RESUMO
Sows from three university research facilities (n = 245) were stratified by parity and initial body weight (BW), and within outcome groups, randomly assigned to fortified corn- and soybean meal-based control or organic trace mineral-supplemented, gestation (3,339 kcal/kg ME; 0.62% standradized ileal digestible [SID] lysine), and lactation (3,374 kcal/kg ME; 0.97% SID lysine) diets. Control gestation and lactation diets were supplemented with inorganic trace minerals (120 ppm Zn from ZnO, 30 ppm Cu from CuSO4, and 50 ppm Mn from MnSO4), and the experimental diets contained the same total level of minerals but complexed organic trace minerals replaced 50% of the inorganic trace minerals. Sows were fed to condition during gestation and on an ad libitum basis during lactation. Sow BW (breeding, d 110 of gestation, 48 h post-farrowing, and weaning) and feed consumed were recorded. During gestation, control sows tended to gain less weight (60.4 vs. 64.6 kg, P = 0.06) and consumed less feed (263.5 vs. 264.8 kg, P = 0.05), and had poorer Gain:Feed (G:F) (0.27 vs. 0.29, P = 0.04) than sows fed the organic trace minerals. Sow average daily feed intake (ADFI) during lactation was similar (P = 0.28) between groups (4.93 vs. 4.74 kg for control and treated sows, respectively). Number of pigs born alive (11.4 vs. 10.9, P = 0.24) and weaned (10.2 vs. 9.8, P = 0.18), and pig pre-weaning average daily gain (ADG) (0.27 vs. 0.27 kg/d, P = 0.77) and mortality (13.1 vs. 12.9%, P = 0.92) were similar for control and treated sows, respectively. Results of the current study demonstrate that sows fed diets supplemented with organic trace minerals displayed similar reproductive performance, but improved weight gain and G:F during gestation compared with sows fed inorganic trace minerals.
RESUMO
AIM: The present study evaluates the effect of different concentrations of antioxidants (catalase - CAT and alpha lipoic acid - ALA) on the follicular activation and morphology, DNA damage, ROS production, and mitochondrial activity in vitrified sheep ovarian tissue. METHODS: This experiment was divided into two steps. First, ovarian fragments were distributed into the following treatments: fresh tissue or control (CTR), incubation (INC), vitrification without antioxidant (VWA), with CAT (10, 20, or 40 IU mL-1) or ALA (25, 50, or 100 µM mL-1). After vitrification/warming, the fragments were additionally incubated for 24 hours and evaluated for morphology and follicular activation, as well as reactive oxygen species (ROS) levels in the culture medium. For the second step, other ovarian fragments were submitted to CTR, VWA, CAT40, and ALA100. After vitrification/warming, the fragments were incubated for 24 hours and evaluated by cell density of ovarian stroma, DNA damage, and mitochondrial and intracellular ROS levels. RESULTS: The percentage of morphologically normal follicles in vitrified ovarian tissue in the presence of ALA in all concentrations did not differ (p > 0.05) from fresh tissue or CTRs. The percentage of activated follicles was higher in ALA100 µM mL-1 than those observed for the treatments INC, CAT (40 IU mL-1), or ALA (25 or 50 µM mL-1). The use of CAT affected (p < 0.05) the density of stromal cells (40 IU mL-1), ROS levels (10 and 20 IU mL-1), as well as DNA damage revealed by ©H2AX (40 IU mL-1). CONCLUSIONS: Although 100 µM/mL of ALA did not alter intracellular ROS, this concentration reduced the levels of ROS in the culture medium, preserved both the follicular morphology, as well as the mitochondrial activity, promoted follicle activation, and protected the follicles from DNA damage.
