Progress 10/01/10 to 09/30/13
Outputs
Target Audience: The target audiences are other investigators working on improving production of bovine embryos in vitro and also companies providing commercial embryo production and technology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? As a PhD student, V.A. Absalon-Medina performed all of the laboratory experiments, data collection, statistical analyses and drafting the journal articles for publication. The work on this project contributed to three chapters in his PhD dissertation which was completed in 2013 and titled: ASSISTED REPRODUCTION TECHNOLOGIES TO IMPROVE DAIRY CATTLE REPRODUCTION; Cornell University Field of Animal Science. How have the results been disseminated to communities of interest? The results have been disseminated to scientific and agri-business audiences through publication of two additional abstracts presented at the national meeting of the American Dairy Science Association during the past year. In addition two journal articles have been submitted for publication in two research journals and a third article is nearing completion. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The research was conducted toward understanding how selection for high milk production has affected functional characteristics of oocytes (ova) in the ovaries of lactating cows. The project investigated and improved reproductive biotechnologies for practical in vitro embryo production using gender-selected semen to produce high quality female embryos for embryo transfer. Addition of chemical regulators for glucose metabolism during embryo culture improved blastocyst rates, but most importantly the rates of expanded blastocysts were higher when compared to control. Cryopreserved embryos are a resource that dairy producers can have available for on-farm use as an alternative to insemination. Supplementation with conjugated linoleic acid (CLA) isomers that is incorporated into cell membranes did not improve embryo production, but inclusion of CLA c9, t11 in vitrification protocols improved post-thaw survival of bovine IVF embryos. Producers applying these technologies will be benefited by maintaining or increasing production or herd size, since each producer is able to improve her/his own genetic base by selection of the best candidates in their herd. NY dairy producers may produce milk with higher components and improve their current herd genetic base. Improved animal reproductive performance benefits the on-farm economy and sustains a viable and more profitable agricultural production system. Objective 1. To compare characteristics of bovine oocytes from virgin heifers and lactating cows for differences associated with embryo development and fertility. Research is ongoing and remains to be completed. Objective 2. To establish standardized in vitro embryo production (IVP) protocols for practical production of high quality transferable female embryos. Metabolic regulators (MR) such as 2,4-dinitrophenol (DNP) and phenazine ethosulfate (PES) can improve glucose metabolism of bovine embryos in vitro via two different pathways viz. glycolysis and pentose phosphate pathways. These metabolic enhancers may be beneficial in stimulating the metabolic switch from low to high glucose utilization at the morula stage as this metabolic adjustment occurs naturally along the oviduct during migration of the preimplantation embryo toward the hypooxygenated environment of the uterus. The objectives of this study were to evaluate the effects of MR, individually or in combination, on development of in vitro produced bovine embryos. In Experiment 1, embryos were supplemented with PES (0.3 μM), DNP (10 μM), the combination, or unsupplemented (Control). In Experiment 2, embryos were supplemented with PES (0.15 μM) in combination with DNP at 3 doses (5, 10 and 30 μM). Two quality control groups were included: one group remained untreated and the other was the combined MR treatment from Experiment 1 (PES 0.3 μM + DNP 10 μM). Experiment 3 evaluated the effects of MR in combination with a conjugated linoleic acid isomer (CLA cis9, trans11) on viability of expanded blastocysts after vitrification using 4 treatment groups: the best treatment from Experiments 1 and 2 (DNP 10 μM + PES 0.3 μM) was supplemented with 0, 50, or 100 μM CLA cis9, trans11 36 hours before vitrification plus a control group that