Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 110 Poster Abstracts was diluted 1:1 with Botu-Semen ® (skim-milk based extender), split in two aliquots and centrifuged at 600xg for 10 minutes. Pellets were ressuspended using Botu-Crio ® (egg-yolk based extender with aminoacids, 1% glycerol and 4% amides - Biotech Botucatu ® , Brazil), or Botu-Crio ® Egg Free ® extender (same basis without egg-yolk - Biotech Botucatu ® , Brazil), to a final concentration of 100x10 6 sperms/mL. Samples were prepared at room temperature. After ressuspension, samples were loaded into 0.5mL straws, stabilized for 20min at 5°C in refrigerator then placed 6 cm above nitrogen level in isopor box (42L) filled with 3 cm of liquid nitrogen for 20 min and finally immersed. Samples were thawed at 46°C for 20s and maintained in 1.5mL plastic tubes in dryblock at 37°C and evaluated for sperm motility by CASA (IVOS 12) and plasma membrane integrity with fluorescent probes (PI and CFDA). ANOVA and Tukey test were performed by GraphPad InStat program. Results obtained for Botu-Crio ® and Botu-Crio Egg Free ® extenders were: total motility (61.4±16.2 x 61.4±15.9%), progressive motility (27.0±9.8 x 26.7±10.6%), VAP (path velocity) (92.6±13.3 x 97.1±13.8µm/s), VSL (straight line velocity) (73.0±8.8 x 75.0±10.3µm/s), VCL (curvilinear velocity) (167.5±21.7 x 176.3±22.9µm/s), ALH (amplitude of lateral head displacement) (6.7±0.5 x 7.0±0.4µm), STR (straightness) (78.1±5.2 x 77.0±2.4%), LIN (linearity) (46.2±5.1 x 43.3±1.9%) and plasma membrane integrity (53.4±11.5 x 57.4±7.0%), respectively. No difference (P>0.05) was observed between extenders. In conclusion, as Botu- Crio Egg Free ® , which is a completely chemically defined media, has shown similar results when compared to Botu-Crio ® , it may be an alternative for freezing equine semen. Further researches are necessary to evaluate in vivo fertility when using this extender. P248 Vaginal biotic flora and its antibiotic resistance profile in terras de miranda jennets Payan-Carreira, RM 1 *, Mota, VR 2 , Barroso, AT 2 , Quaresma, M 3 , Saavedra, MJ 4 1Zootecnia Dept., University of Trás-os-Montes e Alto Douro, Portugal; 2University of Trás-os-Montes e Alto Douro, Portugal; 3 Veterinary Teaching Hospital, University of Trás-os-Montes e Alto Douro, Portugal; 4 Veterinary Clinics Dept., University of Trás-os-Montes e Alto Douro, Portugal In this work authors report the preliminary results on the characterization of the biotic flora found in the vagina of Terras de Miranda jennets. Vaginal swab samples were obtained from the vestibule and transported in transport medium for delivery to the Microbiology Lab. This work was carried in a group of 21 jennets, aged between 4 to 20 years old and the samples were obtained in two retractions (15 animals + 6 animals). For the bacteriological analysis of the vaginal samples different and selective culture media were used, aiming a more significant bacterial growth. For the preliminary identification of the isolated ones a set of biochemical tests was carried through (oxidase, catalase, indol production, use of the citrate, metil red, vogues proskauer and lactose fermentation). Antimicrobial susceptibility testing was performed by disc-diffusion methods to twenty seven antimicrobials (by CLSI recommendations), including different beta-lactams, quinolone and aminoglicoside antibiotics. From the vaginal samples, bacteria’s pertaining to the different generic groups were found: Gram-positive bacteria, such as Staphylococcus spp. and Enterococcus spp.; and Gram-negative bacteria, such as Enterobactereaceae (E. coli, Klebsiella and Enterobacter) and not Enterobactereaceae (Pseudomonas spp.). Regarding to the resistance profile to antibiotics, the most frequently observed resistance was to amoxicillin and ticarcillin (penicillins), even when associated to the beta-lactamases inhibitor (acid clavulanic), as well as the cephalosporin of 1st generation studied (cephalotin). Resistance to aztreonam (antibiotic beta-lactam of the group of the monobactams) was also observed in some cases. P249 Does the microbial flora in the ejaculate affect the freezeability of stallion sperm Pena, FJ 1 *, Ortega Ferrusola, C 2 , Gonzalez Fernandez, L 2 , Macias Garcia, B 2 , Rodriguez-Martinez, H 2 , Tapia, JA 2 , Alonso, JM 2 1Reproduction, University of Extremadura, Spain; 2 Spain In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze, 3 ejaculates from 5 stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The nonparametric Mann–Whitney U-test was used to directly