Innate and humoral immune parameters at delivery in colostrum and calves from heifers experimentally infected with Neospora caninum.

Neospora caninum is a leading cause of abortion in cattle worldwide. The study of the immune response against N. caninum is critical to understand its epidemiology, pathogenesis, diagnosis and, ultimately, in preventing and controlling bovine neosporosis. Herein, we determined the gene expression of innate immune components endosomal RNA-sensing TLRs, BMAP28 cathelicidin, TNF-α and IL-10 and characterized the variation in both IgG ratio and avidity at delivery in N. caninum-infected heifers challenged at day 210 of gestation, colostrum and their calves. Increased BMAP28 expression was observed not only in colostrum but also in peripheral blood mononuclear cells (PBMC) and umbilical cord of calves from N. caninum-infected heifers in comparison with mock-infected control group. In addition, statistically significant decrease of TLR7 and IL-10 expression levels were observed in umbilical cord, suggesting an attempt to avoid an exacerbated immune response against the parasite. At delivery, serum and colostrum samples from infected group evidenced specific IgG anti-N. caninum. Infected heifers showed IgG1/IgG2 ratios <1 and high avidity specific IgG. As expected, colostrum samples of these animals exhibited a high IgG1 concentration and elevated avidity values. Three out of four calves from N. caninum-infected heifers had specific IgG with IgG1/IgG2 ratios>1 and lower avidity values before colostrum intake. Interestingly, both IgG1/IgG2 ratios and avidity values increased in seropositive calves after colostrum intake. Overall, this study provides novel information on neonatal immunity in congenitally infected calves, which is essential to understand how the immune pathways could be manipulated or immune components could be employed in order to improve protection against neosporosis.

Effect of nicotinamide supplementation in in vitro fertilization medium on bovine embryo development.

Increased oxidative stress is one of the main causes of poorly developed embryos in assisted reproductive technologies. Nicotinamide (NAM) has been shown to suppress reactive oxygen species (ROS) production through its potent antioxidative and anti-senescent effects. In the present study, we explored the effects of short-term NAM-treatment (3 and 5 h) during in vitro fertilization (IVF) on the development of bovine embryos. Treatment with 10 mM NAM for 3 h significantly increased the blastocyst formation but extending the treatment to 5 h did not enhance the benefits any further. Immunofluorescence analysis demonstrated that treatment with 10 mM NAM for 3 h decreased the expression of intracellular ROS, 8-oxo-7,8-dihydroguanine, caspase-3, and increased the expression of Sirt1, and incorporation of bromodeoxyuridine in one-cell stage embryos. Similarly, the level of H3K56ac significantly increased in the NAM-treated (3 and 5 h) one-cell stage embryos. Contrastingly, the treatment with 10 mM NAM for 5 h increased the caspase-9 level in blastocysts. Collectively, these findings suggest that NAM possesses antioxidant activity and supplementation of IVF medium with 10 mM NAM for 3 h improves the in vitro developmental competence of bovine embryos.

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.

Supplementation of insulin-transferrin-sodium selenite in culture medium improves the hypothermic storage of bovine embryos produced in vitro.

Hypothermic storage of gametes and embryos at 4 °C can be used as an alternative to cryopreservation, but hypothermic preservation can maintain embryo viability for a short duration only. This study investigated the effect of insulin-transferrin-sodium selenite (ITS) in embryo culture medium on hypothermic storage of bovine embryos at 4 °C. Day 7 bovine embryos were subjected to hypothermic storage in tissue culture medium 199 supplemented with 50% fetal bovine serum and 25 mM HEPES for different time durations. After recovery, the embryos were assessed for survival and hatching rate and gene and protein expression levels. Supplementation of embryo culture medium with ITS significantly increased (P < 0.05) the survival and hatching ability of blastocysts stored at 4 °C for 72 h compared to the control group (100% and 76.3% vs 68.5% and 40.5%, respectively). Furthermore, the beneficial effects of ITS on embryos were associated with greater (P < 0.05) total cell number per blastocyst and lesser apoptotic cells number. Moreover, embryos cultured in ITS had lower intracellular lipid content. The protein expression of sirt1 was greater (P < 0.05) in the ITS group, however, caspase3 protein expression was significantly lesser (P < 0.05) in the ITS group. Quantitative reverse transcription PCR indicated that the mRNA levels of SIRT1 and HSP70 were (P < 0.05) increased upon culture with ITS; however, the mRNA levels of the pro-apoptotic genes BAX and CASP3 were reduced (P < 0.05). Taken together, these data suggest that supplementation of embryo culture medium with ITS improves in vitro bovine embryo quality and survival following hypothermic storage.

