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Beta nerve growth factor (β-NGF) is present in the seminal plasma of some species, including rabbits, acting as an ovulation-inducing factor in camelids. Traditionally, GnRH analogues are used to induce ovulation by intramuscular route when artificial insemination (AI) is performed in rabbit does. A specific rabbit recombinant β-NGF (rrβ-NGF) produced in our laboratory was tested as an alternative method to conventional treatment with GnRH analogues to induce ovulation. In the present work, different concentrations (0, 20, 100 ng/mL and 1, 20 and 100 μg/mL) of rrβ-NGF were added to diluted semen to assess its effect on sperm traits (viability and motility parameters). rrβ-NG was used also, incorporated to the AI dose, to evaluate ovulation response (LH and progesterone plasma concentrations, ovulation rate (OR) and embryo implantation at Day 7) after intravaginal administration. A negative control group stimulated with an empty catheter, and a positive control group inseminated and intramuscularly treated as usual with GnRH were also set up. Results showed that seminal quality was influenced by rrβ-NGF depending on the concentration added, being the highest concentrations tested deleterious for semen. Whereas the highest OR was found in the positive control group (100%), concentrations of 20 ng/mL, 1 μg/mL and 20 μg/mL of rrβ-NGF triggered intermediate OR (30, 60 and 42.9%, respectively), and 100 ng/mL and 100 μg/mL had the lowest OR (20 and 14.3%, respectively). Although LH peak was not observed in the first 2 h after AI in the ovulated females from rrβ-NGF groups, plasma progesterone significantly increased at Day 7, except in those females treated with 20 and 100 μg/mL. Also, 98.4% of ovulated females were pregnant on Day 7. In conclusion, rrβ-NGF added to diluted semen affects seminal quality and provokes ovulation, the development of functional CL and conception by intravaginal route in rabbit does, depending on the concentration added.

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.

In rabbit farms, artificial insemination is usually undertaken using semen preserved around 18 ºC. However, increased reactive oxygen species levels during preservation produce sperm dysfunction. Our aim was to evaluate the effect of adding superoxide dismutase (SOD) antioxidant mimics (Tempo and TempoL) to the extenders of rabbit semen on quality parameters, total lipid peroxidation (LPO) and SOD level. Ejaculates from 12 sexually mature males were diluted with an extender containing 0 (control), 0.5, 1 or 2 mM of Tempo or TempoL, respectively. Semen samples were cooled till 18 ºC and stored for 0, 6 or 24 h. Sperm motility, velocity parameters, viability, plasma membrane integrity, SOD activity and level of thiobarbituric acid reactive substances (TBARS) were determined. Results showed that, in general, SOD antioxidant mimics significantly improved sperm motility compared to control after 6 and 24 h storage. Moreover, at 24 h, Tempo treatments showed motility rates higher than 80% whilst the control group showed the lowest motility rate among all treatments (58.5%, P

[EN] Genetic selection in commercial rabbit lines based on litter size has positively improved the number of kits suckling, presumably to weaning. Although it has been proven that the energetic balance of primiparous does is due to the need to satisfy pregnancy, lactation and growth requirements, litter size adjustment from 7 to 12 kits is applied as a routine in commercial rabbit farms. The suckling stimulus provokes a prolactin (PRL) secretion, which in turn can modulate the preovulatory release of luteinising hormone (LH) and, consequently, the ovulatory and productive responses of the does. This study aimed to determine if litter size of prolific primiparous rabbit does during lactation [Group HL, with high litter density (10-12 kits; n=21) and Group LL, with low litter density (7-9 kits; n=29)] influences plasma concentration of PRL. Blood samples from lactating does were taken weekly throughout lactation starting on day 4 post-partum, until day 32 post-partum, before and immediately after suckling. In addition, the does were re-inseminated after weaning (day 32 post-partum), and sampled at 0 and 60 min after induction of ovulation to determine whether litter size affected the peak of LH, progesterone (P4) concentrations and the main productive parameters of their second pregnancy. All hormones were determined by enzyme immunoassay. Statistical analysis of the results revealed that the PRL concentrations of hyperprolific rabbit does before and immediately after a suckling stimulus from 7-9 or 10-12 kits were significanltly different, as we only detected basal levels, with a rise after weaning in both groups. More studies are necessary, delaying blood sampling to later periods of time after the suckling stimulus, in order to conclude whether the peak release of this hormone is altered or not. There were also no differences in plasma LH and progesterone levels after artificial insemination, or in productive performance of these females after their second pregnancy. In conclusion, the litter size adjustment of prolific primiparous rabbits with 7 to 12 kits determines adequate pituitary, ovarian and reproductive responses at second parturition if the does are inseminated after weaning., This work was supported by the Spanish Ministry of Economy and Competitiveness [Project AGL2015-65572-C2-R1 and Project RTI2018-094404-B-C21].

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., This research was funded by the SPANISH MINISTRY OF SCIENCE (Grant Number: AGL2015-65572-C2-1-R and -2-R, RTI-2018-094404-B-C21 and C22) and UCM funding for RESEARCH GROUPS (Group 920249).

