Axel Wehrend

Ciliary Transport, Gamete Interaction, and Effects of the Early Embryo in the Oviduct: Ex Vivo Analyses Using a New Digital Videomicroscopic System in the Cow

Abstract Using a digital videomicroscopic analysis system in the bovine, we showed that the mechanisms of transport caused by ciliary beating are distinctly different in ampulla and isthmus of the oviduct. The average particle transport speed (PTS) in the oviduct (mean, 133 μm/sec) does not differ in the cycle (metestrus) and during pregnancy after implantation, but it is locally modulated at the site of the embryo. Using videomicroscopy, we were able to document that after entering the ampulla, the cumulus-oocyte complex (COC) is not transported by ciliary beating down the oviduct, but firmly attaches to the ampullar epithelium. This attachment is mediated by the cumulus cells. However, when a COC is degenerated, it is floating in the oviductal lumen. As soon as a vital COC is in the ampulla, the sperm bound in the sperm reservoir of the ampullar isthmic junction leave the reservoir and hurry to the oocyte. When a sperm has penetrated the zona pellucida, the COC detaches and continues its migration. Quantitative measurements showed that the early embryo is able to locally downregulate PTS during its migration down the oviduct. It locally changes the pattern of vascularization and induces the formation of secretory cells. Our studies imply that the oviductal epithelium is able to select vital oocytes. The early embryo is able to induce the formation of secretory cells, modify vascularization, and downregulate speed of transport, thus creating the prerequisite for the first embryo-maternal communication in the oviduct.

Kinetics of meiotic progression, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes: species specific differences in the length of the meiotic stages

The kinetics of nuclear maturation, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes were examined. A further objective was to determine the duration of the meiotic stages during the maturation process. Porcine and bovine cumulus-oocyte complexes (COCs) were incubated in TCM 199 supplemented with 20% (v/v) heat inactivated fetal calf serum (FCS), 0.05microg/ml gentamycin, 0.02mg/ml insulin, 2.5microg/ml FSH and 5microg/ml LH. COCs were removed from the culture media in hourly intervals starting immediately after recovery from the follicle until 24 (bovine) or 48h (porcine) of culture. Oocytes were either fixed to evaluate the maturation status or the activity of MPF, assessed by its histone H1 kinase activity, and MAP kinase were determined by a radioactive assay simultaneously. In oocytes of both species, the MPF activity oscillated during the culture period with two maxima corresponding with the two metaphases: between 27-32 and after 46h (porcine) and between 6-9 and after 22h (bovine). There was a temporary decline in activity after 33-38 (porcine) and after 19h (bovine), which corresponded with anaphase I and telophase I. MAP kinase activity increased during the whole culture period and reached maximum levels after 47 (porcine) and after 22h (bovine). In porcine oocytes, the MAP kinase was activated before GVBD and MPF activation. In bovine oocytes, MPF and MAP kinase were activated at approximately the same time as the GVBD (8-9h of incubation). In average porcine, oocytes remain 23.4h in the germinal vesicle (GV) stage (13h in GV I, 5.7h in GV II, 3.2h in GV III and 1.5h in GV IV), 0.9h in diakinese, 9.6h in the metaphase I, 2.8h in anaphase I and 1.9h in telophase I of the first meiotic division. In bovine oocytes, the temporal distribution of the meiotic stages were 8.5h for the GV stage, 1.2h for diakinese, 8.3h for metaphase I, 1.6h for anaphase I and 1.9h for telophase I. These results indicate that the duration of the meiotic stages differs between the species and that MAP kinase is activated before MPF and GVBD in porcine oocytes.

