Progress 10/01/03 to 09/30/08
Outputs
OUTPUTS: Normal embryonic development depends on the maintenance of a population of normal healthy cells within each embryo. Thus, the disposal of morphologically abnormal cells and cells in excess is crucial during early development. Studies were conducted to evaluate the effect of scrotal insulation on semen samples collected from bulls on embryonic development after IVF; to evaluate the effect of vitrification of oocytes and early embryos on embryonic development after IVF; and to use a combination of apoptotic (programmed cell death) measures to assess differences in embryo quality. Additionally, a serum supplemented and serum free production systems and semen from two bulls of different field fertility and supplementation of Cys during oocyte maturation and as well as growth factors, epidermal growth factor (EGF) and insulin-like growth factor-I (IGF), during embryo culture were evaluated to assess improvement of cleavage and blastocyst rates of in vitro produced bovine embryos. The development competence of vitrified embryos did not differ among the culture environments. Nevertheless, the effect of follicle size on cleavage rate was significant and a higher cleavage rate resulted from oocytes aspirated from greater than 3 mm follicles (71.0%) compared to those collected from less than 3 mm follicles (64.8%). Oocytes vitrified from medium sized follicles had a 9.9% increase in the cleavage rate compared to oocytes from small follicles. The increase in the blastocyst rate was only 5.9% between the oocytes from small and medium-sized follicles. The sires showed significant effects on embryonic developmental rates. The effect of modified droplet vitrification was assessed on cellular actin filament organization, apoptosis related gene expression and the development competence in mouse embryos cultured in vitro. Mouse zygotes, 2-cell embryos and morulae were vitrified in ethylene glycol and ethylene glycol plus DMSO and thawed by directly placing the vitrified drop into 0.3 M sucrose solution at 37 C. A high recovery (93 to 99%) of morphologically normal embryos was evident following vitrification and thawing. No detectable actin filament disruption was observed in the embryos at any development stage following vitrification and thawing and/ or in vitro culture. Culture treatments of bovine oocytes included fetal calf serum (FCS); IGF; EGF; IGF+EGF; 0 h Cys with IGF; EGF; or IGF+EGF; and 12 h Cys with IGF; EGF; or IGF+EGF. Supplementation of Cys during IVM of oocytes, in conjunction with growth factors, excluding IGF, during in vitro culture, resulted in a similar embryonic development to that of FCS, and could effectively be used as a replacement for FCS. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The decrease in embryonic development after IVF when using spermatozoa with morphological abnormalities seems multifaceted. Our results clearly indicated a difference in the embryo quality between embryos obtained after IVF with semen samples from bulls that had an intense response to scrotal insulation. Rapid freezing altered the transcriptional activities of Bax, Bcl2, and p53 genes in vitrified embryos, indicating a strong relationship between reduced developmental competence and the altered transcriptional activities of these genes. The modified droplet vitrification procedure supported better survival of morula stage compared to zygotes and 2-cell mouse embryos. Our results show that differences in development exist in source of oocytes and sires used for IVF after vitrification. No correlation exists between post-IVF outcome and field fertility. Supplementation of Cys during in vitro maturation of oocytes, in conjunction with growth factors during in vitro culture resulted in a similar embryonic development to that of fetal calf serum (FCS), and could effectively be used as a replacement for FCS. These findings will assist producers to increase reproductive efficiency. GH administration increases serum concentration of insulin in cattle and this effect is at least in part due to direct action of GH on insulin gene expression and secretion from the pancreatic beta cells. These studies will allow producers to benefit from understanding endocrine control mechanisms.
Publications
- Pence, K. J., K. F. Knowlton, F. C. Gwazdauskas, R. E. Pearson, and C. O. Wilkes. 2008. Effect of orchardgrass or alfalfa hay in diets of lactating cows during relocation. Prof. Anim. Scientist, (in press).
- Miller, L.E., J.J. Volpe, M.D. Coleman-Kelly, F.C. Gwazdauskas, and S.M. Nickols-Richardson. 2008. Anthropometric and leptin changes in women following different dietary approaches to weight loss. Obesity (in press).
