Fertility analysis of bovine semen by in vitro fertilization.

The aim of the present study was to evaluate the efficiency of using in vitro fertilization to validate semen fertility for artificial insemination. Cryopreserved semen from ten bulls (five Nelore and five Brangus bulls) was evaluated using in vitro production of embryos (IVPE) and via fixed-time artificial insemination (FTAI). There was variation (p < 0.05) in the IVPE (20.9 to 53.7% of blastocyst production) and in the FTAI (42.0 to 56.0% of pregnant cows) results among the semen evaluated. According to the results, there was a positive correlation (rs = 0.8378; p = 0.0001) between the rate of blastocyst production (using IVPE) and the rate of pregnancy (using FTAI) using Nelore bull semen. Variation (p < 0.05) was also found using semen from Brangus bulls, in the rates of blastocyst production (36.5 to 47.0%) and pregnancy (45.6 to 52.2%) via FTAI. There was also a positive correlation (rs = 0.8786; p = 0.0001) between the rates of blastocyst production (IVPE) and pregnancy (FTAI) when using Brangus bull semen. According to the results, IVPE may be used in addition to conventional semen analysis to evaluate and validate the semen fertility of bulls for artificial insemination programs.

Ethanolic Extract of Dried Leaves from the Cerrado Biome Increases the Cryotolerance of Bovine Embryos Produced In Vitro.

In vitro embryo production (IVP) induces excessive production of reactive oxygen species (ROS), which affects blastocyst quality. Therefore, the supplementation of culture media with antioxidants is an alternative to overcome oxidative stress damage. However, there is a growing demand for the use of antioxidant compounds that are more natural and less toxic in cell cultures. The present study is aimed at evaluating the effect of ethanolic extracts from cerrado leaves on IVP. First, the antioxidant capacity and the amount of phenolic compounds of the leaves were evaluated. Then, the best ethanolic extract concentration composed of cagaita (Eugenia dysenterica) and murici (Byrsonima crassifolia) to be used during the in vitro culture of in vitro-produced embryos was determined. Afterward, we evaluated the influence of the extract of both plants on ROS and glutathione (GSH) production, while also evaluating the apoptosis and ROS metabolism gene expression. In a subsequent step, the effect of the ethanolic extracts of dried cagaita and murici leaves during embryonic cultivation on the cryotolerance of expanded blastocysts was studied. The results showed a significant reduction in the proportion of apoptotic cells from embryos cultivated with 0.01 mg/mL of the cagaita ethanolic extract, besides inducing an increase in the GPX4 and PRDX3 transcription levels. The murici ethanolic extract induced an increase in the transcription abundance of these genes but did not reduce the proportion of apoptotic cells. In addition, expanded blastocysts cultivated with extracts at a concentration of 0.01 mg/mL and cryopreserved had higher hatching rates and lower degeneration rates when compared to the frozen group previously supplemented with the extracts. Moreover, the apoptosis rate of embryos cultured for 12 h after cryopreservation was lower in groups previously exposed to extracts during in vitro cultivation. Such extracts may be used as alternatives to increase the cryotolerance of in vitro-produced embryos.

In vitro fertilization: productivity of donors of different bovine breeds.

The objective of the present study was to evaluate efficiency of in vitro fertilization (IVF) in Nelore, Brangus, and Girolando oocyte donors. Ovum pickup (OPU) from the donors was conducted every 15 days to assess oocyte recovery, IVF, and post-transfer pregnancy percentage. For Nelore, the mean numbers of total and viable oocytes recovered (23.5 ± 1.1 and 14.0 ± 1.0, respectively) were higher (p < 0.05) than those for Brangus (12.7 ± 1.9 and 6.6 ± 1.0, respectively) and Girolando (12.5 ± 1.4 and 6.8 ± 0.7, respectively); Brangus and Girolando did not differ from each other (p > 0.05). The percentage of blastocyst production differed (p < 0.05) between Nelore (48.4 ± 2.4%), Brangus (40.3 ± 3.6%), and Girolando (38.9 ± 2.6%), but those in Brangus and Girolando did not differ (p > 0.05). The percentage of blastocysts (transferred) that resulted in pregnancy did not differ (p > 0.05) between Nelore (45.5 ± 3.8%), Brangus (41.7 ± 4.1%), and Girolando (40.7 ± 3.7%). Of the breeds studied, Nelore donors are more efficient for IVF, but conditions of this study.