Mammary Tumor Recurrence in Bitches After Regional Mastectomy

OBJECTIVES To investigate the histologic diagnosis and incidence of new mammary tumor growth in the remaining mammary chain tissue after regional mastectomy. STUDY DESIGN Prospective clinical study. ANIMALS Female dogs (n=99) that had excision of a single mammary tumor. METHODS Female dogs that had regional mastectomy to remove a single tumor were followed for >or=1 year postoperatively. Data regarding tumor type, tumor recurrence, and development of metastasis were recorded. RESULTS Fifty-seven (58%) dogs developed a new tumor in the ipsilateral mammary chain after the 1st surgery; 77% had repeat surgery. There was no significant correlation between the time to new tumor development and the histologic diagnosis for the 1st and 2nd tumor types. In 31 dogs, the histologic diagnosis for initial and subsequent tumors was identical and there was a significant correlation such that dogs with an initial malignant tumor are likely to develop another malignant tumor (P=.0089). The histologic classification of the new tumor was likely to be malignant if it was located close to the side where the initial tumor had been removed (P=.026). CONCLUSIONS Our results show that 58% of dogs developed a new tumor in the remaining mammary glands of the ipsilateral chain after regional mastectomy for removal of a single tumor. CLINICAL RELEVANCE This should be taken into account when deciding on the surgical management (radical or regional mastectomy) in dogs with single mammary tumors.

Treatment of feline mammary fibroadenomatous hyperplasia with a progesterone-antagonist.

KIMBERLIN, R. H. (1993) A scientific evaluation of research into bovine spongiform encephalopathy. A Consultation on BSE with the Scientific Veterinary Committee of the Commission of the European Communities, Brussels, Belgium, September 14 to 15, 1993. Eds R. Bradley, B. Marchant. Dordrecht, Kluwer. pp 455-472 SCOTT, M. R., WILL, R. G., IRONSIDE, J., NGUYEN, H-O., TREMBLAY, P., DEARMAND, S. J. & PRUSINER, S. B. (1999) Compelling transgenetic evidence for transmission of bovine spongiform encephalopathy prions to humans. Proceedings of the National Academy ofSciences 96, 15137-15142 TAYLOR, D. M., FRASER, H., McCONNELL, I., BROWN, D. A., BROWN, K. L., LAMZA, K. A. & SMITH, G. R. A. (1994) Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie. Archives of Virology 139, 313-326 WILL, R. G., IRONSIDE, J. W., ZEIDLER, M., COUSENS, S. N., ESTIBEIRO, K., ALPEROVITCH, A., POSER, S., POCCHIARI, M., HOFMAN, A. & SMITH, P. G. (1996) A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347, 921-925

Clinical efficacy of a GnRH-agonist implant containing 4.7 mg deslorelin, Suprelorin®, regarding suppression of reproductive function in tomcats

The aim of the present study was to test for the efficacy of a slow release GnRH-agonist implant (4.7 mg deslorelin, Suprelorin) in the male cat. Ten toms were implanted sc in the neck. Changes in testosterone (T) secretion, testicular size, body weight and behaviour (mounting, mating, urine marking) were monitored. T concentrations were significantly decreased (P < 0.0001) to basal levels (< 0.1 ng/mL) in 5 of 10 cats after 4 weeks and in all but one tom after 11 weeks (T < 0.1 ng/mL). In this respective tom only partial downregulation with T-values from 0.2 to 0.1 ng/mL was achieved until week 27. In weeks 28 and 32, T concentrations were below 0.1 ng/mL. Compared to pretreatment values, testicular volume was significantly decreased by about 60% in week 12 and about 73% after 36 weeks (P < 0.001). Penile spines disappeared 9.4 ± 1.0 weeks after treatment. Food intake was significantly increased during treatment period (P < 0.001). In all tomcats libido, mating behaviour and urine marking were significantly reduced (P < 0.0001) after an initial stimulation. In one tom, mating an oestrous queen on day 20 after implant administration resulted in pregnancy. Mating of another tom that had T-values between 0.1 and < 0.1 ng/mL since day 24 in week 8 revealed the presence of spermatozoa; however, this mating did not result in pregnancy. Subcutaneous implant administration was well tolerated by all tomcats without sedation or anaesthesia and no treatment related negative effects were observed. These results demonstrate the clinical efficacy of the 4.7 mg deslorelin implants (Suprelorin) in the tom inducing all castration related effects.

