This study evaluated the effects of three maturation systems, namely invitro (MatV) and invivo (MatS) systems, as well as intrafollicular transfer of immature oocytes (IFIOT; MatT), on the accumulation of lipid droplets in bovine oocytes. Lipids were evaluated using confocal microscopy and transmission electron microscopy. The expression of genes related to lipid metabolism, namely acyl-CoA synthetase short chain family member 2 (ACSS2), ELOVL fatty acid elongase 1 (ELOVL1) and fatty acid binding protein 3 (FABP3), was quantified by quantitative polymerase chain reaction. The mean (±s.d.) area occupied by lipids in immature oocytes (13±2%) was similar to those matured invivo (MatS, 16±2%; MatT, 12±2%). However, there was a significant increase in lipids in oocytes in the MatV group (24±2%) compared with all other groups (P<0.001). In the ultrastructural evaluations, MatV oocytes also showed the highest lipid content. The expression of ELOVL1 and FABP3 was similar in the MatS and IFIOT groups. However, transcript levels of ACSS2 were lower in IFIOT than MatV oocytes. These results indicate, for the first time, that oocytes matured by IFIOT are similar to those matured invivo with regard to lipid accumulation, which indicates better quality than those matured invitro.

This study aimed to evaluate the effects of donor age on lipid metabolism during in vitro maturation (IVM) of pigs cumulus-oocyte complexes (COCs). We evaluated transcript levels of genes, the percentage of ooplasm occupied by lipid droplets (LD) and evaluated DNA methylation in COCs from sows and prepubertal gilts. Transcript levels of six genes (ACACA, ACSS2, FASN, FABP3, SLC27A4, PLIN2), which were analyzed in cumulus cells (CCs), increased after 44 h of IVM in the sow group. In the gilt group, only FASN expression increased, while NR3C1 expression decreased after IVM. The measurement of LD in oocytes showed an accumulation of lipids in sow oocytes during IVM, while gilt oocytes showed a decrease in LD. FABP3 and NR3C1 methylation patterns exhibited a demethylation pattern in CCs and oocytes from gilts and sows and showed statistical differences between groups. CCs from sows had a better capacity to change transcription levels of the major genes involved in lipid metabolism during IVM than CCs from gilts. This difference may be involved in accumulation of lipids, acquisition of competence, and maturation of enclosed oocytes. Our results contribute to a better understanding of mechanisms involved in lipid metabolism and acquisition of competence in porcine COCs.

Cows intensively used as oocyte donors for in vitro embryo production (IVEP) are usually kept nonpregnant for prolonged intervals, exposed to successive hormonal treatments, and frequently become overweight. These are all risk factors for the development of endocrine unbalance and, consequently, cystic ovarian disease (COD). The aim of this study was to evaluate the effect of active immunization against gonadotropin-releasing hormone (GnRH) on (1) ovarian follicular population, and (2) development potential of oocytes used for IVEP. Nelore (Bos indicus) cows (n = 14), previously diagnosed with chronic COD (Faria et al. 2017 Anim. Reprod., in press), weighing 620.0 ± 12.8 kg and with body condition score of 4.1 ± 0.2, were assigned to control (n = 6) or treatment (n = 8) group. Cows in the treatment group received 2 SC injections of 1.0 mL of anti-GnRH vaccine (Bopriva, Zoetis, Brazil), 28 days apart (weeks 0 and 4), whereas cows in the control group received placebo on the same schedule. Transrectal ultrasonography was performed weekly from week 0 to evaluate the number and distribution of follicles among size classes, endometrial thickness, and clinical presence of mucometra. Immunization was considered effective (E-IM) when no follicles ≥5.0 mm were observed on the ovaries during a given examination. Cows having E-IM were then used as oocyte donors for IVEP. Cumulus-oocyte complexes (COC) were collected in 5 consecutive ovum pick-up weekly sessions. As a control for IVEP, oocytes from a slaughterhouse were used, with similar procedures performed on the same days and using the same semen batch. The MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) with repeated-measures statement was used to evaluate the effects of treatment, time, and interactions on ovarian endpoints; and the GLM procedure was used to analyse embryo production data. Results are shown as mean ± SEM. There were time and time × treatment effects on ovarian parameters. Treated cows had a decrease (P < 0.05) in the average diameter of the largest follicle and in the number of follicles ≥8 mm, and an increase (P < 0.05) in follicular population after week 6. Nonetheless, individual response to treatment was variable: only 50% of the cows (4 of 8) were E-IM at week 8, whereas 25% (2 of 8) still had COD (largest follicle ≥18.0 mm) at this timepoint. Overall, a negative correlation was detected between follicular population and the diameter of the largest follicle (r = –0.60, P < 0.0001) or the number of follicles ≥8 mm (r = –0.47, P < 0.0001). There was no effect (P > 0.05) of treatment on endometrial thickness or mucometra score. Cows with E-IM produced 22.2 ± 3.6 total and 12.9 ± 2.3 viable COC. Cleavage rate did not differ between E-IM and control (slaughterhouse) oocytes (70.8 ± 7.0 v. 75.1 ± 3.0%, respectively; P > 0.05); however, blastocyst rate was greater in the E-IM group compared with controls (39.7 ± 5.5 v. 20.5 ± 4.7%, respectively; P < 0.02). In summary, our results suggest that active immunization against GnRH leads to variable results in the distribution of the follicular population in cows with COD, but it does not negatively affect IVEP efficiency. This research was supported by Zoetis, CNPq, and CAPES.

