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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

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.

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.

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.

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.

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.

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.

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.

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.

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

J. Block, MS, R. M. Rivera, PhD, F. D. Jousan, MS, F. T. Silvestre, BS, F. F. Paula-Lopes, PhD, O. M. Ocon, BS, H. Rosson, MAg, T. R. Bilby, MS, P. J. Hansen, PhD, Department of Animal Sciences, M. Drost, DVM, Department of Large Animal Clinical Sciences, University of Florida, Gainesville, FL 32611, USA R. L. Monson, MS, J. J. Rutledge, PhD, Department of Animal Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA C. R. Looney, PhD, Ovagenix, Bryan, TX 77868, USA

Structural features of the dairy industry make it well situated to use embryo technologies as tools for enhancing the genetic merit of dairy cattle and improving fertility. Technologies dependent upon embryo transfer have the potential to increase the efficiency of quantitative genetic selection as well as marker-assisted selection, simplify cross-breeding and germplasm conservation procedures and allow incorporation of transgenes into dairy cattle. In addition, embryo technologies may prove useful in improving fertility in infertile populations of lactating cows. The realisation of the promise of embryo technologies has been constrained by suboptimal efficiency in the production of embryos, alterations in embryonic and fetal survival and development associated with in vitro embryo production and cloning, as well as other technical and societal concerns. Solutions to many of these constraints are possible and the use of embryo technologies in both nucleus and commercial herds is likely to increase. Eventually, embryo transfer may compete with artificial insemination as a dominant method for establishing pregnancies in dairy cattle.

Exposure of bovine conceptuses to colony-stimulating factor 2 (CSF2) from days 5 to 7 of development can increase the percentage of transferred conceptuses that develop to term. The purpose of this experiment was to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10 ng/ml CSF2 from days 5 to 7 after insemination, transferred into cows, and flushed from the uterus at day 15 of pregnancy. There was a tendency (P=0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2-treated conceptus (35% for control versus 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-τ (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.08) and keratin 18 (P

This study was designed to evaluate possible mechanisms by which colony-stimulating factor 2 (CSF2) acts during Day 5 to 7 of development to improve embryonic and fetal survival and decrease fetal losses. One hypothesis was that CSF2 causes increased secretion of interferon-τ (IFNT) by the trophoblast of elongated conceptuses. Colony-stimulating factor 2 might also affect elongation of the trophoblast and improve survival of the embryonic disc (ED) because of evidence that treatment of embryo with CSF2 caused differential regulation of a large number of genes involved in developmental processes and a decrease in apoptosis. Bovine embryos were produced in vitro in the presence or absence of 10 ng mL–1 of CSF2 beginning at Day 5 after fertilization. Embryos were transferred at Day 7 to lactating dairy cows using a timed embryo transfer protocol (n = 20 and 15 for control and CSF2, respectively). At Day 15, embryos were recovered and assessed for length, stage, and presence of an ED. The flushings recovered from the first 60 mL were stored for antiviral activity analysis. A total of 4 filamentous conceptuses were divided into 1) the ED and a small amount of adjacent trophoblast and 2) tissue containing trophoblast only. These tissues were analysed for gene expression using the Bos taurus Two Colour Microarray Chip from Agilent (Palo Alto, CA). Quantitative real time PCR analysis of 11 differentially expressed genes and 1 housekeeping gene (GAPDH) was performed to confirm the microarray results. In addition, quantitative real-time PCR of IFNT was performed in all recovered embryos (7 and 10 for control and CSF2, respectively). Results suggest that higher pregnancy rates at Day 30 represents increased embryonic survival at Day 15 (35% for controls v. 66% for CSF2; P

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

One limitation to the use of in vitro-produced embryos in cattle production systems is the fact that pregnancy rates after transfer to recipients are typically lower than when embryos produced in vivo are transferred. Conceptually, the oocyte and spermatozoon from which the embryo is derived could affect competence for post-transfer survival. There are sire differences in embryonic survival after transfer, but there is little evidence that an embryo’s ability to establish pregnancy is determined by sex sorting of spermatozoa by flow cytometry. The role of the source of the oocyte as a determinant of embryonic survival after transfer has not been examined carefully. Conditions for embryo culture after fertilisation can have an impact on the ability of the embryo to establish pregnancy following transfer. Among the specific molecules produced in the reproductive tract of the cow that have been shown to improve competence of in vitro-produced embryos for post-transfer survival are colony-stimulating factor 2, insulin-like growth factor-1 (for recipients exposed to heat stress) and hyaluronan (for less-advanced embryos). There is also a report that embryo competence for post-transfer survival can be improved by inclusion of a carbon-activated air filtration system in the incubator used to culture embryos. Progress in developing culture systems to improve embryonic competence for survival after transfer would be hastened by the development of in vitro assays that accurately predict the potential of an embryo to establish pregnancy after transfer. A group of 52 genes has been identified that are differentially expressed in embryos that developed to term v. embryos that did not establish pregnancy. Perhaps a gene microarray consisting of these genes, alone or in combination with other genes, could be used to screen embryos for competence to establish pregnancy.

