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Abstract: The aim of the current work was to analyze the features of ovarian follicular population and their quality in New Zealand white rabbit does synchronized by 24-h controlled doe-litter separation before artificial insemination (AI) during all their reproductive cycles. Synchronized animals were allocated systematically in two groups. A total of 73 rabbit does (group A) were submitted to a 35-day intensive rhythm (AI on day 4 post-partum [pp] and weaning at 25 days of lactation), and 108 rabbit does (group B) were submitted to a 42-day semi-intensive rhythm (AI on day 11 pp and weaning at 35 days of lactation) during 9 months. At the mid-end of their reproductive life, a total of 26 does (5.4 parturitions), under intensive (n =15) or semi-intensive rhythm (n =11) were either treated in each group with 25 IU eCG 48h before laparotomy to recover their ovaries (n =7 for group A and n =6 for group B) (according to the Bioethics Committee of the University) or not synchronized with the hormonal treatment (n =8 for group A and n =5 for group B). Blood samples were collected at the moment of ovary recovery; morphometrical parameters, number of total follicles and number of follicles ≥1mm in size in the ovarian surface were recorded. Oocytes from follicles of one ovary were recovered and matured in TCM 199 supplemented with 10ng/ml EGF, 100ng/ml IGF-I and 10% FCS. The counterpart ovaries were fixed in paraformaldehyde solution for histological studies. Detection of cell apoptosis was determined using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labelling (TUNEL) technique. Reproductive performance was affected by the rhythm used, with lower reproductive parameters in the intensive group. The average ovary height and width, the mean number of ≥1mm follicles and the number of total follicles were similar between groups. Serum concentrations of estradiol (E2) and testosterone (T) were significantly lower in group A vs. B (E2: 232.4±56.1 vs. 399.7±53.0pg/ml; P <0.05 and T: 1.07±0.10 vs. 1.68±0.23ng/ml; P <0.05). No significant differences were found in follicular population or in the mean follicular apoptosis index between groups. Metaphase II rate was significantly lower in group A vs. B (48.5±3.3 vs. 67.6±3.7%; P <0.01), as well as the migration rate of cortical granules (12.7±2.7 vs. 38.2±6.6%; P <0.001). On the other hand, neither follicular population, nuclear maturation rate nor apoptosis rate were affected by the eCG treatment, but cytoplasmic maturation was higher in animals treated with eCG in group A (29.2% vs. 5.5%; P <0.01). In conclusion, rabbit does under transient litter separation during their reproductive life have both their serum estradiol and testosterone concentrations and oocyte quality influenced by the intensive rhythm, leading to a decrease in reproductive parameters. Also, both intensive and semi-intensive rhythms seem to be less receptive to eCG treatment than expected. [Copyright &y& Elsevier]

Our goal was to elucidate whether acute energy restriction has any influence on ovarian reproductive processes and the relationship with some metabolic markers in the rabbit model. A total of 22 nulliparous rabbits were randomly distributed in 2 groups: control group fed ad libitum(C; n=12) and 72-h fasted group (F; n=10). Before and after the deprivation period, body weight (BW), estimated body composition, serum triglycerides (TG), nonesterified fatty acids (NEFA), and leptin concentrations were measured. After fasting, ovaries were retrieved: one was used to study oocyte in vitronuclear and cytoplasmic maturation (in terms of granule cortical migration; Arias-Alvarez et al.2010 Theriogenology 73, 26–35) and the other one was processed histologically to assess follicle categorization and atresia rate by TUNEL and by immunolocalization of procaspase-3 (PC3). Statistical analysis was carried out by MIXED and CATMOD procedures of SAS program. The BW of control rabbits increased at the end of the experiment (3.96±0.06 v.4.03±0.09kg; P50% of apoptotic cells (31.4±3.6 v.25.4±4.2) did not differ between groups. Cytoplasm staining of PC3 was observed in stroma, follicles of all sizes, and corpora lutea of both groups. Atretic follicles did not exhibit immunoreactivity to PC3. No significant differences were observed in nuclear maturation and migrated cortical granule rates (63.6 v.53.7% and 12.0 v.4.7%, respectively). In conclusion, acute fasting affects BW and some metabolic markers (NEFA and leptin), but follicle and oocyte development is not impaired in our rabbit model. We acknowledge CM, FSE, and MEC for funding. This work is part of an integrated Action between Spain and Italy. [ABSTRACT FROM AUTHOR]

