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The aim of this study was to evaluate the effect of active immunization against GnRH in Nelore (Bos indicus) cows repeatedly used as oocyte donors that developed chronic cystic ovarian disease (COD). In the first experiment, ovarian and uterine features were first compared between COD cows (n = 15) and healthy cows (n = 22, cycling control group) from the same breed and herd. Cows with COD had a greater number of large (P 0.0001) and medium follicles (P 0.01) but lesser small follicles (P 0.05) than cycling controls. Mucometra was diagnosed in 73.3% of COD cows, but in none of the controls. No difference in average thickness of the endometrium was detected between groups; however, endometrial thickness and mucometra score were negatively correlated (R = -0.73, P = 0.0029) in COD cows. In the second experiment, COD cows were randomly allocated into two experimental groups, which received two 1.0 mL SC injections of either an anti-GnRH vaccine (COD immunized group, n = 8) or saline (COD control group, n = 7), given 28 days apart. Cows were examined weekly by transrectal ultrasonography during nine consecutive weeks after the first injection to evaluate the number and distribution of follicles among size classes, endometrial thickness, and presence of clinical mucometra. Vaccination against GnRH resulted in a progressive suppression of follicle growth and a reduction in the average size of the largest follicle, as well as in the number of large follicles (P 0.01) in COD immunized cows compared with COD controls from week 7 onwards. Conversely, the number of small follicles in the COD immunized group increased after week 5 and was greater (P = 0.0023) than controls on week 9. Endometrial thickness and mucometra score were not affected (P 0.05) by immunization against GnRH. In the third experiment, the COD immunized cows with effective suppression of follicle growth four weeks after the 2nd injection (n = 6) were submitted to three consecutive ovum pick-up (OPU) sessions (weeks 10, 11, and 12) for in vitro embryo production (IVEP). Cumulus-oocyte complexes (COC) collected from slaughterhouse ovaries were used as controls for IVEP. COD cows with produced 25.0 ± 3.8 COC per OPU session with no apparent detrimental effect of anti-GnRH vaccine on oocyte developmental potential in vitro, i.e., we observed similar cleavage rate (P = 0.5914) and greater blastocyst rate (P = 0.0177) in immunized cows compared with COC from slaughterhouse controls. Finally, in the fourth experiment wave emergence and follicular dynamics after follicle ablation were compared between COD immunized cows with effective suppression of follicle growth and a subset (n = 6) of the cycling, control group. No follicles grew beyond 4 mm diameter after follicle ablation in the COD immunized group, whereas a normal follicular wave emergence occurred in cycling controls. Antral follicle count was similar between cycling controls and COD immunized groups at 24 h and 96 h post-follicle ablation (P 0.05), but greater in cycling controls at 48 h and 72 h post-follicle ablation (P 0.05). In summary, our results suggest that active immunization against GnRH is effective to induce the regression of follicular cysts as well as increase the number of small follicles growing on the ovaries, in oocyte donors diagnosed with chronic COD, with no apparent negative effect on oocyte developmental potential in vitro.

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

In vitro produced embryos are still sensitive to the freezing process which can be explained, in part, by the high-lipid accumulation that characterizes these embryos. Therefore, we aimed to evaluate the effect of delipidating agents, L-carnitine and the trans-10 cis-12 conjugated linoleic acid (CLA) isomer, on blastocyst development, lipid content, gene expression and cryotolerance when added to embryo culture media. Embryos were cultured in four different media: T1: control (n = 616), synthetic oviduct fluid (SOF) media with 5% foetal bovine serum (FBS); T2: L-carnitine (n = 648), SOF medium with 5% FBS and 0.6 mg/ml of L-carnitine; T3: CLA (n = 627), SOF medium with 5% FBS and 100 μM trans-10 cis-12 CLA; and T4: L-carnitine + CLA: (n = 597), SOF medium with 5% FBS plus 0.6 mg/ml L-carnitine and 100 μM trans-10 cis-12 CLA. Supplementation of culture medium with either or both delipidating agents reduced (p .05) blastocyst rate on D7 (T1 = 49 ± 3.5; T2 = 39 ± 3.0; T3 = 42 ± 3.9 and T4 = 39 ± 3.9), but did not affected gene expression (p .05). Although embryos cultured in the presence of L-carnitine contained fewer (p .05) lipid droplets than the control embryos, they showed a lower re-expansion rate 24 hr post-thaw than those (p .05). In conclusion, although L-carnitine reduced the amount of lipids in cultured embryos, the use of L-carnitine and CLA during in vitro culture was not able to improve the embryo production and the response to cryopreservation.

