Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 64 Poster Abstracts defined follicle classes before and after GnRH application. Ovaries containing periovulatory follicles or new corpora lutea (CL day 1-2) were collected at 0h, 4h, 10h, 20h, 25h (follicles) and 60h (CL) relative to injection of GnRH. The MMP-1 mRNA increased rapidly 4h after GnRH (during LH surge) and remained high during the whole experimental period. In contrast, the MMP-19 mRNA increased significantly only after ovulation. The TIMP-1 mRNA increased 4h after GnRH and again after ovulation. Transcripts of VEGF isoforms (VEGF121, 165, 189) were upregulated 4h after GnRH. All VEGF isoforms and their receptors were upregulated again after ovulation. The VEGF peptide content in follicular fluid decreased 20h followed by an increase in follicle class 25h after GnRH. ANPT-1 mRNA decreased significantly in follicles 4h after GnRH. ANPT-2 decreased 10h after GnRH and in the follicle group around ovulation. Tie-1 and Tie-2 mRNA expression decreased in follicle group around ovulation, with a further increase in early CL tissue. It is likely that the decrease of ANPT-1 and therefore the increase of the ANPT-2/ANPT-1 ratio during the LH surge is a basic mechanism of vascular remodelling in follicles during periovulation. In conclusion, the temporal expression pattern of angiogenic factors ANPT and VEGF as well as member of MMP during periovulation suggests them to be important mediators of the ovulatory process and the early CL formation (angiogenesis) in cow. P109 Changes of the cow’s endometrium, distribution of growth stimulating and degradation factors in postparturition period Sematovica, I 1 *; Jemeljanovs, A 1 ; Pilmane, M 2 1Research Institute of Biotechnology and Veterinary Medicine „Sigra”, LUA Latvia; 2 Institute of Anatomy and Anthropology, RSU, Latvia Introduction Remarkable morphological and physiological changes occur in the uterus tissue, blood vessels, and nerves during the reproductive cycle and gestation. AIM of the research was to reveal inflammatory factors, neuropeptide-containing innervation, distribution of the growth stimulating and degradation factors, and the apoptosis in the cows’ endometrium in after parturition period. Materials and methods Nine cows were biopsied twice – in the first and the fifth week after parturition. At the Institute of Anatomy and Anthropology of Riga, Stradins University were performed analyses: immunohistochemically (IMH) were detected matrix metalloproteinases type 2 and type 9 (MMP–2 un MMP–9, working dilution 1:100, R&D, England), tumor necrosis factor–α (TNF–α, 1:100, Abcam, England), interleukin–10 (IL–10, 1:400, Abcam, England), vascular endothelial growth factor (VEGF, 1:50, DakoCytomation, Denmark), nerve growth factor receptors p75 (NGFR p75, w.d. 1:150, DakoCytomation, Denmark) protein gene product 9.5 (PGP 9.5, 1:1600, DakoCytomation, Denmark), and TUNEL method was used for detection of apoptosis. Also staining for hematoxylin and eosin was performed for each sample. Results There were detected a significant increase of the number of inflammatory cells, TNF–α amount, and expression of VEGF, NGFR p75 (p
16 t h International Congress on Animal Reproduction Poster Abstracts 65 P112 Genomic characterization of Arcanobacterium pyogenes isolates recovered throughout the puerperium of dairy cows Silva, E 1 *; Gaivão, M 1 ; Leitão, S 1 ; Jost, B 2 ; Carneiro, C 3 ; Vilela, C 3 ; Costa, L 1 ; Mateus, L 1 1Department of Reproduction and Obstetrics, C.I.I.S.A., Faculty of Veterinary Medicine, Portugal; 2 Department of Veterinary Science and Microbiology, University of Arizona, USA; 3 Department of Microbiology and Immunology, C.I.I.S.A., Faculty of Veterinary Medicine, Portugal In dairy cows, the establishment and persistence of the puerperal uterine infection is related to the characteristics of the bacteria that colonize the uterus. The clinical signs and reproductive consequences of the uterine disease are generally attributed to the presence of a few pathogens, mainly Arcanobacterium pyogenes (A. pyogenes) alone or in synergy with other bacteria. In order to identify factors that might be associated with the establishment and persistence of uterine infection, we characterized A. pyogenes isolates obtained throughout the puerperal period of dairy cows from two unrelated herds. The isolates were recovered from 26 cows (8 had normal puerperium and 18 developed uterine infection). The genomic characterization of A. pyogenes (n= 57) was performed by BOX-PCR typing and by screening of putative virulence factors through conventional PCR. Considering a genomic similarity of at least 86%, a total of ten different clonal types (5 in each herd) were identified. These clonal types were totally herd-specific and a single type appeared to predominate in each herd. Only one clonal type was not associated with endometritis, while four others were always associated with uterine disease. However, 5 clonal types were present in cases of both normal puerperium and endometritis. Throughout the puerperal period, 1 to 3 clonal types were sequentially or simultaneously isolated from the same animal. In order to identify virulence factors associated with the development of infection, all isolates were tested for the presence of 8 putative A. pyogenes virulence genes: plo (encoding pyolysin), nanH and nanP (encoding neuraminidases), cbpA (encoding a collagen-binding adhesin) and four different fimbrial genes (fimA, fimC, fimE, fimG). The 8 genes were present in 66% of the isolates, 5 of 8 genes (plo, nanH, nanP, cbpA, fimA) were detected in all isolates, fimE in 97%, while fimC and fimG were only present in a subset of isolates. There was no association between BOX-PCR type, presence of the above genes and development of uterine infection. The results of this study suggest that the type of