- Lott, W. M., V. M. Anchamparuthy, M. L. McGilliard, I. K. Mullarky, and F. C. Gwazdauskas. 2008. Influence of cysteine in conjunction with growth factors during in vitro production of bovine embryos. J. Dairy Sci. 91: (E-Suppl. 1) 461.
- Feng, J., F.C. Gwazdauskas, and H. Jiang. 2008. Growth hormone directly stimulates insulin production from the bovine pancreatic islets. J. Dairy Sci. 91: (E-Suppl. 1) 64.
- Anchamparuthy, V. M., A. Dhali, R. E. Pearson, W.M. Lott, and F. C. Gwazdauskas. 2008. Paternal influence on the in vitro embryonic development of vitrified oocytes based on estimated relative conception rate. J. Dairy Sci. 91: (E-Suppl. 1) 461.
- Lott, W.M. 2008. Influence of Growth Factors on Bovine Embryo Development. M.S. Thesis. Virginia Polytechnic Institute & State University. July 28.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Normal embryonic development depends on the maintenance of a population of normal healthy cells within each embryo. Thus, the disposal of morphologically abnormal cells and cells in excess is crucial during early development. Studies were conducted to evaluate the effect of vitrification of oocytes and early embryos on embryonic development after IVF and to use a combination of apoptotic (programmed cell death) measures to assess differences in embryo quality. The concept of ultra-rapid vitrification has emerged in recent years; the accelerated cooling rate reduced injury attributed to cryopreservation and improved post-freezing developmental competence of vitrified oocytes and embryos. The objectives of the present study were to develop a simple and effective ultra-rapid vitrification method (droplet vitrification) and evaluate its effects on post-thaw development and apoptosis-related gene expression in mouse zygotes. Presumptive zygotes were equilibrated for 3 min in
equilibration medium and washed 3 times in vitrification solution. A drop (5 mL) of vitrification solution containing 10-12 embryos was placed directly onto surface of liquid nitrogen, with additional liquid nitrogen poured over the drop. For thawing and cryoprotectant removal, vitrified drops were put into dilution medium for 3 min, followed by M2 medium for 5 min. Although cleavage rate did not differ significantly among the control (90.8%), toxicity control (83.5%), and vitrified (86.2%) zygotes, rates of blastocyst and hatched blastocyst formation were lower in vitrified zygotes (49.7% and 36.0%) and toxicity controls (47.3% and 40.3%) compared with controls (65.5% and 54.2%). Exposure of zygotes to vitrification solution, as well as the vitrification process, down-regulated the expression of Bax, Bcl2, and p53 genes in blastocysts. Although droplet vitrification was efficient and easy, it altered the transcriptional activities of Bax, Bcl2, and p53 genes in vitrified embryos,
indicating a strong relationship between reduced developmental competence and the altered transcriptional activities of these genes. Holstein cows were administered zona pellucida (ZP) DNA vaccine and used to determine the potential of recombinant rabbit ZP glycoproteins (rZP) as immunocontraceptive antigens. Cows were assigned to 1 of 4 treatment groups in which plasmids encoding rabbit ZP proteins were administered, i.d., using a gene gun (ZP55, n = 2; ZP75, n = 2; Hep55, n = 2; and Control, n = 3). Blood samples were taken before initial vaccination, once weekly for 5 wk and at 148 wk post-immunization. An ELISA was developed to assess anti-ZP titer levels in cow serum and ovarian function in cows was monitored using trans-rectal ultrasonography. Four of the six cows in ZP treatment groups developed antibody titer levels with similar linear responses over time. These cows also experienced reduced ovarian function as indicated by decreases in follicular and luteal activity. Estrous
activity was observed in all cows and decreased in ZP treatment cows in comparison to Controls. Further research is needed to determine the relationship between ZP immunocontraception and ovarian function.