Effect of delipidant agents during in vitro culture on the development, lipid content, gene expression and cryotolerance of bovine embryos.

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.

l-carnitine supplementation during in vitro culture regulates oxidative stress in embryos from bovine aged oocytes.

Aging oocytes undergo various molecular, cellular, and biochemical changes. Aging of oocytes results in reduced embryo developmental capacity and blastocyst quality, which is thought to be caused partly by the accumulation of reactive oxygen species (ROS). This study aimed to determine the effect of l-carnitine (LC) on the development of embryos formed from aged oocytes in vitro. The development and quality of the blastocysts in the LC-treated group were significantly higher than those in the untreated aged group after in vitro fertilization (IVF). In addition, after LC treatment, the level of intracellular ROS in aged group significantly decreased, and glutathione (GSH) levels significantly increased compared with those in the untreated aged group. There was no significant difference in the mitochondrial membrane potential among the three groups. Moreover, ROS could induce autophagy and LC3 antibody was widely used as a marker for detecting autophagy. The fluorescence intensity of LC3 in the aged group was significantly higher than that of LC3 in the LC-treated group. Furthermore, Real-time reverse transcriptase-polymerase chain reaction showed that the mRNA levels of antioxidation genes GPX4 and SOD1 were significantly higher in embryos from LC-treated group than in those from the untreated aged group. In summary, our results indicated that LC can improve the developmental capacity of embryos from aging oocytes in vitro by reducing oxidative stress.

Zinc supplementation during in vitro embryo culture increases inner cell mass and total cell numbers in bovine blastocysts1.

Deficiencies in current embryo culture media likely contribute to the poor blastocyst development rates and pregnancy retention rates for in vitro produced (IVP) bovine embryos. Of special concern is the lack of micronutrients in these media formulations. One micronutrient of interest is zinc, an essential trace element involved with various enzyme and transcription factor activities. The objective of this work was to describe whether zinc sulfate supplementation during in vitro embryo culture affects bovine embryo development and blastomere numbers. Either 0, 2, 20, or 40 µM zinc sulfate was supplemented to presumptive zygotes cultured in synthetic oviductal fluid containing AAs and bovine serum albumin for 8 d. None of the treatments affected cleavage rates. Percentage of blastocysts on days 7 and 8 postfertilization was not affected by supplementing 2 or 20 µM zinc but were reduced (P < 0.05) with 40 µM zinc. In blastocysts harvested on day 8, inner cell mass (ICM) and total cell number were increased (P < 0.05) with 2 µM zinc supplementation but not with the other zinc concentrations. Numbers of trophectoderm cells were not affected by zinc treatment. In conclusion, supplementing zinc during bovine embryo culture did not impact blastocyst development but improved ICM cell numbers. This improvement in ICM cell number may have implications for improved pregnancy retention rates after IVP embryo transfer as smaller ICM sizes are associated with poor pregnancy success in cattle.

Magnesium is a critical element for competent development of bovine embryos.

