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Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase-3 (z-DEVD-fmk) could improve post-thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled-rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1-3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 μM) and z-DEVD-fmk (0, 50, 100 or 200 μM), respectively. Post-thaw survival was assessed at 24, 48 and 72 h. For experiments 4-5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post-thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated. Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p .05) re-expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post-thaw cryosurvival was not affected by the addition of either DTT or z-DEVD-fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p .001) levels of intracellular ROS and fewer (p .001) cells with DNA fragmentation. In conclusion, post-thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate.

We consider the following activity recognition task: given a video, infer the set of activities being performed in the video and assign each frame to an activity. This task can be solved using modern deep learning architectures based on neural networks or conventional classifiers such as linear models and decision trees. While neural networks exhibit superior predictive performance as compared with decision trees and linear models, they are also uninterpretable and less explainable. We address this accuracy‐explanability gap using a novel framework that feeds the output of a deep neural network to an interpretable, tractable probabilistic model called dynamic cutset networks, and performs joint reasoning over the two to answer questions. The neural network helps achieve high accuracy while dynamic cutset networks because of their polytime probabilistic reasoning capabilities make the system more explainable. We demonstrate the efficacy of our approach by using it to build three prototype systems that solve human‐machine tasks having varying levels of difficulty using cooking videos as an accessible domain. We describe high‐level technical details and key lessons learned in our human subjects evaluations of these systems.

The objective was to identify the transcriptomic profile of in vitro-derived embryos with high competence to establish and maintain gestation. Embryos produced with X-sorted sperm were cultured from day 5 to day 7 in serum-free medium containing 10 ng/ml recombinant bovine colony-stimulating factor 2 (CSF2) or vehicle. The CSF2 was administered because this molecule can increase blastocyst competence for survival after embryo transfer. Blastocysts were harvested on day 7 of culture and manually bisected. One demi-embryo from a single blastocyst was transferred into a synchronized recipient and the other half was used for RNA-seq analysis. Using P 2-fold or

Metritis is associated with reduced fertility in dairy cows, but the mechanisms are unclear because the disease resolves several weeks before insemination. One hypothesis is that metritis causes persistent changes in granulosa cells during follicle development, which might be evident in the transcriptome of granulosa cells from dominant follicles weeks after parturition. To test this hypothesis, we collected the follicular fluid and granulosa cells from dominant follicles 63 days post partum from cows previously diagnosed with metritis, at least 6 weeks after resolution of the disease and from cows not diagnosed with metritis (control cows). Bacterial lipopolysaccharide was detected in follicular fluid, and concentrations were associated with follicular fluid IL-8 and glucose concentrations. Transcriptome analysis using RNAseq revealed 177 differentially expressed genes in granulosa cells collected from cows that had metritis compared with control cows. The most upregulated genes wereITLN1,NCF2,CLRN3,FSIP2andANKRD17, and the most downregulated genes wereACSM1,NR4A2,GHITM,CBARPandNR1I3. Pathway analysis indicated that the differentially expressed genes were involved with immune function, cell–cell communication, cell cycle and cellular metabolism. Predicted upstream regulators of the differentially expressed genes included NFκB, IL-21 and lipopolysaccharide, which are associated with infection and immunity. Our data provide evidence for a persistent effect of metritis on the transcriptome of granulosa cells in ovarian follicles after the resolution of disease.

The objective of these experiments was to determine the effect of l -carnitine during oocyte maturation or embryo culture on embryo development and cryosurvival. For Experiments 1–3, embryos were produced in vitro using abattoir-derived cumulus-oocyte complexes (COCs). At d 7 after insemination, embryo development was assessed, and blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Post-thaw cryosurvival was determined by re-expansion and hatching rates at 24, 48 and 72 h post-thaw. In Experiment 1, COCs were matured with or without 3.03 mM l -carnitine. There was no effect of l -carnitine supplementation during maturation on embryo development or post-thaw cryosurvival. In experiment 2, presumptive zygotes were cultured in medium supplemented with or without 5% (v/v) fetal bovine serum and l -carnitine at concentrations of 0.0, 0.75, 1.5 and 3.03 mM. There was no effect of l -carnitine treatment on embryo development, but embryos treated with l -carnitine had increased (P ≤ 0.05) post-thaw re-expansion rates, irrespective of concentration. In experiment 3, presumptive zygotes were cultured with or without 0.75 mM l -carnitine from d 1 to d 4, from d 4 to d 7 or for the entire culture period. There was no effect of l -carnitine during culture on embryo development or post-thaw cryosurvival, regardless of the timing of addition. In Experiment 4, COCs were harvested by ovum pick-up from virgin dairy heifers (n = 24) and subjected to in-vitro embryo production with presumptive zygotes cultured with or without 0.75 mM l -carnitine. At d 7 after insemination, morula and blastocyst stage embryos were harvested and subjected to controlled-rate freezing. Lactating Holstein cows (n = 102) were used as recipients and synchronized for timed embryo transfer. At d 7 after anticipated ovulation, a single embryo was thawed and transferred to the ipsilateral uterine horn of each recipient with a corpus luteum. Pregnancy was diagnosed at d 33, 44 and 72 of gestation. l -carnitine had no effect on the percentage of cows pregnant per embryo transfer (P/ET) after transfer of a frozen-thawed embryo. In conclusion, media supplementation with l -carnitine during in vitro embryo production can improve post-thaw cryotolerance as assessed in vitro but had no effect on P/ET after transfer of frozen-thawed embryos.

PURPOSE: Engaging primary care providers (PCPs) in BRCA1/2 testing and results disclosure would increase testing access. The BRCA Founder OutReach (BFOR) study is a prospective study of BRCA1/2 founder mutation screening among individuals of Ashkenazi Jewish descent that sought to involve participants’ PCPs in results disclosure. We used quantitative and qualitative methods to evaluate PCPs’ perspectives, knowledge, and experience disclosing results in BFOR. METHODS: Among PCPs nominated by BFOR participants to disclose BRCA1/2 results, we assessed the proportion agreeing to disclose. To examine PCP’s perspectives, knowledge, and willingness to disclose results, we surveyed 501 nominated PCPs. To examine PCPs’ experiences disclosing results in BFOR, we surveyed 101 PCPs and conducted 10 semi-structured interviews. RESULTS: In the BFOR study overall, PCPs agreed to disclose their patient’s results 40.5% of the time. Two hundred thirty-four PCPs (46.7%) responded to the initial survey. Responding PCPs were more likely to agree to disclose patients’ results than non-responders (57.3% vs. 28.6%, p