In the present study, we evaluated the ovarian response to exogenous FSH stimulation in the absence of endogenous LH, using as experimental model heifers immunized against GnRH. Pubertal, cycling Nelore (Bos indicus) heifers were allocated into three experimental groups: (1) non-immunized, FSH stimulated (B−FSH+, n=5), (2) immunized, FSH stimulated (B+FSH+, n=5), and (3) immunized, nonstimulated (B+FSH−, n=5). Active immunization was obtained by 3 subcutaneous injections of 1.0mL anti-gonadotrophin-releasing hormone vaccine (Bopriva, Zoetis), given at 20-day intervals. Effective immunization was characterised by the absence of growing follicles >4mm or corpora lutea (CL) on the ovaries. Follicular wave emergence was synchronized in groups B+FSH+ and B+FSH− by follicle ablation, and in group B−FSH+ by using of a protocol consisting of an injection of 2mg of oestradiol benzoate and 0.5mg of sodium cloprostenol, and insertion of an intravaginal progesterone (P4) device (1g). Four days later (Day 0), groups B−FSH+ and B+FSH+ received 100mg of NIH-FSH-P1 (Folltropin-V, Vetoquinol), injected twice-a-day in 8 decreasing doses, and group B+FSH− received saline. Transvaginal ultrasonography (7.5MHz) was performed daily from Days 0 to 4 and the number and size of follicles were recorded. P4 devices of group B-FSH+ were removed at Day 3. All heifers underwent ovum pickup (OPU) at Day 4, and the cumulus–oocyte complexes (COC) recovered were graded for quality. Viable COC were used for invitro embryo production. The heifers were re-evaluated at Day 11 (7 days after OPU). The GLIMMIX procedure from SAS (SAS Institute Inc.) with repeated-measure statement was used to analyse the effects of group, day, and interactions; and the Chi-squared method was used to analyse binomial data. The results are shown as mean±s.e.m. A progressive increase in average follicle size was observed in groups B−FSH+ and B+FSH+ (P0.05). Follicle growth rate was similar between groups B−FSH+ and B+FSH+, and both were greater than group B+FSH− (1.2±0.2 and 1.1±0.1 vs. 0.0±0.1 mm/d; P0.05) difference in the number of COC recovered among groups. The group B+FSH+ yielded fewer (P0.05) for B−FSH+, B+FSH+, and B+FSH− (57.1%, 45.9% and 44.2%, respectively). Residual follicles or luteal tissue were observed after OPU only in group B−FSH+, resulting in a significant difference in the size of ovaries between Days 0 and 11, compared with that of groups B+FSH+ and B+FSH− (3.7±1.4 vs. 0.2±0.2 and −0.2±0.2cm2, respectively; P

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 P0.05) 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

