infection status was assessed by an immunofluorescent antibody test (IFAT) and confirmed by immunoblotting (IB). Because of inhibitory components of semen, a protocol was developed to purify N. caninum DNA from bovine semen. Sporadically, N. caninum DNA was detected in non-extended fresh semen samples and frozen extended semen straws of the five seropositive bulls. In all positive samples, specific DNA was consistently found in the cell fraction of semen and not in seminal plasma. The parasite mean load in positive fresh semen samples determined by a real-time PCR was low oscillating between 1 and 2.8 parasites/ml of semen (maximum parasite load detected in one sample was 7.5 parasites/ml of semen). In parallel, another three similar but uninfected bulls acted as controls and no N. caninum DNA was amplified in any of their fresh and straw semen samples assayed. Whether venereal transmission plays a role in the spread of bovine neosporosis needs to be determined.     

Alteration in blood gases in cats naturally infected with Aelurostrongylus abstrusus

Four cats were presented with respiratory signs and first-stage larvae of Aelurostrongylus abstrusus were found in faecal samples. Anthelmintic treatment was given to the infected cats and venous blood gases were analysed during the treatment period. Blood gas analysis suggested hypoventilation and respiratory acidosis in infected cats. Hypoventilation may be the result of airway obstruction by adults and larvae in respiratory bronchioles and the alveolar canals. The blood gas values had returned close to the physiological range by two months after treatment. Assessment of respiratory acidosis may aid development of additional treatment methods in cats infected with A. abstrusus.     

Genital lesions associated with visceral leishmaniasis and shedding of Leishmania sp. in the semen of naturally infected dogs

Although visceral leishmaniasis is primarily transmitted by a biological invertebrate vector, transmission in the absence of the vector has been reported, including venereal transmission in humans. Considering the possibility of venereal transmission, we studied genital lesions in dogs naturally infected with visceral leishmaniasis and shedding of Leishmania sp. in the semen. Approximately 200 dogs were serologically tested for anti-Leishmania antibodies and divided into three groups: 1) serologically negative dogs (n = 20), 2) asymptomatic serologically positive dogs (n = 20), and 3) symptomatic serologically positive dogs (n = 20). Samples from both testes, all segments of both epididymes, prostate gland, glans penis, and prepuce were histologically evaluated and processed for immunodetection of Leishmania sp. Semen samples were obtained from 22 symptomatic serologically positive dogs and processed for detecting Leishmania DNA by polymerase chain reaction. A significantly higher frequency of inflammation was observed in the epididymes, glans penis, and prepuce of dogs with visceral leishmaniasis, which was associated with a high frequency of immunohistochemically positive tissues (up to 95% of tissues from symptomatic dogs were positive by immunohistochemistry). Leishmania DNA was detected in eight of 22 semen samples from symptomatic dogs. Together these findings indicate that genital lesions and shedding of Leishmania sp. (donovani complex) in the semen are associated with visceral leishmaniasis. Additional studies should address the possibility of venereal transmission of the disease in the dog.     

Investigations on the water intake of growing bulls

The voluntary water intake was evaluated in 62 growing Holstein bulls (mean body weight range = 193 to 550 kg) during a whole fattening period. The diet was composed of corn silage and concentrates. Water was offered for ad libitum consumption. A total of 17,772 measurements of water intake were recorded over 282 days. The average daily water consumption was 18 kg/animal (S.D. = 6.7 kg). Applying a multiple regression analysis to the data set yielded the following equation: voluntary water intake (kg/day) = − 3.85 + 0.507  average ambient temperature (°C) + 1.494  dry matter intake (kg/day) − 0.141  roughage part of the diet (%) + 0.248  dry matter content of roughage (%) + 0.014  body weight (kg). The incorporation of the variables body weight gain, relative humidity, maximum ambient temperature, Na intake, and K intake into the equation did not increase the accuracy of the prediction.