Background Colony-stimulating factor 2 (CSF2) is an important maternal regulator of embryonic development. Earlier research indicates that CSF2 can regulate genes involved in cellular stress responses and block apoptosis. Here, we tested whether addition of 10 ng/mL CSF2 at day 5 of development would increase the survival of blastocysts harvested at day 7 and subjected to vitrification. Additional objectives were to determine whether embryo sex affected survival or whether effects of CSF2 interacted with sex. Results Survival after vitrification was measured as the percent of warmed blastocysts that re-established a blastocoele after culture and that underwent hatching from the zona pellucida. In the first experiment, blastocysts were vitrified, warmed, cultured for 24 h, and DNA embryo sexing performed by PCR. There was no effect of CSF2, sex, or the interaction on the percent of blastocysts that re-expanded or that were hatching or hatched. In the second experiment, vitrified blastocysts were warmed and cultured for 24, 48, and 72 h. Treatment with CSF2 increased (P = 0.021) the percent of blastocysts that re-expanded as compared to the vehicle group (overall, 77.8 ± 4.7% vs 73.3 ± 4.7%). Percent re-expansion was highest at 24 h and declined slightly thereafter (P = 0.024). Although the interaction was not significant, the effect of CSF2 was greater at 48 and 72 h than at 24 h because CSF2 reduced the incidence of embryos collapsing after re-expansion. Furthermore, the proportion of re-expanded blastocysts at 24 h that experienced blastocoel collapse by 72 h was lower (P = 0.053) for CSF2 (3.6%; 7/195) than for vehicle (8.2%; 16/195). The percent of warmed blastocysts that were hatching or hatched increased with time (P Conclusion Treatment with CSF2 from day 5 to 7 of development did not cause a significant effect on the percent of blastocysts that re-established the blastocoele after 24 h of culture but CSF2 reduced the collapse of the blastocoele that occurred for a portion of the embryos that had experienced re-expansion at 24 h. Thus, CSF2 can provide protection to a proportion of blastocysts from cryodamage caused by vitrification. Further work is needed to evaluate whether CSF2 increases survival of vitrified blastocysts after embryo transfer.

Choline is a nutrient that plays a role as a precursor for the neurotransmitter acetylcholine, the membrane phospholipid phosphatidylcholine, and the methyl donor betaine. Embryos produced in vitro are usually cultured without an exogenous choline source. We hypothesized that exposure to 1.8 mM choline chloride would increase percent of embryos becoming blastocysts in culture and pregnancy rate after transfer of embryos into recipients. A total of 39 Brahman and Senepol donors were used to produce embryos for transfer into recipient crossbred females. Donors were assigned to have their embryos cultured in either 1.8 mM choline chloride or, as a control, 1.8 mM extra NaCl. The percent of oocytes cleaved were measured 3 days after insemination and percent blastocyst at day 7.5. Embryos were transferred into recipient cows and pregnancy was diagnosed at 28–31 days of gestation and then confirmed at 50–56 days. Data were analyzed using PROC GLIMMIX in SAS. Treatment did not affect cleavage rate (67.3 + 1.6 vs 68.6 + 1.6% for choline vs control; P = .0.5632) or percent of cleaved embryos becoming blastocysts (17.6 + 1.3 vs 18.1 + 1.3%; P = 0.5355). Similarly, there was no effect of treatment on pregnancy days 28–31 [42.5% (48/113 cows) vs 47.3% (54/114 cows) for choline vs control; P = 0.4339] or at days 50–56 [39.1% (36/92) vs 38.5% (32/83); P = 0.5348]. In summary, 1.8 mM choline chloride does not impact embryo development to the blastocyst stage or pregnancy establishment. Further investigation is needed to evaluate the phenotype of the subsequent calves to determine whether early exposure to choline has consequences for postnatal function. Support: USDA-NIFA 2020-67015-30821.

The preimplantation period of embryonic development can be a key window for programming of postnatal development because extensive epigenetic remodeling occurs during this time. It was hypothesized that modification of one-carbon metabolism of the bovine embryo by addition of the methyl-donor choline to culture medium would change postnatal phenotype through epigenetic modification. Embryos produced in vitro were cultured with 1.8 mM choline chloride or control medium. Blastocysts were transferred into females and pregnancy outcomes and postnatal phenotype of the resultant calves determined. Exposure of embryos to choline increased gestation length and calf birth weight. Calves derived from choline-treated embryos were also heavier at weaning and had increased ratio of body weight to hip height than control calves. Choline altered muscle DNA methylation of calves 4 months after birth. A total of 670 of the 8149 CpG examined were differentially methylated, with the predominant effect of choline being hypomethylation. Among the genes associated with differentially methylated CpG were ribosomal RNAs and genes in AMPK, mTOR, integrin, and BEX2 canonical pathways and cellular functions involved in growth and proliferation. Results demonstrate that provision of the methyl-donor choline to the preimplantation embryo can alter its developmental program to increase gestation length, birth weight, and weaning weight and cause postnatal changes in muscle DNA methylation including those associated with genes related to anabolic processes and cellular growth. The importance of the nutritional status of the embryo with respect to one-carbon metabolism for ensuring health and well-being after birth is emphasized by these observations.

The objective was to characterize the transcriptome profile of in vivo–derived female embryos competent to establish and maintain gestation. Blastocysts from superovulated heifers were bisected to generate two demi-embryos. One demi-embryo was transferred into a synchronized recipient and the other part was used for RNA-seq analysis. Data on transcript abundance was analyzed for 4 demi-embryos that established and maintained pregnancy to day 60 (designated as PP) and 3 that did not result in a pregnancy at day 30 (designated as NP). Using a false discovery rate of P

Infection of the postpartum uterus with pathogenic bacteria is associated with infertility months later in dairy cattle. However, it is unclear whether these bacterial infections lead to long-term changes in the reproductive tract that might help explain this infertility. Here we tested the hypothesis that infusion of pathogenic bacteria into the uterus leads to changes in the transcriptome of the reproductive tract 3 months later. We used virgin Holstein heifers to avoid potential confounding effects of periparturient problems, lactation, and negative energy balance. Animals were infused intrauterine with endometrial pathogenic bacteria Escherichia coli and Trueperella pyogenes (n = 4) and compared with control animals (n = 6). Three months after infusion, caruncular and intercaruncular endometrium, isthmus and ampulla of the oviduct, and granulosa cells from ovarian follicles >8 mm diameter were profiled by RNA sequencing. Bacterial infusion altered the transcriptome of all the tissues when compared with control. Most differentially expressed genes were tissue specific, with 109 differentially expressed genes unique to caruncular endometrium, 57 in intercaruncular endometrium, 65 in isthmus, 298 in ampulla, and 83 in granulosa cells. Surprisingly, despite infusing bacteria into the uterus, granulosa cells had more predicted upstream regulators of differentially expressed genes than all the other tissues combined. In conclusion, there were changes in the transcriptome of the endometrium, oviduct and even granulosa cells, 3 months after intrauterine infusion of pathogenic bacteria. These findings imply that long-term changes throughout the reproductive tract could contribute to infertility after bacterial infections of the uterus.

