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β-nerve growth factor (βNGF) plays a crucial role in reproductive physiology and sperm quality. Enzymatic activity of seminal plasma and vaginal fluids reduces available βNGF and it has been demonstrated that chitosan microspheres could protect rrβNGF from degradation. This study examined the effects of microencapsulated rrbNGF with chitosan on rabbit sperm viability, motility and capacitation status. Results showed that 0.5 and 1 μg/mL of microencapsulated rrβNGF, as well as free rrβNGF or empty microspheres, did not adversely affect sperm viability or total motility after 2 h of incubation. However, the highest progressivity kinetic parameters were observed with 1 μg/mL free rrβNGF, while the highest curvilinear velocity (VCL) occurred with 0.5 μg/mL microencapsulated rrβNGF. Empty chitosan microspheres did not induce acrosome reaction (AR), but both concentrations of free and rrβNGFch favoured AR during in vitro incubation. The study suggests that using chitosan spheres did not show any adverse effects on sperm traits, unlike free rβNGF and rrβNGFch promoted capacitation and AR. Further research is needed to explore the potential of rrβNGFch in modifying in vitro capacitation and inducing ovulation during artificial insemination., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

To study retinal and choriocapillaris (CC) alterations using optical coherence tomography angiography (OCTA) in long-term type 1 diabetic (DM1) patients without diabetic retinopathy (DR). Seventy-eight eyes from 78 well-controlled DM1 patients diagnosed at least 15 years prior and 130 eyes of 130 healthy subjects were included in a cross-sectional descriptive study. Six eyes were excluded from the DM1 group. OCTA with Deep Range Imaging (DRI)-Triton swept source (SS)-OCT was performed. Statistically significant differences were found in all areas of the superficial capillary plexus (SCP), with lower values in DM1 patients. Differences were noted in all quadrants of the deep capillary plexus (DCP) except for the central area. Significant changes in CC blood flow were only found in the center. The foveal avascular zone (FAZ) area and diameters in the SCP were significantly different, while the DCP FAZ area was similar in both groups. Disease duration and microalbuminuria correlated negatively with some SCP areas and positively with FAZ values. Anatomical evaluation revealed microaneurysms in both plexuses, FAZ modifications, and areas lacking blood perfusion. Long-term type 1 diabetic patients without DR display microvascular abnormalities affecting retinal and CC blood perfusion, along with anatomical changes in retinal blood vessels.

Background: To evaluate changes in pattern electroretinogram (pERG) and pattern visual evoked potentials (pVEP) in patients with long-lasting type 1 diabetes without diabetic retinopathy (DR)., Methods: Prospective study involving 92 eyes divided into two groups. The diabetic group included 46 eyes of 23 patients with type 1 diabetes (T1DM); the control group included 23 age-matched healthy subjects. pERG and pVEP were assessed using the RETI-port/scan21 recording software (version 1021.3.0.0)., Results: Mean age was 48 ± 9.77 years for the diabetic group and 51.7 ± 4.75 years for the control group. The mean duration of diabetes was 28.88 ± 8.04 years. The mean HbA1c value was 7.29 ± 0.89%. There were no differences in the age or sex distribution. Regarding the pERG, T1DM patients exhibited a significant decrease in the amplitude of the P50 and N95 waves compared to the control group ( p = 0.018 and p = 0.035, respectively), with no differences in the peak time of each component. pVEP showed no significant changes in either peak time or amplitude of the different components., Conclusions: Long-term T1DM patients without DR showed changes in the amplitude of pERG waves with preserved peak times. We did not observe modifications in pVEP. pERG may serve as a subclinical marker of ganglion cell damage in long-term T1DM patients.

To assess full-field electroretinogram findings in long-term type 1 diabetes patients without diabetic retinopathy. Prospective study including 46 eyes of 23 patients with type 1 diabetes and 46 age-matched healthy eyes evaluated by the RETI-port/scan21 and the portable system RETeval following ISCEV guidelines. The average duration of diabetes was 28.88 ± 8.04 years. In scotopic conditions, using the RETI-port/scan21, diabetic patients showed an increase in b-wave implicit time (IT) (p = 0.017) with the lowest stimuli; a diminished b-wave amplitude (p = 0.005) in the mixed response, an increased IT (p = 0.004) with the high-intensity stimuli and an OP2 increased IT (p = 0.008) and decreased amplitude (p = 0.002). Under photopic conditions, b-wave amplitude was lower (p < 0.001) and 30-Hz flicker response was diminished (p = 0.021). Using the RETeval, in scotopic conditions, diabetic patients showed a reduction in the rod b-wave amplitude (p = 0.009), an increase in a-wave IT with the 280 Td.s stimulus (p = 0.005). OP2 had an increased IT and diminished amplitude (p = 0.003 and p = 0.002 respectively). 16 Td.s flicker showed an increased IT (p = 0.008) and diminished amplitude (p = 0.048). Despite variations in values between both systems, nearly all results displayed positive correlations. Long-term type 1 diabetes patients without diabetic retinopathy exhibit alterations in scotopic conditions, as evidenced by both conventional and portable electroretinogram devices. These findings suggest a modified retinal function, particularly in rod-driven pathways, even in the absence of vascular signs., (© 2024. The Author(s).)

