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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A007 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY HIGH FAT DIET AND GENETIC DYSLIPIDEMIA IMPAIRED TESTICLE MORPHOMETRY IN MICE: PRELIMINARY RESULTS Paulo A B Cordeiro, Ana Cristina Silva De Figueiredo, , Miller Pereira Palhão, Iolanda Christina Souza Loyola, Marilu Martins Gioso, Carlos Antônio De Carvalho Fernandes & Jose Antonio D Garcia UNIFENAS, ALFENAS, MG, BRAZIL. High levels of cholesterol and low plasma concentrations of testosterone are associated with a high risk for atherosclerosis. Therefore, cholesterol is the major precursor for sex hormones. Thus, the mice deleted for LDL-receptor gene (LDLr-/-) are related to low tissues influx of cholesterol, which can affect the normal function of the gonads. The aim of this study was to evaluate the effects of high levels of cholesterol on circulating concentrations of insulin and glucose. Additionally, changes in testicle morphometry were evaluated in LDLr-/- mice feeding with different levels of fat. For this purpose, male mice (n = 30) aged 3 months were randomly divided in 3 groups: 1) WT (n = 10) – a wild type mice feeding regular diet; 2) S (n = 10) - gene deleted mice (LDLr-/-) and regular diet; 3) HL (n = 10) - LDLr-/- mice and diet with high levels of fat. Fifteen days after the period of feeding adaptation, blood samples were collected for plasma analysis of the lipids, insulin and glucose. The HOMA index ([Insulin*Glucose]/22,4) was calculated to establish the insulin resistance condition. After testis removal, sectional slices of the tissue were prepared for histological analysis. For each testicle, one microscope slide with 4 sections of the tissue were prepared and stained with Haematoxylin and eosin to assess morphological and morphometric changes. Data were submitted to ANOVA and differences among groups were accessed by Tukey test (5%). The plasma concentrations of HDL were high in S mice, but the insulin resistance was not detected even with moderate hyperlipidemia when compared to WT. For HL mice, very high levels of total lipids were observed (severe hyperlipidemia) and insulin resistance were associated with a decreasing levels of plasma HDL, when compared to S. Pronounced morphological changes of the testicles were observed in HL group. The morphometric analysis had shown an increase in the length and width of the testicles in HL (4.6±0.2 and 7.4±0.2 mm) compared to WT (3.0±0.1 and 5.0±0.2 mm) and S (2.6±0.2 e 5.0±0.2 mm) groups. However, no significant differences were observed for testis (mg)/body (g) weights ratio among groups (4.3±0.1, 4.4±0.1, 4.5±0.2 mg/g for WT, S and HL groups, respectively). At histologic analysis of the testis, the average thickness of the seminiferous tubules was greater in WT (10.0±0.7 µm) when compared to S (6.0±0.3 µm) and HL (6.0±0.4 µm) groups. However, the seminiferous tubule in WT (1.6±0.1 µm) group had smaller diameter of the lumen compared to S (10.7±1.6 µm) and HL (7.5±0.6 µm). In conclusion the genetic dyslipidemia associated with high fat diet induces insulin resistance in mice. Metabolic changes in insulin, glucose and HDL levels affected the testicle structure, mainly in LDLr-/- mice feeding high fat diets. Further analysis of the plasma testosterone concentrations and the seminiferous epithelium can contribute to better elucidate the testicles changes. [Support. FAPEMIG]. Keywords: testoterone, insulin, lipids. A008 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY RECOMMENDED ANTIBIOTIC TIC FOR RAM SEMEN CRYOPRESER OPRESERVATION EXTENDER Elisângela Mirapalheta Madeira, Karina Lemos Goularte, Jorgea Pradieé, Ivan Bianchi, Fábio Pereira Leivas Leite, Rafael Gianella Mondadori, , Arnaldo Diniz Vieira & Thomaz Lucia Jr UFPEL, PELOTAS, RS, BRAZIL. Antibiotics used in ram semen cryopreservation were only tested in bovines, without an assessment of effectiveness in controlling bacterial and the possible impact on ovine spermatozoa. In consequence, this study aimed to determine the bacterial control ability and the influence