Schmallenberg virus detection in bovine semen after experimental infection of bulls.

To study Schmallenberg virus (SBV) excretion in bovine semen after experimental infection, two bulls were inoculated subcutaneously with a SBV isolate (1 ml Vero cell culture 106 TCID50). After inoculation (at day 0), semen was collected daily from both animals for 21 days and samples were tested for SBV by qRT-PCR assay. At 24 days post-inoculation both animals were subjected to necropsy and the genital organs and lymph nodes draining these organs were also tested for SBV RNA (qRT-PCR). After SBV infection both animals in the study showed viraemia (qRT-PCR) with fever and diarrhoea. SBV RNA could be detected in semen from both animals. The highest SBV RNA concentrations in semen were found in the first week (days 4-7 post-inoculation) but concentrations were relatively low (Ct values 30-39). Viable SBV was only isolated from blood samples and not from semen or genital tissues.

Effects of cooling time on membrane integrity and motility of frozen-thawed canine spermatozoa using two different commercial egg yolk-based extenders at two different cooldown equilibration times.

Two commercially available egg yolk-based semen extenders, one marketed for human semen freezing (HEYE) and one marketed for canine semen freezing (CEYE), were used to cryopreserve semen from single ejaculates of 11 different dogs. For each extender, a 30- and a 60-min cooldown period was used prior to the addition of the extender containing glycerol and then immediately frozen in liquid nitrogen vapours. Sperm motility was measured using a computer-assisted semen analysis (CASA) system. Sperm intact membranes were measured using SYBER-14 and propidium iodide. Semen in the HEYE cooled for 60 min had a significantly greater percentage of intact membranes than the semen in the HEYE cooled for 30 min (p = 0.02). Semen in the HEYE cooled for 60 min had significantly greater total motility (p = 0.007) and progressive motility (p = 0.004) than semen cooled for 60 min in the CEYE and semen cooled for 30 min in the HEYE (total motility p = 0.02 and progressive motility p = 0.02). Semen cooled for 60 min in the CEYE did not differ significantly in total (p = 0.6) or progressive motility (p = 0.4) than semen cooled for 30 min in the CEYE. There was no difference in total (p = 0.8) or progressive motility (p = 0.8) between the semen cooled for 30 min in the HEYE and the semen cooled for 30 min in the CEYE.

Cefquinome concentrations in endometrium after intrauterine treatment of cobactan 4.5% in mares and inflammatory response of the endometrium to this treatment.

This study was conducted to measure the concentration of cefquinome in the endometrium of mares after intrauterine treatment and to evaluate associated inflammation. Mares (n = 14) were randomly assigned to one of the following groups: (i) control (n = 4) were either not treated (n = 2) or received (n = 2) lactated Ringer's intrauterine for 1 or 3 days; (ii) treated mares (n = 10) received intrauterine cefquinome for 1 or 3 days. After at least 10 days had passed following the last treatment and ovulation, mares were given Prostaglandin F2alpha (PGF2alpha) and were randomly assigned to an alternate treatment. Endometrial biopsy samples were taken at 2, 8, 24 and 48 h, or at 4, 12 and 36 h, after the last treatment. Biopsy samples were taken at the same time points from control mares (n = 2) and lactated Ringer-treated mares (n = 2). Cefquinome concentrations were quantified using a high-performance liquid chromatography (HPLC) assay and inflammation was assessed using haematoxylin and eosin (H&E)-stained sections. Concentrations of cefquinome [559 (1 day) and 595 microg/g (3 days) at 2 h, and 403 (1 day) and 370 microg/g (3 days) at 4 h] were similar between treatment groups at 2 and 4 h after treatment (p > 0.05). At 8 h, as well as at 24 and 48 h, concentrations were greater in the 3-day group (17 vs 301 microg/g, 3 vs 80 microg/g and 0.1 vs 0.2 microg/g, respectively) (p < 0.05). No significant differences (p > 0.05) in the inflammatory response at 2-48 h after treatment were found between groups.

[Transvaginal aspiration as first treatment of ovarian follicular cysts in dairy cattle under field circumstances]. / Transvaginale aspiratie als primaire behandeling van ovariële follikelcysten bij melkkoeien onder praktijkomstandigheden.