The purpose of this study was to characterize the reproductive physiology, oocyte competence, and chromatin compaction in Nelore calves in the early-prepubertal period (EPP) and the intermediate-prepubertal period (IPP). Calves aged 2–5 (EPP) and 8–11 months old (IPP) were assigned to Trial 1 (morpho-physiological–endocrine evaluations, n = 8) or Trial 2 (oocyte donors, n = 8) vs. the respective control groups of cows (n = 8, each). All morphological endpoints, except the antral follicle count, increased from the EPP to the IPP. The EPP LH-FSH plasma concentrations were similar to cows, whereas LH was lower and FSH was higher in the IPP than in cows. . Cows produced more Grade I (12.9% vs. 4.1% and 1.7%) and fewer Grade III COC (30.1% vs. 44.5% and 49.0%) than the EPP and IPP calves, respectively. The IPP calves’ oocyte diameter was similar to those from cows but greater than those from EPP females (124.8 ± 8.5 and 126.0 ± 7.5 μm vs. 121.3 ± 7.5 μm, respectively). The expression of the chromatin compaction-related gene HDAC3 was downregulated in calves. The proportion of the blastocyst rate to the controls was lower in EPP than in IPP calves (43.7% vs. 78.7%, respectively). Progressive oocyte competence was found during the prepubertal period, which can help to decide whether to recover oocytes from calves.

No abstract

Invitro maturation is a key step in invitro embryo production, since its success will depend on the availability of good quality oocytes. Previous studies have shown that it is during this stage that the greatest accumulation of lipid droplets occurs, which is reflected in the amount of lipid present in embryos produced invitro. However, this is not observed when maturation is performed invivo. Therefore, we hypothesised that lipid accumulation would be avoided if oocyte maturation were carried out in ovarian follicles following intrafollicular transfer of immature oocytes (IFIOT). We compared lipid accumulation in oocytes matured invitro, invivo, and by IFIOT. A total of 90 Nellore heifers were distributed in 3 experimental groups: donors of immature oocytes (D-IMA), ovulators of IFIOT oocytes (D-OV), and superstimulated donors of invivo-matured oocytes (D-FSH). All animals rotated through all groups during the experiment. To obtain immature oocytes, the D-IMA were submitted to ovum pickup (OPU), in which aspiration medium was supplemented with 500μM 3-isobutyl-1-methylxanthine (IBMX), and, after selection, part of the oocytes were cultured invitro for 22h (MatF) and part were used for IFIOT (MatT). To perform MatT, the D-OV had their ovulation synchronized by a progesterone and benzoate oestradiol protocol, in which 30h after the implant removal, the IFIOT was performed on the dominant follicle. The D-FSH oocytes were stimulated with 80mg of FSH (Folltropin; Vetoquinol) over 4 days, every 12h, in decreasing doses. At the same time that the immature oocytes were placed in MatF and IFIOT, ovulation was induced with the gonadotrophin releasing hormone (GnRH) analogue (50µg of lecirelin) in D-OV and D-FSH groups. After 22h, matured oocytes were either removed from culture (MatF) or recovered from follicles by OPU (MatT and MatS). From the recovered oocytes of all groups, only those with a polar body were used for lipid droplet evaluation. To quantify lipid accumulation, denuded oocytes were fixed and stained with boron-dipyrromethene (Bodipy) 493/503 (20 µgmL−1) and evaluated by confocal microscopy. Captured images were evaluated in the ImageJ program (National Institutes of Health), and lipid content was determined by calculating the ratio of the area of the lipid droplets to total oocyte area. Data were analysed by ANOVA with statistical significance set at P&lt;0.05. A total of 95 oocytes were evaluated: 25 immature (CT), 24 invitro (MatF), 30 invivo (MatS), and 16 invivo (MatT). The mean area containing lipid droplets in immature oocytes (14%±0.9) was similar (P&gt;0.05) to that observed in both invivo maturation systems (MatS=17.26%±0.8 and MatT=14.11%±0.9). However, in the MatF oocytes, lipid content (24.34%±1) increased during maturation and was higher than in the other groups (P&lt;0.05). We showed for the first time that oocytes matured by IFIOT are similar to those invivo matured with regard to lipid content, which may imply their superior quality over those matured invitro. This new maturation method opens new possibilities for biotechnologies that need to use mature oocytes, such invitro embryo production, oocyte and embryo cryopreservation, cloning, and transgenesis. This study was supported by FAP-DF and Capes.

No abstract

The demand for calves as oocyte donors for in vitro embryo production is growing. However, Bos indicus have a late puberty, and some aspects of the reproductive physiology during the prepubertal period remain unclear. We characterized endocrine and morpho-functional reproductive features in Nelore calves (n=8) at 2-5 (early prepubertal period, EPP) and from 8-11 months old (mo., intermediate prepubertal period, IPP). The calves’ ovaries and uterus were B-mode transrectal ultrasonography examined, and blood samples were collected every second week. The antral follicles number and size, and ovarian and uterine horn diameters, were recorded, and plasma FSH and LH concentrations were measured (RIA). Non-pregnant, non-lactating cyclic Nelore cows (n=8) were used as controls for endocrine endpoints. Somatic development was monitored by monthly weighing, and 3D scanning of the rump area. The somatic and endocrine endpoints were compared within and between EPP and IPP, and between each period and control cows. Associations were determined by the Spearman correlation method, and the developmental rates were determined by non-linear regression. All morphological endpoints, except antral follicle count, increased (P < 0.001) from the EPP to the IPP. However, within each period differences occurred only at EPP. During the EPP LH and FSH plasma concentrations were similar (P > 0.05), whereas during the IPP LH was lower (P < 0.05) and FSH was higher (P < 0.001) than control cows. The EPP calves showed moderate to high positive correlations among ovarian, uterine, and somatic endpoints. Conversely, the IPP such correlations were mostly weak. In summary, distinct ovarian activity and development patterns of primary and secondary sexual characteristics occurred in Nelore calves at 2-5 mo compared to 8-11 mo.