The objective of this study was to find the optimal proportions of pregnancies from an in vitro-produced embryo transfer (IVP-ET) system and artificial insemination (AI) so that profitability is maximized over a range of prices for embryos and surplus dairy heifer calves. An existing stochastic, dynamic dairy model with genetic merits of 12 traits was adapted for scenarios where 0 to 100% of the eligible females in the herd were impregnated, in increments of 10%, using IVP-ET (ET0 to ET100, 11 scenarios). Oocytes were collected from the top donors selected for the trait lifetime net merit (NM$) and fertilized with sexed semen to produce IVP embryos. Due to their greater conception rates, first ranked were eligible heifer recipients based on lowest number of unsuccessful inseminations or embryo transfers, and then on age. Next, eligible cow recipients were ranked based on the greatest average estimated breeding values (EBV) of the traits cow conception rate and daughter pregnancy rate. Animals that were not recipients of IVP embryos received conventional semen through AI, except that the top 50% of heifers ranked for EBV of NM$ were inseminated with sexed semen for the first 2 AI. The economically optimal proportions of IVP-ET were determined using sensitivity analysis performed for 24 price sets involving 6 different selling prices of surplus dairy heifer calves at approximately 105 d of age and 4 different prices of IVP embryos. The model was run for 15 yr after the start of the IVP-ET program for each scenario. The mean ± standard error of true breeding values of NM$ of all cows in the herd in yr 15 was greater by $603 ± 2 per cow per year for ET100 when compared with ET0. The optimal proportion of IVP-ET ranged from ET100 (for surplus dairy heifer calves sold for ≥$300 along with an additional premium based on their EBV of NM$ and a ≤$100 embryo price) to as low as ET0 (surplus dairy heifer calves sold at $300 with a $200 embryo price). For the default assumptions, the profit/cow in yr 15 was greater by $337, $215, $116, and $69 compared with ET0 when embryo prices were $50, $100, $150, and $200. The optimal use of IVP-ET was 100, 100, 62, and 36% of all breedings for these embryo prices, respectively. At the input price of $165 for an IVP embryo, the difference in the net present value of yr 15 profit between ET40 (optimal scenario) and ET0 was $33 per cow. In conclusion, some use of IVP-ET was profitable for a wide range of IVP-ET prices and values of surplus dairy heifer calves.

Oak-hickory (Quercus spp. and Carya spp.) recruitment and regeneration are negatively impacted by non-natives species. Goats can provide an initial control of non-native vegetation; however, browsing behavior and preference should be studied before their introduction in the forest. Our objective was to analyze goats’ browsing behavior and preference in an eastern oak-hickory forest. We hypothesized that browsing behavior and preference are dependent on browse biomass composition and grazing duration. This study was conducted in Coshocton, Ohio, and contained two units with nine experimental plots each. Vegetation was surveyed during the summer of 2019 for biomass composition. Cluster analyses were performed using species identity and biomass. Vegetation nutritional analysis including fiber, protein, and sugar was completed. Two stocking rate densities were implemented: high (1,019 goat×day×ha-1) and low (509×goat×day×ha-1). Goats browsed for four or two days for a total of 36 days. Continuous observation was implemented to determine browsing behavior. Forage selectivity was measured using Jacob’s Selectivity Index (JSI). A linear mixed model was calculated using clusters, species, and/or grazing duration (fixed effect), experimental plots (random effect), and JSI (dependent variable). Spicebush (Lindera benzoin), multiflora rose (Rosa multiflora Thunb.), privet (Ligustrum spp.), and oriental bittersweet (Celastrus orbiculatus Thunb.) composed more than 70% of goats’ diet. Species identity, including nutritional components and physical defense mechanisms, was the most important factor in browsing selectivity. Goats preferred spicebush and privet and avoided multiflora irrespective clusters (P 0.001). In the first two days of browsing, goats favored spicebush and privet, avoided multiflora, and had a neutral preference for bittersweet (P-value 0.001). In the last two days, privet was selected and other species had a neutral selection (P 0.001). Goats will target spicebush and privet and avoid multiflora; therefore, an extended browsing period is recommended. Future studies should evaluate the effect of chemical defense mechanisms on browsing.

Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n = 12) or vehicle control (n = 11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130 days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16 days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae.

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Uterine infection is associated with infertility in women and dairy cows, even after the resolution of infection. However, the mechanisms causing this persistent infertility are unclear. Here, we hypothesized that induced endometritis in non-lactating dairy cows would reduce the developmental competence of oocytes. Non-lactating Holstein cows received an intrauterine infusion of endometrial pathogenic bacteria (Escherichia coli and Trueperella pyogenes; n = 12) or vehicle control (n = 11) on day 2 of the estrous cycle. Bacterial infusion increased expression of endometrial inflammatory mediators, and a mucopurulent discharge in the vagina confirmed the establishment of endometritis. Oocytes were collected by transvaginal ultrasound-guided ovum pickup on days 2, 24, 45, and 66 following infusion and subjected to in vitro fertilization and embryo culture. Bacterial infusion resulted in fewer cleaved oocytes developing to morulae compared to vehicle-infused controls (30.7 versus 45.0%), with the greatest effect observed in oocytes collected on day 24. Development to morula was inversely correlated with endometrial expression of IL6 on day 6. The expression of genes associated with embryo quality did not differ significantly between morulae from bacteria-infused and control cows. Artificial insemination 130 days after intrauterine infusion resulted in normal, filamentous embryos that produced interferon tau 16 days after conception in both infusion groups. This model of experimentally induced uterine infection successfully resulted in endometritis and a reduction in the proportion of oocytes that developed to morulae following in vitro fertilization. In conclusion, endometritis reduced the capacity of oocytes to develop to morulae.

Uterine contamination with bacteria is ubiquitous in the postpartum dairy cow. Nearly one-half of all postpartum dairy cows develop clinical disease resulting in metritis and endometritis, which cause depressed milk production and infertility. The causative links between uterine infection and infertility include a hostile uterine environment, disrupted endocrine signaling, and perturbations in ovarian function and oocyte development. In this review we consider the various mechanisms linking uterine infection with infertility in the dairy cow, specifically 1) innate immune signaling in the endometrium, 2) alteration in endocrine signaling in response to infectious agents, and 3) impacts of infection on ovarian function, oocyte development, and follicular development. Normal ovarian follicular and oocyte development requires a series of temporally and spatially orchestrated events; however, several of the cellular pathways required for ovarian function are also used during the innate immune response to bacterial pathogens. We propose that activation of cellular pathways during this immune response has a negative impact on ovarian physiology, which is manifest as infertility detected after the clearance of the bacteria. This review highlights how new insights into infection and immunity in cattle are linked to infertility.