A. pyogenes may not be a determinant factor in the development of uterine infection. Host intrinsic factors and/or the synergism between A. pyogenes and other bacteria may play a more relevant role in the establishment of puerperal infections. This research was funded by the grant POCTI/CVT/48773/2002 from “Fundação para a Ciência e Tecnologia”. Maria Elisabete Silva is a post-doc fellow from FCT. P113 Bacteriological and histological studies of cow’s uterus without and with retained fetal membranes Skuja, S 1 *; Antāne, V 1 ; Feldmane, L 2 1Faculty of Veterinary Medicine, Latvian University of Agriculture, Latvia; 2Department of Pathological Anatomy, Riga Stradins University, Latvia Introduction Retention of fetal membranes is economically important disturbances during the postpartum period in cattle in Latvia. Several methods have been suggested for the treatment of retained fetal membranes, and controversial results have been published. The aim of the study was to investigate the postpartum period of cows without and with retained fetal membranes. Materials and methods Bacteriological samples of the cow’s uterus and biopsies of the uterus mucous membranes were collected from 29 Holstein cows. The bacteriological examination was performed in cows with and without retained fetal membranes (RFM) two, 14, 40 – 50 days postpartum (PP). On day 40-50 all cows were biopsied from the uterus endometrium and the uterus status was estimated histologically. According to the 3rd stage of parturition process (expulsion of fetal membranes), cows were divided into the following groups:Control group – fetal membranes were expelled in 6-12 h PP;Group 1 – cows with RFM, removed manually and treated;Group 2 – cows with RFM, it was not removed manually.Group 2 cows with health disturbances (fever, milk fever, mastitis) were treated adequately. Results Bacterial isolates of uterus on day two PP in all cows (100%) contained a wide spectrum of microorganism associations: in 41% of cases there were single isolates – E. coli 33%, Streptococcus spp. 5% and Clostridia spp. 3% , and in 59% there were mixed isolates – E. c. 27%, Streptococcus spp 3%, Clostridia spp 26%, Staphylococcus spp. 3%. On day 14 PP, 93% of cows had microorganisms in the uterus. In addition to the above findings there was one isolate of A. pyogenes in the cow with RFM that was removed manually two days PP. In the next bacteriological examination (40-50 days PP) no bacteria were found, and in 84 days PP after two times of artificial insemination the cow became pregnant. In group 2 cows, which were treated, the number of isolated bacterial species increased on day 14 PP. At the same time cows of group 2, which were untreated, the number of isolated bacterial species remained the previous one, and the number of isolates decreased. On day 40-50 PP, bacterial isolates were found in 86.2% of cows. Cows with a normal parturition process were 50% with single bacterial isolates (Streptococcus spp. and Corynebacteriom spp.). There were no differences between the bacterial species and their number of group 2 treated and untreated cows. In 82.8 % of all investigated cows, histological findings showed evidence of mild to moderate endometritis.At this time, 33.3% ( 8 cows) become pregnant. P114 Identifying AI bulls associated with poor fertility and high rates of embryo loss Sunde, J. 1 * and Refsdal, AO. 2 1Team Semin BA, P.O. Box 8146 Dep., N-0033 Oslo, Norway; 2 Geno Breeding and AI association, N-2326 Hamar, Norway The relationship between fertility and semen quality is not always clear. Occasional AI bulls may show poor fertility even though semen passes quality testing. These bulls represent a potential loss for the dairy farmer, and may even possess undesirable genetic traits that could affect fertility in future generations. It is therefore important that these bulls are identified at an early stage in the breeding programme as soon as insemination data are available for analysis. Norwegian Red (NRF) is the dominant dairy breed in Norway and semen is distributed by Geno, a farmer-owned cooperative. Close to 100% of inseminations are reported back to Geno and 95 % of farmers also report data on calvings, stillbirths and culls through the Norwegian Dairy Herd Recording System. These data allow calculation of accurate non-return rates (NRR) and calving rates (CR) on sire basis. At present, bulls with NRR60 < 65 % after approx. 1000 first inseminations of semen for progeny testing are routinely culled. However, this parameter alone may not be sufficient to identify animals with potential negative impacts on fertility traits. It was therefore investigated whether further analysis of sire fertility data would yield additional information. The data set consisted of 479330 first inseminations on Norwegian Red (NRF) heifers and cows, with semen from 692 qualified NRF test and elite sires. Inseminations were performed between January 2000 and Oct 2007. The first day of return-to-service was registered as a second insemination within 92 days after first insemination (pfi), and a minimum of 1000 first inseminations per bull was required to qualify for the study. NRR curves were modelled using survival analysis, with day of return as outcome parameter, adjusted for confounding factors. Sires were classified as having either‘poor’ or ‘good’ fertility if their NRR92 deviated from the mean by more than 3 standard deviations. Compared to high fertility sires, poor fertility sires showed a more abrupt fall in NRR from 21 days onwards, suggesting a lower rate of fertilisation and/or higher incidence of early embryo death (before maternal recognition). These sires also displayed a higher number of returns-to-service outside of the normal oestral cycle length. This
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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