Ultrastructure of Sheep Primordial Follicles Cultured in the Presence of Indol Acetic Acid, EGF, and FSH

The aim of this study was to investigate the ultrastructural characteristics of primordial follicles after culturing of sheep ovarian cortical slices in the presence of indol acetic acid (IAA), Epidermal Growth Factor (EGF), and FSH. To evaluate ultrastructure of primordial follicles cultured in MEM (control) or in MEM containing IAA, EGF, and FSH, fragments of cultured tissue were processes for transmission electron microscopy. Except in the control, primordial follicles cultured in supplemented media for 6 d were ultrastructurally normal. They had oocyte with intact nucleus and the cytoplasm contained heterogeneous-sized lipid droplets and numerous round or elongated mitochondria with intact parallel cristae were observed. Rough endoplasmic reticulum (RER) was rarely found. The granulosa cells cytoplasm contained a great number of mitochondria and abundant RER. In conclusion, the presence of IAA, EGF, and FSH helped to maintain ultrastructural integrity of sheep primordial follicles cultured in vitro

MANIPULAÇÃO DE OÓCITOS INCLUSOS EM FOLÍCULOS OVARIANOS PRÉ-ANTRAIS E A IMPORTÂNCIA DOS ANTIOXIDANTES – REVISÃO DE LITERATURA

A manipulação de oócitos inclusos em folículos ovarianos pré-antrais (MOIFOPA) vem sendo estudada pensando na perspectiva futura de aplicação direta na reprodução humana, principalmente para mulheres que sofrem de doenças ou que precisam passar por tratamentos que interferem na função ovariana. Nesse contexto, o objetivo deste trabalho é revisar aspectos relacionados com a biotécnica de MOIFOPA e a importância dos antioxidantes no cultivo in vitro de folículos pré-antrais. Foi realizada uma pesquisa na base de dados PubMed, buscando artigos sobre a biotécnica, principalmente relacionados com a necessidade do uso de antioxidantes no cultivo. A grande maioria dos estudos sobre a biotécnica utilizam como modelo experimental os folículos ovarianos de diferentes espécies de animais. A MOIFOPA compreende o isolamento e o resgate de folículos ovarianos pré-antrais provenientes de ovários, seguido da conservação através da técnica de resfriamento ou congelação e o cultivo folicular in vitro, a fim de promover o crescimento, a maturação e a fecundação in vitro (FIV) dos oócitos inclusos nesses folículos, maximizando o potencial reprodutivo feminino e diminuindo a atresia folicular que acontece in vivo. Um aspecto que pode interferir no sucesso do cultivo in vitro de folículos ovarianos pré-antrais é a produção em excesso de espécies reativas de oxigênio (EROs). Os ácidos ascórbico e alfa lipóico vem demonstrando resultados interessantes para reduzir os efeitos que as EROs causam sobre os folículos ovarianos pré-antrais cultivados in vitro

Low versus high antral follicle count on the fertility of timed AI Nelore heifers

