Effect of follicular wave synchronization using a progesterone-releasing intravaginal device-based protocol on in vitro embryo production in Bos taurus cows subjected to 14-day intervals ovum pick-up.

Follicular wave synchronization (FWS) before ovum pick-up (OPU) is one of the strategies used to improve the efficiency of in vitro embryo production (IVP). This study aimed to evaluate the effect of FWS on the total follicular number, cumulus-oocyte complex (COC) recovery, and in vitro embryo development in Angus cows (n = 33) subjected to OPU with 14-day intersession intervals. Additionally, it was also evaluated the presence of carryover effects given the short intersession interval used. The experiment was run as a 2-treatment (FWS vs. Control) x 2-period (1 vs. 2) crossover design. Animals in the FWS group received an intravaginal progesterone implant (1gr), estradiol benzoate (2 mg), and D-cloprostenol (150 µg) on day 0 and the OPU was performed on day 5. Control group animals did not receive any hormone treatment. The FWS increased the number of 6-10 mm follicles (P = 0.05), but it decreased the COC recovery rate (P < 0.01). The FWS did not affect the total or frozen embryo numbers (P = 0.49 and P = 0.17; respectively), but it increased the total blastocyst cell number (P < 0.01). A carryover effect was found on the total and < 6 mm follicles number (P = 0.10 and P < 0.01; respectively), and on the regular, atretic, viable, and total number of COC (P = 0.01, P = 0.08, P = 0.02 and P < 0.01; respectively). We concluded that the FWS increased the quality of embryos after OPU with 14-day intersession intervals in Angus cows and that this kind of OPU/IVP scheme enabled the existence of a carryover effect, especially on the follicle number and COC morphology.

Effects of polyunsaturated fatty acid supplementation on plasma and follicular fluid resolvin D1 concentration and mRNA abundance in granulosa cells in ewes.

The aim of this experiment was to evaluate the effect of increasing dietary omega-3 (n-3) polyunsaturated fatty acid (PUFA) supplementation on plasma and follicular fluid resolvin D1 (RvD1) concentration and the mRNA expression of genes related to RvD1 production, inflammatory response, oxidative stress, hormone receptors and production, and free fatty acid receptors in the granulosa cells of ewes. Dorset × Hampshire ewes (n = 24) aged 2 to 4 yr and with an initial body weight (BW) of 84.08 ±â€…13.18 kg were blocked by body condition score (BCS) and BW, and randomly assigned to 12 pens. Each pen within each block was randomly assigned to one of three treatments: 1) diet without fatty acid supplementation (control), 2) diet with 0.5% n-3 PUFA supplementation (PUFA0.5), and 3) diet with 1% n-3 PUFA supplementation (PUFA1). BW, BCS, and blood samples were obtained on day 1 and every 21 d for 3 mo. Ewes were then synchronized, superstimulated, and ovariectomized. Antral follicles were aspirated to evaluate RvD1 concentration in follicular fluid, and granulosa cells were used to determine mRNA abundance. Data were analyzed as a randomized complete block design using a mixed model (MIXED or GLIMMIX with log as a link function when data presented a nonnormal distribution). A polynomial effect of treatments was used to analyze RvD1 concentration and mRNA expression when there was no interaction. In addition, the correlation between plasma and follicular fluid RvD1 concentration was evaluated. We found no differences in BW (P = 0.28) and BCS (P = 0.29) between treatments. The concentration of RvD1 in plasma and follicular fluid linearly increased (P = 0.03) and tended to increase (P = 0.06) concomitantly to increasing PUFA supplementation. Plasma and follicular fluid RvD1 concentrations were positively correlated (r = 0.61; P < 0.01). The abundance of GPX1 and GPR32 mRNA tended to increase linearly with increasing PUFA supplementation (P = 0.06). In addition, PUFA supplementation linearly decreased and tended to decrease IL-1ß and COX-2 mRNA abundance (P = 0.01 and P = 0.06, respectively). In conclusion, the correlation between plasma and follicular fluid RvD1 concentration indicates a relationship between both compartments. Also, the decrease of IL-1ß and the increase of GPX1 mRNA abundance after PUFA supplementation could have beneficial effects on follicle development.