The study was designed to determine the impact of magnesium (Mg2+) on bovine embryo development. We found that two commercially available sources of bovine serum albumin (BSA) and fetal bovine serum (FBS) contained different amounts of Mg2+ residue: 4 ppm in ICPbio BSA, 114 ppm in Sigma BSA, and 44 ppm in FBS. When CR1 was used as basal medium, PVA and ICPbio BSA produced the lowest blastocyst yield (2.2-2.3%), whereas Sigma BSA increased blastocyst yield to 18.9% (P < 0.05). Supplementation of 1.4 mM MgCl2 into the medium increased the blastocyst rate in the ICPbio BSA group (29.4%) but not in the PVA group (5.4%; P < 0.05) to a level comparable to that of the FBS group (33.7%; P > 0.05). We next found that increasing concentrations of MgCl2 in the culture medium (ICPbio BSA) elevated blastocyst rate from 2.6% (0 mM), 38.4% (0.35 mM) to 50.2% (1.4 mM; P < 0.05), further maintained at 44.9% (2.1 mM) and 43.4% (2.8 mM) (P > 0.05). However, blastocyst rate was reduced to 31.4% (4.2 mM) and 29.4% (5.6 mM) when MgCl2 supplement was increased (P < 0.05). Comparable blastocyst development was achieved in both ICPbio BSA (30.0-33.1%) and Sigma BSA (37.4-38.7%) groups when 1.4 mM Mg2+ was supplemented regardless of its source (MgCl2 vs. MgSO4; P > 0.05). In embryo transfer experiments, higher rates of pregnancy (54.3 vs. 41.5%) and calving (44.3 vs. 32.5%) were achieved in the CR1-Mg2+-supplemented BSA group compared with the FBS group with co-culture, respectively (P < 0.05). These results demonstrate that Mg2+ is a key ion that promotes competent blastocyst and term development. Therefore, a simple and efficient defined medium (CR1-Mg2+-BSA) can successfully replace complex serum and somatic cell co-culture.

L-carnitine prevents bovine oocyte aging and promotes subsequent embryonic development.

L-carnitine (LC) is well known for its antioxidant activity. In this study, we explored the potential mechanistic effects of LC supplementation on aged bovine oocytes in vitro. We showed that in-vitro maturation could enhance the subsequent developmental capacity of aging oocytes, when supplemented with LC. After in vitro fertilization, the blastocyst formation rate in the aged oocytes post-LC treatment significantly increased compared to that in untreated aged oocytes (29.23 ± 2.20% vs. 20.90 ± 3.05%). Furthermore, after LC treatment, the level of intracellular reactive oxygen species in aged oocytes significantly decreased, and glutathione levels significantly increased, compared to those in untreated aged oocytes. Mitochondrial membrane potential, the percentage of early apoptotic oocytes, and caspase-3 activity were significantly reduced in LC-treated aged oocytes compared to those in untreated aged oocytes. Furthermore, during in vitro aging, the mRNA levels of the anti-apoptotic genes, Bcl-xl and survivin in LC-treated aged oocytes were significantly higher than those in untreated aged oocytes. Overall, these results indicate that at least in in vitro conditions, LC can prevent the aging of bovine oocytes and improve the developmental capacity of bovine embryo.

Late embryonic losses in supplemented grazing lactating dairy cows: Risk factors and reproductive performance.