To investigate the ovulatory mechanisms triggered by raw semen (RS) in rabbits, we examined the expression of nerve growth factor (NGF)—a supposed ovulation-inducing factor (OIF)—and cognate receptors in anterior pituitary, ovary, and cervix as well as plasma NGF and luteinizing hormone (LH) concentrations. Six does/group were sham-inseminated with sterile saline (PBS), naturally mated (NM), inseminated with RS alone or after lumbar anesthesia (ARS), or treatment with COX inhibitors (CIRS). Immunohistochemistry revealed positive signals for NGF and receptors in all tissues. RT-PCR confirmed the presence of the target transcripts in the same tissues, except NTRK1 in the cervix. Circulating NGF concentrations rose 3- to 6-fold (P < 0.01) 15 min after semen deposition into the genital tract of NM, RS, and ARS rabbits and remained sustained thereafter. Circulating NGF was 4-fold lower (P < 0.01) in CIRS than in RS does indicating that NGF is mainly synthesized by the uterus. A concomitant rise of LH and NGF concentrations was found in 83.3%, 50.0%, and 16.7% of NM, RS, and CIRS does, respectively, but not in ARS (despite high NGF circulating levels). Seminal plasma NGF concentration was 151.9 ± 9.25 μg/mL. The ovulatory responses were 0%, 83.3%, 66.7%, 16.7%, and 0% in PBS, NM, RS, ARS, and CIRS groups, respectively. Present data confirm that, although RS may induce ovulation via endocrine mechanisms through binding to NGF receptors in the ovary, a novel OIF-mediated neural mechanism facilitates ovulation in rabbits.

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

The neurotrophin b-nerve growth factor (NGF) has been described as an important mediator of seminal plasma-induced ovulation in reflexive ovulators like camelids, but this action has not been evidenced in rabbits. In this study, the mRNA expression of NGF was analysed in rabbit male organs by PCR and in a purified basic fraction of seminal plasma by liquid chromatography mass spectrometry. Besides, the ovulatory response [ovulation rate (OR), corpora lutea number, LH peak and progesterone profile] of 27 rabbit does was recorded after injection with murine NGF, gonadoreline (GnRH) or saline solution (SS). An empty catheter was introduced through the vagina (n ¼ 4 animals per group) to simulate the nervous/mechanical stimulus of coitus. Molecular studies confirmed the expression of b-NGF mRNA in the prostate, seminal gland and testicles of male adult rabbits, and the presence of the protein in seminal plasma. For the in vivo experiment, all GnRH-group does exhibited a significantly higher OR (100%) compared to females treated with NGF (30%) and SS (25%), who showed significantly lower LH and progesterone concentrations at 120 min. However, in the non-vaginally stimulated animals, only the gonadoreline group ovulated properly, since no ovulation was recorded in the SS group, and only 17% of the NGF does ovulated; this was associated to a tendency to a higher rate of anovulatory haemorrhagic follicles. mRNA expression of NGF and its presence in seminal plasma evidence a role in rabbit reproduction. Nevertheless, murine NGF triggered some stimulatory effect in the females but not enough to elicit a significant ovulatory response.

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 (P > 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 (P > 0.05), but perirenal fat was lower (P = 0.020) and skin weight and abdominal fat tended to be lower (P = 0.055 and P = 0.063, respectively) in enriched rabbits than in control ones. Total PUFA content in both LM and perirenal fat was greater (P = 0.021 and 0.255) either pH and NH3-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.

Simple Summary The nutritional quality of fat for human consumption is usually evaluated in terms of the n-6/n-3 polyunsaturated fatty acid (PUFA) ratio (with optimal values ≤4). Moreover, with respect to animal production, the standard feed is unbalanced in terms of n-6/n-3 polyunsaturated fatty acids (PUFAs) with a lower proportion of the latter. Such discrepancy negatively affects the health status of animals, the productive and reproductive performances, and the general quality of their products. Hence, n-3 PUFA intake should be promoted. The increase in n-3 PUFA proportions in animal products would also be in accordance with the human dietary recommendations that often focus on the need of increasing the intake of long-chain n-3 PUFAs. In this regard, two main strategies could be implemented, namely to furnish precursor (α-linolenic acid) or long-chain derivatives (eicosapentaenoic and docosahexaenoic acids). In the present review, the effects of different n-3 PUFA sources on biological activity, physiological/reproductive endpoints, and health implications are compared focusing on the most recent results obtained in the rabbit. Abstract This review compares the effects of different n-3 polyunsaturated fatty acid (PUFA) sources on biological activity, physiological/reproductive endpoints, and health implications with a special emphasis on a rabbit case study. Linoleic acid (LA) and α-linolenic acid (ALA) are members of two classes of PUFAs, namely the n-6 and n-3 series, which are required for normal human health. Both are considered precursors of a cascade of molecules (eicosanoids), which take part in many biological processes (inflammation, vasoconstriction/vasodilation, thromboregulation, etc.). However, their biological functions are opposite and are mainly related to the form (precursor or long-chain products) in which they were administered and to the enzyme–substrate preference. ALA is widely present in common vegetable oils and foods, marine algae, and natural herbs, whereas its long-chain PUFA derivatives are available mainly in fish and animal product origins. Recent studies have shown that the accumulation of n-3 PUFAs seems mostly to be tissue-dependent and acts in a tissue-selective manner. Furthermore, dietary n-3 PUFAs widely affect the lipid oxidation susceptibility of all tissues. In conclusion, sustainable sources of n-3 PUFAs are limited and exert a different effect about (1) the form in which they are administered, precursor or derivatives; (2) their antioxidant protections; and (3) the purpose to be achieved (health improvement, physiological and reproductive traits, metabolic pathways, etc.).

