α-Tocopherol modifies the expression of genes related to oxidative stress and apoptosis during in vitro maturation and enhances the developmental competence of rabbit oocytes.

The developmental competence of invitro maturation (IVM) oocytes can be enhanced by antioxidant agents. The present study investigated, for the first time in the rabbit model, the effect of adding α-tocopherol (0, 100, 200 and 400µM) during IVM on putative transcripts involved in antioxidant defence (superoxide dismutase 2, mitochondrial (SOD2), glutathione peroxidase 1 (GPX1), catalase (CAT)), cell cycle regulation and apoptosis cascade (apoptosis tumour protein 53 (TP53), caspase 3, apoptosis-related cysteine protease (CASP3)), cell cycle progression (cellular cycle V-Akt murine thymoma viral oncogene homologue 1 (AKT1)), cumulus expansion (gap junction protein, alpha 1, 43 kDa (GJA1) and prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclo-oxygenase) (PTGS2)) and metabolism (glucose-6-phosphate dehydrogenase (G6PD)). Meiotic progression, mitochondrial reallocation, cumulus cell apoptosis and the developmental competence of oocytes after IVF were also assessed. Expression of SOD2, CAT, TP53, CASP3 and GJA1 was downregulated in cumulus-oocyte complexes (COCs) after IVM with 100µM α-tocopherol compared with the group without the antioxidant. The apoptotic rate and the percentage of a non-migrated mitochondrial pattern were lower in COCs cultured with 100µM α-tocopherol, consistent with better-quality oocytes. In fact, early embryo development was improved when 100µM α-tocopherol was included in the IVM medium, but remained low compared with invivo-matured oocytes. In conclusion, the addition of 100µM α-tocopherol to the maturation medium is a suitable approach to manage oxidative stress and apoptosis, as well as for increasing the in vitro developmental competence of rabbit oocytes.

In vivo and in vitro maturation of rabbit oocytes differently affects the gene expression profile, mitochondrial distribution, apoptosis and early embryo development.

In vivo-matured cumulus-oocyte complexes are valuable models in which to assess potential biomarkers of rabbit oocyte quality that contribute to enhanced IVM systems. In the present study we compared some gene markers of oocytes and cumulus cells (CCs) from immature, in vivo-matured and IVM oocytes. Moreover, apoptosis in CCs, nuclear maturation, mitochondrial reallocation and the developmental potential of oocytes after IVF were assessed. In relation to cumulus expansion, gene expression of gap junction protein, alpha 1, 43 kDa (Gja1) and prostaglandin-endoperoxide synthase 2 (Ptgs2) was significantly lower in CCs after in vivo maturation than IVM. In addition, there were differences in gene expression after in vivo maturation versus IVM in both oocytes and CCs for genes related to cell cycle regulation and apoptosis (V-Akt murine thymoma viral oncogene homologue 1 (Akt1), tumour protein 53 (Tp53), caspase 3, apoptosis-related cysteine protease (Casp3)), oxidative response (superoxide dismutase 2, mitochondrial (Sod2)) and metabolism (glucose-6-phosphate dehydrogenase (G6pd), glyceraldehyde-3-phosphate dehydrogenase (Gapdh)). In vivo-matured CCs had a lower apoptosis rate than IVM and immature CCs. Meiotic progression, mitochondrial migration to the periphery and developmental competence were higher for in vivo-matured than IVM oocytes. In conclusion, differences in oocyte developmental capacity after IVM or in vivo maturation are accompanied by significant changes in transcript abundance in oocytes and their surrounding CCs, meiotic rate, mitochondrial distribution and apoptotic index. Some of the genes investigated, such as Gja1, could be potential biomarkers for oocyte developmental competence in the rabbit model, helping improve in vitro culture systems in these species.

Reproductive and nutritional management on ovarian response and embryo quality on rabbit does.