Treatment of queens in estrus and after estrus with a GnRH-agonist implant containing 4.7 mg deslorelin; hormonal response, duration of efficacy, and reversibility

Although slow release GnRH-agonist implants have been shown to effectively suppress the estrous cycle in queens, there are still several remaining questions about their use: if the probability and frequency of estrus induction because of initial stimulation is dependent on the stage of cycle when animals are treated, if all effects are reversible, and to what extent fertility is regained after the end of efficacy. The latter is of major interest to cat breeders who want temporary suppression of estrus in breeding animals. Twenty queens (14 with known fertility) were treated with a 4.7 mg deslorelin implant; hormonal changes (progesterone [P4], and estradiol [E2]) and behavioral changes with special respect to estrus signs and subsequent fertility were assessed. Group A cats (N = 10) were treated 3.2 ± 0.8 days after the beginning of estrus and estrus stopped 4.1 ± 2.5 days after treatment. Estrus induction was observed in one queen 6 days after treatment, and one queen showed estrous signs 138 and 155 days after treatment. Progesterone increased significantly after treatment in all animals until day 14, then slowly decreased reaching basal levels on day 56 without any further increase. Group B cats (N = 10) were treated 7 days after the end of estrus; nine cats had P4 > 1.5 ng/mL and basal E2, one cat (B10) had basal E2 and P4. In cat B10 estrus induction occurred after treatment, but in none of the others; however, E2 increased in all group B cats 1 day after treatment but reached pretreatment concentrations on Day 7 again and remained basal. The implant was still effective in one animal of the estrus group with a duration of efficacy >1102 days, in the others duration of efficacy varied between 483 and 1025 days. Eight queens were mated afterwards and gave birth to a healthy litter with 3.3 ± 1.5 kittens. This study proves that (1) the incidence of estrus induction-although very low-is highest after treatment in estrus or postestrus, (2) the duration of efficacy varies between 16 and 37 months, and (3) estrus suppression is reversible and animals remain fertile after the treatment effect has expired.

Polysialic Acid Is Present in Mammalian Semen as a Post-translational Modification of the Neural Cell Adhesion Molecule NCAM and the Polysialyltransferase ST8SiaII

Background: Polysialylated glycoproteins play an import role during numerous biological processes. Results: Polysialylated ST8SiaII and NCAM are components of mammalian semen and are partially associated with spermatozoa. Conclusion: Polysialic acid represents a further glyco-motif in mammalian ejaculates, which is known to influence the immune system. Significance: Administration of polysialic acid during insemination might be useful to increase the number of spermatozoa escaping the female immune system. Fertilization in animals is a complex sequence of several biochemical events beginning with the insemination into the female reproductive tract and, finally, leading to embryogenesis. Studies by Kitajima and co-workers (Miyata, S., Sato, C., and Kitajima, K. (2007) Trends Glycosci. Glyc, 19, 85–98) demonstrated the presence of polysialic acid (polySia) on sea urchin sperm. Based on these results, we became interested in the potential involvement of sialic acid polymers in mammalian fertilization. Therefore, we isolated human sperm and performed analyses, including Western blotting and mild 1,2-diamino-4,5-methylenedioxybenzene-HPLC, that revealed the presence α2,8-linked polySia chains. Further analysis by a glyco-proteomics approach led to the identification of two polySia carriers. Interestingly, besides the neural cell adhesion molecule, the polysialyltransferase ST8SiaII has also been found to be a target for polysialylation. Further analysis of testis and epididymis tissue sections demonstrated that only epithelial cells of the caput were polySia-positive. During the epididymal transit, polySia carriers were partially integrated into the sperm membrane of the postacrosomal region. Because polySia is known to counteract histone as well as neutrophil extracellular trap-mediated cytotoxicity against host cells, which plays a role after insemination, we propose that polySia in semen represents a cytoprotective element to increase the number of vital sperm.