Nutritional status during gestation can influence mother and offspring metabolism. Undernutrition in pregnancy affects women in both western and developing countries, and it is associated with a high prevalence of chronic diseases in later life. The present work was conducted in the rabbit model, as a longitudinal study, to examine the effect of food restriction during early and mid-gestation, and re-feeding ad libitum until the end of pregnancy on metabolic status and body reserves of mother and, its association with development and metabolism of fetuses and female offspring to the juvenile stage. Little changes in live body weight (LBW), compensatory feed intake, similar body reserves, and metabolism were observed in dams. Placenta biometry and efficiency were slightly affected, but fetal BW and phenotype were not modified. However, hyperinsulinemia, insulin resistance, and hypertriglyceridemia were demonstrated in pre-term fetuses. In the juvenile period, these changes were not evidenced, and a similar pattern of growth and serum metabolic parameters in offspring of food-restricted mothers were found, except in serum aminotransferases levels, which increased. These were associated with higher liver fibrosis. Maternal food restriction in the early and mid-pregnancy followed by re-feeding in our rabbit model established a compensatory energy status in dams and alleviated potential long-term consequences in growth and metabolism in the offspring, even if fetal metabolism was altered.

Rabbit does are reflex ovulators such that coitus is needed to release GnRH and elicit the LH surge that triggers the ovulation of mature oocytes. However, the mechanisms eliciting ovulation in this species remain unclear. One of the most promising recently discovered candidates with a role in female reproductive physiology is nerve growth factor beta (β-NGF). This neurotrophin and its high-affinity receptor TrkA and low affinity receptor p75, is present in all compartments of the ovary, oviduct and uterus suggesting a physiologic role in ovarian folliculogenesis, steroidogenesis, ovulation, luteogenesis and embryo development. Besides, evidence exists that β-NGF found in seminal plasma could exert a modulatory role in the female hypothalamus-pituitary-ovarian axis contributing to the adrenergic and cholinergic neuronal stimulus of GnRH neurons in an endocrine manner during natural mating. Probably, the paracrine and local roles of the neurotrophin in steroidogenesis and ovulation reinforce the neuroendocrine pathway that leads to ovulation. This review updates knowledge of the role of β-NGF in rabbit reproduction, including its possible contribution to the mechanisms of action that induce ovulation, and discusses perspectives for the future applications of this neurotrophin on rabbit farms., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Intrauterine growth restriction (IUGR) affects approximately 10% of human pregnancies globally and has immediate and life-long consequences for offspring health. However, the mechanisms underlying the pathogenesis of IUGR and its association with later health and disease outcomes are poorly understood. To address these knowledge gaps, the use of experimental animals is critically important. Since the 50's different environmental, pharmacological, and surgical manipulations have been performed in the rabbit to improve our knowledge of the control of fetal growth, fetal responses to IUGR, and mechanisms by which offspring may be programmed by an adverse gestational environment. The purpose of this review is therefore to summarize the utility of the rabbit as a model for IUGR research. It first summarizes the knowledge of prenatal and postnatal development in the rabbit and how these events relate to developmental milestones in humans. It then describes the methods used to induce IUGR in rabbits and the knowledge gained about the mechanisms determining prenatal and postnatal outcomes of the offspring. Finally, it discusses the application of state of the art approaches in the rabbit, including high-resolution ultrasound, magnetic resonance imaging, and gene targeting, to gain a deeper integrative understanding of the physiological and molecular events governing the development of IUGR. Overall, we hope to engage and inspire investigators to employ the rabbit as a model organism when studying pregnancy physiology so that we may advance our understanding of mechanisms underlying IUGR and its consequences in humans and other mammalian species., (© 2019 Wiley Periodicals, Inc.)

In some induced-ovulating species, beta nerve growth factor (β-NGF) has important roles in ovulation, though data for rabbits are still inconclusive. In this study we first synthesized functional recombinant β-NGF from rabbit tissue (rrβ-NGF) to address the following objectives: 1) to compare rabbit β-NGF amino acid sequence with those of other induced- or spontaneous-ovulating species; 2) to assess the effects of rrβ-NGF on rabbit sperm viability and motility, and 3) to examine the in vivo ovulation inducing effect of rrβ-NGF added to the seminal dose in rabbit does. The NGF gene in rabbit prostate tissue was sequenced by Rapid Amplification of cDNA Ends and annotated in GenBank (KX528686). Recombinant rβ-NGF was produced in CHO cells and purified by affinity chromatography. Once confirmed by Western blotting and mass spectrometry (MALDI-TOF) that the amino acid sequence of the recombinant protein corresponded to β-NGF, its functionality was validated in PC12 cells in a successful dose-response study over 8 days. The amino acid sequence of prostate rabbit NGF differed to that of other species mainly in its receptor binding sites. In all the spontaneous ovulating species examined, compared with rabbit, alanine and proline residues, which interact with the high-affinity receptor, were replaced by a serine. In rabbits, asparagine and methionine were substituted by lysine at the low-affinity receptor binding site. In time- and dose-response experiments, the in vitro addition of rrβ-NGF to the ejaculate did not affect sperm viability whereas sperm motility parameters were enhanced by the addition of 1 μg/mL of the neuropeptide. Addition of this same concentration of rrβ-NGF to the seminal dose administered via the intravaginal route in does induced ovulation with a delayed LH peak, leading to a plasma progesterone increase, gestation and delivery. Our findings suggest that rrβ-NGF could be a useful option for biotechnological and reproduction assisted techniques in rabbits but further studies are needed., Competing Interests: The authors have declared that no competing interests exist.