The objective was to determine whether transfer of two embryos would increase pregnancy rates in heat-stressed dairy recipients receiving an in vitro-produced embryo transferred into the uterine horn ipsilateral to the corpus luteum (CL). Such a treatment would increase the likelihood that the cow receives at least one embryo competent for sustained development. In addition, transfer of two embryos into the ipsilateral uterine horn is likely to increase the amounts of interferon-tau and other embryonic-signaling molecules in the uterus. A total of 32 virgin crossbreed heifers and 26 lactating crossbreed cows were used as timed embryo transfer recipients after being subjected to an ovulation synchronization protocol as follows: GnRH (100 �g) and insertion of previously used progesterone-containing CIDR on Day -10, prostaglandin F2� and CIDR removal on Day -3, and GnRH (100 �g) on Day 0 (day of anticipated ovulation). All recipients had a palpable CL on Day 6 and were randomly selected to receive one (n = 31 recipients) or two (n = 27) embryos on Day 7. At Day 64, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. While not significant, heifers that received two embryos tended to have lower pregnancy rates than those that received a single embryo (20% vs. 41%); there was no difference in pregnancy rate in cows (50% for two embryos vs. 57% for one embryo). Pregnancy losses between Day 64 and Day 127 occurred in one group only cows receiving two embryos. In that group, pregnancy rate was 50% at Day 64 but 17% at Day 127. Overall, there was no difference in pregnancy rates at day 127 between cows and heifers, but recipients that received two embryos (17% for cows and 20% for heifers) had lower pregnancy rates (P < 0.03) than recipients that received one embryo (57% for cows and 41% for heifers). Only one animal, a cow, had twin fetuses at day 127. In conclusion, unilateral transfer of two embryos failed to improve pregnancy rates of dairy cattle exposed to heat stress. The fact that fetal loss occurred sooner for heifers than for cows points out the importance of uterine capacity as a limiting factor for maintenance of fetal development of two conceptuses. The suitability of timed embryo transfer was evident from the high pregnancy rates achieved with crossbreed females that received a single embryo. This work was supported by BARD Grant No. US-1551-14, USDA TSTAR Grant No. 2004-14135-14715, Grant No. 2001-12101-11318 from the USDA-IFAFS Program, and CAPES Grant No. 134202-1).

An experiment was conducted to determine whether pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed cows could be improved by 1) culturing embryos in the presence of IGF-I and 2) treating recipients with GnRH. Lactating Holstein cows (n = 260) were synchronized using a timed ovulation protocol. Embryos were produced in vitro and cultured with or without 100 ng/mL of IGF-I. On d 7 after anticipated ovulation (d 0), a single embryo was transferred to all recipients with a palpable corpus luteum (n = 210). A subset of recipients (n = 164) was injected with either GnRH or placebo on d 11. Plasma progesterone concentrations on d 0 and 7 were used to determine the synchrony of recipients. Pregnancy was diagnosed at d 53 and 81 by rectal palpation. Among all recipients, transfer of IGF-I-treated embryos increased pregnancy rate at d 53 (P0.05) and tended to increase pregnancy rate at d 81 (P0.06). Calving rate also tended to be higher for recipients that received IGF-I-treated embryos (P0.07). Among the subset of synchronized recipients (n = 190), pregnancy rate at d 53 and d 81 and calving rate were higher (P0.05) for IGF-I-treated embryos. The GnRH tended to increase pregnancy rate at d 53 for all recipients (P0.08) and the subset of synchronized recipients (P0.10). There were no effects of GnRH (P0.10) for pregnancy rate at d 81 and calving rate. The overall proportion of male calves was 64.3%. There was no effect (P0.10) of embryo treatment or GnRH on the birth weight or sex ratio of calves. Results of this experiment indicate that treatment of embryos with IGF-I can improve pregnancy and calving rates following transfer of in vitro-produced embryos. Further research is necessary to determine whether the treatment of recipients with GnRH is a practical approach to increase pregnancy rates following in vitro embryo transfer.