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In vitro embryo production is a widely applied technique that allows the expansion of genetics and accelerated breeding programs. However, in cattle, this technique still needs improvement in order to reach quality and pregnancy rates comparable to in vivo-derived embryos. One of the limitations of this technique is related to in vitro maturation, where a heterogeneous population of oocytes is harvested from follicles and cultured in vitro in the presence of gonadotropic hormones to induce maturation. As a result, oocytes with different degrees of competence are obtained, resulting in a decrease in the quality and quantity of embryos obtained. A novel system based on the use of cyclic adenosine monophosphate (cAMP) modulators was developed to enhance bovine oocyte competence, although controversial results were obtained depending on the in vitro embryo production (IVP) system used in each laboratory. Thus, in the present work, we employed a reported cAMP protocol named Simulated Physiological Oocyte Maturation (SPOM) under our IVP system and analysed its effect on cytoplasmic maturation by measuring levels of stress-related genes and evaluating the activity and distribution of mitochondria as a marker for cytoplasmic maturation Moreover, we studied the effect of the cAMP treatment on nuclear maturation, cleavage, and blastocyst formation. Finally, we assessed the embryo quality by determining the hatching rates, total cell number per blastocyst, cryopreservation tolerance, and embryo implantation. We found that maturing oocytes in the presence of cAMP modulators did not affect nuclear maturation, although they changed the dynamic pattern of mitochondrial activity along maturation. Additionally, we found that oocytes subjected to cAMP modulators significantly improved blastocyst formation (15.5% vs. 22.2%, p < 0.05). Blastocysts derived from cAMP-treated oocytes did not improve cryopreservation tolerance but showed an increased hatching rate, a higher total cell number per blastocyst and, when transferred to hormonally synchronised recipients, produced pregnancies. These results reflect that the use of cAMP modulators during IVM results in competent oocytes that, after fertilisation, can develop in more blastocysts with a better quality than standard IVM conditions.

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Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+ ) increases during capacitation. Herein, we studied mitochondrial function by high-resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non-capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2 O2 ) detection was three times higher in CAP than in NC sperm cells. To confirm that H2 O2 production depends on mitochondrial superoxide ( O 2 · - $$ {\mathrm{O}}_2^{\cdotp -} $$ ) formation, we evaluated mitochondrial aconitase (ACO2) amount, activity, and role in the metabolic flux from the sperm tricarboxylic acid cycle. We estimated that CAP cells produce, on average by individual, (59 ± 22)% more O 2 · - $$ {\mathrm{O}}_2^{\cdotp -} $$ in the steady-state compared to NC cells. Finally, we analyzed two targets of oxidative stress: lipid peroxidation by western blot against 4-hydroxynonenal and succinate dehydrogenase (SDH) activity by HRR. We did not observe modifications in lipoperoxidation nor the activity of SDH, suggesting that during capacitation, the increase in mitochondrial H2 O2 production does not damage sperm and it is necessary for the normal CAP process.

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During in vitro maturation (IVM) cumulus-oocyte complexes (COCs) are exposed to conditions that can trigger oxidative stress, thus, reducing oocyte maturation and viability. Aiming to mitigate these detrimental conditions, the effects of IVM medium supplementation with anethole have been tested. Anethole, also known as trans-anethole (1-methoxy-4 [1-propenyl]-benzene), is a naturally occurring phenylpropanoid with various pharmacological properties, including antioxidant effects. However, no study has examined anethole effect on goat COCs during IVM. Thus, the aim of this study was to evaluate the effects of different anethole concentrations on oocyte maturation, oxidative stress, and in vitro development of caprine embryos after parthenogenetic activation. Goat COCs were selected and randomly distributed into the following treatments: TCM-199+ medium (control), or TCM-199+ medium supplemented with 30 µg/mL (AN30); 300 µg/mL (AN300) or 2000 µg/mL (AN2000) of anethole. After IVM, part of the COCs was chosen for oocyte viability and chromatin configuration, intracellular reactive oxygen species levels, and mitochondrial membrane potential assessment. Another part of COCs was parthenogenetically activated, and presumptive zygotes were cultured for 7 days. Results demonstrated that anethole at 30 µg/mL increased oocyte maturation and cleavage rates when compared to the other treatments (P < 0.05), as well as oocyte viability and in vitro embryo production when compared to the control treatment (P < 0.05). Additionally, treatment with anethole at 2000 µg/mL decreased oocyte nuclear maturation and cleavage rates when compared to other treatments (P < 0.05) and embryo production if compared to control and AN30 treatments (P < 0.05). Moreover, anethole at 2000 µg/mL increased mitochondrial membrane potential when compared to the other treatments (P < 0.05). In conclusion, anethole exerts a concentration-dependent effect during goat COCs IVM. For a more desirable outcome of oocyte viability and maturation, and in vitro embryo production, the use of anethole at 30 µg/mL is recommended.