Rabbit does in modern rabbitries are under intensive reproductive rhythms. Females are high milk producers with high energetic expenses due to the extensive overlap between lactation and gestation. This situation leads to a negative energy balance with a mobilization of body fat especially in primiparous rabbit does. Poor body condition and poor health status severely affect the reproductive features (fertility rate and lifespan of the doe as well as ovarian physiology). This paper reviews some reproductive and nutritional approaches used in the last years to improve the reproductive performance of rabbit females, mainly focusing on the influence on ovarian response and embryo quality and with emphasis on epigenetic modifications in pre-implantation embryos and offspring consequences.

Acute fasting before conception affects metabolic and endocrine status without impacting follicle and oocyte development and embryo gene expression in the rabbit.

Food deprivation affects female reproduction. The goal of the present study was to elucidate in the rabbit model the effects of acute energy restriction on ovarian function (follicle development, atresia rate and in vitro oocyte maturation) and embryonic development and gene expression of some candidate genes. Serum metabolic parameters (non-esterified fatty acids (NEFA), triglycerides, glucose, insulin and leptin concentrations) and endocrine markers (oestradiol-17ß and progesterone concentrations) were also studied. A control group of nulliparous does fed ad libitum and a 72-h fasted group were used. At the end of the nutritional treatment, the ovaries of half of the animals were retrieved while the other animals were re-fed and artificially inseminated to recover embryos at 84 h after insemination, during the luteal phase. At the end of fasting, increased serum NEFA and decreased leptin concentrations were observed in the fasted group, but no differences appeared in serum steroid concentrations, follicle population and atresia rate or nuclear and cytoplasmic oocyte maturation. In the luteal phase, insulin concentrations increased notably in the fasted group. The number of recovered embryos per female and the speed of embryo development were reduced in the food-deprived group. Acute fasting altered both metabolic and endocrine markers and embryo development, but follicle and oocyte development and embryo gene expression were not affected.

Effects of feed restriction during pregnancy on maternal reproductive outcome, foetal hepatic IGF gene expression and offspring performance in the rabbit.

Primiparous female rabbits have high nutritional requirements and, while it is recommended that they are subjected to an extensive reproductive rhythm, this could lead to overweight, affecting reproductive outcomes. We hypothesised that restricting food intake during the less energetic period of gestation could improve reproductive outcome without impairing offspring viability. This study compares two groups of primiparous rabbit does in an extensive reproductive programme, one in which feed was restricted from Day 0 to Day 21 of gestation (R021), and another in which does were fed ad libitum (control) throughout pregnancy. The mother and offspring variables compared were (1) mother reproductive outcomes at the time points pre-implantation (Day 3 postartificial insemination [AI]), preterm (Day 28 post-AI) and birth; and (2) the prenatal offspring characteristic IGF system gene expression in foetal liver, liver fibrosis and foetus sex ratio, and postnatal factor viability and growth at birth, and survival and growth until weaning. Feed restriction did not affect the conception rate, embryo survival, or the number of morulae and blastocysts recovered at Day 3 post-AI. Preterm placenta size and efficiency were similar in the two groups. However, both implantation rate (P < 0.001) and the number of foetuses (P = 0.05) were higher in the R021 mothers than controls, while there was no difference in foetal viability. Foetal size and weight, the weights of most organs, organ weight/BW ratios and sex ratio were unaffected by feed restriction; these variables were only affected by uterine position (P < 0.05). Conversely, in the R021 does, foetal liver IGBP1 and IGF2 gene expression were dysregulated despite no liver fibrosis and a normal liver structure. No effects of restricted feed intake were produced on maternal fertility, prolificacy, or offspring birth weight, but control females weaned more kits. Litter weight and mortality rate during the lactation period were also unaffected. In conclusion, pre-implantation events and foetal development were unaffected by feed restriction. While some genes of the foetal hepatic IGF system were dysregulated during pregnancy, liver morphology appeared normal, and the growth of foetuses and kits until weaning was unmodified. This strategy of feed restriction in extensive reproductive rhythms seems to have no significant adverse effects on dam reproductive outcome or offspring growth and viability until weaning.