No reports exist on consequences of in vitro production (IVP) of embryos for the postnatal development of the calf or on postparturient function of the dam of the calf. Three hypotheses were evaluated: calves born as a result of transfer of an IVP embryo have reduced neonatal survival and altered postnatal growth, fertility, and milk yield compared with artificial insemination (AI) calves; cows giving birth to IVP calves have lower milk yield and fertility and higher incidence of postparturient disease than cows giving birth to AI calves; and the medium used for IVP affects the incidence of developmental abnormalities. In the first experiment, calves were produced by AI using conventional semen or by embryo transfer (ET) using a fresh or vitrified embryo produced in vitro with X-sorted semen. Gestation length was longer for cows receiving a vitrified embryo than for cows receiving a fresh embryo or AI. The percentage of dams experiencing calving difficulty was higher for ET than AI. We observed a tendency for incidence of retained placenta to be higher for ET than AI but found no significant effect of treatment on incidence of prolapse or metritis, pregnancy rate at first service, services per conception, or any measured characteristic of milk production in the subsequent lactation. Among Holstein heifers produced by AI or ET, treatment had no effect on birth weight but the variance tended to be greater in the ET groups. More Holstein heifer calves tended to be born dead, died, or were euthanized within the first 20d of life for the ET groups than for AI. Similarly, the proportion of Holstein heifer calves that either died or were culled for poor health after 20d of age was greater for the ET groups than for AI. We observed no effect of ET compared with AI on age at first service or on the percentage of heifers pregnant at first service, calf growth, or milk yield or composition in the first 120d in milk of the first lactation. In a second experiment, embryos were produced using 1 of 2 culture media: synthetic oviductal fluid-bovine embryo 1 (SOF-BE1) or Block-Bonilla-Hansen 7 (BBH7). We detected no difference between cows receiving an SOF-BE1 or BBH7 embryo in gestation length, the percentage of cows in which parturition was induced, or the percentage of cows that experienced calving difficulty, retained placenta, prolapse, or metritis. Among Holstein heifers, birth weight was higher for BBH7 calves than for SOF-BE1 calves. Treatment had no significant effect on calf death. Results indicate that calves born as a result of IVP-ET are more likely to experience alterations in birth weight and increased death in early life but that there were few consequences to the dam of carrying a fetus derived by IVP-ET.

1578 Background: Barriers to population screening for BRCA mutations include access, availability of counseling, and readiness of care providers to participate in this process. The BRCA Founder OutReach (BFOR) study evaluates a digital approach to genetic testing of a defined population using a medical model and risk-adapted follow-up. Methods: The BFOR study (Bforstudy.com) includes web-based enrollment open to individuals in four US cities who are age 25 or older and have at least one grandparent of Ashkenazi Jewish (AJ) ancestry. Participants receive web-based education, provide consent, complete questionnaires, and note their preference for receiving results either from their primary care provider (PCP) or BFOR staff. BRCA AJ founder mutation results are disclosed by (e)mail or phone, depending on need for additional counseling/genetic testing. Participants will be surveyed by email for up to 5 years; a subset of PCPs is also being surveyed. Results: From March 2018 to January 2019, 2562 participants enrolled: 78% female; < 30 years old, 8%; 30-50 years, 39%; > 50 years, 53%. At enrollment, 33% requested disclosure of results by PCP. Among 847 PCPs invited to disclose results, 45% accepted, 50% declined and 5% have yet to respond. 69 (3.2%) participants tested positive for a BRCA founder mutation, of whom 8 (12%) had no significant family history. 2087 participants tested negative, of whom 6% reported a known family mutation, 38% reported a family history of breast/ovarian cancer, and 56% no such history. The most common reason for study participation was referral by a friend. One individual with a distant history of breast cancer tested positive for a BRCA2 mutation and underwent risk reducing surgery that identified an early stage fallopian tube carcinoma. Her daughter then tested positive and underwent prophylactic surgeries. Conclusions: Population screening of individuals at higher risk for cancer-predisposing mutations is feasible and identifies individuals who would not have been tested using clinical criteria. Preliminary findings reveal challenges for engaging PCPs and at-risk individuals, particularly men. Ongoing follow-up and a second phase of the study will address these barriers to testing. Clinical trial information: NCT03351803.

For the 10th experiment on Critical Assessment of the techniques of protein Structure Prediction (CASP), the prediction target proteins were broken into independent evaluation units (EUs), which were then classified into template-based modeling (TBM) or free modeling (FM) categories. We describe here how the EUs were defined and classified, what issues arose in the process, and how we resolved them. EUs are frequently not the whole target proteins but the constituting structural domains. However, the assessors from CASP7 on combined more than one domain into 1 EU for some targets, which implied that the assessment also included evaluation of the prediction of the relative position and orientation of these domains. In CASP10, we followed and expanded this notion by defining multidomain EUs for a number of targets. These included 3 EUs, each made of two domains of familiar fold but arranged in a novel manner and for which the focus of evaluation was the interdomain arrangement. An EU was classified to the TBM category if a template could be found by sequence similarity searches and to FM if a structural template could not be found by structural similarity searches. The EUs that did not fall cleanly in either of these cases were classified case-by-case, often including consideration of the overall quality and characteristics of the predictions. Proteins 2014; 82(Suppl 2):14–25. © 2013 Wiley Periodicals, Inc.

The main objective was to determine the efficacy of using X-sorted sperm to produce embryos in vitro for transfer into lactating dairy cows. Cows were bred by timed artificial insemination (TAI) using nonsorted semen or X-sorted sperm, or they received a fresh embryo produced in vitro by fertilization with X-sorted or nonsorted sperm using timed embryo transfer (TET). Pregnancy rates at approximately Day 32 averaged over all dairies were 39.3 ± 3.2% (least-squares mean ± SEM) for TAI nonsorted, 27.3 ± 3.4% for TET nonsorted fresh embryos, and 30.2 ± 3.3% for TET X-sorted fresh embryos (TAI vs. both TET groups, P < 0.05; 206 to 233 cows per group). Pregnancy losses between approximately Day 32 and term ranged from 16% to 37%, the latter from TET with X-sorted sperm. Pregnancy losses to term were higher for cows receiving embryos produced in vitro than for cows bred by TAI. Calves produced via TET were not substantively different from AI controls in physical measurements or standard blood chemistry profiles.

