Maria Helena Tavares Matos

This study evaluated the effect of epigallocatechin-3-gallate (EGCG) in ovine oocyte in vitro maturation (IVM) medium. Oocytes were assigned to IVM in four experimental groups: CON, composed of TCM-199 supplemented with 500 IU penicillin, 0.5 mg streptomycin, 1.25 µg amphotericin B, 0.2 mM sodium pyruvate, 10 % (v/v) FBS, and 15 IU/mL eCG; and the EGCG25, EGCG50, and EGCG100 groups, where they were matured in the same medium as the CON group, supplemented with 25 µM, 50 µM, and 100 µM EGCG, respectively. At the end of the maturation period, 1.053 oocytes were used for evaluation of cumulus cell expansion, chromatin configuration, and quantification of active mitochondria, reactive oxygen species (ROS), and glutathione (GSH) levels. 391 oocytes was used for in vitro fertilization (IVF) and in vitro embryo culture (IVC). The results showed that the EGCG50 group had higher rates of MII, active mitochondria, and GSH than the other groups, as well as a higher cleavage rate than the CON group. In conclusion, a concentration of 50 µM EGCG improves oocyte maturation rate and in vitro ovine embryo production.

The present study aimed to evaluate the effect of N-acetylcysteine (NAC) on in vitro maturation (IVM) and in vitro embryo production in sheep. Cumulus–oocyte complexes (COCs) were collected and matured in vitro in a control medium or supplemented with different concentrations of NAC (1, 1.5 and 2.0 mM). After maturation, a subset of oocytes was used for morphological assessment and chromatin configuration analysis, as well as for evaluating intracellular levels of reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and DNA fragmentation using the TUNEL assay. Another subgroup of oocytes was subjected to in vitro fertilization (IVF), and the presumed zygotes were cultured in in vitro culture (IVC) medium to assess cleavage rates. Data were statistically analysed, considering significance at P < 0.05. The concentration of 1 mM NAC resulted in lower ROS levels compared to the control and 1.5 mM groups, in addition to reducing DNA fragmentation. No significant differences ( P > 0.05) were observed in GSH levels or mitochondrial activity between treated and control groups. Furthermore, oocytes treated with 1 mM NAC showed higher cleavage rates compared to the other experimental groups. In conclusion, supplementation with 1 mM NAC improves oocyte quality and early embryonic development, as demonstrated by reduced oxidative stress and DNA fragmentation, resulting in increased cleavage rates.

Oxidative stress resulting from the inherent conditions of in vitro culture can compromise oocyte quality. In this regard, there is a growing interest in the use of natural products to prevent oxidative damage and to promote in vitro follicular development. This study was conducted to evaluate the effects of the addition of acidified extract of grape industrial residue (Vitis vinifera cv. Syrah) as a supplement to the base medium for in vitro culture of isolated sheep secondary follicles. Secondary follicles were isolated and cultured for 12 days in α-MEM+ (control medium) or in α-MEM+ supplemented with different grape residue extract concentrations (0.1, 0.2, or 0.4 mg/ml). Follicular morphology, antrum formation, follicular and oocyte diameter, glutathione (GSH) levels, mitochondrial activity, DNA fragmentation and meiotic resumption were evaluated. After 12 days of culture, there was no difference (P >0.05) among the treatments in relation to morphology, antral cavity formation, follicular diameter, DNA fragmentation and the percentage of fully grown oocytes (≥ 110 µm). Furthermore, the GSH levels were similar (P >0.05) among the α-MEM+, 0.2, and 0.4 mg/ml grape residue extract groups at the end of culture. Nevertheless, oocytes from secondary follicles cultured in 0.4 mg/ml grape residue extract showed higher (P <0.05) mitochondrial activity and greater meiotic resumption than oocytes cultured in the control medium. In conclusion, supplementation with 0.4 mg/ml grape residue extract maintained survival, improved mitochondrial activity, and promoted the meiotic resumption of oocytes from sheep secondary follicles cultured in vitro.

