Inactivation of bovine herpesvirus-1 (BHV-I) from in vitro infected bovine semen

Frozen-thawed bovine semen, experimentally infected with bovine herpesvirus-1 (BHV-1) at levels of 10(3) TCID(50)/ml and 10(4) TCID(50)/ml, was treated with a 0.3% trypsin solution to determine the effect of trypsin on the virus and on fertilization using superovulated animals. Virus was not isolated from any trypsin-treated samples using a cell culture assay system. Nor did two calves develop antibodies to BHV-1 following inoculation with trypsin-treated semen pooled from six bulls. Nonsurgical flushing of eight heifers inseminated with trypsin-treated frozen-thawed semen yielded 28 transferable-quality embryos.     

Excretion of lumpy skin disease virus in bull semen

This work was done to establish the incidence and duration of excretion of lumpy skin disease virus (LSDV) in semen of experimentally infected susceptible bulls. Six serologically negative bulls 11-20 months of age were experimentally infected with a virulent field isolate (strain V248/93) of LSDV. Animals were observed for the development of clinical signs, blood was collected until day 90 after infection, and semen was collected every second day until day 18, then twice a week till day 63 and twice a month until three consecutive samples were negative when tested for LSDV by polymerase chain reaction (PCR). An aliquot of each sample which tested positive using PCR was inoculated onto cell monolayers for the recovery of virus. Two bulls developed severe lumpy skin disease (LSD), two bulls showed mild signs and two bulls showed a transient fever only. Multiple samples were positive on PCR from both of the severely affected bulls and one of the mildly affected bulls; between days 10 and 159, days 8 and 132, and days 10 and 21 respectively. Only one sample from each of the other three bulls was positive on PCR. Virus was only isolated from two samples from one of the severely affected bulls and from five semen samples from the other. This study confirmed the excretion of LSDV in bovine semen for prolonged periods, even when obvious clinical signs of the disease were no longer apparent.     

Amplification and characterization of bull semen infected naturally with foot-and-mouth disease virus type Asia1 by RT-PCR

To investigate the security of semen biologically, 15 bull semen samples were collected (of which 5 exhibited clinical signs of Foot-and-mouth disease) and identified by RT-PCR and virus isolation. The results indicated that the semen of the infected bulls were contaminated by Foot-and-mouth disease virus (FMDV), but FMDV was not detected in semen samples from those bulls not showing clinical signs of Foot-and-mouth disease (FMD). This is the first report of the presence of FMDV in bull semen due to natural infection in China. The analysis of the partial sequence of the VP1 gene showed that the virus strain isolated from semen has 97.9% identity with the virus isolated from vesicular liquid of infected bulls showing typical signs of FMD and belonged to the same gene sub-group. Foundation items: State Science and Technology Support Program (2006DAD06A03) and Hi-tech Research and Development Program of China 863 (2006AA10A204).     

Non-infectivity of semen from bulls infected with bovine leukosis virus

Semen and serum were obtained from four bovine leukosis virus (BLV) infected bulls from each of eight bull studs. The samples from the 32 bulls were frozen and stored in liquid nitrogen for subsequent testing. The sera were tested for antibodies to BLV by the agar gel immunodiffusion (AGID) method. Thirty of the bulls were found to be infected with BLV. Pairs of sheep were intraperitoneally inoculated with semen pools of the four bulls from each bull stud. None of the sheep developed antibodies to BLV. A later challenge with BLV infected lymphocytes resulted in the infection of all challenged sheep indicating that they were susceptible to BLV infection. The results provide evidence that transmission of BLV via leukocyte free semen from BLV infected bulls does not occur.     