The aim of this experiment was to evaluate the effects of increasing dietary omega-3 (n-3) polyunsaturated fatty acid (PUFA) supplementation on plasma and follicular fluid resolvin D1 (RvD1) concentration, and the mRNA expression of genes related to RvD1 synthesis, inflammatory response, oxidative stress, reproductive hormone receptors and production, and free fatty acid receptors in ewes. Twenty-four ewes aged 2 to 4 yr were assigned to 12 pens and randomly allocated to one of three treatments: 1) diet without fatty acid supplementation (control), 2) diet with 0.5% n-3 PUFA (PUFA0.5), and 3) diet with 1% n-3 PUFA (PUFA1). Body weight (BW), body condition score (BCS), and blood samples were obtained on day 1 and every 21 d for 3 mo. Ewes were then synchronized, superstimulated, and ovariectomized. Antral follicles were aspirated to evaluate follicular fluid RvD1 concentration, and granulosa cells were used to analyze mRNA concentration. We found no differences in BW and BCS between treatments. Plasma and follicular fluid RvD1 concentration increased concomitantly to increasing dietary PUFA supplementation. There was a positive correlation between plasma and follicular fluid RvD1 concentrations. Omega-3 PUFA increased the mRNA abundance of genes associated with RvD1 synthesis and oxidative damage response. In addition, PUFA supplementation linearly decreased the mRNA abundance of genes associated with inflammatory response. In conclusion, the positive correlation between plasma and follicular fluid RvD1 concentrations demonstrates a relationship between both compartments. Also, changes in gene expression after PUFA supplementation may have a beneficial effect on the follicle and, in turn, on reproduction.

Ghrelin antagonist D-Lys3-GHRP-6 counteract ghrelin effects in bovine cumulus-oocytes complexes matured in vitro.

Ghrelin is a gut hormone related to energy balance and reproductive functions. The aim of this study was to evaluate the effect of ghrelin antagonist D-Lys3-GHRP-6 (GA) as a potential agent that prevents ghrelin effects during bovine oocyte maturation on progesterone production, cumulus cell (CC) viability, CC DNA damage and embryo development and hatching rates. Ghrelin's potential to induce oxidative stress in cumulus-oocyte complexes (COC) was also evaluated. COCs were cultured for 24 hr in medium without supplementation (C) or supplemented with 60 pM ghrelin (Ghrelin60), Ghrelin60 + 20 pM GA (GA20), Ghrelin60 + 60 pM GA (GA60) or Ghrelin60 + 100 pM GA (GA100) for experiment I. For experiment II, C and Ghrelin60 treatments were used. Differences between C and Ghrelin60 and the linear or quadratic association between GAs on Ghrelin60 were evaluated. Results demonstrated that Ghrelin60 increased progesterone concentration, reduced CC viability, induced CC DNA damage and decreased blastocyst and hatching rate compared with C (p < .05). GA20, GA60 and GA100 had a linear effect on CC genetic damage index (p ≤ .05) and a quadratic effect on CC viability (p < .01). GA20 counteracted the low hatching rate produced by Ghrelin60. However, GAs did not counteract progesterone concentration and blastocyst rate (p ≥ .21). GRH60 did not differ from C in the oxidative status (p ≥ .19). Our study highlights that GA could prevent the negative effects of ghrelin during bovine IVM.

Amitraz induced cytotoxic effect on bovine cumulus cells and impaired oocyte maturation.