remained MR and CLA-free. In Experiment 1 and 2, blastocyst rates were significantly increased by MR in combination (DNP 10 μM + PES 0.3 μM; control +) as compared to control and other treatments (> 40% vs. ~30%; P < 0.05). Also the best MR combination resulted in expanded blastocyst rates higher than control (28% vs 15%; P < 0.05). MR resulted in dose-dependent reductions in the triglyceride content of expanded blastocysts (P < 0.05) and increased blastomere number (P < 0.05). Treatment with PES alone, however, resulted in reduced blastomere numbers. In Experiment 3, re-expansion rates after thawing were similar for MR treated embryos when compared to control (71% vs. 60%, respectively); however, treatment with CLA reduced embryo re-expansion rates. Across treatments, vitrified embryos showed no differences in terms of cytoskeleton integrity post-thaw. In conclusion, addition of MR improved blastocyst rates, but most importantly the rates of expanded blastocysts were higher when compared to control. Objective 3. To improve cryopreservation techniques using supplemental conjugated linoleic acid (CLA) for greater viability of oocytes and embryos after embryo transfer. Conjugated linoleic acid (CLA) isomers of linoleic acid (18:2 n-6) and other polyunsaturated fatty acids can affect the lipid profile and signaling of cells and thereby alter their function. A total of 5,700 bovine oocytes were used in this project in a structured series of experiments to test the optimal isomer dose and stage of development for supplementation with CLA, cis 9, trans 11 and CLA trans 10, cis 12, to improve in vitro embryo production. In experiment 1, high doses of CLA during in vitro maturation (IVM) were compared with high or low doses during the entire in vitro culture (IVC) of parthenogenetic embryos. High and low doses of CLA ranged from 50 to 200 μM and from 15 to 50 μM, respectively. In experiment 2, the lowest doses of CLA were tested during IVC after in vitro fertilization (IVF). Experiment 3 compared the effects of low doses of CLA during IVM or IVC on IVF embryos. In experiment 4, resistance to damaging effects of cryopreservation for embryos supplemented with CLA was assessed by post-thaw survival rates and blastomere counts. In experiment 1 when CLA was provided at IVM there were no effects of CLA on overall rates of maturation, cleavage, or blastocysts (92.2 ± 1.6 %, 78.3 ± 4.1 % and 28.9 ± 5.1 %, respectively). However, high doses of CLA, but not low doses, during the entire embryo culture period decreased blastocyst rates (5-20%) in a dose dependent manner. Progesterone concentrations in maturation media were significantly higher for the three high dose CLA groups combined as compared to control (0.77 ± 0.09 ng/ml vs. 0.55 ± 0.09 ng/ml) and low doses of CLAs (15 μM to 50 μM) had no effect. In experiment 2 with IVF embryos, low doses of CLA did not alter cleavage rates (average 84.9 ± 1.9 %) and only 25 μM CLA t10, c12 reduced blastocyst rates below control (38.2 ± 2.3 %). Lipid content of embryos was increased and relative expression of the survivin gene was depressed by CLA t10, c12. In experiment 3 low doses (15 μM) of CLA during IVC significantly reduced blastocyst rates (20.6 ± 2.4 % and 27.7 ± 1.2 % vs. 34.18 ± 1.2 % for CLA t10, c12 and CLA c9, t11 compared to control, respectively) and the effect of CLA was less during IVM. In experiment 4, vitrifying d-8 embryos supplemented with 100 μM CLA c9, t11 during the final 36 hr of culture resulted in the highest survival rates after thawing and the quality was comparable to control embryos not undergoing vitrification. In conclusion, supplementation with CLA isomers did not improve embryo production, but inclusion of CLA c9, t11 in vitrification protocols improved post-thaw survival of bovine IVF embryos.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
V. A. Absal�n Medina, S. J. Bedford-Guaus, R. O. Gilbert, L. C. Siqueira, G. Esposito, A. Schneider, S. H. Cheong, and W. R. Butler. Conjugated linoleic acid supplementation on in vitro bovine embryo production and cryopreservation. J.Dairy Sci. Submitted, 2014.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
V. A. Absal�n Medina, S. H. Cheong, R. O. Gilbert, and W. R. Butler. Enhancing glucose metabolism of bovine embryos in vitro : Preparation for the hypooxygenated uterine environment. Theriogenology Submitted, 2014.
