Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 144 Poster Abstracts immunohistochemistry. Exposure to ewes stimulated an increase in LH pulse frequency (0.19 ± 0.12 vs. 0.75 ± 0.14 pulses/h; P < 0.01), mean concentration of LH (0.16 ± 0.06 vs. 0.53 ± 0.21 ng/mL; P < 0.05) and basal concentration of LH (0.08 ± 0.01 vs. 0.13 ± 0.01 ng/mL; P < 0.05). Exposure did not affect LH pulse amplitude (0.55 ± 0.15 vs. 0.55 ± 0.15 ng/mL; P > 0.1). In Control rams, there was no change (P > 0.1) in LH pulse frequency (0.25 ± 0.10 vs. 0.25 ± 0.10 pulses/h), mean concentration of LH (0.15 ± 0.03 vs. 0.11± 0.01 ng/mL), basal concentration of LH (0.08 ± 0.02 vs. 0.12 ± 0.02 ng/mL) or LH pulse amplitude (0.62 ± 0.09 vs. 0.70 ± 0.09 ng/mL). Preliminary analysis of the histology indicates that Ewe-exposed rams have a greater number of Fos-immunoreactive cells in the ventromedial hypothalamus, and probably other regions, than Control rams. This result corresponds with studies in the ewe that have identified neural activation in the ventromedial hypothalamus of ewes following stimulation by rams (1). 1. Gelez H, Fabre-Nys C. Neural pathways involved in the endocrine response of anestrous ewes to the male or its odor. Neuroscience 2006;140: 791-800. 2. Rosa HJD, Bryant MJ. The 'ram effect' as a way of modifying the reproductive activity in the ewe. Small Ruminant Research 2002;45: 1-16. P354 Plasmatic thyroxine and triiodothyronine concentrations in non-pregnant, pregnant and lactating Saanen breed goats Greco, G 1 *; Baldini, L 1 ; De Paula, M 1 ; Bittencourt, RF 1 ; Maia, L 2 ; Oba, E 1 1Department of Animal Radiology and Reproduction, São Paulo State University -UNESP - Botucatu, Brazil; 2 Department of Veterinary Clinics and Pathology, Fluminense Federal University -UFF, Brazil Thyroid hormone receptors are expressed in most of the animal tissues, being responsible for activating biochemical reactions and organic responses. In pregnant goats, these hormones are essential for normal fetal development and organogenesis and, during the lactational period, they stimulate galactopoiesis. As in sheep, thyroid hormones seem to be involved regulating reproductive sazonality in the caprine species. In comparison to other ruminants, few studies have been published regarding the variations in the concentrations of thyroxine (T4) and triiodothyronine (T3) in goats, according to their reproductive state. The present work had as objective to determine the plasmatic concentrations of T3 and T4 in non-pregnant, pregnant and lactating Saanen breed goats. Forty-three female Saanen goats, aging 24 to 32 months, were assigned into three different groups. Group 1 was composed of 15 non-pregnant goats. Thirteen goats experiencing their last month of pregnancy were assigned to group 2. As for group 3, it was composed of 15 lactating goats, whose kids were at most one month old. Blood was collected from animals through jugular venipuncture into tubes containing heparin, which were centrifugated at 3000 x g for 15 minutes. Plasma obtained was stored at – 20 degrees Celsius. T3 and T4 concentrations were estimated through radioimmunoassay, using Diagnostic Products Corporation® (DPC) kits. The obtained data was statistically analyzed through the Statistical Analysis System®, version 6.1, 1996. Concentrations of T3 and T4 were compared between the groups using the Student- Newman-Keuls test. The correlation between T3 and T4 measured concentrations was established using the PROC CORR procedure. Significance levels were set as P < 0.05. A highly significant (P < 0.01) positive correlation was found between T3 and T4 overall concentrations. Mean T3 concentrations were 150.08 ng/dL +/- 46.7, 108.48 ng/dL +/- 41.1 and 171.55 ng/dL +/- 38.3 in groups 1, 2 and 3, respectively. Pregnant goats had lower (P < 0.05) T3 concentrations than lactating and non-pregnant animals. As for T4, mean concentrations were, respectively, 5.40 μg/dL +/- 1.38, 3.76 μg/dL +/- 1.05 and 6.51 μg/dL +/- 1.43 in groups 1, 2 and 3. Lactating goats had higher (P < 0.05) T4 concentrations than non-pregnant animals. T4 concentrations obtained from pregnant goats were significantly lower (P < 0.05) than those in the remaining groups. In conclusion, T3 and T4 concentrations are lower in pregnant Saanen goats, being the concentration of T4 higher during the lactational period. P355 Salsolinol as a hypothalamic neurotransmitter stimulating prolactin release during suckling in ewes Misztal, T 1 *; Gorski, K 1 ; Tomaszewska-Zaremba, D 2 ; Molik, E 3 ; Romanowicz, K 1 1Department of Endocrinology, 2 Department of Neuroendocrinology, The Kielanowski Institute of Animal Physiology and Nutrition in Jablonna, Poland; 3Department of Sheep and Goat Breeding, Agricultural University in Cracow, Poland Introduction Salsolinol is a dopamine-derived, endogenously synthesized compound associated mainly with dysfunction of dopaminergic neurons. Recent data suggest, however, that salsolinol may