Oxidative homeostasis in oocyte competence for in vitro embryo development.

The objective of this study was to evaluate the levels of reactive oxygen species (ROS) and glutathione (GSH) in oocytes from follicles of different diameters and their relevance in the in vitro production of embryos (IVPE). Bovine ovaries were aspirated according to the diameter of the follicle [2-8 (general), 4-8 (large), and 2 < 4 mm (small)]. The oocytes were evaluated for levels of ROS, GSH, in vitro maturation, and IVPE. Higher levels of ROS and GSH were observed (p < 0.05) in oocytes of the large group (85.6 ± 7.2 and 140.0 ± 9.6) followed by those in the general (81.1 ± 10.5 and 134.3 ± 7.8) and small (73.5 ± 10.1 and 125.0 ± 10.6) groups. However, the proportion of ROS/GSH did not differ (p > 0.05) between the general, large, and small groups. The maturation was higher (p < 0.05) in the large group (87.8 ± 3.0%) than in the small group (72.2 ± 5.8%), but both were similar (p > 0.05) to that in the general group (82.2 ± 2.5%), whereas the IVPE of the large group (57.3 ± 3.0%) was higher (p < 0.05) than those in the general (44.7 ± 4.4%) and small (34.0 ± 4.0%) groups. We report that oocytes from large follicles are more competent for IVPE, whereas higher levels of ROS and GSH appear to be correlated with oocyte competence, as long as oxidative homeostasis is retained.

Effect of single dose follicle stimulating hormone on follicular aspiration, in vitro fertilization and pregnancy rate / Efeito do hormônio folículo estimulante em dose única na aspiração folicular, fertilização in vitro e taxa de prenhez

The present study evaluated Brangus cows treated with single doses of follicle stimulating hormone (FSH) subjected to follicular aspiration after 24 h to assess oocyte recovery, in vitro fertilization and pregnancy rate. Follicles exceeding 3 millimeters in diameter were aspirated, 200 mg of FSH was administered 2 days later, and a new ovum pickup was performed 24 h afterward. These methods were performed 3 times every 3 days. In control, follicular aspirations occurred at intervals of 1-week without FSH administration o. The aspirated oocytes were evaluated, submitted to in v itrofertilization and the embryos were transferred to the recipients. The average recovery of oocytes was higher (p<0.05) in control cows (12.4±1.8) than in treated cows (9.4±1.3). There was no difference (p>0.05) in the mean percentage of viable oocytes (52.0±3.9 and 62.7±4.7%) or the mean percentage of embryos (41.4±4.8 and 41.5±4.2%) among control and treated cows, respectively. The mean percentage of pregnancy did not differ (p>0.05) for control cows (43.8±2.7%), and treated cows (40.9±6.8%). In conclusion, FSH treatment did not improve oocyte recovery, in vitro fertilization, and pregnancy percentage. However, there is possibility of several consecutive ovum pickup every t3 days, concentrating the in vitro fertilization and the pregnancy percentage.

O presente estudo avaliou vacas Brangus tratadas com doses únicas de hormônio folículo estimulante (FSH) submetidas a aspiração folicular após vinte e quatro horas, para avaliação da recuperação oocitária, fertilização in vitro e taxa de prenhez. Folículos superiores a três milímetros de diâmetro foram aspirados, 200 mg de FSH foram administrados dois dias depois e uma nova aspiração folicular foi realizada 24 horas após. Esses métodos foram efetivados três vezes a cada três dias. No controle, as aspirações foliculares ocorreram em intervalos de uma semana sem administração de FSH. Os oócitos aspirados foram avaliados, submetidos à fertilização in vitro e os embriões foram transferidos em receptoras. A recuperação média dos oócitos foi superior (p<0,05) nas vacas controle (12,4±1,8) do que nas vacas tratadas (9,4±1,3). Não houve diferença (p>0,05) na porcentagem média de oócitos viáveis (52,0±3,9 e 62,7±4,7%) ou na porcentagem média de embriões (41,4±4,8 e 41,5±4,2%) entre vacas controle e vacas tratadas, respectivamente. A porcentagem média de prenhez não diferiu (p>0,05) para as vacas controle (43,8±2,7%) e as tratadas (40,9±6,8%). Em conclusão, o tratamento com FSH não melhorou a recuperação de oócitos, a fertilização in vitro e o percentual de prenhez. No entanto, existe a possibilidade de várias aspirações foliculares consecutivas a cada três dias, concentrando a fertilização in vitro e o percentual de prenhez.