Influence of leptin on in vitro maturation and steroidogenic secretion of cumulus-oocyte complexes through JAK2/STAT3 and MEK 1/2 pathways in the rabbit model.

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.

Follicular, oocyte and embryo features related to metabolic status in primiparous lactating does fed with high-fibre rearing diets.

Fertility of primiparous lactating does in the early postpartum (pp) period is very low mainly due to pronounced deficient energy intake, influencing oocyte and embryo developmental competence. The hypothesis used in this work was that high-lignin fibre diet supplied during the rearing period could increase feed intake and, consequently, improve the reproductive physiology and metabolic status of primiparous does in the early pp period. Diets with high-lignin [HL: 15.8% dry matter (DM)] or standard-lignin content (SL: 4.9% DM) were supplied until parturition time. No diet effects in serum oestradiol, progesterone concentrations and follicle categories were found in the histological study. Metaphase II rate of in vitro-matured oocytes was significantly higher in the SL vs the HL group (p < 0.001). Cytoplasmically degenerated oocytes (in terms of abnormal distribution of cortical granules) and follicular atresia rate were significantly lower in the SL group than in the HL group (p < 0.05 and p < 0.005 respectively). In addition, HL-fed does showed lower number of viable embryos and higher rate of retarded in vivo-recovered embryos compared with the SL group (p < 0.05). Neither in vitro embryo development of viable embryos nor conception rate was significantly different between groups. Feed intake increased during the first pregnancy in the HL group (p < 0.05), but not during early lactation. Serum protein, non-esterified fatty acid and leptin concentrations, as well as estimated body composition were similar in does fed with both diets. In conclusion, the enhancement of reproductive management by using highly lignified products in rearing diets does not seem to report physiological reproductive benefits affecting oocyte maturation rate and embryo viability in primiparous lactating does.

Role of nerve growth factor in the reproductive physiology of female rabbits: A review.

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 (ß-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 ß-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 ß-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.

Dietary fish oil and flaxseed for rabbit does: fatty acids distribution and Δ6-desaturase enzyme expression of different tissues.

Standard feeds are imbalanced in term of n-6/n-3 polyunsaturated fatty acids (PUFA) ratio, with a low proportion of the latter. The reproductive system appears to be strongly affected by administration of n-3 PUFA, and ingredients rich in α-linolenic acid (ALA; i.e. vegetable sources) or EPA and DHA acids (i.e. fish oil) can be included in animal diets to balance PUFA intake. The aim of this study was to evaluate the effect of dietary supplementation with flaxseed (ALA) or fish oil (EPA and DHA) on PUFA metabolism in rabbit does. A total of 60 New Zealand White female rabbits were assigned to three experimental groups: control group, FLAX group fed 10% extruded flaxseed and FISH group fed 3% fish oil. Blood, milk, liver and ovaries were collected from the does to assess the lipid composition; furthermore, FADS2 gene expression was assessed in liver and ovary tissues. Reproductive performance of does was also recorded. The fertility rate and number of weaned rabbits improved with n-3 dietary supplementation: does at first parity showed the lowest reproductive results, but the administration of n-3 reduced the gap between primiparous and multiparous does. Feed consumption and milk production were not affected by the feeding regime. The fatty acid composition of milk, plasma, liver and ovaries were widely influenced by diet, showing higher concentrations of n-3 long-chain PUFA (LCP) in does fed with n-3 enriched diets. FISH diet resulted in the highest n-3 LCP enrichment, whereas in the FLAX group, this increase was lower. Blood and milk showed low levels of LCP, whereas liver and ovaries were the main sites of n-3 LCP synthesis and accumulation. Accordingly, although the liver is the main metabolic centre for LCP synthesis, ovaries also have a prominent role in LCP generation. FADS2 expression in liver and ovary tissue was downregulated by FISH administration. In conclusion, the enrichment of diets with n-3 PUFA could be an effective strategy for improving the reproductive performance of does.

Endocrine and ovarian response after a 2-day controlled suckling and eCG treatment in lactating rabbit does.