The aim of this study was to evaluate the genotoxic and cytotoxic effects of amitraz (AMZ) on the primary culture of bovine cumulus cells (CC) and oocyte nuclear maturation. Cytotoxicity was evaluated by assessing mitochondrial activity with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Genotoxicity was estimated using the alkaline single cell gel electrophoresis (SCGE) assay. Apoptosis was detected with the Annexin V-affinity assay. The in vitro maturation test was performed in bovine oocytes. To understand AMZ action, glutathione content, superoxide dismutase enzyme activity, and lipid peroxidation were evaluated in CC. Results showed that AMZ lethal concentration (LC 5024h) for bovine CC was 32.55 µg/mL (MTT assay). A 25 µg/mL induced late apoptosis and necrotic cells (p < 0.05); however, DNA damage was decreased at the same concentration (SCGE assay; p < 0.05). A decrease in metaphase II was observed at 25 µg/mL, and degenerate oocytes were observed at 15 and 25 µg/mL (p < 0.05). None of the oxidative stress parameters evaluated showed significant differences. This study contributes to a better understanding of AMZ in this model, suggesting its potential cytotoxicity and impact on bovine reproduction.

Effect of alpha-lipoic acid during preimplantation development of cattle embryos when there were different in vitro culture conditions.

In many species, alpha-lipoic acid (ALA) is essential for embryo development. There, therefore, was investigation of effects of ALA supplementation to culture media for in vitro development of cattle embryos. In Experiment I, there were assessments of embryo production and oxidative status of cattle embryos derived by in vitro maturation and fertilization (IVM/IVF)that were cultured until the blastocyst stage of development using different ALA concentrations (5, 25 and 100 µM), fetal bovine serum (FBS) and amino acids (aa) as well as 20 % oxygen (O2) in the culture atmosphere. In Experiment II, embryos were cultured without FBS, at different ALA concentrations (2.5, 5 and 7.5 µM) and in the presence or absence of aa when there was a 7 % O2 atmosphere. Embryo development rates and blastocyst quality were evaluated. With 20 % O2 concentration, treatment with 100 µM ALA resulted in lesser hatching rates and development to the blastocyst stage (P < 0.01), while with supplementation with 5 µM ALA there were lesser (P = 0.04) glutathione concentrations and greater protein contents of embryos (P < 0.01). Culturing in the 7 % O2 atmosphere, combined with supplementation with 2.5 µM ALA with FBS and aa resulted in a greater blastocyst cell number (P = 0.03) and lesser hatching rates (P = 0.04). Taken together, results indicate supplementation with the greater ALA concentrations resulted in impairment of embryo development, regardless of the O2 concentration imposed during the culture period, while the relatively lesser supplementation-concentrations with ALA led to improvements in embryo quality.

Effects of EPA on bovine oocytes matured in vitro with antioxidants: Impact on the lipid content of oocytes and early embryo development.

The eicosapentaenoic acid (EPA) is an n-3 polyunsaturated fatty acid (PUFA) present in the lipid composition of bovine oocytes. Little is known about the importance of EPA in bovine oocyte maturation and embryo development in vitro. Although previous work suggest that n-3 PUFAs may inhibit oocyte maturation, the available data are inconsistent. In this study, we evaluated the effect of EPA (1, 10, 100 nM) during in vitro maturation (IVM) of bovine oocytes, alone and in combination with vitamin E (VE) or cysteamine (CYS). EPA treatment in IVM decreased oocyte lipid content and affected lipid droplets pattern (P < 0.05). EPA 100 nM reduced oocytes maturation rate (P < 0.05), without affecting cumulus expansion. At the concentrations tested, EPA did not modify embryo development. However, the addition of antioxidants during IVM reduced the levels of reactive oxygen species in the culture system by increasing intracellular glutathione content (P < 0.05). Besides, the combination of EPA with VE or CYS reduced the percentages of MI oocytes after 24 h of IVM (P < 0.05). EPA reduced oocyte lipid content without any detrimental for embryo development.

Prepartum fatty acid supplementation in sheep. IV. Effect of calcium salts with eicosapentaenoic acid and docosahexaenoic acid in the maternal and finishing diet on lamb liver and adipose tissue during the lamb finishing period1.