Supplementation of bovine embryo culture medium with L-arginine improves embryo quality via nitric oxide production.

Nitric oxide (NO) is a cell-signaling molecule that regulates a variety of molecular pathways. We investigated the role of NO during preimplantation embryonic development by blocking its production with an inhibitor or supplementing in vitro bovine embryo cultures with its natural precursor, L-arginine, over different periods. Endpoints evaluated included blastocyst rates, development kinetics, and embryo quality. Supplementation with the NO synthase inhibitor N-Nitro-L-arginine-methyl ester (L-NAME) from Days 1 to 8 of culture decreased blastocyst (P < 0.05) and hatching (P < 0.05) rates. When added from Days 1 to 8, 50 mM L-arginine decreased blastocyst rates (P < 0.001); in contrast, when added from Days 5 to 8, 1 mM L-arginine improved embryo hatching rates (P < 0.05) and quality (P < 0.05) as well as increased POU5F1 gene expression (P < 0.05) as compared to the untreated control. Moreover, NO levels in the medium during this culture period positively correlated with the increased embryo hatching rates and quality (P < 0.05). These data suggest exerts its positive effects during the transition from morula to blastocyst stage, and that supplementing the embryo culture medium with L-arginine favors preimplantation development of bovine embryos.

Effect of temporary meiosis block during prematuration of bovine cumulus-oocyte complexes on pregnancy rates in a commercial setting for in vitro embryo production.

Ovum pick up (OPU) associated with in vitro production (IVP) of embryos has been shown as an important tool in cattle breeding to increase the number of descendants from animals of high genetic value. In herds maintained distant from the laboratory, collecting cumulus-oocyte complexes (COCs) and transporting them to the laboratory may take several hours and decrease COCs viability, representing a challenge for commercial settings. In this study, a prematuration culture to induce temporary meiosis block was evaluated in a commercial scale IVP setting as a strategy to transport bovine OPU-derived COCs from Nelore and Brangus donors. Effects on embryo yield and pregnancy rates were assessed. Viable COCs from each donor were destined to one of the experimental groups (control, blocks 1 and 2). Control group COCs were placed in cryotubes with 1 mL TCM199-HEPES. In block groups (1 and 2), COCs were placed in cryotubes with 300 µL TCM 199 + 12 µM butyrolactone I (block medium). All groups were gassed and kept in a thermos bottle for 4 hours at 36 °C. Next, COCs in the control group were transferred to IVM medium and block 1 group to block medium, and cultured for 22 hours and 15 hours, respectively, at 38.5 °C and 5% CO2 in air. Block 2 COCs were kept in the cryotubes and in the thermos bottle for another 15 hours at 36 °C to simulate long-term transport conditions. After meiosis block in prematuration culture, blocks 1 and 2 COCs were matured in vitro for 22 hours as for the control group. After IVM, COCs in all groups were submitted to IVF and IVC, and blastocyst rates were evaluated on day 7. Embryos were transferred and pregnancy rates evaluated at 60 days of gestation. The mean total number of COCs retrieved by OPU did not differ between Nelore and Brangus donors (16.8 and 17.2, respectively, P > 0.05), but Nelore donors produced more viable COCs than Brangus (10.1 and 7.6, respectively, P < 0.05) and more embryos/cow (3.8 and 2.7, respectively, P < 0.05). Blastocyst rates were similar for control (40.2% and 36.7%), block 1 (37.3% and 34.5%), and block 2 groups (34.7% and 33.6%) for Nelore and Brangus cattle, respectively (P > 0.05). Pregnancy rates did not differ regardless of breed or treatment (36.7%, P > 0.05). In conclusion, temporary meiosis block during prematuration culture did not affect embryo development or pregnancy rates; therefore, this strategy may be used to transport bovine COCs in a commercial IVP setting.

Expression of PLIN2 and PLIN3 during oocyte maturation and early embryo development in cattle.