compare pairs of values. The Spearman non-parametric test was used to study correlations between microbiological findings (presence and quantity, as number of CFU) and sperm quality post-thaw. The isolated microorganisms were: Staphylococcus spp and Micrococcus spp (in all the stallions), β-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp (in stallions 1, 3-5), Rhodococcus spp (in stallion number 2), Pseudomonas spp (in stallion number 1) and Klebsiella spp in stallions 1, 3 and 5. The presence and richness of Klebsiella and β-haemolytic Streptococcus in the ejaculate were related to two sperm variables post-thaw, namely the proportion of dead spermatozoa (EH+ cells; r=0.55, P
16 t h International Congress on Animal Reproduction Poster Abstracts 111 caused by more than one bacterial clone (64%), whereas this was a less pronounced in infections coused by E. coli (36%). Finally, we observed no difference in the genetic diversity among S. equi ssp. zooepidemicus or E. coli isolated from clitoris of mares from the closed herd when compared to isolates from mares kept as on an individual basis. In conclusion, it appears that no specific strain of S. equi ssp. zooepidemicus or E. coli cause infectious endometritis in the mare. Furthermore, positive cytology is a not a reliable tool to evaluate the infectious status as presence of PMN’s was absent in 33% or 67% of infections caused by S. equi ssp. zooepidemicus or E. coli, respectively. P251 Luteal function and ovulation in mares treated with pgf2α during early and mid-diestrus Holland, BE; Pinto, CRF* North Carolina State University, College of Veterinary Medicine, Raleigh, North Carolina 27606,USA PGF 2α is commonly administered as single injection to mares with a mature corpus luteum (days 5 to 7 post-ovulation. In the present study, it was hypothesized that complete luteolysis (plasma progesterone < 1.0 ng/mL) will be achieved in mares receiving PGF 2α for 3 consecutive days beginning as early as 2 days after ovulation (early diestrus). The specific objectives of the present study were to document ovarian dynamics (follicular growth, ovulation, CL formation) using transrectal ultrasonography, and luteal function determined by concentrations of plasma progesterone (P 4 ). The effect of intramuscular administration of 2.5 mg PGF 2α on luteal function (dinoprost tromethamine) given on day 10 after ovulation (Group 1) and on days 2, 3, and 4 (Group 2) was examined in a switch back design utilizing horse mares (n = 10). Transrectal ultrasonography was performed three times per week or once daily on mares in estrus with follicle diameters ≥ 30mm. Blood samples were taken daily and plasma samples were stored at -20 ˚C until RIA analyses for P 4 . Statistical analyses were done by ANOVA, with significance level set at P < 0.05; data are presented as mean ± SEM. All mares in Group 1 (control) underwent complete luteolysis 2 to 3 days after PGF 2α treatment with a mean of 2.4 ± 0.16 days; all mares in Group 1 ovulated 8.2 ± 0.75 days following PGF 2α treatment. In Group 2 (early diestrus), 6 out of 10 mares underwent complete luteolysis 3.3 ± 0.2 days after beginning of treatment on day 2 post-ovulation; these mares ovulated 9.4 ± 1.36 days after treatment. The remaining 4 mares in Group 2 underwent partial luteolysis followed by resurgence in circulating concentrations of plasma P 4 ; 3 out of these 4 mares ovulated with relatively high circulating concentrations of plasma P 4 at day 9 (P 4 = 3.3 ng/ml), 15 (P 4 = 2.99 ng/ml) and 16 (P 4 = 3.29 ng/ml) following treatment, respectively; whereas the remaining mare underwent natural luteolysis 16 days after treatment with its ovulation occurring on day 19 following treatment. Complete luteolysis followed by ovulation was observed in 60% (5/10) of mares treated during early diestrus. We concluded that the equine corpus luteum could be responsive to exogenous PGF 2α as early as 2 days post ovulation. Increasing the dose or frequency of PGF 2α treatment during early diestrus may provide novel protocols for use in broodmare management. P252 Pregnancy diagnosis in the mare by semi quantitative relaxin quick assay kit Ponthier, J 1 *; Van de Weerdt, M-L 2 ; Deleuze, S 1 1Equine Reproduction, Equine Veterinary Teaching Clinic, Faculty of Veterinary Medicine, ULg, University of Liege, B41, 20, Boulevard de Colonster, B-4000 Liege, Belgium; 2 Lab For Vet, 31 Rue Laoureux, B-4800 Verviers, Belgium Few hormonal assays for pregnancy diagnosis are available in the mare. Progesterone assay is not sensitive, not