The effect of leptin on oocyte maturation and steroidogenic response could be mediated by activation of specific transcription factors. The aim of this work was to establish if the Signal Transducer and Activator of Transcription 3 (STAT3) pathway mediates leptin''s influence on oocyte maturation and steroid secretion of COC by using a rabbit oocyte model. A total of 522 COC from follicles >1 mm were IVM for 16 h (38°C, 5% CO2) in TCM-199 serum free medium plus: 0 or 10 ng mL-1leptin. Each group was further treated with 10 and 100 μM of specific inhibitor for STAT3 pathway (AG490, Sigma, St. Louis, MO, USA). COC were treated progressively with hyaluronidase (2 mM), 0.5% pronase, 4% paraformaldehyde, 0.02% Triton X-100, and 7.5% BSA after IVM. Oocytes examined under a confocal laser-scanning microscope were previously incubated with 100 mg mL-1fluorescein isothiocyanate of Lens culinaris(FITC-LCA) and then with 10 mg mL-1propidium iodide, for cortical granule (CG) and nuclear staining, respectively. Spent maturation media were analyzed for oestradiol and progesterone secretion using Enzimoinmunoassay. Statistical analysis was performed by Chi-square and ANOVA tests. COC supplemented with 10 ng mL-1leptin had significantly higher metaphase IIrate than those IVM without leptin (P< 0.05); but it did not enhance the percentage of oocytes with peripheral migration of CG (Table 1). Leptin-stimulated nuclear oocyte maturation was significantly impaired when the STAT3-inhibitor was added to the media (P< 0.005). CG migration index was not modified when inhibitors were included in IVM media compared to control groups. Estradiol and progesterone secretion by COC were similar in all groups tested; these concentrations were not affected when STAT3-pathway inhibitor was added. The results confirmed that 10 ng mL-1leptin enhances meiotic oocyte maturation but not CG migration, through the STAT3 pathway. In the present rabbit model, this effect is not mediated by an increase of steroid secretion by COC. Table 1.Metaphase II, CG migration rate and steroid production in rabbit COC after use of different concentrations of STAT3-inhibitorWe acknowledge CM, FSE, and MICINN for funding. [ABSTRACT FROM AUTHOR]

Abstract: 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 11dpp. 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 (11dpp) according to a semi-intensive rhythm (n =24), and (b) lactating does euthanized at later post-partum period (25dpp) according to a more extensive rhythm (n =24). Liveweight, body energy content, lipid depots and serum non-esterified fatty acids (NEFA) concentrations decreased from parturition to post-partum period (P <0.05). In addition, serum protein and glucose concentrations increased in the post-partum period (P <0.05). Similar oestradiol and progesterone levels were found in rhythms as well as similar follicle population and nuclear and cytoplasmic maturation rates measured as metaphase II and cortical granule migration, respectively in both post-partum times. However, the number of preovulatory follicles on the ovarian surface was lower (P <0.05) and the atresia rate tended to be higher with a lower percentage of healthy follicles (P <0.1) in ovaries from females of extensive group. In conclusion, the body reserves, serum metabolic parameters and oocyte quality of primiparous non-weaned rabbits does at the late post-partum time (25 days) were not improved. Thus this reproductive management did not present any advantages compared to earlier post-partum (11 days) reproductive rhythm. [Copyright &y& Elsevier]