Esse estudo teve como objetivo determinar o efeito da contagem de folículos antrais (CFA) e da maturidade sexual na fertilidade de novilhas de corte. Neste estudo, 230 novilhas Nelore, com 20 ± 2 meses de idade, foram submetidas a um protocolo de inseminação em tempo-fixo (IATF) a base de estradiol e progesterona. No Dia 0 do protocolo de IATF as novilhas foram examinadas por ultrassonografia transretal e vídeos dos ovários foram gravados para posterior CFA (≥ 3 mm) realizada na sala escura do laboratório de imagens. Trinta dias após a ultrassonografia, as fêmeas que falharam na primeira IATF foram ressincronizadas com o mesmo protocolo hormonal. A prenhez foi avaliada por ultrassonografia 30 dias após cada IATF. A média geral da CFA foi de 22 folículos; assim, as novilhas foram divididas em 2 grupos de acordo com a CFA: CFA baixa (˂ 22 folículos, n=114) e CFA alta (≥ 22 folículos, n=116). A prenhez por IA (P/IA) foi semelhante (P > 0,05) entre os grupos CFA baixa e alta e entre novilhas púberes e pré-púberes. A P/IA não foi diferente entre as novilhas que apresentaram ou não cio (P = 0,2). No entanto, novilhas com CFA alta que apresentaram cio tiveram maior P/IA (P = 0,01) do que novilhas com CFA alta que não apresentaram cio. Em conclusão, a CFA e a maturidade sexual não afetaram a fertilidade de novilhas. Por outro lado, a P/IA das novilhas que apresentaram cio foi maior do que das novilhas não observadas em cio apenas no grupo CFA alta. This study aimed to determine the effect of antral follicle count (AFC), and pubertal status on the fertility of beef heifers. In this study, 230 Nelore heifers, 20±2 months of age, were subjected to an estradiol progesterone-based timed artificial insemination (TAI) program. On Day 0 of the TAI protocol, the heifers were examined by transrectal ultrasound to record videos of the ovaries. Later, in the darkroom of the laboratory of images, the videos were analyzed for AFC (≥ 3 mm) of each ovary. Females who failed the first TAI were resynchronized with the same hormonal protocol. The pregnancy status was evaluated by ultrasonography 30 days after each FTAI. The general mean of the AFC was 22.0 follicles. Thus, the heifers were divided into 2 groups according to AFC: Low AFC (˂ 22 follicles, n = 114), and High AFC (≥ 22 follicles, n = 116). No differences (P > 0.05) in the pregnancy per AI (P/AI) were observed between the Low and High AFC groups, and between pubertal and prepubertal categories. The P/AI was not different between heifers that displayed or did not estrus (P = 0.2). However, considering the estrus response of each AFC group, High AFC heifers that displayed estrus had greater P/AI (P = 0.01) than High AFC heifers that did not display estrus. In summary, AFC and pubertalstatus did not affect the fertility of Nelore heifers. In contrast, the P/AI of heifers that did not display estrus was lower than heifers observed in estrus only in the High AFC group.

Evaluation of Scrotal Temperature in Different Testicular Shapes by Infrared Thermography in Braford Bulls

Background: The mechanisms of testicular thermoregulation may influence the blood flow provided by the testicular artery, where the proper blood supply to the testicle is crucial for promotingspermatogenesis and reproductive function in bulls. The size and shape of the testicles are determined by genetic mechanisms and environmental effects. A better understanding of the relationships between the anatomical characteristics of the testicles and scrotum can support a better reproductive assessment. The purpose of the current study was to evaluate the testicular temperature of different scrotal shapes using infrared thermography in bulls.Materials, Methods & Results: We evaluated 132 Braford bulls with an average age of 24 months. The evaluation of breeding bull semen was performed prior to the beginning of the experiment. Then, animals were selected on the basis of the size of their testes, which was determined by dividing the average width by the average length. The scrotal circumference was measured with a millimeter tape positioned around the largest circumference. Testicular and ocular temperature measurements and analysis were conducted using an infrared thermal camera, Flir T440 with emissivity of 0.98 and thermal sensitivity of 0.05°C. Testicular scrotum temperature and testicular shape were analyzed with one-way ANOVA using Minitab 16, and values of P < 0.05 were considered statistically significant. We observed that 67.42% of testicle shapes were long-oval, and 32.58% were long-moderate. The testicular temperature was higher in bulls with the long-moderate shape compared to those with the long-oval shape (P < 0.05). The mean length was higher in long-moderate shaped testicles compared to those of the long-oval shape (P < 0.01). There was no significant differences in rectal and ocular temperatures or in scrotal circumference between bulls with long-moderate and long-oval shapes (P > 0.05). In addition, the mean width was lower in testicles of long-moderate shape compared to those of the long-oval scrotal format (P < 0.01).Discussion: The results obtained showed that Braford bulls with the long-moderate testicular shape have a higher testicular temperature to maintain proper thermoregulation. The present study demonstrated that IRT can be used to evaluate the testicular temperature in animals with different scrotal conformations. In this study, Braford bulls showed lower length and width values for animals having long-moderate (9.21 and 5.22, respectively) and long-oval formats (8.56 and 5.56, respectively). In contrast, previous reports examining Nellore bulls between the ages of 17-20 months found a predominance of the long oval shape followed by the long-moderate shape, which indicates a change in testicular shape as age progresses, resulting in a rounder testicular shape. Perhaps other factors, such as the external cremaster muscle and tunica dartos, cause the testicles to be retracted towards the body at lower temperatures while at high temperatures, relaxation occurs. The prevailing testicular shape in Braford animals with a mean age of 24 months was the long-moderate shape. Thus, testicles with a larger surface area will have lower temperatures because they can dissipate heat more easily than testicles with lower surface area. The results suggest that the long-moderate scrotum format may influence the testicular temperature in mature Braford bulls