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.

[EN] Selection for growth rate is negatively related with reproductive fitness. The aim of this work was to analyse the causes of fertility failure in rabbit does selected for growth rate and characterised for reproductive deficiencies (line R). In the experiment, 82 does were divided into 2 groups: naturally mated (NM) and artificially inseminated (AI), to relate luteinizing hormone (LH) concentration with ovulation induction and pregnancy rate by laparoscopic determination. Additionally, in 38 of these females ovulation rate and metabolites determination (leptin, NEFA, BOHB and glucose) were analysed and perirenal fat thickness measurement and live body weight (LBW) determined. The results showed that all ovulated does (both NM and AI) presented higher concentrations of LH than non-ovulated females. In addition, non-ovulated females showed high levels of leptin and BOHB, as well as LBW. Females from line R have an inherit reduced fertility due to ovulation failure as a consequence of a reduction in LH release, which could be explained by a heavier body weight and higher leptin concentrations., This work was supported by the Spanish Research Project AGL2011-30170-C02-01 (CICYT). Carmen Naturil-Alfonso was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2013/296). English text version was revised by N. Macowan English Language Service.

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.

Zona pellucida (ZP), the extracellular matrix sheltering mammalian oocytes and embryos, is composed by 3 to 4 proteins. The roles of the three proteins present in mice have been elucidated by KO models, but the function of the fourth component (ZP4), present in all other eutherian mammals studied so far, has remained elusive. Herein, we report that ZP4 ablation impairs fertility in female rabbits. Ovulation, fertilization and in vitro development to blastocyst were not affected by ZP4 ablation. However, in vivo development is severely impaired in embryos covered by a ZP4-devoided zona, suggesting a defective ZP protective capacity in the absence of ZP4. ZP4-null ZP was significantly thinner, more permeable, and exhibited a more disorganized and fenestrated structure. The evolutionary conservation of ZP4 in other mammals, including humans, suggests that the structural properties conferred by this protein are required to ensure proper embryo sheltering during in vivo preimplantation development., eLife digest The egg cells of mammals, called oocytes, are encased in a protective layer called the zona pellucida. This layer is made from proteins called ZP1 to 4. Most studies of the zona pellucida use mice, which do not have ZP4. This means that the research community have limited knowledge of what ZP4 does in humans and other mammals. Scientists can now use a technique called CRISPR to selectively modify the genetics of living things to help us to understand what specific genes and proteins do. The ZP4 protein can be eliminated from rabbit oocytes using CRISPR to help understand its role in egg cell fertilization and development. Lamas-Toranzo et al. examined the effect of losing ZP4 from rabbit oocytes. Without ZP4 the zona pellucida becomes thinner, irregular and more flexible. However, the loss of ZP4 did not affect ovulation (i.e. the release of egg cells from an ovary), fertilization, or the early stages of development of embryos when studied in the laboratory. However, rabbits without ZP4 were much less fertile. Indeed, only one out of 10 female rabbits without ZP4 was able to deliver pups because in most cases the development of embryos in the womb failed. These findings show that ZP4 has a structural role in the zona pellucida. Without ZP4 fertility is reduced. This work lays the ground for further investigation of the role of ZP4. It could also offer new insights into the causes of infertility.

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.

The neurotrophin β-Nerve Growth Factor (β-NGF) is flourishing as a protein with important roles in the ovulation induction process in induced-ovulation species but data in rabbits are still inconclusive, probably due to the species-specificity effect of the neurotrophin to trigger the ovulation. Moreover, β-NGF seems to have a role in sperm function. To clarify these functionalities we aimed, in the present research: 1) to newly synthesize a functional recombinant β-NGF from rabbit (rrβ-NGF), 2) to reveal differences in the amino acid sequence of rabbit β-NGF compared to other sequences of induced and spontaneous ovulator species, and 3) to assess the effects of rrβ-NGF on sperm viability and motility. The nucleotide sequence of NGF from rabbit prostate was sequenced by Rapid Amplification of cDNA Ends (RACE) and annotated in GenBank (KX528686). Then, rrβ-NGF was produced in CHO cells and purified by affinity chromatography. Western blot and MALDI-TOF analyses confirmed the correct identity of the recombinant protein. rrβ-NGF functionality was validated in PC12 cells through a successful dose-response effect along 8 days. The comparison of the amino acid sequences of NGF between rabbit and other species suggested some relevant substitutions at its binding site to both the high-(TrkA) and the low-(p75) affinity receptors. The addition of rrβ-NGF in rabbit sperm, in a time- and dose-response study, did not affect its viability but slightly changed some of its motility parameters at the highest concentration used (100 ng/ml). Thus, it can be considered that this new recombinant protein may be used for biotechnological and reproduction assisted techniques in ovulation-induced species.

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.

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.

