Steven Verberckmoes

Sperm Binding to Epithelial Oviduct Explants in Bulls with Different Nonreturn Rates Investigated with a New In Vitro Model

Abstract A new in vitro method was developed for analyzing the capacity of sperm to bind to oviductal epithelium to determine whether this binding capacity could be used to predict nonreturn rates (NRR). Sperm binding was evaluated by counting 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1)-labeled spermatozoa attached to oviductal epithelium and by measuring the surface area of the oviduct explants by means of an image analysis program. Hepes + Tyrode albumin lactate pyruvate (TALP) was a more useful medium than in vitro fertilization (IVF)-TALP, TCM-199 medium + 10% fetal calf serum, and TCM-199 medium alone for the investigation of sperm binding to oviductal explants. Oviduct explants with a surface area of ;lt20 000 μm2 provided more consistent results than did explants with a surface area of >100 000 μm2. A positive association was found between the loge transformed number of spermatozoa bound to 0.1 mm2 oviductal epithelium and the NRR of the respective sires after 24 h of coincubation, provided that the membrane integrity of the sperm sample was >60%. Determination of the capacity of sperm to bind to oviductal explants could become a reliable in vitro method for predicting the NRR of a given sire.

In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions

In this study, environmental conditions mimicking those prevailing in the epididymis were used for storing ejaculated bull spermatozoa in vitro during 4 days at ambient temperature. These conditions were low pH, high osmolarity, high sperm concentration and low oxygen tension. Hepes-TALP was used as basic storage medium. Fresh spermatozoa were stored at a concentration of 10 x 10(6)spermatozoa/ml in Hepes-TALP of different pH (pH 4, 5, 6, 7 or 8), and osmolarity (100, 300, 400, 500, 600 or 800 mOsm/kg), and under different atmospheric conditions (nitrogen gassed or aerobic). Spermatozoa were also stored undiluted or at different concentrations: 10x 10(6), 100 x 10(6), 500 x 10(6) or 1 x 10(9)spermatozoa/ml. Sperm parameters such as membrane integrity, motility, mitochondrial membrane potential or DNA fragmentation were used to assess semen quality after storage. Adjustment of the pH of Hepes-TALP to pH 6 yielded significantly better results than storage at all other pH values. Isotonic Hepes-TALP (300 mOsm/kg) had a less detrimental effect on spermatozoa than hypo- and hyperosmotic versions. No differences in sperm parameters were observed when spermatozoa were incubated under aerobic or under nitrogen gassed storage conditions. Optimal sperm concentration in vitro is 10 x 10(6)spermatozoa/ml. This is in contrast with the in vivo situation, where spermatozoa are stored at high concentration. However, better results at high sperm concentrations were obtained when spermatozoa were diluted for less than 5 min in Triladyl-egg yolk-glycerol diluent immediately after ejaculation.

Migration of bovine spermatozoa in a synthetic medium and its relation to in vivo bull fertility

Although sperm migration has been extensively refined and validated in human infertility studies, its application to predict bovine fertility has been very limited, and a clear relation between the sperm migration distance and in vivo bull fertility has never been demonstrated. A synthetic medium based upon methyl cellulose (MC) was tested for its suitability to serve as a migration medium for frozen-thawed bovine spermatozoa. The effects of the concentration of MC, the incubation time, and sperm concentration on sperm migration capacity was determined. The relation between sperm migration capacity at different incubation times of the frozen-thawed spermatozoa of five bulls, and their 56 days nonreturn rates (NRRs) was assessed in order to evaluate its suitability as a tool to predict in vivo bull fertility. The highest repeatability of the sperm migration test (CV = 10.7%) was obtained when the sperm migration distance of the five vanguard motile spermatozoa was determined at 30 min incubation at 37 degrees C in a migration medium with 1.35% MC. No significant difference in migration distance was demonstrated when sperm concentrations of 100 x 10(6) and 150 x 10(6) spermatozoa/ml, respectively, were used. Despite the relatively high repeatability of the migration test, no relation was found between the sperm migration distance and the 56 days NRRs of five sire bulls. Therefore, the sperm migration test in 1.35% MC cannot be used to predict in vivo bull fertility accurately.