Synchronization methods are used to obtain higher fertility when artificial insemination (AI) is applied to lactating rabbit does. The most common methods are eCG administration or temporary doe-litter separation. Nevertheless, drawbacks have been reported, such as negative side effects of hormonal treatment in the doe and low litter growth due to absence of suckling, respectively. Recently, improved reproductive performance (without visible consequences on young rabbit growth), has been obtained by applying a 2-day controlled nursing method before AI, by allowing for a 10 min nursing of the litter 24 h of separation. The present study was undertaken to examine the pituitary (PRL, LH, FSH) and the ovarian response (follicle size and number) to those methods. A total of 442 lactating does inseminated on day 11 post-partum were distributed in three experimental groups: 2CN (closing of nest box on day 9, controlled nursing on days 10 and 11), eCG (20 IU administered on day 9 post-partum) and CONTROL (untreated). Blood samples were obtained from 10 does per group at 48, 24 and 0 h before AI, and 1h after AI. Both 2CN and eCG treatments similarly improved sexual receptivity (76.3, 77.5 and 58.2%, respectively; P<0.001) and fertility (63.1, 64.1 and 48.4%, respectively; P<0.05) in lactating does, compared to the CONTROL group. Similar plasma FSH levels in all groups of does and sampling times were observed. Due to the absence of suckling, plasma concentration of PRL on day 10 post-partum in the 2CN group was lower than in the CONTROL group (P<0.05); this endocrine change in PRL levels could explain the better reproductive performances obtained with 2CN treatment. At 1h after exogenous administration of GnRH (at the moment of AI) a high LH response was observed in all groups (P<0.001). Ovaries from 20 rabbits treated in the same way but uninseminated (2CN, n=10; eCG, n=5; CONTROL, n=5 does) were obtained on day 11 post-partum in order to check the morphometric status (weight, width and height) and to make histological and immunohistochemical studies to detect growth hormone receptor (GH-R). As a result, synchronization methods did not show any significant difference in relation to the CONTROL group. However, a small increase in the number of primary follicles was evidenced in the 2CN group with respect to the eCG group, similarly to the CONTROL group (23.0+/-3.7, 9.4+/-4.9 and 14.8+/-4.92 primary follicles, respectively; P=0.1). GH-R immunostaining-presence was more evident in the 2CN and the eCG groups, including primordial follicles and oocytes themselves. Thus, there could have been some direct effects of GH on follicular development, as described in other species. Some ovarian parameters described open new ways to study intra-ovarian mechanism of follicular development in the post-partum period of rabbit does.

Improvements in the conception rate, milk composition and embryo quality of rabbit does after dietary enrichment with n-3 polyunsaturated fatty acids.

This work attempts to confirm the effect of an enriched diet with n-3 polyunsaturated fatty acids (PUFA) trying to mitigate the reproductive performances issues such as low conception rate of primiparous rabbits. A total of 127 does were fed ad libitum throughout their two first cycles with two diets with different fat sources: mixed fat in the control and salmon oil in the enriched one, with 3.19 g/100 g (n=63 does) and 28.77 g/100 g (n=64 does) of n-3 of the total fatty acid, respectively. Feed intake was similar between groups (P>0.05). Plasma progesterone concentration was higher in the enriched females than in control ones at 7 (30.9±2.18 v. 23.9±2.30 ng/ml, respectively; P=0.029) and 14 (38.7±2.18 v. 28.2±2.30 ng/ml, respectively; P=0.001) days of first gestation. Considering both cycles, reproductive parameters of mothers (fertility, duration of gestation and prolificacy) and litter parameters (weight at parturition and weaning, mortality and average daily gain (ADG) of kits during lactation) were similar in both groups. However, individual measurements of neonates of enriched group improved 5.87%, 7.10% and 18.01% (P0.05), but embryo apoptosis rate was higher in control group than in enriched one (31.1±4.56% v. 17.1±3.87%, respectively; P<0.05). In conclusion, dietary PUFA enrichment from the rearing and throughout two productive cycles improved plasma progesterone during pregnancy, fertility, milk fatty acid profile and neonates development of primiparous supporting the beneficial effect of n-3 PUFA supplementation in rabbit does.