The objective of this study was to implement an in vitro-produced embryo transfer (IVP-ET) system in an existing stochastic dynamic dairy simulation model with multitrait genetics to evaluate the genetic, technical, and financial performance of a dairy herd implementing an exclusive IVP-ET or artificial insemination (AI) system. In the AI system, sexed semen was used on the genetically best heifers only. In the IVP-ET system, all of the animals in the herd were impregnated with female sexed embryos created through in vitro fertilization of oocytes collected from animals of superior genetics for different traits of interest. Each donor was assumed to yield on average 4.25 transferable embryos per collection. The remaining animals in the herd were used as recipients and received either a fresh embryo or a frozen embryo when fresh embryos were not available. Selection of donors was random or based on the greatest estimated breeding value (EBV) of lifetime net merit (NM$), milk yield, or daughter pregnancy rate. For both the IVP-ET and AI systems, culling of surplus heifer calves not needed to replace culled cows was based on the lowest EBV for the same traits. A herd of 1,000 milking cows was simulated 15 yr over time after the start of the IVP-ET system. The default cost to produce and transfer 1 embryo was set at $165. Prices of fresh embryos at which an exclusive IVP-ET system financially breaks even with the comparable AI system in yr 15 and for an investment period of 15 yr were also estimated. More surplus heifer calves were sold from the IVP-ET systems than from the comparable AI systems. The surplus calves from the IVP-ET systems were also genetically superior to the surplus calves from the comparable AI systems, which might be reflected in their market value as a premium price. The most profitable scenario among the 4 IVP-ET scenarios in yr 15 was the one in which NM$ was maximized in the herd. This scenario had an additional profit of $8/cow compared with a similar AI scenario that maximized NM$, provided that surplus heifer calves could be sold at a premium price based on the superiority of the EBV of NM$. For the IVP-ET system to be at least as profitable as the comparable AI system during a 15-yr investment period, the surplus calves from the IVP-ET system needed to be sold at the premium prices. The break-even price of fresh embryos was estimated to be $84 for the exclusive IVP-ET system. This resulted in the same profit as the AI system, which maximized NM$ for a 15-yr investment period and in which heifer calves were sold at a premium price.

Inositol phosphates (IPs) regulate vital processes in eukaryotes, and their production downstream of phospholipase C activation is controlled through a network of evolutionarily conserved kinases and phosphatases. Inositol phosphate multikinase (IPMK, also called Ipk2 and Arg82) accounts for phosphorylation of IP(3) to IP(5), as well as production of several other IP molecules. Here, we report the structure of Arabidopsis thaliana IPMKα at 2.9 Å and find it is similar to the yeast homolog Ipk2, despite 17% sequence identity, as well as the active site architecture of human IP(3) 3-kinase. Structural comparison and substrate modeling were used to identify a putative basis for IPMK selectivity. To test this model, we re-engineered binding site residues predicted to have restricted substrate specificity. Using steady-state kinetics and in vivo metabolic labeling studies in modified yeast strains, we observed that K117W and K117W:K121W mutants exhibited nearly normal 6-kinase function but harbored significantly reduced 3-kinase activity. These mutants complemented conditional nutritional growth defects observed in ipmk null yeast and, remarkably, suppressed lethality observed in ipmk null flies. Our data are consistent with the hypothesis that IPMK 6-kinase activity and production of Ins(1,4,5,6)P(4) are critical for cellular signaling. Overall, our studies provide new insights into the structure and function of IPMK and utilize a synthetic biological approach to redesign inositol phosphate signaling pathways.

Technologies for in vitro embryo production have the potential to enhance the efficiency of cattle production systems. However, utilization of in vitro-produced embryos for transfer remains limited throughout much of the world. Despite improvements over the past two decades, problems associated with the production of bovine embryos in vitro still exist which limit the widespread commercial application of this technology. In particular, bovine embryos produced in vitro have a reduced capacity to establish and maintain pregnancy as compared with their in vivo-derived counterparts. Embryo competence for survival following transfer is improved by in vivo culture in the sheep oviduct, thus indicating that standard embryo culture conditions are sub-optimal. Therefore, one strategy to improve post-transfer survival is to modify embryo culture media to more closely mimic the in vivo microenvironment. The maternal environment in which the bovine embryo develops in vivo contains various growth factors, cytokines, hormones, and other regulatory molecules. In addition to affecting bovine embryo development in vitro, recent research indicates that embryo competence for survival following transfer can also be improved when such molecules are added to embryo culture medium. Among the specific molecules that can increase post-transfer embryo survival are insulin-like growth factor-1 (IGF-1), colony stimulating factor-2 (CSF-2) and hyaluronan. This paper will review the effects IGF-1, CSF-2 and hyaluronan on post-culture embryo viability and discuss the potential mechanisms through which each of these molecules improves post-transfer survival.

The objective was to determine whether transfer of fresh or vitrified embryos produced in vitro with sexsorted semen improves pregnancy and calving rates during summer in lactating dairy cows compared with artificial insemination (AI). Lactating dairy cows (n = 722) were enrolled during summer months at 2 commercial dairies in Central Texas and randomly assigned to 1 of 3 treatments: AI with conventional semen (n = 227), embryo transfer-vitrified (ET-V; n = 279) or embryo transfer-fresh (ET-F; n = 216). Embryos were produced in vitro using sex-sorted semen and with Block-Bonilla-Hansen-7 culture medium. For vitrification, grade 1 expanded blastocysts were harvested on d 7 after fertilization and vitrified using the open-pulled straw method. Fresh embryos were grade 1 blastocysts and expanded blastocysts harvested on d 7 after fertilization. Cows were submitted to the Ovsynch56 protocol: d −10 GnRH, d −3 PGF2α, d −1 GnRH and d 0 timed AI; or Select Synch protocol: d −9 GnRH, d −2 PGF2α, and AI following detected estrus (day of AI = d 0). On d 7, all cows were examined for presence of a corpus luteum (CL). A vitrified or fresh embryo was transferred to cows with CL in ET-V and ET-F groups. Cows were considered synchronized if progesterone was

