Comparison between the additive effects of diluted (rFSH) and diluted/dynamized (FSH 6 cH) recombinant follicle-stimulating hormone on the in vitro culture of ovine preantral follicles enclosed in ovarian tissue.

OBJECTIVE: This study compared 2 types of recombinant follicle stimulating hormone (rFSH): diluted and diluted/dynamized, on in vitro development of ovine follicles. METHODS: In experiment 1, ovarian fragments were cultured for 1 or 7 days in α-MEM(+) in the absence or presence of different concentrations of diluted rFSH to determine the best concentration. In experiment 2, the effect of diluted and diluted/dynamized rFSH (rFSH 6 cH--ultradiluted and succussioned), alone or in combination, was studied. RESULTS: In experiment 1, compared to control, 50ng/mL of diluted rFSH induced higher rates of follicular survival after 7 days of culture and higher percentages of growing follicles at day 1 of culture (P<0.05). In experiment 2, compared to control, diluted/dynamized rFSH induced higher follicular diameter and survival rate after 7 days and early follicle activation at day 1 of culture (P<0.05). Compared to diluted rFSH, diluted/dynamized rFSH induced higher rates of follicle activation at day 1 of culture (P<0.05). CONCLUSION: In conclusion, compared to the control medium, diluted/dynamized rFSH promoted survival and early activation of follicles, while diluted rFSH promoted higher activation later in the culture. Thus, diluted/dynamized rFSH may be used as an alternative to diluted rFSH for the in vitro culture of ovine preantral follicles.

Interaction between melatonin and follicle-stimulating hormone promotes in vitro development of caprine preantral follicles.

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM(+)) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM(+) alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM(+) alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.

Induction of reversible meiosis arrest of bovine oocytes using a two-step procedure under defined and nondefined conditions.

The objective was to study the effect of a defined culture system, on nuclear and cytoplasmic maturation of bovine oocytes, using the two-step procedure of IVM to detect possible inhibition and subsequent resumption of meiosis arrest. In the first step, called the prematuration period (PMP), COCs were cultured in T1-non-defined medium (NDM), or T2-defined medium (DM), both for 24 h. In step 2, called the resumption period (RP), COCs were cultured in: NDM (T1); DM + NDM (T3); or DM+DM (T4) for 24 h in each medium. The NDM was composed of TCM-199 supplemented with FCS and FSH. The DM was composed of alpha-MEM supplemented with PVA, insulin, IGF-1, androstenedione, nonessential amino acids, transferrin, and sodium selenium. Oocytes from T2 had a lower (P < 0.05) rate of nuclear maturation (19.8%) than T1 oocytes (83.2%). Also, T2 COCs appeared to be in the process of cytoplasmic maturation, according to the distribution of organelles assessed by transmission electron microscopy (MET). These COCs had characteristics previously described as mature: erect microvilli on the plasmembrane, presence of cortical/evenly distributed mitochondria throughout the ooplasm, and presence of 50% aligned/cluster cortical granules. Immature characteristics such as small PvS, compact cumulus cells, and presence of 50% cortical granule clusters were also observed. The T1 COCs had only characteristics of maturation (P < 0.05). In step 2 (RP), meiosis arrest induced by DM was resumed after an additional 24 h of culture in NDM (T3) with 79.2% mature COCs, whereas in T4, meiosis arrest was maintained, resulting in almost 70% immature COCs (P < 0.05). At the end of RP, T3 COCs had the mature characteristics of mitochondria spread throughout the cytoplasm (P < 0.05), cumulus expansion, and alignment of cortical granules, whereas the T4 group had both immature and mature characteristics. We inferred that DM can be used in lieu of meiosis inhibitors and furthermore, it can provide extra time to study nuclear and cytoplasmic maturation synchrony of IVM.

Effects of alpha-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured / Efeitos dos antioxidantes alpha-tocoferol e ternatina na morfologia e na ativação de folículos pré-antrais caprinos cultivados in vitro

The effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15µM, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2 percent and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture.