Effects of dietary fish oil supplementation on performance, meat quality, and cecal fermentation of growing rabbits.

The objective of this study was to investigate the effect of dietary supplementation with fish oil on growth performance (during all fatening period), carcass characteristics and fatty acid (FA) profile of muscle and fat tissues (at slaughter), as well as cecal fermentation and ileal mucosa morphology of growing rabbits (at 30, 45, and 60 d of age). Two isonitrogenous and isoenergetic diets, only differing in their fat source, were formulated and provided each to 24 does (12 per diet) and their offspring during pregnancy and lactation. The control diet contained 4.59 g of n-3 per 100 g of total FA, and the enriched diet contained 14.9 g of n-3 per 100 g of total FA. From weaning (30 d of age) to slaughter (60 d), the litters (12 per diet; 8 kits each) continued fed the corresponding experimental diet. There were no differences ( > 0.05) between groups in ADFI, ADG and G:F ratio during the growing period. At slaughter, BW, full gastrointestinal tract weight, carcass yield, meat color and pH, drip loss percentage, content of scapular fat and tissue composition of the left hind leg were similar between groups ( > 0.05), but perirenal fat was lower ( = 0.020) and skin weight and abdominal fat tended to be lower ( = 0.055 and = 0.063, respectively) in enriched rabbits than in control ones. Total PUFA content in both LM and perirenal fat was greater ( = 0.021 and < 0.001, respectively) in enriched rabbits, that also showed lower n-6/n-3 ratios in LM (1.61 vs. 5.80; < 0.001) and perirenal fat (4.71 vs. 12.0; < 0.001) than those fed the control diet. Cecal concentrations of total VFA were greater ( < 0.001) in enriched than in control group at 30, 45 and 60 d of age, but diet did not affect ( ≥ 0.332) VFA profile, with the exception of a lower ( = 0.013) proportion of minor VFA (sum of isobutyrate, isovalerate, and valerate) in control group. Diet did not affect ( > 0.255) either pH and NH-N concentrations in the cecum or ileal morphology (crypt depth and villi length). The results showed that dietary fish oil supplementation enhanced beneficial long-chain n-3 FA and decreased n-6/n-3 ratio in rabbit meat and fat, being healthier for human consumption, without having negative effects on growth performance, cecal fermentation, and ileal morphology or carcass characteristics.

A diet supplemented with n-3 polyunsaturated fatty acids influences the metabomscic and endocrine response of rabbit does and their offspring.

The aim of the present study was to evaluate the productive, endocrine, and metabomscic responses as well as oxidative stress of rabbit does and their offspring when fed a diet supplemented with -3 PUFA during their first productive cycle. To this aim, a total of 105 rabbit does were fed ad mscibitum from d 60 to 172 of age 2 isoenergetic and isoproteic diets differing in fatty acid composition. The control diet ( = 52 does) contained 45.9 g/kg of -3 of the total fatty acids and the enriched diet ( = 53 does) contained 149.2 g/kg of -3 of the total fatty acids. Both experimental groups had similar feed intake during rearing, pregnancy, and lactation. The enrichment of diet had no effect on ultrasonographic assessment of does on d 9 and 16 of pregnancy, with an embryonic vesicle number and fetus and placenta size similar between groups ( > 0.05). Even though there were no major effects ( > 0.05) on fertimscity, duration of gestation, and number born amscive and stillborn kits at parturition, mscive kits from enriched does were longer (71.6 ± 2.42 vs. 79.5 ± 2.13 mm; < 0.05) and tended to be heavier (42.5 ± 3.94 vs. 50.8 ± 3.47 g; = 0.07) than those from control does ( < 0.05). The 2 groups had similar milk production and mortamscity values during lactation; consequently, there were no differences between diets in ADG, mscitter weight, and number of weaned kits ( > 0.05). In enriched does, higher plasma leptin and estradiol concentrations than in control does ( < 0.05) were observed. In addition, enriched females also had lower total and high-density mscipoprotein cholesterol (HDL-c) than control females during lactation ( < 0.05). Regarding offspring, the enrichment of diet with PUFA caused a hypermscipidemic status (greater values of plasma triglycerides, total cholesterol, and HDL-c; < 0.05) at 1 d postpartum (dpp), compared with the control group, that disappeared at 32 dpp. Supplemented does before parturition and their offspring at 1 dpp had greater oxidative stress than those in the control group. In conclusion, an increase of -3 PUFA concentration in the diet of rabbit does and, consequently, of their offspring during a productive cycle alters their mscipid profile and the indicators of oxidative stress, without major endocrine modifications or improvements in the productive variables.