The main objective of this study was to evaluate the risk factors for late embryonic loss (LEL) in supplemented grazing dairy cows. Additional objectives were to assess the incidence of LEL and its association with the reproductive performance of cows. A data set containing productive, reproductive, and health records of 13,551 lactations was used. A retrospective case-control study involving 631 cows with LEL (cases) and 2,524 controls (4 controls per case within each study year) was run. A case of LEL was defined when the embryo had no heartbeat or there was evidence of detached membranes or floating structures including embryo remnants by ultrasonography (US) at 28 to 42 d post-artificial insemination (AI), whereas a non-case was defined as a cow diagnosed with positive pregnancy by US 28 to 42 d post-AI and reconfirmed as pregnant 90 ± 7 d post-AI. Four controls per case were randomly selected from the non-cases with a temporal matching criterion (±3 d around the date of the fecundating AI of the case). Multivariable logistic models were offered with the following predictors: year of LEL (2011 through 2015), season of LEL (summer vs. fall vs. winter vs. spring), parity (1 vs. 2 vs. ≥3), uterine disease (UD), non-uterine disease (NUD), body condition score at parturition, body condition score at 28 to 42 d post-AI (BCS-LEL), days in milk (DIM), and daily milk yield (MY). Statistical significance was set at P < 0.05 and a tendency was set at P ≤ 0.10. We found that 4.7, 22, and 23% of cows had LEL, UD, and NUD, respectively. Cases tended to have higher daily MY than controls (32.5 vs. 31.8 kg); also, cases had much longer calving to pregnancy interval (226 vs. 118 d), lower hazard of pregnancy [hazard ratio = 0.39, 95% confidence interval (CI) = 0.35-0.43], and higher odds for non-pregnancy [odds ratio (OR) = 2.89, 95% CI = 2.37-3.54] than controls. We found that the odds for LEL increased with parity number (OR = 2.48, 95% CI = 1.99-3.08 for parity ≥3) and with BCS-LEL <2.50 (OR = 1.81, 95% CI = 1.33-2.47). Conversely, the odds for LEL decreased with BCS-LEL >3.00 (OR = 0.70, 95% CI = 0.53-0.91). The odds for LEL increased with UD (OR = 1.23, 95% CI = 1.01-1.49), NUD (OR = 1.24, 95% CI = 1.01-1.54), DIM (OR = 1.03, 95% CI = 1.00-1.05), and daily MY (OR = 1.14, 95% CI = 1.04-1.25) in univariable models only. Finally, the odds for LEL were not associated with year, season, DIM, and body condition score at parturition. In conclusion, LEL is associated with extended calving to pregnancy interval, and among its risk factors are parity number and BCS-LEL.

The biochemistry surrounding bovine conceptus elongation†.

Conceptus elongation is a fundamental developmental event coinciding with a period of significant pregnancy loss in cattle. The process has yet to be recapitulated in vitro, whereas in vivo it is directly driven by uterine secretions and indirectly influenced by systemic progesterone. To better understand the environment facilitating this critical reproductive phenomenon, we interrogated the biochemical composition of uterine luminal fluid from heifers with high vs physiological circulating progesterone on days 12-14 of the estrous cycle-the window of conceptus elongation-initiation-by high-throughput untargeted ultrahigh-performance liquid chromatography tandem mass spectroscopy. A total of 233 biochemicals were identified, clustering within 8 superpathways [amino acids (33.9%), lipids (32.2%), carbohydrates (8.6%), nucleotides (8.2%), xenobiotics (6.4%), cofactors and vitamins (5.2%), energy substrates (4.7%), and peptides (0.9%)] and spanning 66 metabolic subpathways. Lipids dominated total progesterone (39.1%) and day (57.1%) effects; however, amino acids (48.5%) and nucleotides (14.8%) accounted for most day by progesterone interactions. Corresponding pathways over-represented in response to day and progesterone include (i) methionine, cysteine, s-adenosylmethionine, and taurine (9.3%); (ii) phospholipid (7.4%); and (iii) (hypo)xanthine and inosine purine metabolism (5.6%). Moreover, under physiological conditions, the uterine lumen undergoes a metabolic shift after day 12, and progesterone supplementation increases total uterine luminal biochemical abundance at a linear rate of 0.41-fold day-1-resulting in a difference (P ≤ 0.0001) by day 14. This global metabolic analysis of uterine fluid during the initiation of conceptus elongation offers new insights into the biochemistry of maternal-embryo communication, with implications for improving ruminant fertility.

The effects of crocetin supplementation on the blastocyst outcome, transcriptomic and metabolic profile of in vitro produced bovine embryos.