Os efeitos do α-tocoferol e da ternatina sobre morfologia, ativação e crescimento de folículos pré-antrais caprinos cultivados in vitro, por um ou cinco dias, foram avaliados. Os fragmentos ovarianos foram imediatamente fixados (controle não-cultivado) ou cultivados in vitro, por um ou cinco dias, em Meio Essencial Mínimo (MEM) com ou sem suplementação com α-tocoferol ou ternatina nas concentrações de 5, 10 ou 15µM, formando os tratamentos MEM, TOC5, TOC10, TOC 15, TER5, TER10, TER15. O percentual de folículos pré-antrais normais no controle não-cultivado foi de 73,2 por cento, depois de cinco dias de cultivo, houve redução desse percentual em todos os tratamentos, quando comparados com o controle não-cultivado (P<0,05). O cultivo por cinco dias aumentou a ativação folicular em todos os tratamentos (P<0,05). A análise ultra-estrutural não mostrou folículos pré-antrais íntegros após cinco dias de cultivo em meio contendo antioxidantes. Concluiu-se que o α-tocoferol e a ternatina podem promover a ativação folicular, no entanto a adição desses antioxidantes nas concentrações testadas reduziu a viabilidade folicular após o cultivo in vitro.

Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm.

Freeze-drying sperm is an alternative to cryopreservation. Although sperm from various species has been freeze-dried, there are few reports for bovine sperm. The primary objective of this study was to evaluate the protective effect of various freeze-drying media on the structural and functional components of bovine sperm. The media tested were composed of TCM 199 with Hanks salts supplemented with 10% fetal calf serum (FCS) and TCM 199 with Hanks salts supplemented with 10% FCS and 0.2 M trehalose and EGTA solution. The efficiency of each medium on the preservation of freeze-dried sperm structures was evaluated with conventional and electron microscopy, DNA integrity was analyzed by a TUNEL assay, and fertilizing ability of lyophilized sperm was determined with ICSI. Although the plasma membrane was damaged in all media tested, mitochondria were similarly preserved in all freeze-drying treatments. The acrosome was best preserved in the media that contained trehalose (other treatments also conserved this structure). In contrast, media containing EGTA or trehalose most effectively preserved the nuclei in freeze-dried sperm, with only 2 and 5%, respectively, of cells with fragmented DNA. Furthermore, sperm conserved with these media also had higher (P<0.05) rates of sperm head decondensation (32.5 and 27.5%), pronucleus formation (37.5 and 45.0%) and blastocyst formation (19.4 and 18.3%) than medium supplemented with FCS (15.0, 20.0 and 10.2%, respectively). In conclusion, media with EGTA and trehalose adequately protected bovine sperm during freeze-drying by preserving the viability of their nuclei.

Effects of chronic exposure to soy meal containing diet or soy derived isoflavones supplement on semen production and reproductive system of male rabbits.

Soy and derivative diets deliver large doses of isoflavones to human and animals throughout their lifespan, including gestation. Epidemiologic and experimental data suggest that the consumption of soybean containing foods may protect against cardiovascular disease and decrease breast, prostate and endometrial cancer risk. Based on animal and in vitro studies, however, concerns have been raised that consumption of isoflavones may cause potential adverse effects on the reproductive tract and behavior. The aim of this study was to investigate the effects of chronic consumption of a soy meal containing diet or soy isoflavones supplement on the morphology of reproductive organs, semen quality, age that males reached puberty, and sexual behavior of male rabbits. With this purpose, 16 female rabbits were randomly assigned to receive: (1) a soy- and alfalfa-free diet; (2) a soy- and alfalfa-free diet supplemented with 5mg/kg body wt./day of soy isoflavones; (3) a soy- and alfalfa-free diet supplemented with 20mg/kg body wt./day of soy isoflavones; (4) a diet containing 18% of soy meal, throughout the gestation and lactation. After weaning, male offspring received the same diet, which was given to the respective mother. The age that males reached puberty, semen characteristics and sexual behavior were evaluated in these animals. At 33 weeks of age, the reproductive organs were submitted to histological evaluation. Rabbits, which received large amounts of isoflavones (20mg/kg body wt./day) had a lesser food intake, body weight and semen volume. Spermatogenesis, morphology of male genital organs and sexual behavior did not differ significantly from the control group. We conclude that chronic dietary treatment with soy based diet or soy isoflavones have no adverse effects on the observed reproductive patterns of male rabbits.