On dairy farms covered by the ambulatory service of the Department of Farm Animal Health, 55 Holstein-Friesian cows with suspected ovarian follicular cysts on the basis of rectal palpation underwent confirmatory transrectal ultrasonography. An ovarian follicular cyst was confirmed in 28 cows (50.9%) and was treated by single transvaginal-guided needle aspiration. Cows with a corpus luteum or a luteinized cyst were not treated. After aspiration, 82.1% (n=23) of the cows showed oestrus behaviour at 13.3 +/- 6.0 days. Artificial insemination was performed during the first heat after cyst aspiration and resulted in a pregnancy rate of 64.2%. In conclusion, single transvaginal-guided needle aspiration of ovarian follicular cysts is an easy and good method for the treatment of follicular cysts. Moreover, it is a safe and good alternative method for the manual, active rupturing of cysts during rectal palpation.

[Rectal tears in the horse: malpractice or an unfortunate accident?]. / Rectumscheuren bij het paard: beroepsfout ofeen ongelukkig toeval?

Rectal tears are a relatively rare complication of rectal palpation, mating or dystocia, and idiopathic spontaneous occurrence of rectal perforation has also been described. Rectal tears have been classified in a three or a four-grade system. Immediate recognition of the fact that a tear has been made and prompt action will improve the horses chance of survival, and is the best defence of the veterinarian against legislation. There is no clear explanation why rectal tears sometimes occur. A questionnaire of the Netherlands Equine Veterinary Association revealed that rectal tears occurred both to inexperienced veterinarians and to very experienced colleagues, working with or without a probe for ultrasonography. In the opinion of the authors, the approach in the legislation of rectal tears should be that a rectal tear is only considered 'malpractice' if the veterinarian obviously acted carelessly. However, this would create the ridiculous situation that it seems better for the veterinarian-client relationship when the veterinarian confesses to 'careless' rectal palpation. This point needs further clarification, and perhaps an 'insurance of the horse for rectal palpation' will be the solution. Further, retrospectively over the last ten years, the problem is often not 'making a rectal tear', but 'lacking recognition of making a tear'. Whenever a rectal tear is suspected, the veterinarian must assess its severity, discuss the problem openly with the owner and apply appropriate treatment including referral.

[Enlarged ovary in a mare: review of the literature and a case report]. / Een vergroot ovarium bij de merrie, een literatuuroverzicht aan de hand van een casus.

Mares regularly have an enlarged ovary. The main causes are haematomas, anovulatory follicles, abscesses, and neoplasia. The granulosa-theca-cell tumour is by far the most common neoplasia of the ovary (about 97%) and accounts for 2.5% of all equine tumours. In this article the differential diagnosis of an enlarged ovary and the background of granulosa-theca cell tumours are reviewed. A case is described of a mare with a very large granulosa-theca cell tumour in the left ovary, which was discovered 1 month after delivery of a healthy foal. This case is special not only because the tumour was enormous (diameter 60 cm) but also because the contralateral ovary was functional and there were no behavioural changes. The fact that a haematoma had been found 2 years earlier made the diagnosis even more difficult.

Effect of GnRH and hCG administration on plasma LH and testosterone concentrations in normal stallions, aged stallions and stallions with lack of libido.

Gonadotrophin-releasing hormone (GnRH) (a single intravenous injection with 0.042 mg busereline acetate) was administered to control stallions (n=5), aged stallions (n=5) and stallions with lack of libido (n=5). Jugular blood samples were taken at -10, 0, 10, 20, 40 and 80 minutes after treatment and measured for luteinizing hormone (LH) and testosterone concentrations. A single intravenous injection of hCG (3000 IE) was given 1 day later. Venous blood samples were taken at -60, 0, 15, 30, 60, 120, and 240 minutes after treatment and measured for the testosterone concentration. The experiment was performed in the breeding season. There was a wide variation between stallions in basal concentrations of LH and testosterone. The treatment groups all showed a significant increase in LH and testosterone concentrations after treatment with GnRH. There was a significant difference (P<0.05) between the control, the lack of libido stallions and the aged stallions in the production of LH before and after stimulation with GnRH. The aged stallions had higher basal LH concentrations. GnRH induced a rise in plasma LH in all groups, but the greatest response was observed in aged stallions. No response to GnRH was seen with respect to plasma testosterone. There was an increase in plasma testosterone following hCG; however, this increase was very small in aged stallions. After stimulation with hCG the control and lack of libido stallions had a significant increase (P<0.05) in testosterone production. In conclusion, stimulation with either GnRH or hCG can be a valuable method to test whether the function of the stallion's reproductive endocrine system is optimal.

