João Henrique Moreira Viana

Follicular dynamics in zebu cattle

Objetivou-se neste trabalho estudar a dinâmica folicular em vacas da raca Gir (Bos indicus). Oxa0crescimento e a atresia folicular foram monitorados diariamente ao longo de ciclos estrais completos (nxa0=xa015), utilizando-se um aparelho portatil de ultra-som. Axa0atividade luteal em cada ciclo foi avaliada pela determinacao do nivel de progesterona plasmatica. Foram observados ciclos com duas (6,67%), tres (60,00%), quatro (26,67%) e cinco (6,67%) ondas de crescimento folicular. Nao houve diferenca significativa (P>0,05) nas taxas de crescimento e atresia de foliculos dominantes ou subordinados, entre ondas. Oxa0diâmetro maximo do foliculo ovulatorio foi maior (P 0,05) no comprimento do ciclo (21,11±1,76 e 22,25±1,71 dias) ou na concentracao de progesterona durante o diestro (4,48±1,45 e 5,08±1,40xa0ng/mL), entre ciclos com tres ou quatro ondas de crescimento. Esses resultados demonstraram que a dinâmica folicular na raca Gir e caracterizada pela predominância de ciclos com tres ou quatro ondas de crescimento folicular, associados a baixa persistencia dos foliculos dominantes das ondas intermediarias.

Interrelationships among morphology, echotexture, and function of the bovine corpus luteum during the estrous cycle.

It has been suggested that ultrasound image attributes are a potential indicator of physiological and functional status of the corpus luteum (CL) in several species, including cattle. The aims of this study were to evaluate CL morphological, functional and echotextural characteristics, and also to investigate the hypothesis that those attributes are correlated and change similarly throughout an estrous cycle. Ovaries of crossbred (Bos taurus taurus x Bos taurus indicus) heifers were evaluated using ultrasonography daily throughout an interestrus interval using a B-mode, real-time ultrasound machine equipped with a 5 MHz linear-array rectal transducer, during a natural estrous cycle (Experiment 1; n=12) or during a shortened cycle, with luteolysis induction 10d after estrus (Experiment 2; n=6). Blood samples were collected for assay of plasma progesterone concentrations. Corpora lutea areas were measured and daily images of each CL were videotaped and digitized for computer-assisted analysis using custom-developed software. In Experiment 1, area of luteal tissue increased until a maximum value 10d after estrus (P<0.001), followed by a plateau phase, and then a decline beginning 14 d after estrus. Luteal tissue area was highly correlated to plasma progesterone concentrations (r=0.86; P<0.001). When luteolysis was induced in Experiment 2, loss of CL function (decrease in plasma progesterone concentrations to metestrous values) preceded tissue regression by 48 h (24h compared with 72 h; P<0.001). The mean pixel value of ultrasound images did not change in Experiment 1 (P>0.70), but a day effect on this attribute was observed in Experiment 2 (P=0.052). In contrast, mean pixel value was correlated to plasma progesterone concentrations in Experiment 1 (r=-0.63; P<0.05), but not in Experiment 2 (r=-0.28; P>0.10). In regard to CL heterogeneity, defined as the standard deviation of the mean pixel value of the luteal tissue, a time effect was observed following both natural (Experiment 1; P<0.009) and luteolysis-induced (Experiment 2; P<0.05) estrous cycles (P<0.05). Moreover, this variable was correlated with plasma progesterone concentrations (r=-0.71 and -0.58 in Experiments 1 and 2, respectively; P<0.01), indicating that CL images were more heterogeneous during metestrus and after luteolysis (functional regression). In summary, morphological and echotextural attributes were correlated with CL function and underwent similar changes during the estrous cycle. Luteal tissue heterogeneity, assessed by ultrasonography, is considered a potential indicator of CL functional status, because it is correlated to circulating progesterone concentrations.

Autoclaved, previously used intravaginal progesterone devices induces estrus and ovulation in anestrous Toggenburg goats

