Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 164 Poster Abstracts morphological changes approximately 200 tubule-cross-sections were evaluated and grouped (A-D) according to the most developed germ cell observed. Testosterone (T) and estradiol-17ß (E) were estimated in blood collected during downregulation and at castration. Timing of recrudescence showed distinct individual differences yielding different numbers of dogs per group: Gr. A, spermatocytes (n=4); Gr. B, round spermatids (n=3); Gr. C, elongating spermatids (n=6) and Gr. D, elongated spermatids (n=17). T and E concentrations increased from Gr. A to B (T: 0.14 ± 0.10 to 2.54 ±1.57ng/ml; E: 6.40 ± 2.19 to 9.73 ± 4.16pg/ml) and were constant thereafter. These first results imply that onset of spermatogenesis occurs at low steroid hormone levels, accompanied by expression of the androgen receptor, and is rapidly stimulated by a further increase. To our knowledge, this is the first study giving detailed information about recrudescence of spermatogenesis of the dog following downregulation. P414 Early Detection of Membrane Asymmetry in Frozenthawed Buffalo Spermatozoa Govindasamy, K 1 *; Kumar, S 2 and Sharma, B 3 1PhD Scholar, 2 Principle Scientist, Division of Animal Reproduction; 2 Indian Veterinary Research Institute, Izantnagar, Bareilly (UP), India; 3 Principle Scientist, Division of Animal Biochemistry, Indian Veterinary Research Institute, Izantnagar, Bareilly (UP), India Introduction Mammalian spermatozoa undergo tremendous chemical and physical stresses during cryopreservation. The sperm plasma membrane is one of the key structures affected by cryopreservation. When the cell membrane is disturbed, phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, which is one of the earliest signs of membrane disruption. Annexin V conjugated to Fluorescein isothiocyanate (FITC) fluorochrome retains its high affinity for PS and therefore, serves as a sensitive probe that can be used for flow cytometric detection characterized by the loss of membrane asymmetry. Methods Fifty six semen ejaculates, eight ejaculates from seven healthy Murrah buffalo bulls (4-6 years age) maintained at standard management conditions were used for the study. The standard conventional cryopreservation protocol was adopted. Combination of two fluorescent dyes, Annexin V and propidium iodide (PI) was used to evaluate the membrane asymmetry in fresh and frozen thawed spermatozoa in conjugation fluorescent microscope and flow cytometry. Results Four groups of spermatozoa were identified: (i) viable spermatozoa (Annexin V-negative and PI-negative); (ii) viable spermatozoa with early membrane disruption but integer plasma membrane (Annexin V-positive and PI-negative); and two categories of PI positive, dead spermatozoa (iii) early necrotic (Annexin V- positive and PI-positive); and (iv) Late necrotic cells (Annexin V- negative and PI-positive). The four sperm population varied significantly between fresh and frozen thawed spermatozoa. In fresh semen, the early sperm membrane change was observed only in 17% of the total number of sperm. However, after freezing and thawing, these sperm accounted for more than 31%. After freezing-thawing, there was significant increase in the percentage of live cells with phosphatidylserine externalization (early membrane changes) and early necrotic cells, while reducing the percentage of live normal cells. Conclusions The Annexin V-binding assay is an effective tool to provide early detection of membrane asymmetry in the viable spermatozoa. Further, cryopreservation of buffalo spermatozoa induces translocation of phosphatidylserine as in the early apoptosis of somatic cells. P415 Identification, Isolation and in vitro long term culture of Male Germline Stem Cell in Porcine Su Young, H*; Gupta, MK; Uhm, SJ; Lee, HT Dept of Bioscience & Biotechnology, ARRC, Konkuk University, Seoul 143- 701, Korea Male germline stem cells are the basis of spermatogenesis and reside on basement membrane of seminiferous tubule in testis. Due to the lack of specific markers, identification of male germline stem cells is difficult to study in pig. In this study, we investigated to determine isolation and long-term culture system of male germline stem cell in neonatal pig testis. We used farm piglets 5~10days old, testis were digested by sequential enzymatic system, including 1mg/ml collagenase, 1mg/ml hyaluronidase and 0.25% trypsin/EDTA. Cells were separated by discontinuous density gradient and differential plating to increase of purity. Isolated cells were cultured in DMEM medium supplemented with 15% FBS, and specific growth factors as 1000 IU/ml leukemia inhibitory factor (LIF) and 10ng/ml glial cell line-derived neurotrophic factor (GDNF) at 37℃ incubator with 5% CO2. We have used STO cell line for feeder layer treated by mitomycin C for mitotically inactivation. After 7-8 days culture, three dimensional colonies appeared as original generation. Alkaline phosphatase (AP) staining expressed positively. Also these germ cells expressed stem cell markers OCT-4, SSEA-1, and spermatogonial stem cell markers PGP9.5, Dolichos Biflourus Agglutinin (DBA) in immunocytochemistry. These cells have been investigated by RT- PCR using specific primers, pgp 9.5 and pigvasa, which is expressed specifically in the porcine undifferentiated spermatogonia. We established porcine male germline stem cell lines from neonatal testis and in vitro long-term culture system. These results could be used for identifying the mechanism of spermatogenesis and applying for transgenesis. P416 In vitro effect of sodium nitroprusside (SNP) on sheep sperm motility Hassanpour, H* Department of Basic Sciences, College of Veterinary Medicine, Sharekord University, Iran Nitric oxide (NO) has been recently shown to regulate many functions of sperm such as Acrosome reaction, sperm chemotaxis and motility. The aim of this study is to investigate the effects of sodium nitroprusside (SNP) as nitric oxide donor on sperm motility of sheep. After collecting of normozoospermic samples by artificial vagina from twenty Bakhtiari rams, motile spermatozoa were harvested by the swim-up technique using SOF-HEPES medium then incubated for 120 minutes in the presence of SNP (0.1, 0.5, 0.7 μM). sperm motility assessed in four grades (rapid progressive motility, grade A; slow or sluggish progressive motility, grade B; non-progressive motility, grade C; or immotility, grade D.).In this study, SNP in the concentration of 0.1µM non-significantly increased sperm motility at grades A & C, and decreased at grades B & D. Concentration of 0.5 µM increased grade B & D, and decreased grade C. SNP at the concentration of 0.7 µM significantly decreased sperm motility at grade A (15.1%) and increased at grade D (16.7%), in comparison with their control groups (p