The earliest stages of embryo development are deeply influenced by reactive oxygen species (ROS), byproducts of the mitochondrial oxygen metabolism that play a key role as messengers in normal cell signal transduction and cell cycling. Despite its positive roles, the imbalance caused by the excess of ROS and an inefficient antioxidant system leads to oxidative stress, with negative consequences to the cell such as DNA damage, metabolic changes, mitochondrial stress and cell death. In the present work, crocetin - a natural antioxidant - was added to the culture media of bovine embryos to evaluate the efficiency of its antioxidant capability during embryo culture. Oocytes were in vitro matured (IVM) and fertilized according to standard protocols. Embryos were cultured at 38.5 °C under humidified air with 5% CO2, 7% O2, and 90% N2 in Synthetic Oviduct Fluid (SOF) medium supplemented with amino acids and either 5% of FBS (SOFaa) (control group) or SOFaa supplemented with 1  µM crocetin (crocetin group). After 5 days from the beginning of in vitro culture (IVC) (day 5 - D5), embryos were transferred to individual drops of culture media. At day 7 (D7), embryos were assessed by means of blastocyst rates, morphophysiological analyzes (total cell number, ROS and mitochondrial activity levels), transcript quantitation of 47 genes and metabolomic evaluation of the culture media by Raman spectroscopy. In the crocetin group blastocyst rates were higher and embryos had increased total cell number and decreased intracellular levels of ROS. These embryos also had upregulation of genes related with response to stress and lipid metabolism (ATF4, BAX, FOXO3, GADD45A, GPX1, GPX4, HSF1, SOD2, ACACA, SREBF1 and SREBF2). Raman spectroscopy corroborated these results indicating more active lipid and amino acid production in this group. The absence of crocetin in the culture media resulted in higher ROS level, as well as up regulation of genes related to DNA damage, stress response and energy metabolism (MORF4L2, SOD1, TXN, PFKP, PGK1 and PPARGC1A). In conclusion, crocetin supplementation during culture protects embryos from oxidative stress and influences the adaptive response to stress conditions, leading to an increase in both blastocyst yield and quality, as well as changes in transcriptomic and metabolic profile of in vitro produced bovine embryos.

Early career achievement award: supplementing omega-6 fatty acids to enhance early embryonic development and pregnancy establishment in Bos indicus and B. taurus beef cows.

Our research group investigated the impacts of supplementing Ca salts of soybean oil (CSSO), a source of omega-6 fatty acids (FAs), on reproductive performance of beef cows. Initial studies were conducted with Nelore (Bos indicus) cows grazing tropical pastures. Cows were assigned to fixed-time artificial insemination (AI) and supplemented or not with 100 g/d (as-fed basis) of CSSO, and supplementation regimens ranged from days -11 to 28 relative to AI. Overall, CSSO supplementation during the 21 d after AI increased (P < 0.01) pregnancy rates from 38.1% (623/1,635 as pregnant/total nonsupplemented cows) to 49.0% (843/1,720 as pregnant/total CSSO-supplemented cows), and these outcomes were associated with enhanced early embryonic development and pregnancy establishment when omega-6 FA were supplemented. To verify this rationale, our group compared FA incorporation in grazing Nelore cows (n = 90) supplemented or not with CSSO (100 g/d; as-fed basis) beginning at fixed-time AI until slaughter at day 19 of gestation. Supplementing CSSO increased (P ≤ 0.05) incorporation of linoleic acid and its omega-6 derivatives in plasma, endometrium, corpus luteum, and conceptus, whereas the same responses were not observed (P ≥ 0.25) for omega-3 FA. Complementing these findings, grazing Nelore cows (n = 100) were supplemented or not with CSSO (100 g/d; as-fed basis) beginning at fixed-time AI, and assigned to transcervical uterine flush on day 15 of gestation. Supplementing CSSO increased (P ≤ 0.04) conceptus length (2.58 vs. 1.15 cm) and mRNA expression of interferon-tau (4.1-fold increase) and prostaglandin E synthase 2 (2.6-fold increase), which are critical regulators of pregnancy establishment. These outcomes were recently replicated in B. taurus beef cows consuming temperate forages. Pregnancy rates were greater (P = 0.01) in Angus cows receiving CSSO (100 g/d; as-fed basis) for 21 d after fixed-time AI (60.2%; 226/383 as pregnant/total cows) compared with nonsupplemented cows (51.7%; 193/388 as pregnant/total cows). Supplementing CSSO to Angus × Hereford cows (n = 96) beginning after AI also increased (P = 0.05) mRNA expression of interferon-tau in day 15 conceptuses (1.8-fold increase). Collectively, our research demonstrated that post-AI CSSO supplementation favors incorporation of omega-6 FA into maternal and embryonic tissues, which enhances interferon-tau synthesis by the conceptus and increases pregnancy rates to fixed-time AI in B. indicus and B. taurus beef cows.