Intravaginal progesterone devices are used worldwide for estrus induction in goats. Reused devices are able to induce estrus; however, this can be a health risk within a flock. The objective was to compare new and previously used (and autoclaved) progesterone-releasing intravaginal devices for induction of estrus and ovulation in seasonally anestrous Toggenburg goats. Anestrous goats (n=42) received new intravaginal devices containing 0.3g progesterone (CONTROL), or similar devices previously used for either 6 (USED6) or 12d (USED12) and subsequently autoclaved. All goats received 5mg dinoprost at device insertion and 200 IU eCG 5d later, and all devices were removed after 6d. After device removal, estrus was monitored and females displaying signs of estrus were mated by fertile bucks. Transrectal ovarian ultrasonography was performed after device removal until detection of ovulation. Blood samples were collected for determination of plasma progesterone concentration at different times. There was no difference (P>0.05) among groups CONTROL, USED6 or USED12 for: estrus response (87, 100 or 100%, respectively); duration of estrus (32.3±2.3, 25.2±3.4 or 27.3±4.1h); ovulation rate (100, 88 or 100%); number of ovulations (1.5±0.2, 1.9±0.3 or 1.7±0.3); and pregnancy rate (60, 58 or 67%). Plasma progesterone (P4) concentrations were greater (P<0.05) in CONTROL than in USED6-treated and USED12-treated goats (7.2±1.2, 4.7±0.7 and 4.3±0.6 ng/mL, respectively) at 6h after device insertion; these differences were maintained until 4d after device insertion (3.4±0.4, 2.3±0.2, and 2.5±0.2 ng/mL). Overall, plasma progesterone concentrations were greater (P<0.05) in nulliparous than in lactating goats (3.1±0.8 compared to 2.4±0.6 ng/mL, respectively). In conclusion, autoclaved, previously used intravaginal progesterone-releasing devices resulted in significant lesser plasma progesterone concentrations than new devices, but were similarly effective in inducing estrus and ovulation in anestrous goats.

Gestation length, birth weight and offspring gender ratio of in vitro-produced Gyr (Bos indicus) cattle embryos

In vitro embryo production (IVP) has been suggested to result in a greater proportion of male calves, longer gestation and heavier offspring than artificial insemination in Bos taurus cattle. Despite the increasing use of IVP in tropical countries, its effects upon these traits in Bos indicus have not been conclusively investigated. Gyr is a B. indicus dairy breed with known physiological differences from B. taurus, such as a longer gestation period and lighter offspring. The aim of this study was to evaluate the effect of IVP on gestation length, birth weight and gender ratio in Gyr offspring. Oocytes were recovered from Gyr cows by ovum pick-up and were matured and fertilized with thawed Gyr semen in vitro. Embryos were cultured in CR2aa medium with cumulus cells and 10% fetal calf serum under 5% CO(2) at 38.5 degrees C in air. Seven- to eight-day blastocysts were transferred to synchronized recipients. Data on gestation length and birth weight of calves from in vitro-produced embryos were compared to data obtained from Gyr calves produced by artificial insemination (AI) and natural breeding (NB) during the same period using analysis of variance, and the gender ratio was compared to the expected 1:1 ratio using a chi-square test. IVP increased (P<0.01) the percentage of male offspring (76.9%) compared to the expected 1:1 ratio, while no difference (P>0.05) was observed in the AI and NB groups. Gestation length was similar (P>0.05) between the IVP and AI groups, but IVP-derived offspring were heavier (P<0.05) than AI- and NB-derived ones, mainly for male calves (P<0.05). These data show that in vitro production affects the subsequent development of Gyr embryos, resulting in a skewed sex ratio and increased birth weight.

Developmental competence and expression of the MATER and ZAR1 genes in immature bovine oocytes selected by brilliant cresyl blue.

The objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus-oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 degrees C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB- (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB- group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB- (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB- (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB- groups. Despite the relative expression of MATER in holding control, BCB+ and BCB- were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.

Osmotic challenge and expression of aquaporin 3 and Na/K ATPase genes in bovine embryos produced in vitro

The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900 mOsm) for 5 min following 120 min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (P<0.05) dehydration. Embryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P<0.05). In the experiment 2, the amount of Aqp3 and ATPase1 transcripts were quantified in blastocysts with high or low rehydration after exposure to hypertonic medium. No difference (P>0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72 h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (P<0.05) embryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (P<0.01) amount of Aqp3 transcripts was found in the vitrified-warmed embryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system.

Correlação morfométrica do ovário de fêmeas bovinas em diferentes estádios reprodutivos

The aim of this study was to evaluate morphometric characteristics of bovine ovaries recovered on different reproductive stages. The ovaries used (N=200) were recovered at slaughterhouses and sent to the laboratory, were the size of the ovary, of follicles and of corpus luteum were measured. The pairs of ovaries were classified according to the reproductive stage, and to the estrous cycle or pregnancy stages of the animals from which they were recovered. There was a difference (P 0,05) on organ size between animals presenting estrous cycles or pregnancy, but pregnant cows presented larger corpora lutea and smaller diameter of the largest follicle (P<0,05). Evaluated parameters were affected by estrous cycle and pregnancy stages (P<0,05). There was a significant correlation between ovary and corpus luteum sizes in the animals presenting estrous cycles (R=0.69; P<0,001). In conclusion, ovarian morphologic characteristics changes according to the reproductive stages, could be used as parameters for clinic and functional evaluations of the organ.