Assuntos
Catalase/farmacologia , Criopreservação/métodos , Ovário/citologia , Ovário/metabolismo , Ácido Tióctico/farmacologia , Vitrificação , Animais , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Feminino , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovário/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , OvinosRESUMO
The extract of Auxemma oncocalyx (A. oncocalyx) and its main component, oncocalyxone A (onco A), possess anti-tumoral activity that may affect fertility. There is limited literature on the action of these substances regarding caprine folliculogenesis. In this study, we evaluated the effect of A. oncocaly and onco A on the in vitro culture of isolated secondary follicles (Experiment 1) and on the in vitro maturation (IVM) of oocytes from caprine antral follicles grown in vivo (Experiment 2). Isolated secondary follicles were distributed in six groups: the non-cultured control was immediately fixed in 4% paraformaldehyde. The remaining follicles were cultured for 7 days in α-minimal essential medium (α-MEM+ ) alone (control) or with dimethyl sulfoxide (DMSO), doxorubicin (DXR), A. oncocalyx, or onco A. After culture, the follicles were evaluated for antrum formation, growth rate, apoptosis (TUNEL), cellular proliferation (PCNA), as well as the expression of BCL2 and BAX. In addition, cumulus oocyte complexes (COCs) were aspirated and allocated into five treatments for IVM: control, cultured only in maturation base medium (TCM 199+ ); or supplemented with DMSO; DXR; A. oncocalyx or onco A. After IVM, oocyte chromatin configuration and viability were assessed. After 7 days of culture, a reduction was noted in the percent of morphologically intact follicles, antrum formation, growth rate, and numbers of PCNApositive granulosa cells (P < 0.05). After culture, the DXR treatment showed a higher percent of TUNELpositive follicles and relative BAX:BCL2 mRNA ratios (P < 0.05). After IVM of the COCs, DXR, A. oncocalyx, and onco A treatments showed a greater percent (P < 0.05) of abnormal oocytes and a lower percent of viable oocytes as compared with the control group (P < 0.05)...(AU)
O extrato da Auxemma oncocalyx (A. oncocalyx) e seu componente, Oncocalyxona A (onco A), possui atividade antitumoral, podendo afetar a fertilidade. Entretanto, estudos sobre a ação dessas substâncias em relação à foliculogênese caprina são desconhecidos. O objetivo desse estudo foi avaliar o efeito da A. oncocalyx e onco A no cultivo in vitro de folículos secundários isolados (Experimento 1) e na maturação in vitro (MIV) de oócitos de folículos antrais caprinos crescidos in vivo (Experimento 2). Folículos secundários isolados foram distribuídos em seis grupos, em que o controle não-cultivado foi imediatamente fixado em paraformaldeído 4%. Os folículos restantes foram cultivados durante 7 dias em α-MEM+ sozinho (controle) ou suplementado com DMSO, doxorrubicina (DXR), A. oncocalyx ou onco A. Após o cultivo, os folículos foram avaliados quanto à formação de antro, taxa de crescimento, apoptose (TUNEL) e proliferação celular (PCNA), bem como a expressão dos genes BCL2 e BAX. Além disso, os complexos cumulus-oócitos (CCOs) foram aspirados e distribuídos em cinco tratamentos para MIV: o controle em meio de maturação (TCM 199+), e os demais tratamentos suplementados com DMSO, DXR, A. oncocalyx ou onco A. Depois da MIV, a configuração da cromatina e viabilidade oocitária foram avaliadas. Após 7 dias de cultivo, observou-se redução na percentagem de folículos morfologicamente intactos, na formação de antro, na taxa de crescimento e no número de células PCNA positivas (P < 0,05). Depois do cultivo, no tratamento DXR foi observada maior percentagem de folículos TUNEL positivos (P < 0,05) e também aumento na taxa de RNAm BAX: BCL2 (P < 0,05). Após MIV dos CCOs, nos tratamentos com DXR, A. oncocalyx e onco A, observou-se maior (P < 0,05) percentagem de oócitos anormais e menor de oócitos viáveis quando comparados ao grupo controle (P < 0,05)...(AU)
