Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 8 Workshop Abstracts the number of nuclei, the number of blastomeres was higher in polyspermic embryos due to early fragmentation. Further studies are needed to clarify the phenomenon of ploidy correction in polyspermic zygotes. During IVM, approximately 30% of oocytes fail to reach the M-II stage and about 60-78% of them are arrested at a certain stage characterized by a metaphase plate without polar body. This stage is often referred as “metaphase-I (M-I) arrest”. Our previous studies showed, that unlike real M-I oocytes, “M-I arrested” oocytes obtain cytoplasmic maturation and have the ability to develop to the blastocyst stage resulting in polyploid embryos. Chromosome configurations of such oocytes are different from those of M-I oocytes at 33h IVM and similar to those of M-II oocytes despite of their tetraploid status. It is suggested that in “M-I arrested” oocytes segregation of homologue chromosomes may occur during IVM, however, the extrusion of the first polar body fails and a single metaphase plate is formed again. In conclusion, development to the blastocyst stage is not a perfect indicator of embryo quality in porcine IVP systems, since polyploid embryos can develop to blastocyst stage. Careful selection of M-II oocytes for IVF, regular monitoring of polyspermy rates and further improvements of IVF systems are necessary to reduce polyploidy in porcine IVP systems. WS03-4 Tyrosine kinase receptors (TRK) and bovine early embryonic development Gómez, E*, Rodríguez, A; Caamaño, JN; Díez. C; Facal, N; Muñoz, M Genética y Reproducción, Serida, Gijón, Spain Neurotrophins (NTs) mediate survival, growth and differentiation by binding to two types of cell surface receptors, the anti-apoptotic Trk and the low affinity p75 neurotrophin receptor (p75 NTR ), also termed as p75 NGFR . Subtypes of Trk are NT-specific, such a way NGF binds to TrkA, NT3 to TrkC, and NT4 and BDNF to TrkB; on the contrary, p75 NTR is a common receptor for all NTs. Trk and p75 NTR receptors can form complexes, and this association enhances affinity for ligand binding and neurotrophin discrimination. In previous work, we suggested a role for NTs during early embryonic development, as we detected TrkA (a truncated isoform), TrkB and TrkC proteins in immature oocytes, zygotes, 2-4 cell embryos, morulae, expanded and hatched blastocysts, and the inner cell mass of blastocysts. Using RT-PCR we found mRNA for TrkA, TrkC, p75 NTR and FGFr2 in all the embryonic stages described above; mRNA levels tended to increase during blastulation, and sharply peaked in hatched blastocysts. However, single (NGF, NT3, NT4 or BDNF, at 20 ng/mL each) or pooled NTs in embryo culture with the NT-cooperating factor bFGF (2 ng/mL), did not show relevant effects on development and cell counts. Comparable results were obtained in a recent experiment in the absence of bFGF. bFGF was detrimental compared to controls without bFGF, while NT3, with or without bFGF, increased cell numbers in the ICM as opposed to NT4 and pooled NTs. NT4 enhanced cell counts in the trophectoderm as compared to controls with bFGF and pooled NTs only in the presence of bFGF. Dose-response studies and new experiments to progress in research with NTs and their receptors are in course. We have provided a novel description of Trk proteins and TrkA, TrkC, p75 NTR and FGFr2 mRNA expression, throughout mammalian embryonic development. This research may help to design future research with NTs in bovine embryo culture and embryonic stem cells derivation and maintenance. Grant support: AGL2005–04479. Spanish-Hungarian bilateral project HH2005-0015 (TET E-20/2005). M. Muñoz is sponsored by FICYT. Workshop 04 - Imaging techniques in reproduction Moderator: Gregg P Adams (Canada) WS04-1 Use of 3-D and 4-D ultrasonography in veterinary research Hildebrandt, TB 1 *, Drews, B 1 , Kurz, J 2 , Röllig, K 1 , Hermes, R 1 , Yang, S 3 , Göritz, F 1 1Reproduction Management, Leibniz Institute for Zoo & Wildlife Research, Germany; 2 Institute for Genetik & General Biology, University of Salzburg, Austria; 3 Yunnan Key Laboratory for Animal Reproduction, Kunming Institute of Zoology, CAS, China Three-dimensional-ultrasonography represents an independent (what is meant by independent) imaging modality in human medicine with a wide range of applications, including prenatal diagnosis, gynecology, and oncology. Three-dimensional-ultrasonography allows morphometric measurements in a volume data set and, more recently, the analysis of data in tomographic ultrasound imaging mode (TUI). The advantages of three-dimensional-ultrasonography have also been realized in animal research. Ultrasound equipment used in animals requires additional components and animal-specific scanners for transrectal examination. After substantial system modification transrectal three-dimensional-ultrasonography was applied intensively to characterize embryo/fetal development in Asian and African elephants. Three ultrasound systems were used during this study: a stationary Voluson 530 and a 730 Expert, amd a portable Voluson I (General Electric Inc.). Growth curves for elephant embryo/fetal development were established. In render mode the surface structures were imaged, and used to depict the transition from the oval-shaped embryo to the complete fetus with its characteristic trunk. In inverserender mode, the fluid-filled compartments were displayed separately. This mode was also applied to imaging of ovarian follicular development in rhesus macaques during superovulation. In conjunction with a scientific consultancy for National Geographic, additional investigations were performed in dogs, primates, hares and dolphins. The main purpose was to identify useful morphological parameters of the different developmental stages during pregnancy, and examine intrauterine fetal behavior. For the latter, fourdimensional ultrasound technology (3D in real-time) was applied. We found dramatic differences between species regarding the level of fetal activity, including some interesting behavioral patterns observed from each species throughout their development. According to these preliminary findings, we conclude that intrauterine behavior is an important element during embryogenesis. There is also a strong indication that intrauterine behavior has parallels to known postnatal behavior in the different species. Four-dimensional ultrasonography is, a useful tool for identifying species-specific behavioral elements during intrauterine development. The definition of these key behaviors and the consequences of their presence or absence for the life history of an individual will improve our understanding of evolution and help in clinical diagnosis of developmental disorders. WS04-2 In vivo imaging of sperm transport and survival in the female genital tract using fiber confocal fluorescence microscopy Druart, X 1 *; Cognié J. 1 ; Baril, G. 1 ; Clément F. 2 ; Dacheux JL. 1 and Gatti JL. 1 1UMR 6175 INRA, CNRS-Université de Tours-Haras Nationaux, Nouzilly, France ; 2 INRIA, Paris, France Introduction Fertility of ram sperm is reduced after 24 h of storage at 15°C in milk diluent despite that the in vitro properties, such as viability and motility, are well preserved. This might be explained by a decrease of sperm transport and survival of stored ram sperm in the female genital tract. We studied the influence of in vitro storage of ram sperm on in vivo transport in the female genital tract of the ewe using Fiber Confocal fluorescence Microscopy (FCM, Leica).
