Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 106 Poster Abstracts P235 Prolactin Activity During the Follicular Phase in the Mare: Evidence of an Extra-Pituitary Prolactin Source King, SS 1 *, Roser, J 2 , Jones, KL 1 1Southern Illinois University Carbondale, IL; 2 University of California, Davis, CA Prolactin (PRL) activity in the equine follicle has been documented in the form of PRL receptors on the corpus luteum 1 and PRL in the follicular fluid 2 (FF). The objective of this study was to identify locations and possible sources for PRL in the follicular phase ovary. Circulating PRL concentrations were analyzed from three studies (61 cycles, 17 mares). Plasma PRL was determined by homologous double antibody RIA 3 . PRL concentrations around ovulation (0-1d postovulation) were compared with the early luteal phase (2-10 d). The granulosa/theca layer was removed postmortem from anonymous mares (n=10) and stored frozen (-80°C). Equine pre-PRL mRNA was extracted and prepared for quantitative PCR using primers and reaction conditions previously described 4 . Equine pituitary mRNA was used to generate a standard curve from which concentrations of pre-PRL mRNA transcripts were determined. Presence of PRL in ovarian structures was determined by immunohistochemistry (IHC). Whole ovaries (n=6) from cycling mares were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 5 um sections. Sections were incubated with R4 PRL rabbit anti-porcine first antibody (DL Thompson, Louisiana State Univ) and goat anti-rabbit IgG-biotinylated (avidin-biotin complex) second antibody. Slides were developed with DAB chromagen-Ni and counterstained with nuclear fast red stain. Equine pituitary served as a positive control. Plasma PRL demonstrated a short-term (1-2 d) increase (P=0.001) around ovulation. Equine pre-PRL mRNA was detected in all granulosa/theca samples in concentrations inversely correlated to follicle size (P
16 t h International Congress on Animal Reproduction Poster Abstracts 107 P238 Immunocytochemical localization of leptin (Ob) and leptin receptor (Ob-R) in preovulatory and in vitro matured horse oocytes related to prepuberty and different weight breeds Lange Consiglio, A 1 *, Arrighi, S 2 , Bosi, GP 2 , Aralla, M 2 , Cremonesi, F 2 1Veterinary Clinical Sciences, Reproduction Unit, University of Milano, Faculty of Veterinary Medicine, Italy; 2 Department of Veterinary Science and Technologies, University of Milano, Faculty of Veterinary Med, Italy The onset of puberty in humans and animals is associated with an increase in fat and consequent increase in circulating leptin, suggesting that leptin may be required for normal growth and development of reproductive organs. Moreover, leptin amount in the blood is proportional to body energy stores and/or body mass, so, inadequate nutrition might impair reproductive function leading, for example, to the delayed onset of puberty. A similar relationship between nutrition and reproductive efficiency is not understood in the mare where, besides many reports quantifying the correlation of circulating concentration of leptin with body condition scores, only few informations exist about the presence of leptin (Ob) and leptin receptor (Ob-R) in the ovary or in the oocyte. Taking this into account, we carried out an immunocytochemical study to investigate the presence of Ob and Ob-R in compact cumulus oocytes recovered from fillies and from mares of light or heavy body weight breeds after slaughtering during the autumnal transition. The occurrence of immunofluorescence was determined by scanning laser confocal microscopy in oocytes immediately upon collection and after in vitro maturation (IVM) using monoclonal antibody conjugated to a fluorescent label. IVM was accomplished in TCM199 supplemented with 1 mM pyruvate, 0.1 IU LH, 0.1 IU FSH, 100 ng IGF, 50 ng EGF, 1 µl ITS and 1 µl estrogen per ml. Oocytes were incubated for 40 h at 38°C at 5% CO2. Both Ob and Ob-R, detected in immature oocytes of all kinds of animals analyzed, were uniformly distributed throughout the ooplasm, but the intensity of reaction was lower either in light weight mares or in fillies oocytes, than in oocytes of heavy weight mares. After IVM, heavy breed mares had a higher proportion of oocytes that reached metaphase II than light mares and fillies (35.53±2.71%, 19,84±1.48% and 15,82±1,52 respectively; P
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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