Temporal localization of porcine reproductive and respiratory syndrome virus in reproductive tissues of experimentally infected boars

Porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to be shed in the semen of infected boars. To determine whether the reproductive tissues could be a persistent source of virus and the possible origin of PRRSV found in semen of infected boars, 20 PRRSV-seronegative boars were intranasally inoculated with 5 x 10(6) median tissue culture infective doses (TCID50) of PRRSV and necropsied at different times post-inoculation (p.i.) from Day 2 to Day 37 p.i. Blood samples were collected before experimental inoculation, at necropsy and at different times p.i. At necropsy, epididymal semen and reproductive tissues were collected and the presence of the virus determined by virus isolation. The infection of the boars was demonstrated by the isolation of the virus from the sera of all inoculated boars and by seroconversion. PRRSV was detected in serum samples from Day 2 to Day 23 p.i., although the viremic period was largely dependent on the individual response to infection. Viral replication was proven within different reproductive tissues from Day 2 to Day 23 p.i., being most consistently found in the epididymus. In addition, PRRSV was isolated in semen from Day 4 to Day 10 p.i. The correlation of a diminished viremia and the inability to isolate PRRSV from semen or reproductive tissues may be due to one of two possibilities. First, viremia is responsible for most of the virus isolated from reproductive tissues due to the movement of PRRSV-infected cells out of the blood and into the tissues. Second, viremia may initially seed the reproductive tissues with PRRSV, and then the virus is produced into the reproductive tract and shed into semen at low levels.     

Comparison of electroejaculation and transrectal massage for semen collection in range and yearling feedlot beef bulls

Two experiments were conducted to compare electroejaculation (EE) and transrectal massage (RM) of the ampullary region for semen collection from beef bulls, and to determine the effect of semen collection method on semen traits. In experiment 1, semen was collected either by EE or RM randomly assigned on an alternate basis in 137 range beef bulls unaccustomed to being handled. The maximum time allowed for RM was 4 min and if no semen was obtained, EE was used. In experiment 2, semen was collected from 39 yearling feedlot beef bulls that were accustomed to being handled, by RM followed immediately by EE. The maximum time allowed for semen collection by both methods was 4 min. In both experiments, sperm concentration, percent of progressively motile sperm, percent of sperm staining alive, and sperm morphology were determined. In experiment 1, RM resulted in fewer (P<0.001) successful semen collections and fewer bulls with penile protrusion than EE (80.9% versus 100% and 54.4% versus 91.5%, respectively). The success of RM was not influenced by bull age or breed, or by the veterinarian performing the massage. Transrectal massage required more time (30s, P<0.001) for obtaining a semen sample and resulted in samples with lower sperm concentration (P<0.001), percent motile sperm (P<0.05) and percent live sperm (P<0.001) when compared to EE. In experiment 2, EE and RM were equally effective for obtaining a semen sample (97.4 and 94.9%, respectively), but the proportion of bulls exhibiting penile protrusion during semen collection was lower (P<0.0001) with RM compared to EE. Percent of sperm staining alive was also lower (P<0.01) in samples collected by RM. Sperm morphology (normal sperm, head defects, midpiece defects, proximal cytoplasmic droplets, and detached sperm heads) did not differ between samples collected by EE and RM. In conclusion, semen could be collected by transrectal massage from approximately 80% of range beef bulls and from 95% of yearling beef bulls accustomed to handling. Sperm morphology was not affected by the method of semen collection, but percent of motile sperm and live sperm were lower in samples collected by RM. A reduced ability to stimulate penile protrusion with RM precluded examination of the penis in a large proportion of bulls.     

Detection of bovine papillomavirus type 2 DNA in commercial frozen semen of bulls (Bos taurus)

Papillomaviruses are found in epithelial lesions and are linked to different carcinogenic processes in humans and other animals. Although bovine papillomavirus (BPV) has been characterized as epitheliotropic, the presence of viral DNA has been detected in other sample types, including fresh semen. The aim of this study was to evaluate the presence of BPV DNA in spermatozoa and seminal plasma samples of commercial frozen semen taken from bulls (Bos taurus) and its effects on semen function. PCR assays were conducted with specific primers to detect BPV types 1-6 in 40 semen samples of dairy Gir bulls. The semen quality was assessed by the use of parameters such as motility, vigor, acrosomal integrity and DNA integrity. BPV-2 DNA was detected in all of the sperm cell samples and all the seminal samples; however BPV-1, 3, 4, 5 and 6 could not be detected. The presence of BPV DNA was apparently not a cause of reduced sperm function. This is the first record of BPV-2 DNA the commercial frozen semen taken from dairy Gir cattle by several companies that provide semen. Further studies are needed to assess the viability of the virus and the extent to which it can be spread through semen.     