The objective of this study was to evaluate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation to ewes during late gestation on finishing lamb liver and adipose tissue fatty acid (FA) profile and gene expression. Lambs born from ewes supplemented with Ca salts of EPA + DHA, or palm FA distillate (PFAD) high in palmitic and oleic acid at 0.39% DM during the last 50 d of gestation were used. Lambs were weaned at 61 d of age and adapted to a high concentrate diet for 1.5 mo. After adaptation, 74 lambs (28 pens) were blocked by sex and BW and used in a 2 × 2 factorial arrangement of treatments using the factors of dam supplementation (DS) and lamb supplementation (LS) of Ca salts of EPA + DHA or PFAD at 1.48% DM. Lambs were slaughtered after 42 d and liver and adipose tissue collected for FA and gene expression analysis. Liver concentrations of EPA and DHA were greater (P < 0.01) with LS of EPA + DHA vs. PFAD during the finishing period. In adipose tissue, a lamb × dam interaction was observed for EPA (P = 0.02) and DHA (P = 0.04); LS of EPA + DHA increased EPA and DHA, but the increase was greatest in lambs born from ewes supplemented with PFAD. No lamb × dam treatment interactions were observed for gene expression in liver tissue (P > 0.10). Hepatic mRNA abundance of hormone-sensitive lipase (HSL; P = 0.01) was greater in lambs born from EPA + DHA ewes vs. lambs from PFAD ewes. mRNA expression of stearoyl-CoA desaturase (P < 0.01), fatty acid synthase (P = 0.01), Δ5-desaturase (P < 0.01), and Δ6-desaturase (P < 0.01) were decreased in liver of EPA + DHA lambs. A significant lamb × dam diet interaction was observed for elongation of very long chain fatty acid 2 in adipose tissue (P = 0.01); lambs supplemented with the same FA as their dams had lower expression. Expression of HSL tended (P = 0.08) to be decreased in adipose of EPA + DHA lambs born from EPA + DHA ewes. The changes in mRNA expression suggest that lipogenesis decreased, and lipolysis increased in lamb liver with EPA + DHA vs. PFAD supplementation during the finishing period. In adipose tissue, changes suggest that lipogenesis decreased in lambs born from EPA + DHA supplemented dams and supplemented with EPA + DHA during the finishing period. In addition, these results suggest an interaction between supplementation of FA to dams during late gestation on lamb response of adipose tissue, but not liver, to FA supplementation during the finishing period.

Non-aversive photographic measurement method for subcutaneous tumours in nude mice.

We have developed a new method for the measurement of subcutaneous tumour volume which consists in taking photographs of mice in their home cages, to refine the standard method of measurement with calipers. We consider this new method to be non-aversive, as it may be more compatible with mice behavioural preferences and, therefore, improve their welfare. Photographs are captured when mice voluntarily go into an acrylic tube containing graph paper that is later used as a scale. Tumour volumes measured with the caliper and the non-aversive photographic method were compared to those obtained by water displacement volume and weight. Behavioural and physiological changes were evaluated to assess animal welfare. Significant differences were found between measurements obtained with the caliper and the non-aversive photographic method, v. the reference volume acquired by water displacement (P < 0.001). Nevertheless, there was good consistency for these measurements when tumours were measured repeatedly, with all Intra-Class Correlation Coefficients above 0.95. Mice on which the non-aversive photographic method was employed were significantly less reluctant to establish contact with the experimenter (P < 0.001) and behaved less anxiously in a modified-Novelty Suppressed Feeding test. Particularly, statistically significant differences were found in connection with the latency to eat an almond piece (P < 0.05), the frequency of grooming (P < 0.001) and the frequency of defecation (P < 0.001). Corticosterone concentration in faeces and blood glucose were determined and no significant changes were found. Therefore, we propose the non-aversive photographic method to measure subcutaneous tumours as a way to refine methodologies in the field of experimental oncology.