In vitro-produced embryos store high lipid content in cytoplasmic lipid droplets (LD), and reduction or removal of LD has been demonstrated to improve freeze-thaw viability. The Perilipin Adipophilin Tail-interacting Protein of 47 kD (PAT) family of proteins is involved in the formation and regulation of LD in many cell types, but their presence has not been addressed either in cattle oocytes or preimplantation embryos. Therefore, this study aimed to detect the expression of PAT family transcripts (Perilipin-2 [PLIN2] and Perilipin-3 [PLIN3]) in immature and in vitro-matured (IVM) oocytes, and in in vitro-produced embryos at the stages of two to four cells, eight to 16 cells, morulae (MO), and blastocyst (BL). The expression of PLIN3 was downregulated in response to IVM, and PLIN2 was comparatively more expressed than PLIN3 in IVM oocytes (P < 0.001). During the early stages of embryo development, PLIN2 expression reached its peak at the MO stage (P < 0.001) and decreased again at the BL stage. In contrast, PLIN3 was expressed in low levels during the earliest stages of development, slightly upregulated at the MO stage (P < 0.05), and greatly increased its expression at the BL stage (15-fold; P < 0.001). PLIN3 was comparatively more expressed than PLIN2 during embryo culture in most stages analyzed (P < 0.05), except in eight- to 16-cell embryos. These results indicate that PLIN2 might be involved in the maintenance of lipid stocks necessary to support embryo development after fertilization of IVM oocytes. Also, we hypothesize that PLIN3 is the main PAT protein responsible for stabilization of LD formed in consequence of the acute lipid load seen during embryo development. We confirmed the presence of both PLIN2 and PLIN3 proteins in BL at Day 7 using immunocytochemistry: these PAT proteins colocalized with LD stained with BODIPY. PLIN3 seemed to be more ubiquitously spread out in the cytoplasm than PLIN2, consistent with the pattern seen in adipocytes. These findings suggest that both elderly (bigger) and newly formed (smaller) LD, positive for PLIN2 and PLIN3 respectively, coexist in blastocysts. To our knowledge this is the first report showing that transcripts of the PAT family are present in cattle oocytes and embryos.

Influence of L-arginine during bovine in vitro fertilization.

The objective of this work was to evaluate the effect of using L-arginine during in vitro fertilization (IVF) on in vitro embryonic development using Bos taurus and Bos indicus semen. Effect of different concentrations (0, 1, 10 and 50 mM) of L-arginine, added to the IVF medium, was evaluated on the fertilization rate at 18 h post-fertilization (hpf), NO3(-)/NO2(-) production during IVF by the Griess colorimetric method (30 hpf), cleavage and blastocyst rates (on Day 2 and Day 7 of culture, respectively) and total blastocyst cell number (Day 7 of culture). The results reveal that the addition of 50 mM L-arginine to IVF medium, with either Bos taurus or Bos indicus spermatozoa, decreased the cleavage rate and blastocyst rate compared to the control group. Other concentrations did not affect embryo production. However, 1 mM L-arginine with Bos indicus semen increased the proportion of hatched blastocysts. These results indicate that high L-arginine concentrations may exhibit toxic effects on bovine gametes during in vitro fertilization.

Ovogênese e foliculogênese em mamíferos / Oogenesis and folliculogenesis in mammals

Os ovários são órgãos sexuais femininos que tem como principais funções a gametogênese e a esteroidogenese. A gametogênese caracteriza-se pela produção de células reprodutivas femininas ou óvulos e a esteroidogenese pela produção de hormônios esteróides. Em especial nos ruminantes, a formação dos gametas femininos e dos folículos ovarianos é iniciada no período pré-natal. Esta revisão destaca os principais processos morfofisiológicos envolvidos na ovogênese e na foliculogênese. A ovogênese pode ser definida como o conjunto de processos que compreende o desenvolvimento e diferenciação das células germinativas primordiais até a formação do óvulo e sua fecundação. Já a foliculogênese inclui a unidade morfofuncional do ovário que desempenha a função de produção de hormônios esteróides e a de manutenção da viabilidade do óvulo até a ovulação. Com o crescente avanço das biotécnicas de reprodução assistida, a compreensão das funções da ovogênese e foliculogênese é essencial para eficiência reprodutiva dos animais e para obtenção de descendentes viáveis a partir de ovócitos cultivados in vitro.

