Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 102 Poster Abstracts P223 Effect of a steroidal anti-inflammatory drug on the viability of equine semen cooled for 24 hours Fioratti, EG*; Melo, CM; Villaverde, AISB; Papa, FO; Alvarenga, MA Department of Animal Reproduction and Veterinary Radiology - Faculty of Veterinary Medicine and Animal Science, UNESP, Botucatu, Brazil Post-breeding endometritis in mares is related to exacerbated inflammatory process, which is stimulated by the presence of sperm cells inside the uterus leading to the major cause of infertility. The use of ecbolic drugs associated with uterine flushing and AI with reduced sperm quantity provides the most common treatment. Based on efficiency of steroidal anti-inflammatory drugs in minimizing uterine fluid accumulation by its systemic administration and its possible intra-uterine use, the present study aimed to evaluate the effect of adding dexamethasone to the extender on equine sperm viability during two hours of incubation at 37°C and after a 24 hours cooling period. Four stallions from different breeds were collected twice using an artificial vagina, obtaining a total of eight ejaculates. After evaluation of sperm motility and concentration, two aliquots containing 800 x 10 6 of viable sperm were diluted in a milk-based extender (Botu-Semen®) reaching a volume of 15 mL for each previous diluted aliquot. These samples were re-diluted with 15 mL of Botu-semen® non-supplemented (control group) or supplemented with 2 mg of dexamethasone (treatment group), resulting in a final concentration of approximately 0.067 mg/ mL. One part of the samples from both groups were cooled at 5°C for 24 hours in an equine semen transport box (Botutainer®) and the other part was incubated in a dry-block at 37°C during two hours. Sperm analyses were performed at 0, 30, 60 and 120 minutes following dilution and after a cooling period of 24 hours using CASA and a combination of fluorescent probes to assess plasma (Iodide Propidium) and acrosomal (FITC-PSA) membrane integrity and mitochondrial transmembrane potential (JC-1). Comparing both groups among all evaluated incubation moments, no significant difference (p < 0.05) was observed for total motility, plasma membrane integrity and mitochondrial transmembrane potential. Progressive motility and percentage of rapid spermatozoa values were higher (p < 0.05) in the control group. The treatment group showed lower (p < 0.05) values for VAP at 30 and 60 minutes and higher (p < 0.05) values for percentage of acrosomal membrane integrity at 0 and lower (p < 0.05) at 60 minutes of incubation compared to the control group. The results obtained in 24 hours cooled samples were not different (p < 0.05) between both groups for all parameters mentioned above, except for VAP, which was lower (p < 0.05) for the treatment group. In conclusion, although the treatment group exhibited poor sperm velocity, the steroidal anti-inflammatory drug did not impair sperm viability. P224 The effect of equine growth hormone and its interaction with gonadotropins, estradiol and fetal calf serum on cytoskeleton distribution in equine oocytes matured in vitro Gabriel Pereira, G 1 *, Liu, IKM 1 , Carneiro, GF 1 , Pegoraro, LM 2 , Lorenzo, PL 3 1Population Health and Reproduction, University of California, Davis, UCD, United States; 2 Animal Reproduction, Temperate Climate Research Corporation-EMBRAPA, Brazil; 3 Animal Physiology Department, Universidad Complutense de Madrid, Spain Microtubules and microfilaments are cytoskeletal components that support cell architecture and modulate cyto- and karyokinesis. We hypothesize that the limited success achieved with in vitro maturation (IVM) of horse oocytes is due to abnormalities associated with cytoskeleton structures. The objective of this research was to investigate the effect and the interactions between equine growth hormone (eGH), estradiol (E2), gonadotropins, and fetal calf serum (FCS) on the IVM and cytoskeleton organization of equine oocytes. Equine oocytes aspirated from each follicle