The aim of this study was to evaluate the effect of different water restrictions on the thermoregulation and blood hematological and metabolite parameters of crossbred Santa Inês ewes in a semi-arid climate. Thirty-two ewes were subjected to four water supply levels (100%, 80%, 60%, and 40%), in a completely randomized design with eight replications. The confinement period lasted 77 days, with 14 days allocated for adaptation. Respiratory rate, heart rate, and rectal temperature exhibited a quadratic response. There was an increase in red blood cells and urea. The enzyme alanine aminotransferase decreased linearly with water restriction. Urinary creatinine decreased along with water supply. Regarding urine color characteristics, all groups showed different colors, ranging from clear to cloudy. For the chemical characteristics of urine, a quadratic effect was observed for pH, with the highest value (8.75) at 60%. An increase was observed in total urine proteins and urobilinogen. Crossbred Santa Inês ewes in a semi-arid climate exhibit physiological adaptations to water supply reduction up to 40%. Following an 80% reduction in water supply, animals exhibit mild dehydration, characterized by increased serum urea levels and decreased alanine aminotransferase activity.

Visfatin is an adipokine involved in the regulation of female reproduction. However, there are no studies on the effects of visfatin on the in vitro culture of ovarian tissue in any species. Therefore, the aims of this study were to analyze the effects of visfatin on survival, primordial follicle activation, granulosa cell proliferation, and the immunostaining of tumour necrosis factor-α (TNF-α) in preantral follicles after the in vitro culture of sheep ovarian tissue. Ovarian fragments were fixed for histological analysis (fresh control) or cultured in α-minimum essential medium alone (α-MEM+: control medium) or in α-MEM+ supplemented with different concentrations of visfatin (1 or 10 ng/mL) for 7 days. Subsequently, ovarian tissue was destined to histology (morphology, activation and growth) and immunohistochemistry (granulosa cell proliferation and pro-inflammatory cytokine TNF-α immunostaining). The results indicated that treatments with visfatin (1 or 10 ng/mL) maintained the percentage of morphologically normal follicles at a level similar (P>0.05) to the fresh control and significantly higher than of α-MEM+. A significant increase in primordial follicle activation was also observed in tissue cultured for 7 days at both visfatin concentrations compared to the fresh control and α-MEM+. In addition, only the treatment containing 10 ng/mL of visfatin significantly increased follicular and oocyte diameters, and granulosa cell proliferation compared to α-MEM+, and attenuated inflammatory activity by reducing TNF-α immunostaining after in vitro culture. In conclusion, 10 ng/mL visfatin maintains survival, reduces immunostainig of TNF-α and promotes the activation of primordial follicles by stimulating granulosa cell proliferation after the in vitro culture of sheep ovarian tissue.

Summary: The aims of this study were to evaluate the doxorubicin concentration that induces toxic effects on in vitro culture of isolated mouse secondary follicles and to investigate whether resveratrol can inhibit or reduce this toxicity. Secondary follicles were isolated and cultured for 12 days in control medium (α-MEM⁺) or in α-MEM⁺ supplemented with doxorubicin (0.1 µg/ml) or different concentrations of resveratrol (0.5, 2, or 5 µM) associated with doxorubicin (0.1 µg/ml) (experiment 1). For experiment 2, follicles were cultured in α-MEM⁺ alone or supplemented with doxorubicin (0.3 µg/ml) or different concentrations of resveratrol (5 or 10 µM) associated or not with doxorubicin (0.3 µg/ml) (experiment 2). The endpoints analyzed were morphology (survival), antrum formation, follicular diameter, mitochondrial activity, glutathione (GSH) levels and DNA fragmentation. In the first experiment, doxorubicin (0.1 µg/ml) maintained survival and antrum formation similar to the control, while 5 µM resveratrol showed increased parameters, maintained mitochondrial activity and increased GSH levels compared to the control. In the second experiment, doxorubicin (0.3 µg/ml) reduced survival, antrum formation and follicular diameter compared to the control. Resveratrol at a concentration of 10 µM attenuated the damage caused by doxorubicin by improving follicular survival and did not present DNA fragmentation. In conclusion, supplementation of the in vitro culture medium with 0.3 µg/ml doxorubicin reduced the survival and impaired the development of mouse-isolated preantral follicles. Resveratrol at 10 µM reduced doxorubicin-induced follicular atresia, without DNA fragmentation in the follicles.