Semen and reproductive profiles of genetically identical cloned bulls

In this comparative study, reproductive parameters and semen characteristics of cloned bulls (n = 3) derived from somatic cell nuclear transfer (SCNT) were compared to their original cell donor Holstein-Friesian (n = 2) bulls from the same enterprise to assess the differences in reproductive potential between a donor bull and its clones. The parameters evaluated included motility of fresh, frozen-thawed and Percoll-treated frozen-thawed spermatozoa, as well as in vitro fertilization (IVF) ability, embryo quality, birth and survival of calves following IVF and embryo transfer with frozen-thawed semen. With fresh semen, spermatozoa from one cloned bull had lower motility than its donor. Cloned bulls had higher velocity parameters in fresh semen, but those effects were not obvious in frozen-thawed or frozen-thawed semen selected with a Percoll gradient. Semen collected from cloned bulls had significantly higher IVF rates compared to donors; however, embryo development per cleaved embryo or quality of blastocysts did not differ between donors and cloned bulls. Pregnancy and live offspring rates from one donor and its cloned bull did not differ between fresh (40%, 16/40 versus 46%, 17/37) and vitrified/thawed (13%, 2/16 versus 25%, 4/16) embryo transfer following IVF. A total of 26 calves were obtained from genotypically identical donor and cloned bulls with no signs of phenotypical abnormalities. These preliminary results suggested that the physiology of surviving postpubertal cloned bulls and quality of collected semen had equivalent reproductive potential to their original cell donor, with no evidence of any deleterious effects in their progeny.     

Effect of age and environmental factors on semen quality, glutathione peroxidase activity and oxidative parameters in simmental bulls

Taking into account that semen quality depends on animal age and climate conditions and that oxidative stress has been reported to be a common cause of infertility, the objective of this study was to monitor indicators of oxidative stress and antioxidant protection during four seasonal periods in service bulls of various age to get better insight into the significance of these factors upon evaluating service bull semen. The research was conducted over a year on 19 Simmental service bulls. Animals were divided into two groups according to age; Group I consisted of younger bulls aged two to four yrs (n = 9), and Group II was comprised of older bulls aged five to ten yrs (n = 10). Semen samples were obtained once in the middle of every seasonal period and blood samples for biochemical analysis were collected by jugular venipuncture immediately after ejaculate collection. The activity of total glutathione peroxidase (T-GSH-Px), selenium-dependent glutathione peroxidase (Se-GSH-Px) and selenium-independent glutathione peroxidase (non-Se-GSH-Px), together with the intensity of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) and oxidative protein damage (protein carbonyl content (PCC)) were measured in seminal plasma. In samples of spermatozoa and blood serum, the activity of Se-GSH-Px and TBARS and PCC concentrations were determined. Older service bulls had significantly higher ejaculate volume in summer in comparison with younger bulls, whereas the number of spermatozoa and progressive motility percentage did not significantly vary with age. Younger animals had lower progressive motility percentage during summer than in spring, with more intensive oxidative processes observed in seminal plasma (TBARS) and spermatozoa (TBARS and PCC). Based on the results presented here, it can be concluded that younger bulls are more sensitive to elevated ambient temperatures during the summer, when intensified prooxidative processes in semen plasma and spermatozoa eventually led to decreased sperm progressive motility with consequential semen quality deterioration.     