PARTICIPANTS: Dhali, A., Visiting Professor; Anchamparuthy, V.M., Graduate Student; Butler, S.P., Collaborator; Pearson, R.E., Statistical consultant; Mullarky, I.K., Collaborator; Gwazdauskas, F.C., PI; Foley, C. A., Graduate Student; Boyle, S.M., Collaborator; Wilkes, C. O., Graduate Student; Pence, K. J., Graduate Student; Hurt, A. M., Graduate Student; Becvar, O., Collaborator; Knowlton, K. F., Collaborator; Mcgilliard, M. L., Statistical Consultant; Van Cott, K., Collaborator; Gil, G., Collaborator; Pipe, S. W., Collaborator; Miao, H. Z., Collaborator; Kaufman, R. J., Collaborator; Velander, W. H. Collaborator; Jones, E. T., Graduate Student; Guill, S. G., Graduate Student;Hargens, T.A., Graduate Student; Aron, A., Graduate Student; Butner, K. L., Graduate Student; Mabry, J. E., Graduate Student; Herbert, W. G., Collaborator;
TARGET AUDIENCES: Target audiences include individuals, groups, market segments in academia and the dairy and livestock industry. These groups include populations such as racial and ethnic minorities and those who are socially, economically, or educationally disadvantaged. Our data have been delivered as science-based knowledge to people through formal or informal educational programs such as: formal classroom instruction, laboratory instruction, or practicum experiences; workshops; and national meetings.
Impacts
Rapid freezing altered the transcriptional activities of Bax, Bcl2, and p53 genes in vitrified embryos, indicating a strong relationship between reduced developmental competence and the altered transcriptional activities of these genes. Further research is needed to determine the relationship between ZP immunocontraception and ovarian function.
Publications
- Van Cott, K., Gil, G., Pipe, S. W., Miao, H. Z., Gwazdauskas, F.C., Butler, S. T., Kaufman, R. J., Velander, W. H. 2007. Post-translationally complex therapeutic proteins: expression in milk. UC Davis Conference in Transgenic Research. (Abstract).
- Jones, E. T., Guill, S. G., Hargens, T.A., Aron, A., Butner, K. L., Mabry, J. E., Gwazdauskas, F. C., Herbert, W. G. 2007. Does high-volume physical activity influence insulin resistance and associated cytokines in young men? Amer. College Sports Med. (Abstract).
- Dhali, A., Anchamparuthy, V.M., Butler, S.P., Pearson, R.E., Mullarky, I.K., Gwazdauskas, F.C. (2007) Gene expression and development of mouse zygotes following droplet vitrification. Theriogenology 68: 1292-1298.
- Foley, C. A., Boyle, S.M., Pearson, R.E., Gwazdauskas, F.C. (2007) Evaluation of a DNA Vaccine for immunocontraceptive potential against zona pellucida glycoproteins in cattle. Amer. J. Anim. Vet. Sci. 2: 32-41.
- Wilkes, C. O., Pence, K. J., Hurt, A. M., Becvar, O., Knowlton, K. F., Mcgilliard, M. L., Gwazdauskas, F. C. (2007) Effect of relocation on locomotion and cleanliness in dairy cows. J. Dairy Res. 74: 1-5.
- Anchamparuthy, V. M., Dhali, A., Butler, S. P., Pearson, R. E., Gwazdauskas, F. C. (2007). Nylon mesh vitrification for cryopreservation of bovine oocytes. J. Dairy Sci. 90: Suppl. 1. 530. (Abstract).
- Dhali, A., Anchamparuthy, V. M., Butler, S. P., Pearson, R. E., Gwazdauskas, F. C. 2007. Droplet vitrification method did not induce cytoskeletal damage in mouse embryos. J. Dairy Sci. 90: Suppl. 1. 529. (Abstract).
- Dhali, A., Anchamparuthy, V. M., Butler, S. P., Pearson, R. E., Gwazdauskas, F. C. 2007. In vitro production of bovine embryos in chemically defined serum-free media. J. Dairy Sci. 90: Suppl. 1. 529. (Abstract).