16 t h International Congress on Animal Reproduction Poster Abstracts 165 P417 The effect of centrifugation and dilution on concentrations of reactive oxygen species in canine semen Heaton, L*, Pinto, CRF Population Health & Pathobiology, North Carolina State University College of Veterinary Medicine, United States Reactive oxygen species (ROS) are produced by sperm in small quantities as part of cell signaling cascades that enable the capacitation and acrosome reaction required for fertilization; however, excess ROS production has deleterious effects on DNA and fertility. Semen handling techniques should therefore minimize elevated ROS levels to maintain optimal fertility, but there is limited information regarding what circumstances inadvertently increase ROS. In the present study, it was hypothesized that both centrifuged and undiluted semen samples would have increased ROS concentration and that the addition of semen diluents (extenders) would reduce the concentrations of semen ROS. Canine ejaculates (n = 14) were collected manually and divided into four aliquots, two of which were extended 1:1 using EZ-BF with ticarcillin. Initial ROS concentrations were obtained using chemiluminescence (Lumat LB 9507, Berthold Technology, Oak Ridge, TN). Immediately thereafter, raw semen was analyzed for concentration, motility, viability, morphology, membrane integrity, acrosome integrity, and for the presence of leukocytes in the ejaculate. Following the initial analysis, one extended and one non-extended fraction were centrifuged for 10 minutes at 900 g. After centrifugation, the fractions were evaluated for ROS concentration, motility, viability, morphology, membrane integrity, and acrosome integrity. The ROS concentration, assessed by eleven relative light unit (RLU) readings, was averaged and standardized as RLU/106 spermatozoa. Data was analyzed using two way ANOVA with the significance level set at P = 0.05. The Tukey Test was used for all pairwise multiple comparison procedures of the mean responses to the different treatment levels. There was a significant effect of dilution (P < 0.05); undiluted (raw) semen samples had greater RLU means ROS concentrations than those diluted with semen extenders (320.1 and 87.9, respectively). The effect of dilution within the factor centrifugation was also significant (P < 0.05); there was a consistent trend for greater means of ROS concentrations in centrifuged samples than in non-centrifuged samples. This elevation of ROS did not appear to affect motility or other seminal parameters analyzed, suggesting that evaluating motility or even membrane integrity alone is not sufficient to identify oxidative stress and potential fertility problems. These results emphasize the benefits of using semen extenders prior to laboratory processing to reduce potential ROS-induced detrimental effects on semen quality. P418 Testicular Growth curve of Guzerat (Bos taurus indicus) raised in the savannah region at pasture and supplemented with silage during the dry period Henry, M 1 *, Osorio, JP 1 , Bergman, JAAG 2 , Carmo, AS 1 1Clinics and Surgery Department, Federal University of Minas Gerais, Brazil; 2Animal Science Department, Federal University of Minas Gerais, Brazil Introduction The early detection of superior reproductive traits is desired in any selection program. Unanian and co-workers (2000) stated that scrotal circumference (SC) and testicular volume (VL) need to be evaluated when selecting young males due to the fact that the association of both characteristics improve the confidence in the selection process. The aims of the present study were to characterize some reproductive traits of young males in order to subsidize evaluation and selection of sires of the Guzerat breed as well as to evaluate the correlation between SC and VL. Material and methods Three hundred and thirty Guzerat males were evaluated at three months interval from five to 70 months of age. Data included a total of 1,757 observations. SC, length and width of both testicles were measured and VL was estimated according to Bailey et al. (1996). Electro-stimulation seminal collection was attempted when males had SC above 19cm. Onset of puberty was considered when at least one motile sperm cell was detected in the ejaculate. Descriptive statistics were performed, Pearson correlation between both traits was calculated and SC and VL growth were modeled using Logistic function and General Linear Model Procedures (SAS, 1997). Results and discussion Growth curves for SC and VL reached inflection points, respectively, at 12.8 (18.1cm) and 23.3 months of age (389.4 cm3). Average age at puberty was 20.2 month with mean SC of 22.79cm and a VL of 298.28cm3. Estimated SC and VL for 75 months of age were, respectively, 36.2cm and 778.8cm3. The average growth rates of SC and VL, for the period before and after the inflection point were .58cm/mo, 16,3cm3/mo, .29cm/mo, 7,7cm3/month of age, respectively. Direction and strength of correlation between SC and VL (.91; P
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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