16 t h International Congress on Animal Reproduction Poster Abstracts 103 (r=0.45). There were not multiple linear regressive models with R- square > 0.2. Conclusions No markers of fresh semen quality useful to predict semen quality after thawing were found. P226 Expression of the Cytokines TNFalpha and IFNgamma and their Receptors in the Cyclic Equine Corpus Luteum Galvão, A 1 *; Silva, E 2 ; Mateus, L 2 ; Korzekwa, A 3 ; Skarzynski, DJ 3 ; Ferreira- Dias G 1 1C.I.I.S.A., Department of Physiology, Faculty of Veterinary Medicine, T.U. Lisbon, Portugal; 2 C.I.I.S.A., Department of Reproduction, Faculty of Veterinary Medicine, T.U. Lisbon, Portugal; 3 Department of Reproductive Immunology, Institute of Animal Reproductive and Food Research of PAS, Olsztyn, Poland Corpus luteum (CL) development is characterized by differentiation and proliferation of cells derived from the postovulatory follicle, and changes in microvascularization. It presents an extremely rapid growth and regression dynamics. This process is controlled by different regulatory factors, such as prostaglandins, growth factors, leukotrienes, cytokines (interferon-gamma - IFNγ, tumor necrosis factor - TNFα, Fas Ligand - FasL) and nitric oxide (NO), among others. The cytokines that are produced locally might have an important role in CL function. Therefore, the main objective of this study was to evaluate the expression of TNFα and IFNγ and their receptors, in equine luteal structures from different stages of the luteal phase. Corpora lutea were obtained post mortem from 16 cyclic mares and classified into early luteal phase CL (Early-CL, n= 5), mid luteal phase CL (Mid-CL; n=6) and late luteal phase CL (Late-CL, n= 5), based on plasma progesterone concentration and ovarian structures. Specific primers for target genes TNFα, IFNγ, TNFα receptor 1 and IFNγ receptor 1, and for housekeeping gene (B2MG), were designed and tested by conventional PCR. After primers optimization, relative quantification of the genes expression was accomplished by Real Time PCR ( ΔΔ Ct method). TNFα expression was four fold reduced in Mid-CL, with respect to Early-CL (p≤0.01), and two fold decreased with respect to Late-CL (p≤0.01). IFNγ presented the lowest expression in Early-CL, increasing afterwards. From Early to Mid-CL a four fold increase for IFNγ was observed (p≤0.01), while from Early-CL to Late-CL there was a nine fold increase (p≤0.01). The expression of TNFα receptor 1 was two fold higher during Early-CL, compared to Mid-CL (p≤0.05) and Late-CL (p≤0.01). IFNγ Receptor 1 did not show a significant variation during the luteal phase. These data suggest that TNFα might play a predominant role during early luteal formation and regression, while IFNγ only shows a significant action during luteal regression. The lack of variation in receptors expression might be explained by the fact that they behave as structural proteins. To the best of our knowledge, these data are the first evidence that both TNFα and IFNγ expression varies in the mare’s CL during the luteal phase. These results suggest an important (auto-, paracrine) role of these cytokines in the regulation of CL function including growth and luteolysis. P227 Endocrinology of the estrous cycle in Miniature ponies Gastal, MO 1 *; Neves, AP 2 ; Petrucci, BPL 2 ; Mattos, RC 2 ; Beg, MA 3 ; Gastal, EL 3 ; Ginther, OJ 1,3 1Eutheria Foundation, Cross Plains, WI 53528, USA; 2 Department of Animal Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil; 3Department of Pathobiological Sciences, University of Wisconsin, Madison, WI 53706, USA Information on mare reproductive endocrinology has relied on studies in larger breeds, owing to the lack of scientific study directly in Miniature mares. Considering the small body size (approximately 100 kg) and selective breeding of Miniature mares, direct studies of the endocrinology, as well as other aspects of reproductive physiology, are needed. The purpose of the present study was to characterize the circulating concentrations of FSH, LH, estradiol, and progesterone during the estrous cycle of Miniature mares. Blood samples were taken daily from 4 days before the first ovulation to 4 days after the second ovulation of the interovulatory interval (IOI). Plasma concentrations of FSH, LH, estradiol, and progesterone were studied daily during 12 IOIs and 21 periovulatory periods in nine Miniature