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Progress 10/01/05 to 09/30/06
Outputs
Normal embryonic development depends on the maintenance of a population of normal healthy cells within each embryo. Thus, the disposal of morphologically abnormal cells and cells in excess is crucial during early development. Studies were conducted to evaluate the effect of scrotal insulation on semen samples collected from bulls on embryonic development after IVF and to use a combination of apoptotic (programmed cell death) measures to assess differences in embryo quality. A study was conducted to follow the chronology of pronuclear formation in bovine zygotes after in vitro insemination with a population of spermatozoa having abnormal morphology. Semen samples were obtained and cryopreserved from four Holstein bulls before and after a scrotal insulation period of 48 h (Day 0). A pre- (Day -5) and a Day 20 post-insult semen sample were evaluated for morphology and used for IVF after standard swim-up sperm separation protocols. Pronuclear formation was scored on
subpopulations of presumptive zygotes after they were fixed and stained at 3 h time intervals from 6 to 18 h post in vitro insemination (hpi). Post-thaw morphological evaluation of semen samples previously determined revealed a decrease in the percentages of normal spermatozoa in the post-insult samples compared with the pre-insult samples for Bulls I (74 to 2) and Bull III (68 to 1). The sperm penetration rate (percentage) decreased between the pre- and post-insult samples for Bull I (90 to 76) and III (92 to 70), but was not different for Bull II (92 to 90) and IV (78 to 85). The pronuclear formation rates for post-insult zygotes for Bull II and Bull IV had comparable increases in development over time, while there was no increase in the pronuclear development for the zygotes from the post-insult samples for Bulls I and III and generally a condensed sperm head was observed in the cytoplasm. At 18 hpi the fertilization rate (percentage) between the pre- and post-insult samples for
Bull I (51 to 4), II (88 to 75) and Bull III (94 to 2) decreased, but there was no change for Bull IV (66). Our results suggested that the failure in normal pronuclear formation was associated with an absence of normal decondensation of the penetrating spermatozoon. The role of dopamine in regulation of glucocorticoid and prolactin secretion was investigated in lactating Holstein cows by characterizing serum cortisol and prolactin response to fluphenazine. Twelve anovulatory cows received a bolus injection of either saline or 0.3 mg/kg BW fluphenazine in wk 2 postpartum. There was no difference in serum cortisol concentration between groups before treatments. Fluphenazine increased serum cortisol within 30 min after fluphenazine administration, which remained elevated throughout the sampling period. Prolactin concentration increased after fluphenazine administration (103.1 ng/ml), but did not change in response to saline (18 ng/ml) and remained elevated throughout the sampling period
in treated cows. The data indicate that dopamine antagonist increase cortisol, suggesting that endogenous dopamine may regulate cortisol secretion as well as prolactin in lactating dairy cows during the early postpartum.
Impacts
The decrease in embryonic development after IVF when using spermatozoa with morphological abnormalities seems to be multifaceted. Our results clearly indicated a difference in the embryo quality between embryos obtained after IVF with semen samples from bulls that had an intense response to scrotal insulation.
Publications
- Ahmadzadeh, A., Barnes, M. A., Gwazdauskas, F. C., and Akers, R. M. 2006. Dopamine antagonist alters serum cortisol and prolactin secretion in lactating Holstein cows. J. Dairy Sci. 89:2051-2055.
- Walters, A.H., Saacke, R.G., Pearson, R.E., and Gwazdauskas, F.C. 2006. Assessment of pronuclear formation following in vitro fertilization with bovine spermatozoa obtained after thermal insulation of the testis. Theriogenology 65: 1016-1028.
- Nickols-Richardson, S.M., Beisiegel, J.M., Gwazdauskas, F.C. 2006. Eating restraint is negatively associated with biomarkers of bone turnover but not measurements of bone mineral density in young women. J. Amer. Dietetic Assn. 106: 1095-1101.