Oestrus synchronisation of rabbit does at early post-partum by doe-litter separation or ECG injection: Reproductive parameters and endocrine profiles.

Inseminating rabbit does at early post-partum, in combination with early weaning, can increase prolificacy (total kits born and still born per parturition) and decrease parturition intervals. Oestrus synchronisation increases fertility and prolificacy, while decreasing the number of inseminations required for gestation. However, little is known about the effectiveness of different oestrus synchronisation methods at early post-partum. In this study, does (n = 138) were artificially inseminated nine times (over a period of 1 year, kits weaned at 25 days), on day 4 post-partum after separation from the litter (for 48 or 24 h) or 48 h after 25 UI eCG injection. Plasma levels of prolactin and estradiol were also evaluated in a subsample of 12 multiparous lactating does per treatment, on days 2, 3 and 4 post-partum. The three treatments increased overall fertility of multiparous females compared to controls (which were not synchronised), but there were no differences among treatments in total kits born or stillborn. Does treated with eCG had a higher culling rate. The interval between parturitions and the number of inseminations required for gestation tended to decrease with increasing number of inseminations. In lactating does, there was an interaction between treatment and insemination order. Fertility decreased with increasing inseminations in eCG does but tended to increase above control values in the separated does until the fourth insemination. Control lactating does had significantly less kits per parturition compared to treatments, but eCG lactating does had more stillborn kits. Oestradiol levels increased on day 4 post-partum in all synchronised lactating does (and immediately before artificial insemination in 48 h doe-litter separation), so ovarian activity could be stimulated at early post-partum using all treatments. However, the increase could not be explained by prolactin levels, since there were no effects of suckling absence on plasma prolactin in separated does. In conclusion, separating does from the litter before insemination can be just as effective as eCG treatment, especially during for the first four inseminations.

Expression of nerve growth factor and its receptors in the uterus of rabbits: functional involvement in prostaglandin synthesis.

The aim of the present study was to evaluate: (1) the presence of nerve growth factor (NGF), neurotrophic tyrosine kinase receptor 1 (NTRK1), and nerve growth factor receptor (NGFR) in the rabbit uterus; and (2) the in vitro effects of NGF on PGF2α and PGE2 synthesis and on the PGE2-9-ketoreductase (PGE2-9-K) activity by the rabbit uterus. Nerve growth factor, NTRK1, and NGFR were immunolocalized in the luminal and glandular epithelium and stroma cells of the endometrium. reverse transcriptase polymerase chain reaction indicated the presence of messenger RNA for NGF, NTRK1, and NGFR in the uterus. Nerve growth factor increased (P < 0.01) in vitro secretions of PGF2α and PGE2 but coincubation with either NTRK1 or oxide nitric synthase (NOS) inhibitors reduced (P < 0.01) PGF2α production and blocked (P < 0.01) PGE2 secretion. Prostaglandins releases were lower (P < 0.01) than control when uterine samples were treated with NGF plus cyclooxygenase inhibitor. However, addition of NGFR inhibitor reduced (P < 0.01) PGF2α secretion less efficiently than NTRK1 or NOS inhibitors but had no effect on PGE2 yield. Nerve growth factor increased (P < 0.01) the activity of PGE2-9-K, whereas coincubation with NTRK1 or NOS inhibitors abolished (P < 0.01) this increase in PGE2-9-K activity. However, cotreatment with either cyclooxygenase or NGFR inhibitors had no effect on PGE2-9-K activity. This is the first study to document the distribution of NGF/NTRK1 and NGFR systems and their effects on prostaglandin synthesis in the rabbit uterus. NGF/NTRK1 increases PGF2α and PGE2 productions by upregulating NOS and PGE2-9-K activities, whereas NGF/NGFR augments only PGF2α secretion, through an intracellular mechanism that is still unknown.