β-Nerve Growth Factor (β-NGF) is a neurotrophin which acts through its receptors TrkA and p75, performing important actions in male reproductive physiology and its presence in seminal plasma (SP) has been related to male fertility. The aim of the present study was to evaluate the gene expression profile and the immunolocalization of β-NGF and its high-affinity receptor TrkA in sex organs in rabbits during sexual maturation period. β-NGF concentration for both SP and blood plasma (BP) and BP testosterone levels were determined as well as the seminal parameters during such period. Ten New Zealand White x California young rabbits were trained to semen collection since 20 weeks of age and routinely done once a week with two ejaculations per session. At 22 and 37 weeks of age, semen collection was carried out three times a week and seminal parameters were evaluated. Four males were randomly assigned and slaughtered in each age (n = 8); sex organs (prostate, bulbourethral glands and epididymis) were dissected and collected to determine β-NGF and TrkA gene expression and immunolocalization. SP and BP were also taken at each semen collection session to evaluate β-NGF concentration, and testosterone levels were also assessed in BP. The highest β-NGF mRNA expression was observed in prostate compared to bulbourethral glands and epididymis. These two last tissues showed residual β-NGF mRNA expression and limited localization of the neurotrophin. The prostate epithelial cells and lumen were strongly stained with regard to the other sex organs indicating that immunolocalization of β-NGF rely mainly in the prostate. TrkA gene expression was lower but constant and differentially immunolocalized in the sex organ tissues. Finally, β-NGF concentration in SP and BP remained unchanged in accordance to age, while some seminal characteristics such as sperm concentration, percentage of live sperm and mass and progressive motility were enhanced as endowed by BP testosterone variation. β-NGF and its cognate TrkA receptor are expressed and immunolocalized in the male reproductive tract in the two ages studied, independently of the circulating levels of testosterone and β-NGF., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This study reports the gene expression and immunolocalization of the low-affinity neurotrophin receptor, p75, in accessory glands (prostate and bulbourethral glands) and epididymis (caput and cauda) of male rabbits during sexual maturation. We showed that p75 was expressed in all tissues studied with similar mRNA levels during this period. However, it was differentially immunolocalized in bulbourethral glands and stereocilia of epididymis. These findings may be related with some remodeling processes in the accessory glands during sexual maturation, which could be associated with sperm maturation. The interaction of p75 with neurotrophins in rabbit male reproductive tract suggests the possible implication of this system in sexual maturation in rabbits., (© 2018 Blackwell Verlag GmbH.)