Assuntos
Animais , Bovinos , /anatomia & histologia , Boraginaceae/toxicidade , /anormalidades , Técnicas de Maturação in Vitro de Oócitos , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
The extract of Auxemma oncocalyx (A. oncocalyx) and its main component, oncocalyxone A (onco A), possess anti-tumoral activity that may affect fertility. There is limited literature on the action of these substances regarding caprine folliculogenesis. In this study, we evaluated the effect of A. oncocaly and onco A on the in vitro culture of isolated secondary follicles (Experiment 1) and on the in vitro maturation (IVM) of oocytes from caprine antral follicles grown in vivo (Experiment 2). Isolated secondary follicles were distributed in six groups: the non-cultured control was immediately fixed in 4% paraformaldehyde. The remaining follicles were cultured for 7 days in α-minimal essential medium (α-MEM+ ) alone (control) or with dimethyl sulfoxide (DMSO), doxorubicin (DXR), A. oncocalyx, or onco A. After culture, the follicles were evaluated for antrum formation, growth rate, apoptosis (TUNEL), cellular proliferation (PCNA), as well as the expression of BCL2 and BAX. In addition, cumulus oocyte complexes (COCs) were aspirated and allocated into five treatments for IVM: control, cultured only in maturation base medium (TCM 199+ ); or supplemented with DMSO; DXR; A. oncocalyx or onco A. After IVM, oocyte chromatin configuration and viability were assessed. After 7 days of culture, a reduction was noted in the percent of morphologically intact follicles, antrum formation, growth rate, and numbers of PCNApositive granulosa cells (P < 0.05). After culture, the DXR treatment showed a higher percent of TUNELpositive follicles and relative BAX:BCL2 mRNA ratios (P < 0.05). After IVM of the COCs, DXR, A. oncocalyx, and onco A treatments showed a greater percent (P < 0.05) of abnormal oocytes and a lower percent of viable oocytes as compared with the control group (P < 0.05)...
O extrato da Auxemma oncocalyx (A. oncocalyx) e seu componente, Oncocalyxona A (onco A), possui atividade antitumoral, podendo afetar a fertilidade. Entretanto, estudos sobre a ação dessas substâncias em relação à foliculogênese caprina são desconhecidos. O objetivo desse estudo foi avaliar o efeito da A. oncocalyx e onco A no cultivo in vitro de folículos secundários isolados (Experimento 1) e na maturação in vitro (MIV) de oócitos de folículos antrais caprinos crescidos in vivo (Experimento 2). Folículos secundários isolados foram distribuídos em seis grupos, em que o controle não-cultivado foi imediatamente fixado em paraformaldeído 4%. Os folículos restantes foram cultivados durante 7 dias em α-MEM+ sozinho (controle) ou suplementado com DMSO, doxorrubicina (DXR), A. oncocalyx ou onco A. Após o cultivo, os folículos foram avaliados quanto à formação de antro, taxa de crescimento, apoptose (TUNEL) e proliferação celular (PCNA), bem como a expressão dos genes BCL2 e BAX. Além disso, os complexos cumulus-oócitos (CCOs) foram aspirados e distribuídos em cinco tratamentos para MIV: o controle em meio de maturação (TCM 199+), e os demais tratamentos suplementados com DMSO, DXR, A. oncocalyx ou onco A. Depois da MIV, a configuração da cromatina e viabilidade oocitária foram avaliadas. Após 7 dias de cultivo, observou-se redução na percentagem de folículos morfologicamente intactos, na formação de antro, na taxa de crescimento e no número de células PCNA positivas (P < 0,05). Depois do cultivo, no tratamento DXR foi observada maior percentagem de folículos TUNEL positivos (P < 0,05) e também aumento na taxa de RNAm BAX: BCL2 (P < 0,05). Após MIV dos CCOs, nos tratamentos com DXR, A. oncocalyx e onco A, observou-se maior (P < 0,05) percentagem de oócitos anormais e menor de oócitos viáveis quando comparados ao grupo controle (P < 0,05)...