on sperm viability of different antibiotics for use in freezing extender. Treatments were established using a pool 20x10 9 sperm/mL obtained from the combination of the ejaculates of five Crioula lanada breed rams. In each routine (n = 5), microbiological evaluation and determination of sperm viability were performed before and after freezing. The base extender used was tris-egg-yolk-glycerol without antibiotics (control) or supplemented with antibiotics: gentamicin (500 µg/mL) + tylosin (100 µg/mL) + lincomycin (300 µg/mL) + spectinomycin (600 µg/ mL) = GTLS, penicillin (500 µg/mL) + streptomycin (100 µg/mL) = PENSTREP; sodic ceftiofur (50 µg / mL) = CEFT and enrofloxacin (1000 µg / mL) = ENRO in total dose or concentration 50 and 25% lower (-50 and -25) and higher (+25 and +50) to form the treatments (n = 21). The diluted semen was packaged in 0.25 mL straws, cooled and stabilized at 5° C, frozen in LN2 vapor and stored in a cryogenic container. Sperm viability was determined by evaluating the motility and morphology under phase microscopy and plasma membrane integrity, acrosome and DNA under epi-fluorescence microscopy. Semen samples from each treatment were plated by spreading on agar and brain heart infusion (BHI) and incubated at 37°C/48 h, for colony forming units (CFU) counting. Comparisons between means were made by Tukey test. Plasma membrane integrity, acrosome and sperm DNA, were not affected by treatment (P> 0.05), however, sperm motility was negatively impacted in a dose-dependent manner in ENRO group. The number of CFU in the control group (no antibiotics) was higher (P < 0.05) than in other treatments. The groups PENSTREP and CEFT (in all concentrations used) were less efficient (P < 0.05) than the groups GTLS and ENRO, especially in treatments GTLS+50, ENRO+25 and ENRO+50 which promoted a reduction in the number of CFU. Therefore it is concluded that, despite being effective in reducing the number of CFU, enrofloxacin is not recommended for use in ovine semen extender. The GTLS association, 25% more concentrated than the base, is highly recommended for ram semen extender since controlling bacterial growth without compromising viability. Keywords: viability sperm, antibiotics, ram. s340
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A009 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY QUALIT ALITATIVE TIVE ANALISIS OF THE ESTABLISHED SPERMATOGENESIS IN CAVIA PORCELL CELLUS (LINNAEUS 1758) Adr driana Gradela 1 , Amanda Kar aroline Rodr drigues Nunes 1 , Laura a Flávia Teix eixeir eira Mar artins 1 , Juliana Muniz San antos 1 , Bruna Bor ortolini olini Gouv ouveia 1 , Vanessa Sobue Franzo 2 & Marcelo Domingues Faria 1 1 UNIVASF, PETROLINA, PE, BRAZIL. 2 UFMT, CUIABÁ, MT, BRAZIL. Knowledge of testicular physiology and spermatogenesis are essential for the study of germ line stem cells, which are of great practical value in neoplasms or endangered animals, to safeguard their germ line. In this sense, the Cavia porcellus can be of great importance in animal experiments (Snitkoff. In: Gennaro, A.R.. Remington: a ciência e a prática da farmácia. 20.ed. RJ: G. Koogan, 2004. p.556-68). Our objective was to investigate the development of testicular parenchyma of C. porcellus, evaluating the progress of the process of lumination of the testicular cords. Thirty males from the UNIVASF vivarium – Petrolina- PE were used and divided into six experimental groups with five animals each, within the age of three, five, six, seven, eight and eleven weeks. After fixation and subsequent histological processing of the testicles, were analyzed: the process of seminiferous tubule lumination, the presence of primary spermatocytes, spermatids and the formation of the early stages of the seminiferous epithelium cycle (SEC) according to the tubular morphology method. Animals were classified in stages of testicular development (Courot et al. In: Johnson et al. (Eds.) The testis. NY and London: Acad. Press, 1970. v.1, p.339-432). With three weeks formed gaps of varying sizes even though many testicular cords had persisted with vacuolization in