Os objetivos deste estudo foram verificar o efeito da rutina sobre a maturação in vitro (MIV) de oócitos provenientes de folículos secundários de ovelhas cultivados in vitro e analisar o possível envolvimento da via mTOR na MIV, sob influência da rutina. Os folículos secundários foram cultivados por 18 dias em meio α-Mínimo Essencial (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, glutamina, hipoxantina, transferrina, selênio, ácido ascórbico e leptina (meio controle: α-MEM+). Em seguida, os folículos foram avaliados quanto à morfologia, formação do antro e diâmetro folicular e taxa de oócitos totalmente crescidos (≥110 µm). Oócitos totalmente crescidos foram submetidos à MIV em meio de cultivo de tecidos 199 (TCM199) suplementado com soro fetal bovino (FBS), hormônio luteinizante (LH), hormônio folículo estimulante recombinante (rFSH) (meio controle MIV) ou neste meio com 0,1, 1 ou 10 µg.mL-1 de rutina. Ao final da MIV, os oócitos foram avaliados quanto à atividade mitocondrial, concentração de espécies reativas de oxigênio (ERO) e glutationa (GSH), porcentagem de retomada de meiose, fragmentação de DNA e envolvimento da via mTOR. Após 18 dias de cultivo in vitro, 77,5% dos folículos estavam normais e 77,7% tornaram-se folículos antrais, com 380,41 µm de diâmetro. Além disso, 70% dos oócitos que cresceram in vitro atingiram diâmetro ≥110 µm e foram submetidos à MIV. A concentração de 10 µg.mL-1 de rutina aumentou significativamente a porcentagem de oócitos que retomaram a meiose (47,27%) em comparação ao meio controle (30,43%). Houve um aumento significativo nas concentrações de ROS e GSH em oócitos maturados com 0.1 µg.mL-1 de rutina em comparação com os outros tratamentos com rutina (p < 0,05). Além disso, a maturação de oócitos em TCM199+ aumentou (p0,05). Em conclusão, a concentração de 10 µg.mL-1 de rutina aumentou a retomada da meiose e reduziu os danos ao DNA.

This study was conducted to evaluate the effects of the acidified extract of M. oleifera leaves as a supplement into the base medium for in vitro culture of sheep isolated secondary follicles. Follicles were isolated and cultured for 12 days in α-MEM+(supplemented with bovine serum albumin, insulin, glutamine, hypoxanthine, transferrin, selenium, and ascorbic acid) with or without 0.1; 0.2 or 0.4 mg/ml of the acidified extract of M. oleifera. Follicle morphology, antral cavity formation, follicular and oocyte diameter, glutathione (GSH) concentration, mitochondrial activity and meiotic resumption were evaluated. After 12 days of culture, there was no significant difference among treatments in relation to follicular morphology, antral cavity formation, diameter and mitochondrial activity. Nevertheless, oocytes from follicles cultured in α-MEM+ showed greater GSH concentration than media containing M. oleifera extract. Furthermore, the concentration of 0.4 mg/ml M. oleifera extract significantly increased the percentage of fully grown oocyte (≥ 110 µm) when compared to the other treatments. In conclusion, the concentration of 0.4 mg/ml M. oleifera extract as a supplement of the culture medium, maintained the survival, and increased the percentage of fully grown oocytes.