Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 130 Poster Abstracts integrity (PMI) (propidium iodide and carboxyfluorescein diacetate) and sperm concentration. Samples were divided into three equal aliquots, which were first diluted with TOG and, afterwards, with TOG supplement with 10% of glycerol in proportions that permitted final concentrations of 3, 5 or 7% of glycerol. Sperm samples from the same ejaculate contained equal final volume (100 or 150 µL) and sperm concentration (40 to 60 x 10 6 / mL). After load into 0.25 mL straws, samples were placed in a programmed refrigerator at 5 o C for 60 minutes, then in liquid nitrogen vapour during 15 minutes and immersed. Sperm samples were thawed at 46 o C for 12 seconds and analyzed immediately (CASA and IMP), 30 (CASA) and 60 (CASA) minutes post-thawing. Data were submitted to statistical analysis by ANOVA and Tukey test, with p < 0.05 taken as significant. Among all post-thaw moments, VAP (average path velocity), VSL (straight line velocity), BCF (beat cross frequency) and STR (straightness) were not different between 3, 5 and 7% of glycerol groups. Higher values for TM (total motility), PM (progressive motility), R (percentage of rapid spermatozoa) and PMI (plasma membrane integrity) were obtained in all evaluated moments after thawing for groups 5 and 7% of glycerol, with no statistical difference between them. Values for ALH (amplitude of lateral head displacement) increased after thawing only in group 3% of glycerol. Group 7% of glycerol exhibited lower VCL (curvilinear velocity) values compared to groups 3 and 5%. Since frozen-thawed sperm samples containing 5 or 7% of glycerol showed better results compared to 3% of glycerol and considering that glycerol is known to be toxic for spermatozoa, we can conclude that a concentration of 5% of glycerol is suitable for freezing domestic cat spermatozoa. P310 Preliminary studies on isolation and culture of the epithelial, stromal and endothelial cells from the uterus of domestic cat-technical report Siemieniuch, M., Skarzynski, DJ.* Institute of Animal Reproduction and Food Research, Olsztyn, Poland The most of the previous experiments concerning feline female reproductive regulations, were based on the hormones plasma levels, morphological examination of ovaries and CL during laparoscopy and behavioral changes in the animals. The local, immuno-endocrine events within the feline ovary and/or uterus, and such interaction between different cells of the endometrium have been largely ignored. Thus, we decided to establish methodology for the isolation and culture of different types of endometrial cells (epithelial, stromal and endothelial cells) from uteri of domestic cat. The main goal of the study is to establish the model for further examinations of local, immuno-endocrine regulations in cat uterus and mechanisms of early embryo development. Uteri of queen were obtained by ovariohysterctomy. A polyvinyl catheter was inserted into the uteri horn, and the end of the horn near the corpus uteri was tied shut in order to retain an enzyme solution for solubilizing the epithelial cells. 1-2 ml of enzyme solution (sterile HBSS containing (dispase I, collagenase I, DN-ase IV and 0.1% BSA) was then infused into the uterine lumen through the catheter. Epithelial cells were isolated by incubation twice at 37,5˚C for 45 min and 20 min with gentle shaking. The cell suspension obtained from the first and second digestions was pooled and washed 3 times by centrifugation in the gradient and finally resuspended in culture medium (DMEM/Ham's F-12; supplemented with 10% calf serum). After isolation of epithelial cells, the horns were longitudinally slit and the surface was scratched. The rest of endometrial tissue were digested in 10-20 ml of the above-described enzyme solution. After 20, 40 and 60 minutes stirring, dissociated cells were collected. For isolation of endothelial cells, horns from intact uterus were cut and minced into small pieces (1 mm 3 ). The mix cells were isolated as described for stromal cells isolation. The pure population of endothelial cells was isolated using Dynabeads® M-450 magnetic beads coated by the lectin BS-1. The cells of each cell type were separately seeded at a density of 1 x 10 5 viable cells/ml in 24-well plates. The culture medium was changed every 2 days until confluency was reached. When the cells were confluent the homogenity of cells was estimated using immunofluorescent staining for specific markers of epithelial (cytokeratin), stromal (vimentin) or endothelia cells (von Willebrand VIII factor). The test revealed 95%, 90% and 95 % of purity of the epithelial, stromal and endothelail cells in the culture, respectively. P311 Retroflexion of the urinary bladder in a rottweiler bitch during pregnancy Sontas, BH*; Apaydin, SO; Toydemir, TSF; Kasikci, G; Ekici, H Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Istanbul University, Turkey Clinical case