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Bos taurus indicus bulls are very susceptible to pre-slaughter stress, which directly impacts the decline in muscle pH, leading to darker meat. The aim was to investigate the effect of succinate and atmosphere on the color stability of Nellore (Bos taurus indicus) Longissimus lumborum steaks classified by ultimate pH (pHu): normal pHu (5.40 ≤ pHu ≤ 5.79) and high pHu (pHu ≥ 5.80). The experimental treatment systems were: (i) vacuum packaging without succinate injection, (ii) HiOx-MAP (80 % O2 + 20 % CO2), and (iii) HiOx-MAP (80 % O2 + 20 % CO2) enhanced with sodium succinate injection (pH 5.4). Steaks from all treatment systems were stored at 4 °C for 14 days and tested for instrumental color, myoglobin content, oxygen consumption (OC), metmyoglobin-reducing activity (MRA), lipid oxidation, and microbiological analysis. High and normal pHu vacuum-packaged steaks exhibited greater color stability due to higher MRA. High and normal pHu steaks packaged with HiOx-MAP or HiOx-MAP enhanced with succinate showed improved color due to lower deoxymyoglobin content (%DMb) and OC up to the eighth day of storage. Still, succinate injection promoted increased (P < 0.05) lipid oxidation in normal pHu steaks and reduced MRA after 14 days. These findings emphasize the intricate interplay between pHu and packaging systems on Bos taurus indicus meat quality. Further research in this area could contribute to a better understanding of meat color abnormalities and provide insights into potential meat preservation and enhancement strategies.

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This study aimed to evaluate the use of green microalgae as a nutritional supplement for oocyte and embryo production in goats. Two experiments were performed on adult goats to obtain oocytes (EVO; n = 14) and in vivo embryos (IVD; n = 14). In both, the donors were divided into control (n = 7) and Chlorella (n = 7) groups. All goats received a base diet, and donors were orally supplemented with Chlorella pyrenoidosa (CH) in the Chlorella groups. For EVO, donors received 10 g CH for 14 days, and for IVD, 20 g CH was given for six days before embryo recovery. In EVO and IVD, food intake in the CH group was comparatively low, and it showed relatively high subcutaneous adipose deposition. In addition, the CH group exhibited an increase in triglyceride, cholesterol, and plasma glucose levels. In IVD, a significant increase in peripheral glutathione peroxidase levels was noticed. In EVO, the CH group showed relatively large follicular size and an increase in intrafollicular levels of triglycerides, glucose, and glutathione peroxidase. No differences were observed in the oocyte collected, and CH oocytes showed a low intensity of MitoTracker fluorescence (MT). In IVD, the CH group had a high proportion of transferable embryos, and these structures exhibited high fluorescence intensities for MT and H2DCFDA probes. We concluded that under these conditions, CH did not enhance the quality of the recovered oocytes. However, a daily dose of 20 g CH improved the quality of embryos and stimulated their mitochondrial functionality.

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Pecans (Carya illinoinensis) are considered a functional food due to the high content of polyunsaturated fatty acids, dietary fiber and polyphenols. To determine the effect of whole pecans (WP) or a pecan polyphenol (PP) extract on the development of metabolic abnormalities in mice fed a high-fat (HF) diet, we fed C57BL/6 mice with a Control diet (7% fat), HF diet (23% fat), HF containing 30% WP or an HF diet supplemented with 3.6 or 6 mg/g of PP for 18 weeks. Supplementation of an HF diet with WP or PP reduced fat mass, serum cholesterol, insulin and HOMA-IR by 44, 40, 74 and 91%, respectively, compared to the HF diet. They also enhanced glucose tolerance by 37%, prevented pancreatic islet hypertrophy, and increased oxygen consumption by 27% compared to the HF diet. These beneficial effects were associated with increased thermogenic activity in brown adipose tissue, mitochondrial activity and AMPK activation in skeletal muscle, reduced hypertrophy and macrophage infiltration of subcutaneous and visceral adipocytes, reduced hepatic lipid content and enhanced metabolic signaling. Moreover, the microbial diversity of mice fed WP or PP was higher than those fed HF, and associated with lower circulating lipopolysaccharides (~83-95%). Additionally, a 4-week intervention study with the HF 6PP diet reduced the metabolic abnormalities of obese mice. The present study demonstrates that WP or a PP extract prevented obesity, liver steatosis and diabetes by reducing dysbiosis, inflammation, and increasing mitochondrial content and energy expenditure. Pecan polyphenols were mainly condensed tannin and ellagic acid derivatives including ellagitannins as determined by LC-MS. Herein we also propose a model for the progression of the HF diet-mediated metabolic disorder based on early and late events, and the possible molecular targets of WP and PP extract in preventive and intervention strategies. The body surface area normalization equation gave a conversion equivalent to a daily human intake dose of 2101-3502 mg phenolics that can be obtained from 110-183 g pecan kernels/day (22-38 whole pecans) or 21.6-36 g defatted pecan flour/day for an average person of 60 kg. This work lays the groundwork for future clinical studies.