Addition of insulin-like growth factor-1 (IGF-1) to culture medium increases the proportion of bovine embryos that develop to the blastocyst stage and increases embryo survival follow- ing transfer to heat-stressed, lactating dairy cows. The objective of the present study was to determine molecular and cellular correlates of these actions of IGF-1. Embryos were produced in vitro and cultured for 7 days with or without 100 ng/ml IGF-1. On d 7 after insemination, grade 1 expanded blastocysts were harvested and used to determine total cell number, percent apoptosis, cell allocation to the inner cell mass and trophectoderm, and the relative abundance of several developmentally important gene transcripts. There was no significant effect of IGF-1 treatment on blastocyst cell number, the proportion of blastomeres that were apoptotic, or the number of cells in the inner cell mass and trophectoderm. However, differences in the relative abundance of several mRNA transcripts were observed between control and IGF-1 treated embryos. Addition of IGF-1 increased (P < 0.02) amounts of mRNA for IGF binding protein-3 and desmocollin II and tended (P < 0.08) to increase amounts of mRNA for Na/K ATPase and Bax. More- over, IGF-1 treatment decreased (P < 0.05) steady- state amounts of transcripts for heat shock protein 70 and tended (P < 0.08) to reduce amounts of IGF-1 receptor mRNA. In conclusion, increased survival of embryos treated with IGF-1 does not appear due to effects on cell number, percent apoptosis, or cell allocation. Addition of IGF-1 to culture can, however, alter expression of several transcripts which may be important for embryo development and survival following transfer. Mol. Reprod. Dev. 75: 895- 903, 2008. 2007 Wiley-Liss, Inc.

Use of in vitro produced embryos in commercial settings is limited by alterations in embryo function that result in reduced embryo and fetal survival and increased fetal, placental and neonatal abnormalities. One potential strategy to improve the efficiency of in vitro embryo production systems is to modify embryo culture conditions to more closely mimic embryo physiology in vivo. The milieu of the reproductive tract contains various growth factors and regulatory molecules which can regulate embryonic development. One of these molecules, insulin-like growth factor-1 (IGF-1), has been reported to have beneficial effects on bovine embryo development in vitro. Addition of IGF-1 to embryo culture can increase pregnancy and calving rates in lactating dairy cows. However, recent research indicates that such an effect is only observed when recipients are heat-stressed. In non-heat stressed animals, IGF-1 treatment does not affect conceptus length or interferon-tau secretion; thus, it is not likely that IGF-1 promotes embryo survival by blocking the luteolytic cascade. On the other hand, IGF-1 treatment can alter the relative abundance of certain developmentally important transcripts. Such differences may be important to improve embryo survival during heat stress. Further research into the effect of IGF-1 on gene expression and DNA methylation are warranted. Results indicate that modification of embryo culture with a growth factor can affect embryo survival after transfer. It is important that future research evaluating modifications to embryo culture also includes experiments involving transfer of embryos to recipients. Such experiments are important to demonstrate that proposed modifications actually improve post-transfer embryonic survival.

Administration of recombinant bovine somatotropin (bST) to lactating dairy cows during heat stress increases milk yield, but it also can increase body temperature and may therefore compromise fertility. However, it is possible that bST treatment could increase fertility during heat stress because it has been reported to increase fertility in lactating cows. In addition, bST increases secretion of insulin-like growth factor-I (IGF-I) that promotes embryo survival. The purpose of this study was to determine effects of bST on reproductive function in lactating dairy cows during heat stress. The experiment was conducted in southern Georgia from July to November 2005 using lactating Holstein cows (n = 276 for reproductive traits). For first service timed artificial insemination (TAI), cows were presynchronized with 2 injections of PGF(2alpha) given 14 d apart followed by a modified Ovsynch protocol (GnRH and insemination at 72 h following PGF(2alpha) ). Pregnancy was diagnosed by using ultrasonography on d 29 and reconfirmed by palpation between d 45 and 80 post-TAI. Nonpregnant cows were resynchronized with the modified Ovsynch protocol and received a second TAI. Treatment with bST started 1 wk before the start of Ovsynch and continued at 2-wk intervals. Blood samples were collected from a subset of cows to determine IGF-I profiles immediately before the first bST injection, 1 wk later, and at d 35 of bST treatment. Rectal temperatures were assessed on d 29 of bST treatment. Pregnancy rates (d 45 to 80 post-TAI) did not differ between bST and control cows for first- (16.7 vs. 15.2%) or second-service TAI (14.8 vs. 17.2%). Plasma concentrations of IGF-I and milk yield were greater for bST-treated cows following the initiation of bST treatment and bST increased rectal and vaginal temperatures. Body condition score was less for bST-treated cows. In conclusion, treatment with bST during heat stress increased IGF-I concentrations, milk yield over time, and rectal and vaginal temperatures without affecting first- or second-service pregnancy rates. Thus, at least under certain housing conditions, bST can be used to improve milk yield during heat stress without compromising fertility.

Timed embryo transfer (TET) using in vitro produced (IVP) embryos without estrus detection can be used to reduce adverse effects of heat stress on fertility. One limitation is the poor survival of IVP embryos after cryopreservation. Objectives of this study were to confirm beneficial effects of TET on pregnancy rate during heat stress as compared to timed artificial insemination (TAI), and to determine if cryopreservation by vitrification could improve survival of IVP embryos transferred to dairy cattle under heat stress conditions. For vitrified embryos (TET-V), a three-step pre-equilibration procedure was used to vitrify excellent and good quality Day 7 IVP Holstein blastocysts. For fresh IVP embryos (TET-F), Holstein oocytes were matured and fertilized; resultant embryos were cultured in modified KSOM for 7 days using the same method as for production of vitrified embryos. Excellent and good quality blastocysts on Day 7 were transported to the cooperating dairy in a portable incubator. Nonpregnant, lactating Holsteins (n = 155) were treated with GnRH (100 microg, i.m., Day 0), followed 7 days later by prostaglandin F2alpha (PGF2alpha, 25 mg, i.m.) and GnRH (100 microg) on Day 9. Cows in the TAI treatment (n = 68) were inseminated the next day (Day 10) with semen from a single bull that also was used to produce embryos. Cows in the other treatments (n = 33 for TET-F; n = 54 for TET-V) received an embryo on Day 17 (i.e. Day 7 after anticipated ovulation and Day 8 after second GnRH treatment). The proportion of cows that responded to synchronization based on plasma progesterone concentrations on Day 10 and Day 17 was 67.7%. Pregnancy rate for all cows on Day 45 was higher (P0.05) in the TET-F treatment than for the TAI and TET-V treatments (19.0 +/- 5.0,6.2 +/- 3.6, and 6.5 +/- 4.1%). For cows responding to synchronization, pregnancy rate was also higher (P0.05) for TET-F than for other treatments (26.7 +/- 6.4, 5.0 +/- 4.3, and 7.4 +/- 4.7%). In the TET-F treatment group, cows producing more milk had lower (P0.05) pregnancy rates than cows producing less milk. In conclusion, ET of fresh IVP embryos can improve pregnancy rate under heat stress conditions, but pregnancy rate following transfer of vitrified embryos was no better than that following TAI.