A 2.5-year-old, pregnant rottweiler bitch, weighing 29 kg, was presented with a 24-hour’ history of a large mass of tissue visible through the vulvar cleft and difficulty in parturition. Accompanying complaints were loss of appetite and increased licking of the mass. No previous trauma was reported, but the bitch had been continuously barking through the night because of a snake in the garden. The bitch had mated several times with a mixed-breed dog of the same size two months before presentation and one year before the bitch had whelped five live puppies without requiring any veterinary assistance. On clinical examination a large, firm, non-painful round ball-shaped mass of tissue, approximately 10 cm in diameter, was identified blocking the entrance of the vulva. The tissue was clean with no haemorrhage or ulceration. Abdominal ultrasonography demonstrated several fetuses without heart beats and the absence of the urinary bladder in its anatomical position in the abdominal cavity. Ultrasonography of the mass revealed anechoic appearance with no fetal vesicles inside. Vaginal cytology showed the presence of neutrophils and parabasal cells, typical of a bitch in the luteal phase. Haematology, serum biochemical analysis and radiographic evaluation of the mass or the caudal abdomen were not performed because of the cost. Diagnosis of pregnancy, fetal death and retroflexion of the urinary bladder confined to the vagina were made according to the clinical and ultrasonographic findings. Removal of the gravid uterus by ovariohysterectomy was performed and the bladder was manually repositioned. The bitch recovered well and was sent home after the surgical procedure. One week later, the bitch was healthy with no complaints of dysuria, stranguria or urinary incontinence. Two months after the surgery, the owner reported that the bitch was clinically normal with no recurrence of the retroflexion. To our knowledge, this is the first case of a vaginal mass occurring after the retroflexion of the urinary bladder in a pregnant bitch. Discussion A mass of tissue that is visible from the vulva is the most common sign of vaginal hyperplasia, neoplasia or prolapse (Manothaiudom and Johnston 1991). However, in the present case, the mass was associated with the urinary bladder which was retroflexed into the space between the vagina and pelvic wall because of a perineal hernia. Perineal hernias occur most commonly in the male when degenerative changes develop in the muscles of the pelvic diaphragm as a result of hormonal influences or pelvic fractures (Hayes et al. 1978, White and Herrtage 1986, Risselada et al. 2003, Angeli et al. 2005). References 1) Angeli G, Bellezza E, Arcelli R, Padua S. XII Congresso Nazionale Società Italiana di Chirurgia Veterinaria, Pisa, Italy, june 16-18, 2005. www.vet.unipi.it/clınıca/2005sicv/lavoridef/angeli.pdf. accessed in 02 july, 2007. 2) Hayes H M, Wilson GP, Tarone RE J Am Anim Hosp Assoc 1978, 14: 703-707. 3) Manothaiudom K and Johnston SD. Vet Clin North Am, Small Anim Pract 1991, 21(3): 509-521. 4) Risselada M, Kramer M, Van de Velde B, Polis I, Görtz K. J Small Anim Pract 2003, 44: 508-510. 5) White RAS, Herrtage ME. J Small Anim Pract 1986, 27: 735-746.
16 t h International Congress on Animal Reproduction Poster Abstracts 131 P312 Estrus induction in bitches by using HUMAN MENOPAUSAL GONADOTROPIN & HUMAN CHORIONIC GONADOTROPIN combination Zahedi Abdi, A No 25,Neshat Ave,Bargh St,Urumieh,West Azarbayjan,Iran-Post Code:5715694884 tel:0098-441-3441977 Fax:0098-21-22707955 In this trial,5 leash of bitches were chosen for inducing of estrus by using the combination of Human Menopausal Gonadotropin (H.M.G) & Human Chorionic Gonadotropin(h.C.G).Such bitches were controlled for nutrition and estrus signs in their new home(place) since 2 months before the beginning of trial. To start the trial,H.M.G(H.M.G,75 IU FSH,75 IU LH approx N.V Organon oss Holland)was injected to all of the bitches (5 leash of dogs)during the first 9 days and by the day of 10 th ,H.C.G(intervet Holland)was injected I.M to each bitch. Since 1 month before the beginning of treatment and also during the period of treatment and 1 month after the end of treatment, blood progesterone concentration was detected in such bitches and vaginal smears was obtained to recognize the changes in vaginal cytology. The results showed that,all of the bitches show the signs of proestrus and serosangunis discharge from their vulva,8±0.7 days after the beginning of treatment.These signs were continued upto 9.8±1.6 days.In 2 leash of such bitches breeding was seen 2 days after the end of vaginal bleeding and in other 3 bitches the signs of estrus was appeared in the state of split heat.In 2 leash of bitches,that have matted ,the progesterone concentration increased by the beginning of estrus and rised above a critical plateau(>1ng/ml)and reached upto >20 ng/ml, one month later .But in 3 other bitches the progestrone