Characterization of early changes in fetoplacental hemodynamics in a diet-induced rabbit model of IUGR.

Intrauterine growth restriction (IUGR) is associated with adverse perinatal outcomes and late-onset diseases in offspring. Eating disorders, voluntary caloric restriction and maternal undernutrition can all induce IUGR but a relevant model is required to measure all its possible consequences. In this work, pregnant rabbits were used as an IUGR model. Control females (n=4) received ad libitum diet throughout pregnancy, whereas underfed females (n=5) were restricted to 50% of their daily requirements. Offspring size was measured by ultrasonography and in vivo at birth. Hemodynamic features of the umbilical cords and middle cerebral arteries (systolic peak velocity, end diastolic velocity, pulsatility index and resistance index) were characterized by Doppler ultrasonography. At day 21, maternal underfeeding resulted in a significant reduction of fetal size (occipito-nasal length). At birth, the size of kits from the underfed group was significantly lower (lower crown-rump length, biparietal and transversal thoracic diameters) and a reduced weight with respect to the control group. Feed restriction altered blood flow perfusion compared with does fed ad libitum (significant higher systolic peak, time-averaged mean velocities and lower end diastolic velocity). Fetuses affected by IUGR presented with compensative brain-sparing effects when compared with the control group. In conclusion, the present study supports using rabbits and the underfeeding approach as a valuable model for IUGR studies. These results may help to characterize IUGR alterations due to nutrient restriction of mothers in future research.

Effects of HCG or gonadoreline on seminal parameters and plasma testosterone levels in young male rabbits.

The effect of successive HCG or Gonadoreline injections on some reproductive parameters in 21 young male rabbits, aged from 4 to 4.5 months, has been studied. Animals were randomly allocated in three groups of seven to receive weekly (on each Monday) i.m. injections of 20 mg Gonadoreline, 50 IU HCG, or 0.5 ml Saline solution, respectively. The ejaculate from each male was collected and analysed twice a week (Tuesday and Friday). Higher sperm volumes were observed on the first week after Gonadoreline administration compared to the other groups (p < 0.05), and lower values during the second week in the Control group vs. Gonadoreline group (p < 0.05). Sperm volume increases after the 3rd week in Control animals (p < 0.05). When considering the global period studied, mean sperm volumes achieved after Gonadoreline treatment were higher than in HCG or Control group. Despite the random distribution of the animals to each treatment, and although throughout the experiment more ejaculates were discarded in the Gonadoreline group, the final number of doses obtained per ejaculate was higher in this group followed by HCG treated animals. Globally, the mean sperm concentration of the Control group during the entire studied period was significatively lower compared to the other groups (p < 0.001). No sperm motility differences were detected among the groups (p < 0.1174). HCG injections caused a significative increase in plasma testosterone concentration during the first and second week (p < 0.001) and similar plasma levels were observed in all groups afterwards. No direct plasma testosterone increasing levels owed to Gonadoreline injections were detected, probably due to the frequency of blood sampling (once a week).

Plasma estradiol-17beta levels and gonadosomatic index in tench (Tinca tinca L.) reared in natural and controlled conditions.