Embryonic development, luteal size and blood flow area, and concentrations of PGF2α metabolite in dairy cows fed a diet enriched in polysaturated or polyunsaturated fatty acid.

The objective of this study was to examine effects of sunflower (SO) and palm oil (PO) supplements in the diet on embryonic development, luteal size and blood flow area, PGF2α metabolite (PGFM), and progesterone (P4) concentrations. Prepartum cows (n = 42) were randomly assigned to one of three dietary treatments (control, 4% PO, and 4% SO supplements). Animals were fed diets individually from day 28 prepartum to day 111 postpartum. Luteal size and blood flow area were determined throughout the estrous cycle by Doppler ultrasonography. Oocytes were collected in three ovum pick-up sessions at 2 week intervals for the in vitro embryo production. Oocyte characteristics and embryonic development were not affected by dietary treatments. Cows fed 4% SO had a greater (P < 0.05) concentration of PGFM from day 15 to day 35 postpartum than those cows fed 4% PO and the control group. On day 11 of the estrous cycle (mid-luteal phase), serum P4 concentrations (6.0 ±â€¯0.7, 5.7 ±â€¯0.5, and 4.7 ±â€¯0.6 ng/ml), luteal size (7.0 ±â€¯0.2, 6.5 ±â€¯0.2, and 5.3 ±â€¯0.1 cm2) and luteal blood flow area (1.3 ±â€¯0.2, 1.2 ±â€¯0.1, and 0.9 ±â€¯0.1 cm2) were greater (P < 0.05) in cows fed 4% SO and 4% PO than the control group, respectively. Thus, plant oil supplements in diets affected luteal size and serum P4 and PGFM concentrations, but not early embryonic development. Such changes in secretion of PGF2α and P4 indicate that plant oil supplements during pre- and postpartum may alter uterine and luteal functions.

Oxidative stress differentially impacts male and female bovine embryos depending on the culture medium and the stress condition.

Male and female embryos are known to differ for their metabolism and response to environmental factors very early in development. The present study aimed to evaluate the response to oxidative stress of male and female bovine embryos at the morula-blastocyst stages in terms of developmental rates, total cell number and apoptotic rates in two culture conditions. Embryos where cultured in a medium supplemented with either 5% fetal calf serum (FCS) or 4 mg/mL bovine serum albumin and a mixture of insulin, transferrin and selenium (BSA-ITS). Oxidative stress was applied at Day-5 post insemination (pi) by adding either AAPH or menadione to the culture medium, and blastocysts were analyzed at Day-7pi. The impact on development and blastocyst quality was dependent on the culture medium and the stress inducer but differed between male and female embryos. Male embryos resisted better to oxidative stress in FCS supplemented medium, no matter the stress inducer. Accordingly, the impact on blastocyst cell number tended to be higher in female blastocysts after stress induction with AAPH in FCS supplemented medium. On the other hand, in BSA-ITS supplemented medium, female embryos were more resistant to AAPH induced stress, while menadione had no impact on sex ratio. The weaker resistance of males to AAPH in this medium is in accordance with their trend to show a higher increase in apoptotic rates than females in this condition. In conclusion, this study shows that oxidative stress has differential impact on male and female bovine blastocysts depending on the culture condition and on the way oxidative stress is induced.

Supplementation with sunflower seeds in beef cattle did not impact on oocyte and in vitro embryo production.