For the development of in vitro produced (IVP) as well as in vivo produced bovine embryos, it is extremely important that their energy metabolism works properly because the embryo must be able to metabolize energy substrates that are necessary for producing energy. Lipids play an important role in early embryonic development, acting as source of energy for oocytes and embryos. However, it is known that oocytes and embryos, mainly IVP, accumulate large amounts of lipids in the cytoplasm. Although they are extremely important in embryonic development, lipids have been associated with the reduced survival of bovine embryos following cryopreservation. There is evidence that at least four different categories of lipids affect embryo survival after cryopreservation, including triglycerides (TAG), free fatty acids, cholesterol and phospholipids. Thus, many studies are being conducted to improve the resistance of IVP embryos to the cryopreservation process by reducing the concentration or removing the source of serum from the medium or by reducing oocyte/embryo lipids using mechanical or chemical means. Regarding the use of delipidating agents that reduce the uptake and synthesis of fatty acids (FA) by cells, substances such as phenazine ethosulfate (PES), forskolin, L-carnitine and isomers of conjugated linoleic acid (CLA) have been utilized. This review aims to address important issues related to embryonic energy metabolism, the importance of lipid metabolism and its relation to the cryopreservation of IVP bovine embryos by summarizing the latest research in this field. Para o desenvolvimento de embriões bovinos produzidos in vitro (PIV) e in vivo, é extremamente importante que o metabolismo energético funcione de maneira adequada, pois o embrião precisa ser capaz de metabolizar os substratos energéticos necessários para a produção de energia. Os lipídios desempenham papel importante no desenvolvimento embrionário inicial, atuando como fonte de energia para oócitos e embriões. Entretanto, sabe-se que oócitos e embriões bovinos, principalmente os PIV, acumulam grande quantidade de lipídios no citoplasma. Apesar de serem extremamente importantes no desenvolvimento embrionário, os lipídios têm sido associados com a reduzida sobrevivência após a criopreservação de embriões bovinos. Existem evidências de que pelo menos quatro classes de lipídios afetam a sobrevivência embrionária pós-criopreservação, sendo os triglicerídeos (TAG), ácidos graxos livres, colesterol e os fosfolipídios. Sendo assim, vários estudos estão sendo realizados com o intuito de melhorar a resistência dos embriões PIV ao processo de criopreservação, realizando a redução ou retirada do soro do meio ou a redução mecânica ou química de lipídios. Com relação ao uso de agentes delipidantes que diminuam a captação e síntese de ácidos graxos (AG) pelas células, substâncias como fenazina etossulfato (PES), forskolin, L-carnitina e isômeros do ácido linoleico conjugado (CLA) tem sido utilizadas. O objetivo desta revisão foi abordar aspectos importantes relacionados ao metabolismo energético embrionário, a importância dos lipídios no metabolismo e sua relação com a criopreservação de embriões bovinos PIV sumarizando os estudos mais recentes nessa área.

Objetivou-se com este estudo verificar a existência de associações genéticas entre características de carcaça e características andrológicas em touros Nelore jovens. Realizou-se avaliação andrológica e da carcaça de 223 machos Nelore com média de 21 meses de idade. Para estimação dos parâmetros genéticos utilizou-se a inferência bayesiana por meio do software THRGIBBS1F90. As estimativas de herdabilidade obtidas para DEFMAI, DEFMEN, DEFTOT, AR, AOL, EG e P8 foram 0,33; 0,20; 0,21; 0,35; 0,32; 0,28 e 0,29, respectivamente. As estimativas de correlação genética de DEFTOT com AOL, EG e P8 foram –0,46; –0,28 e –0,19, respectivamente; já de AR com estas mesmas características foram –0,60; –0,03 e –0,12, respectivamente. Os resultados obtidos neste estudo demonstram haver resposta correlacionada favorável entre as características reprodutivas e de carcaça estudadas.

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) 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

Objetivou-se com este estudo verificar a existência de associações genéticas entre características de carcaça e características andrológicas em touros Nelore jovens. Realizou-se avaliação andrológica e da carcaça de 223 machos Nelore com média de 21 meses de idade. Para estimação dos parâmetros genéticos utilizou-se a inferência bayesiana por meio do software THRGIBBS1F90. As estimativas de herdabilidade obtidas para DEFMAI, DEFMEN, DEFTOT, AR, AOL, EG e P8 foram 0,33; 0,20; 0,21; 0,35; 0,32; 0,28 e 0,29, respectivamente. As estimativas de correlação genética de DEFTOT com AOL, EG e P8 foram -0,46; -0,28 e -0,19, respectivamente; já de AR com estas mesmas características foram -0,60; -0,03 e -0,12, respectivamente. Os resultados obtidos neste estudo demonstram haver resposta correlacionada favorável entre as características reprodutivas e de carcaça estudadas.