The developmental competence of in vitro 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 400 µM) 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 100 μM 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 100 μM α-tocopherol, consistent with better-quality oocytes. In fact, early embryo development was improved when 100 μM α-tocopherol was included in the IVM medium, but remained low compared with in vivo-matured oocytes. In conclusion, the addition of 100 μM 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.

Our objective was to analyze the influence of replacing lard (control) with fish oil (FO) rich in long-chain n-3 polyunsaturated fatty acids in the diet of rabbits weaned at 25 days of age on their growth performance, meat quality, cecal fermentation, and ileal morphology. Twenty-four litters (12 control and 12 FO) of nine kits each were fed the experimental diets from weaning (25 days) until slaughter at 60 days of age. Half of the litters (six per diet) were used to monitor productive performance, carcass characteristics, and ileal morphology, and cecal fermentation was assessed in the rest of litters. Diet had no influence on feed intake, meat color, and pH or cecal fermentation, but FO-fed rabbits had lower (p &le, 0.049) average daily gain, chilled carcass weight, and perirenal fat than control rabbits. Fish oil inclusion in the diet resulted in lower morbidity (5.56% vs. 20.4%, p = 0.019) and a healthier fatty acid profile with lower (p &lt, 0.001) n-6/n-3 ratios in both muscle and perirenal fat. In summary, the inclusion of FO in the diet of early weaned rabbits improved the fatty acid profile of rabbit meat and fat and decreased the morbidity, but growth performance was slightly reduced.

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.

The present study evaluated the effectiveness of sildenafil citrate (SC) to improve placental and fetal growth in a diet-induced rabbit model of intrauterine growth restriction (IUGR). Pregnant rabbits were fed either ad libitum (Group C) or restricted to 50% of dietary requirements (Group R) or restricted and treated with SC (Group SC). The treatment with SC improved placental development by increasing vascularity and vessel hypertrophy in the decidua. The assessment of feto–placental haemodynamics showed higher resistance and pulsatility indices at the middle cerebral artery (MCA) in fetuses treated with SC when compared with Group R, which had increased systolic peak and time-averaged mean velocities at the MCA. Furthermore, fetuses in the SC group had significantly higher biparietal and thoracic diameters and longer crown–rump lengths than fetuses in Group R. Hence, the SC group had a reduced IUGR rate and a higher kit size at birth compared with Group R. In conclusion, SC may provide potential benefits in pregnancies with placental insufficiency and IUGR, partially counteracting the negative effects of food restriction on placental development and fetal growth. However, the present study also found evidence of a possible blood overflow in the brain that warrants further investigation.

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

This study evaluated the effect of different feeding regimes from 11 weeks of age to first parturition on feed intake, leptin, non-esterified fatty acids (NEFA) and total protein serum levels, as well as productive performance in young rabbit does. In addition, body composition was estimated by bioimpedance analysis. Thirty-six 11-week-old does were randomly distributed in three groups. The AL-C group was fed ad libitum a control diet containing 350 g neutral detergent fibre (aNDFom)/kg, 11.6 MJ digestible energy (DE)/kg and 173 g crude protein (CP)/kg, and the does were inseminated at 16 weeks of age. The R-C group was fed 150 g/d of the same diet until 16 weeks of age, one week before artificial insemination (AI) at 17 weeks of age, and then fed ad libitum. The AL-F group was fed a diet containing 475 g aNDFom/kg, 9.4 MJ DE/kg and 174 g CP/kg ad libitum, and was inseminated at 17 weeks of age. During rearing (11–16 weeks), does in the R-C group had the lowest DE (1.54 MJ/d; P

This paper reviews the main factors affecting the reproductive performance of rabbit does. In the last 15 years the profitability of rabbit farms has increased mainly due to improvements in management and genetic selection but several problems related to animal welfare have also occurred. The replacement and the mortality rates of female per year are very high and the replaced does often show poor body condition and poor health status. The effect of kindling order, litter size, genetic strain, weaning age and reproductive rhythm on the reproductive performance and welfare of females and mechanisms implicated in these effects are discussed. The rabbit doe of modern strains produces a lot of milk with high energetic value, which leads to a mobilization of body fat resulting in a negative energy balance. In the current reproductive rhythms, there is an extensive overlap between lactation and gestation. The resulting energetic and hormonal antagonism reduces the fertility rate and lifespan of the doe. Strategies to optimize these parameters are discussed. An approach that combines various strategies seems to be required to meet these objectives. Since the factors involved in this system are predetermined (genetic strain, environment) the most powerful way to improve doe welfare is to choose a reproductive rhythm more adapted to the physiology of the does.

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; P0.001), their estimated lipids (16.9+/-1.09%, P0.008), protein (19.7+/-0.07%, P0.0001), and energy (1147+/-42.7MJ/kg, P0.006) body contents were higher and their serum glucose concentrations (158+/-24.5mg/dl, P0.04) 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 folliclesor =1mm was observed compared to NL32 and L32 groups (12.7+/-1.5 vs. 18.0+/-1.45 and 17.6 +/-1.67; P0.05). 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%; P0.05) 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%; P0.05). 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%; P0.05). 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.