Proliferative activity of oocytes in multi-oocyte follicles of bovine ovary

We characterized the proliferative activity of multi-oocyte follicles with anti-nuclear antigen of proliferating cells (PCNA). Ovaries (n = 12) from heifers were processed for histology. From 789 multi-oocytes follicles observed, only 11 were considered appropriated for immunostaining, since they presented all nuclei of the oocytes clearly visible. All multi-oocyte follicles were positive for PCNA, but some oocytes showed no proliferative activity. We conclude that oocytes in multi-oocyte follicles seem to be in different stages of the cell cycle

Puberdade e parto na contagem de folículos antrais de fêmeas Nelore de corte

The objective of this work was to investigate whether puberty and parturition alter the antral follicle count (AFC) in female Nelore (Bos indicus) beef cattle. Two experiments were carried out to compare AFC between the prepubertal and pubertal periods and between the prepubertal and postpartum periods. AFC and follicle concentrations did not differ between the prepubertal and pubertal periods. However, the ovarian area increased after puberty. In addition, AFC and the ovarian area were greater in the postpartum than in the prepubertal period. Therefore, AFC is not affected by the puberty of Nelore heifers, but increases significantly when the prepubertal heifers become cows.O objetivo deste trabalho foi avaliar se a puberdade e o parto alteram a contagem de folículos antrais (CFA) em fêmeas Nelore (Bos indicus) de corte. Foram realizados dois experimentos para comparar a CFA entre os períodos pré-púbere e púbere e entre os períodos pré-púbere e pós-parto. A CFA e a concentração folicular não diferiram entre os períodos pré-púbere e púbere. Entretanto, a área ovariana foi maior após a puberdade. Além disso, a CFA e a área do ovário foram maiores no período pós-parto quando comparado ao pré-púbere. Portanto, a CFA não é afetada pela puberdade de novilhas Nelore, mas aumenta significativamente quando as novilhas pré-púberes se tornam vacas

Inseminação artificial em tempo fixo utilizando progesterona injetável: dinâmica folicular ovariana e taxas de prenhez de vacas Nelore (Bos indicus) com e sem um corpo lúteo