Successful embryonic development is dependent on factors secreted by the reproductive tract. Dickkopf-1 (DKK1), an antagonist of the wingless-related mouse mammary tumor virus (WNT) signaling pathway, is one endometrial secretory protein potentially involved in maternal-embryo communication. The purpose of this study was to investigate the roles of DKK1 in embryo cell fate decisions and competence to establish pregnancy. Using in vitro-produced bovine embryos, we demonstrate that exposure of embryos to DKK1 during the period of morula to blastocyst transition (between d 5 and 8 of development) promotes the first 2 cell fate decisions leading to increased differentiation of cells toward the trophectoderm and hypoblast lineages compared with that for control embryos treated with vehicle. Moreover, treatment of embryos with DKK1 or colony-stimulating factor 2 (CSF2; an endometrial cytokine known to improve embryo development and pregnancy establishment) between d 5 and 7 of development improves embryo survival after transfer to recipients. Pregnancy success at d 32 of gestation was 27% for cows receiving control embryos treated with vehicle, 41% for cows receiving embryos treated with DKK1, and 39% for cows receiving embryos treated with CSF2. These novel findings represent the first evidence of a role for maternally derived WNT regulators during this period and could lead to improvements in assisted reproductive technologies.—Denicol, A. C., Block, J., Kelley, D. E., Pohler, K. G., Dobbs, K. B., Mortensen, C. J., Ortega, M. S., Hansen, P. J. The WNT signaling antagonist Dickkopf-1 directs lineage commitment and promotes survival of the preimplantation embryo.

Physiology of the adult can be modified by alterations in prenatal development driven by the maternal environment. Developmental programming, which can be established before the embryo implants in the uterus, can affect females differently than males. The mechanism by which sex-specific developmental programming is established is not known. Here we present evidence that maternal regulatory signals change female embryos differently than male embryos. In particular, actions of the maternally derived cytokine CSF2 from Day 5 to Day 7 of development affected characteristics of the embryo at Day 15 differently for females than males. CSF2 decreased length and IFNT secretion of female embryos but increased length and IFNT secretion of male embryos. Analysis of a limited number of samples indicated that changes in the transcriptome and methylome caused by CSF2 also differed between female and males. Thus, sex-specific programming by the maternal environment could occur when changes in secretion of maternally derived regulatory molecules alter development of female embryos differently than male embryos.

Physiology of the adult can be modified by alterations in prenatal development driven by the maternal environment. Developmental programming, which can be established before the embryo implants in the uterus, can affect females differently than males. The mechanism by which sex-specific developmental programming is established is not known. Here we present evidence that maternal regulatory signals change female embryos differently than male embryos. In particular, actions of the maternally derived cytokine CSF2 from Day 5 to Day 7 of development affected characteristics of the embryo at Day 15 differently for females than males. CSF2 decreased length and IFNT secretion of female embryos but increased length and IFNT secretion of male embryos. Analysis of a limited number of samples indicated that changes in the transcriptome and methylome caused by CSF2 also differed between female and males. Thus, sex-specific programming by the maternal environment could occur when changes in secretion of maternally derived regulatory molecules alter development of female embryos differently than male embryos.

Cryotolerance of bovine embryos produced in vitro (PIV) can be improved by l-carnitine. The objective of the present study was to determine whether the optimal concentration of l-carnitine is dependent on serum. Bovine embryos were produced in vitro with abattoir-derived oocytes. After fertilization (Day 0), oocytes (n = 2768) were randomly assigned in a 2 × 4 factorial design to culture in SOF-BE1 medium supplemented with or without 5% fetal bovine serum and l-carnitine at concentrations of 0.0, 0.75, 1.5, and 3.03 mM at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. The proportion of oocytes that cleaved was assessed on Day 3, and the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, and hatched) stages was determined on Day 7. Blastocysts and expanded blastocysts (n = 466) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. After thaw, embryos were cultured for 72 h in SOF-BE1 supplemented with 10% (v/v) fetal bovine serum and 50 mM dithiothreitol at 38.5°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 9 times, and data were analysed by logistic regression. There was no interaction between serum and l-carnitine, at any of the concentrations tested, on embryo development or cryotolerance. Cleavage rates were not affected by serum or l-carnitine. Addition of serum during culture increased (P

The aim of the present experiments was to determine whether treatment with the antioxidants ascorbic acid and dithiothreitol, or an inhibitor of caspase-3, Z-DEVD-FMK, during cryopreservation could improve the cryotolerance of bovine embryos produced in vitro. For all experiments, bovine embryos were produced in vitro using abattoir-derived ovaries. At Day 7 after insemination, blastocyst and expanded blastocyst stage embryos were harvested and subjected to controlled-rate freezing following equilibration for 8 to 10 min in freezing medium [Hepes-TALP (Parrish et al. 1986) plus 1.5 M ethylene glycol and 0.5 M sucrose] with treatments as described below. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. For Experiment 1, embryos (n = 578) were equilibrated in freezing medium containing 0, 0.1, 0.3, or 0.5 mM ascorbic acid. For Experiment 2, embryos (n = 243) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM dithiothreitol. For Experiment 3, embryos (n = 227) were equilibrated in freezing medium containing 0, 50, 100, or 200 μM Z-DEVD-FMK. Embryos frozen in freezing medium containing ascorbic acid had increased (P

The objective of the present study was to determine the effect of supplementation of maturation media with L-carnitine and trans-10,cis-12 conjugated linoleic acid (CLA) on embryo development and survival following cryopreservation. Immature bovine cumulus-oocyte complexes (n = 1796) were harvested from abattoir-derived ovaries and randomly assigned in a 2 × 2 factorial design to be matured in maturation medium [TCM-199 with Earle salts supplemented with 10% (vol/vol) bovine steer serum, 2 μg mL–1 oestradiol 17-β, 20 μg mL–1 bovine FSH, 22 μg mL–1 sodium pyruvate, 50 μg mL–1 gentamicin sulfate, and 1 mM glutamax®] supplemented with or without 100 mM CLA and with or without 3.03 mM L-carnitine for 22 to 24 h at 38.5°C in a humidified atmosphere of 5% CO2. The proportion of oocytes that cleaved was determined on Day 3 after insemination, and the proportion of oocytes developing to the blastocyst and advanced blastocysts stages (expanded, hatching, and hatched) was assessed on Day 7. Blastocyst and expanded blastocyst stage embryos (n = 270) were harvested on Day 7 and subjected to controlled-rate freezing following equilibration in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 (Fields et al. 2011) supplemented with 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Post-thaw re-expansion and hatching rates were determined at 24, 48, and 72 h. The experiment was replicated 5 times. There was no effect of supplementation of maturation medium with either CLA or L-carnitine on the proportion of oocytes that cleaved at Day 3 or that developed to the blastocyst and advanced blastocyst stages at Day 7 after insemination. There was no interaction between CLA and L-carnitine affecting cleavage rate or embryo development. Supplementation of maturation medium with L-carnitine did not affect post-thaw re-expansion or hatching rates. In contrast, treatment with CLA during maturation reduced (P