Two-dimensional polyacrylamide gel electrophoresis of bovine seminal plasma proteins and their relation with semen freezability

The objective of this study was to evaluate the low weight (10-30 kDa) protein profile of bovine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine if any of these proteins was associated with semen freezability. Seminal plasma was collected from 16 bulls of high or low semen freezability. Twelve protein spots were identified from the 2D gel (15%); six of these were present in all samples. Of the 12 proteins found, three spots, present in all samples, 3 (15-16 kDa), 5 (16-17 kDa), and 7 (10-12 kDa) had nonsignificant variation among bulls, regardless of their freezability classification. Four proteins were more abundant (P<0.05) in seminal plasma samples collected from bulls with high semen freezability than in samples of bulls with low semen freezability: the spots 3 (15-16 kDa, pI 4.7-5.2), 7 (11-12 kDa, pI 4.8-4.9), 11 (13-14 kDa, pI 4.0-4.5), and 23 (20-22 kDa, pI 4.8-5.2). On the other hand, spot 25 (25-26 kDa, pI 6.0-6.5) was more abundant (P<0.05) on seminal plasma samples from bulls with low semen freezability. The N-terminus sequence of protein 7 was identical to the acidic seminal fluid protein (aSFP). Protein 23 (after trypsin digestion) had structural similarity to bovine clusterin. We concluded that there were differences in the seminal plasma protein profile from bulls with low and high semen freezability; aSFP, clusterin, proteins 3 and 11 may be used as semen freezability markers; and protein 25 was related to low semen freezability.     

Evaluation of duck egg yolk for the cryopreservation of Nili-Ravi buffalo bull semen

This study was carried out to investigate if the substitution of chicken egg yolk (CEY) with duck egg yolk (DEY) in extenders can improve the quality of frozen-thawed semen of Nili-Ravi buffalo bulls and to study if reducing DEY level in extender affects the freezability results. Thirty semen samples collected from three buffalo bulls were diluted in extenders A, B, C, D and E containing tris, citric acid, fructose, egg yolk, glycerol and antibiotics. Extender A contained 20% CEY (control), while extenders B, C, D and E contained 5, 10, 15 and 20% DEY, respectively. After freezing and storage for 24h in liquid nitrogen, samples were evaluated for post-thaw quality. The post extension sperm motility did not differ between extenders A (control) and E (20% DEY). The same was true for post-thaw percentage of sperm with functional plasma membrane and percentage of sperm with abnormal heads or mid pieces. However, extender E showed higher (P<0.05) values for post-thaw sperm motility, livability and absolute index of livability of spermatozoa at 37 °C compared to extender A. Spermatozoa with abnormal tail were lower (P<0.05) in extender E compared to extender A. Values of these parameters of post-thaw semen quality were highest for extender E containing 20% DEY and decreased significantly with decrease in the concentration of DEY, except sperm abnormalities (head, mid-piece and tail) which increased with decrease in DEY level. These results showed that replacement of 20% CEY with 20% DEY in extenders significantly improved post-thaw sperm motility, livability and absolute index of livability of spermatozoa and reduced tail abnormalities. Reduction in the level of DEY in extenders from 20% adversely affected post-thaw semen quality of Nili-Ravi buffalo bulls.     

Occasional detection of Neospora caninum DNA in frozen extended semen from naturally infected bulls