also be involved in the dopaminergic regulation of prolactin secretion. It has been shown that in rats, the brain salsolinol concentration is elevated during situations when pituitary prolactin secretion is increased. In the present study, we hypothesized that salsolinol was present in the infundibular nucleus-median eminence (IN/ME) of lactating ewes and that its extracellular concentration in the IN/ME increased in response to suckling, similarly to the plasma prolactin concentration. The second hypothesis was that exogenous salsolinol, infused into the third ventricle (IIIv) of the brain, could stimulate prolactin secretion in lactating ewes. Material and Methods Stainless steel guide canullae were implanted under stereotaxic control into the IN/ME (n=6) or into the IIIv (n=10) through a drill hole in the skull in the second moth of pregnancy and two experiments were performed during the fifth week of lactation. 1) Perfusions of the IN/ME with Ringer-Locke solution were performed in ewes bilaterally, from 10:00 h to 15:00 h, by the push-pull method and were divided to the non-suckling and suckling periods, both for 2.5 h. At least 9 or 10 perfusates were collected during perfusion. 2) Pulsatile infusions of salsolinol (5 x 1 μg/20 μl, n=5) or vehicle (n=5) into the IIIv were performed from 12:30 h to 15:00 h, corresponding to the suckling period in perfused ewes. The preinfusion period was from 10:00 h to 12:30 h. In both experiments, plasma samples were collected every 10 minutes, through a catheter inserted into the jugular vein. The perfusate concentration of salsolinol and plasma concentration of prolactin were assayed by HPLC and RIA, respectively. Results The presence of salsolinol, but not dopamine, was detected in the perfusates collected from the IN/ME of lactating ewes. Perfusate salsolinol concentrations during the non-suckling period vs. suckling period were 56.82 ± 13.78 vs. 150.08 ± 16.85 pg/50 μl (mean ± SEM, P
16 t h International Congress on Animal Reproduction Poster Abstracts 145 Materials and methods Four group including eight adult male Balb/C mice weighing 30 5g were used in this study. Normal saline administered as placebo to control group and saffron extract in doses of 25 , 50 and 100 were injected intraperitoneally for 20 days to experimental groups. FSH, LH and testosterone in serum samples, were measured by ELISA. Results In comparison with plasebo the level of FSH, LH and testosterone significantly increased in 100 saffron treated group, but no significant differences were observed between other treatments and placebo. The results of this study indicate the efficacy of saffron extract in the male reproductive endocrine function, and showed that it can modify the reproduction activities. Discussion The results of this research show that the extract of saffron in the mass of 100mg/kg has an important effect on level of FSH and LH and testosterone hormone. Saffron can have a key role in increasing the generative power in male mice. Poster 13 - Molecular Biology of Reproduction P357 Goat as model for studying ovarian differentiation in mammals Auguste, A*; Montazer-Torbati, F; Kocer, A; Pannetier, M; Renault, L; Mandon-Pepin, B; Cotinot, C; Pailhoux, E INRA – UMR Biologie du Développement et Reproduction – 78350 Jouy en Josas - France In mammals, once the chromosomal sex XY or XX is set up at fertilization, the presence of SRY gene (Sex-determining Region of Y) will determine the fate of the gonad by initiating testis differentiation. Following SRY expression, SOX9 the key testis differentiating factor is up-regulated in the embryonic XY gonad thus triggering testis differentiation. In the female counterpart, key genes for ovarian differentiation have been isolated from genetics studies of female-tomale XX sex-reversal. Three loci fit in the class of early ovarian differentiating factors, the PIS locus (Polled Intersex Syndrome) discovered in goat, RSPO1 in human and Wnt4 in mouse. In goat, the pleiotropic PIS mutation leads to a phenotype without horn in both sexes (dominant) and to a female-to-male sex-reversal in all XX individuals homozygous for the mutation (recessive). This mutation is a 11.7 kb deletion encompassing any genes, but acting on the transcriptional regulation of at least 3 genes, PISRT1, PFOXic and FOXL2, lying at 25 kb (PISRT1) and 300 kb (PFOXic/FOXL2) apart the deletion. When the PIS region is present (PIS +/+ and PIS +/- individuals), the 3 genes are highly expressed in the developing ovary, from the onset of their differentiation. This expression is abolished in the ovaries of XX fetuses homozygous for the deletion (PIS -/- ). Concomitantly, the expression of SOX9 increased in these mutant gonads and the first histological sign of sex-reversal appeared. So, it seems that one of the PIS-regulated genes have an “anti-testis” effect. Among the three genes, only FOXL2 encodes a conserved protein belonging to the fork-head box family of transcription