Supplementation with compounds rich in linoleic acid, including sunflower seed supplementation, promotes increase in conception rates in cows. We aimed to evaluate whether the sunflower seed (linoleic acid source) supplementation in beef donor females alters the plasma concentrations of cholesterol, triglycerides, HDL and LDL, increases the number and quality of oocytes, increases the cleavage rates and determines an improvement in number and quality of in vitro produced blastocysts. Thus, Nelore females were divided into two groups of 15 animals to receive supplementation with or without sunflower seed for 57 days. Females underwent follicular aspiration and the oocytes were subjected to in vitro embryo production. There was no difference (p > .1) between control group and group supplemented with sunflower seed on the number of displayed follicles; number of aspired oocytes; recovery rate; cleavage rate; number of embryos; number of blastocysts; embryos number of grades I and II; plasma concentrations of total cholesterol, triglycerides; HDL and LDL. Therefore, sunflower seed supplementation in oocyte donors did not increase the number and quality of oocytes, cleavage rates and the number and quality of blastocysts produced in vitro.

Different durations of whole raw soybean supplementation during the prepartum period: Milk fatty acid profile and oocyte and embryo quality of early-lactating Holstein cows.

The objective of this study was to evaluate different durations of whole raw soybeans (WS) supplementation during the prepartum period on nutrient digestibility, milk yield and composition, energy balance, blood metabolites, and oocyte and embryo quality of transition cows. Thirty-one Holstein cows were used in a completely randomized design and assigned to 4 experimental groups (G): G90, G60, G30, and G0 (control), supplemented with a diet containing 12% of WS from 90, 60, 30, and 0 d relative to the calving date, respectively. Cows were dried off 60 d before the expected calving date. After parturition, all cows were fed a diet containing 12% of WS until 84 DIM. Blood samples were collected on d -49, -35, -21, -14, -7, 0, 7, 14, 21, 35, and 70 relative to partum. Ovum pick-ups were performed on d 21 ± 3, 42 ± 7, 63 ± 7, and 84 ± 7 of lactation. Different durations of WS supplementation did not affect DMI and apparent total-tract digestibility in either the pre- or postpartum periods. Duration of WS supplementation had no effect on milk yield and composition nor energy balance of cows. However, the duration of WS supplementation had several effects on milk fatty acid (FA) profile of cows, including a linear decrease in concentrations of cis-9 C18:1, unsaturated C18, total monounsaturated, and unsaturated FA. Further, the milk contents of cis-9,cis-12 C18:2 FA, cis-9,trans-11 C18:2 FA, and total polyunsaturated FA were increased when WS were fed to cows from 30 d but not from 60 or 90 d of the expected calving date. The length of WS supplementation in the prepartum period linearly increased blood cholesterol concentration of cows during the prepartum period, but it had no effect on blood glucose and nonesterified FA concentrations in the pre- and postpartum periods. Duration of WS supplementation during the prepartum period increased the average number of grade 2 oocytes, notably in G60, but it had no effect on embryo production and cleavage proportion of early-lactation cows. The duration of WS supplementation in the prepartum period had no effect on milk yield and energy balance of the subsequent lactation, but it altered milk FA profile in early lactation by decreasing unsaturated FA content, notably when starting to supplement WS at 90 and 60 d from the expected calving date. Our results also showed that the duration of WS supplementation during the prepartum period does not improve oocyte quality in the subsequent lactation of cows.

High doses of lipid-core nanocapsules do not affect bovine embryonic development in vitro.