The ovaries are the female sexual organs whose main functions are the gametogenesis and steroidogenesis. Gametogenesis is characterized by the production of female reproductive cells or ova, and steroidogenesis by the production of steroid hormones. Especially in ruminants, the formation of female gametes and ovarian follicles is initiated in the prenatal period. This review highlights the main morphophysiological processes involved in oogenesis and folliculogenesis. Oogenesis can be defined as the set of processes covering the development and differentiation of primordial germ cells until the formation of the egg and its fertilization. On the other hand, folliculogenesis includes morphofunctional unit of the ovary that serves as the production of steroid hormones and maintenance of the viability of the egg until ovulation. With an increasing breakthrough of biotechnical assisted reproductive functions, understanding of oogenesis and folliculogenesis is essential for reproductive efficiency of animals and for obtaining viable offspring from oocytes cultured in vitro.

Prematuration of bovine oocytes with butyrolactone I reversibly arrests meiosis without increasing meiotic abnormalities after in vitro maturation.

OBJECTIVES: Asynchrony between nuclear and cytoplasmic maturation, and possibly damage to the oocyte meiotic spindle, limits the application of in vitro maturation (IVM) in assisted reproduction. Several studies have suggested that Prematuration with meiosis blockers may improve oocyte quality after IVM, favoring early embryogenesis. Thus, we investigated the effect of Prematuration with the nuclear maturation inhibitor butyrolactone I (BLI) on the meiotic spindle and chromosomal configuration of bovine oocytes. STUDY DESIGN: Immature oocytes obtained from cows slaughtered in a slaughterhouse (n=840) were divided into the following groups: (1) control (n=325), submitted only to IVM in TCM199 for 24h; (2) BLI 18h (n=208) submitted to meiotic blockage with 100 microM BLI for 24h (Prematuration) and then induction of IVM in TCM199 for 18h; and (3) BLI 24h (n=307), pre-matured with 100 microM BLI for 24h followed by 24h of IVM in TCM199. The oocytes were then fixed, stained by immunofluorescence for morphological visualization of both microtubules and chromatin, and evaluated. RESULTS: Meiotic arrest occurred in 90.2% of the oocytes cultured with BLI. Maturation rates were similar for all groups (80.3%, 73.6% and 82.7% for the control, BLI 18h and BLI 24h groups, respectively). We observed 81.3% normal oocytes in metaphase II in the control group, and 80.0% and 81.2% in the BLI 18h and BLI 24h groups, respectively. The incidence of meiotic anomalies did not differ between groups (18.7%, 20.0% and 18.8% for the control, BLI 18h and BLI 24h, respectively). CONCLUSION: Prematuration with butyrolactone I reversibly arrests meiosis without damaging the meiotic spindle or the chromosome distribution of bovine oocytes after in vitro maturation.

Bloqueio da meiose sem aumento da incidência de anomalias meióticas / Blockage of meiosis without increase of meiotic abnormalities incidence

Objetivo: investigar o efeito do pré-maturação com o inibidor de maturação nuclear, butirolactona-I (BLI), sobre o fuso meiótico e distribuição cromossômica de oócitos bovinos, o que poderia melhorar a qualidade oocitária e embriogênese. Material e métodos: oócitos imaturos, obtidos de vacas abatidas em matadouro (n igual 610) foram submetidos nos grupos: Controle (n igual 208) submetidos à maturação in vitro (MI) em TCM 199 por 24 horas; BLI -18 horas (n igual 208) submetidos à pré-maturação com 100 miuM de BLI por 24 horas e posterior indução da MI em TCM 199, por 18 horas; BLI 24 horas (n igual 195), pré-maturados com 100 miuM de BLI, por 24 horas, seguida por 24 horas de MIV em TCM 199. Em seguida, os oócitos foram fixados, corados por imunofluorescência e avaliados. Resultados: o bloqueio meiótico ocorreu em 88,8% dos oócitos pré-maturados. As taxas de maturaçao foram similares (79,3; 73,5 e 82%, respectivamente, para Controle, BLI 18 horas e BLI 24 horas). Observou-se 82,5% oócitos normais em metáfase II no Controle e 80 e 82% nos grupos BLI 18 horas e BLI 24 horas, respectivamente. A incidência de anomalias meióticas não diferiu (17,5; 20 e 18%, respectivamente, para Controle, BLI 18 horas e BLI 24 horas) - p menor que 0,05, teste do x2. Conclusões: a BLI bloqueia a meiose sem promover danos ao fuso meiótico e distribuição cromossômica oocitária após a MIV

Prematuration of bovine oocytes with butyrolactone I: effects on meiosis progression, cytoskeleton, organelle distribution and embryo development.