Prediction of first season stallion fertility of 3-year-old Dutch Warmbloods with prebreeding assessment of percentage of morphologically normal live sperm.

In the selection procedure to acquire a breeding licence, 3-year-old Dutch Warmblood stallions have to undergo a breeding soundness test It is questioned whether this evaluation is predictive of the stallion's fertility results in the first breeding season. Therefore, semen parameters at the beginning of their first breeding season were evaluated and correlated to nonreturn at first cycle and foaling rate of mares bred by stallions (n = 13). The total number of mares inseminated with chilled semen from those stallions was 1055. Semen parameters were recorded on 2 ejaculates, collected 1 h apart. Percentage progressive sperm motility, % morphologically normal from unstained spermatozoa (MNA), % sperm cells with abnormal acrosomes and the total number of spermatozoa were correlated with first cycle nonreturn rate and foaling rate. Mean motility at evaluation was 72 +/- 6%. Mean MNA was 62 +/- 13%. Mean first cycle nonreturn rate and foaling rate were 58 +/- 15% and 69 +/- 12%, respectively. A significantly positive correlation (P<0.05) was found between the MNA and first cycle nonreturn rates. Foaling rates were not significantly correlated with semen characteristics and first cycle nonreturn rates. In conclusion, the breeding soundness test is of predictive value for the breeding results in the breeding season following the test. First cycle nonreturn rates reflect fertilising capacity better than foaling rates.

Reproductive parameters of miniature stallions.

Breeding soundness evaluation (BSE) of stallions is a routine component of stud farm practice. Guidelines for assessing satisfactory breeding potential have been developed using data derived from stallions of full-size breeds. In view of the increasing popularity of miniature stallions, knowledge of normal semen parameters of these stallions is important. Therefore, testicular measurements and semen parameters from 216 sexually rested miniature stallions were obtained. Semen was collected twice, 1.5 to 3 h apart, using an artificial vagina. Values were averaged over the 2 collections because of the sexual inexperience of the stallions. The smaller stallions (Group A, 72 to 86 cm; Group B, 87 to 96 cm) had smaller testicles (P<0.05), and Group A stallions had the lowest ejaculate volume (P<0.05) compared with Group C (97 to 104 cm) stallions. Thus, although there was no difference in the concentration of spermatozoa per milliliter between groups of stallions, Group A stallions had fewer total spermatozoa in their ejaculate than Group C stallions (4.31+/-0.47x10(9) vs. 5.41+/-0.30x10(9), P<0.05). Moreover, miniature stallions had smaller testicles and fewer total spermatozoa in their ejaculate than is commonly accepted as normal in full-size stallions. Average total scrotal width of miniature stallions was found to be 7.13, 7.38 and 7.95 cm for Groups A, B and C, respectively. The average total number of spermatozoa in the ejaculates of miniature stallions in this study was 4.94+/-0.22x10(9) cells, with 1.75+/-0.09x10(9) total normal, motile spermatozoa. When only stallions <96.5 cm in height were considered (conforming to requirements of the American Miniature Horse Association Registry), the average total number of spermatozoa in the ejaculates was 4.59+/-0.30x10(9) cells, with 1.70+/-0.11x 10(9) total normal, motile spermatozoa. Based on these findings, different criteria should be used to evaluate the potential breeding soundness of miniature stallions than are commonly applied to full-size stallions.

[Clinical aspects of fertility in stallions]. / Klinische aspecten van de fertiliteit van de hengst.

The studies described in this thesis investigated the factors that can affect the fertility of stallions. The introduction describes the male gamete and the processes that occur during maturation of sperm and fertilization. Methods to evaluate the quality of sperm and ova are then discussed. Fertility can be expressed in various ways and is also affected by many factors such as the stallion, the mare and management factors. The fertility of stallions is usually assessed a good year after they have served mares, because then the number of foals is known. However, it would be preferable to be able to predict a stallion's fertility before he is put to stud. To this end, it was investigated whether there is a relationship between a number of sperm parameters and the percentage of non-return after the first cycle. The endocrine control of reproduction is briefly described because hormonal factors can also influence the reproductive potential of stallions. The venereal diseases that are important for regulations concerning the international trade in sperm are also described. The relevance of this diseases to reproduction deserves further investigation. The minimum requirement for a stud stallion is that the stallion should produce sperm of adequate quality and should be able to fertilize mares.