In rabbits, modifications in dietary composition may be associated with reproductive benefits. However, the influence of nutrition on ovarian function and embryo development is poorly known in this species. The goal of this work was to study the short-term effects of feeding high-fibre diets with different levels of lignin during the rearing period on productive, endocrine and reproductive parameters of nulliparous rabbit does. A total of 95 New Zealand × California 11-week-old nulliparous white rabbits were randomly allocated in two experimental groups fed ad libitum fibre-rich diets with a high lignin content (HL group: NDF 49.6% of DM, LAD 15.8% of DM; n = 48) or a standard lignin content (SL group: NDF 40.9% of DM, LAD 4.9% of DM; n = 47). All animals were artificially inseminated (AI) at 16 weeks of age. Daily feed intake and live body weight (LBW) were recorded during the rearing period. Conception rate and prolificacy were also determined. In addition, in ten animals of each group, body composition, serum estradiol 17β (E2), progesterone (P4) and leptin levels, as well as reproductive parameters including ovary weight, follicular population, ovulation rate, in vitro oocyte maturation (IVM), blastocyst recovery rate and in vitro embryo development were studied at AI time. Animals fed the HL diet had a significantly higher feed intake until the first AI (P

The effect of neutral detergent-soluble fiber level on gut barrier function and intestinal microbiota was examined in weaned rabbits. A control diet (AH) containing 103 g of neutral detergent-soluble fiber/ kg of DM included alfalfa hay as main source of fiber. Another diet (B-AP) was formulated by replacing half of the alfalfa hay with a mixture of beet and apple pulp resulting in 131 g of soluble fiber/kg of DM. A third diet (OH) was obtained by substituting half of the alfalfa hay with a mix of oat hulls and a soybean protein concentrate and contained 79 g of soluble fiber/kg of DM. Rabbits weaned at 25 d and slaughtered at 35 d were used to determine ileal digestibility, jejunal morphology, sucrase activity, lamina propria lymphocytes, and intestinal microbiota. Suckling 35-d-old rabbits were used to assess mucosa morphology. Mortality (from weaning to 63 d of age) was also determined. Villous height of the jejunal mucosa increased with soluble fiber (P = 0.001). Rabbits fed with the greatest level of soluble fiber (BA-P diet) showed the highest villous height/ crypt depth ratio (8.14; P = 0.001), sucrase specific activity (8,671 mumol of glucose/g of protein; P = 0.019), and the greatest ileal starch digestibility (96.8%; P = 0.002). The opposite effects were observed in rabbits fed decreased levels of soluble fiber (AH and OH diets; 4.70, 5,848 mumol of glucose/g of protein, as average, respectively). The lowest ileal starch digestibility was detected for animals fed OH diet (93.2%). Suckling rabbits of the same age showed a lower villous height/crypt depth ratio (6.70) compared with the B-AP diet group, but this ratio was higher than the AH or OH diet groups. Lower levels of soluble fiber tended (P = 0.074) to increase the cellular immune response (CD8+ lymphocytes). Diet affected IL-2 production (CD25+, P = 0.029; CD5+CD25+, P = 0.057), with no clear relationship between soluble fiber and IL-2. The intestinal microbiota biodiversity was not affected by diets (P >/= 0.38). Rabbits fed the B-AP and AH diets had a reduced cecal frequency of detection compatible with Campylobacter spp. (20.3 vs. 37.8, P = 0.074), and Clostridium perfringens (4.3 vs. 17.6%, P = 0.047), compared with the OH diet group. Moreover, the mortality rates decreased from 14.4 (OH diet) to 5.1% (B-AP diet) with the increased presence of soluble fiber in the diet. In conclusion, increased levels of dietary soluble fiber improve mucosal integrity and functionality.

The aim of the study was to evaluate the effect of the administration of fresh chicory (Chicorium intybus L.) to young pups before and after weaning, investigating the morphological development of two main lymphoid tissues (appendix and Peyer's patch) and the productive performance. The trial was divided in two parts: the first, immediately after kindling, 20 New Zealand White (NZW) multiparous rabbit does were homogenised for litter size (n = 8) and were divided into two groups: control group, in which young rabbits (from 18 to 28 d) fed a standard diet; chicory group, in which young rabbits were fed control diet and fresh chicory, separately. In the second trial the young rabbits pursued the same diet until 77 d old. At weaning, 10 young rabbits/group were weighed and killed. The pH, dry matter, ammonia and volatile fatty acids (VFA) were evaluated on the caecal content. The histological sections were made of the appendix and Peyer's patch. The dietary administration of fresh chicory to young rabbits before weaning, improved biochemical traits of caecum content: the lowered NH3 (0.5 versus 0.6 μmol/g; P