The major goal of animal production is to obtain abundant and healthy meat for consumers. Maternal food restriction (MFR) is often applied in farms to reduce production costs. However, the suitability of MFR in livestock animals is questionable, as this management may compromise maternal fitness due to a severe negative energetic balance and can induce Intrauterine Growth Restriction (IUGR) and prenatal programming in the offspring. Here, we sought to determine, using pregnant rabbits, the consequences of MFR on maternal endocrine and metabolic status and conceptus development. Pregnant dams were distributed into three groups: CONTROL (ad libitum feeding throughout the entire pregnancy; mean pregnancy length being around 31 days), UNDERFED (50% MFR during the entire pregnancy) and EARLY-UNDERFED (50% MFR only during the preimplantation period, Days 0-7). Maternal leptin concentrations and glycemic and lipid profiles were determined throughout pregnancy, whilst conceptus development was assessed ex-vivo at Day 28. Placental parameters were determined by macroscopic and histological evaluations and apoptotic assessments (TUNEL and Caspase-3). The main results of the study showed that, despite MFR altered maternal plasma lipid concentration (P<0.05), there were no effects on maternal bodyweight, plasma leptin concentration or glycemic profile. Fetal crown-rump lengths were reduced in both undernourished groups (P<0.001), but a significant reduction in fetal weight was only observed in the UNDERFED group (P<0.001). Growth in both undernourished groups was asymmetrical, with reduced liver weight (P<0.001) and significantly increased brain: fetal weight-ratio (P<0.001) and brain: liver weight-ratio (P<0.001) when compared to the CONTROL group. A significant reduction in placental weight was only observed in the UNDERFED group (P<0.001), despite both undernourished groups showing higher apoptotic rates at decidua and labyrinth zone (P<0.05) than the CONTROL group. Thus, these groups evidenced signs of placental degeneration, necrosis and stromal collapse. In summary, MFR may encourage the mother to make strategic decisions to safeguard her metabolic status and fitness at the expense of growth reduction in the litter, resulting in enhanced apoptotic and pathological processes at placental level and IUGR., Competing Interests: There is no conflict of interest that would prejudice the information offered in the paper, excepting that AG-B is a PLOS ONE Editorial Board member. However, this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Elevated non-esterified fatty acids (NEFAs) have been recognized as an important link between lipolytic metabolic conditions and impaired fertility in high-yielding dairy cows. However, NEFA effects on the oviductal micro-environment currently remain unknown. We hypothesize that elevated NEFAs may contribute to the complex pathology of subfertility by exerting a negative effect on bovine oviductal epithelial cell (BOEC) physiology. Therefore, the objectives of this study were to elucidate direct NEFA effects on BOEC physiology in three different in vitro cell culture systems. Bovine oviductal epithelial cells (four replicates) were mechanically isolated, pooled, and cultured as conventional monolayers, as explants, and in a polarized cell culture system with Dulbecco's modified Eagle's medium/F12-based culture medium. Bovine oviductal epithelial cells were exposed to an NEFA mixture of oleic, stearic, and palmitic acids for 24 hours at both physiological and pathologic concentrations. A control (0 μM NEFA) and a solvent control (0 μM NEFA + 0.45% ethanol) group were implemented. Bovine oviductal epithelial cells physiology was assessed by means of cell number and viability, a sperm binding assay, transepithelial electric resistance (TER), and a wound-healing assay. Bovine oviductal epithelial cell morphology was assessed by scanning electron microscopy on cell polarity, presence of microvilli and cilia, and monolayer integrity. Bovine oviductal epithelial cell number was negatively affected by increasing NEFAs, however, cell viability was not. Sperm binding affinity significantly decreased with increasing NEFAs and tended (P = 0.051) to be more affected by the direction of NEFA exposure in the polarized cell culture system. The absolute TER increase after NEFA exposure in the control (110 ± 11 Ω.cm(2)) was significantly higher than that in all the other treatments and was also different depending on the exposure side. Bidirectional exposed monolayers were even associated with a significant TER reduction (-15 ± 10 Ω.cm(2); P < 0.05). Cell proliferation capacity showed a decreased cell migration with increasing NEFA concentrations but was irrespective of the exposure side. Bovine oviductal epithelial cell morphology was not affected. In conclusion, in an in vitro setting, NEFAs exert a negative effect on BOEC physiology but not morphology. Ultimately, these physiological alterations in its microenvironment may result in suboptimal development of the pre-implantation embryo and a reduced reproductive outcome in dairy cattle., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: To study how long-term elevated non-esterified fatty acid (NEFA) concentrations, typical in metabolic disorders such as obesity or type 2 diabetes, affect murine follicular development, follicle quality, and subsequent oocyte developmental competence in vitro., Design: Experimental study., Setting: In vitro culture setting., Animal(s): Female and male 13-day old, B6CBAF1 mice of proven fertility were sacrificed for harvesting ovaries and epididymal sperm, respectively., Intervention(s): Early secondary murine follicles were cultured in vitro in the presence of NEFAs until the antral stage (12 days). Treatments consisted of one or a mixture of NEFAs (stearic acid [SA], palmitic acid [PA], oleic acid [OA]) in physiological (basal) or pathological (high SA, high OA, high NEFA) concentrations., Main Outcome Measure(s): Follicular development; follicle and oocyte diameters; secretion of progesterone, estradiol, and inhibin B; and luteinized granulosa cell gene expression patterns were investigated. Oocytes from NEFA-exposed follicles were fertilized in vitro, and presumptive zygotes were cultured until the blastocyst stage., Result(s): Exposure to high SA reduced follicle diameters and day-12 antrum formation. Elevated NEFA concentrations changed luteinized granulosa cell messenger-ribonucleic acid abundance of genes related to energy/fatty acid/steroid metabolism, apoptosis, and oxidative stress. High NEFA and high SA treatments increased progesterone synthesis, compared with high OA follicles. Oocyte developmental competence was substantially reduced in oocytes retrieved from high OA-, high SA-, and high NEFA-exposed follicles compared with basal-treated follicles., Conclusion(s): This study showed, for the first time, that lipolysis-linked, elevated NEFA concentrations can potentially impair fertility, by altering follicular physiology and reducing oocyte developmental competence., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: It has been well documented that the maturing oocyte is very vulnerable to changes in its micro-environment, the follicular fluid (FF). Recent research has focused on different components within this FF, like hormones, growth factors and metabolites, and how their concentrations are altered by diet and the metabolic health of the mother. It has been proposed that fatty acids (FAs) are potential factors that influence oocyte maturation and subsequent embryo development. However, a thorough study of the specific FF FA composition per lipid fraction and how this may be affected by BMI is currently lacking. Therefore, we investigated the BMI-related concentration of FAs in the phospholipid (PL), cholesteryl-ester (CHE), triglyceride (TG) and non-esterified (NE) lipid fraction in the FF of women undergoing assisted reproductive treatment (ART)., Methods: In this descriptive cross-sectional study, the FF of normal weight (18.5 ≤ BMI < 25.0 kg/m(2), n = 10), overweight (25.0 ≤ BMI < 30.0 kg/m(2), n = 10) and obese (BMI ≥ 30.0 kg/m(2), n = 10) women, undergoing ART, was sampled and analyzed for 23 specific FAs in the PL, CHE, TG and NEFA fraction, using a gas chromatographic analysis method. Differences between BMI-groups were studied by means of univariate general linear models and post hoc Sheffé tests., Results: Total FA concentrations in the PL and CHE fraction did not differ between BMI groups. Total TG concentrations tended to differ and total NEFA concentrations differed significantly between BMI groups. Interestingly, 42% and 34% of the total FAs was esterified in the PL and CHE fraction, respectively, while only 10% were present in both the TG and NEFA fraction. Only few individual FA concentrations differed in the PL, CHE and TG fraction between BMI groups, whereas abundant BMI-related differences were found in the NEFA fraction., Conclusions: Our data show that differences in BMI are associated with alterations in the FA composition of the FF, an effect most pronounced in the NEFA fraction. These BMI-related variations could possibly affect granulosa cell viability, oocyte developmental competence and subsequent embryo quality possibly explaining differences in oocyte quality in obese patients described by others.