The improvement of in vitro embryo production by culture media supplementation has been a potential tool to increase blastocyst quality and development. Recently, lipid-core nanocapsules (LNC), which were developed for biomedical applications as a drug-delivery system, have demonstrated beneficial effects on in vitro embryo production studies. LNCs have a core composed of sorbitan monostearate dispersed in capric/caprylic triglyceride. Based on that, we firstly investigated if LNCs supplemented during in vitro oocyte maturation had affinity to the mineral oil placed over the top of the IVM media. Also, the effects of LNC supplementation in different concentrations (0; 0.94; 4.71; 23.56; 117.80 and 589.00µg/mL) during the in vitro maturation protocol were evaluated in oocytes and blastocysts by in vitro tests. LNCs seemed not to migrate to the mineral oil overlay during the in vitro oocyte maturation. Interestingly, LNCs did not show toxic effects in the oocyte in vitro maturation rate, cumulus cells expansion and oocyte viability. The highest LNCs concentration tested (589µg/mL) generated the lowest ROS and GSH levels, and reduced apoptosis rate when compared to the control. Additionally, toxic effects in embryo development and quality were not observed. The LNC supramolecular structure demonstrated to be a promising nanocarrier to deliver molecules in oocytes and embryos, aiming the improvement of the embryo in vitro development.

Supplementation of lycopene in maturation media improves bovine embryo quality in vitro.

This study sought to modulate factors that reduce embryo quality in in vitro culture (IVC) systems. Over eight replicates, 3075 oocytes were cultured in in vitro maturation media containing various concentrations of lycopene, followed by in vitro fertilization and culture. The percentages of MII-stage oocytes, the presumptive zygotes that underwent cleavage and developed into blastocysts were significantly (P < 0.05) higher, the intracellular ROS concentrations reduced significantly (P < 0.05) in oocytes/blastocysts, TUNEL assay demonstrates reduced apoptosis and increased total cell number per blastocyst (P < 0.05), Immunocytochemistry confirmed that diminished protein expression of nuclear factor kappa B (NFκB), cyclooxygenase-2 (COX2), and 8-oxoguanine (indicated by ROS) and relative mRNA expression of the Caspase-3, NFκB, COX2, iNOS and BCL2-associated X (BAX) was significantly (P < 0.05) lower whereas the anti-apoptotic gene BCL2 was significantly (P < 0.05) higher in the 0.2 µM lycopene-supplemented group than the control. In conclusion, lycopene improves blastocyst quality by overcoming unfavorable conditions in in vitro culture systems.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM: Alterations in uteroplacental hemodynamics during melatonin supplementation in sheep and cattle.

Compromised placental function can result in fetal growth restriction which is associated with greater risk of neonatal morbidity and mortality. Large increases in transplacental nutrient and waste exchange, which support the exponential increase in fetal growth during the last half of gestation, are dependent primarily on the rapid growth and vascularization of the uteroplacenta. The amplitude of melatonin secretion has been associated with improved oxidative status and altered cardiovascular function in several mammalian species; however, melatonin mediated alterations of uteroplacental capacity in sheep and cattle are lacking. Therefore, our laboratories are examining uteroplacental blood flow and fetal development during maternal melatonin supplementation. Using a mid- to late-gestation ovine model of intrauterine growth restriction, we examined uteroplacental blood flow and fetal growth during supplementation with 5 mg/d of dietary melatonin. Maternal nutrient restriction decreased uterine arterial blood flow, while melatonin supplementation increased umbilical arterial blood flow compared with non-supplemented controls. Although melatonin treatment did not rescue fetal weight in nutrient restricted ewes; we observed disproportionate fetal size and fetal organ development. Elevated fetal concentrations of melatonin may result in altered blood flow distribution during important time points of development. These melatonin specific responses on umbilical arterial hemodynamics and fetal development may be partially mediated through vascular melatonin receptors. Recently, we examined the effects of supplementing Holstein heifers with 20 mg/d of dietary melatonin during the last third of gestation. Uterine arterial blood flow was increased by 25% and total serum antioxidant capacity was increased by 43% in melatonin supplemented heifers vs. non-supplemented controls. In addition, peripheral concentrations of progesterone were decreased in melatonin supplemented heifers vs. non-supplemented controls. Using an in vitro model, melatonin treatment increased the activity of cytochrome P450 2C, a progesterone inactivating enzyme, which was blocked by treatment with the melatonin receptor antagonist, luzindole. Elucidating the consequences of specific hormonal supplements on the continual plasticity of placental function will allow us to determine important endogenous mediators of offspring growth and development.