Insemination of rabbit does at early post-partum combined with dam-litter separation (DLS) can increase prolificacy and decrease parturition intervals, but little is known about longer-term effects on kit survival and growth. In this study, does (n=138) were artificially inseminated on day 4 post-partum (nine times over a period of 10 months, 35-day reproductive rhythm, kits weaned at 25 days), after DLS (for 48 or 24h) or 48h after 25 UI eCG injection. The litters from each group were adjusted to seven to nine kits per doe and then weighed at 2, 4, 16 and 25 days of age. Mortality was calculated in the pre-weaning period and development of the digestive system was quantified (length of intestinal villi). After DLS24 or DLS48, 4-day-old kits were lighter than eCG or controls of the same age. By 16 and 25 days of age, 48-h weaned kits had regained the same weight as the eCG and control groups (higher daily weight gain). The 24-h weaned kits weighed less, probably because their litter size was larger and daily weight gain was lower. Total kit mortality (day 2 to day 25) was highest in the DLS48 group but percentage mortality varied among treatments depending on the period considered (days 2–4, days 5–16 and days 17–25). In the first period, mortality was higher for DLS48; in the second, mortality was similar for all groups except eCG (which was lower). After 16 days of age, mortality was quite low and similar in all groups. There was a significant effect of the number of parturitions on the parameters considered. Intestinal villi were measured in 12 kits slaughtered at 4, 16 and 25 days of age (DLS for 24 or 48h on days 3 and 2, respectively) and compared with controls (2, 4, 16 and 25 days old). At 4 days (immediately after separation), the stomach and whole digestive system were lighter in DLS48 than controls, due to the absence of the milk, but there were no significant differences among groups by days 16 and 25. Intestinal villi were shorter in 4-day-old DLS kits (24 and 48h) compared to controls of the same age, longer at 16days (possibly due to an intense cellular proliferation) and similar among all groups by day 25. In summary, both 24-h DLS and 48-h DLS can substitute hormonal treatments to synchronize oestrus in

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.

To determine the effect of a transient doe-litter separation (48 h) on milk production and feed intake from 1 to 21 days post-partum, a control group (C) in which litters had free access to nursing, and a biostimulated group (B) in which litters were separated from their does from d 9 to d 11 post-partum were used. Total milk production was higher in (C) than in (B) does (5090 ± 1.1 g vs. 4593 ± 150 g, P < 0.05). On days 12, 13, 14 and 15 of the lactation period, milk production was 40% (P < 0.0001), 18% (P < 0.05), 15% (P < 0.05), and 15% (P < 0.01) higher in (C) than in (B), respectively. No significant differences were observed in feed intake during the period studied (7961 ± 352 g in (C) does, and 7834 ± 329 g in (B) does). After fasting, 11-day-old kits showed lower gut weight, body weight, empty stomach relative weight (RW), stomach content RW and small intestine RW (P < 0.05), but the differences disappeared at 21 days of age. A reduction in villous height (P < 0.065) was observed in separated kits at 11 days old compared to 9- and 11-day-old control kits (579.0 ± 28.4 vs. 664.64 ± 27.6, and 724.33 ± 24.1 µm, respectively), but no differences were observed at either 16 or 21 days of age. Specific jejunal lactase activity increased significantly in 11-day-old separated kits (P < 0.05). A significant increase in sucrase activity at 21 days in both groups (P < 0.05) was detected. In conclusion, a fasting period of 48 h at 9 days of age does not compromise the subsequent development of certain digestive parameters of young rabbits. doe-litter separation / enzyme activities / milk production / villi height

Mammalian oocytes are surrounded by a glycoprotein layer termed the zona pellucida (ZP), which acts as a protective barrier during pre-implantation embryo development and plays essential roles during follicular development and fertilization. This structure is composed of 4 different proteins in several species such as rabbits and humans. However, the oocytes of the only species for which knockout (KO) embryos are readily available, the laboratory mouse, lacks one of these proteins (ZP4), so its function remains elusive. To determine the role of ZP4, we have generated ZP4 KO rabbits by CRISPR and compared their reproductive function to that of heterozygous (Hz) and wild type (wt) rabbits. Following mating with wt males, female KO rabbits showed a clear impairment in reproductive performance compared with Hz or wt females, with only 1 out of 5 KO females producing a small litter of 4 pups, whereas all mated Hz and wt females exhibited normal litter sizes (wt 9.2 ± 0.6; Hz 10.6 ± 0.5; mean ± SEM). In order to elucidate the cause for the impaired delivery rate, ovulation rates and cleavage rates were initially assessed, with no differences observed between groups (oocytes ovulated: wt 11.7 ± 1; Hz 15 ± 2.9; KO 13.3 ± 2.9; cleavage rates (%): wt 81.7 ± 0.1; Hz 95.5 ± 0.1; KO 87.3 ± 0.1), which suggests that ZP4 does not play a critical role in folliculogenesis or fertilization. However, when expanded blastocysts were recovered at Day 6 after mating, embryo development was found to be clearly impaired in embryos lacking ZP4, as none of the embryos obtained from 2 KO females were able to undergo blastocyst expansion. Morphological differences were observed in the zona pellucida from KO rabbits, being irregular, noticeably less elastic and easier to deform, and significantly thinner (thickness: wt 15.2 ± 1.5 µm; Hz 15.3 ± 1.4 µm; KO 10.9 ± 0.7 µm; ANOVA P

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.

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.