factors. In order to better understand the role of these 3 genes, we have restored PISRT1 expression in XX PIS -/- gonad of transgenic goats. As no effect on the sex-reversal phenotype was observed, our current working hypothesis is that FOXL2 is the only gene of the PIS locus responsible for both pathological phenotypes. However, Foxl2 invalidation in mouse leads to an early block of follicle formation but not to sex-reversal. Consequently, we hypothesize that FOXL2/Foxl2 could have additional roles in goat during the early stages of ovarian differentiation, a period that is clearly different between goat and mouse. By contrast to mouse, goat fetal ovaries show an early spatial organization and produce estrogens under the control of FOXL2 before germ cell meiosis. In order to definitely precise the role of FOXL2 in ovarian differentiation of non-rodent mammals, FOXL2 invalidation is currently under process in goat. P358 Identification of genes expressed during sheep ovarian development Baillet, A*; Mandon-Pepin, B; Cabau, C; Poumerol, E; Pailhoux, E; Cotinot, C INRA, UMR 1198; ENVA; CNRS, FRE 2857 Biologie du Développement et Reproduction, Jouy-en-Josas, F 78350, France In mammals, several genes involved in ovarian development have been identified but the molecular cascade leading to a functional female gonad remains poorly documented. Although, in the last decade, progress has been made towards the identification of genes controlling ovarian differentiation using transcriptomic approaches and gene inactivation, most of theses studies have been achieved in mouse. The aim of this work is to identify genes involved in the two main steps of ovarian development in sheep, germ cells meiosis and follicle formation. In contrast to rodents both steps occur during fetal period in several mammals (human, sheep, pig) promoting sheep as a good model to study human ovogenesis and early folliculogenesis. For this study, we used suppressive subtractive hybridization allowing isolation of genes differentially expressed between two developmental stages. This technique was used in order to compare two ovarian developmental stages in sheep: 55dpc (onset of prophase I) and 82dpc (first follicle formation). Two subtractive libraries (55/82; 82/55) were constructed and 7296 clones were recovered. Among them, 6080 clones were sequenced. Using SIGENAE bioinformatics facilities, clones sharing sequence homology were grouped into 2090 unique contigs. Then all contigs were blasted against databases of different mammalian species (human, bovine and ovine) and annotad. In both libraries, 99% of contigs have an Unigene annotation and 1% were unknown. The comparison of the meiosis library contigs with another study of SSH concerning mouse male germ cells revealed 30 genes in common with our library. We have investigated the possible involvement of these 30 transcripts in female meiosis by RT-PCR. Two of them were never described in female and presented a differentially expression pattern. Their expression profile was performed by RT q-PCR in female and male gonads during fetal life in sheep. Both genes showed a high expression level during female meiosis I while their expression remained low in male and during the other stages of ovarian development. Further investigations such as chromosomal localisation in sheep, expression pattern in mouse gonads, cell type origin are currently in progress for these two genes. Moreover, investigations of the folliculogenesis library are in under progress. In parallel, these 2090 genes will be spotted on a nylon membrane to constitute a macroarray dedicated to the ovine ovarian differentiation, in order to evaluate sheep ovarian expression profiles in different physiological or physiopathological conditions. P359 Changes in gene expression of mouse blastocysts treated with high hydrostatic pressure pulse Bock, I 1 *; Mamo, S 2 ; Polgar, Zs 3 ; Pribenszky, Cs 4 1BioTalentum Ltd., Hungary; 2 Genetic Reprogramming Group, Agricultural Biotechnology Center, Hungary; 3 Faculty of Natural Sciences, Constantine the Philosopher University, Slovakia; 4 Faculty of Veterinary Science, St. István University, Hungary High hydrostatic pressure (HHP) treatment of mouse blastocysts before vitrification was reported to increase their post-warming in vitro developmental competence. Similarly, HHP treatment of gametes or embryos has proved to increase substantially the efficacy of the subsequent processes such as vitrification of porcine oocytes, in vitro produced bovine or cloned pig embryos, somatic cell nuclear transfer in pig, bull and boar semen cryopreservation. Proteomic studies have revealed changes in the protein profiles of boar spermatozoa that may relate to increased post-thaw survival and fertility. Here we report the first results of HHP induced alterations in the gene expression of mouse blastocysts.
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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