Recently, the presence of Neospora caninum DNA in semen from naturally infected bulls was reported. In the present work, the presence and quantification of N. caninum by PCR techniques in frozen extended semen straws from naturally infected bulls was investigated. A total of 20 seropositive and five seronegative bulls raised for reproductive purposes in an AI centre were used. Ten extended semen straws from each bull obtained at different time-points during the previous 2 years were selected for Neospora testing. Eight of the seropositive bulls (40%) studied showed at least one positive straw to N. caninum DNA and 14 of their 180 semen straws examined (7.8%) were found to be positive. In all positive samples, N. caninum DNA was consistently detected in the cell fraction and not in the seminal plasma. However, the parasite number in each positive straw was under the detection level of real-time PCR. In parallel, 10 semen straws from each of the five seronegative bulls were also analyzed by the nested-PCR and no N. caninum DNA products were obtained. In order to check the consistent presence of N. caninum in a positive semen batch, three additional semen straws from the same batch of each positive straw from three seropositive bulls were analyzed but N. caninum DNA was only detected in one straw from one bull. In conclusion, we report the sporadic detection of N. caninum DNA in semen straws of naturally infected bulls but the low frequency of contaminated semen straws and the low parasite load observed indicate a minor chance of bovine neosporosis transmission by AI.     

Pyridine nucleosidase in bull semen. II. Biochemical properties.

It is most likely a single enzyme (NAD+ nucleosidase) present in semen from most bulls which hydrolyses the ribosyl pyridinium bond in both NAD and NADP. This conclusion is based on the following results: (i) each of 12 semen samples containing nucleosidase activity hydrolysed NAD at the same rate as NADP (r = 0.99); (ii) other untreated semen samples from different bulls which did not hydrolyse NAD were also inactive against NADP; (iii) enzyme denaturation produced by preliminary heating of semen filtrates for 15 min at varied temperatures or by heating at 55 degrees C for varied time intervals caused similar reductions in the rates of NAD and NADP hydrolysis; and (iv) nicotinamide inhibited enzyme activity to the same degree using either NAD or NADP as the substrate.     

Embryo production from superovulated Holstein-Friesian dairy heifers and cows after insemination with frozen-thawed sex-sorted X spermatozoa or unsorted semen

The aim of this study was to evaluate embryo production in superovulated Holstein-Friesian dairy heifers and cows inseminated with either X-sorted spermatozoa (2 million/dose) or unsorted semen (15 million/dose). Experiment 1 at the research farm involved eight heifers, six cows and semen of one Holstein bull. All transferable embryos were diagnosed for sex. Experiment 2 included embryo collections on commercial dairy farms: X-sorted spermatozoa from three Holstein bulls were used for 59 collections on 28 farms and unsorted semen from 32 Holstein bulls were used for 179 collections on 79 farms. Superovulations were induced by eight declining doses of FSH (total of 12 ml for heifers and 19 ml for cows) starting on days 8-12 of the estrus cycle. Inseminations began 12h after the onset of estrus and were performed two to four times at 9-15 h intervals. Low-dose X-sorted inseminates were deposited into uterine horns and unsorted semen was placed into the uterine body. In Experiment 1, on average 70.3 and 75.0% of embryos recovered from heifers, and 48.4 and 100% of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively. The proportion of transferable female embryos produced approximately doubled when insemination was with X-sorted spermatozoa compared to insemination with unsorted semen (heifers 96.4% versus 41.1%; cows 81.1% versus 39.8%). In Experiment 2, estimated 53.9 and 65.5% of embryos recovered from heifers, and 21.1 and 64.5% of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively. Proportions of unfertilized oocytes were 21.1 and 10.6% for heifers and 56.0 and 14.4% for cows in X-sorted and unsorted groups, respectively. Consequently, cows inseminated with X-sorted spermatozoa produced significantly smaller proportions of transferable embryos (p<0.005) and significantly larger proportions of unfertilized oocytes (p<0.001) than those inseminated with unsorted semen. Proportions of quality 1 or degenerated embryos were similar for the two treatments in both heifers and cows. Within treatments, bulls did not significantly affect the proportions of transferable, unfertilized or degenerated oocytes/embryos. It was concluded that using low-dose X-sorted spermatozoa rather than normal-dose unsorted semen for the insemination of superovulated embryo donors can improve the proportion of transferable female embryos produced but this potential may not be achieved in commercial practice, particularly in cows, because of reduced fertilization rates when using low doses of X-sorted spermatozoa.     