This study evaluated the follicular dynamics and the pregnancy rates of Nelore cows with or without a CL synchronized with FTAI employing injectable progesterone (P4). Multiparous cows (n=44), 45-65 days postpartum were allocated into a 2x2 factorial. Eleven cows with and 11 without a CL received a conventional protocol of FTAI (D0/1 g P4 + 2 mg EB; D8/500 ?g cloprostenol, 300 IU eCG and 24 h later 1 mg EB; D10/FTAI). Other 11 cows with and 11 without a CL received 350 mg of injectable P4 (300 mg sc and 50 mg im) and 2 mg of EB im on D0. On D6, 500 ?g of cloprostenol and 300 IU of eCG were administered im. After 24 h, 1 mg of EB was administered im and FTAI was performed on D8. The results were analyzed by ANOVA and Tukey or by Chi-square test (p?0.05). The presence of follicles ? 5mm on D4, follicular growth rate and CL diameter were similar between groups (50-55%, 1.1-1.2mm/ day and 16.1-17.6mm; respectively). The diameter of the dominant follicle (DF) at FTAI and ovulatory follicle (OF) were similar between groups with CL (10.5±1.8 and 11.4±1.3mm, respectively) and without CL (10.8±2.3 and 11.1±2.2mm, respectively) but device was higher than injectable (11.7±2.0 and 11.8±1.7 vs. 9.7±1.7 and 10.6±2.0mm, respectively). The ovulation and pregnancy rate were similar between groups with CL (59% and 36%, respectively) and without CL (77% and 27%, respectively) but device was higher than injectable (91% and 45% vs. 45.5% and 18%; respectively). The ciclicity did not affect the follicular dynamics and pregnancy rate. The device showed higher diameter of the DF at FTAI, OF and ovulation and pregnancy rates. However, the injectable P4 produced similar percentages of follicles ? 5 mm on D4, follicular growth rate and CL diameter, raising the possibility of further investigation.Este estudo avaliou a dinâmica folicular e a taxa de prenhez de vacas Nelore, com ou sem CL sincronizadas com protocolo de IATF empregando progesterona injetável (P4). Vacas multíparas (n=44), 45-65 dias pós-parto foram alocados em um fatorial 2x2. Onze vacas com e 11 sem CL receberam um protocolo convencional de IATF (D0/1 g P4 + 2 mg BE; D8/500 mg cloprostenol, 300 UI eCG e 24 h depois 1 mg BE; D10/IATF). Outras 11 vacas com e 11 sem CL receberam 350 mg de P4 injetável (300 mg SC e 50 mg IM) e 2 mg de BE IM no D0. No D6, 500 mg de cloprostenol e 300 UI de eCG foram administradas IM. Após 24 h, 1 mg de BE foi administrado IM e IATF foi realizada no D8. Os resultados foram analisados por ANOVA e teste de Tukey ou Qui-quadrado (p?0,05). A presença de folículos ? 5 mm no D4, a taxa de crescimento folicular e o diâmetro do CL foram semelhantes entre os grupos (50-55%, 1,1-1,2mm/ dia e 16,1-17,6mm, respectivamente). O diâmetro do folículo dominante (FD) na FTAI e do folículo ovulatório (FO) foram semelhantes entre os grupos com CL (10,5±1,8 e 11,4±1,3 mm, respectivamente) e sem CL (10,8±2,3 e 11,1±2,2 mm, respectivamente), mas foi maior no grupo dispositivo comparado ao injetável (11,7±2,0 e 11,8±1,7 vs. 9,7±1,7 e 10,6±2,0mm, respectivamente). A taxa de ovulação e de prenhez foram semelhantes entre os grupos com CL (59% e 36%, respectivamente) e sem CL (77% e 27%, respectivamente), mas foi mais elevada no grupo dispositivo comparado com o injetável (91% e 45% vs. 45,5% e 18%, respectivamente). A ciclicidade não interferiu na dinâmica folicular e na taxa de gestação. O dispositivo apresentou maior diâmetro do FD na IATF, do FO e das taxas de ovulação e prenhez. No entanto, a P4 injetável produziu percentagens semelhantes de folículos ? 5 mm no D4, de taxa de crescimento folicular e diâmetro do CL, levantando a possibilidade de novas investigações.

Ovarian follicular dynamics of Nelore (Bos indicus) cows subjected to a fixed-time artificial insemination protocol with injectable progesterone