Ovarian stimulation with equine chorionic gonadotropin (eCG) is largely used in animal reproductive technologies to provide a larger number of oocytes and embryos and to improve the reproductive outcome. However, the consequences of maternal treatment with eCG on embryo gene expression patterns are not widely studied. The aim of this work was to assess the ovarian response (preovulatory follicular population, oocyte maturation, ovulation rate, and serum steroid concentrations), the early embryo survival and gene expression patterns of a panel of quality-genes involved in glucose intake, oxidative stress, apoptosis, proliferation, implantation, and fetal growth in embryos of lactating rabbits treated with eCG. A total of 34 primiparous rabbit does (Oryctolagus cuniculus) were randomly distributed at Day 23 postpartum into a treatment group receiving a unique nonsuperovulatory dose (25 IU) of eCG (eCG group; N = 17 does); or a control group without eCG treatment previously to artificial insemination (control group; N = 17 does). After 48 hours, 8 does of each group were euthanized and their ovarian response was studied. The rest of animals were artificially inseminated and their ovulation was induced with a GnRH analogue. Embryos were recovered 3.5 days later. The oocytes retrieved for in vitro maturation showed no differences in metaphase II rate in both experimental groups, although oocyte cytoplasmic maturation, in terms of cortical granule migration rate, was improved in eCG-treated does (P < 0.05). The mean number of preovulatory follicles was similar between groups but the ovulation rate was significantly higher in eCG-treated does compared with does not stimulated (P < 0.05). No differences were found in serum estradiol and progesterone concentrations of does the day of oocyte and embryo recovery, respectively. However, progesterone:estradiol ratio was slightly increased in eCG group on embryo recovery day (P = 0.1). The percentage of embryos recovered at the blastocyst stage was also increased in eCG-treated does (P < 0.05), nevertheless, there were no differences in the gene expression patterns of candidate genes SLC2A4, IGF1R, IGF2R, SHC1-SHC, TP53, PTGS2, and PLAC8; except for the transcripts of SOD1 mRNA which were downregulated in eCG-derived embryos (P < 0.05). In conclusion, the administration of eCG improves ovulation rate, oocyte cytoplasmic maturation, and blastocyst formation in primiparous rabbit does inseminated on Day 25 postpartum. Although it seems not to influence the gene expression patterns studied, a lower antioxidant defense of embryos developed after the maternal eCG treatment is suggested., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Elevated plasma nonesterified fatty acid (NEFA) concentrations are associated with negative energy balance and metabolic disorders such as obesity and type II diabetes. Such increased plasma NEFA concentrations induce changes in the microenvironment of the ovarian follicle, which can compromise oocyte competence. Exposing oocytes to elevated NEFA concentrations during maturation affects the gene expression and phenotype of the subsequent embryo, notably prompting a disrupted oxidative metabolism. We hypothesized that these changes in the embryo are a consequence of modified energy metabolism in the oocyte. To investigate this, bovine cumulus oocyte complexes were matured under elevated NEFA conditions, and energy metabolism-related gene expression, mitochondrial function, and ultrastructure evaluated. It was found that expression of genes related to REDOX maintenance was modified in NEFA-exposed oocytes, cumulus cells, and resultant blastocysts. Moreover, the expression of genes related to fatty acid synthesis in embryos that developed from NEFA-exposed oocytes was upregulated. From a functional perspective, inhibition of fatty acid β-oxidation in maturing oocytes exposed to elevated NEFA concentrations restored developmental competence. There were no clear differences in mitochondrial morphology or oxygen consumption between treatments, although there was a trend for a higher mitochondrial membrane potential in zygotes derived from NEFA-exposed oocytes. These data show that the degree of mitochondrial fatty acid β-oxidation has a decisive impact on the development of NEFA-exposed oocytes. Furthermore, the gene expression data suggest that the resulting embryos adapt through altered metabolic strategies, which might explain the aberrant energy metabolism previously observed in these embryos originating from NEFA-exposed maturing oocytes.