Effect of site of deposition on the fertility of sheep inseminated with frozen-thawed semen

The widespread use of artificial insemination (AI) in sheep is currently prevented due to the lack of a cost effective insemination technique utilising frozen-thawed semen. The objective of the present study was to determine if the deposition of frozen-thawed semen in the vaginal fornix would result in a pregnancy rate comparable to that achieved following cervical insemination. Multiparous ewes of various breeds were synchronised and inseminated into either the vaginal fornix (n=78) or the cervix (n=79), at 57 h post sponge removal, with frozen-thawed semen. Information on mucus secretion and the depth to which it was possible to penetrate the cervix at insemination (cervically inseminated ewes only) was recorded at the time of AI. Pregnancy rate was subsequently determined either by return to service (oestrus) or after slaughter 30 days post insemination. Insemination site did not significantly influence pregnancy rate using frozen-thawed semen (36.2% compared to 27.6% for cervical and vaginal fornix insemination, respectively; P=0.26). Whilst depth of cervical penetration was positively associated with pregnancy rate (P<0.05), this association needs to be interpreted with caution as none of the ewes where the cervix could not be penetrated (score=0) was pregnant. In conclusion, pregnancy rate following insemination of frozen-thawed semen into the vaginal fornix was within 10% points of that obtained following cervical AI of frozen-thawed semen. As insemination into the vaginal fornix is technically easier than cervical insemination, it may be more practical for use in large scale applications.     

Applications of sexed semen in cattle production

Sexed semen will contribute to increased profitability of dairy and beef cattle production in a variety of ways. It could be used to produce offspring of the desired sex from a particular mating to take advantage of differences in value of males and females for specific marketing purposes. Commercial dairy farmers, those who produce and market milk, could use sexed semen to produce replacement daughters from genetically superior cows and beef crossbred sons from the remainder of their cow population. To increase the rate of response to selection, seedstock dairy cattle breeders could produce bulls for progeny testing from a smaller number of elite dams by using sexed semen to ensure that all of them produced a son. Using sexed semen could then reduce the cost of progeny testing those bulls, because fewer matings would be necessary to produce any required number of daughters. Commercial beef cattle farmers, producing animals for eventual slaughter, could use sexed semen to capitalize on the higher value of male than female offspring for meat production. They could also use sexed semen to produce specialized, genetically superior replacement heifers from as small a proportion of the herd as possible. This would allow the remainder of the herd to produce male calves from bulls or breeds with superior genetic merit for growth, feed conversion efficiency, and carcass merit. Single-sex, bred-heifer systems, in which each female is sold for slaughter soon after weaning her replacement daughter, would be possible with the use of X-chromosome-sorted semen. Use of sexed semen would make terminal crossbreeding systems more efficient and sustainable in beef cattle. Fewer females would be required to produce specialized maternal crossbred daughters, and more could be devoted to producing highly efficient, terminal crossbred sons.     

Individual variations in FSH release after a GnRH challenge and relationship with semen output in young bulls

Blood samples collected from 54 Montbéliarde young post-pubertal bulls were assayed for FSH. In the first trial, 0.25 mg GnRH was given to each of 25 bulls at 12 months of age. In a second group of 29 bulls, each received 20 mg dexamethasone followed by 0.25 mg GnRH 5 h later. Based on measurements of semen output, the bulls were classified into three categories: good, medium and poor semen producers. The total FSH response was significantly different among individuals (P less than 0.05) and repeatable (r = 0.45) only after the combined treatment. The mean total responses did not differ significantly between the 3 categories of semen producers, but individual FSH responses were significantly and negatively correlated to quantitative and qualitative semen production characteristics (r = 0.4-0.7). It was concluded that measurement of FSH after a combined dexamethasone-GnRH challenge permits the demonstration of a significant relationship between FSH release and semen production in individual bulls.     