concentration didn't rise above the 1.5 ng/ml A cytology of vagina was interpretated as superficial intermediate cells after the end of treatment. These results show that the combination of H.M.G & H.C.G can facilitate induction of proestrus in the bitches which have no previous history of parturition. Key words : HMG , HCG , dog, , estrus , proestrus Poster 08 - Reproduction of Zoo and Wild Mammals P313 Sperm sex sorting in Asian elephant Behr, B 1 *, Rath, D 2 , Hildebrandt, TB 1 , Blottner, S 3 , Goeritz, F 1 , Sieg, B 2 , Knieriem, A 4 , Hermes, R 1 1Reproduction Management, Leibniz Institute for Zoo and Wildlife Research, Germany; 2 Institute for Animal Breeding Mariensee, Federal Agricultural Research Centre, Germany; 3 Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research, Germany; 4 Zoo Hannover, Germany Introduction The captive population of the Asian elephant (Elephas maximus) suffers from an insufficient reproductive rate to maintain a self sustaining population. This negative trend leads to an increasing demand for assisted reproduction tools with the artificial insemination as new and promising supplement. As mainly females are affected from premature aging processes and husbandry of several elephant bulls involves challenging management situations, there is a strong need for female elephant offspring in captivity. Application of flow cytometric sex sorted spermatozoa in artificial insemination offers the possibility to predetermine the sex of offspring. Objectives The aims of this study were to determine a suitable semen extender and basic parameters for flow cytometric sex sorting of spermatozoa in the Asian elephant. Methods 18 sperm samples were collected by manual transrectal stimulation from one bull. Sperm quality parameters and sex sortability of spermatozoa were evaluated after dilution in three semen extenders and DNA labelling. Following sex sorting process, samples were stored at 4oC for 12h, imitating the long transportation time to the female designated for insemination. Results Only skim milk containing semen extender was determined as suitable semen extender for sex sorting spermatozoa from Asian elephant, providing repeatable, high resolution between X and Y chromosome bearing sperm populations compared to other extenders. 12 of 18 collected ejaculates could be sex sorted successfully at an average sort rate of 1945.5 ± 187.5 spermatozoa/ sec resulting in a population purity of 94.5 ± 0.7%. Best sortability of spermatozoa was reached after DNA staining for 1.5 - 2h at 37°C. Sperm integrity, progressive and total motility was ≥ 65% after collection, 42.6 ± 3.9%, 48.1 ± 3.3%, 59.4 ± 3.8% after DNA labelling, and 64.8 ± 3.2%, 57.9 ± 5.0%, 70.8 ± 4.4% after sorting process, respectively. After liquid storage of sorted spermatozoa for 12 hours at 4°C, sperm integrity, progressive and total motility was 46.4 ± 5.2%, 33.6 ± 4.9% and 64.8 ± 2.4%, respectively. Discussion This study describes flow cytometric sex sorting of Asian elephant spermatozoa. Quality and purity of sex sorted spermatozoa and a reasonable ability for liquid storage after sorting process provide a promising base for the application of sex sorted spermatozoa in artificial insemination of the Asian elephant. P314 A comparison of three culture media for the incubation of thawed red deer (Cervus elaphus hispanicus) epididymal spermatozoa Domínguez-Rebolledo, AE*, del Olmo, E; Martínez-Pastor, F; Fernandez- Santos, MR, Espinosa, M; Esteso, MC; Soler, AJ and Garde, JJ National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM) and Institute of Regional Development (IDR), University of Castilla-La Mancha, Spain The suitability of incubation media is of capital importance when conducting IVF or other artificial reproduction techniques (ART) in vitro. In this study we tested the effect of three different culture media on frozen/thawed epididymal spermatozoa from red deer (Cervus elaphus hispanicus). Epididymal spermatozoa are available from hunted males, and use of ART on this species is increasing, thus it is important to assess existing techniques for this kind of samples. Frozen epidididymal samples from nine males were thawed, and diluted to 10 7 mL -1 in three common media for IVF and embryo culture (SOF, TALP, BGM) and control (freezing extender without glycerol: Tris-citrate-fructose, 20% egg yolk) at 37 ºC 5% CO 2 and analyzed at 0, 3, 6 and 9 h. Sperm motility index (SMI) and normal acrosome ridges (NAR) were assessed subjectively (phase contrast microscopy); sperm viability according to YO-PRO-1 staining (necrotic and apoptotic sperm detection: VIAB), and mitochondrial status by Mitotracker deep red (spermatozoa with active mitochondria within VIAB: MT) were assessed by flow cytometry. The effects of storage time and incubation media were analyzed by linear mixedeffects models. All parameters decreased with incubation time (P
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