Effects of long-term treatment with the GnrH agonist deslorelin (Suprelorin®) on sexual function in boars

Immunization against GnRH has been proven effective for boar taint removal, and long-term treatment with GnRH analogues has been shown to suppress GnRH dependent reproductive processes in several species. This study was conducted to treat boars (n = 5) with Suprelorin, i.e., an implant that contains 4.7 mg of the long-acting GnRH analogue deslorelin, and to test the effects on sexual function. Insertion of the implant occurred at the age of 5 weeks and animals were observed until market age at 26-27 weeks. Surgically castrated (n = 4) and intact boars (n = 3) served as controls. Testes growth was markedly reduced and steroidogenesis (testosterone, estrone, estrone sulphate, estradiol 17beta) as well as spermatogenesis suppressed in 4 of 5 GnRH treated boars, respectively. The remaining fifth boar resumed testes growth after week 17 of age and had high hormone concentrations when tested at weeks 26 and 27. Restoration of spermatogenesis was observed at 34 weeks of age. There were no effects of treatment on general health, nor were there local inflammatory reactions. Results indicate that suppression of sexual functions in boars due to long-term treatment with the GnRH agonist deslorelin through an implant such as Suprelorin is possible and can last for several months up to market age; thus it has potential as an alternative to other methods used for boar taint removal. Because the maximum duration of suppression seems to vary between boars, further studies are necessary to refine the treatment.

Cytological diagnosis of endometritis in the mare: Investigations of sampling techniques and relation to bacteriological results

Aim of this study was to compare uterine smears made using the Knudsen catheter, the cytology brush and a uterine culture swab with regard to diagnostic usefulness and the occurrence of neutrophils. Additionally correlation between culture results and the occurrence of neutrophils in uterine smears was investigated. Samples were collected from 340 mares, 81.5% of which were in estrus. Smears made using the cytology brush yielded more endometrial cells per high-power field than those made using the other two instruments (p<0.0001), and a larger proportion had PMNs compared with smears made using the uterine swab (p<0.0001). For smears made with the cytology brush, cultures of β-hemolytic streptococci were more often (p=0.002) accompanied by PMNs than cultures of bacteria other than β-hemolytic streptococci, and there was a positive correlation (r(s)=0.2 p=0.01) between the number of PMNs in smears and the number of colonies of β-hemolytic streptococci. The cytology brush was superior to the other methods because it generated a larger proportion of diagnostic useful smears and the occurrence of PMNs in smears was significantly correlated with the occurrence of cultures of β-hemolytic streptococci.

Prevention of pregnancy in cats using aglepristone on days 5 and 6 after mating

The aim of this study was to test for the efficacy of aglepristone treatment for prevention of early pregnancy in the cat. Eleven cats (Gr. 1) were treated with 10 mg/kg aglepristone on days 5 and 6 after mating, 17 cats (Gr. 2) were used as untreated controls. Blood samples for progesterone (P4) determination were collected from 6 cats of Gr. 1 and 9 cats of Gr. 2, respectively. Ultrasound examination on day 25 revealed no pregnancy in any of the treated cats. In both groups, P4 concentrations increased from day 5 (before treatment) to day 20 (P < 0.01). In Gr. 1, the interval between aglepristone treatment and the subsequent estrus ranged from 5-299 d [18.5 (5.2) d]. Mean interestrus interval was 205 +/- 72 d in Gr. 2. Mean duration of subsequent estrus was not different to duration of estrus before treatment in Gr. 1 and in Gr. 2, respectively. Mean time between treatment and next pregnancy was 56.4 (4.7) d, ranging from 5-325 d. Pregnancy rates after the next estrus following treatment were 64 and 82% after the first and second estrus, respectively. No major treatment-related side effects were observed. In conclusion, treatment was found to be highly effective for prevention of early pregnancy.