the central cytoplasmic mass and had appeared early primary spermatocytes and round spermatids, featuring the pre-pubertal testicular development. Within five weeks there was seminiferous tubules formed with elongated spermatids, indicating the puberty stage. However this process occurred asynchronously, verifying testicular cords in different stages of vacuolization between the seminiferous tubules already with lumen. After six to seven weeks the tubular lumen was extensive and after eight weeks there was the expansion of the tubular lumen associated with cell proliferation in the seminiferous epithelium and the increase of testicular volume and weight, indicating the post-pubertal stage, with spermatogenesis showed itself established and the presence of associations of the stages of the (SEC). We conclude that in the C. porcellus the period of three weeks of age corresponded to the pre-puberty stage, five to seven weeks of the puberty stage, and from 8 to 11 weeks of post-puberty. Keywords: Cavia porcellus, spematogenesis, process of lumination. A010 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY GLYCER CEROL AND DIMETHYLFORMAMIDE CRYOPRETECT OPRETECTANS ASSOCIATION FOR THE OVINE SEMEN CRYOPRESER OPRESERVATION Rodrigo Freitas Bittencourt 1 , Antônio De Lisboa Ribeiro Filho 2 , Gabriel Felipe Oliveira De Menezes 3 , Renata Cardoso Andrade 4 , Lais Oliveira Mascarenhas 5 , Priscila Assis Ferraz 6 , Alexandra Soares Rodrigues 7 , Marta Vasconcelos Bittencourt 8 & Marcos Chalhoub 9 1,3,4,5,8 FACULDADE DE CIÊNCIAS AGRÁRIAS E DA SAÚDE - UNIME, LAURO DE FREITAS, BA, BRAZIL. 2,6,7,9 ESCOLA DE MEDICINA VETERINÁRIA - UFBA, SALVADOR, BA, BRAZIL. N Ten semen samples of five rams of the Santa Ines breed were cryopreserved with the objective of verifying the use of cryoprotectants glycerol and dimethyl formamide alone (GL6% e DF3%) or in different levels of association (GL5%+DMF1%, GL4%+DMF2%, GL3%+DMF3%, GL2%+DMF4% e GL1%+DMF5%). The base extender was the Tris-egg yolk (TRIS). After evaluation, the semen was diluted in the different extenders, cooled to 5°C and afterward the samples were frozen in liquid nitrogen vapour. After the thawing, the kinetic sperm parameters were analyzed (total motility-TM, progressive motility-PM and sperm vigor-VIG). Aliquots were collected for the supravital test dye eosin (EOS); the sperm morphology was analyzed and the percentage of bent tails (BT) calculated. The functional integrity of the plasmatic membrane was studied by osmotic shock (OS) followed the dilution: One part semen to 10 (OS 10) parts of the final solution with deionized water. After the diluted semen incubation for five minutes at 37°C, it was fixed with 10 µL of formaldehyde buffered saline. The percentage of reactive spermatozoa to OS was determined by subtracting the percentage of spermatozoa with OS-induced BT, by the BT obtained after thawing. These evaluations were observed in phase contrast microscopy (1000x) and one hundred cells were analyzed per semen sample.All the statistical analyses were performed using the SAS software version 5.0 (1996) (Proceeding MEANS and GLM with P < 0.05). The means (%) of post-thaw TM, EOS and OS were, for the GL6%: 78,0 a ; 44,8 and 42,2; DF3%: 47,0 b ; 41,4 and 33,6; GL5%+DMF1%: 73,0 a ; 37,8 and 37,7; GL4%+DMF2%: 73,0 a ; 48,2 and 47,4; GL3%+DMF3%: 71,0 a ; 43,4 and 44,4; GL2%+DMF4%: 52,0 b ; 40,4; 39,0 and GL1%+DMF5%: 43,0 b ; 31,8 and 47,6.It was observed greatest (P < 0,05) rates of TM and PM for the extenders GL6%, GL5%+DMF1%, GL4%+DMF2% and GL3%+DMF3% in relation to the groups with higher levels of DMF (GL2%+DF4% e GL1%+DF5%) or when this one was alone (DF3%). Despite the wide numerical variations, the rates of EOS, OS and BT did not differ (P > 0,05) among the groups. It can be concluded that the DMF and GL association was effective for the sperm viability maintenance, although high levels of DMF, or in the absence of GL, it had deleterious effect on post-thaw ovine sperm. [Financial support. FAPESB- Brazil]. Keywords: ovine, semen, cryoprotectants. s341
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