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Progress 10/01/04 to 09/30/05
Outputs
Normal embryonic development depends on the maintenance of a population of normal healthy cells within each embryo. Thus, the disposal of morphologically abnormal cells and cells in excess is crucial during early development. Studies were conducted to evaluate the effect of scrotal insulation on semen samples collected from bulls on embryonic development after IVF and to use a combination of apoptotic (programmed cell death) measures to assess differences in embryo quality. Semen samples were obtained and cryopreserved from four Holstein bulls before and after a scrotal insulation period of 48 h. Three types of samples were used for IVF: 1) semen from the bulls collected 5 d prior to scrotal insulation (Pre-insult); 2) semen from Day 13 (2 wk-Post-Insult; 2 wk-PI); and 3) semen from Day 20 (3 wk-PI). The standard protocol for the swim-up sperm separation method was used. After 18 h of sperm-oocyte co-incubation, the zygotes were cultured for 8 d when a developmental
score was assigned to each embryo. The post-thaw morphological evaluation of sperm samples revealed a decrease in the percentages of normal spermatozoa in the 3 wk-PI samples in comparison with the Pre-insult samples for Bulls I and Bull III. The percentage of vacuolated spermatozoa increased significantly for Bull II. There was no apparent change in abnormal sperm populations for Bull IV. The cleavage and blastocyst formation rates and embryo development scores were affected by the interaction of bull by sample collection time. For Bulls I and III (severe responders) the scrotal insulation effects persisted from the time of cleavage through blastocyst formation. In the second study zygotes were cultured and subpopulations were removed from culture at Day 4 and 8 and subjected to either the TUNEL or caspase assay. The apoptotic index and caspase intensity were recorded and no differences in the apoptotic index were found in embryos generated from the semen samples for Bull I. The
apoptotic index for Bull III for the 3 wk-PI embryos (34.9 percent) was significantly lower than the activity for the Control (43.2 percent) and 2 wk-PI embryos (41.4 percent). On Day 8 caspase intensity increased significantly for both Bull I (217) and Bull III (229) for the 3 wk-PI embryo groups compared to the equivalent embryo groups for Bull II (98) and Bull IV (90). The decrease in embryonic development after IVF when using spermatozoa with morphological abnormalities seems to be multifaceted. However, the inability to consistently measure apoptosis in early stage embryos complicates the assessment of differences in embryo quality. Despite the discrepancies, our results clearly indicated a difference in the embryo quality between embryos obtained after IVF with semen samples from bulls that had an intense response to scrotal insulation.
Impacts
Current sperm separation methods used for in vitro fertilization were inadequate in their ability to provide potentially competent sperm for IVF following thermal insult of the testes. The use scrotal of insulation to elevate scrotal temperature continues to be an effective method to obtain semen samples with high percentages of abnormal spermatozoa. The decrease in embryonic development after IVF when using spermatozoa with morphological abnormalities seems to be multifaceted and related to the changes in head shape morphology. Our studies will continue to attempt to evaluate where the weak links are.
Publications
- Foley, C. A. 2005. Immunocontraceptive Potential of Zona Pellucida Glycoprotein Vaccine in Cattle. M.S. Thesis, April 28.
- Walters, A.H., Saacke, R. G., Pearson, R. E., and Gwazdauskas, F. C. 2005. The incidence of programmed cell death after in vitro fertilization (IVF) with morphologically abnormal bovine spermatozoa. J. Dairy Sci. 88: Suppl. 1, 137.
- Hurt, A.M., Gwazdauskas, F.C., Pearson, R.E., Becvar, A., Wilkes, C.O., Pence, K.J., Wilson, S.C., and Harris, L. 2005. Do changes in conductivity measures reflect variation in somatic cell count in bovine milk? J. Dairy Sci. 88: (Suppl. 1) 130.
- Pence, K.J., Knowlton, K.F., Gwazdauskas, F.C., Pearson, R.E., Wilson, C.S., Harris, L., Wilkes, C.O., Hill, S.R., and Hurt, A.M. 2005. The effect of social hierarchy on lactating cows during relocation. J. Dairy Sci. 88: (Suppl. 1) 256.
- Pence, K.J., Knowlton, K.F., Gwazdauskas, F.C., Pearson, R.E., Wilkes, C.O., Hurt, A.M., Hill, S.R., Hollmann, M., and Wilson, C.S. 2005. The effect of diet on lactating dairy cows during relocation. J. Dairy Sci. 88: (Suppl. 1) 256-257.
- Walters, A. H., Saacke, R. G., Pearson, R. E., and Gwazdauskas, F. C. Assessment of pronuclear formation following IVF with bovine spermatozoa having abnormal morphology after thermal insulation of the testis. 2005. Biol. Reprod. 73: (Suppl. 1) 158-159.
- Walters, A.H., Eyestone, W.E., Saacke, R.G., Pearson, R.E., and Gwazdauskas, F.C. 2005. Bovine embryo development after IVF with spermatozoa having bad morphology. Theriogenology 63: 1925-1937.
- Walters, A.H., Saacke, R.G., Pearson, R.E., and Gwazdauskas, F.C. 2005. The incidence of apoptosis after IVF with morphologically abnormal spermatozoa. Theriogenology 64: 1404-1421.