The plasma estradiol-17beta levels (E2-17beta) and gonadosomatic index (GSI) of female tench (Tinca tinca L.) reared outdoors and indoors were studied from December 1996 to April 1997. Water temperature and daylight were gradually increased (from 10 to 25 degrees C and from 9 to 16 hours per day, respectively) in the indoor group. Animals reared in natural conditions had similar plasma levels throughout the experiment but animals under controlled conditions experienced an increase in these levels in March in relation to previous months (4.81+/-0.73 ng/ml in March vs. 0.29+/-0.03 ng/ml and 0.99+/-0.31 ng/ml in January and February, respectively; P < 0.05) and in relation to animals reared outdoors (4.81+/-0.73 ng/ml vs. 0.47+/-0.19 ng/ml, respectively; P < 0.05). In April, a sharp decrease in estradiol-17beta in these animals was observed, probably associated with the end of vitellogenesis. GSI increased in both groups through the experiment and indoor animals showed greater values in March vs outdoor fishes (7.57+/-2.18 vs. 4.05+/-0.63; P < 0.05). No significant correlation was found between E2-17beta and GSI, (r = 0.35). It is concluded that manipulation of the photoperiod and temperature can modify gonadal development in tench, and this is similar to the situation in other Teleostei species. This research is the first step towards achieving, out of season, spawning in tench.

Determination of plasma melatonin levels by enzyme-linked immunosorbent assay (EIA) in turbot (Scophtalmus maximus L.) and tench (Tinca tinca L.).

An enzymoimmunoassay (EIA) kit for plasma melatonin (MLT) measurements was employed in tench (Tinca tinca) and in turbot (Scophtalmus maximus). Tench and turbot plasma samples were purified with a C18 reversed phase extraction columns because this kit is designed for human serum measurements. The lowest detection limit of the technique was 11.48 pg/well with a sensitivity at 50% binding of 100 pg/well. Intra-assay and inter-assay CV (%) were always less than 5% (n=8), and 9% (n=6) in tench plasma samples, and less than 5% (n=8) and 13% (n=5) in turbot plasma samples, respectively. Correlation coefficients between EIA and RIA measurements in tench and turbot plasma samples were 0.93 and 0.89 (p<0.001) respectively. Diurnal and nocturnal plasma melatonin mean levels were 14.7+/-2.1 pg/ml and 87.4+/-11 pg/ml in tench (n=15), and 3.5+/-0.4 pg/ml and 28.1+/-2.1 pg/ml in turbot (n=15). These species showed a melatonin circadian rhythm as in other animals studied. The results suggest that the commercial kit used in this experiment could be a suitable and alternative method to RIA for plasma MLT determinations in tench and turbot although it is necessary to increase volumes (1ml) and concentrate daytime samples.

Endocrine profiles during doe-litter separation and the subsequent pregnancy in rabbits.

This study was carried out to determine the effects of a transient doe-litter separation on plasma prolactin, LH, FSH, estradiol-17beta and progesterone concentrations before artificial insemination and during the subsequent pregnancy. Control does (n=12) had free access to nursing, whereas separated does (n=12) were kept away from their litters for 48 hours before artificial insemination. Both groups were inseminated on day 11 after parturition. Teat stimulation by suckling caused a high increase in prolactin concentrations in separated does (p < 0.0001). Basal prolactin concentrations were observed in both groups on days 8 and 18 of pregnancy. No effect of the treatment was detected on LH and FSH concentrations during the sampling period. A rise of estradiol-17beta concentrations was observed 48 hours after doe-litter separation, compared to control does and to previous values (p < 0.003). Both groups showed low progesterone concentrations before artificial insemination. Pregnant rabbits in both groups showed increased progesterone concentrations on days 8 and 18 of pregnancy. Lower estradiol-17beta concentrations were observed in control does on day 18 of pregnancy compared with separated rabbits (p < 0.003). The results suggest that a transient separation of nursing does from their litters before artificial insemination may promote high follicular steroidogenesis activity leading to increased estradiol-17beta concentrations. This hormonal change could be a result of several stimulatory actions probably triggered by the absence of suckling episodes and may affect the luteotrophic function during the subsequent pregnancy.