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OBJECTIVE: To study the relationship between the seminal sample quality of men with varicocele and sperm capacitation. DESIGN: Cross-sectional observational study. SETTING: Academic hospital. PATIENT(S): Seventy-six men (19 control and 57 with varicocele) were analyzed. INTERVENTION(S): Semen samples were submitted to a discontinuous density gradient for sperm selection. Sperm capacitation was induced using a human tubal fluid medium supplemented with bovine serum albumin. MAIN OUTCOME MEASURE(S): After capacitation induction, the sperm were assessed by capacitation state, computer-assisted sperm motility, mitochondrial activity, membrane integrity, acrosome reaction, and intracellular oxidative stress. RESULT(S): The capacitation period increased sperm motility, showing an increase in the average path velocity and a decrease in the straightness compared with sperm before capacitation (paired analysis). After capacitation, the rate of capacitated sperm, motility, and mitochondrial activity showed differences between groups (control and varicocele). The varicocele group showed lower mitochondrial activity and capacitation than the control group. On the other hand, no significant differences were observed in the other variables evaluated. CONCLUSION(S): Varicocele men showed less viable sperm and mitochondrial activity than control men after capacitation sperm. The induction of capacitation altered motility by increasing path velocity and decreasing straightness in all of the studied groups, evidencing the occurrence of hyperactivation.

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Cardamom seed (Elettaria cardamomum (L.) Maton; EC) is consumed in several countries worldwide and is considered a nutraceutical spice since it exerts antioxidant, anti-inflammatory, and metabolic activities. In obese individuals, EC intake also favors weight loss. However, the mechanism for these effects has not been studied. Here, we identified that EC modulates the neuroendocrine axis that regulates food intake, body weight, mitochondrial activity, and energy expenditure in mice. We fed C57BL/6 mice with diets containing 3%, 6%, or 12% EC or a control diet for 14 weeks. Mice fed the EC-containing diets gained less weight than control, despite slightly higher food intake. The lower final weight of EC-fed mice was due to lesser fat content but increased lean mass than control. EC intake increased lipolysis in subcutaneous adipose tissue, and reduced adipocyte size in subcutaneous, visceral, and brown adipose tissues. EC intake also prevented lipid droplet accumulation and increased mitochondrial content in skeletal muscle and liver. Accordingly, fasting and postprandial oxygen consumption, as well as fasting fat oxidation and postprandial glucose utilization were higher in mice fed with EC than in control. EC intake reduced proopiomelanocortin (POMC) mRNA content in the hypothalamic arcuate nucleus, without an impact on neuropeptide Y (NPY) mRNA. These neuropeptides control food intake but also influence the hypothalamic-pituitary-thyroid (HPT) and hypothalamic-pituitary-adrenal (HPA) axes. Thyrotropin-releasing hormone (TRH) mRNA expression in the hypothalamic paraventricular nucleus (PVN) and circulating triiodothyronine (T3) were lower in EC-fed mice than in control. This effect was linked with decreased circulating corticosterone and weight of adrenal glands. Our results indicate that EC modulates appetite, increases lipolysis in adipose tissue and mitochondrial oxidative metabolism in liver and skeletal muscle, leading to increased energy expenditure and lower body fat mass. These metabolic effects were ascribable to the modulation of the HPT and HPA axes. LC-MS profiling of EC found 11 phenolic compounds among which protocatechuic acid (23.8%), caffeic acid (21.06%) and syringic acid (29.25%) were the most abundant, while GC-MS profiling showed 16 terpenoids among which costunolide (68.11%), ambrial (5.3%) and cis-α-terpineol (7.99%) were identified. Extrapolation of mice-to-human EC intake was performed using the body surface area normalization equation which gave a conversion equivalent daily human intake dose of 76.9-308.4 mg bioactives for an adult of 60 kg that can be obtained from 14.5-58.3 g of cardamom seeds (18.5-74.2 g cardamom pods). These results support further exploration of EC as a coadjuvant in clinical practice.

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BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is predominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

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BACKGROUND: The assessment of oocyte quality is, nowadays, a major challenge in aquaculture, oocyte cryopreservation, and environmental science. Oocyte quality is a determining factor in fertilization and embryo development; however, there is still a lack of rapid and sensitive cellular markers for its assessment. Currently, its estimation is pre-dominantly based on morphological analysis, which is subjective and does not consistently reflect the developmental competence of the oocytes. Despite several recent studies investigating molecular markers related to oocyte quality, methods currently available for their determination pose various technical challenges and limitations. In this study, we developed a novel approach based on fluorescence spectroscopy to assess different intrinsic physiological parameters that can be employed to evaluate egg quality in marine invertebrates that are widely used as animal models such as sea urchins and mussels. RESULTS: Different physiological parameters, such as viability, mitochondrial activity, intracellular ROS levels, plasma membrane lipid peroxidation, and intracellular pH, for egg quality evaluation have been successfully assessed in sea urchins and mussels by using specific fluorescent dyes and detecting the fluorescent signals in eggs through fluorescence spectroscopy. CONCLUSIONS: Based on our findings, we propose these physiological markers as useful predictors of egg quality in marine invertebrates; they can be estimated rapidly, selectively, and sensitively by employing this novel approach, which, due to the speed of analysis, the low cost, and easy use can be considered a powerful analytical tool for the egg quality assessment.

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