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Progress 10/01/03 to 09/30/04
Outputs
Two studies were conducted to evaluate the effect of sperm separation methods for semen samples collected from bulls subjected to scrotal insulation on embryonic development after in vitro fertilization (IVF). Morphologically abnormal semen samples were obtained from Holstein bulls after scrotal insulation was applied for 48 h (d 0). Standard protocols for Percoll gradient separation and the swim-up method were used to separate fractions (A and B). Cleavage rate was significantly different between semen samples and significantly affected by the method of separation. The percentage cleavage rates for the control semen were 74 for swim-up and 55 for Percoll compared to 59 and 26 for swim-up and 44 and 35 for Percoll, for semen from day minus 5 and day 27, respectively. The percentage blastocyst for day minus 5 was 9 for Percoll and 21 for swim-up, while for semen from day 27 there was no blastocyst formation for Percoll separation, with a 4.2 percentage rate for swim-up
separation. In conclusion, our results show that separation methods used were inadequate to provide potentially competent sperm for IVF following thermal insult of the testes. In study two, samples used for IVF were: 1) semen from the test bulls collected 5 days prior to scrotal insulation; 2) semen from day 13 (2 wk-Post-Insult; 2 wk-PI); and 3) semen from day 20 (3 wk-PI). The post-thawed morphological evaluation of sperm samples revealed a decrease in the percentages of normal spermatozoa in the 3 wk-PI samples in comparison with the Pre-insult samples for Bulls I and Bull III (74 to 22.3 and 67.7 to 0.5, respectively). The percentage of vacuolated spermatozoa increased significantly for Bull II. There was no apparent change in abnormal sperm populations for Bull IV. For Bulls I and III (severe responders) the scrotal insulation effects persisted from the time of cleavage through blastocyst formation. In contrast, the percent cleavage and blastocyst formation rates for Bull II
(71.6 and 16.9) and Bull IV (77.8 and 21.4) were unaffected. In conclusion, the use scrotal of insulation to elevate scrotal temperature is an effective method to obtain semen samples with high percentages of abnormal spermatozoa. The decrease in embryonic development after IVF when using spermatozoa with morphological abnormalities seems to be multifaceted and related to the changes in head shape morphology.
Impacts
Current sperm separation methods used for in vitro fertilization were inadequate in their ability to provide potentially competent sperm for IVF following thermal insult of the testes. The use scrotal of insulation to elevate scrotal temperature is an effective method to obtain semen samples with high percentages of abnormal spermatozoa. The decrease in embryonic development after IVF when using spermatozoa with morphological abnormalities seems to be multifaceted and related to the changes in head shape morphology. Our studies will continue to attempt to evaluate where the weak links are.
Publications
- Walters, A.H., Eyestone, W.E., Saacke, R.G., Pearson, R.E., Gwazdauskas, F.C. 2004. Sperm morphology and preparation method affects bovine embryonic development. Andrology 25: 554-563.
- Miller, L. E., Nickols-Richardson, S. M., Ramp, W. K., Gwazdauskas, F.C., Cross, L. H., Herbert, W. G. 2004. Bone mineral density in postmenopausal women. Physician and Sports Med. 32: 18-24.
- Walberg Rankin, J., Goldman, L. P., Gwazdauskas, F. C. 2004. Effect of post-exercise supplement consumption on adaptations to resistance training. J. Amer. College Nutr. 23: 322-330.
- Walters, A. H., Pearson, R. E., Gwazdauskas, F. C. 2004. The effect of different types of morphologically abnormal spermatozoa on bovine embryo development after IVF. J. Dairy Sci. 87: (Suppl. 1) 353.
- Gwazdauskas, F.C., Walters, A.H., Saacke, R.G. 2004. Impact of abnormal spermatozoa used in IVF on early embryonic development in the bovine. Proc. 11th ICBAR. Velke Losiny, Czech Republic. Pp 18-26.
- Walters, A.H., Eyestone, W.E., Saacke, R.G., Pearson, R.E., Gwazdauskas, F.C. 2004. Bovine embryo development after IVF with spermatozoa having abnormal morphology. Theriogenology (accepted).
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