Caracterização de seqüelas subseqüentes à punção folicular em bovinos

Objetivaram-se avaliar lesoes no trato genital em animais utilizados como doadores de oocitos. Foram examinadas vacas da raca Gir (n=20) que participaram de diferentes experimentos envolvendo a tecnica de puncao folicular. Ao termino de cada periodo experimental, os animais foram examinados por palpacao retal, ultra-sonografia e vaginoscopia. Um grupo de vacas (n=8) foi selecionado para a caracterizacao histopatologica das lesoes ovarianas. Os ovarios foram retirados cirurgicamente, fixados e processados, e as lâminas obtidas coradas e analisadas. Foram avaliados animais submetidos a entre nove e 42 sessoes de puncao, e que produziram entre 10 e 719 oocitos. Observou-se uma baixa prevalencia (5,26%) de processos inflamatorios na mucosa do fundo de saco vaginal. A ocorrencia de aderencias apresentou uma correlacao positiva com o numero de sessoes de puncao a que o animal foi submetido (r=0,31; P 0,05). Tendencia oposta foi observada em relacao a fibrose ovariana, que apresentou uma alta correlacao com o numero de oocitos recuperados (r=0,53; P<0,05). A avaliacao histopatologica demonstrou a presenca de tecido cicatricial, infiltrados de celulas inflamatorias e presenca de tecido luteal disperso no estroma ovariano.

Características andrológicas de touros da raça Gir

Gyr bulls were ranked regarding their reproductive potential, following evaluations through the score-based andrological classification system (ACP). For the examinations, three animal age groups were considered: 18 to 24 month-old (G1, n=33), 25 to 31 month-old (G2, n=24), and 32 to 38 month-old (G3, n=12). Scrotal circumference was lower in G1, as compared to G2 and G3 (P<0.05). Percent motility and vigor were not affected by the age groups. Percent rates of major and total defects were higher (P<0.05) in G1 than in G2 or G3. No significant differences were observed when G2 and G3 were compared. Regarding ACP data, G1, G2 and G3 yielded, respectively, 25.7, 58.3 and 61.5% of adequate breeders; 11.4, 20.8 and 15.4% of bulls with undefined breeding capacity, and 57.1, 12.5 and 15.4% of inapt breeders or immature animals. Age group affected scrotal circumference and major and total defects, but did not influence motility or vigor. The highest proportion of adequate breeders was obtained with 24-month-old bulls and older.

Luteal dynamics in goats: morphological and endocrine features

Objetivou-se neste estudo estabelecer as caracteristicas morfologicas e endocrinas da dinâmica luteal em cabras. Foram utilizadas femeas da raca Toggenburg que manifestaram estro natural em um intervalo de 48 horas. Apos o estro, foram realizadas avaliacoes ultrassonograficas diarias dos ovarios durante 21 dias, utilizando-se um aparelho portatil (5 MHz). Amostras de sangue foram coletadas para dosagem de progesterona (P4) no plasma. Os corpos luteos foram detectados pela primeira vez no D5 e aumentaram progressivamente de tamanho ate o D9 (1,26 ± 0,08 cm2), nao havendo variacao nos dias subsequentes. Nas femeas com uma ovulacao, a primeira visualizacao do corpo luteo foi mais precoce que naquelas com ovulacao multipla (4,54 ± 0,18 vs 5,74 ± 0,25 dias). No momento da primeira visualizacao, a area luteal foi menor nos animais com uma ovulacao. A concentracao plasmatica de P4 aumentou progressivamente ate o D9 e nao apresentou aumento significativo ate o momento da luteolise, caracterizada por uma acentuada queda da concentracao de P4, atingindo valores inferiores a 1 ng/mL em um intervalo de 24 horas. A area luteal diminuiu de forma lenta e gradual. Foi observada uma correlacao positiva significativa entre a area e a concentracao de P4 durante a lutegeonese e a luteolise (r = 0,63 e r = 0,50; respectivamente). No dia em que o corpo luteo atinge sua area maxima (D9), as femeas com mais de um corpo luteo, com maior area de tecido luteal, nao apresentaram concentracao de P4 superior a daquelas com uma ovulacao (5,92 ± 0,59 vs 7,04 ± 0,79 ng/mL). Esses resultados indicam que a dinâmica luteal em caprinos da raca Toggenburg segue padroes semelhantes aos observados em outras racas e em outras especies e que o crescimento de tecido luteal refletiu positivamente na funcionalidade do corpo luteo.