O objetivo deste estudo foi avaliar a dinâmica folicular ovariana de vacas Nelore submetidas à inseminação artificial em tempo fixo (IATF) com progesterona injetável (P4) ou dispositivo intravaginal. No grupo dispositivo, 20 vacas receberam um dispositivo intravaginal com 1 g P4 e 2 mg benzoato de estradiol (BE) por via intramuscular (im) no D0. No D8 o dispositivo foi removido e 500 ?g cloprostenol, 300 UI gonadotrofina coriônica equina (eCG) e 1 mg cipionato de estradiol (CE) foram aplicados, im. As vacas foram inseminadas 48h após o CE. No grupo injetável, 16 vacas receberam 350 mg P4 (300 mg, subcutânea e 50 mg, im) e 2 mg BE im no D0. No D6, 500 mg cloprostenol, 300 UI eCG e 1 mg CE foram administradas im. A IATF foi realizada 48h após o CE. No grupo dispositivo, 65% (13/20) e no grupo injetável 75% (12/16) das vacas apresentaram folículos ? 5 mm no D4 (p>0,05). A taxa média de crescimento folicular foi de 0,92±0,25 mm/dia para o grupo dispositivo e 1,06±0,45 mm/dia para o grupo injetável (p>0,05). O diâmetro médio do folículo dominante na IATF foi 11,4±1,4 mm para o grupo dispositivo e 10,8±1,8 mm para o injetável (p>0,05). O diâmetro médio do folículo ovulatório foi 12,1±1,4 mm no grupo dispositivo e 12,3±1,6 mm no grupo injetável (p>0,05). A ovulação ocorreu em 90% (18/20) do grupo dispositivo e 37,5% (6/16) do grupo injetável (p0.05). The average rate of follicular growth was 0.92±0.25 mm/day for the device group and 1.06±0.45 mm/day for the injectable group (p>0.05). The average diameter of the dominant follicle (DF) at FTAI was 11.4±1.4 mm for the device group and 10.8±1.8 mm for the injectable group (p>0.05). In the device group, ovulatory follicles (OF) had an average diameter of 12.1±1.4 mm, and in the injectable group 12.3±1.6 mm (p>0.05). Ninety percent of the device group (18/20) and 37.5% of the injectable group (6/16) demonstrated ovulation (p<0.05). The average diameter of the corpus luteum (CL) was 18.7±4.3 mm for the device group and 17.7±3.6 mm for the injectable group. The injectable P4 showed similarity in the presence of follicles ? 5 mm on D4, follicular growth rate, diameters of the DF, OF and CL. However, the ovulation rate was higher in the device group.

Ultrastructural analysis of bovine oocytes from ovarian follicles with different diameters<br>Análise ultra-estrutural de oócitos bovinos provenientes de folículos ovarianos com diferentes diâmetros

In vitro embryo production is an important technique for facilitating the reproduction of animals with high genetic merit. The greatest challenge for the reproducibility of this technique is the quality of the oocyte that is submitted for in vitro maturation. The aim of this work was to evaluate the ultrastructural characteristics of oocytes from follicles of different diameters using transmission electron microscopy. The animals were divided into 2 groups and were given a single i.m. injection of 250 IU FSH (Pluset, Serono, Italy). To synchronize the follicular growth, all follicles > 2 mm were aspirated during the estrous cycle, which was considered day zero (D0). Group 1 (G1; n = 4) received FSH on day 1 (D1), and the 2- to 5-mm follicles were aspirated on day 2 (D2). The animals in group 2 (G2; n = 5) received FSH on day 2 (D2), and their 10- to 15-mm follicles were aspirated on day 5 (D5). After aspiration, the oocytes from both groups were fixed and prepared for ultrastructural analysis. The oocytes analyzed from both groups had similar ultrastructural characteristics. The presence and distribution of organelles in the cytoplasm of the oocytes did not differ between groups, suggesting that, in relation to the ultrastructural characteristics, oocytes from 2 to 5 mm and 10 to 15 mm follicles are similar.<p><p>A produção in vitro de embriões é uma técnica importante para facilitar a reprodução de animais com elevado mérito genético. O maior desafio para a reprodutibilidade desta técnica é a qualidade do oócito destinado à maturação in vitro. O objetivo deste trabalho foi avaliar as características ultra-estruturais de oócitos provenientes de folículos com diferentes diâmetros por microscopia eletrônica de transmissão. Os animais foram divididos em dois grupos e receberam uma única injeção im de 250 UI de FSH (Pluset, Serono, Itália). Para sincronizar o crescimento folicular, todos os folículos > 2 mm foram aspirados durante o ciclo estral, que foi considerado o dia zero (D0). O Grupo 1 (G1, n = 4) recebeu FSH no dia 1 (D1), e os folículos de 2 a 5 mm foram aspirados no dia 2 (D2). Os animais do grupo 2 (G2, n= 5) receberam FSH no dia 2 (D2), e os folículos de 10 a 15 mm foram aspirados no dia 5 (D5). Após a aspiração, os oócitos de ambos os grupos foram fixados e preparados para análise ultra-estrutural. Os oócitos analisados de ambos os grupos apresentaram características ultra-estruturais semelhantes. A presença e distribuição de organelas no citoplasma dos oócitos não diferiram entre os grupos, sugerindo que, em relação às características ultra-estruturais, oócitos oriundos de folículos de 2-5 mm e 10-15 mm são similares