The effects of prematuration (PM) of bovine oocytes with butyrolactone I (BLI) for 24h on meiosis progression, cell structures and embryo development were assessed. Germinal vesicle (GV) rates decreased (97.4-65.1%, P<0.05) with decreasing BLI concentrations (100-25microM). Without BSA in PM medium, GV rates were similar (98.7-97.2, P>0.05) with low BLI (10-25microM). After in vitro maturation (IVM) for 24h, metaphase II (MII) rates for controls (IVM only) were similar (91.1%, P>0.05) to PM with 10microM BLI in BSA-free medium (B10=91.5%) and 100microM BLI in medium with BSA (B100=92.4%). Meiosis resumption occurred earlier in treated oocytes (71.4-74.3% in GV for B10 and B100, respectively, after 6h IVM compared with 97.3% in controls, P<0.05). By 18h of IVM, most oocytes reached MII (72.0-78.9%, P>0.05). Microtubules and microfilaments were unaffected by BLI. Cortical granules (CG) migration was reversibly blocked by BLI. Mitochondria translocation was partially blocked by PM culture and after IVM more oocytes in B10 and B100 (95.2 and 98.2%, respectively) had mitochondria translocated to a mature pattern (all cytoplasm) than controls (81.5%, P<0.05). Cleavage rates were similar (81-87%, P>0.05), but blastocysts (day 7) decreased in B100 (33.0%, P<0.05) compared with controls and B10 (38.3 and 41.6%, respectively). Day 8 hatching rates (11.0-19.2%) and mean total cell numbers (136-150) were similar (P>0.05). PM did not improve oocyte competence but also did not cause major structural alterations, suggesting that PM may be improved and used to study the mechanisms involved in oocyte differentiation.

Meiotic inhibition with different cyclin-dependent kinase inhibitors in bovine oocytes and its effects on maturation and embryo development.

Cyclin-dependent kinase inhibitors (CDKIs) such as butyrolactone I (BL-I) and roscovitine (ROS) maintain bovine oocytes blocked at the germinal vesicle (GV) stage. Bohemine (BOH), another CDKI, has been used for oocyte activation. The objective of this study was to determine whether BOH blocks meiosis and to compare its efficiency with other CDKIs (ROS and BL-I). Oocytes were cultured for 24 h in 0, 50, 100 and 150 microM BOH to determine the best concentration for blocking meiosis (experiment 1). GV rates were 3.3%, 64.5%, 83.3% and 88.9% (0,50, 100 and 150 microM, respectively). Experiment 2 compared meiotic inhibition efficiency of BOH (100 microM), ROS (25 microM) and BL-I (100 microM). BL-I presented the highest GV rates (97.5%). BOH and ROS were similar to each other (85.4% and 79.9%, respectively). To assess the reversibility of meiotic inhibition (experiment 3), oocytes underwent in vitro maturation (IVM) for 18 h after the 24 h inhibition. Control oocytes were submitted to IVM for 18 h (C18) or 24 h (C24). Maturation rates were either similar to (ROS and BL-I: 96.0% and 93.6%, respectively) or superior to (BOH, 96.9%) C24 (91.0%). All groups were superior to C18 (82.5%). In experiment 4, oocytes were treated as in experiment 3 and then in vitro fertilized and cultured for 8 days. Blastocyst rates for BL-I (32.3%) were similar to C24 (35.0%), while those for BOH (20.2%) and ROS (24.2%) were inferior. All groups were inferior to C18 (43.4%). The results show that: (a) BOH inhibits meiosis resumption; (b) BL-I is the most effective of the CDKIs tested for blocking meiosis; (c) culture of oocytes with meiosis inhibitors is fully reversible in terms of nuclear maturation but they may either decrease (BOH and ROS) or maintain (BL-I) embryo development rates.