Fig 1.
Summary of the experimental design and key findings of this study.
Fig 2.
Pregnant females (F0) were fed ad libitum all through the gestation period (group C) or were feed restricted (105 g/day) from Day 0 (artificial insemination, AI) to Day 21 (group FR) of gestation (gestation length is 31 days). In female offspring (F1), sampling was specially focused when does initiate their reproductive life (16 weeks). The factors examined were body weight (BW), body composition and metabolic status. After induced ovulation with GnRH analogue treatment, ovarian status and, oocyte and embryo quality markers were examined 14 h later (Coc groups) or 84 h after ovulation induction and AI (Emb groups). Also, offspring performance was assessed at parturition, weaning (week 4) and from week 9 to 15 (juvenile phase). Female offspring were fed ad libitum all their postnatal life. FR: feed restriction group; C: control ad libitum feeding group; D0: Day 0 of pregnancy; D21: Day 21 of pregnancy; D31: Day 31 of pregnancy (delivery); LBW, live body weight; GnRH: gonadotropin-releasing hormone; AI: artificial insemination; Coc: cumulus oocyte complex; Emb: embryo; AMH: antimullerian hormone; CCs: cumulus cells.
Table 1.
Primers used to amplify genes of interest in rabbit oocytes, cumulus cells, and blastocysts by quantitative reverse transcription–polymerase chain reaction.
Fig 3.
Body weight, feed intake, and blood glucose levels during the juvenile phase of female offspring (F1).
Data correspond to daughters of dams fed ad libitum (C group, n = 13) or subjected to moderate food restriction during the first two-thirds of gestation (FR group, n = 17). Body weight (a) and feed intake (b) were recorded weekly from 9 to 15 weeks of age, and blood glucose (c) was measured biweekly. Each dot represents one animal; lines indicate least squares means ± SEM. Different superscripts indicate significant differences between groups [a, b (P < 0.05)] or among time points [# (P < 0.0001)].
Table 2.
Body weight (BW) and estimated body composition recorded in F1 sexually mature rabbit females (16 weeks old) born to dams subjected to moderate feed restriction (FR group) for 3 weeks or fed ad libitum (C group) throughout pregnancy. After birth, all offspring were fed ad libitum.
Table 3.
Metabolic serum parameters and hepatic enzyme levels of sexually matured F1 rabbit females (16 weeks old) born to dams subjected to moderate feed restriction (FR group) for 3 weeks or fed ad libitum (C group) during pregnancy at ovulation time point.
Fig 4.
Steroid hormone concentrations in female offspring (F1).
Serum levels of (a) progesterone (P4), (b) estradiol (E2), and (c) the progesterone-to-estradiol ratio (P4/E2) were measured in 16-week-old female offspring of dams subjected to moderate food restriction during the first two-thirds of gestation (FR group) or fed ad libitum throughout pregnancy (C group). Number of does: C group, 14 h (n = 6) and 84 h (n = 7); FR group, 14 h (n = 8) and at 84 h (n = 9) after ovulation. Data are presented as mean ± SEM. Different superscripts indicate significant differences [a,b (P < 0.001)] or a trend [(*) P = 0.07] between time points (14 h vs. 84 h after ovulation), and [c,d (P < 0.05)], or a trend [‡ (P = 0.06)] between experimental groups.
Fig 5.
Relative mRNA abundance of target genes in (a) oocytes, (b) cumulus cells (CCs), and (c) blastocysts from female offspring (F1).
Samples were obtained from 16-week-old female offspring (F1) of dams subjected to moderate food restriction during the first two-thirds of gestation (FR group) or fed ad libitum throughout pregnancy (C group). Group C: n = 41 oocytes with their corresponding CCs, and 32 blastocysts; Group FR: n = 56 oocytes with their corresponding CCs, and 40 blastocysts. Data are presented as the mean ± SEM. Bars with different superscripts indicate significant differences between C and FR groups for each gen [a, b (P < 0.05)] or a trend [(*) P = 0.07].
Fig 6.
Representative images of apoptosis in cumulus-oocyte complexes (COCs),and expanded blastocysts from female offspring.
(a) COCs and (b) expanded blastocysts from F1 females. Apoptosis was evaluated by the TUNEL assay. Confocal images (x40) show maximal projection of a complete COCs (a) and blastocysts (b) illustrating the overlap between nuclei stained with propidium iodide (red) and TUNEL-positive apoptotic cells (green). Graphs (c) and (d) show the apoptotic rate in cumulus cells and blastocysts, respectively, from daughters of dams fed ad libitum (C group) or subjected to moderate food restriction during the first two-thirds of gestation (FR group). COCs: C (n = 11), FR (n = 14); embryos: C (n = 16), FR (n = 27). Each dot represents one COC or embryo; lines indicate least squares means ± SEM.
Fig 7.
Embryos were recovered 84h after GnRH administration and artificial insemination in 16-week-old female offspring (F1) of dams subjected to moderate food restriction during the first two-thirds of gestation (FR, group; n = 9) or fed ad libitum throughout pregnancy (C group, n = 7). The total number of embryos analysed was 58 in the C group and 78 in the FR group. Developmental stages were classified as expanded blastocysts (E-Bl), early blastocysts (Bl), morulae (M), or retarded/degenerate embryos (Deg). Data presented as mean ± SEM. Different superscripts indicate significant differences between experimental groups [a, b (P < 0.005)].
Fig 8.
Rabbit embryos recovered from 16-week-old female offspring (F1) of dams subjected to moderate food restriction during the first two-thirds of gestation (FR, group) or fed ad libitum throughout pregnancy (C group) were cultured in vitro for 48h. The number of embryos cultured was 17 in the C group and 22 in the FR group. Developmental stages were classified as morulae (M), early blastocysts (Bl), expanded blastocysts (E-Bl), or degenerated embryos (Deg). Data are presented as mean ± SEM.