Sperm accumulation in the ampullae and cauda epididymides of bulls

Bulls that appear to have an abnormality of sperm transport accumulate large numbers of senescent sperm in the excurrent ducts of the reproductive tract. Six bulls that accumulated sperm were used to determine the number of physiological ejaculations required to deplete accumulated senescent sperm, semen traits during depletion, the period of time to re-accumulate senescent sperm after depletion, the sites of sperm accumulation, and the effect of sperm accumulation on fertility during natural service. Semen was collected from three bulls (HH, PH1, and CH1) three times daily using internal artificial vaginas placed in cows in estrus. These bulls started to produce semen with >or=70% morphologically normal sperm on the third, fifth, and seventh day, respectively. The percentage of live sperm increased from 5% to 68%, 5% to 63%, and 18% to 68% in HH, PH1, and CH1, respectively. Two weeks later the same bulls were electroejaculated every second day for five electroejaculations to deplete stores of senescent sperm. Each time, electroejaculation was continued until the semen produced had a dilute appearance. The three bulls re-accumulated senescent sperm after 1 month of sexual rest. After re-accumulation of senescent sperm, the total volumes of semen in both ampullae recovered at slaughter from HH, PH1, and CH1 were 5.0, 5.0, and 9.5 ml, respectively. The volumes of semen in ampullae recovered at slaughter from two control bulls (RA and CHC) were 1.7 and 1.9 ml, respectively. The number of sperm recovered from both cauda epididymides of HH, PH1, CH1, RA and CHC was 37.3x10(9), 23.3x10(9), 15.0x10(9), 6.9x10(9), and 7.4x10(9), respectively. Bull CH1 and a fourth bull (LM) that also accumulated sperm, started to produce semen with >or=70% morphologically normal sperm, and >or=60% progressively motile sperm on the third electroejaculation after depletion of senescent sperm by repeated electroejaculations. Pregnancy rates achieved by two bulls that accumulated senescent sperm (CH2 and PH2) were less (P<0.05) during the first week of a 21-day breeding period and there was a tendency for lesser pregnancy rates at the end of the breeding period when compared with two normal control bulls. The present study indicates that bulls that accumulate senescent sperm may achieve greater pregnancy rates approximately 1 week after beginning a period of frequent ejaculation. Re-accumulation of an increased percentage of senescent sperm would likely occur after 1 month of sexual rest.     

Detection of Neospora caninum in the semen and blood of naturally infected bulls

A prospective study was designed to investigate the presence of Neospora caninum in semen and blood of eight bulls seropositive to N. caninum using nested-PCR procedures. Positive semen and blood samples were bioassayed in a BALB/c nu/nu mouse model. Specific anti-N. caninum serological and interferon-gamma (IFN-gamma) responses were also studied. In parallel, five seronegative bulls acted as non-infected controls. All bulls were located in a collaborating AI centre and monitored for 22 weeks. Six of eight seropositive bulls showed N. caninum DNA in their semen and/or blood samples at some time during the course of the study. In all positive semen samples, we consistently found Neospora-DNA in the cell fraction and not in seminal plasma. Parasite load, as determined by a real-time PCR in nested-PCR positive semen samples, ranged from 1 to 10 parasites/ml. We found no association between the presence of N. caninum DNA in semen and blood. N. caninum could not be detected in the BALB/c nu/nu mice inoculated with PCR-positive semen or blood samples. Specific IgG antibody levels in seropositive bulls fluctuated over time, at times falling below cut-off level. The response was predominantly IgG2, with significant differences compared to control bulls (P < 0.05). The overall mean specific IFN-gamma response in seropositive bulls was also higher than those observed in the control group (P < 0.05), although extensive variation in individual responses was observed among bulls and over time. No significant association was found between bulls showing Neospora DNA in semen, blood, or both, and specific IgG, IgG1, IgG2, IgM and IgA levels or IFN-gamma response. This study is the first to report the presence of Neospora DNA in semen and blood of naturally-infected bulls. Our observations indicate intermittent presence of N. caninum in blood and semen and shedding in semen in low numbers.