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.

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.

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.

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.

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.

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.

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.

Induction of ovulation in rabbits by adding Lecirelin to the seminal dose: in vitro and in vivo effects of different excipients.

This study evaluates the effect of Lecirelin (Dalmarelin(®), Fatro, Italy) diluted in different excipients (benzilic alcohol, benzoic acid and paraben) added to a seminal dose on LH concentrations, progesterone concentrations and ovarian status in rabbits. The in vitro effect on spermatozoa was also tested. A total of 100 multiparous female rabbits were divided into 5 groups, which at the moment of AI, received 0.2 mL (5 µg/dose) intramuscular (im) inoculation of Lecirelin (control) or the same Lecirelin dose administered intravaginally (iv) with the seminal dose alone (Lecirelin group) or with benzilic alcohol (Lecirelin BA group), benzoic acid (Lecirelin BAc group) or parabens (Lecirelin PA group) as an excipient. After 7 days, 10 rabbits per group were euthanized to analyze their ovarian status. In the control group, a high LH peak was detected 30 min post AI, while in the iv groups a slight increase in LH occurred after 120 min. The ovulation and fertility rate was similar in control and Lecirelin groups, while the lowest fertility rate was detected in the Lecirelin BA group. In a second experiment, the semen samples collected from male rabbits were diluted in TALP (control) or mixed with the 5 µg of Lecirelin solutions used in the first experiment. The highest percentage of capacitated sperm (68.3%) was recorded in the Lecirelin PA. The lowest percentages were observed in the Lecirelin BA and BAc groups. In conclusion, the iv administration of Lecirelin represents an alternative method for simplifying rabbit insemination procedures.

Reproductive long-term effects, endocrine response and fatty acid profile of rabbit does fed diets supplemented with n-3 fatty acids.

The effect of a diet enriched with polyunsaturated n-3 fatty acids (PUFA) on endocrine, reproductive, and productive responses of rabbit females and the litters has been studied. Nulliparous does (n=125) were fed ad libitum from rearing to second weaning two diets supplemented with different fat sources: 7.5g/kg lard for the control diet (group C; n=63) or 15g/kg of a commercial supplement containing a 50% ether extract and 35% of total fatty acids (FAs) as PUFA n-3 (Group P; n=62). Dietary treatments did not affect apparent digestibility coefficients of nutrients, or reproductive variables of does including milk production, mortality and average daily gain of kits over two lactations. However, on Day 5 and 7 post-induction of ovulation, progesterone of Group P tended to increase to a greater extent than in does of Group C. Total PUFAs, n-6 and n-3 and eicosapentanoic (EPA) contents were greater in adipose tissues of does in Group P than in Group C. Docosapentaenoic acid (DPA), EPA, and docosahexaenoic acid (DHA) concentrations were greater in peri-ovarian than in scapular fat with abdominal fat being intermediate in concentration. In PUFA supplemented does, kit mortality at the second parturition tended to be less than in control does. Also, kits born to does of the PUFA-supplemented group weighed more and were of greater length than from does of control group. In conclusion, effectiveness of dietary intervention on reproductive and performance response is greater in the second parity, which suggests an accumulative long-term beneficial effect of n-3 FA supplementation in reproductive rabbit does.

Influence of source and micronization of soya bean meal on growth performance, nutrient digestibility and ileal mucosal morphology of Iberian piglets.

The effects of inclusion in the diet of different sources of soya bean meal (SBM) on growth performance, total tract apparent digestibility (TTAD) and apparent ileal digestibility (AID) of major dietary components and mucosal ileum morphology were studied in Iberian pigs weaned at 30 days of age. From 30 to 51 days of age (phase I), there was a control diet based on regular soya bean meal (R-SBM; 44% CP) of Argentina (ARG) origin and five extra diets in which a high-protein soya bean meal (HP-SBM; 49% CP) of the USA or ARG origin, either ground (990 µm) or micronized (60 µm), or a soya protein concentrate (SPC; 65% CP) substituted the R-SBM. From 51 to 61 days of age (phase II), all pigs were fed a common commercial diet in mash form. The following pre-planned orthogonal contrasts were conducted: (1) R-SBM v. all the other diets, (2) SPC v. all the HP-SBM diets, (3) micronized HP-SBM v. ground HP-SBM, (4) HP-SBM of ARG origin v. HP-SBM of US origin and (5) interaction between source and the degree of grinding of the HP-SBM. Dietary treatment did not affect growth performance of the pigs at any age but from 30 to 51 days of age, post weaning diarrhoea (PWD) was higher (P<0.001) and the TTAD and AID of all nutrients were lower for pigs fed the R-SBM diet than for pigs fed the HP-SBM or the SPC diets. However, no differences between the HP-SBM and the SPC containing diets were detected for any trait. The TTAD of organic matter (P=0.07) and gross energy (GE) (P=0.05) tended to be higher for the micronized HP-SBM than for the ground HP-SBM and that of GE was higher (P<0.05) for US meal than for the ARG meal. Pigs fed R-SBM had lower villus height (P<0.01) than pigs fed HP-SBM or SPC but no differences in ileal mucosal morphology were detected between SPC and HP-SBM containing diets. It is concluded that feeding the HP-SBM or SPC-reduced PWD and improved nutrient digestibility and ileal morphology as compared with feeding the R-SBM, but had no effect on pig performance. The inclusion in the diet of added value soya products (micronized SBM or SPC) in substitution of the R-SBM increased the TTAD of all nutrients and reduced PWD but had no advantage in terms of growth performance over the use of ground HP-SBM.

