Progress 09/01/99 to 08/31/04
Outputs
Analysis of concentrations of vitamin A (retinol), retinol-binding protein (RBP), cellular retinol-binding protein (CRBP), and ovarian steroids in ovarian follicular fluids and cells revealed that healthy follicles have significantly higher concentrations of vitamin A, estradiol and RBP than atretic follicles. Analysis of RBP and CRBP mRNA and protein demonstrate that vitamin A transport and metabolism are regulated locally within the follicle. A series of studies were performed which demonstrate that administration of vitamin A to superovulated ewes, followed by natural service, resulted in embryos with improved competence to develop in vitro, and survive cryopreservation, embryo transfer, maintain pregnancies and produce viable offspring. In vitro maturation of oocytes, fertilization and embryo culture studies demonstrated that addition of retinol to maturation medium significantly improved blastocyst development compared to controls (25.34% vs 20.11%, p< .05). When
control blastocyst development was less than 20% or oocytes were exposed to heat stress, retinol treatment dramatically improved development (p< .002). Results indicate that retinol treatment may have its greatest positive impact on stressed embryos. Studies were performed to determine the effect of retinol or retinoic acid (RA) administration on embryos cultured in atmospheric (20%) or low (7%) oxygen. Retinol treatment significantly increased embryonic development to the blastocyst stage in high O2 compared to controls (29.6% vs 12.5%). Similar results were observed for RA treatment. Results indicate that retinoid treatment may diminish oxidative stress if embryos cultured in atmospheric O2 and may provide an alternative to the expensive method of culturing embryos in a low 02, three gas system. Metabolism studies performed with sheep oocytes during meiotic matureation demonstrated that retinol treatment had no effect on metabolism but showed ovine oocytes utilize both glycolytic
and oxidative pathways during maturation. Semiquantitative RT-PCR analysis of enzymatic antioxidant genes was performed on 230 individual sheep oocytes from retinol (95) and vehicle-treated (130) ewes. Retinol treatment had no effect on levels of gene expression but for the first time expression of manganese superoxide dismustase (Mn SOD), copper superoxide dismutase (Cu SOD), gammaglutamyl cysteine synthetase (GCS) and glutathione synthetase (GSH) was demonstrated in ovine oocytes.
Impacts
1. Vitamin A is an indicator of ovarian follicular health in cattle. 2. Vitamin A administration improves embryonic developmental competence. 3. Vitamin A treatment during in vitro oocyte maturation improves subsequent embryonic development. 4. Vitamin A protects in vitro derived embryos from oxidative stress. The information gained from this research indicates potential strategies to improve reproductive performance in livestock, thereby increasing productivity and profitability of farms.
Publications
- Lawrence, J.L., Payton, R.C., Godkin, J.D., Saxton, A.M., Schrick, F.N. and Edwards, J.L. 2004. Retinol improves development of bovine oocytes compromised by heat stress during maturation. J. Dairy Sci. 87:2449-2454.
- Livingston, T.E., Eberhardt, D.M., Edwards, J.L. and Godkin, J.D. 2004. Metabolism of sheep oocytes during maturation in vitro. JAVA 3: 174-796.
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Progress 01/01/03 to 12/31/03
Outputs
Analysis of concentrations of vitamin A (retinol), retinol-binding protein (RBP), cellular retinol-binding protein (CRBP), and ovarian steroids in ovarian follicular fluids and cells revealed that healthy follicles have significantly higher concentrations of vitamin A, estradiol and RBP than atretic follicles. Analysis of RBP and CRBP mRNA and protein demonstrate that vitamin A transport and metabolism are regulated locally within the follicle. A series of studies were performed which demonstrate that administration of vitamin A to superovulated ewes, followed by natural service, resulted in embryos with improved competence to develop in vitro, and survive cryopreservation, embryo transfer, maintain pregnancies and produce viable offspring. In vitro maturation of oocytes, fertilization and embryo culture studies demonstrated that addition of retinol to maturation medium significantly improved blastocyst development compared to controls (25.34% vs 20.11%, p< .05). When
control blastocyst development was less than 20% or oocytes were exposed to heat stress, retinol treatment dramatically improved development (p< .002). Results indicate that retinol treatment may have its greatest positive impact on stressed embryos. Studies were performed to determine the effect of retinol or retinoic acid (RA) administration on embryos cultured in atmospheric (20%) or low (7%) oxygen. Retinol treatment significantly increased embryonic development to the blastocyst stage in high O2 compared to controls (29.6% vs 12.5%). Similar results were observed for RA treatment. Results indicate that retinoid treatment may diminish oxidative stress if embryos cultured in atmospheric O2 and may provide an alternative to the expensive method of culturing embryos in a low 02, three gas system. Metabolism studies performed with sheep oocytes during meiotic matureation demonstrated that retinol treatment had no effect on metabolism but showed ovine oocytes utilize both glycolytic
and oxidative pathways during maturation. Semiquantitative RT-PCR analysis of enzymatic antioxidant genes was performed on 230 individual sheep oocytes from retinol (95) and vehicle-treated (130) ewes. Retinol treatment had no effect on levels of gene expression but for the first time expression of manganese superoxide dismustase (Mn SOD), copper superoxide dismutase (Cu SOD), gammaglutamyl cysteine synthetase (GCS) and glutathione synthetase (GSH) was demonstrated in ovine oocytes.