Epidemiologic aspects of Taylorella equigenitalis.

Contagious equine metritis (CEM) is a sexually transmissible disease in mares. Although the disease is commonly diagnosed by culturing the causative bacterium Taylorella equigenitalis (T. equigenitalis) . false negative results do occur. A recently developed Polymerase Chain Reaction (PCR) assay, however, appeared to be much more sensitive, with initial results indicating an unexpected high incidence of the agent in selected horses. In this study, samples from 107 randomly selected mares with no clinical signs of CEM submitted for conventional culture were all negative for T. equigenitalis . but in the PCR-assay 54 (49%) were positive for Taylorella -DNA. Positives in the PCR-assay were found in all breeds tested, even in horses imported from the isolated population in Iceland. These findings suggest that T. equigenitalis was present long before it was first isolated in 1977, The high incidence of Taylorella in horse populations without apparent clinical signs of CEM, the occurrence of incidental clinical case and the known variability between strains, all indicate that Taylorella is endemic in the horse population. In order to explore whether the organism is present in species other than the horse, we also used the PCR-assay on clinically health donkeys (n = 14), zebras (n = 15), Przewalski horses (n = 2) and cows (n = 21). All the animals showed negative results except one of the Przewalski horses, and one cow that was repeatedly found to give positive reaction. We also found that the fertility of 7 stallions with cultures positive for Taylorella (6 used in an AI-program and 1 by natural breeding) was not affected, as shown by the normal range of foaling rates in mares inseminated or bred by these stallions. The overall results may be interpreted to mean that Taylorella is of limited significance in horse breeding.

Reproductive characteristics and semen quality in maiden Dutch Warmblood stallions.

The semen characteristics and testicular size of 398 3-year-old maiden Dutch Warmblood stallions were studied during February and March. Mean values (+/- SD) of age (1030 +/- 88 days) and testicular size (9.8 +/- 0.9 cm) of the maiden stallions were determined as well as the following semen characteristics (mean of two ejaculates, taken 1 h apart): volume (65 +/- 26 ml), sperm concentration (2.061 +/- 1.685 x 10(8) ml-1), total number of spermatozoa (1.129 +/- 0.71 x 10(10)), percentage of progressively motile spermatozoa (68 +/- 9%), percentage of live spermatozoa with normal morphology (66 +/- 15%), total number of progressively motile morphologically normal spermatozoa (5.307 +/- 3.833 x 10(9)) and pH (7.5 +/- 0.2). The morphology of the spermatozoa was studied and data on the most common abnormalities are presented. The first and second ejaculates show significant differences (P < 0.05) in volume, motility, number of spermatozoa, sperm morphology, total number of progressively motile morphologically normal spermatozoa and pH. The original breed has been split up into two different lines. There was a significant difference (P < 0.05) in semen characteristics between the types (riding and carriage horses). The semen quality and quantity of semi-siblings were compared and a significant sire effect (P < 0.05) was shown for the following characteristics: volume, motility (%) and sperm concentration. These results indicate that there are differences of heritability of those factors, which makes it possible to select on semen quality.

Development and evaluation of PCR test for detection of Taylorella equigenitalis.

A PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis, was developed and evaluated. A genus-specific primer-probe set was derived from the 16S ribosomal DNA sequences. The PCR was specific and amplified a 585-bp product from all 64 available T. equigenitalis isolates. This PCR product hybridized with a specific probe in a dot spot assay. A variety of microorganisms from the genital tracts of horses or with a close phylogenetic relationship to T. equigenitalis did not yield a visible PCR product and were all negative in the dot spot hybridization assay. The results of the PCR assay were compared with those of culture by using 191 genital swabs from horses of several breeds. We demonstrate that the sensitivity of the PCR assay is superior to that of culture. The assay is most sensitive when DNA from culture plates incubated for at least 2 days is used. Of the tested samples, 1.5% were positive in the culture assay, whereas 35% were positive in the culture PCR assay. PCR-positive samples were obtained from all breeds tested. This means that many T. equigenitalis-carrying horses go unidentified by the current culturing technique. This affects current views about the spread and control of T. equigenitalis.