Maturation of the oocyte involves nuclear and cytoplasmic changes that include post-translational processing of proteins. The objective was to investigate whether inhibition of proteasomes during maturation would alter competence of the bovine oocyte for fertilization and subsequent development. Cumulus-oocyte complexes were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0–6 h or 16–22 h after initiation of maturation. Treatment with MG132 early in maturation prevented progression to meiosis II and reduced fertilization rate and the proportion of oocytes and cleaved embryos that became blastocysts. Conversely, treatment with MG132 late in maturation improved the percentage of oocytes and cleaved embryos that became blastocysts without affecting nuclear maturation or fertilization rate. Optimal results with MG132 were achieved at a concentration of 10 µM – effects were generally not observed at lower or higher concentrations. Using proteomic analysis, it was found that MG132 at the end of maturation increased relative expression of 6 proteins and decreased relative expression of 23. Among those increased by MG132 that are potentially important for oocyte competence are GAPDH, involved in glycolysis, TUBA1C, needed for organellar movement, and two proteins involved in protein folding (P4HB and HYOU1). MG132 decreased amounts of several proteins that exert anti-apoptotic actions including ASNS, HSP90B1, PDIA3 and VCP. Another protein decreased by MG132, CDK5, can lead to apoptosis if aberrantly activated and one protein increased by MG132, P4HB, is anti-apoptotic. Finally, the pregnancy rate of cows receiving embryos produced from oocytes treated with MG132 from 16–22 h of maturation was similar to that for control embryos, suggesting that use of MG132 for production of embryos in vitro does not cause a substantial decrease in embryo quality.

In this study, we tested the role of colony-stimulating factor 2 (CSF2) as one of the regulatory molecules that mediate maternal effects on embryonic development during the preimplantation period. Our objective was to verify effects of CSF2 on blastocyst yield, determine posttransfer survival, and evaluate properties of the blastocyst formed after CSF2 treatment. In vitro, CSF2 increased the percentage of oocytes that became morulae and blastocysts. Blastocysts that were treated with CSF2 tended to have a greater number of inner cell mass cells and had a higher ratio of inner cell mass to trophectoderm cells. There was no effect of CSF2 on the incidence of apoptosis. Treatment with CSF2 from d 5 to 7 after insemination increased embryonic survival as indicated by improved pregnancy rate at d 30–35 of gestation. Moreover, treatment with CSF2 from either d 1–7 or 5–7 after insemination reduced pregnancy loss after d 30–35. Results indicate that treatment with CSF2 can affect embryonic development and enhance embryo competence for posttransfer survival. The fact that treatment with CSF2 during such a narrow window of development altered embryonic function much later in pregnancy suggests that CSF2 may exert epigenetic effects on the developing embryo that result in persistent changes in function during the embryonic and fetal periods of development.

Two experiments were conducted to determine whether addition of hyaluronan to culture medium could improve survival of bovine embryos after vitrification or following embryo transfer. In Experiment 1, embryos were produced in vitro and cultured for 7 days in modified synthetic oviductal fluid (SOF) containing one of four concentrations of hyaluronan (0, 0.1, 0.5, or 1mg/mL), with or without 4 mg/mL of bovine serum albumin (BSA). On Day 7 after insemination, blastocysts and expanded blastocysts were vitrified using open-pulled straws. At a concentration of 1mg/mL, hyaluronan increased (P

Culture of bovine embryos with insulin-like growth factor-1 (IGF-1) can improve development to the blastocyst stage and embryo survival following transfer to heat-stressed, lactating dairy cows. Two experiments were conducted to determine whether IGF-1 could improve embryo survival and development at Day 14 after ovulation. In Experiment 1, non-lactating Holstein cows (n = 58) were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced in vitro and cultured with or without 100 ng/mL IGF-1. At Day 7 after expected ovulation (Day 0), groups of 7–12 embryos were randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and the presence or absence of an embryonic disc was recorded. Recovered embryos were cultured individually for 24 h to determine interferon-t (IFN-t) secretion. There was no effect of IGF-1 on embryo recovery rate, embryo length or IFN-t secretion. In Experiment 2, non-lactating (n = 56) and lactating (n = 35) Holstein cows were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced as described in Experiment 1. At Day 7 after expected ovulation (Day 0), a single embryo was randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and IFN-t secretion were determined as in Experiment 1. Recovery rate at Day 14 tended (P = 0.1) to be higher for recipients that received IGF-1 treated embryos compared to control embryos (43.2% versus 26.1%, respectively). There was no effect of IGF-1 on embryo length or IFN-t secretion. In conclusion, results suggest that exposure to IGF-1 through Days 7–8 of development does not enhance capacity of embryos to prevent luteolysis. Results of the single embryo-transfer experiment suggested that IGF-1 treatment might affect embryo survival post-transfer as early as Day 14 after ovulation. Further experimentation is warranted to verify this finding.

The objective was to determine the effects of addition of l-carnitine (LC) and trans-10, cis-12 conjugated linoleic acid (CLA) during bovine embryo culture on cryosurvival, lipid content, and gene expression. For all experiments, embryos were produced in vitro using abattoir-derived oocytes. Following insemination, presumptive zygotes were randomly assigned in a 2 × 2 factorial to be cultured in SOF-BE1 supplemented with or without 3.03 mM LC and 100 μM CLA until Day 7. For Exp. 1, blastocyst- and expanded-blastocyst-stage embryos (n = 777) were slow-frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. There was no effect of LC on post-thaw re-expansion rates, but CLA reduced (P

Culture of bovine embryos in the presence of insulin-like growth factor-1 (IGF-1) can increase pregnancy rates following transfer to heat-stressed, lactating dairy cows. The objective of the present experiment was to determine whether the effect of IGF-1 on post-transfer embryo survival was a general effect or one specific to heat stress. Lactating recipients (n=311) were synchronized for timed-embryo transfer at four locations. Embryos were produced in vitro and cultured with or without 100 ng/mL IGF-1. At Day 7 after anticipated ovulation (Day 0), a single embryo was randomly transferred to each recipient. Pregnancy was diagnosed at Day 21 by elevated plasma progesterone concentrations, at Days 27-32 by ultrasonography, and at Days 41-49 by transrectal palpation. Transfers were categorized into two seasons, hot or cool (based on the month of transfer). There was a tendency (P