Impacts
1. Vitamin A is an indicator of ovarian follicular health in cattle. 2. Vitamin A administration improves embryonic developmental competence. 3. Vitamin A treatment during in vitro oocyte maturation improves subsequent embryonic development. 4. Vitamin A protects in vitro derived embryos from oxidative stress. 5. Sheep oocytes maintain an endogenous defense against oxidative stress.
Publications
- Lawrence, JL, Payton, RC, Godkin, JD, Saxton, AM, Schrick, FN and Edwards, JL. 2003. Biol Reprod 68:344.
- Brown, JA, Eberhardt, DM, Schrick, FN, Roberts, MP and Godkin, JD. 2002. Expression of retinol-binding protein and cellular retinol-binding protein in the bovine ovary. Mol Reprod Devel 64:261-269.
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Progress 01/01/02 to 12/31/02
Outputs
Retinoids are recognized as important regulators of vertebrate development, cell differentiation and tissue function. Studies were performed to determine the effects of retinol administration during ovine oocyte maturation in vivo and bovine oocyte maturation and embryonic development in vitro. In addition, the effects of in vivo retinol treatment on in vitro ovine oocyte carbohydrate metabolism were determined. Retinol was administered to sheep, in combination with FSH, followed by natural service. Morula stage embryos were collected from the uterus, frozen in a programmable freezer and stored in liquid nitrogen. After a period of 6-12 months, embryos were thawed rapidly and transferred to the uteri of synchronized recipients. Pregnancy rates of recipients receiving embryos from retinol-treated donors were greater than that of recipients receiving control embryos, resulting in a greater number of viable offspring. Bovine oocytes were matured in the presence and
absence of varying concentrations of retinol. After a 22-24 hour maturation period, oocytes were fertilized, denuded 18 hours later and cultured in modified synthetic oviductal fluid (mSOF) in a humidified atmosphere of 5.5% CO2, 7% O2, 87.5% N2. Cleavage rates as well as the number of 8-cell embryos did not differ between treatments. The addition of 5upsilonM retinol to the maturation medium significantly (p<0.03) increased blastocyst formation, compared to controls. Further analysis revealed that when blastocyst rate of controls were below 20%, retinol treatment dramatically improved embryonic development (p<0.005). The effects of retinol or retinoic acid (RA) addition to embryos cultured in low O2 (7%) and atmosheric O2 (20%) were compared. In the presence of low O2, retinol or RA addition had no effect on embryonic development but significantly (p<0.03) increased blastocyst cell number compared to controls. In the presence of atmospheric O2, retinol or RA dramatically improved
embryonic development to the blastocyst stage (p<0.01) compared to controls. Metabolism studies demonstrated, for the first time, that ovine oocytes use both glycolysis and oxidative pathways throughout in vitro maturation. Retinol treatment had no significant effect on carbohydrate metabolism.
Impacts
Retinoid treatment protocols applied to assisted reproductive procedures improve early embryonic development. Retinoid treatments protect the early embryo against oxidative stress encountered during assisted reproductive procedures.
Publications
- Livingston, T.E., Eberhardt, D.M. and Godkin, J.D. 2002. The effects of retinol on bovine embryo development in vitro. Biol Reprod. 66(supplement 1):104.
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Progress 01/01/01 to 12/31/01
Outputs
In vitro metabolism of oocytes from superovulated, retinol- and vehicle- treated ewes was determined. Oocytes were removed from preovulatory follicles, denuded of cumulus cells, and subjected to metabolic measurements using hanging-drop procedures. Results show that glucose metabolism through the glycolytic pathway changed from early (0-3h) to mid (9-12h) and early to late (21-24h) time periods (p< 0.01). Glucose oxidation through the Krebs cycle changed over time from early to late time periods (p < 0.05). Glucose metabolism via the pentose-phosphate pathway was highest during the early period. Oxidative metabolism, measure by glutamine showed no differences over time. Pyruvate oxidation increased from 0-3 to 21-24h (p< 0.02). Retinol treatment had no effect (p< 0.8) on oocyte metabolism. Addition of retinol to culture medium of bovine oocytes during in vitro maturation, followed by in vitro fertilization, was demonstrated to increase embryonic
development to the blastocyst stage (p< 0.05). Embryo transfer studies demonstrated that embryos from superovulated, retinol-treated ewes have increased competence to maintain pregnancies and develop into viable offspring.
Impacts
The retinol treatment protocols developed during the course of these studies have the potential to significantly improve assisted reproductive procedures in farm animals, endangered species and humans.
Publications
- Livingston, T.E., Eberhardt, D.M., Godkin, J.D. 2001. The effect of retinol on sheep oocyte metabolism. Biol. Reprod. 64 (suppl. 1): 142.