This study was carried out to determine the effects of a transient doe-litter separation on plasma prolactin, LH, FSH, estradiol-17β 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

The effect of 18 mg. melatonin (MLT) implants on thereproductive performance of turbot (Scophthalmus maximusL.) broodstock was determined in 20 females and 12 males. Implantedanimals showed supra-physiological plasma MLT concentrations during threemonthsafter implantation, being at that time about one hundred times higher thandiurnal MLT concentrations. Direct effects of MLT implants were observed ongonadal maturation of turbot females three months after implantation (P

a ´´ Abstract The objective of this study was to explore the effects of a doe-litter separation for 48 h before artificial insemination (AI) on various endocrinological and productivity variables determined in 24 doe rabbits. The control group (C) (n 5 12) had free access to nursing and the separated group (S) (n 5 12) were kept apart form their litters for 48 h prior to AI on day 11 of the lactation period. Lower plasma prolactin (PRL) concentrations were observed 24 h after the doe-litter separation (P , 0.04). The S does had increased plasma oestradiol (E ) concentrations 48 h after the doe-litter separation, compared with 2 preseparation values of the same group and with C does (P , 0.0001). A higher proportion of does showing high sexual receptivity was observed among the S group compared with the C group (P , 0.05). The suckling episode ranged from 3 to 5 min in S does. Teat stimulation by suckling caused an immediate increase in PRL in S does (P , 0.0001). PRL remained high until 7 h after suckling in S does, compared with C does (P , 0.04). The doe-litter separation resulted in a mean (6S.E.M.) reduction in litter weight of 217 ( 622.1) g (P , 0.02). No kits died in either group during the separation period. The litter weight on day 21 after parturition and milk production / doe were not affected by treatment. When analysing the subsequent litters, no differences were observed between S and C does in kindling rate, litter size at birth, number of dead kits / litter, litter size at weaning and number of kits dead from birth to weaning. The results suggest that a transient doe-litter separation causes an increase in the proportion of does showing signs of oestrus and may have a major influence on fertility. These effects may be induced by the absence of suckling episodes together with the higher plasma E and lower PRL 2 concentrations seen in the S does prior to AI. © 2000 Elsevier Science B.V. All rights reserved.

To analyse the effect of pasture availability and genotype on innate immunological status,morphological organisation of lymphoid follicles, behaviour, performance and carcass and meat characteristics, 80 growing rabbits (40 Leprino of Viterbo and 40 New Zealand White, NZW) were reared in bicellular standard cages (control) or in a wired pen, provided with an external grass pasture (pasture). Blood samples and behaviour observations (10 rabbits per group) were performed at different ages (weaning, 49 and 89 d). At the end of the trial, 10 rabbits per group were slaughtered to study meat quality/composition and vermiform appendix structure. Leprino rabbits showed higher serum lysozyme values than NZW for all ages and rearing systems. Leprino rabbits showed the highest values in caged animals (29.6 and 32.4 mg/mL, respectively at 49 and 89 d of age), while NZW animals did so in the pen (2.2 and 0.00 g/mL, respectively at 49 and 89 d of age). Haemolytic Complement Assay increased with age in Leprino (43.7 vs. 48.6 and 51.2 μg/mL, respectively for cage and pen), but decreased in NZW (68.6 vs. 25.8 and 41.1 μg/mL, respectively for cage and pen). Plasma TBARs was always higher in Leprino rabbits (16.0 vs. 7.0 μmol malondialdehyde/L at 29 d of age; 17.9 vs. 8.0 μmol malondialdehyde/L at 49 d of age; 20.0 vs. 11.4 μmol malondialdehyde/L at 89 d of age), whereas plasma tocopherol showed an inverse trend (32.9 vs. 46.6 mg/L at 29 d of age; 32.5 vs. 44.4 mg/L at 49 d of age; 31.2 vs. 42.4 mg/L at 89 d of age). Mortality rate of Leprino rabbits was highest in the cage system, while mortality was highest in NZW housed in the pen. Productive performance, carcass traits and fatty acid composition of meat were strongly affected by genotype and rearing system. Regarding genotype effect, Leprino showed lower daily gains (31.9 and 29.6 vs. 44.2 and 40.0 g, respectively for cage and pen system), live (2563 and 2418 vs. 2902 and 2650, respectively for cage and pen system) and carcass weights (1561 and 1465 vs. 1763 and 1580, respectively for cage and pen system). Pasture availability improved the meat nutritional quality and, in particular, the n-6/n-3 ratio was optimal and the total tocopherol content was suitable to assure a good oxidative stability.

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 0.2mL (5μg/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 30min post AI, while in the iv groups a slight increase in LH occurred after 120min. 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 5μg 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.

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: 7.5 g/kg lard for the control diet (group C; n = 63) or 15 g/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 pro- duction, 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 sup- plemented 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

The effects of different combinations of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on cumulus expansion and meiotic maturation were examined in rabbit oocytes. Selected rabbit follicular oocytes were matured in vitro and were classified as cumulus\p=n-\oocytecomplexes or denuded oocytes. They were cultured in TCM 199, and were treated with growth factors at different concentrations: EGF at 0, 1, 10, 50 and 100 ng ml \m=-\1, IGF-I at 0, 50, 100 and 200 ng ml \m=-\1 and EGF plus IGF-I at 10 + 50; 10 + 100; 50 + 50 and 50 + 100 ng ml \m=-\1, respectively. After 6 h of culture, the oocytes were assessed for nuclear maturation and after 16 h of culture, for cumulus expansion and maturation stage. After culture for 6 h, the incidence of germinal vesicle breakdown was higher (P < 0.05) in all of the growth factor treatments tested compared with controls. After culture for 16 h, EGF enhanced the incidence of cumulus expansion at all of the concentrations tested. Cumulus expansion was greatest with 50 mg EGF ml\m=-\1 plus 100 ng IGF-I ml\m=-\1 (72.0% versus 2.4% in controls). Treatment with IGF-I significantly increased (P< 0.05) the incidence of metaphase II stage, and maximum stimulation occurred at 100 ng IGF-I ml \m=-\1 (84.5% versus 31.1% in controls). However, IGF-I did not affect cumulus expansion. When denuded oocytes were used, no positive effects on nuclear maturation rates were observed for any treatment. These results suggest that: (1) EGF, either alone or with IGF-I, stimulates cumulus expansion; (2) the addition of IGF-I or EGF plus IGF-I significantly enhances nuclear maturation in immature rabbit oocytes; and (3) this effect is mediated by the presence of cumulus cells.