Assessment of a new utero-tubal junction insemination device in dairy cattle

A new artificial insemination device for semen deposition near the utero-tubal junction in cattle (Ghent device) has been developed at the Ghent University (Belgium). In this study, the effect of the new insemination device on sperm quality was evaluated. Moreover, in a field trial 4064 dairy cows were inseminated by 12 inseminators to examine the efficacy of the device under field conditions. The Ghent device is a disposable plastic catheter which can easily follow the curvature of the uterine horns and thus reach the utero-tubal junction (UTJ). After expulsion of the inseminate with 0.7 or 1.7 ml of air, 19.0% of the insemination dose remained in the insemination catheter. Sperm loss can be diminished to 9.0% of the original insemination dose when the insemination catheter is flushed with 0.1 ml of air, followed by 0.6 ml of physiological saline solution. No toxic effect of the insemination catheter on sperm quality or fertilizing capacity was found. In the field trial, sperm were inseminated in dairy cattle which were divided in three groups. The first group was inseminated in the uterine body with the conventional insemination device, the second group in the uterine body with the Ghent device, and the third group in the tip of both uterine horns with the Ghent device. Each insemination was performed with 10 x 10(6) to 15 x 10(6) frozen-thawed spermatozoa. The pregnancy rates (PRs) were significantly affected by the insemination technique (P = 0.02), by the inseminator (P = 0.01), by heifer or cow (P < 0.01), and by the insemination number (P < 0.01). Pregnancy rates obtained with the conventional insemination device (57.6%) were significantly better than those obtained with the Ghent device in the uterine body (52.7%) (P < 0.01), but did not differ significantly from those obtained after deep insemination into both uterine horns (53.8%) (P = 0.27). It can be concluded that the Ghent device is suitable for utero-tubal junction insemination of dairy cattle under field conditions. Whether the Ghent device is also suitable for insemination with lower insemination doses is at present under investigation.

Sampling techniques for oviductal and uterine luminal fluid in cattle

Analysis of luminal fluid microenvironments in the reproductive tract is pivotal to elucidate embryo-maternal signaling mechanisms responsible for successful reproduction in mammals, including cattle. Besides facilitating production of an optimized medium for in vitro fertilization and embryo culture in assisted reproductive technologies, screening of luminal fluid constituents in the oviduct and uterus could also provide critical information for elucidation of mechanisms underlying developmental programming. A key issue in this type of research is the sampling of luminal fluids. In this review we discuss the sampling techniques available for bovine species, including a recent in situ technique developed with the Ghent device, which allows rapid recovery of measurable amounts of pure uterine luminal fluid with minimal disturbance to the donor animal.

Effect of whole blood and serum on bovine sperm quality and in vitro fertilization capacity

Under physiologic conditions, low concentrations of blood may be present in the uterine fluid of the estrous cow at the moment of insemination. To decrease the insemination dose and to obtain good insemination results with less fertile semen, more invasive insemination methods such as utero tubal junction (UTJ) insemination can be used. More invasive insemination methods increase the risk of damaging the hyperemic endometrium, with blood in the uterine fluid as result. In this study, the effect of 0, 0.15 and 1.5% whole blood and serum on bovine sperm quality and in vitro fertilizing capacity was evaluated. Sperm quality as assessed by total motility, progressive motility, membrane integrity and acrosomal status was not affected by the presence of blood or serum (P > 0.05). However, the in vitro fertilizing capacity decreased with increasing concentrations of blood and serum (P < 0.01). The rate of polyspermy increased with increasing concentrations of serum (P < 0.01), but not with increasing concentrations of blood (P = 0.30). In conclusion, no immediate effect of blood and serum was visible on several sperm quality parameters, except for an increased prevalence of head to head agglutination (HHA). However, blood and serum did have a negative effect on in vitro fertilizing capacity.

Intravaginal fibroma in a sheep

was seen immediately after starting therapy with doxycycline, which may be considered evidence for a relationship between drug therapy and the improvement of clinical signs. Spontaneous remission has been previously reported (Scott 1984). However, the number of cats showing spontaneous remission is small and more studies are needed to evaluate the potential and extent of spontaneous remission as well as aetiological factors in feline plasmacytic pododermatitis. The results of this study indicate that doxycline for the treatment of plasmacytic pododermatitis is effective and well tolerated and is thus recommended for cats with histopathologically confirmed plasmacytic pododermatitis as an initial treatment option.