- Type:
Other
Status:
Published
Year Published:
2013
Citation:
V. A. Absal�n Medina, S. H. Cheong, R. O. Gilbert, and W. R. Butler. Enhancing peri-compaction bovine embryo glucose metabolism in vitro in preparation for a hypoxic uterine environment. J.Dairy Sci. 96(E-Suppl. 1):348, 2013. (Abstract)
- Type:
Other
Status:
Published
Year Published:
2013
Citation:
V. A. Absal�n Medina, S. H. Cheong, R. O. Gilbert, and W. R. Butler. Conjugated linoleic acid (CLA) does not improve post-thaw performance of expanded-stage in vitro produced bovine embryos. J Dairy Sci. 96(E-Suppl. 1):348, 2013. (Abstract)
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Obj. 2. In preparation for conducting objective 2 as proposed, further modifications to our bovine IVM/IVF embryo system have been investigated. We have successfully integrated cyclic AMP modulators into our in vitro maturation system. This approach, emulates the gradual physiological degradation of cAMP during oocyte maturation. It requires the presence of cAMP modulators such as forskolin (adenylate cyclase activator), 3-isobutyl-1-methylxanthyne (IBMX [a non-specific phosphodiesterease inhibitor]) and cilostamide (phosphodiesterase 3A inhibitor [oocyte-specific]). The benefit would be to improve oocyte maturation and alter cytoplasmic content in preparation for fertilization i.e. also known as oocyte capacitation. After the addition of this approach to our IVM, our blastocyst rates have consistently increased from ~35% to ~45%. Further experiments were directed at the bovine morula stage. Some in vitro morula embryos do not develop the capacity to increase glucose consumption via glycolysis, a natural event at this stage, and thus become arrested (16-32 cell block). The goal was to use metabolic regulators to improve glucose consumption of compromised embryos that are unable to make this switch and are more likely to undergo embryonic arrest. We tested oxidative phosphorylation uncouplers such as 2,4 dinitrophenol (DNP) that partially inhibits oxidative phosphorylation while enhancing glycolysis and phenazine ethosulfate (PES) that is an electron acceptor for reduced nicotinamide adenine dinucleotide phosphate (NADPH). The objective was to uncouple oxidative phosphorylation while enhancing glucose consumption via glycolysis and the pentose phosphate pathways. Results have demonstrated that supplementation of these metabolic regulators had an additive effect on overall embryonic rates (~30% increase) when compared to our control group. In addition, we were able to observe a decrease in triglyceride content when embryos were supplemented with these chemicals when compared to control (5-20%). Obj. 3. The outputs from the previous year testing with CLA on vitrification procedures for bovine embryos were extended. We found that expanded embryos treated with our best CLA dose were not able to re-expand after vitrification procedures. However, this did not affect developmental rates in terms of blastocyst rates or blastomere count. PARTICIPANTS: PARTICIPANTS: W.R. Butler, Principal Investigator; V.A. Absalon-Medina, PhD student, performed all of the laboratory work. - This project is part of the training and development program for this graduate student and will contribute to his PhD dissertation. TARGET AUDIENCES: The target audiences will be other investigators working on improving production of bovine embryos in vitro and also companies providing commercial embryo production and technology. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Application of select metabolic regulators at specific stages of in vitro bovine embryo development can demonstrably improve embryo viability and production to the blastocyst stage. The resulting improved culture system provides opportunity to dramatically increase efficiency of embryo production, but embryo survival after transfer to recipients and live births requires further testing.
Publications
- No publications reported this period
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Objective. To improve cryopreservation techniques using supplemental conjugated linoleic (CLA) acid isomers for greater viability of embryos after embryo transfer. A bovine in vitro embryo production system has been successfully established. Bovine embryos were incubated with 100 uM CLA cis 9, trans 11 or in control media for 36 hours prior to vitrification to compare cryopreserved embryo performance such as survival and developmental rates post thaw. PARTICIPANTS: W.R. Butler, Principal Investigator; V.A. Absalon-Medina, PhD student, performed all of the laboratory work. - This project is part of the training and development program for this graduate student and will contribute to his PhD disssertation. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Among all treatment groups inclusion of CLA before vitrification procedures resulted in more embryos surviving the vitrification process after thawing. In addition, embryos were more capable to continue with subsequent cell cycles as demonstrated by higher survival and total blastomeric cell counts (p < 0.05). The embryo quality with CLA was comparable to embryos not being vitrified (no cryopreservation stress associated) which would imply substantial benefits, if this technology is adopted.
Publications
- Absalon-Medina,V.A., Bedford Guaus,S.J., Gilbert,R.O., Siqueira,L.C., Esposito,G., Schneider A., Cheong,S.H., Butler, W.R. 2011. Effect of conjugated linoleic acid supplementation on in vitro bovine embryo production and cryopreservation. American Dairy Science Association (ADSA) joint meeting at New Orleans, USA. J. Anim. Sci. vol. 89, E-suppl. 1/J. Dairy Sci. vol. 94, E-suppl. 1.:p. 67 (Abstract).
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