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. © 2013 Elsevier B.V.

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 (P0.01) the expression of estrogen receptor type 1 mRNA. Treatment with CB1 antagonist (rimonabant) inhibited (P0.01) 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.

The present study was designed to determine the influence of several parturition-insemination intervals on the conception rate (CR) and prolificacy in lactating rabbits, as well as the evolution of plasma estradiol-17 β (E2) mean levels and sexual behaviour (SB) during the lactation period. E2 levels and SB were measured daily in 12 lactating rabbits of the California × New Zealand breed with 7–10 pups per litter. E2 levels were determined by immunoassay (ELISA), and SB by means of the turgidity and colour of the vulva. Milk production was predicted in this group by means of the litter weight on Days 21 and 30 of the lactation period, respectively. CR and prolificacy were recorded after parturition in 130 lactating rabbits (7–10 pups per litter) inseminated systematically on 1 postpartum day only, from Days 1 to 11 of the postpartum period (PP). Fresh diluted semen (skim milk + antibiotic) was used in each insemination. Ovulation was induced by administration of an intramuscular injection of 20 μg GnRH at the time of insemination. High means of plasma E2 concentrations were found on Days 1, 5–7, and 23–30 of the PP (P The overall mean CR obtained was 47.4%, suggesting an effect of the parturition-AI interval on the CR (P The general litter size at birth was 7.9 ± 0.2, and the number of born dead per litter was 0.8 ± 0.1 pups. Milk production measured 4228.3 g± 148.4 g and 4019.4 g ± 116.7 g on Days 21 and 30 of the lactation period, respectively. The results suggest the presence of at least three high plasma E2 concentration intervals, and two periods of increased SB in lactating non-pregnant rabbits during the postpartum period. Mean plasma E2 levels differed in rabbits with high SB compared with animals with medium or low SB. No correlation of plasma E2 levels with SB was found. Sexual behaviour and CR appeared to be influenced by the lactation period, probably via prolactin concentrations. Days 1 and 8 of the PP appeared as favourable Al days in lactating rabbits with 7–10 pups per litter.

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, progesteroneestradiol 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. © 2013 Elsevier Inc.

The aim of this study was to compare the pituitary and ovarian responses in rabbit does subjected to different methods of ovulation induction. Forty-eight receptive females were randomly distributed into six groups (N = 8) and were inseminated with standard glass catheters. Buserelin intramuscular (BM) does were inseminated using a pool of fresh heterospermic semen and an intramuscular injection of 1 μg of buserelin acetate to induce ovulation. Buserelin intravaginal (BV) does were inseminated in a similar way, but ovulation was induced with the GnRH analogue (10 μg of buserelin acetate) combined with 0.5 mL of semen extender. The raw semen (R) and saline groups (S) were inseminated with undiluted semen or saline, respectively, without any inducer of ovulation. Another group (A) received lumbar anaesthesia (1.5 mL of 2% lidocaine), and only the empty catheter was introduced into the vagina. The AR does were treated the same way as group A but were inseminated with raw semen instead of an empty catheter. Blood samples were collected to determine the LH concentrations before and after AI (30, 60, 90, and 120 minutes). Ovulation, pregnancy, and conception rates were determined after euthanasia on day 14 post AI. Ovulating does had higher mean LH concentrations than nonovulating does (197.9 vs. 45.9 ng/mL; P < 0.05). The ovulation rates of buserelin intramuscular and intravaginal does were 100%, and the pregnancy rates were 87.5% and 100%, respectively. Rabbit does in groups A and AR did not ovulate and had similar mean plasma LH concentrations after 60 minutes compared with the S group (49.4 and 49.2 ng/mL vs. 41.6 ng/mL, respectively), which reached ovulation and pregnancy rates of 37.5%. Does inseminated only with raw semen had an ovulation rate of 75% and a pregnancy rate of 62.5%; they also demonstrated higher plasma LH concentrations than does of the S, A, and AR groups. In conclusion, ovulation in rabbit does can be induced by exogenous GnRH administration (im and intravaginal). The high plasma LH concentration and ovulation rate in the R group with respect to the S and A groups could weakly indicate the presence of some molecules in the seminal plasma that could act on or be absorbed by vaginal mucosa. Sensory stimulation and "seminal factors" probably exert a synergy on the ovulation response as demonstrated by the comparison of LH release and the ovulation response in the R, S, RA, and A groups.