predictive of foetal viability and is useless after day 120 of pregnancy. PMSG or eCG diagnosis is short-windowed (from day 45 to 100) and can give falsepositive results (in case of embryo mortality). Urinary estrogens assay is only reliable in late pregnancy (after day 150) and requires bladder catheterism. Estrone sulfate measurement predicts foetal viability but is inadequate before day 100. Relaxin, a polypeptid produced by the placenta in the mare, the bitch and the queen, is a candidate for the pregnancy diagnosis and the foetal viability prognosis. Rapid immunological tests have been developed to detect relaxin after day 28 of pregnancy in the bitch and 31 in the queen. In the bitch, circulating relaxin measured by immunological procedure is at 1ng/ml at 4 weeks and peaks at 4ng/ml at 6 weeks. In the queen, relaxin is at 5ng/ml at day 31 and reaches 9ng/ml at day 40. In pregnant mares, relaxin detected by chromatography is basal until day 80 and then reaches 80ng/ml for the mare and 10ng/ml for the pony mare. From there on, concentration will increase in the pony and decrease in the horse. In both cases, concentration falls dramatically at parturition. The aim is to study the validity of a commercially available semiquantitative quick immunological relaxin detecting test for dogs and cats in the mare. Serums of ten mares (pony, saddle or draft) are used for Witness Relaxin ® Test (Synbiotics Corporation, France): an ELISA sandwich test. The sample is mixed with specific primary antibodies, then this complex migrates on a membrane and is captured by secondary antibodies. Positive reaction reveals a coloured band. Proper migration on the membrane is confirmed by a second coloured band (witness). Diagnosis and stage of pregnancy are assessed by transrectal or abdominal ultrasonography. Sensibility and specificity are determined and statistical significance is obtained by Fischer’s exact test. Out of the 10 mares, 4 were non-pregnant and 6 were pregnant over 100 days. No Witness Relaxin ® Test did show a positive result, neither for pregnant nor for non-pregnant mares. Sensitivity is 0% and specificity is 100%. Samples of the 6 pregnant mares were sent to the Synbiotics laboratory for a classical ELISA, but none showed a response for the antibodies used. Antibodies of the Witness Relaxin ® Test have been used successfully in various species (dog, cat, mice). Although equine relaxin has a similar structure with two sub-units (A and B) and a comparable molecular weight, these antibodies fail to recognize its sub-unit B. Further studies are required to develop specific equine antibodies. P253 Clinical Causes for Infertility in Jennets on “Planalto Mirandês” Quaresma, M 1 *, Payan-Carreira, RM 2 1Veterinary Teaching Hospital,; 2 CECAV; Univ. Trás-os-Montes and Alto Douro, Vila Real, Portugal A group of 24 jennets were reported by the local breeder’s association to our Veterinary Teaching Hospital with complains of infertility. Twenty-two animals belong to the breed “Asinina de Miranda” (84,6%), a small portuguese breed of no more than 200 females registered in the Book, and 4 were Miranda’s crossbreed (15,4%). The group showed a wide ages distribution, ranging from 3 to more than 15 years old. The gynaecological evaluation of the jennets included the reproductive anamnesis and examination of the external genitalia, vaginoscopic and digital evaluation of the vagina and the evaluation of the uterus and ovaries by transrectal palpation and ultrasonography. The gynaecological examination detected six jennets having structural anomalies: one with an imperforate hymen, two other animals with severe hymenal constriction and two with cervical hypoplasia. Another one evidenced a polyp in entrance of the uterine body. Ultrasonographic examination evidenced bilateral granulosa cell tumours in four of the jennets. It was also found a female with multicystic uterus resembling “suisse cheese”, and isolated uterine cysts in two of the females. In sixteen animals ultrasonographic evaluation demonstrated an apparent normal ovarian activity, despite that most of them being referred by owners as in anoestrus. It seems that in many of those cases the owners were unable to detect oestrus. Although the data presented here was collected in a relatively small number of animals, it may reflect the high consanguinity that we tend to observe in this breed due to the low number of animals available for reproduction.
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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