Assuntos
Animais , Bovinos , Boraginaceae/toxicidade , Técnicas de Maturação in Vitro de Oócitos , Técnicas de Maturação in Vitro de Oócitos/veterináriaRESUMO
The vitrification of preantral follicles followed by in vitro growth (IVG) could be valuable to produce fertilizable oocytes. However, the meiotic resumption rates of oocytes cultured from vitrified secondary follicles (SF) have been reported as suboptimal. This study aimed to verify two base media (alpha modification of minimum essential medium, α-MEM, and tissue culture medium 199, TCM199) on vitrified SF regarding different requirements during IVG. Sheep ovarian fragments were divided in six groups: (1) Fresh groups (Control α-MEM and TCM199): SF without vitrification; (2) Follicle-Vitrified (Follicle-Vit α-MEM and TCM199): SF vitrified after isolation; and (3) Tissue-Vitrified (Tissue-Vit α-MEM and TCM199): SF vitrified enclosed in ovarian fragments and, subsequently, isolated. The isolated SF were submitted to IVG for 18 days. Thereafter, the recovered cumulus-oocyte complexes (COCs) underwent in vitro maturation (IVM) and evaluation of chromatin configuration. Follicular granulosa cells were analyzed for their gene expression of Bax, Bcl2, and Connexins (CX) 37 and 43. COCs from in vivo antral follicles were used as in vivo control. Data were analyzed by analysis of variance, Tukey, and chi-square tests. Differences were considered significant if p-value is <0.05. Follicle-Vit groups had higher (p < 0.05) percentage of antrum formation compared with Tissue-Vit groups. Vitrification did not affect (p > 0.05) oocyte diameter postmaturation. Oocytes from Follicle-Vit in α-MEM reached metaphase II stage after IVM. Gene expression for CX37, CX43, and Bax was lower in Tissue-Vit groups. For Bcl2, the gene expression was the opposite. In conclusion, during IVG for 18 days, maximal oocyte meiotic resumption was not negatively impacted by vitrification and was greatest for isolated SF using α-MEM as a medium.
Assuntos
Oócitos/citologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Vitrificação , Animais , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Metáfase , Compostos Orgânicos/metabolismo , Ovinos , Fatores de TempoRESUMO
This study investigated the effect of androstenedione (A4) alone or in association with different concentrations of bovine recombinant FSH on the IVC of isolated goat preantral follicles. Follicles were mechanically isolated from ovarian tissue and cultured for 18 days in α-minimum essential medium supplemented or not with A4 (10 ng/mL) alone or in association with fixed (A4 + FixFSH: 100 ng/mL) or sequential (A4 + SeqFSH: Day 0, 100 ng/mL; Day 6, 500 ng/mL; Day 12, 1000 ng/mL) concentrations of FSH. After 18 days, the oocytes were recovered for IVM and fluorescence analysis. At Day 18 of culture, only A4 + SeqFSH treatment showed a lower (P < 0.05) rate of intact follicles, survival probability, and meiotic resumption, as well as higher (P < 0.05) percentage of degeneration and/or extrusion after antrum formation. Taken together, these results reported a positive correlation between fast-growing follicles and follicles that degenerated and/or extruded after antrum formation. When compared with control, the addition of A4 alone or in association of FSH did not increase (P > 0.05) the estradiol production or androstenedione levels on Day 6. However, on Day 18, the androstenedione levels were significantly lower in A4 + SeqFSH treatment when compared with A4 alone or to A4 + FixFSH treatments, whereas the estradiol production did not differ (P > 0.05). In summary, this study found that accelerated follicle growth negatively impacted the morphology of caprine preantral follicle cultured in vitro. In addition, the association of androstenedione with increasing concentration of FSH was detrimental to follicular survival and oocyte meiotic resumption.