The aim of present work was to analyze the body reserves and ovarian features of lactating primiparous rabbit does under extensive reproductive management (artificial insemination (AI) at 25 days post-partum (dpp)) compared with the common insemination rhythm at 11 dpp. A total of 48 primiparous Californian×New Zealand White rabbit does suckling 8 kits were used to assess liveweight, estimated body composition, serum metabolic and endocrine parameters (oestradiol and progesterone concentrations) and ovarian features like follicle population and atresia rate, and oocyte maturation. Rabbit does were randomly allocated in two experimental groups: (a) lactating does euthanized at early post-partum period (11 dpp) according to a semi-intensive rhythm (n=24), and (b) lactating does euthanized at later post-partum period (25 dpp) according to a more extensive rhythm (n=24). Liveweight, body energy content, lipid depots and serum non-esterified fatty acids (NEFA) concentrations decreased from parturition to post-partum period (P<0.05). In addition, serum protein and glucose concentrations increased in the post-partum period (P<0.05). Similar oestradiol and progesterone levels were found in rhythms as well as similar follicle population and nuclear and cytoplasmic maturation rates measured as metaphase II and cortical granule migration, respectively in both post-partum times. However, the number of preovulatory follicles on the ovarian surface was lower (P<0.05) and the atresia rate tended to be higher with a lower percentage of healthy follicles (P<0.1) in ovaries from females of extensive group. In conclusion, the body reserves, serum metabolic parameters and oocyte quality of primiparous non-weaned rabbits does at the late post-partum time (25 days) were not improved. Thus this reproductive management did not present any advantages compared to earlier post-partum (11 days) reproductive rhythm., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Leptin is a metabolic hormone related to body condition and nutritional status that influences fertility in assisted reproductive technologies modulating oocyte and embryo quality. The aim of the present study was to establish the effect of various leptin concentrations (0, 10, 100 ng/mL) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on bovine embryo development and quality in terms of gene expression. The relative mRNA abundance of the genes encoding solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1), Bcl-2-associated X protein (BAX), placenta-specific 8 (PLAC8), aldo-keto reductase family 1 member B1 (AKR1B1) and leptin receptor (LEPR) were determined on Day 7 blastocysts by qRT-PCR. Cleavage rate (P < 0.005) and blastocyst yield (P = 0.05) was significantly lower when cumulus-oocyte complexes (COCs) were matured with 100 ng/mL leptin compared to 0 or 10 ng/mL leptin. No significant effect of different concentrations of leptin added during IVC on blastocyst yield was observed. The presence of 100 ng/mL leptin in both IVM and IVC further decreased cleavage rate (P < 0.005) and blastocyst yield compared to the control group without leptin (P = 0.05) and those supplemented with 10 ng/mL leptin or FCS (P < 0.005). There was no evidence of any leptin-induced difference in the relative transcript abundance of SLC2A1, BAX and PLAC8 genes in Day 7 blastocysts. Expression of AKR1B1 was significantly lower in blastocysts from COCs matured with 100 ng/mL leptin compared to those matured with 0 or 10 ng/mL leptin (P < 0.005). LEPR expression was up regulated when leptin concentration was increased from 0 ng/mL during IVM to 10 ng/mL during IVC, but it was down-regulated in the opposite situation (P < 0.005). In conclusion, high leptin concentrations possibly related to obesity seem to be more detrimental rather than the absence of this hormone for preimplantation embryo survival; this effect is independent of LEPR gene expression and it does not influence expression of SLC2A1, BAX and PLAC8 genes in Day 7 blastocysts., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The aim of this work was to study the effect on body composition, serum metabolic parameters and ovarian status of early weaning at 25 Days post-partum (dpp) as a strategy to decrease energy deficit of primiparous lactating rabbit does prior to insemination at 32 dpp following an extensive rhythm. A total of 34 primiparous lactating rabbit does were used and distributed in three groups: 10 lactating does euthanized at 25 dpp (group L25), 13 does weaned at 25 dpp and euthanized at 32 dpp (group NL32), and 11 non weaned lactating does euthanized at 32 dpp (group L32). No significant differences were observed in live body weight, ovary weight, serum NEFA and total protein concentration among groups. Although NL32 does had a low feed intake (122+/-23.5g/Day; P<0.001), their estimated lipids (16.9+/-1.09%, P<0.008), protein (19.7+/-0.07%, P<0.0001), and energy (1147+/-42.7MJ/kg, P<0.006) body contents were higher and their serum glucose concentrations (158+/-24.5mg/dl, P<0.04) were lower compared to L25 does (11.9+/-1.3%, 18.5+/-0.08%, 942+/-51.3MJ/kg and 212+/-27.9mg/dl) and L32 does (13.4+/-1.03%, 18.5+/-0.1%, 993+/-40.4MJ/kg and 259+/-29.5mg/dl), respectively. In the ovarian surface of L25 does a lower number of follicles > or =1mm was observed compared to NL32 and L32 groups (12.7+/-1.5 vs. 18.0+/-1.45 and 17.6 +/-1.67; P<0.05). Follicular population in the histological ovarian sections and immunolocalization of prolactin receptor were similar between groups. In group L25, both nuclear maturation of oocytes in terms of Metaphase II rate (67.0 vs. 79.7 and 78.3%; P<0.05) and cytoplasmic maturation measured by percentage of cortical granules (CG) totally or partially migrated in oocytes were significantly lower than in groups NL32 and L32 (16.0 vs. 38.3 and 60.0%; P<0.05). Consequently, a higher rate of oocytes with non-migrated CGs was found in group L25 than in groups NL32 and L32 (76.0 vs. 46.8 and 33.3%; P<0.05). In conclusion, even though early weaning at 25 dpp seemed to improve body energy stores of primiparous does, this fact was not well reflected on the ovarian status at 32 dpp, which was similar regardless of weaning time and it could be performed later., (2010 Elsevier B.V. All rights reserved.)

Extreme body mass indexes may impair reproductive outcome in assisted reproductive technologies. Leptin reflects the amount of body fat and could act as a modulator of oocyte quality through activation of specific transcription factors. The aim of this work was to establish whether: 1) leptin influences meiotic and cytoplasmic oocyte maturation; 2) STAT3 and MAPK mediate the effects of leptin and 3) leptin modulates steroid secretion by cumulus-oocyte complexes (COC) during in vitro maturation (IVM). We confirmed immunolocalisation of leptin receptor in oocytes, cumulus/granulosa cells during the peri-ovulatory period. The confocal study showed that COC supplemented with 1, 10 and 100 ng/ml leptin had a significantly higher metaphase II (MII) percentage than those IVM without leptin (P<0.05) and a similar MII index compared to the group supplemented with 10% FCS. Leptin did not increase the percentage of cytoplasmically matured oocytes in terms of cortical granule migration rate, whereas a significantly higher index was found in the FCS group (P<0.001). Oestradiol concentrations in spent media were higher in the FCS group compared to other treatments (P<0.001). Leptin-stimulated nuclear oocyte maturation was significantly impaired when leptin-induced JAK2/STAT3 and MEK 1/2 activation was suppressed by the inhibitors (P<0.001). Steroid secretion of COC was not affected by leptin activation of JAK2/STAT3 or MEK 1/2 pathways. In conclusion, JAK2/STAT3 and MEK 1/2 pathways mediate the enhancement of nuclear oocyte maturation by leptin; however, neither cytoplasmic oocyte maturation nor steroidogenic response of COC were improved in the present rabbit model.