- Nonnecke, B.J., Roberts, M.P., Godkin, J.D., Horst, R.L., Hammemm, D.C., Franklin, S.T. 2001. Influence of supplemental dietary vitamin A on retinol-binding protein concentrations in the plasma of preruminant calves. Dairy Sci. 84:641-8.
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Progress 01/01/00 to 12/31/00
Outputs
Studies were performed to determine the role of retinol and retinoid-associate proteins in oocyte maturation and early embryonic development in sheep and cattle. Immunocytochemical analysis identified cellular retinol-binding protein (CRBP) in granulosa cells of primary, preantral and early antral bovine ovarian follicles. Immunostaining for CRBP was weak or absent in granulosa cells of antral follicles, ovarian stromal cells and blood vessels. Retinol-binding protein exhibited strong immunostaining in granulosa and thecal cells of antral follicles and thecal cells of some preatral follicles. Northern blot analysis of RNA from isolated ovarian cells demonstrated CRBP and RBP mRNA expression in the same cells that produced the proteins. Oocytes from superovulated, retinol and control treated ewes where collected and energy metabolism studies were performed at specific times of in vitro oocyte maturation. Significant time and substrate dependent differences were
observed. Addition of retinol to culture medium of bovine oocytes during in vitro maturation was demonstrated to not effect subsequent fertilization rates or embryonic development to the blastocyst stage. Embryo transfer studies demonstrated that embryos from superovulated, retinol treated ewes are competent to maintain pregnancies and develop into viable offspring.
Impacts
Studies provide novel information on potential mechanisms by which retinol administration positively impacts embryonic development.
Publications
- Nonnecke, B. J., Roberts, M. P., Godkin, J. D., Horst, R. L., Hammell, D. C. and Franklin, S. T. 2000. Influence of supplemental, dietary vitamin A on retinol-binding protein concentrations in the plasma of preruminant calves. Dairy Sci. 83 (in press).
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Progress 01/01/99 to 12/31/99
Outputs
Experiments were conducted to determine the effects of retinoid treatment of superovulated ewes on subsequent in vitro embryonic development. Ewes were treated with all-trans retinol (ROH), all-trans retinoic acid (RA), 9-cis retinoic acid (CIS) or vehicle control on the first and last day of FSH administration. Embryos were recovered at the morula stage of development, cultured for 96h and observed for blastocyst development. Embryos from ROH treated ewes had a higher incidence (p<0.01) of blastocyst formation than embryos from all other groups. In a second experiment, ewes were given ROH or vehicle and treated as above. ROH treatment resulted in an increased percentage of embryos forming blastocysts (70% vs 22%, p<0.05). In the third experiment ewes were treated with ROH or vehicle and embryos were collected at the 1- to 4-cell stage and cultured for 7 days. ROH treatment resulted in increased blastocyst formation (79% vs 6%, p<0.01). ROH treatment of superovulated
ewes dramatically increased embryonic viability and positively impacted embryonic development. Studies were performed to identify RBP expression in the oviduct and to determine the role of ovarian steroids in its regulation. Cell specific expression of RBP was analyzed by immunocytochemical procedures. RBP synthesis was determined by immunoprecipitation of metabolically labeled RBP from the culture medium of oviductal explant cultures and mRNA expression was determined by Northern and Slot Blot analysis. Steroid regulation of RBP expression was evaluated by steroid replacement of ovariectomized ewes. Results demonstrated that RBP is localized, synthesized and secreted from the oviductal epithelial cells. Studies demonstrated that progesterone, applied on an estradiol background, negatively regulates RBP gene and protein expression, whereas, estradiol appears to stimulate RBP synthesis and secretion. The expression of retinoid-binding proteins was identified in the ovaries of sheep and
cattle. Cellular retinol-binding protein (CRBP) and RBP were immunolocalized to the thecal and granulosa cell layers of healthy but not atretic follicles. The thecal layer was shown to be the major site of RBP mRNA and protein production. In sheep, CRBP was expressed most strongly in the theca, whereas in cattle, granulosa cells were identified as the major site of expression. Cellular retinoic acid-binding protein (CRABP) was immunolocalized in the ovum of premordial and primary follicles but not in the ovum or follicle cells of more mature follicles. Results indicate that retinoids are involved in early events associated with oocyte development. A model for retinol delivery from the plasma to the follicle was proposed based on the results.
Impacts
(N/A)
Publications
- Eberhardt, D.M., Will, W.A. and Godkin, J.D. 1999. Retinol administration to superovulated ewes improves embryonic viability. Biol. Reprod. 60:1483-1487.
- Eberhardt, D.M., Jacobs, W.G. and Godkin, J.D. 1999. Steroid regulation of retinol-binding protein in the ovine oviduct. Biol. Reprod. 60:714-720.
- Brown, J.A., Eberhardt, D.M. and Godkin, J.D. 1999. Immunolocalization of retinol binding protein and cellular retinol binding protein in the bovine ovary. Biol. Reprod. 60(suppl 1):51.
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