Embryo gene expression in response to maternal supplementation with glycogenic precursors in the rabbit.

Disturbing maternal metabolism during the first pregnancy and postpartum period is associated with sub fertility in rabbit does. Nutritional strategies can be used during those periods and its effects to improve reproductive management may affect periconceptional events and early embryo development. Our goal was to elucidate if treatment with a glycogenic precursor such as propylene glycol (PG) could affect the maternal metabolic profile, follicular and oocyte quality and gene expression patterns in early embryos. Rabbit does were supplemented with 2.5% (v/v) PG from either mid-pregnancy and for 25 days of lactation (PG-GL group); only during lactation (PG-L group); or were not treated (control group). Ovarian parameters and embryos were studied at the end of treatment. At parturition serum non-esterified fatty acid concentrations increased whilst insulin decreased in all groups. Maternal feed intake was reduced in PG-supplemented does but glycaemia was maintained during the experimental period. When PG was suppressed, blood insulin immediately increased in PG-groups, but no differences in follicular population, follicular atresia, and nuclear and cytoplasmic oocyte maturation were observed compared with non-treated animals. Although embryo development was similar among groups, mRNA of SLC2A4, INSR, IGF1R, PLAC8, COX2 and IGF2R were up regulated in the blastocysts of PG-GL does. Transcripts of SOD1 were lower in PG-L embryos; but NOS3 and TP53 were similar among groups. PG did not affect the maternal metabolic profile during the postpartum period, nor the ovarian response or number of embryos developed. Nonetheless, PG supplied from mid-pregnancy modified mRNA transcripts involved in some important developmental and metabolic events in the blastocysts of those females. More experiments are needed to elucidate the physiological consequence of these results.

Expression of the cannabinoid receptor type 1 in the pituitary of rabbits and its role in the control of LH secretion.

The aim of this study was to elucidate the possible direct regulatory role of the endocannabinoids in the modulation of LH secretion in rabbits, a reflex ovulator species. The cannabinoid receptor type 1 (CB1) was characterized by RT-PCR techniques in the anterior pituitary of intact and ovariectomized does treated with GnRH and primed with estrogen and CB1 antagonist, rimonabant. Cannabinoid receptor type 1 immune reaction was evidenced by immunohistochemistry in the cytoplasm of approximately 10% of the pituitary cells with a density of 8.5 ± 1.9 (per 0.01 mm(2)), both periodic acid-Schiff positive (30%) and negative (70%). All CB1-immunoreactive cells were also immune reactive for estrogen receptor type 1. Ovariectomy, either alone or combined with estrogen priming, did not modify the relative abundances of pituitary CB1 mRNA, but decreased (P < 0.01) the expression of estrogen receptor type 1 mRNA. Treatment with CB1 antagonist (rimonabant) inhibited (P < 0.01) LH secretory capacity by the pituitary after GnRH injection, and estrogen priming had no effect. The present findings indicate that the endocannabinoid system is a potential candidate for the regulation of the hypothalamic-pituitary-ovarian axis in reflex ovulatory species.

Ovarian response and embryo gene expression patterns after nonsuperovulatory gonadotropin stimulation in primiparous rabbits does.

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.

Rabbit zona pellucida composition: a molecular, proteomic and phylogenetic approach.

The zona pellucida (ZP) participates in sperm-egg interactions during the first steps of fertilization. Recent studies have shown that the ZP matrix of oocytes in several species is composed of four glycoproteins, designated as ZP1, ZP2, ZP3 and ZP4, rather than the three described in mouse, pig and cow. In this study, investigations were carried out to unveil a fourth glycoprotein in the rabbit (Oryctolagus cuniculus) ZP. Using total RNA isolated from rabbit ovaries, the complementary deoxyribonucleic acid (cDNA) encoding rabbit ZP1 was amplified by reverse transcribed polymerase chain reaction (RT-PCR). The ZP1 cDNA contains an open reading frame of 1825 nucleotides encoding a polypeptide of 608 amino acid residues. The deduced amino acid sequence of rabbit ZP1 showed high identity with other species: 70% identity with human and horse ZP1, and 67% identity with mouse and rat ZP1. At the proteomic level, peptides corresponding to the four proteins were detected by mass spectrometry. In addition, a molecular phylogenetic analysis of ZP1 showed that pseudogenization of this gene has occurred at least four times during the evolution of mammals. The data presented in this manuscript provide evidence, for the first time, that the rabbit ZP is composed of four glycoproteins.

Metabolic and reproductive status are not improved from 11 to 25 day post-partum in non-weaned primiparous rabbit does.

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.