High-yield lactating does need effective estrus synchronization methods to improve their reproductive outcome by enhancing ovarian function. The aim of the current work was to analyze ovarian follicular and oocyte characteristics of hormonal and nonhormonal estrus synchronization regimes in primiparous lactating rabbit does (Oryctolagus cuniculus) in the early postpartum period (Day 11). Females were randomly treated with either (1) a hormonal standard treatment with 25 IU equine chorionic gonadotropin (eCG) 48h before artificial insemination (eCG group) or (2) an alternative nonhormonal treatment consisting of doe-litter separation 24h before artificial insemination (Bio group). No significant differences were found in serum estradiol and progesterone concentrations between experimental groups. During the histologic study, the Bio group presented a higher number of primordial (P<0.05) and primary follicles (P=0.07) compared with that of the eCG group, whereas secondary and antral follicular populations were similar. Rates of late atretic follicles assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique were not different between treatments, but the eCG group showed a significantly higher number of mid-atretic follicles compared with that of the Bio group. Nuclear in vitro oocyte maturation (IVM), measured as metaphase II rate, and in vitro steroidogenic response of cumulus-oocyte complexes, measured by ELISA, did not show significant differences between treatments. However, confocal study showed that cytoplasmic maturation of oocytes, in terms of cortical granule migration rate, was significantly higher in the Bio group compared with that after the eCG treatment. In conclusion, transient doe-litter separation seems to improve ovarian response in terms of follicular health and oocyte competence compared with that after the eCG treatment. Therefore, a 24-h-long transient weaning could be an alternative nonhormonal method for synchronizing estrus in primiparous lactating rabbit does inseminated in the early postpartum period.

Low reproductive performance of high-yield primiparous animals is closely associated with the metabolic stress caused by a simultaneous gestation and lactation. The aim of this work was (1) to analyze body composition and metabolic environment at three time points along lactation (at parturition time; in the lactation period [Day 11 postpartum]; and in the postweaning period [Day 32 postpartum]) of primiparous rabbit does (Oryctolagus cuniculus) and (2) to investigate the ovarian status at insemination time and the possible link with metabolic environment and with their reproductive performance. To this end, does were either submitted to a semi-intensive reproductive rhythm (Group S, inseminated on Day 11 postpartum) or an extensive rhythm (Group E, inseminated on Day 32 postpartum). Body energy (P<0.05) and protein content (P<0.001) as well as serum leptin (P<0.05) and protein concentrations (P<0.001) increased significantly along the postpartum period. At parturition, body lipid content was significantly lower and serum nonesterified fatty acids concentrations were significantly higher than that on Days 11 postpartum and 32 postpartum. Concerning ovarian status at insemination time, no significant differences were found in mean follicular stages, serum estradiol, progesterone, and prolactin (PRL) concentrations or in prolactin receptor (PRL-R) immunostaining. However, follicles in Group S showed a significantly higher apoptosis index than that of Group E (P<0.001). The nuclear and cytoplasmic oocyte maturation rates of Group S were also significantly lower than that in Group E. In addition, conception rate and prolificacy were improved in Group E (P<0.001 and P<0.05, respectively). In conclusion, in the early postpartum period, metabolic status seems to impact negatively on ovarian follicle and oocyte quality leading to a poor reproductive outcome in primiparous rabbit does.

The aim of the current work was to analyze the features of ovarian follicular population and their quality in New Zealand white rabbit does synchronized by 24-h controlled doe-litter separation before artificial insemination (AI) during all their reproductive cycles. Synchronized animals were allocated systematically in two groups. A total of 73 rabbit does (group A) were submitted to a 35-day intensive rhythm (AI on day 4 post-partum [pp] and weaning at 25 days of lactation), and 108 rabbit does (group B) were submitted to a 42-day semi-intensive rhythm (AI on day 11 pp and weaning at 35 days of lactation) during 9 months. At the mid-end of their reproductive life, a total of 26 does (5.4 parturitions), under intensive (n=15) or semi-intensive rhythm (n=11) were either treated in each group with 25 IU eCG 48 h before laparotomy to recover their ovaries (n=7 for group A and n=6 for group B) (according to the Bioethics Committee of the University) or not synchronized with the hormonal treatment (n=8 for group A and n=5 for group B). Blood samples were collected at the moment of ovary recovery; morphometrical parameters, number of total follicles and number of follicles >or=1mm in size in the ovarian surface were recorded. Oocytes from follicles of one ovary were recovered and matured in TCM 199 supplemented with 10 ng/ml EGF, 100 ng/ml IGF-I and 10% FCS. The counterpart ovaries were fixed in paraformaldehyde solution for histological studies. Detection of cell apoptosis was determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) technique. Reproductive performance was affected by the rhythm used, with lower reproductive parameters in the intensive group. The average ovary height and width, the mean number of >or=1mm follicles and the number of total follicles were similar between groups. Serum concentrations of estradiol (E(2)) and testosterone (T) were significantly lower in group A vs. B (E(2): 232.4+/-56.1 vs. 399.7+/-53.0 pg/ml; P<0.05 and T: 1.07+/-0.10 vs. 1.68+/-0.23 ng/ml; P<0.05). No significant differences were found in follicular population or in the mean follicular apoptosis index between groups. Metaphase II rate was significantly lower in group A vs. B (48.5+/-3.3 vs. 67.6+/-3.7%; P<0.01), as well as the migration rate of cortical granules (12.7+/-2.7 vs. 38.2+/-6.6%; P<0.001). On the other hand, neither follicular population, nuclear maturation rate nor apoptosis rate were affected by the eCG treatment, but cytoplasmic maturation was higher in animals treated with eCG in group A (29.2% vs. 5.5%; P<0.01). In conclusion, rabbit does under transient litter separation during their reproductive life have both their serum estradiol and testosterone concentrations and oocyte quality influenced by the intensive rhythm, leading to a decrease in reproductive parameters. Also, both intensive and semi-intensive rhythms seem to be less receptive to eCG treatment than expected.