16 t h International Congress on Animal Reproduction Workshop Abstracts 9 Materials and methods Semen collected from 3 adult Lacaune rams was diluted in milk at 1.10 9 sperm/ml and stored for 1 h (fresh semen) or 25 h (stored semen) at 15°C. Semen was then fluorescently stained by addition of octadecyl-rhodamine B and MitoTrackerGreen FM, and 75.10 6 sperm were inseminated by endoscopy into the base of each uterine horn of estrus-induced ewes (n = 6 ewes / treatment). In vivo imaging of sperm was done under general anesthesia 4 h after insemination. The uterus and oviducts were exteriorized through a mid-ventral incision and the microprobe of the FCM was inserted into different anatomical regions of the genital tract: the base, middle and tip of the uterine horn, the uterotubal junction and the oviduct. Stilland video-images were recorded for each region of the genital tract, and the distribution of fluorescent motile sperm was determined by counting the number of motile sperm per field. Results The influence of uterine contractions on sperm transport was clearly observed by in vivo FCM. In addition to sperm transport via massive uterine contractions, individual motility of sperm could be imaged at the different regions of the genital tract. When fresh semen was inseminated, the concentration of motile sperm in the lumen 4 h after insemination showed an increasing gradient along the uterus: 10.0 ± 1.7, 15.3 ± 2.2, 22.6 ± 4.1 and 21.0 ± 2.9 sperm / field, respectively, for the base, middle and tip of the uterine horn, and the uterotubal junction (p500µ (bias 0.0). However, the agreement was variable for CL (overall bias -3.72) owing to a less resolvable interface between individual CL by ultrasonography. In conclusion, ultrasound biomicroscopy can be used to accurately assess ovarian follicular dynamics in vivo in the mouse. Research supported by the Natural Sciences and Engineering Research Council of Canada and the Saskatchewan Health Research Foundation. WS04-4 Feeding supplement improves spermatogenetic potential during winter in young merino rams: Preliminary morphometric data Genovese, P 1 , Picabea, N 1 , Vinoles, C 2 , Gil, J 3 , Bielli, A 1 * 1Morphology and Development, Veterinary Faculty, University of Uruguay, Uruguay; 2 Glencoe, INIA, Uruguay; 3 DILAVE Paysandú, MGAP, Uruguay Nutrition influences testicular development before and after puberty. Quantitative histological techniques allow estimation of the actual and potential testicular spermatogenic capacity: greater seminiferous epithelium volume reflects greater sperm producing capacity. The effect of nutritional supplementation on testicular development was examined in Merino rams (n=11) beginning at 17 months of age. The rams divided into 2 groups and fed differentially from March (late summer, breeding season) until the time of castration in July (midwinter, beginning of the non-breeding season) at the Glencoe INIA research station, Paysandú, Uruguay (Latitude, 32 º south). The rams were given free access to native pasture at a stocking rate of 4 rams per hectare. One group (n=5) was not given supplemental nutrition, while the other group (n=6) was supplemented (0.75% of live weight) with sorghum (70%) and soy meal (30%). Testes and epididymides were weighed immediately after castration. Testicular samples were processed for quantitative histological analysis. Paraffin sections (5 µm thick) were stained with hematoxylin-eosin, and 30 images were taken (BX 50 Olympus microscope, CCD camera connected to pc) per ram. The diameter of the seminiferous tubules and the fractional volume of the seminiferous tubules, seminiferous epithelium, seminiferous tubule lumina and testicular interstitium were measured by means of computer assisted analysis with Image Pro Plus® software. Fractional volumes were estimated by counting the percentage of points in a point grid falling over the object of interest. Absolute volumes of seminiferous tubules, seminiferous epithelium, seminiferous tubule lumina and testicular interstitium were estimated by multiplying fractional volumes by testicular volume (estimated from testicular weight). The nutritionally supplemented group of rams had a greater total testicular weight (g, 299.9 ± 24.0 vs 201.9 ± 42.8; P
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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