ponies. Assay of ir-inhibin was done only on Days 10 and 18 to represent the days of expected high and low concentrations of FSH, respectively. The peak of the FSH surge that was temporally associated with emergence of the future ovulatory follicle occurred when the follicle was about 9 mm, compared to a reported diameter of 13 mm in larger breeds. The ovulatory LH surge involved a slow increase between Days 13 to 18 (ovulation = Day 0; 0.6 ± 0.1 ng/day), a minimal increase or a plateau on Days 18 to 21 (0.04 ± 0.1 ng/day), and a rapid increase after Day 21 (2.2 ± 0.4 ng/day; P < 0.0001). The end of the plateau and the beginning of the rapid increase in LH occurred on the day of maximum concentration in the preovulatory estradiol surge. An unexpected mean increase and decrease in LH occurred (P < 0.04) on Days 5 to 9. Changes in concentrations of estradiol and progesterone seemed similar to reported results in larger breeds. Concentration of ir-inhibin was greater (P < 0.0001) on Day 18 than on Day 10, consistent with an FSH/inhibin relationship. Apparently, the largest follicle in Miniature ponies begins to produce adequate ir-inhibin for FSH suppression at a smaller diameter than for larger breeds. Results indicated that in Miniature ponies the peak of the FSH surge associated with emergence of the future ovulatory follicle occurred at a smaller diameter of the future ovulatory follicle than in larger breeds, the ovulatory LH surge increased in three phases, and the ovulatory LH surge was followed by an LH increase and decrease during the early luteal phase. P228 Uterine blood flow and perfusion evaluated by color- and power-Doppler ultrasonography in mares with uterine cysts Gastal, EL 1 ; Ferreira, JC 2 ; Ginther, OJ 1,2 1Department of Pathobiological Sciences, University of Wisconsin, Madison, WI 53706, USA; 2 Eutheria Foundation, Cross Plains, WI 53528, USA Uterine cysts have been described in diverse species of animals, including horses, cattle, ewes, pigs, cats, dogs, elephants, and humans, but the etiopathogenesis of uterine cysts is uncertain. Considering the reports of a high incidence of cysts in older mares and reports of degenerative changes in the uterine artery walls in older mares, there may be a relationship among the presence of uterine cysts and decreased uterine vascular perfusion. Transrectal color- and power- Doppler ultrasonography was used to study uterine blood flow and perfusion in mares with and without uterine cysts. Vascular perfusion of the uterus and blood-flow velocities, vascular perfusion, diameter, circumference, and area of a cross section of the mesometrial attachment were evaluated. To study the effect of internal cysts, two matched groups (cystic and control, n = 21 mares/group) were used. Uterine vascular perfusion in mares with cysts was less (P < 0.0001) in the cystic region than in the noncystic region and less (P < 0.0009) than for controls. The vascular perfusion in the uterine regions without cysts did not differ from the controls. Mares with cysts had lower (P < 0.04) pulsatility index (PI) and greater end diastolic velocity (EDV; P < 0.03) and time-averaged maximum velocity (TAMV; P < 0.05) of the mesometrial vessels than controls. To study the effect of size of internal uterine cystic area, paired mares were arranged in four groups (n = 8-11/group): small uterine cystic area (≤ 275 mm 2 ) versus controls and large uterine cystic area (> 410 mm 2 ) versus controls. A small uterine cystic area did not affect mesometrial blood flow. Mares with large uterine cystic area had lower PI (P < 0.05) and greater PSV (P ≤ 0.05), EDV (P < 0.009), and TAMV (P < 0.005). To study the effect of age in mares that were not bred during the last 10 yr, old versus young mares without cysts were compared (n = 11/group). Old mares had greater EDV (P < 0.02) and TAMV (P < 0.01) than young mares, but uterine vascular perfusion was not affected by age. Results demonstrated, for the first time in any species, reduced uterine vascular perfusion in uterine segments that contained cysts and a positive association between size of the cystic
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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