Objective: The aim of this study is to assess the relationship between birth weight and the lipid profile and blood pressure in adolescents., Patients and Methods: Cross sectional study. We studied seven hundred and forty adolescents aged 18 who attended four different schools located in Madrid. Birth weight (BW) data were obtained from delivery records. All subjects underwent a physical examination, including measurement of blood pressure. Plasma lipids were determined using standardised methods after ten hour fasting., Results: Total cholesterol and cholesterol-LDL in males and females with BW < 3.000 grams were statistically greater than those from subjects with BW > 3,800 grams. Male adolescents also showed a difference in the apolipoprotein B (ApoB) levels. We only found a statistically significant difference in the diastolic blood pressure of male adolescents belonging to the extreme birth weight quintiles., Conclusions: We found an inverse relationship between birth weight and total cholesterol and cholesterol LDL levels in males and females. This association was also noted in Apo B concentrations in male adolescents. The only influence birth weight had on blood pressure was on the diastolic blood pressure of males.

Objective: To determine the influence of socioeconomic status on healthcare demand and resource consumption in our population., Patients and Methods: We performed a cross sectional, retrospective study of socioeconomic status and healthcare and pharmaceutical consumption in randomly selected patients from five primary care pediatric clinics in Madrid. The chi-square test was used to compare percentages. For the remaining analyses, nonparametric tests were used after confirming that the data followed non-normal distribution., Results: We compared 684 questionnaires. Patients in the most disadvantaged socioeconomic levels consumed a greater number of drugs than those in more privileged levels (mean 0.79 vs. 0.47, p = 0.04). The mean pharmacological expenditure was also higher in the lowest socioeconomic levels than in the highest levels (5.28 Euros vs. 2.21 Euros, respectively; p = 0.001). No significant differences were found among socioeconomic levels in the number of consultations or diagnostic tests requested. The number of consultations was higher in younger patients (p < 0.001) or in those with chronic diseases (p = 0.001). Drug consumption was increased in the most disadvantaged levels (p = 0.002) and in patients with chronic diseases (p < 0.001). Lastly, pharmacological expenditure expressed in Euros was also higher in the lowest socioeconomic levels (p = 0.001) and in patients with chronic diseases (p < 0.001) but was lower if one of the parents was a foreigner (p = 0.031)., Conclusions: We found a relationship between socioeconomic level and drug consumption and its attributable cost. These data should be confirmed by broader studies.

Iron deficiency and iron deficiency anemia are very important disorders in childhood because, during this stage of life, the confluence of different factors can lead to iron deficiency. During childhood, there are rapid growth periods in which the need for iron increases and these needs are not always compensated for by a sufficient iron intake. We have studied a sample of 2,224 schoolchildren, both males and females, between the ages of 2 and 18 years, from five different schools in the Community of Madrid. The prevalence of anemia was studied by determining the hemoglobin, MCV and MCHC, whereas iron deficiency was assessed by measurement of serum iron. In our study population, the prevalence of iron deficiency was 4.94% and the rate of iron deficiency anemia was 0.94%. This disorder is predominant in 13 to 15 year olds (3.7% of the males and 3.53% of the females showed hemoglobin values below the reference range). In addition 6.73% of this group were iron deficient. The MCV was decreased in 2.69% of the subjects, after the exclusion of six children with thalassemia trait.

Background: The aim of this study was to evaluate the association between socioeconomic status and the presence of cardiovascular risk factors in children from Madrid., Methods: We studied 2224 boys and girls, ages 2-18 years, attending five different school centers. They were divided into three socioeconomic groups: low class, middle-low class and middle high class, in regard to their parent's occupation and educational attainment. The evaluation included a blood analysis of serum triglycerides (TG), total cholesterol (CT), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), apolipoprotein A1 (ApoA), apolipoprotein B100 (Apo B), and the Apo A/Apo B and LDL/HDL ratios., Results: There was a positive correlation between socioeconomic status and total cholesterol, Apo A, Apo B and Apo A/Apo B, whereas socioeconomic status and triglycerides were inversely related. The LDL/HDL ratio varied according to age., Conclusions: Unlike some previously published studies, we found that children belonging to high socioeconomic status have a more atherogenic lipid profile than those of middle low or low socioeconomic levels.

We have studied 2,224 children and adolescents of both sexes, ranging between 2 and 18 years of age, at five schools in the city of Madrid. We determined the lipid profile: total cholesterol (T-C), cholesterol bound to high density lipoproteins (HDL-C), cholesterol bound to low density lipoproteins (LDL-C), cholesterol bound to very low density lipoproteins (VLDL-C), triglycerides (TG), apolipoprotein AI and apolipoprotein B100 (Apo A1 and Apo B100, respectively). In relationship to age and sex, in males, as age increases, there is an elevation in LDL-C and TG and a diminution in T-C, HDL-C, Apo AI and Apo B100. There are no changes in VLDL-C. In females, there is a diminution in LDL-C, TG, Apo-B100 and an increase in HDL-C, Apo AI and T-C, with the increase in T-C being found only in those older than 15 years.